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JOURNAL OF SHELLFISH RESEARCH
VOLUME 22, NUMBER 1
JUNE 2003
^ . .1 Laboraic,
AUG 1 2003
Wootis loie, r/..\ U25.J3
The Journal of Shellfish Research
(formerly Proceedings of the National Shellfisheries Association)
is the official publication of the National Shellfisheries Association
Standish K. Allen. Jr. (2004)
Aquaculture Genetics and Breeding
Technology Center
Virginia Institute of Marine Science
College of William and Mary
P.O. Box 1346
Gloucester Point. Virginia 23062
Shirley Baker (2004)
University of Florida
Department of Fisheries and Aquatic Sciences
7922 NW 71- Street
Gainesville, Florida 32653-3071
Bruce Barber (2005)
School of Marine Science
University of Maine
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Orono. Maine 04469
Brian Beal (2004)
University of Maine
9 0"Brien Avenue
Machias, Maine 04654
Neil Bourne (2003)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9T 6N7
Andrew R. Brand (2003)
University of Liverpool
Port Erin Marine Laboratory
Port Erin, Isle of Man IM9 6JA
United Kingdom
Eugene BuiTcson (2003)
Virginia Institute of Marine Science
P.O. Box 1346
Rt. 1208 Create Road
College of William and Mary
Gloucester Point, Virginia 23062
Editor
Sandra E. Shumway
Department of Marine Sciences
University of Connecticut
Groton. CT 06340
EDITORIAL BOARD
Peter Cook (2004)
Austral Marine Services
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Simon Cragg (2004)
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Leroy Creswell (2003)
University of Florida/Sea Grant
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Lou D'Abranio (2004)
Mississippi State University
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Christopher V. Davis (2004)
Pemaquid Oyster Company. Inc.
P.O. Box 302
1957 Friendship Road
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Ralph Elston (2003)
Aqua Technics/Pacific Shellfish Institute
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Susan E. Ford (2004)
Rutgers University
Haskin Shellfish Research Laboratory
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Raymond Grizzle (2003)
Jackson Estuarine Laboratory
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Karolyn Mueller Hansen (2004)
1524 Barley Circle
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Journal of Shellfish Research
Volume 22, Number 1
ISSN: 0730-8000
June 2003
Mark Luckenbach (2003)
Virginia Institute of Marine Science
Eastern Shore Lab
P.O. Box 350
Wachapreague, Virginia 23480
Bruce MacDonald (2004)
Department of Biology
University of New Brunswick
Saint John, New Brunswick
Canada E2L 4L5
Roger Mann (2004)
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
Islay D. Marsden (2004)
Department of Zoology
Canterbury University
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Jay Parsons (2005)
Memorial University
Marine Institute
Box 4920
St. John's, Newfoundland
Canada AlC 5R3
Tom Soniat (2004)
Biology Department
Nicholls State University
Thibodaux, Louisiana 70310
J. Evan Ward (2004)
Department of Marine Sciences
University of Connecticut
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Groton. Connecticut 06340-6097
Gary Wikfors (2004)
NOAA/NMFS
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Milford. Connecticut 06460
www.shellfish.org/pubs/jsr.htm
./,)/(/■//((/ „f Slwlljlsh Rcst'tinh. Vol. 22. No. 1. 1-20. 2003.
A REVIEW OF PUBLISHED WORK ON CRASSOSTREA ARIAKENSIS
MINGFANG ZHOU AND STANDISH K. ALLEN, JR.*
Aciiuwulture Genelics ami Breeding Technology Center. Virginia Institute of Marine Science. P.O. Box
1346. Gloucester Point. Virginia
INTRODUCTION
NOMENCLATURE
Field research on the Asian (Suminoe) oyster. C. ariakensis.
began in 1998 at the Virginia Institute (if Marine Science (VIMS)
in response to a resolution from the Virginia Legislature to initiate
investigations on alternative species. All field trials have used
sterile triploids. Initial research indicated promising performances
in C. ariakensis in a variety of salinities for growth and disease
resistance (Calvo et al. 2001). Research on this species cxmtinues
at VIMS today, but in the meantime, the Virginia Seafood Council
has run two commercial trials of C. ariakensis on their own v\ ith
similar promising results. They have proposed a third for 2003
with about a million triploid C. ariakensis. The direction taken by
industry clearly indicates a desire to proceed with larger and larger
scale-ups of aquaculture using triploids. This notion was addressed
in a symposium staged m 2001 (Hallerman et al. 2002) where the
general consensus found that "it is difficult to consider the risks of
aquaculture of triploid (infertile) C. ariakensis as separate from the
risks of diploid (fertile) C. ariakensis. That is. there was consensus
that triploid aquaculture would inevitably lead to some introduc-
tion of reproductive individuals in the Bay. with unknown out-
comes for population growth." Part of the difficulty in assessing
the risk of such a scenario comes from the inherent difficulty of
predicting the consequences of an introduction generally. Another
difficulty of assessing risk, especially for C. ariakensis. is the lack
of information on this species.
The aim of this review was to provide an unabridged overview
of the published works on this species. We may have missed some
references that were obscure or indirectly referred to C. ariakensis.
Many of the works on C. ariakensis were in other languages,
principally Chinese. For Chinese articles, they were translated and
are presented in somewhat more detail than those in English. Some
were obtained while traveling to specific laboratories in China and
would otherwise be difficult to obtain. We were as complete as
possible give the timely need for this review.
We present the information uncritically. That is. we present the
contents of the articles without analysis. Partly this is the result of
space constraints. More importantly, it is unclear that data reported
always apply to C. ariakensis. Morphologic confusion is common
with Crassostrea species. For example, a considerable number of
reports of C. ariakensis occur in west India and Pakistan, geo-
graphically isolated from the main populations in Japan. China,
and Korea. It seems unlikely that this is the same species, but to
judge so a priori would be to leave out this information. We e.xpect
scientists to consider the data critically and test it if appropriate.
The information we collected is organized into general catego-
ries so that one work may be cited repeatedly if it crosses catego-
ries. The content in each category in no way implies the impor-
tance of this information, merely what has been done. Conversely,
categories missing information reflect the absence of data.
*Corresponding author. E-mail: ska@vims.edu
Harry ( 1981 ) described the history of the genus name Crassos-
trea Sacco. 1 897 as follows: Over half a century ago Lamy ( 1929-
1930) surveyed the living oysters and put all species in the genus
Ostrea Linnaeus. 1758. including Crassostrea ariakensis. But
since 1930. other authors, chiefly those interested in the commer-
cial production of oysters (e.g.. Thompson 1954). have separated
Cras.wstrea from Ostrea on the basis that the proniyal passage on
the right side of the excurrent mantle chamber is closed in Ostrea
and open in Crassostrea. Other differences on morphology and
anatomy between these two genera can be found in Ahmed (1971
and 1975). Glude( 1971). and Stenzel (1971). In this review, please
note that Ostrea is cited from many old references.
Nomenclature is confusing for C. ariakensis (Carriker &
Gaffney 1996) because the traditional oyster classification meth-
ods rely mainly on conchological characters, i.e.. external and
internal morphology of the shell, which express high phenotypic
plasticity among environments (Hirase 1930). In addition, oyster
eggs are fertilized in mass spawns that increase the possibility of
hybridization and promote high variation (Guan & Li 1986).
Therefore, species with the same name might be genetically dis-
tinct whereas the ones with different scientific names might be
genetically the same. Species variously called C. rivularis, dis-
coidea. palmipes. or paiiliiceiae in previous literature (Carriker &
Gaffney 1996) might be the same as the species we call C. ariak-
ensis today. In general, it is accepted that rivularis is synonymous
with ariakensis. although it is still possible that rivularis/ ariak-
ensis was misclassified in certain publications. This review in-
cludes all the available publications with the above mentioned
species names.
The authorship of ariakensis has been credited to Fujita (1913).
However, we are confused by the description of Wakiya (1930) on
the origin of the name ariakensis. He wrote his reference as "O.
ariakensis (Wakiya M. S.) Fujita. ... 1913." Harry ( 1981) assumed
that "Fujita proposed the name in 1913. based on a manuscript of
Wakiya." Coan et al. (1995) seemed to agree by giving the refer-
ence in a way of "Fujita. 1913... e.x Wakiya MS." Who proposed
the name ariakensis first, Fujita or Wakiya? We were not able to
locate Fujita (1913), so we cannot answer that question for sure.
According to our publication collection, the species name aria-
kensis was not referred to as frequently as rivularis before mid
1990s, but it has been widely referred to in recent publications.
The history of species name rivularis can be traced back to
1 861 . when Gould described a new species called Ostrea rivularis,
which in Latin means "oysters in small brooks." His original de-
scription was written in Latin. Translated to English, the shells he
observed were "discoid, oblong, slender; inferior valve thick,
purple, with remotely radiate ribs and fortified small mbes: supe-
rior valve simple, with ramosing less purple veins; cavity mini-
mally deep, ovate; white ash-colored broad margin, weak hinge."
He emphasized "the rays of the little tubes below, and the veins
Zhou and Allen
above, are uniiMially clear, distinctive ciiaracters." The dimension
of the observed shells was "Diam. 60; Lat. 10 millim." It "inhabits
the China Seas, as indicated by shells adhering to it."
There is serious ambiguity in the source of Gould's specimen.
The title of his article indicates that his description was based on
the collection of "the North Pacific Exploring Expedition,"
whereas according to Hirase (1930), it was based on a single
specimen from China in Dunker's collection. Hirase did not ex-
plain whom Dunker is except for a reference listed as Dunker
(1882). Several other authors mentioned China as the source of
Gould's specimen (Ahmed 1971, Galtsoff 1964), but no additional
references were offered for further confirmation. Hirase (1930)
also questioned the completeness of Gould's description and its
value for identification because it seems based on a single speci-
men, which seems to be comparatively young according to its size
(60 mm). Gould's description of rividaris and those of others (see
Morphology section) are incompatible. Thus, it is quite possible
that nvitUms of Gould ( 1861 ) is different from the species we call
rividaris or ariakensis today.
O. (C ) rividaris Gould has been widely applied to oysters with
similar conchological characters in many Pacific coastal countries,
such as Japan. China, Pakistan, and India. Its taxonomic status in
each country is still muddled. A review is summarized below.
In Japan, Ariake-gaki, Suminoe-gaki, and Kaki ("gaki" in Japa-
nese means oyster) were some common names for O. rivtilaris
(Amemiya 1928). This species was once classified as O. gii^ax by
Fujimori (1929) but this was refuted by Taki ( 1933) and Imai and
Hatanaka (1949). Wakiya (1930) surmised that O. rividaris of his
in 1915 (Wakiya 1915) and that of Amemiya (1928) was the same
as O. ariakensis. whereas the O. rivularis described by Lischke
(1871) seemed to be the young of O. ariakensis.
In Pakistan, Awati and Rai ( 1931 ) indicated two names lor the
same species, O. discoidea and O. rividaris. Reeve (1871) de-
scribed O. discoidea based on specimens from Fuji Island and New
Zealand, but Ahmed (1971) stated that the figure and the shell
characters published by Reeve were different from that of O. dis-
coidea. According to Ahmed, Reeve's O. discoidea is rounded and
flat to the extent that it looks like the windowpane oyster, Placuna
placenta Linne, 1758, which is abundant in lagoons of Philippines
and South East Asia (Abbott & Dance 1986). Based on his own
experience, Ahmed believed that O. discoidea is not distinguish-
able from C. rividaris.
In China, the common name for O. (C.I rivularis is Jinjiang-
muli ("jinjiang" in Chinese means "close to river" and "muli"
means oyster). One of the long-standing debates on oyster classi-
fication involves two morphologically very similar variants that
occur in the Peari (Zhujiang) River estuary. One is called "white
meat" oyster and the other is "red meat." Very experienced oyster
farmers can separate these two variants by external appearance and
the color of the soft body. Fei ( 1928) believed that both are O.
gigas. However, Zhang and Lou (1956a) identified "white meat"
as O. rivularis and "red meat" as a variant. The "white meat"
oyster is considered better than the "red meat" because of meat
quality and productivity in aquaculture, thus has higher commer-
cial value. The "red meat" oyster is apparently more resistant to
harsh conditions according to observations of it in culture (Guan &
Li 1986). Further investigations by other researchers revealed
other differences. A comparative study on the physiologic and
biochemical indexes (Guan & Li 1986), such as oxygen consump-
tion rate, fatty acid composition, and amino acid composition,
demonstrated sufficient differences in physiology to suspect that
genetic differences are likely. Anatomically, Li (1989) found a
difference in the connection of the body with the gills. In "white
meat" both the left and right epibranchial chamber connect with
the promyal chamber, whereas in "red meat" only the right epi-
branchial chamber connects with the promyal chamber. He be-
lieved the two belong to two different species. A study on genetic
variation using starch gel electrophoresis (Li et al. 1988) demon-
strated that they should belong to different species because their
genetic identity was low (I = 0.548). The estimated divergence
time of the two is 3 x 10" years. The comparison of genetic
similarities and genetic distances suggests that "white meat" is C.
rivularis and "red meat" is probably C. iredalei. Guan and Zheng
( 1990) studied the esterase isoenzyme of the two groups by poly-
propylene amide gel electrophoresis and agreed that they are dif-
ferent species. Above all, it was generally agreed that "white meat"
is C. rivularis. but whether "red meat" is C. iredalei is still un-
confirmed.
MORPHOLOGY
Conchological Characters
References on conchological characters of naturally occurring
C. ariakensis come from three countries: China, Japan, and India.
References from the United States (Pacific Northwest) are also
included because the seed were introduced from Japan. Reports
containing conchological data are listed individually following a
general review to compare and contrast characters of what are
called O. (C.) rivularis, now C. ariakensis. The major conchologi-
cal characters presented in these reports are size; thickness and
shape of the valves; outer structure of the valves; comparison
between the left and right valve; color of outer and inner surface;
size and color of ligament; color, size and position of the muscle
scar; and hinge structure (Table I ).
Review
In China, it is commonly observed that valves of Ostrea (Cras-
saslrea) rivularis are large and thick with varying shapes, basically
round but sometimes elongated into oval, oblong, and even trian-
gular shapes. The right valve is thinner, flatter, and smaller than
the left. Both valves are covered with concentric lamellae (fluted
shell margins on the external shell), with fewer layers of but
stronger, lamellae on the left valve. Density and shape of lamellae
varies by age class, which are thicker and more layered in older
oysters (Zhang & Lou 1956a, Zhang et al. 1960). Color of lamellae
or the outer surface of valves ranged from gray, yellowish brown,
brown, to purple or dark purple. Dark purple coloration is apparent
in C. ariakensis grown in high-salinity areas of Chesapeake Bay
(Zhou & Allen, unpubl.). The inner surface of valves is white or
grayish white, purple on the edge. The ligament area is short and
wide, and the ligament is usually purple black. The muscle scar is
very large, mostly oval or kidney shaped, located in the mid-
dorsal area, purple or light yellow in color.
The coloration of valves and muscle scars of C. ariakensis
described in reports from Japan is different from those from China.
In Japan, the outer surface of the valves was described as cream-
buff or white, streaked with radial chocolate bands, violet bands, or
almost uniformly violet (Hirase 1930, Torigoe, 1981, Wakiya
1929). The inner surface of the valves was strongly lustrous or
partly opalescent (Hirase, 1930, Torigoe 1981). The muscle scar
was usually white or sometimes stained with olive-ocher spots or
CRASSOSTREA ARIAKENSIS REVIEW
TABLE 1.
Characteristics of oysters by citation.
Gould (1861). China. O. rmilahx
Valve shape size: Discoid, ohlong. slender.
Left, righl xalve: Inferior valve thick, purple, with remotely radiating ribs and fortified small lubes; superior valve simple, with ramosing less purple veins;
cavity minimally deep, ovate.
Shell color outer; Purple; white ash-colored broad margin.
Shell color inner; —
Ligament: —
Muscle scar: —
Hinge; Weak.
Zhang and Lou (I'J.Wl. China, O. (C.) nviilaris. includes figurelsl
Valve shape size: Large and thick with various shapes, round, oval, triangle, and oblong; concentric scarce lamellae on outer surface.
Left, right valve: —
Shell color outer; Yellowish brown.
Shell color inner; —
Ligament: —
Muscle scar; —
Hinge: —
Zhang and Lou I I956al. China. O. iC.I rivularis. includes l'igure(s)
Zhang et al. {I960). South China. O. rivuluris. includes figurets)
Similar descriptions from the above two references are combined as Ibllows.
Valve shape size: Valves large and thick with various shapes, round, oval, triangle, and oblong.
Left, right valve; Right valve flatter and smaller than the left one, with yellowish brown or dark purple concentric lamellae on its surface. In 1 to 2-y-old
individuals, lamellae thin, flat, and brittle, sometimes dissociated; on valves older than 2 ys old, flat but sometimes with tiny wavy
shape at the edge; on valves several years old. thickly layered, strong as stone. Left valve is larger and thicker than right valve,
stronger but fewer layers of lamellae. A few samples had inconspicuous radiating ribs or plication.
Shell color outer: Gray, purple, or brown.
Shell color inner; White, grayish purple on the edge.
Ligament: Ligament purple black. Ligament groove shon and wide, like an o,\ horn. The length from the ligament to anterior is one sixth to one
fourth of shell height.
Muscle scar; Muscle scar very large, light yellow, irregular shape, mostly oval or kidney shaped, located in the middle of the dorsal area.
Hinge; —
Cai et al. (1979), China, O. rivularis. includes figure(s)
Valve shape size; Shells large and thick with various shapes, such as round, oval, triangle and oblong.
Left, nght valve; Right shell (latter and smaller than the left shell, with yellowish brown or dark purple lamellae on its surface. The lamellae are thin and
flat, with not much layers and no radiating ribs, but usually with protuberance. The left shell is larger and thicker with irregular shape
and similar lamellae as the right shell.
Shell color outer; Yellowish brown or dark purple.
Shell color inner; White or grayish white.
Ligament: Ligament purple black, ligament groove short and wide.
Muscle scar; Muscle scar large, oval or kidney shaped, located in the middle of the dorsal area.
Hinge: No denticulate on the hinge.
Li and Qi 1 I994i. China. C rivularis. includes figurelsl
Valve shape size; Large variation in shell shape, usually oval or oblong.
Left, right valve; Concentric lamellae tend to coalesce, no radiant ribs.
Shell color outer; Light purple.
Shell color inner: White.
Ligament; Wide ligament groove.
Muscle scar: Light purple.
Hinge; —
Amemiya ( 1928). Japan. O. rivularis. includes figurets)
Valve shape size; It is either circular or oval in form, pronounced elongation as found in O. gigas is absent.
Left, right valve; —
Shell color outer: —
Shell color inner: —
Ligament: —
Muscle scar: —
Hinge: —
Cahn (1950), Japan. O. rivularis. includes riguretsi
Valve shape size: Round, Hat. smooth surfaced, plates thin, almost smooth, shell thick.
Left, right valve; —
Shell color outer; Pale pink, radiating burnt lake strikes on shells.
Shell color inner: —
Ligament: —
Muscle scar; —
Hinge; —
Hirase (I9.WI. Japan. O. (C.) rivularis. includes rigure(s)
Valve shape size: Orbicular, oval, elongated oval, though appearing somewhat subtriangular because of its rather long umbo. There are many intermediate
forms, but on the whole the specimens are oval. The shell is fairly strong and thick, though not to the extent of C. gigas.
continued on next page
Zhou and Allen
TABLE 1.
continued
Shell color outer:
Shell color inner:
Ligament:
Muscle scar:
Left, right vahe: The right valve is somewhat smaller. The conca\il> ot the Icit \alve is larger. The amerior depression of the left valve is very obscure. The
lamellae of the right valve are somewhat thin and almost smooth, and distinct placations are not apparent, but sometimes the lamellae
are covered with somewhat irregularly tubular projections. It is noteworthy that smooth lamellae are more common in the young than in
the adult. The color is cream-buff with many radial chocolate bands, but in adults these bands are fused into larger ones; their
arrangement differs in each individual. In the left valve, the lamellae are generally indistinct, and may be close together or separate.
The common color is pale rhodonite pink with radiating "burnt lake" striae.
The inner shell surface is generally white with strong luster, sometimes with a yellowish central part.
The ligament is "burnt lake" or black.
The muscular impressions, elongated oblong with concave anterior side, are equal in size for the two valves and rather large in porportion
to the inner shell area. The color of the impression is while, or rarely marked with olive-ocher spots; its surface is almost Mat.
Hmge:
Imai (1978), Japan. C. rivularis, includes figure(s)
Valve shape size: Round or elliptical
Left, right valve: The lower shell is shallow and the umbo cavity below the hinge plate is very small.
Shell color outer: The part near the hinge plate m the upper shell is violet-brown in color.
Shell color inner: —
Ligament: —
Muscle scar: —
Hinge: —
Kira (1962). Japan, C rivularis
Valve shape size: Has a large and rather flat shell, oi v\hich the surface bears very coarse and widely spaced concentric lamellae.
Left, right valve: —
Shell color outer: —
Shell color inner: —
Ligament: —
Muscle scar: —
Hinge: —
Torigoc (1981), Japan, C ariakensis, includes Figure(s)
Large sized (height 200 mm x length 1 12 mm, Hirase 1930). Outline orbicular to long spatulate form, mostly tongue form, subequivalves.
Attachment area is small to medium, commonly behind the umbonal area.
Both valves flat, but left valve weakly concave. Both valves have very faint dichotomous radial ribs, left valve more conspicuous than right
valve. Growth squamae flat and stretched parallel to the grow lines. No commissural plication, or very weak even if present.
Commissural shelf small to medium. Umbonal cavity shallow. No chomata. The dorso-ventral section has chalky deposits between soHd
shell layers and no hollow chambers. Both valves are thinner than those of C. gigas. so chalky layers are very thin. The parts of chalky
deposits are often intruded by worms.
"White in ground" (sic) color with pale purple streaks radiating from umbo.
Chalky white or partly opalescent.
Valve shape size:
Left, right valve:
Shell color outer;
Shell color inner:
Ligament:
Muscle scar:
Reniform. dorse -an ten or border concaved and close to ventro-posterior shell margin from the center ot the valve. Lustrous while or
sometimes with purple patches, particularly on nght valve.
Hinge; —
Wakiya (1929). Japan, Osirea ariakensis
Valve shape size;
Shell usually circular or oval in shape. However, its shape varies considerably according to the hardness of the bottom on which it lives.
When found imbedded in soft mud it has an extremely elongated shell so that it is very difficult to distinguish it from that of O.
Inperousi found on a mud bottom of lower salinity, only differing from O. kiperoiisi in having the hinge of lower valve not very long
and subequal to that of the upper one. O. rivularis Gould has. according to the original description, its lower valve provided with
radiating, tube-shaped ribs set distantly. Therefore the species in which the ribs are absent from the lower valve or only very weakly
developed, if present at all. cannot be the species of Gould.
Lamellae imbricated rather compactly, lower valve concave, not provided with ribs; upper valve flat, length of hinge nearly equal to that ot
lower valve. Occasionally, weakly developed ribs are observed on the lower valve of the young of the species, but never on full-grown
ones.
Whitish and streaked with violet, or almost uniformly violet.
Lead white; muscular impression faint, usually not specially colored but sometimes stained purple.
The hinge of the lower valve not so long as. as long as or a little longer than the breadth; no umbonal cavity below margin of hinge.
USA. C. ariakensis. includes fi2ure(s)
Left, right valve:
Shell color outer:
Shell color inner:
Ligament;
Muscle scar:
Hinge:
Coan et al. (1995
Valve shape size; Subtrigonal. flared ventrally, heavier and more rounded than C. gigas.
Left, right valve; Left valve moderately concave, with white to pale pink lamellae; right valve moderately flattend, with many thin commarginal lamellae,
sometimes with dark brown to purple radial color bands. Both valves with densely layered, thin lamellae.
Shell color outer; —
Shell color inner: —
Ligament; —
Muscle scar: White to purple to olive.
Hinge; —
Galtsotf (1964). USA, C. rivularis, includes figurels)
Valve shape size: Orbicular strong and large.
Left, right valve: Left, lower valve slightly concave, upper valve shorter and flat. The left valve has generally indistinct lamellae of pale pink color with
radiating striae. The lamellae of the right valve are thin and most smooth, sometimes covered with tubular projections.
continued on next page
Crassostrea ariakensis Review
TABLK 1.
continued
The color ol the right \alve is LTcaiii hiilt wilh nuiny radial chocolate bands, their arrangements greatly variable.
Situated near the eenler or a little dorsally. is while, occasionally with olive-ochre spots.
Shell color outer;
Shell color inner:
Ligament:
Muscle scar:
Hinge: —
Langdon and Robinson ( 1*^%), USA. C. ariakensis. includes figure{s)
Valve shape size: This species differs from the Pacific oyster morphologically in that the shell is typically more rounded and the edges of shell layers are llal
and no! rippled like those of Pacific oysters (Torigo. 1981 i
Left, right valve: —
Shell color outer: —
Shell color inner: —
Ligameni; —
Muscle scar: —
Hinge: —
Awali and Rai (1931). India. O. discnidea or O. rivularis
Valve shape size:
Left, right valve:
Shell color outer:
Shell color inner:
Ligament:
Muscle scar:
Hinee:
Shell flat and of large size, rounded, foliaceous with conspicuous lines of growth.
Lower valve lightly concave, upper valve of the same size and shape as the lower, slightly convex.
Clear and nacreous.
Ligament area small.
Oblong with a cloudy white or smoky white color.
No denticulations.
Rao (1987). India. C. rivularis. includes figure(s)
Valve shape size:
Left, right valve:
Shell color outer:
Shallow shell cavity
Imai (1978) has slated that the hinge part of the shell of C nvtilaris is violet brown in color. The coloration may be caused by ecological
conditions such as luxuriant growth of seaweeds in the vicinity or other factors and should not be considered of taxonomtc importance.
Shell color inner: —
Ligament: —
Muscle scar: Oblong white.
Hinge: —
Palel and Jetani (1991), India. C. rivularis
Valve shape size:
Left, right valve:
Shell color outer:
Shell color inner:
Ligament:
Muscle scar:
Hinee:
Shell oval, narrow at anterior end and broader with posterior end.
Left valve has deep radial ndges from the hinge and tightly inter locked with upper right valve.
Pink to brownish with tints.
Having narrow hinge-ligament
White.
Having narrow hinge-tigament.
purple patches (Hirase 1930. Torigoe 1981, Wakiya 1929). Rao
(1987) thought the difference in coloration might be caused by
ecological conditions and therefore not considered a character of
taxonomic importance. Reports from the United States are consistent
with reports from Japan for coloration, which indicates that at least
some part of coloration might be caused by genetic factors. O. (C.)
rivularis from India are similarly described. Coloration of the inner
surface of the vahes and the muscle scar are close to Japanese reports.
Reports from Japan were often comparative between C. ariak-
ensis and other species, such as O. (C.) gigas (Amemiya 1928. Hirase
1930. Torigoe. 1981) and O. lopenmsi (Wakiya 1929). O. (C.) gigas
were believed to have stronger, thicker, and more elongated shells
than O. (C.) rivularis. whereas O. rivularis is very difficult to distin-
guish from O. lapennisi foLind on muddy bottom in lower salinity. O.
rivularis differs from O. lapcmusi by having the hinge of the lower
valve not very long and subequal to that of the upper one. Japanese
reports agree that O. IC.) ariakensis has flat valves, with the left one
weakly concave (Cahn 1930. Kira 1962. Torigoe 1981). Wakiya
( 1929) thought the various shapes of O. ariakensis were influenced by
the hardness of the bottom because the ones with extremely elongated
shells were found imbedded in soft mud. This is also a character of
other Crassostrea spp. (Galstoff 1964).
The most confusing character through this review has been
what Gould (1861). who first named O. rivularis. described as
remotely radiating ribs and fortified small tubes on the outer sur-
face of left valve and veins on right valve. He emphasized that
these are usually clear, distinctive characters of this species. His
observation was based on a sample from China. However, no
reports from China agreed with his description of such characters.
Cai et al. ( 1979) and Li and Qi (1994) observed no radiating ribs
in this species. Based on a large-scale investigation of oyster spe-
cies all along the Chinese coast. Zhang and Lou ( 1956a) described
inconspicuous radiating ribs or plication in a few samples of O.
(C.) rivularis. Only one report from India described deep radial
ridges from the hinge on the left valve (Patel & Jetani 1991).
although the origin of the background specimen was unknown.
From Japan, similar characteristics were described as indistinctive
or occurring at very low frequency. Hirase (1930) and Galstoff
(1964) mention that the lamellae are sometimes covered with tu-
bular projections. Hirase (1930) and Cahn (1950) mentioned "ra-
diating burnt lake strikes," which might or might not be the same
feature we are discussing here. Torigoe's ( 1981 ) report said "both
valves have very faint dichotomous radial ribs, left valve more
conspicuous than right valve." Wakiya ( 1 929) is more helpful in
clarifying this confusion. He stated this species was "not provided
with ribs... occasionally, weakly developed ribs are obser\'ed on
the lower valve of young of the species (Ostrea ariakensis). but
never on full-grown ones." Either Gould's original descriptions
6 Zhou and Allen
were inappropriate for adult C. ariakensis. or he described a dif- GEOGRAPHIC DISTRIBUTION
ferent species ( Wakiya 1929). The latter possibility is quite high if
Gould did get his specimen from China because there are around ^''""^ ^" overview of the literature. C. ariakensi.s seems to have
20 oyster species there (Zhang & Lou 1956b. Cai & Li 1990, Li & ^ ^''^^ geographical range. According to Kuroda and Habe 1 1952).
Qi 1994. Guo et al. 1999). and classification based completely on ^^ '■""/<"■" encompassed latitudes 12-34'N. which covers the
morphologic characters is questionable. ^'"'^^ *ro'" southern Japan to southern India. Ranson (1967) listed
sources of C. ahakensis specimens in museums around the world.
ANATOMIC CHARACTERS
coming from Southern Japan to coasts bordering the South China
Sea. including Hong Kong. Vietnam, and Sabah (formerly North
Borneo), Malaysia. Several authors (Wakiya 1929, Cahn 1950,
Review Kira 1962, Coan et al. 1995) mentioned its distribution in Korea.
Anon (1996) mentioned that C. rivutaris was also found from the
Anatomic characters were not studied as broadly and com- Philippines and Taiwan to Thailand. Above all, this species seems
pletely as conchological ones. Reports mainly come froin Japan to occur all along the west coast of the Pacific Ocean, from south-
and China. Researchers had different emphases in their anatomic em Japan to Pakistan (Angell 1986). Sparks ( 1965) even reported
studies. The only character described by more than one researcher that C. rivtilaris was indigenous to Kenya. However, for most
is the mantle. Hirase (1930). Zhang et al. (1960), and Galtsoff areas outside of Japan and China, no references are available to
( 1964) were in agreement that the inner row of the mantle tentacles confinn these observations genetically as C. aiiakensis.
is aligned while the outer row is iiregular. Details of anatomic Quite a few literature reports are available listing specific lo-
characters are given in Table 2. cations in a country where this species occurs naturally. Below we
TABLE 2.
Anatomical characteristics of oysters by citation.
Hirase (1930). Japan, O. (C.) hvulahs
Mantle — In a specimen whose length and altitude are 96 mm and 45 mm. respectively, the mantle is united by the anterior 21 mm. or 0.22 of the
body length. There is no siphon. The mantle margin is dark nigrosine violet or pinkish vinaceous, and the tentacles are arranged in two rows,
the outer consisting of tentacles of irregular size and the inner of slender single tentacles. Fine tendons radiate from the posterior sides of the
adductor muscle as usiLal.
Adductor muscle — The adductor muscle measures 20 mm m altitude and 22 mm in breadth and is suborhicular. with somewhat concave anterior
face and convex posterior face. The distance between the anterior end of the adductor muscle and the anterior end of the body is 52 mm. A
small portion of the posterior part of the adductor muscle is white as usual.
Heiin — The pericardium, continguous to the anterior face of the adductor muscle, is oval and measures 19 mm in altitude and ti m in breadth. The
heart runs obliquely from the antero-dorsal corner of the pericardium to the postero-ventral corner. The ventricle and the auricles are both tlesh
color. The ventricle measures 8 mm m altitude and 6 mm in breadth, while one of the auricles measures 8 mm in altitude and 3 mm in breadth.
Ctenidium — The posterior end of the ctendium curls up along the posterior face of the adductor muscle.
Alimentary system — The palps are as usually found in Crassostrea. The rectum begins at the dorsal region of the pericardium and ends just above
the posterior end of the adductor muscle. About 3 mm of the terminal portion is free, differing from other oysters of this subgenus and shorter
than in Neopycnodonte cochlear, whose free portion is 5 mm. The anal end has a ring. The distance between the mouth and the anus is 55 mm,
its ratio to body length being 0.57.
Imai (1978). Japan. C. rivularis
C. ariakensis differs from C. gigas in that a part of the rectum and anus are away for the soft parts.
Torigoc (1981), Japan, Crassostrea ariakensis
Soft parts are similar to C. gigas but the coloration of soft parts is the palest of Japanese Crassostrea species.
Zhang et al. (1960), South China, 0. rivularis
Mantle — The inner row of the mantle tentacles is aligned while the outer row is irregular.
Heart — Heart chamber is flesh pink.
Li (1989), China. C rivularis
Promyal chamber — The left and right cpibranchial chambers connect with the promyal chamber all together. In the cross section of this type, the
ascending lamellae of the left and right outer demibranch attach to the mantel, whereas the other part of gills are free in the mantel cavity. The
whole epibranchial chamber is connected with the promyal chamber. On the lateral view from the right side of the oyster, the joint of the two
gills attaches to the visceral mass at and below the adductor muscle, while above the adductor muscle, the gills are dissociated so that the two
rows of water tubes on the left as well as the two rows on the right of oyster body can be seen. The "white meat" Jinjiang oyster from
Shenzhen Bay belongs to this group.
Nelson (1938) stated that oysters with a promyal chamber are adapted to low salinity and highly turbid waters, while oysters without it do
better in high salinity, less turbid waters. Thomson (1954) had similar reports. The occurrence of the promyal construction in commonly
cultured oyster species in China and their distribution are consistent with Nelson's statement. Oysters with the chamber inhabit mostly estuary
and intertidal zones, where salinity and transparency are both low and the environmental factors tluctuate. The ones without the chamber inhabit
mosdy shallow seas with higher salinity and relatively stable environments. It is likely that the promyal chamber is an adaptation stemming
from oysters moving into increasingly estuarine habitats.
Galsoff (19641. USA. C rivularis
Mantle — Margin of the mantle is dark \ uilcl; the tentacles are arranged in two rows; those of the outer row are of irregular size; the inner
tentacles in a single row are slender.
Crassostrea ariakens/s Review
summarize this intormation by country, Irom iiortii to south alony
the Pacific west coast.
Japiiii
Kira (1962) reported distribution of C. riviilans roughly from
central Honshu to Kyushu (Fig. 1). Honshu is the largest island of
Japan located in the center of the archipeligo. Kyushu is southern
most. Cahn (1950) reported the restricted range of its distribution
as western Kyushu, mainly in Ariake-kai C'kai" in Japanese means
sea) and Yatsuchiro-wan ("wan" means bay). It is most abundant
in the inner parts of Ariake-kai. the southern coast of Fukuoka and
Saga prefecture. Hedgecock et al. (1999) found a similar distribu-
Honshu Islantd
Pacific Ocean
'Kyushu Island
East China Sea
^
f
Figuri' 1. Locations reported v\ith C. ariakensis popiilutions in .lapan. 1. Ariake-kai; 2. \atsuchiro-\\an; .^. Fukuoka prefecture: 4. Saga
prefecture; 5. Shiranuhi Bay: 6. Kochi prefecture: 7. \ amaguchi prefecture; and 8. Okayama prefecture.
Zhou and Allen
tion in the Ariake Bay. Ariake-kai or commonly called Ariake
Bay, seems to be the most recognized natural habitat and the
namesake of C. ciriakcnsis. as it was mentioned most frequently
(Wakiya 1929. Hirase 1930. Cahn 1950, Galtsoff 1964. Imai 1978.
Hedgecock et al. 1999). In addition. Wakiya (1929) mentioned
Shiranuhi Bay on the northeastern coast of Kyushu, and Cahn
( 1950) listed the Pacific coast of Kochi. the coast of Yamaguchi
and Okayama prefecture.
China
China has an extensive coastline of about 18.000 km extending
from the cold temperate north to the tropical south. Based on an
extensive investigation on oyster species along the Chinese coast
in 1956, O. (C.) liviilaris was identified in each coastal province
(Zhang & Lou 1959: Fig. 2). As Zhang et al. (1960) later stated,
the distribution of this species covers the whole coastal region of
China, with a latitudinal range of 15-40°N and a longitudinal
range of 107-1 24'E. Table 3 lists the names of locations where
O. (C.) vivulaiis has been reported. The locations underlined were
considered by Zhang and Lou (1956b) as major production
areas, which might not be true today. Among those. Xiaoqing
River estuary in Yangjiaogou, Shandong province was specifi-
cally mentioned because a very large population of O. rividaris
was found there. In certain localities, the population was so large
that people call them "oyster hills" because individual oysters
grew attaching to each other (Zhang & Lou 1956b, Zhang et al.
1960). It would be interesting to try to determine whether natural
populations are still available in some locations, having possibly
been shielded from exploitation because of their rarity (Table 3).
India
Although Ahmed (1971) mentioned that C. riridaris was dis-
tributed on both east and west coasts of the Indo-Pakistan subcon-
tinent, other reports maintained that this species was found only on
the west coast of India (Fig. 3). It was first reported along the coast
of Bombay (Awati & Rai 1931). Durve (1986) gave a much wider
range between Ratnagiri and Okha along the coast of Gujarat and
Maharashtra area. Gujarat (Saurashtra) has a long coastline of
1500 km (Patel & Jetani 1991). Specific locations in this range
were described by Mahadevan (1987) as Aramra, Poshetra, Port
Okha, Porbandar, Sikka. Gagwa Creek. Singach Creek. Beet Kada.
Khanara Creek. Laku Point. Gomati Creek (Dwarka), Harsad.
Navibander (Madha Creek). Balapur. and Azad Island. In addition.
Rao (1987) mentioned creeks of Kutch and Aramda Creek in Gu-
jarat and Mahim, Ratnagiri and Jaytapur in Maharshtra. Durve
(1986) also mentioned some trawling areas around Bahrain in the
Arabian Gulf.
Pakistan
This species was found abundant on the coast of West Pakistan
(Ahmed 1971; Fig. 3). The following locations have been men-
tioned in the literature: the coast of Sind (Ahmed 1971 ). Korangi
Creek (18 miles south of Karachi) and Sonari (40 miles west of
Karachi: Asif 1978b). Sandspit backwaters (Qasim et al. 1985.
Barkati & Khan 1987. Aftab 1988), and Port Qasim (Gharo-Phitti
saltwater creek system near Karachi; Ahmed et al. 1987. Barkati &
Khan 1987).
ECOLOGY
Habitat
Below we summarize reports on the nature of the habitat de-
scribed for C. ariakeii\is and the vertical and horizontal ranges of
its distribution.
In Japan, O. riviilaris was only reported from muddy beds
(Ameiniya 1928, Wakiya 1929, Hirase 1930). It generally adheres
to other objects by the umbonal part of the left valve, but many
specimens appear to have lived separately (Hirase 1930), Its ver-
tical range is just above the low tide mark and closely restricted to
the vicinity of the low tide line (Amemiya 1928, Wakiya 1929). Its
horizontal range was determined by water temperature and salinity
(Imai 1978). The salinity range of its natural habitat under ordinary
conditions is 9-30 ppt (Amemiya 1928, Cahn 1950), the lower
range of which is lower than many Crassostrea species. As
Amemiya ( 1928) explained, these conditions are apt to change for
one reason or another. For instance, during ebb tide the exposure
of the beds to the air and sun inevitably inake the surrounding
water more saline due to evaporation. But because this species
lives close to the low tide mark, exposure to high salinities is short.
C. ariakensis can apparently tolerate low salinities as well. O.
rividaris was found in places where the salinity falls occasionally
much below 10 ppt, sometimes even in entirely fresh water
(Amemiya 1928).
In China, this species occurs widely among the river estuaries
along the coast. It is found from the low tide line to 7-10 m below
mean low water (Zhang & Lou 1956b. Zhang et al. 1960, Cai
1966, Cai et al. 1979. Xu et al. 1992). Sometimes it could be found
around the high water mark (Zhang et al. 1960). According to Lu
(1994). the temperature range of C. rividaris is 2-35°C. Normal
salinity range was reported as around 10-30 ppt (Zhang & Xie
1960. Lu 1994) or 9-28 ppt (Zhang & Lou 1956b). Optimum
salinity was reported as 10-25 ppt (Zhang el al. 1960) or 10-28 ppt
(Nie 1991 ). It was observed that C. rividaris could tolerate salinity
as low as 1-2 ppt for a short tenn (Zhang et al. 1960, Zhang & Xie
1960). as Nie (1991) reported its salinity range 1-32 ppt. Pure
fresh water could cause mortality (Zhang et al. I960). An inter-
esting exception to the normal distribution of C. ariakensis was
reported by Chen (1991) for Northern Jiangsu. The silty coast of
Jiangsu province was not originally suitable for O. rividaris. Ac-
tually, few oysters were found in this province. Things changed
when Spanina anglica was introduced. It was planted discontinu-
ously along the coast of Jiangsu province, and by 1991, it occupied
377 km of coastline and 1 80 km^ coastal area of the province. This
plantation changed the local ecology. Chen reported that this plant
kept clay around its growing area and gradually formed small
ridges and backwaters in that area, which he believed was a critical
condition for these oysters. O. rividaris was found at the seaward
boundary of the S. anglica planting area, which was between high
and middle tide mark with one-third to one-half time exposure.
The density of its distribution was as high as 107 per m"^ and the
average shell height of adult O. rivularis was 19.5 cm.
In India, C. rividaris was found on both hard grounds and in
muddy creeks (Mahadevan 1987. Patel & Jetani 1991). Patel and
Jetani (1991) reported its preference of muddy rocks, rocks cov-
ered by 3—4 inches of mud, although we have to think that settle-
ment preceded the mud deposits. This oyster has been found in
groups of four to five large and small individuals attached to
isolated rocks and coral stones that came up in trawl-nets (Durve
Cf<ASSOSTIit:A AR/AKENSIS REVIEW
Figure 2. Locations reported «ith C. ariakensis populations in China. No distinction is made between aquaculture sites and natural populations.
Underlined sites are considered major production areas. I. Xindao (dao: island): 2. Andon); (Dadonggoul; 3. Zhuanghe: 4. Gaipin)>: 5. Fengnan;
6. Ninghe; 7. Beitang; 8. Tanggukou; 9. Yangjiaogou: l(). Ycxian; II. ^antai; 12. Rongchen; 1.1. Dingzigang: 14. Shijiusuo: 15. Sheyang; 16.
Jianggang Bay; 17. Rudong; 18. Huijiao: 19. Daishan: 2(1. Zhenhai: 21. Dinghai; 22. Meilin: 23. Sannien: 24. Wenling; 25. I.ei|ing Bay: 26.
Wenzhou Bay: 27. Xiapu: 28. Ningde: 29. Luoyuan Bay: 3(1. Huian: 31. Tongan: 32. Xiamen: 33. I.onghai: 34. Haiclieng; 35. ^unxiao: 36.
.Shantou: 37. Haimen: 38. Lanbiao. Huilai County: 39. ,)iazi: 4(1. .lieshi: 41. (iaoluo: 42. .Shanwei : 43. Qingcao: 44. Baoan: 45. \iangzhou: 46.
Tangjiahuan: 47. Nanshui: 48. Hengshan: 49. Zhanjiang Bay: 50. Qinzhou\van(Longnien); 51. Baoping Bay: 52. Boao: 53. Qinglangang; 54.
Qiongshan: 55. Lofu Shan: and 56. Deep Bay.
1986) or solitary (unattached) in the littoral zone (Awati & Rai
1931). The vertical range of C rivularis was described as the
littoral zone (Awati & Rai 1931 ). sublittoral low waterline area or
submerged offshore area (Durve I9K6). intertidal (Mahadevan
1987. Rao 1987) or tidal region (Patel & Jetani 1991 ) and also at
9-15 m depth (Durve 1986).
In Pakistan, the preferred habitats of C. rivularis are the back-
waters and creeks along the coast (Moazzam & Rizvi 1983). It
seems that this species thrives in muddy environments (Ahmed
1971, Asif 1978b, Ahmed et al. 1987) and adheres to hard sub-
strate such as stones (Ahmed et al. 1987). It occurs near the low
water mark (Ahmed 1971, 1975, Ahmed et al. 1987, Barkati &
Khan 1987) and the preferred tidal height for spat settlemeni is 0.5
ft mark (Ahmed et al. 1987).
Predators, Harmful Organisms, and Diseases
According to Zhang and Lou (1956b). in China, "led tide" is
generally most hai-mful to oysters. It caused 509r mortality of
10
Zhou and Allen
TABLE 3.
Locations where C. (O.) riviilaris was reported in China.
Province
Locations Where C. (O.) riviilaris was reported
Liaoning Gaiping. Andong (Dadonggou). Xindao. Zhuanghe
( Zhang & Lou 1959)
Hebei Fengnan. Tanggukou. Beitaiig (Zhang & Lou 1959)
Tianjin City Ninghe (Zhao et aL 1991)
Shandong Rongchen (Zhang & Lou 1956b)
Yangjiaogou. Dingzigang (Zhang & Lou I956h. 1959)
Shijiusuo (Zhang & Lou 1959)
Yantai. Yexian (Zhao et al. 1991 )
Jiangsu Sheyang, Rudong (Zhang & Lou 1959)
Northern coast (north of Jianggang Bay; Chen 1991 )
Zhejiang Sanmen (Zhang & Lou 1956b)
Zhenhai, Daishan, Huijiano. Dinghai, Meilin, Wenling
(Zhang & Lou 1959)
Wenzhou Bay (Huang et al. 1981)
Leqing Bay (Zhou et al. 19821
Fujian Xiamen (Zhang & Lou 1956b. 1959)
Tongan. Haieheng (Zhang & Lou 1959)
Luoyuan Bay (.Xu el al. 1992)
Yunxiao. Longhai. Huian. Ningde, Xiapu (Cai 1966)
Guangdong Shanwei. Lanhiao (Zhang & Lou 1956b)
Baoan. Tangjiahuan. Hengshan (Zhang & Lou 1956b.
1959)
Shantou. Jiazi. Jieshi. Haimen, Nanshui (Zhang & Lou
1959)
Qingcao. Gaoluo, Xiangzhou (Zhang et al. 1960)
Zhanjiang Bay (Cai et al. 1992)
Peal River estuary (Guan & Li 1986)
Guangxi Longnien (Zhang & Lou 1959)
Hainan Baoping Ba> (Zhang & Lou 1956b, 1959)
Qiongshan. Qinglangang. Boao. (Zhang & Lou 1959)
Hong Kong Lofu Shan (Ke & Wang 2001 )
Deep Bay (Mok 1974)
cultured oysters in Baoan. Guangdong Province in 19.5.^. Red tide
could be caused by Noctiluca sp. diatom or the more harmful
Dityhun sp, The carnivorous oyster drills Thais gradata (known as
"huluo," which means tiger snail in China) and Naticidae sp.
(known as "yuluo," which means jade snail) are also very harmful
to oysters. Tiger snail can drill through the shell of a spat in 3 min
and in 8 h for a 3-y-old oyster (Wu et al. 1997). Beside these,
carnivorous crabs, such as Scylla. Portunidae. Lithodidae. sea ur-
chin Ecliiiioidea. and sea star Aseroidea. are also harmful to spat.
Below we list the available reports on these subject areas by
publication year.
Harmful organisms to C. riviilaris cultured in Zhanjiang Bay,
Guangdong Province, China (Cai et al. 1992)
The effects of the predator T. gradata and Balanus spp. were
reported in an important estuary for aquaculture. T. gradata was
found harmful to l-y-old oysters. Its density on oyster cultch could
be as high as seven individuals/m". Mortality caused by T. gradata
could be as high as 31%, 14% on average. T. gradata preferred
living in groups, usually hiding in the shaded area of concrete
posts. Its reproductive season was from the beginning of April to
the middle of June peaking from the beginning of April to the
beginning of May. Each female carried .50-100 oospores, with
about 100 eggs in each oospore. Hatchability was very high, al-
most 100%. Incubation period was about 15-30 days. Barnacle
Balanus spp. competed for setting space and food. In the worst
situation, the oyster seed could be smothered with a total covering
of Balanus spp. Balanus spp. set increased from the upper estua-
rine area toward the lower saltier regions. Highest density occurred
in the low intertidal area. Balanus spp. larvae preferred the sunny
side of a setting place.
Mass mortality putatively caused by Proroceiilnim sp. bloom in
Zhanjiang, South China (Zhang et al. 1995)
From late April to late May 1994, an episode of high mortality
occurred at an O. rivularis farm close to the port of Zhanjiang.
Fujian Province. South China. Mortality reached 98% o\er about
25 hectares. Water sampling and histopathological monitoring was
conducted. During the outbreak, the water temperature increased
from 18 to 30°C, pH fluctuated between 6.5 and 7.0. and salinity
ranged 25.6-29.1 ppt. The water was blue-brown in color and all
water samples revealed variable concentrations of phytoplankton.
of which 96% were composed of Prorocentruin sp. with concen-
trations of 201-667 cells/mL over the period of observation. The
temporal association of the mass mortality and a Prorocentrum
bloom suggested that the bloom was probably the cause of the
mortality. This assumption is supported by the histopathological
findings that suggest toxicosis. In particular, the observed lesions
were acute and corresponded with the outbreak.
Affected oysters were gray in color and had a softer than nor-
mal texture. The most outstanding microscopic lesion was intense
accumulation of hemocytes in and around hemolymph channels,
especially in the Leydig tissue. Close examination of the larger
vessels revealed that hemocytes were actively infiltrating the ves-
sel walls, as well as involved in transmigration into the Leydig
tissue and the formation of intravascular thrombi. A diffuse, and
less intense, hemocytosis was present in the interstitium between
the digestive tubules, while a mild hemocytosis was detected in the
gills. Oedematous changes were prominent around the digestive
tubules and in the Leydig tissues where they were accompanied by
tissue necrosis/lysis. The digestive tubules were empty and their
epithelia were dysplastic, varying from low columnar to cuboidal
and in some instances there was necrosis of the tubular epithelium.
Brown cells were pailicularly prominent in the intertubular tissues.
The pathology was consistent with a systemic toxicosis resulting
from absorption of toxins from the digestive gland.
Bouamia-\\V.e parasite found in C riviilaris reared in France
(Cochennec et al. 1998)
C. rivularis was imported from the Haskin Shellfish Research
Laboratory in New Jersey in 1994. Seven months after introduc-
tion, some mortality occurred in quarantine. Histologic examina-
tion revealed the presence of an intracellular protozoan parasite in
the connective tissues of nine dead specimens. Ultrastructure
analysis suggested that the protozoan might belong to the genus
Bonamia. Bonamia was likely transmitted to the experimental oys-
ters from neighboring waters, which are endemic for bonamiosis,
possibly when inlet water treatment lapsed.
An intracellular procaryotic micoorganism associated with lesions in
C. ariakeiisis in Pearl River estuary. South China (Wu & Pan 2000)
A series of mortalities of cultured oysters have occurred in
Pearl River estuary since 1992. usually from February to May. The
mortality peaks at 80-90%' during April and May. The diseased
CRASSdSTREA ARIAKENSIS REVIEW
11
Figure 3. Luculions reported with ('. ariakensis populations in India and Paliistan. India: 1. Ratnagiri (I6N, 73E); 2. Balapur (not locatedl: 3.
Porljander iPorbundar), Navibander (2IN, 69E|; 4. Dwarka (Gomati Creeli) (22N, 68El: 5. Oiiha. Aramda Creek, Posheira, Port Okha, Sikka
(22N, 69E). Pakistan: 1. Korangi Creek (24N, 67E): 2. Karaclii (24N. 64E); and 3. Port Qasini (27N, 68E).
oysters are generally aged 2-7 y. A rickettsia-like iiitracelliilar
microorganism is present in the tissue of diseased oysters.
PHYSIOLOGY
Natural Reproduction
Hermaphroditism and Sex Reversal
Crcissostrea are oviparous and protrandric hermaphrodites (c./..
Coe 1934). The occurrence of true hermaphrodites (both sexes
simultaneously) is rare. Hasan (1960) stated that hermaphrodites
do not exist in O. discoidea ( = C. rividaris). In a study of her-
maphroditism and sex reversal in C. rividaris from the coast of
Karachi, Pakistan, true hermaphrodites were absent (Asif, 1979).
Hermaphrodites observed were actually transitional stages of the
sexes and used to study sex reversal. According to Asif, gonad
generally appeared in C. rivukiris at the age of 2-3 mo at a length
of 0.4-0.6 cm and 62* were male. Protandric hermaphrodites
were found in summer and autumn, which indicates the time of sex
reversal. The percentage of males declines gradually with increas-
ing size as is true for other Cnissostrea spp. Cai et al. ( 1992) also
claimed that sex ratio of C. riviiUiris had an obvious regular change
during the reproductive season (usually summer and autumn) and
the ratio of females to males increased as the oysters got older.
Hasan (1960) also mentioned that individuals with undistinguish-
able sex are fairly common throughout the spawning season. In
Asif s study, the percentage of females increased over males be-
yond the size class 5.0-5.9 cm.
Spawning
Importance of temperature in gonad maturity and spawning of
oysters is well known. Temperature influences the development of
gonad (Orton 1936, Spark 1925. Nelson 1928). Temperature also
directly influences the abundance of food, which is necessary for
the development of gonad (Loosanoff & Engle 1942. Loosanoff &
Tomnier 1948). Periodic examinations of the gonad of O. dis-
coidea showed that normal growth of the reproductive products
was coincident with gradual rise of water temperature and food
abundance in the summer months (Hasan 1960).
The combined effect of temperature and salinity on the start of
12
Zhou and Allen
spawning was discussed by Hornell (1910. cited from Hasan.
1960) and confirmed by Hasan (1960) through an experiment on
O. discoidea in Pakistan. The rise in water temperature helps the
development of gonad, while decrease in salinity stimulates the
gonad for spawning. Cai et al. (1992) also mentioned that oyster
reproduction is closely related to environmental conditions. High
temperature and low salinity could cause mass spawning of C.
rividaiis in Zhanjiang Bay. Guangdong province. Hu et al. (1994)
presented a more detailed and slightly different discussion in his
study of C. lividaris spat collection in Jioulong River estuary.
Fujian province. He agreed that spawning is related to the change
of water temperature and salinity. Water temperature could change
with wind direction or strength. Salinity could be changed by
precipitation, water current, and tides. However, he seemed to
believe that simply a change of water temperature and salinity
could be the trigger for spawning, whether an increase or decrease.
According to his observation, whenever the tide changed from
neap to spring, spring to neap, or during spring tide, oysters would
spawn, as long as their gonad was well developed. If the wind
direction happened to change from northeast to southwest, or cold
air happened to pass by. spawning would increase. He explained
that a temperature change of only about 1-2°C would stimulate C.
rivularis to spawn.
Hasan (1960) studied two natural O. discoidea beds at Wau-
gudar Creek. Pakistan. Spawning starts by the first week of July
when temperature was about 28-29°C and salinity about 24 ppt.
Number of spawning individuals remains almost constant during
August and September, much reduced in November and almost nil
in December
Several authors talked about reproduction of C. rividaiis from
China. According to Zhang and Lou ( 1956a), the optimum salinity
for reproduction of C. rivularis is 10-25 ppt in China. Hu et al.
(1994) reported that in Jiulong River estuary. Fujian province,
gonad maturity reaches its peak from the middle of April until
mid-May. Oysters spawn twice each year: spring spawn is from
May to June and fall spawn, from the end of October to the
beginning of December. During spring spawn, water temperature
fluctuated between 20 and 30°C, salinity 5-25 ppt. Guan and Li
(1986) mentioned that in Zhujiang River estuary. Guangdong
province, the reproductive season is from June to September.
Spawning is mainly during June and July. There might be a second
spawning if appropriate environmental conditions are available.
Guan and Li did not report the environmental conditions associ-
ated with spawning. Cai et al. ( 1992) reported that the reproductive
season is generally from the beginning of April to the middle or
end of June in Zhanjiang Bay, Guangdong. Environmental condi-
tions in the study area (Shimen) are listed as follows: Annual water
temperature ranged from 14 to 31.8°C. Daily water temperature
changed 2 to 4°C. Water temperature was highest in June and
lowest in January. Salinity ranged from 7.52 to 22.18 ppt in sum-
mer (but could drop to 0.00 ppt when flooded). 18 to 30 ppt in
winter. pH ranged from 7.1 to 7.9 in summer and 7.9 to 8.1 in
winter. Zhang et al. (I960) mentioned that reproduction occurred
year round in South China Sea area. The reproductive peak is from
late May to eariy September. Zhang et al. did not report environ-
mental conditions during this time period.
According to Tanaka ( 1954). the spawning season of O. rivu-
laris ranges from late May (20-22°C) to early September (28-
26.5°C) in Ariake Bay, Japan. There are three major spawning
periods during this season: early June (22-23°C). late June to eariy
July (24-26°C). and the beginning to middle of August (30-
28.5"C). The eggs of U. rividtiris measure 49-53 ixm in diameter.
The relation between salinity and developmental condition is
shown in Table 4. The temperature varied from 24 to 27°C
(Amemiya 1928). The above results are neariy identical to those of
Hu/iniori (1920. cited from Amemiya. 1928).
Spalfall
The preferred tidal height of settlement for C. rivularis spat was
reported to be at the 0.5 ft mark in Pakistan (Ahmed et al. 1987).
A broader range was reported from China by Nie ( 1991 ): from the
low tide line to a depth of 10 m. with the maximum setting at
± 0.4 m low water mark. Hu et al. (1994) reported the optmial
water depth for spat collection is from the low tide mark to a depth
of 1 m in Jiulong River estuary. China. Larvae settle 12-18 days
after spawning. In southern China, spatfall occurs from June to
August, the period of highest temperature and lowest salinity (Nie
1991, Cai & Li 1990).
Three reports on spatfall seasons from Pakistan are summarized
below. One study was conducted at Paradise Point situated on the
west coast of Karachi (Moazzam & Rizvi 1983). This is basically
a rockv shore having frequent stretches of boulders and sand. The
subtidal area along this shore is generally more deeply inclined
than the rest of the coast. This is also a power plant site. C.
rivularis occurs in the cooling system of the power plant, which
has been made artificially "protected" and simulates conditions of
a backwater environment. The enxironnient conditions were re-
ported as follows. Temperature dropped to its minimum of 20-
22 C in December-January and reached its maximum of 28-30°C
m June-July. Salinity remained fairly constant (35-36 ppt) except
during the short spell of rains in July-August when salinity
dropped to 28 ppt. The contents of suspended matter fluctuated
between 0.003 mg/L in November and 0.1 16 mg/L in June. Trans-
parency was less than 1 m in June-July. Maximum settlement of
C. rivularis occurred in June and September-October. A consid-
erable number were also observed in July-August.
The second report came from two natural oyster beds (Hasan
1960). One is situated between Korangi and Kadero creeks, south
of the village Vagudar and about 16 miles southeast of Karachi.
The other one is about 6 miles south of Dhabeji. The temperature
and salinity profile were reported from Vagudar creeks. Tempera-
ture profile looks very similar to the one from the above report,
except that it dropped even lower to 16-17°C in January. Salinity
was reported only from April to September, with a maximum of
3(S-37 ppt in April-May and then dropped continuously to 21-22
ppt in September. The pattern of larval settlement of O. discoidea
in this report is different from the one mentioned above. Settlement
at Vagudar Creek occurred from July to December with mid-
TABLE 4.
Relationship between salinity and developmental condition,
accordini> to .\meniiya 1928.
Salinity ppt
Sp. gr. at 0 C
Condition
ca. 7
ca. 1.0056
Minimum salinity
S-14
1.0064-1.0112
Much too low salinity
L'^-IX
1.0120-1.0144
Too low salinitv
1 9-25
1.0153-1.0200
Optimum salinity
26-30
1.0209-1.0241
Too high salinity
31-33
1.0249-1.0256
Much too high salinity
ca. 34
L-a. L0273
Maximum salinity
Crassostrea ariakensis Review
13
September being the peak permd. Moa//aiii and Ri/vi related
setting failure to the presence of high contents of suspended matter
in seawater during the southwest monsoon period (June-
September). This high content of suspended matter is believed to
interfere with larval settlement of many in\ertebrates in this area
(Ahmed et al. 1978).
The third report came form the Gharo-phitti saltwater creek
system (Ahmed et al. 1987). Spat fall occurred from April to
October with peak settlement from April to July. The maximum
settlement occuned during the period June 24 to July 23. No
environmental conditions were given in this report.
Growth
Growth Rate
C. uriakeiisis is well known for fast growth. In Pakistan. C.
rivularis spat reached the si/e of 0.5 mm in about one week and
2.0cm in about I mo (Ahmed et al. 1987). Hasan ( I960) found that
a size of 3.0 cm was reached 2 mo after settlement. In about one
and half years, they become ready for market. Temperature and
salinity data of Hasan's study is shown in the spatfall section. In
China. C. rivularis can growth to 10-16 cm in 2 to 3 y (Zhang &
Lou 1956b). In Japan, it attains full size (20 cm) in 2 or 3 y
(Amemiya 1928). The results of Fujiinori's study (1929) on the
growth rate of O. rivularis was presented in two parts: spat / young
oysters and the sexual adult. Fujimori found that the growth rate of
the spat varies considerably according to their time of attachment.
The size of adult O. rivularis in Kyushu was 5.5 cm shell height at
I y. 9.7 cm at 2 y. 12.4 cm at 3 y. 15.2 cm at 4 y. 17.9 cm at 5 yr.
and 19.7 cm at 6 y. In Japan, growth was most rapid in August and
September (Cahn 1950). Environmental conditions were unavail-
able for the above reports, if not mentioned.
Shell Dimension
C. ariakeiisis reaches a large size. As Cahn ( 1950) mentioned,
the maximum size attained by this species according to the litera-
ture is 257 mm with an estimated age of 20 y. The maximum
length he recorded in Japan was 240 mm. A maximum shell height
of about 200 mm was reported several times from Japan and the
United States (Amemiya 1928. Hirase 1930. Coan et al. 1995).
According to the growth rate of adult O. rivularis determined by
Fujimori (1929). the estimated age of such size is more than 6 y
old. Generally, adult specimens reach 6-7 inches (or 150-170 mm)
in height, as reported from four countries (Hirase 1936. Galtsoff
1964. Ahmed 1971. Rao 1987).
Allometric Growth
A study of the allometric (relative growth) relationship between
shells and tissues of C. rivularis was presented by Barkati and
Khan (1987) from Pakistan. Shell length was defined as the maxi-
mum distance between the tip of the anterior margin and the pos-
terior margin. Shell width was defined as the maximum distance
between the lateral maigins. The following points were reported.
Shell width increased faster than shell length (/■ = 0.85). Shell
length increased faster than dry tissue weight (/■ = 0.52). An
exponential relationship exists between shell length and shell
weight with faster growth in length compared with shell weight
(/■ = 0.84). Dry tissue weight increased faster than shell weight (c
= 0.74). Condition index (the proportion of dry tissue weight to
total dry weight of shell and dry tissue) increased with increasing
shell length (r = 0.41 ). No linear variable was useful to accurately
predict other variables due to low coefficient of correlation (/).
probably due to irregular growth in various shell dimensions
(length and width).
For example. Asif ( 1978b) reported variation in shell growth in
two populations of C. rivularis caused by setting density in Pak-
istan. One population in Korangi Creek was exploited and densi-
ties were low. Another population in Sonari was crowded. In the
Korangi Creek, the oysters are attached to rocks or stones hori-
zontally, whereas those of Sonari grow upward with the umbo
downwards. Generally, the wild stock of C. rivularis of the Kor-
angi Creek are round and shallow whereas the Sonari population is
elongated and deeply cupped. In the majority of the Korangi Creek
population, height plus width varies closely with length of the shell
while in the Sonari population, shell height plus width varies twice
as much as the length.
Feeding
Food .Selectivity
According to Cai et al. ( 1992). C. rivularis (collected in Zhan-
jiang Bay, Guangdong Province. China) is a selective feeder. It
prefened small articles to long-chain groups or large articles. The
majority of its food is composed of phytoplankton such as Cosci-
uihUscus sp., Nitzscliia sp. and Cyclotella sp.
Feeding Habits
Zhang et al. ( 1959) did an extensive study on the feeding habits
of O. rivularis in relation to time, tides, season (change of tem-
perature and salinity) and suspended particles. The experiment was
conducted in the Pearl River estuary and some nearby bays. Most
of the sampled oysters were 3 to 4 y old at the time of examination.
These oysters were collected from the wild as spat and cultivated
in oyster farms. The percent of O. rivularis that are feeding at any
given time (incidence of feeding) was not related to periods of
light and darkness, nor to the periods of tides, or the density of
suspended particles. Salinity and temperature did have certain in-
fluences, as summarized below.
According to examinations at five different times of the year,
the highest average incidence of feeding for O. rivularis was a
little more than 80%. It was also found that feeding time of O.
rivularis adds up to 16-19 h everyday with irregular intervals.
Feeding habits of O. rivularis were not related to change of sea
level or direction or speed of water flow caused by tidal change.
In Pearl River estuary, feeding incidence of O. rivularis was
highest from October to April (50-100%). when temperature
ranges between 10 and 25°C and salinity between 15 and 30 ppt.
During summer, the natural reproductive season of O. rivularis.
when temperature is much higher (22-30°C) and salinity is much
lower (3-26 ppt). feeding incidence is lower (0-70%). Feeding
incidence seems to be more closely related to salinity according to
monthly records. Although O. rivularis is known to tolerate low
salinity, feeding rate was significantly retarded if salinity was
lower than 5 ppt. Above 10 ppt, feeding was active.
Increase in suspended particles in the seawater (higher turbid-
ity) failed to influence feeding incidence of O. rivularis. In this
case, the authors maintained that these suspended particles served
as a food source for the oysters.
Oxygen Consumption
Guan and Li (1988) did an extensive study on oxygen con-
sumption of C. rivularis. A Warburg manometer was used to mea-
sure the oxygen consumption of dissected gill tissue of C rivularis
taken from the Shenzhen Bay Oyster Fann. Oxygen consumption
14
Zhou and Allen
varied with the change of seawater temperature. A negative cor-
relation was found between oxygen consumption and the oyster
age. Tlie older and heavier the oyster, the less oxygen was con-
sumed by its gill tissue. Oxygen consumption differed significantly
in different reproductive periods.
BIOCHEMISTRY
Biochemical composition
Qasim et al. (1985) determined the following biochemical pa-
rameters for C. hvidaris from Pakistan. Water contributes 787r of
soft body wet weight. Of soft body dry weight, 35.7% was crude
protein, 22.5% glycogen, 23% lipid, and 11.2% total inorganic
substances. These are the averages from sampling over a period of
time (sample interval was not stated in the article). Higher value
for lipids (31%) was reported from India (Patel 1979. cited from
Qasim et al. 1985). This difference is probably the result of geo-
graphical variation, seasonal variation, or both.
Qasim et al. ( 1985) mentioned that the ratio between glycogen
and protein changes with reproductive state of an oyster (no spe-
cific information available). Another report on biochemical in-
dexes of C. rivularis from the Pearl River estuary. China (Guan &
Li 1986) showed seasonal change of lipid content and its close
relationship with reproductive physiology of the oysters. As the
authors di.scussed, reproductive season in the Pearl River estuary is
from June to September, of which June and July are primary
spawning periods. There could be a second spawning in September
if environmental conditions were appropriate. In their study, lipid
content was highest in May (2.88% of wet weight), then dropped
dramatically from June until it reached the lowest point 1 .06% in
October, the end of the reproductive season.
For protein, amino acid profile determines the nutritive quality
of tissues. Such a profile of C. rivularis tissue protein has been
reported from the Pearl River estuary. China (Guan & Li 1986) and
Pakistan (Aftab 1988). There are only slight differences between
the two reports. From China, specimens were tested in May, and
the amino acid profiles are presented in Table 5 (Guan & Li 1986).
Glutamine and asparagines are most abundant. From Pakistan, 14
amino acids were analyzed. Methionine and arginine were not
detected. Glycine and aspartic acids were most abundant. Seasonal
variation in bound amino acid content is shown in Table 6 (from
Aftab 1988)
The shells of O. rivularis have been used as traditional Chinese
medicine. Zhao et al. (1991) examined the content of calcium
carbonate, trace elements and amino acids in shells of O. rivularis
collected from Tianjin. Shandong, Zhejiang, and Fujian provinces.
Calcium carbonate in raw shells was 92.0-95.5% and in calcined
shells, 96.4-96.9%. Calcined shells have had organic materials
removed. The raw shells contain large amounts of Ca, small
amounts of Mg, Na, Sr, Fe, Al, Si, and traces of Ti, Mn, Ba, Cu.
etc. Shell decoctions (an extract obtained by boiling the shells)
contain small amounts of Ca, Na, Mg. K. and trace element of Sr,
P, Pb, Zn, Ni, V, Ba. Li. Mn, Ti, Cu, Cr, Mo. As, Hg, etc. The
oyster shells contain 1 7 amino acids. Total amino acid content
amounted to 0.16 to 0.24% in raw shells.
Li et al. (1994) studied the medicinal value of "oyster complete
nutritional tablet," a dietary supplement made from extracts of
both shells and soft body of O, gigas and O. rivularis from South
China Sea. The tablet contains a high content of eighteen amino
acids, especially the eight essential to the human body. Putative
benefits are attributed to the liver, kidney, spleen and intestine to
a certain extent.
TABLE 5.
The amino acid compositions and their contents In C. rivularis
sampled in May, 1984 (Guan & Li 1986).
.\mino Acid
Contents In
Dried Samples ( % )
Alanine
Arginine
Asparagine
Cystine
Glutamine
Glycine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Serine
Threonine
Tyrosine
Valine
2.04
L95
3.30
0.28
4.06
2.15
0.76
1.18
1.87
2.23
0.57
1.05
1.39
1.48
1.34
1.32
Heavy Metals and Toxins
Lu ( 1994) did a preliminary study on the feasibility of using O.
rivularis as a monitoring agent for heavy metals, like Cu, Zn, Cd,
Pb, along the Guangdong coast, China. He found that profiles of
Cu, Zn and Cd content in the oyster correlated with the distribution
of industrial discharge along Guangdong province. Also see Ke
and Wang 2001. Further investigations on the suitability of O.
rivularis as a biomonitor of specific metals or other chemicals are
presented below.
Zn
According to Lu et al. ( 1998a), Zn accumulated continuously in
the tissues of the oyster through 12 days of exposure. Accumula-
tion was linear with time. Loss of Zn from C. rivularis was not
observed over 35 days of depuration. Zn accumulated less readily
with increasing salinity. The author concluded that in general C.
rivularis is a reliable indicator of Zn in marine systems.
TABLE 6.
Seasonal variation in the protein and amino acid composition of
tissue protein hydrolysate of C. rivularis (Aftab 1988).
Component
February
May
August
November
Average
Protein % d.v\.
40.36
41.25
52.50
55.00
47.33
Alanine
6.04
6.91
9.00
9.67
7.90
Aspartic acid
6.78
9.83
12.56
6.58
8.94
Glutamic acid
6.. 30
7.98
11.26
5.08
7.65
GIvcine
13.27
11.88
6.55
9.10
12.10
Histidine
2.07
1.87
2.72
1.91
2.14
Isoleucine
2.38
1.80
3.12
2.08
2.32
Leucine
3.98
2.89
5.34
3.63
3.96
Lysine
1 .59
1.74
1.44
1.28
1.51
Phenylalanine
1.73
1..3()
1.94
1 .55
1.63
Proline
0.46
2.05
0.79
2.40
1.43
Serme
4.07
6.01
7.82
3.51
5.35
Threonine
3.85
5.74
6.92
3.24
4.93
Tyrosine
0.85
0.60
0.91
0.79
0.79
Valine
3.64
3.75
5.55
2.98
3.98
CflASSOSTRKA AK/AKENSIS ReVIHW
15
Cd
Lu et al. (1998b) studied Cd absdiplion in C. riviilaris. The
content of Cd in body tissues of C hviilaris accumulates in linear
proportion to Cd concentration in the water and to exposure time.
Accumulated Cd attenuates slowly with a biologic half-life of 77
days. With increased salinity, rate of accumulation decreases while
rate of Cd loss slows down. C. livuhiris seems to be a reliable
bio-monitor of Cd pollution.
Cu
Cu absorption in C. rivuUiri.s was examined by Lu et al.
(1998c). It continuously accumulated in the tissues of the oyster
through the e.\posure to a concentration of 100 (J-g/L over 12 days.
Accumulation was linear with time and decline of Cu concentra-
tion was slow, with a half-life about 1.^1 days. Rate of Cu accu-
mulation was significantly slower with increased salinity, but rate
of decline in Cu concentration was not signiticantly related to
salinity.
Total Petroleum Hydrocarbons (TPHs)
Lin et al. ( 1991 ) looked at concentration of TPHs in the Pearl
River estuary. China. TPHs in C. rivularis tissues decreased with
time during the period leading to sexual maturity. The rate of
decrease was about 0.24 |jig/g, dry weight. The biologic half-life
was 43 days. Aromatic hydrocarbon compounds with smaller mo-
lecular weight were released sooner from oyster tissues than those
with greater molecular weight. The concentrations of TPHs in
oyster tissues were not significantly related to those in waters and
sediments, and not clearly dependent on the contents of lipids in
oyster tissues during the study period (September 1986 until Feb-
ruary 1987).
GENETICS
Karyotype
So far, research on the cupped oyster species of the genus
Crassostrea shows a common diploid chromosome number of 2;;
= 20, and their karyotypes include only metacentric and submeta-
centric chromosomes. The proportion of these chromosome types
can be different interspecifically (Leitao et al. 1999).
Chromosome number of In = 20 was confirmed in C. aricik-
eiisis (leyama 1975) and in C. rivularis from West Pakistan
(Ahmed 1973) and China (Yu et al. 1993). Yu et al. reported the
karyotype of C rivularis sampled in Southern China had 10 meta-
centric pairs. A more recent karyological study (Leitiio et al. 1999)
on an American population of C. ariakensis originally introduced
from Japan shows that it consists of eight metacentric and two
submetacentric (nos. 4 and 8) chromosome pairs. A variable num-
ber of one to three Ag-NORs (nucleolus organizer regions) was
observed terminally on the metacentric pairs 9 and 10. About 68'7f
of the silver stained metaphases showed Ag-NORs only on pair 10.
Polyploidy
Rong et al. ( 1994) reported their attempts to produce tetraploid
C. rivularis. Newly fertilized eggs of C. rivularis from south Chma
were treated with physical and chemical methods in the first three
minutes before the cleavage of zygotes or at the onset of first
cleavage. Induction rates of tetraploids were 28% for heat shock,
30% for cold shock, 28% for chlorpromazinum treatment and
35,8% for "traditional Chinese medicine" treatment as indicated by
chromosome spreads from larvae. Production of viable spat was
not reported.
Hyhridizaliun
Gaftney and Allen ( 1993) reviewed previous hybridization re-
ports among Crassostrea species and pointed out that most of
reports of successful hybridization suffer from one or more of the
following: I ) ambiguities in classification; 2) possible contamina-
tion during spawning; 3) absence of experimental controls for
assessing the quality of gametes as well as larval viabilities; and 4)
the absence of genetic confirmation of hybrid status. They con-
clude that there was virtually no unequivocal evidence for the
formation of viable interspecific hybrids among Crassostrea spe-
cies.
Early studies on cross-fertilization between C. gigas and C
rivularis gained little success (Miyazaki 1939, Imai & Sakai
1961), but was reported successful by Zhou et al. (1982) and
Downing (I988a,b, 1991). Asif (1978a) reported successful pro-
duction of trochophore larvae 4-5 h for the cross of C. rivularis
with C. glomerata and Saccostrea cuccullata. For the reasons
mentioned above, these should be viewed with caution.
Hybridization of C gigas and C. rivularis was re-examined by
using specimens originally introduced from Japan to the United
States (Allen & Gaffney 1993). Such crosses are of interest be-
cause of the disease resistant properties of these species (Calvo et
al. 1999, 2001). In addition, the hardiness and apparent disease
resistance of C. gigas and the high temperature, low salinity tol-
erance of C. rivularis could lead to promising variants for aqua-
culture, especially if the diploid is sterile. Three replicates of a 2 x
2 factorial mating of C. gigas and C. rivularis were produced to
examine the viability of this cross. Fertilization rate, yield of 48-
h-old larvae, and survival of fertilized eggs was lower in the hy-
brids than in pure crosses. All crosses showed similar larval
growth rates, except C. rivularis (female) x C. gigas, which grew
more slowly. Isozyme electrophoresis and flow cytometry con-
firmed hybridization. Triploid hybrids were produced using tetra-
ploid C gigas and diploid C. ariakeusis (Que & Allen 2002).
Hybridization between C. ariakensis and C. virginica failed
(Alien et al. 1993). Cytogenetic and electrophoretic analysis re-
vealed the formation of hybrid zygotes and larvae between C.
virginica and C. rivularis. but larval survival was limited to a
maximum of 10 days. Larvae stopped growing at about day 4,
reaching a maximum length of about 80 um. Studies on larval
feeding using fluorescent beads indicated that growth limitation
apparently was not caused by an inability to feed. Induced triploidy
did not rescue hybrid failure.
Population Genetics
A number of studies have used molecular markers of various
sorts to distinguish among Crassostrea species, including C. ari-
akensis. Among the earliest was work by Buroker et al. (1979)
who estimated levels of genetic variation for six Crassostrea and
three Saccostrea species based on electrophoretic variation in pro-
teins in about 30 loci, C rivularis among them. Liu and Dai (1998)
used RAPD techniques to differentiate C. talienwhanensis and C.
plicatula froin C rivularis. Li et al. (1988) used electrophoretic
markers to separate four Crassostrea species, and concluded that
the "white oyster" was C. rivularis and the "red oyster," C. ired-
iilai.
C. rivularis was also among those used by Little wood (1994) to
establish the first phylogenetic estimates for this species based on
nuclear DNA. Since then, a number of other studies employing
16
Zhou and Allen
molecular markers have been applied to C. ariakeiisis. mostly to
discriminate among species (O'Foighil et al. 1995. GatTney &
O'Biern 1996. Hedgecock et al. 1999. Francis et al. 20(X)). Hedge-
cock et al.'s study confirmed the occurrence of C. ariakensis in the
northern regions of the Ariake Sea and re-emphasi/cd the need for
genetic confirmation for species identification.
AQUACULTURE
RefeiTences to aquaculture of C. ariakensis come mainly from
Japan and China, and are discussed accordingly.
Aquaculliire in Japan
Of the five edible oysters species in Japan, only O. gii;as and O.
rivuiaris were cultured commercially (Cahn 1950). O. i-i\'nlaris
was second to O. gigas in commercial importance (Amemiya
1928)
According to Amemiya (1928), cultivation of O. rivuiaris be-
gan in Ariake Bay in the late 1890s and seed were later trans-
planted to Kozima Bay in Okayama Prefecture around 1928. An
even earlier report of cultivation in Ariake Bay in the 186{)s was
given by Wakiya ( 1929). Both Wakiya and Langdon and Robinson
(1996) mentioned that the culture of Suminoe oyster were con-
ducted in the Suminoe river. Saga Prefecture from the beginning of
the Meiji period in the mid- 19th century. Discrepancy between
Cahn and Wakiya on the start of C. rivuiaris aquaculture might rest
on their definition of cultivation. Cahn ( 1950) described two types
of culture sy.stems at the mouth of the Suminoe-gawa ("gawa" in
Japanese means river or stream). Ariake Bay. a primitive one and
a more developed one. Cahn did not say when the primitive culture
started, but he implied that the more sophisticated culture started
after 1885. The primitive culture consisted simply of gathering
natural oysters and storing the larger individuals for a short time on
the bottom of the Sumino-gawa. later to be shipped to Nagasaki at
the proper season for sale.
Aquaculture of O. rivuiaris began fortuitously. For some rea-
son during the winter of 1884 these oysters were not shipped for
sale to Nagasaki. The ne.\t year they were considerably larger by
size and weight. From this observation, a new type of culture
evolved in the local area. Young oysters about 2.5 cm in length
were gathered from every possible growing place from July until
March and were placed on oyster beds at the mouth of the river. To
prevent loss, they were heaped close together in masses. They
were washed and cleaned twice or three times each month during
low tide. In April individual oysters were stuck in the mud verti-
cally, hinge down and ventral margins uppermost. As the mud was
very firm, the oysters fared and grew well. As they grew, they were
thinned and replanted to give them more growing space. Growth
was most rapid in August and September.
Aquaculture in China
C. rivuiaris is the most economically important marine shell-
fish species cultured in South China (Zhang et al. 1995), primarily
in Fujian, Guangdong and Guangxi Province. The history of its
culture in Guangdong is over .'^00 y old (Cai et al. 1979). The Pearl
River (Zhujiang) estuary. Guangdong was considered the most
famous cultivation site of this species (Zhang & Xie 1960). Some
other places mentioned in the literature are Yangjiaogou, Shan-
dong Province (Zhang et al. 1960), Leqing Bay, Zhejiang Province
(Zhou et al. 1982) and in Deep Bay, Hong Kong (Mok 1974). In
1996. China produced 2.3 million tonnes of oysters from aquacul-
ture, among which C rivuiaris accounts for 20-30% (Guo et al.
1999). In Guangdong province, C. rivuiaris production was about
40'>f of total sea culture production (Qiu & Li 1983).
The primitive method of oyster culture was to improve growth
and reproduction with procedures like fishing restrictions and pro-
tection from diseases and predators (Zhang & Xie I960). The
advanced method involves collecting natural spat and artificial
grow-out. Modern oyster culture includes larval culture and breed-
ing. Larval culture and breeding of C. rivuiaris larvae has been
successfully accomplished on a research scale in South China (Li-
ang et al. 1983. Cai et al. 1989) but has not been used in large-scale
commercial culture. Hatchery production of seed is seen as a step
to increase the reliability of seed production.
Spat collection and artificial grow-out is still the most popular.
This is composed of four steps: spat collection, grow-out, fatten-
ing, and harvest. For spat collection, cultch material to collect spat
was traditionally oyster shell and gravel (Nie 1991). Since the
1960s, cement plates (17-24 cm x 14-19 cm) or cement bars
(40-80 cm long x 4-6 cm") reinforced with embedded bamboo
stakes were used. Stakes are used increasingly since they are easier
to handle, provide more surface area, and are not so readily cov-
ered by silt. Season and location of spat fall is summarized in
Physiology. Oyster larvae in the water are monitored to ensure the
best time of planting the clutch. Spat collectors are placed in rows
in rectangular blocks, usually 30 to 37.5 x lo' stakes or 100 to 135
X 10' plates per hectare. Further details follow below for specific
culture techniques.
The age of harvest is generally 3.5 to 4 y (Qiu & Li 1983). but
\aries from 2 to 5 y depending on culture location where the
environment, the specific culture technique, and even the expected
market size could be different. For example, Guo et al. (1999)
reported 2 to 3 y in Guangxi where oysters maintain rapid growth
throughout the first 3 y and are usually harvested at a size of 10-15
cm. The culture technique used there is concrete bars or shell
strings hanging on rafts and long lines. In Pearl River estuary,
Guangdong, oysters were usually harvested at 3 y of age by bam-
boo stake culture (Zhang & Xie I960). Cai and Li (1990) reported
the period to be 3 to 5 y in Southern China.
Cai and Li (1990) summarized oyster culture techniques in
China. The ancient bottom culture techniques, including bamboo
stake, stone and concrete culture, are still the major methods, but
farmers are becoming increasingly aware of the advantage of off-
bottom culture, like the rack and raft culture. The various tech-
niques are described below (reproduced from Cai and Li's work,
1990).
Rock (Stone) Culture
Rock culture is usually applied in areas that have hard sub-
strate. Marble flagstones approximately 90 cm x 25 cm wide and
10-cm thick are preferred for this method. Stones may be arranged
one-by-one vertically, resembling tombstones or two stones may
be aiTanged in an "A" shape. Three stones may be ananged to form
a tripod. Average spacing between stone groups is 70 cm. Another
choice of rock is irregularly shaped natural boulders of 4 to 5 kg.
The traditional anangement of the boulders, called "stars in the
sky," involves uniform distribution over the substrate. Two modi-
fications were used along the coast of Guangdong and Hainan
Provinces. One is called "plum blossom" with five or six boulders
grouped together. Another is called "small house" with three flag-
stones aiTanged to form a shed or an upside-down "U." Both kinds
of rocks are thoroughly washed and then covered in limewash 10
davs before use.
Crassostrea ariakensis Review
17
111 Guangdong and [■uiian Proxinces. the rocks are set out in
early May to June or in November. Maxiniuni spatfall is expected
in May. Spat collected in June is usually subject to heavy mortality
due to high temperatures and strong sunlight during attachment.
Spat collected late in the season usually grew poorly because of to
low water temperatures. Oysters are grown to market size at the
site of spat collection.
Approximately 60,000 stones are required for one hectare, and
C. rivularis may be harvested in 3 to 5 y. Production is moderate,
ranging from 750 to 3000 kg per hectare. The oysters grown on
rocks are more subject to predation by starfish and other organisms
than are oysters grown on stakes, so considerable time must be
invested in predator control.
Concrete Culture
Prefabricated posts or tiles are a derivative of the traditional
rock culture technique for the culture of C. rivularis and has been
used since 1930 in Guangdong Province. Spatfall occurs most of
the year, but optimum periods are April and May. To prevent the
tiles or posts from sinking into the mud, they are removed and
reananged around May, September, and December. Concrete cul-
ture requires a 4-y cycle. Spat collection and growth occupies the
first year from June to April. The second and the third years
involve a cultivation period yearly from May to August. Market
size is attained in 2.5 to 3 y and involves a progressive increase in
the spacing of the concrete tiles or posts. The cultivation cycle is
completed by a fattening period extending from September to
January. For fattening, oysters are transferred from the spat col-
lection/grow-out area to prime growing grounds, usually in the low
intertidal zone. For this culture method, in Guangdong, harvest
generally occurs in February to April of the fourth year, when
growth rates begin to decline sharply. Expected production from
the concrete method is 7.5 to 15 tons of meat per hectare.
Rack Culture
Since 1965, rack culture has been used to cultivate C. rivularis
in Guangdong Province. The racks may be constructed of bamboo,
wood, stone or concrete. Because wood and bamboo are rapidly
destroyed by shipworms and stone is heavy and awkward to
handle, concrete is preferred. The forni of the rack varies greatly,
but consists basically of members driven into the substrate to form
a horizontal frame, which supports the oyster cultch 2.5 to 3 m
above the substrate.
Several types of material are used for spat collection. The most
popular one is punched oyster shells, separated by 3 cm bamboo or
plastic spacers, and strung on 2 m lengths of galvanized wire ox
polypropylene line. Concrete tiles, approximately 10 cm" with a
central hole, may be substituted for the oyster shell. Concrete poles
between 70 and 130 cm in length may also be used. The cultch is
suspended from the rack, with spacing proportional to the density
of spat settlement and the character of the growing area. The
number of racks accommodated varies widely between the grow-
ing sites. Production is estimated at 10 to 20 tons per hectare.
Raft Culture
According to Qiu and Li ( 1983). raft culture started in Japan in
1950. Since 1979. the Fisheries Research Institute of the South
China Sea has conducted experimental raft culture of C. rivularis
in Guangdong Province. The fattening period lasts from September
to May. and three crops may be harvested, because 2 mos are
sufficient under optimal seasonal conditions. The ratio of meat
production to shell is some 60'/^ higher in raft-fattened oysters than
in oysters harvested directly from bottom culture.
C. rivularis can be marketed in less than 3 y using rafts, and
that the condition factor will be increased by more that 22% and
the meat quality will be superior to oysters cultivated by the tra-
ditiimal bottom methods (Qiu & Li 1983). Though initial costs are
higher, the increased production and working advantages of float-
ing raft culture are apparent, and it is expected that raft culture will
account for a steadily increasing share of oyster production in
China (Qiu & Li 1983). Nie ( 1991 ) also mentioned that raft culture
gives faster growth and a higher yield. A raft of 84 n\' will produce
in 2 y what 667 nr of bottom culture will in 4 y. Rafts seem to
w ithstand typhoons better than originally thought.
DISCUSSION
C. ariakcusis shares many life history traits with other Cras-
soslrea species. It is clearly an estuarine species v\ith salinity
tolerances similar to C. virginica. Its occurrence in river systems
and apparent responsiveness to salinity changes for spawning cues
suggests that its reproductive strategy is somewhat different than
C. virginica. There are indications that larval behavior differs from
that of C. virginica (M. Luckenbach, VIMS, pers. comm.), perhaps
an adaptation to fluvial existence. Many other questions about its
ecology are unanswered or incomplete and a number of research
priorities have been identified (Rickards & Ticco 2002). One of the
principal problems with extrapolating life history from the avail-
able literature is the uncertainty over species designation. Some
reports are clearly referring to C. ariakensis. e.g.. those from
southeast China where aquaculture activity is concentrated and
there is a long history of working with this species. Other reports
are not so clearly C. ariakensis, especially ones deriving from
western India and Pakistan. Also because of likely morphologic
confusion, the geographic range for C. ariakensis is incompletely
described. For example, it seetns likely that its range should in-
clude the coast of Vietnam, yet there seem to be no direct accounts
of this. There are accounts of its occurrence as far as Borneo, the
Philippines, and Thailand, but these are unconfirmed. Froin a prac-
tical standpoint, C. ariakensis from China are probably an appro-
priate starting stock for an introduction, should that proceed, be-
cause of similarities in latitude. From that respect, this area seems
a most appropriate focus for obtaining more information on the
species. Korea and Japan are possible sources as well. We did not
encounter reports of C. ariakensis from Korea except as casual
remarks. Stocks in Japan seem to be limited in abundance.
It is unclear whether C. ariakensis is a "reef-forming" oyster,
depending on how you define "reef" Clearly, Crassostrea species,
and oysters in general, benefit from aggregation and adults or their
shells provide substrate for recruitment in subsequent generations.
Some accounts of C. ariakensis describe "oyster hills" that would
clearly qualify as reefs (Zhang & Lou 1956b. Zhang et al. I960).
Apparently, it is common knowledge among fishermen in China
that C. ariakensis forms reefs. Other accounts have C. ariakensis
occurring as small aggregates and singles. In our travels to China,
we encountered several sites that had "natural" populations of C.
ariakensis (Allen et al. 2002). There seem to be natural popula-
tions in proximity to Xiamen although we did not observe this first
hand. They were available in the local market and reportedly from
natural populations that were harvested. There are natural sets of
r. ariakensis near Hong Kong on the shores of Deep Bay. but this
18
Zhou and Allen
could be from culture activity in the area. Seed is imported froin
the Pearl River estuary, so there are likely sources of ""natural"
populations in the Pearl River delta system. We observed, first
hand, collection (harvesting) of C. ariakensis adults from sections
of the Shiman River near Guan Du in close proximity to Zhanjiang
Ocean University. According to the diver on hand, they occur in
various assemblages, mostly stuck onto available substrate such as
large rocks. They also occur in the Dafeng River in Guangxi
province near Beihai. There are probably many other natural popu-
lations along the coast of China. By way of caveat, it is difficult to
attest to the "naturalness" of resident C. ariakensis populations.
That is, those that we observed or heard about first hand were
populations that occurred relatively deep (3-10 m) in river sys-
tems. Whether at some time in the past populations of C. ariak-
ensis were distributed in higher reaches of the water column (i.e..
before they were exploited over the millennia) is difficult to es-
tablish. It is also difficult to distinguish whether spat fall is from
natural populations or from aquaculture operations.
There are clearly big questions concerning basic physiology in
the kind of detail that exists for other congeners. C. ariakensis
seems to exhibit growth rates that are extraordinary in head to head
trials with C. virginica. Yet, these trials have been carried out in
disease endemic areas where C. virginica could be sick or dying.
Growth rates of C. virginica in. for example, the Gulf of Mexico,
approach those seen in trials of C. ariakensis in the Chesapeake
Bay or reported growth rates from the literature. Similar knowl-
edge gaps exist for larval biology, reproductive physiology, pre-
dation. competition, etc.
In our opinion. C. ariakensis is an underused resource around
the world. It clearly has aquaculture applications in estuarine areas
that are marginal or unsuitable to C. gigas. the most popular cul-
ture species. It seems hearty, fast growing, and highly marketable.
Of course, utilization of this species would require introduction, as
in the Chesapeake Bay. From that perspective, it would be useful
to have more basic research on C. ariakensis with which to guide
decisions about movement of this potentially valuable oyster spe-
cies.
ACKNOWLEDGMENTS
The authors thank our Chinese colleagues for their warm as-
sistance in compiling many of the papers cited here, particularly.
Dr. KE Cai-Huan, Professor LI Fu-xue, Dr. CAl Lizhe. Dr. WU
Xinzhong, Dr. Catherine Lam. Dr. QILI Dequan. Dr YU Xiang-
yong. Professor CAl Yao-Guo (retired). Director LAO Zan. and
Dr. LIU Zhigang, among others. We also thank S. Shumway for
early editorial assistance. This work was supported by the Camp-
bell Foundation and an award to S. Allen, Jr. from the Virginia
Center for Innovative Technology. Contribution number 2541
from the Virginia Institute of Marine Science, College of William
and Marv.
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Joiinial uj Shellfish Research. Vol. 22, No. 1, 21-3U, 20U3.
CONSUMER RATINGS OF NON-NATIVE (CRASSOSTREA G/GAS AND CRASSOSTREA
ARIAKENSIS) VS. NATIVE {CRASSOSTREA VIRGINICA) OYSTERS
JONATHAN H. (JRABOWSKI.'* SEAN P. POWERS/t CHARLES H. PETERSON,'
MONICA J. POWERS,' AND DAVID P. GREEN"
' University of North Carolina at Chapel Hill. Institute of Marine Sciences, Morehead City,
North Carolina 28557 and 'North Carolina State University; Center for Marine Science and
Technology, Morehead City, North Carolina 28557
ABSTRACT Given suggeslion^ that a non-native oyster be used to replace the depleted native oyster, consumer preference evalu-
ations were conducted to determine how two non-native oysters, Crassostrea gigas and C. ariakensis, when grown in North Carolina
estuaries, were rated by consumers. Tests compared the taste, appearance, and/or aroma of both raw and cooked non-native oysters to
similarly prepared native oysters, C. virginica. In the first series of tests, consumers exhibited a slight preference for raw C. virginica
over raw C. gigas. When cooked, both species were rated equal. In the second series of tests, a larger group of participants ranked the
taste, appearance, and aroma of C. virginica. C. gigas, and C. ariakensis. Participants that tasted raw oysters collectn ely preferred C.
virginica over both non-native species. This preference remained strong regardless of the frequency with which participants consumed
oysters. Preferences for appearance and aroma varied; however, ratings never indicated a preference for either non-native species over
C. virginica. Participants as a whole preferred the taste of cooked C. virginica better than C. gigas. whereas a taste preference did not
exist between cooked C. virginica and C. ariakensisis. Given that participants collectively preferred the taste of both raw and cooked
C virginica to C. gigas. the suitability of C. gigas for substitution in either the raw or steamed oyster market is questionable. For oysters
of similar length (80 to 1 10 mm), dry tissue weight of C. ariakensis was twice that of C. virginica. This higher per-oyster yield suggests
that C ariakensis might be more suitable for a steamed or packaged oyster market where oysters are sold by meat weight rather than
by number. However, these markets often command much lower prices, perhaps rendering unfeasible the aquaculture of this introduced
oyster. Before large-scale introduction of non-native oyster species occurs, consumer preferences should be incorporated into economic
evaluations that include additional economic (oyster prices, market demand and supply functions) and biological information (growth
and survivorship). Profitability expectations generated by the model then need to be weighed against the potential ecological risks and
ecosystem benefits of aquaculture or introduction to the wild for each non-native oyster species.
KEY WORDS: Crassostrea ariakensis. Crassostrea gigas. Crassostrea virginica. economic feasibility, native versus non-native
oysters, raw versus cooked oysters, frequent versus inexperienced consumers, taste test
INTRODUCTION
Landings of the eastern oyster, Crassostrea virginica (Gmelin
1791 ), have declined by over 90'7c during the past century in the
major estuaries of the eastern United Slates (MacKenzie 1983,
Hargis & Haven 1988. Frankenberg 199.S). Habitat degradation
from destructive harvesting techniques (Rothschild et al. 1994,
Lenihan 1999) and mortality induced by bottom-water hypoxia/
anoxia, sedimentation, and parasitic diseases (Seliger et al. 1985,
Ford & Tripp 1996, Lenihan & Peterson 1998, Lenihan et al. 1999)
collectively have contributed to this decline. In North Carolina,
efforts to sustain the oyster fishery over the past several decades
through shell plantings have contributed to but not restored land-
ings, which are less than K/r of historic maxima achieved in the
late 1800s (Frankenberg 1995). Introduction of non-native species
such as C. gigas (Thunberg 1793) or C. ariakensis (Fujita 1913) is
a possible alternative or supplement to continued efforts to restore
native populations, and could resuscitate the oyster industry in the
eastern United States.
The Pacific oyster, C. gigas. accounts for over 9,0'^i of the
world's aquaculture production of oysters (Ayers 1991), and
thrives in shallow, sub-tidal estuaries at higher salinities (Calvo et
al. 1999). Native to Japan and the Korean peninsula (Mann et al.
*Corresponding author. University of Maine at Orono, Darling Marine
Center. 193 Clarks Cove Road. Walpole. ME 04S73. E-mail: jgrabow@
maine.edu
tCurrent address: University of Southern Alabama. Dauphin Island ,Sea
Lab. Dauphm Island. AL 36528
1991). it has been successfully introduced to France, Oregon,
Washington, western Canada. Australia and New Zealand (Shatkin
et al. 1997). C. gigas often establishes populations successfully
when introduced and is successfully cultured in part because it is
highly resistant to the protozoan diseases MSX. Haplosporidiuin
nelsoiii. and dermo. Perkinsus inariiuis (Calvo et al. 1999). MSX
and dermo continue to impede recovery of native oyster popula-
tions along the eastern coast of the US (Ayers 1991. Mann et al.
1991 ). C. gigas also typically reaches harvest size more quickly
than native oysters, leading many culturists to prefer growing C.
gigas over native species (Pollard & Hutchings 1990. Ayers 1991,
Parameswar 1991 ).
In contrast to C. gigas. the Suminoe oyster, C. ariakensis,
currently does not contribute substantially to oyster fisheries of the
world. Despite some taxonomic confusion with C. riviilaris. the
native distribution of C. ariakenis is thought to range from Paki-
stan to Japan, and extends into quite low salinities within the
estuaries that it inhabits (Breese & Malouf 1977, Langdon & Rob-
inson 1996). Like C. gigas. C. ariakensis grows more quickly than
most other oyster species (Byrne 1996. Calvo et al. 2001 ). partly
explaining why many fishermen in North Carolina and Virginia
are advocating its introduction. This species can be grown to mar-
ket size in 12-18 mo in colder waters along the west coast of the
U.S. (Langdon & Robinson 1996). Calvo et al, (2001) also dem-
onstrated that C. ariakensis is resistant to MSX and dermo. Long-
term failure of management to restore native oyster populations
coupled with higher growth rates and disease-resistance of C. gi-
gas and C. ariakensis have created the impetus within industry to
promote triploid aquaculture of and even intentional introduction
21
22
Grabowski et al.
of diploid non-native species along the Atlantic coast of North
America.
Previous intentional and accidental introductions of commer-
cial fishery species have resulted in many well-documented nega-
tive impacts (Naylor et al. 2001 ). For example, the predatory oys-
ter drill, and both MSX and dermo. have been introduced unin-
tentionally through oyster introductions (Carlton 1999, Burreson et
al. 2000). Because of the risks associated with introducing a new
fisheries species, including possible introduction of non-native dis-
eases, competitors, and predators, importation of harmful mi-
crobes, and induction of competition with native species (Ruiz et
al, 2000, Naylor et al. 2001), assessing and contrasting the poten-
tial risks and benefits associated with any proposed introduction
should precede taking action. Here we present results of controlled
trials assessing how oyster consumers rate the palatability of the
two non-native species under consideration for introduction as
compared with C. virginica.
MATERIALS AND METHODS
Two series of tests were conducted to determine consumer
responses to non-native oysters grown in eastern North Carolina
and to compare those responses to native oysters. In both series of
tests, preferences among native, Crassostrea virginica (eastern
oyster) and non-native species, Crassostrea gigas (Pacific oyster),
and Crassostrea ariakensis (Suminoe oyster), were tested sepa-
rately for raw and cooked oysters. Regulations set forth by the
Shellfish Control Authorities in North Carolina mandated that we
inform participants that they were consuming raw or steamed oys-
ters, the location where oysters were grown (non-natives) or har-
vested (natives), and the species of oysters that were being offered.
Participants in the tests were drawn from the local coastal com-
munity surrounding Morehead City, NC and represented a diverse
range of ages (20-81 y old), professions, and knowledge of local
fisheries. Of the 31 individuals that participated in the first taste
test, a few also were among the 96 participants in the second. Each
participant completed and signed a waiver form regarding risk of
raw seafood consumption, completed a deinographic survey, and
provided information on oyster consumption. Finally, participants
were offered water and crackers to assist them to cleanse their
pallets between tasting oysters.
In the first series of tests (conducted on 21 August 2000), we
compared consumer responses to taste and appearance of C vir-
ginica to C. gigas. Triploid C. gigas (approx. 30 mm in length)
obtained from the Virginia Institute of Marine Sciences (VIMS)
had been cultured since February 2000 in plastic mesh vexar cages
held on racks above the sea bottom in Chadwick Bay, Onslow
County, North Carolina. C gigas achieved a length of approx. 80
mm by .August 2000 and were removed from the field and stored
in upwellers at the Institute of Marine Sciences in Morehead City,
North Carolina. Wild C. virginica oysters were harvested in Au-
gust 2000 from both the Newport River and Bogue Sound (Cart-
eret County, North Carolina). Participants were asked to rate un-
labeled raw or cooked oysters in paired contrasts. Separate trials
were performed for raw and cooked oysters. Some participants
were involved in both trials. To begin a trial, two oysters (either
raw or cooked) on the half-shell were presented to each participant,
who then rated each oyster's appearance and (separately) taste on
a scale of I (least desirable) to 5 (Fig. I). Each participant also
specified whether either oyster tasted unappetizing, and, if any
difference was perceived, which one tasted saltier, was more wa-
tery, and was more preferable overall (including an explanation for
any preference). A second pair of oysters was presented to each
participant, who then answered the same set of questions. One of
the pairs of oysters presented a contrast of the two species, whereas
Circle the most appropriate response
1 . Have you eaten raw oyster before? Yes
2. Approximately how many times a year do you eat raw oysters? 0 1 2
1st Test Oyster #
(A) vs. #
JBL
Yes
No
A
B
Yes
No
A
B
Yes
No
A
B
No
5 >6
1 . Rate tlie appearance of eacli oyster on a scale from 1 to 5 with 5 being the best and 1 being the worst.
A=12 3 45 B=12 3 45
2. Rate the taste of each oyster on a scale from 1 to 5 with 5 being the best and ) being the worst.
A= 12345 B= 12345
3. Did one or both of the oysters taste unappetizing?
Ifso whichone(s): A B Both
4. Did one oyster taste saltier than the other?
Ifso which one:
5. Did one oyster taste more watery than the other?
Ifso winch one:
6. If you preferred one oyster over the other briefly explain why.
Figure I, Survey form used in first taste test.
Consumer Ratings oi- Oysthrs 23
OYSTER TASTE PANEL
Panelist Code # Sample: Raw Steamed Date:
Procedure: Three samples of oysters (either raw or steamed) will be placed in front of you. We
would like for you to taste each of the oysters and evaluate them for their quality attributes by
answenng the questions listed below. Please rate each sample accordini; to their four diait code
by placin^ a mark across the unmarked line that best reflects your opinion, e.g.. like greatly (far
right), neither like or dislike (middle) and dislike greatly (far left).
Note that you are not required to chew or swallow the oyster samples. You may spit the sample
out at any time you need to into the cup provided. You are expected to drink (wash mouth)
between samples with water. If you feel a need to be less fatigued in terms of flavors, aromas and
textures blending together between samples, then you should eat crackers and drink some water.
Ql : When you eat oysters either at home or in a restaurant, what quality attributes are most
important to you?
Q2: How does the appearance of the samples appeal to you? What appearance characteristics do
you like? Dislike?
Dislike Greatly Like Greatly
Q3: How does the aroma of the samples appeal to you? What aroma characteristics do you like?
Dislike?
Dislike Greatly Like Greatly
Q4: How does the texture of the samples appeal to you? What texture characteristics do you
like? Dislike?
Dislike Greatly Like Greatly
Q5: How does the flavor of the samples appeal to you? What flavor characteristics do you like?
Dislike?
Dislike Greatly Like Greatly
Q6: What other attributes do you perceive in the samples?
Please dispose of any left over samples in the appropriate trash container. Be sure to turn your
sensory survey sheet to the project assistant when you leave the room. We appreciate your
time in this study! Results will be available from the project coordinator. THANK YOU!
Figure 2. Survey form used for second laste.
the other presented two C. virginica with one from each site to ters (one of each species, either all raw or cooked) on the half-
determine if grow-out location affected the test results. shell, and asked to rate the appearance, taste, texture and aroma of
The second series of taste tests (conducted on 6 and 7 February each oyster. To quantify a participant's ratings of each oyster, we
2002) evaluated consumer responses to appearance, aroma and measured the distance of the mark along the line, creating a scale
taste of C. virginica, C. gigas, and C. ariakensis. Triploid C. gigas from 0 cm (least desirable) to 10 cm (most desirable). We asked
and C. ariakensis (approx. 30 mm in length) had been obtained participants to indicate profession, age group and the frequency
from VIMS and planted at Chadwick's Bay (3 April 2001 ) and in with which they eat oysters (either raw or cooked, depending on
the Newport River (23 March 2001). Oysters were cultured using whether they were tasting raw or cooked oysters) to determine if
the cage and rack method and achieved harvestable size by January these factors influence their ratings.
2002. C. virginica was also harvested in January 2002 from Chad- We also quantified the wet and dry weights of ^0 replicate
wick's Bay and the Newport River in close proximity to culture oysters (80-1 10 mm shell length) for each of the three species to
operations. In this second set of taste tests, we requested more determine whether percent dry tissue or total dry tissue differed
subtle distinctions by asking participants to rate each oyster tasted among the three species. We determined that the shell length of
by placing a mark on a continuous line that ranged from least to oyster specimens did not vary among the three species with a
most desirable (Fig. 2). Each participant was presented three oys- one-factor analysis of variance (ANOVA: F, ,47. 1.06. P = 0.35).
TABLE 1.
Results of Wilcoxon signed rank tests comparing consumer ratings for taste and appearance of Crassostrea virginica with C. gigas in the
first series of taste tests.
All Part
cipants"
Infrequent Oyster Consumers
Taste .\ppearance
Frequent 0\
Taste
ster Consumers
Oyster Feature
Taste
.\ppearance
.\ppearance
Raw oysters
No. of 0 differences"
2
1
1
0
1
1
No. of ranlcs < 0"
4
11
3
8
1
3
No. of ranks > 0"
10
4
5
1
5
3
Z value
-1.38
-1.2.^;
-0.56
-2.19
-1.57
-0.63
P value
(1.17
0.21
0.58
0.03
0.12
0.53
Cooked oysters
No. of 0 differences"
2
2
1
0
1
2
No. of ranks < 0"
6
7
1
2
5
5
No. of ranks > 0"
7
6
3
3
4
3
Z value
-0.21
-1.12
-0.37
-0.27
-0.06
-1.26
P value
0.S.3
0.26
0.72
0.79
0.9.S
0.21
^ Raw data were analyzed collectively and then reanalyzed by subgroup to determine whether those participants who rarely eat oysters have
preferences from those that frequently eat oysters.
" No. of 0 differences indicates the number of participants that rated species equally, no. of ranks <0 indicate participants who rated C. gigns
than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginicn as better than C. gigas.
different
as better
3 1
Participant Category
b. Cooked Oysters
5
Appearance
Participant Category
Figure 3. Results from taste test 1. Taste and appearance ratings of (a) ra« and (b) cooked oysters (Crassostrea virginica vs. C. gigas) are
presented for the following participant categories: I ) all participants, 2 1 infrequent consumers of raw oysters, and 3 1 frequent consumers of raw
oysters. The test in which ('. virginica was ranked significantly lower than C. gigas is marked with an asterisk. Error bars indicate +1 SE.
Consumer Ratings of Oysters
25
Soft tissue was removed from each oyster, placed in a pre-weighed
aluminum pan. and weighed using a Mettler balance (0.001 g).
Tissue was then dried at 60°C in a drying oven for 48 h. and
weighed again to obtain a dry tissue weight (dry weight with pan
minus pan weight). The proportion of each oyster's soft tissue that
is biomass was calculated by dividing the dry weight (tissue
weight minus water weight) by the initial wet weight.
Slatisliial A luilyses
Results from the first taste test were analyzed using the Wil-
co.xon signed rank test. C. virginicci from Bogue Sound were first
compared with C. viri^inica from the Newport River. Because
rankings of native oysters from Bogue Sound and the Newport
River did not differ from each other (in taste: P = 0.97; in ap-
pearance: P = 0..^.^). we concluded that grow-out site did not
affect the taste of native oysters in our study and we analyzed
rankings for C. gigas versus C. virginica from both sites collec-
tively. Separate C. virginica versus C. gigas tests were conducted
for appearance and taste of raw and cooked oysters. Additional
tests were conducted to determine if results varied between groups
that ( 1 ) rarely and (2) frequently (three or more limes per year) eat
oysters to determine if the frequency with which participants eat
oysters affected preferences for native versus non-native oysters.
Results from the second taste test were also analyzed using the
Wilcoxon signed rank test to determine whether participants pre-
ferred the taste, appearance, or aroma of raw and cooked C. vir-
ginica better than C. gigas or C. ariakensis. Each measure of C.
virginica quality was first compared with C. gigas and then to C.
ariakensis for raw and cooked oysters. Two additional series of
Wilcoxon signed rank tests were conducted on the results of the
second series of taste tests (raw and cooked] to determine if rank-
ings of people that eat oysters less frequently differ from those that
often consume oysters. Finally, percent and mean dry tissue
weights of all three species were coinpared using separate one-
factor ANOVA tests. Cochran's test for homogeneity of variance
was perfomied for both response variables (Underwood 1981).
Student-Newman-Keuls (SNK) post hoc tests were conducted on
significant ANOVA results {P < 0.05) to determine which of the
three species differed from each other. The SNK test was selected
because we conducted a balanced experiment with a priori pre-
dictions and a fixed factor (Day and Quinn 1989).
RESULTS
First Series of Tests (C. virginica versus C. gigas)
Collectively, survey participants ranked the taste of raw C.
virginica slightly higher and its appearance slightly lower than C.
gigas, but neither difference was significant (Table 1; Fig. 3). Of
the 16 participants offered raw oysters, 10 preferred C. virginica.
three preferred C. gigas, and three had no preference. Only two of
the 16 considered C. gigas unappetizing and only one replied that
C. virginica was unappetizing. Of the nine raw oyster tasters who
rarely eat raw oysters, the appearance of C. gigas was ranked
significantly higher than C. virginica. but the taste ratings were
similar. Among the seven raw oyster tasters who frequently con-
sume raw oysters, the taste of C. virginica was rated slightly higher
than C. gigas: five of the seven preferred C. virginica. but low
sample size more than likely rendered this difference non-
significant (Table 1). Ratings of the appearance of the two species
did not differ among this subgroup of tasters.
Collectively, tasters of cooked oysters did not distinguish be-
tween species in taste or appearance (Table 1; Fig. 3). Of the 15
TABLE 2.
Results of Wilcoxon signed rank tests comparing consumer ratings for taste, appearance, and aroma of Crassostrea virginica h ith C. gigas
and C. ariakensis during the second series of raw oyster taste tests.
C". virginica vs. C. gigas
C
virginica vs. C
ariakensis
Oyster Feature
Taste
.Appearance
.Aroma
Taste
Appearance
.\ronia
All participants'*
No. of 0 differences"
2
3
15
5
2
16
No. of ranks < O"
22
35
31
32
40
35
No. of ranks > 0''
64
53
44
51
49
39
2 value
-4.75
-2.4
-0.88
-2.96
-0.14
-0.50
P value
<0.0001
0.02
0.38
0.003
0.89
0.62
Infrequent consumers of raw oysters
No. of 0 differences''
0
2
3
0
1
5
No. of ranks < 0"
5
13
15
9
14
15
No. of ranks > O''
24
17
14
20
17
12
Z value
-3.43
-O.ftI
-0.28
-2.32
-0.27
-0.99
P value
0.0006
0.54
0.78
0.02
0.79
0.32
Frequent consumers of raw
oysters
No. of 0 differences''
2
1
12
5
1
11
No. of ranks < O"
16
T)
16
T")
26
20
No. of ranks > O''
40
35
29
31
31
26
Z value
-3.65
-2.48
-1.29
-2.07
-0.35
-1.22
P value
0.()()()3
(1.1)1
0.2(1
0.04
0.72
0.22
■■ Raw data were analyzed collectively and then reanuly/ed by subgroup to delermuic v\hcther participants who rarely eat raw oysters liave dilferenl
preferences from those who frequently eat them.
No. of 0 differences indicates the number of participants who rated species equally, no. of ranks <0 indicate participants who rated the non-native species
as better than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginica as better than the non-native species.
26
Grabowski et al.
■ C. virginica
D C. gigas
■ C ariakensis
All Participants
Rarely Eat Oysters Frequently Eat Oysters
b. Appearance
10 1
■ C. virginica
a C. gigas
■ C. ariakensis
Al! Participants
Rarely Eat Oysters Frequently Eat Oysters
■ C. virginica
a C. gigas
■ C. ariakensis
All Participants Rarely Eat Oysters Frequently Eat Oysters
Figure 4. Results from taste test 2: raw oysters, (a) Taste, (b) appearance, and (c) aroma ratings of raw Crassostrea virginica. C. gigas, and C.
ariakensis for the following participant groups: I) all participants. 2| infrequent consumers of raw oysters, and 3) frequent consumers of raw
oysters. Tests in which C. virginica was ranked higher than non-nati\c oysters are marked with * for C. gigas and # for ('. ariakensis. Error bars
indicate +1 SE.
Consumer Ratings of Oysters
27
TAIU.E 3.
Results of Wilcoxon signed rank tests coniparinj; consumer ratings lor taste, appearance, and uronia of Crassuslrea yirf;iiiica with C. gigas
and C ariakeiisis during the second series of cooked oyster taste tests.
lire
C. virginica vs. C. gigas
C. virginica vs. C. ariakensi.
Oyster Feat
Taste
Appearance
Aroma
Taste
Appearance
Aroma
All participants'
No. of 0 differences''
3
3
15
3
3
14
No. of ranks < 0"
33
49
37
38
40
36
No. of ranks > 0"
54
39
38
47
47
38
Z value
-2.5.'i
-0.89
-0.23
-0.68
-1.22
-0.003
P value
(1.01
0.38
0.82
0.49
0.22
0.99
Infrequent consumers of
cooked oysters
No. of 0 differences"
0
2
4
0
2
3
No. of ranks < 0"
13
19
15
13
16
17
No. of ranks > 0'"
19
12
12
18
14
10
Z value
-1.98
-1.53
-0.32
-0.36
-0.51
-1.59
P value
0.05
0.13
0.75
0.72
0.61
0.1 1
Frequent consumers of cooked
oysters
No. of 0 differences''
3
1
11
3
1
11
No. of ranks < 0"
19
29
21
24
24
18
No. of ranks > 0"
35
27
26
29
32
28
Z value
-1.83
-0.15
-0.57
-0.90
-1.82
-1.26
P value
0.07
0.88
0.57
0.37
0.07
0.21
" Cooked oyster data were analyzed collectively and then reanalyzed by subgroup to determine whether participants v\ ho rarely eat cooked oysters have
different preferences from those that frequently eat them.
'' No. of 0 differences indicates the number of participants who rated .species equally, no. of ranks <0 indicate participants who rated the non-native species
as better than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginica as better than the non-native species.
participants tasting cooked oysters, seven preferred C. virginica.
six preferred C. gigas. and two had no preference. Only one of the
1 .5 considered cooked C. gigas to be unappetizing, whereas two
replied that C. virginica was unappetizing. Splitting participants
out into inexperienced and frequent eaters of cooked oysters failed
to detect any pattern of species preference in taste or appearance of
the cooked oysters (Table 1; Fig. 3).
Second Series of Tests (C. virginica versus C. gigas or C. ariakensis^
In the second taste test, raw oyster tasters collectively ranked
the taste of C. virginica significantly higher than both C. gigas and
C. ariakensis (Table 2: Fig. 4). Appearance of C. virginica was
rated significantly above C. gigas but not above C. ariakensis
(Table 2: Fig. 4). Neither of the paired species contrasts distin-
gLushed native from non-native oysters by aroma. Infrequent oys-
ter eaters ranked the taste of raw C. virginica significantly above
both C. gigas and C. ariakensis. but rankings by appearance and
aroma did not vary among the three species (Table 2; Fig. 4).
Frequent oyster eaters ranked the taste of raw C. virginica signifi-
cantly above both ni)n-native species and the appearance of C.
virginica over C. gigas but not different from C. ariakensis. Aroma
rankings did not differ in either contrast of pairs of oysters (Table
2; Fig. 4).
Tasters of cooked oysters collectively rated the taste of cooked
C. virginica significantly more than C. gigas (Table 3; Fig. 5) but
did not distinguish between cooked C. virginica and C. ariakensis.
Ratings of appearance and aroma did not differ between cooked
native and non-native oysters in any contrast. The subgroup
formed by infrequent consumers of cooked oysters also ranked the
taste of cooked C. virginica significantly better than C. gigas but
failed to distinguish between cooked C. virginica and C. ariakensis
(Table 3; Fig. 5). These relatively inexperienced oyster eaters did
not rate the appearance or aroma of native oysters differently from
non-native species. Finally, frequent oyster eaters ranked the taste
of C. virginica marginally above C. gigas but not significantly
higher than C. ariakensis. For these experienced oyster eaters,
aroma and appearance rankings did not differ significantly be-
tween cooked native and non-native oysters, though the appear-
ance of C. virginica was ranked marginally higher than C. aria-
kensis (Table 3; Fig. 5).
Dry Weight
Percent dry weight of soft tissues (dry weight/wet weight) did
not significantly differ among the three species (Table 4). Prior to
this analysis, percent dry weight data were transformed using a
square root transformation to remove heterogeneity among vari-
ance groups. Total dry tissue weight (g) of C. ariakensis was
significantly greater than that of C. gigas or C. virginica. and the
dry tissue weight of C. gigas was greater than that of C. virginica
(SNK post hoc comparisons; Fig. 6). Because average shell length
did not differ among species, this analysis reflects biomass for
oysters of a fixed range of harvestable lengths (80-1 10 mm).
DISCUSSION
As managers consider use of non-native species to enhance or
restore fisheries, they should weigh carefully the risks and poten-
tial benefits. Decisions on species introductions are driven by a
variety of social and political pressures, often with insulTicient
attention to potential ecological risks or economic benefits (An-
drews 1980). In North Carolina, it is unclear, for example, how
current market prices would adjust to an increase in oyster supply
(Lipton & Kirkley 1994). Oyster and clam markets in the state
have already endured low demand and reduced prices that threaten
the economic viability of both culture operations and wild harvest
28
Grabowski et al.
■ C. virginica
D C. gigas
■ C. ariakensis
All Participants
Rarely Eat Oysters Frequently Eat Oysters
b. Appearance
All Participants
Rarely Eat Oysters Frequently Eat Oysters
c. Aroma
10
o
o
■ C. virginica
' D C. gigas
\ ■ C. ariakensis
All Participants
Rarely Eat Oysters
Frequently Eat Oysters
Figure 5. Results from taste test 2: cooked oysters, (a) Taste, (b) appearance, and (c) aroma ratings of cooked Crassostrea virginica, C. gigas, and
C. ariakensis for the following participant groups: I) all participants, 2) infrequent consumers of cooked oysters, and }) frequent consumers of
cooked oysters. Tests in which C. virginica was ranked higher than non-nati\e oysters were marked with * for C gigas and # for C. ariakensis.
Error bars indicate +1 SE.
Consumer Ratings of Oysters
TABLE 4.
Ki'siills III ANOVA comparison of pcrctnl (lr\ tisMii' Hciyhl ol' Mif(
tissiKs and tiilal dr> tissue weight for Crasso^lrcii \irf;iitica. C. giaas.
and C. ariakeiisis.
Source of Variance
df
MS
F Value P Value
Percent dry tissue weight
Oyster species
Residual
Total dry tissue weight
Oyster species
Residual
2 0.001 1.3X
147 0.001
0.26
2 2.32 28.82y <0.()Oni
147 0.08
fisheries. Although the transport of C. ,?/,i;(i.v from the west coast
for sale in the eastern United States has increased since the col-
lapse of native stocks on the east coast, it is unclear whether
consumers in the eastern United States prefer a particular oyster
species (Lipton et al. 19921 and how such preferences may vary
with targeted market (e.g.. raw on the half-shell versus steamed,
etc.). In this study, we set out to identify (1) whether the taste of
non-nati\e oysters is acceptable to oyster consutners in North
Carolina and (2) whether consumer ratings differ and preferences
exist among raw and cooked C. virginicci, C. gigas. and C. ciria-
kcnsis. Our purpose was to begin the process of evaluating the
market potential of the two non-native species of oyster in North
Carolina and. by extension, the other east coast states where con-
sumers are accustomed to eating native oysters.
Although consumer ratings of taste and appearance provided no
consistent pattern of preference in the first taste test (i.e.. for taste
C. virginicci > C. gigiis. and for appearance C. gigas > C. vir-
ginica). the majority preferred raw C. virginicii more than C. gi-
gas. These findings suggest that consumer preference for raw oys-
ters may be dictated more by taste than appearance. Cooking re-
moved any indication of a difference between species in taste or
appearance, indicating that non-native C. gigcis may be suitable for
local cooked oyster markets. When asked, few participants con-
sidered either C. gigas or C. virginica unappetizing regardless of
C. ariakensis
C. virginica
C gigas
Species
Figure 6. Mean dry tissue weight (g) of Crassostrea virginica. C. gigas,
and C. ariakensis. Error bars indicate +\ SE. Results of SNK post-hoc
mean comparisons are Indicated with letters above the error bars, and
species with different letters above them are signitkantl) different at
P < (1.(15.
preparation (raw or cooked), implying that non-native C. gigas
might be acceptable. Fisheries managers may wish to assess next
whether consumer demand exists for an acceptable but less pref-
erable oyster and if lower preference implies a reduction in market
price before allowing introduction of C. gigas to the east coast.
The larger numbers of participants in the second series of tests
pro\ided greater ability to resolve differences among oysters and
included contrasts with the second non-native species. C. ariak-
ensis. Participants in the raw oyster tests collectively indicated a
strong taste preference for C. virginica over either non-native spe-
cies. This preference held regardless of whether consumers rarely
or frequently eat oysters. Because frequent consumers eat a dis-
proportionately large amount of the raw oysters consumed in
North Carolina, these results raise concern about the suitability of
either non-native species for local raw oyster markets. Though
appearance and aroma preferences were not as definitive, consum-
ers collectively preferred the appearance of raw C. virginica to C.
gigas. which raises further doubt about the marketability of raw C.
gigas on the east coast.
Tasters of cooked oysters in the second test exhibited weaker
preferences among oysters. Yet participants collectively, as well as
the subset who rarely consume oysters, preferred the taste of
cooked C. virginica more than C. gigas. and frequent consumers of
cooked oysters expressed a slight preference for the taste of
cooked C. virginica more than C. gigas. Consumers as a whole, as
well as the subset who frequently eat cooked oysters, did not
exhibit a taste preference for cooked C. virginica or C. ariakensis.
suggesting that C. ariakensis may be more suitable for steamed
and packaged oyster markets. Because the weight of C. ariakensis
oysters was double that of C. virginica of a given length and C.
ariakensis grows to market size much more quickly than the native
oyster (Calvo et al. 2001). the Suminoe oyster might be more
successful in markets that sell by meat weight. However, the high
costs of triploid aquaculture need to be considered in assessing the
economic viability of this industry. On the other hand, our results
show that the most widely marketed and consumed oyster in the
world. C. gigas. is not rated as high by North Carolina consumers
as the eastern native oyster. C. virginica. The alternative non-
native oyster. C. ariakensis. is rated at least as high and in some
contrasts higher than C. gigas. Thus, if the Suminoe oyster could
be produced at sufficiently low cost, then it should compete fa-
vorably with C. gigas for market share.
Because of serious environmental risks associated with intro-
ducing a non-native species as a self-replicating wild population or
even for culture as triploids. we argue that an analysis of economic
viability is necessary for responsible decision making by fisheries
managers. Such an analysis would include our new information on
consumer perceptions, ratings, and rankings of alternative species
of oysters under consideration for use. A complete economic
analysis to follow our study of consumer ratings and preferences
would involve a model to convert these consumer ratings into
prices. Additional costs of each type of culture and impacts on
market supply and demand must also be assessed. Collapsing oys-
ter fisheries along the Atlantic coast and declining water quality
collectively have eroded consumer demand for oysters, such that
current oyster markets are probably less elastic. Therefore, an in-
crease in supply from successful introduction of non-native oysters
in North Carolina could result in a corresponding decrease in oys-
ter prices (Lipton & Kirkley 1994). especially within smaller raw
oyster markets. Biological information on growth and mortality
rates of non-native oyster species must be acquired and compared
with nati\e oysters. Given that non-native oysters were generally
30
Grabowski et al.
less preferable than the native eastern oyster in our study and that
producing cultured oysters from triploid seed is expensive, suc-
cessful culture of triploid oysters would require a substantial bio-
logical benefit in the form of shorter time to market and/or higher
survival. Inclusion of this information into a comprehensive eco-
nomic analysis of potential benefits and costs of introduction
would enable managers to assess whether the environmental risks
are worth taking. Finally, restoration of any oyster will have posi-
tive effects in restoring water quality and compensating for estua-
rine eutrophication (Jackson et al. 2001. Newell et al. 2002), such
that this ecosystem benefit should be included in a complete eco-
nomic evaluation of any potential oyster introduction. If the intro-
duced oyster were to form reefs, then further ecosystem benefits of
habitat enhancement (Lenihan et al. 2001) should also be incor-
porated.
ACKNOWLEDGMENTS
The authors thank Rachael Wagaman, Christina Tallent. David
Gaskill, Hal Sumnierson. and Chris Stewart for culturing the oys-
ters, assistance conducting the two food surveys and quantifying
oyster tissue weights. Stan Allen, Jr., of the Virginia Institute of
Marine Sciences provided disease-free triploid seed and much
guidance. This research was supported by the North Carolina Gen-
eral Assembly through the Rural Development Foundation and the
Fishery Development Foundation and the North Carolina Depart-
ment of Natural Resources.
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marine biology and ecology. Oceanogr. Mar Biol. Anna. Rev. 19:513-
605.
Jo:inml of Slicll/isl, Rcscairh. Vol. 22. No. 1. .M-.^S. 20(13.
TAXONOMIC STATUS OF FOUR CRASSOSTREA OYSTERS FROM CHINA AS INFERRED
FROM MITOCHONDRIAL DNA SEQUENCES
ZINIU YU,'"* XIAOYU KONG,' LIUSUO ZHANG,' XIMING GUO.- AND JIANHAI XIANG'
^College of Fisheries. Ocean University of Qingihio. Qingclao 266003. Peoples Republic of China:
-Haskin Shellfish Research Laboratory. Institute of Marine ami Coastal Sciences. Riitiiers University.
Port Norrls. New Jersex 0S.U9: and ''Institute of Oceanology. Chinese Academy of Sciences, Qingdao
266071. Peoples Republic of China
ABSTRACT It has been presumed ihat there are tour eoaiiiion Cra\snstrea oyster species along the eoast ol China; the Pacitic oyster
(Crassostrea gigas), Zhe oyster (C plicatula). Suminoe oyster (C ariakeiisis). and Dalianwan oyster (C. talienwbanensis). Classifi-
cation and species identification of these Crassostrea oysters have been difficult because of morphologic plasticity. In this article,
phylogenetic analysis was performed to clarify taxonomic status of these species using mitochondrial DNA sequence data. Nucleotide
sequences of a 443-bp fragment of ribosomal RNA gene and a 579-bp segment of cytochrome c oxidase I gene were obtained through
sequencing and used for analysis. Genetic distances among the four species, using C. virgiiiica as outgroup, were computed based on
the sequence data, and phylogenetic trees for the five species were generated. The divergence between C. gigas and C. talienwhanensis
was very low. as was that between C. pticaiula and C ariakeiisis. Phylogenetic analysis showed that haplotypes of C. gigas and C.
lalieimhaiieiisis clustered in one clade and those of C. plicaluta and C ariakeiisis in another one. Our data suggest that C. gigas and
C latieimlumensis may be the same species. However, the lack of divergence between C. plicaltila and C. ariakeiisis samples may
indicate that the C. plicaliila specimen we sampled could actually be a morph of C. ariakeiisis living in high salinity habitats. More
work is needed for confirmation.
KEY WORDS: Crassostrea oysters, taxonomy, phylogenetic analysis, 16S rDNA, COI gene, nucleotide sequences
INTRODUCTION
Ainotig the over 20 species of oysters recorded in China, four
Crasso.strea species are most cotnmon and of commercial impor-
tance; the Pacific oyster (Crassostrea gigas), Zhe oyster (C. pli-
catula). Suminoe oyster (C. ariakeiisis). and Dalianwan oyster (C
talienwhanensis; Zhang et al. 1956, Qi I9S9). The Pacific oyster,
which occurs naturally along the coast of China, is a well-
recogni/ed species. However, most of the Pacific oysters cultured
in China were originally introduced from Japan or Korea (Wang et
al. 1993). The Zhe oyster is commonly found along the entire coast
of China. It is relatively smaller in body size than the Pacific and
Suminoe oysters and thin-shelled (Qi 1989, Guo et al. 1999).
Suminoe oysters are also distributed along most of the coast of
China with two major populations, one in the estuaries of Yellow
river and the other in Guangxi and Guangdong in southern China.
It can tolerate a wide range of salinity but prefers low-salinity
estuaries and riverbeds (Torigoe 1981. Li & Qi 1994). The Dalian-
wan oyster occurs mainly in areas along the coast of Liaoning and
Shandong provinces in the North (Zhang et al. 1956, Qi I9S9).
Because of the morphologic plasticity, there have been dis-
agreements about the taxonomic status of the four Crassostrea
types and difficulties in their identification. Some believed that the
Pacific and Dalianwan oysters are different species (Zhang et al.
1956, Qi 1989), whereas others argued that the Dalianwan oyster.
described by Zhang et al. ( 1956), is the Pacific oyster, or a variety
of Pacific oyster (Torigoe 1981, Li & Qi 1994). In addition, some-
times the discrimination of Pacific and Suminoe oysters was am-
biguous with shell morphology, although it is distinguishable w ith
some body anatomic features (Li & Qi 1994). The most common
oysters found in the rocky intertidal zone and extensively cultured
in the south are generally believed to be the Zhe oyster, although
♦Corresponding author. Tel: 856-785-0074; Fax: 856-7S5-I544; E-mail:
carlzyu @ hsrl.rutgers.edu
Li and Qi (1994) assumed it was the Pacific oyster. Liu et al.
{ 1 998 ) compared RAPD data from several Crassostrea species and
concluded that the Dalianwan oyster, Zhe. and Pacific oysters were
sister species with each other.
Because of this confusion, further study, especially with DNA
markers, is needed. DNA polymorphisms are useful tools for eco-
logical, genetic, and evolutionary studies of both terrestrial and
marine organisms, and DNA sequences can be used to detect dif-
ferences among species, populations, or individuals. Proper iden-
tification of oyster stocks will assist management, including con-
servation and the sustainable use of these resources. Past efforts to
investigate and identify differences among populations and species
of oysters along the coast of China have provided useful but in-
conclusive information {Liu et al. 1998, Yatig et al. 2000).
Because of its fast sequence evolution and inaternal. nonrecom-
bining nature of inheritance in animals, mitochondrial genes have
proved a powerful tool in phylogenetic studies and species iden-
tification (Banks et al. 1993, Littlewood 1994, Jozefowicz et al.
1998, Lapegue et al. 2002). The I6S rRNA and COI gene frag-
ments are popular choices for phylogenetic analysis (O'Foighil et
al. 1995, O'Foighil et al. 1998. Canapa et al. 2000). In this study,
mitochondrial 1 6S rRNA and COI gene fragments from these four
putative species were amplified and sequenced for phylogenetic
analysis.
MATERIALS AND METHODS
Sampling and Polymerase Chain Reaction (PCR) Amplifications
Crassostrea gigas samples (eight specimens) were obtained
from a hatchery broodstock in Shandong province; C ariakeiisis
samples (seven individuals) were collected from estuaries of the
Yellow River, in Yantai, Shandong province, which is a typical
habitat of this species in north China. C talienwhanensis was
sampled from Dalian (five individuals). Liaoning province and
Rongcheng (five individuals). Shandong province. C. plicatula
31
32
YU ET AL.
samples were collected from Qingdao (five specimens). Shandong
province and Wenzhou (five specimens). Zhejiang province. Sam-
pling sites are showed in Figure 1 . C. virf^iiiica was collected from
Delaware Bay in the United States. Morphologic identification was
made according to that described in Zhang et al. (1956). Qi (1989),
Torigoe (1981). and Li and Qi (1994).
Total DNA was e,xtracted from mantle tissue using an extrac-
tion kit (Pure Gene, Centra, USA). Fragments of the 16S rDNA
and COI gene were amplified using two pairs of universal primers:
1 6sar-L/ 1 6sbr-H: 5 ' -GCCTGTTTATCA AAA ACAT-3 75 ' -
CCGGTCTGAACTCAGATCACGT-3'(Palumbi 1991 );
COIL 1 490/CO1H2 1 98: 5 '-GGTCAACAAATCATAAAGATAT-
TGG-37 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'
(Folmer et al. 1994).
Amplification of the products was performed using a PTC- 100
thermal cycler (MJ Research. USA). The 100-p.L amplification
reaction contained 2.0 niM MgCK; 200 (j.M of each dNTP: 0.2 (xM
each primer; 2.5 p.L of template DNA; and 2.5 units of Taq poly-
merase (Sangon. Canada) with supplied buffer. For all amplifica-
tions, hot-start PCR was initiated by addition of polymerase and
primers after an initial 2-min denaturization at 80°C. The PCR
cycling profile was as follows: 35 cycles at 94'C/45 sec. 48°C
(COI) or 50°C ( I6S)/1 min and at 72°C/1 mm. with a final exten-
sion at 72"C for 7 min.
Sequencing
PCR products were purified using UNIQ-5 Column PCR Prod-
uct Purification Kit (Sangon. Canada), ligated into pMD18-T Vec-
tor by following instniction of Takara DNA Ligation Kit ver.2
(Takara. Japan) and used to transform competent JM109 Escheri-
chia coli cells using standard protocols. Recombinant colonies
were identified by blue-white screening. Inserts of the correct size
jr^
VJliaoning J'
'NortK
KorSa
BEIJING^JJ
(^^^ 'On Inn y
"X
/hebei
/SHANDONG 1 j/
/fiurwo
1
o^^-i«'Ss4^ a fi y u 03 s cmj
^
r^JIANGSU
, anhV?^*''"'"
^^/>
'^'i/^-y ZHEJIANG
UIAN
K /^^ •
IC hii IJ
GXI^ ^/
Figure \. .V map nt sampling area »ith sampling sites underlined.
were detected via restriction enzyme digestion by EcoRI and
HiiicHU. Vector DNA containing the desired insert was further
purified using Pharmacia EasyPrep Kit. Sequencing was per-
fonned for both strands of every sample on an ABI PRISM 377XL
DNA Sequencer using ABI PRISM BigDye"^"^ Terminator Cycle
Sequencing Ready Reaction Kit w ith AmpliTaq DNA Polymerase.
FS (Perkin-Elmer. USA).
Dala Analysis
The 16S and COI sample sequences, along with those already
obtained for C. gigcis and C. ariakensis (0"Foighil et al. 1995.
1998; courtesy of Dr. D. OToighil) were aligned with CLUSTAL
W (Thompson et al. 1994). For clarity and convenience in com-
paring with other published sequences, the sequences were
trimmed to the same length as published sequences after align-
ment. Parsimony analysis was made with Phylip (Ver.3.56C.
Felsenstein 1989) using the program DNAPARS with C. virginica
as the out-group. Bootstrap analysis with 1000 replication was
performed by the SEQBOOT and CONSENSE programs. Consen-
sus phylogenetic trees were drawn with DRAWGRAM program in
the Phylip package. Pair-wise sequence divergence between hap-
lotypes and species were estimated by the DNADIST program of
Phylip according to Kimura's two-parameter model (Kimura
1980).
RESULTS
A PCR fragment of 488 bp from the mitochondrial IdS ribo-
sonial gene and a fragment of 649 bp from the mitochondrial COI
gene were obtained and sequenced for 37 individuals of five spe-
cies (including two C. virginica specimens). Figure 2 shows the
alignment of 1 68 sequences of the seven haplotypes detected
among all specimens in this study, along with those of C. gigas and
C. ariakensis from O'Foighil's study. Eight specimens of C. gigas
and 10 of C. plicatitla exhibited only one genotype, whereas seven
C. ariakensis and 10 C. lalienwhanensis individuals had two hap-
lotypes each. The two haplotypes of C. taliemvhanensis came from
different sampling locations. Including the outgroup. 80 nticleotide
positions were variable in the 16S data set. Six insertion/deletion
sites were detected between C. virginica and all other haplotypes.
Similarly, the alignment of the seventeen COI haplotypes de-
tected in our study and those two of C. gigas and C. ariakensis
from O'Foighil's study are shown in Figure 3. The 17 haplotypes
in our study included one for C. gigas (gigas 1. 8 individuals),
seven for C. plicatula (plical. 2. 3. 6 and 7. one individual for
each; plica4. three individuals; plica5. two individuals), three for
C. ariakensis (ariakenl. 4 individuals; ariaken2. two individuals
and ariaken3. one individual), five for C. ralienwhanensis
(talienwl. 2 and 3. one individual each; talienw4. four individuals;
talienw5. three individuals), and one for C. virginica (virgl. two
individuals). Including the outgroup. 170 positions are variable.
No insertions/deletions were detected for this protein-coding gene
fragment.
Pair-wise genetic distances of 16S sequences among all nine
haplotypes and those of COI sequences among all 19 haplotypes
were computed, then the mean genetic distances were obtained
(Table 1 ). In the 16S sequence, the genetic divergence between C.
gigas and C. talienwhanensis was low. 0.81%, and so was that
between C. ariakensis and C. plicatula. 0.13%. The sequence di-
vergences between C. gigas or C. ralientvhanensis and C. aria-
kensis or C. plicatula were higher, ranging from approx. 1.74 to
Taxonomic Status of Cr.assostrea Oysters
33
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
gigasl
talienwl
gigasO
talienw2
plical
ariakenl
ariaken2
ariakenO
virgl
80
GCAATACCTG CCCAGTGCGA AATATTACTG TAAACGGCCG CCCTAGCGTG AGGGTGCTAA GGTAGCGAAA TTCCTTGCCT
C . ATAAGTC . . C T .
160
TTTGATTGTG GGCCTGCATG AATGGTTTAA CGAGGGTTTG ACTGTCTCTA AATTTTTTAT TGAAATTGTA CTGAAGGTGA
.A . .
.A . .
.A . .
.A . .
.A . .
.T G.
240
AGATACCTTC ATTTAAAAGT TAGACAAAAA GACCCCGTGC AACTTTGAAA A--TTAACTT TATTCAGGAG TAAAAGATTT
. .A. .
. .A. .
. .A. .
. .A. .
. AAG.
.GC.A.G. .G A.
320
TTAGGTGGGG CGCCTAGAAA GCAAG-TCTA ACCTTT-CTG AATAACT--A ACTCTTTCCG GATTTGACCC GATTATATTC
. -C.
. -C.
. AA . T C . GT .
. C.TT.--.
. .T. .ATA.
GT
.T. . .AA.TA
400
GATCATAGGA GAAGTTACGC CGGGGATAAC AGGCTAATCC TTTAGTAGAG TTCGTATTGG CTAAAGGGAT TGGCACCTCG
443
ATGTTGAATC AGGGATAATA GCTTCAAGGC GTAGAGGCTT TGA (8)
(5)
(7)
(5)
(10)
(5)
(2)
(5)
(2)
Figure 2. Mignnu'rit of seven oyster haplotypes of a 443-bp fragment of the mitochondrial I6S rDNA obtained in this study (C virgiiiica as
oulgroup) «ith published sequences for (. gigas and ('. ariakensis (O'Foighil et al. 1995, 1998). gigasO and ariakenO designate the sequences
of C. gigas and C. ariakensis from O'Foighil's study, respectively. Haplotype names are abbreviated as: gigas for C gigas. talienw for ('.
talienwhaiiensis. plica for ('. plicaliilu. ariaken for C. ariakensis. and virg for C. rirginica. Additional haplotypes per species are numbered
consecutively. Dots indicate nucleotide identity to the first sequence presented, gigasl. Dashes indicate inferred nucleotide indels relative to C.
rirginica. The number of individuals observed for each haplotype is indicated in parentheses at the end of sequence.
2.45%. The same pattern appeared in the COI data set: the coire-
sponding numbers were 1.08% between C. gigas and C. talien-
whanensis. 0.59% between C. ariakensis and C. plicaluUi. and
approx. 10.72 to 11.43% for the same comparisons mentioned
above. It is worth noting that the COI sequence was more variable
than the 16S sequence.
Consensus phylogenelic trees based on a parsimony analysis of
the 16S and COI fragments sequenced are presented in Figures 4
and 5. respectnely. Two groups (clades) in the 16S tree were
clearly distinguishable: C. ariakensis and C. pliiatida vs. C. gigas
and C. talienwhancnsis. whereas three groups (clades) were ap-
parent in the COI tree: (1) C ariakensis and C. plicatula: (2) C.
gigas and C. laliemvhanensis: (3) C. ariakensis from O'Eoighii's
study.
DISCUSSION
Oysters are among the most extensively studied and morpho-
logically variable marine invertebrates. However, our knowledge
of oyster phylogeny and systematics is still limited. There had been
over one hundred recorded species of oysters until 1970s, but two
thirds of them could be synonymous w ith each other according to
34 YU ET AL.
80
gigasl GCTGTTCTTG CGGGAACTAG GTTTAGGTCT CTTATTCGTT GGAGACTTTA TAACCCTGGA GCTAAGTTTT TAGACCCCGT
talienwl
gigasO
talienw2
talj.enw3
talienw4
talienw5
plical
ariakenl
plica2
plica3
plica4
ariaken2
plica5
ariakenS
plica6
plical
ariaKenO
virgl
gigasl
talienwl
gigasO
talienw2
talienw3
talienw4 C. .G.
G
r
G
r
-G
A
G
.A. .
r
r
G
. .C.
.G
.A.
G
.A. .
r
r
G
. .C.
.G
A
G
.A. .
r
r
G
. .c.
T
p^
.G
A
G
.A. .
r
r
G
. .c.
T
ft
.G
A
G
.A. .
r
r
G
. .c.
T
A
.G
C. . . .
A
G
.A. .
r
T
r
G
. .c.
.G
A
G
.A. .
r
r
G
. .c.
.G
.A.
G
.A. .
r
T
c
G
c
T
ft
.G
A
G
.A. .
r
T
r
G
. .c.
.G
A
G
.A. .
r
T
r
G
. .c.
. .c
TA.
.T.
GCA
. . .C. .A
.A.
TT
_G. .
r
A
G
c
T
. .A. . .T.A.
GACTTATAAT
.G. .C. .
GTTGTAA
T
CTAGGCATGC GTTGGTTATG
.A. .T. .A. .
ATTTTTTTCT
. .CT G
TTGTTATACC
A
TGTAATAATT
G. .T. .
160
GGGGGGTTTG
C
talienwS C. .G.
plical A G. . A. .G.
ariakenl A G. . A. .G.
plica2 A G.. A..G.
plica3 A G.. A..G.
plica4 A G. . A. . . .
ariaken2 A G. . A..G.
plicaS A G. . A. .G.
.A,
A.
. .G. .
A.
.G. . .
.A.
A.
. .G. .
A.
.G. . .
A. .C. . .
. .C
.C.
.A.
A.
.A.
A
C.
,T.
G.
. .C
TGTG. . .
. .c
. .T. .
,G. .
.G.
.T.
, .C. .
.A.
.A. .
.C. .
. .G. .
A.
.T. .
240
GTAACTGGCT
TATCCCTTTG
ATGCTTCTAG
TAGCAGACAT
GCAATTTCCT
CGATTAAATG
CATTTAGATT
TTGAGTTTTG
A ' '. . .
_ A
. . .T.
A
.A. .
. .T.
0
r
. . GC . .
, . C
c
. . .T.
A
.A. .
. .T.
A
r
. . GC . .
. .C .
c
.T.
A
.A. .
. .T.
B
r
. .GC. .
. C
c
. . .T.
.A
.A. .
. .T.
A.
. .GC. .
. .c.
.c. . .
. . .T.
A
.A. .
T
A
r
. .GC. .
. .c.
.c. . .
. . .T.
A
.A. .
T
A
r
. . GC. .
. .c.
.c. . .
. . .T.
A
.A. .
T
A
r
. . GC. .
. .c.
.c. . .
. . .T.
A
.A. .
T
A
r
. . GC . .
. .c.
.c. . .
. . .T.
.A
.A. .
A.
.c.
. .GC. .
. .c.
.c. . .
. . .T.
A
.A. .
. .T.
A
r
. . GC . .
. .c.
c
.A. .T. .
.A.
.A
.A. .
. .T.
.G.
. G. . .
C.
.G.
T. .
. . .T.
.GC
.T
. .A,
GA. .
.G.
.G.
.0.
.T.
C.
.A. . .
ariaken3 A G. . A. .G.
plica6
plica7
ariakenO
virgl
gigasl
talienwl
gigasO
taiienwZ
taJ ienw3
talienwj
talienwS
plical
ariakenl
plica2
plica3
plica4
ariaken2
plicaS
ariaken3
plica6
plica7
ariakenO
viryl
320
gigasl CCAGGGTCTC TTT.ATCTTAT GCTTATGTCT AACATTGTAG AAAACGGAGT TGGGGCAGGG TGAACAATTT ACCCTCCTTT
talienwl
gigasO
talienw2
talienw3
talienw4 C G
talienwS C
plical A.. A T G.
ariakenl A. .A T G.
plica2 A. .A T G.
plica3 A.. A T G.
plica4 A. .A T G.
ariaken2 A. .A T G.
plicaS A. .A T G.
ari3ken3 A.. A T G.
plicae A. .A T G.
plica7 A T G.
ariakenO C..A TC GT..G.. C A
virgl AT .GCTG..A.. AT . G A . . T . . . . CT . . G . GA T....A C GC .
Figure 3. Alignment of 17 oyster haplot.vpe.s of a 579-bp fragment of the mtCOI gene obtained in this study (C virginica as outgroup) with
published sequences for C. gigas and C. ariakensis (O'Foighil et al. 1995. 1998). gigasO and ariakenO designate the sequences of C. gigas and
C. ariakensis from O'Foighil's study, respectively. Haplotype names are abbreviated as: gigas for C. gigas. talienvv for C. talienwhaneiisis, plica
for C. plicalula, ariaken for C. ariakensis and virg for C. virginica. Additional haplotypes per species are numbered consecutively. Dots indicate
nucleotide identity to the first sequence presented, gigasl. The number of individuals observed for each haplotype is indicated in parentheses at
the end of sequence.
GG
C
GG
_ _ . . . r. _
GG
GG
. . .c
GG
C
GG ....
r
GG
GG
GG
GG
Taxonomic Status of Crassustrea Oysters 35
400
gigasl ATCAACTTAC TCTTATCATG GAGTTTGTAT AGACCTTGCA ATTCTAAGCC TTCACCTTGC TGGTATTAGC TCTATTTTCA
talienwl
gigasO
talienw2
talienw3
talienw4 C T C.
talienwS C T C.
plical G..G C. G T TT .A A.. ..
ariakenl G..G C. G T....TT .A A
plica2 G..G C. G T....TT .A A
plica3 G..G C. G T....TT .A A
plica4 G..G C. G T....TT .A A
ariaken2 G..G C. G T....TT .A A
pllcaS G..G C. G T....TT .A A
ariakenB G..G C. G G T....TT .A A
plicae G..G C. G T....TT .A A
plica7 G..G C. G T....TT .A A
ariakenO G..C..C TT.A A.
virgl G TT C C.. G..TT....C . . . T . . . . GT .A...T.A.. A....
480
gigasl GGTCAATTAA TTTCATAGTA ACGATTAGAA ATATGCGATC TGTTGGGGGC CATTTACTAG CACTATTCCC TTGATCTATT
talienwl
gigasO
talienw2
talienw3 G
talienw4 T T.. C
talienwS T.
plical T A.
ariakenl T A A T.G. .G..G..T.. C
plica2 T A A T.G. .G..G..T.. C
plicaB T A A T.G. .G..G..T.. C
pllca4 T A A T.G. .G..G..T.. C
ariaken2 T A A T T.G. .G..G..T.. C
plicaS T A A T.G. .G..G..T.. C
ariakenS T A A T.G. .G..G..T.. C
plicae T A A T.G. .G..G..T.. C
plica7 T A A T.G. .G..G..T.. C
ariakenO T C..T..G GT.G. .G T.. A..G C
virgl ....T T C C T ..CA..T T G..A...
560
gigasl AAGGTTACTT CATTCTTGCT TTTGACTACT CTCCCAGTGT TAGCTGGAGG TCTTACTATA CTTTTGACTG ATCGTCATTT
talienwl
gigasO
talienw2
talienwS
talienw4 G
talienwS G
plical TC.A A T G.. C G C
ariakenl TC.A A T G. . C G C
plica2 TC.A A T G.. C G C
plicaS TC.A A T G. . C G C
plica4 TC.A A T G. . C G C
ariaken2 TC.A A T G.. C G
plicaS TC.A A T G. . C G
ariakenB TC.A A T G.. C G
plicae TC.A A T G.. C G
plica7 TC.A A T G.. C G
ariakenO ..A..C..A T..A A..A..C ..T..G..AC C..G C..G
virgl ..A..G..A C... GC.T..C..G ..A..T..TC C. G G . . CC . T A
579
gigasl TAATACCTCT TTTTTTGAC (8)
talienwl ( 1 )
gigasO (20)
talienw2 ( 1 )
talienw3 (1)
talienw4 C . . . ( 4 )
talienwS C. . . (3)
plical . . .C. .G T (1)
ariakenl ...C..G T (4)
plica2 . . .0. .G T (1)
plicaS . . .C. .G T (1)
plica4 ...C..G T (3)
ariaken2 ...C..G T (2)
plicaS ...C..G T (2)
ariaken3 ...C..G T (1)
plicae . . .C. .G T (1)
plica7 . . .C. .G T (1)
ariakenO ...C..G T (5)
virgl A. .G (2)
Figure 3. (Continued)
Harry ( 1985 ). The inability to clearly classify closely-related oys- proven to be a powerful tool for oyster identification and discrinii-
ters has created problems for classification and species identifica- nation between closely related species or between nati\e and non-
tion worldwide. native species. Banks et al. (1993) discriminated closely related
Although morphologic identification of oysters often turned out oyster species, C. gigas and C. sikamea. via mitochondrial I6S
to be unreliable or ambiguous. mtDNA sequence analysis has rRNA gene sequencing and PCR/RFLP analysis. O'Foighil et al.
36
YU ET AL.
TABLE I.
Pair-wise sequence divergence (mean genetic distances! according to Kiniura's two-parameter model iKimura 198(1) among the five species
based on 443-nucieotide 16S rDNA and 579-nucleotide COI sequences.
16S
COI
Species
1
2
3
4
5
ft
1
2
3
4
5
6
C. gigas
C. lalienwhanensis
0
0.0()81
0
0
0.0108
0
C. plicanda
0.0233
0.0174
0
0.1113
0.1072
0
C. ariakensis
0.024?
0.01 S5
0.0013
0
0.1143
0.1 100
0.0059
0
C. ariakensisO
0.0450
0.04S7
0.0444
0.0462
0
11.1619
U.1639
0.1652
0.1691
0
C. virginica
0.1636
0.1 60S
0.1654
0.1673
0.1937
0
0.2569
0.2573
0.2510
0.2513
0.2849
0
C. ariakensisO indicates S. ariakensis sequence from OToighil's studies (1995. 1998).
Pair-wise comparisons yielding low genetic distances estimates are showed in boldface.
(1995) succeeded in distinguishing C. viri>iiuco from two closely
related oysters. C. gigas and C. ariakensis. and C. gigas from C.
ariakensis by employing sequencing and PCR/RFLP analysis of
pan of a fragment (443 bp) of the 16S rRNA gene. Sequence data
revealed that C. gigas and C. ariakensis showed higher levels of
similarity to each other (95%) than to C. virginica (84-86%).
Comparison of a 579-nucleotide fragment of the COI between the
Portuguese oyster. C. angiilala. and several Japanese oysters were
made by OToighil et al. (1998). showing that Portuguese oyster
haplotypes clustered firmly within a clade of Asian congeners and
were closely related to C. gigas (but not identical). This result
supports an Asian origin for the Portuguese oyster.
Reportedly, there are over 20 recorded species of oysters oc-
curring along the coast in China (Zhang et al. 1956, Qi 1989). and
for some of them classification and identification have been prob-
lematic or uncertain. Based upon extensive anatoinic studies of
almost all oyster species in China. Li and Qi ( 1994) concluded that
there were 15 species of oysters, and claimed that identification of
a few oyster species was clarified. Most of the species are rare and
found in South China Sea. However. Even for the four common
species (the Zhe oyster. Pacific oyster. Suminoe oyster, and
Dalianwan oyster), it is often not empirically easy even for marine
zoologists sometimes, to distinguish them clearly. This has caused
inconveniences and difficulties in broodstock management and
aquaculture practices. If the Dalianwan oyster is a discrete species.
ariakenO
— ariakenl
— ariaken2
— plical
talienw2
talienwl
gigasi
— gigasO
separate stock conservation and management should be applied.
Accordingly, clarification of the Zhe oyster's status would also
help oyster aquaculture practices. These are widespread concerns
for the oyster fishery along the coast of China
The molecular data provide some clarification on the species
status and phylogenetic relationships of these four species. For
Dalianwan and Pacific oysters, the 16S data show close similarity
between the samples of these two species, and the haplotypes of
Dalianwan and Pacific oyster formed a clear clade in the phylo-
genetic tree. This relationship is strongly supported by the COI
data set. in which all five haplotypes of the Dalianwan oyster and
the only haplotype of the Pacific oyster clustered closely. This is
also supported by the evident similarity in moi-phology between
these two species. The Dalianwan oyster samples were collected
from t> pical distribution areas, identified carefully according to the
plica5
— virgl
Figure 4. A consensus phylogenetic tree based on parsimony analysis
of 443-nucleotide mt I6S rDNA fragment according to Kimura's
model with C. virginica as an outgroup.
talienw4
talienwS
■ virgl
Figure 5. .\ consensus phylogenetic tree based on parsimony analysis
of 579-nucleotide mt COI gene fragment according to Kimura's model
with ('. virginica as an outgroup.
Taxonomic Status of Chassostrea Ovstbrs
37
descriptions of Zhang et al. ( 1956) and Qi ( 1989). Although there
are some morphologic differences compared with the Pacific oys-
ter, Dalianwan oysters share some morphologic characteristics
with Pacific oysters as described by Zhang et al. ( 1956) and Qi
(1989). A similar situation exists in scallops Pecten imiximus and
P. jacoheiis, where they share highly similar morphologic features
but have a surprisingly close genetic distance based on 16S se-
quences (Canapa et al. 2000). Our molecular data suggest that
Dalianwan and Pacific oysters belong to the same species, which
supports Li and Qi"s (1994) conclusion based on anatomy studies.
Results for the Zhe and Suminoe oysters are rather surprising.
The divergence between the two is much less than expected. The
genetic distances between them are as low as 0.1 39i- (for 16S) and
0.59% (for COl), even lower than that between the Dalianwan and
Pacific oysters (0.81 and 1.08'^H. They share a high degree of
similarity in these two gene fragments. In contrast, they showed
higher divergence from the Pacific and Dalianwan oysters in both
the 16S and the COl sequence data, though more strongly in the
latter. Also, haplotypes of the Zhe and Suminoe oysters clustered
in a single clade in both trees. This result is different from that
generally concluded from morphologic data. Morphologically, the
Zhe and Suminoe oysters are easy to distinguish in most cases.
Therefore, caution should be taken for the concern of status of
these two species. A possible explanation could be as follow, the
"Zhe oysters" we sampled could actually be a morph of Suminoe
oysters living in high salinity habitats. Because ecologically the
Suminoe oyster has a wide distribution and can tolerate a wide
range of salinities, morphologies could vary in different habitats.
Samples collected from the habitats other than an estuary may look
different from the Suminoe oysters from a typical habitat. It is
possible that Suminoe oysters from high salinity area and on rocky
shores are mistakenly classified as Zhe oysters because of mor-
phologic plasticity. It has been shown that the Zhe-like small oys-
ters found in the rocky intertidal zones of northern coast, once
removed to more productive waters, could grow to a bigger size,
which resemble the Suminoe oysters from an estuary habitat (R.
Wang, personal comm.). To confirm either of these possibilities, a
more extensive sampling and sequence analysis throughout their
natural range are needed.
An interesting finding from this study is that O'Foighil's COl
sequence of the Suminoe oyster showed a significant divergence
not only from that of the Dalianwan-Pacific oysters, but also of the
Suminoe-Zhe oysters. The divergence may be due to the fact that
mt protein-coding genes like COl are usually more variable than
iDNA (Hixson & Brown 1986) and the fact that OToichil's Sumi-
noe oyster samples, which came from a hatchery stock originated
from Japan, may represent a different population that is genetically
isolated from the Chinese population (our samples). However,
analysis of more specimens from Japan or other parts of their
natural range is needed for confirmation.
Li and Qi (1994) suggested that the Zhe-like oysters most com-
monly found in the rocky intertidal zone were Pacific oysters
instead of Zhe oysters as most people assumed. If so, the iiitDNA
sequences of these (Zhe) oysters should have higher similarity to
(or low divergence with) those of the Pacific oysters or Dalianwan
oysters we presented here and that of O'Foighifs. Actually this is
not the case. Our sequence data show that these smaller oysters
from rocky shores could be Suminoe oysters, rather than Pacific
oysters.
Additionally, in this study the COl sequences showed more
variations, as expected, than the 16S sequences. For instance, in
the 16S data, we detected only one haplotype for Zhe oyster, two
tor each of the Dalianwan and Suminoe oysters; but in COl data,
the numbers of haplotype are seven, five and three for these three
species, respectively. Also, the divergence between C. gigas and
C. plicatida or C. ariakensis is three times as high as that between
C. gigas and C. taUt'imhanensis in the 16S data, whereas the
divergence is eleven times higher in the COl data. The COl se-
quence is more sensitive in discriminating closely related species,
supporting the observation by Boudry et al. ( 1998) where no vari-
ability was detected with nine endonucleases among 253 individu-
als of C. gigas and C. angiilata with 16S rDNA, but reasonable
polymorphism was detected with four enzymes with COL Other
works have also proved that CO! sequence is a good choice for
similar purposes (Meyran et al. 1997. O'Foighil et al. 1998).
In summary, the mtDNA sequence data strongly suggest that C.
laliemvlianensis is not a discrete species and should be considered
as synonymous with C. gigas. Our data also indicate that the "Zhe
oyster" is different from the Pacific and Dalianwan oysters, but is
genetically very close to Suminoe oyster, at least for the ones we
sampled.
ACKNOWLEDGMENTS
This work was financially supported by National Science Foun-
dation of China (39600113) and Research Foundation (2001) of
Institute of Oceanology, Chinese Academy of Sciences, Qingdao
266071, P. R. China. Yu and Guo are partly supported by grants
from US Sea Grant and New Jersey Commission on Science and
Technology.
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Jniiniul ,>f Shclirish Rcsi-anh. Vol. 22, No. I. 34-19. 2()().V
INCREASED BIOMASS YIELD FROM DELAWARE BAY OYSTERS (CRASSOSTREA
VIRGINICA) BY ALTERNATION OF PLANTING SEASON
JOHN N. KRAEUTER,' SUSAN FORD,' AND WALTER CANZONIER"
^Haskin Shellfish Research Lahnniiory. Iiistiliite of Marine and Cixistal Sciences. Rutgers University,
6959 Miller Avenue. Port Norris. New Jersey US349: and 'Aquarius Associates. Manasijuan. New Jersey
ABSTRACT The practice of moving oysters from low-salinity to high-salinity areas for improving growth and meat quality has been
practiced for well over a century. In the Delaware Bay. the practice was abruptly changed when MSX [Haplosporidium nelsoiii) caused
large-scale oyster mortality in the higher salinity portions of the bay. Similar disruptions occurred in Chesapeake Bay and other areas.
In lime the Delaware Bay. the oyster industry learned how to operate around the disease, but in early 1990s. Dermo (Pert^innis mariims)
began to cause serious mortality on transplanted oysters. Despite the historic and continuing movement of oysters within and between
estuaries, there is little published scientific literature indicating optimum conditions for transplantation. We investigated the effects of
transplantation from a low-salinity seed bed to a typical higher salinity leased ground. The transplants were designed to evaluate an
early, the traditional spring, and two fall transplant dates on the subsequent disease levels, growth, and survival of the oysters in three
size classes: market, submarket. and small. Environmental and oyster disease data suggest we conducted the experiment under nearly
worse-case conditions, high Dermo. and low food (chlorophyll). There were no significant differences associated with the timing of
transplant. We did not record significant growth on any size oyster and disease caused mortality exceeded 50% for early transplants.
Smaller oysters experienced greater mortality than market size individuals. Despite these conditions, meat dry weight nearly doubled
within 1 to 2 mo after transplant in all but the March transplant. Under these di.sease and environmental conditions the only economic
gain would be from the doubling of the meat weight and associated better meat quality. No gain can be expected from submarket
oysters growing into the market size classes.
KEY WORDS: oyster. Cnisso.strea. Delaware Bay. season, disease, growth
INTRODUCTION
In the Delaware Bay oysters have been transplanted from upper
bay low-salinity seed producing areas to lower bay higher-salinity
growing beds for more than 150 years (Ford 1997; Fig. 1 ). Similar
transplantation strategies have been used by oyster growers in
Chesapeake Bay (Andrews & McHugh 1957) and New England
(Ingersoll 1881, Goode 1887). Further, to increase production and/
or to supplement local seed as resources became depleted, oysters
were imported from distant sources. Despite these historic and
continuing large scale movement of oysters within and between
systems, there is little scientific literature indicating the optimum
conditions for transplantation.
Hopkins and Menzel ( 1952) developed a framework for study-
ing the transplantation of oysters based on the biomass yield of the
product, and Andrews and McHugh (1957) used biomass yield
estimates from trays of oysters to evaluate the effectiveness of
transplantation strategies. Reliance on biomass as a means of as-
sessment in both of these studies was based on the assumption that
the majority of oysters were destined to be shucked, and thus meat
yield was the most important aspect of production. This may not be
the case for those oysters that are grown to be sold for the half-
shell trade. In this latter case, assuming adequate meat quality,
numbers at market size are more important than total bicmiass.
Haskin et al. ( 1983) and Hargis and Haven (1988) both indicate
that the oyster planting industry in the Delaware Bay and the
Virginia portion of Chesapeake Bay, respectively, operated under
the assumption that transplanting was profitable if one bushel of
seed oysters yielded one bushel of market oysters. In the late
1950s, the parasite MSX, Haplosporidium nelsoni. caused epi-
zootic mortalities in both estuaries and forced major changes in
oyster industry practices. In the Virginia portion of Chesapeake
Bay, growers abandoned higher salinity grounds and concentrated
efforts in areas that historically produced higher than the 1:1 yield
(Hargis & Haven 1988), Despite H. nclsoni-c-Msed losses, the
Delaware Bay oyster industry continued to transplant oysters
based on the system developed in the KSOOs. Oysters were left on
the planted grounds, where high salinity favored the H, nelsoni
parasite, but for no more than 1 y (Ford 1997), and yields contin-
ued to be about 1:1 (Haskin & Ford 1983). After the 1950s H.
nelsoni epizootic, the importation of seed from out of state into the
New Jersey portion of Delaware Bay was banned.
In 1990, an outbreak of Dermo disease caused by Perkinsiis
niarlniis prompted a further change in strategy by the Delaware
Bay oyster industry. After 1990. P. inarinns infected most of the
oysters in the seed bed areas (Ford 1997), and oysters planted in
the spring of 1991 suffered high mortality in the late summer. The
oyster industry and the State of New Jersey responded by devel-
oping a program to market oysters directly from the seed beds.
This strategy produced oysters that had poorer meat quality and a
lower value than those from higher salinity waters.
At the same time, it was realized that although Powell et al.
( 1997) modeled the effect of transplant time, disease, and preda-
tion on market oyster populations, there were no real data on which
to base transplantation decisions in the presence of this new para-
site. The model predicted that fall (November) transplants left for
1 y yielded the best survival of market oysters compared with
transplants in January, March, or May that were harvested in No-
vember. In all cases the number of market oysters declined from
July to November. The model did not include an August transplant
with immediate harvest that fall, a strategy that would minimize
disease-caused mortalities while still taking advantage of typically
good fall "fattening" conditions. The industry requested data on
the following: 1) the best time of the year to transplant oysters;
2) the survival of transplanted oysters at various times after trans-
plant; 3) the numbers of market oysters expected from the net
result of growth and mortality; and 4) the gains that could be made
in iTieat quality and the length of time after transplant this gain
might take.
The industry, through a nonprofit foundation, collaborated with
39
40
Kraeuter et al.
NEW JERSEY
DELAWARE
CAPE HENLOPEN
Figure 1. Delaware Bay showing locations of the seed beds and Shell
Rock bed, leased grounds, and the ground used for transplant
studies.
state New Jersey Department of En\ironmental Protection
(NJDEP) and Haskin Shellfish Research Laboratory (HSRLl per-
sonnel to conduct an initial test of alternative planting dates. This
study (Canzonier 1998) moved oysters from the Shell Rock seed
bed to higher salinity grounds (527 D) in December. February.
May, and August. The effort clearly established that transplanting
in months different from the historical spring period was economi-
cally feasible, but cautioned that a single year's result could not
provide sufficient background for assessing year-to-year variation.
In addition, all months but the traditional spring transplant period,
represented by the May transplant, gave nearly identical results.
The May transplant had significantly less market oysters produced
than the other months (Canzonier 1998).
The information at the onset of the current study suggested that
transplantation strategy would depend on several factors: oyster
population size frequency distribution, source stock disease level,
seed bed used as a source, environment of the planted ground,
disease pressure, and harvest timing. In addition to biological vari-
ables, market factors, and industry seasonal work cycles affect the
economic impact of alternative planting seasons. The present study
builds upon earlier efforts and evaluates the effects of varying the
timing of transplanting oysters from one seed bed to a lower bay
planting ground.
MATERIALS AND METHODS
Experimental Design
Oysters from Shell Rock Bed were transplanted to ground 354
D (Fig. 1 ) in March, May, September, and October of 1999. Shell
Rock was selected because it represented a central seed bed source,
had a significant number nearly market size oysters, and pro\ ided
the oysters for the Can/onier ( 1998) study.
The transplant ground was subdivided into experimental plots.
each marked with navigation coordinates. A preliminary sampling
indicated that only a small number of large residual oysters (mean
99 mm) were present (mean 2.4 oysters bu~' from 8 one-bushel
samples). Approximately 1800 US Standard bushels (36.4 L;
herein after referred to as bushels or abbreviated as bu.) of oysters
were planted on each 24.4 x 91.4 m plot each transplant time
(3.200 bu.acre"' or 90.000 oysters hectare"').
At each transplant time, triplicate bushels of oysters were re-
moved from the deck load of the boat and analyzed in a manner
similar to the techniques used for the subsequent monthly samples
(see below). In addition, oysters were processed for disease diag-
nosis.
After planting, at least three dredge samples were collected
each month from each planting. All material was placed in the
bushels so that triplicate composite bushel samples of material
were examined from each planting each inonth. These were ex-
amined in the same manner as the source oysters, but with special
attention to growth, meat condition. P. inarintis level, and mortal-
ity (apportioned by oyster size). In the latter months, additional
oysters were set aside after the samples had been collected to be
sure enough material was available in all size classes to process P.
marinus and condition index samples. H. nelsoni levels were not
detemiined on the monthly samples, but were evaluated on the
fmal samples from each plot in No\'ember, as well as on the initial
transplants.
Sample Processing
All live oysters >20 mm, old, new boxes, and gapers in the
entire sample were counted. All oysters >20 mm were measured
and divided into market (>76 mm) and submarket (35-73 mm) and
small (<55 mm) classes. All parameters were normalized to a
standard bushel for comparison with other samples. Mortality was
estimated by calculating the percentage of new boxes and gapers in
each sample. This was considered recent mortality. Recent mor-
talities were accumulated to provide an estimated cumulative mor-
tality at the end of the study (Ford & Haskin, 1982).
Twenty oysters (six or seven from each of the 3 bu.) of each
size class were set aside for evaluation of condition index and an
additional group of similar size was examined for P. inariiuis
infection. Condition index was derived from the ratio of meat dried
at 50°C, and greatest shell dimension (height). P. marinus was
diagnosed after incubation of the rectum and a piece of mantle in
Ray's fluid thioglycollate medium. Infection intensity was scored
from 0 to 5 (Ray 1954) and a weighted prevalence calculated as the
mean intensity, including zeros, of all oysters in a sample. Oysters
in the initial planting and final sampling were diagnosed for H.
nelsoni by tissue section histology. Infection intensities were rated
from 0 to 4 (Ford 1983) and a weighted prevalence calculated as
for P. marinus.
Individual Oyster Growth and Mortality Study
To evaluate production requires size-class-specific growth and
mortality data. This was approximated from the bushel samples,
but a second method was utilized to provide a more precise evalu-
ation of individual oysters. A group of experimental oysters rep-
resentative of the source bed was deployed at the time of trans-
plant. This group consisted of five replicates of 20 oysters from
each of three size classes (63.5 to 69.9 mm, 70 to 75.9 mm, and
>76 mm) for a total of 300 oysters. Fishing leader tethers were
glued to the top valve of each oyster with Marine Tex. The tethers
Increased Biomass Yield ok Oysters
41
were then attached with cable ties along the side of a square
reint'orcing rod frame square (~l m on each side) that was held
approximately 5 cm above the bottom by a centrally located ce-
ment anchor. The entire array was attached to a surface lloat. Each
individually identified oyster was measured (height) and the array
deployed so that the oysters would lie on the bottom. Each month
each oyster was measured and mortality or loss noted. In this
instance, mortality was calculated directly because the history of
each oyster was known.
Environmental Data
The following environiuenlal data were collected on bottom
water on at least an every other week basis: temperature, salinity,
dissolved oxygen, pH, total suspended solids. Chlorophyll a. and
suspended organic material. In addition, temperature was moni-
tored continuously with an electronic recorder. Salinity was ob-
tained with a refractometer. All grab sample temperature and dis-
solved oxygen data were measured with a YSI oxygen meter, and
pH data were obtained with an electronic pH meter. Suspended
solids, chlorophyll and particulate nitrogen samples were obtained
from at least 500 ml of water filtered through Whatman GF/C glass
fiber filters, which were stored on ice until they were returned to
the laboratory. Chlorophyll samples were immediately placed in
buffered acetone and refrigerated. Particulate samples were dried
at 50°C. All en\ ironmental data were analyzed according to Strick-
land and Parsons ( 1968).
Data Analysis
Size frequency data were normalized by adjusting the base live
and recent dead (gapers and new boxes) frequency distributions
from all individuals collected in the three bushel samples (in 5-mm
increments) to 100 individuals. These frequencies were then ad-
justed to the number of live or dead bu."' by multiplying the
frequency of occurrence in all sizes by the average number of live
or dead bu."' Data were summarized and significant tests were run
using one-way analysis of variance, I tests, or other descriptive
techniques. Percentages were transformed using an arc-sine trans-
formadon before performing analysis.
RESULTS
Envirnnnicntal linla
Temperature on the transplant ground was 3.5°C in March, at
the beginning of the study, and peaked in August at 27.5"C. Sa-
linity was generally between 21 and 23 ppt.. with a low of 19 ppt
in April and a high of 26 ppt in October and December. pH
remained relatively stable, ranging from 7.8 to 8.6 with the excep-
tion of a low value of 6.9 on September I. Dissolved oxygen
ranged from a high of 13.5 mg L"' in March to a low of 5.6 mg L^'
on July 14. In general dissolved oxygen levels remained near or
above saturation at temperatures below 2()"C and near or slightly
below saturation above those temperatures. Total suspended solids
were typically between 30 and 55 mg L"'. with highest and lowest
values of 86 and 1 8 mg L~ ' on August 1 8 and May 5, respectively.
Chlorophyll a showed a typical spring (late March to early April)
bloom followed by generally lower vales in summer (Fig. 2).
There was an increase in Chlorophyll a in fall (October to early
November). Highest Chlorophyll a levels were found March 25,
April I, May 18 and November 5 with values of 54, 46, 38 and 39
mg m~' respectively.
60
50
^40
30
D-
O
U
20
10
Mar 9 Apr 1 May 5 May 18 Jun 18 July 14 Aug 18 Sept 23 Oct 8 Oct 29 Dec 15
Mar 25 Apr 18 May 1 1 Jun 7 Jun. 30 Aug 4 Sept I Oct 5 Oct 22 Nov 5
1999
1996/1997
Figure 2. Buttuni water chlorophyll a In samples taken from bottom water over ground 554 1) in Delaware liay in 1999 compared with similar
data taken over ground 527 D In Delaware Bay in 1997. Data are in mg per m'. 1997 data from Canzonier (1998).
42
Kraeuter et al.
Oyster Data
Because the samples taken at the time of transplant represented
the source bed and culHng machinery on the boat, not the ground
to which the oysters were transplanted and monitored, time 0 (7",,)
for subsequent analyses was the first sample after transplant. The
samples taken from the deck at the time of transplant were utilized
to estimate the size, condition and numbers of oysters transplanted.
Numbers of Live and Dead Oysters
The numbers of oysters being transplanted, based on the initial
samples for each transplant period, suggests that all groups, with
the exception of the October transplant, received uppro.ximately
the same number of individuals per unit volume of material
moved. The October samples had fewer oysters than those groups
transplanted in March and September, but was equivalent to the
May transplant (Table I ). It seems likely that more live oysters
were moved in the May transplant than in October, but the high
variance in May precludes making a definite statement.
The total numbers of live oysters significantly decreased from
Tf, to the fmal samples {T,) in November. The numbers in the
March and May transplants fell approximately 50* from 200 in
initial post-planting samples to <I00 bu.~' in the final November
sample (Table 1 ). The mean oysters bu.~' in October and Novem-
ber, traditional harvest months, were greatest for the September
transplants, but the difference was statistically significant only in
October. The decrease in oysters from planting to November was
least in the September transplants, but the time between 7",, and T,
was only one month. No calculation can be made for the October
planting because Tq = Tf (Table 1).
Live oyster numbers were also analyzed by size (Table 2). Data
from dredged samples show that numbers of marketable oysters
declined about 50% for March transplants, but that subsequent
transplants experienced little or no loss. Submarket and small oys-
ter numbers also declined, and. with the exception of the Septem-
ber transplant, these declines were usually greater than for market
size oysters and often more than 50Vc. Despite large losses of
oysters, there were no statistically significant differences in No-
vember in the number of market size, or submarket size oysters in
any transplant period. Numbers of small oysters in the March and
May transplants had declined appreciably by November and there
were about half as many small oysters per bushel as in the other
two size classes, even though small oysters were most abundant at
the time of transplant. Numbers of small oysters remained high in
the final sample of the September transplant, but not in the October
group.
Recent mortality, for all size classes, was greatest in the fall
(Fig. 3). These losses occurred across all size classes, but, with the
exception of the October transplant, losses were greatest in the
smallest size classes. Estimated cumulative mortality from trans-
planting to the final sample of all size oysters was 54, 55, 15, and
9% for March, May, Septeinber and October transplants, respec-
tively. Total losses of small oysters were greater than those of
market or submarket oysters for the March and May transplants
(Table }). There were no differences between the market and
submarket oyster losses in any transplant group.
Disease Levels
H. nc'l.sdiii was detected, in initial and final sainples. only at
very low levels. There was no association with size or transplant
time. The highest infection level (prevalence) was 30%, but most
infections averaged <15%. The highest weighted pievalence (0.4)
was found in the fall samples.
In contrast. P. marinus levels were high in all plantings and all
size classes (Fig. 4). Infections were nearly as heavy and abundant
on the source bed as they were in oysters already transplanted to
the higher salinity experimental site. Percent infection (prevalence)
for the March and May transplants exceeded 80% by July and was
usually 90 to 100% until it dropped below 80% in November. For
the later transplants, P. marinus levels usually increased to 90 to
100% within 1 mo after transplant. Weighted prevalence was rela-
tively high in the March transplants, but underwent a typical drop
in April/May (Bushek et al. 1994). The same drop occurred on the
source bed as the May transplants had a weighted prevalence simi-
lar that of the March transplant at the same time. Intensities in both
groups then increased over the summer until September, when
levels in all size categories decreased concurrent with an increase
in mortality (compare Fig. 3 to 4). Levels increased again in the
October sample and then dropped by nearly 50% in November. At
TABLE L
Mean numbers of live oysters >20 mm bu."' by month with 95% conndence limits (h = 3 for each monthly sample).
March
May
September
October
Mean
95% Conf
. Limits
Mean
95% Conf.
Limits
Mean
95% Conf. Limits
Mean
95% Conf. Limits
M
323
363
283
A
212
124
156
105
119
80
108
76
230
189
209
155
144
120
131
87
195
59
103
56
93
40
84
65
M
296
403
188
J
J
A
I8,S
121
76
90
92
79
239
169
154
143
104
113
137
73
0
38
79
45
S
307
355 259
0
169*
146
203 136
205 87
243
254 232
N
78
101 56
Bold numbers indicate a significant difference from the prior month. The area in gray indicates samples removed from the deck of the transplant vessel.
These were not used in subsequent calculations.
* Significantly more oysters than in other transplants during the sample period.
Increased Biomass Yield of Oysters
43
I ABIE 2.
Mean number of live market (>7f) ninil, subniarket (75-55 mm), and small (55-20 mm) oysters bu. ' of dredfjcd material from transplants
in March, May, September, and October 1999.
March 1999
May
1999
September 1999
October 1999
Market
Submark
Small
Market
Submark
Small
Market Submark
Small
Market Submark
Small
M
58
115
150
(^
63
48
61
24
40
30
32
76
32
42
37
41
30
38
29
75
45
54
40
38
21
38
15
M
78
104
114
.1
J
34
38
23
25
38
34
64
34
25
37
33
33
87
49
28
39
21
16
s
56
86
167
54 70
o
29
27
47
35
94
84
120
N
25 26
28
Oys(ers were transplanted from Shell Rock to Ground 554D on the Delaware Bay leased grounds. Areas of gray indicate samples from deck loads of
transplanted oysters. All other samples were dredged from transplant plots. Submark = submarket.
this linic. heavy (iiortality was observed in the March transplants to those transplanted earher. but, unhke the former, infections
only (Fig. 3) and the drop was probably the beginning of the retnained at very high levels in these oysteis into November. The
overwinter loss of infections (Bushek el al. 1994). Oysters trans- persistence of high infection levels was as.sociated with low mor-
planted in September and October had weighted prevalence siniilar tality in both fall groups.
Mar >75mm
Mar 55 to 74mm
Mar <55mm
I Li
.....III!
Mil
Apt
Mav June Iul> Aug
May >75mm
Scpl
Oci
Nov
*7 1
30-L--
1
1
1"
1
1
1
■
■ - ■■
"
Apt Miy June July Aug Sept Do Nov
Sept >75nun
Hi
Mm Apr May June July Aug Sep( Oci Nov
Oct >75mm
II
Apr May June July Aug Sep! Ocl Nov
Sept 55 to 74 mm
li
Apt M»y June July Aug Sept Oct Nov
Oct 55 to 74 mm
h
Mai Api Mdy June July Aug Scpi Oct Nov
May <55mm
Mar Apr May June July Aug Sepi Oct Nov
Sept <55 mm
■ I
Ms Apr Miy tunc luly Aug Sepi Oa Nav
Oct <::55 mm
Apr Miy June July Aug Sept CJti Nov
Mat Apt Miy June July Aug Sepi Oci Nov
Mat Apt Miy June July Aug Sept Oct Nov
Figure 3. Interval percent mortality by month of market (>75 mm), submarket (55 to 74 mm), and small {<55 mm) oysters transplanted from
Shell Rock to Delaware Bay ground 554 D in 1999. Transplant months were March {top graphs), May (middle top graphs), September (middle
bottom graphs), and October (bottom graphs).
44
Kraeuter et al.
TABLE 3.
Estimated cumulative percent mortality, from plantin}< to November 19')9. by size category of dredged oyster samples collected in Delaware
Bay by transplant month.
March 1999
May 1999
September 1999
October 1999
Market
Submark
Small
Market
Submark
Small
Market Submark
Small
Market
Submark
Small
46
48
65
45
4,S
XS
19 16
14
11
11
5
Market (>76 iiini). Mibniarket (75-55 mm), and small (55-20 mml.
Growth and Condition
With the exception of the March transplants, there were no
differences in the sizes of oysters in the subniarket and small
categories through time. Mean dry meat weight of market oysters
for the March and May transplants increased significantly in June,
after .^ and I mo, respectively (Table 4). That of markel-si/e Sep-
tember and October transplants rose in November after 2 and I mo.
respectively. There were no significant differences in meat weight
among any of the transplanted groups by November. While not
statistically significant, there was a consistent increase in meat
weight in all transplants of market-si/e oysters between October
and November. In general, meat weight increases of submarket
and small oysters mirrored those of the market-size individuals.
Reflecting the increase in meat weight without increased shell
size in market oysters, the condition index increased during the
study period. With the exception of the March transplants, oysters
required one month after transplant to the lower bay to improve
condition, and they typically retained this condition throughout the
summer and into the fall. While not statistically .significant, there
Mar > 75 mm
Mar 55-75 mm
March April May June July Aug Sept Oct Nov
irxll
March April May June July Aug SepI Oct Nov
Mar < 55 mm
4-
3-
2-
0-
Mafch April May June July Aug SepI Oct Nov
May > 75 mm
5 :
llili
mil
T r
March April May June July Aug Sept Ocl Nov
May 55-75 mm
III T .
illi
1
Oct
nil
i
JUU
1
March April
May June July Aug Sept
Nov
May < 55 mm
March April May June July Aug SepI Ocl Nov
Sept > 75 mm
March April May June July Aug Sept Ocl Nov
4—]
Sept 55-75 mm
T i
[
1 1 I 1 1 !
March April May June July Aug
1 1 1
^ept Oct Nov
Sept < 55 mm
i
i™i™i ™i
— I — i — I — I —
March April May June July Aug Sept Oct Nov
Oct > 75 mm
5„
1 1 1 1 1 1 r
March April May June July Aug Sept Oct
i
Oct 55-75 mm
1 1 1 1 1 1 r
March April May June July Aug Sepl Ocl
I
Oct < 55 mm
t
1 1 1 1 1 1 r
March April May June July Aug SepI Oct
i
Figure 4. Monthly weighted prevalence of Dermo (/'. nuirinu\) infections in market (>75 mini, subniarket (55 to 74 mini, and small (<55 mml
oysters transplanted from Shell Rock to Delaware Bay ground 554 D in 1999. Transplant groups were March (top graphs). May (middle top
graphs), September (middle bottom graphs) and October (bottom graphs). For each transplant group, the llrst sample represents that on Shell Rock
bed when the oysters were moved. All subsetpient samples represent infection levels on ground 554 D. Error bars represent 95 Cr confidence interval.
Increased Biomass Yield of Oysters
45
TABI.K 4.
Mean dry meal Heijjht (jjl of markel-size (ijsterv b> month with 'tS'c conlldence Mmit.s.
March
May
September
October
Mean
95 "/f Conf.
Limits
Mean
95% Conf.
Limits
Mean
95% Conf. Limits
Mean
95% Conf. Limits
M
1,1
1.2
0.9
A
1..^
1..5
1.2
M
1,?
1..S
1,2
l.s
1,7
1.3
.1
2.4
2.8
2.0
2..^
2.6
1.9
J
2.5
2.8
T 1
2.3
2.7
2.0
A
-> 1
2.4
2.0
1 T
2.4
2.0
S
2.4
2.7
2.1
2.5
2.8
T 1
1.6
1.8 1.4
O
2 2
2.6
1.8
2.3
2.7
2.0
1.9
2.3 1.6
I.I
1.3 1,0
N
2.9
.V4
T 1^
3.1
3.6
2.6
2..S
2.9 2.1
2.7
3.2 2.2
Bold niinihers indicate a significant difference from the previous month.
was a general trend for market oysters to improve in condition
from October to November.
Condition index for submarket and small oysters generally fol-
lowed the same trends as for the market oysters with no significant
change from June to Nmember. In general, there was a significant
increase in condition w ithin 1 mo after transplant for all submarket
and stnal! oysters with the exception of the March transplants and
small oysters transplanted in October.
By November, the meat condition index of all si/e classes in
the March and September transplants was statistically the same.
Among the October transplants, condition of submarket and mar-
ket oysters was the statistically similar, and greater than that of
small oysters, while the condition of market oysters in the May
transplants was greater than that of either submarket or small oys-
ters.
(iriiHlli ami Mdilalily of hulividually Marked Oysters
For calculations of mortality, the data from the tethered oysters
were corrected for oysters lost during the experiment by reducing
the numbers of oysters present from the initial counts. A few
oysters were lost because of detachment of the adhesive, but one
entire rack was lost.
Mortality of tethered oysters mirrored that of oysters trans-
planted at similar times, with a few notable exceptions (Fig. 3). It
IS evident from the cumulative mortality data (Table 5) that the
tethered oysters (particularly those put out in May and September)
liad substantially more mortality than that estimated from exami-
nation of boxes and gapers in dredged samples. At times, shells on
one section of an airay were observed to have become blackened.
This suggests that some silting had taken place around these oys-
ters and may have elevated the mortality above that experienced by
the planted oysters, but we have no independent measure to evalu-
ate if some planted oysters were silted in and not adequately
TABLE 5.
Cumulative percent mortality of tethered oysters, and oysters in
dredged samples as a function of transplant time.
Month of Transplant
Method
March
May
September
October
Tethered
Dredged
76
,54
93
55
59
15
38
9
sampled with the dredge. There were no significant differences in
recent or cumulative mortality based on si/e of the tethered oys-
ters.
Because all tethered oysters were large and the growth incre-
ment was small relative to the potential error, the monthly growth
increment of tethered oysters was difficult to measure. This diffi-
culty is evident in the fluctuations in increment growth for the
various size classes (Fig. .5) and the negative growth measured for
some months. Growth, as indicated by new shell being accreted to
the oysters, was observed on some oysters in all but the coldest
months.
Because individual oysters were followed, cumulative growth
is the difference between the initial measurement and the measure-
ment of surviving oysters at any time period (Fig. 5). Because not
all oysters survived through all time periods, cumulative growth
reflects both survival and growth of individuals.
By November there were no differences in growth of surviving
tethered oysters classed as market-sized in March and May, but
individuals in both groups had grown more than those tethered in
September and October. There was no statistically significant
growth for either of these latter two periods. Growth of submarket
size oysters was also at the limits of detection. The 70- to 75-mm
size class showed >0 growth only for the May and September
groups when the mean were 4.8 and 1.8 mm. respectively. With
the exception of the March tethered individual (only one oyster
survived to October) oyster classed as small did not show tnea-
surable growth.
DISCUSSION
Hopkins and Men/el ( 1952) indicated that the major difficulty
in deriving estimates of production was not related to measurement
of growth, but to measurement of losses due to mortality. In our
case, where only large oysters were being evaluated and growth
was poor; it was also difficult to assess growth.
The dominant themes of Delaware Bay oyster transplantation
in 1999 were related to high Dermo (P. niciriiiiis) levels and the
associated high mortality and low chlorophyll and the associated
poor growth. There is a general hypothesis that mortality of trans-
planted, market-sized oysters, due to disease or other factors, can
be made up for by oysters growing from smaller sizes to the
market classes during the year Powell et al. (1997). This can hap-
pen in some years (Canzonier 1998), but in periods such as 1999
with high P. iiiarinus levels and relatively low food, growth may
46
Kraeuter et al.
Mar 70-75 mm
Apnl Nby June July Aug Sepi Oct Nov Dec
May 70*75 mm
-\ 1 ! ! ! '■ \ \ 1
Apnl May June July Aug Scpi Ocl Nov Dec
Sept 70-75 mm
1^1 May lunc July Aug SqX On Nov Dec
Oct >75 mm
i
1
■
1
■
1 1 1 1
Alril
Mv
luoe
July
Am
Sq«
On
Nov
Dec
Oct 70-75 mm
7J " —
1
S
i; 1 1
III
■ ! ' 1 1
Mar 63-70 mm
1
a
E
g ,5
■ ■ -
m
■■
^Hll
■
1
1
1 1 1 1 1 1 i
Apnl
my
June July Aug Sept
May 63-70 mm
Oci
Nov
!
Dee
1
g jj^
■ ■
3
.II.B
[ ; \
t
Apnl
Miv
June July Aug Scpl
Sept 63-70 mm
OCL
Nov
Dec
!
1
= 2S
s
■
t ----- - ■ -"'
Ap«l
Miy
1 1 !
June July Aug Sept
Oct 63-70 mm
Oci
Nov
Dee
i
i
^
! ! 1
1
i
^
1 } 1
!
!
- ^-4—1 ! I I H ! I I
Apnl May June luly Aug Scpi Oci Nov Doc Apnl May lunc luly /wg Scpl Oci Nov Dec Apnl May lunc July Aug Scpl Oci Nov Dk
Figure 5. Cumulative growtli of >75 nini, 7(1-75 mm, and 63- to 7(l-mni tetiiered oysters transplanted from Shell Rock to Delaware Bay ground
554 D in 1999. Transplant months Here March (top graphs), Ma_\ (middle top graphs), Septemher (middle hottom graphs), and October (bottom
graphs). Negative growth is due to measurement error. All oysters were followed as individuals and growth is the summation of all oysters alive
in that size class at the time of measurement.
be reduced to the point that this hypothesis is not valid. Neither the
tethered oysters nor the transplanted oysters in the dredged
samples, of any size class, in the present study showed statistically
significant growth.
The data did not show statistically sigiuficant differences in
numbers, based on month of transplant, of market, submarket. or
total oysters per bushel in final sampling in November. This sug-
gests that in periods of high P. imiriinis. high i-i-)ortality. and low
food the timing of transplantation is not a major consideration
from the point of view of the numerical yield of market oysters. In
addition to the nearly 50% losses of submarket and market oysters,
losses of si-nall oysters exceeding 65% suggest that transplantation
of small oysters with the expectation that they will grow into the
market-size category is not an efficient use of the resource under
high P. mariiuis conditions.
In view of lack of significant differences in the numbers of
i-i-)arketable oysters associated with transplant month, possible dif-
ferences in meat quantity need to be considered. In all cases (ex-
cept the March transplants when water temperatures were low)
total meat weight improved within one month following transplan-
tation (Table 4). Beyond this initial improvement in there was no
change durinc the summer months, but in all cases there was a
trend (not statistically significant) toward further improvement in
between the October and the November samples. Clearly the im-
provement in meat weight in the May to June period could be due
to the increase in gonadal tissue, but the weight did not decrease in
the summer or fall, after the spawning period, indicating that some
of this weight gain was more than gonadal production. The im-
provement in meat quality occurred in 1999 despite the high dis-
ease levels, high mortality and lack of shell growth.
Comparison with Previous Studies
Powell et al. (1997) modeled the effect of transplanting Dela-
ware Bay seed bed oysters in November. January. March. April,
and May on the number of market size oysters available the fol-
lowing July to November. The model predicted that a November
transplant with a November harvest provided the best yields, and
that growth of submarket sized oysters compensated for the losses
of market sized individuals. Mortality of submarket oysters was
less than for larger ones because the added scope-for-growth offers
these individuals some disease protection. Simulated P. nmrinus
levels peaked slightly above four weighted prevalence a level
nearly reached in the present study. The model simulated that
Increased Biomass Yield of Oysters
47
TABLE 6.
Comparison of niimbcrs of marktl and siihniarkil ovslurs hu.
plantt'd on leastd fjrounds in IMMft to IW7 and IWy.
Year of
Transplant
Market
95 '7r
Confidence
Limit
Submarket
Confidence
Limit
1999
1996/97
62
?6
tl3
232
576
±5?
tl05
Data from 1996 to 1997 are from Canzonier (199S). Data arc from samples
removed from the deck of the transplant vessels.
submarket size oysters were less susceptible to mortality from P.
mciriiuis than the market-sized oysters, which allowed them to
grow to market size and replace larger, individuals with lethal
infections. This simulation was not verified in the present studies.
One reason is that, in contrast with the model simulation, the
smaller oysters did not grow. Thus, they did not increase in bio-
mass fast enough to "outgrow" the parasite and maintain parasite
burdens below lethal levels. It is important to emphasize that the
food present in 1999. as indicated by Chlorophyll a. was lower
than that used in the model of Powell et al. ( 1997). It seems likely
that the low food concentrations in 1999 reduced the potential for
compensatory growth of submarket oysters to replace market oys-
ters that died during the study period. The lack of growth may also
have been a consequence of high disease levels (Men/el & Hop-
kins 19.'i.'i. Paynter 1996). Further, many of the assumptions of the
Powell et al. ( 1997) simulations were based on age/size relation-
ships observed in the Gulf of Mexico, which do not apply to
Delaware Bay. In Delaware Bay. for instance, submarket-sized
oysters (35-75 mm) obtained from seed beds are at least 3 years
old and many of the small oysters (<55 mm) are at least 2 y old.
All sampling of oysters in the Bay indicate that by age 2, oysters
have P. marimis infection levels that are equal to that of older
oysters. Thus, it is not surprising that cumulative mortality for our
submarket and small oy.sters was equal to. or greater than, that of
market-sized oysters. A second major difference between our
study and the model simulations is that significant numbers of
submarket oysters did not grow into market individuals in 1999.
Canzonier et al. (1998) reported on a similar transplant. He
moved oysters from the same seed bed (Shell Rock) in December
1996, and February, May and late August 1997. and sampled them
until November 1997. Growth of oysters into the market size cat-
egory was clearly evident in the 1996 to 1997 period (Canzonier
1998). The number of oysters bu. ' transplanted differed signifi-
cantly between this study and the present one (Table 6). There
were no differences (P = 0.43) in the numbers of market oysters
bu. ' from the deck loads of the two studies, but there were neariy
twice as many submarket oysters in the earlier trial (Table 6). In
1996 to 1997. the percentage of market oysters bu. ' ranged from
8 to 10% whereas in 1999 market oysters were between 18 to 26%
of the total. Canzonier (1998) found the number of market oysters
from dredge samples remained relatively constant throughout the
test period in spite of the substantial mortality. Thus despite twice
as many submarket size oysters and growing conditions that were
better than in 1999. there were no changes in the number of market
size oysters in any month of transplant in 1996 to 1997. Growth of
submarket oysters made up for the loss of older oysters.
As opposed to the 1999 results, in which a 21% decrease in the
numbers of market oysters was observed in all transplants. Can-
zonier ( 1998) reported an insignificant 4% decrease in the number
of market-size oysters at the end of the experiment in November.
P. mariniis levels were generally lower in 1996/97 when compared
to both the model and the 1999 data (Table 7). Cumulative mor-
tality was less for December and February transplants but appar-
ently higher for May and August transplants in 1996/97 when
compared with roughly similar transplant months in 1999 (Table
8). Chlorophyll ii in 1997 showed a slight peak in the spring, a
second peak in June and continued high levels (relative to 1999)
throughout the summer, but a general decline from late August to
November (Fig. 2). In this latter condition. Chlorophyll a in the
earlier period was similar to those in the Powell et al. (1997)
model. The presence in 1996 to 1997 of high summer food con-
centrations, lower P. mariiuis. and consequently lower moitality
than in 1999 suggests that the 1999 conditions may be nearly a
worst-case representation. The only exception would be the pres-
ence of the fall bloom in 1999 that would have allowed the oysters
to enter the winter in better condition. This may or may not be
important because there was no difference between the dry meat
weights in 1996 to 1997 when there was no fall bloom and 1999.
Canzonier (1998) reported that market oysters moved from
Shell Rock in December. February. May, and August averaged the
same dry meat weight (1.2 to 1.3 g) as those at the time of trans-
plant in the present study. His final product in November had a
meat weight of 2.8 g. the same weight as oysters in 1999.
How the increase in meat quality in transplanted oysters, vs.
those marketed directly Iriim the seed beds, would affect profit-
TABLE 7.
Initial and selected months.
December
February
May
.August
Market
Submark
Market
Submark
Market
Submark
Market
Submark
D
l.S
1.3
F
0.8
0.7
A
0.2
0.1
0.1
0.1
M
0.1
0.1
A
2.1
1.4
1.2
1.3
0.7''
1.1
1.0
0.6
S
1.2
1.3
1.9
1.3
1.3
2.3
1.7
1.8
N
0.4
0.6
0.2
1.2
0.5
1.2
0.5
0.9
Weighted prevalence oi P. marimts (Dermo) in oysters transplanted from Shell Rock to 527D m 1996 to 1997. Market >75 mm. Submark = Submarket
(55-75 mm). (From Canzonier 1998).
48
Kraeuter et al.
TABLE 8.
Cumulative percent mortality from planting to November of oysters
from Can/.onier (IWSt and present study.
Study
Month of
Transplant
Present study
March
54
May
55
September
15
October
4
Canzonier (I99SI
Decemtier
43
February
45
May
30
August
15
ability is dependent on the relationship among the following pa-
rameters: 1 ) the number of market oysters bu.~' and/or the amount
of meat bu."' that could have been harvested directly from the seed
beds; 2) the number of market oysters and/or the amount of meat
bur' that could have been harvested from the transplanted oysters;
3) the cost of re-harvesting the transplanted oysters; 4) the added
value that is derived from post-shucking processing (washing with
fresh water and blowing with air to help remove shell materials) a
higher salinity oyster; and 5) the value of the bushel of oysters to
the market. The latter \ alue is dependent on the season of harvest,
competing product and whether the oysters are shucked or sold in
the shell.
If oysters are used as shell stock, there would be little gain in
value to the harvester from an increase in meat yield, because in
current conditions, there is little chance (hat additional price would
be paid (S. Fleetwood. Bivahe Packing, pers. comm.). The best
that could be expected would be a longer term value increase
because of better market acceptance. Before the disease infesta-
tions. Delaware Bay oysters received a premium price because of
their high meat yields. Thus for shell stock oysters, in years of high
or moderately high P. marinus disease-caused mortality, there
would be little to gain from transplantation.
For oysters that are to be shucked, results of both the 1996 to
1997 and 1999 studies indicate a significant increase in meat yield
after transplantation. It is important to note that the meat yield
increase, during months with warm water, can be obtained in one
or at most two months. In 1999 the average meat yield increase by
November was about 1 13'-^. and in 1996 to 1997 the meat yield
increased by about 1339}- (Table 9).
Given that there was no difference in the number of oysters
available for market in November (Table 9) associated with trans-
plantation time, it would appear that there was no value added
from transplantation in any month or tor the average of all months.
It should be emphasized that under current conditions, market
oysters are culled on board. This means that nearly equal numbers
of oysters bu."' would be delivered to the packing house from both
the seed beds and the planted grounds. Under these conditions the
meat from oysters harvested from the planted grounds in both trial
periods would weigh approximately 1249^ more that of oysters
from the seed beds. In both cases the use of oysters for shucking
stock would result in increased yields. The higher salinity on the
planted grounds and the added meat weight, will provide addi-
tional gains during the washing and blowing of the meats during
processing.
CONCLUSIONS
When combined with the Canzonier (1998) study the data
cover two of a myriad of possible cases. In 1996 to 1997 there
were slightly elevated summer chlorophyll levels, moderate
growth and moderate P. marinus. whereas in 1999 there were low
or typical Delaware Bay sunmier chlorophyll levels, no growth and
high P. iiniriiuis. The month of transplant did not have a significant
effect on the numbers of market oysters available at the end of the
year. When P. marinus levels were elevated and food supply was
low. transplanted small oysters were lost at a higher rate than
market or submarket oysters. The data from both studies suggest
that food levels on the planted grounds in the warmer part of the
year are generally sufficient to support increases in meat yield 1 to
2 mo after transplant, but may not be sufficiently high to support
shell growth in all years. Under high to moderate P. marinus
conditions, exclusive of tiiarkel timing, meat weight or shucked
meat volume gain were the most important factors for economic
comparison of market oysters between the seed beds and the
planted grounds.
TABLE 9.
Estimated dry meat yield (g) of market oysters (>76 mm) bu. ' of dredged material at time of transplant (Shell Rock) and in November
1997 and 1999.
Shell Rock
Transplants
Transplant Month
Oyster/bu.
Dry Meat Wt
Dry Meat/bu.
Oyster/bu.
Dry Meat Wt
Dry Meat/bu.
1999
March 99
63
1.1
69
32
2.9
92
May 99
34
1.5
51
34
3.1
105
September 99
29
1.6
44
27
2.5
68
October 99
25
1.1
28
25
2.7
68
Average
38
1.3
49
30
2.8
84
1996/1997
December 96
110
1.1
121
108
2.8
302
February 97
92
1.2
110
110
2.5
275
May 97
95
1.5
143
93
2.7
251
August 97
133
1.3
174
106
3.0
318
Average
108
1.2
130
104
2.8
291
Oyster numbers for Shell Rock have been adjusted by using data from the first month of post transplant sampling to accommodate for differences culled
deck load samples and dredge samples. Oysters transplanted from Shell Rock by month of transplant.
Increasbu BioMASS Yield of Oysters
49
ACKNOWLEDGMENTS
The study was funded through funds supphed by the State of
New Jersey for evaluation of the Delaware Bay oyster resources,
and allocated through the Oyster Industry Science Committee of
the Delaware Bay Shellfish Council. The present study could not
have been completed without the on-the-water efforts of Royce
Reed and Russell Babb of NJDEP— Shellfisheries. Staff of the
Haskin Shellfish Research Laboratory (Bob Barber. Beth Brewster
and Meagan Cummings) were instrumental in carrying out much
of the sampling and sample processing efforts. The NJ Agriculture
Experiment Station also pro\ided support.
LITERATURE CITED
Andrews. J. D. & J. L. McHugli. \95T. The sLir\i\al and giowlh of South
Carolina seed oysters in Virginia waters. Pidc. Nur. Shclljlsh As.s<ic.
47:.V17.
Bushek, D., S. E. Ford & S. K. .Mien. 1994. Evaluation of melhods using
Ray's fluid thioglycollate medium for diagnosis of Perkinsiis mariniis
infection in the eastern oyster. Cnissostiva virginicn. Ann. Rev. Fi.sh
D/sraiM 4:201-217.
Canzonier. W. J. 1998. Increased oyster production hy alteration of planing
season. Commercial scale project in Delaware Bay — 1996 to 1998.
Ford. S. 1997. History and present status of molluscan shellfisheries from
Bamegat Bay to Delaware Bay. In: C. L. MacKenzie. Jr.. V. G. Burrell.
Jr., A. Rosenfield & W. L. Hobart. editors. The history, present con-
dition, and future of the molluscan fisheries of North and Central
America and Europe. Volume 1. Atlantic and Gulf Coasts. US Dept.
Comm. NOAA Tech. Rept. NMFS 127. pp. 119-140.
Goode. G. B. 1887. The fisheries and fishery mdustries of the United
States. Washington. DC: in ."i sections.
Hargis. W. J.. Jr. & D. S. Haven. 1988. The nnperilled oyster industry of
Virginia. A critical analysis with recommendations for restoration. Spe-
cial Report 290 in Applied Marine Science and Ocean Engineering,
Virginia Institute of Marine Science. Gloucester Point. VA. 130 pp.
Haskin. H. H.. R. A. Lutz & C. E. Epifanio. 1983. Ch. 13. Benthos (shell-
fish). In: J. H. Sharp (ed.). The Delaware Estuary: Research as hack-
ground for estuarine management and development. A report to the
Delaware River and Ba> .Authority. Unnersity of Delaware. Lewes.
Delaware. 326 pp.
Haskin. H. H. & S. Ford. 1983. Quantitative effects of MSX disease iha-
plosporidium nelsoni) on production of the New Jersey oyster beds in
Delaware Bay. USA. Int. Counc. E,\plor. Sea. CM 1983/Gen:7/Mini
Symp.. Goteborg. Sweden.
Hopkins. S. & R. W. Menzel. 1952. Methods for the study of oyster plant-
ings. Convention Addresses NaL Shellfish. Assoe. 1952:108-112.
IngersoU. E. 1881. The oyster industry. In: The history and present con-
dition of the fishery industries: Tenth Census of the United States.
Department of the Interior. Washington. DC 251 pp.
Menzel. R. W. & S. H. Hopkins. 1955. Growth of oysters parasitized by the
fungus Dermocystidium marinum and by the trematode Bucephalus
cueiihis. J. Parasitol. 41:333-342.
Paynter. K. T. 1996. The effects of Perkinsus mariniis infection on physi-
ological processes in the eastern oyster. Cnissosrren virginicn. J. Shell-
fish Res. 15:119-125.
Powell. E. N.. J. M. Klinck. E. E. Hoffman & S. Ford. 1997. Varying the
timing of oyster transplant: implications for management from simu-
lation studies. Fish. Oceanogr. 6:4. 213-237.
Ray. S. M. 1954. Biological studies of Dermocystidium mariiuim. Rice
Institute Pamphlet. Special Issue. (The Rice Institute. Houston. Texas).
Strickland. J. D. H. & T. R. Parsons. 1968. A practical handbook of sea-
water analysis. Fish. Res. Bd. Canada. Bull. 167. 311 pp.
.loiinuil oj Slu'ltfisk Rcscanh. Veil. 22. No. I, 51-59. 200.^.
U.S. CONSUMERS: EXAMINING THE DECISION TO CONSUME OYSTERS AND THE
DECISION OE HOW FREQUENTLY TO CONSUME OYSTERS
LISA HOUSE,'* TERRILL R. HANSON," AND S. SURESHWARAN'
^ Fo(xl and Resource Economics Di'purtnwnt. University of Florida. P.O. Box 1J024U, Gainesville.
Florida 32611: 'Department of Agricultural Economics. Mississippi State University, PO Box 5187.
Mississippi State, Mississippi 39762: and Higher Education Programs, Cooperative State Research,
Education and Extension Sen'ice, USDA, Mail Stop 2251, 1400 Independence Ave, SW, Washington, DC
20250-2250
ABSTRACT Oyster consumption has been decreasing in the United States. Investigating consumer attitudes and preferences can help
identify factors involved in this decrease. This study used data obtained through a nationwide survey in a douhle-hurdle regression
model to determine factors that influence both the decision to consume oysters and frequency of consumption. Results uidicate there
is a significant difference in the reasons people choose to eat oysters or not and the reasons oyster consumers choose how frequently
to eat oysters. Concern for product safety significantly influenced the decision of how frequently to consume but not whether to
consume oysters. Consumers also indicated a potential willingness to pay for measures that would increase product safety.
KEY WORDS: consumer preference, double-hurdle model, food satety . marketing, oyster industry
INTRODUCTION
METHODS
Overall per capita fresh shellfish consumption in the United
States has increased from 2.5 pounds in 1980 to a high of 4.7
pounds in 2000 (Fig. 1). Per capita consumption of oysters, how-
ever, has decreased from an average of 0.35 pounds per year
(average of 1980-1989) to 0.25 pounds in 1990 to 0.20 pounds in
1999 and 2001 (USDOC 2001; Fig. 2).
Food safety is a factor often blamed for decreases in consump-
tion of oysters. In a 1993 news release, a multi-state outbreak of
viral gastroenteritis related to consumption of oysters occurred iti
Louisiana. Maryland, Mississippi, and North Carolina (Centers for
Disease Control and Prevention 1993). In 1998. bacteria-tainted
oysters from Texas were identified as the cause of sickness for 368
people, and in the preceding summer, 209 laboratory-confirmed
cases of illnesses were linked to consumption of raw oysters har-
vested in the Pacific Northwest (ABC News 1998). The Center for
Science in the Public Interest has asked FDA "to take immediate
action to protect consumers from raw oysters contaminated with
deadly bacteria" (Center for Science in the Public Interest 2000).
They cite 36 deaths in the previous 2 years and 1 19 deaths since
1989 associated with Vibrio viiliiifuus — contaminated raw oysters
and other shellfish. In 1990. Billups (2001) showed only 9% of
respondents considered oysters "not at all safe" compared with
31% rate in a similar survey conducted 5 years later.
Although food safety is suspected to be a major factor in the
decision to consume oysters, other factors may be involved. Re-
gional and national oyster consumption can be affected by many
determinants that may vary across geographical region, ethnicity,
income levels, and perceptions of nutritiim (Wessells et al. 1994.
Gempesaw et al. 1995. Wessells & Anderson 1995. Manalo &
Gempesaw 1997, Wessells & Holland 1998, Holland & Wessells
1998). The goal of this study was to investigate the decision to
consume oysters and the decision of frequency of oyster consump-
tion.
*Corresponding author. E-mail: lahouse@'utl.edu
The data for this study was obtained through a mail survey.
After conducting a number of focus groups of seafood consumers
and nonconsumers (in three locations in the United States), and
conducting survey pretests, a questionnaire designed to elicit in-
formation on seafood consumption, specifically consumption of
oysters, shrimp, tuna, and catfish, was mailed to a sample of 9(J00
households in the United States, with 1000 mailed to each of the
nine major census regions (shown in Fig. 3: Hanson et al. 2002).
The stratified sample was chosen as the region is expected to be a
significant determinant of both the choice to consume and the
choice of how often to consume oysters. The surveys were mailed
in late 2000 and early 2001, with households receiving a second
copy of the survey if they did not return the first. This approach
resulted in a return of 1 790 surveys or a response rate of 20. 1 %
(after accounting for "return-to-sender" surveys). Because of the
length and complexity of the survey, a large number of respon-
dents did not answer all of the questions in the survey, therefore,
a total of 874 observations are included in this study.
Table 1 shows descriptive statistics for the responses used in
this study. Compared with U.S. Census data (United States Census
Bureau 2000), the results showed a larger percent of Caucasians
responded to the survey (89% in the survey compared with 75% in
the 2000 US Census). The survey results also contained a sample
slightly older than the US population, with 69% of survey respon-
dents over the age of 45. compared with 53% of the US adult (over
25) population. The tnean response for income in the survey was
in the S50,000-$59,999 category, compared with a US mean of
$42,148. Religious composition of the survey respondents corre-
sponds to that presented in the World Almanac and Book of Facts
(1999), i.e., 85% of the US population practices Christianity, in-
cluding 23% Catholic, and approximately 2% and 1% of the US
population practices Judaism and Islam, respectively. Our survey
results indicated 83% Christianity with 25% Catholic, and 3%'
practicing Judaism.
In a series of six questions, respondents were asked to indicate
how often they consumed oysters for breakfast, lunch, and dinner,
both at home and away from home. This differs from most previ-
ous studies (including Cheng & Capps 1988. Yen & Huang 1996)
52
House et al.
o>
o
^—
CN
CO
■V
lO
CD
h-
oo
CJ)
o
^—
oo
O)
CD
cn
cn
O)
O)
CT)
cn
o>
cn
o
o
a>
o>
O)
O)
cn
OJ
cn
<3)
cn
cn
cn
o
CM
o
CM
Figure 1. I'liited States per capita fresh and liozen shellllsh consiimplicin (Source: IISDA, ERS. 1999).
that analyze at-home consumption only. Overall. 56.9% of the
respondents indicated that they never ate oysters. The means and
ranges of the responses are shown in Table 2. As expected, con-
sumption of oysters, as well as other seafood products, differed by
region of the respondent's residence (Fig, 3),
Additionally, respondents were asked to identify and rank the
top three reasons they consumed and did not consume oysters.
Results from the question on reasons nonconsumers do not con-
sume oysters and why consumers do not consume more oysters
provide an interesting insight into the data (Fig. 4). Visual inspec-
tion of the results from this question may provide support for a
double-hurdle regression model because it appears nonconsumers
have different reasons for not consuming compared with consum-
ers decision on frequency of consumption.
A number of factors were hypothesized to be relevant to the
consumption and frequency of consumption decisions. The same
set of variables was used as regressors in both equations as theory
provides no guidance for differences and to allow for a specifica-
tion test. The dependent variable was constructed from responses
to a set of six questions regarding frequency of consumption of
oysters for breakfast, lunch, and dinner at-home and away-from-
home. If a respondent indicated they never consumed oysters for
each of the six questions, the value of the dependent variable was
set to zero. For the sample, 56,9% of the responses were zero. For
the remainder of the sample, the responses were summed to de-
termine the frequency of consumption in one month. For example,
if a respondent answered they consumed oysters once per month
for dinner at home and once per month for dinner away from
home, but never for lunches and breakfasts, their frequency of
consumption for the month was two. Those who did eat oysters
consumed oysters on an average of 2.2 times per month. Quantity
of oyster consumption was not obtained in this survey because
respondents were not asked how much was consumed (or by how
many in the household) because of time and space limitations of
the survey. Additionally, because the survey was asking for all
consumption, including away from home and recreational catch, it
was determined from the focus groups and test surveys that re-
spondents were having difficulty answering in terms of quantity
(i.e,, pounds or ounces — other quantities, such as number of oys-
ters, were not considered because of the fact other species were
considered and did not have comparable measures).
Independent variables included demographic variables (age,
gender, ethnicity, religion, household income), variables relating
to the respondents geographic location and variables relating to
slated preference. For geographic location, a dummy variable was
included representing the census region the respondent belonged
to, as well as one variable that represented how close the respon-
dent currently lives to a coast. It was hypothesized that persons
li\'ing closer to the coast would have a higher probability of con-
suming shellfish. Other expected explanatory variables included
perceptions of safety and top reasons for eating and not eating
oysters as indicated by the respondent. Descriptive statistics for all
variables are shown in Tables 1 (demographic) and 3 (other).
Model
Cheng and Capps (1988) and Yen and Huang (1996) recog-
nized the restrictions of using a tobit model in demand analysis for
finfish and shellfish. The tobit model assumes the factors that
affect level of consumption are the same as those that determine
the probability of consumption. Cheng and Capps (1988) used a
Heckman two-step procedure and Yen and Huang (1996) used a
generalized double hurdle model to analyze household demand for
finfish. As a result of information obtained in focus groups and the
preliminary visual appearance of the data, we have chosen to use
Cragg's ( 1971 ) double-hurdle model, similar to the model u.sed by
Yen and Huang (1996).
The double-hurdle model has separate participation and con-
sumption equations that are related in the following manner:
= 0
if V,* > 0 and </, > 0
otherwise
(1)
(2)
U.S. 0\sTtR C0N.SUMPT10N - To Eat or Not to Eat
53
0.30 1
0.20
1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001
Figure 2. I'nited Slates per capita consumption of oysters (Source: L'SDOC7N0.4.4/NMFS, 'Fisheries of the L.S., 2001,' September 2002).
where v,* represents the consumption decision and i/, is a latent
variable describing participation as shown below:
= .V,'P + £,
(3)
the same explanatory variables appear in all three equations, the
following value will be distributed as a x" random variable with
degrees of freedom equal to the number of explanatory variables
under the null hypothesis that the Tobit specification is correct:
a + Tii
(4)
where .v, and -, are vectors of explanatory variables and \i and a are
vectors of parameters. Estimation o( the double-hurdle model is
straightforward. Maximum likelihood estimation of a probit equa-
tion is used to evaluate the censoring rule (r,'a). whereas maxi-
mum likelihood estimates that account for a truncated normal dis-
tribution are used for the subsample of uncensored obser\ alions. A
specification test that evaluates the restrictions imposed by the
tobit specification (assumption that the decisions are based on the
same parameters) is obtained through a comparison of the log-
likelihood function \ alues of the tobit. probit. and truncated nor-
mal regression models (Greene 1993). Specifically, assuming that
'^ — -VTdhit ./pr.ihil ./Truncalcd'- ^-'j
where the /,s represent the respective log-likelihood function val-
ues.
RESULTS
Using the double-hurdle model with frequency of oyster con-
sumption as the dependent \ ariable. the model was estimated with
the variables described in Table 4. The coefficients from the probit
and truncated tobit equations, as well as the marginal effects (cal-
culated at the means) are reported in Table 5. The probit model
correctly predicted a consumer's likelihood to consume or not
1.00
0.80
0.60
0.40
0.20
0.00
T3
n I
III
I I I IIJUULIJ
-a c
^1
(/I
CS
C
0
CO
SI
0
2
3
0
2
4-»
z
^
4-1
C
U^
c
3
■t^
^mi
u
0
cd
u
0
«a
U
UJ
^
UL)
o 2
1) (J
c
2
c
3
O
2
a.
Figure 3. Percent consumption of oysters by region.
54
House et al.
TABLE 1.
Summary of demographics.
Oyster
Nonconsumers ( % )
Oyster
Consumers ( % )
Overall
Sample (%)
Age of Respondent
Greater than 6?
Between 50 and 65
Between 35 and 50
Under 35
Gender
Percent female
Household Income
Less than $29,999
Between $30,000 and $59,999
Between $60,000 and $99,999
100.000 or greater
Region of Residence
New England
Mid-Atlantic
Southeast Atlantic
East North Central
East South Central
West North Central
West South Central
Mountain
Pacific
Lives within 50 miles of Coast
Religion
Catholic
Christian
Other
Ethnicity
Caucasian
Noncaucasian
Education
High school or less
Some College
College degree(s)
17.3
34.0
39.4
9.3
52.7
16.3
37.2
29.2
17.3
13.1
10.7
9.3
14.7
8.2
13.9
7,0
13.5
9.7
29.4
26.4
56.1
17.5
90.5
9.5
17.1
32.2
50.7
19.6
39.3
33.7
7.4
67.4
11.4
34.0
28.1
26.5
9.8
8.8
14.6
8.0
12.2
9.3
13.8
13.0
10.6
30.0
23.6
59.4
17.0
87.3
12.7
14.9
30.0
18.3
37.0
36.3
8.5
59.0
14.2
35.8
28.7
21.3
11.7
9.8
11.6
n.8
10.0
11.9
10.0
13.3
10.1
29.6
25.2
57.6
17.3
89.1
10.9
16.1
31.2
52.6
consume oysters 87% of the time (incorrectly predicted consump-
tion 49c of the time and no consumption 9% of the time). The
results of the test shown in equation (5) indicate the double-hurdle
model is a better specification than the traditional tobit (\ =
264.9, df = 431. The results indicated that different variables
affected the decision to consume versus the decision of frequency
of consumption, as expected. A set of variables was included to
determine if the location of purchase of seafood affected either
decision. Results indicated that if a person bought seafood (any
seafood, not just oysters) at grocery stores (GRSOURCE) or spe-
cialty stores (OTHERCS; such as fish markets or gourmet stores),
they were more likely to be oyster consumers. However, these
variables did not significantly influence frequency of consumption.
The variables indicating if a person consumed seafood purchased
from restaurants (RESTSC) or obtained through recreational catch
(RECCATCH) were not significant in determining if a person
would consume oysters, but significantly decreased the frequency
of consumption. A potential explanation for these results is that if
a person purchases seafood (again, any seafood) from grocery
stores or specialty stores, they are a different type of seafood
consumer than someone who purchases from a restaurant or eats
recreational catch. Perhaps they are more "dedicated" seafood con-
sumers than those who eat at restaurants, hence more likely to eat
oysters, as well as consume different types of seafood than those
who eat recreational catch (unlikely to be oysters). Following this
line, a person who does eat oysters, but is a restaurant or recre-
ational catch consumer is likely to consume oysters less frequently.
Our results indicate the average oyster consumer consumes oysters
2.21 times per month. Respondents who purchased seafood from
restaurants were likely to consume oysters 1.16 times per month
and those who indicated recreational catch as a source of seafood
were likely to consume 1.84 times per month.
Respondents were asked to identify the top three reasons they
consumed oysters. These reasons give insight to the type of person
that both consumes oysters and what influences a person to con-
sume more or less frequently. If the person indicated they enjoyed
the flavor (FLAVOR) of oysters, as expected, they were both more
likely to consume oysters (66.5% more likely) and consume oys-
ters more frequently (0.46 more times per month). Tradition
(TRAD) plays a part in determining how frequently a consumer
eats oysters, but did not influence whether the person was a con-
sumer. In other words, those who indicated they eat oysters out of
tradition, or habit, were likely to eat oysters 0.62 times more often
per month. Importance of availability was shown in the probit. but
U.S. OvsThR Consumption - To Eat or Not to Eat
55
TABLE 2.
Statistics on frfqutiK> of ojsttr consumpliun (H = 1(167).
Mean
Mode
(Times
Consiinii
dAIonthl
(% Frequency)
Range
Breakfast at home
(1.0.^
Never (93.0%)
Never to less than weekly
Breakfast away from home
(1.01
Never (97.1%)
Never to less than 1 /month
Lunch at home
(1.14
Never (84.0%)
Never to 1/week
Lunch away from home
11.2(1
Never (74.8%)
Never to 1/week
Dinner at home
(1.21
Never (73.8%)
Never to 1/week
Dinner away from home
(1.34
Never (63.0%)
Never to 1/week
Respondents used a scale of 0 to 6 to indicate frequency where 0 = Never; 1 = Infrequently (<I/month); 2 = l/moiilh. 3
1/week .
Daily.
tiot truncated tobit equation. Consumers who believed availability
was an important reason for consumption were 22.4% more likely
to consume oysters. This may be reinforced by the results from the
regional variables. Additionally, those who indicated variety in
diet (VDIET) was an important factor were 30.3% more likely to
consume oysters. Although insignificant, it is interesting to note
the sign on the coefficient for VDIET in the results from the
truncated tobit equation was negative. Intuitively this is attractive,
as someone interested in adding variety might eat oysters, but not
that frequently. Factors that were indicated as a reason for con-
sutning oysters, hut were not significant, included health reasons
(HEALTH), price (PRICE), convenience (CONVl. preparation
knowledge (KNOWHOW). and aphrodisiac properties (APHROD).
Respondents were also asked to identify the top three reasons
they did not consume oysters, or did not consume oysters more
frequently. Three of these reasons significantly influenced the de-
cision to consume oysters: price (NOPRICE). allergic reaction
(ALLERGY), and taste (TASTE). Consumers who indicated they
did not like the taste of oysters or were allergic to oysters were
significantly less likely, 16.3% and 38.7%. respectively, to con-
sume oysters. Those who indicated price was a reason for not
consuming oysters were 17.9% more likely to be oyster consum-
ers, but were likely to consume 0.39 times less frequently than the
average oyster consumer. Oyster consumers who lacked prepara-
tion knowledge (LPKLDGE) were likely to consume 0.62 times
less frequently per month than average.
Perhaps the most interesting result is that "concerns about prod-
uct safety" (PRODSAFE) did not influence a person's decision
whether to eat oysters. Additionally, a variable that indicated the
respondent believed oysters were the least safe of all seafood prod-
ucts (UNSAFE) was not significant in the decision to consume.
Concern about product safety did. however, decrease frequency of
o
^^ ^ CC
(J- < J
c <u
S
c
o
O M
o
•♦-•
a "^
rt ^
o
s
(30
c
a>
Prepar
Know
3
u
D
H
o
o
H
S
C
o
a.
0-
CO
X
tj
INon- Consumers D Consumers
Figure 4. Reasons given for not consuming oysters or not consuming more oysters.
56
House et al.
TABLE 3.
Statistics on factors included in the double-hurdle model.
Mean.
Mean.
Overall
Nonconsumers
Consumers
Mean
Frequency of oyster consumption (dependent variable)
U/monlh (4M7 observations)
2.21/month (377 observations)
U.y5/month
Indicated oysters were the least safe of all
shellfish and fintlsh
products
34.6%
44.5%
39.0%
Indicated the following was a source of seafood for con^
iumption:
Grocery store
86.1%
89.4%
87.5%
Restaurant
86.3%
90.7%
88.2%
Recreational catch or fish farms
15.7%
27.1%
20.6%
Fish market or gourmet store
17.5%
37.1%
26.0%
Indicated the following was one of the top
three
reasons
for
consuming oysters
Enjoy flavor
4.4%
65.6%
31.8%
Variety in diet
2.2%
31.6%
15.3%
Availability
1.5%
21.9%
10.6%
Tradition/habit
2.2%
16.6%
8.6%
Health/nutrition
1.0%
16.4%
7.9%
Know how to prepare
0.5%
8.2%
3.9%
Convenience
0.5%
7.2%
3.5%
Price
1.0%
5.9%
3.2%
Aphrodisiac properties
0.3%
4.8%
2.3%
Other
0.3%
4.0%
2.0%
Indicated the followmg was one of the lop
three
reasons
for not
consuming oysters
Taste
49.7%
8.8%
31.5%
Texture
43.8%
10.1%
29.1%
Smell
26.7%
5.5%
17.2%
product safety concerns
20.9%
25.3%
22.9%
Price
12.7%
37.9%
23.9%
Fresh not available
5.1%
20.4%
11.9%
Lack of preparation know ledge
9.8%
12.0%
10.8%
Custom
4.2%
4,4%
4.3%
Health/nutrition
2.5%
6.3%
4.2%
Too time consuming to prepare
3.0%
5.9%
4.3%
Other
8.2%
3.2%
5.8%
consumption for oyster consumers, from the average of 2.21 to
1.63. a 0.58 per month decrease.
Demographics did have an effect on both the choice to con-
sume and the frequency decision. Persons living in the Southeast
Atlantic (SEATL) and West South Central (WSC) regions of the
country were more likely (17.891- and 33.29f respectively) to con-
sume oysters than persons living in New England. Other regions
did not significantly differ from the New England region. Persons
in the East South Central (ESC). West South Central (WSC). and
Pacific (PACIFIC) regions were likely to consume irtore fre-
quently (0.90. 1.08. and 0.80 times per month, respectively) than
those in the New England region. In the United States. 67% of
oyster landings come from the Gulf of Mexico and 23% from the
Pacific region (USDOC 2002). Given the three regions that con-
suined oysters significantly more frequently are closest to oyster
production, these results make intuitive sense.
All income categories above the base category of $30,000 or
less consumed oysters significantly more frequently. However,
income was not a factor in the decision to consume. Birlhdate (BD)
was a factor in both decisions, with younger ages significantly less
likely to consume oysters, or if they were oyster consumers, sig-
nificantly likely to consume less frequently. Education levels, re-
ligion, gender, and ethnicity did not significantly infiuence either
the participation or consumption decisions in this study. However,
the sample did not include a representative portion of the nonCau-
casian population in the United States. Future studies might benefit
from specifically targeting these populations for information on
seafood consumption.
DISCUSSION
The two main goals of this study were to determine whether the
factors that infiuenced the decision to consume oysters differed
from the factors that influenced the decision of how often to con-
sume oyster and to see what factors were significant that could be
used to develop marketing strategies for the oyster industry. Re-
sults showed that the two decisions were based on significantly
different factors, as suspected. Though food safety is often credited
as a reason why people do not consume oysters, this was not. in
fact, the case. Concerns about food safety did influence how often
oyster consumers ate oysters, but did not significantly influence
whether a person was an oyster consumer. In fact, the belief that
oysters are the least safe of all fish and seafood products did not
influence this decision either. Somewhat surprisingly, nearly 45%
of oyster consumers identified oysters as the least safe of all sea-
food products, while only 35% of nonconsumers identified oysters.
U.S. Oyster Consumption - To Eat or Not to Eat
57
\ariate
Source cil purchase
Reasims lor eating oysters
Reasons tor not eating oysters, or
not consuming oysters more
frequentls
TABI.F, 4.
Description ol independent \ariables.
\ ariable Name
Safety perception
Region of residence (U.S. Census
GRSOURCE
RESTSC
RECCATCH
OTHERSC
FLAVOR
HEALTH
TRAD
PRICE
AVAIL
CONV
VDIET
KNOWHOVV
APHROD
NOPRICE
NOFPAVAI
NOCUSTOM
LPKLDGE
TOOTIME
TEXTURE
SMELL
TASTE
TRAUMA
PRODSAFE
ALLERGY
UNSAFE
Description
I if seafood is purchased at a grocery store
1 if seafood is purchased at a restaurant
I if seafood is from recreational catch
I if seafood is purchased at specialty fish markets or gourmet stores
The following variables are 1 if this reason was listed as one of the top three reasons for
consuming oysters:
Enjoy flavor
Health/nutrition
Tradition
Price
Availability
Convenience
Variety in diet
Know ledge of how to prepare
Aphrodisiac properties
The following variables are I if this reason was listed as one of the top three reasons for
NOT consuming oysters, or not consuming MORE oysters:
Price
Lack of availability of fresh products
Custom
Lack of preparation knowledge
Too time consuming to prepare
Dislike texture
Dislike smell
Dislike taste
Traumatic experience
Product safety concerns
Allergic reaction
I if respondent believes oysters are the least safe of all seafood products
Religion
Race/Ethnicity
Income
Education
Proximity to Coast
Age
Gender
NEWENG New England (omitted category)
MIDATL Mid-Atlantic
SEATL Southeasit Atlantic
ENC East North Central
ESC East South Central
WNC West Nonh Central
WSC West South Central
MOUNTAIN Mountain
PACIFIC Pacific
CHRISTIA Christian (omitted category)
CATHOLIC Catholic
OTHERREL Other religions
CAUC 1 if Caucasian, 0 otherwise
EMCI <$30.000 (omitted category)
INC2 $30.000-$.59.999
INC3 $60.000-S99.999
INC4 SIOO.OOO or above
EDUCATI High School degree or less
EDUCAT2 Some College
EDUC.AT3 At least one degree from College
PROXCST I if currently lives within 50 miles of a coast
BD Birth date
GENDER 1 if female
However, 25% of oyster consLimers indicated they ate oysters less
frequently due to product safety concerns.
Results indicated that people did not consume oysters, and did
not consume oysters as frequently, if they indicated price was an
inhibiting factor. Future studies are needed to address the issue of
willingness to pay for safer oyster products. Consumers who in-
dicated price was a reason they did not consume oysters more
frequently were likely to consume oysters 0.39 times per month
58
House et al.
TABLE 5.
Empirical results from double-hurdle model.
Variable
Probit
Truncated
Name
Coefficient
F(z)/X
Coefficient
E(Y*)/X
Source of seafood for consumption
GRSOURCE
0.391**" (0.197)"
0.155
1.949(2.054)
0.263
RESTSC
0.005(0.196)
0.002
-7.783* (2.142)
-1.050
RECCATCH
0.249(0.164)
0.099
-2.711*** (1.509)
-0.366
OTHERSC
0.699* (0.155)
0.277
2.039(1.362)
0.275
Top three reasons
for consuming
oysters
FLAVOR
1.682* (0.181)
0.665
3.434*** (2.016)
0.463
HEALTH
0.155(0.324)
0.061
-2.771 (2.968)
-0.374
TRAD
-0.223(0.241)
-0.088
4.579* (1.746)
0.618
PRICE
-0.201 (0.340)
-0.080
3.124(2.134)
0.422
AVAIL
0.566** (0.261)
0.224
-0.821 (1.445)
-0.111
CONV
0.411(0.467)
0.163
1.210(2.034)
0.163
VDIET
0.766* (0.223)
0.303
-1.576(1.361)
-0.213
KNOWHOW
0.164(0.417)
0.065
1.819(2.147)
0.245
APHROD
0.569(0.517)
0.225
-2.500 (3.286)
-0.337
Top three reasons
for not consuming oysters, or not consuming more oysters
NOPRICE
0.454* (0.155)
0.179
-2.852** (1.473)
-0.385
NOFPAVAI
0.172(0.209)
0.068
0.402(1.749)
0.054
NOCUSTOM
-0.217(0.296)
-0.086
-3.530(3.464)
-0.476
LPKLDGE
0.065(0.184)
0.026
-4.618** (2.170)
-0.623
TOOTIME
-0.314(0.307)
-0.124
0.008 (2.556)
0.001
TEXTURE
-0.030(0.175)
-0.012
3.312(2.523)
0.447
SMELL
-0.215(0.192)
-0.085
-3.531 (3.511)
-0.477
TASTE
-0.412** (0.169)
-0.163
-5.850** (3.054)
-0.790
TRAUMA
-0.727(0.519)
-0.288
14.509(9.523)
1.958
PRODSAFE
-0.145(0.152)
-0.057
-4.311* (1.708)
-0.582
ALLERGY
-0.977** (0.589)
-0.387
-4.596(7.728)
-0.620
BeMe\'ed oysters to be least safe of all seafood products
UNSAFE
-0.048(0.1.%)
-0.190
1.889(1.354)
0.255
Demographics
MIDATL
0.152 (0.279)
0.060
3.535 (3.207)
0.477
SEATL
0.450** (0.270)
0.178
2.263(2.918)
0.305
ENC
-0.118(0.290)
-0.047
4.071 (3.470)
0.549
ESC
0.480 (0.299)
0.190
6.632** (3.211)
0.895
WNC
0.040 (0.297)
0.016
3.991 (3.438)
0.539
WSC
0.840* (0.308)
0.332
8.017* (3.151)
1.082
MOUNTAIN
0.246(0.290)
0.097
3.851 (3.367)
0.520
PACIFIC
0.139(0.274)
0.055
5.927** (3.044)
0.800
CATHOLIC
0.039(0.150)
0.015
-1.259(1.538)
-0.170
OTHERREL
-0.008(0.168)
-0.003
1.183(1.688)
0.160
CAUC
-0.266(0.190)
-0.105
-0.944(1.796)
-0.127
INC2
0.151 (0.193)
0.060
7.973* (2.601)
1.076
INC3
0.099(0.210)
0.039
6.859* (2.634)
0.926
INC4
0.224 (0.229)
0.089
6.105** (2.701)
0.824
EDUCAT2
0.081 (0.190)
0.032
2.545(2.070)
0.343
EDUCAT3
-0.077(0.191)
-0.031
-0.831 (2.014)
-0.112
PROXCST
-0.220(0.185)
-0.087
2.112(1.705)
0.285
BD
-0.008* (0.0002)
-0.003
-0.007* (0.003)
-0.001
GENDER
0.106(0.130)
0.042
1.461 (1.453)
0.197
Log-likelihood function
-281.04
-635.67
Percent of correct
predictions in
prohit model
87.1%
■" One. two, and three asterisks indicate significance at the 0.01,
'' Standard errors of the coefficients are reported in parentheses.
0.05. and 0.10 levels, respectively.
less frequently than the average oyster consumer. However, con-
sumers who indicated concern for product safety was a reason for
not consuming were likely to consume oysters 0.38 titties per
month less frequently. The tradeoff between an increased price due
to increases in costs of implementing safety programs and in-
creases in consumption if consumers believe oysters to be safer is
an area for future investigation.
Overall, this study does identify characteristics that the oyster
industry can use to segment consumers for marketing purposes. As
expected, people living in regions nearest to oyster production are
U.S. O'l'STBR Consumption - To E.m or Not to Eat
59
more likely to consume oysters and more likely to consume more
oysters. Avuilubility ot fresh products also significantly increased
the likelihood of the respondent to consume oysters. Consumers
who purchase seafood products at grocery stores or specialty stores
may be a segment that could be targeted, as they are more likely
to consume oysters.
ACKNOWLEDGMENTS
This research was supported by the Florida Agricultural Ex-
periment Station and the following grants and approved for pub-
lication as Journal Series No. R-09388. This work is a result of
research sponsored in part by the National Oceanic and Atmo-
spheric Administration, U.S. Department of Commerce under
Grant #GMO-99-24. the Mississippi-Alabama Sea Grant Consor-
tium. Mississippi State University, and University of Florida. The
U.S. Government and the Mississippi-Alabama Sea Grant Consor-
tium are authorized to produce and distribute reprints notwith-
standing any copyright notation that may appear hereon. The views
expressed herein are those of the author(s) and do not necessarily
reflect the views of NOAA or any of its subagencies. This material
is based upon work supported by the Cooperative State Research.
Education and Extension Service, U.S. Department of Agriculture,
under Agreement No. 99-.388 1 4-8202. Any opinions, findings,
conclusions, or recommendations expressed in this publication are
those of the author(s) and do not necessarily reflect the view of the
U.S. Department of Agriculture.
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REHABILITATION OF THE NORTHERN QUAHOG (HARD CLAM) (MERCENARIA
MERCENARIA) HABITATS BY SHELLING— 11 YEARS IN BARNP:GAT BAY, NEW JERSEY
JOHN N. KRAEUTER,' MICHAEL J. KENNISH," JOSEPH DOBARRO/
STEPHEN R. FEGLEY/ G. E. FLIMLIN JR.'
^Haskiii Shellfish Research Laboratory. Institute of Marine and Coastal Sciences. Rutgers University:
6959 Miller Avenue, Port Norris. New Jer.'iey 08349. 'Institute of Marine and Coastal Sciences. Rutgers
Utiiversity. 71 Dudley Road. New Brunswick. New Jersey 08901. ^Marine Field Station. Institute of
Marine Coastal Science. 132 Great Way Blvd. Tuckerton, New Jersey. '^Department of Oceanography
Castine, Maine 04421. ^ Maine Maritime Academy. Rutgers Cooperative Extension. 1623 Whitesvllle
Road. Thomas River. New Jersey 08753
.ABSTRACT The use of shell or other coarse material to enhance sur\ ival of newly set hard clams (Mcnciiana incrccnariu) has been
suggested as a management strategy to increase clam stocks. Barnegat Bay, New Jersey and surrounding areas supported a large clam
fishery throughout the 1950s and 1960s, but this resource has declined in recent years. We established replicate 20 x 70 m plots of high
shell densitv, low shell density, and no shell (control) in a Latin Square design in 1990 and have obtained periodic samples since that
time. The shell, obtained from ocean quahog processing plants, had been broken into a variety of sizes. High-density shell received
900 bu per plot, and low -density shell received .^00 bu per plot. Plots with high shell density had significantly more clams after 10 years
than those with low-density shell or controls. High shell density significantly increased hard clam recruitment, but this exceeded 1 m""
in only one year, from the years 1990 to 2000. In plots with low shell or in controls, recruitment never exceeded 0.4 nr-. and in half
or more of the years no recruitment was found. Some individual plots with shell did not enhance recruitment, indicating that factors
not investigated must be important as well. In spite of the low recruitment density, there appears to be an increase in survivorship when
the shell content is greater than 8000 gm"".
KEY WORDS: Merccnaria meicfiniha. shelling, hard clam recruitment, quahog
INTRODUCTION
Methods of increasing natural abundance of hard clams {.Mer-
cenaria mercenaria) are important to state resource managers and
the shellfish industry. There are several approaches a manager can
use to improve shellfish stock abundance: ( 1 ) increasing the num-
bers of spawners (spawner sanctuary); (2) reducing harvests or
providing alternate areas in some cycle so the stocks last longer;
(3) adding hatchery produced clam seed to a selected area; and (4)
protecting naturally set clams (shelling or other substrate modifi-
cation and use of chemicals to eliminate predators).
The theoretical concept underlying a "spawner sanctuary" is
that increasing the number or density of clams in an area will
increase the number of eggs, larvae, and, set clams. The potential
for an increased number or greater concentration of clams to pro-
duce more larvae when conditions are favorable is suspect because
it depends on the existence of a spawner-recruit relationship (more
spawners = more recruits) over a wide range of clam densities. In
addition, there are large numbers of clams in most bays even at low
densities, and thus the numbers of clams that inust be transplanted
to have even a small probability of significantly increasing the
number of active spawners in the region is extremely large. Fi-
nally, of those sanctuaries that have been created in New Jersey
and New York, preliminary evidence indicates that little detectable
enhancement of natural hard clam stocks may be expected (Kass-
ner & Malouf 1982, Barber et al. 1988).
Reducing harvests allows clams to be harvested over a longer
period of time while waiting for the next surviving .set. While this
appears to be attractive, hard clams are different than most species
harvested from the wild. Smaller sizes of hard clams (liltlencck)
command a premium price. Econoinic considerations suggest that
most of the clams should be harvested in the smaller sizes and that
larger clams should only be taken as a last resort. Growth rates in
most areas are such that clams remain in these premium si/c
classes only a few years. This suggests that the best economic
returns would be from intense harvest on these sizes. The only way
to manage the fishery for maximizing economic benefit would be
through an extensive monitoring program to delineate areas with
maximum concentrations of appropriate sizes (McHugh 1991).
The third option, the use of hatchery seed to enhance hard clam
production is well established in aquaculture (Manzi & Castagna
1989). In general, predation rates on high-density plantings of seed
without protection devices are too high to recoinmend this option
(Kraeuter & Castagna 1989). Preliminary experiments using low
density seeding of hard clams suggest this may yield higher sur-
vival rates than would be expected from dense plantings [Macfar-
lane (Orleans, MA), and Relyea (F. M. Flowers and Sons, pers.
comm.)]. These observations are supported by the work of Paulsen
and Murray (1987). They conducted a number of short-term (less
than one year) experiments using three seed sizes, at high and low
density, planted both on and below the sediment surface. They
reported that survival (58 days) of clams planted below the sedi-
ment surface at high densities was no greater than if seed were
broadcast. Low-density plantings of hard clams below the surface
significantly increased long-term survivorship when compared
with similar high-density plantings. Peterson et al. (1995) have
provided additional evidence indicating that low-density plantings
of large (>20 mm) seed may be an economically viable means of
increasing hard clam stocks in isolated basins.
The fourth option, modifying the substrate to increase post
settlement survival of juvenile hard clams, has been shown to
work. MacKenzie ( 1977, 1979) demonstrated that treating areas of
bay bottom with various pesticides significantly increased juvenile
hard clam survivorship by eliminating arthropod predators such as.
shrimp and crabs. Siinilar techniques provided additional protec-
tion to seed clams planted in mesh and gravel protected aquacul-
ture plots (Kraeuter & Castagna 1985). The use of this technique
61
62
Kraeuter et al.
is considered to be unacceptable because it requires introducing
toxic chemicals into tlie environment, and these may produce long-
term detrimental effects. Parenthetically, it is plausible that the
massive use of pesticides during the 1950s and 1960s, to control
insects in the coastal marshes of New Jersey, was the proximal
cause of the high abundance of hard clams in some of these shal-
low, poorly flushed systems.
An alternative of the fourth option, that has also been shown to
increase survival of juvenile hard clams in different habitats, is
"shelling" the bottom (Parker 1975. Kraeuter & Castagna 1977,
Kraeuter & Castagna 1989. Kassner et al. 1991). This practice
involves broadcasting pieces of broken shell or stone aggregate
over the bottom to increase the percent composition of larger par-
ticles (stone or shell) in the sediments. This technique was devel-
oped from the many studies revealing that hard clams are more
abundant in areas with a higher percentage of shell in the bottom
(Pratt 1953. Wells 1957. Saila et al. 1967. Walker & Tenore 1984.
Craig & Bright 1986. Papa 1994). The larger particles have two
mechanisms by which they can affect hard clam abundance. Wells
( 1957) suggested that shell might create areas of low current speed
in which small clams either collect {sensu Carriker 1961) or at
least are not swept away. He also proposed that the hard substrates
provide a byssal attachment point for newly set clams. A large
body of evidence indicates that coarser material can interfere with
the ability of many hard clam predators to detect or manipulate
small clams (Arnold 1984, Kraeuter 2001). Any or all of the.se
mechanisms can have a positive effect on natural set resulting in
greater numbers of clams surviving to market size.
The shelling option can be used, but it cannot he used with
confidence. Kassner et al. (1991) found no significant enhance-
ment of a clam area with low abundance in Great South Bay. New
York, one year after placing 12.5L shell m"" on a mud bottom.
Several important variables associated v\ ith construction of shelled
plots are unknown. For example, the amount of shell added must
fall within a bounded region; too little shell may not effectively
deter predators while too much shell may serve as a haven for the
same predators. There is uncertainty regarding the amount of shell
needed to afford protection. For example, most studies and surveys
of natural populations indicate a positive effect of larger particles,
but Day (1987) has observed in the laboratory that mud crab pre-
dation was greater in gravel and gravel and sand mixtures than in
sand alone. She suggests that the gravel substrates offer hiding
places for these small predators and thus increase the predation
rate. Further, little information exists on: density of shelling, shell
size, plot size, substrate type (grain size, percentage of organic
matter, redox discontinuity level, water content, etc.) and their
interactions (density of shell x shell size, density of shell x plot
size, density of shell x substrate type, etc.) relative to clam sur-
vival. This information is essential to allow some predictive capa-
bility concerning whether the increased numbers of clams avail-
able for harvest will justify the cost of the original shelling. In
addition to the effects of shelling on the clams, infonnation con-
cerning the shell size, shelling density, substrate type, and their
interactions is required to evaluate the increased effort that might
be required to harvest the potentially increased numbers of clams
(shell fragments could interfere with the harvest).
METHODS
This study was designed to determine whether shelling the
bottom, at a spatial scale large enough to be meaningful to habitat
management, produces significant increases in hard clam abun-
dance. A subset of the design examined two densities of shell
cover: low density and high density. The major uncontrolled vari-
ables were the sporadic nature of hard clam spat set and predator
populations.
The experimental design was a Latin Square matrix of 20 x 70
m plots (slightly more than 0.15 ha). A rectangular shape was
chosen because a boat was used to place the shell into the plots.
Plots were arrayed in a 3 x 3 Latin Square design with 30 m buffer
zones between each of the separate plots. The entire matrix was
surveyed using sextants: comers were marked with stakes and
buoys. Three treatments were arrayed within the plots: (I) 10
high-density shelling — 900 bushels per plot (15 L/m"); (2) 20 low-
density shelling — 300 bushels per plot (5 L/ni"); and (3) control —
no shell added. Most of the shell consisted of broken pieces (2-8
cm") of A rctica islandicci. although some Spisiila soliilissima shell
and the shell debris of other offshore species could be seen. This
shell is available in large quantity from several local clam-
processing plants.
Shell Spreading
The experiment was located approximately 200 m east by
northeast from Gulf Point in Barnegat Bay. New Jersey. The co-
ordinates of the matrix are: NE corner — 39^44. 23 'N by
74°9.05'W, NW comer— 39°44.23'N by 74°9.I2'W, SE comer
— 39°44.04'N by 74°9.05'W, and SW corner— 39°44'N by
74"9.I2'W. The site, characterized by sandy sediments with rela-
tively low silt-clay content and few naturally occuning shells,
experienced only moderate tidal currents. It had a fairly uniform
bottom composition and water depth (4 m). was protected from the
longest fetches that occur in the bay. and had long been a hard
clam habitat. The latter was determined through discussions with
several local watermen who aided in the site selection process and
designated this area as the best location for our project and least
disruptive to their activities.
Shell was spread onto the experimental plots during the week
of April 23 to April 27. 1990 using the Ocean County Bridge
Department's LCM. the Beujamin H. Mahie. The shelling required
2 days to complete.
The shell was stored in the middle of the ship and transferred
to a hopper with a small catloader. The viilume of the scoop of the
catloader was calibrated previously so that the shell volume going
overboard could be estimated. The shell moved from the hopper
via a conveyor belt to a highway salt spreader located in the bow
approximately 4 m above the water. This procedure produced an
evenly dispersed spread of shell on the bay bottom. SCUBA ob-
servation subsequent to spreading confirmed the even nature of the
shell on the bottom.
During the second day. it became apparent that the volume of
shells delivered was short. To accommodate this, we reduced the
size of the last high-density plot to 20 x 50 m to maintain the same
density of shell. To ensure that all plots received as nearly identical
disturbance as possible, the LCM was powered over each control
plot as if it was being shelled.
Sampling
Samples were retrieved from each plot using a diver-operated
suction sampler. Each plot was located with sextant coordinates (or
later GPS); the center was marked, and a diver was deployed
Rehabilitation of Mercenaria mercenaria
63
approximately 9 ni from the center mark. Diiriny the first year of
sampling (May 1991). a ring made from a bottomless galvanized
bucket was used to mark the area to be sampled. Samples were
collected approximately 1 m apart by removing all material from
the ring to a depth of 10 cm with a suction sampler. All materials
were collected in a .^ mm mesh bag. brought to the surface, and
preserved. During the first year of sampling. 9 samples were col-
lected from each plot each sample covering 0.043 m". Samples
were returned to the laboratory and numbers of clams removed and
the volume of the material was recorded. All hard clams were
measured in length, height and width.
Subsequent samplmg followed the same protocol except thai
the ring was modified and size of the area sampled was increased
to 0.25 m". The number of samples was reduced to five or six
during 1996 and increased to 10 during partial sampling in 1998 (.3
plots) and in 2001. The procedure in the laboratory remained the
same except that that the weight of the dried shell material was
measured rather than its volume. A factor of approximately 850 g
dry weight is equivalent to IL of this material. We chose <15 mm
as the size limit for seed clams (0 y class). For the 1996, 1998. and
2001 samples, we sectioned one of the valves of each clam that
was older than seed to determine approximate age. We counted the
annual growth rings in the valves to determine if the clams were
those that might have set since the 1990 shelling. We could not
accurately age animals older than 10 years; therefore, we consid-
ered these individuals to have been the residual population, even
though by 200! they may have recruited after the experiment
started.
RESULTS
Samples were retrieved from plots on May 2 1 to May 28. 1991 ;
September 30 to October 2, 1992; November 24, 1993; June 23 to
June 25, 1996: August 10 to August 12. 1998; and November 14
to November 15, 2001 . During the first year of sampling, only one
hard clam was found in the 72 samples that were sorted in the
laboratory. Because of insufficient numbers of hard clams in the
samples, these data were not analyzed further.
During the second year, we increased the sample size to 0.25
m~ and reduced the numbers of replicate samples per plot (Table
1). In general, setting was sparse. Data from the second year in-
dicated that on one heavy shelling treatment there was enhanced
setting. Shell weight data indicated that the other heavily shelled
plots were not sampled, and control plots were over represented.
None of the control plots had seed clams, and there were seed
clams on two of the three low-density treatment plots. Because of
the sampling difficulties, no Latin Square analysis was attempted
on the 1992 data. A linear regression of the effect of shell mass on
total clams and seed clams collected showed that shell density had
a significant positive effect on the presence of both total clams and
seed clams (Table 2).
Considerable effort was directed toward surveying the plots for
the third year, and weights of shell indicate we were successful in
sampling the stations in all but one case. Even with this effort, the
low-density plot (2-3). based on shell weight data (Table 1) ap-
pears to have had high-density shell. We conducted an ANOVA
with that plot characterized both "as-sampled" and "corrected". In
TABLE I.
Numbers of replicate samples removed from Barnegat Bay, NY shell plots by year.
Sample (Jrid
1-1
2-2
3-3
1-2
2-3
3-1
1-3
2-1
3-2
Shell Density
H
H
H
L
L
L
C
C
C
Year
1991
# replicates
Mean Shell DW
9
9
9
9
9
9
9
9
9
Total Clams
1
0
0
0
0
0
0
0
Recruits
0
0
0
0
0
0
0
0
1992
# replicates
6
6
6
5
5
8
5
5
4
Mean Shell DW
4994
1676
59.9
1059
0.2
1315
409
2.5
14.6
Total Clams
13
1
0
3
0
3
1
0
1
Recruits
11
1
0
2
0
0
0
0
0
1993
# replicates
5
5
5
5
5
.s
5
5
5
Mean Shell DW
5892
5305
3904
1463
4256
1538
89
27
23
Total Clams
10
6
2
9
3
5
0
1
1
Recruits
8
6
2
7
2
3
0
0
0
1996
# replicates
5
5
5
5
5
5
5
5
5
Mean Shell DW
6279
3966
4264
312
2986
1893
10
56
154
Total Clams
5
4
7
2
11
2
1
0
3
Recruits
5
4
6
1
5
2
0
0
2
1998
# replicates
Mean Shell DW
Total Clams
Recruits
13
4031
11
10
10
288
5
1
10
1004
4
4
2001
# replicates
10
10
10
10
10
10
10
10
10
Mean Shell DW
3285
2358
5095
639
1104
1039
24
->1
280
Total Clams
15
12
20
5
1
4
2
1
4
Recruits
12
11
IS
3
0
.^
">
0
1
H = High density shell. L = low density shell, and C = control. In 1991 replicates were 0.(143 m" all subsequent samples were 0.25 ni-
ls in grams. Clams and recruits are the totals for all samples.
. Shell drv wei.sht
64
Kraeuter et al.
TABLE 2.
Intercept, regression coefficient and correlation coefficient for the
effects of shell density (g) on total clams and those that have
recruited since 1990 (clams 0.25 m"").
Year
Intercept
Regression
IT
1992
Total Clams
0.005 NS
4.028 E-04***
0.47
Recruited Clams
-0.082 NS
3.338 E-04***
0.48
1993
Total Clams
0.297 NS
2.152 E-04**
0.18
Recruited Clams
0.1 IONS
2.080 E-04***
0.26
1996
Total Clams
0.363 NS
1.827 E-04**
0.19
Recruited Clams
0.118NS
1.876 E-04***
0.31
1998
Total Clams
0.314 NS
1.476 E-04*
0.14
Recruited Clams
0.073 NS
1.624 E-04**
0.26
2001
Total Clams
0.129 NS
3.713 E-04***
0.45
Recruited Clams
-0.003 NS
3.703 E-04***
0.53
NS = not sisnificant. *0.05. **0.01.
*0.001.
both cases the results with respect to treatments were similar, and
we have presented the as-sampled data (Table 3). High and low
shell density plots had similar numbers of total clams and seed;
both had significantly more total clams and seed than the controls.
We arrayed the data according to shell density and used linear
regression. Both total numbers of clams and seed clams (Table 2)
were significantly correlated with shell density.
Latin Square analysis of the 1996 data on shell weight indicated
that column 2 had significantly less shell than the other two. These
differences negated further use of the Latin Square. We evaluated
the total clams and recruited clams with ANOVA based on the
three treatments (high density shell, low density shell, and con-
trol): a linear regression for all satiiples (total clams and recruited
clams vs. shell weight) was then computed. There were no sig-
nificant differences in total clams with treatment (Table 3); how-
ever, the regression line showed a significant positive effect of
shell density (Table 2). In contrast, the ANOVA analyzing the
effect of shell on clams that had recruited since 1990 was signifi-
cant. A Tukey (HSD) test found that clam density in high-density
shell and low density shell were not significantly different, and low
TABLE 3.
Tukey (HSD) results (number 0.25 m"" for total number (Total) of
hard clams {Mercenaria mercenaria) and those that had recruited to
the population (Recruit) since the beginning of the
experiment ( 1990).
1993
High Low Control
1996
High Low Control
Total
Recruit
1948
Total
1.29
1.14
High
0.85
Recruit 0.69
1.13
0.73
Low
0.50
0.30
0.07
0.00
Control
0.40
0.10
Total
Recruit
2001
Total
Recruit
1 .07
1.00
High
1.53
1 .00
0.53
Low
0.33
1,40 0.20
0.27
0.67
Control
0.23
0.10
High = those areas covered with high density of shell, low = those areas
covered with low density shell, control = those areas that did not receive
shell. Underlines indicate those treatments that were not significantly dif-
ferent a (a = 0.05).
density shell and control areas had similar clam density (Table 3).
High density shelling increased clam recruitment over that ob-
served in the control areas.
In 1998, only 3 plots were sampled, and ANOVA results were
similar to 1996. There was no difference in total clams between
treatments, but the clams that had recruited since 1990 were more
abundant in high shell plots. There were no significant differences
between low shell and control (Table 3). Again, linear regression
indicated a positive effect of shell density on total and recruited
clams (Table 2).
In 2001. as with previous sampling, Latin Square analysis of
the shell distribution revealed significant differences between all
columns and some rows. The total numbers of clams and clam
recruitment were evaluated relative to shell weight and treatment
type with general ANOVA and linear regression techniques. After
I \+ years, most plots remained intact, but the increasing differ-
ences between rows and columns suggest that the shell is gradually
being dispersed. In contrast to 1996, when total clams were not
significantly different by treatment, both the total and recruiting
clams since 1990 exhibited significant differences by treatment. In
both the total clams and recruited clams, the Tukey (HSD) test
found that high shell density plots had significantly more clams
than either the low-density shell or the control. The latter two
treatments were not significantly different from each other. The
similarity between total and recruiting clams after I l-l- years may
have been greater than indicated by the base data. We were unable
to distinguish ages of clams >10 y. Thus, some of the clams in this
class may have recruited to the area since the shell was placed on
the bottom. In 2001. 20.7% of the sampled clams were in the age
10 or older category. As a comparison in 1996, 31.4% of the clams
were from classes that had recruited before the shell was placed on
the bottom).
Recruitment
We considered clams <15 mm in shell length to be seed clams.
Relatively few of these clatns were found (Table 4), and never in
the control areas. In some years, seed can be as large as 20 mm.
We found only one clam of this size in a control plot (Table 4).
We have attempted to evaluate annual recruitment (long-term
survival) of clams at this site by back calculating from the age data
to determine when particular clams had set (Fig. I). We have
averaged the data from the 1996, 1998, and 2001 samples, but,
because so few animals were obtained by sampling, have not at-
tempted to place error bars around these estimates. With the ex-
ception of 1993, there is a relatively good correspondence between
the back calculated data and that from animals recovered. The
TABLE 4.
Mean number of seed clams m'" bv treatment.
.Seed <15.1 mm
Seed <20.1 mm
Year
High
Low
Control
1992
1993
1996
1998
2001
2.00
2.67
(1
0
0
0.50
0.53
0.53
0
0
High
Low
Control
2.25
0.75
2.67
0.80
0.27
0.53
0.27
0
0
0
0
Seed = <15.1 nimor<20.1 mm Shell Length. Numher of 0.25 nr samples
is given in Table 1.
Rehabilitation of Mercenaria mercenaria
65
1.40
-High Shell
-Low Shell
-Control
Figure 1. RiTiuitnieiit of hard clams {Mercenaria mercenaria) into hi)>h-densit> shell. l<iH-dc'nsit\ shill and cdnlrol plots in Barnegeat Bay, New
Jersey. Data represent the average estimated recruitment, based on live animals collected in 1996, 1998, and 2001.
scarcity of animals precluded meaningful statistical analysis of
these data. For both estimates, areas with high shell density had
more recruiting clams than areas of low shell, and these in turn
receive more recruits than control areas. Based on these estimates,
clam recruitment to this area has generally been very low for the
past decade. Annual average recruitment, based on aged shells,
exceeded 1 ni"" only on the high shell plots sampled in 1992.
These same plots approached 1 m'" again in 1994. Recruitment in
the low shell density and control plots was below 0.5 m^" in all
years and has been 0 since 1997. Average annual recruitment in the
high shell plots shows a general trend toward less recruitment from
1998 to present when it reached 0. Data from clams <20. 1 mm also
suggest there has been little or no recruitment since 1996.
Growth
Size-at-age was computed for clams from the 1996, 1998. and
2001 samples. These data were compiled and averaged to yield an
estimate of growth (Fig. 2). Although we have few clams of age 1
and 2, the data indicate that growth is rapid until age 3, and then
abruptly slows. Growth is sporadic after age 5. The largest clam
found at this site was 82.8 mm shell length. In addition, when the
clam meat was being removed to prepare the shell for sectioning it
appeared very dark brown, black, or gray in color, e.xcept in small
clams. This condition has existed in Barnegat Bay and Little Egg
Harbor clams for a number of years.
DISCUSSION
Age (Years!
Figure 2. Growth of hard clams based on average size-al-age of live
clams collected in all experimental plots in 1996, 199S, and 2001, and
all clams <20.1 nun collected in all plots from 1992, 199.V 1996, 1998,
and 2001. ,\ll plots were in Barnegat Bay, New Jersey. Data are mean
length (mm) and the 95'7f confidence limits. Bars lacking conlldence
limits are based on one individual, .\nimals older than 9 could not be
aged, thus all data for ages 10 and 11 come from animals collecled in
1996. .Animals >10 y are based on the average of all data from all
animals aged in all years.
We have demonstrated that shelling increased ihe number of
hard clams on the bottom at an experimental site in lower Barnegat
Bay. These data are consistent with observations about the effects
of shell on the bottom and wild hard clam populations. At this site,
the shell has persisted for 1 1 years and appears to continue to
support hard clam recruitment. After 1 1 years, linear regression of
both total and recruited clams shov\ed the positi\'e effect of shell
density, but the effect of shell on clam recruitment was not sig-
nificant until shell exceeded 8 kg m^" (Fig. 3). The larger numbers
of recruits between 1 992 and 1 994. as well as Ihe lack of difference
between clam abundance in high and low density shell during this
period, suggests that the shell continued to enhance recruitment.
Beginning with the 1996 samples, there was no statistical differ-
ence in clam abundance between the high and low shell density
plots. There was also no significant difference between the low
shell density and the control sites, and by 2001 the high-density
plots were significantly different from the low-density shell and
the control. This appeared to be coupled with a general loss in
overall recruitment at the sites. The low density shelling may have
started to lose its effectiveness, but we cannot determine whether
this reflects a drop in actual recruitment or some loss of effective-
66
Kraeuter et al.
Oto2.0 2IIO4.0 41108.0 8 I to 120 i:i(ol60 l6 1to26-CI
Shell Density (Kg/sq meterl
Figure 3. Numbers of surviving clams m"" based on survival in higli-
densitj shell, low density shell, and control plots placed in Barnegat
Bay New .Jersey in 1990. Data represent back calculated (from regres-
sion equations) mean and 95 "/r confidence limits of the number of live
clams and clams <10 y of age. Numbers of the latter clams are based
on shell sections and ages of animals. These represent the animals
recruited since 1990.
ness of the shell caused by its protracted residence time on the
bottom.
Our study indicates that in areas experiencing low recruitment,
several years of data may be required to thoroughly evaluate the
effectiveness of shelling on the survivorship of hard clam seed.
Similar experiments, perhaps of significantly smaller scale, should
be conducted on different types of bottom to ascertain how much
shell is required.
Economics is one of the many important factors to consider
before any large-scale shelling program commences. It clearly
costs more to add more shell to the bottom, but we do not have
sufficient data to determine full costs per unit of shell spread. The
cost of the shell is a direct multiple of the amount to be spread (3
X more shell will cost 3 x more), but the cost of spreading the
higher density shell will be somewhat less per unit on the bottom
than will the lower density shelling. Shell costs are not insignifi-
cant, and transportation adds to these costs. In New Jersey there
are large quantities of shell produced by the surf clam and ocean
quahog processing plants and these can be purchased for about
$0.50 bu~'. The logistics of handling the shell on a regular basis
have precluded it being available free for repletion. Private con-
tractors remove the shell and store it for roads and other purposes.
Oyster shell repletion, utilizing large boats (3,000 -l- bu load) cost
about $1,000 day"', for the boat. Smaller boats (1.000 bu. load)
cost about $600 day"'. Extrapolating from these basic data, it
would cost between $2,300 and $3,100 acre"' to spread shell at the
highest density used in this experiment, but boat availability, trans-
port of shell to the sites, and other logistical costs may make these
data unreliable. We know that this particular shelling lasted at least
1 1 years without substantial loss of shell. Figure 2 also makes it
clear that high-density shell increased the clam population from a
mean of 0.7 m""-7.6 m"". nearly a factor of 10 increase, during the
first few years. This population generally persisted throughout the
course of the experiment. It is impossible to know whether the
sporadic nature of the recruitment was due to changes in recruit-
ment, shell effectiveness, or a combination of the two.
It is also unclear how long a plot can continue to enhance clam
set. It is certain that high shell density continued to support more
clam, even after 1 1 years, but there has been a noticeable decline
in the number of clam .seed (those <15 mm) through time. This is
true in both the shelled and unshelled areas. As noted above,
whether this is due to loss of effectiveness of the shell or lack of
recruiting individuals cannot be determined, but there was a gen-
eral tendency for low-density shell to be somewhat effective at the
beginning. By 2001, low-density shell had clearly reduced capac-
ity to sustain clam recruitment, but high density shelling continued
to retain recruited animals. The different rates of loss of effective-
ness make it tempting to conclude this is a function of the shell
density; however, under conditions of low recruitment, other fac-
tors may be operative and the interpretation remains uncertain. It
will require placing shell out for a number of consecutive years on
different bottom types to allow evaluation of the length of time
shell remains effective. This requires differentiation of recruitment
processes on freshly planted shell and shell placed out for a num-
ber of years.
Disturbance of the shell either by natural physical forces, such
as burial by sediments, or human activities, such as clam harvest-
ers working within an area, could alter the effectiveness of the
shell. We have no data regarding the effects of increased clam
harvesting on the enhancement capability of each shell density.
The density of marketable hard clams was low in this area; there-
fore, we do not believe disruption of the shell or sediment by
harvesting was high during the study. Pieces of shell were covered
with fouling organisms so at least some of the material remained
near the sediment surface for the duration of the study.
A 2002 survey of hard clam populations by the New Jersey
Department of Environmental Protection in Little Egg Harbor Bay
stopped just south of our experimental area, but it reported a nearly
two thirds reduction in hard clam standing stocks since the last
survey in the middle 1980s (Joseph pers. Comm.). Commercial
clam harvesters working throughout the area also indicated that
they believe that clam populations have declined significantly in
recent years.
Low levels of recruitment made it dilTicult to detect statistically
significant effects, even with 0.25 m" samples. It was only through
time and repeated sampling that we were able to evaluate the
effectiveness of the shell in this low clam density, low recruitment
area. It is also clear that in the 1 1 years of this experiment that the
control areas had just sufficient recruitment to maintain the popu-
lation al the 1990 levels. This study only covered one type of
substrate and the results could be very different under different
substrate, depth, and current regimes.
While the relationship between shell in the bottom and in-
creased hard clam density occurs wherever studies of natural popu-
lations have been conducted (Gulf of Mexico to New England), the
types of predators and their effects are substantially different. Dur-
ing 1996. we enumerated other organisms in the samples. There
was an increase in species, mainly epifauna. on the shelled areas
relative to the controls. This clearly indicates that other species are
enhanced as well. The nature of the sampling (suction sampler and
a 3-mm mesh collection bag) precluded examination of the effects
on infauna. Many of the epifauna we found are known to prey on
hard clam seed (Kraeuter 2001 ). The "reef effect" from mounds of
shell may cause an increase in epifaunal predators. It is important
to spread the shell evenly and not allow mounds to form that would
attract and retain these organisms. The best combination is for
shell to become an integral part of the bottom with only a small
portion protruding above the sediment surface. In other areas, par-
ticularly where oyster setting is high, the effect of shelling on the
establishment of oyster populations needs to be carefully evalu-
ated. Extrapolation of shell density recommendations to different
Rehabilitation of Mercenaria mercenaria
67
environments should be examined carefully before large-scale at-
tempts are made.
The slow growth rate of clams after 3 to 5 years and the small
size oi clams >I0 years old. the small size of the largest clam
collected (82.8 mm shell length), and the dark color of the meat on
most clams suggests that conditions at this site are not optimal for
hard clam production at present.
CONCLUSIONS
Shelling the bottom of Barnegat Bay. New Jersey increased the
abimdance of hard clam seed by nearly a factor of 10. The shell
remained on the plots for at least 1 1 years and continued to en-
hance the set throughout that period. Settlement was 0.5 clams m"^
on the control plots and exceeded 1 m"" only once in the high shell
areas. Clams <I3 mm in shell lenizth were never found in control
plots. This method presents a potentially viable protocol for in-
creasing survivorship of small clams from natural set. but more
thorough evaluation is needed before it can be used on a variety of
bottom types.
ACKNOWLEDGMENTS
This study would not have been possible without a large num-
ber of volunteers, and the Ocean County Board of Chosen Free-
holders who allowed the use of their LCM, and its crew from the
Bridge Department. The initial grant to provide for shelling and
sampling in 1992 came from the New Jersey Department of En-
vironmental Protection. Intermediate sampling was based on vol-
unteer effort and limited fund from the New Jersey Agriculture
Experiment Station and the New Jersey Commission on Science
and Technology. The final sampling was provided by funds from
the Fisheries Information Development Center.
LITERATURE CITED
Arnold. W, S. I9S4. The effects of prey iv/e. prediitor size, and sediment
compositicm on the rate of predation of the blue crah {CcilUnecles
sa/Hdiis Rathhun) on the hard clam {Mercenaria mercenaria Linnel. J.
Experimental Mar. Biol. Ecol. 80:207-220,
Barber. B. J.. S. R. Fegley & B. J. McCay. 1988. The Lmie Egg Harbor
hard clam spawner sanctuary: a reproductive evaluation (report). Tren-
ton. New Jersey: The New Jersey Fisheries Development CommLssion.
Carriker. M. R. 1961. Interrelation of functional morphology, behavior,
and autecology in early stages of the bivalve Mercenaria mercenaria.
J. Eli.slm Mitchell Sci. Sac. 77:168-241.
Craig. M. A. & T. J. Bright. 1986. Abundance, age distributions and
growth of Texas hard clam. Mercenaria mercenaria texana in Texas
Bays. Contrih. Mar. Sci. 29:59-72.
Day, E. A. 1987. Substrate type and predatory risk: effects ot mud crah
interaction with juvenile hard clams. State University of New York,
Marine Environmental Science (MSc thesis). Marine Science Research
Center. 1 12 pp.
Kassner. J. & R. Malouf 1982. An evaluation of "spawner transplants" as
a management tool in Long Island's hard clam fishery. ./. Sliellfrsh Res.
2:165-172.
Kassner. J., R. Cerrato & T. Carrano. 1991. Toward understanding and
improving the abundance of quahogs (Mercenaria mercenaria) in east-
ern Great South Bay, New York. In: M. A. Rice, M. Grady & M. L.
Schwartz, editors. Proceedings First Rhode Island Shellfish Confer-
ence. Rhode Island Sea Grant, Kingston, RI. pp 69-78.
Kraeuter, J. N. 2001. Predators and predation. In: J. N. Kraeuler & M.
Castagna, editors. Biology of the hard clam. Developments in Aqua-
culture and Fisheries Science, pp. 441-589.
Kraeuter. J. N, & M. Castagna. 1977. An analysis of gravel, pens, crab
traps and current battles as protection for juvenile hard clams. Merce-
naria mercenaria. Proc. World Marie. .Soc. 8:581-585.
Kraeuter. J. N. & M. Castagna. 1985. The effect of clam size, net size and
poisoned bait treatments on survival of hard clam. Mercenaria merce-
naria. seed in field plots. J. World Marie. Soc. 16:337-385.
Kraeuter, J. N. & M. Castagna. 1989. Factors affecting the growth and
.survival of clam seed planted in the natural environment. In: J. J. Manzi
& M. Castagna. editors. Clam mariculture in North America. Devel-
opments in .'\quaculture and Fisheries Science, vol. 19. New York:
Elsevier Science, pp. 149-165.
MacKenzie. C. L. 1977. Predation on hard clam Mercenaria mercenaria
populations. Trans. Amer. Fish. Soc. 106:530-536.
MacKenzie. C. L. 1979. Management for increasing clam abundance. Mar.
Fish. Rev. 1979:10-22.
Manzi. J. J. & M. Castagna. (editors.) 1989. Clam mariculture in North
America. Developments in Aquaculture and Fisheries Science. Vol. 19.
New York: Elsevier Science 461 pp.
McHugh. J. L. 1991. The hard clam fishery past and present. In: J. R.
Shuhel. T. M. Bell & H. H. Carter, editors. The Great South Bay.
Albany: State University of New York Press. 107 pp.
Papa. S. T. 1994. Distribution and abundance of the hard clam in relation
to environmental characteristics in Great South Bay New York (MSc
Thesis). Stony Brook: MSRC SUNY. 147 pp.
Parker. K. M. 1975. A study of natural recruitment of Mercenaria merce-
naria. Report to North Carolina Division of Marine Fisheries. Wrights-
ville Beach. North Carolina.
Pratt, D. M. 1953. Abundance and growth of Venus mercenaria and Cal-
locardia morrhuana in relation to the character of bottom sediments. /
Mar. Res. 12:60-74.
Paulsen. R. & P. Murray. 1987. Test of hard clam seed survival as affected
by subsurface planting. Final report the New York State Urban Devel-
opment Corporation. AquacuUure Innovation Program. 31 pp.
Peterson. C. H., H. C. Summerson & J. Huber. 1995. Replenishment of
hard clam stocks using hatchery seed: combined importance of bottom
type, seed size, planting season, and density. / Shellfish. Res. 14:293-
300.
Saila, S. B.. J. M. Flowers & M. T. Cannario. 1967. Factors affecting the
relative abundance of Mercenaria mercenaria in the Providence River.
Rhode Island. Proc. Natl. Shellfish. Assoc. 57:83-89.
Walker. R. L. and K R. Tenore. 1984. The distribution and production of
the hard clam. Mercenaria mercenaria in Wassaw Sound. Georgia.
Estuaries 7:19-27.
Wells. H. W. 1957. Abundance of the hard clam Mercenaria mercenaria in
relation to environmental factors. Ecology 38:123-128.
Jouniiil I,] Slicllfhh Research. Vol. 22, No. I, 69-7.^, 2()().V
SPATIAL VARIATION IN THK BODY MASS OF THE STOUT RAZOR CLAM,
TAGELUS PLEBEIUS: DOES THE DENSITY OF BURROWING CRABS,
CHASMAGNATHUS GRANULATA, MATTER?
JORGE L. GUTIERREZ* AND OSCAR O. IRIBARNE
Deparhimento cle Biologia. FCEyN, Uuivcisidad Nacinncd de Mar del Plata. CC 573.
B76UUWAG Mar del Plata. Arqeiitina
ABSTR.ACT A series of functional-group hypotheses proposed for marine soft-sediment systems predict that either deposit-feeders
or hijihly mobile bioturbators exclude low-mobile supension feeders because of their sediment reworking activity. However, a
low-mobile suspen.sion-feeder — the stout razor clam Tagelus plebeius — coexists with highly mobile deposit-feeding burrowing crabs.
CImsmagnalhus gramduta, in several Southwestern Atlantic estuaries. In this study, we compared the body mass (as relationship
between shell length and dry weight of flesh) of the stout razor clam between replicated patches showing contrasting densities of
burrowing crabs. Spatial variation was observed in the slope of the relationship between shell length and dry weight of tlesh of T.
picbeiiii in the three samplmgs dates (July 1999, January 2(J00. and April 2000). However, the pattern of spatial variation in the slope
of this relationship was not consistent with the pattern of spatial variation in crab density. In addition, the pattern of spatial variation
in the slope of the relationship between shell length and dry weight of tlesh of the stout razor clams was not consistent between the
three sampling dates. These results suggest either that ( 1 ) body mass of the stout razor clam is affected by habitat features other than
crab density, or (2) effects of burrowing crabs on body mass of the stout razor clam are masked by spatial variation in other habitat
features that affect body mass of stout razor clams or the extent to which crabs are able to affect clams.
KEY WORDS: bioturbation. body mass, Cluisnuignailnis Kiuiudata. spatial variation, Tagelus plebeius
INTRODUCTION
The stdLit ra/or clam Tagelus plebeius Soiaiider (Veneroida:
Solecurtidae) is an euryhaline species that occurs in estuarine en-
vironments from North Carolina (34°N. United States) to the San
Matias Gulf (41' S, Argentina; see Holland & Dean 1977a, 1977b,
Viegas 1981. Gutierrez & Iribame 1998. 1999. Gutietrez & Valero
2001 1, This is a suspension-feeding species that construct perma-
nent burrows (up to 50 cm deep) lacking lateral mobility (Holland
& Dean 1977a. 1977b. Gutieirez & Valero 2001), In several
Southwestern Atlantic estuaries, this species coexists with the bur-
rowing grapsid crab Cluisimignathus gramduta Dana (Gutierrez &
Iribame 1998. Gutierrez & Valero 2001), C, granulata is one of
the dominant macroinvertebrates in tidal flats and salt marshes of
Southwestern Atlantic estuaries from Rio de Janeiro {23°S. Brazil)
to the San Mati'as Gulf (41' S. Argentina; Bo.schi 1964. Spivak et
al. 1994. Iribame et al, 1997), This is a gregarious species that
excavate and maintain semipermanent open burrows in the inter-
tidai, from soft bare sediment flats to areas vegetated by the
cordgrass Spuriina densiflora (Spivak et al. 1994. Iribarne et al.
1997). At sediment Hat areas, individuals of C. gruiuilata behave
as deposit-feeders, showing large (up to 1.4 1 volume) and mobile
burrows (up to 5 cm day"'; Iribame et al. 1997),
Coexistence between these two species, however, must not be
expected according to any of the functional-group hypotheses that
were proposed to predict species assembly in soft-substrate envi-
ronments. For instance, the trophic-group amensalism hypothesis
(Rhoads & Young 1970) predicts that deposit-feedeis, such as
Chasmagnalhus granulata. exclude suspension feeders, such as
Tagelus plebeius. by increasing the amount of sediment resus-
pended in the water column, which clogs the filtering appendages
of suspension-feeders. The adult-larval interaction hypothesis
(Woodin 1976) predicts that sediment reworking by deposit-
feeders kill the larvae of recently settled suspension-feeders be-
*Corresponding author. E-mail: jlgutie@mdp.edu. ar
cause of direct damage or burial to unsuitable depths. The mobil-
ity-mode hypothesis (Brenchley 1981. 1982) proposes that mobile
benthic species, such as C. gramdata. exclude more sedentary
forms, such as T. plebeius. by continually burrowing trough the
sediment. The coexistence between C. granulata and T. plebeius,
however, illustrates that sedentary suspension-feeders are not al-
ways excluded from areas inhabited by mobile burrowing deposit-
feeders. In fact, the latter is not a novelty: much evidence support-
ing the occurrence of the mechanisms predicted by the functional-
group hypotheses often refer to negative but non-lethal effects (see
Posey 1989 for a review). Therefore, regardless the lack of exclu-
sion between both species, we are still in conditions to expect for
negative, but nonlethal effects of C. granulata on stout razor
clams.
The patchy distribution of bun'owing crabs in the tidal flats of
several Southwestern Atlantic estuaries (see Botto & Iribarne
1999. 2000) provides a good opportunity to explore this possibility
at a realistic scale. In this study, we compare the body mass (the
relationship between dry weight of tlesh and shell length) of the
stout razor clam in patches with high and low density of burrowing
crabs. We recognize that this comparative approach does not allow
to address cause-effect relationships between the presence of crabs
and the body mass of stout razor clams, but comparing the body
mass of stout razor clatns among replicated areas with high and
low density of burrowing crabs allow to discern between the fol-
lowing logical possibilities:
( 1 ) The body mass of the stout razor clam vary between habi-
tats depending on crab density, which may indicate (a) that
burrowing crabs affect body mass of the stout razor clam
or. (b) that the habitat features that affect crab density also
affect body mass of stout razor clams.
(2) The body mass of the stout razor clam vary between habi-
tats but irrespective of crab density, which may indicate (a)
that body mass of the stout razor clam is affected by habitat
features other than crab density, or (b) that effects of bur-
rowing crabs on body mass of the stout razor clam are
69
70
Gutierrez and Iribarne
T.\BLE 1.
Mean (SD) density (burrows m~-) of burroHing crabs Chasinagnalliiis granulata in the locations under study and results of one way ANOVA
(df = 114) evaluating differences in crab density between locations.
Location
ANOVA
Sampling Date
1
2
3
4
5
6
MS
F
July 1999
January 2000
April 2000
l..\5 (0.81)
3.15 (1.35)
1.70(0.86)
1,.^0 (0.86)
3.35 (1.50)
1.60(0.99)
1.40 (0.75)
3.60 (1.43)
1.80(0.83)
0.15 (0.37)
0.45 (0.51)
0.60 (0.60)
0.25 (0.44)
0.50(0.61)
0.45 (0.51)
0.20 (0.52)
0.60 (0.50)
0.55 (0.51)
2.64
6.85
1.97
24.36*
53.87*
14.68*
' P < 0.01. Tiikey tests: (1 = 2 = 3) * (4 = 5 = 6) in all sampling dates
overwhelmed by spatial variation on other habitat features
that affect body mass of stout razor clams or the ability of
crabs to affect clams.
(3) The body inass of the stout razor clam did not vary between
habitats, which may indicate (a) that burrowing crabs does
not affect body mass of stout razor clams, or (b) that effects
of burrowing crabs are being compensated by spatial varia-
tion in other habitat features that affect body mass of the
stout razor clam.
MATERIALS AND METHODS
This study was conducted at the Mar Chiquita coastal lagoon
(37°S. Argentina), which is a 46-km" body of brackish water af-
fected by semidiurnal low amplitude (<l m) tides and character-
ized by mudflats and large surrounding marshes dominated by the
halophyte Spartina densiflora (Spivak et al. 1994, Iribarne et al.
1997). Samplings for crab density and collections of stout razor
clams were conducted in July 1999 and January and April 2000. in
an area approximately located 2.5 km upstream from the lagoon
inlet, which comprises about 700 m of shoreline. At this area, six
locations were selected; three of them characterized by high bur-
row densities of Chasina\(iiatluis f;raiu(lata (locations I. 2. and 3).
and the others by very low bun'ow densities (locations 4. .5. and 6).
Crab density at each location was estimated by random sampling
using a 1 X 1 m sampling unit (;; = 20). Single factor analysis of
variance followed by Tukey test (Zar 1984) was used to test for
differences between locations in the density of burrowing crabs.
Locations grouped under the same level of crab density did not
differed significantly in the density of crab burrows in all sampling
dates (see Results and Table I). Sixty clams per location were
collected at each sampling date by excavating the sediment using
hand shovels. The length of the clams was measured along the
anterior-posterior axis to the nearest 0.01 mm and their flesh was
removed from the gaping shell after a short immersion in boiling
water. The flesh was dried separately at 70°C for 48 h before their
dry weight was determined. Correlation analysis (Zar 1984) was
used to evaluate the existence of a significant relationship between
shell length and dry weight of flesh in clams at each location and
sampling date. Once significant relationships between the shell
length and the dry weight of flesh of the clams were observed at all
locations and sampling dates (see Results and Table 2), parallelism
tests followed by Tukey tests (Zar 1984) were used to compare the
slope of this relationship between locations at each sampling date.
Gi\ en that clams smaller than .50 mm occurred in low numbers and
not in all locations, we excluded these data from the analysis of
correlation and parallelism to cover the same range of sizes in all
locations. After removing these data, we also randomly discarded
some data from clams larger than 50 mm to attain an equal sample
size between locations (July 1999; /; = 57; January 2000. n = 56.
.A.pril 2000. n = 52).
RESULTS
Single-factor analysis of variance indicated that the density of
burrowing crabs significantly differed between locations in the
three sampling dates (Table I ). Tukey tests revealed that the six
locations can be subdivided in two clearly defined groups: loca-
tions with relatively high crab density (locations 1. 2. and 3) and
locations with low crab density (locations 4. 5. and 6): being this
pattern consistent in the three sampling dates irrespective of tem-
poral variations in the density of crab burrows (Table 1). Corre-
lation analysis indicated a significant linear relationship between
the dry weight of tlesh of stout razor clains larger than 50 mm and
their shell length in all locations and sampling dates (Table 2).
Parallelism tests indicated that the slope of the relationship be-
tween shell length and dry weight of flesh of T. pleheiiis differed
TABLE 2.
Site-specific regression equations and determination coefficients (between brackets) observed for the relationship between dry weight of flesh
and shell length of the stout razor clam Tageiiis pleheiiis in the three sampling dates.
Location
Julv 1999
January 2000
April 2000
y = 0.019X-0.467 (0.386)
y = 0.030X-1.135 (0.562)
y = 0.027x-^.897 (0.361)
y = 0.026X-0.927 (0.446)
y = 0.013X-0.286 (0.285)
v = 0.032X-1.169 (0.548)
y = 0.026X-0.739 (0.383)
y = 0.034.X-1.191 (0.282)
y = 0.014X-0.126 (0.182)
y = 0.014X-0.082 (0.086)
y = 0.033X- 1.088 (0.331)
y = 0.018X-0.458 (0.265)
y = 0.()21.x-0.509 (0.383)
y = 0.019X-0.427 (0.244)
y = 0.028.x- 1.024 (0.540)
y = 0.016x-0.3(.17 (0.333)
y = 0.033x^1. 144 (0.317)
V = 0.O26X-O.870 (0.416)
P < 0.05 in all cases.
Spatial Variation in Bod\- Mass of Tagelus flebeius
71
significantly between locations in the three sampling dates (Table pattern of spatial variation in crab density in any of the sampling
3, Fig. 1 ). Tukey multiple comparison of slopes indicated that the dates (Table 3).
patterns of spatial variation in the slope of the relationship between
shell length and dry weight of tlesh of T. pkbeius was not con-
sistent between sampling dates. In addition, spatial variation in the Recalling the logical possibilities established in the introduc-
slope of the dry weight-shell length relationship did not match the tion. our results suggest either ( 1 ) that body mass of the stout razor
DISCUSSION
3
h-
O
LU
>-
Q
15^ LOCATIOIMS
♦ 1 a2 m^ o4 a5 o6
0.5
July 1999
1
1-5 1
0.5
1.5
0.5
45
»
0.8
0.6
0.4
0.2
45 50 55 60 65 70 45
January 2000
«"o
1.2
1
0.8
0.6
0.4
0.2
45 50 55 60 65 70 45
April 2000
1.1
0.9
0.7
0.5
0.3
50 55 60 65
70
45
50 55
60
65 70
50 55
60 65
50
55
60
SHELL LENGTH (mm)
70
5C
65 70
Figure 1. Relationship between dry weight (if flesh and shell length of the stout razor elani Tardus pkbeius at each location and sampling date.
Left: Dry weight data points plotted against shell length. Right: Cur\es corresponding to the linear fit of data points in the figures on the left.
Locations are denoted by numbers beside each curve (locations 1, 2, and 3: High crab density: locations: 4, 5, and 6: low crab density). Curves
showing the same letter beside their respective location numbers have slopes that are not significant different [P > 0.(15) after Parallelism tests
followed by Tukev tests.
72
Gutierrez and Iribarne
TABLE .V
Results of tests for parallelism and Tukey tests used to e\aluate
differences between locations in the slope of tlie relationship
between dry weight of flesh and shell length of Tageliis pleheius
Sampling
Parallelism Test
Date
MS
F
Tukey Test
July 1999
0.015
24.12(5*
(1,2,4.6) (3.6) (5)
January 2000
0.021
20.418*
(1.3.4) (3.4.6) (1.2) (2.5)
April 2000
0.014
14.194*
(2.3.4,6) (1.2.4) (5l
Numbers between brackets indicate locations that did not significantly
differed in the slope of the dry weight-shell length relationship after Tukey
tests.
*P<0.01.
clam is affected by habitat features other than crab density, or (2)
that effects of burrowing crabs on body mass of the stout razor
clam are masked by spatial variation in other habitat features that
affect body mass of stout razor clams or the extent to which crabs
are able to affect clams. This is reasonable to occur because the
locations encompassed in this study differ in many features -as
sediment characteristics and orientation-iirespective of the pres-
ence of crabs (personal observation). Sediment characteristics rnay
directly affect clam body mass (e.g., by determining the costs of
burrowing: see Swan 1952. Newell & Hidu 1982) as well as the
nature and extent of habitat tnodifications derived from crab bur-
rowing that may be detritiiental for stout razor clams (e.g., sedi-
ment resuspension; see Turner & Miller 1991). Differences in the
orientation of the locations in relation to winds determine, for
example, the degree to which clams are exposed to events of
environmental disturbance by waves and cun'ents (see Turner &
Miller 1991, Bock & Miller 1995) as well as the degree to which
sediment reworking by crabs might be overwhelmed or not by
physical reworking (see Grant 1983).
The overall conclusion of this study is that crabs alone do not
promote a spatial pattern in body inass of the stout razor clam at
the scale of crab patches. It is uncertain, however, whether effects
of crabs on the body mass of stout razor clams are occurring at
locations with high density of crabs but overwhelmed by other
sources of spatial variation that affect clams. Several lines of evi-
dence suggest that crabs might have iinportant local effects on the
body mass of stout razor clams. For instance, organisms that are
known to exclude low-mobile suspension-feeders, such as calli-
anassid shrinips (see Posey 1989) excavate sediments at rates of
2.7-3.5 kg (dry) nr- d"' (Vaugelas 1984. Swinbanks & Luter-
nauer 1987. Witbaard & Duineveld 1989). whereas burrowing
crabs excavate sediments even at higher rates [5.9 kg (dry) m""
d"': Iribarne et al. 1997]. Consequently, the detrimental effects of
sediment reworking by crabs on the stout razor clam predicted by
the functional-group hypotheses are still possible.
However, considering the rates at which burrowing crabs and
callianassid shrimps remove sediments, the question at this point is
why burrowing crabs does not exclude stout razor clams as calli-
anassid shrimps do with a variety of suspension-feeders. The an-
swer is. perhaps, in the different modes by which callianassid
shrimps and burrowing crabs rework sediments. Callianassid
shrimps burrow and sift the sediments continuously for food, de-
stabilizing them and increasing water turbidity (Aller & Dodge
1974. Murphy 1985). Ht)wever, C. granulata reworks sediments
mostly during low tide eventually depositing mounds of fine, co-
hesive sediment above the surface, which are not likely to be easily
resuspended by tidal cuirents (e.g., Iribarne et al. 1997, Botto &
Iribarne 2000), This implies that some mechanisms predicted to
exclude suspension-feeders from areas dominated by deposit-
feeders, such as sediment resuspension (see Rhoads & Young
1970) might not take place in the case of buiTOwing crabs. Further,
the latter suggests that sediment reworking is not a good predictor
of the actual effect of burrowing deposit-feeders on suspension
feeders.
ACKNOWLEDGMENTS
This project was supported by grants from Universidad Nacio-
nal de Mar del Plata. CONICET. FONDECyT. and Fundacion
Antorchas. J.L.G. is supported by scholarships from CONICET
and this article is part of his Doctoral thesis.
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Jmiriuil III Shellfish Research. Vol. 22, No. I. 75-X.^. 2()().^,
MARICULTURE SITING— TIDAL CURRENTS AND GROWTH OF MYA ARENARIA
WILLIAM R. CONGLETON, JR..' BRYAN R. PEARCE," MATTHEW R. PARKER,' AND
ROBERT C. CAUSEY'
DcjHiriiui'nt of Animal ami Veterinary Science. Univer.sin of Maine. Orono. Maine 04469 'Depanment
of Civil ami Enviroiuiiental Enginecrini>. Univer.'iity of Maine. Orono. Maine 04469
ABSTRACT Mariculture of the soft-shell clani Myii uienaria L. involves seeding juvenile shellfish on nitertidal niudtlats for
grow-out. Laborator>- studies have shown that constant current velocity affects shellfish growth. Few studies have determined the effect
of tidal currents on shellfish growth in siiii. Spot estimates of tidal currents can be generated with portable current meters and by
measuring the erosion of Plaster of Paris hemispheres called clod cards placed in the current. Current velocities for Geographical
Information System (CIS) coverages for entire estuaries can be estimated using numerical flow models. Although these different types
of measurement have different relative advantages of cost, ease of describing large areas, and accuracy, each can be potentially used
in evaluating sites for shellfish grow-out. Current velocities averaged over the flood tide were estimated by a numeiical flow model
and by clod cards for 16 locations at the same elevation in a bay in Eastern Maine and were compared with the annual shell increment
of clams collected at the same locations. Statistical models included main effects and interactions between initial shell size, year of
sample, and high-low current category estimated by clod cards or a numerical model. Models explained 57-58% of the variability in
growth increment with initial shell size and year affecting growth more than current. Faster tidal currents resulted in 22-24% greater
shell growth. Sites categorized as low flow had means for tidal currents {±SD) of 4.35 ± 0.37 cm/s and 2.99 ± 0,43 cm/s using the
numerical model and clod cards, respectively. Least squares means (±SE) for the annual increment in shell length increment was 9,56
+ 0.247 mm for the low flow sites identified using the numerical model and 9.5 1 ± 0.274 mm for the low flow sites idenfified using
clod cards. Sites categorized as high flow had current means (±SD) of 5.86 ± .62 cm/s using clod cards and 5.84 ± 0.46 cm/s using
the numerical model and least squares means (±SE) for growth increment of 1 1.90 ± 0.32 and 1 1.70 ± 0.33 mm, respectively. The
stimulatory effect of tidal currents on clam growth could be used in mariculture siting. Placing clod cards at specific intertidal locations
at the same elevation could be used to estimate relative current velocities. Current velocities estimated using numerical models and
displayed as CIS grids of entire regions will not have the same resolution as spot estimates from current meters or clod cards. However,
grids can be used for siting if the grid cells are comparable in si/e to area to be seeded.
KEY WORDS: numerical model. Geographical Information System (GIS), current, growth, Mya iireiuirui
INTRODUCTION
Seed planting and transplanting has been an integral part of the
hard clam and oyster industries (Malouf 1989). With hundreds of
miles of mudflats in Northeastern Maine and a 457f decline in state
landings over the past 13 y (DMR, 1997). mudflats with low
densities oi Mya arenaria L, are being seeded with juvenile clams.
Site-specific characteristics must be evaluated in selecting sites for
shellfish seeding (Beal et al. 2001. Peterson et al, 1995, Newell
1996). but determining environmental parameters capable of sus-
taining populations of bivalve seed is difficult in most cases (Mal-
ouf 1989).
Among a variety of biologic and environmental that influence
growth of bivalves in situ, sufficient current speed is recognized as
an important factor. Water velocity, horizonlal adveclion, and ver-
tical mixing in the water column influence the availability of phy-
toplankton to mussels (Frechette et al. 1989), Currents are needed
to avoid depletion of oxygen and food particles to suspension
feeders, especially at high-density levels (Jorgensen 1990). Newell
(1990) suggested a minimum current speed (about ,3 cni/s) below
which bottom culture of mussels may not be cost effective. An
actual reduction in food intake of bivalves was found when current
rates are not kept high enough (Bayne et al. 1976). Faster flow
results in a greater flux of organic particles (Peterson & Skilleter
1994). Shell growth rates for hard clams over a 15 wk period
increased by 10.7% in fast relative to slow current sites in coastal
lagoon in New Jersey (Grizzle & Morin 1989). Soft-shell clams
were found to orient perpendicular to the principal component of
current direction potentially to optimization energy acquisition
during an entire tidal cycle (Vincent et al, 1988),
The effect of water flow on growth varies with species of
bivalve. For infauna, northern quahogs displayed a consistent in-
crease in shell growth with higher flow speed in the range of
stream velocities between 0 to 4 cm/sec (Grizzle et al. 1994).
Growth response in the soft-shell clams was similar to that ob-
served in hard clams with a proportional increase in shell length
for 4 y old, 40 mm clams with flow and no evidence of growth
inhibition between free-streatn velocities of 0.1 to 5.8 cin/sec (Em-
erson 1990),
For epifauna species, it has been speculated that growth is
maximized at water flows that match inhalant pumping speed.
Mussels grown in multiple flume trials at flow velocities of 0. I. 2.
4. and 8 cm/sec had a statistically nonsignificant increase in shell
growth at a flow of 2 cm/sec. which matched the approximate
inhalant pumping speed (Grizzle et al, 1994). Eastern oysters in-
creased growth at a flow of 1 cm/sec relative to tlows of 0 and
>1 cm (Grizzle et al. 1992). The constant flow in flume studies,
however, is different from tidal currents, which vary in magnitude
and direction. Flume experiments with ascending and descending
flows have found clearing or grazing rates of scallops differed by
307f (Pilditch & Grant 1999).
Currents may affect shellfish growth, but estimating current
velocities can be difficult. A device coinmonly used to determine
flow rates is a current meter. However, collecting time series ve-
locity profiles with current meters over large areas is time con-
suming with conventional instrumentation, particularly in inter-
tidal waters. When current rates and flow patterns are needed for
large regions being considered as potential shellfish grow-out sites,
the use of current meters becomes impractical.
Two- and three-dimensional numerical computer models can
be used to describe the direction and magnitude of currents for
individual cells in grids covering coastal areas. The output data
from numerical models can then be used to create thematic maps
for Geographical Information System (GIS) coverages (Congleton
75
76
CONGLETON ET AL.
et al. 1999). Numerical models are supported by data for bottom
elevations for each cell in the grid and tidal amplitude at the ocean
boundaries of the models. They simulate time series estimates of
velocity vectors for grid cells covering the model domain. Veloc-
ities may be estimated for discrete layers in individual grid cells or
may be vertically averaged, as in this study. Model output can be
analyzed in the GIS to identify sites with optimum conditions for
shellfish growth. The major drawback, however, is the difficulty of
initializing and running a numerical model.
An alternative method for estimating currents is by measuring
a process, which is affected by the current magnitude. A physical
analog measurement of current velocity is the dissolution of cal-
cium sulfate (Plaster of Paris or gypsum) blocks or hemispheres,
called clod cards, placed in moving water (Muus 1968. Doty 1971,
Peterson & Skilleter 1994). Thompson and Glenn (1994) devel-
oped an equation for calculating mean water speed from field
deployed clod cards using clod cards from the same batch for
laboratory calibration in quiescent water of the same salinity tem-
perature as in the field. They concluded that proper execution of
field and calibration tests result in a simple and practical method
for measuring water motion over a wide range of temperatures,
salinities, and current speeds. Clod cards are inexpensive and
simple to construct, but the difficulty of deploying large numbers
limits their usefulness for estimating cunent magnitudes o\ er large
areas.
The objective of this study is to evaluate the relationship be-
tween ( 1 ) field measurements of tidal currents made with clod
cards; (2) average current estimates generated by a numerical flow
model; and (3) growth of soft-shell clams on a mudfiat in Eastern
Maine. The appropriateness of incorporating current estimates
from a numerical model into a GIS for the selection of sites for
grow-out of juvenile shellfish will then be considered.
METHODS
The study was conducted in Mason Bay in Eastern Maine on
the western side of Englishman Bay, which bounds the Gulf of
Maine. The bay (Fig. I A) is 2.39 km long by 1.03 km wide,
oriented in an east-west direction, and is located 9.7 km north of
Jonesport, Maine (44°61.80'N, 67°56.23"W). At low tide (mean
low water = -1.875 m nisi), mudflats are exposed along the entire
length of the bay with two channel inlets from Englishman Bay
joining on the west side of Spar Island and running the length of
the bay (Fig. IB). Water temperatures vary from 5°C in April to
I6'=C in September (Beal et al. 2001).
Soft-shell clams were collected at 15 sites at the same water
line spaced 40 m apart to the south of Spar Island and west of
Flake Point Bar (Fig. IB) at an elevation of -2.0 m msl in Spring.
1 996. These sample sites were close to one of the inlets of the bay
with a maximum separation of 485 m from the most easterly site
to the most westerly site. A sixteenth site between the tip of Flake
Point Bar and Spar Island was sampled during spring of 2000 to
increase the range of water velocities sampled. One of the low flow
sites in the center of the earlier sampling array was also sampled
the second year. Sites were relocated in the second year using their
global positioning system (GPS) coordinates.
Location of the 16 sites was determined by caiTier-phase GPS
measurements made with a Trimble GeoExplorer™ GPS receiver,
and post-processed. Carrier-phase GPS is commonly used for sur-
veying with sub-decimeter accuracy for measurements in the ho-
rizon plane. Measurements in the vertical plane are less accurate.
The range in the elevation measurements for the 10-min carrier-
phase GPS readings at the 16 sites was -1.5 to -2.7 m msl with
95% confidence range of ±0.55 m for individual measurements
(Congleton et al. 1999). Because inaccuracy in GPS measurements
alone could have resulted in a difference in elevation between
sites, locations were selected with simultaneous flooding and dry-
ing times.
Sample site coordinates were then imported into the Maplnfo'^'
GIS creating a layer of sampling site locations (Fig. IB ). Sediment
cores from four of the sites were analyzed for composition by the
Analytical Laboratory of the Maine Soil Testing Service using the
hydrometer method for particle size and 1050'"C combustion ana-
lyzer for total carbon. Fifty clams were dug with a clam rake at the
1 5 sites South of Spar Island at the end of the first growing season.
A single low flow site (sixth site counting from the most easterly)
and the high flow site SE of Spar Island were sampled in the
second growing season.
External annual rings were used to determine the increase in
shell length during the preceding summer. Brousseau ( 1979) found
winter rings to be a reliable method of determining age in soft-
shell clams from Gloucester. Massachusetts. However. Mac-
Donald and Thomas ( 1980) found external growth rings to be less
reliable for age determination than thin shell sections, and Lewis
and CeiTato (1997) found shell increment might be temporarily
decoupled from soft-tissue growth by high temperature or starva-
tion. However, external growth rings have been used for long-term
estimation of growth (Kube et al. 1996) and growth and age
(Jacques et al. 1984. Evans & Tallmark 1977) of /;; situ Mya
arenaria.
Because of limitations of using growth rings for measuring age.
length between the last shell check marks were used to measure the
size at the beginning of last growing season. Initial size was then
used as a covariate in the statistical analysis instead of age. Annual
growth increment was then calculated by subtracting the final shell
length from the initial size. The problem of lengthy shell abrasion
limiting the usefulness of external rings in aging was minimized by
taking measurements of growth only in the last growing season.
NUMERICAL MODEL OF TIDAL CURRENTS
Estimated currents for Mason Bay were obtained from the Ma-
son Bay Model (MBM). which is an adaptation of Princeton Ocean
Model (Mellor 1992. Blumberg & Mellor 1987) modified to de-
scribe intertidal areas (Congleton et al. 1999). Input bathymetry
data for the model were processed in the Maplnfo GIS including
sublidal depths from NOAA nautical chart no. 13325, the shoreline
boundary traced frotn an aerial photograph and 27 high accuracy
canier phase GPS measurements made at the waterline near low
water on a single Spring tide. To increase the accuracy of the
description of the bottom in the study area, fourteen of the GPS
measureinents were in the region, which is enlarged in Figure lb.
These data were used to generate a 100 by 76 grid covering the bay
composed of square cells with 36.125 m sides. The 7600 cells in
the grid gave increased resolution of depths between points with
known elevations without unnecessarily increasing computing
time for a run describing a tidal cycle. Grid cells (36 m sides) were
smaller than the distance between the clam sampling locations
(40 m) resulting in a different estimate of current velocity at each
sample site.
The model generated estimates of vertically averaged cuirent
velocities for each grid cell flooded by the tide at one-second
Tidal Currents and Clam Growth
77
O Sample site
+ High flow.
- Low flow i^^°del
H High flowj _
L Low flow*
Water displacement
per minute
-2 3 Depth m msl
Shoreline mean
high water
Figure 1. (A) Location of Mason Bay in Eastern Main near Jonesport. Maine with Englisliman Ba> and the Atlantic Ocean to the east connected
by channels north and south of Dunn Island. Lines and labels show locations and extent of 7.5 niin L'S(;S quadrangles. (Bl Aerial photos of
Mason Bay. Right image is the rectangular area in SE image of the entire Bay. The array of sample locations (-2 m msl) are spaced 40 m apart
except for the site nearest Spar Island. Vectors show water displacement/minute at maximum flood tide.
78
CONGLETON ET AL.
intervals for an average 2 m amplitude tide (Congleton et al. 1999).
A vertical average of the current velocity for each time step was
used because tidal amplitude and shallow water depths would in-
hibit stratification. Bottom friction was proportional to the square
of the veilically averaged bulk flow. Vectors showing the current
magnitude and direction estimated by the model for each grid cell
were imported into the GIS. Layers of cuirent vectors at different
times in a tide cycle described flow throughout the bay.
For the statistical analysis of clam growth, the time series of
velocities were averaged over the flood phase. The layer showing
the sample site locations was placed over a layer of average cuirent
velocities to estimate velocities at each site. Because the sample
locations were not centered on the grid used by the numerical
model, the mean velocity of adjacent grid cells with the same
approximate elevation were averaged.
FIELD MEASUREMENT OF CURRENTS— CLOD CARDS
Plaster of Paris hemispheres (clod cards) were used for mea-
suring relative water motion at each of the fifteen sampling sites.
In previous studies, rectangular clod cards were used (Doty 1971,
Thompson & Glenn 1994). Clod cards used in this study (Fig. 2)
were molded in hemispheric plastic capsules (32.36 cm'), creating
a uniform surface area exposed to the current regardless of card
orientation.
Commercial Plaster of Paris or gypsum was mixed two parts
powder to one part water. The slurry was poured into the capsules
and leveled off with a straightedge and left at room temperature for
a week to insure thorough drying. After attachment to a 9 x 6.5 cm
sheet of plastic with silicone epoxy. initial dry weights for each of
the clod cards were measured and recorded.
For field deployment, the backing sheet of each clod card was
attached to a brick with rubber bands. One clod card was placed at
each of the 16 clam sample sites (Fig. IB), and a total submersion
time was estimated for the period that included air exposure at low
tide. Because all clod cards were deployed on a spring tide in April
(-0.7 m mllw), they were recovered after 4 days while the loca-
tions were still accessible at low tide. After recovery, cards were
lightly rinsed to remove mud and were left to dry at room tem-
perature for one week and weighed. The percentage loss and the
change in weight were calculated.
The calibration of clod cards in quiescent water or under free
convection conditions is necessary for the overall calculation of
integrated field water speed. Four clod cards from the same lot as
those used in the field trial were suspended 5 cm below the surface
of a 22-1 cylindrical container containing seawater (30-32 ppt
salinity). The container was placed in a larger recirculating tank
maintained at 7''C. which corresponded to the average water tem-
perature during the field trial.
Every 24 h, the water inside the container was replaced with
fresh salt water and the dissolved Plaster of Paris on the bottom of
the container discarded. After the calibration period of four days,
each card was dried at room temperature for a week and then
weighed. The average of the initial weights and average of the final
weights for the four calibration cards were used in the water ve-
locity calculations.
The scalar arithmetic mean velocity of the water in the field (V)
was estimated for the 16 sites following the methods of Thompson
and Glenn (1994):
V = 4.31 (W,„„„„/A,„,„,,)"-^(S,„,'-^/S,,„„„„,,„)
(1)
where W,,,,,, ,, is the initial clod card weight of the field deployed
card: Ai,,,,,.,, is the initial exposed surface area: Si,^,,,, and S^^,|,„„on
are calculated as
|i-(W,-„,„/w„„„.„)"-'i/e
(2)
where W,,,,^^, and W„„„_,| are the final and initial weights of the
field and calibration tests, and H is time submerged in the field and
calibration tests.
Theta for the field trial 0 was total time between deployment
and recovery even though the clod cards in the field experienced
air exposure during low tides. During periods of air exposure, field
clod cards remained wet and continued to dissolve. On average,
the cards were subjected to aerial exposure for approximately 1 h
during each low tide.
STATISTICAL ANALYSIS
Tidal velocities for each site were categorized as high or low
using estimates from the clod cards and numerical model. Because
the mean (±SD) velocity estimated using the clod cards (4.96 ±
0.88 cm/s) was higher than the mean estimated by the numerical
model (3.85 ± 1 .34 cm/s), high flow sites were identified as having
flow s greater than 5 cm/s using clod cards and 4 cm/s the numeri-
cal model. Mean flow at the seven high flow sites identified using
clod cards a\eraged 5.87 ± 0.63 cm/s and at the five high flow sites
identified using the numerical model averaged 5.85 ± 0.47 cm/s.
Mean flow at the nine low flow sites identified by clod cards
averaged 4,35 ± 0.37 cm/s and at the 1 1 low flow sites identified
by the numerical model averaged 3.02 ± 0.33 cm/s. High and low
flow means categorized using either clod cards or the numerical
model were statistically different {P < 0.001) using pooled vari-
ance f-tests.
Variability in shell growth increment during the preceding
growth season was analyzed by analysis of variance (ANOVA)
Plastic backing sheet
Figure 2. (Jypsum clod cards constructed from a plastic mold cemented to a 9 x 6.5 cm backing stieet of plastic.
Tidal Currents and Clam Growth
79
using the GLM procedure in SYSTAT v. 10. Shell length at the
beginning of the growing season (distance between the last most
anterior and posterior margins of the last growth check), years of
sampling (1996 and 2000), and water velocity category of either
high or low as indicated by either clod card weight loss or the
numerical model were the independent factors. The initial model
included main effects and all interactions.
y, = B,, + BiX, + B.X, + B^X, + B4.V1.Y, + fijA-iX, + B,,^,^,
+ fiyXiA'.A', + e
Where y, is the annual growth increment; B„ is the intercept; B,X,
are the coefficient and categorical variable for year; fi-,X, are co-
efficient and value for initial size and 5,^", are the coefficient and
categorical current estiinate (H,L) either from clod cards or the
model: B,X,X„ S^X^X, and fi^XiX^X, are coefficients for two and
three way interactions; and e is the random error term.
Terms that were statistically insignificant iP > 0.05) were de-
leted from the model using the backward elimination procedure
(Draper & Smith 1466).
To ensure independence of residual errors in predicting growth
increment of spatially proximate observations, the Durbin- Watson
Test Statistic was used to test for the existence of autocorrelated
errors. Because 50 clams were collected at each location, residual
eiTor terms remaining after fitting the GLM model might not be
independent if there is a site effect independent of local cun-ents.
First-order autocorrelation (lag = 1 ) results in the error term con-
sisting of a fraction of the previous error term plus a new random
disturbance tenn (Neter et al. 1996). Error terms are uncorrelated
only at the time the autocorrelation term (p) is statistically equal to
zero.
RESULTS
Composition of the sediments at the 16 sites ranged from 47-
55% sand. 29-tl<7f silt. 12-16% clay and 1.15 to 1.27% carbon.
Shell lengths at the start of the two years ranged from 6.9 mm to
55.7 mm. with average (±SD) of 23.1 ± 10.8 mm. After excluding
the juveniles or individuals without a growth check mark, sample
size was 724 with an average (±SD) growth increment of 9.4 ± 4,0
mm with clams sampled once.
Trends in estimated water velocities from the numerical model
and clod cards were similar. Velocities estimated with clod cards
were highest at the site nearest Spar Island and at the sites near
Flake Piiint Bar. Velocities decreased at the sites near the center
and increased at the western end of the cove (Fig. IB). Estimates
from the numerical model displayed a similar trend generally de-
creasing moving westward from Flake Bar. but without the in-
crease at the most western locations.
The correlation coefficient between the 16 estimates of current
velocity from the numerical model and Eq. I was 0.74 (P < 0.05).
Velocity averages over the flood tide at the sixteen sites ranged
from 2.2 cm/sec to 7.14 cm/sec as estimated by the numerical
model and ranged from 3.8 cm/sec to 7.52 cm/sec as estimated by
Eq. I. The estimation of current velocities by a numerical com-
puter model and Eq. 1 were similar although the estimates from the
numerical model were lower. The velocities estimated by the clod
cards were during a spring tide, which would be expected to be
higher than the velocities predicted by the numerical model during
an average tide. Clod card measurements, however, were near the
bottom where velocities are decreased by bottom shear.
The maximum water speed on the flood tide was also estimated
for the sixteen sites. Maximum velocities predicted by the numeri-
cal model ranged from 4.0 cm/sec for some of the western and
central sites to 21.4 cm/sec at the site closest to Flake Point Bar.
All linear models used growth increment as the dependent vari-
able, year ( 1996 vs. 2000) and flow (high vs. low as categorized by
either clod cards or the model) as categorical variables and in-
cluded initial size as a continuous variable. The Durban-Watson
Statistic indicated that the GLM models had statistically signifi-
cant first order autocorrelations. An inspection of autocorrelation
plots of correlation versus lag indicated significant but diminishing
positive autocorrelations up to lag 10 (Fig. 3). Autoconelation
significance (P < 0.05) was deterniined from the 95% confidence
interval for the sampling distribution of the autocorrelation of lag
k or i-f.. which is normal with (x^^ = 0 and a^^- = l/n"" with a
sample size of n (Lin et al. 1995).
A difference transformation replaced values for the dependent
variable (growth increment) with the difference between it and the
preceding value. Differencing is a popular and effective method of
removing trend from spatial (location effect) and time series (tem-
poral effect) data. Autoconelation plots following the transforma-
tion had no trend because as lag increased there was a random
distribution of positive and negative autocorrelations (Fig. 3). To
ensure validity of significance tests using the transformed data, a
linear regression with a hierarchical layout with clams (or trial)
nested or stacked within site was used. The trial or clam within site
effects was insignificant for hierarchical models tested (P = 0.87).
Consequently, independence of error terms could be assumed and
significance tests based on the diffei-ence-transformed data would
be valid.
The ANOVA tables for the difference transformed growth in-
crement as the dependent variable and high-low current category
estimated by clod cards or the numerical model are in Tables 1 and
2. Both models explained 57-589^ of the variability in growth
increment. Estimates from both models indicated clams grew
slower the first year of sampling ( 1996) and that larger clams grew
less with -0.26 mm and -0,28 mm decrease in the growth incre-
ment for each mm increase in initial size depending on whether
1.0
0.5-
0 0 Jljrthm..Tnrrnii
0.0
-0.5
+0.5
0,0
-0,5 -
-1.0
f
10
20
30
Lag
40
50
60
Figure 3. Autocorrelations between residual linear model errors with
lags from 1 to 50 for predicting shell increment (top) and difference
transformed measurements of shell increment ( bottom ). Lines above
and below zero baselines are 95% amlldence Intervals for autocorre-
lation = 0.0.
so
CONGLETON ET AL.
TABLE 1.
ANOVA of growth increment with a difference transformation resulting from fitting a complete model reduced until only statistically
significant effects remain. Current categories were average current <5 cm/s or average current 55 cm/s as estimated from clod cards using
Eq. 1. R- of 58%.
Source
Sum-of-Squares
df
Mean-Square
F-Ratio
Year
Initial size
Clod card current
Current * size
Current * year
Error
39:.378
513S.024
143.122
33.430
77.765
435.^,1 OQ
392.378
513S.024
143.122
33.430
77.765
6,032
65.049
851.793
23.727
5..542
12.892
0.000
0.000
0.000
0.019
0.000
cuiTents were described with the numerical model or clod cards
(Fig. 4). Larger average currents also stimulated growth although
the effect on growth increment was less than that of year or initial
size (Table .3). The adjusted least squares mean (±SE) for the
growth increment at the sites identified by clod cards as low flow
was 9.6 ± 0.25 and at the high flow sites was 1 1.9 ± 0.32 (Table
.3). The least squares means (±SE) for growth increment at sites
identified by the numerical model as low flow was 9.51 ± 0.274
cm/s and at the high flow sites was 1 1.70 ± 0.33 cm/s.
There was a significant interaction between year and current
(Tables 1 and 2l. Increased growth for high flow was expected
during the second year because the highest flow site was only
sampled in the second year. There were also significant two-way
(clod card analysis) and three-way (numerical model analysis) in-
teractions involving the effect of initial size indicating an incon-
sistent stimulatory effect of current on growth for animals of dif-
ferent size. However, interaction terms involving initial size made
the smallest contribution to the model Sum of Squares or R".
DISCUSSION
A previous study (Congleton et al. 1999) also reported general
agreement between water velocities estimated by the numerical
model and measured by a portable current meter. The conelation
between flows estimated by the numerical model and Eq. 1 in this
study were lower than reported in Congleton et al. 1999. The 16
sites in this study, however, were a subset of the 25 sites in the
previous study and had a smaller range of current velocities.
Numerous factors affect the accuracy of using clod dissolution
in measuring currents. Mean current velocities estimated with the
clod cards were higher than the velocities estimated using the
model (Table 3). As previously noted, cards were deployed during
a Spring tide when cunents were stronger than an average tide that
is simulated by the model. High estimates of currents using clod
cards compared with other techniques ha\e been previously re-
ported with dissolution rates in field experiments 16-18% high
(Porter et al. 2000) compared with measured flows. Although flow
estimates using cards in this study were higher than estimates
using the model, there should also be some negative bias in the
clod card estimated flows because 9 in Eq. 1 included the time
when the cards were air exposed at low tide while the H used for
calibration was total emersion time. Clod card accuracy could be
increased by calibration in known steady flows rather than using a
diffusion index factor as in this study (Porter et al. 2000).
Flows were anticipated to be greatest at the most easterly and
most westerly sample locations because the flood tide entered the
cove on either side of Spar Island. This anticipated pattern was
seen in the flow rates estimated by the clod cards, but not the
numerical model. The failure of the numerical model to predict
increased currents west of Spar Island may be caused by the av-
eraging of flow rates of the surrounding grid cells, because sample
sites were not centered on the grid. Also, velocity estimates were
an average for a cell with an area of 1305 m". A model with greater
spatial resolution would show flow patterns in greater detail.
With a significant correlation between the current velocities
estimated by the clod cards and numerical model, the similarity in
the statistical analysis for the two sets of current measurements
was not unforeseen. As expected, initial size had a significant
effect on the grow th increment of M. arenaria. resulting in slower
growth in larger individuals (Fig. 4).
In an earlier study (Beal et al. 2001 ) placed clams at the same
intertidal locations in Mason Bay and measured increment in shell
length between time of removal from the hatchery and seeding on
the flats in April and removal from the flats at monthly intervals
until December. Mean shell length increased from 14.1 mm to 21.9
mm resulting in a 7.S mm increase between June and August to
December. Growth increment for the entire srowins season was
TABLE 2.
ANOV.\ of growth increment with a difference transformation resulting from fitting a complete model reduced until only statistically
significant effects remain. Current categories were average current <4 cm/s or average current >5 cm/s as estimated from the numerical
model. R' »{S19c.
Source
Sum-of-Squares
df
Mean-Square
F-Ratio
Year
Initial size
Model current
Current * year
Current * year * size
Error
304.81(1
3524.739
155.673
142.192
62.972
4458.564
I
1
1
1
1
722
,W4.810
3524.739
155.673
142.192
62.97
6. 1 75
49.360
570.781
25.209
23.026
10.197
0.000
0.000
0.000
0.000
0,001
Tidal Currents and Clam Growth
81
Annual Shell Increment
E
E.
*•>
c
0)
E
S.
u
c
<u
CO
10
20
30
40
50
Initial Size (mm)
Figurt 4. Imrement in shell length for the year 2(1(11) for clams In low
and high How sites as categorized using clod cards. (Card L. Card Hi
and the numerical model (Model L. Model H).
slightly less than 12 mm. Although juvenile clams without an
initial growth check were excluded from the sample in this study,
the growth increments predicted for 10 mm clams in Figure 4 is
similar to the value reported by Beal et al. (2001). Brousseau
(1979) predicted an asymptotic size of 108.12 mm and individuals
in age class 5 reaching a harvestable size on Georgetown Island.
Maine. Growth increments from both studies in Mason Bay would
also result in a market size of 51 mm being reached in approxi-
mately 5 y. Results from this study also indicate market size would
be reached earlier by clams at sites with average flows >5 cm/s
than flows <.'i cm/s.
Walne ( 1972) concluded that water current is a significant fac-
tor affecting filtration rates of bivalves, leading to higher growth
TABLE 3.
Adjusted least squares means for annual shell growth increment in
low and high flows as estimated b> clod cards and a numerical How
model. ANOV.A and signincance tests are in Tables 1 and 2.
Mean Flow
G
-owth Increment (mm)
Least
Flow Estimate
(cm/s)
Sq
uare Mean
SE
Clod card
Low flow
4.357 + 0.370
9.565
.247
High flow
5.860 ±0.618
11.899
.323
Numerical model
Low tlow
2.994 ± 0.428
9.505
.274
High flow
5.838 ± 0.457
1 1 .699
.327
rates. The relationship, however, varies with species of bivalve. As
velocities increase, an increased supply of particles corresponds to
increased consumption rates in mussels (Frechette et al. 1989).
Higher currents would afso cause sediment resuspension. Both
frequency of sediment resuspension and sediment food value were
found to be adequate to provide a nutritional benefit to scallops on
George's Bank (Grant et al. 1997). However, filtration and growth
rates were observed to be inhibited at higher flow levels. Mussels
reduce filtration rates on average by 4.8% at velocities >25 cm/sec
(Wildish & Miyares 1990). At a specified algal concentration,
Cahalan et al. (1989) found that growth rates of bay scallops
peaked at an intermediate fiow velocity of 6.5 cm/sec. Sea scallop
feeding is inhibited at currents >10 cm/sec (Wildish & Saulnier
1992. Wildish et al. 1987), and growth may even cease at 12
cm/sec (Kirby-Smith 1972).
Species differences in the stimulatory effect of water currents
on growth were explained by an "inhalant pumping speed" hy-
pothesis that predicts maximum growth at ambient flow the same
as the inhalant pumping speed of the species. Siphonate taxa gen-
erally ha\e greater inhalant pumping speeds. Hard clams (Grizzle
et al. 1992) and mussels (Grizzle et al. 1994). however, increased
growth rates over a wider range of currents.
Although year and initial size had more effect on clam grov\ th
in Mason Bay than did water velocity (Tables 1. 2), clams at high
flow sites did have a larger growth increment than the low flow
sites (Table 3). The results from this study show increasing shell
increments of Mya arenaria of 23-24% at higher average current
velocities. It is possible that the site closest to Flake Point Bar with
a inaximum estimated free stream flow 2 1 .4 cm/sec could have had
feeding inhibition at maximum flood tide. However, preliminary
data (Turner 1991 ) found no decrease in average pumping velocity
of Mercenaria mercenaria in flows between 20 to 30 cm. Addi-
tional studies need to be completed to identify the current velocity
at which physiologic inhibition of feeding occurs in clams and
other siphonate bivalves and also to determine the effect of a wider
range of tidal flows on feeding and growth.
The R" values for the linear models accounted for 57-58% of
the variability in the annual growth increment with differences in
initial size responsible for most of this variability in growth. The
range of water velocities across the study sites was not large. Some
of the unexplained variability inay have been partially caused by
error in counting external growth lines particularly for older indi-
\iduals as was reported for Geiikensia demissa (Brousseau 1981).
Error in predicting current velocities would also decrease R~
for the statistical models. Clod cards were wet and dissolving, but
air-exposed during part of the tidal cycle resulting in overestinia-
tion of 9 in Eq. 2 and a possible underestimation of current speed
in Eq. 1. Field deployed clod cards could be eroded by waves and
cuirents. Shallow water waves result in a local "to and fro" water
motion on the bottom increasing gypsum erosion resulting in over-
estimation of tidal currents using clod cards.
Different calibration techniques for clod cards could increase
accuracy of their use. Calibration of gypsum dissolution in flumes
with known flows was superior to still water calibration (Porter et
al. 2000) as used in this study. Porter et al. 2000 also found that the
gypsum dissolution method should not be used to compare flows
in different flow environments or to measure flows in an environ-
ment different from the calibration environment. These consider-
ations limit the usefulness of clod cards in tidal environments
because the flow environment changes during a tidal cycle. How-
ever, gypsum dissolution experiments should be interpreted as
82
CONGLETON ET AL.
measuring mass transfer relationships rather than flow speed. Bio-
logic response variables such as shell growth in this study may be
directly influenced by mass transfer of nutrients and indirectly
affected by flow.
Another limitation to the predictive capability measured in this
study is the bivalves in the present were not maintained in a con-
trolled environment. Numerous factors could cause stress and af-
fect growth. In a mariculture operation, trampling, predation. and
reburial after digging could be eliminated. Under these conditions,
the impact of water movement on variation in growth may be
greater.
Differences in clam density could also affect growth. Clam
density was not controlled in the present study. Beal et al. 2001
varied seed clam densities between 330 m"" and 1320 m"" at the
same location in Mason Bay without significantly affecting the
growth increment in shell length (Beal et al. 2001). Low clam
densities at all study sites were apparent during field sampling
from the digging effort required to collect the clams. Density was
also found not to have a significant effect on final shell length of
Mercenaria mc'rcenaria grown in bags (Fernandez et al. 1999).
Application to Mariculture Siting
The relationship between bivalve growth and the clod card
erosion should be useful in evaluating mariculture sites. Although
the contribution of cunent magnitude to the R" of the linear model
of growth was small relative to year and initial size, the increase in
growth predicted for clams of uniform size that are seeded at the
same time (or year) would be increased by 22-249f in high fiows
sites relative to low flow sites.
Relative water flow can be estimated by measuring percentage
weight loss of cards deployed at different sites. The use of Eq. 1
for calculating an estimated velocity requires laboratory measure-
ment of clod card loss in quiescent water, but determining the
percent weight loss of cards should be sufficient for estimating
relative flow rates at locations with the same air exposure and
water temperature.
The number of cells required in a grid with sufficient resolution
to estimate local tidal currents is a possible limitation on using a
numerical model. Grid scale is an important aspect of tide mod-
eling in the Gulf of Maine (Sucsy et al. 1993). For use in mari-
culture siting, grid cells should be of the same size or smaller than
the location where the clams are to be seeded. Ramming and
Kowalik ( 1980) considered using a grid with iiTegular steps with
the smallest grid distance in the region of primary interest with
larger grid cells away from the region of high resolution. The
solution for the irregular grid, however, is much more complicated
compared with an equidistant grid with spurious effects decreasing
the accuracy expected from grid refinement. Despite these limita-
tions. Kowalik and Murty ( 1993) gave a number of examples of
models using a combination of coarse and fine grids in their con-
sideration of the problem of using nested and multiple grids to
describe tidal flats.
A frequently used approach is to use the solution from a model
using a coarse grid as input for the boundary conditions for a fine
mesh grid for the area where higher resolution is required. The
development of multiple models at different scales would be fa-
cilitated by using an object-oriented approach. The object-oriented
feature of inheritance allows a general description of model com-
ponents in a base class to be inherited by a child or derived class
with the specific components to be added for a specific implemen-
tation. An object-oriented, two-dimensional landscape model with
biologic components has been pre\ iously developed (Congleton et
al. 1997).
For time series descriptions of current magnitude and direction
over large areas, obtaining estimates from a numerical model
would be the most practical. The incorporation of current estimates
from a numerical model in a GIS, as described by Congleton et al.
(1999), would make the information readily retrievable for use in
aquaculture siting and other applications.
ACKNOWLEDGMENTS
This project was supported by the Maine Agricultural Experi-
ment Station (MAES Pub. No. 2630). Assistance of Brian Beal in
digging clams and identifying growth checks is greatly appreciated.
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MATURITY AND GRO\VTH OF THE PACIFIC GEODUCK CLAM, PANOPEA ABRUPTA, IN
SOUTHERN BRITISH COLUMBIA, CANADA
A. CAMPBELL AND M. D. MING
Shellfish Section. Stock Assessment Divisio?) Science Branch. Fisheries and Oceans Canada. Pacific
Biologiccd Station. Nanaiiuo. British Columbia. Canada WT 6N7
.ABSTRACT Measurements were made to determme size and age at maturity and growth of the Pacific geoduck clam. Punopea
abnipm. from two areas in southern British Columbia. Canada. Growth rates were slower for P. ahrupm from Gabriola Island than
those from Yellow^ Bank. Histological examination of gonads indicated that at sizes <90 mm SL considerably more males matured than
females, but at sizes a90 mm SL the sex ratio was similar for males and females. Size at 50% maturity was similar for P. ahnipta
from both areas (58.-3 and 60.5 mm SL. respectively), but age at 50% maturity was slower for geoduck from Gabriola Island (3 y) than
those from Yellow Bank (2 y). Although one hermaphrodite was recorded, P. ahrupm was considered basically gonochoristic
(dioecious).
KEY WORDS: Pacific geoduck. Panopca ahrupia. maturity, sex ratio, hermaphrodite, reproduction
INTRODUCTION
The Pacific geoduck clam, Panopea abrupta (Conrad, 1849)
(Pelecypoda: Hiatellidae). is distiibuted along coastal areas from
southern California to Alaska and west to southern Japan (Bernard
1983. Coan et al. 2fW0). Geoduck are found buried up to 1 m deep
within soft substrates (e.g.. mud and sand) from the low intertidal
to at least 100 ni (Jamison et al. 1984. Goodwin & Pease 1989).
There are commercial fisheries for geoduck in Alaska, British
Columbia, and Washington State (Campbell et al. 1998, Bradbury
& Tagart 2000, Hand & Bureau 2000). Geoduck are long-lived,
reaching ages up to 168 y (Bureau et al. 2002). Adult geoduck
have separate sexes and broadcast spawn annually, usually during
summer (Andersen 1971. Goodwin 1976. Sloan & Robinson
1984). Planktonic larvae settle on substrates within 47 days, and
juveniles burrow into the substrate (Goodwin et al. 1979, Goodwin
& Pease 1989). Geoduck juveniles and adults feed by filtering food
particles (e.g., phytoplankton) from seawater (Goodwin & Pease
1989). Geoduck growth is variable but most rapid in the first 10 y:
thereafter, although growth in shell length is greatly reduced, shell
thickness and meat weight continue to increase at a slow rate
(Bureau et al. 2002).
Andersen (1971 ) found SO'^r maturity occurred at about 75 mm
SL in geoduck sampled in the Hood Canal. Washington State, but
little is known about the rate of sexual tnaturity for P. ahrupta.
especially in British Columbia. (Sloan & Robinson 1984). The
purpose of this paper is to present information on the sexual ma-
turity and growth rates of P. abrupta from two areas in southern
British Columbia.
MATERIALS AND METHODS
Samples from as wide a range as possible of P. ahnipia were
obtained from Yellow Bank, near Tofino on the west coast of
Vancouver Island, (Lat. 49°14.18'. Long. 125"55.48') during 28
May, 1991 and Gabriola Island, near Nanaimo in Georgia Strait,
(Lat. 49°07.6'. Long. 123°45.05') during 22 to 23 May, 1991, at
depths between 5-15 m for both areas. The clams were transported
to the laboratory in coolers (2°C) and kept in running sea water
(ambient temperature) until processed within 48 h of capture.
For each geoduck, shell length was measured as the straight-
line distance between the anterior and posterior margins of the
shell to the nearest mm with vernier calipers. The age of each
geoduck was estimated using the acetate peel method of Shaul and
Goodwin ( 1982). Each right valve was sectioned through the hinge
plate, the cut surface polished, etched with a \% hydrochloric acid
solution for 1.5 min. washed with distilled water, dried, and an
acetate peel made by applying an acetate sheet on the hinge surface
with acetone. Growth rings imprinted on the acetate peel were
counted on a digitizing table after x40 magnification using a Neo-
Promar projector. Although most individuals had their SL and age
ineasured. there were some that had only the SL or only the age
measured; these latter individuals were included in the analysis
where appropriate. Reproductive condition of each geoduck was
determined by removing a sample from the central portion of the
gonad and preserving the tissue in Davidson's Solution (Shaw &
Battle 1957). Histological slides were prepared with sections of the
gonad stained with heniatoxylin-eosin. Histological sections of the
gonads were classified into six stages according to Andersen
( 1971 ). Stage 0 was immature (no differentiation in gonadal tissue:
loose vesicular connective tissue in gonad). The other stages were
for mature geoduck (connective tissue well developed, primary
cells evident on follicle walls or eggs or sperm development evi-
dent) and classified as: ( 1 ) early active: (2) late active: (3) ripe: (4)
partially spent: and (5) spent.
Average von Bertalanfy growth curves were fitted to all data
points of size at age using the equation;
L, = L fl
')
where t is age in years. L, is shell length (mm) at age t, L,, is
theoretical maximum size, k is a constant, determining rate of
increase or decrease in length increments, t„ is the hypothetical age
at which the organism would have been at zero length. The pa-
rameters L^ . k, and t^, were estimated using a non-linear Gauss-
Newton least squares method (SYSTAT 2000).
The proportion of mature geoduck (P) at shell length or age (X)
was estimated using the equation;
Px = X/(X -t- e'*-''^')
where A and B are parameters estimated using a non-linear Gauss-
Newton least squares method (SYSTAT 2000). Data for both sexes
were combined for each of the growth and maturity curve analyses
since sex could not be distinguished in the immature sizes.
85
86
I-
O
LU
200
150-
100-
LU
^ 50H
Campbell and Ming
200 n
o
o
O O On
OO OCPO (5>
n
oO Q?' O O
o
20 40 60
AGE (YEARS)
80
100
)J!i^
>^
T
T
20 40 60 80
AGE (YEARS)
100
Figure I. Growth curves for P. abnipla collected from (Al Gabriola Island, and (Bl Yellow Bank. Curves calculated from the von Bertalanfy
growth parameters (Table ll.
RESULTS
Growth
The oldest P. ahruphi collected was 77 y (146 mm SL| from
Gahriola Island, and 1 17 y ( 154 mm SL) from Yellow Bank. The
smallest and largest geoduck, respectively, was 10 mm SL (age
unknown, probably 1 y) and 163 mm SL (42 y) from Gabriola
Island, and 43 mm SL (2 y) and 180 mm SL (58 y) from Yellow
Bank. Growth was fastest in the first 10 y followed by slow growth
thereafter for geoduck from both areas (Fig. I). There was con-
siderable variability of size within each age group. Growth rates of
P. ahrupui from Gabriola Island were slower than those from
Yellow Bank (Fig. 1. Table I).
Gonadal Condition
Immature gonads comprised 10.85% and 12.10% of the total
geoduck gonads sampled from Gabriola Island {n = 129) and
Yellow Bank (n = 124, includes three individuals without SL
measurements), respectively (Fig. 2). The largest immature geo-
duck was 80 mm SL (5 y) and 72 mm SL (4 y) from Gabriola
Island and Yellow Bank, respectively. There were Insufficient data
to determine spawning periods because seasonal monthly samples
were not collected. However, most mature gonads were in the
ripe or partially spent condition for geoduck collected from both
areas (Fig. 2). There were no gonads that were spent (gonadal
TABLE I.
Von Bertalanfy growth parameters for P. abnipla from Ciabriola
Island and Yellow Bank during May 1991. Values in brackets are
approximate 95% confidence intervals.
Area
Lx
Gabriola Island
Yellow Bank
129.6 (±4,1)
147.7 (±5. Si
0.146 (±0.020)
0.189 (±0.055)
-1.02 (±0.951
-1.42 (±1.17)
120
108
condition 5). This suggested that geoduck spawning had begun at
both areas during mid to late May 1991.
Sex Ratio
For geoduck <90 mm SL. in both areas combined. 41.1 8% were
immature, and 54.41% were males (Table 2). The sex ratio for
mature geoduck <90 mm SL was predoniinantls (92.5%) male
70 n
0 12 3 4
GONADAL CONDITION
Figure 2. Frequency of gonadal condition stages found in gonads of all
/'. ahrupta collected from Gabriola (black bars) and Yellow Bank
(hatched bars). Gonads classified as 0 = immature, and mature stages
that are I = early active: 2 = late active: 3 = ripe: and 4 = partially
.spent.
Geoduck Maturity
87
TABLE 2.
Pericnl of total gonads differentiated into mature males and females
and immature /'. ahnipla from (iahriola Island and \ eiloM Bank
during .Ma> IVMI. One 91 mm SI, hermaphrodite was found. N =
total nuniher. Includes onlv individuals with SL measurements.
Percent of Total
Area
Male
Female
Immature Hermaphrodite
N
<y() mm SL
Gahricila Island
56.76
5.40
.37.84
.37
\elk)W Bank
.51.61
.■i.2.^
45.16
31
Total
.54.41
4.41
41.18
68
>90 mm SL
Gabriola Island
57.61
42..^9
92
Yellow Bank
45.56
53..^-^
1.11
90
Total
5 1 .65
47.80
0.55
182
uith few (7. 3%) females for both areas combined. In contrast.
geoduck s90 mm SL had generally a more equal se,\ ratio, al-
though males were slightly more abundant than females in the
Gabriola Island sample, whereas there were slightly more females
than males in the Yellow Bank sample (Table 2),
Figure 3. Ph()liiriiicni;;ra|)lis iil /' ahnipla gonadal tissue cross-
sections of (.\l Male (x4(Mt magnilkation) showing spermatozoa-filled
follicle surrounded by connective tissue, (B) Female (x4()0) showing
oocyte-filled follicle surrounded bv connective tissue.
Hermaphroditism
.Although most of the histological material of mature P. ahnipta
gonads allowed differentiation between females (follicles with oo-
cytes) and males (follicles with .spermatozoa) (Fig. 3) there was
one individual that was a hermaphrodite, with a gonad showing
both male and female characteristics (Fig. 4). This gonad had some
follicles containing only either female or male gametocytes per
follicle, and other follicles, which contained spermatozoa and oo-
cytes in the same follicle. The geoduck was 91 mm SL (age was
not determined).
Malurity
Mean size at 50% maturity was similar for geoduck from
Gabriola Island, 58.3 mm SL (55.2-59.4 mm SL, lower and upper
95% confidence intervals, CI), and Yellow Bank, 60.5 mm SL
(51.1-64.0 mm SL. 95% CI) (Fig. 5, Table 3). Mean age at 50%
maturity was about 1 y slower for geoduck from Gabriola Island,
3.09 y (2.68-3.25 y, 95% CI), than at Yellow Bank, 2.04 y ( 1 .72-
2.16 y. 95% CI) for Yellow Bank geoduck (Fig. 6. Table 3). The
smallest mature male was 45 mm SL (2 y) and 60 mm SL (2 y),
the smallest mature female was 59 mm SL (4 y) and 88 mm SL
(2 y), and the largest immature geoduck was 80 mm SL (5 y) and
72 mm SL (4 y), respectively, in the samples from Gabriola Island
and Yellow Bank.
B
*■ ■
. *•
s-
Figure 4. Photomicrographs of hermaphrodite I', ahnipta gonadal tis-
sue cross-sections of (.\) (x250 magnification), and (B) (xl60) showing
single follicles containing oocytes and spermatozoa.
88
Campbell and Ming
1.0
UJ
Q: 0.8H
Z)
0.6-
01 0.4
O
CL
o
a: 0.2H
Q_
0.0-
I I I I
T
0 50 100 150
SHELL LENGTH (MM)
200
1.0
LU
Qi 0.8-
I-
<
^ 0.6-
z
o
fe 0.4-
O
Q.
o
01 0.2-
CL
0.0-
O OC
OO QCO
0 50 100 150
SHELL LENGTH (MM)
200
Figure 5. Size at maturity curves for P. abntpta collected from (A) Gabriola Island, and (B) Yellow Bank. Symbols indicate number of individuals
per shell length: "O" = I; "X" = 2; "+" = 3. See text for equation for the predictive curve and Table 3 for parameter values.
DISCUSSION
Our findings indicated that growtli rales were faster for geo-
duck from Yellow Bank than those from Gabriola. Results were
similar to those of Burger et al. (1998) and Bureau et al. (2002)
who found that geoduck from Georgia Strait were generally
smaller than those from the west coast of Vancouver Island. Rea-
sons for the differences in P. abntpta growth rates between areas
could be attributed to a variety of environmental and biological
factors associated with different habitats (e.g.. substrate type, tem-
perature, exposure to water surge activity, pollution, food avail-
ability, and geoduck density or genetic characteristics) (Breen &
Shields 1983. Harbo et al. 1983, Goodwin & Shaul 1984, Goodwin
& Pease 1991, Noakes & Campbell 1992. Hoffman et al. 2()0().
Bureau et al. 2(X)2).
Our examination of gonadal condition suggested thai the
spawning period for geoduck from both study areas was just be-
ginning in mid to late May 1991. Results agree with other gonadal
studies of geoduck, which found the main spawning period was
TABLE 3.
Parameter estimates for equation indicating relationships betv\een
proportion that are mature with shell length (SI. in mm) or age
(years! of P. abriipta from (iabriola Island and Yellow Bank during
May 1991. See text for equation formula. \ alues in brackets are
approximate 95% confidence intervals.
Parameter Estimates
Variable
.\rea
X
Gabriola Island
SL
Yellow Bank
SL
Gabriola Island
Age
Yellow Bank
Age
8.512 (±2.741) 0.076 (±0.044) 79
7.224 (±2.. ^14) 0.052 (±0.0.^3) 80
2.956 ( ± 1 .55 1 ) 0.59 1 ( ±0.435 ) 1 5
2..397 (±1.540) 0.828 (±0.644) 14
during June and July (.Andersen 1971. Goodwin 1976. Sloan &
Robinson 1984).
The male:female sex ratio of mature P. abntpta found in this
study (52:48) was similar to that reported by Goodwin (1976)
(.53:47) and Sloan and Robinson ( 1984) (37:43). The high percent-
age of males in the small sizes (young ages) in this study was
1.0-
LU
cn O.BH
3
0.6-
q: 0.4-
O
Q.
o
q: 0.2
Q.
0.0-
6 6 5 12 2 1 3
XM^^:^!^'^ ig^ O » O
, ' " "^/Al 4 5 6 3 11
-1 1 1 \ 1 1 1 r
0
5 10
AGE (YEARS)
15
Figure 6. Age at maturity curves for P. abriipta collected from
Gabriola Island ("O" solid curve), and bellow Bank ("X" and dashed
curve). Number by each symbol indicates numlier of individuals per
age group. See text for equation for the predictive curve and Table 3
for parameter values.
Geoduck Maturity
89
similar to Andersen's ( 1971 ) findings of94.4'7i- males among geo-
duck with <100 mm SL.
Our findings indicated the first recording of a P. cibnipui her-
maphrodite. Most bivahe species are dioecious (sexes are sepa-
rate) although hermaphroditism does occur in some species of this
group (Coe 1943, Coan et al. 2000). Factors causing hermaphro-
ditism in P. ubnipia are unknown. Whether the "simultaneou.s"
hermaphroditism (Coe 1943. Eversole 1989) in this geoduck was
fully functional in producing viable eggs and sperm is unknown.
However, sexuality of different sizes (or ages) In F. nhntpta has
not been studied extensively. We estimated thai only -1.200 indi-
vidual gonads have been histologically examined to date from
mature P. dhniprn sampled in Washington State and British Co-
lumbia (Andersen 1971. Goodwin 1976. Sloan & Robinson 1984.
this study). Andersen (1971) and Goodwin (1976) suggested that
P. ahnipia might be gonochoristic where sex is determined by
development with males maturing at a smaller size (earlier age)
than females. Although we suspect that hermaphroditism is rare in
P. ahriipta. the probability that some level of protandry. sex re-
versal, or "simultaneous"" hermaphroditism in P. nhnipla (espe-
cially for sizes <I00 mm SL) ma\ occur and should be in\esti-
gated further.
Sexual maturity was variable between P. ahniplu individuals
and sexes. Males started to mature at an earlier age than female
geoduck in Yellow Bank than Gabriola Island. Although size at
SC/f maturity was similar for P. ahnipia from both areas (58.3 and
60.5 mm SL. respectively) age at 50% maturity was slower for
geoduck from Gabriola Island (3 y) than Yellow Bank (2 y).
Andersen ( 1971 ) found sexual maturity of geoduck to be variable,
the smallest sexually mature geoduck to be 45 mm SL, and 50%
size at maturity to be 75 mm SL (which Andersen estimated to be
an age of 3 y). Our study is the first to show that although size at
maturity may be similar for geoduck from two different areas,
differences in growth rates may influence the age at which geo-
duck matures sexually. These findings are siinilar to some studies
of other bivalve species, which suggest that onset of maturity may
depend more on size than age (e.g.. Nakaoka 1994). However, size
and age at sexual maturity can also vary between populations in
the same bivalve species (Ponurovsky & Yakovlev 1992. Sato
1994). Variation in environmental (e.g.. temperature, current pat-
terns, substrate type, and depth) and biological (e.g., genetics, food
supply, growth and mortality rates, predation. and parasitism) fac-
tors may affect maturity rates w ithin bivalve populations at differ-
ent locations (Thompson et al. 1980, Ponurovsky & Yakovlev
1992, Nakaoka 1994, Sato 1994, Taskinen & Saarinen 1999).
ACKNOWLEDGMENTS
The authors thank M. Boudreau, G, Hickie, D. Larson, M.
Lanoie. and N. Sorenson for the geoduck collections. S. Bower. W.
Carolsfeld. B. Clapp. S. Dawe. L. Lee. and T. White for technical
assistance, and J. Blackbiiurne, N. Bourne, S. Bower, and G.
Gillespie for helpful comments on early drafts of this manuscript.
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Andersen, A. M. 1971. Spawning, growth, and spatial distrihution of the
geoduck clam Punope generosa (Gould), in Hood Canal, Washington.
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Bernard, F. R. 1983. Catalogue of the living Bivalvia of the eastern Pacific
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Bradbury. A. & J. V. Tagart. 2000. Modeling geoduck. Panopea abrupta
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Breen, P. A. & T. L. Shields. 1983. Age and size structure in five popu-
lations of geoduc clams (Panope generosa) in British Columbia. Can.
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2002. .Age, size structure and growth parameters of geoducks [Panupca
ahnipia. Conrad 1849) from 34 locations in British Columbia sampled
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Burger, L.. E. Rome. A. Campbell. R. Harbo. P. Thuringer, J. Wasilewski
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Campbell. A., R. M. Harbo & C. M. Hand. 1998. Harvesting and distri-
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bia. Can. Spec. Publ. Fish. Aquat. Sci. 125:349-358.
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terly Rev. Biol. 18:154-164.
Eversole, A. G. 1989. Gametogenesis and spawning in North American
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gna, editors. Clam mariculture in North America. New York: Elsevier
Science Publishing Company Inc. pp. 75-109.
Goodwin, C. L. 1976. Observations of spawning and growth ol suhlidal
geoducks {Panope generosa. Gould). Proc. Natl. Shellfish Assoc. 65:
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Goodwin, C L. & B. C Pease. 1991 . Geoduck, Panopea abrupta (Conrad,
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parameters in Paget Sound, Washington. / Shellfish Res. 10:65-77.
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geoduck clam (Panope generosa. Gould) in Puget Sound Washington.
Wash. Dept. Fish. Prog. Rep. 215:30.
Goodwin, L., W. Shaul & C Budd. 1 979. The larval development of the
geoduck clam [Panope generosa. Gould). Proc. Nat. Shellfish. .Assoc.
69:7,V76.
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abrupta) fishery in British Columbia for 2001 and 2002. Ottawa: Can
Stock Assessment Secretarial Res. Doc. 53 pp.
Harbo, R. M.. B. E. Adkins. P. A. Breen & K. L. Hobbs. 1983. Age and
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(Bivalvia: Protobranchia). Mar. Ecol. Prog. Ser. 114:129-137.
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the Japanese littleneck, Ta/ws phitippmarium (A. Adams and Reeve,
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Joiinuil ,>/ Shellfish Ri'search. Vol. 22, No. I. 91-94. 2(K).\
THE EFFECTIVENESS OF N-HALAMINE DISINFECTANT COMPOUNDS ON PERKINSUS
MARINUS, A PARASITE OF THE EASTERN OYSTER CRASSOSTREA VIRGINICA
M. A. DELANEY,'* Y. J. BRADY,- S. D. WORLEY,' AND K. L. HUELS-
^ Aquatic Animal Health Research Laboratory. USDA-ARS. P.O. Bo.\ 952. Auburn. Alabama 36831:
'Department of Fisheries and Allied Aquaeultures. Auburn University. Auburn. Alabanui 36H49:
Department of Chemistry. Auburn University, Auburn, Alabama 36849
ABSTRACT The pathogenic protozoan Perkinsus marinus (Mackin. Owen and ColHer) is the cause of extensive mortalities in
Eastern oyster. Cwssosirea virgiiiicci. populations along the Gulf and East Coasts of the United .States. A series of experiments was
undertaken to determine the effect of N-hakutiine disinfectants on this protozoan parasite. The organic N-halamine disinfectants.
1.3-dichloro-2.2.5.5-tetramethyl-4-imidazolidinone (DC) and l-chloro-2,2.5,5-tetramethyl-4-imidazolidinone (MC). apparently dam-
age the permeability of the parasites outer membrane and alter the osmoregulatory functions of the cell. Damaged parasites were unable
to reproduce at concentrations as low as 14.9 mg/L DC at 8 h exposure, or for the chemical MC at 24.9 mg/L at 12 h exposure. The
chemical compounds appear to lyse the larger meronts first, followed by lysis of the daughter spores. These studies strongly suggest
that the chemical compounds DC and MC can be u.sed to disinfect seawater allowing the production of specific pathogen-free stock
in oy.ster hatcheries, and having the potential to prevent the spread of these parasites froin contaminated oysters to uninfected oysters.
KEY WORDS: ovster. Pcikinsii.\ marimis. disease, disinfection. N-halamine
INTRODUCTION
The Eastern oyster. Crassostrea vir^inica (Gmelin 1791 ) natu-
rally occurs in North America frotn the Gulf of St. Lawrence in
Canada to the Gulf of Mexico. It is common in estuaries in coastal
areas of reduced salinity, and is an important commercial species.
Once considered the most abundant source of oysters in the world,
eutrophication, overharvesting and the parasites Haplosporidiiim
nelsoiii and P. nuu'iiuis have caused the Chesapeake Bay oyster
population to be reduced to a critically low level (Andrews 1988.
Haskin & Andrews 1988, Hargis & Haven 1988). The parasites
inhibit growth, reduce fecundity, and lower the oyster's condition
and glycogen content (Menzel & Hopkins 1953, Newell 198.3,
Barber et al. 1988. Crosby & Roberts 1990). Oyster populations
that have incurred high infection prevalence and intensities typi-
cally have low mortalities during their first year, but suffer higher
mortalities in the following years (Paynter & Buneson 1991 ). The
parasite does not have the same drastic effects on the oyster popu-
lation in the Gulf of Mexico as it does in the Chesapeake Bay. An
oyster requires three or tnore years to reach marketable size in the
cooler waters of the Atlantic; however, only two years are required
in the warmer waters of the Gulf of Mexico. In the Gulf of Mexico,
this parasite infects over 80% of Eastern oysters with annual mor-
talities typically 50% of the adult oyster population. Transmission
of the parasite occurs through the water by release of infective
stages from the feces of living oysters, the tissues of dead oysters
(Ray 1932. Mackin & Hopkins 1962). and by the gastropod ecto-
parasitic snail, Boonea impressa (White et al. 1987).
Perkin.sus marinus has several life stages in the host oyster
(Mackin & Boswell 1956. Perkins 1969). These include immature
thalli. mature unicellular thalli (trophozoites), and presporangia.
When released into seawater. presporangia develop a resistant cell
wall, and then enlarge to become hypnospores. Under aerobic
conditions, hypnospores differentiate into sporangia and produce
*Corresponding author: E-mail: mdelaney@vetmed.auburn.edu
This study was funded by Mississippi-Alabama Sea Grant Consortiimi.
motile zoospores (aplanospores in Mackin & Boswell 1956) within
the hypnospores cell wall. One sporangium of P. marinus is ca-
pable of releasing approximately 354.700 zoospores (Chu &
Greene 1989). Zoospores are released from hypnospores and un-
dergo free-living stages in seawater.
Eradication of these pathogens in the wild is not possible be-
cause of the widespread nature of the diseases and the lack of
knowledge regarding other species that might carry the disease
(Elston 1990). Resistance to H. nelstmi. but not to P. marimis
(Barber & Mann 1991 ) has been achieved through selective breed-
ing of C. virgiiiicci (Ford & Haskin 1987. Foid et al. 1990. Bur-
reson 1991).
Developing and maintaining hatcheries to produce larval oys-
ters for grow out for co)nmercial production or to repopulate de-
pleted areas is one approach to alleviate the lack of natural repro-
duction. This method, however, requires the incoming seawater to
be specific pathogen free. The traditional methods of using ozone
and ultrafiltration are expensive for continuous production. Chlo-
rine is an inexpensive alternative for water disinfection; however.
Its chemistry changes when combined with seawater.
Observations of oyster larvae exposed to chlorine-treated sea-
water indicate a lethal concentration for 50% of the test organisms
(LC 50) for C. virgiiiica larvae of 0.005 mg/L free chlorine (CI+),
regardless of whether static or intermittent addition of chlorine was
used (Roberts et al. 1975, Bellanca & Bailey 1977. Roberts &
Gleeson 1978). Concentrations as low as 0.05 mg/L of bromate,
broiTtoform and chloroform caused some C. virgiiiica 48 h larval
mortality (Stewart et al. 1979). Galtsoff (1946) noted a 46% de-
crease in pumping action at a dose of 0.2 mg/L chlorine. He and
other workers concluded, however, that chlorine was an effective
means for disinfecting shells of contaminated oysters and that the
oxidant would not interfere with depuration if chlorine levels were
kept at a minimum. Later studies agreed with this finding but
cautioned that oysters reduce pumping when chlorine concentra-
tions exceed 0.01 mg/L. At chlorine concentrations above 1.0 ing/L.
pumping cannot be maintained; thus, the use of chlorine as an
effective means of depuration is limited by the tolerance of the
species. The ability of adult shellfish to respond to low concen-
trations of total residual oxidant and to cease pumping may be
91
Delaney et al.
beneficial because it allows the animal to survive chlorine-
produced oxidant (CPO) concentrations as high as 10 mg/L for 30
days (Galtsoff 1964). The corresponding decrease or cessation,
however, of shell growth and feeding is disadvantageous. The
most severe restrictions to chlorine use arise from the formation of
chemical compounds from adding this to seawater. Halogenated
organic compounds are formed that display complex chemistry.
The products of chlorination of seawater are complex and not fully
understood (Carpenter & Macalady 1973. Davis & Middaugh
1977, Wong & Davidson 1977, Carpenter et al. 1980). In seawater
and brackish water, chlorine replaces some of the bromine in hy-
pobromous acid releasing the bromine cation that is considered the
disinfecting compound. Full strength seawater has a bromide ion
concentration of 65 mg/L, and chlorine reacts with it to produce
hypobromous acid and hypobromite ion. Bromamines and
chloramines may be formed in the presence of ammonium ion. For
normal seawater of pH 8, the initial products of chlorination are a
mixture of hypobromous acid and hypobromite ion that are un-
stable with respect to decomposition and disproportionation
(Macalady et al. 1977).
The N-halamine compounds used in this study were 1,3-
dicliloro-2,2,3.5-tetramethyl-4-imidazolidinone ( DC; dichloro)
and 1 -chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC;
monochloro). Both compounds were synthesized at Auburn Uni-
versity in the laboratory of S. D. Worley. Department of Chemis-
try. The compound MC can be produced in the laboratory and as
a result of the hydrolysis of the compound DC. The compounds
will be marketed by Vanson/HaloSource Corporation, Seattle
WA'. These compounds are more stable in water and dry storage
than free chkirine and other commercial products, such as the
hydantoins and isocyanurates (Tsao et al. 1991). The N-halamine
compounds do not produce trihalomethanes or react with bromide
in seawater and should be more stable and more effective than free
chlorine. The compound DC is the faster acting compound and the
amide N-Cl moiety is more labile than the amine N-Cl group,
providing a small amount of free chlorine. The hydrolysis decom-
position product MC. having only the more stable amine N-Cl
moiety, acts more slowly as a disinfectant.
In this series of experiments, the parasites were exposed to the
chemical compounds in sterile artificial seawater (SASW) to de-
termine the effectivity of the compounds. A related compound,
3-chloro-4,4-dimethyl-2-oxa/olidinone, has been shown to kill
Giaidia lanihlia more effectively than free chlorine (Kong el al.
1988), and it was speculated that DC or MC would penetrate oyster
tissues and the thick parasite walls at a reduced level of chlorine.
A previous study using Anadara trapezia (blood cockles) and
Haliotis laevii-ata (greenlip abalone) showed that free prezoospo-
rangia of Peikinsus sp. (remo\ed from oyster tissues) died within
30 min in chlorine solutions of 40 mg/L (Goggin et al. 1990);
however, within tissues the parasites presumably are more pro-
tected and survived at least 2 h. Their study was concerned pri-
marily with disinfecting meats of abalone. The objective of this
study is to determine if the parasite P. iiniriiuis could be eliminated
in the water column. The possibility of controlling P. inariiuis in
an oyster hatchery by treating incoming water, or as an interim
control preventing the spread of the parasite between oysters,
could mean economic gains associated with increased health and
growth characteristics.
Use of trade or manufacture's name does tuh imply endorsement.
METHODS
A series of three experiments were conducted to evaluate the
effectiveness of these compounds on P. marinus.
Perkinsiis mannus cultures were obtained from the American
Type Culture Collection (ATCC), and cultured according to La
Peyre and Faisal (1995). In experiment one, an aliquot was re-
moved from culture, vortexed briefly to break up cell clumps, and
then centrifuged at 5(.)0i; for 5 min. These cells were rinsed twice
with 15 ppt sterile artificial seawater (SASW), then resuspended in
SASW at a concentration of approximately 5 x lO'' cells niL"'.
The chemicals DC and MC, which were synthesized according to
the method of Tsao et al. (1991), were prepared in three concen-
trations: 0.3. 14.9 and 29.8 mg/L and 0.5, 24.9 and 49.8 mg/L,
respectively. These concentrations are based on molar equivalents
of chlorine. Four replications of each chemical at each concentra-
tion were prepared in sterile. 50 niL. polypropylene centrifuge
tubes. Approximately 5000 parasites were added to tubes contain-
ing 50 mL of each chemical concentration. The same amount of
SASW with and without parasites served as the positive and nega-
tive controls. Contact time consisted of eight time intervals: 0.5. 1,
2, 4, 8, 12. 18. 24. and 48 h. At the appropriate time, the samples
were mixed and I niL removed from each tube. Sodium thiosulfate
(0.02 N) was added to neutralize the chlorine (i.e., to quench
disinfecting action! and the cells were observed microscopically at
xlOO with and without staining with Lugols Iodine.
A second experiment was initiated to determine the percent
mortality at \ arious concentrations and time intervals using a vital
dye, trypan blue, which distinguishes between living and dead
cells. This viability test evaluates the breakdown of membrane
integrity determined by the uptake of the dye to which the cell is
normally impermeable. Cell and chemical preparation was the
same as previously described. Contact time consisted of three time
intervals: 1. 2. and 8 h. At the appropriate time, the samples were
mixed and 1 niL removed from each tube. The cells were washed
with Hanks Balanced Salts Solution (HBSS) (Sigma, St. Louis,
MO) and resuspended in 0.3 niL HBSS to which 0.5 niL trypan
blue was added. The cell suspension was mixed and allowed to
stand at room temperature for 5-15 min. Living and dead cells
were counted and enumerated using a hemacytometer at xlOO.
Dead cells stained a dark blue, but living cells were able to exclude
the dye. Cells with an intermediate blue color stain were consid-
ered dead.
A third experiment was performed to detemiine the viability of
the cells after exposure to the two chemicals, targeting the cells
that lightly stained indicating damage to the membrane. It was
important to know whether these damaged cells would be able to
recover and initiate a new infection.
Cells were removed from culture, centrifuged to pellet the para-
sites then resuspended in SASW. Four concentrations of DC (7.4.
14.9. 29.8. 44.6 mg/L) and 4 concentrations of MC (12.9. 24.9.
49.8. 76.6 mg/L) v\ere prepared in sterile, polypropylene centri-
fuge tubes, and then 2 niL were transferred to individual wells of
tissue culture plates. Three replications of each chemical con-
centration were prepared. Approximately 20 |j.L of the P. marinus
(4.5 X lO'* parasites mL~') cell suspension were added to each
disinfectant chemical. The same amount of SASW with and with-
out parasites was added to the positive and negative controls.
Contact time consisted of four time intervals: 1. 2. 8. and 12 h. At
the appropriate time, the chlorine in the samples was neutralized
with 20 |jiL of 0.02 N sodium thiosulfate and the cells resuspended
Effectiveness of N-Halamine Compounds
93
TABLE 1.
Experiment 2: the etTect of DC and MC concentration and exposure
time on mortality of P. mariniis.
TABLE 2.
Experiment 3: the effect of DC and MC concentration and exposure
time on mortality and replication of P. mariniis.
'Jc Staining
'?c Staining
mg/L
I hour
2 hours
8 hours
DC 0..^
0.3
1.3
DC 14.9
11.4
77.4
DC 29.8
80.0
80.8
MC0.5
0
0.2
MC 24.9
.VI
16.3
MC 49.S
19.2
22.9
in 3 mL of culture media. A portion of the cell suspension was
removed and evaluated with typan blue staining as previously
described. The remainder of the samples were incubated in the
dark at 25°C and evaluated at 24 and 48 h.
RESULTS
In the first experiment, no visible effects on P. marimis were
observed for DC or MC treatments at any tested concentration up
to 4 h. At 8 h exposure to either DC or MC. all parasite cells
appeared to decrease in size, and at 18 h all cells were completely
lysed at all concentrations. The negative controls appeared free of
debris and bacterial contamination during the test. The positive
controls appeared unchanged and did not exhibit any decrease in
size, nor did they lyse.
The second experiment attempted to refine the earlier one by
determining viability at various contact times. The viability of the
cells exposed to DC has been reduced by 80% at I h at a concen-
tration of 29.8 mg/L (Table 1). At a concentration of 49.8 mg/L
MC at 1 h, a reduction of only 19.2% was observed. At the end of
8 h. 99.8% mortality was observed at 29.8 mg/L DC. as compared
with 25% with 49.8% MC.
In the study addressing the viability and the ability of the para-
site to recover from exposure to the DC and MC compounds
showed a trend towards more rapid deactivation of the parasites by
DC as compared with MC. at similar concentrations (Table 2).
Cells in the positive control treatment exhibited normal growth and
development.
DISCUSSION
Results of this study demonstrated that the compounds MC and
DC eliminated the pathogen P. mariniis in 15 ppt seawater under
laboratory conditions. It is important to kill all parasites because a
single sporangium of P. mariniis is capable of releasing approxi-
mately 354.70(J zoospores (Chu & Greene 1989).
Mortalities of 100% of P. mariniis can be achieved using the
faster acting chemical DC at concentrations of 14.9 mg/ for 8 or
12 h. 29.8 mg/L for 8-12 h or 44.6 mg/L for a minimum of I hour.
mg/L
I hour
2 hours
8 hours
12 hours
12.9
DC 7.4
4.1
11.0
6.9
13,4
88.2
DC 14.9
12.7
34.8
83.0"
33.0"
99.8
DC 29.8
10.4
29.8
36.0"
98.6"
0.2
DC 44.6
98.1"
99.6-'
100-'
100"
16.0
MC 12.4
0
3.4
6.2
7.1
25.0
MC 24.9
MC 49.8
10.9
17.2
14.3
14.6
20.7
82.0"
70.0"
87.2"
1 was
MC 76.6
0
0
98.6"
90.3"
" Indicates cultures in which all parasites died without producing viable
offspring when observed 48 hours after chemical treatment.
The slower acting chemical MC can achie\e 100% mortality at
concentrations of 24.9 mg/L for 12 h, 49.8 mg/L for 8 or 12 h. or
76.6 mg/L for 8-12 h. Additional testing would be desirable to
determine lower concentration effectivity against this pathogen.
Both DC and MC are effective against the oyster parasite P.
mariniis in vitro at concentrations less than the estimated LDg,, of
the oyster larvae exposed to these same chemicals (Delaney et al.
2002). Histologic and physiologic information would be required
on the long term effects of chemical exposure to oyster larvae;
however, either compound has the potential to be used in oyster
hatcheries to prevent infections of P. marimis from occurring, or to
prevent the spread of the disease through the water column if the
contact time is sufficient. Electron microscopy would provide ad-
ditional insight on the mechanism of damage to the parasite's cell
walls at different stages in the life cycle of the parasite.
N-halamines DC and MC at concentrations of total chlorine
within the lar\ al and adult oysters range of tolerance, are effective
for the control of a protozoan pathogen. P. marimis. of Eastern
oysters. These compounds have the potential to be used in oyster
hatcheries and in recirculating based systems to produce specific
pathogen free oysters. The use of these compounds as a substitute
for free chlorine or chloramines would mitigate deleterious physi-
ologic effects currently observed on oyster recruitment and sur-
vival in estuaries receiving chlorinated discharges.
ACKNOWLEDGMENTS
The authors thank Dr. David D. Rouse, Dr. Sharon R. Roberts,
and Dr. George W. Folkerts for their technical assistance and
Dr. Thomas McCaskey, for his attention to details which improved
this manuscript. Additional thanks to Dr. Jeffrey Williams of the
Vanson/HaloSource Company for providing the chemicals used in
this study and technical assistance, and Dr. John Supan for pro-
viding larval oysters.
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Water Research 11:971-978.
Jounnil of Shellfish Research. Vol. 22, No. 1, 95-W. 2003.
HATCHERY REARING OF THE BLACK SCALLOP, CHLAMYS VARIA (L.)
A. LOURO. J. P. DE LA ROCHE. M. J. CAMPOS, AND G. ROMAN*
lusiituto Espauol de Occauograjia. Centra Uceaiiognifico de A Conirui, FO Box JJU,
15080 A Conma. Spain
ABSTRACT Thi.s work describes methods used for conditioning, spawning, and growing larvae of Clihiiny. vuria in hatcheries and
the results obtained. Conditioning in winter results in fast ripening. Oocytes are easily obtained by injecting serotonin. Different
antibiotics were tested and the results compared. Different systems for setting were compared. C.vuria prefers flat surfaces rather than
monofilament as settlement substrate.
KEY WORDS: Chlaniys vuiiii. hatchery, conditioning, spawning, larval culture, settlement, antibiotics
INTRODUCTION
Worldvv ide production of pectinids has increased spectacularly
in recent years, rising from 200.000 t in 1970 to 1.7 million t in
1996. The rise is largely the result of an increase in production of
these shellfish by aquaculture. which accounts for W/e of the total
production (Bourne 2000).
In Spain, as in the rest of Atlantic Europe, Pectcn nui.xiiniis is
the most commercially valuable of the pectinid species exploited;
however, experiments have recently been peiformed to assess the
possibility of cultivating smaller pectinids. such as Aequipecteii
opercuUihs (Roman et al. 1999) and Chlaniys varia (Acosta &
Alvarez 1990. Acosta et al. 1990. Roman 1991),
Chlamys varia is found in the eastern Atlantic, ranging from
southern Norway to Senegal and also in the Mediterranean (Ansell
et al. 1991, Brand 1991). It displays rhythmic consecutive her-
maphroditism; most younger/smaller specimens are males that un-
dergo a gradual sex change so that most older animals are females
(Lubet 1956. Lucas 1965. Reddiah 1962. Burnell 1983),
This species is relatively scarce in Spain. It is therefore rarely
sold commercially, and there is very little information available
about its biology and ecology. However, the potential for culturing
the species in Galicia is presently being considered. Methods of
obtaining gametes have been determined (Roman & Fernandez
1990). and spat have been cultivated in suspension from rafts
(Acosta et al. 1990); Parada et al. (1993) provide information on
the reproduction of C varia cultivated in suspension. In Galicia
the use of collectors to capture spat in natural environments has
proven unsuccessful (Roman et al. 1987. Ramonell et al. 1990) and
therefore spat production must be conducted in hatcheries. Hatch-
ery cultivation of this species has been described by Burnell
(1983), Le Pennec and Dis-Menguss (1985, 1987), Acosta and
Alvarez (1990), and Roman (1991).
The aims of the present study were to investigate ( I ) the larval
behavior of Chlaniys varia under the standard conditions estab-
lished at the Centro Oceanografico de A Coruna (COAC), for the
culture of P. nia.\imiis larvae, as summarized below; (2) the effect
of different antibiotics on larval growth and survival: and (3) the
behavior of the larvae at settlement, with the aim of optimizing the
culture methods to increase the yield of spat.
At the COAC. culture of P. ntaxinuis larvae has been conducted
intermittentlv since 1976. with some modifications to the tech-
niques described by Roman and Perez (1979) and Roman (1986.
19911,
The use of antibiotics in larval cultures is controversial. In
general in Europe pectinid larvae cannot be consistently cultivated
without chloramphenicol (Gonzalez & Roman 1983. Samain et al,
1992, Torkildsen et al. 2000). the use of which is presently pro-
hibited by the EU. Other antibiotics must therefore be used com-
mercially.
During settlement of pectinid larvae, mesh bottomed cylinders,
or collectors made of different materials are often used. Pearce and
Bourget ( 1996) have reviewed the use of different materials for the
settlement of competent spat of various pectinid species, although
no reference is made to hatchery rearing of C. varia. Only Rod-
house and Burnell (1979) mention settlement preferences of C.
varia on undersurfaces or on shaded areas in sections, of PVC
slats, in laboratory experiments.
MATERIALS AND METHODS
Conditioning
*Corresponding author. Tel: +34 981 205362; Fa.\: -i-34 981 229077;
E-mail: guillermo.roman@co.ieo.es
Adult C varia of between 30 and 50 mm in height were trans-
ported from the sea to the COAC and conditioned from the end of
December 2000 until March 2001. The trial started when scallops
were totally spent. Scallops were placed in tanks (180 x 50 x 30
cm), through which sea water flowed at a rate of 6 L min"' at
ambient temperature ( 12-14°C), An average number of 21.8 x 10''
cells day"' of Skelelonema costatiini. 13.7 x 10'' cells day"' of
Tahitian I.uichiysis ajf. galbana and 14.0 x 10'' cells day"' of
Pavlova lutheri were added to the circulating sea water using a
dosing pump for density of 183 cells/|jiL. Males and females were
kept separately after their sex was established by microscopic ex-
amination of gonad samples taken by needle puncture.
Stimulation
When females were observed to have well-developed gonads
(swollen appearance and color range between white, cream or
yellow), they were injected intramuscularly with 0.2 mL of 0.2
mM serotonin (Roman & Fernandez 1990). Once spawning began.
8 to 10 males were injected, and the sperm suspension from vari-
ous specimens was mixed. The oocytes were sieved (100-(JLm
mesh) to remove large particles and feces. The number of oocytes
shed by each female was counted using a l-mL Gallemkamp
counting cell ( Sedge wik-rafter S50). Sperm suspension was added
to the containers in which the oocytes were held, so that there were
approximately five sperm per oocyte (Gruffydd & Beaumont
1970).
95
96
LOURO ET AL.
Inctihalion
Incubations were performed in 130-L conical-bottomed fiber-
glass tanks containing 0.45-|jim filtered sea water at 16-18°C with
slight aeration, for 3 days. Food was added on the second day (25
cells fj-L"' of a 1:1 mixture of Tuhitian /. aff. galhiuui and P.
lutheri) and on the third day the tanks were emptied and the larvae
collected in 60-|jim mesh sieves. Larvae of normal appearance
were counted and the hatch yield was calculated. Three ranges of
incubation density (<6; 6-10; >10 eggs/niL) was tested.
Lanal Culture
Larvae were cultured in 150-L tanks containing 0.45-p.m fil-
tered sea water at ambient temperature (16-18°C) at an initial
density between 0.5 and 8 larvae niL^'; 8 mg L~' of chloramphen-
icol was added, and a mixed diet of 50 cells |xL"' of Tahitian /. aff.
gathana. and P. Iiillieri (1:1) was provided. The water was changed
three times a week and the mesh size of the sieve used to retain the
larvae was increased depending on the si/e of the larvae; each time
the water was changed a sample of larvae retained was measured.
Larvae reached a final density of less than 1 larva niL"' at the time
of settlement. When competent pediveliger larvae appeared, the
culture was 140-|j.m mesh sieved. If the number of pediveligers
with eye spots was greater than 509c. they were placed in settle-
ment systems.
Effect of Different Antibiotics
Larvae were cultivated at three different treatments: chloram-
phenicol (8 mg L"' ). penicillin plus streptomycin (.^0 mg L"' -i- 50
mg L" ' ). and erythromycin { 8 mg L ' ) and no antibiotic as control
from hatching until settlement. The number of settled larvae was
counted for each treatment. All treatments were carried out in
duplicate.
Larval Sultleiiunt Systems
Three trials were perfonned with C. varia using two settlement
systems, i.e.. the traditional and the modified system. These two
settlement systems were compared in the first experiment. The
traditional system, consisting of a PVC cylinder that was 43 cm in
diameter and 40 cm in height with a 140-|ji,m mesh base through
which water was circulated in an upwelling system, was placed in
a 150-L tank. The method developed at the COAC (modified
system) using artificial seaweed as a settlement substrate was pre-
pared in another tank of the same size. A total of 172.500 pedi-
veliger larvae were added to each tank. Water was changed by
displacement. Food was added daily according to larval culture
and 5. costatum was included in the diet.
The effect of the substrate and the density of pediveligers on
settlement was investigated in a second trial. The traditional sys-
tem was used, but with a settlement substrate also provided. Nine
140-p.m mesh-bottomed cylinders 25 cm in diameter and 19 cm in
height (1983 cm~ internal surface area) were placed in 200-L ca-
pacity tanks (180 x 50 x 30 cm). Three larval densities (10.000.
20.000. and 30,000 larvae/mL) and two settlement substrates (ny-
lon monotllament, artificial seaweed, no substrate control) were
used.
In the third trial, different settlement substrates were tested. For
this, collectors comprising of artificial seaweed, nylon monofila-
ment filling and scallop shells were placed in a 400-L tank along
with 312.125 pediveliger larvae. The numbers of spat on each
substrate and on the tank walls were determined after approxi-
mately 45 days.
RESULTS
Conditioning
After 6 or 7 wk on the conditioning system, scallops were
observed to have swollen gonads, from which viable gainetes were
obtained after stimulation of spawning.
Stimulation
Scallops were artificially stimulated by serotonin injection, in
January. February, and March, and gametes were obtained on each
occasion. A total of 58.3'7f of the females and 80.0Vr of the males
responded to serotonin stimulation. The time needed to obtain
sperm and oocytes ranged between 7 and 43 min and 9 and 52 min,
respectively. An average number of 0.6 x 10^ (range: 0.05 x 10'-
2.4 - 10'\ n = 16) oocytes were obtained from each female; the
mean diameter of the oocytes was 68.8 ixm ± 1.9 (SD).
Incubation
Incubation yields for three eggs density ranks were 25.5% (0-5
eggs/mL, 11 = II): 34.1% (5-10 eggs/niL, n = 5); and 31.8%
(>I0% eggs/mL, /; = 6). Statistical differences were not found
between them (analysis of variance, P > 0.05). Mean size of larvae
D obtained was 1 10.28 |xm ± 2.61.
Standard Culture
Larval development (until 50% of the larvae developed eye
spots) lasted an average of 19.3 days ± 2.0 (/? = 16): 8 days after
the .spawning (larvae size = 134 (xm ± 1 a purple spot, which is
characteristic of this species, appeared on the dorsal posterior re-
gion of the larvae. Although larvae with eye spots may appear after
13 days, the proportion did not reach 20% until day 17 (larvae size
= 194. 1 p.m ± 13. 1 ). At the end of the culture period, the average
yield of pediveliger larvae was 31.2 ± 17% (larvae size = 21 1.8
240
5 10 15
Days after spawning
20
Figure 1. Larval growth of Clilainys varia (mean ± SD of 16 lartal
cultures).
Hatchery Culture of Black Scallop
97
TABLE 1.
Elfett of different antibiutics on lar>al yields.
Percent
Pediveliger
Percent
Settlement
Cloramphenicol
Penicillin + streptomycin
Erythromycin
Control
73.2 ±5.1
71.7 ±9.3
83.6 ± 4.5
78.0 ±8.3
10.1 ±4.9
10.8 ± 1.4
10.0 ±3.3
1.7 ± 1.0
|xm ± 9.9). The rate of growth from hatching initil the final day of
culture was 5.3 (j.m day"' (Fig. 1 ).
Effect of Different Antibiotics
The percentage .survival of the larvae, at the lime of recording
50% Vi'xlh eye spots, exceeded 709(- in all treatments, including the
control in which no antibiotics were used (Table I). However.
during settlement, \.19c larvae settled compared >\(Y/c for the
antibiotic treatments.
Settlement
First Trial
Similar spats settlement was recorded in the tanks in which
artificial seaweed and mesh bottomed PVC cylinders were used
(30.1% and 30.6%, respectively) and 31.907 and 52,7(10 spat were
obtained, respectively. More spat settled on the sides of the cyl-
inder than on the mesh bottoin. In the tank containing artificial
seaweed, most spat settled on the walls of the tank. Although the
spat on each substrate were not counted, there was a marked pref-
erence for vertical walls in both cases.
Second Trial
Effect of substrate and density of pediveligers on settle-
ment. The number of spat settled in each cylinder was deter-
mined, the numbers that settled on the walls and the substrates
provided were counted separately. The results are shown in Table
2. Most of settlement took place on the walls.
Third Trial
Settlement in 400 L capacity tank with various sub-
strates. The results are showed at Table 3. A total of 19.7% spat
settled were recorded. The higher settlement was on tank walls
(12.7%) with preference on bottom (Table 3).
DISCUSSION
Cultivation of C. variii larvae was performed using the tech-
niques developed over se\ eral years at the COAC for cultivating P.
inaximus (Roman, unpublished data). However, C. varia behaves
differently from P. inii.xiiiiiis. The most important differences were
associated with settlement and effect of antibiotics. At the COAC,
P. niaxiiniis larvae have not been successfully cultivated without
antibiotics (Gonzalez & Roman 1983. Ruiz 1996), and to date,
artificial seaweed has been found to be the best settlement sub-
strate for this species (Roman, personal communication). In con-
trast, C. varia can be cultivated to pediveliger successfully without
antibiotics and artificial seaweed was not a particularly good
settlement substrate for this species, the larvae preferring to settle
on the tank walls.
Part of the standard cultixation method of C. varia involves
discarding batches in which the oocytes are not spherical or in
which there is a low hatching rate (<10%'). Not all times of the year
are suitable for obtaining good quality larvae and hatcheries do not
have unlimited space, therefore when larvae are available the best
possible production rates must be obtained. Early removal of
batches of poor quality larvae allows culture of other batches ob-
tained from different spawns. With this method, time and money
are saved and better average yields are obtained, as cultures that
would probably die are eliminated.
Conditioning of C. vcirin during the winter months allows vi-
able gametes to be obtained from January onwards, thereby bring-
ing forward the natural spawning times, which usually take place
in spring and early summer (Parada et al. 1993). Unlike other
pectinid species that have been cultivated at the COAC (P. maxi-
iniis. P. jacobaeiis. and Aeqiiipeclen opcrciilaris) C. varia matures
quickly during the conditioning period (4-5 wk) and gametes are
obtained using serotonin, allowing the timing of the larval cultures
to be planned. Furthermore, there is no risk of self-fertilization and
polyspermy is easily avoided.
The result of the response of C. varia to stimulation by sero-
tonin was similar to those described by Roman and Fernandez
(1990) although complete emptying of the gonads was not always
observed in this study.
The average number of oocytes per female obtained in the
present study (0.6 x 10". ma.ximum 2.4 x 10") was less than those
previously reported: 1.54 x 10" (Roman & Fernandez 1990). 4.5 x
10" (Le Pennec & Diss-Menaus 1985) and 5 x lO" (Burnell 1983).
TABLE 2.
Effect of larval density and settlement substrates on yield of spat of C. varia (Trial 2),
Settlement Substrate Provided
Percent of Settlement
Number of Pediveligers
Kl.OOd
2(1,(1011
30,(100
Control (mesh bottomed cylinder only)
Mesh bottomed cylinder + monofilament
Mesh bottomed cylinder + artificial seaweed
Cylinder (Vr)
Cylinder (%)
Monofilament (%)
Total (%)
Cylinder ( a )
Artificial seaweed
Total (%)
35.1
32.2
1.5
33.7
9.1
9.9
19.0
52.3
4L4
9.2
50.6
37.7
20.8
58.5
48.3
19.1
4.1
23.2
13.8
8.4
22.2
98
LOURO ET AL.
TABLE 3.
Effect of settlement substrates on yield of spat of C. varia (Trial 3)
Collector Substrate
No. of Spat
Percent Settlement
Tank wall;,
39500
Standard net filling
10455
Scallop shell
9722
Artificial seaweed
1S85
Total
12.7
3.3
3.1
0.6
19.7
However, these authors used larger adult stock than in the present
study (30-50 mm; Roman and Fernandez used specimens of be-
tween 50-75 mm and Bumell, specimens >50 mm).
The mean diameter of the oocytes was similar (average range,
68-72 |jim) to those found by Bumell (1983; 65-70 |jim) but larger
than those found by Le Peiinec and Diss-Mengus (1985; 50-60
|xm).
The density of eggs incubated did not appear to affect the yield
of larvae. This is consistent with the results of Roman and Fernan-
dez (1990). who found no significant effect of density (using be-
tween 1 and 50 eggs mL"' ) on the yields. O'Connor and Heasman
( 1995) obtained yields of up to MV/r with cultures of C. asperrlma
using a density of 100 eggs mL^' and 48% with a density of 1 egg
mL"'. Le Pennec and Diss-Mengus (1987) obtained hatching
yields of 77.7% after a period of incubation of 2 days (density 2.3
eggs mL"'), after which D larvae of 90 jxm were collected (using
sieves of mesh size 43 |jLm). Roman and Fernandez (1990) also
incubated the eggs for 48 li and obtained a yields of 17.9%.
O'Connor and Heasman ( 1 995 ) reported that 54% of C. aspenima
eggs hatched, and veliger larvae were obtained, following 2 days
incubation. With the culture technique developed at the COAC,
larvae were incubated for 3 days, then 60 |jLm mesh sieves were
used to remove small or abnormal larvae. Although the yield of D
larvae (29.2%) was lower than that reported by the authors men-
tioned above, better results were subsequently obtained because
dead or abnormal larvae, which usually appear at the end of the
incubation period, have already been removed.
The duration of the larval period of C. varia has been reported
as 22 days at 18°C (Bumell 1983). 19 days at 16-18°C (present
.study and Acosta & Alvarez 1990), and 15 days at 17°C (Le
Pennec & Diss-Mengus 1985).
The characteristic purple spot that occurs in this species, has
been reported to appear at different ages and in different sizes of
larvae: on day 4, in larvae of 120 jxm (Le Pennec & Diss-Menguss,
1985); on days 10-12, in larvae of 130-140 [j.m (Bumell, 1983);
and on day 8, in larvae of 134 |j.iii, (present study).
Larvae with eye spots appeared from day 13 onwards. In the
present study, 20% of the larvae had eye spots on day 17 (average
size of larvae, 194.1 p.m). Acosta and Alvarez (1990) detected the
pigmentation on day 14 (161.7 fxm). whereas Burnell (1983) de-
tected it in 2()-day-old larvae (200 iJiml.
Similar growth rates have been reported: 5.3 (xm day"' (present
.study), 4.8 |j.m day"' (Acosta & Alvarez 1990), and 5.3 |j.m day"'
(Burnell, 1983). all of which are much lower than that reported by
Le Pennec and Diss-Mengus (1987; 10 (jliii day"').
The larval culture yield obtained (31.2%' pediveliger larvae,
average size 211.8 (jim) was lower than those obtained by Le
Pennec and Diss-Mengus (1985, 1987; of between 65.5% and
70%. of larvae of 210 ixm). Using the same conditions, Burnell
(1985) did not obtain more than 4% survival of larvae of size
215 |j.m.
Despite the fact that few studies have been made of this species,
there is considerable variation in the results obtained by different
authors. This may be because of genetic differences or more prob-
ably, to different culture conditions, such as the quality of the
gametes or the diet. De la Roche (pers. com.) cultivated C.voria
larvae obtained from adults originating from Malaga and from
Galicia simultaneously and did not observe any differences in the
diameter of the oocytes, the age and size at which the pigmented
mark appeared, size at the time of appearance of the eye spot or
growth rate. Of the studies compared, the best results (in terms of
growth rale and yields), were obtained by Le Pennec and Diss-
Mengus (1985, 1987), possibly because of the diet provided, which
included diatoms, and to better conditioning conditions.
It appears that antibiotics are necessary for successful cultiva-
tion of pectinid larvae but not all give good results. Chloramphen-
icol appears to give the most consistent results. Uriarte et al.
(2001) reported higher growth and survival rates in Argopecten
purpunitiis using chloramphenicol at doses of 2 and 8 mg L" than
without the antibiotic. Mendes et al. (2001 ) obtained survival rates
of 20-25% in cultures of Nodipecten nodosus using clorampheni-
col. in contrast with almost total mortality on using florphenicol.
Ruiz (1996) reported high mortality in Pecten maximiis larvae
cultured with erythronnycin and high rates of survival with tetra-
cycline and triniethoprim plus sulphamethoxazole. Gonzalez and
Romi'in (1983) reported no yield of Pecten maximus larvae cul-
tured without antibiotics, in contrast to cultures in which chloram-
phenical was used at a concentration of 2,5 mg L"'. Samain et al.
(1992) found much higher survival and growth rates einploying
antibiotics and Torkildsen et al. (2000) obtained larval yields of
30% when chloramphenicol was added to the cultures.
The percentage of settlement was variable in the different cul-
tures [30% (trial I), between 19 and 58% (trial 2). and 20% (trial
3); approximately 10% in the cultures conducted with different
antibiotics). This variability may have been due to intrinsic factors,
but there were also variations within the same culture batches,
depending on the quality of the substrates provided (extrinsic fac-
tors). It is clear that C. varia prefers to settle on the tank walls than
on nylon monofilament. O'Connor and Heasman (1994) found that
Clilamys asperrima also preferred the tank bottom and walls to the
collectors provided for settlement. C. varia showed a preference
for the more sheltered, poorly lit areas of the collectors (Rodhouse
& Bumell 1979). However, in experiment 3 of the present study,
we found a very low settlement rate on the scallop shells, despite
the fact that they were hung with the concave part of the shells
facing downwards, an arrangement which should have provided
the most sheltered conditions in the tank.
Although improvements in conditioning (quality of gametes),
larval diet and the substrate and settlement conditions must be
made, hatchery culture of C. varia larvae is possible, and com-
mercially viable numbers of spat can be obtained, which would
allow development of an industry dedicated to the production of
this species.
ACKNOWLEDGMENTS
This work was financed by FEDER, project IFD 1997-0201-
C()3-()l. The autliors thank .luan Feniandez-Feijoo and Carmen
Vazquez.
Hatchkry Culture of Black Scallop
99
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Journal of Shellfish Research. Vol. 22. No. I. I()1-I(W, 2()(1.V
EFFECT OF DEPLOYMENT DATE AND ENVIRONMENTAL CONDITIONS ON GROWTH
RATE AND RETRIEVAL OF HATCHERY-REARED SEA SCALLOPS, PLACOPECTEN
MAGELLANICUS (GMELIN, 1791), AT A SEA-BASED NURSERY
LORELEI A. GRECIAN,' G. JAY PARSONS,'* PATRICK DABINETT,^ AND
CYR COUTURIER'
'Fisheries and Mciriiw Inslittite. Memorial University of Newfoundland. P.O. Bo.x 492U. St. John's,
Newfoundland, Canada AIC 5R3 and 'Department of Biology. Memorial University of Newfoundland,
St. John's. Newfoundland. Canada AIC 5S7
ABSTRACT The effect of date of deployment on jti'owth ;ind subsequent retrieval of hatchery-reared scallop spat from a land-based
hatchery to a sea-based nursery was studied to provide information for management of juvenile-size scallops, ranging from 1.4-7.0 mm
ui shell height. The objective of this study was to determine the optimal time period for spat deployment to a sea-based nursery to yield
commercially acceptable growth rates and retrieval (scallops reinaining after mortality and loss through nets). Spat of the same size
class and stocking density were deployed over five consecutive 16-23 day intervals beginning in August 19^7. Environmental factors
were monitored weekly. Scallops were sampled after each deployment period for determination of shell height and retrieval. Scallops
were then re-deployed and sampled before (November! and after (June! the winter season. Results demonstrated that there were
significant differences in scallop growth and retrieval among the t~ive consecutive deployments. Only scallops that had been deployed
in August were greater than 7 mm by November and could be sorted and transferred to larger mesh equipment for ongrowing prior
to winter. The findings of this study demonstrated that early deployment (August! to sea-based nursery yielded high growth rates and
retrieval. Deployment later than eariy September required over-wintering in nursery culture before transfer to ongrowing. Significant
correlations were found between both growth rates and retrieval and some of the environmental parameters (e.g., temperature,
chlorophyll-a, particulate organic matter!. Acclimation to the new farm conditions inay be necessary for nursery-sized .scallops to adjust
physiologically without a major lag in growth following transfer from the hatchery to the sea.
KEY WORDS: growth, nursery culture, Phuupeelen mai;ellanieus. scallop spat, sea star
INTRODUCTION
The aim of a nursery stage in bivalve aquaculture is to foster
the development of young postmetamorphic settled animals to an
optimal size for ongrowing and handling. For scallops, the nursery
stage starts with the transitional period between a planktonic larval
phase in a well-maintained hatchery setting and a benthic postlar-
val phase where the settled spat are deployed to a sea-based nurs-
ery or to a semicontrolled land-based growout environment. Sea-
based nursery culture can be improved by determining the varia-
tion of environmental factors at the nursery site and by
manipulating the liming of the deployment of spat to nursery cul-
ture to coincide with optimal conditions.
Determining the timing of deployment at the sea-based nui'sery
is necessary to optimize growth rates of hatchery-reared Pati-
nopecleii yessoensis (Bourne & Hodgson 1991 1. Spat deployed
during optimal food density and temperatures have higher growth
rates and survival.
The window of opportunity of deployment to the sea-based
nursery can be assessed by determining growth rates and retrieval
as functions of measurable natural factors, such as water quality,
food availability, and the presence of potential predators over time.
When adequate nursery conditions are provided, growth rates and
survival are maximal, and the time scallops spend in the nursery
stage exposed to other risk factors decreases.
Growth rates of scallops vary seasonally as a result of tluctua-
tions in food supply and temperature (Kirby-Smith & Barber 1974,
Vahl 1980, Grecian et al. 2000). Growth rates of cultured P. ma-
gellanicus are highest in the summer and lowest in the winter
•■^Corresponding author. Tel: 709-778-0331; Fax: 709-778-053.'^; E-mail:
Jay.Parsons@mi.mun.ca
(Dadswell & Parsons 1991. 1992, Cote et al. 1993. Kleinman et al.
1996. Parsons et al. 2002) and show no increase during the autumn
bloom compared with summer (Emerson et al. 1994). Sea scallops
in some areas of Atlantic Canada are able to naturally produce two
cohorts annually of which the summer (June to July) cohort grows
faster than the autumn (September to October) cohort over the
entire culture period (Dadswell & Parsons 1992). Dadswell and
Parsons (1992) proposed that the higher growth rates of the first
cohort were caused by the initial exposure of spat to the summer
food conditions in the water column and a longer, more favorable
period of warmer water. Thus, in bi\al\e hatcheries and nurseries,
the early production of scallop spat is important for deployment to
nursery culture early in the summer, as is the practice for oysters.
This may result in the growth of scallop spat to a size of 7 mm or
greater by the autumn, at which time spat would be large enough
to transfer to intermediate culture gear as well as for sale to com-
mercial growers. This growing period is much shorter than waiting
until the following summer, which is the current protocol in the sea
scallop industry (Dadswell & Parsons 1991, Couturier et al. 1995).
Salinity, temperature, and predation impact survival of scal-
lops. Salinity concentrations below 13 psu and 18 psu cause mass
mortality in scallops in short-term and long-term exposures, re-
spectively (Bergman et al. 1996, Frenette & Parsons 2001). As
well, sea star predation on scallops can be significant in wild or
bottom seeded scallops (Dickie & Medcof 1963, Scheibling et al.
1991, Barbeau & Scheibling 1994a). Sea star predation on scallops
is limited in suspended nursery culture gear, unless the nursery
gear is deployed prior to the settlement and growth of sea stars
(Dadswell & Parsons 1992, Parsons 1994). Survival of post larval
scallops, Pecten ma.ximus. transferred from hatchery to nursery
was dependent on the immersion time during transfer, temperature
differential and spat acclimation to the thermal regimen of the
101
102
Grecian et al.
sea-based nursery (Christophersen 2000. Christophersen &
Magnesen 2001).
Timing of deployment of nursery-sized spat at the sea-based
nursery is critical for optimizing growth rates and survival. The
objective of this study was to determine the window of opportunity
for deployment of hatchery-reared sea scallops at a sea-based nurs-
ery that enhances growth rates and retrieval and provides avail-
ability of spat for intermediate grow-out. Based on previous re-
search on sea scallops, the hypotheses for this study are: ( 1 ) growth
will be highest in scallops deployed earliest in the summer (Au-
gust) when temperature and food availability are highest and (2)
retrieval of scallops will decline with the onset of sea star settle-
ment.
MATERIALS AND METHODS
Study Site
Scallops were deployed on a scallop farm. Shell Fresh Farms
Ltd.. based in Poole's Cove. Newfoundland. Canada. The inain
study site was located in North Bay, head of Fortune Bay. NL at
the Ladder Garden lease (47°42'N, 55°26'W).
Experimental Design and Sampling Protocol
This experiment was designed to determnie the optimal period
for the deployment of nursery-size, post larval scallops at a sea-
based nursery. Scallops were deployed over consecutive treatment
intervals from the time they were first available from the hatchery
and were large enough to be handled Ol .4 mm shell height) until
no new cohorts of spat were available in the autumn. The spat were
reared at 15°C from several spawnings undertaken at the Belleo-
ram Sea Scallop Hatchery. Belleoram, Newfoundland (47^32 'N.
55°25'W). Spat were sorted by screening and those between 1.4
and 2.0 mm in shell height were used in the study. A sample of
spat was obtained for initial shell height measurements {n = 30)
for each deployment.
Scallops were counted and deployed on five occasions at 500
spat/collector in 1.2-mm-mesh collector bags on August 4, August
22. September 7. September 26. and October 19. 1997. Two col-
lector bags, each filled with 1 m of NetronT*' (34 g). were held in
individual plastic bread trays (69 cm x .'i7 cm x 15 cm) at a 5 m
depth (Grecian et al. 2000). The number of replicate bags varied
from two to four depending on scallop spat availability. The initial
"short-term" interval duration between successive deployment and
retrieval dates ranged from 16 to 23 days and depended on site
accessibility. Each short-term deployment interval ended when the
next set of collector bags was deployed and the final short-term
deployment interval ended on November 8, 1997.
Scallop retrieval (defined as number remaining after mortality
and any potential loss through the mesh of the nets) was assessed
by counting scallops remaining at the end of each interval and
scallops were measured for shell height (/; = 30). All scallop
treatments were then redeployed and again counted and measured
for shell height before and after the winter season on November 8.
1997 and June 24, 1998, respectively. During the experiment, all
scallop treatments were handled in a similar manner.
Water samples were pumped from a 5-m depth for phytoplank-
ton identification, density and determination of total particulate
matter (TPM), particulate inorganic matter (PIM). particulate or-
ganic matter (POM), and chlorophyll-iv concentration. Tempera-
ture and salinity were measured through the water column to a
depth of 10-m using a YSI Model No. 30 S-C-T meter. Sea star
settlement was also determined (see below). Each parameter was
sampled approximately weekly during the short-term intervals
(August to November).
Immediately after water samples were collected, the phy-
toplankton samples were fixed with Lugol's Iodine and 1% form-
aldehyde. These samples then sat undisturbed for at least two
weeks to allow the seston particles to settle. The top 909^ of water
was siphoned off and its volume was measured. The remaining
volume, which contained all settled algal particles, was also mea-
sured. This concentrated volume was mixed thoroughly and 10 mL
were transferred to a 10-mL Utermohl settling chamber for over-
night settlement. The sample was analyzed visually for total num-
ber of cells and species composition using a Zeiss Axiovert 35
microscope under phase contrast at 400x magnification.
The total plankton assemblage was categorized into 8 major
groups (McKenzie. 1997). Seven of these were on the basis of size
while the final group comprised "unidentified species." The size
categories included microzooplankton including tintinnids and
ciliates (>20 iJim in diameter), autotrophic and heterotrophic di-
notlagellates (12 to 60 |jLm). prymnesiophytes comprising small
(2 to 12 (xm in diameter) spherical nanofiagellates. auto-nano-
flagellates comprising spherical flagellates from 2 to 20 |j.m in
diameter, cryptophytes comprising small (8 to 18 |j.in in length)
tear-drop shaped biflagellates. centric diatoms (12 to 30 (jim in
diameter, connected in long chains), and pelagic pennate diatoms
(30 (jLin in length, single cells). Phytoplankton were identified
according to Rott (1981).
For TPM and chlorophyll-a samples, 15 L of seawater were
pimiped from a depth of 5 m and pre-screened at 300 p.m into
separate 20-L buckets and taken to the hatchery. Water samples (4
L) for TPM were filtered onto Whatman GF/C 45-mm diameter
glass microfiber filters, which had been previously combusted in a
muffle furnace at 500°C for 4 h to remove organic matter and were
then weighed. The filters were then stored frozen at -20'C and
ultimately oven-dried at 80°C for 24 h, weighed for TPM, trans-
ferred to a muffle furnace for 4 h at 500°C, and reweighed to
determine PIM. From these weights, ash-free dry weight or POM
was calculated according to the formula TPM = POM + PIM.
An additional 4 L of seawater was filtered onto Whatman GF/C
filters for chlorophyll-n and pheopigment determination. Filters
were frozen (-20°C) for later processing according to the fluoro-
metric methods of Strickland and Parsons ( 1968) and Parrish et al.
(1995).
Sea star settlement was monitored weekly from July 15 to
November 8, 1997. by deploying strings of eight empty pearl nets
(34-cm X 34-cm square base pyramidal-shaped nets. 6-mm mesh)
weekly at the farm with retrieval after approximately two weeks.
Individual pearl nets were washed and all material greater than 250
(xm was collected on a mesh screen and preserved in 40% metha-
nol. Samples were analyzed using a dissecting microscope for
determination of numbers of sea stars present.
Data Analysis
Data were analyzed using the SPSS statistical package (Version
8.0). All percent data were arcsine-square-root transformed before
statistical analysis (Sokal & Rohlf. 1995). Differences in growth
rates and retrieval were analyzed using an analysis of variance
(ANOVA) and the post hoc Tukey's b test was used to test for
differences among treatments. Equality of means was analyzed
Effect of Deployment Time on Sea Scallops
103
using an Independent sample Mest. Pearson correlation analyses
were also performed on growth and retrieval data with the envi-
ronmental parameters. The le\el of sisznificance was set at a =
0.05.
RESULTS
Grow til Rales
Initial shell height among the replicates was not significantly
different for all dates {P > 0.01) except September 7 (One-way
ANOVA;F = 9.735. df= 2, 87, P< 0.001). This was because the
scallops in one of the replicates were from a slow-growing batch
of larvae and they were not randomly assigned among the repli-
cates for that date, hence this replicate was not used for further
analysis or in figures. The initial mean size ranged from 1.41 to
1.62 mm shell height (Fig. I).
The mean shell heights of .spat at the end of each short-temi
deployment interval were significantly different from the initial
mean shell heights (Hests. P < 0.05. Fig. I). As well, mean shell
heights at the end of each short-term interval were significantly
different among the different deployment dates and decreased
from 3.54 mm to 1.51 mm shell height (one-way ANOVA; F =
556.621, df = 4. 445. P < 0.001 ).
Growth rates declined over the short-term intervals (Fig. 2).
Significant differences were found among growth rates for the
different intervals (one-way ANOVA: F = 95.162; df = 4. 1 1. P
< 0.001). Highest growth rates occurred during the first deploy-
ment interval at I 18 |jLm d"' (SE ± 1 .3). whereas the lowest growth
rates occurred during the last interval at 3.3 p.m d"' (SE ± 0.7).
The mean growth rate of all spat deployed between August 4 to
November 8. 1997 was 43.2 pim d"' (SE ± 0.8).
Growth rates of scallops from the earliest deployment were
higher in the autumn and over winter than those from the subse-
quent deployments (Fig. 3). For scallops deployed on August 4 and
22. growth rates were high until November 8. For the same scal-
lops, growth rates from November to June 24. 1998. declined to a
level similar to that of scallops deployed from September 7. 1997.
Scallops deployed on September 26 and October 19. had lower
overall giowth rates to November [1 1.4 |xm d"' (SE± 1.1) and 3.3
p.m d"' (SE ± 0.7). respectively] and to June |2I.5 [xm d~' (SE ±
1.4) and 7.2 fjim d~' (SE ± 1.3). respectively].
04-Aug
22-Aug
07-Sep
26-Sep
Initial deployment date
Figure 1. Mean shell height of scallops deployed over five consecutive
2-Heek intervals in 1997 and on November 8, 1997, and June 24. 1998,
at Shell Fresh Farms Ltd., Poole's Cove, NL. The initial date of an
interval was the final date of the previous short-term interval, t'oni-
mon letter denotes no significant difference among mean shell heights
for each sample period iTukey's b test). Vertical bars are ±SE.
I5U
120 -
90 ■
60 ■
30 ■
0 ■
'd -■■■■'-1.
^^* Short-term Growth i
""■•■"' Short-term Retneval 1
-3
5.
1
"■•-.T i
2
M
li
b
r *
1
n
a
100
90
80
70 '
60 S
50 1
40 i
30 0
20
10
0
04-Aug
:-Aus; 07-Scp 2b-Sep 19-Ocl
Initial deployment date
Figure 2. .Mean growth rates and retrieval of scallops over consecutive
deployment intervals at Shell Fresh Farms Ltd., Poole's Cove, NL. The
initial date of an interval is the final date of the previous interval.
Common letter denotes no significant difference in growth rates or
retrieval among intervals (Tukey's b test). \ ertical bars are ±SE.
Retrieval
Retrieval of spat at the end of each deployment interval (num-
ber remaining after mortality and loss) declined over time (Fig. 2)
and was significantly different among the different short-term de-
ployment intervals (one-way ANOVA; f = 47.129, df = 4. I I, P
< 0.001 ). Highest retrieval was obtained from spat deployed during
the first interval (97%), whereas lowest retrieval was for spat
deployed on September 26 (53%). Retrieval of spat from their
initial deployment to November 8 was not significantly different
than their retrieval after the short-term intervals (Paired t-test; t =
0.013. df = \4.P = 0.990; Fig. 3).
En vironmeiital Characteristics
Water temperature declined over the deployment periods (Au-
gust-November. Fig. 4A). Mean temperatures for the five con-
secutive deployment intervals were 14.7, 13.6. 11.3. 11.2. and
7.9°C. respectively. Spat were not acclimated from 15 C in the
hatchery to ambient seawater temperatures before sea-based de-
ployment. Salinity increased over the study period (Fig. 4A). Mean
salinity was 28.3 psu whereas the range was from 26.5 to 31.5 psu.
Chlorophyll-fl concentrations (one-way ANOVA; F = 0.544.
df = 14. 24. P = 0.881). pheopigment concentrations (one-way
-^ .Autumn
'Spring
" November Retneval
04-Aug 22-Aug 07.Scp 26.Sep 19-Ocl
Initial deployment date
Figure 3. Mean growth rates and retrieval of scallops deployed at a
sea-based nursery at Shell Fresh Farms Ltd.. Poole's Cove. NL, on five
dates in 1997 and sampled on November 8. 1997, and June 24, 1998.
Common letter denotes no significant difference in growth rates or
retrieval among intervals (Tukey's b test). Vertical bars are ±SE.
104
Grecian et al.
u
3
«
u
u
a.
20
16
12
4 ■
■»- *-
Jul
22- 5- 19- 2- 16- 30- 14- 28-
Jul Aug Aug Sep Sep Sep Oct Oct
35
30
""i
B.
a
71)
,>i
IS
c
lU
"3
5
C/2
Nov
8- 22- 5- 19- 2- 16- 30- 14- 28- 11-
Jul Jul Aug Aug Sep Sep Sep Oct Oct Nov
=5.
C
O
o
c
o
U
20
16
12
8
4
0
8-
Jui
• Chlorophyll
" Phaeopignients
A.
A-A A-
Jul
5-
Aug
19-
Auti
2- 16-
Sep Sep
Date
30-
Sep
14-
Oct
Oct
11-
Nov
Kisure 4. Water quality at Ladder Garden site. Shell Fresli Farms Ltd., Poole's Cove, NL, from July IS to November 8, 1997. A) Temperature
and salinity (±SE: ;i = 3), Bl seston, C) chlorophyll and pheopigments at 5 m. (TPM, total particulate matter; POM, particulate organic matter).
ANOVA; F = 0.500. df = 14. 24. P = 0.910), and POM (one-
way ANOVA; F = 0.71.5. df = 14. 21. P = 0.737) were not
significantly different o\er the duration of the study.
TPM remained constant al Ladder Garden (Fig. 4B) with
weekly mean TPM being 5.6 mg L '. POM was also constant at
Ladder Garden with a mean of 1.9 mg L '. Chlorophyll-o and
pheopigments averaged 2.4 and 10.1 mg L"', respectively (Fig. 4C).
There was a significant difference in total phytoplankton den-
sity among the weekly samples (one-way ANOVA; F = 7.084. df
= 13. 28. P < 0.001; Fig. 5). The total phytoplankton density
peaked around the middle of August, followed by a decline. The
decline was also evident when the mean total phytoplankton den-
sity was calculated for each interval (Fig. 6). The autotrophic
nanoflagellates, pelagic pennate diatoms, and dinotlagellates were
the numerically dominant groups present (Fig. 7A and B). The
species that contributed to the peak abundance were Naviciila sp..
Cliluinydoinoiuis sp.. Ochronxnias sp.. Microinonas sp. (Fig. 8A
and B). Percent abundance of phytoplankton size groups indicated
that species <5 p,m had the greatest contribution to phytoplankton
biovolume (Fig. 9).
Sea star settlement at the Ladder Garden site peaked between
September 19 and October 23 (Fig. 10). There were significant
differences in sea star settlement over the different sampling dates
(ANOVA; F = 99.674. df = 13. 336. P < 0.001). Maxiinum
i
S-Jiil 2:-Jul 5-Aug 19-Aiig 2-Sep 16-Sep 30-Sep 14-Oct 28-Ocl 1 1-Nov
Date
Figure 5. Total phytoplankton density at Ladder Garden site of Shell
Fresh Farm, Poole's Cove, NL, from ,luly 15 to November 8, 1997.
Effect of Deployment Time on Sea Scaelops
105
Deploynicnt inlenai
Fij"ure 6. Mtun density of total phvtoplankton over five intervals of
scallo|) deplovMunt on a sea-based nursery at Shell Fresh Farm,
Poole's Cove, Nl.. Intervals hejjan on Ausiust 4 and ended on Novem-
ber S. IW7. \ertieal bars are ±SE.
setllenieiit was 311) sea stars per collector per day ami mean sea
star settlenienl was 79 sea stars per collector per day.
Most environmental factors were highly correlated with growth
rates and retrieval (Tables 1 and 2). TPM and dinoflagellates were
not correlated with growth rates and TPM and PIM were not
correlated with retrievals.
DISCUSSION
Effects of Deploymiiil Dale on Growth Rates anil Retrieval
The date of transfer or deployment of scallop spat from hatch-
ery to nttrsery was a useful predictor of growth and retrieval. The
higher growth rates and retrievals in the earlier deployments were
related to several parameters in this study, where ambient tem-
5-
19-
T.
16-
30-
14-
28-
11-
Aug
Aug
Sep
Sep
Sep
Oct
Oct
Nov
A
2500 ■
2000 ■
1500 ■
1000 ■
500 ■
ri ■
/ .♦■■♦■
--■•■-
" Pelagic pennale diatoms
~ Dinoflagellates
~ Unidentified phytoplankton
~ Autotrophic nanoflagellates
U
— e—
Q
V
^fe
*f»==«^«-,.>-*.T^,
8- 22- 5- ly- 2- 16- 30- 14- 2S- 11-
Jul Jul Aug Aug Sep Sep Sep Oct Ocl Nov
J
80
60
4U 1
20
c
D
0
• Microzooplankton
* Prymnesiophytes
- - o - - Centric diatoms
/\
.'^ A
/^^^:^:i\
8-
Jul
22- 5- 19- 2- 16- 30- 14- 2S- 11-
.lul Aug Aug Sep Sep Sep Oct Oct Nov
Date
Figure 7. Mean density of "(.V)" four dominant and "(B)" three less
dominant groups of major plankton at Shell Fresh Farms Ltd., Poole's
Cove, NL, from July 15 to November 8, 1997.
■ Rluzosolenia sp.
- Coccolithophore sp.
Prorocentruin sp.
Choanoflagellate sp.
Sirohilidnim innninuin
Dinophysis non'egica
-J
C
Q
8- 22- 5- 19- 2- 16- 30- 14- 28- 11-
Jul .lul Aug Aug Sep Sep Sep Oct Oct Nov
Date
Figure 8. Mean den.sity of "(A)" dominant and "(B)" less dominant
plankton species that showed a declining trend over intervals of scallop
deployment at a sea-based nursery at Shell Fresh Farms Ltd., Poole's
Cove, NL, from .luly 15 to November 8. 1997.
perature and food availability and quality (species composition,
organic content and lipid characteristics inferred from literature
reports) were higher initially, then declined after early August.
Predator (sea star) abundance peaked near the second deployment
date before declining. Spat growth and retrieval from the initial
deployment demonstrated that there is an optiinum time or window
of opportunity, which could be used to maximize nursery growth.
After this period, scallops face increasing adversity in terms of
declining temperature and food quantity and quality, and increas-
ing predation and temperature shock (the difference between
hatchery and ambient temperatures). In a similar study in Southern
Norway, Pecten inaximus spat transferred from hatchery to sea-
based nursery from March to August showed increased growth and
survival during the summer when water temperatures were >10°C
and when temperature differences between the hatchery and nurs-
ery were minimal (Christophersen & Magnesen, 2001).
Temperature and food availability declined from August to
November while sea star settlement began in mid-September.
Variations in temperature and food availability were similar to
those found in other areas of Atlantic Canada, including Concep-
tion Bay, NL, and Bedford Basin, NS (Mayzaud et al. 1989, Na-
varro & Thompson 1995). In earlier studies of sea scallop aqua-
culture, temperature and food availability were the main predictors
of growth (Parsons & Dadswell 1992, Cote et al. 1993. Emerson et
al. 1994. Kleinman et ul. 1996). Likewise, sea stars are a signifi-
cant predator of scallops (Barbeau & Scheibling 1994a. b).
Changes in these parameters may best explain the variation in
growth and retrieval of the scallops over the different deployment
intervals.
A negative correlation of salinity with growth and retrieval of
scallops in the deployinent study was probably a coincidence as
106
Grecian et al.
04-Aug 22-Aug 07-Sep 26-Sep
Initial deployment date
19-Oct
H <5 Hill
■ <10nm
D <20 urn I
D<50nm
O<100nm
■ <2()0 urn
D >200 urn
B Unidentified
Figure 9. Particle size frequency distribution of planlvton at Ladder Garden, Sliell Fresh Farms Ltd., Poole's Cove, NL, over five consecutive
deployment intervals of scallops at a sea-based nursery.
the salinity tolerance range for wild juvenile sea scallops is >25
psu (Frenette & Parsons 2001), which is lower than the salinity
during the present study. The increase in salinity over the study
period reflects the decreased runoff and the increased upwelling
that occurs in the autumn in this area.
Decreases in metabolic processes due to declining temperature
may explain why reduced growth rates were observed in scallops
deployed on different dates in this study as has been found for
Pecteii fiimatiis (Cropp & Hortle 1992). Respiration rates in sea
scallops decrease with declining temperature (Shumway et al.
1988), but clearance rates are coirelated with ambient temperature
in sea scallops (MacDonald & Thompson 1986) as well as in the
eastern oyster, Crassostrea virginica and the bay scallop. Ar-
gopecleii irradians (Rheault & Rice 1996). In the present context,
reduced clearance rates would be expected to decrease food intake
and result in reduced growth.
Declining retrieval over time was correlated with deployment
temperature. This however, does not indicate that scallops died as
a direct result of decreasing temperature. Scallops are able to live
within a temperature range of-2' C to 22"C (Dickie 1958). Hence
their survival should not have been influenced by decreasing tem-
peratures per se. Christophersen and Magnesen (2001 ) found that
when Pecten maximus spat were deployed at water temperatures
>10°C, spat had up to a 4-fold increase in survival compared with
scallops deployed at temperatures <10°C. The sea scallops were
likely influenced more by the temperature difference from the
hatchery to the sea-based nursery environment than their physi-
ologic condition or predation by sea stars.
Effects of Food Variation on Growth Rates and Retrieval
Scallops deployed when Prnrocciilninu DiiuipltYsis and Nav-
icitla spp. densities were elevated exhibited higher growth rates
than scallops deployed when densities of these phyloplankton spe-
cies had declined. All these mircoalgae have been found in gut
analyses of adult scallops (Shumway et al. 1987). We found all
three species in high abundance and the first two species are con-
sidered to add greatly to the energy uptake of scallops (Shumway
et al. 1987). Cryptophyte densities also peaked during August
when growth rates were highest. Cryptophytes are rich in the fatty
acids. 22:6w3 and 20:5w3 (Volkman et al. 1989. Viso & Marty
1993) and are important for a good diet and membrane fluidity in
bivalves (Enright et al. 1986, Napolitano et al. 1992). Crypto-
phytes are a preferred alga in mixed diets and are related to growth
350
8-Jul 22-Jul 5-Aug 19-Aug 2-Sep 16-Sep 30-Sep 14-Oct 2S-0ct 11-Nov
Date
Figure 10, Mean sea star settlement al Ladder Garden lease of Shell Fresh Farms Ltd., Poole's Cove, NL, from .July 15 to November 8. 1997
(;i = 8). Vertical bars are ±.SH
Effect of Deployment Time on Sea Scallops
107
TABLE 1.
Pearson's ciirrelalion coefficients of shorl-lcrni };ro"th rates and retrieval of nursery-size scallops «ilh mean water quality parameters at a
sea-based nursery at Shell Fresh Farms ltd.. I'oolc's Cove, NL. from August 4 to November 8, 1997.
%
Sea Star
Temperature
Salinity
Chlorophyll-a
Phaeopigments
TPM
PIM
POM
POM
Settlement
Growth rate
/■ value
0.840
-0.826
0.901
0.940
-0.043
-0.573
0.700
0.773
-0.796
Signifieancc (two-tailed)
<0.001
<0.0()1
0.001
<0.001
0.4.19
0.013
0.002
0.001
<0.001
Retrieval
/• value
0.828
-0.698
0.849
0.870
0.2.1.1
-0.358
0.714
0.644
-0.890
Significance (two-tailed)
<0.001
0.002
<().()01
<0.001
0.201
0.095
0.001
0.005
<0.0()1
15 for all parameters.
in sea scallops (Shumway et al. 1985. Pairish et al. 199.5). It is
expected that scalkips exposed to a higher quality diet allowitig
adaptation to declining conditions would peifomi better than scal-
lops exposed to a lower quality diet (see Shunnvay et al. 1997).
MacDonald and Thotnpson ( 1985) found that shell growth was
higher under favorable conditions of food and tetiiperature, and
that this was site specific. Location of sea-based nursery sites
should consider food quantity and quality. However, because there
have been so few growth studies of juvenile bivalves with respect
to natural phytoplankton composition, the actual quantity and qual-
ity of food required is not l<nown (Newell et al. 1989. Parrish et al.
1995, Grant 1996). Phytoplankton is a major component of the diet
of adults (Shumway et al. 1987, Cranford & Grant 1990); however,
further research is necessary to determine the actual quantity and
quality that allow juvenile scallops to perform optimally (Shum-
way et al. 1997).
Predation Effects on Retrieval
There was an increased negative correlation between retrieval
of scallop spat and sea star settleinent during the short-term inter-
vals. Increasing sea star settlement coupled with declining sea
scallop retrieval was expected (Dadswell & Parsons 1991. 1992.
Barbeau & Scheibling 1994a. Parsons 1994). Successful predation
may be due to the similar size of the settling sea stars and scallop
spat as well as debilitation caused by significant temperature
changes between hatchery and nursery environments (Dickie 1958.
Barbeau & Scheibling 1994a). In the present study temperature
difference between hatchery and nursery progressively increased
with deployment date from 0 to 7.1°C. Although sea star predation
may be reduced with decreasing temperature (Barbeau & Scheib-
ling 1994a). the temperature shock may have rendered spat more
susceptible to sea star predation.
Dickie ( 1958) observed a lack of mobility of scallops for about
a month when they were exposed to drops of 4-7°C in ambient
temperature, which he speculated may be detrimental if predators
are unaffected. Temperature debilitation may have coincided with
highest mortality of scallops in the present deployment study.
which was during the period of peak sea star settlement on the
culture gear.
Importance of Acclimation on Growth Rales and Retrieval
The effect of increasing differences between hatchery and at
sea nursery conditions on the performance of scallops raises con-
cerns over handling protocols. Although acclimation to different
conditions was not specifically examined in this study, a few gen-
eral observations can be made regarding its importance. Sea-based
nursery conditions were within the natural environmental ranges
for scallops; however, scallops performed increasingly poorer with
each consecutive deployment interval. Other studies have found
that sudden changes in the environmental or rearing conditions can
decrease survival and growth (Thompson 1984. Cranford & Grant
1990. Cote et al. 1993. Christophei'sen 2000. Christophersen &
Magnesen 2001 ). Acclimation of Fccleii inaximiis to lower ambi-
ent water temperature did confer a small increase in survival in
TABLE 2.
Pearson's correlation coefficients of short-term growth rates of nursery-size scallops \\ith mean phytoplankton densities at a sea-based
nursery at Shell Fresh Farms Ltd., Poole's Cove, NL, from August 4 to November 8, 1997.
.Autotrophic Centric Rhizosolenia
Total Microzooplankton Choanoflagellates Dinotlagellates Pryninesiophytes Crjptophytes nanoflaj^ellales Diatoms t'nidentifled sp.
r value
0.994
(LS.S8
0.772
-O.O.'iS
0.895
0.789
0.991
-0.635
o.yyi
0.891
Significance
(two-tailed)
<().(XII
0.0.1 1
0.001
0.837
<0.001
<0.00l
<0.(K)l
0.011
<o.ooi
<0.(XJI
Navictila Chlamydomtmas Ochromoiias Micromoilas Prnrocenlruin Choanodajiellatc Strnmbilium Pelagic Pennatf
sp. sp. sp. sp. Coccollthophore sp. sp. minimum Diatoms
/■ value 0.726
Significance
(two-tailed) 0.(K)2
0.987
<0.001
0.687
0.005
o.y 1 1
<0,00l
0.980
<0.00l
0.895
<0.00l
0.944
<0.001
0.772
0.(X)l
0.974
<0.(X)1
" = 15 lor all parameters.
108
Grecian et al.
juvenile scallops (Christophersen & Magnesen 2001 ). Mylilus cJu-
lis requires 14 days to acclimate oxygen consumption, filtration
rates and assimilation efficiency (Widdows & Bayne 1971). Hall
( 1999) observed that in sea scallops 15-21 days were required for
membrane fluidity to adjust to a temperature decrease from 13 to
5°C. The temperature and diet differentials between hatchery and
nursery may have been too great or too abrupt for scallops to
maintain optimal peiformance without the opportunity to accli-
mate, pai'ticuiarly later in the deployment season.
Implications for Halcheiy, \'iirsery and Grow out
The findings of this study provide growers with a protocol for
working with animals in a dynamic environment, under optimal
and suboptimal conditions. Hatchoy tnanagers may be able to use
our results to improve decisions on when to deploy sea scallops
and nursery managers may now have the ability to optimize
growth and retrieval of sea .scallops reared in a sea-based nursery
system and to better plan for transfer to grow out when scallop spat
reach the desired target size,
CONCLUSIONS
Growth rates and retrieval of nursery-sized scallops were in-
fluenced by time of deployment at a sea-based nursery during a
period that spanned early summer to late autumn. Highest growth
rates and retrieval of nursery-sized scallops were observed during
August and early September when the nursery site water column
was characterized by high food densities, high temperature and
low sea star settlement. However, scallops deployed in late Sep-
tember and October had low retrieval as well as low growth rates
until the following spring or later.
The ability of nursery-sized scallops to grow and survive may
be related to the differences between hatchery and sea-based nurs-
ery environments in terms of food quality and temperature differ-
entials. There is a need to detennine the nutritional requirements of
nursery-sized scallops and to practice acclimation protocols.
ACKNOWLEDGMENTS
This research was supported by the Canadian Centre for Fish-
eries Innovation and the Canada/Newfoundland Economic Re-
newal Agreement - Aquaculture Componoit. Special thanks to
staff and management at Belleoram Sea Scallop Hatchery and
Shell Fresh Farms Ltd., where research was conducted. The au-
thors thank Dr, Cynthia McKenzie from the Ocean Sciences Cen-
tre, Memorial University for assistance in plankton identification
and enumeration, Elizabeth Hatfield, Ocean Sciences Centre for
assistance in chlorophyll analysis and Guilherme Rupp and Dr.
Michael Dadswell for reviewing the manuscript.
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.loiiiiNil <-/ Slwllfish Rcscanh. Vol. 22, No. I. 111-117, 2003.
DEVELOPMENT, EVALUATION, AND APPLICATION OF A MITOCHONDRIAL DNA
GENETIC TAG FOR THE BAY SCALLOP, ARGOPECTEN IRRADIANS
SP:IFU SEYOUM,' THERESA M. BERT,'* ami WILBUR.- WILLIAM S. ARNOLD,' AND
CHARLES CRAWFORD'
'F/.s7( ((/((/ Wilillife Conservation Commission. Florida Marine Research Institute. 100 Eighth Avenue SE.
St. Petersburg. Florida 33701-5095: and 'Department of Biologic Sciences. University of North
Carolina-Wilmington. 601 S. College Road. Wilmington. North Carolina 28403
ABSTRACT As a component of an aquaculuirc-based, bay .SLuiiop stock-restoration program in west-central Florida nearshore
waters, we developed a genetic tag for the bay scallop, Argopecien irradians. Using the polymerase chain reaction technique, we
amplified segments of highly purified scallop mitochondrial DNA using 10-base-pair random primers and generated fragments that we
investigated for use as genetic tags. We excised and cloned the amplicons obtained from six individuals to assess them for nucleotide
variability. We chose one, highly polymorphic umplicon of 1049 base pairs and designed a set of sequence-specific polymerase chain
reaction primers for it. We used these primers to sequence portions of the fragment, from both the 5' and 3' ends, respectively,
effectively dividing the fragment into two distinct segments separated by 66 nucleotide base pairs. The two segments contained
sufficient polymorphism such that 729^ (segment 1) and HO'f (segment 2) of the individuals were unique in a sample of 97 wild
individuals; 979f of these individuals were unique when both segments were considered. Nucleotide sequences appeared to be faithfully
transmitted from one parent to its presumed offspring; indications of heterozygosity and heteroplasmy were not observed for any
individual throughout the study. Our analysis of this DNA fragment suggested that it is an mtDNA component, but we were unable
characterize the gene region that it encompasses. To test our genetic tag, we used these two segments to preliminarily assess the
contribution of the stock restoration program to the bay scallop population at a single area targeted for stock restoration. Our analysis
suggests that the stock restoration effort either did not contribute or contributed at a low level to the local population, but our
postrestoration sample sizes may have been too small to detect very small contributions. Our work demonstrates the utility of using
random primers to develop mtDNA genetic tags for species for which little is known about the nucleotide sequence or gene order of
the mtDNA molecule and the potential for application of that tag as a preliminary evaluation tool in a stock restoration or stock
enhancement program.
KEY WORDS: Argopecten iiradicm.s. bay scallop, DNA sequencing, Florida, genetic tag, mitochondrial DNA, stock enhancement,
stock restoration, random primers
INTRODUCTION
Throughout the world, many commercially and lecreationally
valuable species of shellfish and finfish are declining as a result of
overfishing, pollution, habitat degradation, and disease (e.g., Har-
gis 1999, Marelli et al. 1999. Hutchings 2000). Management of
these fisheries through quotas and closures has not always been
effective in preventing further decline or allowing natural recovery
to take place. For this reason, aquaculture-based stock restoration
and enhancement are now accepted methods for restoring depleted
fishery stocks (Tettelbach and Wenczel 1993. Peterson et al. 1995.
Southworth and Mann 1998. Svasand et al. 2000. Ai-nold 2001 ).
The bay scallop, Argopecten irradians (Lamarck. 1819), a spe-
cies valuable to the people of Florida as both a commercial and
recreational resource, was once plentiful in west-Florida nearshore
waters and high-salinity embay ments. By the early 1960s, popu-
lation numbers and abundances had severely declined in many
regions, due in part to dwindling seagrass beds and pollution
during the 1950s (Haddad 1988, Blake et al. 1993, Arnold et al.
1998). Later, concerted efforts by state and federal governments
and environmental groups led to water-quality improvement and
restoration of habitats suitable for scallop propagation (Blake
1998), In the 1990s, the commercial fishery was closed, and area-
specific restrictions or prohibitions on harvesting were imple-
mented for the recreational fishery (Arnold el al. 1998). Despite
these efforts and those of a small-scale bay-scallop stock-
enhancement project ongoing throughout the 1990s (Blake 1998),
*Corresponding author. E-mail: theresa.bert@fwc. state. fi. us
bay scallop population numbers and abundance continued to de-
cline in west-central Florida waters. Therefore, in 1997, a multi-
year, aquaculture-based. stock restoration program was initiated in
west-central Florida nearshore waters to re-establish extirpated bay
scallop populations and enhance depleted populations.
One way to assess the success of a stock restoration endeavor
is to estimate the contribution of the original aquaculture bi'ood-
stock to the local or regional population. For bay scallops, the most
reliable method is a genetic tag, generated from nuclear or mito-
chondrial DNA (mtDNA). A genetic tag can be used to estimate
the success of a stock restoration or enhancement program because
an assessment of the contribution of hatchery-reared or hatchery-
derived individuals to the recipient population can be made (Bert
et al. 2002). The genetic tag must be sufficiently powerful to
discriminate aquaculture-derived individuals from wild individu-
als, or at least the tag should be composed of genotypes of suffi-
cient rarity to allow detection of the stock restoration contribution
through changes in the percentages of these genotypes in the popu-
lation. In either case, the contribution of the restoration effort must
be sufficient to enable detection by sampling.
There are several methods of genetic tagging (Palsboll et al.
1997, Palsb0ll 1999, Bert et al, 2002). but one of the easiest is to
find and use a highly variable portion of mitochondrial DNA
(mtDNA). Nontranscribed regions of the mtDNA molecule serve
as excellent genetic tags (Simon et al. 1994) because they typically
mutate more rapidly than most other DNA segments (Meyer
1993), In addition, if the mode of inheritance is uniparental, track-
ing it in first-generation offspring is straightforward.
In invertebrates, the mtDNA molecule can vary greatly in both
1 II
112
Seyoum et al.
gene order and nucleotide sequence, even among closely related
taxonomic groups (e.g.. Boore & Brown 1994. Boore et al. 1995.
Wilding et al. 19991. Thus, the universal mtDNA primers de\ el-
oped tor vertebrates (Palumbi 1996) are not always successful in
amplifying invertebrate mtDNA segments. Here, we report on the
development of a compound mtDNA genetic tag for the bay scal-
lop using an unidentified bay scallop mtDNA fragment initially
amplified by 10-base-pair (bp) random primers obtained commer-
cially. We evaluate its genetic diversity and applicability through
a preliminary assessment of the contribution of our slock restora-
tion program to the bay scallop population at one location (Ho-
mosassa Bay. Florida; Fig. 1 ). Last, we discuss the general utility
of single-gene genetic tags.
MATERIALS AND METHODS
Dcrelopineiit uf the Mitochiindrial DNA Genetic Tag
To search for an mtDNA fragment that could serve as a genetic
tag, we first obtained highly purified mitochondrial DNA from the
gonad tissues of sexually mature bay scallops from Homosassa,
Florida (;i = 6); mature bay scallops contain both male and female
reproductive tissues. We used a modified homogenization buffer
containing 100 (jlM sucrose and the standard extraction method of
cesium-chloride density gradient ultracentrifugation (Lansman et
al. 1981). The mtDNA band was collected in a 1-mL syringe h\
side puncture with a hypodermic needle and purified by dialysis.
The purified mtDNA yielded a single fragment of approximately
16.000-20.000 bp when run through a 29K ethidium-bromide-
stained. low-EEO. agarose gel (Fisher Biotechnologies. Pittsburgh.
PA). According to the methods described by Williams et al.
(1990), we amplified portions of the highly purified mitochondrial
DNA of these individuals using the twenty 10-bp random primers
supplied in RAPD Kit A (Qiagen Operon Technologies. Inc.,
Alameda. CA). Five microliters of each polymerase chain reaction
(PCR) product was run in a low-EEO agarose gel to view the
amplifications. Multiple bands were obtained for most primers
except for OPA-3 (AGTCAGCCAC) and OPA-18 (AGGTGAC-
Atlantic
Ocean
Gulf of Mexico
Figure L Collection locations for bay scallops {Argopecten irradians)
in Florida to estimate the frequencies of original-bruodstock haplo-
types in the wild prior to the restoration effort (sample sizes are given
in Table IB). HO was the location of the stock restoration evaluation
presented in this report. .Abbreviations: ST = Stcinhatchee; CK =
Cedar Key; HE = Hernando; HO = Homosassa Bay; AN = .Anclote
Estuary; TB = Tampa Bay; SS = Sarasota Bay.
CGT). OPA-3 gave a single band of approximately 1.000 bp and
OPA-18 gave two intense bands of approximately 1.000 and 1.6(X) bp.
The remaining 45 (xL of the OPA-3 and OPA-18 PCR reactions
were run in a gel of 2% low-melting-point agarose (NuSeive,
FMC. Rockland, ME). According to the standard method of Sam-
brook et al. (I9S9). the fragments were excised and cloned in a
plasmid vector (Bluescript PBC KS. Stratagene. La Jolla. CAl that
was initially cleaved with EcoRV and tailed with 2-mM dTTP
(Marchuk et al. 1990). Afterplating the transformed cells, the three
fragments were amplified from two colonies of each of the six
individuals using the T3 and T7 primers (Stratagene. La Jolla.
CA), which annealed to the Bluescript vector on either side of the
insert. The PCR products were electrophoresed in a 1.5%, low-
EEO, agarose gel, excised, and purified using a Strata Prep DNA
Gel Extraction Kit (Stratagene, La Jolla, CA). The purified DNA
was resuspended in 50 (xL of sterile distilled water.
Cycle sequencing was performed from both the 5' and 3' ends
of each fragment using 0.5 (xL of the purified DNA, two |j.l of
Big Dye'^' Terminator Cycle-Sequencing Ready-Reactions with
AmpliTaq FS DNA polymerase (PE Biosy stems, Foster City, CA)
and 0.5 |xL of 3.2-pM solutions of the T3 and T7 primers, in a total
volume of 5 p.L. The reaction product was then ethanol precipi-
tated and resuspended in 20 |j.L of Template Suppression Reagent
(PE Biosystems, Foster City. CA). according to the manufacturer's
instructions.
The resuspended product was analyzed by using an ,^BI
PrismT^' 310 Genetic Analyzer (PE Biosystems. Foster City, CA).
The sequences obtained were aligned and edited using the
AutoAssembler^"^ DNA Sequence Assembly Software (PE Bio-
systems. Foster City. CA); further electropherogram editing was
perfomied using Chromas v. 1.6 (Technelysium Pty. Ltd. Heles-
ville. Queensland. Australia). The two OPA-18 fragments cloned
and sequenced for the six individuals were composed of multiple
sequences, most of which matched very poorly when aligned and
were therefore considered nonhomologous. Some sequences
aligned very well and were thus presumed to be homologous, but
they were invariable in this fragment. However, sequences of the
OPA-3 fragment for the six individuals were homologous and
differed among all six individuals at one or more nucleotides. This
L049-bp fragment was named SCAOPA-3 and was identified as a
possible genetic tag. A representative sequence of the SCAOPA-3
fragment has been deposited in GenBank under accession number
AF261938. Highly specific primers for the fragment were de-
signed: SCA-I, composed of 5'-AGTCAGCCACCCACTAAA-
TTAGATCTCA-3' and SCA-2, 5'-AGTCAGCCACTGGTT-
TATAGTGGAATAGTT-3'. The first 10 bp of these primers con-
stituted the sequence of the 10-bp primer that was initially used to
amplify the SCAOPA-3 fragment.
Using each custom-made primer, we sequenced the
SCAOPA-3 fragment from the two ends toward the center, thereby
effectisely dividing it into two segments. The sequences for the
first portion (termed segment 1) consisted of 471 bp beginning at
position 33 and ending at position 503; the second segment
(termed segment 2) consisted of 450 bp beginning at position 569
and ending at position 1018. These segments did not overlap and
66 bp between these segments were not included.
For the remaining genetic analyses, bay scallops were obtained
alive and from each individual, a section of adductor muscle was
excised, labeled, and stored at -80°C. For each individual, we .
purified total DNA from the adductor muscle using the modified
Mitochondrial DNA Genetic Tag for Bay Scallop
113
PureGene DNA Extraction protocol for small tissue samples, ac-
cording to the manufacturer's instructions (Centra Systems. Min-
neapolis. MN).
To identify the origin of the SCAOPA-3 fragment (i.e.. mito-
chondrial or nuclear DNA). we used our broodstock bay scallops.
The bay scallop stock-restoration project involved two generations
of broodstock (an "original-broodstock" |parental| generation and
a '"restoration-broodstock" [F,] generation). The offspring of the
restoration-broodstock generation constituted the aquaculture-
derived "brood" (F^) generation that should supplement the wild
population. We determined the nucleotide sequences of 26 resto-
ration broodstock raised from eight original broodstock collected
from the wild Homosassa Bay population in 1997 and 23 restora-
tion broodstock raised from five original broodstock collected
Ironi the wild Homosassa Bay population in 1998. We examined
these sequences for among-individual heteroplasmy and for
within-individual heterozygosity. We also compared the sequence
of SCAOPA-3 to published sequences and to those a\ailable in the
computer database GeneBank.
Testing the Generic Tag
To assess the natural level of polymorphism of the SCAOPA-3
fragment and the potential of each segment to serve as an inde-
pendent component of a compound genetic tag, we sequenced
from one direction each of the two segments for 97 individuals
collected in 1997 and 1998, prior to the time of potential input
froin the stock restoration program. Twenty-three of these were
from Homosassa. of which 13 were the original-broodstock scal-
lops used in the Homosassa Bay stocking effort; the remainder of
these were collected from Tampa Bay (/; = 50) and the Anclote
Estuary (h = 24) (Fig. 1). Scallops from these nearby sites were
used because the Homosassa bay scallop population had collapsed
and thus individuals from that location had to be used with dis-
cretion To estimate the frequencies of the original-broodstock
haplotypes in the wild population, we collected and analyzed 54
individuals from Homosassa and 271 individuals from six other
west-Florida nearshore locations (Fig. I ). These individuals were
collected prior to 1999. the first year that aquaculture-derived in-
dividuals could ha\e contributed to the population.
To test the utility of our genetic tag, we examined the
SCAOPA-3 sequences from bay scallop recruits collected from
Homosassa Bay during appropriate years, determined as follows.
In west Florida, bay scallops, which are hermaphrodites, com-
mence spawning in October and generally cease by December
(Barber and Blake 1983; Arnold et al. 1998). Therefore, from
September through early October of each year, the original-
broodstock scallops were collected from wild populations at loca-
tions targeted for restoration, brought into the laboratory, and
spawned under controlled conditions. Their offspring (the restora-
tion broodstock) were reared in containment through the winter
and the following spring until they attained approximately 20-30
mm shell height. These scallops were then planted in cages in the
vicinities of collection of the original broodstocks. There, they
were to complete their growth through the summer and. hopefully,
contribute to the spawning stock when they sexually matured in
the fall. Their recruits (the brood generation), along with wild
recruits also inhabiting the restoration locations, would be of suf-
ficient size to be collected and tested for parentage in summer of
the year after they were spawned by the restoration broodstocks
and 2 y after collection and breeding of the original broodstocks.
Bay scallops can live for 2 y (Orensanz et al. 1991). but it is not
known whether they contribute to the spawning stock in the second
year of their lives. To insure that we accounted for this possibility,
we collected bay scallop recruits from restoration sites and assayed
them for the genetic tag for two years after the planting of the
restoration broodstocks. if those broodstocks survived for 2 y.
Thus, a single cycle of bay scallop stock restoration, including the
genetic monitoring, was a 3- or 4-y process.
We searched for haplotypes that could be from the offspring of
the restoration broodstocks that were planted in Hoinosassa Bay in
1998 and 1999; these were derived from original broodstocks col-
lected in 1997 and 1998. We removed the 1998 restoration brood-
stock after the 1998 spawning sea.son because most of those indi-
viduals died. However, we left the 1999 restoration broodstock in
their cages through both the 1999 and 2000 spawning seasons.
Therefore, we assayed bay scallop recruits collected from Homo-
sassa Bay in 1999 for genotypes that matched the 1997 original-
broodstock genotypes and assayed bay scallop recruits collected
from the bay in both 2000 and 2001 for genotypes that matched the
1998 original-broodstock genotypes.
To obtain these post-restoration "assessment" collections of
bay-scallop recruits, we randomly allocated 20 sampling stations
within an area of Homosassa Bay defined by the 0.7 m and 2.0 m
depth contours and by somewhat arbitral^ latitudinal borders that
were selected based upon our knowledge of the area. Using
SCUBA, at each station we .searched within I m on each side of a
300-m transect line and collected ail scallops within that zone (600
m~ per transect, 12,000 m" total). We also collected scallops using
vessel-deployed rollerframe trawling gear. Those samples were
obtained from deeper water sites (approximately 1.5-m to 3.5-m
depth). The Global Positioning System locations (available upon
request) of these collections were recorded. All assessment collec-
tions were potentially composed of an admixture of wild recruits
and hatchery-derived recruits, the latter of which could have as
parents either two restoration-broodstock indi\ iduals or one resto-
ration-broodstock and one wild individual. (We recognize that, if
mtDNA is maternally inherited in scallops, any recruit generated
by the union of an egg from a wild individual and a sperm from a
restoration-broodstock individual would not be identified as a pos-
sible aquaculture-derived bay scallop.)
Bert and Tringali (manuscript in preparation) describe the
samples needed to perform a complete assessment of a stock res-
toration or enhancement effort. Following their suggestions, we
analyzed the following individuals for their genetic-tag nucleotide
sequences. After they completed spawning, we assayed the eight
original-broodstock individuals used in fall 1997 and the five
original-broodstock individuals used in fall 1998 for both seg-
ments 1 and 2 of our genetic tag. Because bay scallops are her-
maphroditic, any or all of the original-broodstock individuals may
have passed their mtDNA on to the restoration broodstocks. We
did not assay the restoration broodstocks because many individuals
died before we could collect them. We assayed the following
numbers of bay scallop recruits: 199 collected in 1999. 253 col-
lected in 2000, and 242 collected in 2001. To detect individuals
with aquaculture-derived mtDNA haplotypes in these assessment
collections, we first compared the SCAOPA-3 segment-2 haplo-
type of each recruit to that of each original-broodstock scallop
from the appropriate year. We then sequenced for segment I any
recruit that had a segment 2 haplotype that matched that of an
appropriate-year, original-broodstock scallop. We used the sag-
114
Seyoum et al.
merit 2 component of our genetic tag first because it was slightly
more variable than segment 1 (see below).
Data Analyses
To examine the level of genetic diversity of our SCOPA-3
fragment, provide baseline data for estimating the contribution of
our stock restoration effort to the Homosassa bay scallop popula-
tion, and estimate the sensitivity of our genetic lag, we first cal-
culated a number of standard measures of genetic diversity for
each segment using the Arlequin statisfical package (Schneider et
al. 2000) on the 97 bay scallops collected from the three west-
Florida locations. We then estimated the frequencies of original-
broodstock haplotypes in the seven wild bay-scallop collections
and used the AMOVA program in Arlequin to obtain a baseline
estimate of the distribution of the original-broodstock haplotypes
in those collections, which included the Homosassa Bay collec-
tion. We also used Arlequin to quantify the genetic diversity of the
segment 2 haplotypes in each of the wild bay-scallop collections
and in the collective wild population. In addition, we searched for
original-broodstock haplotypes in the collections of wild bay scal-
lops. We tested our ability to detect original-broodstock haplotypes
in the wild population by calculating the minimum detectable fre-
quency iMDF) of the original-broodstock haplotypes, using the
basic binomial sampling equation
MDF= 1 -cxp
ln(a)
(1)
where a = 0.05 and /; = number of individuals, and defined as
the frequency below which the probability of detecting at least one
individual bearing an original-broodstock haplotype would be
<0.05. We assumed that our sampling and the distribution of the
haplotypes in the wild population were random.
To estimate the contribution of our stock restoration effort to
the Homosassa Bay scallop population, we first examined the ap-
propriate assessment collections for the presence of original-
broodstock haplotypes. Then we used Equation I to calculate the
probability of detecting those haplotypes in those assessment col-
lections. (Detailed mathematical and statistical approaches for the
overall assessment of the restoration effort will be described in a
later manuscript [Wilbur et al. in preparation]).
We further explored the limitations of our genetic tag by con-
ducting probability assessments on simulated data based on hap-
lotype frequencies observed in individuals from the 1999 and the
2000 + 2001 assessment collections. For the simulations, we ran-
domly eliminated 5%, 10%, 15%, 20%, or 25% of the individuals
in the collections and substituted at the designated frequency a
single, randomly chosen haplotype from the 1997 or 1998 original
broodstock, as appropriate for the assessment collection(s) under-
going the simulation analysis (Table 2). We then calculated hap-
lotype diversity, nucleotide diversity, and the percentage of differ-
ent haplotypes in the population for these simulated collections
and compared these statistics to those calculated for the corre-
sponding actual assessment collection without the hypothetical
stock restoration contribution. If the stock-restoration program was
successful, we would expect to see significant shifts in the fre-
quencies of haplotypes possessed by the original broodstock in
populations following restoration efforts. To determine the mini-
mum post-restoration frequency differences that would be needed
to detect contributions from restoration broodstock. we used the
V-test (DeSalle et al. 1987) to compare the haplotype frequency
distributions of our simulated as.sessment collections with the ap-
propriate actual assessment collection. For the 5% increment
within which we detected significance, we simulated assessment
collections for each 1 % increment of stock restoration contribution
and tested each of those simulated collections for significant dif-
ferences in haplotype distribution compared with our actual as-
.sessment collections.
RESULTS AND DISCUSSION
Evaluation of the SCAOPA-3 mtDNA Fragment
Our characterization the origin of the SCAOPA-3 fragment
suggested that it is of mitochondrial DNA origin. Each of the
SCAOPA-3 sequences from our 49 restoration-broodslock scal-
lops strictly matched only one of the haplotypes in the appropriate
pool of original-broodstock haplotypes. The DNA sequencing pro-
tocol that we used allowed for detection of heterozygous individu-
als if they were present; that is, heterozygous sequences charac-
teristically appear as two peaks of approximately equal intensity at
a given nucleotide site. However, none of these bay scallops were
heterozygous, and we found no heterozygous individuals in any of
our subsequent analyses. Therefore, we conclude that SCAOPA-3
is transmitted from parent to offspring as a haploid molecule and
we presume that it is mitochondrial DNA. At present, we cannot
say if SCAOPA-3 is inherited maternally: paternal mtDNA inher-
itance occurs in other bivalves (e.g., Mytilius: Liu et al. 1996.
Zouros et al. 1994). Despite comparing its nucleotide and pre-
sumptive amino acid sequences to those reported for other organ-
isms, including other mollusks (Hoffmann et al. 1992, Boore and
Brown, 1994) and to unpublished sequences, (e.g., GeneBank ac-
cession numbers AB055625, AB065375), we were unable to
characterize with certainty the gene region it encompasses. How-
ever, this will not affect the study, provided that SCAOPA-3 is
faithfully transmitted as a haploid molecule from parent to off-
spring.
Sensitivity and Application of the SCAOPA-3 Fragment
The eight 1997 original-broodstock individuals had only seven
different segment 2 haplotypes. However, the two individuals that
were identical for segment 2 differed for segment 1 . Thus, each of
our broodstock individuals had a SCAOPA-3 haplotype that was
unique in the aquaculture hatchery.
All differences among individuals in segment 1 and segment 2
of our mtDNA fragments were in the form of single bp substitu-
tions. In Table I A, we present estimates of genetic diversities for
the two segments as determined by sequencing the individuals
used to characterize the fragment. Separately, these segments dis-
tinguished high percentages of individuals: collectively, they dis-
tinguished nearly all of the individuals.
Results of the AMOVA analysis suggest that the bay scallops
comprising the west-Florida pre-restoration collections were ge-
netically homogeneous with respect to the SCAOPA-3 mtDNA
fragment. In Table IB, we summarize the segment 2 genetic di-
versities for these collections and for the west-Florida population.
Both the percentage of individuals with different haplotypes and
the proportion of haplotypes that were unique were very high in
the individual samples and high in the combined data. Eighty-five
individuals (26%) were defined by four haplotypes in the propor-
tion 42:19:18:6: thus, the most common haplotype present in the
population occurred in only 13% of the individuals. Conespond-
Mitochondrial DNA Genetic Tag for Bay Scallop
115
TABLE 1.
Kstiniates of bay scallop {Argopecten irradiaiis) genetic divcrsitifs for the SCAOPA-3 mitochondrial DNA genetic tag in (A) ')! wild
individuals from Tampa Bay, Homosassa. and Anclote, Florida, (segments I and 2 are defined in Materials and Methods! and (Bl western
Florida collections made prior to the stock restoration effort (segment 2 only).
Segment
No. bp
HN
HQ
RS
h
P
K
A.
1
451
72
^)l
0.19
(1.97 ±(1.(11
(1.86 + 0.48
3.9 ± 2.0
2
450
80
S6
(1.2(1
(1.49 ± (1.(1(1
L.Vi ±0.07
5.6 ±2.7
Total
901
97
98
U.20
1.0(1 ±(1.(10
L10±()..S6
9.5 ± 4.5
Location
Year
N
HN
HQ
h
P
K
B.
ST
1997, 1998
61
75
89
0.97 ±0.01
1.22 ±0.66
5.4 ± 2.6
CK
1997
20
75
87
0.94 ± 0.04
1.07 ±0.61
4.7 ±2.4
HO
1997. 1998
54
81
89
0.99 ±0.01
1 .38 ± 0.74
6.2 ±3.0
HE
1997, 1998
32
81
85
0.98 ±0.01
1.12 ±0.63
4.8 ±2.4
AN
1997. 1998
67
84
93
0.99 ±0.01
1.30 ±0.70
5.4 + 2.7
TB
1997
65
80
90
0.98 ±0.01
1.30 ±0.70
5.6 ± 2.7
SS
1998
26
85
86
0.98 ± 0.02
1.80 + 0.60
4.8 ±2.4
Total
325
62
85
0.98 ± 0.00
1 .26 ± 0.68
5.3 ± 2.7
Abbreviations: No. bp = number of base pairs; HN = percentage of individuals with different haplotypes: HQ = percentage of haplotypes unique to
single individuals; RS = number of polymorphic sites per nucleotide site; li = haplotype diversity; p = nucleotide diversity in %; K = number of
pairwise nucleotide differences; N = number of individuals; ST = Steinhatchee; CK = Cedar Key; HO = Homosassa; HE = Hernando; AN =
Anclote; TB = Tampa Bay; SS = Sarasota Bay.
h. />. and K are mean values ± standard deviations.
ingly. all standard measures of genetic diversity were conipura-
tively high (e.g., haplotype diversity ranged 0.94-0.99).
Twenty-four wild-individual haplotypes matched five 1997
original-broodstock haplotypes for segment 2. However, none of
the individuals that matched original-broodstock segment-2 hap-
lotypes also matched the same broodstock individual for seg-
ment 1. No wild individuals collected in 1998 matched any of the
original-broodstock segment-2 haplotypes. If our assumptions as-
sociated with Equation I were vahd, we could expect to obtain a
match between a wild-individual haplotype and an original-
broodstock haplotype if the broodstock haplotype was present in
our wild-population sample at a frequency of approximately \% or
greater {MDF,,^ = 0.00917). Thus, the estimated prerestoration
frequency of each of the 1997 and 1998 broodstock haplotypes in
the wild population probably was less than 1%.
Ten of the assessment scallops collected in 1999 matched three
of the 1997, segment-2, original-broodstock haplotypes. Eight of
those were identical to the single original-broodstock scallop with
the haplotype that was the second most common in the wild popu-
lation. However, the haplotypes of all of those individuals differed
from that original-broodstock individual's segment 1 haplotype.
No segment-2 haplotypes from assessment bay scallops collected
in 2000, and only one segment-2 haplotype from an assessment
bay scallop collected in 2001, matched any 1998, original-
broodstock, segment-2 haplotype. That individual did not match
for segment 1 the original-broodstock individual that it matched
for segment 2. Thus, our collective sample size of 694 individuals
gave no indication that the bay scallop restoration project contrib-
uted to the local Homosassa bay scallop population during 1999-2001.
The MDF^,^ for detection of an original 1997 or 1998 brood-
stock haplotype in the appropriate assessment collection(s) was,
respectively 0.015 (1999 assessment collection) or 0.0060 (2000 +
2001 assessment collections). Original-broodstock haplotypes that
were present in the putative admixed Homosassa population at
frequencies near or below the MDFg^s were at statistical risk of not
being detected. However, these frequencies were so low that stock
restoration contributions at or below these levels may essentially
be inconsequential.
Although haplotype diversity and nucleotide diversity in our
hypothetical assessment of stock restoration contribution were pro-
portionally reduced with increasing stock restoration contribution,
they were not as sensitive to the input of stock restoration contri-
bution as was the percentage of different haplotypes (Table 2).
Nevertheless, our simulations indicate that a stock restoration con-
tribution of at least 15% in the 1999 assessment collection and
\0% in the 2000-2001 combined assessment collection would be
needed to generate a significant difference between those assess-
ment collections with versus without stock restoration contribu-
tions.
Genetic Tags and Molluscan Stock Restoration
The general strategy in a stock restoration program is to collect
animals from the targeted restoration site, produce large quantities
of aquaculture-reared or. in the case of our bay scallop program,
aquaculture-derived (one generation removed 1 individuals, and use
them to supplement or replenish the population at the same site.
Determining the success of such an effort depends on the ability to
detect the contribution (in numbers or percentages) of hatchery-
reared or hatchery-derived offspring in the post-restoration re-
cruits. In supplemented populations, the frequency of aquaculture-
generated individuals can range from undetectable to a complete
swamping of the admixed population. A single-gene genetic tag
such as ours can indicate whether restoration effort has resulted in
essentially undetectable input, substantial input, or a complete
swamping of the local population. However, the capacity of this
tag to estimate the contribution of the stock restoration effort be-
tween the extremes of essentially no input and very high input is
116
Seyoum et al.
TABLE 2.
Hypothetical analysis of stock restoration contribution in the
assessment collections from Honiosassa with levels of contribution
varying from 0% (original assessment collection) to 25% (see
Materials and Methods for method of simulating stock restoration
contributions). (A) 1999 assessment collection (A' = 199
individuals). (B) 2I)(II) + 20(11 combined assessment collections
(A' = 495 individuals).
SRC(%)
Nl
N2
HNl
HN2
A.
0
199
0
0.72
0.72
5
189
10
0.73
0.70
10
179
20
0.73
0.66
15
169
30
0.72
0.62
20
159
40
0.75
0.60
25
149
50
0.77
0.5S
B.
0
495
0
0.69
0.69
5
470
35
0.69
0.66
10
445
69
0.69
0.62
15
421
104
0.70
0.73
20
396
139
0.72
0.57
25
370
174
0.73
0.55
Abbreviations: SRC = hypothetical stock restoration contribution; Nl =
number of individuals taken from the specified year assessment collection;
N2 = hypothetical number of individuals contributed from the stock res-
toration program (within a single percentage, all of which were taken from
a single, randomly chosen broodstock individual); HNl = percentage of
individuals with different haplotypes without stock restoration contribution
(calculated based on Nl only); HN2 = percentage of individuals with
different haplotypes with stock restoration contribution (calculated on Nl
+ N2).
related to the degree of statistical uniqueness, as measured by
statistical probability, of the tag in each application. To precisely
define an intermediate-level contribution from a stock restoration
effort, the assessment collection must consist of a very high num-
ber of individuals; the genetic tag must be complex (e.g., com-
posed of our compound mtDNA genetic tag plus several micro-
satellite loci), or. if it is a single-gene tag. extremely variable; or
the method for determining the contribution must differ from ours.
Because we found no original-broodstock haplotypes in either
the wild population or the assessment collections, we can combine
all of these collections to estimate the uniqueness of our original-
broodstock haplotypes and calculate the MDF above which we
might expect to encounter one of these haplotypes. We can esti-
mate with 95'7r probability that v\e would have detected at least
one original-broodstock haplotype in this combined sample ( 1,019
individuals) if the frequency of any of these haplotypes was 0.003
or greater. Clearly, frequencies below this MDF would represent
inconsequential contributions from a stock restoration effort. Thus,
our single-gene genetic tag should be useful for assessing the
success of our entire bay scallop restoration effort.
In many cases, a single-locus, preliminary genetic tag such as
ours could be useful in assessing the contribution of stock resto-
ration efforts. Multi-locus genetic tags can be laborious, time-
consuming, and expensive to develop, test, and apply. Fuilher-
more. in our case, the potential for reproductive mixing between
restoration broodstock and wild scallops limits the ability for
nuclear DNA-based assignment of individuals to either the brood
generation or to the wild population. Our genetic tag can be used
to preliminarily evaluate the success of a bay scallop stock en-
hancement or restoration effort and thereby to evaluate whether it
is worth the expense and effort to develop a more definitive ge-
netic tag. Then, if it appears that the stock restoration effort may
have contributed a potentially significant fraction of the recruits to
an area, a high-resolution, multi-gene tag can be developed. How-
ever, under certain conditions, the type of genetic tag presented
here may be sufficient for an entire study.
The advantages of using a single-gene genetic tag composed of
more than one hypervariable segment and in which the segments
can be used sequentially are increased resolution and reduced ef-
fort. In our genetic tag. both segment 1 and segment 2 had ample
and nearly equivalent variation. By sequencing first for segment 2.
the expense and time required were reduced significantly because
only the individuals that had segment 2 haplotypes identical to
those of the original-broodstock haplotypes also needed to be se-
quenced for Segment 1 ,
The utility of a single-gene genetic tag such as that presented
here is enhanced if the broodstock used possesses essentially
unique haplotypes or genotypes. However, there are limitations to
this type of approach. A large number of wild individuals or a high
percentage of the wild population must be assayed to establish the
frequencies of the genetic-tag haplotypes in the pre-restoration
population, and individuals with "unique" haplotypes should be
used as broodstock. Threatened or depleted populations can be
further endangered if they are flooded with aquaculture-derived
individuals that collectively possess only a few naturally rare
genotypes or haplotypes, if those individuals interbreed exten-
sively and successfully with the remnant wild population. Never-
theless, for some applications, the procedure that we described
here provides researchers with a method for finding an mtDNA
genetic tag in organisms for which little is known about their
mtDNA. This type of genetic tag can be used to screen individuals
and derive parentage or group associations for stock restoration
efforts, conservation biology, or other suitable applications.
ACKNOWLEDGMENTS
We thank M. Tringali for assistance in the designing of the
primers and notable suggestions in many aspects of the analysis.
We also appreciate the assistance of D, Marelli. M. Parker, M.
Harrison, and S. Peters with the field collections and C. Lund, T.
Thompson, and D. Warner for various types of assistance. We
additionally thank M. Tringali, A. McMillen-Jackson, and two
reviewers for valuable comments on our manuscript. This study
was funded by a grant from the National Oceanic and Atmospheric
Administration (NOAA), grant NA76FK0426 and project FWC
2234 and by the state of Florida. The views expressed herein are
those of the authors and do not necessarily reflect the views of
NOAA or any of its sub-agencies.
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GAMETOGENESIS IN A SYMPATRIC POPULATION OF BLUE MUSSELS, MYTILUS EDULIS
AND MYTILUS TROSSULUS, FROM COBSCOOK BAY (USA)
A. P. MALOY,* B. J. BARBER, AND P. D. RAWSON
School of Marine Sciences, University of Maine, Orono, Maine 04469
ABSTRACT To lest the hypothesis that a temporal variation in species-specific spawning times is the mechanism Hmiting hybrid-
ization and maintaining genetic integrity in a Mylilits ediilis (L.) and M. irossiihi.s (Gould) hybrid /one in eastern Maine, mussels from
a low intertidal site in Cobscook Bay were histologically examined at monthly to semi-monthly intervals throughout the year 2000.
Analysis of gamete volume fraction and oocyte area measurements detected no difference in the timing of gametogenesis and spawning
between M. edulis and M. trossuliis. Differences in mature oocyte area measurements, however, indicated that M. ediilis spawned larger
eggs than M. trossuhis. At this location, low frequency of hybridization and maintenance of genetic identity for these two species is
unlikely the result of temporally distinct spawning times.
KEY WORDS: Myiihis. gametogenesis. hybridization, mussels
INTRODUCTION
The Mylilus species complex is composed of three closely re-
lated blue mussel species. M. edulis. M. trossuius, and A/, gullo-
provincialis. In the northern hemisphere, M. edulis occurs princi-
pally in the eastern and western Atlantic; M. trossuius is found in
the Baltic Sea. the northwestern Atlantic Ocean, and the northern
Pacific Ocean; and M. galloprovincialis occurs in the Mediterra-
nean Sea. the Atlantic coa.st of southern Europe, northern Africa,
and the Pacific coast of North America (Gosling 1984, 1992.
Koehn 1991, McDonald et al. 1991, Suchanek et al. 1997). An
early survey of Mytilus spp. on the east coast of North America
indicated the presence of only a single species, M. edulis (Koehn
et al. 19761, but in a later study, Koehn et al. (1984) identified two
genetically distinct taxa inhabiting Atlantic Canada. These two
genetically distinct groups (M. edulis and M. trossuius) form a
zone of sympatry from northern Newfoundland south to the east-
ern coast of Maine (Varvio et al. 1988. McDonald et al. 1991.
Bates and Innes 1995. Comesana et al. 1999, Rawson et al. 2001 ).
Hybridization is commonly reported wherever members of the
Mylilus complex are sympatric (Gosling 1992). In the Baltic Sea.
M. edulis and M. trossuius hybridize so readily that they are con-
sidered semi-species (Viiinola & Hvilsom 1991 ). M. edulis and M.
galloprovincialis hybridize extensively in a zone of sympatry that
extends from the coast of Spain through the British Isles. The
frequency of hybrid genotypes varies significantly among loca-
tions but can reach values as high as 80% in some populations
(Hilbish et al. 1994. Cotnesafia & Sanjuan 1997. Sanjuan et al.
1997). In contrast, the frequency of hybrid genotypes formed by
interspecific matings between M. edulis and M. trossuius in the
northwest Atlantic is much lower, ranging from 12 to 26% (Koehn
et al. 1984. Varvio et al. 1988, Bates & Innes 1995. Mallet &
Carver 1995. Saavedra et al. 1996. Comesana et al. 1999. Rawson
et al. 2001 ). Although variation among sampling locations and the
use of different methodologies (e.g., morphologic analysis, allo-
zyme electrophoresis, mitochondrial, and nuclear DNA-based
markers) may be partly responsible for the variation in the fre-
quency of hybrids observed, these studies suggest that hybridiza-
tion is less prevalent among blue mussels on the Atlantic coast of
North America than in the Baltic or European hybrid zones.
Mate choice, habitat specialization and differential environ-
mental tolerance, spawning asynchrony, and gamete incompatibil-
ity are processes that can initiate and maintain reproductive isola-
tion between closely related species in sympatric populations
(Palumbi 1994). In free-spawning marine invertebrates, mate
choice, per se. is unlikely to play an important role in limiting
hybridization. Increasing evidence, however, suggests that gamete
interactions can affect reproductive isolation. For example, rapid,
divergent evolution in sperm proteins (bindin and lysin) limits
interspecific hybridization in sea urchins and abalone (Swanson &
Vacquier 1998. Palumbi 1999). respectively. The existence of
similar mechanisms in bivalves has not been confirmed.
Additionally, any habitat-specific selection that creates patchy
species distributions may also limit hybridization because fertil-
ization is more likely among close neighbors. Gardner (1996) has
suggested that blue mussel hybrid zones occur in regions of envi-
ronmental discontinuity so that the general patterns of species
distribution are determined by differential adaptation. Several
studies have observed that the distribution of blue mussel species
is conelated with changes in environmental parameters, both in the
contact zone between M. edulis and M. galloprovincialis in west-
ern Europe (Hilbish et al. 1994, Gardner 1996, Gilg & Hilbish
2000. Hilbish et al. 2002) and between M. trossuius and M. gal-
loprovincialis on the Pacific coast of North America (Sarver &
Foltz 1993). In the northwest Atlantic, research has focused on
differences in salinity and wave exposure in structuring the species
composition of blue mussel populations. There has been little evi-
dence to directly link any of these factors with either the distribu-
tion, or the relatively low frequency, of hybrids within the region
where M. edulis and M. trossuius are sympatric.
Reproductive isolation and maintenance of genetic identity
may also be dependent on temporal variation in spawning events.
In sympatric populations of M. galloprovincialis and M. edulis in
southwestern Europe, low hybridization is observed when spawn-
ing periods are out of phase, whereas sites with a greater degree of
synchrony have a higher degree of hybridization (Gardner 1992,
Seed 1992). The objective of the present study was to determine
whether the relatively low rate of hybridization occurring between
M. edulis and M. trossuius in eastern Maine could be attributed to
temporal variation in spawning.
*Corresponding author. Department of Biochemistry. Microbiology, and
Molecular Biology. University of Maine, Orono. ME 04469. Fax: 207-
581-2801; E-mail: aaron.maloy@umit.maine.edu
MATERIALS AND METHODS
Adult mussels (35 to 50 mm in shell length) were collected by
hand from a sympatric. low intertidal population in East Bay (lati-
119
120
Maloy et al.
tilde 44°56'30"N; longitude 67°07'50"W: Cobscook Bay. Maine)
throughout 2000 (Table 1 ). Samples of 120 mussels were obtained
monthly from January through April. October through December,
and semi-monthly between 4 May and 14 September. Mussels
were transported on ice to the University of Maine, and a piece of
mantle tissue approximately 0.5 cm" was removed and preserved
in 95'/f ethanol for DNA extraction. The remainder of the mussel
was preserved in Dietrich's fixative (Gray 1954) for subsequent
histologic preparation. All preservation was completed within 24 h
of collection.
DNA was extracted from gonadal tissue following the protocol
of Rawson et al. (2001). Three polymerase chain reaction-based
nuclear markers, polyphenolic adhesive protein (Glu-5'). internal
transcribed spacer. Mytihis anonymous locus-I. and one mitochon-
drial marker (mtl6s-F: Rawson et al. 2001), were used to identify
mussels with M. edulis and M. trossidus genotypes from each
sampling period. Initially, the Glu-5' marker was run on al!
samples and used to identify 30 (n = 40 on 17 and 30 August)
individuals homozygous for both M. edulis and M. trossidus Glu-
5' alleles. These 60-80 mussels were subsequently genotyped at
the remaining three markers. Individuals not scored as inultilocus
homozygotes for M. edulis or M. trossulus alleles at all markers
(i.e., hybrids) were eliminated from further anal
bined results of all four markers were used to pick
(;; = 30 on 17 and 30 August) of each species for assaying re-
productive condition.
Preserved individuals were transversely sectioned (2- to 3-mm
thick) anterior of the byssal gland, dehydrated in an ascending
alcohol series, cleared with Xylenes, and embedded in Paraplast
(Howard & Smith 1983). Cross sections (5 ixm) of each block were
cut on a rotary microtome, placed on glass slides, stained with
Shandon instant hematoxylin and eosin Y, and permanently
mounted. Slides were examined using a compound microscope
(Nikon LABPHOT-2) equipped with a video camera (Dage CCD
72). Images were digitized with a fraine grabber (Flash Point 128,
vsis. The com-
20 individuals
Integral Technologies Inc.) and measurements made using image
analysis software (Image Pro Plus; Media Cybernetics).
Reproductive state was measured by two separate methods.
First, the gamete volume fraction (GVF) of all indixiduals was
calculated as the area of reproductive tissue present in one micro-
scopic t~ield divided by the entire area (Bayne et al. 1978). Thus,
estimates of GVF indicate the proportion of mantle that is com-
prised of reproductive tissue. The mean of five random fields
(300x) was calculated for each individual and used in subsequent
statistical analysis. In addition to the GVF. mean oocyte area was
estimated for each female from 50 measurements ( 1 200x ) of the
cross-sectional area of oocytes with a clearly visible nucleolus
(Garrido & Barber 2001).
GVF data were analyzed using a three-way ANOVA for
sample date, species, and gender. Oocyte data were evaluated with
a two-way ANOVA across sample date and species. Both data sets
were evaluated at a = 0.05 using simultaneous BonfeiToni pair-
wise comparisons of sample level means. Statistical analyses were
performed using Minitab 13.0. which automatically adjusts the
Bonferroni a lev el to compensate for the total number of possible
pairwise comparisons. Because all possible combinations of pair-
wise comparisons were not of interest, the a level was manually
readjusted to account for the appropriate number of comparisons
used in the analysis.
RESULTS
Gametogenesis in M. edulis (mean length 44.8 mm ± 3.7) and
M. trossulus (mean length 44.3 mm ± 3.5) was highly synchronous
at the East Bay site throughout 2000. Species-specific mean ga-
mete volume fractions (estimated for both male and female mus-
sels) were relatively low in February and increased steadily in both
species from February to June. The peak mean GVF of 0.89 in M.
edulis was identical to the 0.89 estimated for M. trossulus mussels
sampled on 4 June. GVF remained high in both species throughout
TABLE L
Mytilus edulis, Mytilus trossulus: relative number of males, females, and undifferentiated mussels sampled In East Ba>, 20(10.
Mytilus edulis
Mytilus trossulus
Males
Females
LndifTerentiated
Males
Females
Undifferentiated
Totals
19 Jan
7
9
4
5
5
1
31
20 Feb
9 .
6
5
8
11
1
40
21 Mar
11
7
2
11
8
1
40
17 Apr
7
11
2
9
10
1
40
4 May
8
10
2
8
12
40
1 8 May
12
8
-
11
9
40
4 Jun
8
12
8
11
39
18 Jun
11
9
13
7
40
30 Jun
8
11
9
11
39
17 Jul
12
8
12
8
40
1 Aug
10
10
9
11
40
17 Aug
19
10
1
17
11
58
30 Aug
15
14
13
16
58
14 Sep
7
10
3
13
4
3
40
15 Oct
9
11
7
5
8
40
17 Nov
10
9
1
7
7
4
38
9 Dec
6
12
1
6
8
6
39
Totals
169
167
21
16(1
154
25
702
Undifferentiated individuals were not used in statistical analysis.
Gametogenhsis in Sympatric Blue Mussels
121
June and July and then declined precipitously between 1 7 July and
1 August samples among mussels of both species. Following this
initial dramatic decline, a less pronounced decrease in GVF was
observed up to the 15 October sampling date, after which GVF
estimates were constant and nearly equal to those observed m
February (Fig. 1 ).
Analysis of gender-specific patterns of GVF \ariation indicated
that while gamete development in the females of both species was
comparable to that of males, it lagged behind that of the males. For
example, mean GVF estimates for females were consistently lower
than those observed in males from February to April but by June
these differences had disappeared. In addition, spawning in fe-
males resulted in a greater loss in GVF relative to males. Overall,
males had an average yearly GVF approximately lO'/r higher than
(enialcs for both Mytilus ediilis and M. trossuliis, Bonferroni pair-
wise comparisons (a = 0.05) indicated a significant difference in
GVF between males and females on 30 August (Fig. 2 A and B).
Consistent with the graphic analysis, a three-way ANOVA re-
vealed that significant differences in GVF occurred between date
and gender but not between species. Significant interactions oc-
curred between date and species and between date and gender
resulting from the seasonality of gamete development. Gametoge-
nic cycles (as defined by GVF) were the same for both species and
there were no significant interactions between species and gender
or date*species*gender (Table 2). With respect to the shaip de-
crease in GVF. Bonferroni analysis indicated that significant de-
creases in GVF at both the species and gender levels corresponded
with the initial spawning period between 17 July and 1 August.
Though differences occurred between sexes because of the high
postspawn variation, spawning times were still highly synchro-
nous.
Similar results were obtained using mean oocyte areas to assess
gametogenic cycles. Mean oocyte areas increased sharply for both
species from 21 March through 4 June. After 4 June, oocyte areas
gradually increased until maxima were observed on 17 July [Myti-
lus cdulis 678.6 ^JLm" and M. trossuliis 530.1 |j,m"). A sharp de-
crease in mean oocyte areas occurred between 17 July and 1 Au-
gust. After I August, there were increases in oocyte area until 30
August for M. ecluHs and 14 September for M. trossuliis. followed
by a less pronounced and protracted period of decline until 9
December (Fig. 3).
The two-way ANOVA for oocyte areas indicated a significant
l.U -
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Figure 2. (A) Mytilus edulis. Mean (±1 SDl jjaniete volume fraction for
male vs. female mussels from East Bay, Maine. I'SA. (B) ,Mytilus tros-
suliis. Mean (±1 SD) gamete volume fraction for male vs. female mus-
sels from East Ba>, Maine.
interaction between date and species (Table 3). The difference
between species was caused by variation in mean oocyte size
rather than a variation in the timing of gametogenic events; aver-
age yearly oocyte area was 338.2 \i.m~ forM. edulis and 308.2 p,m"
for M. trossuliis. Significant declines in species-specific oocyte
area were observed between 17 July and 1 August, corresponding
with a period of spawning indicated by GVF analysis. Additional
TABLE 2.
Gamete volume fraction of Mytilus eilulis and Mytilus trossulus:
results of a three-«a> .\NO\ .\ testing the effects of date, species,
and gender on the gametogenic cycle.
Figure 1. Mytilus edulis, Mytilus trossulus. Mean (±1 SD) gamete vol-
ume fraction for mussels from East Bav, Maine, USA.
Source
df
Mean Square
F Value
Date
16
4.4869
142.53***
Species
1
0.0003
0.01
Gender
1
1.8650
59.24***
Date X species
16
0.1061
3.37**
Date X gender
16
0.0827
2.63**
Species x gender
1
0.0407
1.29
Date x species x
gender
16
0,029.1
0.93
Error
."^S?
** P< 0.01.
*p<o.m\.
122
Maloy et al.
800
*N
700
E
a
600
a
SOO
<
n
400
>,
(J
o
o
300
200
— ■ — M edlilis
T*" M :rossiiltis
C -= OU OU 5JJ C- T1
= ^ 3 3 3 O ^
g -g I £. ^ ^ g
V "r 5 < 5 S 3
2 c - r- ■ ' T ^ - _ _ r^ o • - r- •-..
— r. ^, _ -f =c — ^, _ „ — — _
Sample Date
Figure 3. Mytiliis ediilis. Mytilus troxsiiliis. Mean (±1 SD) oocyte area
for mussels from East Bav, Maine.
significant decreases between dates were slightly out of phase,
with the mean oocyte area of M. edulis decreasing from 14 Sep-
tember to 15 October and that of A^. trossidits from 15 October to
17 November. A significant difference in oocyte area (t = 7.24,
P < 0.001) was observed just prior to spawning on 17 July indi-
cating that M. edulis spawned larger eggs than did M. trossiihis.
Mean shell length of females sampled on this date was not sig-
nificantly different (t = 1.29, P = 0.220).
DISCUSSION AND CONCLUSIONS
The reproductive cycle of mussels in this population was highly
seasonal which is typical of many benthic marine invertebrates in
northern temperate zones. In this study, gonadal development was
minimal through the winter as indicated by low GVF and small
oocyte diameters. Increased gametogenic activity in spring corre-
sponded to increasing water temperature and presumably food
a\'ailability. A significant decrease in GVF and oocyte diameters,
indicative of a major spawning event, took place in late July and
involved a large proportion of the population. Interestingly, GVF
for females increased slightly in samples collected after this initial
spawning event. Such an increase could be caused by redevelop-
ment of the gonad in preparation for a second spawning. However,
we observed little histologic evidence of redevelopment in indi-
vidual mussels that had already spawned. The predominant histo-
logic feature at this time was empty follicles containing a few
refractory oocytes. Thus, the few individuals that did not spawn or
had only partially spawned after the peak-spawning event in late
July were responsible for the observed increase in GVF.
TABLE 3.
Mean oocyte area of Mytilus edulis and Mytilus trossulus: results of
a two-way ANOVA testing the effects of date and species on the
gametogenic cycle.
Source
df
Mean Square
F Value
Date
16
1.6398
53.67***
Species
1
0.1236
4.04*
Date X species
16
0.0665
2.18**
Error
285
0.0306
* P < 0.05. **P < 0.01, ***P < 0.001,
More importantly, the reproductive cycles of Myliliis edulis and
M. trossulus sampled from this population were highly synchro-
nous. For the year 2000 at the East Bay site, the results of this
study indicate that interspecific fertilization between M. edulis and
M. trossulus is possible based on spawning times. Similar findings
have been reported elsewhere. Freeman et al. (1992) and Mallet
and Carver ( 1995) observed synchronous reproductive patterns in
populations of M. edulis and M. trossulus from Lunenburg Bay,
Nova Scotia. Additionally, Toro et al. (2002) found that the ini-
tiation of spawning was coincident between these species and their
hybrids in Trinity Bay, Newfoundland; although M. trossulus dis-
played a more protracted period of spawning at this location the
variation alone was not sufficient to explain the limited numbers of
hybrids observed. Thus, four studies covering a wide geographic
region from Maine to Newfoundland have observed similar results
all suggesting that hybridization is not limited solely by species-
specific differences in spawning times.
It is possible that genetic identity is maintained between M.
edulis and M. trossulus by a factor other than different spawning
periods. Gamete recognition proteins have been shown to drasti-
cally reduce the hybridization potential between closely related
taxa of marine invertebrates. Interestingly, molecular phylogenies
suggest that M. trossulus is the most divergent of the blue mussel
taxa (Rawson & Hilbish 1995). It has been recently shown that M.
edulis and M. trossulus have also diverged significantly with re-
spect to amino acid sequence at a spenn lysin locus (C. Riginos.
pers comm). Divergence in gamete recognition proteins such as
sperm lysin could act to limit hybridization between M. trossulus
and other blue mussel taxa. Though no evidence of functional
differentiation has been documented as yet, preliminary data indi-
cates that cross-fertilization of M, edulis and M. trossulus is lim-
ited except at very high sperm concentrations (Rawson unpub-
lished). Thus, future effoils should focus on more detailed obser-
vations of the spawning behavior of these two species as well as
the potential for functional variation in gamete recognition pro-
teins.
The present study found that M. trossulus had smaller mean
oocyte size at maturity and presumably spawned smaller eggs than
M. edulis. Given that M. trossulus has a higher reproductive output
(Toro et al. 2002), it follows that similarly sized M. trossulus
produced more (but smaller) eggs than M. edulis. which might
provide a selective advantage for the more fecund M. trossulus.
Similarly, M. galloproviiicialis has a higher fecundity per unit
length than M. edulis at Croyde in S.W. England, but genotypic
ratios between these two species have not changed over time be-
cause of large numbers of small M, edulis (Gardner & Skibinski
1990). Smaller oocytes may also represent a response to environ-
mental stress. Cobscook Bay in eastern Maine is near the southern
distributional limit of M. trossulus (Rawson et al. 2001) and as
such, may be a less than optimal environment for this species.
However, M. tros.sulus from Newfoundland also produces smaller
eggs, has a smaller size at first maturity than M. edulis (Toro et al,
2(J02), as well as a population structure containing a higher fre-
quency of small M. trossulus individuals (Comesana et al. 1999).
Given that a difference in oocyte size has been observed in both
Maine and Newfoundland it is more likely that this difference is
the result of a difference in life history strategy rather than a
response to environmental stress. Additional data are needed on
extrinsic factors such as population structure, size at first maturity,
reproductive output, and size dependent mortality to draw coiiclu-
Gametogenesis in Sympatric Blue Mussels
123
sions concerning the intrinsic factors sinaping (he lite history evo-
lution of M. cdiilis and M. trossiiliis.
ACKNOWLEDGMENTS
Funding for this project was provided through a Maine Aqua-
cuhure Innmation Center crant to B. J. Barber and P. D. Rawson.
Maine Sea Grant, and Experiinent Station Hatch Funds to
P.D. Rawson. We are also grateful to D. Beane for histologic
preparations, and S. R. Fegley and P. A. Haye for helpful
comments on earlier versions of this manuscript. This is Maine
Agricultural and Forest Experiment Station external publication
#2627.
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Journal of Slwllfish Reseanh. Vol. 22. No. I. 125-134, 2003.
MODELING OF FILTER-FEEDING BEHAVIOR IN THE BROWN MUSSEL, PERNA PERNA (L.
EXPOSED TO NATURAL VARIATIONS OF SESTON AVAILABILITY IN SANTA
CATARINA, BRAZIL
F. M. SUPLICY,'* J. F. SCHMITTr N. A. MOLTSCHANIWSKYJ,' AND J. F. FERREIRA'
'School of Aquacuhiire. Tasmanian Aciuaciiluivc and Fisheries Institute. University of Tasmania,
Lockcd-Bag 1-370. Launceston. Tasmania. 7250. Australia: 'Lahoratorio de Ciiltiro dc Mohiscos
Marinhos iLCMM). Departamento de Aquicultura. Universidade Federal de Santa Catarina. P.O. Box
1 0-1. IS. Floriandpolis. Santa Catarina. CEP S,S062-6()I. Brazil
ABSTR.ACT The aim of this -.tiidy is lo quanlity and model the filter-feeding beha\ ior of the mussel Pcnui penui feeding on natural
seston. Models were generated that described each step of the feeding process and produced a predictive model of rates of food uptake
by P. perna in culture areas from Southern Brazil. Feeding experiments using the hiodeposition approach were conducted with mussels
ranging in shell height from 3.94 to 9.22 cm of three sites, including turbid and clear water environments. Organic content of the seston
tOCS. fraction) decreased as total particulate matter (TPM, mg L"') increased. The maximum filtration rate (FR. mg L~') measured
for an individual mussel was 156.7 mg h^' and was recorded when TPM was 33.9 mg L"' and OCS was 0.18. Rejection rate of particles
had a strong positive relationship with TPM, and an inverse relationship with OCS. Maximum rejection rate recorded was 124.1 mg
h ' and was measured under the same seston conditions as maximum filtration rate. Net organic selection efficiency by mussels (NOSE,
fraction) was related to the amount of particulate organic matter (POM. mg L"') and particulate inorganic matter (PIM, mg L"')
available in the water. NOSE was positive below PIM values of 2 mg L"', but had negative values when POM was above 3 mg L~'
and PIM between 2 and 15 mg L"', and positive values when POM was below 3 mg L~' and PIM above 15 mg L"'. Maximum NOSE
was 1.71. when PIM was 1.02 mg L"' and POM was 0.67 mg L"'. Organic content of ingested matter (OCI. fraction) had a positive
relationship with NOSE and TPM. Maximum OCI was 1.24 and was measured when TPM was 33.9 mg L"', OCS was 0.18, FR was
151.30 mg h~', and NOSE was 1.30. The net absorption efficiency of ingested organics (NAEIO) increased with increasing OCI in a
hyperbolic relationship. The net organic absorption rate (NOAR, mg h"') increased with both FR and OCI, The coupling of the
equations that described filter-feeding processes for P. pema in the STELLA software environment produced a robust model with
relatively low complexity and specificity. The model can predict the P. perna feeding behavior in turbid or clear water and can be used
with different species if the correct coefficients are used. The coupling of this feeding model with future models of energy budget,
population dynamics, seston hydrodynamics, and primary production will be valuable for the evaluation of shellfish carrying capacity,
KEY WORDS: mussel physiology, model, Perna perna. STELLA
INTRODUCTION
Assessing carrying capacity, the environmental capacity for
shellfish culture is generally approached using ecophysiological
modeling (e.g., Brylinsky & -Sephton 1991, Newell & Campbell
1998, Schcilten & Smaal 1998). The inclusion of processes relative
to rates of selectivity, rejection, and absorption by molluscan filter
feeders is of primary importance for both ecosystem and local
scales models (Smaal et al. 1998). Sessile suspension-feeders ob-
tain energy by selectively feeding on seston, which includes a
variable mi.xture of algae, detritus, and silt. Not only does the
seston have a small fraction with nutritional value f Smaal & Haas
1997), but also the composition changes on time scales of minute
to tnonths (Grant 1993). The available organic content of the
seston ranges from 5 to 809^ (Bayne & Hawkins 1990). Such
nutritional variability in the seston forces sessile organisms like
mussels to maximize their energy intake and ultimately their net
energy balance, by varying rates of feeding and digestion in re-
sponse to seston concentration and organic content (Bayne el al.
199.3).
The literature describing bivalve rates of filter feeding and
digestion is extensive (see reviews by Bayne & Newell 1983.
Griffiths & Griffiths 1987, Bayne 1993). However, recent findings
suggest that previous studies have limited application because they
used artificial diets, and it is unclear to what extent using artificial
diets provides a realistic representation of "(/; .■iitu" feeding behav-
*Corresponding author. Fax: -1-61-3-6324-3804; E-mail: fsuplicy@utas.edu.au
ior (Bayne & Hawkins 1990). Normal feeding processes and be-
havior are better measured in experiments where the animals are
allowed to feed on natural seston (Hawkins et al. 1996a. Wong &
Cheung 2001. Gardner 2002),
Most research on the ecophysiological processes in shellfish
has focused on temperate species (e.g., Mytihis ediilis). and there
has been limited work on tropical species and their environments
(Hawkins et al. 1998a, Wong & Cheung 2001 ), Although bivalves
use the same general selective mechanisms for food acquisition
(Hawkins et al. 1998b), there are both intra- and inter-specific
differences in feeding rates (Navarro et al. 1991). Describing the
physiologic responses characteristic of each species is needed,
rather than extrapolating data from other species (Gardner &
Thompson 2001, James el al. 2001). There are likely to be a
number of significant differences in tropical environments. Our
understanding of the feeding physiology of Perna perna (Lin-
naeus. 1758) (Berry & Schleyer 1983, Bayne et al, 1984, van
Erkon Schurink & Griffiths 1992) is limited to laboratory experi-
ments using microalgae monocultures or a mix of microalgae spe-
cies and silt. Furthermore, these studies were carried out in South
Africa where cold south Atlantic currents are predominant; in con-
trast, the Brazilian coast has warm waters brought by central At-
lantic currents. Such differences in temperature and productivity,
and consequently in food availability and its organic content, will
be reflected in ecophysiological differences of these filter feeders.
The aim of this study is to generate a model to predict food
uptake by P. perna in culture areas of Southern Brazil, based on
measurements of the filter-feeding process using natural seston.
123
126
SUPLICY ET AL.
The model reproduce the sequential passage of food through the
feeding steps of filtration, selection, rejection, ingestion, and ab-
sorption, and the calculation of each step is based on relationships
either with quantity and quality of seston or with some of the
preceding steps on the food processing sequence. Mussel aquacul-
ture is a fast growing industry in Brazil and problems regarding the
environmental capacity of this industry may occur in the near
future. This research will have the capability to deliver information
that can be incorporated into models of energy budget and growth
as a function of stocking density, for use in planning and managing
strategies of growing areas.
METHODS
Feeding experiments were conducted at three sites within mus-
sel farms in Southern Brazil; Bnto Cove (48°37'W, 27'46'S),
Porto Belo (48°33"W, 27°8'S). and Arma?ao de Itapocoroi
(48°38'W, 26°58'S). Rope-cultured P. perna were collected from
mussel farms at each site immediately before the experiments. All
experiments were done on one to three occasions at each site and
were exposed to natural differences in concentration and organic
content of seston at each site and time (Table 1 ). Each site was
arbitrarily classified as turbid or clear, based on total particulate
matter (TPM). The clear site had TPM <3 mg L"' (Porto Belo).
while the turbid sites had TPM between 10-40 mg L"' (Brito Cove
and Armagao do Itapocoroi).
The experiments were conducted on a raft containing a tray
with 10 individual 330-mL plastic chambers. Eight individual
mussels, cleared of epibiotic growth, were placed in separate
chambers, with two chambers left empty to act as blanks. Seawater
was pumped into the chambers with flow rates in each compart-
ment between 150 and 200 niL min"'; these were adjusted at the
beginning of the experiment. A battle between the mussel and the
inflow water provided a homogeneous distribution of water flow
inside the feeding chambers (Fig. 1). The mussels were initially
left undisturbed for 1 h to acclimate, after which time all biode-
posits on the bottom of the chambers were removed. Once the
experiment started the mussels were allowed to feed for four hours,
during which time all feces and pseudofeces for each mussel were
separately collected using a pipette immediately after being re-
leased. For each individual mussel the feces and pseudofeces col-
lected in each hour were stored in separate test tubes on ice. A 2-L
sample of inflow seawater was collected every 20 min for the
determination of seston concentration and organic content. Water
temperature and salinity were monitored every hour during the
experiment.
After 5 hours of feeding the experiment was terminated and the
mussels and samples were transported back to the laboratory on
ice. The biodeposit samples were homogenized by repeat pipetting
and filtered onto pre-ashed and weighed Whatman glass microfi-
bre (serie C) 1.2 p-m (GF/C) filters (25 mm or 47 mm diameter).
The samples were rinsed with 15 mL distilled water to remove
salts and dried at 60°C for 48 h before re-weighing and calculation
of the total sample dry weight. Each sample was then ashed at
450°C for 4 h prior to final weighing, allowing calculation of both
of the ash (inorganic) and ash-free (organic) mass of each filtered
sample. To account for settled material in the chamber, the mean
organic and inorganic weight of sediment material collected from
the blank chambers was subtracted from the mean organic and
inorganic weight of the collected feces and pseudofeces. To de-
termine seston concentration and organic content, three 300^00
niL samples from the 2 L of inflow seawater collected were fil-
tered onto pre-ashed and weighed Whatman GFC filters (25 mm
diameter) and dried, ashed, and weighed in the same way as the
biodeposit samples. The mean of the three values was calculated.
The seston concentration and organic content for each hour was cal-
culated as an average of the three 2-L samples taken during that hour.
To determine the lag time between when the mussels consumed
food and when feces and pseudofeces production occurs, mussels
starved for one day in the laboratory were fed green microalgae.
Green feces were observed within an hour of feeding therefore we
assumed the gut transit time to be 1 h. Green pseudofeces were
seen within minutes of the microalgae being added. Therefore, in
the analysis of the field data the quantity and content of the feces
was correlated with seston concentration and organic content in the
preceding hour. No time lag was assumed in correlation with
pseudofeces production. Feeding and absorption parameters were
defined and calculated (Table 2) using procedures outlined in
Hawkins et al. (1996a, 1998b), and using the mean of the hourly
feeding rate obtained for each mussel throughout the experiment.
For the regression analysis, seston concentration and organic con-
tent were the means of the hourly values obtained during each
experimental run.
From each mus.sel used in the experiments, total length was
measured and soft tissue removed, dried at 60°C for 48 h, and
weighed. To standardize findings and allow comparison of results
with other studies, feeding responses were expressed per 1 g dry
weight using Y„ = (W^AV^)*" * Y^„ where Y„ is the coiTected
TABLE L
Summary of envirunmental parameters and mussel size range for each day the experiments were run. Data of environmental characteristics
are the mean ± SD. TPM: total dry particulate mass; POM: total particulate organic matter; OCS = organic content of TPM; ND = no data.
Enviror
imental Characteristics n = 12
Mussels
Experiment
TPM
POM
OCS
Temperature
Turbidity
Shell Length
Dry Weight
Days
Location
(mg L"')
(mg L"')
(fraction)
(°C)
(NTU)
(cm)
<S>
14/().V0I
Brito's Cove
29.6 ± 11.9
4.7 + 3.7
0.15 + 0.05
25.7 ± 0.5
ND
5.05-8.90
0.398-3.522
14/04/01
Brito's Cove
12.4 ±3.0
1.2 ±0.3
0.10 ±0.02
25.5 ± 0.5
7.7 ± 1,7
5.70-8.16
0.485-2.034
05/06/01
Brito's Cove
9.8 ±3.1
1.0 + 0.1
0. 1 1 ± 0.03
22.2 ±0.3
4.5 ± 1.6
5.72-8.27
0.628-2.517
07/02/01
Porto Belo
1.7 + 0.3
0.7 + 0.3
0.41 ±0.17
29.0 ±0.4
0.5 + 0.2
5.74-8.28
1.177-3.257
31/O.VOl
Piirto Belu
1.6 ±0.4
0.3 ±0.1
0.20 ± 0.08
26.5 ± 0.4
1 .0 ± 0. 1
5.05-9.22
0.618-3.103
07/07/01
Porto Bell)
1.2 + 0.3
04 + 0.1
0.36 + 0.09
18.3 ±0.0
tl.3 + 0.1
4.11-8.22
0.343-2.757
26/0,';/01
A. Itapocoroi
4.6 ±0.7
2.3 ± 0.4
0.10 ±0.08
21.3 ±0.2
2.8 ± 0.8
6.00-8.49
0.857-3.087
Modeling Feeding Behavior in Perna fekna
127
Secondary tap
i
Water sample
outflow
Inflow
^
Main tap
Pump
lndi\ idual chamber
Figure 1. Schematic diagram ol' the feeding tray used in the biodeposition experiments.
parameter. W^ is the standard weight ( 1 g). W^, is the weight or
length of the experimental animal, Y^. is the uncorrected parameter,
and b is the average size exponent (Hawkins et al. 2001 ). However,
given the absence of spawning synchronicity (Marques et al.
1991), there is high variability in mussel dry weight within the
same population in every time of the year. Therefore, we used the
shell length equivalent of 1 g dry weight (6.26 cm) and the power
exponent that scales the feeding rates with SL (b = 1.85). The
power exponent has previously been used for Myltlus gcdlopnnin-
ciiiUs (Perez Camacho & Gonzales 1984, Navarro et al. 1996) and
for P. perna (Berry & Schleyer 1983).
All statistical analysis was done using SPSS for Windows,
Version 10 (SPSS Inc.. Chicago, ID and Sigma Plot. Multiple
regression models were fitted using the step-wise technique, en-
tering the most significant independent variable at the first step and
then adding or deleting independent variables until no further vari-
ables could be added to improve the overall fit. The coupling of the
equations to produce an integrated feeding model and the posterior
TABLE 2.
Dennitions and descriptions of the calculation of separate components of feeding behavior.
Parameter
.Acronym
Units
Calculation
Purticulated inorgunic matter
Particuluted organic matter
Organic content of seston
Clearance rale
Total filtration rate
Organic filtration rate
Inorganic filtration rate
Organic content ot tillered matter
Rejection rate
Inorganic rejection rate
Organic rejection rate
Net organic selection efficiency
Ingestion rate
Organic ingestion rate
Inorganic ingestion rate
Net organic ingestion rate
Organic content of ingested matter
Net absorption efficiency from
ingested organics
Net organic absorption rate
PIM
mg L-'
POM
mg L-'
OCS
fraction
CR
1 h-'
FR
mg h~'
OFR
mg h"'
IFR
mg h-'
OCF
fraction
RR
mg h"'
IRR
mg h-'
ORR
mil ir'
NOSE
fraction
IR
mg h"'
OIR
mg h"'
IIR
mg h*'
NOIR
mg h"'
OCI
traction
naeio
traction
NOAR
mg h
Asli tree dry weight of TPM
TPM-PIM
POM/TPM
(mg inorganic matter egesteU both as true feces and pseudoteces h~' -h (mg inorganic
matter available T' seawater)
(mg inorganic matter egested both as true feces and pseudoteces h~') -^ (I-OCF)
CR X mg total particulate organic matter r' seawater
CR X mg total particulate inorganic matter 1"' seawater
OFR ^ FR
mg total pseudoteces egested h~'
RR-ash free mg total pseudoteces egested h" '
RR-IRR
I (-(organic fraction within pseudoteces) -^ (OCS)l
FR-RR
OFR-ORR
IFR-IRR
(FR X (OC.S)|-|RR + (organic fraction within pseudofeces)]
NOIR ^ (FR-RR)
NOAR ^ NOIR
N01R-[(mg total true feces egested h"') x (organic fraction within true feces)]
128
SUPLICY ET AL.
sensitivity analysis was done using STELLA research software
(High Performance Systems, Inc.. Hanover. USA).
RESULTS
Organic content of seston (OCS) decreased as TPM increased
(Fig. 2, Table 3). Clearance rate of mussels decreased froin 10 to
5 L h"' as TPM increased from <3 to 30 mg L"' and OCS in-
creased from <0. 15 to 0.40. The parabolic relationship (Fig. 3A).
suggests that P. perna pumps more water under low TPM (<10 nig
L"') and OCS «0.20) conditions.
Filtration rate (FR. mg h"'). rejection rate (RR. mg h"'). in-
gestion rate (IR. mg h"' ). and net organic absorption rate (NOAR.
mg h"') were all related to TPM and OCS (Table 3. Fig. 3B, C. D.
and E). The nia.ximum filtration rate measured was 156.7 mg h''
when TPM was 33.9 mg L" ' and OCS was 0. 1 8. Rejection rate had
a strong positive relationship with TPM and inverse relationships
with OCS. The maximum rejection rate recorded was 124.1 mg
h"'. which represented 83% of filtered matter, and was measured
under the same seston conditions as the maximum filtration rate.
Pseudofeces production was observed when TPM levels were as
low as 2 mg L"', suggesting a very low threshold for pseudofeces
production in this species.
Net organic selection efficiency (NOSE, fraction) was con-
trolled by the proportion of particulated organic and inorganic
matter in the water (POM. mg L"' and PIM. mg L"' respectively).
Higher NOSE values were observed on the lower and higher ex-
tremes of PIM. Negative NOSE values, a minimum of -0.56, was
recorded at intermediate values of PIM and POM. and positive
values were recorded when POM was below 3 mg L"' and PIM
above 15 mg L"'. Maximum NOSE was 1.71 when PIM was 1.02
mg L"' and POM was 0.67 mg L"' (Fig. 3F. Table 3). Organic
content of ingested matter (OCI. fraction) had a positive relation-
ship with NOSE and it was not strongly affected by TPM. Maxi-
mum OCI was 1.24 when TPM was 33.9 mg L"', OCS was 0.18,
FR was 151.3 mg h*', and NOSE was 1.30 (Fig. 4A, Table 3). The
net organic ingestion rate (NOIR, fraction) was below 10 mg h"'
when mussels were feeding on TPM levels below 5 ing L"'. but
this increased to 25 mg h"' when TPM was above 30 mg L"' and
ingestion rate was ca. 50 mg h"' (Fig. 4B. Table 3).
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Modeling Feeding Behavior in Perna perna
129
of eiivironmeiits. Model predictions and observed data of FR. RR.
IR, NOSE. OCI. and AR of mussels in a range of TPM between 2
and 40 mg L '. are shown in Fig. 7 A. B. C, D. E. and F. respec-
tively, showing that predicted values satisfactorily reproduce the
main trends of feeding behavior observed in P. perna.
As bivalve feeding behavior is mainly controlled by concen-
tration and organic content of seston (Hawkins et al. 1998b). it is
likely that this model is sensitive to these forcing functions (TPM
and OCS). To verify the model sensitivity to changes in the coef-
ficients of the equation that predicts OCS as a function of TPM. we
ran the model three times, varying the coefficients values. Each
coefficient (EQ. ( I ). Table -■^. Fig. 2| was varied by ±10"^* from its
standard value, and the sensitivity was measured by the following
equation:
S = [x/x|/IP/P]
where (S) is a measure of sensitivity, x refers to model outputs at
the end of the integration period in the standard model, and r'»x is
the change in the value of x brought about by varying the model
Figure 3. Perna perna. The relationship between total particulate mat-
ter (TPM, mg !,"') and organic content of seston (OCS, fraction I and
(Al clearance rates (C'R I h '). (I?) filtration rate (FR, mg h"'), (C)
rejection rate (RR. mg h 'l, (I)) Ingestion rate (IR. mg h"'l, (E) net
organic absorption rate (N().\R, mg h 'l. Net organic selection effi-
ciency (NOSE, fraction) is plotted against particulated organic and
inorganic matter (PIM and POM, mg L"') (F). Refer to Table 3 for
equations and statistics.
Both the net absorption efficiency of ingested organics
(NAEIO, fraction) and the net organic absorption rate (NOAR. mg
h'') had a hyperbolic relationship with the organic content of
ingested matter (Fig. 4C and 5. Table ?}. NOAR was essentially
controlled by quantity (filtration rate) and quality (OCI) of food
passing through the digestive system (Fig. 4C, Table 3). The ab-
sorption rate across the experiments varied from 21.84 mg h"
(TPM .3.3.18 mg L '. OCS 0.18) to -0.69 mg h"' (TPM 10.09 mg
L"'. OCS 0.10).
The differential equations, logical functions, and starting values
of the state variables used to couple the equations describing the
filter-feeding processes for P. perna in STELLA are listed on
Table 4. We produced a robust model with relatively low com-
plexity and specificity. Figure 6A depicts the conceptual diagram
of the P. perna feeding process as a function of TPM and OCS.
The sub-model inserted inside the "ingested matter" variable (Fig.
6B ) reproduces the absorption of organic matter and the passage of
inorganic matter as inert material through the gut. As the model
was based on natural seston in both turbid and clear environments
and feeding rates measured in these environments, we believe that
it has incorporated feeding adaptations by P. perna for both kinds
B
Figure 4. Perna perna. The relationship between (.A I net organic se-
lection elTiciency (NOSE, fraction), total particulate matter (TPM. mg
L"') and organic content of ingested (OCI. fraction): (B) ingestion rate
(IR, mg h"'), TPM and net organic ingestion rate (NOIR. mg h'); (C)
net organic absorption rate (NO.\R, mg h"'), filtration rate (mg h"')
and OCI. Refer to Table 3 for equations and statistics.
130
SUPLICY ET AL.
O
• •
-0,2
0.0
02
1.0
12
1.4
04 06 08
OCI (fraction)
Figure 5. Penia perna. The relationship between the organic content
of ingested (OCI, fraction) and the net absorption efficiency from
ingested organics (NAEIO, fraction). Refer to Table 3 for equaliiins
and statistic.
coefficient. Similarly, the denominator measures the variation in
the coefficient of interest divided by its standard value. This equa-
tion compares the percentage change in the model outputs with a
given percentage change in one of the model parameters. The
value of (S) was averaged for positive and negative variations and
the results of the model outputs (absorbed matter, pseudofeces, and
feces produced) for the coefficients relating TPM and OCS are
shown in Table 5. The output most sensitive to variation in the
relationship between seston TPM and OCS was pseudofeces pro-
duction, as a result of increased or decreased rejection rate.
DISCUSSION
This study showed that P. perna, like other mussels, controlled
its feeding mechanisms to achieve an optimum organic absorption
rate independent of fluctuations in seston concentration and qual-
ity. It is important to note that the range of TPM recorded was
within normal values during the year for other bivalve aquaculture
locations in Southern Brazil (Suplicy, unpub. data). Therefore, the
TPM range experienced in the experiments and included in the
model are directly applicable to Brazilian shellfish famis condi-
tions. Although seasonal changes in feeding physiology were not
examined in this study, time series data of TPM, POM, and OCS
from 1998 to 2002 do not suggest strong seasonal changes in food
availability in the sub-tropical waters of Santa Catarina, (Suplicy et
al. unpublished data). Similarly, the condition inde.x of P. perna
does not follow a seasonal trend, as seen in Mylilus echtlis (Navanxi
& Iglesias 1995), because spawning occurs throughout the year
with small peaks in summer, autumn and spring (Marques et al.
1991 ). Therefore, we believe that the findings reported here can be
used to predict feeding physiology throughout the year.
Food availability (TPM and OCS) was the main forcing func-
tion of the models produced, therefore characterizing the available
seston is of primary importance to generate a model to predict food
uptake by P. perna. Data for Southern Brazil showed that the
organic content of available food decreased as TPM increased, a
common pattern in many estuaries and sheltered bays both in
teiTiperate and tropical waters (Hawkins et al. 1996a, 1998b). This
reduction of the organic proportion is a function of the dilution of
organic particles when resuspended silt increases particulate inor-
ganic matter on the water column (Frechette & Grant 1991. Wid-
dows et al. 1979)
The methods used in this study to estimate clearance rates of
filter feeders were less accurate than the methodology proposed by
Hawkins et al. (1998a, 1999) for measurements using natural
seston. The most appropriate method to accurately measure clear-
ance rates by bivalves is controversial (Cranford 2(M1. Riisgard
2001. Widdows 2001 ). As new methods are being developed, new
models about how these animals control their food uptake are
being produced. It is agreed that mussels do not always filter at
their maximal rate in their natural environment (Riisgard 2001.
Widdows 2001 ). This may be due to a regulation of feeding pro-
cesses in response to changes in quantity and quality of suspended
particles, salinity, temperature, and the presence of pollutants in
the water (Widdows 2001 ). In this study only ll^c of the variation
clearance rates of mussels using TPM and OCS as independent
variables was explained, and the significant proportion of the re-
maining variance in clearance rate in POM was not. In their ex-
periments, however, Hawkins et al. (1999) increased the amount of
the variability in clearance rate explained from 13-,').^'/f when they
included Chi and TPM as independent variables instead of only
POM. Although all precautions proposed by Iglesias et al. (1998)
in the use of the biodeposition method for suspension-feeding
TABLE 4.
Equations used in the formulation of feeding physiology model
in STELLA.
TPM = GRAPH (time-series)
OCS = 1/(2.55 -hO.47 * TPM)
PIM = 0.22 -1-0.81 * TPM
POM = TPM-PIM
PR = 68.77-0.12 TPM-370.10 OCS -i- 0.07 TPM" -i- 565.80 OCS"
Fillered matter (t) = Filtered matter (t - dt) + (FR - RR - IR) * dt
INIT Filtered matter = 219.81
RR = 52.43 + 0.97 TPM-362.47 OCS -i- 0.02 TPM" + 589.79 OCS"
Pseudofeces (5) = pseudofeces (t - dt) -f (rejection) * dt
IR = filtration-rejection
Ingested (t) = ingested (t - dt) + (ingestion' - NIIR - NOIR) * dt
INIT ingested - 36.46
NOIR = 1.37 - (.1.23 TPM -i- 0.1 1 IR -i- 0.01 TPM- + 0.004 IR"
NIIR = mgested-NOIR
Inorganic (t) = inorganic (t - dt) ■(- (NIIR - IM on gut) * dt
Organic (t) = organic (t - dt) + (NOIR - OM on gut) * dt
OM on gut = organic
Im on gut = inorganic
INIT organic = 13.17
INIT inorganic = 23.29
Ingested matter = food on gut + organic + ingested -i- inorganic
Food on gut (1) = food on gut (t - dt) -i- (OM on gut -i- IM on gut -
absorption' - egestion') * dt
INIT food on gut = 0
NOSE = 0.30 - 0.21 PIM -i- 1.03 POM + 0.01 PIM" - 0.20 POM-
OCI = 0.13 - 0.001 TPM + 0.27 NOSE -t- 0.0002 TPM" -i- 0.19
NOSE-
NOAR = -2.62 -I- 0.012 RF -i- 15,73 OCI -i- 0.0001 FR- - 9.22 OCI"
Ahsorption' = NOAR
Absorption = absoiption'
Absorbed matter (t) = absorbed matter (t - dt) + (absorption) * dt
INIT absorbed matter = 0
Egestion' = IM on gut + (NOIR-NOAR)
Egestion = egestion'
Feces (t) = feces il - do -i- (cgestioni * dt
Modeling Feeding Behavior in Perna perna
131
absofbed matter
-^
pseudofaeces
B
organic
organic matter
Figure 6. (A) Diagram of the feeding processes of a general niter-fceding bivalve, used on the modeling of P. perna feeding physiology. (B)
Diagram of the sub-model of a mussel gut showing the absorption of organic matter and feces production. Refer to Tables 2 and 3 for variables
and acronyms and Table 4 for logical and differential equations.
measurements were taken in this study, it seems that the new
methodology proposed by Hawkins et ai. (1998b, 1999) is more
appropriate for studies using natural seston. It seems that qualita-
tive features of seston may be just as important as availability of
food in mediating feeding responses (Hawkins et al. 1998b). The
general trend for decreasing clearance rates as seston concentra-
tions increase, however, is seen in other studies (Hawkins et al.
1999, Hawkins et al. 1998b, Wong & Cheung 2001). There are
many methods to quantify concentration and organic content of
seston in feeding experiments. Most use mass measurements of
total particulate matter available in the seston (TPM, mg L"'),
pailiculate organic matter available in the seston (POM, mg L"'),
and the ratio between these two variables, which is the organic
content of seston (OCS. fraction). Recent findings suggest that
clearance rate is primarily dependent on seston availability mea-
sured in terms of total volume, rather than mass. This helps to
explain the confusing variation in clearance rate reported by many
studies and stresses a need to consider volumetric constrains in
bivalve feeding studies (Hawkins et al. 2001 ). More detail about
the seston organic fraction can be obtained if the carbon;nitrogen
ratio is measured, which can vary from <4 to >26 (Bayne &
Hawkins 1990). The measurement of the biologically available
132
SUPLICY ET AL.
160
140
-. 120
E
— 80
cr
^ 60
1 0
08 -
I"'
o
m
^ 04
UJ
to
O 02
00
E,
a: 40
TPM (mg I"')
0 10 20 30 40 50
TPM (mgr')
Figure 7. Predictions of the P. pcnia filter-feediii}; model produced on
STELLA. (A» nitration rate IFR, nig h '), (B) rejection rate (RR, nig
h"'). (CI ingestion rate (IR. mg h '), ID) selection efficiency (NOSE,
fraction), (E) organic content of ingested matter (OCL fraction), and
(F) net organic absorption rate (NOAR, mg h~'), in the range of total
particulate matter (TPNL mg L ') observed in this study.
organic carbon and nitrogen in the water and in associated biode-
posits can provide, not only more accurate measurements of the
clearance rate, but also important information about the absorption
of these elements by filter feeders.
The biodeposiljon approach demands that the gut residence
time is correctly calculated to generate accurate physiologic feed-
ing rates. As starved animals were used to estimate gut passage
time this may have over-estimated the normal passage time. How-
ever, our estimates are comparable to those from other biodepo-
sition studies using Penui canaliculus, in which the gut passage
time for non-starved mussels was 80 min. and no delay time was
assumed for Perna viridis (Hawkins et al. 1998al.
Perna perna appeared to selectively enrich the organic content
of ingested matter by rejecting particles of higher inorganic con-
TABLE 5.
Sensitivity analysis of absorbed matter, pseudofeces and feces
production for the coefficients a and h in the equation OCS = l/(a +
b * TPM).
Absorbed
Matter
Pseudofeces
Feces
0.2(12
0.2-^2
0.(1.^7
0.7-14
0.118
0. 1 36
tent before ingestion. This selection efficiency was a function both
of filtration rate and the proportion between inorganic and organic
particulated matter available in the water. The increase in selection
efficiency at higher filtration rates is important, because this helps
to maintain nutrient acquisition independent of fluctuations in
seston organic content (Hawkins et al. 1998a). Extreme values of
net organic selection efficiency measured in this study (NOSE >l
or <0) must be considered with caution as they are probably mea-
surement errors associated inadvertently with collecting settled
sediment when collecting biodeposits. This would effectively alter
the organic ratio of pseudofeces. Extreme values were observed in
15% of measurements. Nevertheless, NOSE values recorded in
this study (>0.7) suggest that P. perna is efficient in selecting
organic particles available in the seston. Hawkins et al. (1996a)
recorded NOSE values of up to 0.5 in M. edulis. and Hawkins et
al. ( 1998b) report maximum NOSE of 0.7 for P. viridis.
Maximum net organic ingestion rate (NOIR) recorded for P.
perna was 24.05 mg h"' and occurred when TPM was 33.93 mg
L"' and OCS was 0.18. This is similar to values obtained for P.
canaliculus in New Zealand, that showed maximum organic in-
gestion rate of 27.3 ± 6.3 mg h"' (Hawkins et al. 1999), and for P.
viridis in Malaysia with a recorded rate of 24.8 ± 3.6 mg h"'
(Hawkins et al. 1998a). These rates are considerably higher than
the maximum organic ingestion rate of 6.5 mg h"' reported for M.
edulis (Hawkins et al. 1997). The growth rates of P. perna in
southern Brazil are among the fastest reported for mussels in the
Perna genus, reaching commercial size (80 mm) in 8-10 mo (Su-
plicy. unpub. data). This rapid growth is probably related to higher
weight-specific rates of energy acquisition and higher water tem-
peratures in the sub-tropical waters of southern Brazil.
Data from this study suggested that P. perna takes advantage of
the abundant organically rich seston available in Brazilian waters
throughout the year by maintaining high ingestion rates. There is
evidence that when ingestion rate is high absorption efficiency is
high and gut residence time is short (Bayne et al. 1988). Fuilher-
more, the proportion of gut volume occupied by ingesta may vary,
thereby facilitating an increase in absorption efficiency with little
change in the gut passage time (Bayne et al. 1987). Widdows et al.
( 1979) report that absorption efficiency declines as ingestion rate
increases and food progresses from the digestive gland to the in-
testine. However, this pattern may be counterbalanced by elevated
organic content of ingested matter due to selection processes (this
study, Hawkins et al. 1999) that positively increase the absorption
efficiency and ultimately the absorption rate. Similarly to the con-
siderations raised for NOSE values, negative absorption rate val-
ues are not biologically meaningful and must be considered with
caution as these could be caused by collection of inorganic sedi-
mented material together with mussel feces. Negative absorption
rates were measured in 7% of measurements.
The integration of all equations from Table 4 with STELLA
software resulted in a reductionistic and deterministic non-linear
model that reproduces the feeding processes of P. perna in both
clear and turbid environments. The general conceptualization of
the diagram was based on the description of the bivalve filter-
feeding process provided at the TROPHEE workshop (Bayne
1998. Hawkins et al. 1998b), and final equations were based on
intensive measurements that enabled calibration of the outputs.
This feeding model may not be a perfect reproduction of the bi-
valve feeding process, but the objective is to provide a useful tool
to understand and predict feeding processes of this species. The
model includes a complete sequence of steps in the feeding process
Modeling Feeding Behavior in Pekna perna
133
that may cause an accumulation of predictive error (Grant &
Baciier 1998). Its value lies in the ability to provide an understand-
ing of the interaction between a mussel farm and the environment,
for example, the amount and organic content of biodeposits re-
leased into the water column and sediment beneath the farm.
Sensitivitv analysis indicated that model predictions of ab-
sorbed matter and feces production were less affected by changes
in the relationship between TPM and OCS than model prediction
of pseudofeces production. This analysis suggests that predicted
absorption would stay reasonably invariable if the model is applied
to environments with different seston concentration and organic
content. Therefore, mussels maintain a reasonably constant or-
ganic ingestion rate in varying seston conditions by compensating
for low organic content of the seston through adjusting selection
efficiency and rejection of inorganic matter as pseudofeces.
This feeding model can be used as an important tool for the
understanding of how P. pcniu interact with the culture environ-
ment. Current studies are under way to integrate this feeding model
w ith energy budget and population dynamics of P. perna. Further
coupling of the P. perna biologic models with physical models of
seston hvdrodynaniics and models of primary production are also
planned, and this approach will allow the development of cairying
capacity analysis for suspended mussel culture in sub-tropical en-
vironments like the southern Brazilian coast.
ACKNOWLEDGMENTS
The research was supported by CNPq, a Brazilian govcrnnienl
agency for scientific and technologic development. The authors
thank two anonymous reviewers for their valuable criticism and
comments of the original manuscript.
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Joiinuil „f Shflllhh Research. Vol. 22. No. 1, 135-140. 200.^.
PHENOTYPES OF THE CALIFORNIA MUSSEL, MYTILUS CAUFORNIANVS, CONRAD (1837)
JORGE CACERES-MARTINEZ,'* MIGUEL A. DEL RIO-PORTILLA.'
SERGIO CURIEL-RWIIREZ GUTIERREZ,' AND IGNACIO MENDEZ GOMEZ HUMARAN"
^ Departamento de Aciiicultura del Centra de Investigacion Cientifica y de Ediicacion, Superior de
Ensenada. A. P. 2732 C. P. 22860 Ensenada. Bajci California. Mexico: 'Instiruto Nacional de la Pesca,
Pitdi^oras 1320 6° Piso, Col. Sia. Cruz Aroyac. C.P. 03310. Mexico D.F.
ABSTRACT The morphological variability of Mytilii.s ediilis complex species has been the subject of a variety of studies. However,
the morphological variability of Mytiliis califomiumis has not been studied. We found that there are some M. californiamis without
some of the shell characteristics mentioned by Conrad ( 1837) in the original description of this species. The most remarkable difference
was the absence of radial ribs on the exterior of the shell: thus, we tested the presence of at least two phenotypes in M. ealiforniainis.
Six hundred ninety five M. ealiforniainis of different sizes were collected from the locations La Mina del Fraile. La Bufadora, and La
Salina in Baja California. For comparison, 58 M. i^atloprovincialis were collected from an aquaculture facility at Rincon de Ballenas
in Bahi'a de Todos Santos. Baja California. Fourteen morphometric measures and the weight of the shell were measured and a principal
coinponent analysis (PCA) and a logistic regression (LR) were carried out to tlnd differences between mussels studied and for obtaining
a prediction to assign the phenotypes. The presence of ribs, small ligament margin, a narrow posterior byssal retractor muscle scar, and
shell weight were the discriminating characters between two groups in M. californiamis. These findings confirm the presence of at least
two phenotypes in this species, in all mussel sizes and the studied locations. The LR correctly assigned 99.28% of the shells to each
phenotype. and it considered only eight out of the fifteen morphometric measures. The PCA showed a clear morphologic difference
between both phenotypes of A/, ealifornianiis and A/, gulloprovineialis. The original description of this species by Conrad in 1837 was
done taking into account only the phenotype with ribs.
KEY WORDS: Mytilus ealifornianiis. Mytiliis eihilis complex species, morphological variability, phenotypes
INTRODUCTION
MATERIALS AND METHODS
The marine mussels of the genus Mytilus are widely distributed
in boreal and temperate waters of the Northern and Southern
Hemispheres (Soot-Ryen 1955), Prior to protein separation and
molecular genetics, about nine species of the genus Mytilus were
recognized (Gosling 1992). Today, about five species are consid-
ered belonging to this genus: Mytilus californiamis. Mytilus cor-
».«■;(.? (Gould 1861), Mytilus edulis (Linne 1758). Mytilus gallo-
provincialis and Mytilus. trossulus (Gould 1850) (Seed 1992), The
three later species are considered to be the M. edulis complex
species because they are very close in their external shell mor-
phology. These species have caused a variety of studies for their
differentiation, taking into account shell morphology, allozyme,
and molecular genetics (Beaumont et al, 1989. Figueras &
Figueras 1983, McDonald & Koehn 1988. Koehn 1991, McDonald
etal. l99l,Gelleretal. 1994. Inoue et al, 1995. Rawson & Hilbish
1995. Ohresser et al, 1997), Mytilus californianus has never been
questioned as a separate species from the Mytilus edulis-comp\ex
because of its characteristic radiating ribs, strong growth lines, and
heavy shell in larger specimens: these characters allow easy dif-
ferentiation from the other species in adult stage (Soot-Ryen 1955.
Koehn 1991). During a field study of Mytilus californianus in an
exposed rocky shore of the West Coast of Baja California, Mexico,
we found some specimens with typical external characteristics of
the shell described by Conrad in 1837. Other individuals, however,
showed a smooth shell without coarse ribs, similar to the M. edulis
complex form, but with heavy shells. A question arises from this
observation, are there two or more phenotypes of M. califor-
nianus'l This study focused on answering this question.
*Corresponding author. Mailing address: Department of Aquaculture.
CICESE. PO Box 434844, San Diego, CA 92143
In March 1997, 129 M. californianus (size range from 16,8-
1 13,5 mm, mean size 59,1 mm) were collected from an exposed
rocky shore along the intertidul zone during low tide in La Mina
del Fraile. B. C, Mexico, In August 2()(X), 278 mussels were col-
lected from La Salina (size range from 27.6-98.1 mm, mean size
56.9 mm) and 288 from La Bufadora (size range from 44.7-88.1
mm. mean size 54.1 mm). B. C. Mexico, both areas exposed rocky
shores, and the mussels were collected during low tide along the
intertidal zone. Additionally. 58 M. galloprovincialis were ob-
tained from culture long-lines placed at Bahi'a de Todos Santos,
B,C. (size range from 47.2-85.3 mm. mean size 61.4 mm) and they
were used to compare the morphological characteristics with M.
californianus (Fig. 1 ).
The shells of ail mussels were cleaned with a brush and water
stream and dried in an oven at 40"C overnight. The following
morphometric dimensions were measured for differentiation
among mussel groups and species (Fig. 2): number of ribs on the
external shell (rib), maximum shell length (si), height (sh) and
width (sw), the position of maximum shell width (a) along the
dorso- ventral axis, the maximum dimensions of the anterior (aams)
and posterior (pam) adductor muscle scars, the maximum length
(Ibr) and width (wbrs) of the posterior byssal retractor muscle scar,
the location of the center of the posterior adductor muscle scar
along both the anterior-posterior (pam-pm) and dorso ventral
(pam-vm) axes, the size of the hinge plate (hp) and number of
hinge teeth, the distance between the palial line and the \entral
shell margin (pl-vm) midway along the shell, and ligamentary
margin (Im), All measurements were taken with an electronic digi-
tal caliper to the nearest 0. 1 mm and were in accordance with those
taken by Beaumont et al, ( 1989), The dry shell weight (w) was also
measured for all mussels and it was included in the analyses, A
principal component analysis (PCA) was carried out to discrimi-
nate between phenotypes, followed by a logistic regression (LR)
135
136
Caceres-Martinez et al.
_32"3r
Pacific Ocean
V La Salina
\ Baja California
_32«
^^ <^\ Ensenada
Baja V
California'
Mussel culture facilityN.
• a]
La Bufadora \
_30°3r
^- — f
1 1 ,„ /La mina del Fraile
1 4
Figure 1. Map showing the three exposed rod^y shore localities where
Mytihis californiaiius was collected: La Mina del Fraile. La Bufadora.
and La Salina. The blue mussel yfyliliis f;allopr(niiiciali\ was collected
from a culture facility at Bahia de Todos Santos, Baja California.
Mexico.
(Sokal & Rohlf 1995) to fit the mussel phenotypes. A two way
ANOVA was used to determine possible differences between mus-
sel size among locations and phenotypes. A comparison through
the PCA between both phenotypes of M. culifonmimis with M.
gaUoprovincialis was carried out. These analyses were done using
the JMP statistical package by SAS Institute Inc.
RESULTS
Fifteen piincipal axes were extracted from the morphological
and shell weight data of M. califomiamts (Table I). The first
component explained 16% of total variance and was considered as
a size axis. A low correlation of size with number of ribs sug-
gests that the number of ribs does not change w ith mussel si/e. The
second component accounted for 1% of the variation indicating
morphological differences. Mussels with a high number of ribs
were correlated with this second component separating two groups
(Fig. 3). Also, in the second component, mussels with higher shell
weight (w). but with small ligament margin dm) and a naiTow
posterior byssal retractor muscle scar (wbrs) were coiTelated. The
rest of the components had eigen values smaller than the unit
accounting for about 139<^ of the total observed variance and thus,
no further explanation is necessary (Table 1 ). These data provide
statistical support to validate the presence of two phenotypes in M.
californiwms: A (with ribs) and B (without ribs), and they were
visually differentiated in mussels of different sizes (Fig. 4). After
separating both groups in all locations. 689f of the total mussels
belong to phenotype A and the rest to phenotype B.
Both phenotypes of M. caUfornianus were present in the three
locations. The two way ANOVA showed size differences among
mussel from different locations. (F -.f,^^ = 6.58. P = 0.001 ). but
the phenotype mean size was similar (F , ^^1) ~ 0.02. P = 0.892)
without interaction (F ,f,gg = 2.11. P = 0.122).
Once the PCA differentiated two phenotypes. the LR (Sokal &
Rohlf 1995) was used to determine whether it was possible to
assign any M. ctiUfonuanus to a particular phenotype. taking into
account morphological \ariables. excluding the number of ribs.
The LR considered only eight morphological measures from the
original fifteen to assign any mussel to a particular phenotype.
(X-
1 84.73. P < 0.0001 ; Lack of fit: x"
684.5. P
= 0.51). The coefficients of the eight morphometrical variables
were positive for: shell length (si = 0.104) and height (sh =
0.247). posterior adductor muscle scar (pain = 0.483). the dis-
tance between the palial line and the ventral shell margin (pl-vm
= 0.708). and weight (wO. 145); while the shell width (sw =
-0.281). the position of maximum shell width (a = -0.333). and
the ligamentary margin (Im = -0.348) were negative. After ap-
plying the LR we found that 99.28% were correctly assigned to
each phenotype. Thus, the visual. PCA and LR confirm the pres-
ence of two phenotypes in the Californian mussel.
Results of the PCA between morphometric data and v\eight of
Figure 2. Morphometric dimensions measured for Mytilus califoniiamis and Myliliix galldprovincialis: number of ribs on the external shell (ribi,
maximum shell length (si), height (sh) and width (sw), the position of maximum shell width (al along the dorso-ventral axis, the maximum
dimensions of the anterior (aams) and posterior (pam) adductor muscle scars, the maximum length (Ibrl and width (wbrs) of the posterior byssal
retractor muscle scar, the location of the center of the posterior adductor muscle scar along both the anterior-posterior (pam-pm) and dorso
ventral (pam-vm) axes, the size of the hinge plate (hp), and number of hinge teeth, the distance between the palial line and the ventral shell
margin (pl-vm) midway along the shell and ligamentary margin (Im).
Phenotypes of the California Mussel
137
TABLE 1.
Eigenvalues, explained variance (^rl. cumulative explained variance I "^i- 1 and eigenvectors (rounded to luo decimal places) from the
principal component analysis of Myliltis califoniUiiiiis niorphometric data from the Facillc coast of Baja California.
Principal Components
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
Eigenvalue
11.45
1.07
0.59
0.45
0.29
0.25
0.22
O.IS
0,14
0,11
0.08
0.08
0.05
0.03
0.02
Variance!*)
76.31
7.12
3.93
3.02
1.94
1 .69
1.46
1.17
0.96
0,71
0.51
0.50
0.35
0.21
0.12
Cum. var.(%)
76.31
83.43
87.36
90.37
92.31
94.00
95.46
96.64
97.59
98.3
98.81
99.32
99.67
99.88
100.00
Eigenvectors
rib
0.02
0.95
0.17
-0.02
0.02
-0.02
-0.06
0.06
0.20
0,07
0,04
0,09
0.04
-0.03
0.01
si
0.29
-0.04
0.02
-0.15
-0.06
-0.10
-0.10
-0.03
0.20
0.03
-0. 1 3
-0. 1 2
0.19
0.16
-0.86
sh
0.28
0.05
-0.11
-0.01
0.04
0.01
0.03
0.32
0.07
-0.23
-0.16
-0.12
-0.82
0.15
-0.03
sw
0.29
-0.06
0.04
-0.09
0,02
-0.12
-0(16
-0.12
0,04
-0,01
0,24
0, 1 7
-0.17
-0.86
-0.11
a
0.27
-0.06
-0.27
0.09
0.14
-0.09
0.15
0.62
0.22
-0.30
0.07
0.26
0.42
-0.02
0.12
aams
0.26
0.01
0.05
-0. 1 1
-0.04
0.92
-0.14
0.03
-0.13
-0.09
0.06
0.01
0.10
-0.04
-0.01
pam
0.27
0.05
0.14
-0.33
0.29
-0.08
0.48
0.17
-0.39
0.34
0.24
-0.35
0.06
0.05
0.04
Ibr
0.28
-0.04
0.00
0.03
-0.08
-0.05
-0.20
-0.13
0.45
-0.04
0.00
-0.70
0.16
-0.07
0.35
wbrs
0.21
-0.13
0.76
0.59
0.04
-0.01
0.07
0.10
0.00
0.01
-0.05
0.06
0.00
0.04
-0.01
pam-pni
0.28
0.04
0.14
-0.31
0.00
-0.13
0.03
-0.17
-0.24
-0.20
-0.75
0. 1 3
0. 1 5
-0.08
0.22
pam-vm
0.28
0.00
0.15
-0.22
-0.04
-0.21
-0.17
-0.33
-0.12
-0.45
0.5 1
0.20
-0.01
0.38
0.11
hp
0.25
0.05
-0.34
0.35
0.61
0.09
0.16
-0.48
0.11
0.09
-0.07
0. 1 3
-0.02
0.10
-0.01
Im
0.27
-0. 1 8
0.04
-0.23
-0.16
0.00
-0. 1 3
0.04
0.33
0.64
-0.01
0,41
-0.10
0.21
0.23
pl-\iii
0.26
0.10
-0.27
0.32
-0.07
-0.17
-0,57
0.16
-0.54
0.23
0.00
-0.08
0.06
0.02
0.02
u
0.25
0.12
-0.25
0.28
-0.69
0.02
0.51
-0.20
-0.08
0.01
0.03
0.02
0.02
0.01
0.00
M. ccdifornianus and M.gaUoprovincialis are shown in Table 2.
The fist component explained 71% of the total variance and was
also considered a size axis. The number of ribs had a low corre-
lation with this axis. The second component explained 10% of the
total variance. The number of ribs (rib) and the shell width (sw)
were positively correlated with this component whereas the width
of the adductor muscle scar (wbrs) was negatively correlated.
Components 3 to 15 accounted for 19.4% of the total variance, but
their eigen values were smaller than one and they are not explained
further. The graphic presentation of the component scores shows a
Figure 3. Principal component scores plots between PC2 vs. PCI for
M. calif ornianiis. Phenotype A. open circles; Phenotype H, bold
squares.
clear difference between phenotypes of M. californianus and
among these phenotypes and M. gaUoprovincialis (Fig. 5).
DISCUSSION
Figure 4 shows different shell characteristics among M. cali-
fornianus specimens, and the PCA and LR support this visual
perception confirming that there are two phenotypes in M. cali-
fornianus. one with ribs and the other with a smooth shell, and
Figs. 4 and 5 show a morphological differentiation between both
phenotypes of M. californianus and M. galloprovincialis.
The original description by Conrad (1837) for Mytilus califor-
nianus was done from specimens collected by Thomas Nuttal in
upper California. Conrad describes "shell ovate elongated, in-
flated; anterior margin straight; posterior side emarginated; ribs
not very numerous, slightly prominent broad, rounded; lines of
growth very prominent"". This description agrees with phenotype A
studied here, where the rib number goes from 4 to 14 and they are
very prominent. In phenotype B. however, the ribs are not distin-
guishable and the growth lines are very prominent. Intraspecific
differences in shell sculpture on specimens from different habitats
have been noted in several gastropod species from the genus Lil-
torina (Struhsaker 1968. Johannesson et al. 199.3. Rush 1997).
These differences have been related to the degree of wave expo-
sure— extreme ribbed and with nodes forms live on dry raised
benches, not generally subject to horizontal water swash; while
extreme smooth forms predominate on low. moist benches subject
to strong wave swash. It is probable that a similar relation occurs
among M. californianus phenotypes and wave action or their po-
sition along the intertidal zone. We are carrying out a field study
to explore this. The presence of ribs has been conelated with shell
strength; the ribbed mussel Geukensia emissa has a stronger shell
than M. edulis. this strength was correlated with shell mass, shell
curvature and valve thickness (Majewski 1995). This could also be
138
Caceres-Martinez et al.
Figure 4. Mytiliis califoniianiis of different sizes showing the two phenotypes found in this study: ( Al with rihs and (B) no ribs. For comparison,
Mytilus gallopruvincialis of similar sizes also were included in figure (C). Note that different phenotypes appeared since young specimens.
TABLE 2.
Eigenvalues, explained variance ( 7r ), cumulative explained variance I '''< ) and eigenvectors from the principal component analysis between
Mytiliis califoniianiis and Mytilus galloprorincialis morphometric data from the Pacific coast of Baja California.
Principal Components
10
II
12
13
14
Eigenvalue
VarianceCvJ- )
Cum. var.(%l
rib
si
sh
sw
a
aanis
pam
Ihr
wbrs
pam-piTi
pam-vm
hp
Im
pl-vm
10.587
VCSSI
70.581
0.015
0.301
0.278
0.229
0.281
0.266
0.275
0.292
0.184
0.286
0.276
0.259
0.281
0.258
0.25.^
1.502
10.013
80.594
0.621
-0.043
-0.205
0.^94
0.016
0.000
0.05 1
0.037
-0.476
0.007
-0.201
0.030
-0.169
0.253
022.^
0.751
5.009
85.603
0.737
-0.010
0. 1 82
-0.339
-0.163
0.036
0.074
-0.083
0.428
0.091
0.190
-0.035
-0.094
-0.144
-0.101
0.459
3.063
88.665
0,001
-0.139
0.182
-0.291
0.220
-0.102
-0.334
-0.023
0.145
-0.326
-0.166
0.553
-0.223
0.325
0.274
0.3 I I
2.075
90.741
-0.044
-0.017
-0.268
0.279
-0.222
-0.114
-0.228
0.144
0.582
-0,061
-0.074
-0.373
-0.007
0.187
0437
0,277
1.844
92,584
-0,011
0,014
-0.142
0.269
0.073
-0.722
0.298
0.022
0.237
0.107
0.034
0.294
-0.128
0.074
-0,339
0.263
1 .755
94.139
-0.030
-0.118
-0.265
0.277
-0.169
0.592
0.048
0.030
0.287
-0,122
-0.197
0.336
-0. 1 .%
0.044
-0.432
0.216 0.173
1.442 1.155
95.781 96.937
Eigenvectors
-0.077
-0.125
-0.074
0.040
0.028
0.046
0.454
-0.179
0.094
-0.002
-0.211
0.222
-0.138
-0.585
0.516
0.020
-O.IOI
0.I7I
0.000
0.588
0.087
0.317
-0.216
0.185
-0.170
-0.344
-0.453
-0.075
0.215
-0.147
0.143
0.956
97.893
0.215
0.169
0.016
0.126
0.294
-0.100
-0.264
0.459
0.05 1
-0.318
-0.258
0.031
0.341
-0477
-0.129
0.100
0.667
98.560
0.052
0.007
-0.034
-0.211
-0.383
-0.094
0.344
-0.098
-0.020
-0.257
-0.309
0.093
0.652
0.268
0.027
0.079
0.527
99.086
0.062
-0.004
-0.077
0.077
0.166
-0.010
-0.388
-0.480
0.102
0.580
-0.317
0. 1 35
0.329
-0.053
-0.018
0.074
0.496
99.582
0.086
-0.161
-0.156
0.273
0.198
0.019
-0.080
-0.467
0.02!
-0.432
0.573
0.055
0.283
-0.062
0.037
0.045
0.299
99.882
-0.032
-0.209
0.764
0.467
-0.322
-0.065
-0.092
-Olio
-0.004
-0.073
-0.109
-0.019
-0.016
-0.074
-0.040
0.018
0.118
100.000
0.013
-0.866
-0.047
-0.074
0. 1 1 3
-0.006
0.036
0.353
-0.016
0.222
0.098
-0.012
0.210
0.016
0.000
Phenotypes of the California Mussel
139
CM
O
Q.
2
0
-2
4
■ ■
^ TtTT ▼
t'V
▼ ' T
-10
-5
0 5
PC1
10
15
Figure 5. Principal component scores plots between PC2 vs. PCI for
M. californiaiiiis Phenotype A, open circles; Phenotype B, bold
squares; and M. galloprmincialis bold triangles.
the case for M. caUfoniiiiniis where the presence of ribs might
indicate a stronger sheH.
In accordance with Seed (1968), variations in the M. cJiilis
shell form can be attributed to differences in age, habitat, growth
rate, and density. Old mussels have heavier shells, down-turned
divergent innboes, and varying degrees of incurvature of the ven-
tral shell margin than the young ones do. In this study, small and
large individuals showed similar morphometric characteristics;
therefore, the age or size of these mussels (which grow in the same
habitat) seems to have little influence on the variability of the
studied morphological characters. In relation to the habitat. Seed
(1968) comments that in areas free of predators (like the upper
shore) old individuals are common, whereas in areas where the
mussel turnover is rapid there is a predominance of young mussels.
Also, the presence of predators can affect shell morphology. M.
edulis has been found to ha\e a smaller shell lencth. heii;ht and
width with larger posterior adductor muscle, thicker shell, and
more meat per shell volume when a starfish was present (Reimer
& Tedengren 1996). In the Baja California region. M. califor-
niunus is the dominant species where there is high wave action,
whereas M. gallopravincialis is the dominant species in protected
bays with thinner shell and more meat than M. californianus
(Harger 1970. Harger 1972). It has been observed that shore level
has an influence on the morphology and physiology of M. gallo-
provincialis in the Adriatic see (Dalla Via et al 1987). Low shore
level mussels have higher and narrower shells and a higher dry
weight ratio whereas high shore mussels have a higher o.xygen
consumption rate. When cultivated Mytihis edulis was transplanted
between two different locations there were some morphological
differences that were considered to be due to genetic variation
(Stirling & Okumus. 1994). The same characters found in parents
of distinct ecotypes also occurred in progeny raised in the labora-
tory thereby indicating that the phenotypic differences have a ge-
netic basis (Struhsaker 1968). The presence of two phenotypes and
similar morphometric characteristics of the shell in small and large
M. californianus in all three locations indicates not only some
similarity among environments but it also strongly suggests that
the presence of ribs is genetically produced. To our knowledge.
there is no record on hybridization between M. californianus and
M. gallopravincialis. which could result in a heavy shell without
ribs. Our morphological results showed a clear difference between
both phenotypes of M. californianus and M. galloprovincialis,
which may suggest that phenotype B of M. californianus, is not the
result of hybridization with M. galloprovincialis. Further studies
using genetic markers will help to discard whether there has been
any degree of introgression between these two species due to hy-
bridization, which has been found in other Mvtilus species (Geller
et al 1994).
ACKNOWLEDGMENTS
The authors thank Antonio Figueras Jr., Antonio Figueras
Montfort. Andy Beaumont; for encouraging us to finish this study,
and Biologist R. Vasquez Yeomans from CICESE for his help with
the sample analysis. This work was supported by projects numbers
623106 and 6231 13 of CICESE.
LITERATURE CITED
Beaanuiiil. A. R.. Seed R. & P. Garci'a-Martfnez. 1989. Electrophorelic
and morphometric criteria for the identification of (he mussels Mytilits
edulis and M. gcdloprovincialis. In: J. S. Ryland & P. A. Tyler, editors.
Reproduction. Genetics, and Distribution of Marine Organisms. Den-
mark: Olsen and Olsen. pp. 251-258
Conrad T. A. 1837. Description of new manne shells, from upper Califor-
nia. Collected by Thomas Nuttall Esq. J. Acad. Nal. Sci. Phila. 7:227-
242
Dalla Via G. J.. U. Tappemer. & G. Bilterlich. 1987. Shore-level related
morphological and physiological variations in the mussel Mytiliis gtd-
loprinincialis (Lamarck. 1819) (Molusca Bivalvia) in the north Adri-
atic Sea. Mond. Zool. Ilal. 21:293-305
Figueras A. & A. J. Figueras. 1983. Variabilidad ecomorfica del mejilkin
silvestre y cultivado en Espafia (gen. Mytilii.s) y relacion con su
posicion sistematica. Investigacion Pesquera Al:yi-li
Geller J. B.. J. T. Carlton & D. A. Powers. 1994. PCR-based detection of
m( DNA haplolypes of native and invading mussels on (he nor(heas(ern
Pacific coast. Lati(udinal pauern of invasion. Mar. Biol. 117:243-249
Gosling. E. M. 1992. Systematics and geographic distribution of Mytiliis.
In: E. Gosling, editor. The mussel Mytihis: ecology, physiology, ge-
netics, and culture. A(ns(erdam: Elsevier Science Publishers B.V. pp.
1-20
Harger, J. R. 1970. Comparisons aniong grow(h charac(eris(ics of (wo spe-
cies of sea mussel. Mytiliis edulis and Mytiliis califoniianiis. The Ve-
liger 13:44-56
Harger. J. R. 1972. Compe(i(ive coexistence: maintenance of interacting
associations of the sea mussel. Mytiliis edulis and Mytihis califor-
nianus. The Veliger 14:387-410
Inoue, K.. S. Odo. T. Noda. S. Nakao. S. Takeyama. E. Yamaha. F. Ya-
masaki & S. Harayama. 1995. A possible hybrid zone in the Mytihis
edulis complex in Japan revealed by PCR inarkers. Mar. Biol. 128:91-
95
Johannesson, K., B. O. Johannesson & E. Rolan-Alvarez. 1993. Morpho-
logical differentiation and genetic cohesiveness over a microenviron-
mental gradient in the marine snail Littorina sa.\atihs. Evolution 47:
1770-1787
Koehn. R. K. 1991. The genetics and taxono(ny of species in the genus
Mytihis. Aquaculture 94:125-145
Majewski, M. 1995. Comparahve mechanical shell strength of the blue
mussel a Mylilus edulis and the ribbed mussel Ceukensia demissa. In:
J. P. Grassle. A. Kelsey. E. Dates & P. V. Snelgrove. editors. 23rd
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Caceres-Martinez et al.
Benthic Ecology Meeting. New Brunswick, New Jersey. (Abstract
only).
McDonald, J. H. & R. K. Koehn. 1988, The mussels Mylilus gallopiovin-
cialii and M. trossulus on the Pacific coast of North America. Mar.
Biol. 99:111-118
McDonald. J. H., R. Seed & R. K. Koehn. 1991. Allozyme and morpho-
metric characters of three species of Mylilus in the Northern and South-
em hemispheres. Mm: Biol. 1 1 1:323-335
Ohresser, M., P. Borsa & C. Delsert. 1997. Intron-length polymorphistn at
the atin gene locus mac-1: a genetic marker for population studies in
the marine mussels Mytihis galloproviniiulis Lmk and M. ('(/»//,v L.
Mol. Mar. Biotech. 6:123-130
Rawson, P. D. & T. J. Hilbish. 1995. Distribution of male and female
mtDNA lineages in population of blue mussels, Mytilus trossulus and
M. galloprovincialis. along the pacific coast of North America. Mar.
Biol. 124:245-250
Reimer, O. & M. Tedengren. 1996. Phenotypical improvement of morpho-
logical defenses in the mussel Mytilus ediilis induced by exposure to
the predator Asterias rubens. Oikos 75:383-390
Rugh, S. N. 1997. Differences in shell morphology between the sibling
species Liltorina scutulata and Litlorinu plena (Gastropoda; Piosobran-
chia). The Veliger 40:350-357
Seed. R, 1968. Factors influencing shell shape in the mussel Mylilus edulis.
J. mar. bid Ass. U.K. 48:561-584
Seed, R. 1992, Systematics evolution and distribution of mussels belonging
to the genus Mylilus: An overview. Am. Malacol. Bull. 9:123-137
Soot-Ryen, T. A. 1955. A report on the family Mytilidae (pelecypoda).
.Mlait Hancock Pacific 20:1-175
Sokal, R. R. & F. J. Rohlf. 1995. Biometry. 3rd ed. New York: W. Freeman
and Company. 887 pp.
Stiriing, H. P. & 1. Okumus. 1994. Growth, mortality, and shell morphol-
ogy of cultivated mussel (Mytilus edulis) stocks cross-planted between
two Scottish sea lochs. Mar. Biol. 119:115-124
Struhsaker, J. W. 1968, Selection mechanisms associated with intraspecific
shell variation in Liltorina picia (Prosohranchia: Mesogastropoda).
Evolution 22:459-480
Jiiiiival oj Shellfish Research. Vol. 22. No. 1. 141-140. 2()(U.
ADJUSTMENTS OF LIMNOPERNA FORTUNEI (BIVALVIA: MYTILIDAE) AFTER TEN YEARS
OF INVASION IN THE AMERICAS
G. DARRIGRAN,' C. DAMBORENEA.' P. PENCHASZADEH," AND C. TARABORELLi'
^Division Zoologia Inveitehnulos. FCN v Miiseo, UNLP. Paseo del Bosque s/ir ( IWO) La Plata.
CONICET Argentina: -Dep. C. Bioldgicas. FCEyN. USA. Cnulad Univcrsitaria. Pah II. Niii'ic:.. Piso 4°.
Buenos Aires. MACN-CONICET. Argentina
ABSTRACT Limnoperna fortunei (Dunker, 1857) or golden mussel invaded South America through the Ri'o de la Plata estuary in
1991. Ten years later, the golden mussel lives in the main rivers of the Plata Basin. The gonadal cycle and the population settlement
in a temperate climate are discussed in this article. This basic knowledge is needed to assist industries that may suffer the effects of
macrofouling and also increment the ability to predict potential invasions of other countries. The study of population density and
reproductive cycle was performed in Ri'o de la Plata estuary. Argentina. The temporal variation of population density from data of
settlement and age structure collected between 1991 and 2001 is presented. The reproductive cycle between August 1998 and March
2000 was analyzed. Through the analysis of oocyte percentages four gonad spawning events were observed. The spawning events
appear regulated by temperature changes. After the initial increase in population density following the invasion, there was a decrease.
The population appeared stabilized at one third of the initial peak.
KEY WORDS: in\asion. Limnoperna foriimei. freshwater, bivalve, reproductive cycle. Neotropical Region
INTRODICTION
Limnoperna fortunei (Dunker 1857). or golden mussel, is a
freshwater invasive bivalve, from the southeast of Asia. It invaded
South America in 1991. through the Rio de la Plata estuary. This
represents the first record of L. fortunei for the American conti-
nent. Ten years later, the golden mussel lives in the main rivers of
one of the most itnportant Basins of the Neotropical Region
(Bonetto 1994). the Plata Basin (the Rio de la Plata, and the Uru-
guay. Parana, and Paraguay rivers). Since 1999. this species in-
vaded the Guaiba Basin in the south of Brazil (Mansur et al. 1999).
The golden mussel spreads 240 km/year, upstream along the Plata
Basin. (Darrigran & Ezcurra de Drago 2000).
The golden mussel attaches to every available hard substrate.
This lifestyle (epifaunal) is atypical in local freshwater bivalves.
The attachment capability and the great adaptability and reproduc-
tive capacity of these mussels make this species very effective
invaders (Darrigran 2000). The mussels impact on the natural en-
vironment (displacement of native species — Darrigran et al.
1998b, Darrigran et al. 2000 — or change of native fish diet —
Penchaszadeh et al. 2000) as well as on human activities (macro
fouling in fresh water (Darrigran 2000. Darrigran & Ezcurra de
Drago 2000).
Detailed infomiation about the life cycle of this harnitiil inva-
sive species provides a basis for the development and application
40
30
20
10
liC
Au Oc IDe Fe Ap Ju Au Oc I3e Fe
1998 1999 2000
Figure 1. Monthly variation of mean air tenipiTature (line) and water
temperature (bars) during sampling period in Bagliardi Htach. Rio de
la Plata. HVithout data.
of control strategies. The impact caused by this species in human
activities (plugging of water intake for industrial cooling, power
generation, and potable water) resembles what happened in the
north hemisphere with the zebra mussel Dreissena polynwrplia
(Pallas. 1771 ). The study of reproductive cycle, age structure and
temporal density variation, is essential to generate sustainable
techniques for golden tnussel prevention and control.
Details of the reproductive cycle, and the population settlement
in temperate climate are discussed in this article. This type of
knowledge is not only essential to assist biologists and ecologists
in the industries which tnay suffer frotn this new economic-
environtnental problem in the Neotropical Region, but it is also
necessary for predicting potential invasions of other countries in
the north hemisphere such as USA (Ricciardi 1998) and southern
Europe.
TABLE 1.
Date and number of specimens histologically processed per sample.
Date
Size
range (mm)
Males
Females
2.V08/98
27
0.6-2.5
17
10
25/09/98
30
0.6-2.6
23
17
30/10/98
29
0.4-2.5
18
11
27/1 1/98
17
0.5-2.6
14
13
23/02/99
14
0.5-2.9
13
11
19/04/99
20
0.8-2.2
7
13
15/05/99
24
0.7-2.2
14
10
30/06/99
29
0.7-1.9
13
16
26/07/99
25
0.7-2.1
10
15
27/08/99
28
0.5-1.8
19
9
21/10/99
32
0.6-2.1
22
10
27/11/99
34
0.5-1.7
23
11
16/12/99
31
0.5-1.7
14
17
26/01/00
27
0.6-2.1
16
11
22/02/00
35
0.7-2.1
25
10
12/03/00
29
0.6-2.2
18
11
Total
431
266
195
141
142
Darrigran et al.
DENSITY (ind/m-
200,000-
150,000-
100,000
50,000H
a
r^^ rh
r+i
I I I I I I I I I I I
Oct Oct March Oct March Oct March Oct Oct Oct Oct Oct
1991 1992 1993 1993 1994 1994 1995 1996 1997 1998 1999 2000
Oct
2001
Figure 2. Temporal variation of mean density (bars) and standard deviation (lines) oX Limnuperna fortumi in Ba^liardi Beach, Rio de la Plata.
• 4-5 ind/m". *\Vithout data.
MATERIAL AND METHODS
To study the golden mussel population density and reproduc-
tive cycle, samples were collected along the rocky banks of Ba-
gliardi Beach (34°55'S: 57°49'W). Rio de la Plata estuary. Ar-
locality has a temperate regimen ranging from approximately 1 I °C
to 3I°C (Fig. I). The physicochemical features of the Rio de la
Plata may be found in Darrigran (1999). The density data were
obtained partly from Darrigran. et al. (1998b) and through sam-
pling carried out ad-hoc (October 1998 and October 2001) in
gentina. South America. — where the mussel was found for the first Balgliardi Beach. Samples of mussels were collected for density
time in 1991 (Pastorino et al. 1993). The water temperature in this analysis from the fringes with macrobenthos from a rectangular
20
15
10
5
0
October 1992
n = 481
III...
March 1995
n= 1059
■.■iiMMlii
>N
15
u
c
OJ
in
J
cr
Ol
5
.1
October 1 993
n = 677
I..
I.ll
October 1998
n = 289
I
20
15
10
5
0
October 1 994
n = 631
llllll...
October 2001
n = 289
Illllllllll.llil..
length (mm)
Figure 3. Size frequency (%) ot Limnoperna fortunei in Bagliardi Beacli. Rio de la Plata.
L. FORTUNEi Ten Years After the Invasion
143
area, variable in size, according to Darrigran et al. ( 1998b). For the
age structure analysis, the niaxinium shell length was measured
and the length frequency distribution was made at 1 mm class
intervals (see Fig. 3 later).
The dates of sampling for reproductive cycle analysis, per-
formed at low tides, may be observed in Table 1. The maximum
shell length of the 431 collected individuals was taken. The ma-
terial was fixed in Bouin solution and the histologic processing
was performed according to Darrigran et al. (1999).
Approximately 25 oocytes with conspicuous nucleolus, both
free in the follicular lumen and attached to the follicle wall, for
each gonad were measured. The percentage of males with sper-
matozoids and the percentage of follicular occupation on the
mantle were calculated for each sample. The latter was calculated
using magnification (x200) in three different sections of the
mantle, (upper, middle, and lower) through the visual estimation of
field. The lysis periods were detemiined by microscopical analysis.
RESULTS
The temporal variation of population density found on the
rockv litoral zone of Baszliardi Beach between 1991 and 2001 is
given in Figure 2. From 1991 to 1995, the density increase was
remarkable (from four to five individuals/nr to over 100.000
ind/m~). The population density then decreases and stabilizes at
approximately 40,000 ind/nr. In Figure 3 it is shown that since
1 994 the population has had an age structure where most size class
intervals are represented.
The female and male tollicles grow in the mantle and in the
visceral mass. During this study 0.25% hermaphrodite specimen,
with female, male, and mixed follicles were recorded.
The gonad growth is characterized by growing follicles. In this
stage the follicles are small and there exists an abundant connec-
tive tissue between them. A more developed stage shows young
oocytes on the wall, many stalked oocytes (Fig. 4A) and abundant
spermatogoniums in the males (Fig. 4D). In a later stage the fol-
licles are bigger and the follicular lumen contains abundant oo-
cytes half-grown and also almost fully grown oocytes (60-80 p.).
When fully mature, the female and male follicles reach the maxi-
mum size. Male follicles are packed with spermatozoa (Fig. 4E)
and females" follicles with fully-grown (80-100 p.) oocytes.
When the gonads are spent and partially spent, the follicles
contain large spaces. Partially spent gonads retain genital products.
Figure 4. Female and male tollicles in different development stages. (A) Female follicle partiallj gro«n with voiing oocytes on the wall and many
stalked oocytes, scale bar = 1(10 fi. (B) .Spawned female follicles with abundant yellow bodies (arrows!, scale bar = 5(1 p. (C) Female follicles partly
spawned, scale bar = 100 p. (Dl Developing male follicles, scale bar = 50 fi. (E) Fully developed male follicles, scale bar = 100 \i. (Fl Male follicles
partly spawned, scale bar = 50 jj.
144
Darrigran et al.
IL
23/08/98
)(= 57 06
DS = 57 06
n = 224: N =
10
Ij.
23/02/99
X = 75 22
DS = 21 14
= 262, N=11
il
26/07/99
X = 45 19
DS = 45 19
n = 267, N
lll^
16/12/99
X = 43 02
DS = 17 93
n = 353, N=17
llx.
25/09/98
x = 56 91
DS = 20 27
n = 400. N= 17
III] L_ Liilijili.
19/04/99
X = 60.57
n = 225, N=13
27/08/99
X = 38 99
DS = 14 95
n= 177, N=9
I
L.
26/01/00
X = 47 65
OS = 16 £
n = 292; N=ll
31/10/98
X= 51.67
DS = 5167
n = 300. N = 1 1
u
15/05/99
X=46 52
DS = 46 52
n= 170. N=10
21/10/99
X = 51 92
DS= 17 91
n = 248; N
22/02/00
X = 49 94
n = 225. N=10
j1 iIL
LJ.
30/06/99
X = 44 87
DS = 21 72
n = 352. N=16
Jul
110 1» 130 0 10 K Ti
oocyte diameter (um;
12/03/00
X = 55 03
DS = 22 83
n = 320. N=11
K X) 40 so K 70 so so ICO 110 120 130
Figure 5. Frequency I in percentage) of oocyte sizes (ji) in different samplings, x, mean oocyte size; DS, standard deviation; n, number of oocytes;
N, number of females.
In males spennatozolds and spermatocites are observed (Fig. 4F|.
Partly developed oocytes, oogonies, and young oocytes are re-
tained on the female follicle walls (Fig. 4C). Oocitary lysis phe-
nomena (Fig. 4B). with yellow bodies are evident for a short time
after spawning is completed.
The body length at which the follicle, either female or male,
development is completed, varies seasonally. The smallest shell
length at which follicles differentiate is 5.5 mm. for both males and
females (Fig. 5). During this study (August 1998 to March 2000).
oocyte growth was always recorded. Froin May 1999 until August
1999, the oocytes smaller than 20 p. were 309f of the total oocytes
examined.
The change in frequency of oocytes <20 p, and >60 p, indicates
two reproductive peaks each year. The first peak occurs at the end
A
B
oocyte (%) S.
JJrlrl J rlbll.rhftrllJ
De I Fe
1999
* *
¥
males (%)
De I Fe
2000
n = 266
Figure 6. Temporal variation. (.\) Percentage of oocytes bigger than 60 fi (full bars) and smaller 20 yt (empty bars). The arrows indicate moments
of gamete liberation. (B) Percentage of males with sperm. ■ Without data, n, number of male individuals.
L. FOHTUNEi Ten Years After the Invasion
145
90
80
70
60
50
40
30
occupation of the mantle (%)
n = 431
1
M
Au Oc De Fe Ap Ju Au Oc De
1998 1999 2000
Finiirt' 7. Temporal variation of mantlt occupation. Female follicles
(full barsi and males (empt> bars), n, total number of considered males
and females. 'Without data.
of winter or beginning of spring (August to September of 1998.
October to November of 1999) and the second peak is recorded
during the summer (February of 1999. March of 2000). During
these periods in the female follicles the oocytes bigger than 60 |ji.
dominate, while smaller oocytes are scarce (<20%). During the
period of study gonad recuperation were observed (October 1998
and May to June of 1999). Through the analysis of oocyte per-
centages present in the gonad, four spawning events were observed
(Fig. 6A):
( i ) From September to October 1998.
(2) February 1999 to May 1999. It is the most important for its
duration and magnitude.
(3) in July to August 1999, the least important.
(4) between October and December 1999,
Figure 6B shows the percentage of males with sperm through-
out the period considered. The pattern agrees in general with that
observed for females.
The spawning pattern mentioned is similar to the follicular
occupation of the mantle (Fig. 7). The percentage of occupation
decreases during the spawning periods and stays low during the
recuperation period (June. July, and August 1999).
Lysis phenomena were observed in several samples (Fig. 8).
They are more important during May to August 1999, and coincide
with recuperating follicles or in partial evacuation.
DISCUSSION
The bivalve sexual processes are generally related to ambient
temperature (Lubet 1983). The results presented here for a popu-
lation of L. fiirliiiu'i. as well as those observed in the first study
(Darrigran et al. 1999), those performed for a Hong Kong popu-
lation (Morton, 1982), and the analysis of larvae density in the Ri'o
de la Plata (Cataldo & Boltovskoy 2000) show the strong relation-
ship between ambient water temperature and the reproductive
cycle. The spawning events are regulated by changes in tempera-
ture, and increases and decreases of temperature rule the gameto-
genesis in this species.
During the initial study (Darrigran et al. 1999). we found that
oocytes were always present in the mussels even during the resting
period. Periods of scarce proliferation were recorded from Decem-
ber 1993 to May 1994. This study was performed a short time after
the first record of L. forlimei in the Americas (Pastorino et al.
1993). The analysis of reproductive biology at that time differen-
tiated numerous spawning events (five were recorded), many of
them of low magnitude. Between September 1992 and January
1993 (the first period), two spawnings of reduced intensity were
recorded and between February 1993 and November 1994 (the
second period) three spawnings were recorded (two of these of
higher magnitude). During the first period, the oocytes bigger than
60 |xni and those smaller than 20 |jLm are always present and their
proportion is similar (about 309H. the spawnings are low in mag-
nitude but the proportion of oocytes bigger than 60 \vm is always
larger than 20%. During the second period, the spawnings are
more intense and result in a diminution of the bigger oocytes
proportion (by 10%). In contrast to the first period, the oocytes
bigger than 60 (xm reach more than 60% (Darrigran et al. 1999).
The population analyzed here shows a predictable reproductive
pattern. Only two major spawnings are observed throughout the
year, one when summer temperatures are recorded and the other
with spring temperatures. A small winter spawning is also ob-
served. This pattern, after 10 years of settlement in America, is
similar to that described by Morton (1982) for the population of
Hong Kong where the spawnings take place between May to June
and November to December. The pattern shown during the first
study [only after a year of settlement in the location considered
(Darrigran et al. 1999)] could be due to the recent invasion.
Morton (1982) describes short spawnings for a month in spring
and a month in autumn. In this study in South America, mainly in
autumn, the evacuation continues from April 1999 to May 1999.
The presence of larvae in the Ri'o de la Plata, between August and
April (Cataldo & Boltovskoy 2000). also indicates that the spawn-
ing periods are longer than those described by Morton (1982).
Similar to what was found in the first study of the golden
mussel reproductive cycle (Darrigran et al. 1998a) 0.25% of the
population was hermaphrodite.
According to the variation of population density, this species, at
the beginning of the invasion in temperate climate, presents a
noticeable increase of density. Then, it decreases its density to a
third part and stabilizes. At the same time, it presents an age
structure with most class intervals represented. These facts would
indicate a stable settlement of the population to the en\ ironment.
%
Ll
* *
Au Oc De
Ap Ju Au Oc De
I Fe
Figure 8. Percentage of females with follicles where lysis phenomenon occur. *Withoul data. n. number of considered females.
146
Darrigran et al.
The initial increase recorded in a temperate climate could also
be observed in a subtropical climate. Despite the preliminary stud-
ies of this species invasion in the south of Brazil, subtropical
climate (Mansur et al. 1999). the golden mussel presents an in-
crease in its population density similar to that observed in this
study. Two years after its first record (Mansur et al. 2001 a. Mansur
et al. 2001b). the maximum density is 62.100 ind/m".
The golden mussel, like other invasive species, is opportunistic.
This fact makes it difficult to relate the reproductive pattern with
environmental variables and to determine the different facts that
might be modified in the reproductive cycle. L. forliinei. for its
great adaptability and reproductive capacity, increases its distribu-
tion permanently by occupying environments of particular fea-
tures.
ACKNOWLEDGMENTS
The authors thank Renata Claudi for her comments on a draft
version of the manuscript. This work was partly financed by grants
BID 1201 OC/AR PICT 01-03453 from the Agenda Nacional de
Promocion Cientffica y Tecnologica, Argentina; Facultad Ciencias
Naturales y Museo, Universidad Nacional de La Plata (UNLP) and
Fundacion Antorchas.
LITERATURE CITED
Bonetto. A. A. 1994. Austral rivers of South America. In: R. Margalef.
editor. Limnology Now: a paradigm of planetary problems. Amslerdan:
Elsevier Science, pp. 425-472.
Cataldo, D. H. & D. Boltovskoy. 2000. Yearly reproductive activity of
Umnoperna fortuiu'i (Bivalvia) as inferred from the occurrence of its
larvae in the plankton of the lower Parana river and the Rio de la PUiia
estuary (Argentina). Aqiialic Ecology 34:307-317.
Darrigran, G. 1999. Longitudinal distribution of molluscan communities in
the Rio de la Plata estuary as indicators of environmental conditions.
Malacological Review- (Suppl.) 8:1-12.
Darrigran, G. 2000. Inva,sive Freshwater Bivalves of the Neotropical Re-
gion. Dreissena 11:7-13.
Darrigran, G. & I. Ezcurra de Drago. 2000, Invasion of Limnopenm for-
timei (Dunker, IS.'i7) (Bivalvia: Mytilidae) in America. Nautilus 2:69-
74.
Danigran, G., M. C. Damborenea & P. Penchaszadeh. 1998a. A case of
hermaphroditism in the freshwater invading bivalve Limnoperna for-
timei (Dunker. 1857) (Mytilidae) from Rio de la Plata. .Argentina.
Iberus 16:99-104.
Darrigran, G., S. M. Martin, B. Gullo & L. Armendariz. 1998b. Macroin-
vertebrates associated to Limnoperna fortunei (Dunker. 1857) (Bi-
valvia, Mytilidae). Ri'o de La Plata, Argentina. Hxdrobiologia 367:
223-230.
Darrigran, G., P. Penchaszadeh & M. C. Damborenea. 1999. The life cycle
oi Umnoperna forlunei (Dunker, 1857) (Bivalvia:Mytilidae) from a
neotropical temperate locality. J. Shellfish Res. 18:361-365.
Darrigran, G.. P. Penchaszadeh & M. C. Damborenea. 2000. An invasion
tale: Limnoperna fortunei (Dunker, 1857) (Mytilidae) in the neotropics.
International Aquatic Nuisance Species and Zebra-Mussels Confer-
ence. 10:219-224.
Lubet. P. 1983. Experimental studies on the action of temperature on (he
reproductive activity of the mussel (Myliliis eilnlis L. Mollusca, Lamel-
lihranchia). J. Mollusc. Studies (Suppl.) 12A: I(J0-I05.
Mansur, M. C. D., L. M. Zanirichinitti & C. Pinheiro dos Santos. 1999.
Limnoperna fortunei (Dunker, 1857), Molusco Bivalve invasor, na ba-
cia do Guafba, Rio Grande do Sul, Brasil. Biociencius 7:147-150.
Mansur, M. C. C. Santos, G. Darrigran, G. Heydrich, C. Quevedo & L.
Iranco. 2001a. Preferencias e densidades do mexilhao dourado Lim-
noperna fortunei (Dunker, 1857), em diferentes subtratos da bacia do
Guai'ba, Rio Grande do Sul. Brasil. RESUMOS V Congresso de Eco-
logia do Brasil: 246.
Mansur, M. C. D., C. Pinheiro dos Santos, G. Darrigran. 1. Heydrich. C.
Barbosa Quevedo & L. Bernades Iranco. 2001b. Densidade e cresci-
mento populacional do mexilhao dourado Limnoperna fortunei
(Dunker, 1857), na bacia do Guaiba e novos registros na Laguna dos
Patos, Rio Grande do Sul. Brasil. RESUMOS XVII Encontro Brasileiro
de Malacologia: 61.
Morton, B. 1982. The reproductive cycle in Limnoperna fortunei (Dunker,
1857) (Bivalvia: Mytilidae) fouling Hong Kong's raw water supply
system. Oceanologia el Limnologia Sinica 13:312-324.
Pastorino. G.. G. Darrigran, S. M. Martin & L. Lunaschi. 1993. Limno-
perna fortunei (Dunker, 1857) (Mytilidae), nuevo bivalvo invasor en
aguas del Ri'o de la Plata. Neotropica 39:34.
Penchaszadeh, P., G. Darrigran. C. Angulo, A. Averbuj, N. Brignoccoli, M.
Brogger, A. Dogliotti & N. Pirez. 2000. Predation on the invasive
freshwater mussel Limnoperna fortunei (Dunker, 1857) (Mytilidae) by
the fish Leporinus obtusidns Valenciennes, 1846 (Anostomidae) in the
Rio de la Plata, Argentina. J. Shellfish Res. 19:229-231.
Ricciardi. A. 1998. Global range expansion of the Asian mus.sel Limno-
perna fortunei (Mytilidae): Another fouling threat to freshwater sys-
tems. Bu'fouling 13:97-106.
J<:nniiil ofSlwllfish Research. Vol. 22. Nci. 1. 147-lh4. 2(KI,V
QUANTITATIVE EVALUATION OF THE DIET AND FEEDING BEHAVIOR OF THE
CARNIVOROUS GASTROPOD, CONCHOLEPAS CONCHOLEPAS (BRUGUIERE, 1789)
(MURICIDAE) IN SUBTIDAL HABITATS IN THE SOUTHEASTERN PACIFIC
UPWELLING SYSTEM
WOLFGANG B. STOTZ,* SERGIO A. GONZALEZ, LUIS CAILLAUX, AND JAIME ABURTO
Universidad Catolica del Norte, Facultud de Ciencias del Mar, DepariaiueiUo de Biolo^iia Marina,
Casilla 117. Coquiinhd. Chile
ABSTRACT Landings of Concholepas coinholepas. a carnivorous gastropod and valuable fishery resource, appear disproporlion-
ately high compared with herbivores or suspension feeding mussels. The species has been previously described as feeding on a great
variety of prey, the most important being barnacles, mussels, and tunicates. To quantitatively evaluate published information on the
diet of C. comluilepas. an analysis of the stomach contents of 92.'i individuals was performed, representing a wide size-range, broad
geographical distribution (29''30'S to 32°08'S), and different community types (variety of potential prey choices). The diet was based
principally on suspension feeders, such as barnacles iBaUiiui.s Uicvis and juveniles of .Aii.\tii)nu'.i;iihcilanus fi.siikicii.s) 175%) and the
ascidian Pyiira chilensis (16%). An additional sampling, in which abundance of prey in the habitat and microhabitats occupied by the
gastropod was determined, showed that the gastropod positively selects these prey species, the ascidian being the most preferred. The
rest of the diet was made up of Calyptraea Irochifoniii.s and mytilid bivalves. According to the literature, intertidal individuals of this
species only feed at night. To confirm this behavior for subtidal populations, two 24-h samplings (analyzing digestive tract contents)
were performed at a single location. No distinct circadian cycle of feeding for subtidal populations was found, most animals feeding
most of the time. This, together with the characteristics of diet, made mainly by suspension feeders, which transfer energy from primary
productivity in the water column which varies along the coast, to benthic carnivores, help to explain the high productivity of the
gastropod and its variability along the coast of Chile.
A'£l' WORDS: feeding beha\ ior, circadian rhythm, selectivity, carnivorous gastropod, Chile, subtidal, upwelling system
INTRODUCTION
The muricid gastropod Concholepas concholepas (Bruguiere
1789) ("Chilean abalone") is distributed from 12°S to 55°S along
the Peruvian and Chilean coasts and is an important predator oc-
cupying rocky shores (Castilla 1981. Castilla &. Paine 1987). It is
a valuable product in artesanal fisheries (Castilla & Jerez 1986)
along its entire distribution. In Chile, the highest landings ranged
between 6,369t and 25,()()Ot between 1978 and 1988 whereas the
fishery was unregulated; the maximum value was recorded in 1980
(SERNAP 1999). In region IV (between 29' 30'S and 32°08'S, 320
km coast) in the period between 1985 and 2000 landings fluctuated
between 258 and 2,2 19t for this carnivorous gastropod species. In
the same period and along the same stretch of coast the herbivo-
rous gastropods FissurcUa spp. (eight different species that are
fished) and Tegula aira (Lesson), which share the habitat with C
concholepas. together registered landings between 695 and 1525t.
The aim of this work was to investigate what kind of food sustains
the comparatively important production of this high trophic level
carnivore, the ecological position to which C concholepas is usu-
ally assigned, Stotz (1997) has shown that within management
areas the abundance of C. concholepas is related to the amount of
food, the species overexploiting its food source when not fished,
and then migration to other areas. Thus, the knowledge of diet and
feeding behavior is also of importance in developing a manage-
ment strategy of the species within management areas.
According to published literature, C. concholepas has been
observed feeding on a variety of prey, the most often mentioned
being barnacles, mussels, and tunicates (Viviani 1975, Castilla &
Cancino 1979, Castilla & Guisado 1979, Castilla et al. 1979.
DuBois et al. 1980, Castilla 1981, Guisado & Castilla I98.\ Sotn-
*Corresponding author. E-mail: wstotz@socompa.cecun.ucii,cl
mer 1991. Sommer & .Stotz 1991 ). Bui quantitative feeding infor-
mation is scarce; the number of published observations for indi-
viduals feeding in their natural subtidal habitats was less than 96,
observed at two localities (Castilla et al. 1979, Guisado & Castilla
1983, DuBois et al. 1980, Sommer 1991). These did not represent
the entire spectrum of subtidal communities in which the gastro-
pod lives. There are also qualitative observations (Viviani 1975,
Castilla et al. 1979, Castilla 1981 ) that increase the data regarding
the prey diversity of C. concholepas but do not allow evaluation of
the relative dietary importance of the different prey species of this
gastropod.
The published quantitative information on food types con-
sumed by C concholepas was obtained by feeding behavior ob-
servations (Castilla et al. 1979). DuBois et al. (1980) stated "an
individual is feeding when one observes an unusual extension of
the foot over a potential prey species or when the individual shows
movements to remove a prey." This includes lifting individuals to
check for empty shells, direct observations of ingestion of prey,
empty spaces on the substrate in front of the mouth or of the "shell
teeth", which the species has on the anterior border of the shell,
proboscis introduced into the prey, or prey held by the propodiuni
and directed to the mouth (Castilla ct al. 1979). This method gath-
ers information on the specific prey being consumed at the mo-
ment of observation. Thus, those prey species that are more diffi-
cult to consume and for which the process of ingestion lasts longer
will have a higher probability of being observed. Also, in order not
to disturb animals and thus record observations of natural feeding
behavior, observations have been limited to individuals found on
open surfaces. Feeding by individuals found in crevices or on the
undersides of boulders, including most juveniles and medium-
sized individuals of C. concholepas (Castilla & Cancino 1979,
Guisado & Ca.stilla 1983, Sommer 1991, Stotz & Lancellotti 1993)
cannot be easily observed. Thus, observations of C. concholepas
on open surfaces will focus only its feeding on prey abundant on
147
148
Stotz et al.
such places and food composition described using this method
may not necessarily reflect the relative importance of the different
prey species in the diet of C. concholepas.
In contrast, the analysis of digestive tract contents provides a
quantitative measure of food consumption over a certain time in-
terval, representing the range of prey species and their relative
importance in the diet of the predator. Only in case digestion rates
for different prey species differ greatly, some bias may occur. This
is the first work in which feeding of C. concholepas has been
studied through the analysis of the contents of the digestive tract.
According to published information. C. concholepas feeds only
at night (Castilla & Guisado 1979. Castilla & Cancino 1979.
Castilla et al. 1979. Guisado & Castilla 1983). However, this has
been conckided mainly from laboratory experiments mostly using
individuals collected in the intertidal zone. Only DuBois et al.
(1980) have made observations in the subtidal. recording the feed-
ing activity of 96 individuals of this species.
Intertidal gastropods search out and consume food mainly at
night to avoid desiccation (Underwood 1979, Branch 1981, Hawk-
ins & Hartnoll 198.^. Lowell 1984). Subtidal populations of C.
concholepas, not exposed to this stress, may feed mainlv at night
for other reasons: ( I ) to avoid visual predators active during da> -
time (Castilla & Cancino 1979) and/or (2) to capture prey that
respond to visual stimuli and may be able to escape predation by
C. concholepas during the day.
Visual predators, which are known to include C. concholepas
in their diet, such as the sea-otter Lmrafelina (Molina) (Castilla &
Bahamondes 1979), the sea lion Otariaflavescens (Shaw) (Aguayo
& Maturana 1973) and the fishes Pimelometopon macidatus
(Perez) and Sicyases sanguineus Miiller & Troschel (Viviani
1975). do not figure prominently in the monality of this gastropod
species. L. felina has been suggested to be highly specialized on
fish and Crustacea as prey (Sielfeld 1990); O. flavescens does not
appear to prey on gastropods firmly attached to substrates, as is the
case for C. concholepas (George-Nascimento et al. 1983); and the
fish species prey mainly on juveniles of C. concholepas which,
according to our observations, are hidden in crevices in the sub-
tidal. Prey selection is an unlikely factor promoting night time
feeding, as the main prey of C. concholepas are sessile species,
such as the barnacles Auslromegabalanus psittacus (Molina).
Balanus laevis Bruguiere, and Jehlius cirratus (Darwin); the tuni-
cate Pyura chilensis (Molina); the mitilid Peruniytilus puipuratus
(Lamarck); and the hemisessile gastropod Calyptraea trochifonins
(Boml (Castilla & Guisado 1979. Castilla et al. 1979. DuBois et al.
1980, Guisado & Castilla 1983, Castilla & Durdn 1985, Moreno et
al. 1986, Sommer 1991. Sommer & Stotz 1991). Therefore, there
appears to be no strong argument that subtidal populations of C.
concholepas feed exclusively at night. Nevertheless, this needs to
be investigated, which is one aim of this work.
This work reports food composition and feeding behavior (cir-
cadian feeding rhythm and food selection) for C. concholepas
based on the analysis of the food content in the digestive tract. A
greater variety of habitats than in previous studies were sampled,
including open surfaces, crevices, the undersides of boulders, hold-
fasts of the subtidal kelp Lessonia trabeculata (Villouta & San-
telices), and under the canopy of this algae along an extensive
stretch of coast from 29°30'S to 32°08'S (ca. 320 km). On one site
the sampling and analysis of the digestive tract contents of a large
number of individuals collected over a 24-h cycle was conducted.
For some of the individuals sampled along the coast and in dif-
ferent communities, the abundance of potential prey in the envi-
ronment is quantified to establish to what degree the food in the
gut represents the availability of prey. This allows us to study
whether there is some kind of preference for some prey species.
MATERIALS AND METHODS
Study Sites
Individuals of C concholepas were collected at several sites
along the ca. 320 km of coast of the Coquimbo Region, between
Pichidangui (32°08'S) and Punta Choros (29°30'S) (Fig. 1 ). The
sites were chosen considering accessibility and being representa-
tive of different coast and community types. A qualitative de-
scription of subtidal communities of each sampling site is provided
in Table I . Quantitative data of communities in which the gastro-
pod was sampled are provided in Tables 3 and 4. For the 24-h
sampling the site at Punta Lagunillas (30°05'S; 7|-26"W). located
ca. 15 km south of Coquimbo, was chosen. It is a rocky point
forming the northern border of Bahia Guanaqueros (Fig. I ). .-M-
though it is an exposed coast, it has an irregular configuration that
creates sheltered ponds that allow for safe diving through the surf
and at night. The substrate is formed by different sized boulders
that are covered by a dense kelp forest formed by small and bushy
(many blades, short stipes) individuals of Lessonia trabeculata. It
corresponds to community type I (Table I). Quantitative data for
the community at this site are given in Table 3. Larger individuals
of C. concholepas are found mostly within the kelp forest, w hereas
smaller individuals are mainly hidden in crevices or on the under-
sides of boulders.
PACIFIC
29
" S
OCEAN A
ELTEMBLADOR
Punla Choros
\
TOTORAULLO ^^S^
NORTE
\
P LINT A
LAGUNILLAS
J
30^
s
PUERTO X.'S
ALDEA ^
Coquimb
o
—
DEVACA --^^^BahlaTongoy
SAN y]
LORENZO
1
3,o
s
PUERTO 1
OSCURO >\
HUENTELAUQEN \
ISLA ^ I
HUEVO ^^^ /
LASTINICUNAS \ T
Vilos
32
s
TOTORAULLO ^V
SUR ""^f
7 Bah
aPichidan^
ui
Figure I. Location of the study sites along the coast in the region of
Coquimbo (region IV).
Diet and Feeding Behavior of C. concholepas
149
TABLK 1.
Siibtidal fommunitifs »herf ('. coiuhiilepus was collected a general description ol' each community is given.
Type
Communities
Localities
I Kelp hed nf Lcssonia traheciiUita El Tenihlador, Punla Lagimillas. Puma Lengua de Vaca, San Loren/o. Caleta Las Conchas. Totoralillo
Sur (isle and bay)
II Barren gniund Toralillo Norte (rock). Puerto Oscuro
III Barnacles and seaweeds Toioralillo Norte (isle)
IV Colonies of Pxiini chilen.\ii Puerto Aldca
General description of the subtidal community types
Type tcimmunity
General Description
Kelp bed of Lessonia traheculata
Barren ground
Barnacles and seaweeds
IV Colonies of Pxiira chileusis
Community characterized by the kelp Lessonia traheculata. Under the canopy, dense patches of
barnacles (e.g.. Balaims laevis) and to a lesser extent the ascidian Pyiiru chilensis are found. In
crevices and on the underside of boulders are observed aggregations of the gastropod Calyptraea
trocliifdiinis. sponges and small patches of barnacles.
Community characterized by an high cover of calcareaus crustose algae and high densities of the black
urchin Tetrapygiis niger. In crevices and on the underside of boulders are observed aggregations of
Pyiira chilensis, of C. trochyfonnis and patches of barnacles.
Community dominated by extensive patches of barnacles, specially by Austmmegabalaniis pssittacus,
which can be covered by a dense mat of the red algae Gelidiiim chllense. Also aggregations of the
ascidian Pyura chilensis may be present in crevices.
Community formed mainly by aggregations of the ascidian Pyiira chilensis. which covers most of the
surface. The ascidians could be partly covered by the algae Glgartina chamissoi. On the underside of
boulders aggregations of Calvpiniea trochiformis can be observed.
Sampling of C. concholepas Along the Coast to Describe Diet
Individtials were collected by Hookah di\'ing from the intertidal
down to a maximum depth of 25 m. At each site two divers
collected all C cimcholepa.t that they were able to find within
approximately 1 h of diving, which allows the inspection of an area
of about 200-500 m". Individuals of all sizes were collected and
the searches included the undersides of boulders. Table 2 summa-
rizes the number and size range of individuals collected at each site
of the samplings undertaken between January 1994 and December
1995.
Experiments for the Identification of Prey and Food Retention Time
in the Gut
The identification of each prey item was aided by a simple
experiment in which known prey were offered to individual C.
concholepas. Three groups of 10 adult individuals (70-110 mm
peristomal length) were collected at Punta Lagunillas and main-
tained in tanks with running seawater. Each group was offered one
of the most important prey items described in the literature (Som-
mer & Stotz 1991): the barnacles Aiistromegahulanus psittacits
and Bdhiiuis laevis. the gastropod Calyptraea trochiformis. and the
TABLE 2.
C. concholepas: Number of individuals collected in the field, number of entire digestive tracts analyzed in the laboratory, size range, number
of individuals with food in their tracts, and number of individuals with recognizable prey in their digestive tracts are given.
Sample
Sample
Individuals
Recognizable
Size
Size
Size
\>itb Food
Prey
Field
Labor.
Range
Localities
(N")
(N")
(mm)
No.
%
No.
Vf
Playa EI Teniblador
76
74
24-122
54
73.0
47
87
Totoralillo Norte (rock)
21
21
37-93
13
61.9
10
76.9
Totoralillo None (isle)
9
8
15-122
4
50.0
4
1 00.0
Punta Lagunillas (August)
166
166
2I-I2I
122
73.5
110
90.2
Punta Lagunillas (January)
282
260
7-125
235
90.4
225
95.7
Puerto Aldea
13
13
102-129
13
1 00.0
11
84.6
Punta Lengua de Vaca
52
45
51-116
33
73.3
22
66.7
San Lorenzo
188
158
16-126
105
66.5
93
88.6
Puerto Oscuro
7
7
59-100
5
71.4
5
lOO.O
Isla Huevos
54
54
24-131
52
96.3
52
1 00.0
Totoralillo Sur (isle)
95
72
69^7
65
90.3
62
90.4
Totoralillo Sur (bay)
51
47
26-125
40
85.1
39
97.5
Total
ini4
925
7-131
741
80.1
680
91.8
150
Stotz et al.
ascidian Pyiira chilensis. Individuals were maintained continu-
ously with food, sampling after the initial 48 h, and then daily, two
individuals. Sample animals were dissected and their stomach and
gut contents exaiuined. The physical characteristics of each prey
item after ingestion by C. conclwlepas were recorded and then
used as a reference in the analysis of stomach and gut contents
from individuals sampled in nature.
To measure the time the food is held in the digestive tract, a
field experiment was performed at Punta Lagunillas on October
25-26, 1995. Therefore, all the individuals collected during a
30-min period at 1800 h and again at 0600 h of the next day were
maintained in a mesh bag in the water in the study site, without
food. Every 2 h, six individuals of this mesh bag were sampled and
sacrificed, fixinc the visceral mass in 10% saline formalin. In the
laboratory, the proportion of individuals with food in the stomach
or gut in each sample was determined.
Samplings to Compare Diet with the Food A railable in
the Environment
At seven sites (El Temblador. Punta Lagunillas. Punta Lengua
de Vaca. Huentelauquen, Isla Huevos. Tinicunas. Totoralillo sur)
(Fig. 1), between January 1996 and March 1997. samplings were
repeated, but this time recording also abundance of prey in the
environment. For each C. conclwlepas individual collected, the
density and percent cover of species present on the spot, was
recorded. A 0.25-m" quadrant with 100 regularly distributed points
STOMACH
Pyura
chilensis
INDETERMINATE
89,6 CIRRIPEDIA
Q 3 Calyptrsea
''■^ ' trvctiiformis
INTESTINE
83 9 CIRRIPEDIA
INDETERMINATE 73
8,3 ^"^
chilensis
TOTAL
Pyura
chilensis 15,88
INDETERMINATE
Calyptraea trochifonvis o,65
74 67 CIRRIPEDIA
0,05 MYTILIOAE
m
CIRRIPEDIA
MYTILIDAE
^v8i Pyura chilensis
INDETERMINATE
Calyptraea trochiformis
Figure 2. Dietary composition of Conclwlepas conclwlepas.
Diet and Feeding Behavior of C. concholepas
151
was used. The quadrant was loL-ated with its center on the spot
were the C. conclioU-pas indi\ idual was captured.
For four of these seven sites (Isla Huevo. Punta Lengua de
Vaca. Punta Lagunillas. and El Temblador) (Fig. 1). a general
quantitative description of communities present on the site was
done. A 50-ni long and 2-m wide transect was placed parallel to
the coastline. For less frequent species their abundance in the
entire transect area (100 m") was counted, whereas for smaller,
more frequent species five 0.25-ni" quadrants, distributed regularly
along the transect, were used. To quantify the laminarian algae
Lcssduia inilicculata. the transect was divided into 2.^1 areas of 2 x
2 m. estimating percent cover within each of these areas. Within
these same areas the percent cover of each substrate type was
estimated in those cases in which the bottom was a mixture of sand
and rocks. This estimate was used to correct abundance and per-
cent cover estimates of species, in order that they refer only to
rocky bottom.
24-b Sampling al Punta iMgiiiiillas
The 24-h sampling was accomplished twice: on October 24 and
2.S. 1994 and August .S and 6. 1996. Dives took place at 1700.
STOMACH
>
u
c
(D
<1>
00
i
fe
^
EES
i
m
60 1
x^-'
:|;
40 i
5 - CN CM
i ^ ^ 1
1 Z 2 c
o
(A
Surl
Sur2
Total
o
o
o
O
illo
illo
i- - - m
n to
uj 2 2 -^
as
5
2 2
a
o
£
^—
INTESTINE
>
u
c
0)
3
a
u.
100 .
80 1
60
40
m
iiizza^
m
TOTAL
100
m
CIRRIPEDIA
MYTILIDAE
Pyura chilensis
Calyptraea trochlformis
INDETERMINATE
Figure 3. General dietary composition of Cnnchnlepas concholepas from each sampling site.
152
Stotz et al.
2100, 0100, 0500, 0900, and 1300 h. On each dive, two divers
sampled the subtidal at depths between 4 and 10 m, collecting each
C. concholepas they were able to tlnd within a half-hour dive.
Searches were concentrated beneath the canopies of L. trabeciilata
and included the undersides of boulders. At night searches were
conducted using underwater flashlights. Diving was conducted us-
ing a compressor on the beach that provided air to the divers
through a hose (Hooka diving). In the 1996 sampling, the indi-
viduals collected by each diver were considered as replicate
samples.
Processing of Samples
All samples of C. concholepas were processed immediately
after collection. Peristomal length of individuals were measured
with calipers and grouped into se\ en si/e classes from <30 mm to
>130 mm (see Figs. 4 and 5). Each specimen was taken out of the
shell and the visceral mass dissected and fixed in 10% saline
formalin. Visceral masses of all individuals from each size class
were stored together in a single container and transported to the
laboratory.
In the laboratory, the digestive tract of each individual was
dissected; the contents emptied separately for stomach and gut in
two Petri dishes, diluted with tap water, and spread on the bottom
of the dish. The relative abundance of each prey item was recorded
for each individual using a dissecting microscope. Therefore the
dish was put over a point matrix, recording the food item over each
point, and calculating its proportion to all the points covered by the
sample. Also the presence of prey species, which were present, but
not registered over any point, were annotated.
For C. concholepas from the 24-h sampling a measure of full-
ness was recorded. Fullness and digestion level was determined
Using the following scale:
Fullness:
Full: contents occupy ca. 100% of the volume of the stomach or gut.
Medium: contents occupy around 50% of the \ olume of the stom-
ach or gut.
Presence: contents occupy around 10% of the volume of the stom-
ach or gut.
Empty: no contents registered.
Digestion level:
Some digestion: entire structures are observed, such as pieces of
cirri, 2ills, muscles, etc.
STOMACH
>
o
z
LU
a
UJ
100
80 -
60
40
20
^^^
^
m
INTESTINE
>
o
z
tu
r>
o
lU
a:
u.
100
80
60
40
20
0
i^*c/j
m
CIRRIPEDIA
MYTILIDAE
^vvi Pyura chilensis
INDETERMINATE
Calyptraea trochiforwis
Figure 4. Dietary composition of Concholepas concholepas in different size classes (length of peristomal opening).
Diet and Feeding Behavior of C. concholepas
153
STOMACH
INTESTINE
>
o
z
lU
o
ai
u.
>-
o
z
lU
a
Ul
a:
u.
100)
sa
so
4a
20^
100
80i
6o^
4a
2a
I
0 +
^S3^^^SSS
80i
I
2(y
10Q
80,
601
4a
2a
EL
TEMBLADOR
^?
LAGUNILLAS
O
z
LU
a
UJ
(t
LL
o
z
UJ
o
UJ
100|
801
■^1
61
4a
2a
10Q
!
so]
40*
2a
0
^^
100|
8a
60
4a
20
'' 0
Wi
100
80
60
40
20
LAS
CONCHAS
^^
TOTORALILLO
SUR
T- CO
SIZE CLASSES (Cm)
CIRRIPEDIA
Pyura chilensis
INDETERMINATE
Figure 5. Dietary composition of Concholepas concholepas in different size classes (lengtli of peristomal opening) Iroin lour sampling localities.
mate significance levels, using the following relations:
Species A Other spp. Total
Medium: structures could still be identified, but already with some one degree of freedom (Sokal & Rolf 1969, Pearre 1982) to esti-
digestion.
Total digestion: soft parts are completely digested, only pieces of
shells or hard skeletons can be identified.
Prey Selection Analysis
To determine the degree of selection of prey by C. cdiuhdlepas
an index proposed by Pearre (1982) was used. This allows the
estimation of the selection index C. but also using a x' tc'st with
In the diet
A,
«./
\
+ e.
= c
In the
environment
.4,,
fi„
.4,
+ s„
= D
■\,
+
.4,,
= ,4
«,,
+
«,,
= H
■\
+ ■'>„
+ Bj + B„
= N
154
Stotz et al.
10 12
18:30
Starvation period (hours)
Figure 6. Percentage of Individuals v\ith contents In the stomach and
Intestine during the starvation periods beginning In the morning (A)
and In the afternoon (B).
Where:
Aj = Proportion of species A in the stomach
/4,, = Proportion of species A in the environment
Bj = Proportion of the rest of species in the stomach
fi„= Proportion of the rest of species in the environment
The index "C" is obtained from the followina relation:
Where:
N
X'
N
(A,rB,,-A„- BJ--
A- B- CD)
The index C varies between -1 and +1. A significant positive
value indicates that the prey species was preferred and rejected
with a significant negatise value. Values around zero means that
the prey species is consumed in the same proportion it appears in
the environment.
For estimation of the index only those species found
in the diet of C. concluilepus where considered. For the cal-
culations, the density of invertebrates present in the quadrant
was transformed into percent cover to have all the values on
the same scale. For this, the area occupied by an average indi-
vidual was estimated, calculating its proportion within the
2.?00 cm" of the sampled area. This proportion was multiplied by
the number of sampled individuals, thus obtaining their percent
cover.
Once this proportions where estimated, a correction for poten-
Degree of
Fullness
n Empty
^ Presence
B Medium
■ Full
Digestion
Level
D Empty
^ Total
B Medium
■ Some
0 2 4 6 8 1012 1416 0 2 4 6 8 10 12 14 16
STARVATION PERIOD
Figure 7. Prey digestion level (first column: A, C) and degree of fullness (second column: B, D) of stomach and intestine during the starvation
periods beginning In the morning (first line: A, B) and in the afternoon (second line: C, D).
Diet and Feeding Behavior of C. concholepas
155
MORNING
SAMPLE
Cirripedia
(principally Balanus laevis)
Indeterminate
Totally digested
AFTERNOON
SAMPLE
Mollusca
Pyura chilensis
Calyptraea
trochiformis
Cirripedia
(principally Balanus laevis)
Mollusca
Indeterminate
Totally digested
Pyura chilensis
Figure 8. Prey composition of Concholepas concholepas in the starva-
tion experiment at Punta Lagunillas.
tial prey species was done. Therefore, the percent cover values for
algae and empty space was eliminated, calculating a new propor-
tion considering that potential prey species cover 100% of the
substrate.
For these analyses, only the content of the stomach was used
because this represents the most recently ingested food, most prob-
ably from the sampled spot. Also, empty or destroyed stomachs
were not considered.
RESULTS
Diet
Of the 1.014 individuals of C. concholepas collected at nine
sites (Table 2) visceral masses of 925 individuals were examined.
Of these, only 741 individuals (SO.I^r), covering a size range from
7-131 mm peristomal length, had food in their digestive tracts
(Table 2).
Only 8.2% of the digestive tracts had contents that could not be
identified because the process of digestion was already too ad-
vanced (Table 2). About 98% of the individuals examined fed on
one prey type. Only 18 individuals (2%) had more than one prey
item in the digestive tract.
The most important prey items were barnacles, representing
89.6% of the stomach contents, and 83.9% of intestinal contents
(Fig. 21. The second most important prey item, the ascidian P.
chilensis. represented 5.47r and 8.3% of the stomach and gut con-
tents, respectively. The remainder of the prey was Calyplraea
trochiformis, mitilids. and unidentified materials. Differences be-
tween stomach and intestine were produced by more advanced
digestion in the latter. That favored recognition of the ascidian in
the intestine because its remains were recognized mainly by color,
which was not affected by digestion. C. trochiformis was not found
in the intestine. But these different digestion rates of the various
prey did not change the general dominance of barnacles in the diet.
The dietary importance of barnacles was most pronounced at
Caleta Las Conchas, where they represented the only prey. In
contrast, at Puerto Aldea, where C. concholepas was introduced by
fishermen, barnacles were entirely replaced by P. chilensis (Fig.
3). With only two exceptions (Puerto Aldea and Lengua de Vaca).
in all sites the barnacles were the predominant prey (Fig. 3). even
though the basic community structure varied (Table I ).
Prey composition did not differ among the different size groups
within the pooled sample, where barnacles were always the dom-
inant prey item (Fig. 4). The same analysis made at selected sam-
pling sites, also showed in general, with only two exceptions (El
Temblador 9-1 I cm; Totoralillo Sur 5-7 cm) (Fig. 5) that the
barnacle was the predominant prey. Although in all cases the
smallest and the biggest indi\iduals only fed on barnacles, interme-
diate-sized individuals showed a slightly more varied diet (Fig. 5)
Identification of Prey and Food Retention Time
The feeding experiments with known prey items allowed gen-
eral descriptions of the prey after ingestion by the gastropod. Skel-
etal plates, cirri, and eggs were observed in the stomach and gut
when C. concholepas fed on barnacles. When the ascidian Pxuru
chilensis was the prey, an orange or red mass sometimes contain-
ing syphons was observed. In the case of Calyptraea trochiformis.
while-colored muscular tissue and egg capsules could be recog-
nized. Comparison of these characteristics with those observed in
the digestive contents of individuals collected in the field allowed
the identification of most prey items.
Regarding food retention, the percentage of individuals with
content in the digestive tract is highest (83.3%) in the morning
(0630 h) and in the evening (1830 h) when just sampled. As the
starvation period increases, the proportion of individuals with con-
tent in the digestive tract fluctuates, decreasing after 12 h of star-
vation (Fig. 6). The decrease is more evident and regular for the
stomach, not so much for the intestine. The stomach appears com-
pletely empty after 16 h of starvation. Accordingly, the percentage
of full stomachs or those with the content showing some digestion
decreases as the starvation period increases (Fig. 7). Nevertheless,
the tendency is not that clear, close to the end of the experiment
appearing again individuals with full stomach or intestine, and
showing just some digestion (Fig. 7). This suggests that some
contamination of the experiment may have occurred. The problem
probably stems on the fact that the shells of the individuals put
together in the mesh bag were not cleaned. Thus the barnacles,
which normally are attached to the shell, might have been con-
sumed by some of the experimental indi\ iduals. Considering this
possible contamination, the experiment suggests that the retention
time in the stomach is around 6 h. whereas in the intestine the food
seems to be retained up to 16 h. The prey species the experimental
individuals had ingested were the same as described above for the
individuals sampled along the coast (Fig. 8).
Prey Selection by C. concholepas
The most important prey species are not the most abundant
species in the habitat (Table 3). Barnacles appear in small patches.
156
Stotz et al.
TABLE 3.
Abundance of macroalgae and invertebrates (percent co>er and density, mean and standard deviation) in the rocky subtidal in which
Concholepas concholepas was collected at lour sites.
Temblador
Lagunillas
Lengua de Vaca
Isla Huevo
Percent cover (%)
Algae
Rhodophyta
Mesophytlwn sp.
Corallina officinalis
Gelidium chilense
Calcareus algal crusts
Phaeophyta
Glossophora kiinthii
Lessonia irtibccnUilii
Porifera annellida
Phnii^inalopoma sp.
Roiiunuiiellii piisudata
Spionidae
Crustacea
BaUmus laevis
Httluiut\ flosciitii\
Austromei>ubalaiuis psitlacus
Bryozoa
Bugula sp.
Briozoa indeterminated
Hemichordata
Pyura chilensis
Free space
Density (ind.m"')
Mollusca
Nassarius gayii
Crassilabnim crassilnhrwn
Tegula sp.
Mitrellii iinifasiiura
Crepiilula sp.
Tegula Iridentala
Calyptnwa Iroclnformis
Density (ind. 1 00m"-)
Cnidaria
Anemonia alicemartiinie
Phymactis clematis
Phymanlhea pluvia
Mollusca
Concholepas concholepas
Fissurella cosrara
Fissurella ciimingii
Crustacea
Paroxanthiis barbiger
Taliepus denumis
Homalaspis plana
Rhynchncinetes typiis
Echinoderniata
Aelionidiwn chilensis (Holoduiroidea)
Meyenaster gelalinosus
Stichaster strialus
Heliaster helianihus
Tetrapygiis niger
19.8 ±25.07
4.6 ± 10.29
5.0+ 11.18
2.4 ±2.51
68.0 ±28.72
29.8+ 17.04
0.2 ±0.45
5.6 ± 12.52
0.2 ± 1.79
0.8 ± 1.79
9.8 ± 10.43
21.2 ± l,V81
15.2 ±25.52
1.6±2.19
0.8 ± 1.79
20
361
16
2
45.6 ± 27.57
0.4 ± 0.55
4.0 ± 6.42
10.0± 11.16
70.0 ± 15.55
2.2 ±4.92
3.6 ± 5.68
1.6 ±2.30
10.6 ± 10.67
0.4 ± 0.8
1.2 ± 1.64
20.8 ± 23.86
0.6 ± 1 .34
1.6 ±3.58
1.0 ± 1.00
0.4 ± 0.89
95
2
55
1
3
17
1
5
57.0 ± 19.46
4.6 ±4.67
6.2 ± 8.90
1.2± 1.79
60.8 ±21.78
0.2 ± 0.45
6.6 ± 7.47
3.0 ±6.7 1
6.0 ± 7.04
15.2 ± 15.32
20.8 ± 29.04
4.8 ± 10.73
0.8 ± 1.79
1.6 ±2.19
0.8 ± 1.79
1
25
23
6
1
1
5
1
584
49.8 ± 23.22
0.4 + 0.55
10.0 ± 20.20
!3.8± 13.18
49.2 ±28. 12
1.0 ± 1.73
17.4± 13.92
0.4 ± 0.89
7.2 ± 16.10
124.8 ±265.85
164.0 ±257.74
125.6 ±265.38
26.4 ± 36.40
26
2
1
mostly associated to the area immediately around the holdfast of
Lessonia Irabeciilata. where fronds do not wipe the rock. Pyura
chilensis is mostly restricted to crevices. Percent cover of both
prey species together lluctuates between 10 and 20% cover. But C.
concholepas within the habitat selects microhahitats in which his
prey species, mainly barnacles, are more abundant. In those mi-
crohahitats percent cover of barnacles may increase up to almost
80% (Table 4). The polychaeta Phragmatopoma sp.. which con-
Diet and Feeding Behavior of C. concholepas
157
TABLE 4.
Proportion (%) of potential prtj in the different microhuhitats In Hhlcli Concholepas loiichohpas was captured on seven study sites.
lA-nj^ua de Totoralillo Las
El Temblador \ aca Huentelauquen Isla Huevo Sur Tinicunas Laguniiias
Main prey species
Pxura chilensis
14.34
6.16
1.01
Cirripedia
24.75
8.52
21.14
68.04
74.63
Phragmatopoma sp.
Other potential prey
Porifera annellida
45.66
8.76
43.74
6.88
8.13
27.68
2.52
4.88
14.63
Polvchaeta indeterniined
10.16
56.91
Romunclu'lla pusUilaui
3.68
6.47
Mollusca
Calyplniea InKJiifonnis
0.29
1.75
Fix.surella spp
0.81
0.19
0.49
Timiciii elegans
0.31
Brachiodomes granulaia
0.22
2.26
Crassilahnim crassiUihnim
0.22
0.10
0.56
Tegiila spp
Naisariiis gayii
0.51
0.41
0.41
5.37
Bryozo
Brvozoa
L59
8.42
9.76
Cnidaria
Hydriv.oa indeterminate
Echinodermata
Tetnipygus niger
Hemichordata
4.41
3.25
1.96
9.82
38.34
1.75
6.88
5.58
0.67
1.38
5.90
0.46
0.34
3.66
0.67
0.92
0.40
0.55
34.09
6.64
structs tubes of sediment attached to the rock surface, in some
areas gets very important, covering together with the barnacles
most part of the space in some sites (Table 4).
The digestive tracts of C. concholepas from the sampled sites
contained mainly barnacles and P. chilensis. Although barnacles
are the most abundant prey species in the environment. P. chilensis
was only rarely found, mostly in very low abundance. Only in one
site the ascidian was important in the environment (El Temblador,
Table 4). Barnacles appear in four of the seven sites as being
positively selected (Table 5. Fig. 9). In the remaining three sites
barnacles are consumed proportionally to their abundance in the
environment. P. chilensis was present only in four of the seven
sites (Table 4), being always positively selected (Table 5 1. On one
site (Las Tinicunas) P. chilensis did not appear registered in the
environment (its proportion less than 1%), but was in the digestive
tract of the gastropod. When the data from all the sites are grouped
and analyzed together, it is shown that only P. chilensis is posi-
tively selected, the rest of preys being consumed proportionally to
their abundance in the environment (Fig. 9H).
Circadian Feeding Rhythm
A total of 275 individuals were collected, representing a size
range between 29 to 120 mm of peristomal length in the first 24-h
sampling period. For the second period 88 and 84 individuals were
sampled by each diver, representing a size range between 2(1 to 1 19
mm of peristomal length (Table 6). Numbers collected during
individual sampling hours varied from 13 individuals at 2100 h to
71 individuals at 1300 h in the first sampling period and seven
individuals at 2100 h to 28 individuals at 1700 h for the second
sampling period (Table 6). As some of the samples were destroyed
during the transport to the laboratory, the analysis is based on 254
individuals for the first sampling period, and on 66 and 81 indi-
viduals respectively for the two replicate samples of the second
sampling period.
TABLE 5.
Selection index C and x" for main prey species of Concholepas concholepas on seven sites.
Lengua
de
El Temblador
Vaca
Huentelauquen
Isla Huevo
Totoralillo Sur
Las Ti
nicunas
Lagu
C
nillas
C
X'
C
X^
C
X-
C
X^
C
x'
C
x'
X^
Pxura chilensis
0.18*
6.64
0.47*
43.89
0.26*
13.39
0.34*
20.47
0.28*
16.22
Cirripedia
0.19*
6.95
0.05
0.45
0.80*
128.54
a 10
2.15
-0.(39
1.53
-0.43*
32.35
0.23*
10.37
Phragmatopoma sp.
-0.39*
30.42
-0.53*
.55.98
-0.63*
79.55
-0.32*
20.33
-0.16*
5.00
-0.09
1.76
Other species
0.03
0.21
0.01
0.0 1
-0.34
23.30
-0.13
3.23
0.14*
4.19
0.25*
11.11
-0.40*
3 1 .59
Values with * show significant positive or negative selection.
158
Stotz et al.
TABLE 6.
Date and time of 24-h samplings, number of individuals collected in
the field, number of entire digestive tracts analyzed in the
laboratory, and inditiduals with food in their digestive tracts
(number and percentage).
Sample
Individuals
Indi
viduals
Size
Field
Analyzed
in the
with Food
Date
Time
(No.l
Lab (No.)
(No.)
(%l
24 OCT 1994
17:00
44
42
31
73.8
21:00
13
13
11
84.6
01:00
46
40
31
77.5
05:00
44
44
37
84.1
09:00
57
52
41
78.8
13:00
71
63
45
71.4
Total
275
254
196
77.2
5 AUG 1995
17:00
28
19
15
78.9
(Replicate 1)
2 1 :00
7
5
5
100
01:00
S
7
6
85.7
05:00
15
7
6
85.7
09:00
15
15
14
93.3
13:00
15
13
11
84.6
Total
8S
66
57
86.4
5 AUG 1995
17:00
21
21
21
100
(Replicale 2)
21:00
7
7
7
100
01:00
12
12
9
75.0
05:00
16
13
12
92.3
09:00
15
15
13
86.7
13:00
13
13
10
76.9
Total
84
81
72
88.9
Considering all the individuals analyzed for the entire 24-h
sampling, the individuals with food in their digestive tract (stom-
ach and/or gut), represent 77.2% for the first sampling period and
86.4% and 88.9%. respectively, for the two replicate samples for
the second sampling period (Table 6. Fig. 10). During the different
sampling hours the proportion of individuals with food in their
digestive tract for all sampling hours represented at least 71.4%.
Although no clear pattern appears, in all sampling periods, the
highest values were always registered at the late afternoon and
early morning, thus suggesting that feeding intensity increases
during the afternoon and in the second half of the night, or at dawn
and dusk. Nevertheless, no statistical difference was detected be-
tween day (individuals sampled at 0900. 1300. and 1700 h) and
night (individuals sampled at 2100. 0100. 0500 h). as well as
between the different replicate samples (sampling in October 94.
and each diver in August 96) (3 x 3 G test, x" = 1 1.714: df = 7:
P > 0. 1 ). Neither statistical difference was detected between dif-
ferent hours (Contingency Table 6*3*2; x" = 32.7304; df = 27;
P > 0.05).
At the different sampling hours different degrees of fullness
were observed (Fig. 10). Although no clear pattern can be identi-
fied, the stomach shows a slight tendency of greater fullness in the
afternoon or late afternoon hours, decreasing during night, with the
same tendency repeating during the early morning hours. For the
intestine it is observed, that as the stomach empties, the intestine
increases in fullness (Fig. 10). Thus, again the data suggest that
intake of new prey tends to increases at dawn and dusk.
In all sampled individuals during both 24-h samplings, bar-
nacles appear as the main prey species, with proportions ranging
from 41.9% to 75.2%. with a mean value of 57.9% (Fig. 11). The
second most important prey was Pyiini chilensis, which comprised
17.7% to 40.3% of the digestive tract contents. The remaining
individuals had other preys of minor importance, such as Ciilrp-
tniea trochifonnis (Fig. 11). The food composition also did not
vary greatly with sampling time, and barnacles were always the
dominant prey item.
DISCUSSION
Concholepas concholepas fed almost exclusively on barnacles
and the ascidian Pyiini cliileiisis. The similarity in diet composi-
tion among individuals from different localities and among differ-
ent size classes, suggests that this is a general characteristic for
subtidal populations of this species.
These data support corresponding literature data (Castilla et al.
1979. DuBois et al. 1980. Sommer 1991 ), but show quantitatively,
that barnacles were usually the most consumed prey in the differ-
ent community or microhabitat types where C. concholepas was
found. The smaller individuals of C. concholepas live on the un-
dersides of boulders or in crevices (Stotz 1997. Guisado & Castilla
1983. Sommer 1991). where potential prey is probably different
from that present on the rock surfaces where larger individuals
live. Nevertheless, all size groups had consumed very similar food
types. This suggests a strong feeding preference for barnacles,
which nevertheless seems not always supported by the analysis
with the selection index. With the pooled data. P. chilensis appears
as the most preferred prey species. However, the preference is
better shown by the fact that the gastropod is always found in
microhabitats in which the barnacles predominate. And within
such microhabitat the index is not any more able to show a pref-
erence. Considering all the prey species described, the preference
extends in general to suspension feeders. A similar behavior has
been described for Acanthina lugubris angelica, the diet of which
was restricted exclusively to sessile suspension feeders (Vermeij
et al. 1994). The diet based on suspension feeders seems to be a
general pattern for benthic predators, such as diverse gastropods
and seastars (Table 7).
The most common barnacles in subtidal communities are Baki-
nus laevis and Aiistromegabalantis psituiciis. Individuals of the
latter species are mostly small individuals with 0.5-1 cm basal
diameter, while the species is able to growth to sizes of ca. 5-cm
basal diameter. But in the I'egion. barnacles of such big size are
seldom observed.
Feeding based on barnacles that are small, sessile, and form a
uniform cover on the substrate makes C. concholepas conceptually
resemble a grazer. The feeding of C. concholepas is similar to the
"grazing" of hydroid colonies by nudibranchs. or even to grazing
gastropods, for example, the keyhole limpets Fisurella spp.
(Moreno & Jaramillo 1983, Moreno et al. 1984. Godoy & Moreno
1989). This observation applies to many gastropods and starfishes
(Table 7). It is a well-described characteristic for intertidal whelks
(Dayton 1971. Paine 1966. Menge & Sutherland 1987). habitat in
which the sessile suspension feeders are the main space occupiers,
but less known for species living in the subtidal. where a wider
variety of potential prey species may be expected. In fact. C.
concholepas makes use of a wider variety of prey in such habitats,
including mobile predators as crabs and even fishes (personal ob-
servations), but quantitatively only the suspension feeders are im-
portant.
The feeding behavior of C. concholepas, not showing a clear
circadian rhythm, differs from what has been published previously
Diet and Feeding Behavior of C. concholepas
159
.2
Q
1U0
N=18
A
>.
o
50
**
*
r-1 n
S "-
<u
a
P. Cirr
orrzr
50
100
10O
50
TJ
Phrag. Other
N=7
Cirr Phrag. Other
50
10O
100
50
0
0
50
100
100
50
N=7
JZL
n
n
p. Cirr Phrag. Other
TJ
N=3
n
p.
Cirr
Phrag. Other
u
50
00
—
100
50
50
100
100
50
50
100
100
50
100"
Figure 9. Frequency of prey in the diet and In the niierohnhilut in which Concholepas concholepas was captured in xarious localities: (.\)
Teniblador, (B) I.engua dc Vaca, (C) Huentelauquen, (D) Isia Huevo, (E) Las Tinicunas, (F) Lagunillas, (G) Tutoralilio Sur, (H) Pooled Sample.
N=22
B
p. Cirr Phrag. Other
N=14
*
XIL
D
p. Cirr Phrag. Other
TJ
N=26,,
F
50
*
0
n
n
P Cirr Phrag. Other
50
100
100
50
P. Cirr Phrag. Other
0 T— rn \ZT-
N=
=97
H
*
.. n
for this species by Castilla and Guisado (1979), Castilla and Can-
cino (1979), Castilla et al. (1979). Guisado and Castilla (198.^).
and DuBois et al. (1980). Differences in the methodological ap-
proach may explain this. Previous studies have been based in the
intertidal zone, or in the laboratory, but using individuals collected
from the intertidal. Environmental characteristics of the intertidal
zone, principally dessication stress, often cause circadian rhythms,
with activity periods at night and resting periods during the day
(Underwood 1979, Branch 1981. Hawkins & Hartnoll 1983. Low-
ell 1984). Pino et al. (1993) compared the activity periods of the
intertidal gastropod FissureUa crassii Lamarck and the subtidal
species F. kitimurpnuta Sowerby and observed that the intertidal
species had a distinct day-night activity cycle whereas the subtidal
species did not. The novelty for C. concholepas is that in this case,
the difference is between different populations (intertidal and sub-
tidal) of the same species. However. DuBois et al. (1980) has also
reported a day-night activity cycle for a subtidal population of C.
concholepas.
DuBois et al. (1980). as all the published work done before on
the feeding of C. concholepas. based his conclusion on the direct
160
Stotz et al.
B
Individuals with Food(%)
Sample Size (N°)
100
Stomach
Intestine
Tide
]Day>Night
17 21 01 05 09 13
17 21 01 05 09 13
17 21 01 05 09 13 Time
FULL
i
MEDIUM
SOME
PREY
EMPTY
Figure 10. Circadian variations: Percentage of individuals witii contents in tlieir digestive tracts, corresponding sample sizes, and percentage of
individuals with different degrees of fullness of the stomach or intestine for each of the three replicate samplings (A) October 1994; (B) August
1996. replicate I: (C) August 1996, replicate 2.
observation of capture and ingestion, using criteria defined by
Castilla ( 1979). If the prey is small and the predator is positioned
directly over it, no sign of feeding will be seen. This may often be
the case when C. concholepas feeds on barnacles, its main prey
species. Study results may also be influenced by different condi-
tions of observation (day and natural light, night and artificial
light). For example, it is possible that at night the field observa-
tions are made mainly on more active individuals located on the
surface of rocks, whereas during the day individuals found in
crevices and between the algae might be included, and for these
individuals it would be more difficult to establish if they were
active or resting. Moreover, depending on the light conditions,
animals could react differently to the presence of the diver. Finally,
DuBois et al. (1980) also mention that some of the animals
included in their observations from Caleta Hornos were intro-
duced to the study site prior to the experiment. The behavior of
these individuals might differ from that of resident (subtidal)
animals.
In the approach used by DuBois et al. (1980). if capture and
ingestion of prey occurs rapidly and is of short duration, it is less
likely that observations will be recorded. The study of digestive
tract contents also includes the process of digestion, thus covering
a much longer time period, being less likely that a individual which
has been feeding is missed. But on the other hand, the long reten-
tion time shown by C. concholepas, may obscure the e.xistence of
a circadian feeding rhythm. Nevertheless, if no ingestion of food
took place over the day (or over the night), at the end of the day
(or night) most of the stomachs should be empty, as seen in the
experiment in which the individuals where starved. And this is not
A B
Total
Calyptraea
trochiformis 0.3%
Gastropoda 1.2%
ndetarminate
8.9%
Pyura chilensis
31.8%
Figure 11. Prey composition of Concholepas concholepas sampled
over 24 h at Punta Lagunillas. Composition of each replicate sampling
(A) October 1994: (B) August 1996, replicate 1: (C) .August 1996,
Replicate 2; and total diet are shown.
Diet and Feeding Behavior of C. concholepas
161
TABLE 7.
Summary of prey species for several gastropods and starflsh.
Predator
Main Prtv
Site
Author
Gastropods
Thais c'likiifiuuila
Thais tlaviiit'ta
Thais hi serai is
Acanthina hrcxideuuita
Thais emarginaia
Thais canaliculata
Thais lamellosa
Niicella lapilUis
Nmtlla lapiUns
Nmclla emargimna
Chicoseus capucinus
Siramonila haemastoma
Strtinionila liaemasloma
Concholepas concholepas
Starfishes
Leplasterias polaris
Astehas vulgaris
Asrerias rubens
Asterias vulgaris
Asterias forhesi
Aslcrias vulgaris
Crossasrer pappusus
Lepraslerias polaris
Coscinas calainaria
Cosmasieria luriila
Pisasier ochraceus
Asterias vulgaris
Stichaster australis
Leptasterias hexaclics
Pisasier ochraceus
Balanus gluiulula
Telractita squamosa
Balanus amphitrite
Siphonaria japonica
Barnacles
Bivalves
Barnacles
Semihalanus balanouUs. Balanus
creniUns.
Mytilus edulis and cither bisalves
Mytilus edulis. Seniihaltunis
halanoides
Bivalves
Barnacles
Bahuuts iunplurrite
Modiolus sp
Crassosrrea virginica
Brachiodomes pharatniis
Barnacles
Barnacles, tunicates
Mytilus edulis
Mytilus edulis
Mytilus edulis
Mytilus edulis
Balanus crenatus
Balanus balanoide
Mytilus edulis
Chlamys islandica
Mytilus edulis
Chlamys islandica
Ascidea sp.
Didemnum albidum
Mytilus edulis
Mya spp
Hiatella artica
Balanus sp
Halocvnthia pxrifinmis
Ascidea sp
Chlamvs asperrinius
Ascidaceas
Podoclavella cvlindrica
Botrylloides leachii
Stolonica australis
Aulacomya ater
Balanus spp.
Tunicata:
Styella melincae
Colonial tunicate
Mussels
Mussels
Mussels
Balanus cariosiis
Balanus glandula
Mytilus edulis
Cluhamahis dalii
Washington. USA
Cape d' Aguilar. Hong
Kong
Costa Rica
Washington. USA
New England. USA
Maine, Anglesey
Canada
Singapur
Gulf of Mexico
Israel
Chile
Canada
Canada
German Bight. North Sea
Outer Brewster Island
(Massachusetts)
Gulf of St. Lawrence
Gulf of St. Lawrence
St. Lawrence Estuary
Rapid Bay (Australia)
Puerto Toro (Chile)
Temperate NE Pacific
Temperate NW Atlantic
Temperate SE Pacific
San Juan Island,
Washington
Paine 1%6
Blackmore 2000
Paine 1966
Dayton 1971
Menge & Sutherland 1976.
1987
Hughes 1992. Dietl. 2000
Gosselin & Chia 1996
Koh-Siang Tan 2000
Brown & Stickle 2002
Rilov, Gasith & Benayahu
2002
This study
Gaymer et al. 2001
Gaymer et al. 2001
Saier 2001
Menge 1979
Himmelman 1991
Himmelman 1991
Himmelman & Lavergne
1985
Keough & Butler 1979
Vasquez& Castilla 1984
Menge 1992b
Menge 1974
162
Stotz et al.
TABLE 7.
continued
Predator
Main Prev
Site
Author
Heliasrer helUmllius
Pxcnopinliii hflitinthiiitles
Asterias vulgaris
Leptasterias polaris
Meyenaster gehitinosus
Meyenaster gelatinosus
Semimytitus algosus
Perumytilus purpuratus
Brachiodomes sp
Cbamidae sp
Jehlius cirratus
Chlhamalus scabrosus
Mylilus edulis
Bivalves
Balanus spp
Mytitus edulis
Macoma spp
Mya tiuncara
Mytilus edulis
Mya tiuncara
Mya arenaria
Macoma spp
Brachiodontes graiudara
Semele solida
Balanus sp
Aulacomya ater
Megahahmus sp
Pyura sp
Ancon Bav. Peru
Tokeshi el ul. 1989
Torch Bay. Alaska
Golf of St. Lawrence
Golf of St. Lawrence
El Frances. Chile
Golfo de Penas, Chile
Duggins 1983
Himmelman & Dutil 1991
Himmelman & Dutil 1991
Vasquez 1993
Dayton et al. 1977
the case. Although no statistical differences was detect between
individuals with food at different hours, a slight indication of the
existence of greater ingestion is suggested to happen at dawn and
dusk, at least in two of the three replicates.
Thus, the high percentage of individuals with stomach contents
throughout the day and night, show ing no distinct pattern of varia-
tion which could be associated with the circadian rhythm, suggests
that most animals are feeding at all day and night hours. Thus. C.
concholepas invests most of its time to feeding, as has been de-
scribed by Bayne and Scullard ( 1978) for the snail Thais (Nucella)
lapilhis. They estimated that this species spends between 45 and
63% of its time feeding.
The conclusion that C. concholepas feeds almost over the entire
24-h cycle is important for the validation of our study of the food
composition of this species because sampling time does not appear
to be an impoilant factor. Although our results show some minor
variation in the prey composition with time, this can probably be
attributed more to normal variability of the diet, rather than to
circadian rhythms of feeding.
The high production described for C. concholepas (Stotz &
Perez 1992) can be explained by its feeding on the lowest con-
sumer level, which shortens the energy pathway frotn the primary
producer level (Whittaker 1975). By feeding on barnacles and
ascidians, this benthic gastropod effectively shortens the food
chain. Through the consumption of suspension feeders C. con-
cholepas accesses the much larger energy pool of primary produc-
tion in the water column. For some coastal environments it has
been calculated that 509^ of the net primary production of the
water column is used by benthic animals (Grahame 1987). which
is the process C. concholepas is taking advantage of. By this
feeding habit, C. concholepas is taking advantage of the high
productivity provided by upwelling processes along the coastal
zone of the southeastern Pacific coast of South America (Raymont
1980. Bakum & Nelson 1991, Thomas et al. 1994).
Upwelling processes, being localized in certain coastal areas,
generate a spatial variability of primary production along the Chil-
ean coast (Fonseca & Fari'as 1987, Acufla et al. 1989). The possible
relation of this variability and the different production levels of C.
concholepas along the coast, as shown by variable landings in
different regions along the Chilean coast (Stotz 1997) is a hypoth-
esis of much interest for this valuable fishery resource. Stotz
(1997) showed that average landings for the period 1985-1995
along the entire coast of Chile, expressed as t per km of rocky
coast, shows two patterns: (1 ) a general trend of decreasing land-
ings from the south to the north, and (2) spots with higher landings
than observed in surrounding areas (see Fig. 10 in Stotz, 1997).
The first trend may be related to a similar trend for primary pro-
ductivity described by Thomas et al. ( 1994), who integrated infor-
mation for 8 y ( 1979-1986). These authors describe high primary
productivity year around for the area close to the coast (0 to 100
km from the coast) in front of region X (43°S)(see Fig. I for
location of regions). In front of region VIII (37°S) there are periods
of high primary productivity only during autumn and winter. In
front of region IV (29°S) the period of high primary productivity
is restricted to a short period in winter. Further north primary
productivity is year around low. The second pattern suggests
a close relation to upwelling centers located in the regions VIII
and IV. At a smaller geographic scale, for region IV, Stotz
(1997) also shows a similar pattern, with the highest landings
registered in the areas around the local upwelling center located
in front of Punta Lengua de Vaca (Fig. I). Variability of landings
may be produced by variations in productivity of the gastropod,
which, as shown by Stotz and Perez ( 1992) and Perez and Stotz
(1992) differs between sites along the 320 km of coast of the
Coquimbo region (region IV). Greater production of C. conchole-
pas associated to upwelling would be evidence for the hypothetical
alternative interaction webs in sites with differences in primary
production in the water column, as postulated by Menge (1992a).
Diet and Feeding Behavior of C. concholepas
163
In places with higher primary production, filter feeders get
more important, and consequently small carnivores, the category
to which C. concholepas would correspond, also increase. The
understanding of this variability and its causes are essential for
the management of this important fishery resource. The estima-
tion of catch quotas for different regions should consider this
variability. Knowledge of the quantitative relation between
primary production, production of suspension feeders and con-
sequent production of this gastropod, would improve predictive
capabilities, thus greatly aiding proper management of this
resource.
ACKNOWLEDGMENTS
We are grateful to the Servicio Nacional de Pesca for facilities
given special permission for the sampling as well as to the differ-
ent fishemien's organizations that allowed diving within their
management areas at Caleta Totoralillo Sur. Caleta Las Conchas,
Caleta San Pedro in Los Vilos. Caleta Huentelauquen, Caleta Puer-
to O.scuro. and Caleta Puerto Aldea. Thanks are given also to
Raymond Bienert and Louis DiSalvo, who improved the English
of the manuscript. This study was funded by Project FONDECYT
N° 1941146/1994.
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Stotz, W. & D. Lancellotti. 1993. Asentamiento del loco Coiuholepas
concholepas (Bruguiere. 1789) en la zona intermareal: Excepcion o
regla. XIII Jornadas de Ciencias del Mar. Vit'ia del Mar (Chile). 26 al
28 de Mayo
Thomas. A.C.. F. Huang. P.T. Strubb & James, C. 1994. Comparison of the
seasonal and interannual variability of phytoplankton pigment concen-
trations in the Peru and California Current systems. J. Geophysical Res.
99:7355-7370.
Tokeshi. M.. C. Estrella & C. Paredes. 1989. Feeding ecology of a size
structured predator population, the South American sun star Heliaster
heliamhus. Marine Biol. 100:495-505.
Underwood. A. J. 1979. The ecology of intertidal gastropods. Adv. Marine
Biol. 16:111-210.
Vasquez. J. 1993. Abundance, distributional patterns and diets of main
herbivorous and carnivorous species associated to Lessonia traheculata
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Catolica del Norte. Cocpnmho Chile Serie Ocasional 2:213-229.
Vasquez. J. & J. Castilla. 1984. Some aspects of the biology and trophic
range of Cosmasleria lurida (Asteroidea. Asteriinae) in belts of Mac-
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Vermeij. G. J.. H. A. Lescinsky, E. Zipser & H. E. Vermeij. 1994. Diet and
mode of feeding of the muricid gastropod Acanthinucella liigubris
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Viviani. C. A. 1975. Las comunidades marinas litorales en el Norte Grande
de Chile. Iquique. Chile: Publicacidn ocasional Laboratorio de
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.loiiriHil ,.f Shellfish Research. Vol. 22. No. I. I6.';-I64, 200.^.
FEEDING AND GROWTH IN THE KEYHOLE LIMPET, FISSURELLA FICTA (GMELIN, 17911
D. A. LOPEZ.* M. L. GONZALEZ. AND M. C. PEREZ
Ltihoiaiono de Cultivos Marinas. Dcpariumeiiio dc Aciiiculliira. Univcisidad de Los Liigos, Casilla 933,
Osorno. Chile
ABSTRACT The feeding habits and growth relationships ol the keyhole limpet ("lapa") hissiirelhi pieki were analyzed in the field
and under laboratory conditions. This species is of significant commercial value and considerable ecological importance in southern
Chile. F. picta is not strictly a herbivore, although it prefers algae; the quantity of vegetable items con.sumed compared with animal
items did not vary seasonally. The items most commonly found in the stomach of F. picta were the algae Ulva sp, Polysophonia sp
and Gelidhim .tp. The abundance pattern of the principal items did not vary seasonally. However, there was greater diversity in the
summer. The relative abundance of items in the diet was closely associated with their relative abundance in the environment. Under
laboratory conditions, adults showed a higher consumption rate for the alga Gracilaiia chileii.\is (artificial diet) than for Ulva sp
(natural diet). The preferred alga is not usually found in the natural habitat of F. picta and has a lower caloric value than that of Ulva
sp. C. chilensis proved to be the best source of energy available for growth in juveniles. Keyhole limpets feeding on the chlorophyte
alga Ulva sp show a negative energy balance. Specimens maintained in suspended systems and fed with the artificial diet (G. chilensis)
reached the average commercial size of 5.^ mm in -3 y; the average survival rate was 90"/?. The results suggest that keyhole limpets
prefer food with a high energetic scope for growth, although in field conditions they consume food with a lower energetic content but
high in abundance. Factors such as morphology or palatability of food are more important than caloric value or presence in the natural
habitats of keyhole limpets. This information is important for the culture of the keyhole limpet.
KEY WORDS: feeding, scope for growth, keyhole limpet. Fi.'.siirella picia
INTRODUCTION
Keyhole limpets ("lapas") of the genus Fissurella are grazing
molluscs that consume a wide variety of macroalgae in the inter-
tidal zone (Branch 1981, Hawkins & Hartnoll 1983). Previous
studies indicate that they also ingest other types of food, such as
crustaceans, small molluscs, coralline algae, ostracods, and
sponges, although they remain preferentially herbivores (Ward
1966. Bretos 1978, Santelices & Coirea 1985, Osorio et al. 1988).
Among Chilean species of lapas, Fissurella crassa is classified
as a generalist herbivore, which prefers to consume foliacious
algae, such as Ulva sp.. Emeromorpha sp and Porphyni sp (Bretos
1978, Santelices et al. 1986). Data available on F. maxima, based
on studies of its stomach contents, indicate that this species is
euriphycophagous (Osorio et al. 1988). Experimental field studies
on F. picta suggest that this species is a nocturnal herbivore, which
migrates during the night to the middle intertidal zone (Jara &
Moreno 1984. Moreno et al. 1984), to feed on the algae Iridaea
horycma and Ulva rigida.
F. picta. has an important commercial value, and over-
harvesting has resulted in the depletion of natural stocks in south-
ern Chile (Bretos 1978. Bretos 1988). In addition, human exploi-
tation of other species has, indirectly, had a negative effect on
keyhole limpet recruitment (Lopez et al. 1999). This species also
has ecological importance given that it can modify the spatial and
temporal distribution patterns of intertidal macroalgae (Mi)reno et
al. 1 984). Knowledge of the diet and dietary preferences of F. picta
is necessary to evaluate its growth rate in artificial cultures and to
interpret the ecological role of the population under field condi-
tions.
Published literature suggests that the interaction between quan-
tity and quality of food with factors such as pH. temperature and
salinity, influences growth in mobile marine invertebrates (Newell
1979. Frantzis & Gremare 1992). The effect of type of food in-
gested on growth can be determined by measuring the increase in
*Conesponding author. Fax: -1-56-6-420-5271; E-mail: dIopezCfl'ulagos.cl
weight or size of the animals, or in terms of energy through scope
for growth, established by evaluating the components of the energy
balance (Paine 1971, Bayne & Newell 1983. Gonzalez et al 1990,
Gonzalez et al. 1993, Thompson & MacDonald 1991, Navarro &
Torrijos 1994, NavaiTO & Torrijos 199.S). The aims of this study
are to determine the feeding habits of the keyhole limpet, F. picta
(Gmelin) in the field and under laboratory conditions and to es-
tablish the relationship between feeding and growth.
MATERIALS AND METHODS
Stomach Conteiil in the Wild
The feeding habits of keyhole limpets were observed in the
intertidal and subtidal zone of Metri Bay (4r36'S, 72°42'W), in
southern Chile. The stomach contents of 40 F. picta specimens
(between 32.9 and 64.8 mm total length) were analyzed per season.
Specimens collected at high tide were immediately injected with
formalin dissolved in seawater to stop digestion. The stomach
contents were analyzed over a 100-point grid (81 mm~). Thus, it
was possible to determine ( I ) the relative frequency of vegetable
and animal items; (2) the relative frequency of empty and full
stomachs; and (3) the quantity and frequency of each item in the
diet. A reference collection of all fronds of alga species present in
different habitats and at different periods of the year was estab-
lished to facilitate the identification of alga species consumed by
lapas. Analysis was carried out under a dissecting scope. The
relative abundance of sessile species present in the study area was
verified during each season, based on coverage, using a 100-point
grid 0.0625 m~ along ten linear transects of 15-18 m in the inter-
tidal zone (Bumham et al. 1980).
The statistical comparison between vegetable and aniinal con-
tent in keyhole limpets was carried out by the x" test. The differ-
ences in dietary preference and energy consumed and lost in ani-
mals feeding on Ulva sp. and G. chilensis were analyzed with a
r-tesl. Using correlation analysis, the relative abundance of algae in
the diet was associated with the food supply of algae available in
the environment.
165
166
Lopez et al.
"Scope for growth" with Natural and Artificial Diets
Juveniles of F. picta (length between 25.0-32.3 mm) were
collected from the rocky intertidal zone in Metri Bay. The animals
were separated into two groups and acclimated in the laboratory at
10°C ± 1°C for 20 days. During the experimental phase, each
group was fed ad libitum with Ulva sp (Chlorophyla) or C. chil-
ensis (Rodophyta).
All the parameters of energy balance were standardized as
joules per day per gram of shell-free dry weight (J • d~' • gdw~').
(using 1 cal = 4.18 J) (Lucas & Beninger 1985). Animal dry
weight was obtained using the regression equation for length ver-
sus dry weight, calculated for 150 keyhole limpets with lengths
between 20.0-36.0 mm.
The experimental procedures for the two groups were as fol-
lows:
To evaluate the effect of natural and artificial diets on ingestion
rate, 40 F. picta specimens of 42.2 ± 9.5 mm total length, collected
in the middle and lower rocky intertidal zone of Metri Bay, were
transferred to aquaria for an acclimation period of 13 days at 15°C
± 1°C. The specimens were permanently submerged and the water
was changed every 5-7 days. The ingestion rate of two types of
macroalgae was compared: Ulva sp, which is the most frequent
item found in the habitat of F. picta (natural diet) and G. chileiisis.
a rhodophycean species of alga, not present in the keyhole limpet's
natural habitat (artificial diet). G. chilensis is the principal species
used in artificial culture with an average annual production of
821 19.5 ton y~' (Semap 1998). The two alga species have distinct
forms: Ulva sp is foliaceus and G. chilensis is ramified. Each alga
species was offered ad libititm to two groups of twenty animals
of similar sizes kept in 1-L individual aquaria. The ingestion rate
was measured gravimetrically, at 7-day intervals. An aquarium
containing only alga samples was used as a control. The inges-
tion rate was obtained by comparing differences in alga weight
at the beginning and end of the experiment, expressed in grams
of dry weight of algae consumed per individual per day (gdw •
ind"' • d"'). Measurement of alga consumption was adjusted ac-
cording to percentage weight variation of algae in the controls. No
animal items were used as food because F. picta feed principally
on algae and an important fraction of animal items in its diet are
epiphytic organisms. The caloric contents of the Ulva sp and G.
chilensis used in the experiments was measured with a Parr bomb
calorimeter. Energy consumed (C) was determined using the ca-
loric value of the algae.
The energy loss due to metabolism (R) was measured in 39
animals as the standard oxygen consumption in a 145-mL hermetic
flask using a WTW-530 oxygenometer (0.01 mg 0,/l accuracy).
For conversion into energy, the Thompson and Bayne ( 1974) oxi-
caloric value of 1 niL O, = 19.95 J was used.
The excretion rate of ammonia (U) was determined in 40 in-
dividual keyhole limpets measuring the concentration of ammonia
accumulated over a period of 15 min in 200 ml aquaria, using the
Solorzano method (Solorzano 1969). Conversion into energy units
was carried out using the Elliot and Davison ( 1975) constant of 1
mg NH4* = 24.85 J. The energy loss through feces (F) was
measured in 15 keyhole limpets that were placed individually in
1-L aquaria containing filtered seawater (mesh size: 1 |j,m) that
was changed daily and with a constant supply of air. The feces
were collected every 1 2 h according to methods described by
Navarro and Thompson (1996). rinsed with isotonic solution of
ammonium formate, kept in containers, and dried in a Memmert
500 furnace at 75°C until a constant weight was reached. The
caloric value of the feces was determined in a Parr adiabatic bomb
calorimeter. Energy loss through mucus (M) was evaluated by
filtering water through 120-|jLm mesh.
The energy values of scope for growth were calculated accord-
ing to the following equation, using above average calculated val-
ues:
P = C-(F-^R + U-fM)
where P = scope for growth; C = energy from food consumed:
F = fecal energy loss; R = metabolic energy loss; U = energy
loss due to excretion and M = mucus.
Determination of Absorption Efficiency
Absorption efficiency was calculated using the Conover equa-
tion (Conover 1966):
AE =
(F-E)
(I -E)x F'
100
where AE = absorption efficiency (9<-); F = ash-free dry weight
food/total dry weight food and E = ash-free dry weight feces/total
dry weight feces.
To determine the algal and fecal organic matter content, algae
and feces were carefully rinsed with distilled water and then dried
in a Memmert 500 furnace at 75°C. until constant weight was
reached. The samples were then incinerated in a muffle furnace at
450°C for 4 h. The organic matter was obtained by establishing the
difference between the constant weight and the weight of the ash
of each sample after incineration.
The results of all the above determinations were then compared
(differences between animals fed with a diet of G. chilensis or
Ulva sp), using one-way ANOVA after logarithmic transformation
(Sokal & Rohlf 1979).
Dietary Preference — Natural and Artificial Diets
The same quantity of Ulva sp and C. chilensis (volume and
weight) was supplied simultaneously to a group of 20 individuals
of 46.7 ± 9.5 mm total length. The amount of algae consumed by
each specimen was determined daily, based on the biomass varia-
tions, with an electronic balance (±0,01g accuracy). A control was
also set up.
Growth of Keyhole Limpets in Suspended Systems Feeding on an
Artificial Diet
The direct effects of the artificial diet on keyhole limpets'
growth and mortality were determined in ailificial cultures.
This study was carried out over 12 months in Metri Bay. At this
location, average water temperature varies between 9.6°C (winter)
and I8.2"C (summer); salinity fluctuated between 28%f and 32%t
during the study period.
Two hundred and forty specimens of F. picia collected from the
intertidal zone were placed in trays ("lintemas") that were sus-
pended from a raft. Specimens were fed ad libitum with the red
alga G. chilensis. Four size categories were used. Initial average
size and the standard deviations of keyhole limpets placed in ex-
perimental growth systems (n = 20 per group) were; group 1 : 25.9
+ 1.3 mm; group 2: 31.9 ± 1.9 mm; group 3: 37.8 ± 0.7 mm and
group 4; 45.0 ± 0.9 mm. The experiments were replicated three
times. Total weight and maximun length were measured monthly.
Feeding and Growth in Fissurella picta
167
n Without gasinc content
■ With gasdic content
100
80
60
40
20
0
lL
Ax
s
w
Sp
Figure
without
(Sp).
SEASON
1. Relative seasonal frequencj of Fissiirella picta with and
gastric content. Summer (Si; Autumn (A); Winter (W); Spring
RESULTS
D = Vegetable items
80 n
J 1 = Animals iteins
Aimjr^
? «°
« 40
01
3
T
« 20 .
0
i
><
o
80
60
40
20
Sloinaih Contents in the Wild
WINTHR
The relative frequency of F. picta specimens with empty stom-
achs was less in autumn and winter than in summer and spring
(Fig. 1). The percentage of vegetable items was always signifi-
cantly higher than the animal items {P < 0.05), with no variation
between different periods of the year (Fig. 2).
The most frequent items present in F. picta stomachs were the
algae Ulva sp, Pohsiplioitia sp, and Gelidiuin sp, especially during
autumn. The main animal items were cirripedes and juvenile bi-
valves (Fig. 3). There was a positive correlation between the rela-
tive abundance of food items present in the stomachs throughout
the year and the relative abundance of these items in the environ-
ment (r = 0.891; n = 65: P < 0.05).
Scope for Growth
The diets used in scope for growth measurement had different
energy values. The energy content of Ulva sp ( 1 3,990.5 J • gdw^' )
was higher than that of G. chilensis ( 1 1.101 2 J • gdw"' ). The type
of food intluenced the energy balance and the scope for growth.
The scope for growth was highest when F. picta consumed G.
chilensis (Table 1 ). The negative energy balance in specimens fed
with Ulva sp was due to energy loss (Table 1 . 1 The amount of
energy consumed by F. /nrfcv juveniles did not vary significantly in
animals fed with G. chilensis and those fed with Ulva sp (t =
100
5- 80
5" 60
c
s «
e
ll 20
0
D Vegetable items
■ Animals items
S
w
Sp
SEASON
Figure 2. Relative seasonal frequency of vegetahle and animal items in
gastric content o( Fissiirella picta. Summer (S): Autumn (A): Winter
(W); Spring (Sp).
><
u
ou -
60-
SPRING
40
20
0
T
n 1.
80
£ 60
>t
u
S 40
20 4
U
SlMJBi
a
Ex
Ch
Jb
Items
Figure 3. Seasonal frequency (average ± standard deviation) of food
items in the stomachs of Fissurella picta. Ulva sp (U); Chondrus sp
(Ch); Gelidiuin sp ((i); I'olysiphonia sp (P); Fnteroinorpha sp (E); Cir-
ripeds (C); juvenile hivalves (Jb); Sodilittorina araucana (I.).
0.098; df = 28; P< 0.005) (Table 1.). The quality of food affected
the metabolic losses in F. picta (Fig. 4A). Oxygen consumption
was significantly higher in animals fed with Ulva_sp than in those
fed with G. chilensis (t = 5.48: df = 37; F < 0.001 ). The energy
loss due to excretion was significantly higher in animals fed with
G. chilensis, 23.0 J • d"' • gdw~', than in those fed with Ulva sp,
5.4 J • d"' • gdw-' (t = 8.10; df = 13; P < 0.001). The fecal
energy loss was also affected by the quality of food (Fig. 4B).
Specimens fed Ulva sp had significantly higher fecal energy los.ses
than those fed G. chilensis (ts = 6.56; df = 13; P < 0.001 ), Since
no mucus was found in the aquaria, and given that this value would
only represent IVc of the energy ingested in herbivorous molluscs
(Paine 1971 ). energy loss through mucus (M) was not considered.
168
Lopez et al.
TABLE 1.
Energy ingested, energy loss and scope for growth in Fissiirella piciii
juveniles fed with L'lva sp (natural dietl or Gracilaria chitensis
(artificial diet) in joule/day/gram dry weight of soft parts.
Food
Parameter
Ulva sp
Gracilaria
chileiisis
Range of energy ingested
(J ■ d"' gdw"')
Total energy loss (J • d~' ■ gdw"
Average scope for growth
(J -d"' -gdw-')
605.3-1.504.3
740-1.920.3
803 ±238.6 4(.)y ± 140.2
-10.4 390.6
Absorption Efficiency
Absorption efficiency was highest in specimens fed Ulva sp.
83.4%, and lowest in those fed G. cliilensis. 74,6% (x" = 0.49;
df = 1; 0.05).
Dietary Preference — Natural and Artificial Diets
In specimens of F. piclci. consumption rates of C. chilensis
(artificial diet) were higher than those of Ulva sp (natural diet)
(t = 76.12; df = 27; P < 0.001 ) and they also presented a greater
preference for C. chilensis than for Ulva sp (t = 19.89; df = 28;
P<0.00\).
Growth of Keyhole Limpets in Suspended Systems
The alga G. cliilensis proved to be suitable food for growth
and survival in keyhole limpets. The annual average survival rate
was 90'7(- under these experimental conditions. The growth rates of
the animals varied according to size. Using these data it was cal-
culated that F. picta reached 26.0 mm in about 14 mo. Thus, the
average commercial size of .53 mm would be achieved in approxi-
mately 3 y (Table 2).
DISCUSSION
The results obtained indicate that F. picta is preferentially a
herbivore, as has been described for other species of this genus.
(Osorio et al. 1988. Santelices et al. 19861. However, it also con-
sumes animal items. Similarly, the high consumption of foliaceus
species such as Ulva sp (Jara & Moreno 1984) was also confirmed.
This can be associated with the food supply available in the envi-
TABLE 2.
Growth in four groups in = 20) of Fissurella picta in suspended
cultures, feeding on Gracilaria chilensis (artit'icial diet).
Initial Length
Final Length
Time
Group
( mm 1
(mml
(Month)
1
25.9 ±1.31
38.6 ± 4,4
12
48.2 ±0.1
2!
2
31.98+ 1.97
46.3 ± 5.7
12
3
37.86 ± 0.76
50.0 ± 5.3
12
4
45.03 ± 0.95
54.6 ± 1.2
X
55.3 ± 2.3
14
<B —
600
500 <.
400
O O) 300
■S '
f 3 200
s
100
_^
B 600 n
0)
» 500
c
O — 400
t3 s
= E, 300 -
|'5 200-
01 100 -
u
0)
u. 0 -
G U
Figure 4, P'nergy loss through metabolism (,\) and feces (B) in Fis-
surella picta feeding Gracilaria chilensis ((J) or Viva sp (Li I,
ronnient, as has been verified in other species of Fissurella (San-
telices et al. 1986). Ulva sp and Polysiphonia sp, the most frequent
items in the keyhole limpets" stomachs, are opportunist algae spe-
cies in the field. They densely colonize the intertidal zone of Metri
Bay (Buschmann 1991).
The higher consumption rates, trophic preference, and scope
for growth obtained with G. chilensis. which is not usually found
in the natural habitat of F. picta. compared with those for Ulva sp.
indicate that food items might not be selected due to their energy
characteristics. The trophic preference is not related to the caloric
value of food, given that Ulva sp has a higher caloric value than C.
chilensis, and the energy budget was not associated with the food
availability in the field. Although the laboratory results cannot be
reliably extrapolated to the natural habitat, it can be assumed that
the preference for macroalgae consumption may be associated
with their digestibility, morphology, or palatability (Lowe &
Lawrence 1976. Tugwell & Branch 1992). Although the chemical
defenses of algae are lower than in terrestrial plants, the secondary
compounds related to the plant-herbivore relationship, cannot be
discarded (Hay & Fenical 1992). Further research is required to
test these hypotheses.
The scope for growth in juvenile limpets varied according to
the algal food offered. Specimens fed with G. chilensis (artificial
diet) presented a positive energy balance. Considering the fact that
the specimens studied were juveniles that had not yet reached
sexual maturity, the balance of the energy budget can be consid-
ered as energy available for growth. In species such as the gastro-
pod Concholepas concholepas (Bruguiere 1789) and the echino-
derm Lo.xechinns alhiis (Molina 1782), it has been shown that the
type of food offered greatly influences both the "sign" of energy
balance and the amount of energy available for growth (Gonzalez
et al. 1990, Gonzalez et al. 1993). These results coincide with
those obtained in F. picta.
Feeding and Growth in Fissurella picta
169
Keyhole limpets maintained in suspended cultures and fed ex-
clusively on G. chilensis. had high sur\i\al rates. This study in-
dicates that the type of food offered can have a considerable in-
fluence on the growth rate of juvenile F. picta. Our data mdicate
that, under artificial conditions, it can be possible to maximize
reproduction and growth by selecting the food items offered. This
finding could ha\e significant consequences for cultivation of this
important resource. Other factors, however, such as culliire den-
sity, must be investigated to obtain higher growth rates.
ACKNOWLEDGMENTS
The authors thank FONDECYT for the financial support
through Grant 040-93. University of Los Lagos for pro\iding
the facilities. Dr. J. Jimenez and anonymous referees for the
critical review. J. M. LIribe. J. Castro, and C. Pino for the collabo-
ration in field and laboratory measurements. S. Mancilla for pro-
viding secretarial assistance, and S. Angus for translating the
manuscript.
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Joiimal oj Shellfish Research, Vol. 22. No. 1. 171-175. 2003.
A COMPARISON OF THE DIGESTIVE CAPACITY OF BLACKLIP {HAUOTIS RUBRA) AND
GREENLIP {HAUOTIS LAEVIGATA) ABALONE
MEEGAN E. VANDEPEER'* AND ROBERT J. VAN BARNEVELD"
'Soiith Australian Research and Development Institute. PO Box 120. Henley Beach. South Australia
5022 and 'Barneveld Nutrition Pty. Ltd.. 19-27 Coonan Rd. South Maclean.
Queensland. Australia 42S0
ABSTHACT In this study, the digestive capacity of blacklip ahalone. Haliolis nihni Leach, was compared whh that ol the greenlip
ahalone. Halioris Uicvigaw Donovan. This was performed by assessing each abalone species ability to digest the protein and energy
from 12 ingredients; semolina, defatted soytlour. fishmeal. casein, pregelatini/ed maiz.e starch, mung beans, whey powder, skim milk
powder, whole lupins [LiipifiKS anxKstifoliKs and Liipiiuis hileiis). dehulled lupins [L. cmfiiisiifolii(s). and bull kelp iDiinillea potci-
tonim). Significant differences were found between the two abalone species in their capacity to digest the protein and energy from some
of the ingredient.s assessed. Based on the differences observed, it was hypothesized that blacklip abalone are more efficient at digesting
protein and cellulose than greenlip abalone and greenlip abalone might have a greater capacity to digest soluble nonstarch polysac-
charides.
KEY WORDS: abalone. greenlip. blacklip. digestibility, protein, energy. Haliuiis rubra. Huliotis hievigiite
INTRODUCTION
Greenlip abalone [Huliotis laevigata) and blacklip abalone
{Huliotis rubra) are the predominant species commercially farmed
in Australia. Moratoriums on the collection of macroalgae for use
in commercial abalone production necessitate the use of manufac-
tured diets in these systems. To date, a significant amount of
research has been completed to characterize the nutritional quality
of ingredients and the nutritional requirements of greenlip abalone.
It is uncertain, however, whether this information is relevant to
blacklip abalone. If similarities exist between the digestive capac-
ity of greenlip and blacklip abalone, then a large proportion of the
research completed on the nutritional quality of ingredients for
greenlips need not be replicated for blacklips.
Studies investigating the feeding preference of blacklip and
greenlip abalone have shown that when given a choice, both spe-
cies prefer to eat red algae (Hone & Fleming, unpublished data;
Shepherd & Steinberg 1992, Fleming 1995). In the wild, however,
abalone are forced to eat what algae is available. For example.
along the coasts of Victoria blacklip abalone feed extensively on
the fronds of the large kelp Phyllopsara comosa whereas on Tas-
manian coasts they often feed on drifting blades of the giant kelp
Macrocxstis pyrifcra as well as on red algae (Shepherd 1975).
The structural and storage polysaccharides present in red and
brown algae are quite different. The storage polysaccharides in
brown algae are mannitol, a sugar alcohol, and laminaran, a glu-
can, whereas the storage polysaccharide for red algae is a starch
known as tloridean starch. The cell wall of brown algae are two
layered with an inner matrix of cellulose and microfibrils and outer
layer of alginic acid and sulphated fucans (Stewart 1974). The cell
walls of red algae consist of an inner rigid component made up of
microfibrils and an outer tnore amorphous component consisting
of mucilage or slime. The characteristic amorphous inucilages that
make up most of the rest of the cell wall (up to 709^) are usually
sulfated galactan polymers (Schweiger 1978). The two largest
groups are the agars and the carrageenans.
Because they differ in their structural and storage carbohy-
*Corresponding author.
Phone: -t-61 8 8 200 2466; Fax: -h61 8 8200 2481; E-mail: vandepeer.
meegan@saugov.sa.gov.au
drates, it is reasonable to suggest that different en/.ymes would be
required to digest red and brown algae. If, as the result of living in
different habitats, blacklip abalone consume different or a broader
range of algae than greenlip abalone, then it would be expected
that they might have a different digestive enzyme profile. If this
were so, then they may also differ in their capacity to digest the
nutrients from the ingredients that are used in manufactured diets,
particularly different carbohydrate sources.
Results from comparative studies conducted on other abalone
have shown there are differences between species in their nutri-
tional requirements or physiology. Mercer et al. ( 1993) examined
the nutritional value of eight algal diets for H. tuherctdala and H.
discus hannai by comparing feeding rates, growth rates, and bio-
chemical composition of the animals. The algae A. esculenta. L
saccharina. and U. lactuca were found to have different dietary
values for the two abalone species with quite different feeding
rates and feed conversion efficiency values being reported for
each. Significantly different responses in growth rates were also
recorded when fed particular diets. The lowest growth rates re-
corded for H. tuberculata occurred when it was fed with L. sac-
charina or C. crispus whereas the lowest growth rates recorded for
H. di.tcus hannai occurred when it was fed with U. lactuca. The
differences in dietary values of the algae to the two abalone species
were attributed to differences in their specific nutritive require-
ments and/or digestive physiology (Mercer et al. 1993).
Given that differences have been observed between other aba-
lone species in their ability to use the same algal diets (Mercer et
al. 1993), then it is possible that greenlip and blacklip abalone
differ in their digestive capacities and/or nutrient requirements.
This has important implications as feed costs represent a large
proportion of farm running costs in Australia and our current
manufactured diets are formulated based on results from research
done on greenlip abalone. The objective of this experiment was to
compare the protein and energy digestibility of a range of ingre-
dients for blacklip and greenlip abalone and thus establish whether
they differ in their digestive capacity.
MATERIALS AND METHODS
Diet Formulation and Manufacture
Twelve diets were fomiulated (Table 1 ) to evaluate the protein
and energy digestibility from semolina, defatted soyflour. Tasma-
171
172
Vandepeer and Van Barneveld
TABLE 1.
Composition of experimental diets (g/lig, air dry basis).
Diet
Ingredient
1
2
3
4
5
6
7
8
9
10
11
12
Semolina
400.0
_
_
-
-
-
-
_
_
-
_
-
Defatted soyHour
-
625.0
-
-
-
-
-
-
-
-
-
-
Tasmanian t'ishmeal
-
-
420.8
-
-
-
-
-
-
-
-
-
Casein
-
-
347.6
-
-
-
-
-
-
-
-
Pregelled starch
189.4
214.4
418.6
200.0
489.4
158.7
289.4
150.0
150.0
374.8
100.0
100.0
Mung beans*
-
-
-
-
-
630.7
-
-
-
-
-
-
Bull kelpt
-
-
-
-
-
-
500.0
-
-
-
-
-
Whey
-
-
-
-
-
-
-
600.0
-
-
-
-
Skim milk powder
-
-
-
-
-
-
-
-
600.0
-
-
-
Lupin Ij
-
-
-
-
-
-
-
-
-
389.6
-
-
Lupin 2§
-
-
-
-
-
-
-
-
-
-
421.1
-
Lupin 31
-
-
-
-
-
-
-
-
-
-
-
500.0
Jack Mackerel oil"
-
-
-
-
-
-
-
-
-
-
-
20.0
Mineral premix**
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
2.0
Vitamin premix**
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
3.0
Vitamin C
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
Vitamin E
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
Sodium alginate
-
-
-
-
-
-
-
-
-
5.0
-
-
Kaolin
400.0
150,0
1 50.0
441.8
500.0
200.0
200.0
2.W.4
239.4
200.0
448.4
369.4
Chromic oxide
?.o
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
5.0
* Whole Vigna radiala.
t Dun'illea potatorum.
± Whole L liiteus.
§ Dehulled L angusiifoliKs.
']1 Whole L angusiifolius.
" Trachunis deciivis (Triahunna Fish Oils, Triabunna. Tasmania).
** Vitamin and mineral premi.xes as described by Uki et al. (1985).
nian fishmeal, casein, whey powder, skim milk powder, whole
mung beans {Vigna nidiata). pregeiatinized waxy maize starch,
bull kelp (Durviltea potatorum), and lupins (whole L. luteiis.
whole L aiigii.stifoIiK.s and dehulled L aiigKstifoliiis) by greenlip
and blacklip abalone. The crude protein and gross energy of each
of these ingredients is given in Table 2. Because of the wide range
in crude protein levels of the ingredients being evaluated, it was
TABLE 2.
Protein (g/kg, air-drj basis) and energ) (MJ/kg, air-dry basis)
content of the 12 ingredients used in the experimental diets.
Ingredient
Crude Protein
(.V X 6.25)
Gross Energy
(MJ/kg)
Semolina
104.0
Defatted sovtlour
480.0
Fi.shmeal
713.0
Casein
863.0
Pregelled starch
3.1
Mung beans
253.7
Bull kelp
69.0
Whey
135.0
Skim milk powder
361.0
Whole L Inteiis
385.0
Dehulled L cingiistifotius
380.0
Wht)le L. angiistift'
'lilts
320.0
15.51
17.45
18.71
22.00
15.65
16.54
10.77
15.20
17.26
18.03
18.28
17.74
not practically possible to formulate the diets to be isonitrogenous.
It is desirable for the diets to be isonitrogenous as it means that
unbiased comparisons can be made among the different ingredi-
ents in regard to the digestibility of their protein.
Before incorporation into diets, the mung beans and lupins
were cnjshed into a fine powder (<500 (xm) using a hammermill.
Each diet contained an equivalent amount of vitamin C (ascorbic
acid) and E (DL-alpha tocopherol) and vitamin and mineral pre-
mixes as described by Uki et al. (1985). Sodium alginate was
included in some diets to aid in binding. Kaolin and pregeiatinized
waxy maize starch were used in the diets as fillers. Chromic oxide
was included at 0.57r for use in subsequent digestibility calcula-
tions.
All diets were initially hand mixed and then mixed in a spiral
action dough mixer Clmpastrice". Hill Equipment and Refrigera-
tion. Adelaide, South Australia). The mixture was then fed through
a commercial pasta machine (La Prestigiosa medium. IPA. Vi-
cenza. Italy) where it was made into 300-mm long strips using a
die with slots 18 mm x 1.5 mm. The strips were dried on mesh
trays overnight in a forced draft oven at 55°C. They were then
broken into three pieces before feeding.
Diet Allocation
Each diet was randomly allocated to three digestibility tanks to
prmide three replicates per diet. Because there was only 18 tanks
in total, this meant that there were four separate collection periods.
Digestive Capacity of Abalone
173
Ahalone and Feeding
Juvenile greenllp and blacklip abaUme (shell length 40-60 mm)
were used in the experiments. The abalone had been obtained Irom
commercial hatcheries and raised on manufactured abalone feed.
The abalone were preconditioned for 1 week on the test diet as-
signed to their tank. During both the preconditioning and experi-
mental periods, the animals were fed lo excess every day at ap-
proximately 1700 h.
Tanks and Collcclion Syslem
Conical-shaped digestibility tanks were used. Abalone were
housed in 20-L buckets {approximately 80-100 per bucket) that
fitted inside the tanks. All the buckets were fitted with plastic mesh
bottoms ( 1 .3-cm x 1 .3-cm mesh ) allowing containment of the aba-
lone while permitting feces to drop into the collection tube at the
base of the tank. Three 25-cm lengths of PVC pipe (8 cm in
diameter) were placed in the buckets as shelters for the abalone.
Attached to the bottom of each digestibility tank was a screw-on
collection tube (11-cm long. 15-mm diameter). Tanks were on a
flow-through water system at a rate of about 2 L/min. The seawater
was filtered to 30 |jim by primary sand filters, then to 10 (xm by
secondary composite sand filters before entering the tanks. Aera-
tion was supplied at 0.5 L/min to each tank at all times by an air
stone. Water temperature and lighting were controlled during the
experiment with temperature maintained at I8.0°C ± 1.0 and a
light regime of 12-h light: 12-h dark. Salinity was 35-36SJ(
throughout the experiment.
Fecal Collection
Feces were collected by settlement every day until 5-6 g of
feces (dry weight) was collected for each replicate sample. This
took approximately 2 weeks. On each day of fecal collection the
buckets containing the abalone were removed and the digestibility
tanks were drained of water and all fittings were cleaned of feces
and uneaten feed. After cleaning, the tanks were refilled and the
buckets replaced. Collection tubes were fitted by 0900 h. A small
foam container was placed underneath each tube and filled with ice
to keep the collecting feces cold and reduce degradation by mi-
crobes. The feces were collected from the tubes at 1 630 h by gently
pouring the contents onto a 1-nim diameter mesh. The mesh was
then placed into a petri dish and frozen at -30°C. The following
day the frozen fecal sample was scraped off the mesh, pooled into
a composite sample, and stored in the freezer until required for
analysis. Before analysis, the samples were freeze-dried and
ground with a mortar and pestle.
Chemical Analyses
Gross energy was determined by a PaiT 1281 bomb calorimeter
(Parr Instrument Company, Moline, ID. Crude protein was deter-
mined by the combustion method using a LECO* CN-2000 Car-
bon and Nitrogen Analyser (RACI 1999).
Chromic oxide was determined using atomic absorption spec-
troscopy based on a modification of the methods described by
Hillebrand et al. (1953). The modified methodology involved pre-
liminary ignition of the sample at 500''C to remove organic ma-
terial and the dissolution of the sample in hydrochloric acid instead
of sulphuric acid (M. Frith, personal communication. University of
Tasmania. Launceston. Australia).
Digesliliility Delerminiilion
The apparent digestibilities of nutrients in the diets were cal-
culated using the following formula (Hardy 1997):
Apparent digestibility = 1
Cr^,.., X Nutriein,,
Cr,
X Niilrii'iil,,
where C, is chromium content and Niitriciil is nutrient or energy
content of the diet.
Statistical Analysis
The data were analyzed by analysis of variance using a gener-
alized linear model (SAS Institute Inc. 1988). Before analysis,
residuals were plotted to establish that the data were in fact nor-
mally distributed, which was the case. Within species treatment
means for nutrient digestibility of the twelve ingredients were
compared by least significant difference.
RESULTS
Significant differences were found between blacklip and green-
lip abalone in their apparent fecal digestibility of protein and en-
ergy of some of the ingredients evaluated (Table 3). Significant
differences in protein and energy digestibility were also found
among ingredients within each species (Table 3).
With respect to gross energy digestibility, blacklip abalone di-
gested the energy from whole L. aiii;iislifoliii.s. fishmeal. and skim
milk powder significantly better than greenlip abalone. and green-
lip abalone digested the energy from whey, bull kelp, and dehuUed
L. angustifoliiis significantly better than blacklip abalone (Table
3). No significant differences were found between the two species
in their ability to digest energy from semolina, defatted soyfiour.
casein, pregelatinized maize starch, mung beans, and L. luieiis
(Table 3).
Greater differences were found between the two species in their
capacity to digest protein from the ingredients with statistically
similar protein digestibility values only being obtained for mung
beans, whey and L. luteus (Table 3). Blacklip abalone digested
significantly more protein from defatted soyfiour, fishmeal. casein,
bull kelp, and skim milk than greenlip abalone. whereas greenlip
abalone digested significantly more protein than blacklip abalone
from semolina and dehulled and whole L. aiigustifolins (Table 3).
Comparisons among ingredients within species showed that
there were significant differences in their apparent protein and
energy digestibility for both species of abalone (Table 3). Whey
was the most digestible ingredient, having significantly higher
protein and energy digestibility than all other ingredients exaluated
for both blacklip and greenlip abalone I.P < 0.05). Bull kelp con-
tained the least-digestible protein for both species of abalone iP <
0.001), while semolina contained the least-digestible energy for
both species of abalone (P < 0.001 ).
DISCUSSION
The results from the current experiment demonstrate that black-
lip and greenlip abalone differ in their digestive capacity. Signifi-
cant differences were found in their ability to digest the protein and
energy from se\'eral ingredients.
With regard to protein digestibility it is interesting to note that
blacklip abalone can digest significantly more protein from, in
general, nonplant-derived proteins (excluding soyfiour and bull
174
Vandepeer and Van Barneveld
TABLE 3.
Comparison of the apparent faecal protein (PD) and energy (GED) digestibility coefficients obtained for 12 different ingredients fed to
blacklip and greenlip abalone.
PD
PD
GED
GED
Blacklip
Greenlip
Blacklip
Greenlip
Ingredient
Abalone
Abalone
Fu4
P
SEM
.Abalone
Abalone
f..4
P
SEM
Semolina
0.62''
0.84"
441
***
0.762
0.30''
0.34'
5.49
NS
1.265
Defatted soytlour
0.83'
0.82'
18.38
**
0.730
0.83"
0.78'
0.73
NS
1.507
Fishmeal
0.56'
0.46'
27.72
**
1.382
0.63^
0.52'
48.09
*
1.144
Casein
0.828
0.77"
27.42
**
0.624
0.79'=
0.78"
4.02
NS
0.579
Pregelled starch
-
-
-
-
-
0.92"
0.93"
1.80
NS
0.647
Mung beans
0.89''
0.9 1*"
5.13
NS
0.630
0.658
0.67'
2.40
NS
0.986
Bull kelp
0.46'
0.23'
105
»**
1.600
0.75'
o.sr
29.45
*
0.805
Whey
0.96"
0.95-"
1.46
NS
0.373
0.99''
LOO-*
43.20
*
0.106
Skim milk powder
0.94"
0.85'
510
***
0.286
0.95"
0.89"
1338
***
0.101
Lupin It
o.9r
0.91"
0.03
NS
0.804
0.79'
0.83'
2.83
NS
1.780
Lupin 2t
0.85=
0.92"
723
***
0.211
0.70'
0.82'
66.19
**
1.169
Lupin 3§
0.84"='
0.91"
371
***
0.284
0.63^
0.50'
202
:!=**
0.682
Within a species, superscripts have been used to identify Mgniticant differences among ingredients for their nutrient digestibility (within column
comparisons). Between species comparisons of nutrient digestion of each ingredient are made across rows and indicated by *.
NS, not significant
* P < 0.05
** P< 0.01
***/>< 0.001
"'8 Within a column, ingredient digestibility coefficients with different superscripts differ significantly (P < 0.05).
t Whole L luteiis.
± Dehulled L ungustifolius.
§ Whole L. anguslifolius.
kelp) than greenlip abalone. In contrast, greenlip abalone can di-
gest significantly more protein from plant-derived sources (lupins
and semolina) than blacklip abalone. This finding is in agreement
with that of Wee et al. (1994). who reported that blacklip abalone
digested significantly more protein than greenlip abalone from a
manufactured diet containing 50'7c fishmeal. It appears blacklip
abalone may not be able to digest the soluble nonstarch polysac-
charides found in terrestrial plants as efficiently as greenlip aba-
lone and that soluble nonstarch polysaccharides may actually in-
terfere with and reduce blacklip abalone's ability to digest nutri-
ents (both protein and energy I from plant feedstuff's which contain
them. As a consequence, use of exogenous enzymes that cleave
soluble nonstarch polysaccharides may improve the digestive ca-
pacity of blacklip abalone.
Dehulling had no effect on the digestibility of protein from L.
aiigHstifoHus when fed to blacklip abalone. Although a significant
increase was found in the digestibility of its energy for blacklip
abalone after dehulling it was much less than was found for green-
lip abalone (0.63 to 0.70 for blacklips compared with 0.50 to 0.83
for greenlips). After removal of the hull the energy from L. an-
guslifoliiis changed from being significantly less to significantly
more digestible for greenlip compared with blacklip abalone. The
hull of the lupin is composed primarily of cellulose. It appears that
blacklip abalone have a greater capacity to digest cellulose than
greenlip abalone given that the removal of the hull had a much
smaller effect on the capacity of blacklip abalone to digest energy
from this lupin compared with greenlip abalone.
Milk-based products (casein, skim milk powder, and whey) are
very digestible sources of protein and energy for both blacklip and
greenlip abalone. In particular, the sugar component of milk (lac-
tose) is very digestible for abalone given the extremely high gross
energy digestibility coefficients obtained for whey (the residue
from milk after removal of the casein and most of the fat). Lactose
is a disaccharide composed of galactose and glucose. Thus, it is a
much simpler carbohydrate than those found in many terrestrial
plant-based feedstuffs, such as lupins, which are composed of
complex structural and storage polysaccharides, p-galactosidase
(lactase) activity, needed for the hydrolysis of lactose, has been
found in abalone (Oshima 1931, Bennett et al. 1971). Obviously
P-galactosidase activity in wild abalone would not be for the di-
gestion of lactose, but probably for the breakdown of galactose,
one of the major components of carrageenan which is found in the
cell walls of red algae.
Pregelatinized waxy maize starch was also found to be a highly
digestible source of energy for both species of abalone. Again, this
is not surprising because the starch found in red algae, termed
floridean starch, is essentially the same as waxy starches found in
terrestrial plants in that it consists almost entirely of amylopeetin.
In addition Elyakova et al. (1981) found evidence for amylase-a-
1.4-glucanase activity against amylopeetin in extracts from the
hepatopancreas of W. asinina and H. vaiia. The fact that the starch
has been gelatinized, whereby the application of moist heat brings
about swelling and rupturing of the starch granules facilitating
amylolysis, would also increase energy digestibility.
The low protein digestibility of bull kelp by both species could
be caused by the presence of tannins, naturally occurring polyphe-
nols present in plants to protect them against herbivory. Their main
characteristic is that they bind and precipitate proteins. In vivo
studies have shown that protein digestibility is greatly reduced
when tanniniferous feeds are part of animal diets (Reed 1995).
Polyphenols are predominant in brown algae (Ragan & Glombitza
1986, Steinberg 1989). It should be pointed out that bull kelp has
Digestive Capacity of Abalone
175
a ver\' low crude protein content (69 g/kg) and that e\en though it
was included in the diet at a le\el of 500 g/kg the crude protein
content of the diet was onl\ 3.45 g/kg. Thus the endogenous N
contribution would ha\e had a much larger effect on the apparent
protein digestibility of kelp than for other ingredients, resulting in
these values being reduced as a result of an experimental artifact.
Neither species were able to digest the energy from semolina
very well, particularly blacklip abalone. In another study semolina
was found to affect the digestibility of other ingredients within a
diet (Vandepeer. unpublished data). The poor digestibility of
semolina and its effects on the digestibility of other ingredients is
a concern given that it is currently one of the major ingredients
used in manufactured diets in Australia. Further research is re-
quired lo establish the reasons why energy from semolina is so
poorly digested, however, it is possible that the starch component
significantly influences these results.
The results from this experiment demonstrate that greenlip and
blacklip abalone have different digesti\e capacities and thus a
different basis should be used for the formulation of manufactured
diets. Further comparisons of the nutritional requirements of
greenlip and blacklip abalone may also be justified.
ACKNOWLEDGMENTS
The authors would like to thank Dr. Ann Fleming for reviewing
and commenting on the manuscript. This research was funded by
a grant from the Fisheries Research and Development Corporation.
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S. A. Shepherd. M. J. Tegner & S. A. Guzman del Proo. editors. Aba-
lone of the world. Biology, fisheries and culture. Oxford: Blackwell
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Stewart, editors. Oxford: Blackwell Scientific Publications, Osney
Mead. 989 pp.
Wee. K. L.. G. B. Maguire & S. Hindrum. 1994. Methodology for digest-
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JoiiiiKil ,>f Shellfish Research. Vol. 22. No. I. I77-IS4. 2()()_V
revip:vv of techniques to prevent introduction of zebra mussels
(dreissena polymorpha) during native mussel (unionoidea)
conservation activities
W. GREGORY COPE,'* TERESA J. NEWTON," AND CATHERINE M. GATENBY'
^ North Carolina State University, Department of Environmental and Molecular Toxicology, Box 7633.
Raleigh. North Carolina 27695; ^United States Geological Siiney. Upper Midwest Environmental
Sciences Center, 2630 Fanta Reed Road, La Crosse, Wisconsin 54603: Academy of Natural Sciences,
Patrick Center for Environmental Research. I'-MM) Ben Franklin Parkway. Philadelphia. Pennsylvania 19103
ABSTRACT Because ot the declines in diversity and abundance of native freshwater mussels (superl'amily Unionoidea). and the
potential decimation of populations of native mussels resulting from the rapid spread of the exotic zebra mussel Dreissena polymorpha.
management options to eliminate or reduce the threat of the zebra mussel are needed. Relocating native mussels to refugia (artificial
and natural) has been proposed to mitigate the threat of zebra inussels to native species. Relocation of native inussels to refugia such
as t~ish hatchery facilities or natural habitats within their historic range, which are unlikely to be infested by zebra mussels, necessitates
that protocols be developed to prevent the inadvertent introduction of zebra mussels. Several recent studies have developed such
protocols, and have assessed their effectiveness on the health and survival of native mussels during subsequent relocation to various
refugia. The purpose of this project is to synthesize and evaluate the current protocols and to develop a set of procedures that resource
managers and researchers should consider before conducting conservation activities in zebra mussel infested waters. We found that the
existmg protocols have many common points of concern, such as facility modification and suitability, zebra mussel risk assessment
and management procedures, and health and disease management procedures. These conservation protocols may have broad appli-
cability to other situations and locations. A summary and evaluation of the mformation in these main areas, along with recommended
guidelines, are presented in this article.
A'£)' WORDS: relocation, Unionidae, Dieissenu polymorphu. conservation, refugia
INTRODUCTION
Native freshwater mussels of the families Martiariliferiihw and
Unionidae (supeifamily Unionoidea) are one of the most rapidly
declining fauna! groups in North America. About 67% of the
nearly 300 native species found in North America are considered
vulnerable to extinction or already extinct (Bogan 1993, Williams
et al, 1993). The decline of native mussel populations in Noilh
America has occurred steadily since the mid 1 800s and has been
attributed to overharvest, construction of dams and impoundments,
sedimentation, navigation, pollution, and habitat degradation
(Fuller 1974, Bogan 1993, Naimo 199?, Brim Box & Mossa 1999,
Vaughn & Taylor 1999). An additional recent threat to the native
fauna has come from the introduction of the zebra tnussel Dreis-
sena piilyiiiorpha. This species colonizes native mussels and im-
pedes their movement, reduces the ability to feed and eliminate
wastes, and coinpetes for food and space ( Mackie 1 99 1 , .Schloesser
et al. 1996. Strayer 1999).
Because of the declines in diversity and abundance of native
mussels and the rapid and severe impacts of zebra inussels on
native mussels (Gillis & Mackie 1994. Nalepa et al. 1996). a
national strategy for the conservation of native freshwater mussels
was developed to provide a framework for preventing further
population declines and species extinction (National Native Mus-
sel Conservation Committee 1998). This document identified a
number of conservation needs and outlined goals, strategies, and
tasks to address these needs. Listed among these was the recom-
mendation to develop management options for eliminating or re-
ducing the threat of zebra mussels to native mussels. These options
included relocating native mussels to artificial and natural refugia.
Although tiiany mussel relocations have had poor success (e.g..
*Corresponding author. E-mail: greg_cope@ncsu.edu
Cope & Waller 1995), recent studies conducted with improved
techniques, experimental design, and monitoring programs, have
been successful (Dunn et al, 2000, Cope et al, 2003). Thus, with
the increased likelihood of successful relocation efforts, and the
continued range expansion and adverse effects of zebra mussels on
native tnussel populations, any relocation done to conserve native
mussels necessitates that protocols be developed to prevent the
inadvertent introduction of zebra mussels.
Several recent studies have developed protocols to ensure that
zebra mussels would not be inadveHently introduced during native
mussel conservation activities and have assessed the health and
survival of native mussels during subsequent relocation (Patterson
et al. 1997, Patterson et al, 1999. Gatenby et al, 2000, Nichols et
al. 2000. Hallac & Marsden 2001. Newton et al, 2001), The pur-
pose of this project was to synthesize and evaluate the current
protocols and to develop a set of procedures that resource manag-
ers and researchers should consider before conducting native tnus-
sel conservation activities in zebra mussel infested waters.
RESULTS AND DISCUSSION
Almost all of the recent native mussel salvage and relocation
projects have used some type of quarantine to prevent the inciden-
tal introduction of zebra mussels. The exceptions are those studies
intended to remove zebra mussels from fouled native mussels and
replace them back to their original location (e.g., Schloesser 1996,
Hallac & Marsden 2000), By necessity, most of the quarantine
protocols have been location and facility specific. For example,
Gatenby et al. (2000) reviewed procedures for relocating native
mussels from the Ohio River. Likewise, Newton et al, (2001)
developed a specific set of procedures for relocating native mus-
sels from the Mississippi River to artificial ponds and to fish
hatchery facilities. However, these and other protocols developed
for specific studies have many common points of concern, such as
177
178
Cope et al.
TABLE 1.
Summary of collection and quarantine-related conditions and procedures, and recommended guidelines for preventing introduction of zebra
mussels during native mussel conservation activities.
Condition or Procedure
Reference
Gatenbv et al. (2000)
Newton et al. (20011
Recommended Guidelines
Collection setting
Time of collection
July. September. October \W5 May 1W5
Species of native mussels
No. of native mussels
Native mussels analyzed for
disease and pathogens
before relocation
Air temperature (X)
Water temperature (°C)
Mechanism for removing
zebra mussels from native
mussels
Method for holding scrubbed
native mussels at collection
site
Emersion time (min) during
collection and processing
Transportation to quarantine
facility
Quarantine facility
Type
Mussel density (no./ni")
Water source
Water temperature ("O
Dissolved oxygen (mg/L)
pH
Potas.sium (mg/L)
Alkalinity (mg CaCOj/L)
Hardness (mg CaCO,/L)
Total ammonia nitrogen
(mg/L)
Unionized ammonia (|ji.g/L)
Total residual chlorine (p.g/L)
Nutrition/feeding
Amhiema plicata, Quadnila
ptistulosa, ElUptio
crassidens. Pleurobema
cordatum. Obliquaria
reftexu, Ponmnhis ulanis
27(»
No
20-28
Hand scrubbed vMth plastic-
bristled brushes
Mesh bacs in river*
20
Between moist burlap in
coolers with ice (no direct
contact of mussels and ice)
Above-ground tanks, l-t-500 L
Well water
LS0-2.'S()
2-28
6-14
7.2-8..'i
1.6
90
90
£1.0
2-66
■1 X 10'' cells/mL three times
per week in quarantine;
relocation ponds were
fertilized with a
nitrogen;phosphorous (N:P)
ratio of 10:1 (1.0 mg/L N.
0. 1 mg/L P) with NH^NO,
and NaHPOj salts
Early spring, before zebra mussel
spawning begins (water temperatures
<15°C) or mid to late fall when
natives have greater energy reserves
and juvenile zebra mussels are
visible (>2-5 mm shell length)
Amblema plicata, Fusconaia
flava, Leptodea fragilis,
Obliquaria reflexa. Quadrula
qiiailnihi
768
Yes
6-18
11-14
Hand scrubbed with plastic-
bristled brushes under x2
magnification
Hatchery truck with aerated
well water
Between moist burlap in
coolers with ice (no direct
contact of mussels and ice)
Pond (0.04 ha), mussels held in
8-2720 L mesh baas
If possible
Early spring or late fall temperatures;
minimize differences between air and
water temperature
Early spring or late fall temperatures;
minimize differences between air and
water temperature
Hand scrub with plastic-bristled brushes
under magnification
Hold in zebra mussel-free water after
scrubbing
Keep to minimum, but <20
Between moist burlap in coolers with
ice in plastic bags for transport
durations <12 h; no direct contact of
mussels and ice bags
-19-159
Keep
to
minimum, hut <1.50
water
Well
water
1.1-27
<28
6-20
>6
7.8-10.6
6.5-9.0
2.6
<4
110-160
>15
180-200
>50
0.03-0.2
<1.0
2-20
.3 g/m" of 10:10:10 N:P:K
fertilizer added to quarantine
pond 2 weeks prior to adding
unionids; relocanon ponds
were not fertilized
<25
<17
X 10' cells/mL or 4.0 mg dry wt./L
twice daily or 2.0-5.0 x lO"* cells/mL
or 1.9 mg dry wt./L on a continuous
basis (Gatenby 2000, 2002); suitable
algal species include Neochloris
oleoabimdans. Bracteacoccus
grandis. and Pliaeodactylum
tricormHum
continued on next page
Preventing Zebra Mussel Introduction
179
TABLE 1.
continued
Reference
Condition or Procedure
Gatenbv et al. (2(H)0)
Newton et al. (2001)
Recommended Guidelines
Da\s in quarantine Minimum of 30. but up In 120;
re-inspected under 4x
niagnirieation
Disinfection of equipment and Chlorine solution of 25 mg/L
supplies
Monitoring
Temperature, dissolved
oxygen, and pH
All other water quality
variables
Disease and inortalit\
Dessication for up to 4 d
Twice daily
Daily to weekly
Not specified
35; re-inspected under 2x
magnification
Not specified
Daily
Daily to weekly
Not specified
Minimum of 30; re-inspect under
magnification
Chlorine solution of 25-250 mg/L,
depending on type of material;
dessication in warm dry air for 3-5 d
At least daily
Daily to weekly
At least weeklv
' All native mussels were rinsed with a high pressure hose before being placed into the quarantine facility.
facility modification and suitability, zebra mussel risk assessment
and management procedures, and native mussel health and disease
management procedures, that may have broad applicability to
other situations and locations.
Facility-Specific Concerns and Procedures
The availability of aquatic facilities for long-term captive care
of freshwater mussels is limited. Thus, most of the salvage and
quarantine facilities have involved the short-term use of state and
US Government owned fish hatchery ponds and raceways or simi-
lar research aquaculture facilities (Dunn & Layzer 1997. Pinder et
al. 1999. Gatenbv 2000. Newton et al. 2001). The main facility
concerns have focused on the type of rearing or holding system
(e.g.. ponds, raceways, or above-ground tanks capable of housing
hundreds to thousands of mussels), the facility's proximity to the
source of relocated mussels (to reduce transportation time and
handling stress), on-site water quality for maintenance of mussel
health, and production of an algal-based food supply. The objec-
tives of any given conservation project will likely dictate the type
of facility or holding system used and any modifications that may
be required. Nonetheless, whether used for short-term quarantine
or for long-term captive care, all facilities should be able to pro-
vide space for isolation and quarantine, water quality characteris-
tics to meet requirements for shell growth and metabolic processes,
and food quantity and quality to support growth and reproduction
(Table 1).
Specific isolation and containment modifications are probably
necessary at most facilities to control and contain source water
inflow and potentially contaminated outflow. For example, the
outflow of water from quarantine units may need to be passed
through filtration or disinfectant treatments to remove or kill po-
tential zebra mussels before the water is discharged through nor-
mal routes. Containment procedures commonly used at facilities
conducting zebra mussel research have included filtration of out-
flow water through small mesh bags ( 100 (xm or smaller), chlorine
treatment tanks (230 mg/L for I h). and sand filtration units (J. J.
Rach, U.S. Geological Survey. Upper Midwest Environmental Sci-
ences Center. La Crosse, WI, pers. com.). Additional facility pre-
cautions may include the capping of all exterior drains to prevent
the release of potentially contaminated water from the affected
areas and the development of a flood risk assessment, if the facility
is within a designated floodplain.
The type of facility selected, however, may influence the rela-
tive success of the conservation project. Success could depend on
its use only as a short-term quarantine facility for subsequent re-
location to a natural or artificial system, or its use for long-term
captive care. For example. Newton et al. (2001) relocated five
species of native mussels (1,392 mussels total) from the Upper
Mississippi River to a fish hatchery pond after 35 d of quarantine
in an artificial pond (81% of mussels survived during quarantine).
Mussel survival in the hatchery pond averaged SO^c after 1 y. but
only 35% 3 y after relocation. Of the mussels in a handling-control
treatment that were placed back into the Mississippi River after
quarantine, survival was 80% after 1 y and 75% after 3.3 y. The
authors attributed the differences in survival between the hatchery
pond and riverine relocated mussels to inadequate nutritional re-
sources in the pond. This study illustrates the potential utility of
natural or managed refugia over artificial refugia for long-term
conservation (Nichols et al. 2000. Cope et al. 2003). Gatenby
(2000) observed similar decreases in survival of six large river
species relocated to pond refugia after a 30-d quarantine in above-
ground tanks. Mean survival of native mussels during quarantine
was 97%. Mean survival after 1 y in the ponds ranged between 82
and 93%. depending on species. Despite an abundance of a suit-
able algal food supply and adequate water quality conditions in the
ponds, however, the survival of relocated mussels decreased to
44%- after 2 y and to 5% after 3 y. Gatenby (2000) attributed the
mortality to high water temperatures in July and August during
years 2 and 3 of that study. Large river species of mussels relo-
cated (with no quarantine period) to fish hatchery raceways with
flowing water and sediment also showed high survival (95%) after
1 y (Dunn & Layzer 1997). but their long-term (3-5 y) success in
this type of system is unknown.
The relocation of native mussels after quarantine to natural
refugia or raceway systems supplied by natural river water will
likely have greater success for long-term preservation of the mus-
sels than retention in artificial pond refugia for two key reasons:
water temperature and food quality. These two cotnponents are
critical to the livelihood of any aquatic organism. Rapid fluctua-
tions in temperature, unnaturally high temperatures, and inad-
equate food supplies are known to cause stress in aquatic organ-
180
Cope et al.
isms, and can lead to mortality (Bayiie et al. 1973). Thus, tem-
peratute. food quality, and food quantity will also be key
components to the success of native mussel captive care programs.
Zebra Mussel Risk Assessment and Management Procedures
Because the threat of zebra mussels to native mussels has been
the primary causal factor for initiating most mussel conservation
activities, special precautions have been necessarily incorporated
into the collection and handling protocols where native mussels are
relocated. These precautions taken during collection, transport,
processing, and quarantine of native mussels are of utmost impor-
tance. Only the careful collection and handling of native mussels
from zebra mussel-infested waters will ensure that hatchery fish,
native mussels, and other aquatic species in the ecosystem are
protected from the incidental introduction of zebra mussels.
In situations where there is unceitainty in the co-existence of
zebra mussel populations in the watershed, the most prudent and
conservative approach is to treat all native mussels as if they
originated from zebra mussel-infested waters. A review of zebra
mussel range distribution and population dynamics in the particu-
lar river basin is also warranted. Particular items of interest in-
clude, the nearest known reproducing population of zebra mussels
to the native mussel collectiiin site, the relative density and poten-
tial spawning periods of zebra mussels at that site, and the likeli-
hood of an undetected presence at the native mussel collection site
(e.g.. lack of an active monitoring program).
The optimum time for collection of native mussels for a given
conservation project is largely unknown. Conservation projects,
however, should strive to select periods that reduce the stress
associated with handling as much as possible. Potential criteria
include choosing a period that coincides with the absence of zebra
inussel larvae in the water column, minimizes the temperature
differential between air and water, and does not inteiTupt the re-
productive cycle for most of the species being relocated. Zebra
mussel contamination can be minimized by collecting native mus-
sels during early spring or late fall periods when zebra mussel
larvae are likely not present in the water column (e.g., water tem-
peratures <15' C. Mackie 1991 ) or when the settled juveniles are of
a sufficient size to be easily seen (e.g.. 2-^ mm in shell length),
respectively. Freshwater mussels are categorized as either long-
term (bradytictic) or shon-term (tachytictic) brooders. Long-term
brooders, like many species of lampsilines and anodontines. be-
come gravid in late summer, retain the developing glochidia in the
gill marsupia throughout winter, and spawn in early spring (Mc-
Mahon & Bogan 2001 ). In contrast, short-term brooders, like many
species of amblemines. become gravid in early spring and spawn
in late summer (McMahon & Bogan 2001).
Newton et al. (2001) collected native mussels in early spring
when water temperatures ranged between 1 1 and 14°C. a period
before zebra mussel spawning, which generally occurs when water
temperatures reach 15 to 17°C (between May and June), in north-
em temperate regions of the United States and Canada (Mackie
1991 ). The collection of native mussels in early spring also has an
added potential benefit of reduced energetic stresses associated
with handling because of the cooler water temperatures (Jokela
1996, Newton et al. 2001). For example, glycogen concentrations
in Amhleina plicata were highest between May and July and
dropped precipitously thereafter — a pattern that closely paralleled
reproduction in this short-term brooder (Monroe & Newton 2001 ).
Similarly, Jokela et al. (1993) observed that glycogen concentra-
tions decreased substantially between July and October in An-
(uldiita pisciinilis. a long-term brooder. Ftirthennore. Jokela ( 1996)
suggested that transplanting females before fertilization or during
the early development of the brood had no detectable effect on
reproductive output.
Data on energetic reserves in marine bivalves contradict the
recently reported data in freshwater bivalves. In the marine envi-
ronment, it has been suggested that mussels collected in fall may
be able to better withstand handling stress because of their higher
energy reserves and because their metabolism is slowed by the
cooler water temperatures (Bayne et al. 1973). For example, by
mid to late fall, the marine species Mytihis edulis and M. trossulus
had accumulated abundant carbohydrate energy reserves (Hawkins
& Bayne 1985. Kreeger 1993, Kreeger et al. 1995). The differ-
ences between marine and freshwater species may be caused by
differing reproductive strategies. Results from a recent study with
native freshwater mussels, however, suggest that some species of
native mussels may build up their energy reserves in fall (Gatenby
2002). Obviously, this is an area where additional research is
needed.
When native mussels are collected from multiple sites in a
watershed with a known or suspected gradient in zebra mussel
density, working from the least infested site to the most infested
site will reduce potential zebra mussel contamination of boats and
other equipment. Optimally, boats used to collect or deploy native
mussels in zebra mussel infested areas should be cleaned (before
and after) by a high-pressure hot-water wash and diver wet suits,
supplies, and equipment (e.g.. ropes, buckets, etc.) used in the
study should be disinfected with a mild solution of chlorine bleach
(25 mg/L) or air dried (3-5 d) before use (Gatenby et al. 2000).
If the quarantine or relocation facility is also an operational fish
hatchery or aquaculture center, precautionary measures to protect
endemic wild species and cultured fish species should be consid-
ered. Before entrance into the facility, a subsample of native mus-
sels should be obtained from the collection site and submitted to a
United States Fish and Wildlife Service. National Fish Health
Center (Newton et al. 2001 ) or similar laboratory, to assess poten-
tial disease and pathogen presence (see section later on native
mussel health and disease management procedures).
After screening for diseases and pathogens, collection of native
mussels should proceed with procedures to minimize contamina-
tion from adult and larval zebra mussels. These include scrubbing
individual native mussels with plastic bristled brushes, visual in-
spection of all exterior surfaces of the shell with magnifying
lenses, and holding cleaned natives in zebra mussel-free water
(Table 1 ). Care should be taken during scrubbing and inspection to
avoid overlooking small zebra mussels that may be attached in
crevices, in areas of shell erosion (native mussels with severely
eroded or damaged valves should be discarded), or along the hinge
line (Gatenby et al. 2000, Newton et al. 2001). Only personnel
experienced in mussel biology should conduct the inspections to
ensure accuracy and efficiency of these procedures.
During collection and processing of native mussels, emersion
(exposure to air) and thermal stress should be kept to a minimum.
Recent studies have shown that handling mussels over a range of
emersion air temperatures ( 15-35°C) and emersion durations ( 15-
60 min) did not acutely impair survival, behavior, or biochemical
composition (Bartsch et al. 2000, Greseth et al. 2003). A minimal
emersion time (<20 min). however, is generally recommended
from recent efforts (Table 1 ). Moreover, water temperature and
Preventing Zebra Mussel Introduction
181
dissolved oxygen concentrations in the holding \esscls during col-
lection should be measured frequently (at least once per hour) and
maintained at or near (±2 'O the ambient stream conditions at the
time of collection with non-chlorinated ice and external aeration, if
possible (Gatenby et al. 2000).
Depending on the proximity of the native mussel collection site
to the quarantine facility (a transport time generally <12 h). mus-
sels should be transported in coolers covered with moist burlap and
kept cool (within ±2°C of the water collection temperature, if
possible) w ith ice in plastic bags without direct contact of ice bags
and mussels (Gatenby et al. 2000. Newton et al. 2001. Cope et al.
200.^). This method is advantageous over the use of water-filled,
aerated tanks (Chen et al. 2001) because of the reduced need for
costly and cumbersome trucks and equipment and of miniinizing
potential problems associated with maintaining stable dissolved
oxygen concentrations in water during transport.
At the quarantine facility, native mussels have generally been
held for a minimum of 30-35 d (Gatenby et al. 2000, Newton et al.
200 1 ) to allow any small or previously undetected zebra mussels to
become visually apparent on re-inspection. The 30-35 d quaran-
tine period is based on reported zebra mussel growth rates of
0.06-0.15 mm/d (Mackie 1991. Martel 1995. Chase & Bailey
1999), which would allow a newly settled zebra mussel to reach a
visible shell length of about 2-5 mm during quarantine. During
this time, basic water quality measurements (e.g., temperature,
dissolved oxygen, and pH) should be taken at least daily. Other
water chemistry variables such as alkalinity, hardness, potassium,
total ainnionia nitrogen (TAN), and unionized ammonia should be
measured at least weekly to ensure that water quality conditions
for minimum life requirements are met (Table 1 ). In addition,
mussels in quarantine should be monitored at least weekly for
disease (see section below on native mussel health and disease
management procedures) and mortality.
Isolation of native mussels from other aquatic species, their
contact water, nets, or other equipment at the quarantine facility is
necessary to protect organismal health and the physical facility.
These concerns can largely be addressed by applying standard best
practices for maintaining fish health. Disinfection of equipment
and supplies for native mussel quarantine should be guided by
National Fish Health Policy and Procedures, Part 713, sections
FWI and FW 3 (USFWS 1995): chlorine (200-250 mg/L for 1 h),
.sodium or potassium salts (saturated solutions) or other chemical
treatments (e.g., benzalkonium chloride at 100 mg/L for 3 h) and
desiccation (3-5 d) have been successfully used or recommended
(Reid et al. 1993, Waller et al. 1996. Gatenby et al. 2()()()).
After the minimum quarantine period (30-35 d). individual
mussels are thoroughly re-inspected by hand with magnifying
lenses to evaluate the presence of zebra mussels. If zebra mussels
are not found, the mussels are deemed zebra mussel-free and can
be relocated elsewhere (e.g.. to natural or artificial systems or to
other facilities for long-term captive care). Because no zebra mus-
sels were found after quarantine in the study of Newton et al.
(2001). the mussels were subsequently relocated to fish hatchery
ponds. In contrast. Gatenby et al. (2000) found zebra mussels on
initial re-inspection and consequently held native mussels in quar-
antine for additional 30 d intervals each time zebra mussels were
found, up to a total of 120 d. Because of declines in mussel health
and condition over time during quarantine (Patterson et al. 1997.
Newton et al. 2001). Gatenby et al. (2000) recommended re-
inspection of mussels at 7 d intervals after the initial 30 d period
when zebra mussels are found, and to hold them onlv for 30
additional days after the last zebra mussel is found, to shorten the
overall quarantine time. However, the added stress of handling
native mussels more frequently must be weighed against the prob-
ability of earlier detection of zebra mussels.
Additionally, native mussels could be treated with chemical
disinfectants. Certainly, the benefit of this type of treatment must
be weighed against the risk of added stress and reduced fitness in
the native mussels, but a study by Waller and Fisher (1998) found
that limited application of specific chemicals (e.g., 20,000 mg
NaCl/L for 6 h) may be feasible for certain tolerant native species.
They cautioned, however, that chemical disinfectants cannot guar-
antee the elimination of all zebra mussels from native mussel
shells and stated that pre-treatnient or multiple treatment (e.g.,
once per week) of native mussels and their holding tanks may be
most valuable for reducing the time held in quarantine. Many fish
hatchery and aquaculture facilities may already be using various
chemical treatments (Waller et al. 1996. Edwards et al. 2000.
Edwards et al. 2002) or hazard analysis protocols such as the
Aquatic Nuisance Species-Hazard Analysis Critical Control Point
(ANS-HACCP) approach (Gunderson & Kinnunen 2001) to pre-
vent the spread of zebra mussels and other aquatic nuisance spe-
cies during their activities, which may be adapted to the collection,
transport, and quarantine of native mussels.
,\ative Mussel Health and Disease Management Procedures
Although liltle is known about the diseases of native freshwater
mussels, recent studies have shown the potential for pathogen
transmission among native mussels and fish (Starliper et al. 1998,
Starliper & Morrison 2000). The primary concern for fish hatchery
or aquaculture facilities that contain native mussels is the potential
for transmission of disea.se and pathogens between host mussels
and hatchery fish. Transmissions from hatchery fish to mussels and
from mussel to mussel are also important vectors to control for
maintaining mussel health. Therefore, a pathogen and disease
monitoring plan for native mussels, similar to that commonly used
for hatchery-reared fish, should be considered. Hatchery personnel
are routinely trained in fish health protocols and record keeping:
these procedures could easily be adapted for monitoring mussel
health. The United States Government standards and protocols
currently exist for a disease control and classification system for
coldwater fish (salmonid) pathogens — similar guidelines for
warmwater fish or native mussels do not exist (USFWS 1995).
Revisions to the United States Fish and Wildlife Service, Fish
Health Policies and Procedures are currently underway to include
warmwater fish and other aquatic organisms (Richard Nelson,
United States Fish and Wildlife Service, La Crosse Fish Health
Center, Onalaska, Wl, pers. com.). Until those changes are imple-
mented, however, native mussels may only be screened in the near
term for reportable coldwater pathogens and diseases. On a posi-
tive note, a recent study evaluating the effect of depuration on the
transmission of the bacterial fish pathogen Aeromonas salmoni-
cicla (the causative agent offish furunculosis) between the unionid
Anihic'ina plicata and two strains of Arctic char Scilveliniis alpinus
found that the minimum 3()-d quarantine of native mussels recom-
mended for preventing the spread of zebra mussels was sufficient
for depuration of the fish pathogen and eliminating transmission of
the disease (Starliper 2001 ). Therefore, when adequate safeguards
and standard best practices for fish health are used in combination
with a 30-d quarantine, disease and pathogen transmission risks
should be minimal. Native mussels held in quarantine should be
182
Cope et al.
screened before being placed in tlie quarantine facility and moni-
tored monthly throughout the duration of their captive care to
document disease and pathogen incidence and history. More re-
search and policy development is needed in this area to ensure
protection of fish and native mussels.
Maintaining the physiologic condition of native mussels during
quarantine is difficult because diet and nutritional requirements are
poorly understood. Although the specific time course for changes
in biochemical indices of mussels caused by quarantine is un-
known, recent studies have shown that substantial decreases in
glycogen concentrations occur in as little as 7-35 d after quaran-
tine. For example. Patterson et al. (1997) found that glycogen
concentrations in mantle tissue in Amhieinii plicata and Quadriila
pustidosii dropped significantly after 7 d in quarantine and by day
30. concentrations had declined to only 15-31% of that measured
in wild-caught specimens. Likewise, glycogen concentrations in
foot tissue of A. plicata decreased 44% from 279 ± 191 mg/g dry
weight at day 0 to 178 ± 105 nig/g dry weight after 35 days in
quarantine (Newton et al. 2001 1.
Based on the poor physiologic condition of native mussels after
quarantine shown by previous studies, it is critical to provide the
best source of nutrition during quarantine. Previous studies have
relied on an algal-based diet, either produced //; situ by stimulating
algal growth with fertilizers in ponds or cultured indoors on site
and added directly to mussel holding tanks (Gatenby et al. 1997,
Patterson et al. 1997. 1999, Gatenby 2000, Gatenby et al. 2000,
Newton et al. 2001 ). A number of algae have been tested as food
for juvenile and adult mussels (Gatenby et al. 1997, Gatenby 2000,
Beck 2001). Recent biochemical analysis of three algae (Neochlo-
ris pleoahmulans. Bnuteacticciis gnmdis. and Phacodactyliiiu lii-
ainuttuiii) indicate that these could be nutritionally suitable for
maintaining freshwater mussels in captivity (Gatenby et al. 2002).
If mussels are to be quarantined or relocated to ponds, the follow-
ing should be kept in mind: ( 1 ) standard commercial pond fertil-
izers should not be used to stimulate growth of algae; (2) the
potassium levels in commercial fertilizers are toxic to freshwater
mussels (Imlayl973); (3) the nitrogeniphosphorous ratio (N:P) of
the standard 10:10:10 nitrogen:phosphorous:potassiuni (N:P:K)
fertilizer will not promote suitable algae for mussels that typically
require an N:P ratio of 10:1 (McCombie 1953); and (4) an unsuit-
able, or indigestible filamentous blue-green algal bloom will result
when 10: 10: 10 N:P:K is used. Therefore, we recommend using the
fertilizers indicated in Table I, following Gatenby et al. (2000).
Although feeding requirements for native mussels will likely de-
pend on the species involved, temperature conditions, and meta-
bolic activity, Gatenby et al. (2000) recommended that native mus-
sels be fed 1 x 10'' cells/niL or 4.0 mg dry weight/L twice daily
(Table 1 ). This was a conservatively high recommendation based
on initial feeding studies and assimilation efficiencies. This con-
centration resulted in the greatest assimilation of organic carbon,
but a significant amount of this ration went unused by the animals
(Gatenby 2000). More recent data indicate that a diet ration of
2.0-5.0 X lO'* cells/niL or 1.9 mg dry weight/L per feeding cham-
ber should maintain mussel condition during summer growth pe-
riods (Gatenby 2002). Particle concentrations should be monitored
and not allowed to drop below 60% of this recommended ration.
Feeding frequency will depend on the species and total biomass
being held in captivity (Gatenby 2002). Thus, monitoring the par-
ticle concentration on a daily basis is necessary. Initially, particle
concentration may need to be monitored two to three times daily
until the manager is familiar with the particle depletion rate or
clearance rate of the native mussels held in captivity.
CONCLUSIONS AND RECOMMENDATIONS
Native freshwater mussels should only be relocated from ex-
isting areas as a la.st resort (Cosgrove & Hastie 2001 ). Other op-
tions to relocation and salvage, such as periodic cleaning of zebra
mussels from native mussels and replacement (Hallac & Marsden
2000, Hallac & Marsden 2001 ), and the use of natural or managed
refugia (Nichols et al. 2000), should be considered as first alter-
natives where practical. For example, Hallac & Marsden (2000,
2001 ) suggested that periodic cleaning and replacement might be
a viable option for conservation of native mussels, especially in
areas where food is not limiting and where collection and cleaning
are logistically feasible. If, however, freshwater mussel relocations
are required to conserve localized populations from zebra mussels
or other catastrophic events, the concerns and procedures de-
scribed in this article should provide general guidance for devel-
oping plans to prevent the incidental introduction of zebra mussels
during these activities and for maintaining the health of the native
refugees while under captive care.
In addition, procedures for ensuring long-term viability of na-
tive mussel populations need to be considered throughout the plan-
ning and Implementation process. For example, similarities in wa-
ter quality, substratum characteristics, food, and necessary fish
hosts among the systems are critical elements in a native mussel
relocation strategy. Additional ecological and evolutionary con-
cerns, such as retention of genetic diversity of the mussel popula-
tions, need to be carefully considered before relocating native
mussels to natural refugia, especially if the mussels are to be
relocated between river basins or between sub-basins of the same
river system (Villella et al. 1998, Storfer 1999).
Because of costs and limited availability of facilities for quar-
antine and captive care of native mussels, the United States Fish
and Wildlife Service and its resource conservation and manage-
ment partners may wish to designate several facilities within re-
gions of the United States that can accept, hold, and screen mussels
for disease and pathogens. These facilities may include state or
national fish hatcheries, research or aquaculture centers, and fish
health centers.
To our knowledge, this synthesis represents the "state-of-the-
science"" for minimizing the incidental introduction of zebra mus-
sels during native mussel conservation activities and for ensuring
their short-term and long-term health and viability. Readers of this
article should be cautioned that the information presented is only
recommended guidelines and that future improvements to proce-
dures will be made through research and policy development.
ACKNOWLEDGMENTS
This project was funded by the United Stales Fish and Wildlife
Service, through a contract with the Freshwater Mollusk Conser-
vation Society. Linda Drees and Tina Proctor provided valuable
insight on the relevance of the project to resource managers. Steve
Ahlstedt, Arthur Bogan, Heidi Dunn, Jerry Fairis. Doug Jensen,
Patricia Morrison, Pam Thiel, and Kurt Weike provided informa-
tion critical to preparation of the document. The authors thank
Robert Anderson, Heidi Dunn, Richard Neves, Jeixine Nichols.
Tom Watters. and Kurt WeIke for reviewing a draft of the docu-
ment.
Preventing Zebra Mussel Introduction
183
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A COMPARISON OF THE PARASITE AND SYMBIONT FAUNA OF COHABITING NATIVE
{PROTOTHACA STAMINEA) AND INTRODUCED (VENERUPIS PHILIPPINARVM AND
NUTTALIA OBSCURATA) CLAMS IN BRITISH COLUMBIA
W. L. MARSHALL, S. M. BOWER,* AND G. R. MEYER
Fisheries and Oceans Canada. Biological Sciences Brancli Pacific Biological Slalion Nanainm.
British Columbia. Canada. V9T 6N7
ABSTRACT Native littleneck clams iPronnlnii a sitiiiiincti). Manila clams {Vcncnipls pliilippiminim. inadvertently introduced in the
iy3().s), and varnish clams (NtittaUiu obiciiiaia, inadvertently introduced in the 1980s and lyyOs) were collected from the same
microsite at two different locations and examined for parasites and symbionts using histology and light microscopy. Varnish clams are
currently being assessed for their long-term fisheries potential but there is little knowledge of their parasite and symbiont fauna. This
study initiates the documentation of parasites and symbionts of varnish clams and adds to the continuing documentation of organisms
found within native littleneck clams and Manila clams. Host exposure to potential parasites and symbionts that were prevalent in at
least one of the clam species was assumed lo be similar for all clams due to their close proximity. This close association in the natural
environment allowed for the comparison of host specificity and response of the clams to multiple invasive species. All three of the clam
species had a different assemblage of parasites and this pattern was mostly consistent for both sites. Host preferences of each type of
parasite or symbiont v\'ere also consistent between sites and they were often restricted to a single host species. The most common
parasites of varnish clams were Nemaropsi.s-Wki! spores, pea crabs (Pinnixa fciha) and parasitic copepods (Mylilicolu sp.) and less
frequently a turbellarian inhabiting the kidney tubule. An undocumented eimeriorin-like kidney coccidian was found in 4% of Manila
clams and two previously undescribed inclusions bodies were found in native littleneck clams at low frequencies.
KEY WORDS: hixalve. Pniiotliaca suimiiwu. Vciicnipis philippiiuiniiu. Nuttullia (ihscitrala. parasites, symbionts
INTRODUCTION
In .Itnic of 2002 three species of clams (one native and two
introduced) were chosen for a survey of parasites and symbionts.
The native littleneck cluni \Prounhaca staminea: (Conrad 18.^7);
= Paphia suiininca. = Venus staininea] was the most important
fresh-market clam until the advent of the Manila clam [Venenipis
philippinanim: (Adams & Reeve 1850); = Riiditupes philippi-
nariim. = Tapes japonica. = Tapes philippinanim. = Tapes
semideciissata. = Venenipis japonica. = Venenipis semideciis-
satu\. another member of the family Veneridae with similar mor-
phology to the native littleneck clam but with a longer market
shelf-life. The Manila clam, also known as the Japanese littleneck
clam, was first observed in British Columbia near Ladysmith Har-
bour in 1936 (Quayle 1964). Introduction presumably occuned
during transplantation of Pacific oyster (Crassostrea gigas) .seed
from Japan, when young Manila clams of several millimeters in
shell length may have been trapped in the oyster shells (Quayle
1964). The dispersal of Manila clams was rapid, and by 1941 they
formed a significant proportion of the commercial catch and were
the doniniant lamellibranch of many beaches (Quayle 1964). They
are now established along both coasts of Vancouver Island, al-
though less abundant in the northern parts, and along similar lati-
tudes on the mainland coast (Bourne 1982).
Varnish clams {Niittallia obscurata (Reeve 1857); = Sole-
lellina ohsciirala, = Psammobia olivacea. = Satelettina japimica].
also known as purple mahogany or Savory clams, belong to the
family Psammobiidae. Originally native to Korea and the Japanese
Islands of Kyushu, Honshu, and Shikoku (Coan et al. 2000), they
have been recently introduced to the Georgia Strait, probably via
ballast water (Gillespie et al. 1999). They have since spread north
into Johnstone Strait, along the west coast of Vancouver Island
north to Checleset Bay. along the mainland coast, south into Puget
*Corresponding author. E-mail: BowerS@dfo-mpo.gc.ca
Sound and along the Oregon Coast to Port Townsend (Dinnel &
Yates 2000. Gillespie et al. 2001). There have been some trial
fisheries but the long-term potential of the fishery is currently
under investigation (Gillespie et al. 1999, 2001).
The purpose of this study was to compare the parasites and
symbionts found in each of the clam species at two different sites.
Clams from each site were gathered at close proximity to each
other and were assumed to have had similar exposure to the spec-
trum of parasites enzootic to that site. This sampling regimen helps
minimize suspicions that observed differences could be the result
of temporal or spatial variations, thereby increasing the interpre-
tative value of negative results. This survey is the first to examine
varnish clams for parasites and symbionts using histological meth-
ods and also contributes to the continuing documentation of para-
sites and .symbionts found in Manila and native littleneck clams.
MATERIALS ANU METHODS
On 10 June 2002. Manila clams, native littleneck clams, and
varnish clams (n = 25) were collected from each of two locations
within the Strait of Georgia on the coast of British Columbia for a
total of 150 clams. The first 75 clams were collected from Crofton
at a beach below a sewage outfall located between the ferry ter-
minal and pulp mill, the others were gathered 2 h later from Boul-
der Point. Ladysmith. At each location clams between 40 and 57
mm in length were dug from a single site (2.0-2.5 m" in area,
approximately 15 cm deep) within the mid-intertidal zone, away
from evidence of eutrophication and fresh water runoff, where
none of the target species were more than 1.5 times more abundant
than another. All clams appeared healthy and were held in tanks
(one tank per site) with flowing ambient seawater for 3—4 days.
Each clam was then shucked, the shell length and wet weight of
soft tissue recorded, superficially examined and pool fixed (5 per
jar) in Davidson's solution. Pea crabs were collected, preserved in
Davidson's solution and held for identification. After at least 24 h
in the fixative two cross sections, one through the region of the
185
186
Marshall et al.
stomach and digestive gland and the other through the kidney and
heart were made. The labial palps, siphon and posterior adductor
muscle were also sampled and processed with the cross-sections
using routine histological techniques. Sections (3-(jLni thick) were
cut and stained with Harris's modified hematoxylin and 0.5% al-
coholic eosin. Additional sections from selected speciinens were
stained with Brown and Hopps Gram stain and also tested for the
presence of DNA using the Feulgen stain reaction. All sections
were examined under a compound microscope (100 to lOOOx).
RESULTS
Average shell lengths of each clam species varied little between
sites but clams collected from the Crofton site had lower wet
weight to shell length ratios (Table 1 ). Native littleneck clams
ranged in length between 41.6 to 50.1 mm from Boulder Point and
41 .6 to 49.4 mm from Crofton; their wet weights were between 5.6
to 11.9 g from Boulder Point and 5.8 to 10.4 g from Crofton.
Manila clams ranged between 41 .4 to 56.4 mm from Boulder Point
and 40.2 to 55. 1 mm from Crofton: wet weights were between 6. 1
to 14 g from Boulder Point and 4.7 to 1 2.9 g from Crofton. Manila
clams showed the least difference in wet weight to shell length
ratio (Table 1 ). Varnish clams ranged between 41 .4 to 53.4 mm in
length from Boulder Point and between 40.0 to 5 1 .7 from Crofton,
wet weights ranged between 4.6 to 1 1 .6 g from Boulder Point and
3.9 to 7.8 g. from Crofton. The average wet weights to shell length
ratio was much less in varnish clams collected from the Crofton
site (Table 1 ).
Pea crabs (family Pinnotheridae) were collected from both Ma-
nila and varnish clams during the shucking process. Only one
immature Piiinixa fabci was found in the Manila clam sample,
however 16-24% of varnish clams contained one pea crab (Table
2). These were also identified as P. faha and were either immature
or male; the largest measured 13 mm across the carapace. The
presence of pea crabs had no obvious pathological effects and did
not affect the wet weight to shell length ratio. For example, the wet
weight to shell length ratio of the six varnish clams from Boulder
Point containing a pea crab was 0.18 g/mm whereas this ratio for
the 19 varnish clams from the same location without pea crabs was
0.17 g/mm. All other organisms were found during histological
examinations.
Colonies of intracellular prokaryotes (Rickettsiae or Chlamy-
diae) were observed within the epithelial cells of the gills and
digestive gland tubules in both Manila and native littleneck clams
(Fig. 1). Gill infections in Manila clams were less frequent (8-
20%) and were considered to be of light intensity (<80 colonies)
compared with native littleneck clams where there was a higher
prevalence (>88%) and many examples of moderate and high
(>200 colonies) intensities (Table 2). Infections within the diges-
tive gland were also more prevalent in native littleneck clams than
in Manila clams (Table 2). The digestive gland was the most
frequent site of infection in Manila clams whereas the gill infec-
tions greatly outnumbered digestive gland infections in native
littleneck clams. Most digestive gland infections were light (<10)
to moderate (10 to 24) in both species except for two cases of
heavy infection in native littleneck clams from the Crofton site
where as many as 55 colonies were counted. The identity of the
intracellular prokaryotes is unknown and may be representative of
more than one species. The colonies within the digestive gland
tubules appeared to be denser than those found within the gill
tissue where it was often possible to see the individuals within the
colony. Between hosts, the colony moiphologies were consistent
and appear to be the same agents as those described by Bower et
al. ( 1992). No associated host response was observed, however the
infected cells (especially gill epithelium) were often swollen be-
yond their normal size (Fig. 1 ). In many cases, host cells of gill
infections were ruptured and the prokaryotes were leaking out into
the water channel.
Colonies of large intracellular rod shaped bacteria (Fig. 1 ) were
obser\ ed at low intensities within gill epithelial cells of 4-52% of
native littleneck clams. The maximum size of these bacteria was
6.3 |xm long by 1 .4 (a,m wide but there were also smaller variants.
Staining characteristics ranged from strongly to very weakly ba-
sophilic and were predominantly gram positive, however, there
were also Gram-negative representatives throughout the entire size
range. Colonies were often 28 |xm in diameter but did not appear
to incite any hemocytic response or otherwise show any indication
of pathology. There was a weak correlation between intensity of
Rickettsia or Chalmydia-like infections and the number of colonies
of rod shaped bacteria observed, clams containing colonies of rod
shaped bacteria were usually infected with moderate to high num-
bers of Rickettsia or Chahnyilia-Wke colonies.
Another inclusion body, also unique to native littleneck clams,
was found in low intensities with 12% prevalence at both sites
(Table 2). These bodies were large, with an average diameter of 65
|j.m. and bound by hemocytes that appeared to have flattened
against the infected cell forming a thick eosinophilic membrane
(Fig. 2). The material within was basophilic, Feulgen positive and
Gram negative, it was of a very fine matrix and denser near the
edges of the colony. The infection was found in nearly every tissue
(heart, kidney, gonad, gill, and palps) and appeared to be the result
of an infected, extremely hypertrophied hemocyte.
Apicomplexan spores resembling Nematopsis sp. were ob-
served at least once in all three species, however, mainly in Manila
and varnish clams collected from the Crofton site (Table 2). The
prevalence in native littleneck clams was very low (4% and 12%)
and there were never more than two spores within an infected
clam. One spore was in the gill epithelium and the others were
found within the gill connective tissue, those found within the
TABLE 1.
.Average shell length and «et weight to shell length ratios of nati>e littleneck clams {Prnlolhaca slamiiiea). Manila dams iVenenipis
philippinarum). and varnish clams {Nitttallia obscurala) examined from two locations in British Columbia, Canada in = 25 for each species at
each location).
Native Littleneck Clams
Boulder Pt./Crofton
Manila Clams
Boulder Pl./Crofton
\ arnish Clams
Boulder Pt./Crofton
Average Shell length (nimi
Wet weight to shell length ratio (g/mm)
4,^.2 / 44.9
0.20/0.17
A5J /4i^.5
().[4/0.1S
47.7/44.7
0.17/0.12
Parasites of Three Bivalves in British Columbia
187
TABI.K 2.
Pre\ak'nce* and intensityt of parasites and synihionts in native littleneck clams {I'mtathaca stainiiuat. Manila clams {Venerupis
philippinarum), and varnish clams [Sutlallia iihsciirata) from two localities in British Columhia. Canada.
Parasite/Svmbiont
Native Littleneck Clam
Boulder Pt. / Crofton
Manila Clam
Boulder Pt. / Crofton
Varnish Clam
Boulder Pt. / Crofton
Rickellsia or Chlamydia in gill
Rickettsia or ChUiiii>dia in
digestive gland
Large intracellular rod shaped
bacteria
Fine-matrix inclusion bodies
Apicomplexan spores
Nemiirnpsis-like
Trichikliiki spp.
Order Rhynchodida
Einieriorin-like coccidian
(Apiconiplexal
Copepods, (Myiilicola-hke)
Other copepods
Tremalode metacercariae
Turbellarians
Pinnotheridae
88%; 19L, 3M (1-130)/ 100';;:
6L. 7M. I2H (12-600)
48%: 9L. 3M { 1-241 / 52%: 7L.
4M. 2H (1-35)
4%: L (l)/52%; L (1-16)
12%: L (1-9)/ 12%: L (3-5)
4%: L(l)/ 12%: L (1-2)
0% / 0%
0% / 0%
0% / 0%
4%: L(l)/8%: L(I)
4%: L(l)/0%
0% / 0%
0% / 0%
0% / 0%
8%: L (1-2)/ 20%:
L(l-
30)
0% / 0%
36%: 7L. 2M ( 1
-24
/ 32%: 5L.
0% / 0%
2M(l-20)
0% / 0%
0% / 0%
0% / 0%
0% / 0%
0%:/80%: 17L
2M
IH
4%; L(l)/ 100%: 16L. 6M. 3H
(3-200)
(3-230)
20%: L (1-3)/ 56%:
L(l
-11)
0% / 0%
20%: L (1-5)/ 44%:
L(l
-15J
0%/0%
4%:L(4)/4%:
L(3)
0% / 0%
4%:L(l)/0%
64%;L(1^)/60%:L(1^)
0% / 0%
4%; L(l)/0%
8%: L(l)/0%
0%/0%
0%/4%;L(l)
8%;L(l)/0%
4%;L(l)/0%
24%:L(1)/16%:L(1)
* Recorded as the percentage of each clam species infected with a given organism at each location.
t Recorded as the number of clams with heavy (H), moderate (M). or light (L) infections (as defined in text), followed by the range of colonies or
individuals of each parasiie/symbiont observed in parenthesis.
conneL'ti\e tissue were acctimpanied by a mild hemocytic re-
sponse. Manila clams were only infected at the Crofton site; the
majority of these infections were light (<60 spores per histological
section) with only a few cases of moderate or high (>150) inten-
sity. Gill connective tissue was the primary focus of infection but
was accompanied by a light infection (one to five spores) of the
palps in 289^ of the clams. There was also one instance where a
single spore was found in the gonadal tissue. The spores appeared
opaque, with no visible internal structures or nuclei, and were
usually accompanied by a focal hemocytic response, identical to
those described by Bovver et al. (1992). Spores found in varnish
clams also occurred predominately within the gill connective tis-
sue but there were also a few spores in the palps (three clams) and
kidney (two clams). A little over half of the spores were of the
same morphology typical to the Manila clams (Fig. .3) and usually
showed a hemocytic response. The remaining spores (Fig. 4) often
contained a nucleus and were clustered within clumps of
hemocytes. making them difficult to discern and accurately count.
These variations appeared to be part of the host immune response
since there was no evidence to suggest that the spores were alive
and capable of progenesis. There also appeared to be a size dif-
ference between the two spore morphologies with one having an
average length of 9.26 ± 1.33 p.m (n = 30) and the other an
average length of 7.91 ± 1.31 (jtm (/; = 30). However the si/e
differences were not statistically significant.
The remaining protistan parasites detected were an eimeriorin-
like coccidia (Apicomplexa) and two ciliates, a Sphenophyra-\\k&
ciliate of the order Rhynchodida and a Trichodina spp., and all
were found exclusively in the Manila clams. The coccidian was
observed within the kidney tissue in one Manila clam from each
location (Table 2) but only the macrogamont stage was observed
(Fig. 5). The macrogamonts were spherical, with a granular cyto-
plasm and a large central nucleus. These macrogamonts have not
been previously observed in Manila clams but ones with similar
morphology have been observed in native littleneck clams (Desser
and Bower 1997a). Although the large size of the macrogamonts
(32-33 |xm in diameter) was sufficient to stretch the kidney tu-
bules there appeared to be very little impact on the host due to the
low intensity and lack of other life stages. The Rhynchodyda-like
ciliate was attached by a stalk between the cilia of the gill epithe-
lium in IfWc and 44% of Manila clams (Table 2). They had a large
prominent nucleus and appeared to be the same as those described
by Bower et al. (1992). There was no evidence of a hemocytic
response and the intensity of infection appeared too light to have
a significant pathological effect. Trichodina spp. (similar to those
described by Bower et al. 1992) were found attached to or closely
associated with the foot, inner surface of the siphon and in one case
the mantle. The prevalence of these organisms was 209f and 56%
and the intensity was light (Table 2). There was no evidence of
tissue disruption or hemocytic response indicative of a pathologi-
cal impact.
Copepods resembling Mytdkola spp. (commonly called red
worms; Fig. 6) were observed at least once in all three clam species
(Table 2), although predominately in varnish clams (60% and 64%
infected) and rarely in the other species (4%^ to 8%). They were
usually found w ithin the lumen of the stomach or intestine but one
was found in the digestive gland duct of a native littleneck clam
(Fig. 7). Intensity was recorded as the number of cross sections and
therefore the same organism may be represented more than once.
In cases where there was more than one cross section in one part
of gut. the lumen was somewhat distended (Fig. 6). otherwise there
was no indication of serious pathology. These copepods have been
observed previously in Manila clams and native littleneck clams as
well as other bivalves (Bower et al. 1994).
188
Marshall et al.
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Figures 1-5. Inclusion hiidies and protozoa «bser>ed in histological sections of clams from British Columbia, Canada (hematoxylin and eosin
stain, scale bars arc 2(t Mm).
Figure 1. Two strongly basophilic rod-shaped bacteria colonies (B) next to a Rickettsia or Chalmydki-WVx inclusion (R) in the gills of a native
littleneck clam [Pioldllima slamiiiea). Note the size difference in individuals in each type of colony. Both types of inclusions cause considerable
distortion of the host cell.
Figure 2. Large inclusion bound by hcmocytes within the gonad of a native littleneck clam {P. skiminea). The thick membrane surrounding the
inclusion appears to be the result of layers of flattened hcmocytes.
Figure 3. Four Nematopsis-Vke spores (arrowhead) surrounded by hcmocytes within the water channel of a varnish clam {Nuttallia obscuraui).
Figure 4. Three \iinalopsh-\\V.e spores (arrowheads) in the water channel of a varnish clam (,V. obscurata) gill. Note the smaller spore size and
greater number of responding hcmocytes compared with Figure 3.
Figure 5. Three macrogamonts of an eimeriorin-like coccidia in the kidney tubule of a Manila clam (Venerupis pbilippinarmn). The tubule is
greatly distended as a result of the large size of the macrogamonts.
All other metazoaiis observed wei'e copepods. turbellarians. or
trematode metacercariae and all occurred at low frequencies
(Table 2). Two different copepods were found, one in the gill of a
native littleneck clam and the other in the gonad of a varnish clam
(Table 2). The gill copepod (Fig. 8) was large, nearly 750 (im long
in the tissue section, and was observed within the water channel of
the gill. It did nol appear to be attached and despite its size there
was no significant tissue disruption. Two metacercariae were
found in Manila clams, one in the digestive gland (Fig. 9) and
another unencysled one within the pericaidial space. The metacer-
caria within the digestive gland was sunounded by a thick layer of
hcmocytes that caused some local tissue disruption. One turbellar-
ian was found within the intestine of a Manila clam (Fig. 10) and
two turbellarians were found in the kidney tubules of varnish
clams (Fig. 1 1 ). The turbellarians found in the varnish clams both
appeared to be of the same species and were quite large, one was
over 200 |xm in diameter, and therefore caused considerable swell-
ing of the tubule, otherwise no pathological effects were observed.
DISCUSSION
Comparisons of parasite and symbiont prevalences between
Manila, native littleneck. and varnish clams provide strong evi-
dence that there are host preferences. Each parasite/symbiont had
the same order of host preference at both locations except in the
case of Nematopsis-Wke spores. Nematopsis-Wke spores were
rarely observed at the Boulder Point site but were common in
clams from Crofton. Because Nematopsis spores do not reproduce
once they are inside the molluscan host (Sprague and Orr 1955) the
clams from Crofton had a significantly higher rate of invasion.
This may be related to the fact that known species o{ Nematopsis
require a decapod host to complete their life cycle (Lauckner
1983); possibly the Crofton site was more suitable for the alternate
host(s). Another possibility may be related to differences in expo-
sure, the Crofton beach was in a bay and had more protection from
waves and current due to the nearby ferry dock and marina. The
infectious agents may have been washed away from the Boulder
Point site before they reached the filtration field of their potential
bivalve host. These data are limited by time of year and are rep-
resentative of a small geographic area. Whether these patterns of
host specificity are constant throughout seasonal fluctuations and
at different locations is unknown. The assumption that clams of
similar sizes dug from the same micro-site have similar exposures
to potential parasites and symbionts does not work as well for
parasites that are accumulated at low intensities over a long period
of time. Because size is not an accurate measurement of age. clams
of similar sizes cannot be assumed to have the same exposure
times, also clams found in the same micro-site one year may have
been more widely separated in previous years.
Parasites of Three Bivalves in British Columbia
189
\
n % w^^ j- ,}■!
i •,
^v.
y*
* • V -^ f r "•/':
»•. v^
Figures 6-11. Metazoa in clams from British Cuiumbia Canada (hematoxylin and eosin stain).
Figures 6-8. Copepods found during histological examination (scale bars are 10(1 nm).
Figure 6. Mylilicola spp. in intestine of >arnish clam {\iillallia ohsciiiala). Multiple sections maj represent the same organism folding back on
itself. Damage to intestine wall (D) appears to be a sectioning artifact.
Figure 7. Mytilicola spp. in a duct of the digestive gland of a native littleneck clam [Prnlnlhaca slaminea). Note damage to intestinal wall in upper
left of photo between appendages of the copepod.
Figure 8. Section shown is through the appendages and abdomen of a copepod within the water channel of a native littleneck clam iP. slaminea)
gill-
Figure 9-11. Metacercaria and turbellarians (scale bars are 50 pm).
Figure 9. Metacercaria (arrov\ I within the digestive gland of a Manila clam iVenerupispliilippinanim) surrounded b> a focal hemocvtic response.
Figure 10. Turbellarian in the intestine of a Manila clam (\. philippinanim).
Figure 1 1. Turbellarian within a kidney tubule of a varnish clam (,V. ohscurala). The kidney tubule is grossly distended to accommodate the large
size of the turbellarian.
Nemalopsis-Vike spores are able to gain entry into many species
of bivalves (Sprague & Orr 1955, Bower et al. 1994) but do not
always remain viable (Bower et al. 1992). None of the Nemalop-
j/.T-like spores observed in these clams appeared to be alive and
were probably within the wrong host. Viable interactions between
bivalve host and Neiiiatopsis spp. are likely to be highly specific
(.Sprague & Orr 1955). There also appears to be some inhibition of
infection because native littleneck clams were not infected to the
same degree as varnish or Manila clams. Native littleneck clams
have been known to contain Nematopsis-Wke spores (Bower et al.
1994) but these may represent a different species than those en-
countered in this study. The few spores observed in native little-
190
Marshall et al.
neck clams here were slightly smaller and may have represented a
different species that was less abundant. It is uncertain whether the
spores of two different sizes found in the varnish clams were the
same species. However, both spore types were found in the same
tissues and were proportional in abundance so could represent
different stages of host response.
There were many instances where a parasite or symbiont was
unique to only one host, for example, Trichodina spp., Rhyn-
chodida-like and eimeriorin-like protizoa were only found in Ma-
nila clams. Trichodina spp. and Rhynchodida-like ciliates have
been observed on other bivalve species (Bower et al. 19941 and
have a worldwide distribution. Both of these ciliates can be found
in association with Manila clams throughout their range (Bower et
al. 1992); the particular species found on Manila clams may be
enzootic and introduced to British Columbia along with their host.
Both are belie\ed to be benign, large numbers of Rhynchodida-like
ciliates have been reported with no obvious host response or mor-
talities (Bower et al. 1994).
The presence of eimeriorin coccidia in Manila clams and not in
native littleneck clams was unexpected. An eimeriid coccidian
parasite from the kidney of the native littleneck clam has been
described in Washington State, USA (Morado et al. 1984). A
similar, presumably the same, parasite was described and named
(Maii>olisieIla liabatai) by Desser and Bower (1997a) in a low
percentage of native littleneck clams from Southern Vancouver
Island. The macrogamonts observed in the Manila clam appeared
similar to those described in native littleneck clams, however M.
kabatai shares many ultrastructural similarities to coccidian mac-
rogamonts found in California abalone (Hidiolis spp.; Friedman et
al. 1995). Because macrogamonts were the only stage obser\ed it
is impossible to determine whether this is a different species or if
M. kabatai is also able to invade Manila clams. More than one host
species is not unknown in eimeriorin coccidia (Leger 1897. Leger
& Duboscq 1915); however, a survey of 994 Manila clams (Bower
et al. 1992) came across no evidence of this parasite. A possible
explanation may be related to geographic distribution of the para-
site. The Manila clam survey performed by Bower et al. (1992)
only sampled 80 clams south of Nanaimo and those were sampled
in early spring. All records of the kidney coccidia lie further south
than the boundaries of the Manila clam survey, it is possible that
M. kabatai may only be infecting Manila clams from more south-
em populations. Although heavy infections of kidney coccidia in
native littleneck clams can da)iiage the architectural integrity of the
kidney due to lethal hypertrophy of parasitized cells containing
maturing macrogamonts (Morado et al. 1984), the intensity of
infection observed in this study probably had minimal effect on the
host. No link between clam beha\ ior and coccidian infection has
been established in British Columbia, unlike those reported in
Washington by Morado et al. ( 1984). Possibly this parasite has a
greater impact at lower latitudes.
Native littleneck clams weie the only clams infected with fine
matrix inclusion bodies and colonies of large rod shaped bacteria.
Both of these infections are previously undocumented and may be
unique to native littleneck clams. Native littleneck clams have not
been surveyed as intensively as inti'oduced and farmed species of
shellfish so these infectious agents may have escaped detection
until now. Those native littlenecks that have been surveyed were
collected at different locations (Bower et al. 1992), so range or
annual fluctuations may be an explanation. The fine matrix inclu-
sions have potential to be harmful to the host due to their extreme
size if they multiplied or accumulated in vast numbers.
The rod shaped bacteria were at first reminiscent of Rickettsia
or Clialinydici-hke prokaryotes but these individuals were larger
than others described from those groups (see review in Elston and
Peacock 1984). Most of the colonies were much more basophilic
and were usually Gram positive, unlike the paler Gram negative
colonies of what were more typical of Rickettsia-like prokaryotes.
The variations in Gram staining may be related to stages in devel-
opment; there was a tendency for the larger individuals to be Gram
positive but this was not always the case. The conelation between
the intensities of infection of colonies of typical Rickettsia-like
prokaryotes and rod shaped bacteria may be a function of clam
filtering activity or maybe some individuals are more susceptible
to gill infections than others. Unfortunately it was impossible to
compare clam size to infection intensity because the clams had
been pool-fixed. Although this paper separates these bacteria from
the more typical Rickettsia-like colonies it is not unusual to find
variations in the sizes of individual prokaryotes in bivalve inclu-
sions (Elston & Peacock 1984). However, the differences are not
usually as great as those observed here. The taxonomy of intra-
cellular prokaryotes from bivalves is very poorly understood and is
based on morphological observations as opposed to biochemical,
infective or taxonomic relationships with similarly named organ-
isms in higher animals.
Parasitic or commensal crustaceans are common within most
bivalve species; however, those encountered in this survey were
predominantly in varnish clams. Manila clams can be host to more
than one species of pea crab (Bower et al. 1992) but all accounts
to date have found only one species (P. faba) in varnish clams
(Gillespie et al. 2001 ). Immature P. faba are found in many species
of clams in British Columbia but mature pairs are most often found
in the horse clam, Tiesiis capa.x (Hart 1982). Pea crabs are usually
harmless to their host however one study of Manila clams in Japan
found that the presence of pea crabs was related to a decrease in
the ratio of wet weight to shell length compared with unexposed
clams (Sugiura et al. 1960). This relationship has not been ob-
served in any bivalves examined as such in British Columbia. The
prevalence of pea crabs found in the varnish clams is consistent
with a more extensive count by Gillespie et al. (1999) but the
reason varnish clams have so many is unknown.
None of the clams in the present survey were examined fresh;
thus, the specific identity of the Mylilicola-Wke copepod was not
determined. However the most common Mytilicola spp. encoun-
tered in British Columbia is Mytilicola orientalis. which was in-
troduced via Pacific oyster seed (Bernard 1969). It is improbable
that these copepods are enzootic to varnish clams and introduced
at the same time since varnish clams are presumed to have arrived
here in a larval form within ballast water. Rates of infestation of
Mytilicola intestinalis between individuals of the same bivalve
species is passively determined by the host's field of filtration
(Gee & Davey 1986) and are often found in greater abundance in
larger sized hosts (Goater and Weber 1996). This does not explain
their predominance in varnish clams since they are less dependent
on filter feeding and were not significantly larger. Either more
larvae are entering varnish clams or the survival rate is lower in
Manila and native littleneck clams. Varnish clams are deposit and
pedal feeders in addition to filtering (Gillespie et al. 1999), this
action may stir up the sediment more, re-suspending larvae and
increasing the incidence of infection. Some experiments using M.
intestinalis in Europe have been linked to poor growth, tissue
damage and gut metaplasia in oysters and mussels (Koringa 1952,
Parasites of Three Bivalves in British Columbia
191
Odlaug 1946. Sparks 1962) however no pathology has been re-
ported in British Columbia as a result of M. oricntalis (Chew et al.
1965, Bernard 1969).
Both gill and digestive gland Riekettsia or Chalm\dia-\\kc in-
fections showed the same order of host preference with a complete
absence from varnish clams. .Mthough there was no correlation
between numbers of gill colonies compared with number of di-
gestive gland colonies in infected individuals this trend in host
specificity may indicate a close relationship between these two
types of infections. Possibly they are the same species and only
appear different because they are found in different host cells. The
similarity in appearance between species supports this theory and
suggests that one agent may be responsible for these infections.
However, detailed ultrastructural observations, serological or ge-
netic analysis is necessary to make these distinctions. A greater
dependence on filter feeding does not completely explain why
nati\e littleneck and Manila clams have these colonies while var-
nish clams do not as the prokaryotes are not picked up indiscrimi-
nately by passive filtration. Gulka and Chang ( 1984) tried infecting
other bivalves with a rickettsia isolated from a scallop (Pla-
copeclen magelUinicus) but were unsuccessful. This suggests that
these organisms are fairly host specific and those found here were
not able to infect varnish clams. It is possible that these intracel-
lular prokaryotes are a natural parasite/symbionts of native little-
neck clams and are able to successfully colonize Manila clams at
a lower rate due to certain similarities between the hosts. The
prevalence found in Manila clams from this study is similar to that
found by Bower et al. (1992), in comparison the prevalence and
intensity found in native littleneck clams was very high. Infections
of this degree have been observed in farmed scallops without any
indication of pathology, in this case the intensity decreased after
the scallops were moved from contained aquaculture ponds to the
open environment (S. Bower & G. Meyer, personal communica-
tion). This was another case in which location had a pronounced
effect on frequency and intensity of infection, possibly related to
the differences in wave and current exposure between the two
locations. In general these types of prokaryotic infections are not
linked to a pathological response but it has been suggested that
heavy infections may reduce the metabolic efficiency and reduce
the nutritional status of the host (Otto et al. 1979. Elston 1986).
There are a few cases linking intensity of Rickettsia or Chalmydia-
like infections to mortality (Gulka & Chang 1983. Le Gall et al.
1988. Leibovitz 1989) but no detrimental effects have been re-
ported in British Columbia.
The low prevalence or absence of some organisms is also worth
noting. Native littleneck clams collected by Bower et al. (1992) in
1986 and 1990 and by Desser and Bower (1997b) in 1995 were
infected with the elongate sporozoites of a Coccidia-like Apicom-
plexan (37% to 100% prevalence), these organisms were also
found in Manila clams near the Northern end of their distribution.
Some of these samples were taken at the same time of year as the
samples in this study, so seasonal fluctuations are probably not the
cause. These parasites may have been in low abundance in 2002 or
possibly the unknown alternate host does not occur in the Georgia
Strait. There were also fewer turbellarians observed than expected,
this is may be due to an annual fluctuation since they are usually
common in both Manila and native littleneck clams.
ACKNOWLEDGMENT
A heartfelt thank you to J. Blackbourn for technical assistance
and help with staining procedures.
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Desser. S. S. & S. M. Bower. 1997a. Margolisiella kahatai gen. et sp. n.
(Apicomplexa: Eimeriidae). a parasite of native littleneck clams. Pm-
lotluieea siaminea. from British Columbia, Canada, with a taxonomic
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Desser. S. S. & S. M. Bower. 1997b. The distribution, prevalence, and
morphological features of the cystic stage of an apicomplexan parasite
of native littleneck clams (Protolhaca siaminea) in British Columbia. ./.
Parasitol. 83:642-646.
Dinnel. P. A. & E. Yates. 2000. Biological and ecological as.sessments of
Niiltallia obsciinua in north Puget Sound. ./. Shellfish Res. 19:630.
Elston. R. 1986. Occurrence of branchial rickettsiales-like infections in two
bivalve molluscs. Tapes japaniea and Patinopecten yessoensis. with
comments on their significance. J. Fish Dis 9:69-71.
Elston. R. A. & M. G. Peacock. 1984. A Rickettsiales-like infection in the
Pacific razor Clam. Siliqua patula. J. Invert. Pathol. 44:84-96.
Friedman. C. S.. G. R. Gardner. R. P. Hedrick. M. Stephenson. R. J.
Cawthom & S. J. Upton. 1995. Pseudoklossia haliotis sp. n. (Apicom-
plexa) from the kidney of the California abalone. Haliotis spp. (Mol-
lusca). J. Invert. Pathol. 66:33-38.
Gee, J. M. & J. T. Davey. 1986. Experimental studies on (he infestation of
Mxtilus edulis (L.) by Mylilicola intestinalis Steuer (Copepoda. Cyclo-
poida). / Con.seil 42:265-271.
Gillespie, G. E.. M. Parker & W. Merilees. 1999. Distribution, abundance,
biology and fisheries potential of the exotic varnish clam (Nuttallia
obsenraia) in British Columbia. Can. Stock Assess. Secret. Res. Doc.
99/l93:39p.
Gillespie, G. E., B. Rusch. S. J. Gormican. R. Marshall & D. Munroe.
2001. Further investigations of the fisheries potential of the exotic
varnish clam (.Nuttallia obscurata) in British Columbia. Can. Stock
Assess. Secret. Res. Doc. 143:59p.
Goater. C. P. & A. E. Weber. 1996. Factors affecting the distribution and
abundance of Mytilicola orientalis (Copepoda) in the mussel. Mytilus
tro.tsulus. in Barkley Sound. B.C. / Shellfish Res. 15:681-684.
Gulka. G. & P. W. Chang. 1983. Prokaryote infection associated with a
mass mortality of the sea scallop. Placopecten magellanicns. J. Fish
Dis. 6:355-364.
Gulka. G. & P. W. Chang. I9S4. Pathogenicity and infectivity of a rick-
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ettsia-like organism in tlie sea scallop. Placopecten magellaniciis. J.
Fish Dis. 8:309-318.
Hart. J. F. L. 1982. Crabs and their relatives of British Columbia. British
Columbia Provincial Museum. Victoria. 267 pp.
Koringa. P. 1952. Epidemiological observations on the mussel parasite
Mytilicola intestinulis Steur. carried out in the Netherlands. 1951. ,4/;;).
Biol. Copenhagen 8:182-185.
Lauckner, G. 1983. Diseases of mollusca: Bivalvia. In: O. Kinne. editor
Diseases of marine animals, volume II: Introduction. Bivalvia to
Scaphopoda. Hamburg: Biologische Anstalt Helgoland, pp. 542-548.
Le Gall. G.. D. Chagot, E. Mialhe & H. Grizel. 1988. Brachial Rickettsi-
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Pecren nia\inni.s. Dis. Aqiiat. Org. 4:229-232.
Leger. L. 1897. Sur la presence des coccidies chez les mollusques lamel-
libranches. C R. Soc. Biol. 49:987-988.
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coccidie de lamellibranche. Arch. Zool. Exp, Gen. 55:7-16.
Leibovitz. L. 1989. Chlamydiosis: a newly reported serious disease of
larval and postmetamorphic bay scallops, Argopecren irraiiians (La-
marck). /. Fish Dis. 12:125-136.
Morado. J. F.. A. K. Sparks & S. K. Reed. 1984. A coccidian infection of
the kidney of the native littleiieck clam. Prototlimca staminea. J. In-
vert. Pathol. 43:207-217.
Odlaug. T. O. 1946. The effect of the copepod Mytilicola orienlalis upon
the Olympia oyster. Oslrea liirida. Trans. .Am. Microscop. Soc. 65:3 1 1-
317.
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mclusions containmg obligate prokaryote parasites, in commercial bi-
valve molluscs from Maryland estuaries. Haliotis 8:285-295.
Quayle. D. B. 1964. Distribution of introduced marine Mollusca in Bntish
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(Thunberg) caused by infection with the copepod Mytilicola orientalis
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(Eugregarinina: Porosporidae) with special reference to the host para-
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Jounuil i>f Shcllt'ish Research. Veil. 22. No. I. 19.^203. 2003.
POPULATION DYNAMICS OF THE ASIATIC CLAM, CORBJCULA FLVMINEA (MULLER) IN
THE LOWER CONNECTICUT RIVER: ESTABLISHING A FOOTHOLD IN NEW ENGLAND
D. P:. MORGAN, M. KESER, J. T. SWENARTON. AND J. F. FOERTCH
Millslone Enviniiiiiicnkil Lab. DominiDii Nuclear Connecticut. Inc.. Wciterford. Connecticut 06.^85
ABSTRACT The founding population of Corhicula flwnmea ni the Lower Conneeticut River, discovered in 1990. was studied for
ten years ( 1991-2000). Seasonal abundance of si.x size classes was monitored near three electric power plants. Corhicula abundance
varied seasonally as well as annually, but peaked in 1992. Winter survival of clams was positively correlated with the average winter
water temperature and negatively correlated with frequency of daily mean water temperatures s 1 °C and with frequency of daily mean
April spring freshet flows ^1700 m'/s. Higher winter survival at Middletown Station sites during most years, when compared with
survival near Connecticut Yankee, was attributed to the influence of the Middletown Station thermal discharge. Thermal discharge did
not support a permanent population at Connecticut Yankee because of temperature extremes during power plant operation in summer.
Clam growth under ambient river temperatures began in May when water temperatures exceeded I0°C and ceased in December when
temperatures fell below this threshold. Cooling water discharges altered this seasonal growth pattern; growth began in November, as
temperatures fell below 35"C. and ceased in the summer, when discharge temperatures exceeded this upper thermal threshold.
Reproduction occurred in the river when water temperatures were between I7"C and 28'C. typically from June to October. Peak
spawning occurred in August. Discharge temperatures shifted clam reproduction back to spring (March to May). The key to Cor-
Ivcula's success in establishing a population in the Connecticut River is its ability to colonize refugia from winter temperature and
spring freshet flow extremes that often cause high clam mortality.
KEY' WORDS: Asiatic clams, Corhicula flumiueu. thermal discharges, electric power plants, winter survival, thermal tolerance,
reproduction, growth, invasive species
INTRODUCTION
The Asiatic clam {Corhiculu Jiuininea) is a freshwater bivalve,
native to southeast Asia, that is now common in Europe, Africa,
the Pacific Islands, and North and South America. Early evidence
of Corhicula in Noith America was empty shells collected in 1924
at a British Columbia site (Counts 1981 ) and at a Columbia River
site in Washington. United States in 1938 (Burch 1944). Today.
Corhicula is reported in 37 US states including, most recently.
New York and Connecticut (McMahon 1983; Foehrenbach &
Raeihle 1984; Morgan et al. 1992). The rapid spread and persis-
tence of Corhicula throughout North America is related to its rapid
growth rate, early onset of maturity, high fecundity, and its ability
to tolerate a wide range of environmental conditions (Mattice &
Dye 1976, Aldridge & McMahon 1978, Graney et al. 1980, Mc-
Mahon & Williams 1986a, McMahon & Williams 1986b, McMa-
hon 2002).
While Corhicula is considered an economically important food
species in its native range (Chen 1990), it is recognized as a
nuisance in North America (Ingram 1959, Sinclair 1964, Prokop-
ovich 1969, McMahon 1977, McMahon 1983, Isom 1986). Its
ability to clog water systems makes Corhicula a serious and costly
problem for the electric generating industry (Goss & Cain 1975,
Mattice 1979, Page et al. 1986). Thus, the discovery of Asiatic
clams in water systems at Connecticut Yankee Nuclear Power
Station (CY) on the Connecticut River in May 1990 (Morgan et al.
1992) received considerable attention. The range extension of Cor-
hicula to the Connecticut River, the northemtnost population in the
eastern United States, was not expected because river temperatures
frequently drop below 2''C, the minimum temperature tolerated by
this clam (Mattice & Dye 1976). This study was initiated in 1991
as a condition of a Connecticut Department of Environmental Pro-
tection (CTDEP) permit to allow CY to continuously chlorinate its
service water system to prevent Corhicula biofouling. Monitoring
was later expanded upriver to the Middletow n and South Meadow
power plant sites. This study examines the abundance, growth, and
reproductive phenology of Corhicula under ambient Connecticut
River conditions and under thermal discharge conditions at the
Connecticut Yankee and Middletown power plant sites.
SITE DESCRIPTION
The Connecticut River originates in northern New Hampshire
near the Canadian border and flows south for 660 km, dropping
800 m in elevation by the time it reaches the mouth at Long Island
Sound (LIS) (Merriman & Thorpe 1976 and Fig. 1). Annual av-
erage water fiow. measured at Thompsonville CT (102 km from
LIS), during the period 1991 to 2000 ranged from a low of 410
mVs in 1995 to a high of 735 mVs in 1996 (USGS 2002). Daily
maximal rates usually occur in April, often exceeding 1700 m /s.
The focus of this study is the lower Connecticut River extend-
ing downstream from Hartford, Connecticut to a point 30 km
above the mouth of the river (Fig. 1 ). The survey area extends over
a 51 kin section of river and encompasses three electrical power
plant sites: South Meadow Station (SM), a 68.5 megawatt, solid
waste-to-energy plant; Middletown Station (MS), an 856 megawatt
oil fired power plant; and Connecticut Yankee (CY), a 582 mega-
watt nuclear power plant (Fig. 1 ). River width varies between -400
m and 600 m over the study area. Depths at sampling sites were
1-6 m below mean low water. Semidiurnal tides affect river fiow,
bringing on average 425 mVs of additional flow to the lower
Connecticut River in the vicinity of CY (Merriman & Thorpe
1976), causing periodic fluctuations in river height of ~l m (NSI
1995, Rozsa 2001). The tidal influences are large in relation to
natural flow during periods of low river discharge, and absent or
nearly absent during freshet conditions (Boyd 1976, Rozsa 2001).
The study areas at CY and farther north at MS and SM are above
any seawater incursion. Daily average ambient water temperatures
were similar for all three power plants and ranged between -1.7
and 30.6°C during the 10-year study period (Fig. 2). The river
frequently freezes over during the winter in our study area, but the
duration of ice cover varies from year to year. Discharge water
temperatures at CY during plant operation were 8 to 12"C above
ambient river temperatures at a maximum flow rate of 25 m /s.
193
194
Morgan et al.
HARTFORD
South Meadow
■Station (SM)
VERMONT \NEW HAMPSHIRE
/ i
1 MASSACHUSETTS
cc
o
>-
I
z
OSPRtNGFIELD
HARTFORD pi \
Q
Z
<
CONNECTICUT
":°'^'r-"^->=^
NEW YORK y"^"^
Middletown
Station (MS)
Connecticut
Yankee (CY)
Figure I. Location of Asiatic clam study area and sampling sites on
the Connecticut River, showing the three electric power station sites
(SM. MS, and CY).
The CY cooling water discharge flows through a man-made canal
1 km long before mixing with ambient Connecticut River waters.
Connecticut Yankee ceased operation on July 22. 1996. At MS. the
average sustained discharge temperatures from 1992-1994 ranged
between 7 and 10"C above ambient river conditions with an av-
erage discharge of 3.6 niVs. At MS and SM. the cooling water is
discharged directly to the river.
MATERIALS AND METHODS
This study was conducted between August 1991 and November
2000. Data at CY were collected during the entire study at four
sampling sites located in the river near the power plant and one site
in the discharge canal (CY discharge). The four CY river sites
were similar in Corbuiiki abundance and the data from each were
combined for data analysis (CY). Sampling was extended to three
sites at MS in May 1992 and continued through November 1994;
two sites were grouped for data analysis as river sites (MS), and
the third, adjacent to the cooling water discharge (MS discharge),
was analyzed separately. At SM. a single river site downstream of
the cooling water discharge (minimal thermal influence) was
sampled between August 1993 and November 1994.
In the first year of the study ( 1991 ), field sampling was con-
ducted in August and November. For the remainder of the study
period (1992-2(M)). field sampling was conducted three times
each year, in May, August, and November. To collect Corbicula.
five 0. 1 ni" bottom sediment samples were obtained at each sam-
pling site using a weighted Peterson grab (Wildlife Supply Com-
pany. Buffalo. NY). Sample processing techniques were similar to
those of Gardner et al. (1976). Grab samples were sieved in the
field by passing the sample through a series of three screens (6.3.
2.0. and 1 .0 mm mesh size). Clams and sediment retained on the
I -mm screen were subsampled in the field by placing a well-mixed
1-L sample in an elulriator (Magdych 1981) for 3 min al a water
flow of 20-30 L/min. The overflow from the elutriator was col-
lected on a I -mm mesh sieve and sorted in the laboratory under a
dissecting microscope (lOx). Sediment and clams retained on the
6.3 and 2.0 mm screens were taken to the laboratory and washed
through a series of five US Standard Testing Sieves (19.0. 12.5.
6.3. 3.4. and 2.0-mm mesh sizes). Size classes were determined
based on the mesh size on which clams were retained. Clams
Figure 2. Intalve (-
91 92 93 94 95 95 97 98 99 00 01
and discharge (----) water temperatures at CV from January 1. 1991 to January 1, 2(M>0. Horizontal reference lines
represent upper and lower lethal temperature limits for Ciirhiculii Jluminea.
CORBICVLA IN THE LOWER CONNECTICUT RiVKR
195
c/)
E
O
0)
u
c
CD
■D
C
3
<
Month
Year
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
5
8
11
399 ±131
807 ±387
55 ±40
2,568 ±1,538
5,209 ±2,630
4.0 ±8.5
35 ±24
206 ±91
0
68 ±22
225 ±118
124+56
1,828+622
1,522 ±565
0
56 ±22
80 ±38
8.0 ±7.5
57 ±28
350±136
38 ±33
291 ±130
649 ±272
94 ±35
2.148 ±617
1.758 ±635
78+58
366±138
412±189
ANNUAL
-
2,610 ±1,136
89 ±40
98 ±45
1,158 ±328
45 ±16
138 ±59
326 ±116
1,334 ±362
286 ±85
Figure 3. Average abundance (# clams/m-) of CorbUiila fliiminea by size class (graph) and total (table. ±95% CI) at CV river sites.
retained on the 1.0-nim sieve averaged 2.0 mm in shell length: on
the 2.0-mm sieve. 4. 1 mm; on the 3.4-mm sieve, 6.7 mm; on the
6.3-mm sieve, 14.1 mm; on the 12.5-mm sieve, 19.3 mm; and on
the liJ-mm sieve, 31.1 mm.
Individual clam growth was monitored monthly in 1993 and
1994 using shell length measurements to the nearest 0.1 mm. In the
river near the CY plant intakes, marked clams maintained in lan-
tern nets were used for monitoring growth. In the CY discharge
canal. 12 clams collected randomly from lantern nets were mea-
sured monthly to assess growth.
Clam fecundity was determined monthly using techniques of
Aldridge and McMahon ( 1978). Several hundred adult clams (>8.0
mm in shell length) were collected from the river in May/June of
1991 through 1994 and held in lantern nets placed at two locations,
one in the river near CY plant intakes, the other in the CY dis-
charge canal. Clams were collected monthly from river nets until
winter, when no live clams remained in lantern nets. In the CY
discharge canal, all clams were dead by June (when water tem-
peratures at this site exceeded 37°C). In this study, data for fecun-
dity in the discharge canal were collected from November 1 992 to
July 1993, and June and July fecundity data were at ambient river
temperature due to a power plant shut down. Twelve clams were ANNUAL 11,482 ±4,416 616,.±227 555±253
subsampled monthly from each net. In the laboratory, each clam Figure 4. Average abundance (# clams/m") oi Corbiciila fluminea by
was held under static conditions at 20 "C for 24 h in a lOO-ml size class (graph) and total (table. ±95% CI) at MS river sites.
Month
Year
1992
1993
758 +688
10,413 ±7,233
23,275 ±5,494
43 ±36
878+451
928 ±339
1994
i'38"±68
786 ±577
533 ±295
196
Morgan et al.
beaker filled with filtered Connecticut River water. The number of
juveniles released during this period, determined with a lOx dis-
secting microscope, was recorded as an index of spawning activity.
Additional fecundity assessments were made by dissecting these
clams and noting the presence of brood. Maturity of gametes was
assessed by removing egg and sperm cells from the gonadal tissues
and examining the cells under a compound microscope (400x),
Statistical analyses were performed using SAS version 8 soft-
ware (SAS Inc., Cary. NC). Abundance data in figures are pre-
sented using arithmetic means and non-transformed data. Statisti-
cal comparisons of abundance data were always carried out after
log transformation. The relationships between winter clam survival
(detlned as the ratio of May clam abundance to November clam
abundance from the previous year, expressed as a percentage) and
temperature or river tlow indices were assessed using the rank-
order Spearman correlation. Growth and reproduction data were
not transformed prior to statistical testing.
RESULTS
Abundance
Corhiciila abundance exhibited high intra- and inter-annual
variability. Year to year abundance fluctuations were considerable
at all ambient temperature river sites (Figs. 3, 4. 5; note different
vertical scales). At CY. mean annual clam abundance in 1992,
1995, and 1999 (range 1.158-2,610 clams/nr) was significantly
higher (P < 0.05) than in all other years (range 45-326; Fig. 3). At
MS, mean annual abundance in 1992 (11.482 clams/m") was sig-
nificantly higher (f < 0.05) than in 1993 or 1994 (616 and 555
clams/nr, respectively. Fig. 4). At SM, mean annual abundance
was low, with 82 clams/nr in 1993 and 67 clams/nr in 1994
(Fig. 5).
Of ambient temperature river sites, seasonal abundance at CY
Month
Year
1993
1994
5
8
11
112 ±30
52 ±21
0
114 ±103
88+75
ANNUAL
82 ±27
67 ±43
Figure 5. .Average abundance (# clanis/ni") of Corhiciila ftuminea by
size class (graph) and total (table. ±95'7f CD at S.M,
over a 10-year period was significantly higher (P < 0.05) in No-
vember than in May or August. November abundance at CY
ranged from 80 clams/nr in 1996 to 5,209 clams/m" in 1992. By
contrast, over the 3 years surveyed at MS ( 1992-1994) and 2 years
surveyed at SM ( 1993 and 1994), abundance was not significantly
different (P > 0.05) between August and November samples. No-
vember abundance at MS in 1992 (23,275 clams/m~) was the
highest observed during the study. Lowest November abundance
occurred at SM in 1993 (52 clams/nr). At all sites, clam abun-
dance in May was significantly (P < 0.05) lower than that in either
August or November.
Of thermally infiuenced sites, seasonal clam abundance in the
CY discharge canal had significant differences (P < 0.05) among
the three sampling periods (Fig. 6). May abundance ranged from
0-92 clams/m". August abundance ranged from 0-12.174 clams/
m". November abundance ranged from 24 to 880 clams/m". At the
MS discharge. August and November abundance estimates were
not significantly different (P > 0.05). ranging from a low of 322
clams/nr in November 1993 to a high of 7.100 clams/m" in No-
vember 1992 (Fig. 7). As with river sites. May abundance at both
CY and MS discharge sites was significantly lower (P < 0.05) than
that in .August and November.
Annual abundance was variable at the CY discharge site. A
pooled f-test of total abundance during operational (1991-1996)
vs. post-operational years (1997-2000) indicated that clam abun-
dance increased significantly (P = 0.007) during post-operational
years. This increase was the result of higher abundance of larger
size class clams (7-14 mm and 19-31 mm) following power plant
shutdown. At the MS discharge site, total clain abundance was
significantly higher (P < 0.05) in 1992 (3.322 clams/nr) than in
1993 and 1994 (496 and 549 clams/nr. respectively: Fig. 7). Clam
abundance was not significantly different (P > 0.05) between the
river and discharge sites at MS, except for the largest clams (31
mm size-class), which were most abundant at the MS discharge
site. In fact, the largest clam measured during the entire study (37.6
mm) was collected at MS in August 1992.
Winler Snnival
Declines in clam abundance from November of one year to
May of the next were used to determine winter survival; values at
CY ranged from 0% in 1994 and 1996 to 55% in 1995 (Fig. 3). The
effects of winter water temperatures and peak river fiows on clam
winter survival were examined using Spearman-ranked correlation
(Table I ). The severity of winter water temperatures, as indicated
by the number of days with average water temperature <2°C, was
not significantly correlated (r^, = -0.65, P = 0.081) with clam
winter survival. The number of days, however, sl°C was nega-
ti\ely correlated (r., = -0.73. P = 0.040) with winter survival, and
average December through April water temperature was positively
correlated (r.,= +0.87. P = 0.004). Highest average monthly flow
in the Connecticut River typically occurs in April (Fig. 8). Ac-
cordingly, the number of days each year exceeding 1.700 mVs in
April was used as an index of spring freshet severity. This index
was negatively correlated with winter clam survival (r.. = -0.91.
P = 0.002). Data from 1993 were omitted from this analysis
because a single storm in March caused total mortality of clams at
our sampling sites.
Growth
Corbicida growth rates under ambient river conditions exhib-
ited seasonal cycles, and growth of marked clams was size-
E
m
O
c;
u
c
ro
■D
c
<
CORBlCfLA IN THE LOWER CONNECTICUT RlVER
12,096
197
95 11 5 8
96 11 5 8
97 11 5 8 r-
98 11 5 8
00
Month
Year
1991
1992
1993
1994
1995
1996
1997
1998
1999
2000
5
8
11
34 ±58
178±181
2 ±5.0
12, 174 ±30.269
96 ±121
32 ±22
0
42 ±26
0
60 ±54
880 ±1254
24 ±29
38 ±50
90 ±187
8.0 ±5.1
48 ±75
44 ±70
92 ±62
6 ±15
24 ±6.3
20 ±22
62 ±83
212 ±227
76 ±37
243 ±172
210 ±73
4±10
30 ±35
268 ±181
ANNUAL
-
4,091 ±8,454
25 ±14
313 ±397
51 ±53
33+28
41 ±27
98+78
173 ±63
101 ±83
Figure 6. .Average abundance (# liams/m'l of Cnrhiciiki Jhiiniiiea b> size class (graph) and total (table, ±95'7f CI) at C\ discharge.
dependent (Figs. 9 and 10). In 1993, clams with an initial shell
length of -14.5 mm had a higher growth rate (0.54 mm/wk) from
June to October than those starling at -17.5 mm (0.41 mm/wk).
and -21.7 mrt) (0.35 mm/wki. A similar size-dependent relation-
ship was also observed in the 1994 study; clams with an initial
length of -12 mm grew fastest from June lo October (0.51 mm/
wk), followed by -20 mm (0.32 mm/wk) and -30 mm (0.14 mm/
wk) clams. Growth rates were significantly different [P < 0.05)
among the three size classes through August. In September
through December, however, mean monthly growth rates for all
size classes were generally low and not significantly different from
each other.
Clam growth rates in the CY discharge canal from November
1992 to February 1993 were £0.18 mm/wk. when water tempera-
tures were 13-19°C, I0-12°C above ambient river temperatures
(Table 2). As these clams were not marked, negative growth rates
could occur as a result of mortality of large individuals. Growth
rates were as high as 0.27 mm/wk from March to May when water
temperatures ranged from I3-27°C. Maximum growth rates at this
site occurred during June (0.38 mm/wk) and July (0.33 mm/wk),
when canal temperatures were similar to those at ambient river
conditions because of a power plant outage. All clams died after
the power plant restarted and discharge water temperatures ex-
ceeded 37"C (July).
ReprodiictiDii
Microscopic examination of gametic tissues of clams held un-
der ambient river and CY discharge conditions show that eggs and
sperm were continually present as long as clams were alive (Fig.
1 1 ). For clams held at ambient river temperatures, the presence of
embryos and veligers in the demibranchs (brooding) and the active
release of juveniles occurred primarily over a 4-month period
(June to September). By October, only one clam out of 48 exam-
ined was still spawning. The maximum number of juveniles re-
leased per clam per day typically occurred in August across all 4
years in which reproduction was monitored (2.862 juveniles/clam/
day; Fig. 12). This pattern of juvenile release allowed maximum
recruitment to occur just after the period of maximum river water
temperature (July, with a 4-year average of 27.5°C). The number
of juveniles released per adult in August was positively correlated
with the size of the clam (r,= 0.77; P < 0.01; Fig. 13).
The reproductiv e cycle of Corbicuta in the CY discharge canal
was seasonally shifted (Fig. II). Brooding and releasing of juve-
niles first occurred in November 1992 when discharge tempera-
tures averaged I8.3°C, and ceased from December through Feb-
ruary when temperatures averaged <I4°C. Spawning began again
in March and increased through April when discharge tempera-
tures averaged I7'C. The sharp decrease in May was the result of
198
Morgan et al.
Month
5
8
II
1992
206"±i"22
2.666 ±836
7.100 ±1.896
ANNUAL 3,322 ±1,721
Year
1993
"326±14l'
840 ±450
322 ±69
496 ±186
1994
———
340 ±116
860 ±93 1
549 ±283
Figure 7. Average abundance (# clams/m") of Corbicula fluminea by
size class (graph) and total (table, ±95% CI) at MS discharge.
a power plant outage beginning on May 1 3, which dropped cooling
water temperatures from 30°C to 18°C in a single day (Fig. 14).
Spawning activity recovered and peaked in June and July as the
plant outage continued, similar to the pattern observed at ambient
river temperatures (17-27°C). On July 21. 1993 the power plant
restarted and temperatures increased to >35°C in 4 days. By Au-
gust 18. 1993 all clams held in the CY discharge were dead.
DISCUSSION
Corbicula fluminea was first documented in the Connecticut
River in May 1990 (Morgan et al. 1992). the first report of this
nonindigenous clam in New England waters. Before this discov-
ery. Coiiiicula was not expected to colonize the Connecticut River
because water temperatures routinely fall below 2^C for prolonged
periods. It is commonly accepted among researchers that the lower
lethal temperature limit for Corbicula is ~2°C (Homing & Keup
1964. Bickel 1966. Mattice & Dye 1976. Rodgers et al. 1979.
Cherry et al. 1980).
Corbicula abundance varied seasonally as well as annually, but
3500
3000
2500-1
to
5 2000'
I 1500-
1 1000'
<
500-
95 96
Year
Figure 8. Connecticut River daily flow rates (mVs) at the Thompson-
ville, CT gaging station in April from 1991 to 2000.
clearly peaked in 1992. Survival of clams from one year to the next
is positively coirelated with the average December to April water
temperatures and negatively correlated with the number of days
the river water temperature was below I °C and the number of days
that river flows exceeded 1700 mVs in April. For example, no
clams were observed in May at our Connecticut Yankee sampling
sites following the two coldest winters (1993-1994 and 1995-
1996). when river water temperatures dropped below 2^C for 12-
15 weeks and the highest winter survival occurred in 1995 when
daily average river flow in April never exceeded 1700 m /s.
Low survival at Connecticut Yankee and Middletown Station
during the winter of 1992-1993. when water temperature did not
drop below 2°C, was attributed to winter storm Joshua (March 13.
1993). This storm produced low water levels ( 1-2' below normal)
and left shoal areas, specifically our sampling areas, exposed to air
temperatures as low as -8°C. freezing sediment and clams
(NUSCO 1994).
Higher winter survival at Middletown Station sites, when com-
pared with those around Connecticut Yankee, was attributed to the
influence of the Middletown Station thermal dischaige. River wa-
ter temperatures seldom dropped below 2°C in the Middletown
Station discharge mixing zone (NUSCO 1994). Other over-
wintering populations likely exist in the river in refugia provided
by other industrial thermal discharges or in areas of the river
receiving regular influxes of groundwater that maintains a tem-
perature of 9.0 ± 2°C (R. Lewis. State of Connecticut Geologist;
pers. comm.). Graney et al. ( 1980) and Kreiser and Mitton ( 1995)
suggest that warm water refugia such as these were assisting the
Asiatic clam in expanding its geographical range northward.
Clam densities in the Connecticut Yankee discharge canal were
TABLE 1.
Spearman correlations coefficient ( rj for percentage winter survival of Cnrhicula fluminea at CY \ersus indices of winter temperatures and
Connecticut Rixerflow.
Variable
r^
Prob >lrl
n"
Mean
Std Error
Min.
Max.
Percentage Survival''
_
_
8
12.7%
6.23%
0%
54.9%
Ave. Winter Temp.*^
-1-0.87
0.004
8
2.93
0.37
1.32
4.86
No. Days S1°C
-0.73
0.040
8
54.9
8.75
17
93
No. Days s2°C
-0.65
0.081
8
70.6
8.12
28
103
Flow a 1 700 ni'/s''
-0.9 1
0.002
8
6.4
1.54
0
13
' 1993 data were omitted because of the mortality caused by the March storm Josliua (see text).
'% Survival = (May abundance/prior November abundance) x 100,
' Average Winter Temperature = the annual December to April mean daily Connecticut River temperature at CY.
' Number of days in April when the Connecticut River flow equaled or exceeded 1700 m'/s.
CORBICULA IN THE LOWtR CONNECTICUT RlVER
199
Jun
1.1 -
1.0-
^ 0.9-
-0.8-
^ 0.7-
Ld 0.6-
< 0.5-
^ 0.4-
|0.3.
g 0.2.
0.1 -
1 <-
-^^
V
^^-.
"^^
0.0-
Jul
Aug
Sep
Nov
Dec
Figure 9. Corhiciila Jhiminca growth rates (mni/«k) in 1993 for marked clams within initial size classes based on shell length. Vertical bars
represent two standard deviations around the mean growth rates for three individuals in each size class.
most \ariable. Large numbers of small (2 mm) clams that appar-
ently survived passage through the power plant cooling water sys-
tem characterized transient populations in the canal. A permanent
population, however, was not established during power plant op-
eration because summer water temperatures often exceeded 37°C,
the upper lethal temperature limit for Corbicula in our study. Mc-
Mahon and Williams (!986b) reported similar findings for Cor-
bicula living in the themial discharge of the Handley Power Sta-
tion in Texas. Following Connecticut Yankee closing in 1996, size
range of clams collected in the discharge canal has increased with
shell lengths now ranging from 2-19 mm. These results indicate
not only that clams are successfully over-wintering in the canal
under ambient river temperatures, but also surviving for >1 year.
The canal essentially has become a cove where circulation is de-
pendent on semidiurnal tidal exchange, and not \ ulnerable to high
spring freshet water Hows.
Clam abundance in the Middletown Station discharge area also
fluctuated, but was consistently higher than abundance at CY dis-
charge during the same period. Similar to the CY discharge, the
population near the MS discharge was dominated by clams 2 mm
in size. In contrast, however, to the CY discharge, clams of all size
clas.ses, including those in the 31 mm class, were regularly col-
lected at the MS discharge. The presence of larger size clams
suggests that this area provided a more stable refugium. The 37-
mm clam collected at this site in 1992. along with growth rates
observed during our study, suggests that Corbicula has been
I
n 4-
1—
5
o
0.3-
(r
o
0.2-
0.1 •
0.0-
Aug
Sep
Figure 10. Corbicula fluminca growth rates (mm/wkl in 1994 for marked clams within initial size classes based on shell length. Vertical bars
represent two standard deviations around the mean growth rates for two to five individuals in each size class.
200
Morgan et al.
TABLE 2.
Corbicula fluininea growth in the C\ discharge canal from November 1992 to July 1993.
Date
Growth
Weeli
Average Length
(mm)
SE
Minimum Length
(mm)
Maximum Length
(mm)
Growth Rate
(mm/wk)
1 1/10/92
0
20.20
12
0.69
15.3
1 2/22/92
6
20.04
12
0.38
18.1
01/26/93
11
20.97
12
0.59
17.7
02/23/93
IS
20.89
12
0.48
18.6
03/23/93
19
21.96
12
0.43
19.2
04/29/93
24
23.04
12
0.42
20.6
05/18/93
27
22.57
12
0.31
20.4
06/24/93
32
24.57
11
0.40
21.5
07/22/93
36
25.89
12
0.39
24.2
24,0
21.5
26.5
23.8
24.4
24.9
24.1
26.1
27.6
-0.026
0.185
-0.019
0.267
0.205
-0.172
0.378
0.330
.Ian i Feb Mar Apr May -lun
.Aug Sep
A
fnhicn
River Conditions
WINTER
MORTALITY
Uts
Sp.rn,
Brooding
Releasiim Ju\cniles i i i
1 1 i
Cooling
Water Discharge Conditions
1 1 i
Fees
SUMMER
MORTALITY
Spcnn
Brood inj^
Rclcasmc.luvcnilc.s
Figure 11. Summarization of the 1991 to 1994 annual reproductive
cycle of Corbicula Jhiiniiiea under ambient Connecticut River condi-
tion,s and the thermallv elevated conditions of the CV cooling water
discharge.
present in the river since 1988. Winter water temperatures were
moderated by the Middletown Station thermal discharge, and sum-
mer thermal stress was reduced because of rapid dilution of dis-
charge waters with ambient river water. In addition, the MS ther-
mal discharge flow was only ~\59r that of CY.
Ciiibicida growth in the Connecticut River under ambient wa-
ter temperatures is consistent with reports by other researchers in
North American (Morton 1977. Britton et al. 1979. Eng 1979.
Mattice 1979, McMahon 1983. Welch & Joy 1984, Joy 1983.
Matlice & Wright 1986. McMahon & Williams 1986b. Doherty et
al. 1990, French & Schloesser 1991). and was primarily influenced
by water temperature. Growth began in May when water tempera-
tures rose above IO°C and continued until December when water
temperatures dropped below this threshold. Other researchers re-
ported 9-15°C to be the lower temperature threshold for growth of
Corbicula fluininea in their studies (Hall 1984. Mattice & Wright
1986, McMahon & Williams 1986a, French & Schloesser 1991).
o
o
Q
40
30
5
o
;;^ 20
LU
>
3
O 10-
.
>^.
■
^
/
p
\
N.
-
1
^
.
/
1
/
I
\
\
\
/
1 \
\
,
/
1 \
\
■
9
/ \
\
\
■
1
1
I \
■
1
1 I
1 ^
\
*
/
■
\ «
-
A 1
\
.
y /
\ \
.
/
\ ^
■
■' 1
V
> -
=^ , 1 y . .
, ^> ^ 9 ^
30
25
2
<
20 3
15 73
4 5 B 7
MONTHS OF THE YEAR
10 11 12
10
Figure 12. Corhiciila fluininea fecundity
from 1991-1994.
(- -) and water temperature (- - : - -) for clams held in ambient temperature Connecticut River water
COHHICULA IN THE LoWhR CONNECTICUT RlVER
201
o
o
>-
<
<
_J
O
•\
>
3
O
d
75
50
25-
n =47
r=0.77
p<0.0001
.^<'-€>
100-
.■■0^-^^'
75;
a .^<^>>" ,„-'-
50-
o
o ,
Q' ^^^ , '' o
.'.^ ,-' o
yh-
^^^'' °
:
^^
'' rx.^
-■d' o
■
'--°'c
■^^-'
ol
T 1 1—
ro^
1 1
°o
1 1 1 1 1 1 1 1 1 1 —
12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42
LENGTH OF CLAM (mm)
Figure 13. Linear regression with 95 '7r CI on mean predicted \aliies for the number of Juveniles released per day in relation to shell length (mm)
of the spawning Corbktila jlumiiwa during the peak spawning month of August in the mainstem Connecticut River.
MONTHS OF THE YEAR
Figure 14. Corhiciila Jhiminea fecundity (-0-) and water temperature (- - 0 - -) for clams held in the discharge canal at CV from November
1992 to August 1993.
202
Morgan et al.
Highest growth rates occurred in July and August, when river
water temperatures pealced (25-30°C). and growth rates were sig-
nificantly higher for the smaller clam sizes.
The upper temperature tolerance of Corhicithi determined in
this study is within ranges reported by other researchers in labo-
ratory and field experiments (Mattice & Dye 1976. Dreier 1977.
Mattice 1979. Cairns & Cherry 1983. McMahon & Williams
1986a). Corhuula growth in the CY thermal discharge canal was
initiated in November 1992 when water temperatures dropped to
<35°C. Growth continued until August 1993. when water tempera-
tures were >37°C and clams died.
Seasonal water temperatures also control reproductive cycles of
the Connecticut River Corbiciila population. The presence of eggs
and sperm was continuous in the Connecticut River population of
this species as long as water temperatures supported its survival.
Brooding and releasing of juveniles occurred when water tempera-
tures were between 17-28^C. typically from June to October.
Spawning temperatures of 14— 27°C were reported by other re-
searchers in North America (Eng 1979. Mattice 1979. Hall 1984.
Cherry et al. 1986. Foe & Knight 1986; McMahon & Williams
1986a: Doherty et al. 1987; Rajagopal et al. 2000).
A single annual spawning peak for the Corhicithi population in
the Connecticut River occurred in August. Others reported two
Corbiciila spawning peaks, one in spring and one in fall (Heinsohn
1958. Aldrige & McMahon 1978. Eng 1979. McMahon 1983. Foe
& Knight 1986. McMahon & Williams 1986a). Several others
have reported a single spawning peak (Bickel 1966. Homback
1992, Mouthon 2001). The presence of a single reproductive peak
in the Connecticut River population may be related to longer pe-
riods of cold-water conditions, more severe spring Hooding, and
the quantity and quality of available food.
The altered thermal regimen within the CY discharge canal
shifted the period of reproduction from the ambient river period of
June through September to November and March through May
when water temperatures in the canal ranged between 16-30'C.
Spawning during July and August 1993 occurred because the
power plant was off-line and the discharge water temperatures
were not elevated. These results demonstrate that thermal dis-
charges can alter the reproduction cycle of Corbiciila. Aldridge
and McMahon (1978) and Dreier and Tranquilli (1981) reported
that Corbicula fliiminea spawning activities stopped at tempera-
tures of 30-34°C. most likely due to thermal stress. Graney et al.
( 1980) speculated that elevated temperatures in thermal discharges
may e.xtend the spawning season into the winter.
In conclusion, this study showed that the Connecticut River
has supported a fluctuating Corbiciila population for at least 10
years. Cold water temperatures (<2°C) for several weeks, and
high water flow in the spring caused high mortality of clams in the
river during the winter and early spring. Growth and reproduc-
tion for Corbiciila in the Connecticut River peaked in July and
August when river temperatures ranged between 24-30°C and
only one spawning peak occurred each year. The key to Corbicii-
la's unexpected success in establishing a population in the
Connecticut River is its ability to colonize refugia from cold win-
ter water temperatures and spring freshet flows that cause high
clam mortality. Following the closing of (he CY power plant.
Corbiciila continued to populate the CY river sites establish-
ing a more mature population in the discharge canal. Based on
our observations of Corbiciila in the Connecticut River, we ex-
pect that this species will continue to successfully colonize other
rivers and lakes in New England, where similar winter refugia
exist.
LITERATURE CITED
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Bickel. D. 1966. Ecology of Corbicula manileiuis in the Ohio River at
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Boyd. W. A. 1976. Hydrology. In: D. Merriman & L. M. Thorpe, editors.
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Cherry. D. S.. J. H. Rodgers. Jr.. R. L. Graney & J. Cairns, Jr. 1980.
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CELCO Plant. Virginia. In: J. C. Britton & R. S. Prezant. editors.
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Doherty. F. G., D. S. Cherry & J. Cairns. Jr. 1990. Multiseasonal tissue
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abundance of Corbicula in an Illinois cooling lake. In: R. W.
Lawrimore & J. A. Tranquilli, editors. The Lake Sangchris Study: case
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Eng, L. L. 1979. Population dynamics of the Asiatic clam, Corbicula
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Texas Christian University, pp. 39-68.
Foe, C. & A. Knight. 1986. A thermal energy budget for juvenile Corbicula
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Joiinuil oj Shclllhh Rcwiinli. Vol. 22, No. I. 205-2()S. 201)3.
QPX, A PATHOGEN OF QUAHOGS (HARD CLAMS). EMPLOYS MUCOID SECRETIONS TO
RESIST HOST ANTIMICROBIAL AGENTS
ROBERT S. ANDERSON,'* BRENDA S. KRAUS,' SHARON MCGLADDERY," AND
ROXANNA SMOLOWITZ'
^Chesapeake Bi(>loi>ical Laboratory. University of Maryland. Center for Environmental Science. P.O.
Box .^(S. Solomons. Maryland 206HS: ^Department of Fisheries and Oceans. Canada. Gulf Fisheries
Center. P.O. Box 5030. Moncton. N.B. EIC 9B6: "'Marine Bioloi;ic Laboratory. 7 MBL Street. Woods
Hole. Massachusetts 0254.1
.ABSTRACT The thraustochytrid protist quahog parasite unknown (QPX) has caused mass mortalities of hard clams (Mercenaria
nicneiuiria) in Atlantic Canada and Massachusetts. It typically secretes copious mucus in vivo and in vitro. M. mercenaria plasma
contains naturally-occurring agents that modulate growth of QPX cultures. This activity was shown by exposing washed, mucus-free
QPX (wQPX) to filter-sterilized M. mercenaria plasma. Low plasma protein concentrations (<10 |xg/ml) in the medium tended to
stimulate QPX growth; higher concentrations (10-50 (jLg/ml) produced dose-dependent inhibition. If wQPX were incubated for various
times before exposure to an inhibitory concentration of M. mercenaria plasma, a time-dependent protection from the plasma was
observed; total protection was seen after -24 h preincubation. This effect was probably a result of the re-establishment of the mucoid
coats around the wQPX during preincubation. These data suggest th;it ihe mucoid secretion of QPX may represent an important
virulence factor.
KEY WORDS: quahog parasite unknown (QPX). Mercenaria mercenaria. virulence factors, clam diseases
INTRODUCTION
Whyte et al. (1994) described a protistan parasite that caused
high mortalities in a hard clam (Mercenaria mercenaria) hatchery
on Prince Edward Island, Canada; the causative agent was named
quahog parasite unknown (QPX). This organism was similar or
identical to the clam pathogen first observed by Drinnan and Hen-
derson ( 1963) in New Brunswick, Canada. Subsequently, QPX has
been cited as the cause of mass mortalities of M. mercenaria in
Massachusetts (Smolowitz et al. 1998) and has been reported in
several Virginia coastal embayments (Ragone Calvo et al. 1998).
Molecular phylogeny studies based on sequencing of I8S riboso-
mal RNA suggest that QPX is a member of the phylum Labyrin-
thulomycota (Maas et al. 1999. Ragan et al. 2000). in the thraus-
tochytrid phylogenetic group (Stokes et al. 2002).
A medium developed by Kleinschuster et al. (1998) has per-
mitted in vitro cultivation of QP,\. In culture, thalli were shown to
grow and mature into sporangia containing numerous vegetative
endospores. The endospores were released on rupture of the spo-
rangia and in turn matured to form thalli. and the stages of the
vegetative life cycle were repeated. Whyte et al. ( 1994) and Klein-
schuster et al. ( 1998) reported conversion of endospores to motile
zoospores in sterile seawater. Later studies (Brothers et al. 2000).
however, were unable to replicate these findings. The vegetative
life stages of QPX have been observed in the tissues of infected M.
mercenaria. In many instances, the QPX cells were seen in histo-
logic sections to be enclosed by a translucent space; this was
initially attributed to lysis of host tissue by enzymes secreted by
the parasite (Whyte et al. 1994). Subsequently. Smolowitz et al.
( 1998) determined that in live animals, the space is occupied by a
muco-fibrillar substance produced by the parasites; and that this
substance is removed by histologic processing. It was suggested in
that study that phagocytosis of the parasite in the clams' tissues is
inhibited by the mucofibrillar secretions of the parasite.
The disease caused by the Canadian strain (CA QPX) as de-
scribed by Whyte et al. ( 1994) is similar to that described for the
Massachusetts strain (MA QPX) by Smolowitz et al. (1998). MA
QPX. however, primarily infected the mantle and gill and some-
times produced nodules; CA QPX infections were more commonly
seen in the connective tissue of the foot and were rarely associated
with nodules. Areas of infection by CA QPX and MA QPX trig-
gered inflammatory responses involving extensive infiltration of
adjacent host tissues by hemocytes. with some evidence of phago-
cytosis and/or encapsulation of the parasites. Inflammatory foci
caused by MA QPX sometimes contained phagocytic multinucle-
ated giant cells similar to those produced /;) vitro by Anderson
(1987). Apparently QPX infection elicits a vigorous cellular re-
sponse, but this activity is insufficient to control the disease. Hu-
moral QPX modulatory agents in M. mercenaria plasma are de-
scribed for the first time in this article, and Ihe role of QPX mucoid
secretions in protection from them.
MATERIALS AND METHODS
*Corresponduig author. Tel.: -^ 1-4 10-326-7247; Fax; +1-410-326-7210;
E-mail; andersonts'cbl. umces.edu
QPX
These studies were carried out using MA QPX obtained from
Dr. R. Smolowitz, Marine Biologic Laboratory, Woods Hole, MA.
They were propagated in the medium of Kleinschuster et al.
(1998). The initial seeding density was 10"'/ml and the cultures
were maintained at 23°C and were har\ested at 7 d ( 168 h) while
still in exponential growth phase. The QPX cells were enveloped
by a heavy mass of mucoid secretion, which was routinely washed
off the cells by dilution with a saline solution. lO (25 ppt. Instant
Ocean®, Aquarium Systems Inc.; Mentor, OH), followed by re-
peated centrifugations (300 x g, 10 min, 21-0, x3). Washed QPX
(wQPX) were >90'7f viable by the trypan blue exclusion assay
(Hanks & Wallace 1958) and almost immediately resumed mucus
secretion. The numbers of QPX cells in particular cultures and cell
numbers required for subsequent experiments were quantified
spectrophotometrically using a standard curve of the numbers of
205
206
Anderson et al.
wQPX (as determined in a Ineniacytometer) as a function of tlieir
absorbance at 560 nm.
C9G
Another thraustochytrid. C9G. closely related to QPX (Ander-
son et al., in press) was isolated from gill tissues of Canadian M.
meirenaria and provided by Mr. G. S. MacCallum and Dr. S.
McGladdery, Gulf Fisheries Center. Moncton. Canada. Like QPX.
C9G was maintained in the medium of Kleinschuster et al. ( 1998)
at 25°C and subcultured at 7 d.
M. mercenaria Plasma
M. mercemiria. collected from the Ware River. VA by a com-
mercial supplier; were maintained with recirculating water (25 ppt.
10. 1 1°C). Hemolymph samples were withdrawn by syringe from
an adductor muscle hemolymph sinus and held on ice in polypro-
pylene tubes. The hemocytes were centrifuged out of suspension
(300 X g. 10 min. 4°C). The pooled supernatant (plasma) was
sterilized by filtration (0.2 |j.m pore size), and assayed for protein
content (BCA kit. Pierce Co.. Rockville. IL). Individual plasma
samples from three to four hard clams were pooled and were
frozen (-20°C) in aliquots. The frozen samples were used soon
because the QPX-modulatory activity declined after -2 mo in stor-
age. In one series of experiments, plasma was heat-treated by
exposure to 65°C for 10 min. the plasma was cooled to room
temperature (~25°C) before use.
Immediate Exposure of Thraustochylrids to Plasma
QPX cells from 7d cultures were washed, as described above,
and resuspended (2.5 x lUVml) in 25 ppt lO. Plasma protein con-
centration was standardized (usually to 0.2 mg/ml ) by dilution with
10 and serial dilutions prepared. Replicate culture flasks for each
protein concentration tested were prepared with experimental (1.9
ml Kleinschuster" s minimal essential medium (KMEM), 0.1 ml
QPX suspension, and 0.5 nil plasma dilution), control (1.9 ml
KMEM. 0. 1 ml QPX suspension, and 0.5 ml lO). and the necessary
blanks. After 7 d incubation at 24°C. the contents of each flask
were removed, and the QPX washed thoroughly and quantified, as
described previously. In related experiments. QPX or C9G were
incubated for 2 h in lO containing plasma, washed, and resus-
pended in KMEM. Percent inhibition was determined using the
following formula:
% inhibition = 1
experimental value
control value
X 100
Delayed Exposure to Plasma
In the delayed exposure experiments. wQPX were permitted to
incubate in KMEM for various time intervals <24 h before expo-
sure to 40 jjLg/ml M. mercenaria plasma proteins. The QPX cells
resumed typical secretory activities during these pre -exposure pe-
riods, as seen by microscopic examination. This plasma protein
concentration was selected because it had been shown in previous
immediate exposure experiments to inhibit -95% of the growth of
QPX cultures.
Viability Assays
QPX viability tests were carried out using viability/cytotoxicity
kit #1 (Molecular Probes, Eugene, OR). The test is based on the
differential permeability of live and dead cells to a pair of fluo-
rescent stains. Cell populations exposed simultaneously to both
dyes become differentially stained: live cells are stained green and
dead cells appear red. This assay was used to check wQPX viabil-
ity after exposure to 10 or M. mercenaria plasma.
RESULTS
Effects of M. mercenaria Plasma on Washed QPX
At the lower plasma concentrations tested, inhibition was low
and variable, with some pools actually stimulating growth (Fig. 1 ).
However, at plasma protein concentrations s 10-50 jxg/ml, a dose-
dependent inhibition was consistently recorded (-100% inhibition
was seen at >50 (j.g/ml). The inhibitory EC^,, was calculated to be
-19 p-g/nil. When this procedure was carried out with heat-treated
(65"C. 10 min) plasma, the stimulatory effects of the lower con-
centrations were not evident (Fig. 2). The inhibitory EC^,, for
heated plasma was -32 (xg/ml; therefore, this heat treatment only
partially inactivated (-40%) the growth inhibitory factor(s).
The inhibitory effects of M, mercenaria plasma were exerted in
a short period. When wQPX were exposed to 40 p.g/ml plasma for
2 h, washed free of plasma and cultured for 7 d in plasma-free
medium, the resultant QPX cell numbers were 80.7 ± 13.3% (n =
3) reduced as compared with untreated controls. A similar degree
of inhibition (94.3 ± 5.\%. n = 4) was seen when 40 |j,g/ml
plasma was left in the medium for the entire duration of the assay.
No significant difference was found between these means by way
of a 2-tailed, unpaired Mest. The inhibition produced by 2-h ex-
posure of wQPX to 40 p,g/ml plasma protein did not result from
QPX-cidal activity. Plasma-treated and untreated wQPX were
similar (treated: 94.0 + 1.7%r, n = 3; and untreated: 94.0 ± 3.0%-,
// = 3 viable). A degree of specificity for M. mercenaria plasma
is also indicted because exposure of wQPX to 40 |xg/ml produced
>90%i inhibition, whereas under the same conditions. C9G was
minimally inhibited (Fig. 3).
Reactions of M. mercenaria plasma with mucus-enveloped QPX
The typical response obtained by exposing wQPX immediately
to M. mercenaria plasma (Fig. 1) was not seen after comparable
100
75
50
25
0
-25
-50
-75
-100
c
o
10 20 30 40
— I 1
50 60
Protein Cone. (Mg/ml)
Figure 1. QPX-modulatory activity of M. mercenaria plasma ex-
pressed as percent inhibition of cultures after 7 d incubation. Final
plasma protein concentration in tlie medium is indicated. Linear re-
gression ly = lll..^[log xl - n.M: r- = 0.7497) of log-transformed
concentrations was used to calculate tlie inhibitory EC;,, = 18.99 (ig/ml.
QPX Mucoid Secretions
207
C
o
!c
c
100 n
75
50
25
0
-25
-50 H
-75
■100
0 10 20 30 40 50 60
Protein Cone, (pg/ml)
Figure 2. QPX-modulatory activity of heat-treated (65'C. 10 niin) M.
inerccnaria plasma expressed as in Figure I. Linear regression (\ =
48.22|log \| - 22.71; r" = 0.67151 of log-transfornied concentrations
was used to calculate the inhibitory V.C=,„ = 32.20 Mg/ml.
exposure of vvQPX that was incubated for 24 h before the addition
of plasma (Fig. 4). The lowest dose tested (3.75 |jLg/ml) apparently
produced some inhibition, whereas all other doses (:£60 jjig/ml)
seemed to stimulate the QPX cultures. The apparent inhibition
produced by the lowest concentration tested was not significantly
different from zero (P > 0.05. one sample Mest. 2-tailed). The
higher concentrations tested were all stimulatory. (P > 0.05. one
sample ;-test. 2-tailed). wQPX cells were either immediately ex-
posed to a highly inhibitory plasma concentration (40 |jig/ml) or
allowed to incubate in plasma-free medium for 2-24 h before
exposure; in these delayed exposure experiments, a time-
dependent linear decrease in growth inhibition was ob.served (Fig.
5 1. Unlike QPX. C9G cells in culture secreted no mucoid material
visible in preparations examined under the microscope. Preincu-
bation of washed CQG cells for 24 h before exposure to 40 p.g/ml
plasma had no significant protective effect as compared with cells
immediately exposed.
100
c
o
c
75-
50-
25
C9G
QPX
Figure 3. The effects of 40 pg/nil M. merciiiaria plasma proteins on
growth of 7 d cultures of QPX and C9G, a closely related thraus-
tochytrid also isolated from hard clams. The protists were exposed to
the plasma for 2 h. washed, and cultivated (7 d) in KMEM. Mean
percent inhibition and standard deviations are indicated.
o
luu-
•
•
n
•
u
t
•
•
100
•
•
•
•
•
•
•
•
•
•
200
r-
•
r
0 10 20 30 40 50 60
Protein Cone, (pg/ml)
Figure 4. (Irowth of wQPX preincubated lor 24 h in plasma-free me-
dium before exposure to 40 (ig/ml M. menenaria plasma. The QPX
cells continuously secreted mucoid material during the preincubation
period.
DISCUSSION
When wQPX cells were introduced into media containing vari-
ous concentrations of M. meixenahu plasma, their subsequent
growth was altered according to plasma concentration. This may
be seen in Figure 1 where 7 d QPX culture growth was often
stimulated in the presence of low plasma levels but consistently
suppressed at >I0 jjig/ml. These effects could be explained by the
presence of two QPX-modulatory agents in the plasma. Stimula-
tion at low protein levels might be caused by a factor with high
QPX-affinity and low to moderate activity. The effect of this
stimulator would be lost at higher protein levels if a low QPX-
affinity. higher activity inhibitor were present. The presence of two
growth modulators was also suggested by the differences in ther-
mal sensitivity (Fig. 2). Heat treatment of 65°C for 10 min seemed
to eliminate all stimulatory activity: however, the inhibitory effects
persisted with somewhat reduced activity. The growth modulating
activity of M. menenaria plasma takes place rapidly after inter-
action with wQPX. If wQPX was exposed to an inhibitory con-
centration of plasma (40 |jLg/ml) for 2 h. and then washed free of
plasma proteins before growing the culture in plasma-free me-
dium, culture growth was inhibited to about the same extent be-
cause it would have been if the cells had been continuously ex-
100
-25
-50
8 12 16 20 24 28
Time (hrs)
Figure 5. Effects of length of wQPX preincubation before exposure to
40 ng/ml M. menenaria plasma. Mean percent inhibition and standard
deviations are indicated.
208
Anderson et al.
posed to 40 ^Lg/ml plasma. These experiments could not establish
whether the inhibitory effects produced by M. mercenaria plasma
on the cell density of 7 d QPX cultures were caused by growth
inhibition or by cidal activity. Direct killing was ruled out by the
fact that 40 |xg/ml exposed, (potentially highly inhibited) wQPX
and unexposed wQPX were -95% viable.
Figure 3 presents evidence that the QPX-inhibilory plasma fac-
tor shows target specificity; C9G growth was hardly affected by 40
p.g/ml. Sequence analysis of C9G placed it in the thraustochytrid
phylogenetic group as a sister taxon to Thnnistocliytiium pachy-
denniim. and these sequences were grouped with QPX with a
parsimony jackknife support value of 100 (Anderson el al. in
press). Clearly. QPX sensitivity to low (<40 |xg/ml) plasma con-
centrations exceeds that of C9G; however, C9G growth was in-
hibited (-60%) by exposure to >180 |Jig/ml plasma (Anderson et
al. in press). Because the pathogenicity of C9G for M. Dierceinirici
has yet to be established, it is not known whether inhibition dif-
ferences caused by clam plasma between QPX and C9G reflect
differences in pathogenicity.
Incubation of wQPX in plasma-free medium allowed the cells
to resume mucus secretion. The cells underwent minimal division
for the first 48 h in culture, then proceeded lo grow w ith a doubling
time of -3 d (QPX growth curve not shown). The wQPX cells
were suspended in a loose gelatinous mass by 24 h. This mucoid
secretion often infiltrated the entire culture medium by 7 d in
culture. When the cells were permitted to develop their mucoid
covering for 24 h before the addition of plasma (Fig. 4). concen-
tration of -7-60 p-g/ml failed to inhibit QPX growth in 7 d cul-
tures. Unexpectedly, the lowest concentration tested (3.75 jjig/ml)
seemed to have inhibitory activity, but the mean of these experi-
mental values were not significantly different from zero. These
data suggested that the mucus material might protect QPX from A/.
iiicrceiuiria humoral defense mechanisms such as antimicrobial
factors. This hypothesis was supported by the results of the de-
layed exposure experiments, where protection from growth inhi-
bition was dependent on the time of incubation before exposure to
40 |j.g/ml plasma protein (Fig. 5). Because QPX cells in clam
tissues are typically enveloped by mucus, a role of this secretion as
a virulence factor seems likely. This is supported by a recent report
that clams injected with wQPX did not develop infections or dis-
ease (Smolowitz et al. 2001).
ACKNOWLKDGMENTS
This study was supported by Maryland Sea Grant, NOAA,
grant number NA06RG010I. This is Contribution No. 3642 of the
University of Maryland Center for En\ ironmental Science, Chesa-
peake Biological Laboratory.
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Ragan. M. A., G. S. MacCallum, C. A. Muiphy, J. J. Cannone. R. R. Gutell
& S. E. McGladdery. 2000. Protistan parasite QPX of hard-shell clam
Mercenaria mercenaria is a member of Lahyrinthulomycota. Dis.
.Aqual. Org. 42:185-190.
Ragone Calvo. L. M.. J. G. Walker & E. M. Burreson. 1998. Prexalence
and distribution of QPX. Quahog Parasite Unknown, in hard clams
Mercenaria mercenaria in Virginia, USA. Dis, Aquat. Org. 33:209-
219.
Smolowitz. R.. D. Leavitt. B. Lancaster. E. Marks. R. Hanselmann & C.
Brothers. 2001. Laboratory based transmission studies of quahog para-
site unknown (QPX) in Mercenaria mercenaria. J. Shellfish Res. 20:
555.
Smolowitz, R., D. Leavitt & F. Perkins. 1998. Observations of a protistan
disease similar to QPX in Mercenaria mercenaria (hard clams) from
the coast of Massachusetts. J. Inverlehr. Pathol. 71:9-25.
Stokes. N. A.. L. M. Ragone Calvo. K. S. Reece & E. M. Bun-eson. 2002.
Molecular diagnostics, field validation, and phylogenetic analysis of
Quahog Parasite Unknown (QPX), a pathogen of the hard clam, Mer-
cenaria mercenaria. Dis. Aquat. Org. 52:233-247.
Whyte, S. K.. R. J. Cawthom & S. E. McGladdery. 1994. QPX (quahaug
parasite X), a pathogen of northern quahaug Mercenaria mercenaria
from the Gulf of St. Lawrence. Canada. Dis. Aquat. Org. 19:129-136.
J.Hinuil ofSlwllJhh Re.scanh. Vol. 22. No. 1. 209-212, 200.^.
A PORTABLE AND PRACTICAL METHOD TO MONITOR BIVALVE FEEDING ACTIVITY IN
THE FIELD USING TIME-LAPSE VIDEO TECHNOLOGY
BRl'CE A. MACDONALD* AND LISA M. NODVVELL
Dcpcinniciit of Biology. Centre for Coiisral Stiullcs and Aquaciihnrc. University of New Briins\vu± Saint
John. P. O. Box 5050 Saint Jolin. New Brnnswicl<. Canada. E2L 4L5
ABSTRACT We developed a simple iiielhod to measure leeding activity of Mylilii.s filiilis using a canicorder placed inside an
underwater housing, a plastic frame for holding mussels and time lapse videography. Exhalant siphon area, indicative of feeding
activity, was monitored in laboratory mussels exposed to filtered seawater and various concentrations of microalgae, including Pavlova
lulheri or TetraseUnis suecica. Exhalant siphon area increased as algal concentration increased from zero to -25-30 x 10' cells ml"',
hut declined again at higher concentrations. Advantages of this method include portability and relatively low cost, high resolution of
data over shon and long temporal scales, potentially large sample sizes, and minimum logistics required for deployment in a variety
of different environments. Once relationships between exhalant siphon area and other indicators of feeding such as filtration rate have
been established, this method could greatly miprove our understanding of bivalve feeding in situ and how they respond in dynamic
natural conditions.
KEY WORDS: Mvrilus etliilis. bivalve feeding, time-lapse recording, exhalant siphon area, particle concentration
INTRODUCTION
There have been numerous studies on measuring feeding ac-
tivity in a variety of suspension-feeding bivalves over the last
several decades. There has recently been much discussion and
debate on whether or not bivalves have the capability of physi-
ological regulation or are pumping at full capacity all the time
(Jorgensen 1996, Bayne 1998, Hawkins et al. 2001 ). This includes
numerous comments on the proper interpretation of the published
literature and diverse opinions on the reliability of some of the
methods used (Cranford 2001. Riisgard 2001. Widdows 2001),
One such method considered to have good potential for assess-
ing feeding activity remotely with little interference by the ob-
server and minimal disturbance to the bivalve is the estimation of
valve gape and siphon area in mussels (Newell et al. 2001 ). Posi-
tive relationships have been reported between pumping rates of
mussels, valve gape and the exhalant siphon area (Jorgensen I960.
Riisgard & Randlov 1981. Famme et al. 1986. j0rgensen et al.
1988. Jorgensen 1990) and between exhalant siphon area and mus-
sel filtration rates (Newell et al. 2001).
Filtration rates of mussels have been shown to be linked to
particle concentration with low levels observed for filtered water
but increasing with natural levels of seston before decreasing again
at higher seston loads (Foster-Smith 197.5. Winter 197.^. Bayne
1993). Riisgard and Randl0v ( 1981 ) found comparable reductions
in filtration rates and valve gape of blue mussels at densities of
Plmeodactylum trieonmutwn lower than 1.500 cells ml" and
higher than .W.OOO cells ml"'. Newell et al. (2001) found a similar
apparent threshold for the filtration response to particle concen-
tration to occur at 2.000-6,000 particles ml"' in a Hume environ-
ment. Dolmer (2000a, 2000b) observed that high algal concentra-
tions may lead to decreases in valve gape as well as estimates of
filtration in the field.
There is ample evidence to suggest that exhalant siphon area is
a useful indicator of feeding activity in mussels and it is responsive
to variations in the concentration of suspended particles. The pur-
pose of this study was to develop a ponable and reliable method to
*Corresponding author. E-mail: bmacdon@unbsj.ca; Fax: +1-5U6-648-581 1.
remotely estimate exhalant siphon area for numerous undisturbed
mussels simultaneously. It would be particularly advantageous if
the method could be deployed to the field where mussel response
could be continuously evaluated while natural seston and flow
conditions are monitored. The combination of time lapse capabili-
ties and high resolution image of a digital camcorder, a portable
underwater housing, a plastic frame for holding mussels, and
readily available image analysis software provides an effective
tool for studying mussel feeding activity. Exhalant siphon area was
monitored in this study in mussels exposed to various concentra-
tions of cultured microalgae in the laboratory en\ ironment.
MATERIALS AND METHODS
Mussels [Mytilus edidis Linnaeus 1758) were collected from an
inlet in the Pasamoquoddy Bay. New Brunswick and transported to
University of New Brunswick in Saint John. New Brunswick,
Canada. Mussels were acclimated to laboratory conditions for a
minimum of 2 d and a maximum of 7 d. Experiments were per-
formed in a 530 I (244 cm long, 66 cm wide, and 33 cm deep) tank
with well mixed recirculating seawater. flowing approximately
5-10 cm s"'. Experiments were performed in full room light and
temperature and salinity were maintained at I2°C and 35-36'^f.
respectively. Water was prc-filtered in the tank with three inline
filters of 20. 5. and I p.m. Mussels were exposed to filtered sea-
water and cultured microalgae ranging in initial concentration
from 5.000-85.000 cells ml"' while siphon area was monitored
over periods of hours using time-lapse videography. Mussels were
exposed to experimental conditions for 30-60 min prior to mea-
surements to ensure feeding activity had resumed. With a few
exceptions experiments for each series of mussels typically ran tor
2_t h to ensure a good time series of measurements and a detect-
able change in particle concentration. Algal concentration was
measured using an electronic particle counter (Coulter Multisizer
II) with a 100 p-m tube orifice diameter. Algal diets provided in
experiments were one of Pavlova lutlieri (Provasoli-Guillard
CCMP1325) or TetraseUnis suecica (Provasoli-Guillard
CCMP904) or a mussel spat formula of Nanocliloropsis ocidata.
Chaetoceros-B, and Phaeodaelyltim iricorniaum (Innovative
Aquaculture Products Ltd.).
209
210
MacDonald and Nodwell
At least one day prior to the experiments Velcro was attached
to the mussel shell using cyanoacrylate cement and. after drying,
mussels were attached to individual plastic posts also covered in
velero. The posts containing the mussels were secured to a plastic
plate and attached to a frame connected near the lens of a video
recording device (Fig. 1 A). The number of mussels observed (usu-
ally 9-12 adults) in the video frame depended on the size of the
mussels and the efficiency of arranging mussels to adequately
view the external siphon. A Sony Mini DV (model DCR-TRV900)
three ccd camcorder was enclosed in an Amphibico 900 underwa-
ter housing and set to an interval recording mode of 2 s every
30 s over the entire period of each experiment to capture siphon
activity.
Multiple images from the mini DV tapes were collected using
the photo feature of the camcorder and stored on memory cards
before being transferred to a personal computer (Fig. IB). Varia-
tion in siphon area was estimated for individual mussels using the
program Image J (NIH public domain Java image processing pro-
gram— URL: http://rsb.info.nih.gov/ij). Siphon area was calibrated
using a 1 cm mark on the mussel posts. The inherent variation in
measuring exhalant siphon area was 2.4-3.8%. To standardize
individual responses for different sizes of mussels to different algal
concentrations, exhalant siphon area data were converted to per-
cent of maximum values observed for each mussel.
RESULTS
There was a consistent decline in algae over time in all the
experiments, indicating removal of microalgae by the mussels in
the course of the experiments (Fig. 2). Exhalant siphons were
opened, confirming feeding activity by the mussels. The fitted
lines for the uptake rates of algae typically had r" values exceeding
0.90-0.95 in all examples.
The percent maximum exhalant siphon area in individual mus-
sels exposed to filtered seawater (no algae) was consistently lower
than the siphon areas reported for the same mussels exposed to
microalgae (Fig. 3A). A similar trend of greater exhalant siphon
area was akso observed for groups of mussels exposed to different
concentrations of microalgae compared to those held in filtered
seawater (Fig. 3B). Note that mus.sel exhalant siphon area was still
approximately 20-309f of the maximum when exposed to filtered
seawater.
The percent maximum exhalant siphon area in mussels in-
creased with increasing particle concentrations to a maximum of
near 90-95% at concentrations approaching 25-30.000 cells mP'
(Fig. 4) Further exposure to concentrations above 30.000 cells
ml"' resulted in a decline in percent maximum exhalant siphon
area.
DISCUSSION
By modifying an underwater housing and combining it with a
high resolution camcorder capable of time-lapse videography we
have developed a simple and relatively inexpensive method to
remotely study bivalve feeding behavior. There have been other
devices developed to remotely monitor bivalve activity but, for
various reasons, they have not been readily adopted by scientists
working on bivalves. This includes The Musselmonitor* devel-
oped as a biological early warning system containing sensors to
record shell opening and closing while mussels are exposed to
various pollutants (Baldwin & Kramer 1994). Manuel and Lob-
siger ( 1999) de\eloped the MarineCanary'^' as a biomonitoring
tool using an underwater camera and a time-lapse system to assess
the marine environment through changes in bivalves" valve gape
and mantle activity.
Using this new method we have established a positive relation-
ship between exhalant siphon area and the concentration of cul-
tured microalgae, also observed by Newell et al. (2001) in their
study. Feeding activity is this study was confirmed by the con-
tinuous decline in the concentration of microalgae in the experi-
y = -976.76x + 6671
R' = 0.9487
Figure I. {\). .\n adjustable plastic frame attached to the front of an
underwater video housing containing a high resolution camcorder
with time-lapse capabilities. Mussels are secured with \ elcro to move-
able posts inserted into a plate positioned in front of the video lens. (B)
A tvpicai black and while photo made from a video frame captured
from the mini DV tape. Exhalant siphons are clearly visible for several
mussels simultaneously.
Elapsed Time (h)
Figure 2. An example of variation in declining algal concentration,
attributable to mussel feeding, during a typical medium — low concen-
tration experiment.
Time-Lapse Video Technique to Estimate Mussel Feeding
w
<
e
o
a
R
0)
Mussel #2 Mussel #3 Mussel #5 Mussel #8
100
■ No Algae
=1 10-20000 cells ml
B 20-30000 cells ml
n 30-45000 cells ml"'
*
i
Figure 3. (A) Variation in individual mean percent maximum exhal-
ant siphon area of representative mussels held in filtered sea« ater and
exposed to microalgae in different algal concentrations (5-45.000 cells
nil"'). (B) Mean response for groups of mussels exposed to filtered
seawater and three different experimental concentrations of microal-
gae (5—15.000 cells ml"'). Values are means ± 1 SE.
mental tanks (Fig. 2). The shape of the line when fitted to semi-log
transformed data (i.e.. rate of clearance) was comparable to the
reduction observed by Riisgard (1991) when Myliliis edutis was
grazing on Rhodomonas baltica. The positive relationship between
particle concentration and exhalant siphon area was apparent until
concentrations reached 25.000-30.000 cells ml"' and exhalant si-
phon area appeared to decrease with further increases in concen-
tration (Fig. 3B). Clausen and Riisgard (1996) also observed that
mussels partly closed their valves and reduced the opening of the
exhalant siphon at high algal concentrations but they found this
reduction to occur at around 13-24.000 cells ml"'. Note that we
did observe some moderately high values for siphon area for mus-
sels at the highest algal concentrations. This may have been an
artefact of the experimental design where a group of starved mus-
sels were exposed initially to \ery high concentrations of microal-
gae.
There are several advantages to the time-lapse videography
method for the observation of feeding activity in bivalves. This
includes its size. cost, portability and readily available components
including public domain software. A variety of underwater hous-
ings are available today for most commercial camcorders capable
of using time-lapse technology. Because of the small size of the
housing, they can be placed unattended in a wide variety of habi-
tats for extended periods of time — up to 10-15 hours with the new
3 <
It
(/)
fli TO
LU
100
90
SO
70
60
50
40
30
20
10
z
i
i
s
10000 30000 .50000 70000
Algal concentration (cells ml'^)
Figure 4. Variation in percent maximum exhalant siphon area of mus-
sels exposed to different concentrations of microalgae. The closed dia-
mond represents an experiment where 8 mussels were subjected to
algal concentrations from no algae to 45.000 cells ml"'; the open circle,
13 mussels subjected to algal concentrations of 0-85.000 cells ml"': the
open triangle, 8 mussels subjected to algal concentrations of 0-17,000
cells ml', \alues are means ± 1 SE.
generation of long-life batteries. Short-term bivalve feeding re-
sponses will be estimated more accurately //; sUu by monitoring
their activity continuously and unintenaipted rather than relying on
measurements at regular intervals or convenient points in time. It
is not necessary, as with more traditional methods to measure
feeding activity, to confine the bivalve in any kind of experiment
chamber, which may facilitate measuring the change in particle
concentration over tune but exposes the bivalve to unrealistic flow
conditions. Harrington et al. (2002) have successfully used this
method to compare feeding activity in mussels held near salmon
cages to mussels held in adjacent reference sites. We have ob-
served between 8 and 12 mussels simultaneously, an obvious ad-
vantage for sampling rate and statistical power o\er methods that
observe a single bivalve at a time. However, there exists a trade-off
between the number of mussels that can be observed and the
resolution of the siphon area for individuals obtained from the
video tape.
Filtration rate by mussels is a function of pumping rate, particle
concentration and filtration efficiency, such that control over
pumping rate is viewed as a major factor contributing to energy
acquisition by bivalves. As any one of these factors changes, there
may be an uncoupling between exhalant siphon area and filtration
rate. In order for this method, or any other method that measures
exhalant siphon area, to be used to estimate a rate of feeding the
variation in relationships between exhalant siphon area and filtra-
tion or pumping rate must be established in future studies. We are
proposing that this method, using a camcorder in an underwater
housing, a plastic frame for holding mussels and time lapse
videography, is a practical and potentially useful tool to address
many questions on how bivalves respond, in real time, to changes
in a naturally dynamic environment.
ACKNOWLEDGMENTS
This research has been supported in part by funds from AquaNet.
the Network of Centres of Excellence for Aquaculture. Financial
!i:
MacDonald and Nodwell
support was also provided through a NSERC research grant held by for constructing the mussel posts and frame and for technical assistance
B. A. MacDonald. The authors would like to thank Wayne Armstrong and Kelly Barrington for assistance in conducting the experiments.
LITERATURE CITED
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tems (BEWS). In: K. J. M. Kramer, editor. Biomonitoring of Coastal
Waters and Estuaries. Boca Raton: CRC Press, pp. 1-28.
BaiTingtom. K. A.. B. A. MacDonald & S. Robinson. 2002. Assessing the
feeding behaviour of blue mussels (Mytilus ediili.s). living within an
Atlantic salmon (Salmo salarl aquaculture site, with time-lapse videog-
raphy. Aquaculture Association Of Canada Meeting. Charlottetown.
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Berlin: Springer-Verlag. pp. 1-24.
Bayne. B. L. 1998. The physiology of suspension feeding bivalve mol-
luscs: an introduction to the Plymouth "TROPHEE" workshop. J. Ex/i.
Mar. Biol. Ecol. 219:1-19.
Clausen, I. & H. U. Riisgard. 1996. Growth, filtration, and respiration in
the mussel Mytihis editlis: no regulation of the filter-pump to nutri-
tional needs. Mar. Ecol. Prog. Sen 141:37-45.
Cranford. P. J. 2001. Evaluating the "reliability" of filtration rate measure-
ments in bivalves. Mar. Ecol. Prog. Ser. 215:303-305.
Dolmer. P. 2000a. Algal concentration profiles above mussel beds. ./. Sea
Res. 43:1LVII9.
Dolmer, P. 2000b. Feeding activity of mussels Mylilii.s edulis related to
near-bed currents and phytoplankton biomass. J. Sea Res. 44:221-231.
Faninie, P.. H. U. RiisgSrd & C. B. J0rgensen. 1986. On direct measure-
ments of pumping rates in the mussel Myliliis edulis. Mar. Biol. 92:
323-327.
Foster-Smith, R. L. 1975. The effect of concentration of the suspension on
the filtration rates and pseudofecal production for Mytilus edulis L.,
Cerasloderma edule L. and Venerupis pulla.stra (Montagu). J. E.xp.
Mar. Biol. Ecol. 17:1-22.
Hawkins. A. J. S.. J. G. Fang. P. L. Pascoe, X. L. Zhang & M. Y. Zhu.
2001. Modelling short-term responsive adjustments in particle clear-
ance rale among bivalve suspension-feeders: separate unimodal effects
of seslon volume and composition in the scallop Chlmiiys farreri. J.
E.xp. Mar. Biol. Ecol. 262:61-73.
Jorgensen, C. B. 1960. Efficiency of particle retention and rate of water
transport in undisturbed lamellibranchs. J. Cons. Cons. Int. E.xplor.
Mer. 26:95-116.
Jorgensen. C. B. 1990. Bivalve filter feeding: hydrodynamics, bioenerget-
ics. physiology and ecology. Fredensborg. Denmark: Olsen and Olsen.
Jorgensen. C. B. 1996. Bivalve filter feeding revisited. Mar. Ecol. Prog.
Ser 142:287-302.
Jorgensen, C. B., P. S. Laren. F. Mohlenberg & H. U. Riisgard. 1988. The
mussel pump: properties and modelling. Mar. Ecol. Prog. Ser. 45:205-
216.
Manuel. J. L. & U. Lobsiger. 1994. The MarineCanary'^': Using time-lapse
observation of mussel behaviour to assess the marine environment.
Bull. Aquacul. Assoc. Canada. 99:23-30.
NeweU. C. R., D.J. Wildish & B. A. MacDonald. 2001. The effects of
velocity and seston concentration on the exhalant siphon area, valve
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Ecol. 262:91-111.
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Riisgard, H. U. 2001. On measurement of filtration rates in bivalves — the
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Jounmi of Shcllfisl, ficscanli. Vol. 22. No. I. 2I.V223. 2(J03.
PARALYTIC SHELLFISH TOXINS IN PUGET SOUND, WASHINGTON STATE
VERA L. TRAINER.'* BICH-THUY L. EBERHART.' JOHN C. WEKELL.'
NICOLAUS G. ADAMS,' LINDA HANSON," FRANK COX," AND JUDY DOWELL"
Mariiie Biotoxins Prngniin. Emiroiimcutid Consenaiion Division. Northwest Fisheries Science Center.
National Marine Fisheries Service. National Oceanic and Atmospheric Administration. 2725 Montlake
Boulevard East. Seattle. Washington 9H1 12 and ~\Vasliini>ti>n State Department of Health, Food Safety
and Shellfish Programs. 7171 Cleanwater Lane. Olympia. Washington 98504
ABSTRACT The first illnesses and only deaths in Washington State resulting from paralytic shellfish poisoning were documented
in the 1940s, resulting in the establishment of one of the longest monitonng programs for paralytic shelltlsh toxins in commercial and
recreational shellfish in the United States. An analysis of the Washington Department of Health's monitoring data for the Puget Sound
area has allowed us to examine temporal changes in shellfish toxin levels and geographical distribution of shellfish harvesting closures.
The values of toxins in shellfish were normalized to control for variable levels of toxin accumulation in different shellfish species by
dividing individual values by the yearly average for a given species. These normalized values increased significantly over the past five
decades, indicating that the ob.served increase in paralytic shellfish toxin levels in Puget Sound shellfish was not caused by the shift
in species monitored. A geospatial map of the first shellfish closures or paralytic shellfish-poisoning event in each Puget Sound basin
suggests that over time, toxigenic .Mexainlriiim cells have been transported from northern to southern Puget Sound. Shallow sills that
restrict the exchange of water between adjacent basins have hindered the transport of toxic dinofiagellates. especially because these
cells generally do not prosper in mixing conditions that are characteristically found at sills. Large-scale events, such as the bloom that
occurred in the Whidhey and Central basins in 1978. may have been induced by global climate changes or shifts, such as the Pacific
Decadal Oscillation. Although greater numbers of closures have been observed over time in basins of Puget Sound, closures as a
percentage of total samples analyzed have decreased or remained constant in all basins, indicating that the Washington Department of
Health has established an effective monitoring program to protect public health while allowing for maximum harvest potential.
KEY WORDS: paralytic shellfish poisoning, saxitoxin, Puget Sound
INTRODUCTION
Background
Paralytic shellfish poisoning (PSP) is an acute illness in hu-
mans caused by eating bivalve shellfish (e.g.. mussels and clams)
that have ingested dinoflagellates that produce neurotoxic com-
pounds. The dinofiagellate. Alexandriiim catenella (Whedon and
Kofoid) Balech, previously described as belonging to the genus
Conyaidax Whedon and Kofoid or Protogonyaulax Taylor, has
been identified as the primary causative organism on the west
coast of North America, but recent evidence indicates that at least
five known species oi Alexandrium can produce PSP toxins (PSTs)
in Northwest waters (Homer et al. 1997). These dinofiagellates
occur either as single cells or as chains of cells. Their two flagella
enable them to vertically migrate to the surface during the day and
to depth at night, giving them advantages over nontlagellated phy-
toplankton. Generally, dinofiagellates thrive in stratified water be-
cause of their motility and ability to move to nutrient-rich areas
within the water column. When conditions for growth become less
favorable, A. catenella cells form resting cysts that settle to the
sediments, where they await the return of fa\orable growth con-
ditions (Anderson 1980).
Historically, PSP has been known in the Pacific Northwest and
Alaska for centuries. Records of PSP events date back as early as
June 15, 1793 (Vancouver 1798), when a member of Captain
George Vancouver's exploration team died after eating contami-
nated mussels harvested in the uncharted coastline of what is now
known as British Columbia. In 1799. 100 Russian hunters died
after consuming toxic mussels near Sitka. Alaska (Halstead 1965).
The first recorded outbreak of PSP on the eastern shore of Van-
*Corresponding author. E-mail: Vera. L.Trainer(S'noaa. gov
couver Island. Canada, in October 1957 caused serious illness in a
number of people (Waldichuk 1958) and resulted in a mandatory
monitoring program for PSTs in Washington State.
The PSTs include saxitoxin and at least 12 structurally related
chemical compounds (see. for example. Baden I9<S3). The record
level of PSTs in shellfish ever measured along the Pacific coast of
North America was 31,000 (xg saxitoxin equivalents (STXeq)/100
g shellfish in October 1989 in the inside passage of British Co-
lumbia, just north of the US and Canadian border (Bricelj & Shum-
way 1998).
PSP ill Washington Slate
The Washington State Department of Health (WDOH) initiated
a shellfish toxicity surveillance program in the early 1930s (Lilja
1978) as a joint effort between WDOH and the George Williams
Hooper Foundation for Medical Research in San Francisco. This
initial monitoring by WDOH focused on commercial shellfish and
was expanded to include recreational shellfish in the early 1990s
when the Puget Sound Water Quality Authority gave WDOH the
authority to monitor recreational species. Washington State's only
three fatalities due to PSP were recorded in 1942 (Quayle 1969)
near the entrance to the Strait of Juan de Fuca (Fig. 1 ). Since then,
the Washington Department of Fisheiies has imposed annual har-
vesting closures for all shellfish except razor clams from April 1 to
October 31 in the area west of Dungeness Spit (near Port Angeles.
WA; Fig. 1 ) along the Strait of Juan de Fuca and southward along
the coast to the Columbia River (Nishitani & Chew 1988). In
general, razor clams do not retain high levels of PSTs but are now
known to accumulate domoic acid (Wekell et al. 1994). The shell-
fish surveillance program in Washington State was terminated in
1946 when it was believed that this seasonal closure was effec-
tively protecting public health. In June 1957. PST monitoring was
reestablished to include all species of commercial shellfish in areas
of north Puget Sound and the outer coast after WDOH was advised
!I3
214
Trainer et al.
49 N
48 N-
47 N
46 N
125 W
124 W
123 W
122 W
Figure 1. Map of western Washington. Puget Sound basins described in the text are outHncd. Locations of sills less than 70 ni deep are noted
byXs.
of the prevalence of PSTs in British Columbia shellfish. The moni-
toring of recreation and sport harvesting on the outer coast and in
Puget Sound was sporadic until the early 1970s, when closures
caused by PSTs in shellfish above the FDA regulatory limit of 80
fjLg STXeq/100 g shellfish occuned in the Bellingham area (Fig. 1 )
for the first time.
Physical Oceanography
Puget Sound is a complex fjord made of several distinct envi-
ronments that are each influenced by different forces and condi-
tions, including river runoff controlled by dams, free flowing riv-
ers that undergo flooding due to snow-melt or heavy mountain rain
and tidal flushing (Strickland 1983). Because of these distinguish-
ing environmental factors. Puget Sound can be partitioned into a
series of basins or environments using the descriptions and chart
developed by Strickland (1983). The North basin extends from the
Canadian border and includes the Strait of Georgia. San Juan
Islands and Samish bay. In the North basin, the San Juan Islands
are partially bounded from the Northwest basin by a sill at their
southern edge (Fig. 1). In addition, the waters in Bellingham Bay
are partially separated from the San Juan Islands by sills in the
Rosario Strait. The Northwest basin is comprised of two semi-
enclosed bays. Sequim and Discovery bays, with oceanic influence
from the Strait of Juan de Fuca. This basin has the longest recorded
history of PSTs in the Puget Sound with frequent blooms of varied
intensity and duration. The Central. Whidbey. and South basins
are partially bounded from the Strait of Juan de Fuca by a sill at
Admiralty Inlet to the north and west (Fig. 1 ). The Whidbey basin
is relatively shallow and strongly influenced by high volumes of
fresh water from the Skagit River, controlled by a series of hy-
droelectric dams on its upper reaches. The Central basin fronts the
high population center of Seattle and contains the deepest waters
of Puget Sound. While the Central basin receives fresh water in-
puts from u number of rivers to the north and south, the volume of
its salt water mass is enormous compared w ith the other basins. Its
circulation is influenced by sills at both the northern (Admiralty
Inlet) and southern (Tacoma Narrows) ends. The sill at Tacoma
Narrows also borders the South basin that extends to the south-
ernmost reach of Puget Sound as a series of small, finger-like
shallow fjords. The eastern and western finger inlets of south Puget
Sound are believed to be two dynamically distinct water bodies
with separate circulation (Ebbesmeyer et al. 1998). The primary
freshwater influence in the South basin is the Nisqually River, fed
by melting snows from Ml. Rainier and the surrounding mountain
ranges. Currents in the South basin are strongly influenced by
tides, due largely to the shallowness of this area. Finally. Hood
Canal is partially isolated by a sill near its entrance that limits the
transport of deep marine waters in and out of the canal (Burns
1985). Currents in Hood Canal are slow, perhaps because the basin
is a closed-ended fjord without large volume rivers. It is the most
poorly flushed of all inlets in Puget Sound (Strickland 1983). but
the strongest currents tend to occur near the entrance at the north.
Paralytic Shellfish Toxins in Puget Sound
215
In summary, all Puget Sound basins are strongly influenced by
fresh water input, resulting in density-dependent stratification, es-
pecially in the summer months.
The spread of PSTs into previously unaffected areas, such as
south Puget Sound (Nishitani & Chew 1988) has raised an aware-
ness of the significant and expanding threat to human health and
economics of some of the most productive recreatioual and com-
mercial shellfish regions on the US west coast. An analysis of PST
data for the Puget Sound areas collected over the past five decades
has allowed us to examine changes in PST levels and geographical
distribution over the past five decades. This assessment will allow
us to evaluate whether modifications of the current monitoring
program or additional preventive measures are needed to effec-
tively protect seafood consumers as well as assist aquaculturists.
METHODS
WDOH Database
Shellfish toxHi data were provided by the WDOH Office of
Food Safety and Shellfish Programs that routinely mouitors PSTs
throughout the state in both commercial and recreational shellfish.
The data have been collected over a period of more than 40 y from
samples submitted by commercial growers and local health agen-
cies as required by federal and state regulations. In some cases,
local health agencies have collected samples directly from beaches
in their jurisdictions but have also relied on samples submitted by
volunteers.
In the last 20 y. mussels have been selected as a sentinel species
for PSTs because they bioaccumulate the toxins at a faster rate
than other shellfish. However, in the early years of monitoring
(1960-1980), Pacific oysters {Cnissosrreo gigas) and butter clams
(Saxidomus giganteus) constituted the major species sampled for
PSTs (Table I ). Since 1989, WDOH established a sentinel mussel-
monitoring program (Nishitani 1990) in which the blue mussel,
Mytilus edulis. generally was sampled; however, M. galloprovin-
cialis and M. ccdifornianiis were collected at a few Puget Sound
sites (Determan 2000). At most sites, mus.sels were sampled every
2 wk during the year from wire mesh cages suspended about one
meter deep below floats and docks. These cages were periodically
restocked with mussels. About 100 mussels provided the 100 g of
TABLE 1.
Number of shellfish samples collected by the Washington State
Department of Health during each decade.
Mvtiliis
Saxidomus
Protothaca
Crassoslrea
Decade
edulis
giganteus
staminea
gigas
Other*
1050s
4
127
20
169
69
1960s
0
208
146
362
157
1970s
649
1248
471
684
422
1980s
2361
3977
2712
2327
1773
1990s
9246
2498
4237
7078
779
*Other species (not all shellfish) include: Cancer imigister. Chioite sp..
Chlainys nibida. Cliiuicarditim nnriallli. Cnissadoma gigantea, Cnisso-
streci sikamea. Eiisis americiiiuis. Fusitriton oregonensis. Hcdiolis kwnt-
scliatkiiiui, Macoina nasula, Mucoma secta. Modiolus modiolus. Mya
arenaria. Mytilus califomianus. Mylilus gaUopiovincialis, Ostrea edulis.
Ostrea lurida. Panopeii ahrupta. Paraslichopus californicus. Parinopeclen
cauriinis. Polinices Icwisii. Tapes philippinarum. Tiesns nullallii.
tissue needed for toxin analysis. Mussels were collected, packed
with frozen gel packs, and shipped to WDOH for analysis.
WDOH performed all testing for PSTs using the standardized
mouse bioassay. The procedure has been modified since its incep-
tion in the 1920s by the inclusion of a saxitoxin standard provided
by the US Food and Drug Administration (FDA), and expression
of results in saxitoxin equivalents. STXeq (AOAC 1990). Early
data from the 1950s and 1960s expressed as "mouse units,"" were
converted to the newer designation by multiplying the mouse units
(MU) by the factor 0.2. Thus 400 MU/lOO g shellfish tissue is
e(|uivalent to 80 p-g STXeq/lOO a, the current "action level" speci-
fied by the FDA (AOAC 1990).
Data collected over the years by WDOH were not intended for
establishing trends but rather were collected solely to protect the
health of shellfish consumers. In other words, there was increased
sampling during a toxic event, to characterize the extent and se-
verity of the event, resulting in a greater proportion of tests that are
positive for toxin. For the purpose of this study, we included all
data for shellfish collected from 1957 through 1999. Blue mussels,
butter clams, littleneck clams (Protothaca staminea), and Pacific
oysters make up the largest number of samples analyzed (Table 1 ).
During the 20-y period from 1957 to 1977, sampling by WDOH
was relatively constant, averaging about 145 samples per year.
After the record-breaking PST level measured in 1978 (30,360 (j.g
STXeq/lOO g), the agency increased its sampling.
Data Analysis
A shellfish toxin database was constructed from indi\ idual PST
test data sets from the WDOH for each year from 1957 through
2000. These data sets were formatted and imported into a data
table in Microsoft Access (Microsoft Corp. Bellevue. WA). The
sample numbers that were assigned by WDOH were used as
unique identifiers for each record. A table containing latitude and
longitude coordinates along with sampling site descriptions was
linked to the PST data table through a field containing a code that
uniquely identified each sampling site. Similar methods were used
to link tables containing common names for the samples and the
names of the counties in which the sampling sites were located.
Queries were constructed that allowed fields in any of the tables in
the database to be searched.
RESULTS
Data Reduction for Trend Analysis
Sampling intensity throughout Puget Sound has been variable
over the past 40 y, primarily because of budgetary constraints of
the WDOH monitoring program. A variety of edible shellfish spe-
cies with different toxin accumulation and retention capabilities
was selected for monitoring purposes primarily because of avail-
ability. The major species used for monitoring in each basin since
1957 were oysters (North basin), littleneck clams (Northwest ba-
sin), blue mussels (Whidbey basin), littleneck clams (Central ba-
sin), and blue mussels (South basin; Table 2). These shellfish have
different rates of accumulation and depuration of PSTs. For ex-
ample, butter clams are known to retain high levels of toxin for
months, whereas mussels are known to depurate toxins over a
period of days (Bricelj & Shumway 1998). Additional variabilit>
in the data is caused by a disproportionate increase in sample size
over time in certain basins relative to other basins. During recent
decades, more reports of PSP illness, especially in south Puget
216
Trainer et al.
TABLE 2.
ShellHsh collected by the Washington State Department of Health (1957-1999).
Mytiltis ediilis
Saxidomus gigaiUeus
Protothaca staminea
Crassostrea
gigas
Other*
Basin
Total
(%)
C^f)
(%»
{%)
(%)
North
10.175
IS
19
11
31
21
Northwest
5.%!
12
22
38
13
15
Whidbey
3.696
55
29
7
1
8
Central
13.673
25
25
26
8
16
South
5.644
43
4
4
33
16
* Other species (not all shellfish) include: Ccuiccr iDUiiistcy. Chmuc sp., Clilamys nihida. ClinociirJiiim nultallii. Cnissadoma giganlca. Cnissasireu
sikumea. Ensis americwms. Fusirriton oregoneiisis. Haliotis kamlSLluitkana. Mcicomii nasuhi. Macoimi sectu. Modiolus modiolus. M\a aienaria. Mvtihis
californiaiuis. Mytihis galloprovincialis. Ostrea edulis, Ostiea hirida. Panopea abruplu. Purastichopus califomicus, Patinopcclen caurinus. Polinices
lewisii. Tapes philippiiuiriini. Tn'siis niilhillii.
Sound, have required an increase in PST testing. The different
sampling intensity as well as the shift in shellfish species collected
over time has necessitated data reduction for the purpose of trend
analysis. Because we examined the data for trends in PST activity,
only samples having quantifiable levels (S32 ixg STXeq/100 g) of
PST by mouse bioassay were included. All the quantifiable PST
data for San Juan Island shellfish are shown in Figure 2A. San Juan
Island was chosen because one of the longest historical records in
Puget Sound is available from this site. Data were simplified by
showing only the highest annual level of PST (Fig. 2B). Averages
per decade (Fig. 2C) of those maximum annual levels were cal-
culated in all shellfish from the San Juan area from the 1950s to the
1990s. Finally, data were normali/ed to control for different rates
of uptake and depuration of PSTs in all shellfish tested by dividing
individual PST values by the average for that species. The maxi-
mum normalized PST values were determined for each year then
averaged for the decade (Fig. 2D). When the nomialized maxima
per decade for the 1950s through 1970s were compared with the
past two decades ( 1980s and 1990s), the more recent two decades
were significantly higher (/-test. P < 0.001 ). The rise in PST values
over the past several decades is clearly seen in Figures 2C and D.
PST ill Basins of Puget Sound
A series of environmental factors such as the presence of
bounding sills, river input, and unique bathymetry were used to
divide Puget Sound into distinct basins (Strickland 1983; Fig. 3).
Sites that show typical PST le\els within a given basin were se-
lected for this study upon recommendation by WDOH. Because
central and south Hood Canal shellfish have remained essentially
free of PSTs. this arm of water west of the Central basin was not
included as part of this analysis. A summary of averages by decade
of maximum PSTs in all defined basins in Puget Sound showed
increasing magnitude of toxins in all shellfish monitored at all sites
with the exception of Whidbey and Central basins (Fig. 3). In the
North basin. Samish Bay had relatively low levels of PSTs during
the past three decades, whereas San Juan Island and Georgia Strait
had more intense toxic events with the average by decade of an-
nual maximum levels increasing from the 1970s to the 1990s. In
the Northwest basin we observed obvious increases in levels of
PSTs in both Sequim and Discovery bays over several decades. In
the Whidbey basin, PST levels remained relatively low, except for
an anomalously high level of toxin (30,360 jxg STXeq/100 g) in
1978 at Holmes Harbor. Levels of this magnitude had never before
(and have not yet again) been observed in Washington State. Rec-
ord levels of shellfish toxin measured from Whidbey Island south
to central Puget Sound in 1978 were responsible for the anomalous
peaks seen in Holines Harbor and Agate Pass in the 1970s (Fig. 3).
In the Central basin. Quartermaster and Kilisut harbors, as well as
7000
6000
5000
4000
3000 -
2000
1000
0
ii^jkiiiiiiiLliLii
I ''58 1^63 1968 1973 1978 1983 1988 1993 1998
1958 1963 1968 1973 1978 1983 1988 1993 1998
Figure 2. PST levels (^g STXeq/100 g) in all shellflsh from San Juan
Island, collected from Jan 1958 to Nov 1999. All data (A), maximum
annual PST levels (B), and average per decade of annual maximum
PST levels (C», and normalized average per decade of annual maxi-
mum PSTs (U) are shown.
Paralytic Shkllfish Toxins in Puget Sound
217
North K:Wm
Wliidlx'x Basin
■ <;i'iir};ia Strait (.^)
D Saiiiish Bay (4)
. . ^ Sanjuaii Maud (?
a
% 500 ■ •
50s 60s 71)s «0s 90s
tfl
Northwest Basin
■ DKuiM'O lla< III
□ Scquim Hay l2l
50s t)Ob 70s 80s W:
47 N
123 W
122 W
■ Iliilme', II^irlK>rl6l
^ 1 501) •
SlVimCi.yc-
I7l
-a KMMI '
J 50(1 •
^ „.
i
. 1
■
s(K 60s 70s SOs lOs
Central Basin
2000
■ \Bati- l'assi»i
□ Kilisulllarlxin'll
^ 1500+ E3 yuartt-niLislvr HarlwnlO)
S 1000 • •
SOs 60s 70s SOs 90s
South Basin
ICarrlnlcl llli
3 Cast InU't |12|
SOs 60s 70s mis i)Os
Figure 3. A^ erages per decade of maxinium PSTs (pg STXeq/100 g) in each Puget Sound basin. The locations of representative sites in each basin
are numbered.
Agate Pass, showed clear increases in average of annual niaximinii
levels over the past two decaiJes. In the South basin. PST levels
have recently reached record highs. Carr Inlet had its first shellfish
harvesting closures in 1988. although monitoring had been done at
this site since 1957. Before 1988. PSTs had only occasionally been
measured in the South basin but at levels below regulatory limit.
Nearby Case Inlet had its first closure in 1991. Since the 1991
event, this area has experienced more frequent toxic events and
higher levels of PSTs. reaching a maximum of 1.^.769 fxg STXeq/
100 g in blue mussels in 2000.
Frequency of PST Closures
The frequency of PST closures over time in each Puget Sound
basin is shown in Table 3. Although the number of samples col-
lected over time has increased, closures as a percentage of total
TABLE 3.
Number of closures in Puget Sound basins, also as a percentage of total samples analyzed during each decade.
Northwest
North
VVhidbey
Central
South
Decade
Closures
%
Closures
%
Closures
9(
Closures
%
Closures
%
1950s
32
25
1
2
0
0
0
0
0
0
1960s
195
45
2
1
0
0
0
0
ND*
ND
1970s
227
27
260
20
165
39
109
18
(1
0
1980s
610
34
827
22
119
7
912
23
238
22
1990s
387
14
486
10
31
1
1088
12
998
22
*ND =
No data
218
Trainer et al.
samples analyzed in each basin were variable. However, in general
a decrease in percentage of closures in each basin during the 1990s
relative to previous decades was evident, except in the South basin,
where 22% of the samples analyzed resulted in closures in both the
1980s and 1990s.
Seasonal Duration of Closures
The greatest number of closures during each decade occuired
from July through No\ ember with 81'^ of all closures occurring
during these months in the 1950s. 69% in the 1960s, 63% in the
1970s, 65% in the 1980, and 73% in the 1990s (Table 4).
Spread of PSTs
The historical record of PSP events causing illness and death in
humans and initial shellfish closures in the different regions of
Puget Sound is shown in Figure 4. The death of three people and
illness of two others after their consumption of mussels and butter
clams from the beach in Sekiu in 1942 was the first evidence of
high levels of PSTs in Washington State. The death of three mem-
bers of the Ucluelet Tribe after eating mussels containing PSTs on
the west coast of Vancouver Island, British Columbia, Canada,
was recorded three days prior to the mortalities in Sekiu (L. Han-
son, pers. comm.), indicating that this event was probably wide-
spread in the Pacific Northwest. From 1942 to 1957, Washington
State monitoring was sporadic and was actually temporarily
stopped in 1946 because of blanket closures that were in effect at
this time (Lilja 1978). Monitoring for PSTs in Washington became
formalized in 1957 after a large outbreak of PSTs occurred in
British Columbia, Canada (Waldichuk 1958). During this year, the
first shellfish closure occurred in Sequim Bay when a level of 162
jxg STXeq/100 g was measured in butter clams. The first shellfish
closure in the San Juan Islands occurred in 1958 when a level of
122 |j.g STXeq/lOO g was measured in butter clams. In the early
1970s, when WDOH monitoring efforts increased, shellfish con-
taining PSTs were found further east in Lummi Bay (Fig. 4) when
465 (ig STXeq/lOO g was measured in Pacific oyster in 1973. In
1978, anomalously high PST levels (up to 30,360 (jig STXeq/lOO
g) caused the first shellfish closures in both Whidbey and Central
Puget Sound basins. Over a period of several weeks, the contami-
nation spread southward in Puget Sound to an area between Seattle
and Tacoma in south-central Pucet Sound. In 1987. levels of PSTs
49.0 N
47.0 N
125,0 W
124.0 VV
22.0 \V
First Shellfish Flarvesting Closures and PSP Event in Each Region
Record ^'c.ir Rccn.)n Location ot first closure and/or PSP event
Five cases of PSP in Sekiu. three deaths
Sequim Bay/Discovery Bay
San Juan Islands
Lummi Bay
Whidbey Basin/Central Basin. 9 cases of PSP
Northern Hood Canal
Carr Inlet, I case of PSP
Case Inlet
Totten and Eld Inlets
Figure 4. First recorded PSP events and shellflsh harvesting closures
in each Puget Sound basin. Locations of each event are numbered on
the map of Puget Sound,
in northern Hood Canal were measured above the closure limit for
the first time since WDOH sampling began (234 (xg STXeq/lOO g
in Pacific oyster). The first closures of shellfish harvesting in south
Puget Sound in 1988 were due to PST levels up to 10,982 |xg
STXeq/lOO g in Carr Inlet. One person was hospitalized after
ingesting oysters from Minter Bay, Carr Inlet in September 1988
(F. Cox, pers. comm.). In 1991, the first incidence of shellfish
1
1942
NW
1
1957
NW
3
1958
N
4
1973
N
5
1978
C
6
1987
C
7
1988
S
8
1991
S
9
1997
sw
TABLE 4.
Number of monthly closures, also as a percentage of total closures during each decade.
1950s
1960s
1970s
1980s
1990s
Month
Closures
%
Closures
%
Closures
%
Closures
%
Closures
%
Januarv
1
■^
10
5
40
5
80
3
82
4
February
0
0
11
5
30
4
49
2
66
3
March
0
0
8
4
35
5
97
4
53
1
April
4
9
14
7
65
8
173
7
60
3
May
2
4
1
0
33
4
155
6
70
3
June
2
4
2
1
42
5
238
9
147
7
July
3
7
24
12
101
13
442
17
353
16
Ausiist
13
28
20
10
109
14
449
18
337
15
September
5
11
27
13
98
13
408
16
321
14
October
6
13
41
20
115
15
260
10
393
18
November
10
■>■>
29
14
65
8
III
4
204
9
December
0
0
I.S
7
37
5
94
4
150
7
Paralytic Shellfish Toxins in Puget Sound
219
closures occurred in Case Inlet, with levels of 779 ixg STXeq/100
g in blue mussels. In the fall of 1997. PST levels up to 6799 (j.g
STXeq/lOO g were measured in Eld and Totten inlets, causing the
first shellfish closures in these small southv\'estern finger inlets of
south Puget Sound. Pre\ious routine monitoring, necessitated by
ihe presence of commercial shellfish operations at these sites, de-
tected only low levels of PSTs that were below the regulatory limit
of SO (xg STXeq/lOO g (Saunders et al. 1982. Determan 2000). For
example, the first measurement of PST in Carr Inlet was in 1981
at a level of 51 |xg STXeq/lOO g in blue mussels.
When the highest annual PST levels exceeded 80 ixg/lOO g
e\en once at a particular monitoring site during a given decade,
that site was shown to have a closure during that decade (Fig. 5).
Although samples were tested in several areas throughout Puget
Sound, in the 1950s and 1960s the only areas with shellfish clo-
sures were in the Northwest and North basins. In the 1970s, the
number of sampling sites increased substantially, and closures
were seen in central Puget Sound. During the 1980s, the first
closures were seen in the eastern inlets of the South basin; shellfish
closures occurred throughout much of south Puget Sound in the
1990s. An increase in the number of monitoring sites sampled over
the decades is evident. Data from the 1970s indicated the high
number of closures in 1978 in the Whidbey basin, however by the
1990s, few closures were observed here. The actual numbers of
samples tested for toxins and closures in each basin as a percent of
the total closures in all of Puget Sound are shown in Table 5. The
greatest number of closures occurred in the Northwest basin in the
1950s (977f of all closures) and 1960s (999J- of all closures), in the
North basin in the 1970s (34% of all closures), in the Central basin
in the 1980s (34% of all closures), and in the Central (36% of all
1950s
1960s
49.0 N
48.5 N -
48.0 N
47.5 N
47.0 N
123.5 W 123.0 W 122.5 W 122.0 W
1970s 1980s
1990s
Figure 5. Closures because of PST in shellflsh at all Puget Sound monitoring sites for each decade. Symbols represent maximum values for each
cittade sIhih n as open circles (below 80 (ig STXeq/lOO g) or solid circles (greater than or equal to 80 jig STXeq/lOO g). Data for the 19S0s include
only 1957-1959.
220
Trainer et al.
TABLE 5.
Number of samples analyzed for PSP toxins in each basin and closures in each basin as a percentage to total closures in Pugct Sound.
Northwest
North
Widbey
Central
South
Total
Total
Closures
as a % of Total
Decade
Number
C* )
Number
(^n
Number
['^r)
Number
(1)
Number
C?-)
Measurements
Closures
Measurements
1950s
130
97
53
3
1
0
13
0
4
(1
201
33
16
1960s
433
99
202
1
1
0
17
0
ND"
ND
653
197
30
1970s
841
30
1302
34
423
Tl
607
14
24
0
3197
761
24
1980s
1793
23
3759
31
1704
4
3966
34
1080
9
12302
2706
22
1990s
2764
13
4859
16
1566
1
9070
36
4536
33
22795
2990
13
Highest level*
(dale)
3074(9/17/901
5968 (7/27/99)
30360(9/28/78)
4822(7/11/90)
10982(10/22/98)
* Highest level = (ig STXeq lOOg"
* ND = no dala.
. all in blue mussels.
closures) anti South basins (339f of all closures) in the 1990s.
Whereas the highest percentage of total closures occurred in north-
ern Puget SouncJ in the 1950s, the greatest percentage has nwie
recently occurreiJ in the central and south Puget Sound regions.
Closures as a percent of total measurements made have decreased
since the 1960s.
DISCUSSION
There is speculation that harmful algal bloom events are in-
creasing in intensity, frequency, duration, and geographical loca-
tion; however, the long-term monitoring data needed to support
these ideas are often insufficient for trend analysis. Because of
documented illnesses and deaths due to PSP beginning in the
1940s. Washington State has one of the longest monitoring histo-
ries for PSTs in the United States with the State of Maine ha\ ing
the next oldest monitoring program, established in 1958 (Shum-
way et al. 1988). Data collected in Washington State are compi-
lations of PST measurements at shellfish harvest sites designated
by the WDOH to have the greatest risk for human exposure to PSP.
Although the location and frequency of monitoring at these sites
have changed substantially over the years, we were able to use tlic
data to establish trends for Puget Sound shellfish closures due to
PSTs.
Spread of PSTs into Central and Southern Panel Sound
Until the last decade, the only Puget Sound basins with no
measured PSTs were southern Hood Canal and the southernmost
inlets of Puget Sound (Rensel 1993, Determan 2000). Since the
1980s, the frequency of PST detection has increased in southern
basins of Puget Sound, an area that contains the region's most
productive shellfish-growing beaches. Shallov\ sills that restrict the
exchange of water between adjacent basins (Strickland 1983; see
also Fig. I ) have likely hindered the movement of toxic dinotlagel-
lates, especially because these cells generally do not prosper in
mixing conditions that are characteristically found at sills. AUw-
aiidriiim cells thrive in stratified environments, presumably due to
the supply of nutrients, trace minerals, and natural humic sub-
stances that may serve as growth stimulants at the density interface
(see. for example, Anderson 1997). Therefore, sills, which are
found at several sites in Puget Sound (Fig. I ), have likely delayed
the spread of Alexandriiim cells to the South basin.
A geospatial map showing the first accounts of shellfish clo-
sures or PSP in each region of Puget Sound (Fig. 4) suggests that
over time, toxigenic Alexamiriwn cells, cysts or both have made a
slow progression from northern Puget Sound to the south. The
numbers of cysts and cells likely have increased over the decades
in the areas near sills, eventually reaching a critical mass that
enabled their survival during transport over these natural barriers.
Conditions for Alexandriuin cell growth are ideal in south Puget
Sound because of the many shallow, poorly flushed bays and inlets
where thermally-caused stratification occurs during summer
months, allowing ideal grow th conditions for dinollagellate cells to
persist for weeks (Rensel 1993). However, the initial population of
Alexwulnum cells or cysts probably entered south Puget Sound
only in recent years. The first detectable PSTs in south Puget
Sound were noted in Carr Inlet (57 |j.g STXeq/100 g in blue
mussels) in 1981. Some anecdotal evidence from the epidemio-
logic record also supports the gradual spread of toxigenic Alexan-
driuin cells into south Puget Sound. Of the nine people who be-
came ill after eating mussels from Carr Inlet in the suinmer 2000,
one woman who was sick during that event pre\ iously had eaten
shellfish from the same beach in Carr Inlet for more than 50 y with
no PSP symptoms (Cox 2000).
A possible pathway of cells into Puget Sound was through the
Strait of Juan de Fuca. into the Northwest basin and western San
Juan Islands, then past the sill to the south of the San Juan Islands
and Rosario Strait into Bellingham Bay. From the Northwest ba-
sin, cells may have been transported southward to the Whidbey
basin, past the sill at Admiralty Inlet to central Puget Sound, and
also past the sill at the entrance to Hood Canal to northern Hood
Canal. Finally, from the Central basin, cells spread into south
Puget Sound past the sills at Tacoma Narrows and the Nisqually
(Fig. 1 ). The hydrographic separation of the ea.stem and western
inlets of south Puget Sound (Ebbesmeyer et al. 1998) can explain
the temporal lag in the first documented shellfish harvesting clo-
sures in Case Inlet in 1991 compared with Totten and Eld inlets in
1997 (Fig. 4).
The Initial Population of .\lexandrium in Washington State
The first recorded PSP event in Washington State, at Sekiu in
1942 (Fig. 4), coincided with three deaths on the western coast of
Vancouver Island, Canada. The next PSP episode in British Co-
lumbia was in the inland waters of the Strait of Georgia in 1961
when 61 people fell ill (Taylor & Homer 1994). It is possible that
the source of the "seed"" population of toxigenic A. catenella cells
in Washington State originated from the inland or coastal waters of
Canada. Indeed, the first documented PSP event in all of North
America dates back to 1793, when four members of Captain
PARAL^■TIC' Shellfish Toxins in Puget Sound
221
George Vaneoiiver's erew beeame sick and one died of PSP during
exploration of present day British Columbia (Quayle 1969). Unlike
its neighbor to the north. Washington State had no recorded ill-
nesses or deaths of humans with descriptions of PSP symptoms
before 1942. Alexainlriiim calenella is the chief source of PSP off
the west coast of British Columbia and eastern Vancouver Island
(Taylor & Harrison. 2002) and evidence suggests that the earliest
recorded PSP outbreaks were at least partially because of blooms
of this dinotlagellate species (Quayle 1969). Because prevailing
winds and currents are from the north during the summer months
(Mickey 1989), when growth conditions for Alexandrium are op-
limal. and because the inlet to Puget Sound is at the north end of
this tjord. a north to south transport would support the natural
dispersal of algal cells from Canada. The routes of toxigenic cell
dispersal in the Pacific Northwest could be defined in the future by
a study of population genetics of A. catenella isolates from both
British Columbia and Washington State.
Incriasid I'ST Levels
Because of increases in aquaculture activity as well as the
measurement of PSTs in new areas of Puget Sound, the number of
samples taken annually for PST testing has increased steadily from
1988 to the present time (Table 5). However, increased sampling
frequency has not resulted in a higher percentage of closures dur-
ing the latter decades (Table 3). The majority of closures during
each decade was in July through November; a shift to more clo-
sures in earlier or later months has not been observed in recent
years. In addition, no correlation between the highest toxin levels
and total number of samples collected annually was observed
(Table 5), suggesting that apparent increases in PST intensity are
not due to increased sampling. Because mussels can accumulate
higher levels of PSTs, the shift of reliance on oyster and clam
samples in the inonitoring program in the 1960s to mussel samples
in the I99()s (Table 1) may account for some of the observed
increase in toxin intensity. However, the normalized maximum
\alues of PSTs in all shellfish have also increased over the past
fi\e decades (Fig. 2D), showing a statistically significant increase
during the 1980s and 1990s compared with the three previous
decades, suppoiling the fact that the increase in PST levels in
Puget Sound shellfish was not due to the change in shellfish spe-
cies monitored over the years,
PST Inlensily Versus Human Population Growth
Over the last four decades, modern human development has
extensively altered the shoreline habitats of Puget Sound (see the
Department of Ecology, Water Quality Monitoring web page,
http://www.ecy.wa.gov/programs/eap/mar_wat.html ). A compari-
son of maximum PST averages per decade and population esti-
mates (of all counties bordering Puget Sound) over the last 40 y
shows a high level of correlation (r = 0.987; Fig. 6). Although
statistical correlation does not establish a causal link, it does sug-
gest that some factor(s) associated with population growth may
influence the magnitude of PSTs at any given site. Increased nu-
trients to our coastal environment may provide more favorable
growth conditions for Alexandrium cells that populate a given
basin. It has been speculated that the lack of nitiogen in surface
and subsurface waters of Puget Sound has been a major factor
limiting the further spread of PSTs into bays and inlets otherwise
suitable for A. calenella (Rensel 1993). Land clearing, logging,
aerial forest fertilizing by timber companies, direct sewage out-
5(M)()
4(M)()
3000
2000 ,
1(M)0
1,0
1.5
2,0
3,0
4,0
Population (Millions)
Figure 6. Maximum PST average per decade versus population esti-
mates. Census data for counties Ijordering Puget Sound were obtained
from the rollowing site: http://H«\v, census.gov/population/cencounts/
«aiy009().txt
falls, agricultural runoff, and e\'en aquaculture operations have
increased the amounts of nutrients, including nitrogen, that are
supplied to the coastal ecosystems of Puget Sound (Howarth
2001). Inlets and fjords with low flushing rates that adjoin urban-
ized shorelines have the greatest sensitivity to nutrient addition
(Mackay & Hairison 1997). The increased levels of PSTs in the
.semi-enclosed bays of south Puget Sound in recent years may, at
least partially, be explained by increased eutrophication and gen-
erally poor circulation. Indeed, south Puget Sound is described by
the Washington State Department of Ecology as one of the areas
most susceptible to impacts of eutrophication (Cusimano 2002).
Because the depth of south Puget Sound inlets is much shallower
and flushing time is slower, nutrient inputs to surface waters pro-
vide ideal growth conditions for A. catenella.
Natural Events
Although the intensity of PSTs in shellfish has increased with
time (Fig. 2), toxic events do not occur in each basin in every year.
For example, shellfish closures have occurred in northern Hood
Canal only in 1991. 1996. and 1997-1999. What sets those years
apart from all other years'? Environmental conditions such as water
temperature, mixed layer depth, sunlight, and nutrients all work
together to increase the chance of a toxic event in a particular basin
and in any given year (Rensel 1993. Nishitani et al. 1988). In
addition to microscale, basin-specific environmental factors that
result in a periodicity of Alexandrium blooms, large-scale occur-
rences, such as the bloom that occurred in the Whidbey and Cen-
tral basins in 1978, may have been motivated by global climatic
events or shifts. In 1977. a large shift to a positive Pacific Decadal
Oscillation occurred, with a resulting ecological response to the
environmental changes. This period was marked by an enhance-
ment of overall productivity that appeared to be closely related to
changes in upper ocean mixed-layer depths and temperatures
(Mantua et al. 1997). Indeed, an exceptionally deep surface layer
of warm water was believed to have exacerbated the 1978 Whid-
bey basin bloom (Erickson & Nishitani 1985), Toxin levels of that
magnitude have not been measured since that year in Whidbey
basin, giving credence to the possibility that some unique, large-
scale environmental factors influenced the occurrence of this
event. The linkage of harmful algal bloom magnitude and fre-
quency to climatic regime shifts has been suggested in recent
222
Trainer et al.
studies (Epstein et al. 1998, Hayes et al. 2001). The specific co-
variance of levels of PSP toxins in shellfish with strong El Nino/
Southern Oscillation events (Erickson & Nishitani 1985) and other
environmental parameters such as the condition of oysters in Wil-
lapa Bay (Ebbesmeyer et al. 1995) has been suggested.
Effective Monitoring
Although greater numbers of closures have been observed over
time in many of the basins of Puget Sound, the percentage of
closures relative to the total sites monitored in a given basin has
decreased in all but south Puget Sound (Table 3). Although PSP
toxins pose a serious threat to commercial and recreational shell-
fishing operations, the large number of sites monitored by WDOH
allows the agency to pinpoint areas within a basin that are safe for
harvest. This rigorous monitoring has resulted in a greater propor-
tion of open than closed sites for shellfishing in the Puget Sound
region where the risk for PSP is extreme. Increased HAB events
and interest in commercial shellfish operations in all regions of
Puget Sound and wide-scale, year-round recreational harvest op-
poilunities will likely result in a mandate for the WDOH to sustain
its rigorous sampling efforts. In the future, improved monitoring
methods (e.g.. molecular probes for cells and rapid analytical as-
says for toxins) will be essential for cost-effective and timely
management of the fishery in Puget Sound.
CONCLUSIONS
The following conclusions can be obtained from our study. 1 )
There has been a significant increase in the magnitude of PSTs in
Puget Sound shellfish with time. 2) The geographical scope of
shellfish closures caused by high levels of PSTs in Puget Sound
has increased over the past four decades. The first recorded shell-
fish closures in the Northwest basin in the 1950s, the Central basin
in the 1970s, and the South basin in the 1980s are likely due to the
spread of A. calenella cysts and/or cells from north to south. 3)
Shellfish closures in south Puget Sound may have been delayed
until recent years by the physical blockage of cell movement by
sills to the north. Hydrographic blockage may also explain the
delayed appearance of PSTs in the southwestern finger inlets of
south Puget Sound. 4) Increased shellfish closures caused by PSTs
over the past few decades are not just the result of greater numbers
of samples collected over time. 5 ) Global climate changes, such as
the Pacific Decadal Oscillation and increased eutrophication in
nearshore areas, are possible explanations for the increased mag-
nitude of PSTs in shellfish today.
ACKNOWLEDGMENTS
Thanks to the numerous volunteers who have collected shell-
fish samples for WDOH over the years. The authors thank Rita
Homer and Tim Determan for their constructive comments on an
earlier version of this manuscript. Funding for database construc-
tion and analysis was provided by the National Ocean Service,
NOAA, through the Environmental Services Data and Information
(ESDIM) program, project 0I-4I4F, "Access to Pacific Region
Harmful Algal Bloom (PACHAB) Data." The authors thank
Michelle Tomlinson for assistance with the database.
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FEEDING SOUTHERN ROCK LOBSTER, JASVS EDWARDSII HUTTON, 1875.
PHYLLOSOMATA IN CULTURE: RECENT PROGRESS WITH LIPID-ENRICHED A^reMM
MATTHEW M. NELSON,'* BRADLEY J. CREAR,-t PETER D. NICHOLS,' AND DAYID A. RITZ'
^Department of Zoology, University of Tasiuania. GPO Box 252-05, Hobart, TAS 7001. An.stralia:
~Marine Re.searcli Laboratories, Tasmanian Aijiiaciibure and Fisheries Institute, University of
Ta.smania, Taroona, TAS 7053. Australia: and 'CSIRO Marine Research, GPO Box 1538, Hobart,
TAS 7001, Australia
ABSTRACT Jasus fchvanlsii phylloMnna larvae were successfully grown in static culture with antihiotics Ironi newly hatched to
stage V with high survival. Feeding phyllosomata on Artemki salitia Linnaeus, 1758. enriched with ( 1 ) a triacylglycerol (TAG)-rich
Al DHA Selco-Chaeroceros inuelieri Lemmermann. 1898. nutrient source or (2) a formulated ethyl ester (EE)-rich nutrient source was
compared with the more novel approach of using a formulated mussel powder-polar lipid diet attached to mesh. Individuals showed
an increase to stage V in dry mass (0.1-1.5 mg) and total length (2.1-6.1 mm). Survival o( Anemia-'iti phyllosomata was high
(92-98'5'f from stages II-III; 497? mean total survival). Animals fed the mussel powder-polar lipid diet had low molt success, although
the presence of faecal trails confirmed they were consuming the diet. Total lipid remained generally constant in .4(Vfm(V;-fed phyllo-
somata from newly hatched to stage V ( 155 mg g ' dry mass): this was notably higher than observed for previous feeding trials. The
major lipid class in all phyllosomata samples was polar lipid, followed by sterol, with TAG as a minor component only, and EE not
detected. The main fatty acids were 18;l(n-9)c. 18:2(n-6), 16:0. eicosapentaenoic acid [20:5(n-3l]. 18:0. 18:l(n-7lc. arachidonic acid
(20:4(n-6)]. and docosahe.xaenoic acid |DHA; 22:6(n-3)]. Levels of the essential polyunsaturated fatty acids (PUPA), namely, arachi-
donic acid, eicosapentaenoic acid and. in particular. DHA. decreased, on both a relative and absolute basis, from newly hatched to stage
V. although phyllosomata fed the EE-rich enriched Artemia diet showed higher essential PUFA content together with oil content. This
experiment further \alidates that lipids and fatty acids are important nutritional component in rock lobster larvae and that feeding
phyllosomata with lipid-enriched Artemia maintains excellent growth and survival in early stages. Strategies will be needed, however,
to either overcome the issue of low DHA. in particular, delivered by Arieinia (because of retroconversion), or to supply DHA by
alternate means at later stages.
KEY WORDS: Arleiuiu. enrichment, fatty acids, Ja.su.s I'dwanlsii, lipids, lobster, phyllosoma
INTRODUCTION
Rock lobster in Australasia has recently attracted the interest of
a number of research institutions for its potential as a valuable
aquaculture species. The fishery for southern rock lobster, Jasus
edwardsii Hutton, 1875, boasts a value of over A$2()0 million in
Australia (Punt & Kennedy 1997) and NZ$100 million in New
Zealand (Breen & Kendrick 1997). As wild fishing pressure esca-
lates (Booth & Phillips 1994). future exploitation of the rock lob-
ster marketplace will logically be realized through aquaculture
(Phlegeret al. 2001).
As an aquaculture species, rock lobster possesses the allure of
potentially high financial reward. Equally great is the challenge for
research scientists because the larval phase, including metamor-
phosis from phyllosoma to puerulus, is extensive (Phillips & Sas-
try 1980. McWilliam & Phillips 1997), currently requiring close to
a year in culture (Tong et al. 2000). To conquer this challenge,
several vital aspects of culture of rock lobster phyllosomata can be
identified as follows: (1) exploration of feeding capabilities of
phyllosomata (Johnston & Ritar 2001, Nelson et al. 2002a) to
determine appropriate format of feed presentation; (2) determina-
tion of nutritional requirements to focus further the feed format;
(3) a suitable aquarium design (Kittaka & Booth 2000. Ritar 2001 )
to optimize exposure of animals to the food source while mini-
mizing microbial loading (Igarashi et al. 1990, Diggles et al.
2000).
This study examines the second aspect (noted above), nutrition,
and in particular the requirements for lipids. To focus this aspect.
*Corresponding author. E-mail: mmnelsonC^utas. edu.au
tCurrent address: Geraldton Fishermen's Co-operative. P.O. Box '
aldton. WA 6531. Australia.
features of lipid nutrition under examination include: (1 ) total lipid
content, the mg g ' of the lipid provided in the diet and that
incorporated into larvae; (2) the lipid classes, examination of the
delivery, and incorporation of types of lipids, such as triacylglyc-
erol (TAG), polar lipid (PL) and ethyl ester (EE); and (3) the
profile of fatty acids (FA), which are components of lipid classes.
Building on the studies of lipids and FA in wild phyllosomata
(Phlegeret al. 2001) and potential prey items (Nichols et al. 2001 ).
we have examined enrichment of Arlcniici with essential polyun-
saturated fatty acids (PUFA) (Phleger et al. 2001, Nelson et al.
2002b, Smith et al. 2002) and feeding of these TAG-enriched
Artemia to phyllosomata (Nelson et al. 2003). The evidence
amassed to date from these studies indicates that wild phylloso-
mata largely obtain, and therefore may require, lipid in a PL form
rather than in a TAG form. However, Artemia store their lipid
enrichment as TAG (McEvoy et al. 1996, Sorgeloos et al. 1998,
Harel et al. 1999). With this in mind and because phyllosomata do
consume static food items (e.g., mussel pieces) (Kittaka 1997b,
Matsuda & Yamakawa 2000, Nelson et al. 2002a). the present
study was performed to provide phyllosomata a diet presented at a
feed station (i.e., formulated diet attached to aquaria), a format
cunently receiving attention (Cox & Johnston 2003), A compari-
son was made for feed-station fed larvae to animals fed Anemia,
enriched with either a TAG-rich product or with a novel docosa-
hexaenoic acid (DHA)-rich EE product, by examining the effects
on J. edwardsii phyllosomata survival, growth and lipid composi-
tion.
METHODS
Artemia Enricliiiieiit
3, Ger-
Decapsulated Anemia cysts (INVE, Great Salt Lake Prime
Gold) were hatched at 28 ± IC in .50-L white fiberirlass cones in
225
226
Nelson et al.
0.2-|jim filtered brackish water (27 ± 1 g leg"'), with vigorous
aeration and a 150 W light suspended 0.5 m above the water. After
24 h, Arteinia nauplii were removed from the hatching cones.
rinsed in freshwater for 2 min and transfened into 1000-L tanks of
filtered seawater (0.2 (xm. 34 ± 1 g kg"', 27 ± \°C). Anemia were
fed twice daily with a rice pollard-soy flour-wheat flour brine
shrimp diet (Eyre Peninsula Aquafeeds. South Australia) at a rate
to maintain a Secchi depth of 25-30 cm. The environmental pa-
rameters remained stable for the duration of the on-growing pe-
riod; salinity (35.7 ± 0.2 g kg"' ), pH (8.3 ± 0.0), dissolved oxygen
(7-7.2 mg L"'). and temperature (26.9 ± O.TC). After 5 days,
80.000 /4rte/)?/n with a total length of 1.5 ± 0.2 mm were removed
from the on-growing container, rinsed in freshwater for 2 min and
transferred to the 50-L white fiberglass cones containing 10 L of
filtered seawater to achieve a density of 4 niL"'.
Anemia were enriched for 24 h with 0.6 g L"' of three nutrient
sources (i.e.. Aireniia enrichment diets):
(DAI DHA Selco (INVE Group, Belgium).
(2) The microalga Chaetocems nuielleri Lemmermann 1898.
(3) Ethyl ester-mussel: a mixture of New Zealand Greenshell
mussel (Pema canaliculus Gmelin. 1791 ) powder (NIWA
Research. Auckland. New Zealand)-DHA (66'7f) EE oil
(CSIRO Marine Research. Hobart. Australia)-AA (39%)
TAG marine oil (Sun-TGA40S, Suntory Limited, Osaka,
Japan )-Greenshell mussel polar lipid (NIWA Research,
Auckland, New Zealand) (56:30:10:4 by mass).
C. muelleri were cultured and the cell density was measured
daily as described in Wilkinson (2000). The nonalgal enrichment
diets were prepared daily by homogenizing ingredients suspended
in seawater.
Experimental Aquaria
Three-hundred phyllosomata aquarium"' were grown from
newly-hatched to stage V in 3-L plastic static aquaria on three diet
treatments; each treatment was conducted in triplicate. The diet
treatments consisted of:
(1) Anemia enriched with Al DHA Selco and Anemia en-
riched with C. muelleri (1:2 v/v).
(2) Anemia enriched with the Ethyl ester-mussel nutrient
source (as described above).
(3) Mussel powder-polar lipid feed station diet [Greenshell
mussel powder-Greenshell mussel polar lipid-lyprinol
(from Greenshell mussel) (NIWA Research, Auckland,
New Zealand)-sodium alginate (81:10:5:4 by mass)] af-
fixed to 8 X 17 cm meshes (bird netting) with 10% CaCK
solution.
The water in aquaria was changed daily. After recording any
molts/mortalities, the contents of each aquarium were poured
through a l.000-|xm screen, retaining the phyllosomata while the
uneaten feed and debris went to waste. The aquaria were cleaned,
refilled with seawater, and larvae were washed back in. Phylloso-
mata were provided with new diets once daily in the afternoon.
Anemia were fed to phyllosomata at a rate of 3 Anemia niL"'.
Oxytetracycline was added to the water at 20 mg L"' daily. After
each molt, all animals were counted and 10 phyllosomata
aquarium"' were measured for total length, carapace length and
carapace width utilizing a dissecting microscope, digital camera
and Scion Image Beta 4.0.2 software (Scion Corporation, Freder-
ick, MD).
Lipid Extraction
Anemia and phyllosomata samples were filtered through 4.7-
cm Whatman glass filters (GF/F) and rinsed with 0.5 M ammo-
nium formate. Sample numbers of phyllosoma taken for lipid
analyses were as follows: 400 newly hatched (sampled at start
before distribution of larvae to aquaria); from each aquarium 50
stage II. 35 stage III, 25 stage IV, and 15 stage V; all midstage.
Samples were lyophilized to determine dry mass and quantitatively
extracted overnight using a modified Bligh and Dyer (1959) one-
phase methanol:chloroforni:water extraction (2:1:0.8 v/v/v). The
phases were separated by the addition of chloroform: water (final
solvent ratio, 1:1:0.9 v/v/v methanol:chloroform:water). The total
solvent extract was concentrated using rotary evaporation at 40°C.
Lipid Classes
An aliquot of the total solvent extract was analyzed using an
latroscan MK V THIO thin-layer chromatography-flame-
ioni/ation detector (TLC-FID) analyzer (Tokyo. Japan) to quantify
indi\idual lipid classes (Volknian & Nichols 1991 ). Samples were
applied in duplicate to silica gel SIII chromarods (5-|xm particle
size) using 1-p.L micropipettes. Chromarods were developed in a
glass tank lined with pre-extracted filter paper. The primary sol-
vent system used for the lipid separation was hexane:diethyl ether:
acetic acid (60:17:0.1), a mobile phase resolving nonpolar com-
pounds such as wax ester (WE). TAG, free fatty acids (FFA) and
sterols (ST). A second nonpolar solvent system of hexane:diethyl
ether (96:4) was also used to resolve hydrocarbons, WE, TAG. and
diacylglyceryl ether (DAGE). After development, the chromarods
were oven dried and analyzed immediately to minimize absorption
of atmospheric contaminants. The FID was calibrated for each
compound class (phosphatidylcholine, cholesterol, cholesteryl ole-
ate, oleic acid, squalene, TAG [derived from fish oil[, WE [derived
from orange roughy oil], and DAGE [derived from shark liver oil];
0.1-10 |jig range). Peaks were quantified on an IBM-compatible
computer using DAPA Scientific software (Kalamunda, Western
Australia). TLC-FID results are generally reproducible to ±5-10%
of individual class abundances (Volkman & Nichols 1991).
Fatty Acids
An aliquot of the total lipid was /)i:7«,s-methylated to produce
fatty acid methyl esters (FAME) using methanol:chloroform:conc.
hydrochloric acid (10:1:1, 80°C, 2 h). FAME were extracted into
hexane:chloroform (4:1, 3 x 1.5 ml) and treated with /V.O-bis-
(trimethylsilyl)-trifluoroacetamide (BSTFA 50 p.L. 70'-C, over-
night) to convert ST and alcohols to their corresponding TMSi
ethers.
Gas chromatographic (GO analyses were performed with a
Hewlett Packard 5890A GC (Avondale, PA) equipped with an
HP-5 cross-linked methyl silicone fused silica capillary column
(50 m X 0.32 mm i.d.). an FID, a split/splilless injector, and an HP
7673A auto sampler. Helium was the cartier gas. After addition of
methyl nonodecanoate and methyl tricosanoate internal injection
standards, samples were injected in splitless mode at an oven
temperature of 50''C. After 1 min, the oven temperature was raised
to 150°C at 3()X min ', then to 250'C at 2°C min"', and finally
to 300°C at 5°C min '. Peaks were quantified with Waters Mil-
lennium software (Milford, MA). Individual components were
identified using mass spectral data and by comparing retention
time data with those obtained for authentic and laboratory stan-
dards. GC results are subject to an error of ±5% of individual
Feeding Soui-hbrn Rock Lobster in Culture
227
cnmpiment area. GC-mass speL-trometric (GC-MS) analyses were
pertoniied on a Fiiinijjan Thermoquest GCQ GC-mass spectrom-
eter (Austin. TX) fitted with an on-column injector. The GC was
fitted with a capillar)' column siinilar to that described above.
RESULTS
Miiri>liiiiiiilncs
The increase in total length and mass in phyllcsomata was
similar between the Iwo Artemia diet treatments, with a good fit of
exponential trend lines observed (R' > 0.999; Fig. 1 ). Phylloso-
mata fed the A 1 DHA Selco-C mitellch-Artcmia diet treatment
showed a greater increase in total length (2.1 to 6.1 mm) and mass
per individual (0.1 to 1.5 ing dry mass) from stages 1 to V than did
larvae fed ethyl ester-mussel-enriched Arteinia (total length: 5.9
mm; mass per individual: 1.2 mg dry mass; Fig. 1). Percentage
survival was >68% between each stage and was highest from
stages II-IIl (92-98'^f-: Table 1 ). Total survival to stage V was high
for animals fed Anemia enriched with either the Al DHA Selco-
C. miwllcii (57%) or ethyl ester-mussel (42%) nutrient sources.
There were no differences in intermolt period for Artemia-itd
phyllosomata among treatments. Intermolt periods were 9. 11, 12,
TABI.K 1.
Intermoll period (days) and pfrci'nlayt survival of phyllosomata fed
different diets.
Diet
Al
Mussel
Selco-C.
Ethyl
Powder-Polar
muellerf'
Ester-Mussel"
Lipid'
Survival''
I-II
81.8 ±4.8
76.2 ± 15.9
-
II-III
97.5 ± 2.2
92.3 ± 3.5
93.5 + 7.2
III-IV
87.3 ± 6.0
86.0 ± 6.7
5 1 .3 ± 3.6
IV-V
81.1 ± 14.5
68.3 ± 6.8
-
Total
56.5 ± 12.6
41.5+ 11.2
-
Intermolt
period
I-II
9
9
-
II-III
II
11
10
III-IV
12
12
-
IV-V
1.^
15
-
" Presented as mean
" Enriched Anemia.
■^ Feed station.
± SD: It = 3
L5 -
1 -
0.5
» Al DHA Selco-C. muelleri
O Ethyl ester-mussel
= 0.0884e"
y = 0.I101e"
R^ = 0.9998
St II
Total Length (mm)
Fijjure I. Dry mass as a function of total length of y. edwardsii phyllosomata from stages I to V on two diet treatments of Artemia enriched with
either A I DHA Selco-C muelleri or ethyl ester-mussel nutrient sources. Presented as mean ± SD: filled with exponential trend lines.
228
Nelson et al.
and 13-15 days to commencement of molt for stages 1-11. 11-111.
III-IV. and IV-V, respectively.
Phyllosomata fed the Mussel powder-polar lipid diet failed to
molt to stage II on the feed station diet alone. Two animals re-
mained alive at stage I for 30 days, at which time they were put on
the Ethyl ester-miissel-enriched Anemia diet. At day 41 they suc-
cessfully molted to stage 11. and were sampled at day 36. After
sampling stage II animals at day 15. phyllosomata fed A I DHA
Selco-C. »i»e//cn'-enriched Artemiu were divided and half were
put on the Mussel powder-polar lipid diet. After 10 days, these
animals molted to stage 111. were sampled at day 30, but failed to
molt to stage IV. After sampling stage IV animals at day 37.
phyllosomata fed ethyl ester-mussel-enriched Anemia were di-
vided and half were put on the Mussel powder-polar lipid diet.
They did not molt to stage V. but were sampled concurrently with
Anemia-fed phyllosomata at day 56.
Lipid Content and Classes
The two nutrient sources were lipid-rich with Al DHA Seico
higher than EE-mussel (960 and 410 mg g"' dry mass, respec-
tively; Table 2). Al DHA SeIco was dominated by TAG (88%)
and ethyl ester-mussel by EE (55%), with TAG the second most
abundant lipid class (28%). Lipid content of Anemia enriched with
A I DHA Selco-C. muellcri and ethyl ester-mussel was identical
(250 mg g ' dry mass). TAG was the major lipid class (46-51% of
total lipid), followed by PL (37-40%). ST (5-6%), FFA (4-10%),
diacylglycerol (DG; 0.7-1.9%). and WE (0.1-0.3%) were minor
components.
In Anemia-fed phyllosomata. although lipid per individual gen-
erally increased from newly hatched to stage V (8 to 180 |a.g), the
absolute lipid content remained generally constant in Anemia-fed
phyllosomata from newly hatched to stage V (Table 2). Total lipid
was 155 mg g"' dry mass in newly hatched phyllosoma and in-
creased slightly from stage 1 to stage II ( 207 and 1 73 mg g ' for A I
DHA Selco-C. miielleri and ethyl ester-mussel Anemia-fed phyl-
losomata, respectively) and to stage IV ( 157 and 176 mg g"' ). By
stage V. total lipid decreased to the starting (newly hatched) value
in ethyl ester-mussel Anemia-fed phyllosomata ( 156 mg g ' ) and
was slightly lower in animals fed Al DHA Selco-C. miielleri-
enriched Anemia ( 128 mg g ' ). PL comprised the major lipid class
in all phyllosoma samples (73-87% of total lipid), followed by ST
(4-8%; mainly cholesterol), FFA (2-8%). DG (2-i%), and WE
(0-0.5%). Minor TAG was detected (0-0.4%).
Stage IV phyllosomata fed the Mussel powder-polar lipid diet
were similar to those fed either A/7e)?!(a diet, with 156 mg g"' dry
mass of lipid and PL the dominant lipid class (83%; Table 2). ST
were comparatively higher (10%). Compared with other phyllo-
soma, PL (73%) and DG (1%) were lower in stage 111 Mussel
powder-polar lipid feed station-fed animals, with a proportionate
TABLE 2.
Percentage lipid class composition of nutrient sources, enriched Artemiu. feed station, and phyllosomata.
Free
Wax Ethyl Fatty
Ester Ester Triacylglycerol Acid Diacylglycerol
Sterol
Lipid
Lipid as
Mass
nigg"'
Indiv'
Polar
Drv
Dry
Lipid
Mass
Mass (pg)
Nutrient souces
Al DHA SeIco
Ethyl ester-mussel
Anemia
Al DHA Selco-C.
muelleii
Ethyl ester-mussel
Feed station
Mussel powder-polar
lipid
Phyllosomata
Newly hatched
Al DHA Selco-C.
inuelletT^
II
IV
V
Elhyl ester-mussef
II
IV
V
Mussel powder-polar
lipid*'
III
IV
0.0 + 0.0
0.7 ±0.0 .S?.! ±."1.8
U. 1 ± 0.0
0.3 ± 0.2
2.S±0.1
l.7±0.1
0.3 ± 0.0
0.3 ± 0. 1
O.-'S ± 0.3
0..'i±().l
2.7 ±0.2
1.0 ±0.3
88.2 ±0.8 1.6 ±0.1 - 0.8 ±0.0 9.5 ± 0.9 958.5 ±18.7
27.9±0.7 5.4±1.6 0.1 ±0.0 1.4±0.4 9.3±3.1 411.9±44.2
45.9±().4 10.0±0.2 l.y±().3 .';.6±0.0 .m5±1.0 2.';9.7±12.8
50.5 + 0.2 3.5 ±0.5 0.7 ±0.1 5.2 ±0.0 39.7 + (19 2-'i4.2 + 0.3
12.5±0.3 26.2±0.1 0.4±().l 3.5±0.3 54.6±0.9 191.0±11.4
0.2±0.1 9.9±0.1 - 9.9±0.1 78.3±0.1 l.M.6±9.4 8.0+1.1
5.5 ±5.0 1.5 ±0.4 4.3 ±1.7 88.7 ± 6.8 207.5 ±19.6
0.2 ±0.1 4.7 + 0.6 3.9 + 0.7 6.9 ± 0.2 84.1 ± 1.4 157.2 ±13.9
0.3 ±0.1 2.1+0.2 3.0 ±0.3 7.1 ±0.6 87.2 + 0.9 127.8 + 9..S
8.1 ±3.4 1.5 ±0.0 5.5 ±0.4 84.8 ± 3.7 173.0 ±17.5
0.2 ±0.1 4.9 + 0.8 3.0 + 0.4 6.3 ± 0.4 85.1 ±1.1 175.6+18.4
0.4±0.1 2.3±0.5 3.2±().2 7.5 ± 0.4 86.1 ±1.3 l.S6.(l±19.3
1.1 ±0.1 9.2 ±1.2 0.8 ±0.3 12.8 ±0.8 73.4 ±0.9 .S4.2 ± 7.5
0.5±0.3 2.7 + 0.3 2.2±0.9 10.1 ±1.9 83.4±2.5 155.5 + 86.4
72.7 + 6.1
LS6.0±8.0
188.9 ±25.2
61.3 ± 13.3
137.3 ±8.3
180.0± 13.3
46.7 ± 10.8
133.3 ±57.0
Presented as mean ± SD; /( = 3; (-), below detection.
" Enriched Anemia.
'' Feed station (molted from staae 11 lo 111 unlv).
Feeding Southern Rock Lobster in Culture
229
iiiL-rease in ST (L^'/M and WE {i9c). Lipid content was a third that
of other Artciiiia-t'eiS samples (54 mg g"' dry mass).
Fatly Acids
The FA ill the two nutrient sources differed markedl) (Table 3).
hi Al [)HA Selco. dominant FA in decreasing order of propor-
tional abundance of total FA were: palmitic acid ( 1 6:0; 1 79r ). EPA
(159^). oleic acid [18:l(n-9)c; 14%]. palmitoleic 1 16:l(n-7)c. 97r],
DHA (8%). myristic acid (14:0; 7%) and linoleic acid |18:2(n-6):
5'/f]. The ethyl ester-mussel nutrient source was doniinated by
PUFA (75%). with major FA as DHA (37%). AA (13%). EPA
(12%) and 16:0 (7%).
The major FAs in enriched Anemia were as follows: I S: 1 ( n-9 )c
(32-36%). l8:2(n-6) (23-27%). 16:0 (9-1 I7f). m-vaccenic acid
|18:l(n-7)c: 4%|. stearic acid (18:0: 4%). and 16:l(n-7)c (2-4%:
Table 3). Anemia enriched with ethyl ester-mussel had higher
essential PUFA (3% AA. 6% EPA. 7% DHA) than those enriched
with Al DHA Selco-C. miielleri (1% AA. 2% EPA, 1% DHA).
The Mussel powder-polar lipid diet was dominated by 16:0 ( 19%).
EPA ( 14%). and DHA (14% ). with A A at 3% of total FA. Com-
pared with Anemia, levels in the Mussel powder-polar lipid diet of
18:0 fatty aldehyde (6%), 20:l(n-9)c (4%) and minor C„ PUFA
(3%) were elevated, and levels of 18:l(n-9)c (3%) and 18:2(n-6)
(2%) were lower.
In ,4)7<7/;/(/-fed phyllosomata, the major FA were similar to
those found m the enriched Anemia and in decreasing order of
abundance were: 18:l(n-9)c (23-27% of total FA). 18:2(n-6) ( 17-
22%). 16:0 (9-11%). 18:0 (7-9%). EPA (7-11%). 18:l(n-7)c (4-
6%). DHA (4-6%). and AA (2-5%%: Table 4). These phylloso-
mata experienced a decrease in essential PUFA, on both a relative
(Table 4) and absolute basis (Fig. 2). from newly hatched to stage
V. In phyllosomata fed ethyl ester-mussel-enriched y4rfe/»(rt, there
was a concurrent drop in levels of AA (5^%. 4 to 3 mg g"' dry
mass). EPA (21-9%. 14 to 6 mg g" ' ). and DHA ( 14-5%. 9 to 3 mg
g''). with a similar, although more pronounced, decrease in ani-
mals fed Al DHA Selco-C. mnelleri-enrkhed Anemia (AA: 3%,
2 mg g ' dry mass; EPA: 8%. 5 mg g"'; DHA: 4%, 2 mg g~').
Conversely, levels increased in 18:l(n-9)c (8 to 23-24%) and 18:
2(n-6) (1-18%). The FA profile of animals fed the Mussel pow-
der-polar lipid diet closely reflected the diet, being dominated by
16:0 (12-15%). EPA (8-14%). DHA (6-9%). and AA (5-6%).
Compared with Anemiu-fcd phyllosomata. levels in the Mussel
powder-polar lipid-fed animals of 18:l(n-9)c (9-18%) and 18:2(n-
6) (5-13%) were lower, while in stages 111 and IV Mussel powder-
polar lipid-fed animals, levels of 20:2(n-6) (2-3%) and 22:l(n-9)
(3^%) were higher.
DISCUSSION
A major feature of previous Australian feeding trials with
southern rock lobster phyllosoma has been comparatively poor
survival. This trial, however, represents a turning point in Austra-
lian rock lobster phyllosomata nutritional research, with greater
than 80% survival of Anemia-fed phyllosomata through each stage
from newly hatched to stage V. We believe that a primary differ-
TABLE 3.
Percentage fatty acid composition of nutrient sources, enriched Artemia. and feed station.
Nutrient Sources
Artemia
Feed Station
Al DHA Selco
Ethyl Ester-Mussel
Al DHA Selco-C. muelleri
Ethyl Ester-Mussel
Mussel Powder-Polar Lipid
14:0
6.9 ±0.2
0.7 ±0.1
1.3 ±0.1
0.5 ± 0.0
3.7 ± 0.3
16:l(n-7lc
8.8 ± 1.0
0.8 ±0.1
3.9 ±0.1
2.0 ± 0.0
5.3 ± 0.4
16:0
16.6 + 0.0
7.2 ±0.8
11.0 ±0.2
9. 1 ± 0. 1
18.5 ± 1.2
lS:4(n-3)
2.7 ±0.0
1.5 ±0.0
0.2 ± 0.0
0.3 ±0.0
2.1 ±0.1
lS:2(n-6)
5.1 ±0.2
3.6 ± 0.3
27.2 ± 0.3
22.9 ± 0.3
1.9 ±0.1
lS:l(n-9)c/18:3(n-3)
14.1 ±0.8
3.5 + 0.7
35.8 ± 0.2
31.8 ±0.6
2.6 ±0.1
18:l(n-7)c
3.0 ±0.1
0.5 ± 0.0
4.1 ±0.0
3.7 ±0.1
2.6 ±0.1
18:0
3.5 ± 0.2
3.4 ± 0.3
4.2 ± 0.0
4.4 ±0.1
5.1 ±0.0
18:1 Falde
0.1 ±0.0
-
-
0.7 ±0.8
6.4 ±0.1
20:4(n-6)
0,9 ±0.0
1.^.2 ±0,6
0.5 ± 0.0
3.4 ±0,0
2.7 ±0.1
20:-'i(n-3)
14.9 ±0.6
12.3 ±0.3
2.4 ± 0.3
6.3 ± 0.0
13.9 ±0.4
2():4(n-3)
0.1 ±0.0
0.1 ±0.0
0.9 + 0.0
1 ,4 ± 0. 1
2.1 ±0.1
20:l(n-9)c
0.4 ± 0.4
0.4 ± 0.5
0.0 ± 0.0
0.8 ±0.1
3.6 ± 0.2
22:6(n-3)
7.9 ± 0.3
37.1 ±0.7
0.8 ±0.1
7.0 ±0.6
13.5 ±0.9
C,, PUFA
-
0.7 ±0.0
-
-
2.9 ±0.3
Ottier
14.8
15.0
7.6
5.7
13.0
Sum SFA
31.8 + 0.2
15.9 ±0.4
18.9 ±0.3
16.5 ±0.1
31.6± 1.6
Sum MUFA
30.7 ±1.5
8.5 ± 1.2
47.0 ± 0.6
40.8 + 0.1
18.1 ±0.3
Sum PUFA
37.4 ±1.3
74.7 ± 0.2
33.7 + 0.6
42.4 ± 0.5
43.1 + 1.8
Sum (n-3l
28.3 ± 1.0
52.9 ±0.9
3.7 ± 0.3
14.0 ±0.7
32.8 ± 1.5
Sum 01-6)
7.0 ±0.1
20.6 ± 1.0
28.2 ± 0.5
27.5 + 0.5
6.5 ± 0. 1
Ratio (n-3)/(n-6l
4.1
2.6
0.1
0.5
5.0
Ratio EPA/AA
15.8
0.9
5.2
1.9
5.1
Ratio DHA/EPA
0.5
3.0
0.3
1.1
1.0
Presented as mean ± SD: n = 3; (-). helow detection. AA, arachidonic acid; EPA. eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated
fatty acids; MUFA, monounsaturated fatty acids; PUFA. polyunsaturated fatty acids; Other includes components pre.sent at <2%: il5:0. al5:0. 15:0. il6:0.
C|„ PUFA, I6:l(n-9)c, I6:l(n-7)t/16;2. 16:l(n-5)c. 16:0 Falde (fatty aldehyde), 16:1 Falde, 117:0, al7:0, 17:1. 17:0, 18;3(n-6), il8:0, 18:l(n-7)t,
I8;l(n-5)c, 18:0 Falde, il9:0. 19:1. 20;3(n-6), 20;2(n-6), 20:l(n-ll)c. 20:l(n-9)c. 20:l(n-7)c, 20:0. C,, PUFA. 21:0. 22:5(n-6). 22;4(n-6). 22:5(n-3).
22:l(n-lll. 22:lin-7), 22:0. 24:1. 24:0,
230
Nelson et al.
TABLE 4.
Percentage fatty acid composition of phyllosomata from feeding trial.
Diet
Al DHA Selco-C n
iielleri"
Kthyl Ester-Mussel"
Mussel Power-Polar Lipid''
Newly
Hatched
II
IV
V
II
IV
V
11
III
IV
16;l(n-7)c
4.1 +0.2
2.7 ±0.1
1 .8 + 0.0
3.1 ±0.2
1.6 ± 0.0
0.9 + 0.0
1.6 ±0.2
2,1
2.1 ±0.4
0.9 + 0.0
16:0
12.2 ±0.4
10.7 ±0.5
10.0 ±0.0
11.3 ±0.1
10.4 ±0.0
9.0 ± 0.2
10.1 ±0.3
15,4
15.1 ±2,2
12.2± 1.0
17:0
1 .6 ± 0.0
0.8 ±0.0
0.8 + 0.0
0.9 ± 0.0
0.8 + 0,0
0.6 ± 0.0
0.7 ±0.1
4.6
1,3 + 0.2
0.9 ± 0. 1
18:2(n-6)
0.7 + 0.0
17.4 + 0.4
21.7 + 0.2
18.3 ±0.3
17.1 ±0.1
20. 1 + 0.0
18.3 ±0.3
9.7
5.4 ± 0.8
13.0 ±2.4
18:l(n-9)c/18:3(n-3)
8.1 ±0.2
26.5 ± 0.4
27.4 ±0.2
23.5 ± 0.3
24.8 ± 0.3
24.9 ± 0. 1
22.7 ±0.5
14.5
9.4 ± 1.8
17.6 ±3.4
18:l(n-7)c
4.9 ±0.1
5.9 ±0.1
4.7 + 0.0
5.8 ±0.1
5.6 ±0,1
4.0 ± 0.0
4.5 ±0.1
3.9
4.1+1 .0
3.9 ± 0.5
18:0
8.3 ±0.0
7.4 ±0.1
7.2 ±0.1
8.5 + 0.2
8.3+0.1
7.5 ±0.1
8.7 ±0.1
13.3
10.9+ 1.2
1 1 .6 ± 1 .0
20:4(n-6)
5.1+0.1
2.4 + 0.1
2.0 ±0.0
2.6 ± 0. 1
4.7 ±0.2
4.3 ±0.1
4.3 ± 0. 1
6.1
5.2 ± 0.8
5.4 ± 0.3
20:5(n-3)
21.0±0.2
10.1 ±0.4
6.9 ±0.1
8.2 ±0.2
10.8±0.1
8.8 ±0.3
8.6 ±0.2
10.2
14.1 ±2.4
8.7 ± 0.5
20:2(n-6)
1.7 ±0.0
0.0 ± 0.0
-
1.5 ± 1.3
0.0 ± 0.0
0.8 ± 1.5
1.8 ± 1.6
-
2.6 ±0.2
2.1 + 1.8
20:Un-ll)c
0.3 ± 0.0
1 .7 ± 0. 1
2.3 ± 0.0
0.8 ± 1.3
1 .8 ± 0.0
1.7 ± 1.5
0.8 ± 1.4
-
-
1 . 1 ± 1 .9
22;6(n-3)
13.5 + 0.3
5.3 ± 0.3
4.0 ±0.1
3.5 + 0.1
5.4 ±0.4
6.3 ± 0.2
4.9 ± 0.2
5.6
8.6 ± 1.5
5.9 ± 0.2
22:l(n-9)
0.8 ± 0. 1
0.1 ±0.0
1.6 ±0.2
1.8 ±0.3
0. 1 ± 0.0
1.7 ±0.2
1.9 ±0.1
0.5
3.9 ± 2.6
3.2+ 1.8
Other
17.5
9.0
9.7
10.3
8.6
9.2
10.8
14.0
17.1
13.4
Sum SFA
27.6 + 0.3
22.2 ± 0.3
21.1 ±0.2
24.6 ± 0.4
22.8 + 0.1
20.1 ±0.3
23.7 ± 0.9
35.6
.34.8 ± 5.0
29.9 ± 3.4
Sum MUFA
23.8 ± 0.3
40.2 ± 0.4
40.8 ±0.1
38.0 ± 1.0
37.0 ± 0.4
35.8 ± 1.4
34.4 ± 2.0
24.8
23. 1 ± 0.7
29.8 ± 2.0
Sum PUPA
44.4 ± 0.7
37.3 ± 0.7
37.9 ±0.2
35.7 ± 0.3
39.8 ± 0.6
43.0 ± 0.4
39.8 ±0.8
39.6
37.6 ±4.8
37.5 ± 1.6
Sum (n-3)
36.6 + 0.5
16.5 + 0.8
11.8 + 0.2
12.4 ±0.1
16.9 + 0.5
15.9 + 0.5
14.3 ±0.3
18.3
24.0 ± 4.0
15.6 ±0.3
Sum (n-6)
8.5 ±0.1
20.4 ± 0.3
25.4 ± 0.3
24.1 + 1.4
22.5 ±0.1
27.2+ 1.6
26.8 ± 1.9
21.3
15.2 ± 1.3
23.2 ± 2.6
Ratio (n-3)/(n-6)
4.3
0.8
0.5
0.5
0.8
0.6
0.5
0.9
1.6
0.7
Ratio EPA/AA
4.1
4.3
3.5
3.1
2.3
2.0
2.0
1.7
2.7
1.6
Ratio DHA/EPA
0.6
0.5
0.6
0.4
0.5
0.7
0.6
0.6
0.6
0.7
Presented as mean ±
SD. /) = 3.
" Enriched Artemia.
^ Feed station (molted from stage II to III only).
'/! = 1.
(-). below detection.
AA. arachidonic acid; EPA. eicosapentaenoic acid: DHA. docosahexaenoic acid: SFA. saturated fatty acids: MLTA. monounsaturated fatty acids: PUFA,
polyunsaturated fatty acids: (-), below detection: Other includes components present at <2'7c: 14:0, iI5:0, al5:0. 15:0. il6:0. C,,, PUFA. 16:l(n-9)c.
16:l(n-7)t/16:2, 16:l(n-5)c. 16:0 Falde (fatty aldehyde) il7:0, al7:0. 17:1. 17:0. 18:3(n-6). 18:4(n-3). il8:0, I8:l(n-7)t. 18:l(n-5)c. 18:0 Falde. i 19:0. 19:1.
20:3(n-6l. 20:4(n-3). 20:l(n-lllc. 20:l(n-7)c. C,, PUFA. 2 22:4(n-6). 22:5(n-3). 22:5(n-6), 22:l(n-n). 22:l(n-7). 24:1. 24:0.
ence between this and the majority of previous trials has been the
daily use of antibiotics in static culture. Although static culture and
antibiotics are less appropriate for medium to large-scale culture of
phyllosomata (Ritar 2001 ). they have been used in raising phyllo-
somata to pueruli (Matsuda &. Yamakawa 2000). Additionally, the
growth results from this trial, although similar to a previous trial,
had much tighter standard deviations (Nelson et al. 2003). This
suggests that larvae from this trial had more similar environmental
parameters resulting from both aquarium design and use of anti-
biotics. The successful use of antibiotics in static culture in this
experiment highlights the fact that because the vital aspects of
phyllosomata culture (i.e.. feeding capabilities, nutritional require-
ments, aquariutn design and microbial loading) are intrinsically
linked, advances cannot be readily made sequentially, and should
ideally be performed concurrently. In previous trials, it has been
difficult to test the effectiveness of feeding phyllosomata on Ar-
temia. including using different enrichments, when experiments
may be confounded by the adverse effects of microbial loading and
aquarium design. The higher survival and good growth in this trial
sugge.st that enriched Artemia may be adequate for early stage
phyllosomata.
The dominance of TAG in enriched Artemia illustrates the
propensity for readily incorporating TAG from nutrient sources, as
well as metabolizing EE to TAG for assimilation into their tissues.
Similar results were observed when providing Artemia with a
high-PL diet (Nelson, unpublished). These lipid class results are
comparable to previous trials using 5-day old Artemia (Nelson et
al. 2002b, Smith el al. 2002, Nelson et al. 2003).
A distinction between this trial and an earlier trial (Nelson et al.
2003) is the detection of TAG in phyllosomata, albeit at low
amounts and the higher relative proportion of DG. Although the
difference is small, the presence of these short-term energy storage
molecules is consistent with unproved larval health. In a prior
feeding trial, total lipid content dropped markedly in phyllosoma to
below 100 mg g"' by stage IV, and was also accompanied by
poorer survival (Nelson et al. 2003). This result contributed to the
hypothesis that phyllosoma, like puerulus (Jeffs et al. 2001 ), may
be better served by use of PL, rather than TAG (Nichols et al.
2001, Nelson et al. 2003). Animals in the present trial did not
experience the same marked decrease in lipid content. This finding
may be the result of a number of reasons. First, if lipid is critical
to survival, a drop in total lipid is associated with the poorer
survival in previous trials. Maintenance of lipid at above 100 mg
g ' in the present trial may therefore be linked with good survival.
Second, because animals had high survival, but still did not have
total lipid equal to wild phyllosomata (250 mg g ' lipid dry tnass
Feeding Southern Rock Lobster in Culture
231
New hatch
IV
IV
Al DHA Selco-C muellen
Ethyl estei -mussel
Diet & Stage
Figure 2. Content (mg g') uf the essential long chain-polyunsaturated FA arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosa-
hexaenoic acid (DHA) in / edwardsii phyllosoniata from stages I to V on two diet treatments o( Artemia enriched with either Al DHA Selco-C.
miwlleri or cth>l ester-mussel nutrient sources. Presented as mean ± SD.
at stage V) (Phleger et al. 2001). the class of lipid provided (i.e..
TAG in feeding trial versus largely PL in wild) was less effective.
Thirdly, aquarium design and microbial loading can affect metabo-
lism of lipids in larvae. For example, in previous trials, conducted
in flow-through aquaria without antibiotics, and the present trial,
conducted in static aquaria with antibiotics, Artemia that were
similarly enriched with DHA Selco-C. nnielleri were fed to phyl-
losomata. Animals from the present trial had 189 mg g"' lipid dry
mass al stage V. while animals in the previous trial had 50 mg g '
lipid dry mass at stage V (Nelson et al. 2003). The animals in the
previous trial either did not store lipid, or used more lipid as
energy, while under the strain of microbes and/or swimming. Al-
though Artemia supported excellent survival for larval stages I-V
in the present trial, the Artemia diet may still not sufficiently
condition phyllosomata for later stages; Artemia may not be pro-
viding adequate total lipid for growth and high survival, especially
if the aspects of aquarium design and microbial loading are not
addressed.
The current emphasis in phyllosoma culture in Australia is the
use of Artemia for feeding stages I-V. This concept stems from
developments with other aquaculture species, such as marine fin-
fish, where it has been impossible to grow them during the early
part of their life cycle without using live, motile feed (Olsen 1997.
Castell et al. 1998). With rock lobster, complete rearing of phyl-
losoma to puerulus was achieved by feeding on Artemia. fish
larvae and/or iriussel tissue (Kittaka 1997b. a. Kittaka & Abrun-
hosa 1997. Matsuda & Yamakawa 2000). Mussel gonad has been
identified as the key to this success (Kittaka 1997b), used exclu-
sively after the third instar (Kittaka 1997a). In culture, phylloso-
mata have been observed ingesting inanimate food particles, such
as lobster, prawn and mussel pieces at late stages (Thomas, un-
published). Early stage animals have likewise been observed con-
suming pieces of mussel, jellyfish and other inanimate foods
(Mitchell 1971. Nelson et al. 2002a, Cox & Johnston 2003). Phyl-
losomata in the present study were no exception. The larvae were
observed consuming the Mussel powder-polar lipid feed station
diet, a diet with which we attempted to build on the success of
using mussel gonad. Additional evidence of feeding was the pres-
ence of faecal trails, and molting, considering that phyllosomata do
not molt when not feeding (Abrunhosa & Kittaka 1997). Never-
theless, since phyllosomata fed the Mussel powder-polar lipid diet
failed to molt properly beyond more than one stage, there is per-
haps a necessary component either not present in sufficient
amounts, or lost by leaching, in the feed station diet that contrib-
utes to molting. Therefore, the use of Artemia up to the third instar
(Kittaka 1997a) remains valuable for phyllosomata. However, to
improve conditioning of larvae, the potential use of co-feeding of
Artemia (Dhert et al. 1999), along with a PL source, should be
examined, particularly for later stage animals.
Of note is the decrease in essential PUFA from newly hatched
to stage V phyllosomata. On a relative basis, Artemia-fed phyllo-
somata and wild-caught animals at stage V had similar levels of
232
Nelson et al.
AA (trial. ?,-i'7r: wild. 2-3%) and EPA (trial. 8-9%; wild, 7-9%),
with markedly lower DHA in cultured animals (trial, 4-5%: wild.
16-17%) (Phleger et al. 2001). Results from a previous feeding
trial are similar for relative levels of these FA (3-6% AA: 8-9%
EPA; 2^% DHA) (Nelson et al. 2003). However, because the
larval lipid remained above 189 nig g ' lipid dry mass at stage V.
on an absolute basis this trial represents a marked improvement for
incorporation of essential PUFA. The fact that the amount of total
lipid remained the same to stage V. but there was a drop in the
level of essential PUFA. in particular DHA. highlights the impor-
tance of these FA. Higher absolute concentrations of these FA may
be associated with enhanced survival and growth in this feeding
trial compared with previous trials (Nelson et al. 2003, Hart et al.,
unpublished). Total lipid and levels of essential PUFA in phyllo-
somata fed ethyl ester-mussel-enriched Anemia were higher than
in larvae fed A I DHA Selco-C. nuielleri-emiched Artemia. Be-
cause there was no direct association of enhanced FA profiles with
survival and growth for phyllosomata from the two Artemia diet
treatments, and the majority of lipid provided to phyllosomata
through enriched Anemia was TAG. we propose that the impro\ed
survival and growth in this trial may result from the presence of
lipid in the diet (as described above) in combination with better
health. Furthermore, we suggest it is likely that the PUFA profiles
will have a more significant effect if provided in a PL form. These
results also support the suggestion that co-feeding ai Anemia and
a PL source should be trialed to improve larval condition.
In conclusion, the use of antibiotics and static culture has en-
abled a clearer picture of the effects of nutrition on larval health.
Our experiment demonstrated that lipid-enriched Anemia support
excellent growth and survival in early stages of phyllosomata. and
we are now better placed to take nutrition of phyllosonia forward.
The results suggest that the class of lipid provided \\\i Anemia may
not adequately condition larvae, nor supply sufficient quantities of
the essential PUFA. in particular DHA. for later stages. Thus, for
successful culture of phyllosomata. the development of a formu-
lated diet, which can provide the nutritional requirements in the
right form to enhance long term conditioning of the larvae, is likely
to be vital, particulariy for later stage larvae.
ACKNOWLEDGMENTS
We are extremely grateful to B. D. Mooney. G. G. Smith. A. J.
Ritar. and C. W. Thomas for their invaluable expertise and assis-
tance during the experiment. The Greenshell mussel products
(powder, polar lipid and lyprinol) were kindly provided by Dr. A.
G. Jeffs. NIWA Research. Auckland. New Zealand. D. Hold-
sworth and B. D. Mooney managed the CSIRO GC-MS and GC
facility. M. M. Nelson gratefully acknowledges a University of
Tasmania Thomas A. Crawford Memorial Scholarship. This work
was supported in part by the FRDC RLEAS Subprogram (2000/
214) and FRDC project 1999/331.
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Journal of Shellfish Ken'cinh. Vol. 22, No. I, 235-239, 2003.
THE RELATIONSHIP BETWEEN HEMOLYMPH CHEMISTRY AND MOULT INCREMENT
FOR THE SOUTHERN ROCK LOBSTER, JASUS EDWARDSII HUTTON
R. J. B. MUSGROVE'* AND P. J. BABIDGE"
^SARDl Aquatic Sciences. P.O. Box 120. Henley Beach.
GPO Box 397. .Adelaide. SA 5001. Aiisrralia
SA 5044. Australia and -SARDl Biochemistry.
ABSTRACT Growlh data are essential to rock lobster fisheries stock assessment. At present, predictions of growth tor a given year
are based on data from previous years with the accuracy of the estimates being unknown until measures of growth are obtained in the
year in question. This article tests the hypothesis that premoult hemolymph lipid concentration is a predictor of moult increment for
the southern rock lobster. Jasus edwardsii. in the laboratory. The study was undertaken to develop a nonlethal means of moult
increment prediction, which could then be used in the field. Premoult carapace length had no effect on percent moult increment (P >
0.05) in the laboratory. Both phospholipid and triglyceride were significantly correlated with percent moult increment. Phospholipid
showed the highest coefficient at r^ = 0.66. Our data suggest that hemolymph phospholipid level has the potential to predict moult
increment. However, the hemolymph lipid/moult increment data were gathered over a short time period and within a relatively
controlled en\ironment. Further field studies are essential to better understand the relationship between hemolymph lipid level and
moult increment in w ild populations of this species.
KEY WORDS: Jasus ednardsii. lipid, moult increment, moulting, growth
INTRODUCTION
Growth data are essential to rock lobster fisheries stock assess-
ment. At present, predictions of growth for a given year are based
on data from previous years with the accuracy of estimates being
unknown until measures of actual growth are obtained for the year
in question.
The shedding of all hard parts at ecdysis complicates measure-
ments of growth in rock lobsters and other crustaceans. No struc-
tures are retained (sensu fish otoliths) from which age at size, and
therefore growth information, may be gathered. During recent
work in South Africa, Cockcroft (1997) suggested that growth may
be estimated from hepatopancrealic lipid level having found a
significant relationship between moult increment and percent
hepatopancreatic lipid during premoult in Jasus kdandii. The find-
ing of this relationship was a significant advance, although it was
still necessary to kill the animal to gather the data, a step that tnight
be avoided by isolation of an equally useful hemolymph compo-
nent. During a recent study. Musgrove (2001) found that
hemolymph protein, in combination with hemolymph pigment
level and moult stage, was useful in distinguishing between lob-
sters at high and low growth sites within the South Australian
fishery. He was able to show that grouping serum protein data by
pigment stage with reference to the major pigment, astaxanthin.
allowed the differentiation of lobsters at the beginning and those at
the end of intermoult. Given the correlation between serum protein
and 9^ dry weight, differences in lobster condition between high
and low growth sites could be examined more thoroughly using
this method. Hemolymph protein has been used successfully in
other studies as a measure of condition (Leavitt & Bayer 1977.
Musgrove 2001 ) but has not been shown to be useful in predicting
moult increment. Given Cockcroft's work, premoult hemolymph
lipid appeared to be the most likely to show a predictive relation-
ship with moult increment. If hemolymph lipid could be used in
place of total hepatopancreas lipid to predict moult increment, the
necessity to kill the lobster would be avoided and multiple samples
may be taken over time from the same individual.
*Corresponding author. E-mail: musgrove.richard@saugov.sa.gov.au
Phospholipids are the major circulating lipid and triglycerides
the major storage lipid in crustaceans. Both are found in the
hemolyinph and hepatopancreas (Chang & O'Connor 1983). The
hepatopancreatic lipid component of Jasus lalandii is largely tri-
glycerides (neutral lipids) with phospholipids (polar) of less im-
portance (<14<7f) Cockcroft (1997). In the hepatopancreas, in-
gested neutral lipids are cleaved to mono or diglycerides, which
are then converted to phospholipids. These are expelled into the
hemolymph and transported to various tissues, either for use as
membrane components or conversion to triglycerides and storage
(Chang & O'Connor 1983).
The hepatopancreas of rock lobsters increases in size and lipid
content through the moult cycle, reaching a maximum just before
ecdysis (Musgrove 2000b, Cockcrof, 1997) it may also be ex-
pected that other chemical compounds would show similar pat-
terns. Thus, as the hepatopancreas reached maximum storage dur-
ing late premoult (Musgrove 2001, Mercaldo-AUen 1991). so
hemolymph lipid would reach maximum concentration. Further-
more, as Cockcroft ( 1997) found that hepatopancreas lipid was an
indicator of moult increment in the field, so may hemolymph lipid
be, as phospholipid would be used for both the cell membranes of
the expanding hepatopancreas and, after conversion to triglyceride,
as the main lipid store.
This article tests the hypothesis that premoult hemolymph lipid
concentration is a predictor of moult increment for the southern
rock lobster. Jasus edwardsii. in the laboratory and examines the
relationship between hemolymph and hepatopancreatic lipid con-
tent and tissue weight. The study was undertaken to develop a
non-lethal means of moult increment prediction, which could then
be used in the field.
MATERIALS AND METHODS
iMhoratory Experiment I: Relalinnship Betneen Moull liieremeiil and
Premoult Hemolymph Level
Forty lobsters (mean CL: 89.88 ± 0.60 mm, mean weight 364.6
± 6.54g) were individually housed in 30-L plastic tanks in a flow-
through system (0.4 L/h/tank) for 185 days. Each tank was inde-
pendently supplied with air and water of a constant temperature
235
236
MUSGROVE AND BaBIDGE
(18°C, which was similar to the average summer temperature in
the area ot capture). Day length was set at 12 h and the lights
covered with red cellophane to minimize disturbance. Lobsters
were fed ad libinim daily on a mixed diet of artificial pellets (four
pellets/feed. Geddes et al. 2000) and cockles (four cockles/feed.
Donax deltoicles) in a rotation. Daily consumption was assessed by
eye from day 52 and categorized as 0. <257r. 25-50%. and >507f.
Excess food was removed and tanks cleaned each mornmg. taking
care to minimize disturbance to the lobsters.
Hemolymph samples (0.5 mL) were taken fortnightly from
each lobster by pericardial puncture for analysis of hemolymph
serum. Once the pigment stage of each hemolymph sample had
been noted (Musgrove 2001 ) it was snap-frozen (-196°C) for later
analysis. Pigment stage refers to the color of the hemolymph.
which changes from light blue through beige to deep orange during
the moult cycle, the beige becoming visible during intermoult
(Musgrove 2001). If the lobster was immediately premoult.
samples were taken before and after ecdysis. Pleopod samples
were also taken periodically to track moult stage by examination of
setal development (MusgroNC 2000).
Laboratory Experiment 2: Relationship Between Moult Increment and
Premoult Hemolymph Level in a Less-Controlled Environment
Seven premoult lobsters were selected from animals that had
been kept in an outside tank for several months with other species
(echinoderms. other decapods) and fed two to three times a week
on blue mussels (Mytilus sp.). The tanks were at ambient tempera-
ture (about 16°C) and contained abundant limestone rocks, Mac-
rocystis sp.. Ulva sp. and other aquatic macrophytes. The selected
lobsters were measured (range. 65.3 to 103.8 mm CL). moult
staged (after Musgrove 2000) and placed in plastic cages within
the aquaria. They were fed mussels ad libitum 3 to 4 times a week.
Pleopods were taken regularly to keep track of the moult stage and.
during late premoult (Stage D,) a 0.2-mL hemolymph sample was
taken, pigment staged, then snap frozen. Once each lobster had
hardened (i.e.. at intermoult) it was re-measured. The data were
then compared with those collected from laboratory experiment 1.
For experiments 1 and 2. blood was taken during the afternoon
to standardize postprandial effects on hemolymph lipid (sensii Dall
1981). Lobsters were fed after extraction was completed. In both
cases, lobsters were not observed to feed during daylight.
Field Study: Relationship Between Tissue Lipid and Hemolymph
Lipid Level
One hundred and thirty nine rock lobsters were collected from
the wild fishery as described by Musgrove (2001) and hemolymph
samples taken as described above within 3 h of capture, the pig-
ment stage noted and the sample snap-frozen (-196"C) for later
serum lipid analysis. A pleopod was also taken for moult stage
determination by examination of setal development (Musgrove
2000). The lobsters were then frozen (-30°C) and retained for
dissection and tissue analysis.
Within two weeks of collection, lobsters were rapidly thawed
and the abdominal tissue and hepatopancreas removed, weighed
then dried to constant weight (60°C. 72 h). The tissue was then
allowed to cool to room temperature in a desiccator over silica gel.
reweighed (to nearest 0.1 mg) and dry weight and percent dry
weiaht calculated.
Hemolymph Serum Analysis
All whole hemolymph samples from the laboratory study and a
random selection of samples from the field collection (n = 139)
were analyzed for triglyceride and phospholipid. The clotted
hemolymph was thawed then broken up gently with a glass stirring
rod and the sample centrifuged (Hettich EBA12 centrifuge, 15
min. 17.280g) to extract the serum. Serum aliquots were analyzed
on a Cobas Mira Autoanalyser for triglyceride and phospholipid
using commercially produced test kits (Roche). To test for phos-
pholipid the triglyceride kit (Roche. No. 07 3679 1 ) was modified
as follows. 250 units phospholipase C (Sigma No. P4014) were
added to a 30-mL bottle of triglyceride reagent. The modified
reagent was then incubated with the serum sample for 15 min at
37 "C (cf 6 min for ti-iglyceride) to convert the serum phospholipids
to diglycerides, which were then converted to glycerol by the
lipase in the kit. The incubation time was chosen by incubating a
lecithin solution (2 mM) to give a result equivalent to 2 mM
triglyceride. Accuracy was maintained for all tests using commer-
cially available quality controls (Nycomed Farmer).
Data Analysis
If data were normally distributed or could be normalized analy-
ses were performed using analysis of covariance (ANCOVA) or
analysis of variance (ANOVA) with the GLM module (General
Linear Models) on SPSS. If data could not be normalized, the
Kruskel Wallis nonparametric ANOVA or the Wilcoxon Rank
Sign were used. In all cases significance was accepted at P = 0.05.
RESULTS
iMhoratory Experiments I and 2
Percent Moult Increiiient, Tank Placement, and Feeding Regimen
Premoult CL had no effect on % moult increment (P > 0.05,
ANCOVA). and there was considerable overlap between the
ranges of 9c moult increment recorded in the outside tanks (2.4 to
8.0% of premoult CL. n = 1) and those inside (0.8 to 5.2%. n =
9). The slopes of the percent moult increment; lipid regressions
were the same for inside and outside tanks (P > 0.05. ANCOVA).
For this reason, data from inside and outside tanks were pooled for
further analyses.
Hemolymph Serum Lipid and Moidt Increment
Both lipid tractions were significantly correlated with percent
moult increment (Table 1). Phospholipid showed the highest co-
efficient at r = 0.66. Both phospholipid and triglyceride showed
a progressive increase with pigment stage (Fig. 1 ) until PS3.0 to
4.0 then declined to PS4.5.
TABLE 1.
Relationship between percent moult increment and haeniolyniph
lipid (Mmol 'l for phospholipid, triglyceride, and TP (triglyceride
+ phospholipid).
Parameter
P
Phospholipid
Triglyceride
TP
0.0715
0.1140
-0.0786
2.072
2.248
1.765
0.66
0.40.1
0.641
.10.062 <0.001
II . 1 2,1 0.005
27.781 <0.001
The regression model is Log % moult increment = allog (lipid)]^. n
16.
Hemolymph Chemistry and Moult Increment in Lobsters
237
35
30
25
o
E 20
1.5
1.0
05
0.0
■
' ^^ E
\ E
i \ "5
T 5
T i !_i
T " '\
0.5
1
1.5
4.5
2 25 3 3.5
Pigment Stage
Figure 1. Laboratory: mean hemolymph serum triglyceride (mmol/I.)
±SE, phospholipid Immol/L) ±SE, and triglyceride plus phospholipid
(TPl (mmol/I, I ±SF, vs pigment stage. Closed dianKmd, TP; closed
square, triglyceride; closed triangle, phospholipid.
1.8
1.6
1.4
1.2
1
0.8
06
0.4
0.2
0
0.5 1
1.5
3.5
4.5
2 25 3
Pigment stage
Figure 3. Field: mean hemolymph serum triglyceride (mmol/l,( ±SE.
phospholipid Immol/lj +SK. and triglyceride plus phospholipid (TPl
(mmol/L) ±SE vs pigment stage. Closed diamond. TP: closed square,
triglyceride: closed triangle, phospholipid.
Feeding Rales
Feetiing rate increased 4-fol(i after the moult in those lobsters
for which there was data (Fig. 2). There were no se.xual differences
in feeding rate (P > 0.03).
Field Study
Hemolymph Serum Lipid
The field serum lipid data showed a progressive increase in
lipid content with pigment stage (Fig. 3) in a similar fashion to that
found in the laboratory, although in this case the peak occurred at
PS4.
Hemolymph Lipid and Hepatopancreas Weight
Hemolymph lipid increased with hepatopancreas dry weight on
both a total weight and a percentage basis (Fig. 4a and b) up to
PS4. However, although hemolymph lipid was significantly cor-
related with tissue weight during intermoull (Table 2). the rela-
tionship declined after PS2-2.5.
ImI'ihiiIoiv Fxperimeiits and Field Study Comparisons
Moult Stage and Pigment Stage
The relationship between moult stage and pigment stage was
similar in the laboratory and the field (Fig. 3). In the following
30
25
20
15
10
05
00
fVt*
987654321
Weeks before moult
2 3 4 5 6 7
Weeks after moult
Figure 2. Mean feeding rates (±SE| for 10 weeks before and after
ecdysis. Four daily consumption categories (0, <25, 25-50, >50'!'r ) were
used and assigned numbers from I to 4 (h = 18).
analysis, comparisons are made between pigment stage-specific
laboratory and field hemolymph lipids. Before this was done,
analysis was undertaken to check that the same pigment stages had
similar distributions of moult stages in the laboratory and the field.
To facilitate the analysis each moult stage was assigned a number
(1-11).
The laboratory distribution of moult stages within each pigment
stage was similar to that in the field (Mann-Whitney U. Zar 1984).
The only significant difference was in PS 4 (U = 37.3. P = 0.014,
mean moult stage|.,b„„,„ry = 9.46 ± 0.3Q. mean moult stage,-,^,j =
8.13 ± 0.249). otherwise P > 0.212.
Hemolymph Serum Lipid
Pigment stage-specific total lipid of laboratory animals was
greater than that in the field until PS3.3 (Mann-Whitney U. P <
0.03: Fig. 6). The patterns in the relative importance of the two
lipid fractions were also different. In the field, the proportion of
phospholipid increased until PS 2.5 (Fig. 7) then fell until PS 4.5.
in contrast to the laboratory where the peak was reached during
PS 1 . Both laboratory and field showed the same trends after PS2.3.
DISCUSSION
The key result to come out of this study is the potential use of
hemolymph lipid in the prediction of percent moult increment.
Although further field studies are needed to be sure of the result,
this outcome is potentially very useful because hemolymph lipid
measurement does not require killing the lobster. Questions remain
as to whether higher growth sites, showing higher serum protein
content v\ould also ha\e higher moult increments. In this regard,
significant differences were reported in mean serum protein level
between sites by Musgrove (2001). The differences occurred
mainly during intermoult, which is the period when hemolymph
lipid is significantly correlated with both serum protein and
hepatopancreas percentage dry weight. This may suggest a rela-
tively higher degree of lipid accumulation at those sites, pointing
to a higher moult increment. Dall (1981) suggested that the prin-
cipal function of digestive gland lipid in Nephrops noii'egicus was
238
MUSGROVE AND BaBIDGE
a)
TABLE 2.
25
1
2'
y 1
).
:V^
■^
TP (mmol/l)
-*■ bi
J
■^
^A
r
^\
1 — i
►
1
i
05
w
7^ ' I
/
i^
1
^ \ H \ 1
1.5 2 2.5 3 3.5
Pigment Stage
4.5
o
E
E
P- 1
s \
39
ss
In
; -37
*•*
- 35
1
•^
>.
33
■D
\
C
r3l
0)
o
6)
a.
Pigment Stage
Figure 4. Comparison of (a) hepatopancreatic mean dry weight (g,
±SE). (b) mean percent dry weiglil {±SE). and triglyceride plus phos-
pholipid (TPl \s pigment stage. Mean dry weight (g) standardized for
carapace length using GLM analysis of SPSS. Data are displayed for
a 97.9-mm CL lobster. Closed diamond, TP; closed square, weight (g).
in the moulting process so one might expect that lipid accumula-
tion in Jasiis edwordsii would be similarly focused.
Cockcroft ( 1997) also found a significant relationship between
moult increment and hepatopancreas lipid level, for Jasus lalaiulii.
He reported that moult increment was positively related to peak
"/flipid values occurring during late premoult in the hepatopan-
creas, similar to the present study, where the significant relation-
ship was between heiiiolymph lipid (|j.mol/Ll and '/<- moult incre-
ment. Furthermore, he suggested a "window" period of reserve
accumulation, essential for growth. This occurs from intermoult to
early premoult. especially the former, as suggested for/ edwardsii
by the relative increase in feeding rate after ecdysis. The period of
reserve accumulation (PRA) would probably lead up to a "reserve
saturation point" as suggested by Anger (1987) for crustacean
larvae. Cockcroft found that lobsters starved during PRA, then
feed during premoult. showed severely reduced growth rates and
10
Field data regression statistics for pigment stage-specific percent
dry
9.5
weight and total dry
weight
versus total
lipid (T + Pl.
9
Percent or
8.5
S
£
Total Weight
PS
r
F
P
n
8
Percent
1
0.722
17.33
<0.()01
25
7.5
1
l-.S
1
0.887
0.599
118.29
29.93
<0.001
<0.001
17
24
7
r^
2.5
0.685
41.39
<0.()01
21
6.5
o
3
0.-U8
S.IO
,017
12
>3
0.067
1.93
0.176
29
6
Total Weight
1
0.532
24.96
<0.001
25
5.5
1.5
0.767
49.50
<0.001
17
2
0.389
13.37
0.001
24
5
2.5
0.282
7.47
0.013
21
3
0.000
2.9"*"
0.987
12
>3
0.078
2.29
0,142
29
Regression model is Lipid = a Weight'^ except for percent dry weight at
pigment stage 1. where the best fit was given by the cubic model (lipid =
a -I- Pi weight -I- p, weight^ + p, weight')
even shrinkage. Those starved prior to moulting but led during
PRA moulted with similar growth increments to those of control
lobsters, which were fed throughout. Therefore, it is this PRA that
is critical to future growth, influencing both moult increment and
intermoult period (Cockcroft, 1997).
The question is, why should the percent moult increment be
correlated with the lipid level in the hemolymph at PS4.3, when it
was not related to the hepatopancreas percent dry tissue at that
stage? At PS4.5 about 95% of lobsters were beyond D,'". The
rigorous investigation of this question is outside the framework of
this study but it may be that the apparent decoupling of the rela-
tionship between hepatopancreas weight and hemolymph lipid at
the later pigment stages is due to a mobilization of lipid reserves
from the hepatopancreas to the hemolymph in preparation for the
energetic demands of ecdysis. The correlation may arise because
the higher the level of stored lipid in the hepatopancreas, the
creater the reserve that may be mobilized in readiness for ecdysis.
D3
D,
D,'"
D,-
0/
Do
C3
C2
B/C,
A
0.0 1.0 1.5
3.5 4.0 4.5 5.0
2.0 2.5 3.0
Pigment Stage
Figure 5. Mean moult stage (±SF) within each pigment stage for labo-
ratory experiments (pooled, n = 40) and field study (n = 135). There
were no lobsters at PS3.5 in the laboratory sample. Closed diamond,
laboratory: closed square, field.
Hemol\mph Chemistry and Moult Increment in Lobsters
239
2,5
? 15
E
E
05
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5
Pigment stage
Figure 6. Mean triglyceride and phospholipid (TP: nimol/I>) ±SE:
field vs laboratory data by pigment stage. Significant differences
(Mann-Whitney U testi between laboratory and field data are indi-
cated by asterisks {**P < 0.001 1. Closed square, laboratory TF; closed
diamond, field TP.
The importaiK-e of phospholipid in the relationship fits in with
Chang and O'Connor's (1983) contention that phospholipid is the
main circulating lipid in crustaceans. Bligh and Scott (1966) re-
ported that 65'7f of the total lipid in the hemolymph of the lobster,
Hdiiiaiiis (iniericciiuis. was phospholipid, with the remainder al-
most equally divided between triglycerides and sterols. The latter
has a primarily structural role (Fraser, 1989). Free fatty acids com-
prised only about 2.4% of the total lipid. O'Connor and Gilbert
( 1969) reported similar results for the land crabs. Gecarcinns lat-
eralis and Canliosoma giianhmiu.
Finally, the relative levels of hemolymph protein and lipid in
the laboratory and the field suggest that the rate of accumulation
differs, particularly in the early stages of the moult cycle. One
would assume that these differences occur because captive lobsters
did not have to hunt for food, more nutrients being directed to
muscle accumulation and lipid storage earlier in the moult cycle.
Our data suggest that hemolymph phospholipid level has the
0.7
065
0.6
Q.
H
•B
0 55
a.
o
0.5
,c
a.
o
045
s:
Q.
0.4
0.35
0.3 ' -
0 0.5 1 1.5 2 2,5 3 3.5 4 4.5 5
Pigment Stage
Figure 7. Hemolymph scrum phospholipid/! triglyceride plus phospho-
lipid, nimol/L( ±SE: field vs laboratory data by pigment stage. .Signifi-
cant differences (Mann-Whitney I' test) between laboratory and field
data are indicated by asterisks (**/" < O.OUl). Closed square, labora-
tory TP; closed diamond, field TP.
potential to predict moult increment. However, the hemolymph
lipid/moult increment data were gathered over a short time period
and within a relatively controlled environment. Further field stud-
ies are essential to better understand the relationship between
hemolymph lipid level and moult increment in wild populations of
this species.
ACKNOWLEDGMENTS
The authors would like to thank the South Australian Rock
Lobster Industry for their support, for supplying the lobsters for
this study, and for allowing us the use of their facilities for the
initial data collection. We wiuild also like to thank Dr Stephen
Mayfield and Dr Jason Tanner for critically reviewing the manu-
script. Financial support for this study came from Fisheries Re-
search and Development Corporation (Project 96/160).
LITERATURE CITED
Anger, K. 1987. The D„ threshold: a critical point in the larval developnienl
of decapod cru.staceans. J. Exp. Biol. Ecol. 108:15-30.
Bligh, E. G. & M. A. ScoU. 1966. Blood lipids of the lobster. Hoimuus
americcmus. J. Fi.sh. Res. Bd. Can. 23:1629-1631.
Chang, E. S. & J. D. O'Connor. 1983. Metabolism and transport of car-
bohydrates and lipids. In: L. H. Mantel, editor. The biology of Crusta-
cea. Vol 5: Internal anatomy and physiological regulation. New York:
Academic Press, pp. 263-287.
Cockcroft. A. C. 1997. Biochemical condition as a growth predictor in
male west-coast rock lobster {Jasiis lalandii). Mar. Fresh. Res. 48:845-
856.
Dall. W. 1981. Lipid Absorption and utilisation in the Norwegian lobster.
Nephrops non'ei>iciis (L.). J. E.xp. Mar. Biol. Eeol. 50:33— 15.
Fraser. A. J. 1989. Triacylglycerol content as a condition index for fish,
bivalve and crustacean larvae. Can. J. Fish. Aqiial. Sci. 46:1868-1873.
Geddes. M. C. S. R. Bryars. C. M. Jones. B. J. Crear, P. R Hart. C. Thomas
& L. Linton. 2000. Determination of the optimal environmental and
system requirements for juvenile and adult rock lobster holding and
grow-out. Fisheries Research and Development Corporation Report
98/305: 141 pp.
Leavitt D. F & R. C. Bayer. 1977. A refractometric method of determining
serum protein concentrations in the American lobster. Aquaciilture 12:
169-171.
Mercaldo-Allen. R. 1991. Changes in the blood chemistry of the American
lobster, Homarus amerieanus. H. Milne Edwards. 1837. over the moult
cycle. J. Shellfish Res. 10:147-156.
Musgrove. R. J. B. 2000a. Moult staging in the southern rock lobster y</.vH.v
edwardsii (Hutton. 1875). J. Crust. Biol. 20:44-53.
Musgrove, R.J.B. 2000b. Condition and its assessment in the southern rock
lobster Jasiis edwardsii. ii. Field application of the techniques for con-
dition assessment and moult staging developed in the laboratory. Fisheries
Research and Development Corporation Project Report 96/160: 48 pp.
Musgrove. R. J. B. 2001. Interactions between the haemolymph chemistry
and condition in the somhem rock lobster, .lasits eduurdsii. Mar. Biol.
139:891-899.
O'Connor, J. D. & L. 1. Gilbert. 1969. Alterations in lipid metabolism
associated with premolt activity in a crab and a freshwater crayfish.
Comp. Biochem. Physiol 29:889-904.
Zar. J. H. 1984. Biostatistical Analysis. Second Edition. Englewood Cliffs.
NJ: Prentice-Hall Inc.. 718 pp.
JoKimil .'/ Slicllfish Rc.uanh. Vol 22. No. I. 24I-24M, 2(103.
BLUE CRAB IVIORTAIJT^ IN THE NORTH CAROLINA SOFT-SHELL INDUSTRY:
BIOLOGICAL AND OPERATIONAL EFFECTS'
JUAN C. CHAVES* AND DAVID B. EGGLP:STONt
Niinh Ciirolliici Stale Uiiivcrsiry Dcparlimiit nf Marine. Earth and Atiuasplicric Sciences.
Ralcif^li. North Carolina 27695 -H20H
ABSTRACT The rapid grow th ol the soft-shell blue crab ( Calliiwcles supuhis) industry in North Carolina and elsewhere has outpaced
the generation of certain information to address key management and operational issues concerning this fishery. The specific objectives
of this study were to quantify: I ) mortality rates of white-line versus red-line peeler crabs; 2) size-specific mortality rates of crabs in
shedding systems; 3) mortality rates of peeler crabs as a function of crab source (purchased or self-caught), system type (closed versus
open), and gear (hard crab pot vs. peeler pot); 4) any relationship between peeler crab mortality and water quality parameters, such as
di.s.solved oxygen, temperature, salinity, and nutrients; 5) the effects of crab se.x on peeler mortality rates; 6) the effects of feinale crab
presence or absence and molt stage on time-to-molt and survival of male peelers; and 7) the effects of crab density on time-to-molt
and survival of male peelers. We addressed these objectives during May-October 2001 through collaboration with II different
commercial crab shedders located throughout coastal North Carolina. Study locations represented a broad spectrum of water quality
while simultaneously providing replicated closed and open systems and replicated use of purchased versus self-caught crabs. Both
large-scale seafood producers and small backyard operations were represented in this study. The key findings were 1 ) significantly
higher mortality of white-line than red-line peelers; 2) relatively high mortality rates of peelers in shedding systems ranging from an
average of 10-3O'7r per tank per day, but no effect of crab size on mortality rales; 3) no relationship between mortality of peelers and
water quality parameters, such as dissolved oxygen (DO), temperature, salinity, and nitrates; 4) significantly higher mortality of peelers
purchased by crab shedders than peelers caught by shedders; 5) no significant difference in peeler mortality between closed and open
systems or between those crabs captured by hard crab pots or peeler pots; 6) decreasing peeler mortality with increasing density of
peelers in holding tanks; 7) significantly higher mortality rates for male than female peelers and significantly lower time-to-molt for
males than females; 8) no significant increase in male peeler mortality or time-to-molt in the presence of red-line females; and 9) a
significant decrease in a male red-line peeler's time-to-molt in the presence of a red-line female and interniolt male. Implementing best
management practices in the soft crab industry could encourage crabbers to take better care of peeler crabs by always placing them
in a cooler on ice immediately after capture or underneath wet burlap sacks. The benefits of best management practices will likely
include a reduction in the mortality rate of peeler crabs in shedding systems, increased financial profits for crabbers who sell peelers
that are now more likely to survive in shedding systems, and improved profits of shedding system operators who purchase peeler crabs.
It is important to reduce mortality in North Carolina's soft-shell blue crab industry because I ) soft-crab landings are increasing rapidly
and becoming a larger component of overall landings. (2) approximately 23'7f crabs placed in shedding systems die. and 3) there is an
urgent need to conserve the blue crab spawning stock given the recent SO'/r decline and a highly significant stock-recruitment
relationship for the blue crab in North Carolina. The information from this study should lead to improvements in shedding technology,
better fishery management, and improved profits.
KEY WORDS: blue crab, Callinectes SLq^iJiis. crab mortality, density-dependence, management issues, peeler crab, soft-shell
industry
INTRODUCTION
Soft-shell blue crabs (Callinectes sapidus. R;ilhbun 1884) are
prodticed by aquacuitLire operations ("shedding operations") that
hold preinoh ("peeler") crabs until they molt (Ary and Poirrier
1989), Commercial fi.shermen sell soft-shell ("soft crabs") crabs
for nearly seven times more per pound than intermolt ("hard")
crabs (Oesterling 1993). In North Carolina, the soft-shell crab
indtistry has become an increasingly important component of the
blue crab fishery, the state's most valuable fishery in terms of total
landings, value, processing, participation, employment, and
amount of gear used (Henry and Mckenna 1998). Recently, a
significant decline in the state's blue crab population and commer-
cial landings (Eggleston et al, 2002) has increased financial pres-
sure on commercial fishermen, who look to production of soft
crabs as a means of economic survival. Although most shedding
operations are profitable, large financial losses are also common
*Current address: Center for Marine Science and Technology, NC State
University, 303 College Circle, Morehead City, North Carolina 28557.
tCorresponding author. Tel: 919-515-7840; Fax: 919-515-7802; E-mail:
eggleson@ncsu.edu
because of the high mortality of peelers (Dell Newman, crab shed-
der and commercial fisherman. Swan Quarter, NC, personal com-
munication; Connie Ingraham, crab shedder. Wilmington, NC,
personal communication). Thus, the value of the soft crab industry
is directly dependent on mortality rate of peeler crabs. Several field
and laboratory studies have documented the effects of water qual-
ity on crab survival and molting success (Ary and Poirrier 1989,
Das and Stickle 199,3, Lakshmi et al. 1984, Weis et al. 1992):
however, few. if any. published studies have quantified mortality
of peeler crabs in the soft crab industry or identified sources of
mortality. This study quantified how blue crab mortality in soft-
shell shedding operations varied with biologic and operational fac-
tors.
Blue Crab Molting
The time period between molting in blue crabs varies froin days
to inonths depending upon crab size. For example, the smallest
juvenile stages of crabs (6-10 mm carapace width, CW) molt on
the order of days, whereas sub-adult crabs with a carapace width
of 80-100 mm CW molt on the order of weeks, and crabs > 100
mm CW molt on the order of months (Miiliken and Williams
1984). Shedding of the exoskeleton (i.e.. ecdysis) occurs when
241
242
Chaves and Eggleston
crabs secrete a new exoskeleton within the old one. The old exo-
skeleton then cracks along suture-lines and the crab exits the old
shell with a sofl-shell that is larger than the old one. Four or five
hours after molting, the soft shell gradually hardens. Crabs in the
soft-shell industry are collected shortly after molting and before
the shell hardens. When crabs begin to secrete their new shell, a
white-line becomes visible inside the cuticle of the crab's last
appendage or swimmeret. This white line indicates that the crab
will molt within two weeks. As molting time nears. the indicator
line gradually changes color: a pink line peeler will molt within 1
wk. and a red-line peeler will molt within 3 days (Oesterling
1984).
North Carolina 's Soft-Shell Blue Crab Industry
Soft crab landings in North Carolina ha\e made up l.69r of the
total blue crab landings for the past 8 y (-6,166.160 lbs), but the
value of this fishery has averaged 6.6% of the total during that
same time and increased to nearly 10% during 2001 (-$3,336,990;
North Carolina Division of Marine Fisheries 2002). The increase
in value of the soft crab fishery in North Carolina may be attrib-
utable to drastic declines in the blue crab population and in hard
crab catch (Eggleston et al. 2002). and increasing local, regional,
and worldwide demand (Oesterling 199.'i).
In North Carolina, peeler crabs are trapped as by-catch in the
hard crab fishery using hard crab pots or targeted directly using
peeler pots. Hard crab pots are constructed of 3.8-cni wire mesh,
fitted with at least two escape rings of 5.9-cm inside diameter
(North Carolina Division of Marine Fisheries 2002). and are baited
with dead fish. Peeler pots are constructed of 2.54-cm mesh and
are not fitted with escape rings because there is no size limit on
peeler crabs (North Carolina Division of Marine Fisheries 2002).
Peeler pots are either unbaited or are baited with a mature male
crab whose urine may attract prepubertal female peeler crabs
(Ryan 1966). Prepubertal female peeler crabs are attracted to male
blue crab urine because they are only able to copulate during a
brief period of 2-3 h after ecdysis.
Two types of shedding systems, open and closed re-circulating,
are used primarily in North Carolina. In open systems, water is
pumped into shedding tanks from a nearby source such as a creek
or bay. and drains back into the same water source. In closed
systems, tanks are either tilled with well water and aquarium salt
added, or water is trucked in from the nearest suitable source.
Water drains into a biologic filter tank and is continuously pumped
back into shedding tanks. Nitrogen fixing bacteria in filter tanks
reduce the toxicity of ammonia in the water by reducing it to nitrite
and then to nitrate (Wheaton 1977).
Management and Operational hsues
The rapid growth of the soft-shell blue crab industry in North
Carolina and elsewhere has outpaced the generation of certain
information to address key management and operational issues
concerning this fishery. Input on key management and operational
issues concerning the soft-shell crab industry in North Carolina
were provided through direct communication with the North Caro-
lina Division of Marine Fisheries (NC DMF) and through a series
of public workshops that sought input from commercial crabbers
as a part of the North Carolina Fisheries Resource Grant Program.
administered through North Carolina Sea Grant. Specific manage-
ment questions are described below.
1. Do white-line peelers in the soft-shell blue crab industry
suffer relatively high mortality rates caused by long holding
periods (e.g., held for weeks) compared with red-line peeler
stages (e.g.. held for days)'? (Henry and McKenna 1998).
2. Are overall mortality rates of crabs in shedding operations
relatively high, and does crab mortality vary with crab size?
(S. McKenna. NC DMF, personal communication). Cur-
rently, there is no size limit on peeler crabs in North Caro-
lina.
Specific questions raised by soft-shell crab shedders in NC
during public workshops are described below.
1. Do peelers purchased by shedders suffer higher mortality
than those they caught?
2. Is peeler crab mortality caused by low dissolved oxygen and
high temperatures?
3. Is peeler crab mortality higher in closed than open systems?
4. Is peeler crab mortality elevated for crabs captured in hard
crab pots as opposed to peeler pots'
5. Does peeler mortality increase with the crab density in hold-
ing tanks?
6. Is peeler crab mortality and time-to-molt higher in males
than females? Is male peeler crab mortality disproportion-
ately high in the presence of female peelers?
Such untested questions are the basis of at least one current
regulation in North Carolina. For example, the NC DMF prohibits
harvest of white-line peelers after June I each fishing season be-
cause of assumed high mortality during summer months. More-
over, much of the hypothesis testing in the present study was
driven by the collective observafions of commercial crab shedders.
The overall objectives of this study were to address the man-
agement and operational questions raised above by quantifying: 1 )
mortality rates of white-line versus red-line peelers; 2) size-
specific mortality rates of crabs in shedding systems; 3) mortality
rates of peelers as a function of crab source (purchased or self-
caught), system type (closed versus open), and gear (hard crab pot
vs. peeler pot); 4) the relationship, if any, between peeler mortality
and water quality parameters such as dissolved oxygen, tempera-
ture, salinity, and nutrients; 5) the effects of crab sex on peeler
mortality rates; 6) the effects of female crab presence or absence
and molt stage on time-to-molt and survival of male crabs; and 7)
the effects of crab density on time-to-molt and survival of male
crabs. This information should lead to improvements in shedding
technology, better fishery management, and improved profits.
METHODS AND MATERIALS
Study Locations
Data were collected in collaboration with 1 1 different commer-
cial crab shedders throughout coastal North Carolina (Fig. I ) from
May until October 2001. Study locations were selected to represent
a broad spectrum of water quality while simultaneously providing
replicated closed and open systems and replicated use of purchased
versus self-caught crabs (Chaves 2002). Both large-scale .seafood
producers and small backyard operations were represented in this
study. Seven locations used closed systems, three used open sys-
tems, and one location used both an open and a closed system
(Chaves 2002).
Crab Collection
Crabs were captured by commercial fishermen using hard crab
and peeler crab pots. After capture, peeler crabs were either stored
Blue Crab Mortality in North Carolina
243
Chaves^ Egglesion. Moruliiy of sofl crabs
\
Elizabeth City ■, 1^
Albemarl
Vv
ar/e SountT ^
** ^^
w
• 4^
Pamlico
Sound Cape
Hatteras
:»i^W
t
N
Wilmington
Cape Lookout
A a an tic Ocean
Cape Fear
Figure 1. Map of North Carolina showing study locations (stars).
in wooden baskets on the deck of a boat or were placed in coolers
on ice. Once landed at the dock, crabs were placed in nearby
shedding tanks or trucked to shedding operations up to 200 km
away. At each of the 1 1 crab shedding study locations, premolt
crabs were sexed and their carapace width measured (mm CW).
Crabs were separated according to peeler stage (red-line vs. white-
line) and then equally distributed among four experimental tanks
measuring 1 .2 m wide x 2.4 m long and 20 cm deep. The crab sizes
used ranged from 5-17.2 cm CW. A total of 49 experiments were
conducted. A single experiment could last for -6 days or -21 days
for red-line or white-line peelers, respectively, to allow crabs
enough time to molt. Some shedders conducted experiments in
either closed or open systems, using only purchased or self-caught
crabs. Other shedders would conduct simultaneous experiments
with red-line and white-line peelers that were self-caught. Others
might sw itch systems and crab source from one month to the next.
For example, a shedder might conduct an experiment with only
purchased crabs in a closed system in one month, followed by an
experiment using only self-caught crabs in an open system the next
month. Each experiment at a specific location was treated as a
single independent replicate, since a new grouping of crabs from
varying sources was placed in the shedding tanks at the initiation
of each experiment, and the experimental methods were standard-
ized across locations. In the following Methods and Results .sec-
tions, the objectives of the study are described within the topics of
operational (system, crab source, gear, water quality) and biologic
(crab molt stage, sex, density, size) considerations.
Operational Cuiisideratiom
Kffiits ot \\ ater Quality on Crab Mortality
To quantity the effects of water quality on crab mortality, the
following water quality parameters were measured daily at -0800 h;
dissolved oxygen (DO) (mg/L), temperature (°C), salinity (parts
per thousand; ppt), pH. and concentrations of nitrite (mg/L), nitrate
(mg/L). and ammonia (mg/L). A weighted mean calculated from
the number of crabs that died in each experimental tank per day
divided by the number of crabs in the tank on that day was used.
Thus, the response variable in all cases dealing with crab moilality
was a weighted percent mortality/day. Tanks with red-line crabs
were monitored for 6 days and tanks with white-line crabs were
monitored for up to 21 days. If all crabs in a tank shed or died
before the 6- or 2 1 -day period, the experiment was terminated. The
experimental unit was each tank, and four leplicate tanks were
used at each of the 1 1 sites.
Statistical analyses used a multiple regression model with crab
source (purchased vs. self-caught) as the independent variable and
water quality parameters as independent continuous variables. In
this case, crab source was highly significant (see below), which
confounded mortality associated with the source of crabs and wa-
ter quality parameters. Thus, in subsequent statistical analyses, the
data were first divided into separate categories of self-caught ver-
sus purchased crabs. In assessing the effects of water quality on
crab mortality, we then used a backward, stepwise multiple regres-
sion model. Alpha to enter and remove factors from the model was
0.10. A Levene's Median test assessed constant variance among
the responses and a Kolmogorov-Sminiov test tested for normal-
ity. In cases where the data failed to meet the assumptions, the data
were transformed using ArcSine or log 10 transformations, which
were successful in all cases.
Effects of Shedding System, Crab Source. Gear Type, and Crab
Density on Crab Mortality
The mean daily crab peicent moitality in closed versus open
systems, between self-caught versus purchased crabs, and between
crabs caught in hard crab pots versus peeler pots was compared
with three separate one sample / tests. The relationship between
mean daily crab density and mean daily crab proportional mortal-
ity pooled across 23 experiments using self-caught crabs and 26
experiments using purchased crabs was examined with a linear
least squares regression model.
Biologic Considerations
Effects of Crab Sex, Size, and Peeler Stage on Crab Mortality.
To quantify the effects of crab sex and peeler stage on crab
percent daily mortality, we compared the mortality of male versus
female crabs, and white-line peelers versus red-line peelers using
an analysis of covariance (ANCOVA) model with crab sex and
peeler stage as factors and crab size as a covariate. The data were
noi'iiially distributed and variances were homogeneous.
Effects of Female Crab Presence on Mortality and Time-to-Molt in
Male Crabs
The effects of female crab presence and their molt stage on the
time-to-molt and moilality of male crabs were examined in a
closed shedding system in Swan Quarter, NC. Twenty tanks, mea-
suring 1.2 m wide x 2.4 m long and 20 cm high were filled with
estuarine water from Albemarle Sound to a height of 15 cm. Once
the tanks were filled, the pump was turned off and water was not
allowed to circulate between tanks, thereby preventing any poten-
tial pheromone contamination across tanks. Tanks were aerated by
aquarium air pumps. Crabs were purchased from several fishermen
and randomly assigned to one of the following three treatments: 1 )
one red-line male per tank (control |; 2) one red-line male and one
intermolt female per tank; and 3) one red-line male and one red-
line female per tank. All crabs were visually examined houriy to
record the time that thev molted or died. When a red-line male crab
244
Chaves and Eggleston
molted or died, the trial was stopped. Each male red-line peeler
was an experimental unit and each treatment was replicated seven
to nine times. We tested whether there was a treatment effect on a
male crab's time-to-molt and percent daily mortality with a one-
way analysis of variance (ANOVA). The data were log-
transformed to meet assumptions of normality and homogeneity of
variance.
Effects of Increasing Male Crab Density in the Presence of a
Red-Line Female on Mortality and Time-to-Molt of Male Crabs
The effect of increasing male crab density on percent daily
mortality and time-to-molt in male crabs was also tested at Swan
Quarter, North Carolina. Four treatments were randomly inter-
spersed among tanks: 1 ) one red-line male per tank [control]; 2)
one red-line male and one red-line female per tank: 3) one red-line
male, one red-line female, and one intermolt male per tank; and 4)
one red-line male, one red-line female, and three intermolt males
per tank. Each male red-line peeler was an e.xperimental unit and
the response variables were time-to-molt and percent daily mor-
tality. Each treatment was replicated five to se\en times.
Effects of Crab Sex on Time-to-Molt
Time-to-molt of male versus female crabs in the absence of
other crabs was quantified in separate experiments at Swan Quar-
ter. North Carolina. This experiment was conducted to determine
if males simply took longer to shed than females regardless of any
other factors such as presence of females or increasing crab den-
sity. Each tank contained a single male or female red-line crab.
The response variable was time-to-molt in hours. Each crab was an
experimental unit and each treatment was replicated 10 times.
The LIFETEST procedure in SAS was used to compare the
distribution of male's time-to-molt in the presence and absence of
red-line females and other male crabs. The data was right censored
(experiments ended before a response could be observed) due to
the early termination of several trials when male crabs died before
molting. The censored data points can not be left out of the analy-
sis because crabs that take longer to molt are also more likely to
die. The LIFETEST uses both censored and uncensored times to
molt when comparing distributions of times to molt for various
treatments. An uncensored data point is an actual observation of
the time-to-molt. but the time-to-molt for censored data points is a
calculation based on the distribution of times to molt among non-
censored data points. Chi-Square tests were used to detect differ-
ences in mortality between treatments, and ANOVA was used to
detect differences in the time-to-molt between male and female
crabs.
RESULTS
Operational Considerations
Effects of Water Qualit> on Crab Mortality
Water quality was somewhat poorer in closed than open recir-
culating systems (Table 1). For example. DO was lowest (2.9
nig/L) and nitrates highest (77.4 mg/L) in closed systems. Never-
theless, most of the water quality values were well within tolerance
limits of blue crabs (Manthe et al. 1983). The percent daily mor-
tality of self-caught and purchased peeler crabs did not vary sig-
nificantly with any of the water quality parameters recorded (mul-
tiple regression; self-caught: all P > 0.08. purchased: all P > 0.16).
Effects of Crab Source, Shedding System. Gear Type, and Crab
Density on Crab Mortahty
Mortality of peeler crabs was significantly higher for purchased
than self-caught crabs (f test; t = -2.22, df = L.'iO, P = 0.03; Fig.
2). Shedding system type (i.e.. open vs. closed) did not signifi-
cantly affect the mortality of self-caught it test; t = 1.23. df =
1,48, P = 0.22) or purchased crabs (r test, t = 0.32. df = 1.44,
P = 0.15). For self-caught crabs, there was no difference in crab
mortality between crabs caught by peeler pots or those caught by
hard crab pots (peeler pots: mean daily percent mortality = 2%,
SE = 0.007, /; = 16; hard crab pots; mean daily percent mortality
= 3%, SE = 0.006, /I = 8; r = 0.54, df= 1,22, P = 0.60). We
were unable to test the effects of gear type on mortality of pur-
chased crabs because all purchased crabs came from hard crab
pots.
Surprisingly, the percent daily mortality of red-line male peel-
ers decreased with increasing density of peelers held in shedding
tanks for both self-caught and purchased crabs (Fig. 3). The de-
clining trend in percent daily peeler mortality with density was
significant for self-caught crabs (linear least-squares regression: F
= 14.27. df = 1.17. P<0.01; Fig. 3A). and marginally significant
for purchased crabs {F = 4.05. df = 1.19. P = 0.06; Fig. 3B).
Biological Considerations
Effects of Crab Sex, Size, and Peeler Stage on Crab Mortality
Mortality rates of self-caught crabs were unaffected by crab sex
and the covariate of crab size (two-way ANCOVA; sex: F = 3.06.
TABLE L
Means and ranges of water quality parameters measured.
DO (mg/L)
Temp ( C»
Sal ippt)
Nitrite
Nitrate
.\nimonia
pH
Closed systems
Mean
5,9
25.4
20.1
0.2
21.7
0.5
7.4
Low value
3,0
17.4
3.7
0
0
0.1
7.1
High value
8.2
30.8
39.8
0.5
77.4
3.2
8.0
Open systems
Mean
6.7
26.4
17.3
0.1
3.6
0.3
7.4
Low value
3.6
19.5
5.0
0.0
0.0
0.2
6.9
High value
9.7
31.8
35.9
0.2
8.3
0.5
7.8
Nitrate, nitrite and ammonia are presented as mg/L.
Blue Crab Mortality in North Carolina
245
0.20 1
0.15
•^ 0.10
o
E
0.05
0.00
(A) Self-caught
self-caught purchased
Crab source
Figure 2. Mean daily proportional mortality (+ SE) of self-caught
(A' = 24) and purchased (A' = 27l peeler crahs. .Asterisk denotes sig-
nillcant difference. See text for details of statistical test.
df = 1.20. P = 0.10: size; F = 3.03, df = 1.20. P = 0.10);
however, self-caught white-line peelers experienced significantly
higher mortality rates than self-caught red-line peelers (molt-stage:
F = 3.4, df = 1.20, P = 0.03; Fig. 4A). Mortality rates of
purchased crabs were not affected by crab size (one-way
ANCOVA; F = 0.02. df = 1.44, P = 0.88): however, purchased
male peelers experienced significantly higher mortality rates than
purchased female peelers (F = 10.04, df = 1.44, P < 0.01: Fig.
4B). It was not possible to determine the effect of crab stage on
purchased crabs because no white-line peelers were purchased.
Effects of Female Crab Presence on Mortality and Time-to-Molt in
Male Crabs
There was no significant difference in time-to-molt between
male red-line peelers held alone, held with intermolt females, or
held with red-line females (ANOVA; F = 0.13: df = 3.23; P =
0.718; Fig. 5A). There was also no significant difference in mor-
tality between males that were held alone, held with intennolt
females, or held with red-line females (Chi-square test: x""" = 4.14.
df = 1,44, P = 0.13).
Effects of Increasing Male Crab Density in the Presence of a
Red-I.ine Female on .Mortality and lime-to-Molt of Male Crabs
Time-to-molt in male peelers varied significantly according to
whether a red-line female and intermolt male were also present
(ANOVA: F = 13.06; df = 2.10: P < 0.01). In this case, time-
to-molt was significantly shorter among males held with one red-
line female and one green male compared with the control group
of a single red-line male held alone (Ryan's Q-multiple compari-
son test: Fig. 5B). There was no significant difference in daily
percent mortality of male peelers in the presence or absence of
red-line female and intermolt male crabs (Chi-square test; x" =
3.06, df = 3.50, P = 0.38).
Effects of Crab Sex on Time-to-Molt
The average time-to-molt for male crabs was significantly
shorter than for female crabs (ANOVA; F = 14.21, df = 1,19. P
<().01. Fig. 5C).
0.4 n
•
y=0.27-0.002'
R^=0.44
p<0.0001
►x
0.3
•
\^^
ro
0.2
• ^\
o
\^^
E
0.1
0.0 -
• • ^^
•• •
0 20 40 60 80 100 120
mean density (crabs/tank)
(B) Purchased
y=0.21-0.0007*x
R'=0.14
p=0.0594
140
0.00
50 100 150 200
mean density (crabs/tank)
250
Figure 3. Relationship between mean daily proportional mortality of
(.\) self-caught (,V= 18) versus (15) purchased (A" = 20) peeler crabs and
mean density per tank.
DISCUSSION
The soft-shell blue crab industry is one of the fastest growing
fisheries in North Carolina. In this study, we collaborated with a
team of commercial crab shedders across 1 1 different locations
spanning the entire North Carolina coast to address key manage-
ment and operational questions intended to better manage the blue
crab resource, improve shedding technology, and increase profits.
The key findings were as follows; 1 ) significantly higher mortality
of white-line than red-line peelers; 2) daily mortality rates of 10-
30% per tank (primarily poorly handled red-line peelers) in shed-
ding systems, but no effect of crab size on mortality rates: 3) no
relationship between moilality of peelers and water quality param-
eters, such as DO, tempeiature, salinity, and nitrates: 4) signifi-
cantly higher mortality of peelers purchased by crab shedders than
peelers caught by the shedders: 5) no significant difference in
peeler mortality between closed and open systems, or between
those crabs captured by hard crab pots or peeler pots: 6) decreasing
peeler mortality with increasing density of peelers in holding
tanks; 7) significantly higher mortality rates for male than female
246
Chaves and Eggleston
(A) Self-caught
0.20 n
0.15
(A)
^ 0.10
o
E
0.05
0.00
white-line red-line
Molt stage
(B) Purchased
U.4
0.3
J
*
^
^ 0.2
O
T
E
0.1
males females
Crab sex
Figure 4. Mean daily proportional niorlality (+ SE) of (A) self-caught
white-line (h = 15) versus (B) red-line peelers (A' = 37), and (B) pur-
chased males (A' = 21) versus females (// = 25). Asterisk denotes sig-
nificant difference. See text for results of statistical tests.
peelers, and significantly lower time-to-molt for males than fe-
males; 8) no significant increase in male peeler mortality or time-
to-molt in the presence of red-line females; and 9) a significant
decrease in a male red-line peeler's time-to-molt in the presence of
a red-line female and inlermolt male.
Blue Crab Management Issues
White-Line Peelers
Mortality of self-caught white-line peelers was significantly
higher than self-caught red-line peelers. We were unable to assess
the effect of molt stage on purchased peelers because commercial
shedders do not shed white-line peelers for fear of high mortality
rates. White-line peelers in this study probably experienced higher
mortality rates than red-line peelers because of the relatively long
periods of time required for them to molt, in which they are more
likely to suffer from accumulated stress, as compared with red-line
peelers. Most crabbers will feed white-line peelers if they will eat.
100
~ 80
e
3
o
S 60
40
o
E
o
0)
E
■^ 20
20
T
T
control
1 green
female
1 red-line
female
(B)
120
100
O 80
I 60
I
o
% 40
E
*
*
T
control 1 red 1 red 1 red
female female female
1 green 3 green
male males
(C)
120 1
-. 100
(A
o 80
o 60
o
Z 40
E
20
0
*
male
female
Crab sex and molt stage
Figure 5. Mean 1+ SE) time-to-molt for male red-line peelers as a
function of crab sex and molt stage. Green = intermolt crabs. N = 7-9.
Asterisk denotes significant differences between treatments. See text
for results of statistical tests.
Blue Crab Mortality in North Carolina
247
Depending upon temperature and where a crab is in the moh cycle,
white-hne peelers cease to feed somewhere between 10-21 days
hetore moltinL' (pers. obs.). The peeler crab fishery in NC is regu-
lated on the assumption that male white-line peeler mortality is
very high during summer months, and that to keep them would be
a wasteful practice. Our results support this management practice;
however, it is important to note that the mortality of self-caught
w hile-line peelers was similar to that of purchased red-line peelers
(compare Fig. 2 and 4A), highlighting the importance of crab
source as a key determinant of peeler mortality. Oesterling (1984)
suggested that it is not economical to keep white-line peelers for
more than 10 days, and various coastal Sea Grant extension agents
have urged crabbers that harvest peelers to take better care of them
after capture.
OMTiill and Size-Specific Crab Mortality Rates.
Mortality rates of red-line peelers placed in shedding tanks in
this study averaged \59( per day. This daily mortality rate is ex-
tremely high when compounded over the typical 5-day duration of
shedding, and is dramatically higher than natural mortal it\ rates.
For example, the annual mortality rate for sub-adult and adult blue
crabs is 50% (Eggleston 1998). For a hypothetical, yet realistic
example of how mortality in shedding systems is compounded
over time, assume that 100 crabs are placed in a shedding tank on
Day 1. On Day 2, -15% of the crabs would have died and 507r
would have molted successfully and been sold. This would leave
35 crabs on Day 2. Assuming these same daily percentages of
death and successful molting, we would have five dead crabs and
17 crabs nu)lting successfully on Day 3, a total of two dead crabs
and seven molting successfully on Day 4, and one dead crab and
two molting successfully on Day 5, after which there would be one
crab left. Thus, out of a starting population of 100 peelers in the
shedding table, an average of 23 (23% ) would die iiver 5 days. The
cumulative mortality for white-line peelers would likely be even
higher since they are generally held two to four times longer than
red-line crabs before they molt. Depending upon method of harvest
and crab source, a high mortality of peelers could be expected
mimediately after crabs are stocked, with a large decrease in mor-
tality as stronger crabs shed.
We did not detect any effect of crab size on crab mortality rates,
contrary to popular belief that male peeler crab moilality increases
with crab size, especially when males become very large. There
may not have been enough contrast in our data to reveal a positive
relationship between crab size and mortality since male peelers
>16 cm CW were rarely observed. The belief among crabbers that
male peelers experience high mortality is so prevalent that many
have given large male soft-shell crabs the nickname "miracle
crabs."
Operational Considerations
Source of Peelers
Crab source was always the single greatest source of variation
in crab mortality rates, with highest inortality in purchased peelers.
Variability in mortality because of crab source is likely caused by
different handling methods used by crabbers who shed their own
peelers versus crabbers who sell peelers. Crabbers who shed their
own peelers put forth great effort to ensure the survival of their
peeler crabs, such as carrying a cooler with ice and wet burlap bags
in their boats, which keep crabs cool and moist. Crabbers who sell
peelers usually place peeler crabs in a wooden basket on the deck
of their boat, unprotected from bright sun. wind, and extreme heat.
Purchased peeler crabs may also experience significantly higher
mortality rates than self caught peelers because they are more
likely to travel greater distances from the point of capture to shed-
ding systems. Long travel times may cause crabs to dehydrate, but
also increase the likelihood that crabs will experience large
changes in salinity from the point of capture to shedding tanks.
Although blue crabs are euryhaline organisms that can survive in
a broad range of salinities from 0 ppt to over 40 ppt, sudden large
changes in salinity (i.e.. > 10 ppt in less than 24 h) may exceed a
crab's ability to osmoregulate the tissues in its body, causing mor-
tality (Engel and Thayer 1998). Although we were unable to record
changes in salinity from the point of capture to a given shedding
system, these salinity changes may be another source of stress, in
addition to poor handling, leading to significantly higher mortality
rates of purchased than self-caught peelers and is worthy of further
research. If large changes in salinity prove to be an important
source of stress for peelers, then shedders capable of regulating
salinity in their systems could alter the salinity to accommodate the
peelers they buy. A crabber who sells poorly treated peelers is
unaffected by high mortality rates that may occur in shedding
systems because market demand for peeler crabs ensures that they
will receive top dollar for these peelers, despite their relatively
poor treatment.
One of the most obvious ways to reduce crab mortality in North
Carolina's soft-shell crab industry is for shedders to capture their
own crabs rather than rely on purchasing peelers. The majority of
all peeler crabs in North Carolina shedding systems, however, are
purchased crabs (Tony Roughlon, Seafood dealer and commercial
fisherman, Columbia, NC. personal communication), and the data
in this study indicate that purchased peeler crab mortality is 1 1%
greater than self-caught crabs. The 11% difference in mortality
rates of self-caught and purchased peelers could easily equate to a
financial loss of over $776,044 per year for crab shedders alone
(see Appendix for calculations). The relatively high mortality of
purchased crabs could probably be reduced greatly by taking better
care of peelers on the boat, such as using coolers or wet burlap
sacks to keep crabs cool, inoist, and out of the sun and wind, and
reducing the travel times between initial harvest, and placement in
shedding systems. If the mortality rate of purchased crabs were
reduced to that of self-caught crabs, the soft crab industry would
increase in value by 11% ($776,044) without any increases in
harvest size.
Effects of Crab Density on Crab Mortality
Mortality of both self-caught and purchased crabs decreased as
crab density increased, in contrast to expectations. It is possible
that crabs abandon aggressive behavior that causes mortality once
density is increased to a certain level, as evidenced by some spe-
cies of fish that abandon territorial behavior if density surpasses a
certain threshold (Dr. Jon Shenker, Fl. Inst. Tech., personal com-
munication). Many shedders feel that peelers can be stocked at
extremely high densities (>250 crabs/tank) without any hannful
effects as long as good aeration is maintained in the tanks and all
crabs placed in the tank are red-line peelers. Failure to carefully
examine the molt stage of each crab placed in tanks allows the
248
Chaves and Eggleston
accidental entry of both intermolt and white-line peelers, which are
known to cannibalize red-line peelers and soft crabs.
Isolating crabs from each other may be a highly effective
method of reducing crab mortality. One of the shedders in this
study reduced mortality in his system by placing 100 plastic mesh
cylinders in each tank to isolate crabs from each other. The cyl-
inders were originally designed to eliminate cannibalism, but the
shedder felt that the reduction in mortality was much greater than
what would have been caused by cannibalism alone. Crab mortal-
ity at the study location where the cylinders were used was con-
sistently the lowest in the entire state during our study. Eliminating
physical interaction between crabs may greatly reduce crab stress.
and therefore reduce mortality. Although the use of mesh cylinders
in shedding tanks also reduced the density of crabs that could be
held in a shedding table, the lack of a decline in mortality with
decreasing crab density in this study suggests that creating physi-
cal barriers between crabs by using mesh cylinders explains the
reduction in mortality of peelers. Most shedders that we spoke with
were reluctant to try isolating crabs in tanks because they felt that
cylinders would greatly reduce the capacity of shedding tanks
during large runs of peeler crabs when tanks are stocked at den-
sities of 200-300 crabs. These peeler runs only occur two or three
times each year, however, and it is not likely that peeler supply
would exceed the capacity of 100 crabs per tank during the rest of
the shedding season.
Biologic Considerations
Effects of Crab Sex
Crab sex had a significant effect on the mortality of purchased
crabs but not on the mortality of self-caught crabs. Commercial
crab shedders that purchased crabs report that they always ob-
served higher male mortality than female mortality, whereas shed-
ders that caught their own peelers reported that they never ob-
served higher male mortality than female mortality. Several crab-
bers have suggested that high male mortality associated with
purchased crabs is the result of aggressive encounters that males
experience in hard crab pots. We could not statistically test for a
significant interaction between crab sex. crab source, and gear type
on crab mortality because nearly all purchased crabs were caught
in hard crab pots, and nearly all self-caught crabs were caught in
peeler pots. Nevertheless, relatively high male crab mortality did
coincide with the use of hard crab pots and not with peeler pots.
Female peeler crab mortality may not be affected by the use of
hard crab pots because female peeler crabs that enter hard crab
pots are usually cradled with a pre-copulatory embrace by a male
crab immediately after entry into a crab pot (personal observation i.
The male crab protects the female from other crabs and attempts to
mate with her (Dell Newman, commercial fisherman. Swan Quar-
ter. NC. personal communication). Alternatively, when a male
peeler crab enters a hard crab pot. he is not protected from ag-
gressive encounters with intermolt crabs, and may experience in-
juries or sub-lethal stress that will not become manifest until he is
placed in a shedding system and dies. In a peeler crab pot. inter-
molt crabs are rarely present, so males and females do not encoun-
ter aggressive intermolt crabs. Whether peeler crabs face sub-lethal
aggressive encounters with aggressive intermolt crabs in hard crab
pots is unknown, but further research in this area may explain the
higher mortality of purchased male than female peelers.
Effect of Female Crab Presence and Increasing Male Density on
Mortality and Tinie-to-Molt in Male Crabs
Male red-line peelers held with a relatively low density of
intemiolt male crabs experienced signit~icantly shorter times to
molt than control crabs, but time-to-molt did not differ signifi-
cantly between male red-line peelers held v\ith a relatively high
density of intennolt males and the control treatment of no conspe-
cifics. This result was contrary to the expectation that increasing
male density would lead to longer times-to-molt. The biologic
explanation for the decreasing time-to-molt of male peelers with a
low density of intermolt male crabs is unclear and warrants further
experimentation.
Male red-line peelers experienced significantly shorter times to
molt than female red-line peelers, contrary to our expectation that
time-to-molt would be equal among males and females or that
males might experience longer times to molt than females. The
findings in this study differ from the opinions of many crab shed-
ders that report male red-line peeler crabs in shedding tanks take
longer on average to molt than female red-line peeler crabs. Fur-
ther investigation might reveal the extent to which intermolt peri-
ods differ between male and female blue crabs. In this study, male
peeler crabs showed no ability to regulate their time-to-molt in
response to different situations (i.e.. the presence of red-line fe-
male, different levels of conspecific density).
CONCLUSIONS
Our study revealed sources of mortality in the North Carolina
soft-shell blue crab industry that fisherman are capable of elimi-
nating. The survival of self-caught peeler crabs is significantly
higher than for purchased crabs. Implementing best management
practices in the soft crab industry could encourage crabbers to take
better care of peeler crabs by always placing them in a cooler on
ice immediately after capture or underneath wet burlap sacks. The
benefits of best management practices will likely include a reduc-
tion in the mortality rate of peeler crabs in shedding systems,
increased financial profits for crabbers who sell peelers that are
now more likely to survive in shedding systems, and improved
profits of shedding system operators who purchase peeler. It is
important to reduce mortality in North Carolina's soft-shell blue
crab industry because 1 ) soft-crab landings are increasing rapidly
and becoming a larger component of overall landings; 2) approxi-
mately 23% crabs placed in shedding systems die; and 3) there is
an urgent need to conserve the blue crab spawning stock given the
recent 80% decline and a highly significant stock-recruitment re-
lationship for the blue crab in NC (Eggleston et al. 2002).
ACKNOWLEDGMENTS
We are extremely grateful to the blue crab shedding operators
that participated in this project: Bob Austin, Murray and Kristina
Bridges. Russel and Gerry Howell. Connie and Luke Ingraham,
Santa Klotz and Jim Messina. Pam Mason. Dell Newman. Willy
and Jake Phillips. Scott and Patti Rader. Tony Roughton and Vir-
ginia Phelps, and Phillip Smith. We also thank Dr. David Dickey,
Dr. Joe Hightower. Dr. Steve Rebach. Sean McKenna, David
Nadeau. Geoff Bell. Eric Johnson. Todd Kellison. and Ashton
Drew for scientific and editorial input. The authors thank the NC
Sea Grant/Blue Crab Research Program (grant Ol-FEG-03) for
funding this project, and Bob Hines. Dr. Steve Rebach. Marc
Turano for their enthusiastic administration of this project.
Blue Crab Mortality in North Carolina
249
LITERATURE CITED
Arv. R. D.. Jr. & M. A. Poirrier 1989. Acute Tiwicity ot Nitrile In the Blue
Crab iCcillinecle.s sa/'idiis). Progr. Fish Culiurisl 51:69-72.
Chaves, J. C. 2002. Biological and operational factors causing mortality in
North Carolina's soft shell blue crab industry. MS Thesis, North Caro-
lina State University, Department of Marine. Earth & Atmospheric
Sciences. Raleigh, NC 27695-8208 USA. 32 pp.
Das, T. & W. B. Stickle. 1993. Sensitivity of crabs CaUmecU-s sapiihis and
C similis. and the gastropod Siicmwnini luiemasumui to hypo.xia and
anoxia. Mar. Ecol. Prog. Ser 98:263-274.
Eggleston, D. B. 1998. Population dynamics of the blue crab in North
Carolina: statistical analyses of fisheries survey data. Final Report for
Contract M-6053 to the NC Department of Environmental Health and
Natural Resources, Division of Marine Fisheries, 66 pp.
Eggleston, D. B.. J. Hightower & E. Johnson 2002 Population dynamics of
the blue crab Calliiwctes sapidus in North Carolina, Fishery Resource
Grant report 99FEG-10 and 00-FEG-l I. 22 pp.
Engel, D. W. & G. W. Thayer. 1998. Effects of habitat alteralion on blue
crabs. / Sliellfr'.h Res. 17:579-585.
Henry. L. T. & S. McKenna. 1998. Status and management of the blue crab
fishery in North Carolina. / Shellfish Res. 17:465-468.
Lakshmi. G. J.. C. M. Trigg. H. M. Perry & A. Venkataramiah. 1984. The
effects of ammonia accumulation on blue crab shedding success, Mis-
sissippi-Alabama Sea-Grant Consortium, Project R/RD-2, Ocean
Springs, Mississippi.
Manthe, D. P., R. F. Malone & H. M. Perry. 1983. Water quality tJuctua-
tions in response to variable loading in a commercial, closed shedding
facility for blue crabs. J. Shellfish Res. 3:175-182.
Milliken. M. R. & A. B. Williams. 1984. Synopsis of biological data on the
blue crab. Callinectes sapidus Rathbun. NOAA Technical Report Na-
tional Marine Fisheries 1. FAO Fisheries Synopsis Number 138.
North Carolina Division of Marine Fisheries 2002. Web-site (http://
www.ncfisheries.net)
Oesterling. M. 1984. Manual for handling and shedding blue crabs {Calli-
iieeles sapidus>. Virginia Institute of Marine Science, College of Wil-
liam and Mary, Special Report 271, Gloucester Point, Virginia.
Oesterling. M. 1995. The soft crab lishery. Virginia Marine Resource
Bulletin 27:13-14.
Ryan. E. P. 1966. Pheromonc: evidence in a decapod crustacean. Science
151:340-_341.
Weis. J. S.. A. Cristini & K. Ranga Rao. 1992. Effects of pollutants on
molting and regeneration in Crustacea. Am. Zool. 32:495-500.
Wheaton, F. W. 1977, Aquacultural engineering. Wiley-Interscience. New
York.
APPENDIX
Estimation of Annual Financial Loss for North Carolina's Soft-Shell
Bine Crab Industry I sing Purchased Peeler Crabs
Assume 2.565.434 purchased peelers are placed in shedders
each year in North Carolina. Assume each dead crab represents a
loss of $2.75 (purchase price = $0,75; lost revenue = $2.00). If
one assumes a mortality rate of 16% (this study), then 410.469
dead crabs die at a cost of -$1,128,790 per year.
Jouniiil of Shellfish Research. Vol. 22, No. I, 251-254, 2003.
SEX-SPECIFIC RESPONSE TO DISTURBANCE IN A FIDDLER CRAB
PABLO D. RIBEIRO.'"^* CAROLINA G. LUCHETTI," AND OSCAR O. IRIBARNE"
^UniversidaJ Nacional dc Mar del Plata. CC 573 Corrco Central. B7600WAG Mar del Plata.
Argentina: and ^Universidad de Btieno.s Aires. Argentina CONICET. Argentina
ABSTRACT Fiddler crabs are organism with an extreme sexual dimorphism. Male crabs have an enlarged claw used for sexual
display and combat but not for feeding, which place them in foraging disadvantage when are compared with females. Given that
avoiding disturbance (e.g., predators or human activity), courting, and feeding are incompatible behaviors, males should have different
time budget to balance all the activities. In this study we experimentally evaluated the hypothesis that males of the Southwestern
Atlantic fiddler crab Uca unif;i(arensis have a sex-specific response to disturbance. We performed an experiment where we applied
an artificial disturbance (created by addition of flags). During a tidal cycle we found that males were more affected by disturbance than
females. During the ebb tide, more males than females remained into their burrows because of the artificial disturbance. After
disturbance (i.e.. when flags were removed) the male-to-female sex ratio on the surface increased in disturbed plots. However, once
disturbance was interrupted the male-to-female sex ratio on previously disturbed plots differed from the observed in control plots, being
smaller during the ebb tide and larger dunng the flood tide. The latter might indicate that male crabs increase their foraging effort to
compensate the time they loss for feeding as consequence of disturbance. Disturbance also affected the proportion of courting males,
but when disturbance was removed courtship returned to initial values of activity, which indicates that the cost of stop courting may
be higher than cost of stop feeding. However, after 27 days of experimental disturbance comparison of body condition (dry weight in
relation to their carapace width) showed no effect of disturbance, suggesting that males were able to compensate the decrease in feeding
time.
KEY WORDS: Uca unigiiayensis. fiddler crabs, disturbance, sex-specific response
INTRODUCTION
Fiddler crabs are interesting animals for studying the effect of
sexual dimorphism on their behavior. Male tiddler crabs show an
enlarged claw used for courtship displays (Crane 1975. Christy &
Salmon 1984); its size gives them an advantage in combats, bur-
row acquisitions (see Hyatt & Salmon 1979). and probably in mate
acquisition. However, given that the enlarged claw is not used for
feeding, male feeding efficiency is lower, which leads to different
foraging strategies in males and females (Vaiiela et al. 1974.
Weissburg 1993). Furthermore, the color of the claw makes males
more conspicuous and visually detectable than females (Crane
197.5), and its size hinders escape from predators (Iribame and
Martinez 1999). Thus, if the visual detectability by predators is
related to predation rate (Utne-Palm 2000) male fiddler crabs may
have higher predation insk than females because of their conspicu-
ousness and their longer time they spend on the surface (although
some field studies show the opposite situation; e.g.. Bildstein et al.
1989. P. Ribeiro. unpublished data).
Fiddler crabs are intertidal organisms with surface activity
(feeding or courtship) only during low tide remaining inside their
burrows during high tide (Crane 1975. Wolfrath 1993). During the
courtship season both sexes feed mostly during the first hours after
the tide ebbs and then, males court (by waving their large chelae)
until the tide start to fiood when they go back to feed (Crane 1975.
Wolfrath 1993). However, males usually keep feeding longer than
females before sheltering into their burrows while tide is flooding
(Wolfrath 1993). This difference may be the product of lower
feeding efficiency and/or higher energetic investment by males
during waving.
When a disturbance occurs (i.e.. predators, such as shorebirds.
human activity) most tiddler crabs shelter into their burrows as a
generalized antipredatory response (Frix et al. 1991. Iribame &
Martinez 1999). However, the trade off between feeding, mating.
*Corresponding author. E-mail: pdribeirCsmdp.edu.ar
and survival may not be the same for males and females. There-
fore, we expect the response to disturbance to be sex specific. For
example, given the visual conspicuity of the enlarged claw . males
should show an earlier response than females to disturbances or
potential predators. However, given that males need to feed longer
(Vaiiela et al. 1974), receding early inside buiTOWs may involve a
higher cost in terms of food acquisition and loss of mating oppor-
tunities.
In this work, we experimentally evaluate the hypothesis that
fiddler crabs show a sex-specific response to disturbance by study-
ing the SW Atlantic fiddler crab Uca unignayen.sis (Nobili 1901 ).
We predict the following responses to disturbance: ( I ) more males
than females will shelter into their buiTows during ebb tide, given
that the time available for feeding is still long and they may be. for
their conspicuousness. at a higher risk of predation than females;
(2) more females than males will shelter into their burrows during
flood tide because time available for feeding is now short; (3) there
will be an overall reduction in the time allocated to courtship
during the whole tidal cycle; and (4) if males do not reduce the
time allocated to courtship, their body condition will be affected.
MATERIALS AND METHODS
The study was conducted at the Mar Chiquita coastal lagoon
(Argentina. 37°32' to 37''45"S and 57''19' to 57°26'W) from Feb-
ruary 4 to March 3. 2000 (Austral summer). Uca uruguayensis
occurs in the upper levels of the tidal flats, adjacent to the border
of extensive marshes dominated by the cordgrass Spariiiia densi-
flora (Spivak et al. 1991 ). We marked 16 plots (each 2 m long and
6 m width) parallel to the shoreline with four 50-citi height (30-
mm diameter) iron stakes. Plots were arranged at the same tidal
level and were separated from each other by 2 x 2 m areas. Eight
plots were subjected to disturbance (disturbed) and the remaining
eight plots were kept as controls (control). Treatments were sys-
tematically assigned. Disturbance was applied by means of thirty
flags consisting in iron stakes (30 cm high, homogeneously dis-
251
252
RiBEIRO ET AL.
persed) with black and red nylon stripes (30 cm long. 2 cm width)
added on their tips. Nylon stripes were easily waved by the wind
and when approached to a crab induced it to shelter into its burrow.
Control plots were without these nylon stripes but we walked on
them to keep the same effect of setting up the nylon stripes as in
the treatment plots. In all cases observation of crab behavior (focal
census) were conducted using a 10 x 50 binoculars. 8 m from the
plots and 5 min after the setting or extraction of the nylon stripes,
to allow crabs reinitiate their activities after disturbance caused by
the experimental setup.
To assure that crabs were entering inside their burrows in re-
sponse to disturbance, we quantified in 12 plots the density of
crabs on the surface. In each plot we sampled one transect of 2-m
long by 0.2-m wide counting the number of crabs. Then, in six of
these plots we placed the nylon flags and quantified the number of
crabs again following the same procedure. Finally, the nylon
stripes were extracted and crabs were quantified again. Data was
square root transformed to comply with the assumptions and two-
factor repeated measures ANOVA (Neter et al. 1991 ) was used to
evaluate the density of crabs on the surface in relation to treatment
(disturbed-control) and the disturbance state (before-during-after;
as the repeated measures factor).
To evaluate the effect of disturbance on the sex ratio (males to
females) and on the proportion of courting males we carried out an
experiment encompassing a complete diurnal tidal cycle. The ex-
periment began 4 h before low tide and finished 4 h after low tide.
In disturbed plots, we applied two intervals of disturbance (from 4
to 2 h before low tide and from 0 to 2 h after low tide: thereafter
during disturbance) and two intervals where disturbance was re-
moved (from 2 to 0 h before low tide and from 2 to 4 h after low
tide: thereafter after disturbance). To measure the sex ratio and the
proportion of courting males we performed focal censuses. For this
we started by randomly taking a male crab and then we succes-
sively located the most near crab, which was sexed (a simple task
because of the sexual dimorphism). This procedure was system-
atically performed to reach a minimum quantification of 20 male
crabs. For males we noted if they were feeding or courting (de-
noted by the waving movement of the enlarged claw ). These ob-
servations were performed for both periods of disturbance and for
both periods where the disturbance was removed. Given that 4
hours since low tide represent the moment where crabs unplug
(when ebbing) or plug their burrows (when flooding), the 4 h of
disturbance affected the 50'/r of the available surface time. To fit
parametric assumptions (Neter et al. 1991 ) the sex ratio was trans-
formed to the square root of data and the proportion of courting
males was transformed with the arc-sin of the square root of data.
A three-factor repeated measures ANOVA (Neter et al. 1991 ) was
used to evaluate the effect of treatment (disturbed-control), tidal
state (ebb-tlood: repeated measures factor) and disturbance state
(during-after: repeated measures factor) on 1) sex ratio on the
surface and 2) the proportion of couiling males.
We conducted a 27-day experiment to evaluate the effect o\'
disturbance on the body condition of crabs. For this, during the
diurnal tidal cycle of all days of this period we applied two inter-
vals of disturbance and two intervals where the disturbance was
removed (similarly as was explained before). After 27 days, 10
adult males and 10 adult females (carapace width larger than 9
mm) were sampled from each plots (a total of 80 crabs of each
sex), measured (maximum carapace width, precision 0.02 mm),
and then dried at 70°C for 48 h and weighed (precision 0.001 g).
Carapace width was log transformed to fit linearity of model
(Neter et al. 1991). Differences in dry weight between treatments
(disturbed-control) in relation to carapace width were e\aluated
with ANCOVA (Neter et al. 1991 ). Given the allometric growth of
the enlarged claw of males, regression equations of dry weight in
relation to carapace width of males and females are not parallel
(f i,.ii4= 59.0.\ MS,,,,,, = 0.1534. P < 0,01 ). thus we made the
analysis for each sex separately.
RESULTS
There was an interactive effect of treatment and disttirhance
state on the density of crabs (f, .„ = 10.13. MS^.„^.^.^ = 12.14,
P < 0.001 ). The density of crabs on the surface before disturbance
was applied did not differed between the plots to be disturbed and
the plots to be maintained as controls (disturbed plots: .v = 20.83,
SE = 3.71: control plots: x = 19.58, SE = 5.35). However, the
density of crabs on the surface in disturbed plots was lower than in
control plots during the disturbance (disturbed plots: .v = 1.25, SE
= 0.51: control plots: .v = 18.33, SE = 2.26). Once disturbance
was interrupted, crab density on the surface returned to initial
values (disturbed plots: .v = 16.67, SE = 2.55: control plots: .v =
23.75. SE = 1.93).
An interactive effect between Treatment. Disturbance State and
Tidal State affected the male to female ratio on the surface (F, u
= 9.32,M5„„„ = 0.2535, /*< 0.01; Fig. 1 A). During disturbance
the male to female sex ratio was higher in control plots than in
disturbed ones. Nevertheless, the male to female sex ratio in-
creased in disturbed plots after disturbance. During the ebb tide.
10
a a
Z LU
t- a:
<> =>
X LU
LU I
CO h-
DURING
AFTER
u
J
EBB
FLOOD
Figure I, F.ffects of disturbance on the behavior of the fiddler crab
Uca uriiguayinsis. (\) Male-lo-feniaie sex ratio on the surface and (B)
proportion of courting males. Limits of boxes represent the (1.75 and
0.25 percentiles, lines represent the 0.01 and 0.99 percentiles, and the
line inside boxes is the median. Different lowercase letters indicate
differences from multiple comparisons for three factors interaction.
Different numbers indicate differences from multiple comparisons for
the Treatment X Disturbance State interaction. Significance is at P <
0.05. C = control plots, D = disturbed plots, EBB = ebb tide. FLOOD
= flood tide, DURING = during disturbance, and .AFTER = after
disturbance.
Sex-Specific Response in a Fiddler Crab
253
the increase in the male to female ratio after disturbance was not
large enough to surpass the \ alues observed at control plots. Dur-
ing the flooding tide, however, the male to female ratio observed
after disturbance exceeded the value observed at control plots.
The proportion of courting males was higher during the ebb
tide than during the flood tide iF, ,^ = 25.19. M5^.„,,, = 0.70Q4.
P < 0.001 : Fig. 1 B ). However, it was lower during the disturbance
at disturbed plots than either after the disturbance or at control
plots (interaction effect between Treatment and Disturbance state
f , ,4 = 22.18. MS^,,^.^., = 0.5884. />< 0.001; Fig. IB). There were
not sianificant interactions between Treatment and Tidal State
^F,
0.06. MS,
0.001 6. P > 0.8) nor between Tidal State
and Disturbance state (f ,.,4 = 0.02. M5,ff,„ = 0.0002. P > 0.9)
nor between Treatment. Tidal State and Disturbance state (f j ,4 =
2.36. MS^.„,^, = 0.0370. P > 0.1) on the proportion of courting
males.
There were no effect of disturbance on the dry weight of both
males (F,
0.43. MS,,
0.0019. P > 0.5; Disturbed plots.
Slope = 2.20. SE = 0.04. Elevation = 2.03. SE = 0.04; Control
plots. Slope = 2.48. SE = 0.03. Elevation = 2.34. SE = 0.04)
and females (F,;„ = 2.60. MS,,,,,, = 0.0020. P> 0.1; Disturbed
plots. Slope = 0.91. SE = 0.01. Elevation = 0.80. SE = 0.01;
Control plots. Slope = 0.99. SE = 0.01. Elevation = 0.88. SE =
0.01).
DISCUSSION
Artificial disturbances are useful for the study of behaxioral
responses of organisms by simulating natural environmental con-
ditions (e.g.. Bell 2001. Sloman et al. 2001). Responses to distur-
bances are helpful for understanding how organisms face critical
trade offs under changes in their envii-onment. Our disturbance
e.vperinients show that the fiddler crab Uca iiniguayensis has a
sex-specific response. Disturbance decreased the male to female
sex ratio on the surface, indicating that more males shelter into
iheir burrows in response to disturbance. However, during the ebb
tide and after the disturbance the male-to-female sex ratio in dis-
turbed plots was lower than in control plots. This pattern was the
opposite to the observed during the flood tide, which suggest that
the proclivity of crabs to shelter and stay inside their burrows may
depend on the time available for feeding before the tide flood their
habitat. Given that males need to feed for longer periods as a
consequence of the sexual dimorphism ( Valiela et al. 1974. Weiss-
burg 1992). the cost of stop to feed may be higher for them when
the remaining feeding time is short.
Our study encompassed the effect of disturbance at a teinporal
scale of days in relation to the body weight and at a temporal scale
of hours in relation to the behavioral avoiding response. Other
works analyzed the effect at a lower, practically immediate, tem-
poral scale where they look at the direct effect of the disturbance
in the avoidance response of crabs. Frix et al. (1991). found that
both male and feinale fiddler crabs Uca piigiUitor and U. pugiui.y
shelter into their burrows at similar rates when simulated predators
approach them indicating, in fact, that both sexes may perceive a
similar risk of predation. However, females descend further into
their burrows than males. This pattern could be expected it the
female is the most preyed sex. as is recognized to happen in the
Frix et al. ( 1991 ) study case (see also Bildstein et al. 1989). For the
case of Uca iiniguayensis. we did not investigated if feinales and
males shelter at similar rates, but instead, we know that during the
disturbance males spend less time on the surface. The dispropor-
tionate effect on males that we have observed may be expected
from a high predation rate on male crabs. However, there are not
evidences of high shorebird predation in our study site (Bogazzi et
al. 2001): but in nearby population of U. unigiuiyensis (Sambo-
rombon Bay; 36°22'S. 56°45'W) predation by migratory shore-
birds is intense (Iribarne and Martinez 1999). Nevertheless, the
occurrence and nature of sex-specific predation pressure is likely
to be dependent on the predatory species present at the locale and
their abundance because some predators prefer females whereas
other prefers males (Iribarne & Martinez 1999). In any case, it was
observed that the overall effect of predation is not male-biased
(Ribeiro et al. unpublished data). Thus, the male-biased response
to disturbance in this species is not related with the extant sex-
specific predation pressure. This response, therefore, might have
evolved under other selective forces than extant predation pres-
sure. This scenario can occur with a higher relative abundance of
shorebirds that specialize on males, such as the Ruddy Turnstone
Aremina imcipres (Iribarne & Martinez 1999).
Disturbance also decreased the proportion of courting males,
which after disturbance returned to values similar to those ob-
served in controls. This is contrary to the expectation that males
may increase their foraging effort if they loss the opportunities to
do it by evading the disturbance (or potential predators). This
response suggests that courtship is risky when disturbance is in
action, given that the male waving display may enhance their
vulnerability to predator (P. Ribeiro. unpublished data)
However, despite males lose a larger proportion of time avail-
able for feeding than females, their body condition was not af-
fected as consequence of disturbance. This might be because dis-
turbance was not so severe or the experimental period was not long
enough. Alternatively this result may indicate that crabs were able
to successfully compensate in some way for the time they loss as
consequence of disturbance, which is potentially available for
feeding. Given that males are less efficient foragers than females
(Valiela et al. 1974. Weissburg 1992) the mechanism solving this
trade off should incorporate changes in their foraging effort and
changes in the mechanisms of food delivery and extraction (Weiss-
burg 1993). The fact that the proportion of males increased after
disturbance and that it was higher in disturbed plots than in control
ones during the flooding tide strongly suggest that males are in-
creasing their foraging effort after disturbance.
ACKNOWLEDGMENTS
This project was partially supported by grants from the Uni-
versidad Nacional de Mar del Plata. IFS-Sweden (A2501-2F).
Fundacion Antorchas (Argentina AO 13672). National Geograph-
ic Explorafion Grants (#6487-99). CONICET (PIP2851). and
ANPCyT (#1-7213). all granted to O. I.). P. D. Ribeiro is sup-
ported by a scholarship from CONICET. This work is part of the
doctoral thesis of the first author.
Bell. A. M. 2001. Effects of an endocrine disrupter on courtship and
aggressive behaviour of male ihree-spined stickleback. GiisrcmsifKs
aculealus. Anim. Behav. 62:775-780.
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Bogazzi. E.. O. Iribame. R. Guerrero & E. Spivak. 2001. Wind Pattern may
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Christy, J. H. & M. Salmon. 1984. Ecology and evolution of mating sys-
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Crane. J. 1975. Fiddler crab of the world, Ocypodidae: genus Uca. Prince-
ton. NJ: Princeton University Press, 736 pp.
Frix. M. S., M. E. Hostetler & K. L. Bildstein. 1991. Intra- and interspecies
differences responses of Atlantic sand (Uca pugilator) and Atlantic
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Biol. 11:523-529
Hyatt. G. W. & M. Salmon. 1979. Comparative statistical and information
analysis of combat in the fiddler crabs. Uca pugilator and U. [nigiw.x.
Behaviour 9S:\-2?>.
Iribame, O. O. & M. M. Martinez. 1999. Predatlon on the southwestern
Atlantic fiddler crab {Uca uruguayensis) by migratory shorebirds [Plii-
vialis dominica. P. squatarola. Arenaria imerpres and Nwnenius
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Neter. J.. W. Wasserman & M. H. Kutner. 1991. Applied linear statistical
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Sloman, K. A., A. C. Taylor. N. B. Metcalfe & K. M. Gilmour. 2001.
Effects of an environmental disturbance on the social behaviour and
physiological function of brown trout. Aiiim. Behav. 61:325-333.
Spivak. E. D.. M. A. Gavio & C. E. Navarro. 1991. Life history and
structure of the world southernmost Uca population: Uca uruguayensis
(Crustacea. Brachyura) in Mar Chiquita Lagoon (Argentina). Bull.
Mar. Sci. 48:679-688.
Utne-Palm, A. C. 2000. Prey visibility, activity, size and catchability's
(evasiveness) influence on Gobiusculus flavescens prey choice. Sarsia
85:157-165.
Valiela. 1., D. F. Babiec, W. Atherton, S. Seitzinger & C. Krebs. 1974.
Some consequences of sexual dimorphism: Feeding in male and female
fiddler crab. Uca pugna.x (Smith). Biol. Bull. 147:652-660.
Weissburg. M. 1992. Functional analysis of fiddler crab foraging: sex
specific mechanism and constraints in Uca pugna.v (Smith). J. Exp.
Mar. Biol. Ecol. 156:105-124.
Weissburg. M. 1993. Sex and the single forager: gender-specific energy
maximization strategies in tiddler crabs. Ecology 74:279-291.
Wolfrath. B. 1993. Observations on the beha\'iour of the European fiddler
crab Uca langeri. Mar. Ecol. Prog. Ser. 100:1 11-118.
Journal ,yf Shfllfish Research. Vol. 22. No. 1, 255-262. 2003.
GEOGRAPHICAL EXPANSION OF A NONINDIGENOUS CRAB, CARCINUS MAENAS (L.),
ALONG THE NOVA SCOTIAN SHORE INTO THE SOUTHEASTERN GULF OF
ST. LAWRENCE, CANADA
DOMINIQUE AUDET,' DEREK S. DAVIS," GILLES MIRON,'* MIKIO MORIYASU,^
KHADRA BENHALIMA,' AND ROBERT CAMPBELL'
' Universite de Monclon. Depurteiucnt tie biologie. Monctun. Nuitvemi-Bnmswick El A 3E9 Canada;
'Nova Scotia Museum of Natural History. 1747 Summer Street, Halifax Nova Scotia B3H 3A6 Canada;
Department of Fisheries and Oceans Gulf Rei^ion. Science Branch P.O. Box 5030 Moncton. New
Brun.'iwick EIC 9B6 Canada
ABSTRACT The European green crab. Cuniiui.s maenus. was first observed in the western Atlantic in the I9th century (from New
Jersey to Massachusetts, USA). A northward expansion along the coast of New England has been observed in the first half of the second
century. The green crab was ob.served in Canadian waters in Passamoquoddy Bay in 1951. The species has gradually invaded the Bay
of Fundy in the 1950s, and the Atlantic coast of Nova Scotia from the 1960s to the mid 1990s, and reached the southern Gulf of St.
Lawrence in the mid 1990s. Further westward expansion in the southern Gulf of St. Lawrence has been confirmed along the eastern
coast of Prince Edward Island in 1997 and more recently in the Northumberland Strait at the border between Nova Scotia and New
Brunswick.
KEY WORDS: Carciiiii.', iiiacnas. green crab, geographical expansion, nonindigenous crab, northwestern Atlantic, southern Gulf of
St. Lawrence
INTRODUCTION
Accidental and voluntary introduction of species has occuired
as a result of expanded human settlement and international trade.
Over the past 200 years, the invasions were mainly due to shipping
activities. Various species of invertebrates with free-swimming
larvae were accidentally introduced into many coastal areas when
ships using ballast water appeared around 1880 (Carlton 1985).
Rui/ et al. (2000) suggested that about 298 species of invertebrates
and algae have been introduced in marine and estuarine regions in
North Ainerica. Crustaceans and mollusks constitute ca 50% of the
intruders. The green crab. Carciiius niaenas (Linnaeus. 1758), is a
good example of a species that is now well established in estuarine
habitats around the world.
Carciniis inaeiias was originally distributed along the eastern
Atlantic coast, from Norway to Mauritania including southern Ice-
land (Broekhuysen 1936. Crothers 1968, Grosholz & Ruiz 1996).
This species was recorded on the northeastern American coast in
1817 (Say 1817). Sporadic introductions in Brazil, Hawaii and
Panama Bay were also recorded in the second half of the 19th
century (Smith 1880). Australian occurrences were first docu-
mented about a hundred years ago in Port Phillip Bay. Victoria
(Thresher 1997). The crab has since expanded its distribution from
South Australia to New South Wales in the late 1970s (Zeidler
1978) and on the east coast of Tasmania in 199."^ (Gardner et al.
1994). The green crab was first recorded in South Africa near Cape
Town in 1983 and is now well established (LeRoux et al. 1990).
The species also colonized the San Francisco Bay area (California.
USA) in 1989 to 1990 (Cohen et al. 1995). The present green crab
distribution on the eastern Pacific coast lies between Morro Bay
(South California. USA) (Grosholz et al. 2000) and Esperanza
Inlet on the west coast of Vancouver Island (British Columbia,
Canada) (Glen Jamieson, pers. comm.). This rapid and irregular
expansion, which occurred from 1997 to 1999. could be related to
'Corresponding author. E-mail: mirongfe'unioncton.ca
an El Niiio event during the saine period (Behrens Yamada & Hunt
2000). According to these investigators, the green crab's range
expansion is limited off the northwestern American coasts since
then because of a declining recruitment.
On the northeast American coast, the green crab was first docu-
mented in New York and New Jersey in 1817 and slowly migrated
northward towards New England where it was reported in Casco
Bay (Maine. USA) in the early 1900s (Rathburn 1905). Through
the following 50 years, the species has colonized various estuarine
habitats along the coast of Maine up to the Bay of Fundy in Canada
(Scattergood 1952, Glude 1955. MacPhail et al. 1955).
The green crab is a voracious predator of a wide range of
invertebrates (Elner 1981) with preferences for bivalve species
(Ropes 1968) (e.g.. American oysters [Cras.sostrea virginica],
soft-shell clams |A/v(( arenciria]. blue mussels [Myliliis eihilis] and
northern quahogs \Mercenaria mercenaria]). Aquaculture stake-
holders in the southern Gulf of St. Lawrence (SGSL) expressed
serious concerns about a potential threat to cultured and wild shell-
fish populations in the Canadian maritime provinces.
The purpose of this paper is to document the northeast expan-
sion of the green crab in eastern Canadian waters, from the Pas-
sainaquoddy Bay area in New Brunswick (NB) along the shores of
Nova Scotia (NS) to the SGSL. The possible effects on the shell-
fish aquaculture industry are also discussed.
MATERIALS AND METHODS
Musium .\rclnvcs and liiWrvietts
Unpublished museum records were examined from the Nova
Scotia Museum of Natural History (NSMNH) (Halifax. NS). the
Atlantic Reference Centre (ARC) (St. Andrews, NB) and the Ca-
nadian Museum of Nature (CMN) (Ottawa. Ontario) to complete
the history of occurrence of C. macna.', along NS and NB coasts.
Interviews were carried out among twelve eel fishermen and
four fishery officers in the fall of 1998. Eel fishermen were chosen
because they were fishing in potential green crab habitats, and
fishery officers for their frequent contacts with various fishermen.
255
256
AUDET ET AL.
Interviews were held in northern NS and western Cape Breton
Island (CBI) to obtain information on the year and location of the
first green crab occurrence in commercial catches.
Survey
Annual observations on the presence and absence of green
crabs were made during the summer period (June to September)
from 1997 to 2001. Forty-six stations (estuary and river systems)
were chosen at an interval of 30-50 km along (1) the coast be-
tween the southwestern region of Bras d'Or Lakes and the tip of
CBI: (2) between the western coast of CBI and Shippagan along
the NB coast; and (3) around Prince Edward Island (PEI). (Table
1 and see Fig. 2). A frozen mackerel was placed in a modified
pearl lantern net (30 x 30 cm with two openings) and immersed to
the bottom at each observation site for a duration of 15-30 min to
determine the green crab presence.
RESULTS AND DISCUSSION
Northward Expansion of C. maenas Along the .Xew England
Coast, USA
Rafmesque (1817). as stated in Fowler (1912). reported the
presence of the green crab off the coasts of Long Island. New York
and New Jersey in 181 7. and Say (1817) confirmed the presence of
the green crab in estuarine habitats off the Atlantic coast of the
United States in 1817. Smith (1880) stated that the range of C.
maenas was limited in northwestern Atlantic waters in 1871 and
1872. At the time, the crab seemed to be found in great numbers
and well established in Great Egg harbor (New Jersey), on the
southern coast of Long Island (New York), in Long Island Sound
(Connecticut), and in Vineyard Sound. Buzzards Bay and Proxince-
town (Massachusetts) (Fig. 1). Rathburn ( 1905) reported that the
crab reached Maine's Casco Bay area (Eagle Harbor. Haipswell
and New Meadows River) in 1905. Green crab observations, how-
ever, in Maine at that time were scarce and the species was not
considered a regular member of the community before 1935 (Scat-
tergood 1952). According to Scattergood ( 1952), the northern limit
of its distribution was near Winter Harbor (Maine) from 1939 to
1942 (Fig. 1 ). Dow and Wallace ( 1952) reported that the presence
of green crabs at Lakeman's Harbor on Spruce Island near
Jonesport was observed in 1919 by a lobster fisher. There were
no further reports until 1948 and by 1951, green crabs were abun-
dant in Jonesport and also found in Lubec in Passamaquoddy Bay
(Fig. I).
Expansion of C. maenas /;/ the Bay of Fundy
A specimen of C. maeiuis was discovered in 1 95 I m the estuary
of the Digdeguash Ri\er (Fig. 1) in Passamaquoddy Bay near
Oven Head (NB) (Scattergood 1952. MacPhail 1953). Five green
crabs were also collected the same year at the mouth of Magagua-
da\ic Ri\er. near St. George (ARC unpublished records). A small
number of crabs were found a year later in the western Bay of
Fundy. Crabs were observed, however, in great numbers in the
entire Passamaquoddy Bay in Pocologan Harbor and in the Le-
preau Basin (NB) by spring of 1953 (MacPhail et al. 19551. They
then spread eastward in the Bay of Fundy (Welch 1968) where it
was reported in Sandy Cove on the northern shore of St. Marys
Bay (NS) and at the mouth of the Pereau River in the Minas Basin
(NS). both in 1953 (MacPhail et al. 1955) (Fig. 1). By 1958. green
crab populations were established in Minas Basin (Bousfield &
Leim 1960. NSMNH unpublished records). The eastward range
expansion in NS was confirmed with the presence of one crab in
Wedgeport in 1954 (MacPhail et al. 1955). A survey made from
Cape Fourchu to Three Fathom Harbor (between Lawrencetown
TABLE 1.
Confirmed sighting of green crab iCareinus maenas) in the southern Gulf of St. Lawrence and adjacent area during the summer period
(June to September) between 1997 and 2001. The station number corresponds to the numbers in Figure 2.
St.
Site
97
98
99
00
01
St.
Site
97
98
99
00
01
1
NB Shippagan
3
Tracadie
}
Escuminac
4
Richibuctou
5
Pointe-du-Chene
6
Murray Comer
7
Baie Verte
8
NS Pugwash
9
Sand Point
10
Caribou River
II
Meri2oniish
12
Malignant Cove
13
Bayfield
14
Strait of Canso
15
Mabou
16
Inverness
17
Maraaree Harbor
18
Petit Etans:
H)
Pleasant Bav
20
South Harbor
:i
Ingonish
Tl
Indian Brook
23
Baddeck
-
-
-
24
Wagmalcook
+
+
+
+
-
-
-
25
Dundee
+
+
+
+
-
-
-
26
PEI Cap Traverse
-
-
-
-
-
-
-
27
Clyde River
-
-
-
-
-
-
-
28
Vernon Bridge
-
-
+
+
-
-
-
29
Belle River
-
-
-
-
-
-
-
30
Murray Harbor
-
+
+
+
-
-
-
31
St. Mary's Bay
-
+
+
+
-
-
-
32
Cardigan River
+
-
+
+
+
+
+
33
Bay Fortune
-
+
+
+
+
+
+
34
East Lake
-
-
+
+
+
+
+
35
Naufrage
-
-
+
+
+
+
+
36
St. Peters
-
-
-
-
+
+
+
37
Winter Bay
-
-
-
-
+
+
+
38
North Ruslico
-
-
-
-
+
+
+
39
French River
-
-
-
-
+
+
+
40
Indian River
-
-
-
-
+
+
+
41
Bideford River
-
-
-
-
+
+
+
42
Cascunipec Bay
-
-
-
-
-
-
-
43
Anglo Tignish
-
-
-
-
+
+
+
44
Miminegash
-
-
-
-
+
+
+
45
Baie Egmont
-
-
-
-
+
+
+
46
Summerside
-
-
-
-
Green Crab Expansion in Eastern Canada
257
Figure 1. Historical records of sighting
contlrmcd sighting.
of Carciiiiix moeiuis from the eastern I SA to the eastern Canadian coasts. Date indicates the earliest
and Petpeswick Inlet) hy Bousfield (1958) in 1956. revealed that
C. maenas was present only in the Cape Fouichu and West Puh-
nico areas in few numbers (CMN unpublished records). According
to the NSMNH records, the presence of the green crab was con-
firmed in Westport on Brier Island in I960. MacPhail et al. (1955)
reported a low catch rate of green crabs (i.e.. an average of two
crabs a day) at the mouth of the Sissiboo River in St. Marys Bay
in the mid 1950s. From the first green crab sighting in the Bay of
Fundy, the species expanded its range more than 400 km in 2
years. The crab density increased significantly from 1952 to 195.^
in Passamaquoddy Bay (MacPhail et al. 1955).
FiirOwr Xorlhward Expansion Along the Eastern Coast of
A'oia Scotia
The spread of the green crab around the southwestern end of
NS began at the latest in 1954 to 1956. The presence of the crab
was reported in Lockeport on the southeastern coast of NS in 1960
(Anonymous 1961 ). Green crabs were considered hy fishermen to
be abundant startnig from 1964 in the La Have Islands area only 4
years after their arrival on the southeastern coast of NS (ARC
unpublished records). They were collected from Peggys Cove to
Prospect Bay from 1964 to 1966. respectively (NSMNH unpub-
lished records). After reaching Peggys Cove in the mid 196()s. the
abundance of green crabs decreased considerably and the rate of
expansion further north seemed to have diminished possibly due to
the influence of the cold Nova Scotian coastal current (Davis &
Browne 1996).
During a survey made in Passamaquoddy Bay in 1954. about
300 crabs were caught per baited trap with a 24-h soak time. In
1958, the catch rate was recorded at 53 crabs per trap per day. It
then dropped to 7.5 crabs in 1960 (Anonymous 1961). This de-
crease of crab abundance in the Bay of Fundy seems to coincide
with a general cooling period, which was reported from 1953 to
1962 (Lauzier & Hull 1962). A significant diminution of crab
abundance was also observed in Trenton (Maine, USA) where
catches decreased from 27 1 crabs per trap per day in 1953 to a total
absence in 1958 to 1965 (Welch 1968). Welch (1968) suggested
that this rapid decrease in crab abundance might be caused by
severe winter conditions along the New England coast between the
late 1950s and mid 1960s. Similarly, on the other side of the
Atlantic, winter was particularly cold in 1962 to 1963 around the
British Isles and large adult crabs did not survive the cold weather,
resulting in a major drop in density (Clay 1967). However, juve-
niles and smaller adult crabs survived and repopulated the British
Isles. The recently established population in the Bay of Fundy was
probably smaller than the one from the British Isles. Genetic varia-
tions may explain both populations" response to a cold environ-
ment. For instance, the Canadian green crabs may be less cold
environment-adapted. It then took a longer period of time for the
Maritime green crab populations to adapt to new environmental
conditions through the cold period.
258
AUDET ET AL.
Collections and records of intertidal animals made by NSMNH
(unpublished) in Halifax Harbor and at Lawrencetown in 1965 to
1966 did not include C. imwiuis. No record of the species was
made during an extensive study in Petpeswick Inlet near Halifax
Harbor in 1971 (Davis 1972. NSMNH unpublished records) or in
the St. Marys River estuary in 1973 (Davis 1976).
No surveys were conducted during the period when the north-
ern limit of the green crab distribution progressed toward the
Canso area, CBI and the northern coast of NS. The green crab
sampling program carried out by the NSMNH on the eastern shore
of NS since the late 1970s was rather sporadic. No direct study has
been conducted on C inaenas in this area until today. Green crabs
were collected in Marie Jo.seph in 1982 and in Tor Bay (NS) in
1983, the species being most likely established at these localities
before those dates. It was not observed in Guysborough Harbor in
1983. Green crab probably entered Chedabucto Bay around 1985,
which potentially provided access to the Northumberland Strait
through the Strait of Canso, and to the Bras d"Or Lakes through St.
Peter's Canal. Anecdotal information suggested the presence of
this species in the Bras d"Or Lakes before 1995 (Kara Paul & John
M. Tremblay, pers. comm.). The species is widely distributed in
the main lake since 2(J00.
Expansion of C maenas /row Cape Breton Island Toward the
Southern Gulf of St. Lawrence
The westward expansion of this species within the last 20 years
was rapid (Fig. 1). This species, considering that it was not re-
ported frequently in northeastern CBI, may have invaded the
SGSL through the Strait of Canso in the early 1990s. Squires
( 1990) misinterpretation of Bousfield and Laubitz's ( 1972) records
led him to conclude that the species was present in Northumber-
land Strait in 1 960. This result was due to the duplication of station
number series (S-series) used for studies in the SGSL in 1960 and
southwestern NS in 1963. Bousfiels and Laubitz ( 1972), however,
did not record C. maenas in the Northumberland Strait during their
studies. Eel fishermen interviewed from the western side of CBI
caught green crabs in their nets for the first time in 1998. One
fisherman from Margaree Harbor mentioned that he has been col-
lecting green crabs since either 1994 or 1995. He latter stated that
the abundance had increased in 1998. The earliest green crab re-
port concerning St. Georges Bay was from an eel fisherman in
Pomquet in 1997. The occurrence of green crabs in eel nets is
directly related to the fishing effort during the eel fishing season.
Most fishermen from Caribou up to Port Hastings have not en-
countered crabs within the years preceding the survey. In this area,
the fishing effort increased when fyke nets were first used in 1993.
As a decreasing trend in eel density and fishing effort was ob-
served in 1995 (Chaput et al. 1997, Paulin 1997), the chance of
encountering green crab might also have decreased after 1995.
Crabs were observed in great numbers in Antigonish (St. Georges
Bay) in 1999 (Jim Williams, pers. comm.).
A qualitative survey carried out along the coast of the SGSL
(from NB to CBI and around PEI) from 1997 to 2001 (Table 1,
Fig. 2) revealed that the green crab was present in estuaries along
the northeastern shore of CBI and in the Bras d"Or Lakes in 1997.
The survey also confirmed that C. maenas was present in Malig-
nant Cove (NS) from at least 1997, which was the mo.st advanced
expansion in the SGSL at the time. In 2000, the abundance pos-
sibly became greater (the catching method used was greatly influ-
enced by the abundance) and the distribution reached the eastern
opening of the Northumberland Strait. The western limit of the
green crab gradually moved from Merigotnish in 1998 to Caribou
River in 1999, indicating that the crab has been moving westward
along the coast of NS. Shellfish aquaculturists started to express
their concerns regarding green crabs off the northern coast of NS
when crabs were reported near Sand Point in Tatamagouche Bay
(J. Mark Hanson & Andrea Locke, pers. comm.) and in Wallace
Gulf of St. Lawrence
^4f Magdalen
Q"^ Island
Chedabucto Bay Bras
d'OrLakes
Figure 2. Sampling location for the survey ol the occurrence of Carcinus maenas conducted in the southern Gulf of St. Law rence and adjacent
area during summer (June to September) between 1997 and 2001. Station number corresponds to the sampling sites described in Table 1.
Green Crab Expansion in Eastern Canada
259
Bay (Marc Ouellette. pers. comm.) in 2000 and 2001 . respectively.
The crab has recently (June 2002) been collected near Port Elgin
in Bale Verte (NB) (J. Mark Hanson & Andrea Locke, pers.
comm.).
Green crabs were present in Tor Bay in 1983 and probably
invaded Chedabiicto Bay around 1985. They then spread into St.
Peters Bay to possibly reach the Bras d'Or Lakes before 1995 (D.
Davis, unpublished). A lobster fisherman reported the presence of
the species in Port Hastings, along the Strait of Canso in the early
1990s (John M. Tremblay. unpublished). It is difficult however, to
trace the pathway of the species" expansion around CBL as little
information was collected in the late 1980s and early 1990s. The
species did not .seem to reach the SGSL through the Strait of Canso
first because it was reported in St. Georges Bay only in 1997. The
presence of C. inaenas was rather first observed in the SGSL in
1994. on the western coast of CBI. Still, there is no evidence of
in\asion pathway into the western CBL
Invasion of new habitats may be due to natural larval transport
and migratory patterns, but may also be the result of transfer with
other species (e.g., oysters, blue mussels, scallops [Placopecten
nuii^ellwiicus]. American eels \Angiiilla msrnita]. and American
lobsters) from already invaded regions. Roff et al. ( 1984) studied
brachyuran larvae off the Scotian Shelf in 1977 to 1978 and re-
ported that zoeae and megalopae of C. maenas were common, but
restricted off the coast of southwestern NS. A blue mussel grower
from Whitehead. 600 km northeastward (Fig. 1). collected green
crabs in mid to late 1970s (John M. Tremblay. unpublished). This
report is the only case of a simultaneous occurrence of the species
at such distant locations throughout the northern geographic inva-
sion history of this species in the western Atlantic. A low research
effort on this species at that time may be the reason why we
observe punctual invasions (i.e., not being observed in the White-
head area for a long period after 1970). There is no reason to
presume however, that the northeastward invasion of green crab
along the NS coast is continuous and initiated by a single source
from a southwestern area. The invasion of green crab could be the
result of multiple invasions as suggested by Geller et al. (1997) for
C. aestiiarU in Japan and in South Africa. Further comparative
studies have to be carried out on the genetic characteristics of the
species along the coast of NS.
Invasion of Prince Edward Island
The geographic distribution of green crabs in PEI was limited
to the Cardigan River system, in the suinmer of 1997 (Table 1 . Fig.
2). In 1998, crabs were reported from Fortune Bay to Murray
Harbor along the eastern coast of PEI. Our survey, held from 1997
to 2001, indicated that the geographical expansion from 1999 to
present did not exceed Naufrage and Vernon Bridge on the north
and south shores, respectively. Intensive surveys conducted by the
PEI Department of Fisheries. Aquaculture and Environment
(PEIDFAE). however, showed that C. maenas was mainly re-
stricted to the southeastern coast in 1999. the distribution including
North Lake on the north shore and Gascoigne Cove on the south
shore. In 2000 crabs were detected in the Charlottetown Harbor
area, and in 2001 the western limit of the distribution moved
toward Victoria on the south shore and Savage Harbor on the
northern shore. Ovigerous females were observed in samples col-
lected in eastern PEI in the summer of 1999 (Gillis et al. 2000).
This observation suggests that this species is locally self-
reproductive. There have been isolated reports of green crabs in the
blue mussel and American oyster culture sites in Cascumpec and
Malpeque Bays in the northwestern part of PEI in 2000 (Neil J.
MacNair. pers. comm.). We consider that these crabs might have
been accidentally introduced around the Island by way of aqua-
culture activities, as no further report on the presence of green crab
in this area was made since then.
According to an investigation carried out in 1998 and 1999 by
the PEIDFAE. the green crab likely arrived as a result of natural
larval transport from NS (Gillis et al. 2000). There is no factual
data, however, to support the arrival of this species in eastern PEI
by larval transportation. If true, the megalopal settlement would
have occurred as early as the mid 1990s, shortly after the known
introduction of the crab on the western coast of CBI. Zoea larvae
can travel with cuirents in the open sea. Larvae from CBI could be
the source that fed the southeastern shores of PEI. Oceanographic
conditions between PEI and CBI appear to support this hypothesis.
Lauzier 1965 and Koutitonsky & Bugden (1991) showed that a
gyre is induced by wind and internal wave activity at the mouth of
St. Georges Bay. Currently, the green crab is observed from Pleas-
ant Bay on the northwest coast of CBI down to Baie Verte at the
NS-NB border and from Savage Harbor to Victoria, PEI. The
gradual westward progression of green crabs is taking place at a
similar rate on both sides of the Northumberland Strait.
Potential Expansion in the Gnlf nf St. iMwrence
The coastal habitats of PEI are rich in estuaries and are sur-
rounded by the warm summer waters of the Magdalen Shallows
and the Northumberland Strait. The environmental characteristics
in the SGSL are ideal for a rapid and effective proliferation of the
green crab. Warmer coastal temperatures in the summer and
shorter winters would allow the species to grow faster and to
expand their habitats as observed in the last 10 years in NS.
Lauzier and Hull ( 1 962 ) showed that the Bay of Fundy area was
under a general warming period in the 1940s and 1950s (the mean
water temperature increased by 1.8°C from 1940 to 1953). Ac-
cording to Pocklington et al. (1994). the warmest years were ob-
served from 1951 to 1953. This was followed by a cooler period
from 1953 to the mid 1960s and 1970s. The sea surface tempera-
ture followed a siinilar trend. Temperatures were above normal
from 1930 to 1960 and reached a maximum in the late 1950s.
These investigators suggest, however, that there was a general
cooling period from the 1 960s to present. In fact, water tempera-
tures between 1981 and 1990 in eastern Canada seem to be near
the long-term average and significantly colder than recorded dur-
ing the warm conditions of the 1950s (Pocklington et al. 1994).
The green crab may have invaded the Bay of Fundy during the
warming period and this species has now reached the SGSL. Good
seasonal environmental conditions in this area may contribute to a
northwestern geographical expansion in the SGSL. Cohen et al.
( 1995). for instance, predicted that C. maenas could establish itself
from California to Alaska, considering the wide range of tempera-
tures and salinities the species can tolerate in the Pacific Ocean.
At this point, the abundance of green crabs in PEI is lower than
what is observed on the northern coast of NS. A mean catch rate
of 10 ± 5 crabs per trap with a 24-h soak time was recorded in
Basin Head between 2000 and 2002 on the eastern coast of PEI
(Audet et al. in prep.). The same fishing gear captured hundreds of
crabs in a few hours in Antigonish (NS) (Jim Williams, pers.
comm.). As the green crab appears to be well established on the
western coasts of CBI through the last decade, an increase in
abundance and a westward expansion of the species are expected
in PEI and NB in the near future. A close monitoring program is
260
AUDET ET AL.
needed to follow the progression of green crabs on a possible
northwestward expansion from the edge of the Northumberland
Strait to the Chaleur Bay. where coastal temperatures (Savoie &
Lanteigne 2002) are fa\orable to the species.
Potential Impacts on the Aquaculture Industry
The green crab is an omnivorous species. Its diet includes
polychaetes, crustaceans, mollusks, and green algae (Crother 1968.
Ropes 1968). Juvenile crabs are considered, among all, as green
algae grazers, using the sea lettuce ( Ulva lactuca) beds as a refuge.
Large adult males prey on various species including commercially
exploited molluskan species (e.g., blue mussels and dogwhelks
(Nucella lapilhis). flat oysters (Ostrea ediilis). Pacific oysters
iCrassostrea gigas). the soft-shell clams and the northern quahog)
(Glude 1955, Kaiser et al. 1993. Feare 1970. Marin et al. 1973,
Mascaro & Seed 2000, Walton & Walton 20011. Naylor (1962)
and Miron et al. (2002) observed that the feeding activity of the
green crab varied considerably depending on water temperatures.
They suggested that green crabs would cause certain damage to
molluskan species during the summer period. Case studies from
NS (MacPhail et al. 1955. Ropes 1968) and Maine (Smith 1954.
Glude 1955) demonstrated a high vulnerability of molluskan spe-
cies to green crab predation. Blue mussel and American oyster
aquaculture are the most lucrative industries on PEI (Boghen
1995). Natural populations of soft-shell clams are currently heavily
exploited and a trial production of northern quahogs is underway
in PEI (Brown et al. 1995). The industry in the SGSL has an
increasing interest in the cultivation of native shellfish. The rapid
expansion of the green crab population in the same area may
threaten the shellfish aquaculture industries. Some protective mea-
sures could be used, such as fencing aquaculture sites to prevent
intrusion of the green crab as practiced in Norway to protect scal-
lops (Pecteii Diaxiiniis) against the brown crab (Cancer pagunis)
predation (Strand et al. 1999).
Physiological Adaptation and Limitation
The green crab has a high reproductive potential (e.g., 200,000
eggs per female) (Broekhuysen 1936). They are also known to be
tolerant to extreme environmental conditions (Broekhuysen 1936,
Wheatly 1981, Abello et al. 1997). The green crab population
established itself quickly in the North Pacific (Jamieson et al.
1998) by colonizing the intertidal habitat (0.7-1.4 m above mean
lower low water) in sheltered areas. Green crab inhabits depths
down to 10 m in the SGSL (Gillis et al. 2000). This is probably due
to their physiological tolerance to low water temperature condi-
tions during the winter period. Preliminary results obtained by
Audet et al. (in prep.) revealed that key biological events (e.g..
molting, mating, and egg bearing) occur later in the SGSL com-
pared with similar events occurring in the southern Atlantic (Ber-
rill 1982). Temperatures are warmer during summer periods,
reaching 26 C in lagoons on the eastern coast of PEI and -2°C
during the winter season. Water temperature remains <10°C for at
least 8 months of the year. Although the embryonic stages are
vulnerable to fluctuating water temperatures and salinities (Naga-
raj 1993, Anger et al. 1998), the species possibly adapted to a
naiTow breeding time frame during the warmer months. Zoeal
larvae, which prefer high salinities, probably migrate offshore dur-
ing ebb tides and re-invade the estuarine habitats as euryhaline
megalopae (Queiroga 1998). Nagaraj ( 1993) reported that the four
planktonic stages of C. niaenas developed successfully in tempera-
tures ranging from 10"C-25°C and salinities from 20 to 35'3f. This
may be the reason why the green crab has successfully established
itself in the Bay of Fundy and off the southeastern coast of NS
during the last 50 years despite low mean surface water tempera-
tures (12°C-14°C) (Harding et al. 1983).
A threat to the ecological equilibrium is also possible. Long
term effects are still difficult to identify at the moment, but may
have great consequences. Carcinus maenos. with its high fecun-
dity, high capability to tolerate a wide range of environmental
conditions, and omnivorous feeding behavior, appears as an ex-
cellent invader and can certainly displace endemic species. La-
goons and estuaries around PEI that have been colonized by green
crabs are also used by various commercial crustacean species such
as the American lobster. Competition for space and food may be
foreseen (Moody & Steneck 1993). The American lobsters, rock
crabs {Cancer irroratus). and various mud crabs (Ritluopanopeiis
harrisii and Drspanopeiis sayi) represent potential species that
might have to compete with the green crab in the SGSL. On the
North American Pacific coast, inter-specific competition forced
juvenile Dungeness crab (Cancer magister) to emigrate from their
natural oyster shell habitat. Green crabs also seem to be able to
dominate equal size Dungeness crabs during altercations (Mc-
Donald et al. 2001 ). It is therefore important to closely monitor the
ecology of non-indigenous species, as their ecological effects are
not well known (e.g., alteration of food webs, displacement of
other resident crustacean species).
ACKNOWLEDGMENTS
The authors thank all eel fishermen and fishery officers from
NS. NB and PEI (Canada) for the collection of valuable informa-
tion on green crab sighting and Mr. Neil J. MacNair and his team
(PEIDFAE, Charlottetown, PEI) for providing us with up to date
information regarding the PEI green crab distribution. Special
thanks are directed to Mrs. Kara Paul (Eskasoni Wildlife Com-
mission, Eskasoni, NS, Canada) and Leslie E. Pezzack (Nova
Scotia Museum of Natural History, Halifax Canada) and Drs. Glen
Jamieson (DFO, Pacific Biologic Station, Nanainio, BC, Canada),
Andrea Locke and J. Mark Hanson (DFO Gulf Fisheries Centre,
Moncton, NB. Canada). John M. Tremblay (DFO Bedford Institute
of Oceanography, Bedford. NS. Canada). Jim Williams (St. Fran-
cis Xavier. Biology Department, Antigonish, NS, Canada), Hubert
J. Squires (Paradise, NEED, Canada), and members of the Atlantic
Reference Centre (St. Andrews, NB) and Canadian Museum of
Nature (Ottawa. Ont.) for providing us valuable unpublished in-
formation on the occurrence of the green crab in eastern Canada.
We also thank Drs. J. M. Hanson and A. Locke (DFO Gulf Fish-
eries Centre, Moncton, NB, Canada) who patiently reviewed the
manuscript.
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MINIMUM ENVIRONMENTAL POTASSIUM FOR SURVIVAL OF PACIFIC WHITE SHRIMP
LITOPENAEUS VANNAMEI (BOONE) IN FRESHWATER
WILLIAM J. MCGRAW* AND JOHN SCARPAt
Harbor Branch Oceaiiogruphic Instinuion. Inc. Acjiuiciilturc Division 5600 US 1 North, Fori Pierce,
Florida 34Q46
ABSTRACT The effect of three essential osiiioregiilalory ions (Mg"*, K*. and SOj"") on the short-term survival of Pacific white
.shrimp Litopemieus vunnamei in freshwater (<l ppt total ion concentration) was e.xamined in several experiments. Shrimp posllarvae
(PL-18 and -28) were acclimated from seawater (32 ppt) to freshwater (700 ppm TDS; 280 ppm CI") over 48 h and held for an
additional 24 h before being placed in treatment solutions. Treatments consisted of various Mg-*. K*, and SO4-" concentrations in Na*
and Ca"* solutions that were all derived from chloride or sodium based chemicals added lo distilled water. Ten shrimp were placed
in triplicate 4-L plastic containers holding 2 L of treatment solutions for 24—48 h. Potassium was found to significantly (P < 0.05)
increase shrimp survival, whereas Mg"* and SOj"" had no effect. Solutions with K* exhibited an average increase in survival of 20%
and 42% above solutions without K* at 24 and 48 h. respectively. This study demonstrates the necessity of K* in "freshwater" at a
minimum concentration of 1 ppm for the survival of this euryhaline marine shrimp. The regulatory aspects involved in maintaining K*
in crustaceans under hypo-osmotic conditions are discussed.
KEY WORDS: Liuipenaeii.s rannamci. shrimp, osmoregulation, potassium, ions
INTRODUCTION
US seafood imports have steadily increased over the last sev-
eral years. Shrimp imparts alone for the year 2000 were valued at
$3.8 billion (Harvey 2002). accounting for approximately 80% of
the total shrimp consumed in this country. Although marine shrimp
is the highest seafood import in terms of dollar value, the high cost
of coastal land, user conflict, and strict requirements regarding
effluent discharge have, at least in part, prevented the expansion of
shrimp aquaculture in the U.S. (Hopkins et al. 1996). An oppor-
tunity exists to expand US shrimp culture through the use of inland
well water with low concentrations of ions (700 -3000 ppm total
dissolved solids (TDS)]. Saline well water exists under two thirds
of the United States (Feth 1970) and some catfish fanners already
use this water source for aquaculture (Teichert-Coddington, Green
Prairie Aquafarm. personal communication. 2()()()) as it provides
an added benefit of reducing the toxicity of nitrite in catfish blood
(Boyd 1990).
The use of well water from inland locations for shrimp culture
faces many challenges for development. The shrimp species best
suited for low salinity or freshwater culture is the species most
used for aquaculture in the western hemisphere: Litopenaeus van-
iiaiuei (Ogle et al. 1992. Scarpa & Vaughan 1998). Information on
essential environmental ions and minimum concentrations neces-
sary for survival and growth of this shrimp is lacking, although
salinity tolerance has been examined (Ogle et al. 1992. Scarpa &
Vaughan 1998, Laramore et al. 2001, McGraw et al. 2002). L
vannamei is being grown successfully in freshwater (700-1000
ppm TDS) at Harbor Branch Oceanographic Institution (Van Wyk
et al. 1999) and in low salinity water in other areas of the United
States (Samocha et al. 1998. Ednoff 2001. Samocha et al. 2002).
Concentrations of the major ions involved in shrimp osmoregu-
lation (Na*. Ca'*, Mg-*. K*. CP. SO4-': Schmidt-Nielsen 1990)
and total salinities of ground waters vary widely in the United
States (Saoud et al. 2002). A knowledse of which of the essential
*Current address: Taste of the Ocean Pty Ltd.. PO Box 8.52. Sydney NSW
1230. E-mail: wjm.toto@bigpond.com
tCoiresponding author. Tel: 772-465-2400. Ext. 404; Fax: 772-4601857;
E-mail: JScarpa@hboi.edu
ions and their concentrations are necessary for survival and growth
of marine shrimp in freshwater (<1000 ppm TDS) can help deter-
mine the suitability of well water sources for inland aquaculture of
L. vannamei.
During investigations of the environmental ionic requirements
of marine shrimp cultured in freshwater (McGraw & Scarpa 2002),
it became apparent that K* had a significant effect on survival. The
following work describes a series of experiments that examined
the effect K* had on the short-term survival of the Pacific white
shrimp Litopenaeus vannamei in freshwater.
MATERIALS AND METHODS
Experimental Design
Postlarval Pacific white shrimp (Litopenaeus vannamei Boone)
(PL-9: 9 days after larval metamorphosis), were obtained from a
commercial hatchery (Shrimp Improvement Systems. Islamorada.
FL). Shrimp were cultured in seawater (local source. -32 ppt) and
fed a prepared diet (44% protein. Bonney. Laramore & Hopkins.
Inc.. Ft. Pierce, FL) ad libitum three times per day until the be-
ginning of salinity acclimation (PL-L5 and -25). Postlarval shrimp
were acclimated from 32 ppt seawater to HBOI freshwater (-280
ppm chloride. -700 ppm TDS) al a rate of 50% reduction in
salinity per 8 h over a 48-h period (Van Wyk et al. 1999). Accli-
mation was stopped after 48 h. Shrimp were held in HBOI fresh-
water for another 24 h before being placed in triplicate 4-L plastic
containers filled with 2 L of the various treatment solutions, which
included HBOI freshwater (well water) as an outside control.
Shrimp density was 5 PLs/L. Diffused air was used to aerate all
treatment and control solutions.
Treatment solutions were prepared by adding reagent-grade
chloride-based chemicals (NaCl. CaCK*2H,0. MgCU*6H,0 and
KCl. Sigma Chemical. St. Louis. MO), except sulfate (Na^SOj). to
distilled water. All ions in treatment and control solutions, except
sodium and chloride, were measured using Hach DR/3 spectro-
photonietric methods (Hach Company, Loveland, CO). Chloride
was measured using a titration kit (Lamotte Co., Chestertown.
MD). Sodium concentrations were calculated. All measured ion
concentrations were within 5% of the listed treatment values. Tem-
263
264
McGraw and Scarpa
perature and pH were measured using a standard mercury ther-
mometer and pH meter (pH, Engineered Systems and Designs.
Newark. DE). respectively. Survival of shrimp was checked at 24
and 48 h after placement into treatment solutions. Shrimp were not
fed during the 48-h test peiiod.
Statistical tests [general linear model (GLM), analysis of vari-
ance. Dunnets and Student-Newman-Keuls] were used to com-
pare survival between ion treatments and control versus ion treat-
ment waters iLentner & Bishop 1993). All percentage survival
data were transformed (arcsine*square root) before statistical
analyses (SuperAnova. Abacus Concepts Inc.. Berkeley. CA). Dif-
ferences were considered significant if P < 0.05.
Effect of Mg'* and K* Inns
The effect of different Mg"* and K* concentrations on short-
term survival of postlarval (PL- 18) shrimp in Na* and Ca"* solu-
tions was e.xamined. Sodium and calcium concentrations were held
constant at .^00 ppm ( 1 .i niM) and 60 ppm ( 1 .5 niM). respectively.
To this base solution. Mg~^ and K* were added at 75 ppm (.^1
mM) and 10 ppm (0.26 mM). respectively, alone, in combination,
or not at all (Table 1 ).
Ion concentrations of treatment solutions were based on HBOI
freshwater ion data taken from an alternate well water source that
was different in ionic composition than the well water used in the
present study as a control. Chloride concentrations of ion treatment
solutions ranged from 568 to 796 ppm (16 to 22 mM) with cal-
culated total ion concentrations varying from 928 to 1241 ppm.
Temperature of all solutions was 25-27°C and pH values of treat-
ment water ranged from 6.5 to 6.7. whereas the HBOI water pH
was 8.2.
Effect of SOj-' Ions
The effect of sulfate ions on short-term survival of postlarval
(PL-28) shrimp was examined because sulfate is: 1) present in
HBOI freshwater; 2) considered to be an essential ion; and 3) was
not tested in the previous experiment. Sodium, Ca"*, and Mg"*
concentrations were held constant at 290 ppm ( 12.6 mM). 54 ppm
(1.3 mM). and 53 ppm (2.1 niM). respectively (Table 2). To this
base solution. K* and SO4"" were added at 15 ppm (0.39 mM) and
140 ppm (1.4 mM). respectively (Table 2). Sulfate was added to
treatment solutions as Na^SOj with all sodium ions accounted for.
All treatment solutions were prepared as described previously with
one exception; NaHCO^ was added to produce 16 mg/L alkalinity
as CaCOj (with all additional Na* ions accounted for. causing a
decrease of 15 ppm of CP). Chloride concentrations of treatment
solutions ranged from 592-682 ppm (17-19 niM) with calculated
total ion concentrations ranging from 1079 to 1 144 ppm (exclud-
ing bicarbonate ions). Temperature and pH of treatment solutions
were 26-27'^C and 7.3. respectively.
Effect of K* Ions
Results from the previous experiments indicated that K^ had a
major effect on short-term postlarval shrimp survival. Therefore,
the effect of various K* concentrations on short-term survival of
postlarval (PL-28) shrimp was examined. Sodium. Ca"*. and Mg^*
concentrations were held constant at 290 ppm ( 12.6 mM), 54 ppm
( 1.3 niM), and 53 ppm (2.1 mM), respectively. To this base solu-
tion. K"^ (as KCl) was added at graded levels ( 1-50 ppm. 0.02-1 .3
niM; Table 3). Sodium bicarbonate (NaHCO,) was added to pro-
duce 80 mg/L alkalinity as CaCO,. This increased pH values from
6.5 to 6.7 to between 7.4 and 7.6, closer to that of the control (8.2).
Temperature was maintained at 26-27°C. Chloride concentrations
of treatment solutions ranged from 626-694 ppm (17-20 mM)
with calculated total ion concentrations ranging from 1023 to 1141
ppm (excluding bicarbonate ions).
RESULTS
Effect of Mg'* and K* Ions
Mean survival of postlarval shrimp (PL- 18) in treatment solu-
tions ranged from 73-97% for 24 h and from 43-83% for 48-h
survival periods (Table 1 ). Potassium had a significant effect (P =
0.023) on 24-h survival of L. yaniiuiiwi postlarvae but not so on
48-h survival (P = 0.075). Magnesium did not significantly affect
shrimp survival for either time-period (24 h: P = 0.092; 48 h: P
= 0.789). There were no significant interactions for either time
period (24 h: P = 0.171; 48 h: P = 0.491). Survival in the full
complement ion solution (containing all five ions: Na*, CI", Mg"*,
Ca"*, K*) was not significantly different than HBOI water for the
24- and 48-h periods; (24 h: P = 0.673; 48 h: P = 0.899). The
highest 24- and 48-h survivals were obtained w ith treatment 3 and
the HBOI water, which contained all of the treatment ions.
Effect of SOj- Ions
Mean survival of postlarval shrimp (PL-28) in treatment solu-
tions ranged from 43-86% for 24 h and from 26-86% for 48 h
(Table 2). Among the four individual treatments there was no
statistical difference, however, there was significantly (P < 0.01)
lower survival between treatments I and 2 (without K"^) compared
with treatments 3 and 4 (with K*). Survival for the treatment
TABLE I.
Mean (± SE, n = i) 24- and 4S-h survival (%) of PL-18 L. vannaniei in different ion solutions (ppm). Potassium had a significant effect
(P < (1.05 level) only for the 24-h survival period.
ppm"
Solution
Na"
Ca-*
Mg-*
K*
cr
Total Ions
24-h % Survival
48-h f^f Survival
1
.^00
60
.■^68
428
73(8.8)
60(17.3)
2
.^0(1
60
7.S
786
1221
77(6.7)
43(13.3)
3
3()(.)
60
7.S
10
796
1241
97(3.3)
83(12.0)
4
300
60
10
."^77
447
83 (5.8)
77(12.0)
Control
IKl
44
31
10
2X0
."^46
93 (5.8)
87 (8.8)
" Total ppm ion value for control does not include SOj'^ (106 ppm
) or trace elements.
Pacific White Shrimp Survival
265
TABLE 2.
Mean l±SE. n = i) 24-iin(l 48-h siirxJMil ['ft I of PL-28 /.. raiinaimi in different icin solutions (ppnil.
ppm
Solution
Na*
Ca-*
Mr*
K*
SO4--
CI"
Total Ions
24-h Ci Survival
48-h % Survival
1
240
54
542
SS6
43(12.0r'
30(15.3)-'
2
290
54
53
682
1079
53(1 3.3 )■'
26(13.3)"
3
29(1
54
53
15
696
iins
S6(3.3)-'
86(6.7)''
4
29(1
54
53
15
140
592
1144
X3(I2.0)"
76(18.6)"
Control
ISl
44
31
10
106
280
652
90 (5. SI
90(5.8)
Total ppni Ion value for control does not include trace element.s.
Survival values followed by a different superscript are significant at the P < 0.05 level
containing all the major essential ions (#4. Table 2), although less,
was not significantly different for either time period {P = 0.92.
0.78) compared with HBOI water.
Effect of A* Ions
Mean sur\i\al of postlarval shrimp (PL-28) at different potas-
sium concentrations ranged from 47-93% for 24 h and from 37-
90% for 48 h (Table 3 ). At 24 h. there was no significant difference
among treatments, but after 48 h. survival was significantly re-
duced at 0 ppm K-'' (P = 0.01, Table 3). The HBOI water treat-
ment showed interiTiediate survival compared with the other ion
treatments.
DISCUSSION
Theie is u dearth of information regarding minimum environ-
mental concentrations of indi\ idual ions necessary for the survival
of marine shrimp species cultured in freshwater (<1000 ppm TDS).
Preliminary experiments at HBOI have shown that CP is neces-
sary at concentrations >200 ppm for L. vannamei survival (Scarpa,
unpublished data). Chloride and Na* have been determined by
Chen and Chen (1996) to be the major ions contributing (88.4%)
to heniolymph osmolality in marine shrimp. The addition of Ca"*
to freshwater is thought to be necessary for the survival of shrimp
because this ion is needed to form the exoskeleton. which is shed
repeatedly during molting (Villalon 1991. Wyban & Sweeny
1991 ). Shrimp exuvia is composed mainly of CaCO, (99% of the
inorganic portion; Richards 1951). L. vannamei does not possess
internal Ca"* reserves like some freshwater crustaceans (McWhin-
nie 1962). Therefore Ca"* must be continually absorbed from the
environmental medium (Robertson 1953. Greenaway 1983).
In the present study. Mg"*. SO4"". and K* ions were examined
for their effect on survival of postlarval L. vannamei in "artificial"
freshwater (i.e.. distilled water with sodium, chloride, calcium and
carbonate). Magnesium and SO4"" were not found to have a criti-
cal effect on short-term survival. Magnesium and Ca"* have been
linked to membrane integrity (Douglas & Home 1997) and Mg"*
concentrations in heniolymph have been conelated with crusta-
cean activity (Mcfarland & Lee 1963). Sulfate is the third most
prominent ion in seawater. but it has been shown to be nearly
undetectable in shriinp heniolymph at low salinities (Dall & Smith
1981).
Ti"eatnient solutions without |ii)tassium in the present study had
lower survivals compared with solutions with K*. Potassium was
shown in all three experiments to be a significant factor contrib-
uting to the short-term survival of L. vannamei. The addition of 1
ppm of potassium doubled survivals over treatment waters with
only Na*. Ca"*. and Mg-*.
Compared with the other essential ions, K* is a minor constitu-
ent in brackish and fresh water (Home 1969). but this ion plays a
major role in metabolism of invertebrates (Schmidt-Nielsen 1990).
Potassium was suggested by Robertson (1953) to be important in
the maintenance of neroniuscular efficiency in decapods, whereas
other authors have discussed the iinponance of K* in cmstacean
metabolism (Gross 1958. Bursey & Lane 1971. Dall & Smith
1981. Schmidt-Nielsen 1990). Enzyme activity is directly depen-
dent on K* concentration, which is maintained within narrow lim-
its in the heniolymph of penaeids despite changing environmental
salinity (Gross 1958. Bursey & Lane 1971. Dall & Smith 1981).
In the marine environment, K* was constantly regulated in the
heniolymph of P. dnnraiiim as the salinity of the extemal medium
TABLE 3.
Mean (±SE, n = }) 24- and 48-h survival CXr 1 of PL-28 /,. vannamei at different K* concentrations (ppm).
Solution
Na*
Ca-*
Mg=*
K*
ci-
Total Ions
24-11 % Survi
1
290
54
53
0
626
1023
47(14.5)"
2
290
54
53
1
627
1025
83(11.5)"
3
290
54
53
10
635
1(342
90 (5.8)"
4
290
54
53
25
649
1071
93 (3.3)"
5
290
54
53
50
694
1141
93 (3.3)"
Control
181
44
31
10
280
546
80(14.1)
48-h ^, Survival
37(3.33)"
80(6.7)"
77 (8.8)"
77 (3.3)"
90(10.0)"
60(5.0)
Total ion value for control does not include trace elements.
Survival values followed by a different superscript are significant at the /•' < 0.05 level.
266
McGraw and Scarpa
changed (Bursey & Lane 1971 ). Potassium concentrations of 9-10
meq/L in the hemolymph were maintained between salinities of 7
to 35 ppt. whereas CI" and Na* concentrations were similar to that
of the surrounding medium. Four Australian shrimp species stud-
ied by Dall and Smith ( 1981 ) showed hemolymph K* concentra-
tions were maintained between 5 and 1 5 meq/L over a range of 1 0
to 30 ppt salinity, with a trend of K* accumulation with increasing
salinity. Potassium ions in P. monodon hemolyinph were strongly
regulated during changing environmental salinity (Lin et al. 2000).
Shrimp transferred from 45 to 1 5 ppt showed K* levels reached a
steady state after 4 h. Euryhaline penaeids sampled from Gulf
Coast waters showed higher K* concentrations in muscle tissue
compared with stenohaline species taken from the same area (Mc-
Farland & Lee 1963).
The ability of K* to be stringently regulated in the hemolymph
can be partly explained by the regulation process of this ion. Gross
(1958) stated that hemolymph Na* and K* concentrations were
maintained in an intertidal crab {Pachygrapsus crassipe.s) via in-
tracellular pools as well as active uptake under hypotonic condi-
tions. Changes in the Na* and K* concentrations between the
hemolymph and surrounding medium were 84 and 68%. respec-
tively, of the total hemolymph ion change while the additional 16
and 32% were assumed to come from internal salt pools.
Hemolymph K* concentrations were maintained within narrower
limits than Na* concentrations, despite the change in ion concen-
tration of the surrounding medium. These salt pools were thought
to be an ecological adaptation to buffer the ionic change between
incoming and outgoing tides in an estuarine environment (Gross
1958). Gilles and Pequeux ( 1983) made a similar determination. A
large decrease in the intracellular K* concentration of crustaceans
appears to occur immediately following the application of hypo-
osmotic conditions. The decrease in intracellular K* concentration
being inversely proportional to the extracellular K* concentration,
with lower extracellular K* concentrations producing a greater
release of K* from isolated cells.
The increase in pH between the first, second and third experi-
ments of the present study did not appear to increase survival of
shrimp. Optimum pH values of 6.6-8.5 for L. vannamei have been
reported by Tsai (1990) and the pH values for all experiments
listed here were within that range (within 0.1 pH value). Pillai and
Diwan (1999) did not find any correlation of pH (7.04-7.84) with
ion concentrations in the hemolymph of the shrimp Metapeiuieiis
inonoceros taken from a tropical estuary over an 18-nio period.
Wickens ( 19841 observed good growth and survival of P. monodon
at pH values ranging from 6.7 to 7.9.
Although the present study used PLs of slightly different ages
for each experiment (PL- 18 to -28), it is unlikely that osmoregu-
latory ability differed between these age groups. McGraw et al.
(2002) found that PL- 10 L. vannamei had significantly lower sur-
vival than PL- 15 and -20 when subjected to various acclimation
rates, however, survival of PL- 15 and -20 age groups were not
different from each other. This is probably caused by the full
development of gills and osmoregulatory capacity of postlarval L.
vannamei. which occurs at approximately PL- 12 (Lucu 1990. Pe-
queux 1995, Van Wyk et al. 1999). Similar results have been ob-
served for other penaeid species postlarvae (Olin & Fast 1992,
Tsuzuki et al.. 2000).
It is also unlikely that major environmental ion deficiencies
may be compensated through dietary supplementation and. there-
fore, feed supplementation during the trial would have had little
impact. Dietary calcium supplementation for catfish cultured in
calcium-free water had little effect on body calcium levels or spi-
nal deformities (Scarpa & Gatlin 1993).
The present study establishes the importance of potassium to L
vannamei survival in freshwater solutions. Potassium addition to
ponds as potash (K,0) has been recommended for pond fertiliza-
tion (Boyd 1990) and ion supplementation (Boyd 2002). Potash
has been used as a source to increase potassium concentrations in
ponds for growing shrimp (Teichert-Coddington. Green Prairie
Aquafarm, personal communication, 2000); however, the eco-
nomical feasibility of this practice for shrimp culture in inland
freshwater locations has yet to be determined. Because of this,
decisions regarding potential inland sources of saline well water
for growing L. vannamei should focus in part on the presence and
concentration of potassium in water sources.
ACKNOWLEDGMENTS
We thank the Harbor Branch Institution post doctorate fellow-
ship program for providing funds for this research. Special thanks
to the HBOI library personnel for providing many invaluable li-
brary searches and interlibrary loan documents. Gratitude is also
expressed to those who have critically reviewed this manuscript.
This is HBOI contribution 1495.
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PHYSIOLOCxICAL AND GENETIC VARIATIONS IN DOMESTICATED AND WILD
POPULATIONS OF LITOPENAEUS VANNAMEI FED WITH DIFFERENT
CARBOHYDRATE LEVELS
LETICIA ARENA.' GERARD CUZON." CRISTINA PASCHAL,' GABRIELA GAXIOLA.'
CLAUD SOYEZ,- ALAIN VAN WORMHOUDT.' AND CARLOS ROSAS'*
^Lahoratorio cle Biologi'a Marina Expcriincntiil. Apclo. Post 69. Cd. del Carmen. Camp.. Mexico:
'Centre Oceanologic/tic dii Pacifiqiie. BP 7004. Taravao. Taliiti, French Polynesia: and Station du
Biologic Marine du Mti.seum National d'Histoire Naturelle el du College de France. BP 225. 29900.
Concarneau. France
ABSTRACT The relationship between polymorphism of a-amylase and physiologic and hiochemical hehaMur of /.. vniiiuiiiici was
used to determine whether artificial selection based on body weight and body size affect the adaptation ability of shrimp to use dietary
carbohydrates as a source of energy. Shrimp fitness was addressed by measurement of energy balance using growth (P), oxygen
consumption (R). and ammonia excretion (U) of juveniles from wild. 7th, and 25th generations of cultured shrimp. Hemolymph
glucose, digestive gland glycogen, amylase activity, and amylase polymorphism was also evaluated in the three shrimp populations.
Heterozygosity, amylase activity, and starch metabolism were affected by artificial selection of L vaiinamei. Shrimp from a 25th-
cultured generation had less heterozygosity and physiologic alteration than did wild shrimp. Shrimp from a 7th-generation cultured
shnmp population showed an intermediate state of genetic and physiologic alteration. Although a statistical comparisons cannot be
made between the three studied populations, it is evident that there is a reduction in amylase activity related to shrimp domestication,
with high values in wild shrimp (between 24 to 39 lU mg"' protein), intermediate in 7th-generation cultured shrimp (between 16 to
25 lU mg'' protein), and low in 25th-generation cultured shrimp (between 3.6 to 15.8 lU mg"' protein). A reduction in the frequency
of alleles of amylase genes possibly related to domestication of shrimp was also demonstrated. It appears that the reduction of allele
frequency of ainylase genes affected the adaptative ability of shrimp to use dietary carbohydrates as a source of energy and molecules
and caused farmed populations to be protein dependent. Results of energy balance studies indicate that there are differences in
production efficiency (P/AS) between populations: a reduction in P/AS as a function of generations of farmed shrimp suggests that
efficiency with which shrimp transform energy into biomass is reduced with artificial selection.
A'£l' WORDS: Liiupciiaciis vuimamei. physiology, genetics, populations, domestication, bioenergetics. blood parameters
INTRODUCTION
The Pacific white shrimp L. vuwuiiiici (Boone) is the most
important shrimp species cultivated in the Antericas and the sec-
ond in word production (Ben/ie 2000). More than 90% of the
shrimp cultivated in 1998 on the American continent were L. van-
luiinei (132 000 t; Rosenberry 1998). For that reason, shrimp farm-
ers are establishing selective breeding programs for L. vannamei
throughout the natural range of the species, as well as the US
Atlantic coast and Bra/il (Sunden & Davis 1991. Paiva-Rocha
2001. Garci'a-Calleja 2000). These programs are motivated in pail
by the serious disease problems caused by uncontrolled farmed
population movements (Wyban et al. 1993. Bedier et al. 1998) and
are focused to obtain better profitability through the selection of
body weight or body size for optimal harvest. Although a better
growth rate has been observed in breeding programs with L. van-
namei. the impact of reported reduction of genetic diversity
(Sunden & Davis 1991 ) on the general physiology of shrimp is not
known (Benzie 1998). In a recent study Xu et al. (2001 1 showed a
reduction in genetic diversity in cultured P. numodon compared
with wild populations. That genetic differentiation pattern among
populations was related to the prevalence of IHHNV viral disease
in the same populations, indicating that the change in genetic
diversity of shrimp could change the disease susceptibility of cul-
tured or wild shrimp, affecting their fitness.
Assimilation (As) is the key characteristic of living organisms
because it is a direct index of the energy allocated to body weight
or cametes or to maintain homeostasis. According to Lucas ( 1993).
*Corresponding author. E-mail: cr\'@hp.fciencias.unam.mx
As = P -I- R, where P is the energy allocated to production of
biomass or gametes and R is the metabolizable energy. Although
the fitness of a population has reproductive consequences, in a
practical sense many researchers have been using the energy bal-
ance on juvenile forms to determine how the environmental fluc-
tuations or types of food affect the energy allocation in Crustacea
trying to predict the environmental or nutritional consequences in
energy pailitioning (Mayzaud & Conover 1988. Stickle et al. 1989,
Du-Preez et al. 1992. Koshio et al. 1992. Hopkins et al. 1993,
Rosas et al. 1993, Rosas et al. 1995, Guerin & Stickle 1997, Rosas
et al. 1998, Rosas et al. 2001).
The energy derived from food depends on mechanisms of
transformation of dietary components that, in turn, depends on the
ability of organisms to hydrolyze. absorb, and assimilate those
dietary nutrients (Ceccaldi. 1998). In a series of recent articles, we
have demonstrated that energy allocation derived from dietary car-
bohydrates (CHO) has been found to be a limiting factor in L.
stylirostris. L. vannamei. and L. seliferus (Rosas et al. 2000a,
Rosas et al. 2000b. Rosas et al. 2001 ). In these works, we reported
that glucose uptake in metabolism was limited because of satura-
tion of a-amylase when shrimp are fed with diets above 33%
CHO. At the same time, the digestive gland was saturated with
glycogen in shrimp fed with diets >33% CHO, affecting nutrient
absorption and in consequence growth rate and biomass produc-
tion. Shrimp fed without dietary CHO can produce their own CHO
using the gluconeogenic pathway, demonstrating that shrimp pro-
tein metabolism is well adapted to produce its own metabolic
energy despite energy lost through ammonia excretion.
Shrimp a-amylase is one of the best-studied polymorphic di-
gestive enzymes in shrimp. Two allelic forms were measured in
Aselhis aquaticus, four isoforms in Palaemonetes varian.'i, seven
269
270
Arena et al.
isoforms in P. elegans three isoforms in P. serratiis and L. van-
nainei. and three in Farfaiuept'iiaeiis nolialis. in L. schmitti. and in
L. setifems (Lomholt & Christensen 1970. Christensen & Lomholt
1972. Van Womihoudt 1983. Van Wormhoudt & Favrel 1988.
Diaz et al. 1995, Le Moullac et al. 1996, Ball et al. 1998. Arena
1999. Garci'a-Machado et al. 2001). This enzyme can be induced or
repres.sed by dietary CHO. protein levels, or by circadian. annual,
or moult cycles (van Wormhoudt 1974. van Wormhoudt 1977).
Van Wormhoudt et al. (1980) reported a reduction in amylase
activity in Palaemon serratiis as a function of the increase in
dietary glucides. Rosas et al. (2000a) showed an increase in
a-amylase of L. stylirostris as a function of an increase in dietary
CHO levels. Lovett and Felder (1990) stated that a significant
increase in amylase activity of L. setifems postlarvae might be a
response to low levels of CHO in the postlarval diet. Le Moullac
et al. (1996) reported a reduction of amylase activity in L. van-
nainei when the amount of this protein increa.sed in diets, showing
that a-amylase gene expression could be repressed by casein, re-
flecting the control that diet has on activity of amylase isoforms. In
the present research, a relation between polymorphism of a-amy-
lase and physiologic and biochemical behavior of L. vannamei was
used to study whether artificial selection based on body weight and
body size affected the ability of shrimp to use dietary CHO as a
source of energy. Shrimp fitness was addressed through measure-
ment of energy balance using growth, oxygen consumption, and
ammonia excretion of juveniles from wild, 7th, and 25th genera-
tions of cultured shrimps. Hemolymph glucose, digestive gland
glycogen, amylase activity, and amylase polymorphism was also
evaluated in the three shrimp populations.
MATERIAL AND METHODS
The study was divided between two experiments. The first was
conducted in Mexico where comparisons were made wild and
7th-generation specimens of L. vaniuiniei. The second experi-
ment was conducted at the French Marine Research Institute
(IFREMER) Tahiti facilities with 25th-generation specimens of L.
vannamei. Both experiments were conducted under the same basic
conditions and with the same experimental diets.
Experimeiilal Conditions
For experiment I , live wild L. vannamei {n = 200; 0.8 ± 0. 1 g
wet weight) were collected from Huizache and Cainianero Lagoon
on the Pacific Coast of Mexico. Shrimp were transported by plane
in plastic bags with cool sea water {30%c salinity; 20°C) to the
Experimental Marine Biology Laboratory of National Autono-
mous University of Mexico in Cd. del Carmen. Campeche.
Mexico. Shrimp were acclimated to laboratory conditions for 2 wk
before any experimental procedure was initiated. During this pe-
riod shrimp were maintained in a circular external pond (20 nr)
with aerated (O^ > 5.0 mg/L) natural seawater (32%c; 29 ± 2°C).
During acclimation, shrimp were fed twice each day on a com-
mercial shrimp diet containing 45% protein (Api Aba camariin
ultra, Malta Clayton SA"' ). At the same time, a sample of 7th-
generation cultured shrimp in = 200; 0.03 ± 0.03 g live weight)
from a farm located in Sisal. Yucatan, was transported to the
laboratory in cool sea water (35%c salinity, 24°C) and acclimated
under identical conditions to those described above.
After 2 wk of acclimation, a sample of each population was
removed and distributed in 90-1 plastic tanks. For experiment one,
shrimp were reared for 55 to 58 days in a flow-through sea water
system (32%r salinity) at a density of 10 shrimp per tank. For
experiment 2. we used 1600 postlarvae (0.009 ± 0.001 wet weight)
of 25th-generation L vannamei obtained in the IFREMER hatch-
ery facilities. In IFREMER shrimp were reared in 800-L tanks for
36 days in a flow-through sea water system (36%o salinity) at a
density of 100 shrimp per tank.
In both experiments shrimp were fed three times a day (0800,
1400, and 2000 h), uneaten food particles were removed twice a
day (0730 and 1700 h) and water quality variables were main-
tained as temperature 28 ± 1°C, dissolved oxygen >5.0 mg/L, and
pH >8.2 ± 0.3. In both locations the photoperiod was set at
12h/12h. Samples of digestive gland for biochemical and genetic
analysis from experiment 1 were stored at -80'"C and then freeze-
dried until analysis. Digestive glands from 25th-generation culti-
vated shrimp were freeze-dried at the IFREMER facilities in Tahiti
before analvsis.
Diets
L. i</;;/!(////(7 juveniles were fed practical diets, formulated with
two levels of carbohydrate (CHO); 3% and 44%. Experimental
diets were prepared by thoroughly mixing dry ingredients with oil
and then adding water until a stiff dough resulted. The dough was
passed through a mincer with a 2-mm die, and the resulting spa-
ghetti-like strings were air dried at 60°C. After drying, the strings
were broken up and sieved to a convenient pellet size and stored at
-4°C.
Growth and Survival
The growth rate was evaluated as the difference between wet
weight at the beginning and end of the experiment and calculated
as daily growth coefficient {DGC.%; Cho 1992):
DGC = ia)x ||finalweight{g)]"'-[initialweight(g)'"])/time(days)
The DGC measure was chosen to make comparisons in growth
tests because initial weights were different between treatments
(Bureau et al. 2000. Cho 1992). The sur\ ival rate was calculated as
the difference between the number of live animals at the beginning
and end of the experiment.
Amylase Activity
At the end of growth trials, digestive glands from fasting ( 1 2 h)
shrimp (40 per treatment) were dissected immediately, quickly
frozen in liquid nitrogen, and then kept at -80°C for subsequent
analysis. Frozen samples were homogenized in 500 p-L of ice-cold,
deionized water. Honiogenates were centrifuged at 16000 i; for 6
niin at 8°C. Part of the supernatant was diluted in 10 volumes of
ice-cold deionized water. Honiogenates (crude or diluted) were
immediately used for enzyme analysis (Brito et al. 2001). The
soluble-protein content was measured in diluted honiogenates by
the Bradford (1976) method using the Sigma Micro Protein De-
termination Kit (Procedure No. 610). Samples were read in a Bio-
Rad model 550 microplate reader at 495 nni. Duplicate assays for
each sample were made. Amylase activity was assayed in diluted
homogenates according to the method Bemfeld (1955) with 1%
oyster glycogen (Sigma G8751) as substrate in 10 niM phosphate
buffer, pH 7. One unit of amylase activity was defined as I mg of
maltose liberated in I niin at 30^C. Each sample was assayed in
duplicate. Activity was expressed in units of |xM substrate cleaved
Physiologic, Genetic Variations in L. vannamei
271
per minute, based on an extinction coefficient Kj,,, = ISOOO L
nior' cm"'. Each sample was assayed in duplicate.
HeiiKilymph (iliuiise
Blood glucose measurements were made in the same shrimp
sampled for amylase activity. Before sampling, shrimp were
placed in chilled (1S°C) and aerated seawaler for .S niin to reduce
the effect of manipulation before the hemolymph extraction (Rosas
cl al. 200()a). Only shrimp in intemiolt stage (C stage) were used.
Hemolymph (approximately 200-300 jxL per shrimp) was indi-
vidually sampled through a chilled syringe needle inserted at the
base of the fifth pereiopod after the shrimp had been dried with a
paper towel. The individual weight (±0.05 g) was measured.
Molting stages were identified by uropod examination (Drach &
Tchernigovtzeff 1967). Commercial kits were used for glucose
(GH: GOD-PAD. Merck- 740393 ) determinations and were read
with a microplate using 20 p-L of plasma (obtained after 8000 g
centrifugation) and 200 p,L of enzyme chromogen reagent. Absor-
bance was recorded in a microplate reader (Bio-Rad model 550)
and concentrations were calculated from a standard solution of
substrate.
Glycugeii Concentration in Digestive Gland (DGG)
Glycogen was extracted in the presence of sulfuric acid and
phenol (Dubois et al. 1965). The digestive gland was first homog-
enized in trichloroacetic acid (5%) for 2 min at 6.000 rpm. After
centrifugation (7000 g), the supernatant was quantified. This pro-
cedure was done twice. One milliliter of trichloroacetic acid was
pipetted into a tube and mixed with 5 volumes of y5'/r ethanol. The
tubes were placed in a oven at 37^0°C for 3 h. After precipitation,
the tubes were centrifuged at 7000 g for 15 min. The glycogen
(pellet) was dissolved by addition of 0.5 mL of boiling water and
then 5 niL of concentrated sulfuric acid and phenol (5%) were
added and mixed. The content of the tubes was transferred to a
cu\etle and read at 490 nm in a spectrophotometer.
Amylase Allozyme Analysis
Digestive glands from each population were homogenized in
500 p.L of TRLS-phosphoric acid buffer (0.06 Mol /L. pH7) and
centrifuged at 12000 rpm (4'C, 20 min). We used conventional
10% vertical polyacrylamide gel electrophoresis with TRIS-
glycine as the running buffer (Davis 1964). Polyacrylamide gels
were run at 250 V for 4 h. Band staining was done using an agar
gel ( 1 % ) with 1 % amylose. 1 M Ca. and I M Mg. phosphate buffer
(pH 7). and 250 mM NaCl. The acrylamide-agar gel matrix was
incubated at 37°C for 20 min and then stained with lugol solution
( 1:5| to obtain the bands. Alleles were coded by letters according
their relati\'e migration on gels (Garci'a-Machado et al. 2001). A
locus was considered polymorphic when the frequency of the most
common allele in the population did not exceed 95% and rare
when the frequency was <0.005%. Genetic variability deviations
of Hardy-Weinberg (H^.) expectations were determined using
Wright's F statistics (Wright 1965).
Energy Balance
Energy balance was estimated using the equation of Lucas
(1993):
Ah = R + U + P
where Ah is the absorbed energy (joules day"' gww~' ). R is routine
respiration. U is the energy lost through ammonia excretion, and P
is the energy invested in production of biomass. Assimilated en-
ergy (AS) was estimated using the equation (Rosas et al.. 1998):
AS = P + R
Production (P) was obtained from the growth rate of the shrimp.
The mean value of 4900 ± 147 J gdw"' was used to transform the
growth data into production units (P; J g"'dw d"'). This value was
obtained from analyzing the energy content of the muscle of 25
shrimp by means of a calorimeter (Parr), previously calibrated with
benzoic acid.
Respiration (Rrout) or basal metabolism (Hem) was obtained
through oxygen consumption measurements in nine fasting (12 h)
shrimp on each dietary regimen. Oxygen consumption was mea-
sured on individual shrimp in a continuous flow respirometer (Ro-
sas et al. 1998). Oxygen consumption was calculated as follows:
VO, = O,, - 0,„ X Fr
where VO, is oxygen consumption (mg O, h~' animaP'). O,^
indicates oxygen concentration at the entrance to the metabolic
chamber (mg L"'), Ojex 's oxygen concentration at the exit (mg
L"'), and Fr is the flow rate (L h"'). Oxygen concentration was
measured using a digital oximeter (YSl 50B digital. Dayton. OH)
with a polarographic sensor (±0.01 mg L"'). previously calibrated
with oxygen-saturated seawater at 28''C. The shrimp were then fed
food pellet fragments of 0.06 ± 0.002 g each in the respirometric
chambers. The same amount of food was placed in a control cham-
ber without an organism to estimate the oxygen lost by food de-
composition. Oxygen consumption of fed shrimp was measured
every hour for 4-6 h between 0800 and 1300-1500 h. Once the
experiment was concluded, the shrimp were weighed. Specific
routine oxygen consumption rate (mg g ' h"' ) was estimated from
the VO, of the unfed shrimp. The specific rate of the apparent heat
Hicrease (AMI), mg g"' h"'. was estimated from the difference
between VO, of the unfed shrimp and the maximum value attained
after feeding. A 14.3 J mg"' conversion factor of oxygen con-
sumption was used to transform the unfed and fed VO, to J g"' dry
weight (dw; Lucas 1993).
Along with the oxygen consimiption measurements, water
samples for ammonia excretion were obtained. Ammonia excre-
tion was determined as the difference between the ammonia con-
centration at the entrance and the exit of each respirometric cham-
ber and multiplied by the rate of water flow. The concentration of
ammonia (total ammonia; NHj* -i- NH,) was measured using a
flow injection-gas diffusion system (Hunter and Uglow, 1993).
This technique consists of a canier stream of NaOH (0.01 M)
separated from an indicator solution (bromolhymol blue 0.5 g L"')
by a gas permeable membrane (PTFE). All ammonia in the sample
is converted to gaseous NH,. which diffuses across the membrane
and reacts with the indicator to produce a pH-dependent color
change that is detected by a photometer. A calibration curve was
made using different concentrations of (NHj)-,S04. The ammonia
excretion of unfed and fed shrimp (postprandial nitrogen excre-
tion; PPNE) was converted to energy units using the value of 20.5
J per mg N-NH, excreted (Lucas 1 993 ) and defined as U^„^^ for the
energy lost before feeding and f 'p|,„^. the energy lost after feeding.
Total ammonia excretion was defined as C/j,,,;,,.
Rf^Hi snd C/ppn(, (J g~' WW day"') were estimated considering
the time needed for peak oxygen consumption after feeding and
the number of rations fed to the shrimp per day {Rt = 3),
272
Arena et al.
TABLE 1.
Daily growth coefficient of L ra«Ha»ifi juveniles from wild and cultivated populations: experiment I: wild vs. 7th-cultured
generation comparisons.
Wild
HCHO
LCHO
7th Generation
HCHO
LCHO
0.06 ± 0.05
0.07 ±0.01
4.10 ±0.34
6.0 ± 0.22
68 ± 10"
68 ± 6-'
55
58
2.20 ±(1.2"
2.42 ± 0.2'
fi
6
Initial weight, g
Final weight, g
Survival, %
Time, days
DGC. 9c
l..^l ±I).(I2
8.42 ±0.19
78 ±6"
55
1.71 ±0.4'
6
i,.^4±(),()2
8.54 ± 0.38
60 ±6-'
57
1.63 ±0.4"
6
Different letter means statistical differences, P < 0.05.
Values are mean ± SE.
HCHO, high dietary carbohydrates; LCHO, low dietary carbohydrates.
^.AHi = ['VO2 af - VO, hf X 14.3 J mg"' ) x (7" x Rr\] Amylase Activity
t/pp_^^, = |(N - NH, af - N - NH, bf x 20.5 J mg"') x {T x Rt)]
where VO, or N-NH, are the oxygen consumption after (af) and Exptnment 1
before (bf) feeding. 14.3 J mg"' and 20.5 J mg"' are the constant
to convert VO, or N-NH3 in energy units. Tis time (h) to reach the
pealc after feeding.
/?roui and '^n.ui 'J g~' ^'*' day"') were estimated as:
^rm,( = 11 VO, bf X 14.3 J mg"') x (7^, x Rr)]
f^Mu. = [(N - NH, bf X 20.5 J mg"' ) x (T,,, x Rr)]
where 7^^,, is the difference between time of one day (24 h) and
iTx Rt).
Statistical Analysis
Statistical analyses were used separately in each expeniiienl.
Analysis of growth rates was performed independently for each
population to emphasize dietary influence. Student Mests were
used on final average weight gains. The effect of dietary carbo-
hydrate was analyzed for physiologic and genetic data using 2-way
analysis of variance in E.xperiment 1, and one-way analysis of
variance in Experiment 2. Arc sine transformation was used prior
to analysis of survival data expressed in percentages. Homogeneity
of variances of all distributions was verified with Cochran's test.
Means obtained during the treatment were compared by using
Duncan's multiple range test (Zar 1974).
RESULTS
The amylase activity was affected by dietary CHO and was
higher in wild shrimp than in 7th-generation shrimp (Fig. 1 A; P <
0.05). A higher amylase activity was observed in wild shrimp fed
with high dietary CHO (35 0.1 lU/mg protein) than in wild shrimp
fed with low dietary CHO (26.8 lU/mg protein). A significantly
lower mea» value of amylase activity was obtained in shrimp from
the 7th-generation population (2i lU/mg protein) than in wild
shrunp (Fig. lA; P < 0.05).
Experiment 2
Dietary CHO levels significantly affected the amylase activity
with high values in shrimp fed with low dietary CHO (13.5 lU/mg
protein) and low values in shrimp fed with high dietary CHO (4.1
lU/mg protein) (P < 0.05; Fig. IB).
Hcmolympli Glucose
Experiment 1
A lower glucose hemolymph level was measured in wild
shrimp fed with low dietary CHO (0.13 mg/niL) compared with
that measured in wild and 7th-generation cultured shrimp (mean
value of 0.28 me/niL; Fisi. 2A, P < 0.05).
Growth and Survival
Experiment I
The daily growth coefficient (DGC/r ) was affected by dietary
CHO and was higher in shrimp from the 7th generation than in
wild shrimp (Table I: P < 0.05). The DGC of 7th-generatit>n
shrimp was higher in shrimp fed with low dietary CHO than that
in shrimp fed with high dietary CHO (P < 0.05). No differences
were observed between wild shrimp fed with high or low dietary
CHO levels (Table I; P > 0.05). Survival was not affected by
dietary CHO in either of the shrimp populations. A mean value of
69% survival was obtained in all treatments (Table 1).
Experiment 2
L. iY((i;!(»)/c/ juveniles from Tahiti population (25th generation)
were not affected bv dietary CHO (Table 2; P > 0.05).
TABLE 2.
Dailj growth coefficient for juveniles of L vannamei from
25th-cultured generation: Experiment 2.
HCHO
LCHO
Initial weight.
0
0.009 ±0.001''
0.009 ±0.001"
Final weight.
0
0.72 ± 0.04
1.02 ±0.05
Survival, %
85 ± 5
88 ±6
Time, days
36
36
DGC, 9c
1.91 ±0.7''
2.21 ±0.78"
N
8
S
Different letter means statistical differences, P < 0.05.
Values are mean ± SE.
HCHO. hiah dietary carbohydrates; LCHO. low dietary carbohydrates.
Ph^ .sioLOGic, Genetic Variations in L. vannamei
273
45- A
1 40-
1
2 35
c
'o) 30
5 25
1 20-
1 15-
Amylase
CJl o
Wild HCHO Wild LCHO 7th HCHO 7th LCHO
Population origin
18 B
b
'|> 12 -
5 10-
activity,
O) C»
a
Amylase
!
1
25th HCHO
25th LCHO
Dietary CHO level
Figure 1. Amylase ac'ti>it> h> wild and 7th-f;eneration cultured /,.
vannamei (A) and 25th-generatlon cultured L. vannamei (Bl. Mean ±
SE. Different letter means statistical differences at f < 0.05 level.
Experiment 2
A significantly high glucose hemolymph level was measured in
25th-generation shrimp fed with high CHO ( 1 mg/niL) that was 2.6
times the value in shrimp fed with low CHO (0.39 mg/mL) (Fig.
2B; P < 0.05).
Digestive Gland Glycogen
Experiment 1
Digestive gland glycogen concentration was affected by dietary
CHO and the origin of shrimp (Fig. 3A). In wild and 7th-generation
cultured shrimp, a high glycogen concentration was measured in
shrimp fed with low dietary CHO (P < 0.05).
Experiment 2
In 25th-generation cultured shrimp, the high dietary glycogen
level was measured in shrimp fed with high dietary CHO (2.0
mg/g) that was 30% higher than that in shrimp fed with low dietary
CHO (1.4 mg/g: P < 0.05; Fig. 3B).
Pattern of Allozyme Variation
An eight-band pattern was observed in the electrophoretic
analysis of amylase. These patterns were classified into two sys-
tems; system 1 with alleles a, b, and c and system two with five
alleles: a, b. c. d. and e (Fig. 4). Both systems were polyinorphic
(Table 3). System 1 was inore conservative than system 2. In such
a system, alleles a. b. and d were rare with an allelic frequency
<0.05. A reduction in H in system 2 was observed into domesti-
cated populations, with high values in wild shrimp (H = 0.29) and
low values in 25lh-generation cultured shrimp populations (H =
0.08). reflecting a high percentage of homozygosity. Amylase loci
from wild and 7th-generation cultured shrimp were in equilibrium.
Locus from the 25th-generation of cultured shrimp showed sig-
nificant deviation from Hardy-Weinberg proportions (heterozygo-
sis deficit: P < 0.05) (Table 4).
Energy Balance
Experiment 1
Oxygen consumption of 12-h fasting shrimp was affected by
dietary CHO in both wild and cultured populations (Table 5). The
1,20 1
A
1 00 -
1 0 80 -
cn
^ 0 60
o
^0 40
b
O
1
0,20 -
0 00 -
a
b
S
b
f
WHCHO WLCHO 7 HCHO 7 LCHO
Population Origin and Dietary CHO level
cn
E
o
o
O
1 z -
B
b
1 -
0 8 -
0 6 -
a
0 4 -
¥
0.2 -
0 ■
i
1
HCHO
LCHO
Dietary CHO. level
Figure 2. Glucose hemolymph level of wild and 7th-generation cul-
tured L. vannamei (A) and 25th-generation cultured shrimp (B). Mean
± SE. Different letter means statistical differences at f < 0.05 level.
274
Arena et al.
6 T
o) 4
E
c
0)
O)
o
o 2
3 -
1 --
T '^
i
:
b
F
a
^ !
\ 1
WLCHO WHCHO 7LCH0
Dietary CHO level
7HCH0
TABLE 3.
Genetic diversity {Hj in wild and cultured populations of
L. vannamei.
Population
System I
He
System 2
He
Wild
7th generation
25th generation
0.66
0.51
0.51
0.29
0.27
COS
weight). Ammonia excretion increased after feeding, reaching a
ma.ximum value between 0.5 to 3 h after feeding depending on
shrimp group (Table 6). The highest postprandial amtnonia excre-
tion value was recorded in 7th-generation shrimp fed with low
dietary CHO and the lowest in wild shrimp fed with high dietary
O) 4
E
£=" 3
O)
O
O
LCHO HCHO
Dietary CHO level
Figure 3. Digestive gland glycogen of u ild and 7th-generation cultured
L. vannamei {.\) and 25th-generation cultured shrimp iB). Mean ± SE.
Different letter means statistical differences at P < 0.05 level.
highest oxygen consumption was measured in 7th-generation cul-
tured shrimp (0.63 mg 0-,/h/g wet weight) fed with high dietary
CHO (P < 0.05). The lowest oxygen consumptio// value was in
wild shrimp fed with low dietary CHO (0.19 mg 0,/li/g wet weigh;
P < 0.05). The oxygen consumption rate increased after feeding in
each treatment (Table 5). Oxygen consumption of shrimp during
feeding followed either of two patterns: one for wild shrimp fed
with low dietary CHO and the other for the remaining shrimp
groups. During feeding, oxygen consumption of wild shrimp fed
with low dietary CHO was significantly lower than in w ild shrimp
fed high dietary CHO shrimp and 7th-generation shrimp fed with
high or low dietary CHO. In each, oxygen consumption increased
rapidly after feeding and decreased afterwards until reaching levels
similar to those at the start of experiment. The time required to
achieve oxygen consumption peak was higher in 7th-generation
shrimp fed with high dietary CHO (2 h; than in all remaining
shrimp groups (0.5 to 1 h).
Ammonia Excretion
Ammonia excretion in fasting wild shrimp (mean value of 0.06
mg N-NH^/h/g wet weight) was significantly lower than in 7th-
generation shrimp (mean value of 0.15 mg N-NHj/h/g wet
System 1
0.6
>. 05
u
o 04
£ 03
< 0 1
0
7th generation cultured shrimp
DHCHO
BLCHO
System 1
a b
System 2
25th generation cultured shrimp
DHCHO
SLCHO
b c
System 1
b c
System 2
Figure 4. .Allele frequencies of wild, 7th-, and 25th-generation cul-
tured I., vannamei fed with high dietary CHO (HCHO) and low dietary
CHO (IXHOl.
Physiologic, Genetic Variations in L. vannamei
275
TABLE 4.
Allelic fri'i|ui'ncies comparison among different populations of/., ramitimci from wild (Mexico), 7th-generation cultivated shrimp and
25th-!;eneratiun cultivated shrimp.
Wild
7th Generation
25th Generation
Population
SI
S2
SI
S2
SI
S2
Wild
7th generation
25th t>eneralion
NS
NS
NS. without significant statistical difference. *Statistical differences aX P < 0.05 level.
CHO. Intermediate values were recorded in tlie remaining shrimp
groups ( P < 0.05 ).
The respiratory energy (/^y,,,.,,) varied between populations and
was affected by dietary CHO (Table 7). Of the /?Toiai. 1'7'7<- was
wasted in /J^,,, in wild shrimp fed with low dietary CHO in com-
parison with the .^.4-4'^ waste as R.^y,, in the remaining shrimp
groups (Table 7). R„^, was observed between S.^-y7Vr of /^x,,,,,,
with the lowest value in wild shrimp fed with low dietary CHO.
There were statistical differences between C/Totai between popula-
tions and between treatments in 7th-generation cultured shrimp
(Table 7; P<0.05).
The percentage of Lfj^,,.^, that was t/^„„, varied between shrimp
populations with the lowest value in wild shrimp fed with low
dietary CHO (37%) and the highest (82%) in 7th-generation
shrimp fed with low dietary CHO. The energy wasted after feeding
(t/pp) was higher in wild shrimp fed with low dietary CHO (6.^%
of t/y,„^|) than that in 7th-gene]ation cultured shrimp fed with the
same diet (18% of U-y^,^,). Absorbed energy {Ah = P + R + U)
showed differences between shrimp groups and was affected by
distary CHO with high values in wild shrimp fed with high dietary
CHO (S24J"' g"' WW day"') and low values in 7th-generation
cultured shrimp fed with same diet (598 J"' g"' ww day"': Table
7). Ut-„,^| varied between 5-1 1% of Ab with low values in wild
shrimp fed with high dietary CHO and high values in 7th-
generation cultured shrimp fed with high and low dietary CHO
(11% and 10% ). Between 89 and 95% of Ab was assimilated. The
energy assimilated {AS) was the result of adding R to P. The .4.v
value was affected differently in each shrimp population. In wild
shriinp the highest value was observed in shrimp fed with high
dietary CHO whereas in 7th-generation cultured shrimp the high-
est value was observed in shrimp fed with low dietary CHO (Table
7). Respiratory efficiency (R/AS) was lower in wild than in 7th-
generation cultured shrimp and was affected by dietary CHO in
each shrimp group (Table 6). Inversely, growth efficiency (P/AS)
was higher in wild than in 7th-generation shrimp and highest in
shrimp fed with low dietary CHO in both shrimp groups.
Experiment 2
Oxygen Consumption
No difference was measured in 12-h fasting oxygen consump-
tion values between treatments (mean value of 0.23 mg O^/h/g wet
weight; Table S: P > 0.05). A similar ma.ximum oxygen consump-
Uon value was observed in both dietary shrimp groups (0.32 mg
Oi/h/g wet weight). The time to reach the peak was different
between treatments with 1 h for shrimp fed with high dietary CHO
and 2 h for shrimp fed with low dietary CHO (Table 8).
Ammonia Excretion
In 25th-generation shrimp, 12-h fasting shrimp had similar val-
ues of ammonia excretion between treatments (mean value of
0,022 mg N-NH,/li/g wet weight; P > 0.05; Table 9). After feed-
ing, the ammonia excretion increased. The time to reach the peak
was similar in both treatments with high values in shrimp fed with
high dietary CHO (0.040 mg N-NH,/h/g wet weight) and low
values in shrimp fed with low dietary CHO (0.035 mg N-NH,/h/g
wet weight; P < 0.05).
Dietary CHO affected /^y^,,^,, (Table 10). Shrimp fed v\ith high
dietary CHO had the higher proportion of energy from /?t„,„, that
was channeled to /^^^,^, (96%) and at the same time the lower
proportion of ^x„,.,i that was used in R^h, (4%). In contrast the
higher proportion of energy of f/^,,, ,, that was lost as f/^..^, was in
TABLE 5.
Oxygen consumption (mg ();/li/g«vv) of L. vannamei after 12-h fasting (time = 0) and at lime increments after feeding: Experiment 1.
Time After
Feeding, h
Wild
HCHO
ECHO
7th Generation
HCHO
ECHO
0
0.5
1
2
3
4
0.44 ± 0.09-'
0.59 ±0.11"
0.61 ±0.11"
0.58 ± 0.07"
0.52 ± 0.08"
0.46 ± 0.06"
0.19±0.(1V'
0.44 ± 0.04"
0.46 + 0.07"
0.28 ± 0.05"
0.39 ± 0.04"
0.22 ± 0.03-'
0.65 ± 0.06"
0.61 ±0.09"
0.65 ± 0.09"
0.72 ± 0.04"
0.48 ± 0.05"
0.35 ± 0.04-
0.54 ± 0.05^
0.57 + 0.08"
0.70 ± 0.09"
0.59 ±0.11"
0.59 ± 0.06"
0.61 ±0.14"
Different letter means statistical diflerences. P < 0.05.
Values are mean ± SE.
HCHO, high dietary carbohydrates; LCHO, low dietary carbohydrates.
276
Arena et al.
TABLE 6.
Ammonia excretion (mg N-NH/li/gww) of L vannamei after 12-h fasting (time = (l( and at time increments after feeding: Experiment 1.
Time After
Feeding, h
Wild
HCHO
ECHO
7th Generation
HCHO
ECHO
0
0.5
1
2
3
4
0.067 ± 0.002'
0.18 ±0.004"
0.12 ±0.003''
0.19 + 0.003''
0.08 ± 0.003°
0.11 ±0.003"
0.05 + 0.008'
0.15 ±0.02"
0.19 ±0.03"'^^
0.21 ±0.03*^^
0.22 ± 0.06"
0.07 ± 0.009-'
0.15 ±0.02"
0.15 ±0.05"
0.16 ±0.007"
0.16 ±0.03"
0.25 ± 0.03"^
0.06 ± 0.0 r
0.14 ±0.02"
0.34 ± 0.03''
0.35 ± 0.04"
0.24 ± 0.03"
0.33 ± 0.05'-'
0.14 ±0.04"
Different letter means statistical differences, P < 0.05.
Values are mean ± SE.
HCHO, high dietary carbohydrates; ECHO, low dietary carbohydrates.
shrimp fed with low dietary CHO (87'7r ) in comparison to 15% lost
in C/f„„, in shrimp ted with high dietary CHO. Inversely the pro-
portion of t/xotai 'hat was lost as U^p was higher in shrimp fed with
high dietary CHO (25%) than in shrimp fed with low dietary CHO
{M^c: Table 10). In both treatments QSVr of energy absorbed {Ab)
was assimilated [AS). Dietary CHO affected AS and growth and
respiratory efficiencies. Shrimp fed with low dietary CHO showed
the higher AS and growth efficiency (72%) compared with shrimp
fed with high dietary CHO (61%; Table 10).
DISCUSSION
In L. vannamei shrimp from the 2,5th-cultured generation ex-
hibited less heterozygosity than did wild shrimp. From results
obtained, the 7th-generation cultured shriinp showed an interme-
diate genetic and physiologic alteration. Although results demon-
strate significant genetic differentiation among cultured and wild
populations when based upon only an amylase allozyme marker,
we acknowledge the necessity to confirm such differences at the
mtDNA level through sequence variation of the amylase gene as
recommended by Xu et al. (2001) and Garcia-Machado et al.
(2001). A more detailed study involving molecular biology and
genetic alterations by domestication of L. vannamei is in process.
As a consequence of selection in cultured populations, carbohy-
drate metabolism routes (hydrolysis, absorption, and synthesis) in
shrimp fed with different dietary CHO was affected. A different
enzyme activity-dietary CHO relation was observed depending on
population characteristics; wild shrimp amylase activity was in-
duced by high dietary CHO whereas low dietary CHO induced a
high amylase activity in cultured shrimp. If reduction of heterozy-
gosis means a reduction in amylase genes, then amylase activity
induction was a compensatory response to obtain the highest pos-
sible glucose from the diet, increasing enzyme synthesis when
shrimp are fed with low dietary CHO. On the contrary, in wild
shrimp an excess of dietary CHO induced amylase activity because
those shrimp have all the isoforms of the atnylase enzyme to
respond directly to the dietary starch. If atnylase production in
domesticated shrimp is efficient enough to process dietary CHO, it
can be analyzed in a general context. Although a statistical com-
parison cannot be done among the three studied populations, it is
evident there is a reduction in amylase activity as a function of
dotnestication. with high values in wild shrimp (between 24 to 39
lU mg"' protein), intermediate in 7th-generation cultured shrimp
(between 16 to 25 lU mg~' protein), and low in 25th-generation
cultured shrimp (between 3.6 to 15.8 lU mg"' protein; Fig. 1).
Such reduction indicates that the reduction of allel frequency of
amylase genes affected the adaptative ability of shrimp to use
TABLE 7.
Energy balance in juveniles of L. vannamei: Experiment 1.
Wild
HCHO
ECHO
7th Generation
HCHO
ECHO
167.3 ± 18.8
162.1 ±26.7
6.0 ± 0.8
6.8 ±0.8
173.3
168.9
46.1 ±6.1
60.3 ± 8.6
18.5 ±2.4
12.9 ± 1.6
64.6
73.2
359.9 ± 46
500.9 ± 65
597.8
743.0
533.3
669.8
89.2
90.1
32.5
25.2
67.5
74.8
/?„„. J/day/gww
S^Hi- J/day/gww
/?To>ai- J/day/gww
(/„,, J/day/gww
f PPNE. J/day/gww
^Tniai- J/day/gww
p. J/day/gww
Absorption (Ah) J/day/gww
Assimilation (As) J/day/gww
Ef assimilation. As/AI^
Respiratory efficiency, '7r R/As
Production efficiency, % P/A.s
147.1 ± 1.9
5.2 ±0.8
152.3
24.7 ± 0.73
13.8 ±1.9
38.5
633.4 ± 70
842.2
785.7
95.3
19.4
80.6
57.1 ± 10.7
11.6± 1.1
68.7
18.5 ±2.70
31.4 ±4.7
49.9
614.6 ±73
732.6
683.3
93.3
10.1
89.9
Mean ± SE.
HCHO, high dietary carbohydrates; ECHO, low dietary carbohydrates.
Physiologic, Genetic Variations in L vannamei
277
TABLE 8.
Oxygen consumption (mg 0,/h/g\v\M of/,, ycninamei (2Sth
generation) 12 h lasting (time = 0( and at time increments after
feeding: Experiment 2.
Time .\fter
Feeding, h HCHO I.CHO
0.24 ± 0.02'
0.31 ±0.02*"
0.29 ± 0.02"
0.28 ± 0.02"
0.26 + 0.02""
0.25 ± 0.02"
0.27 ± 0.02"
0.21 ±0,01'
0.31 ±0.02"
0.33 ± 0.02"
0.27 ± 0.02'-'
0.27 ± 0.02''
0.28 ± 0.02"
0.25 ± 0.02"
Dit't'erent letter means statistical differences. P < 0.05.
Values are mean ± SE.
HCHO. liicli dietary carbohydrates; LCHO. low dietary carbohydrates.
dietary CHO as a source of energy and molecules, which could
cause farmed populations to be protein dependent.
Juveniles of Uloiwiiaeiis vaimainei can synthesize their own
glucose from protein through a gluconeogenic pathway (Rosas et
al. 2001). Shrimp fed with low dietary CHO had digestive gland
glycogen levels that were higher than when fed with high dietary
CHO because the enzymatic system is induced to synthesize CHO
from protein (Cuzon et al. 2001 ). In the present study, an increase
in digestive gland glycogen was measured in wild and 7th-
generation shrimp fed with low dietary CHO indicating that an
induction mechatiism is working. In contrast, in the 2,5th-
generation farmed shrimp, that mechanism appears to be working
in the opposite direction, producuig more digestive gland glycogen
in shrimp fed with high dietary CHO than in shrimp fed with low
dietary CHO. If Amylase genes are repressed after 25th genera-
tions of selection then a high probability exists that other genes
could be repressed also, producing changes and reducing the glu-
coneogenic route in shrimp.
This indicates that artificial selection of shrimp favored more
than size and harvest weight, as it also favored protein metabolism
bv acting on shrimp digestive capacity. The use of high levels of
animal protein in shrimp feeds in all phases of shrimp culture, from
larvae to broodstock (including Anemia, krill, Cyclops, high qual-
<
100
90
80
70
60
50
40
30
20
10
0
HCHO-W LCHG-W HCHO-7 LCHO-7 HCHO-25 LCHO-25
Diet and group shrimp
Figure 5. Growth eniciency (P/AS,%) of wild (W) 7lh (7)-. and 25th
(25)-generation cultured L. vannamei fed with different carbohydrates
levels. HCHO. high dietary carbohydrates; ECHO, low dietary car-
bohydrates.
ity fish meal, and squid) is responsible for activation and repres-
sion of genes. For amylase, Le Moullac et al. (1996) reported a
reduction of enzyme activity in L. vannamei after an increase in
dietary protein, which was related to a regulating role of amino
acids on amylase expression. They observed a disappearance of
one amylase mRNA associated with a high protein level suggest-
ing that a regulation of amino acids would take place at the tran-
scriptional level. Because, in selected shrimp, protein metabolism
was favored and growth rate depended on dietary protein (An-
drews et al. 1972). one can explain why 7th and 25th-generation
farmed shrimp possess a higher growth rate than wild shrimp
(Tables 1 and 2).
There are several costs that are necessary to take into account
with the breeding programs that only take into account the size of
shrimp at harvest, which is also related to growth efficiency. From
results on energy balance, there are differences in production ef-
TABLE 10.
Energy balance of L. vannamei (25th generation): Experiment 2.
TABLE 9.
excretion (mg N-NH,/h/gHH ) of L
vannamei (25th
Dietary CHO
Ammonia
HCHO
LCHO
generation)
12-h fasting (time = 0) and at time
feeding: Experiment 2.
/e„„. J/day/gww
72.07 ± 6.0"
54.1 ±2.6"
Ri,n,. J/day/gww
/?j^„ji, J/day/gww
U,^,. J/day/gww
t/ppNE, J/day/gww
(/j„,^i, J/day/gww
3.0 ± 0.85"
75.1
7.4 ±0.7"
2.5 + 0.3"
10.3 ±1.7"
Time .\fter
Feeding, h
HCHO
LCHO
64.4
8.9 ±0.2"
1.4-^0.1"
0
0.020 ± 0.002"
0.024 ± ().()004"
9.84
10.2
1
0.030 ± 0.002"
0.028 ±0.001"
P. J/day/gww
96.78 ± 14"
137.6 ±20.6"
2
0.040 ± 0.002'
0.035 ± 0.00 r
Absorption (AIj). J/day/gww
181.7
212.2
3
0.030 ±0.001"
0.029 ±0.001"
Assimilation (As). J/day/gww
171.8
202.0
4
0.037 ± 0.002"-^
0.026 ±0.001"
Ef assimilation As/Ah
94.6
95.2
5
0.029 ±0.001"
0.030 ± 0.0009"
Respiratory efficiency
%
R/As
43,7
31.9
6
0.03 ±0.001"
0.027 ± 0.0008"
Production efficiency.
9c
P/As
56.3
68.1
Different letter means statistical differences. P < 0.05. Different letter means statistical differences. P < 0.05.
Values are mean ± SE. Values are mean ± SE.
HCHO, high dietary carbohydrates; LCHO. low dietary carbohydrates. HCHO, high dietary carbohydrates; LCHO, low dietary carbohydrates.
278
Arena et al.
ficiency between populations (Fig. 5); a reduction of the P/AS ratio
depending on the generations of farmed selected shrimp indicate
that efficiency with which shrimp transform energy into biomass is
reduced with artificial selection. That situation has several impli-
cations on coastal ecology. When selected shrimp are lost by pond
break caused by floods or hurricanes they could be liberated to
surroundings environment. If those shrimp are from a breeding
program based on size only, they shrimp could growth faster and
consume more protein than wild shrimp, wasting energy due to its
reduced assimilation efficiency and wasting other nutrients offered
by the natural environment in the form of CHO and in conse-
quence changing the relation between nutrients and consumers. In
this same sense a reduction in PIAS ratio could have implications
on the shrimp industry if is considered that a reduction in produc-
tion efficiency could means the use of foods with more and more
fish meal to satisfy the protein requirement of shrimp provoking
that the shrimp industry to compete with other industry that use
fish meal to produce meat for human consumption.
On the other hand, selection shrimp programs could have rel-
evance for the health of farmed shrimp. Recently. Xu et al. (2001)
showed that there is a relation between genetic diversity and
IHHNV sensitivity of P. moiwdon from Philippines. Although
such relation is not understanding at all it could means that at the
same time that shrimp are selected for size some other genes
related with virus tolerance could be selected as well, provoking a
segregation of the genes involved in virus resistance. If such con-
cepts are applied to L. vannamei from breeding programs we could
help to develop an industry based on rapid growth, low efficiency
and vulnerable shrimp. It will necessary change looking for an
shrimp based in the conception of breeding program that try to
select shrimp that have wider adaptative ability to respond de-
mands including all that are related to feed composition, produc-
tivity, and sustainability (Fenucci et al. 1982, Boureau et al. 2000).
and biosecurity (Xu et al. 2001 ).
ACKNOWLEDGMENTS
Thanks to Ellis Glazier for editing this English-language te.xt.
The authors thank the ECOS Mexico-France program for its sup-
port to researcher exchanges during this study. Special thanks are
given to Adriana Paredes. Ariadna Sanchez. Manuel Valenzuela.
Gabriel Taboada. and Gabriela Palomino for help during the ex-
periments. The present study was partially financed by CONACYT
through proyect .^1 137B to Carlos Rosas. Special thanks are given
to Industrias Pecis for its support.
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Joiunul of SlwUthli Research. Vol. 22. No. 1. 2S1-2S4, 2I)().\
EFFECT OF TEMPERATURE ON POST-PRANDIAL METABOLISM OF BROWN SHRIMP
FARFANTEPENAEUS CALIFORNIENSIS
LUCIA OCAMPO,'* CARLOS ROSAS,' AND HUMBERTO VILLARREAL'
'Ccntro de liivcsrigacioncs Bioldi^ica.s del Noroeste (CIBNOR) P.O. Bo.\ I2H. La Paz. B.C.S. 23000.
Mc.xico and 'Gnipo de Muhcultura. Lah. Ecoflsioloi^fa. Faciiltad de Cleiuia.s. UN AM. P.O. Box 69,
Ciiidad del Carmen, Canipeelie 24140. Mexico
ABSTRACT The effect of three temperatures 09. 23. and 27°C) on the postprandial metaboHsm (apparent heat increment) of
iu\cnile larfantepenaeus californiensis was evaluated. The unfed metabolic rate and post-prandial metabolic rates were determined
with an intermittent-tlow respirometer during 5 h. A peak in oxygen consumption was found 2 h after feeding at 19 and 27"C whereas
at 23°C the peak was found after I h. The unfed metabolic rate at 23°C was not different from that at 27°C . The maximum metabolic
rates of fed animals were 2.1, 1.6, and 1.7 times that of unfed animals in order of increasing exposure. The highest apparent heat
increment was found at 27°C. Energy loss varied from 4.11 to 11.43 J. Calculated Q,„ thermal coefficients indicate metabolic
overcompensation for temperature changes between 19 and 27°C, and between 19 and 23"C, except at the maxmium metabolic rate.
In contrast. Q|,,s for temperature changes between 23 and 27°C indicate compensation.
KF.Y WORDS: energy loss. Faiiaiuepemieus califoruicnsis. oxygen consumption, postprandial metabolism, temperature
INTRODUCTION
Rtihner ( 1902) defined the heat increment resulting from bio-
chemical reactions to ingestion of a meal as specific dynamic
effect. Since then, various terms, such as specific dynamic action,
heat of nutrient metabolism, thermogenic action, calorigenic effect
of food, postprandial respiration, and heat increment, have been
used widely to represent energy losses associated with feeding in
ectotherms (Johling & Davies 1980. Beamish & Trippel 1990).
The physiologic basis of this increased heat production includes
postabsorptive processes related to ingestion, particularly of pro-
tein-rich food, the metabolic work required for formation of ex-
cretory nitrogen products, and the synthesis in the tissues of pro-
teins and fats from the newly absorbed food derived substrates like
amino and fatty acids. The energies required for grasping, chew-
ing, and swallowing food are technically distinct from the heat
increment but are difficult to separate experimentally (Beamish &
Trippel 1990). The apparent heat increment (AHI) is the energy
required for the mechanical processes of feeding and the ingestion
and digestion of food (Hewitt & Irving 1990). In homeotherms.
heat increment has multiple influences, including tiine spent in
eating, muscular work, secretion of saliva, fermentation heat,
transport of the absorbed nutrients, hormonal effects, and pharma-
cokigical effects of food constituents, and is related to the enthalpy
change associated with the generation of ATP (Blaxter 1989). In
fish, there is ample evidence that AHI is influenced by meal size
and feeding frequency, temperature (Bret 1976); size of the ani-
mal (Beamish 1974); quantity, quality, and proportions of the di-
etary energy components (Smith et al. 1978); and the nutritional
status (Hart 1980). Despite the amount of information published on
AHI. experimental techniques have varied greatly among the stud-
ies, and observations of the effects of temperature on AHI have not
been consistent. In addition, there is little information on AHI in
Penaeids (Hewitt & Irving 1990. DuPreez et al. 1992. Rosas et al.
1996). DuPreez et al. ( 1992) reported that the AHI for P. immodon
*Corresponding author. Tel +6i;
E-mail; locampo@cibnor.mx
-125-36-33; Fax: -1-612-125-36-2.5;
Fabricus ranged from 2-17% when fed commercial pellets and
from 2.4 to 19.5% when fed shrimp flesh. Rosas et al. (1996)
reported that the highest AHIs were found for P. duoranim
Burkenroad and P. notialis Perez Farfante feeding on a 65%
diet, whereas the lowest were found for P. setifeiiis Linnaeus and
P. schmitti Burkenroad fed a 40% protein diet. The authors con-
cluded that AHI varied with diet protein content for all these
species.
Brown shrimp Faifantepenaeus californiensis Holmes is cur-
rently being evaluated as a cold-tolerant species with potential for
aquaculture at our center. Studies of nutritional and metabolic
aspects that are influenced directly by factors such as temperature
are important to better understand the physiology of this species.
This study presents information about the effect of temperature on
the AHI of juvenile F. californiensis. Some physiologic responses
and possible mechanisms of adaptation are discussed.
MATERIALS AND METHODS
Juvenile F. californiensis from the Centro de Investigaciones
Biologicas del Noroeste experimental shrimp farm were selected
randomly, fed a commercial diet containing 35% crude protein
(RANGEN®) with filtered seawater at a salinity of 37 ppt. A
photoperiod of 12-hL:12-hD was maintained throughout the study.
Shrimp were acclimated ( 1' C/day) at three different temperatures
(19, 23, and 27°C) for a period of 5 days. After a 24-h starvation
period. 12 animals of each temperature treatment were placed
indi\ idually in an intermittent flow respirometer system similar to
the one described by Villaireal (1989) 2 h before commencing the
test to minimize the effect of handling and previously calibrated at
each experimental temperature. The fasting metabolic rate was
determined for 2 to 3 h thereafter. Next, shrimp were allowed to
feed on commercial pellets for 1 h. Uneaten food was siphoned out
completely and collected, and water was replaced completely.
Oxygen intake was recorded hourly for 5 h after ingestion of the
meal with an oxygen electrode (Yellow Spring Instruments, Model
58). Water was replaced completely after each record to prevent
accumulation of ammonia. At the end of the experiment, shrimp
were weighed on a digital balance after blotting. Data were cor-
281
282
OCAMPO ET AL.
■c
O
00
o
S
S
£^
o
u
oi
0.9 r
0.8 -
19°C
23°C
■27°C
0.7
0.6 -
0.5 -
= 0.4 -
0.3 ■-
0.2
0.1
Time after feeding (h)
Figure 1. Respiratory metabolism (mgOi/g shrimp/h) of juvenile Farfantepenaeus califontiensis after feeding on a 35% crude protein diet
(RANGEN®) at different temperatures (°C|. Unfed respiration is sliown as the respiration at time 0. n = 12 shrimp/temperature. *Signifieant
differences.
reeled for oxygen consiiniption with u control respirometer with no
shrimp. AHI (J) at each temperature wa.s calculated as:
AHI = (maximun postprandial rate - unfed rate)(20.06)
over the period studied (Rosas et al. 1996, Lucas 1993). Differ-
ences between treatments were defined by one-way ANOVA and
the Tukey multiple range test.
RESULTS
Respiration (mg O^/g shrimp/h). as a function of time, is shown
in Figure 1 . Time 0 was defined as the end of the 24-h starvation
period. The highest unfed metabolic rate occurred at 27°C, but it
was not significantly different from that at 23°C {P > 0.05). The
lowest unfed metabolic rate was at 19°C and represented approxi-
mately 55% of the value at 23 and 27''C.
A tendency to increase metabolic rates at all temperatures after
feeding was observed, but this increase was signitlcant only at
19°C. At 19 and 27°C, the highest rate was reached after 2 h. At
23°C. the maximum was observed after 1 h, and was sustained
over 2 h. The highest overall increase in metabolic rate after feed-
ing of 677c occurred at 27°C. whereas at 23 and 19 C. the meta-
bolic rate increased 59% and 110%, respectively (Table I). The
time after commencement of feeding until the appearance of the
first feces varied from 30 to 60 min at 27 and 23T, whereas at
19°C the time was approximately 90 min.
AHls are shown in Table 1. The highest AHI was at 27"C and
the lowest AHI was at 23°C. When AHI was expressed as energy
lost, values varied from 4.1 1 to 11.43 J. These values were cor-
rected for the time needed to reach the peak and represent the
metabolic efficiency of heat loss. The highest value was at 27°C
and the lowest was at 23°C.
Qii, coefficients were calculated for temperature increments
between 19 and 23°C, and 19 and 27 ^C, and are shown in Table 2.
A Qui value of 2 indicates a doubling of the metabolic rate with an
increase in temperature of 10°C. Q,,, for 23-27 for the unfed
period showed adaptation, whereas Q^s for 19-23 and 19-27
showed overcompensation. Q„, for 23-27 for the feeding period
showed compensation for almost the entire trial except during the
second hour. Qm for 19-23 showed overcompensation, except for
the second hour when there was adaptation. Little compensation
was observed between 19 and 27°C in this experiment.
TABLE L
Mean effect of temperature on unfed and postprandial metabolism (mg02/g shrimp/h). apparent heat increment (AHL J), and energy lost
(J) in ju>enile Farfantepenaeus calif orniensis.
Temp. Unfed Rate
(°C) (mgO,/g Shrimp/h 1 ± SD
Maximum Postprandial Rate Increase AHI Time to Reach Energy Lost
(mgO,/g Shrimp/h) ± SD (%) (J) Peak (h) (J)
19
().I42±0.()68-'
23
0.349 ± 0.066''
27
0.428 + 0.091''
0.403 ± 0.063"
0.554 ± 0.077"
0.713 ±0.142'
no
4.2.^
">
39
4.11
1
67
5.72
2
S.46
4.11
1 1 .43
N = \2 shrimp/temperature. Entries with the same letter are not statistically different (P > 0.05)
Post-Prandial Metabolism of F. cauforniensis
283
TABLE 2.
Calculated Q,,, values for unfed and postprandial metabolic rales in
juvenile Farfanlepenaeus californiensis.
Time After
Q,„
Q,„
Q,„
Feeding
(hi
(19-2.Vt)
( 19-27 C)
(23-27 C)
0
4.49
2.7.^
1.6(1
1
6.02
2.61
1.L1
2
L95
2.04
2.14
3
3.02
L67
0.92
4
5.19
2.54
1.24
5
3.5
2.09
1.2.';
Values for Q,,, were calculated using the formula Q,,, = iRJR,) exp"""-""'.
where ft, ;ind /?, are the metaholic rates at temperatures r, and /,, respec-
tively.
DISCUSSION
DuPreez et al. (1992) conciucJed that the magnitude and dura-
tion of oxygen consumption peaks could be influenced by diges-
tion rate, environmental temperature, and activity of the animal. In
our study, a peal^ was seen 2 h after feeding at 19 and 27°C. and
decreased thereafter, showing that oxygen consumption of F. cali-
forniensis was affected only by food in the experimental device
used. Perhaps the amount of food consumed or the digestion rate
were responsible for sustaining maxiinum post-prandial rate over
2 h at 23°C.
The induction in oxygen consumption for F. cuUfomiensis was
approximately 43% higher at 19°C than at 27°C (Table 1). Fur-
thermore, the maximum metabolic rate at 19°C equaled the pre-
feeding rate at 23'C. Juveniles at I9"C appeared lethargic, and v\e
observed a lower ingestion rate at this temperature. Although pre-
liminary trials indicated that 5 h was adequate for complete diges-
tion of food, we noticed that the digestive tract of some shrimp at
\TC still contained food at the end of the experiment. The slower
appearance of feces showed that more time is needed to finish
digestion at 19°C. and could be related to a decrease in the appetite
and the general movement of shrimp. DuPreez et al. (1992) found
two peaks for P. monodon. 30 min and 6 h after feeding. Perhaps
we would have found a second peak if the trial had continued past
5 h at 19 C, when the shrimp reinitiated digestion.
Villarreal and Ocampo (1993) concluded that F. californiensis
postlarvae and juveniles exposed to a temperature range from 19 to
3rC could adjust their normal metabolic rate with gradual, short-
term temperature modifications. Similar results were obtained in
this study, in which the unfed metabolic rate at 23°C was close to
that at 27"C. It seems that organisms at 23°C have a mechanism
for metabolic compensation or adaptation to temperature varia-
tions at this stage of life (Q,„ for 23-27 = 1.66). This equivalence
in metabolic rate could be explained as a modification in enzyme
kinetics. Ocampo and Ezquerra (2002) found that the effect of
temperature on total //; vivo protease activity of F. cciliforniensis at
23°C was 58% higher than that at 21°C, for shrimp that had been
acclimated for 50 days to the temperatures, and suggested that
different digestive protease enzymes arise as an adaptation mecha-
nism to temperature and dissolved oxygen variations.
In general, homeostatic regulation of enzymatic catalysis in
animals can be accomplished in two ways: by modifying enzyme
concentration, or by modifying catalytic efficiency (Hochachka &
Somero 1973). When enzymatic concentration is increased, the
rate of reaction increases. At 23°C. juveniles might increase their
reaction rate as a quantitative strategy for temperature compensa-
tion (see QioS). However, this quantitative strategy might be less
efficient during cold adaptation since less time is required for
changing enzyme concentration via synthesis of new protein. This
metabolic reduction enhances the resistance of F. cuUfomiensis to
low temperature stress. The process seems to involve controlled
decreases in metabolism and organelle function, coupled with si-
multaneous controlled stabilization of macromolecule and or-
ganelle structures (Hochachka 1990).
DuPreez et al. ( 1986) calculated AHl as the increase in oxygen
consumption over the time until oxygen consumption decreased to
the prefeeding level. In our study, AHl was expressed as the dif-
ference in oxygen consumption between unfed and fed animals
(Rosas et al. 1996). AHl is best expressed as percent metaboli/able
energy (Blaxter 1989). Taylor et al. (1987) stated that most ani-
mals displayed maximum metabolic rates that were 5 to 20 times
the normal rates. However, some experiments did not take into
account stress caused by handling, and the need for a "resting"
period in the chamber before initiating the trial. Rosas et al. (1996)
reported the maximum metabolic rate of fed P. schmitli was 2.6 to
3.6 times that of unfed. In our study, maximum metabolic rates
found were 2.1. 1.6. and 1.7 times that of unfed animals, with
increasing temperature.
We emphasize that the heat increment AHl corresponds to the
production of ATP (maintenance heat increment) and tissue energy
deposition (production heat increment). Cho and Kaushik (1990)
estimated the heat increment of feeding for a maintenance ration is
approximately one third of the total heat increment, and the rest is
used for productive gain. In general, AMI might range from 1 1-
24% of digestible energy (6-19% of gross energy intake: Beamish
& MacMahon 1988), and is a more or less constant fraction of
dietary energy (Brody 1964). Further research is needed to relate
protein intake with AHl in F. calif imiensis. However, the results
of this experiment show that F. californiensis juveniles present a
metabolic strategy to digest food efficiently at 23°C, leaving more
time to consume food and saving heat energy loss. This strategy is
not related to their optimum aquacullure temperature, but is related
to their physiologic optimum, which would indeed be a good tem-
perature to maintain the animals.
ACKNOWLEDGMENTS
Luci'a Ocampo was a student-fellow of CONACYT, Mexico.
Thanks to Jean-Charles Guillaume for observations and sugges-
tions and the CIBNOR editing staff.
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ABSTRACTS OF TECHNICAL PAPERS
Presented at The 23rd Annual
MILFORD AQUACULTURE SEMINAR
Milford. Connecticut
February 24-26, 2003
285
Milt'ord Aqiiaciilture SL-inmar. Milford. Coiiiiecticul Abstracts. February 2003 287
CONTENTS
Walter J. Bloguslawski
Overview. 23rd Milfiird Aquaculture Seminar 289
Kathleen Becker and Kim Tetrault
Photo doLiinienlation as a vital element in community based shellfish restoration programs 289
David Berry
Insuring your aquaeulture erop 289
Don Bishop
Economies, marketing and how they relate to growers husbandry methods 289
Diane Broiisseau, Sara Brady, and Allison Schaffer
Preliminary investigations of shelter competition among the Asian shore crab and native mud crabs 290
Susan Biinsick
Governing offshore aquaeulture: Progress and challenges 290
Joe Buttncr and Dale Leavitt
Augmenting the lobster catch: Oyster aquaeulture in modified lobster traps 290
Lisa Calvo. Eugene Burreson, Susan Ford, John Kraeuter, Dale Leavitt. and Roxanna Smolowilz
Variation in QPX susceptibility w ith host genetic origin 291
Julie Coininsky, Maureen Mikos, and Katie Sicona
The potential of heat shock treatment for imprcned salinity tolerance of Siiliitd India 291
Todd Corayer
Deep water, loiigline shellfish farming in NaiTagansett Bay 291
Barry A. Costa-Pierce
The Rhode Island Aquaeulture Initiative 292
Yvonne Coursey, Nina Ahmad, Barbara McGee, Nancy Steimel, and Mary Kimble
Embryonic blood cell formation in Liiiiulus polyphciuiis ( horseshoe crab) 292
Peter DeSanctis and Kim Tetrault
Preliminary lindnigs on the effect of manipulating photoperiod on gonadal index of the bay scallop (Argopecten
irrailiaiis iiiadians ) 292
Mark Dixon and Gary Wikfors
Rotifer production on microalgal diets: Defining parameters for optimal production 293
Gef Flimlin, Michael Celestino, John Kraeuter, Robert Macaluso, and Michael Kennish
Raritan Bav hard clam fishery management: Getting the data to make decisions 293
Tessa Getchis, Cori Rose, John Volk. Peter Francis, Robin Bray, Mark Johnson, and R. Michael Payton
Aquaeulture policy in Connecticut — Constructing a permitting roadniap for stakeholders 293
Jack Grundstrom. Bonnie McAneney, Scott Weston, Mark Fregeau, and Joe Buttner
Community efforts to restore local clam Hats 294
Edward Jaskolski, Michael Rice, and Karin Taninii
Growth of Rhode Island quahogs. Meirenaria incrcenaria, in experimental upwellers as a part of the North Cape Oil
Spill Restoration Project 294
Richard Karney and Enid Sichel
In search of labor saving culture strategies for the bay scallop. Argopecten irradians irradians 295
Dale Leavitt. Brad Morse, Scott Soares, and Keith Wilda
There is something fishy about that cranberry bog! 295
Clyde MacKenzie. Jr.
The spread of sea lettuce in estuaries of North America and Europe and its potential effects on shellfish culture 295
Christopher Martin, Dean Perry, David Nelson, Robin Katersky, Stephen Metzler, Fu-Lin Chu, and Eric Lund
Ciyptlu'cddiiiiiiiii cohnii. heterotrophic marine dinotlagellate: Is it a good alternate source of essential fatty acids for
first-feeding larval finfish? 296
Paul Mangle
Urban aquaeulture in Connecticut 296
Mary Morgan, Kathleen Becker. Marion Maino, and Kim Tetrault
The first 1 8 months of a community-based shellfish restoration project for eastern Long Island. NY 296
Jessica Miische and David Bengtson
Effects of weaning strategies on growth and survi\al of ju\enile summer flounder. Paraliclitliys dciiiatiis 297
288 Abstracts. February 2003 Milford Aquaculture Seminar. Milford. Connecticut
David Nelson, Dean Perry, and Edward Baker
Natural spawning of blacis sea bass. Centroprislis striata, at the NMFS Milford Laboratory and the UMASS
Dartmouth Laboratory w ith observations on spawning beha\ ior 297
David Nelson, Dean Perry, Robin Katersky, and Stephen Metzler
Grow th of juvenile black sea bass, Centropristis striata, in a recirculating seawater system 298
Christopher Parkins
The potential of polychlorinated biphenyls contamination of aquaculture products through feed 298
Dean Perry, David Nelson, Robin Katersky, Mark Dixon, and Stephen Metzler
Effects of high levels of ammonia. pH, and salinity in algal feeds on the mass production of rotifers 299
Cori Rose, Peter Francis, Robin Bray, and Tessa Getchis
Evaluation factors for aquaculture gear applications 299
Anthony Rossomando. Ryan Kilmartin, John Roy, and Richard Cooper
A comparison of mortahtv m the American lobster. Hoinaiiis americanus. using two methods of tagging 300
Otto Schmid, Armand DeLuca, and Kim Tetrault
It takes a communitv to build a hatchery 300
Laurie Stafford, Jessica Miische, and David Bengtson
Effects of container size on growth and metamorphosis of larval summer flounder. Paralichtliys deiuatus 300
Sheila Stiles, Joseph Choronianski, and Dorothy Jeffress
Genetic strategies for culture and stock enhanceinent of bivalves 301
Amandine Surier and Richard Karney
Oyster triploidy trials on Martha's Vineyard 301
John Wadsworth, Tessa Getchis, and Nancy Balcom
Razor clam, Ensis directits, growth rates in Niantic River, Connecticut 302
Bill Walton
The long and winding road; Towards sustainable fisheries management and meaningful shellfish restoration
( Wellfleet. MA ) 302
Scott Weston, Bonnie McAneney, Mark Fregeau. and Joe Biittner
Mo\ ing tow ards cominerciali/ation of softshell clam culture on Massachusetts' Northshore 302
James Widman, Jr. and David Veilleiix
Demand feeding of bav scallops. Artiopecteii irradians irradiaiis using an automated control system 302
Gary Wikfors, Barry Smith, Shannon Meseck, Mark Dixon, and Jennifer Alix
A decision tree for designing a process to produce microalgal feeds for aquacultured animals 303
William Wilcox and David Grunden
Initial in\ estigation of an annual Proioccmntm bloom in Lagoon Pond, Martha's Vineyard 303
Lawrence Williams, Tessa Getchis, and hike Sunila
An update on blue mussel culture in Long Island Sound 304
Milford Aqiuicultuie Seminar, Miltord, Connecticut
Abslrach. February 2003 289
OVERVIEW. 23rd MILFORD AQUACULTURE SEMINAR.
Walter ,|. Blogoslawski, United States Department of Commerce.
National Oceanic & Atmospheric Administration, National Marine
Fisheries Service. Northeast Fisheries Science Center, Miltord
Laboratory. 212 Rogers Ave.. Milford. CT 06460.
There were 162 registrants for the 2.^rd Milford Aquaculture
Seminar, a gathering of industry, research, and academic interests.
By blending both the theoretical and practical aspects of aquacul-
ture. the meeting permitted attendees an exchange of technology in
aquaculture methods outside their own expertise and provided a
forum where the latest innovations were mtroduced and discussed.
Forty-two formal papers and posters were presented by attend-
ees from eleven US states, the District of Columbia and Canada.
Meeting attendees represented three vocational aquaculture high
schools. !.■? universities, five marine labs, and se\eral state and
federal institutions involved in shellfish and finfish aquaculture. A
highlight of the meeting was a set of papers reviewing the aqua-
culture research activities at the NMFS Milford lab in algae and
tlsh culture, fish feeds, scallop culture, and the role of genetics in
culture and enhancement of aquacultured products. Other papers
co\ered crop insurance, fish fanning in cranben'y bogs and how pol-
lutants can bioaccumulate in culture feeds. Mr. Tim Keeney. NOAA
Deputy Assistant Secretary for Oceans and Atmosphere, descnbed
NOAA's position on aquaculture during a luncheon address.
The Seminar has de\eloped a tradition of offering the latest
information available in the field in an informal atmosphere. This
has succeeded in promoting a free exchange among all with an
interest in the success and future of aquaculture. This Seminar
continued that approach which allowed all attendees to enjoy and
learn from the formal presentations and afforded informal oppor-
tunities to di.scuss the latest developments pertinent to this impor-
tant expanding field.
At this year's seminar thirty-three separate aquaculture com-
panies met in an evening session for their annual industry group
meeting of the East Coast Shellfish Growers Association. The
Association's goals are to promote and protect shellfish members"
needs in state and regional contexts and involve all stakeholders in
the task of enhancing the shellfish aquaculture industry. In addi-
tion, federal and state agencies involved in regulation of offshore
aquaculture described the new permitting system and how it might
affect the indu.stry's development.
The meeting was sponsored by the National Marine Fisheries
Service. Northeast Fisheries Science Center. Milford Laboratory.
Milford, CT. Abstract printing was courtesy of the U. S. Depart-
ment of Agriculture, Northeastern Regional Aquaculture Center,
N. Dartmouth, MA.
PHOTO DOCUMENTATION AS A VITAL ELEMENT IN
COMMUNITY BASED SHELLFISH RESTORATION PRO-
GRA.MS. Kathleen Kmet Becker, and Kim Tetrault. Cornell
Cooperative Extension of Suffolk County Marine Program, Marine
Environmental Learning Center, Southold. NY 11971.
Community nnoUenient in local programs dedicated to vari-
ous aspects of shellfish restoration has grown dramatically in re-
cent years. Documentation is increasingly important and expected
and can be used as a powerful tool to benefit any program. The
Special Projects in Aquaculture Training (SPAT) program has.
from its onset in January of 2001, compiled an extensive library of
photographic images as one pail of its documentation process.
The photo documentation provides an ongoing chronology of
the program's projects and growth. It is being used to document
scientific data collection and community involvement in restora-
tion and stewardship activities. It is useful in the grant application
and subsequent reporting process. As a visual aid and information
sharing tool, it is being used to educate and to communicate to a
broader public through posters, marketing and Power Point® pre-
sentations.
Photographic recognition of individual volunteer participation
in restoration activities highlights the grass roots efforts and helps
illustrate the social dynamics of a program.
® The use of trade names is to identify products and does not
imply endorsement by the National Marine Fisheries Service.
INSURING YOUR AQUACULTURE CROP. David Berry,
Hartford Company, 2625 S. 158th Plaza, Omaha, NE 68130.
As an aquaculturist, you face various inherent financial risks.
Among them are the loss of the initial investment in the crop, loss
of the investment in any growing facilities, and loss of income
associated with the finished crop. This session will help you better
understand these risks, and ways to minimize them. It will explain
how insurance can diminish your financial losses, and some of the
other functions it performs. An overview of some risks for which
insurance can be purchased will be given, such as power failure,
disease, and windstorm. Attendees will also be given an outline of
the underwriting and claims process involved with an insurance
policy, and a brief review of the Federal Clam Insurance Program.
ECONOMICS, MARKETING AND HOW THEY RELATE
TO GROWERS HUSBANDRY METHODS. Don Bisliop,
Bishop Aquatic Technologies Inc., Fukui North America, P.O.
Box 669. IIO-B Bonnechere Road. Eganville, Ontario, Canada
KOJ IT.
The current Shellfish production in the United States and
Canada has a wholesale trade of approximately 243 million USS.
There is a substantial amount of imported shellfish that when
added to this further creates a serious economic sector of the
seafood industry. It is estimated that with an increased supply of
safe, quality, branded product that the market place could be in
excess of 325 million USS over the next decade.
Consumer taste and consumption patterns are in constant
change in our brand conscious society, the understanding of this
and the relationship to social class structure and the buying habits
290 Abstracts. February 2003
Milford Aquuculture Seminar, Milford. Connecticut
present evidence and opportunity for the shellfisli industry to grow
very profitably.
To address and take advantage of these factors, shellfish grow-
ers have to deliver what the customer wants and not just what the
shellfish grower can supply. Technology and strategies have been
developed from the larval stage though husbandry practices to
point of sale marketing that will attract and develop new and repeat
customers.
The challenge the industry will face will be the supply of a
"Safe, Quality, Branded product" that can be sold at a premium
price; this means that farm yield and efficiency is an equally im-
portant part of the equation.
The information presented will allow growers and industry spe-
cialists in attendance to learn what is available in production tech-
nology and marketing initiatives as well as the direction that they
may take to develop a more profitable shellfish business for them-
selves or their specific regional area now and in the future.
PRELIMINARY INVESTIGATIONS OF SHELTER COM-
PETITION AMONG THE ASIAN SHORE CRAB AND NA-
TIVE MUD CRABS. Diane J. Brousseau. Sara Brady, and
Allison Schaffer, Biology Department, Fairfield University, Fair-
field. CT 06824.
This study examined the potential impact of the recently intro-
duced Asian shore crab, Hemigrapsus sanguineus, on shelter uti-
lization by two native species of mud crabs. Euiypaiwpeus de-
pressus and Paiu)peus herbstii. using laboratory experiments and
field sampling at two sites in western Long Island Sound (Black
Rock Harbor, BRH: Milford Harbor, MH). Abundance and distri-
bution patterns of these species differed at the two sites. Similar
numbers of mud and Asian crabs were found under rocks at BRH,
but Asian crabs outnumbered mud crabs 15:1 at MH. Asian crabs
were most abundant at mid-tide level, whereas 90% of the mud
crabs occurred low in the intertidal. This is likely due to the low
tolerance for desiccation exhibited by xanthid crabs (Grant & Mc-
Donald 1979). At low tidal elevation, where most of the overlap
occurred, between-site differences in under-rock microhabitat uti-
lization were present. Only mud crabs were found beneath 75% of
the rocks sampled at BRH. but at MH, mud crab species alone
were found under only 5% of the rocks. Relative crab densities
likely affect competitive outcomes and ultimately space utilization
patterns. Results of shelter competition experiments conducted in
the laboratory did not support the hypothesis that H. sanguineus
affects shelter utilization by native mud crabs. The percentage of
mud crabs occupying shell shelters remained unchanged when
Asian crabs were present, but the percentage of Asian crabs oc-
cupying shell shelters decreased relative to controls in trials where
mud crabs were present. These findings suggest that E. depressus
and P. Iicrhstii may affect patterns of habitat use by H. sanguineus.
especially in the lower intertidal, where these species occur to-
gether. However, direct experimental manipulations in the field
coupled with long-term monitoring are needed to fullv understand
the role of competitive interactions in determining the local dis-
tribution of these species.
GOVERNING OFFSHORE AOLIACULTURE: PROGRESS
AND CHALLENGES. Susan M. Bunsick, Marine Policy Con-
sultant. 3 1 14 Wisconsin Ave., NW, #702, Washington, DC 20016.
Six key components of a governing framework for offshore
aquaculture are identified, and used as benchmarks in assessing
progress toward the development of offshore aquaculture policy
for the U.S. Exclusive Economic Zone. From an aquaculturist's
perspective, the most important components of a governing system
for offshore aquaculture are mechanisms for ( 1 ) granting a range
of rights to the aquaculturist and (2) protecting those rights. From
the broader perspective of a national government, there is a need
for mechanisms that (3) protect the rights of other legitimate users
of public waters and (4) consider a range of other important na-
tional interests and policy priorities. There is also a need to (51
develop administrative systems that are fair, effective, and effi-
cient. This may include a requirement that (6) the aquaculturist
provide some form of compensatii)n in exchange for the right to
locate and operate an aquaculture operation in public waters. Fed-
eral agencies, the research community, and others have begun to
address the development of a governing framework for offshore
aquaculture in the United States. While these initiatives have re-
sulted in some progress, challenges remain.
AUGMENTING THE LOBSTER CATCH: OYSTER AQUA-
CULTURE IN MODIFIED LOBSTER TRAPS. Joe Buttner,
Northeastern Massachusetts Aquaculture Center and Department
of Biology. Salem State College, Salem, MA 01970; Dale Leavitt,
Roger Williams University, One Olde Ferry Rd., Bristol, RI
02809.
Traps used by commercial fishers to capture the American
lobster {Hoinarus americanus) are constructed and fished in ways
that approximate technologies commonly employed to culture the
eastern oyster (Ciassostrea virginica). By modifying traditional
lobster traps to incorporate trays for oysters it was hypothesized
that oysters would survive, grow, and augment the income of
lobstermen while promoting acceptance of aquaculture among
commercial fishers, local cominunities, and regulatory agencies.
To explore the biological feasibility and practical integration of
oyster aquaculture in modified lobster traps a 2-y. cooperative
study involving commercial lobstermen, regulatory agencies, and
research/extension personnel was initiated in 2001.
Ten lobstermen, six from Massachusetts" Northshore and four
from Massachusetts" Southshore/Cape Cod/Islands were identi-
fied, trained, and provided with modified traps and oysters from an
approved source. Modified traps were fished adjacent to or in the
same line as unmodified traps between May/June and October/
Milford AquacultLire Seminar, Milt'ord, ConneLticut
Absuacls. February 2003
November in 2001 and 2002. Lohstermen recorded information
(date, deptli fished, capture rale, and handling elTorl) in standard-
ized journals.
Results indicated that oysters in modified lobster traps sur-
vived, grew, and can be managed without excessive handling or
interfering with lobster removal. Lobster capture rales varied
widely and it is unclear whether the modified lobster traps fish
differently from traditional traps.
Oyster growth was temperature dependent and integration of
oyster culture in lobster traps seems most appropriate on the
Southshore, Cape Cod, and the Islands. It is likely that the tech-
nology can be transferred to other areas and applied to other bi-
valves, providing supplemental income to lobsterfishers while nur-
turing acceptance of aquaculture and perpetuating an important
New England tradition, commercial fishing.
VARIATION IN QPX SUSCEPTIBILITY WITH HOST GE-
NETIC ORIGIN. Lisa M. Ragone Calvo. and Eugene M. Bur-
reson, Virginia Institute of Marine Science, College of William
and Mary, Gloucester Point. VA 23062; Susan E. Ford and John
N. Kraeuter, Haskin Shellfish Research Laboratory, Rutgers Uni-
versity, 6959 Miller Ave.. Port Norris, NJ 08349; Dale F. Leavitt,
Roger Williams University. One Olde Ferry Rd.. Bristol, RI
02809; and Roxanna Snioiouitz, Marine Biological Laboratory,
Woods Hole, MA 02543.
In recent years epizootics of quahog parasite unknown (QPX),
a protistan pathogen of hard clams, Merceiuiria mercenaria. have
occurred in Massachusetts, New York, New Jersey, and Virginia.
Although it has been found in wild hard clam populations, this
parasite has most seriously affected cultured hard clams suggesting
*^at aquaculture practices may promote or predispo.se clams to the
disease. In this investigation we examined the influence of host
genetic origin and geographic location on QPX disease suscepti-
bility. Five clam strains, originating from Massachusetts, New
Jersey, Virginia, South Carolina, and Florida were produced at a
single hatchery and evaluated during a 3-year period for growth,
survival, and QPX susceptibility at three QPX endemic sites (Mas-
sachusetts, New Jersey, and Virginia). Severe winter-associated
clam losses occurred at the Massachusetts site precluding comple-
tion of the study at that location. At the Virginia site, mortality at
the termination of the experiment was 79% in Florida clams and
52% in South Carolina clams, as compared to 369f in Virginia.
33% in Massachusetts, and 209c in New Jersey clams. Differences
between stocks were significant with mortality in the Florida and
South Carolina clams being significantly higher than in the north-
em clams. QPX prevalence in the South Carolina and Florida
stocks ranged from 19-21% and 27-29% respectively in the sec-
ond and third year of the study, while QPX prevalence in the
Virginia, New Jersey, and Massachusetts slocks was 10% or less.
Mortality was significantly correlated with QPX prevalence during
the second and third years of the investigation.
A similar trend was observed at the New Jersey site. Mortality
al the termination of the experiment was estimated to be respec-
tively 53%. 40%, 20%. 6%, and 4% in the Florida, South Carolina,
Virginia, Massachusetts, and New Jersey clam stocks respectively.
QPX was first detected in the clams 14 mo after planting. At 17
and 22 mo after planting. pre\alences ranged from 13-18% in the
Florida stock. 20-38% in the South Carolina stock. 0-18% in the
Virginia clams, and 0-5% in the New Jersey and Massachusetts
clam stocks. These results suggest that genotype-environment in-
teractions are important determinants of QPX disease. As such,
hard clam culturisis should consider the geographic origin of clam
seed an important component of their QPX disease avoidance/
management strategies.
THE POTENTIAL OF HEAT SHOCK TREATMENT FOR
IMPROVED SALINITY TOLERANCE OF SALMO
TRUTTA. Julie Cominslvy. Maureen Mikes, and Katie Sicona.
Bridgeport Regional Vocational Aquaculture School. 60 Saint
Stephens Road, Bridgeport, CT 06605.
Heat shock treatment has been applied to cross protection stud-
ies, including salinity, ammonia, and nitrogen compounds. Brown
trout. Sciliiio iniiici. were selected for this study to determine the
potential of heat shock treatment for improved tolerance of salinity
stresses. The heat shock was conducted in 10-gallon freshwater
tanks for 10 minutes. Visual observations were conducted at 30-
sec intervals. These visual observations included swim patterns
and orientation, mucus excretion, respiratory motion rate, and
scale loss. The fish were then removed from the heat shock treat-
ment tanks and transported to the post shock recovery tank system.
The post shock recovery tank system consisted of four 10-gallon
tanks in a cold-water bath. The heat shock treatment range was
23-29°C set at 2°C intervals. As the shock temperatures increased,
negative behavior patterns were observed, with mortalities occur-
ring at 29°C. Based on these observations 27°C was determined
the optimal temperature to perform the heat shock treatment in the
salinity applications of 5 ppt to 20 ppt at 5 ppt intervals. Noticeable
disparities between the control set and the heat shock set were not
realized until the 20 ppt concentration was conducted. At 20 ppt
the heat shock data showed a 100% survival rate over 96 h of
salinity exposure, while, the control set showed a 70% survival
rate over 96 h of salinity exposure.
DEEP WATER, LONGLINE SHELLFISH FARMING IN
NARRAGANSETT BAY. Todd Corayer, Salt Water Farms
LLC, 30 George St., Wakefield, RI 02879.
Salt Water Farms is developing a multi-species aquaculture
business specifically sited to make use of an underutilized water
column, and excellent environmental factors in an effort to estab-
lish responsible, large-scale shellfish aquaculture in waters with
many historical users.
292 Abstracts. February 2003
Milford Aquaculture Seminar, Milford. Connecticut
Anchored with two different custom mooring configurations
for this dynamic, open water site and serviced by our 36' vessel,
the New Hope, we have deployed both vertical cage assays for
Crassostrea vir}>inica culture and blue mussel drop-socking, in
conjunction with spat collectors. Approximately 300,000 oysters,
at an average size of 25 mm were confined in cages at densities
that reflected market size spatial requirements. Mytilus edidis spat
collectors, both svnthetic and recycled pot waip. were set to iden-
tify spawning patterns and preferences. Blue mussels were also set
into socking and have been examined throughout their grow-out.
In cooperation with the University of Rhode Island, seasonal grab
samples are being conducted, to determine any effects the farm
may have on the benthic environment.
Despite the usual learning curve, we experienced sufficient
growth rates to enable a reasonable percentage of oysters to reach
market size by the end of the growing season. Positioned mid-
water, the design is an effort to establish a prototype large-scale
farm that can operate successfully in the midst of other recreational
and commercial users. Our main goal is to build a farm business
where both animal and gear stocking densities have been thor-
oughly tested and analyzed to have minimum environmental and
social impact, while operating profitably.
THE RHODE ISLAND AQUACULTURE INITIATIVE.
Barry A. Co.sta-Pierce, Rhode Island Sea Grant College Program,
Graduate School of Oceanography. University of Rhode Island,
Narragansett. RI 02882.
In an attempt to ele\ ate Rhode Island from last place among the
50 states in aquaculture production. Senator Jack Reed obtained
1.5 million USS for developing aquaculture in the Ocean State.
The Rhode Island Aquaculture Initiative (RIAI) is a unique col-
laboration that unites federal and state interests as well as aca-
demic, regulatory, and industry resources.
Funding from the National Oceanic and Atmospheric Admin-
istration was awarded to the Rhode Island Coastal Resources Man-
agement Council (CRMCl, the states lead regulatory agency for
aquaculture. CRMC, in turn, enlisted the Rhode Island Sea Grant
College Program to administer the project. In 2002, the RIAI di-
rected 600,000 USS of that money toward aquaculture research
and development in the state through a series of multi-year re-
search grants and 1-y "mini-grants", awarding funding for projects
that seek to improve the health and longevity of farmed shellfish,
educate students and coinmunities about aquaculture, address con-
cerns about aquaculture"s effects on the environment, help re-
searchers and aquaculturists access aquaculture data, and reduce
conflicts between aquaculturists and traditional capture fishermen.
Funding for new capacity-building projects and industry-relevant
aquaculture research has been made to help jump-start a new era ot
aquaculture development in Rhode Island — a place where every-
one says our collective challenges are among the greatest any-
where— and help Rhode Island become a world-class aquaculture
research and development center.
EMBRYONIC BLOOD CELL FORMATION IN LIMULUS
POLYPHEMUS (HORSESHOE CRAB). Yvonne Coursey,
Nina Ahmad, Barbara McGee, Nancy Steimel, and Mary
Kimble, Department of Biology. University of South Florida,
4202 E. Fowler Ave., SCA 1 10. Tampa. FL 33620.
Invertebrates produce blood cells, but unlike vertebrates where
blood cell production (hemopoiesis) takes place primarily in the
bone maiTow, hemopoietic sites in invertebrates vary from species
to species. The blood cells (amebocytes) of Limulus polyphemus
Linnaeus are among the most widely studied of any invertebrate.
Despite having received an enormous amount of attention the
site(s) of blood cell forination in Limulus have remained elusive.
The primary goals of this research were to determine where horse-
shoe crabs (Limulus polyphemus) produce their blood cells, and
when during embryogenesis blood cell production begins.
To distinguish Limulus amebocytes froin other tissue, a poly-
clonal antibody was raised against purified coagulogen protein, the
major protein found in the amebocyte granules. The anti-
coagulogen antibody allowed the identification of maturing em-
bryonic blood cells from all other embryonic cells. Blood cell
production begins in the developing embryo at stage 18, approxi-
mately half way through embryonic development. Embryonic
blood cells are located in body cavities. Blood cells mature in
circulation, as seen by the increase in granulation of blood cells
comparing stage 1 8 to stage 20 embryos. The presence of coagu-
logen in the granules was confirmed using the anti-coagulogen
antibody.
PRELIMINARY FINDINGS ON THE EFFECT OF MA-
NIPULATING PHOTOPERIOD ON GONADAL INDEX OF
THE BAY SCALLOP (ARGOPECTEN IRRADIANS IRRADl-
ANS). Peter N. DeSanctis, and Kim Tetrault, Cornell Coopera-
tive Extension of Suffolk County-Marine Program. Marine Envi-
ronmental Learning Center, Southold, NY 11971,
Using gonadal index as a measure of fecundity, a preliminary
experiment was performed in an attempt to demonstrate the effect
of light on the reproductive capacity and rate of conditioning of the
bay scallop. Populations of scallops were exposed to a regime of
ambient light, continuous light, or continuous dark. All other vari-
ables, such as water temperature and feed were held constant for
the three test populations. In this initial and abbreviated study, it
was observed that differences in gonadal index varied with pho-
toperiod. It was found that scallops subjected to continuous light
showed in a higher gonadal index throughout the test period as
compared to the other treatments. Subsequent investigations will
address fecundity, egg size and quality and minimizing condition-
tiine to spawning.
Milford AquacLillure Seminar, MiU'ord, Coniieclicut
Absnaas. February 2003 293
ROTIFER PRODUCTION ON MICROALGAL DIETS: DE-
FINING PARAMETERS FOR OPTIMAL PRODUCTION.
Mark S. Dixon and Gary H. VVikfors, USDOC. NCAA. National
Marine Fisheries Ser\ ice. Northeast Fisheries Science Center. Mil-
ford Laboratory. Milford. CT 06460.
Production of live feeds can be a bottleneck in finfish aquacul-
lure. Producing sufficient numbers of rotifers on live microalyac
can be an especially problematic step in this process. A balance of
producing large volumes of suitable quality algae, maintaining
appropriate growth paraineters for rotifers, and timing is required
for success. Rotifer production at the Milford National Marine
Fisheries Service Lab has had \aried levels of success and failure
during fintlsh aquaculture prtijects. These e.xperiences. and a re-
view of literature, suggest that a narrow set of parameters in both
microalgae culture and rotifer culture must be met to assure con-
sistent live feed production. Previous work at the Milford lab
identified the Tetraselmis strain. PLY429. of microalgae as the
best food for rotifer production of ten algae tested. It was also
found that maintaining algal densities of six million cells per mil-
liliter yielded the highest conversion efficiency of algal biomass to
rotifer biomass. This study focuses on identifying the specific param-
eters required to re;ir rotifers successfully on a moderate scale.
Six. 30-liter, round-bottom drain vessels were used to test a
single parameter at two different levels (each level in triplicate) at
a time. Parameters tested included: ammonia level, light level,
algal cell density, and mixing method. Rotifers were stocked at
50/ml to begin each trial. Total rotifers produced, percent of roti-
fers with eggs, and various water/culture quality parameters were
measured during each trial. Maintaining ammonia levels below 1
ppm in the algae and rotifer cultures was essential to rotifer
growth. Illuminating the "green" rotifer cultures to levels of 1000
microeinsteins/square meter/second PAR at the surface led to
higher rotifer production and reduced ammonia levels compared to
room light alone. Maintaining algae densities at a constant high
level (2-3 million cells/ml) produced more rotifers than letting
rotifers graze down the algae population. Adequate bubbling to
keep dissolved oxygen levels over 5 mg/1 throughout the "green
water" culture was also essential to rotifer production. When all
tested parameters were optimized, and with full 30-liter vessels, it
was possible to consistently produce 500 rotifers/ml (15 million
per tank) from 50/ml in 5-7 days.
RARITAN BAY HARD CLAM FISHERY MANAGEMENT:
(JETTING THE DATA TO MAKE DECISIONS. Gef Flinilin.
Rutgers Cooperati\e Extension. 1623 Whitesville Rd.. Tt)ms
River. NJ 08755: Michael Celestino, NJ DEP Bureau of Shell-
fisheries, Nacote Creek Research Station. P.O. Box 418. Route 9.
Milepost 51. Port Republic. NJ 08241 : .John N. Kraeuter, Haskin
Shellfish Research Laboratory. Rutgers L!ni\ersity. 6959 Miller
Ave.. Port Norris. NJ 08349: Robert J. Macaluso, Brookdale
Communitv Colleize. Sandv Hook Field Station. Building 53.
Sandy Hook. Highlands. NJ 07732: and Michael Kennish. Rut-
gers Institute of Marine and Coastal Sciences. Cook College. 71
Dudley Rd.. New Brunswick. NJ 08901.
The hard clam fishery in the Raritan and Sandy Hook Bays was
a \iable entity until a hepatitis outbreak in the early 1960s closed
the fishery. In 1983. a state-sponsored relay program and private
depuration allowed the fishery to re-open in Monmouth County. At
this time there are about 200 full and part-time clammers working
those areas. They supply two depuration plants or move their catch
to relay beds in appixned water in Ocean County for a 30-days
purging process. They harvest about 35-40 million clams a year
that have a dockside value of over 5 million USS. Some clammers
can gross over $100,000 in this operation.
Although a stock assessment was done by the State in 1983 a
lack of funds prevented more surveys until 2000 when the New
Jersey Department of Fish and Wildlife Bureau of Shellfisheries
surveyed the same area again. At the same time. Rutgers Haskin
Shellfish Lab. Rutgers Institute of Marine and Coastal Sciences,
and Rutgers Cooperative Extension along with Brookdale Com-
munity College conducted studies to determine the age and growth
of the shellfish and a natural mortality study. Since the fishery
appears to be very lucrative, there is increased interest by others to
open depuration plants. The information from these three studies
can allow the industry and the state to work better together to
manage the harvest pressure and the participation in the area.
Fortunately, the data show that the stocks are at higher levels than
when harvest restarted in 1983. possibly allowing for further ex-
ploitation.
AQUACULTURE POLICY IN CONNECTICUT-
CONSTRUCTING A PERMITTING ROADMAP FOR
STAKEHOLDERS. Tessa S. Getchis. Connecticut Sea Grant.
University of Connecticut. 1080 Shennecossett Road. Groton. CT
06340: Cori M. Rose, United States Army Corps of Engineers.
New England District. 696 Virginia Road. Concord. MA 01742:
John Volk. Connecticut Department of Agriculture. Bureau of
Aquaculture. P.O. Box 97. Milford. CT 06460; Peter Francis and
Robin Bray, Connecticut Department of Environmental Protec-
tion. Office of Long Island Sound Programs, 79 Elm Street. Hart-
ford. CT 06106: Mark Johnson, Connecticut Department of En-
vironmental Protection. Fisheries Division. P.O. Box 719. 333
Ferry Road, Old Lyme, CT 06371; R. Michael Payton, Connecti-
cut Department of Environmental Protection, Boating Division,
P.O. Box 280, 333 Fen^ Road. Old Lyme. CT 06371.
The permitting system for marine-based aquaculture in the
State of Connecticut has had a complete overhaul in the past 2 y.
As floating and submerged shellfish structures (longlines. cages,
bags, racks, etc.) have been shown to be an efficient and produc-
tive method for growing shellfish, their use has grown dramati-
cally. The implementation of these types of gear has raised a
number of permitting issues concerning; navigation, boater safety,
aesthetics, environmental effects, liabilitv. etc.
294 Abstracts. February 2003
Milford Aquaculture Seminar, Milford, Connecticut
A new aquaculture permitting policy was set up in Connecticut
in October of 2001. The Connecticut Department of Agriculture.
Bureau of Aquaculture (DA/BA) has collaborated with the United
States Army Corps of Engineers (US ACE) and the Connecticut
Department of Environmental Protection (DEP) to develop the
Connecticut General Programmatic Permit for Aquaculture. The
extensive new permitting process requires review of the above
listed issues and others by a number of state (DA/BA. DEP).
federal (USACE. National Marine Fisheries Service. United States
Fish & Wildlife Service, United States Environmental Protection
Agency), and in some cases, local officials.
The Connecticut Sea Grant Extension Program (SGEP) has
sponsored a workshop series on aquaculture policy and the per-
mitting process. SGEP's partners include USACE, DA/BA, CT
DEP, and municipal shellfish and harbor management commis-
sions that aid in workshop development. The series includes work-
shops specialized for various stakeholders including growers,
policy-makers, extension services, researchers, educators, and the
general public. The intent of the.se workshops is to provide stake-
holders with information on Connecticut's aquaculture permitting
process from local, federal and state perspectives, and to address
the questions or concerns of these stakeholders.
The goal of this workshop series is to facilitate communication
and information transfer among stakeholders in the aquaculture
permitting process. A list of objectives or ""needs" was developed
at the first planning meeting. The immediate needs from the policy
makers' standpoint were:
(1) To develop a roadmap for aquaculture permittmg in Con-
necticut.
(2) To develop an online ""Guide to Aquaculture in Connecticut."
(3) To develop a new strategic plan for aquaculture in Con-
necticut.
COMMUNITY EFFORTS TO RESTORE LOCAL CLAM
FLATS. Jack Grundstrom. Shellfish Constable. Rowley. MA
01969; Bonnie McAneney, Scott Weston, Mark Fregeau, and
Joe Buttner, Northeastern Massachusetts Aquaculture Center and
Department of Biology, Salem State College. Salem. MA 01970.
Since May 1999. officials and volunteers (primarily shellfish-
ers) have released or redistributed millions of wild-caught and
hatchery-reared softshell clams (Mv(( areiiaria) onto approved
tidal flats in Rowley, Massachusetts. Initially, 6 capture nets (35'
X 8' nets with a 1/4" x 1/8" mesh) were installed on flats in the
Rowley River. Only two nets successfully collected wild clam
seed. In 2000, 20 capture nets were set and all nets retained seed;
some nets collected thousands of clams per square foot. Most
clams caught in 2000 were distributed among local flats. -200,000
were transferred to the Northeastern Massachusetts Aquaculture
Center's (NEMAC) Cat Cove Marine Laboratory and over-
wintered. Concun-ently, the same number was held using spat bags
in the Rowley River. These clams were seeded in the spring of
2001 and covered with predator exclusion netting (35' x 14' or 50'
X 14' with a 1/4" x 1/4" mesh). In 2001, over 60 capture nets were
deployed and all collected softshell clam seed with maximum den-
sity reaching a few hundred per square foot. High densities were
reduced by replacing the capture nets (35' x 8' ) with larger preda-
tor exclusion nets (50' x 14').
Between 1999 to 2001 natural recruitment yielded large
numbers of clam seed; however, in 2002 almost no seed was col-
lected under capture nets in Rowley (and nearby towns such as
Gloucester and Ipswich). Poor recruitment was partially mitigated
by hatchery production. The town of Rowley received over
800,000 hatchery-reared clams from NEMAC. Clams were cul-
tured in a Floating Upwelling System (FLUPSY). In the fall, clams
were planted and covered by predator exclusion nets, to be har-
vested when they attain market size. To restore and maintain
healthy clam flats requires broad community support that includes
monitoring and record keeping, facilitating wild recruitment, pos-
sibly a hatchery, creative networking, and a lot of work!
GROWTH OF RHODE ISLAND QUAHOGS. MERCE-
NARIA MERCENARIA. IN EXPERIMENTAL UPWELLERS
AS A PART OF THE NORTH CAPE OIL SPILL RESTO-
RATION PROJECT. Edward Jaskolski and Michael A. Rice,
Department of Fisheries, Aniinal and Veterinary Science, Univer-
sity of Rhode Island, Kingston, Rhode Island 02881; Karin
Tamnii, Department of Environmental Management Coastal Fish-
eries Laboratory. 1231 Succotash Rd. Wakefield. RI 02879.
The growth of northern quahogs, Mercenaiia mercenaiia.
spawned from native Rhode Island non-notata broodstock was
evaluated in experiment upwellers for the purpose of evaluating
seed production methods for shellfish restoration projects. Down-
wellers were constructed to accommodate 1.2 million small (400
fxm to 1 mm) hatchery-reared seed. Seed were moved to upwellers
once they reached an average valve length of -2 mm. Upwellers
were purchased from commercial sources and deployed at two
sites. The primary location for the study was the Rhode Island
Department of Environmental Management Coastal Fisheries Lab,
Jerusalem, Rhode Island, vsith a secondary location at Roger Wil-
liams University, Bristol, Rhode Island. Growth of the quahog
seed was determined as a function of location, stocking density,
le\'els of biofouling. and water tlow through the upweller silos.
The quahog seed reached a maximum size of 13 mm at the end of
the 20()2-growing season. To minimize overwintering and preda-
tion loss the quahogs were overwintered in benthic cages. The seed
will be field planted in designated shellfish restoration sites in the
2003 season when they reach an average valve length of 20 mm.
This is publication number 3972 of the College of the En\ironment
and Life Sciences, University of Rhode Island.
Milford Aquacultuie Seminar, Milford. Coiiiicclicut
Abstracts, February 2003 295
IN SEARCH OF LABOR SAVING CULTURE STRATE-
GIES FOR THE BAY SCALLOP. ARGOPECTEN IRRADl-
ANS IRRADIANS. Richard C. Karney, Marthas Vineyard
Shellfish Group. Inc.. P. O. Box 1352. Oak Bluffs. MA 02557;
Enid K. Sichel. Woods Hole Oceanographie Institution. Woods
Hole. MA 02543.
Farming the bay scallop. Argopecteii inadians inadians. is a
labor-intensive proposition, primarily due to biofouling control on
the netting of culture structures. Attempts to field culture small,
early juveniles (2 mm) requires the use of small-mesh nettings { 1.5
mm) that require almost daily brushings to maintain adequate wa-
ter flow to support survival and growth. Larger mesh nettings used
to grow older scallops require less frequent cleaning, however, the
number of cages required increases dramatically as the scallops
grow. Several culture strategies including, reduced densities, cage-
less culture methods using artificial eelgrass, biodegradable burlap
nurseries, and adhesives were investigated as possible means of
avoiding the labor costs associated with net cleaning.
Juvenile scallops were cultured in spat bag nurseries at four
densities (-3,000. 5,000. 7,000. and 11,000/bag) to determine if
simply lowering the density could reduce the requirement for fre-
quent bag brushing. Although growth correlated inversely with
density, growth at even the lowest density was poor.
"C-weed®". an artificial polyethylene (HOPE) eelgrass at-
tached to a weighted aerated pipe, was investigated for its potential
as both a spat substrate for setting scallops and a cageless field
nursery system. Seed scallops that had set on the C-weed® grew
well in the field but the initial set on the artificial eelgrass in the
hatchery was poor. The use of biodegradable burlap to set and
field-culture juvenile scallops remains a superior method.
Twenty commercially available adhesives were tested for pos-
sible application in a cageless culture methodology that involves
attaching juvenile scallops to polyethylene netting with the adhe-
sives. Several promising adhesives have been identified for further
investigation.
® The use of trade names is to identify products and does not
imply endorsement by the National Marine Fisheries Service.
THERE IS SOMETHING FISHY ABOUT THAT CRAN-
BERRY BOG! Dale Leavitt. Roger Williams University. One
Olde Ferry Rd.. Bristol, Rl ()2S09; Brad Morse, DoubleM Cran-
berry Company, 980 Walnut Plain Rd., Rochester, MA 02770;
Scott Soares, Mass Department Food & Agriculture. 251 Cause-
way St., Boston, MA 021 14; Keitli Wilda. Western Mass Center
for Sustainable Aquaculture, University of Massachusetts. Am-
herst. MA 01002.
Over-production and limited market development ha\e de-
creased the farmgate value of cranberries to the point where it is
not covering production costs. Cranberry farming in southern New
England is an economically important industry that also controls
vast amounts of undeveloped land in an area that is rapidly ap-
proaching build-out. It is imperative to develop alternate crops to
permit cranberry farmers to stay in business thereby protecting an
important sector of the local economy and protecting the land. We
ha\e been involved during the past 2 y in modifying a new fish
farming technology, the partitioned aquaculture system (PAS), to
allow its use within a cranberry bog system while not changing the
overall physical structure of the cranberry bog. A demonstration
bog/PAS fish farm has been operating for part of one stimmer
growing largemouth bass, yellow perch, and brown bullheads. Al-
though a full grow-out season has not been reali/.ed. preliminary
growth data suggest that the bog/PAS fish farm has potential to
allow the cranberry farmer to produce an alternate crop within the
bog system. While some water quality parameters were not com-
pletely controlled (i.e., pH and dissolved oxygen) we were able to
grow fish at a rate comparable to other fish farms. At this time we
are planning to further develop the farm next summer to enhance
phytoplankton production, the means of removing soluble nitrogen
from the fish waste, by way of better control of water quality. In
this presentation, we plan to introduce the concept of the parti-
tioned aquaculture system and demonstrate its application w ithin a
cranberrv bog.
THE SPREAD OF SEA LETTUCE IN ESTUARIES OF
NORTH AMERICA AND EUROPE AND ITS POTENTIAL
EFFECTS ON SHELLFISH CULTURE Clyde L. Mackenzie.
Jr., USDOC. NCAA. Northeast Fisheries Science Center. James J.
Howard Marine Sciences Laboratory. 74 Magruder Road. High-
lands. NJ 07732.
In recent decades, the distribution of sea lettuce, Ulva sp.. has
spread due to increasing loads of nutrients in estuaries in North
America and Europe. The sea lettuce covers vast areas of shallow
fiats in some years. A 2000 study in New Jersey and a 2001 study
in Italy show that sea lettuce has a detrimental effect on macro-
fauna. In New Jersey, small invertebrates were 2% as abundant on
the surface of sea lettuce, U. lactuca, sheets and 25% as abundant
under the sheets as they were on unvegetated sand bottoms nearby.
In the Venice Lagoon in Italy the presence of sea lettuce U. rigida
substantially changed the species composition of macrofauna and
lowered their density from what it was 30 y earlier. The results
suggest that the presence of sea lettuce substantially decreases
abundance of small invertebrates and changes their species com-
position. Sea lettuce crowds out eelgrass. Zostera marina, softshell
clams, Mya arenaria. and forces northern quahogs. Mercenariu
mercenaria. to emerge from the bottom. Aquaculturists who grow
softshell clams and quahogs should remove sea lettuce from their
planted beds. This can be done with a haul .seine: a 50- or 100-foot
minnow seine is suitable. Removal needs to be done twice a sum-
mer, initially about the first of June and again in late July or eariy
August. Controlling sea lettuce also improves the condition of the
overall ecosvstem in estuaries.
296 Abstracls. Febniary 2003
Milford Aquaculture Seminar. Milfoid, Connecticut
CRYPTHECODINWM COHNII, HETEROTROPHIC MA-
RINE DINOFLAGELLATE: IS IT A GOOD ALTERNATE
SOURCE OF ESSENTIAL FATTY ACIDS FOR FIRST-
FEEDING LARVAL FINFISH? Christopher Marthi. Dean
Perry, David Nelson, and Robin Katersliy, USDOC. NCAA.
National Marine Fisheries Service. Northeast Fisheries Science
Center. Milford Laboratory. Milford. CT 06460; Stephen
Metzler. End to End. 415 Port Centre Parkway. Portsmouth. VA
2.^704; Fu-Lin Chu and Eric Lund, Virginia Institute of Marine
Science, College of William and Mary, Gloucester Point. VA
23062.
Traditionally, fish culturists have turned to autotrophic microal-
gae for enrichment of larval prey (i.e.. rotifers and brine shrimp
nauplii). For this puipose. algal strains have been selected for their
essential fatty acid composition. Two long-chain polyunsaturated
fatty acids (PUFAs) have received special attention since they
have been shown to improve growth and survival of larval fish.
These are docosahexaenoic acid (DHA. 22:6n-3) and eicosapen-
taenoic acid (EPA. 20;5n-3). Their role in the normal development
of the brain and visual system of young fish is of particular inter-
est. The amount of DHA and EPA varies widely among microal-
gae with some strains containing more of one than of the other.
Consequently, it is usually necessary to enrich with a mixture of
two or more different algal strains in order to achieve the desired
fatty acid level in larval prey. Our objective is to demonstrate that
a commonly occun'ing heterotrophic dinotlagellate. Crxpthccod-
liiitiiii cohiiii Biechler. could serve as a source of DHA in this
application.
C. cohnii is a small, colorless dinotlagellate commonly found
in association with decaying macroalgae (especially Fuais spp.) in
the littoral /.one of temperate and tropical waters. It is readily
isolated from its fa\ored substrate and cultured on seawater en-
riched with simple sugars and yeast extract. Having a short gen-
eration time (8-10 hrs). cultures attaining a density of ca.lO'' cells/
mL may be achieved in 3—1 days. It grows best in the absence of
light and. while it is not fussy, seems to produce more fatty acids
at 30'C than at lower temperatures. C. cohnii is an excellent source
of DHA. Purification of DHA from this organism has led to com-
mercial production of enrichment products for use specifically in
aquaculture.
We chose American Type Culture Collection strain 30772
\Ciyptliecodinium cohnii Biechler] for this work based on its re-
ported fatty acid composition. We demonstrated the ease with
which it could be grown in axenic batch culture on simple media.
We showed that live cells of this heterotrophic dinotlagellate are
acceptable to both rotifers and brine shrimp. Moreover, we docu-
mented the transfer of PUFAs from C. cohnii to larval fish via
enriched rotifers. Finally, we confirmed that larval tautog fed
twelve days on rotifers enriched with live cells of C. colinii accu-
mulated DHA and EPA in the near-optimum ratio 3.31 ± 0.02. In
contrast, larvae fed rotifers enriched with a mixture of Isoclirvsis
sp. and Tetraselmis sp. accumulated DHA and EPA in the ratio
1.42 ±0.07.
URBAN AQUACULTURE IN CONNECTICUT. Paul D.
Maugle, Mohegan Aquaculture LLC. .'i Crow Hill Rd.. Uncas-
ville. CT 06382.
Aquaculture in Connecticut has for the last 130 years tradition-
ally harvested native-set shellfish from the bottom. Connecticut's
oysters are among the most valued oysters reared in the United
States. Knowing that bottom harvesting of native-set shellfish is
not inherently sustainable in Eastern Connecticut waters. Mohegan
Aquaculture LLC has chosen to have, at its core, a shellfish hatch-
ery, coupled with mid-water and surface rearing of shellfish. The
hatchery will be based on the systems installed by the Garbo
Lobster Company in the village of Stonington. Connecticut. This
hatchery when complete is projected to have the productive capa-
bility of more than 200 million shellfish seed each year. This
would make it one of the most productive on the east coast of the
United States.
The company's goal is to become a leading North American
aquaculture producer of premium marine shellfish. With that goal
in mind Mohegan Aquaculture will look to control the entire pro-
duction process from culturing the microalgae that form the live
feed for the larval shellfish to packing the finished product. This
ensures that the company can deliver a superior quality eating
experience to both ethnic and white tablecloth markets.
When completed, the Stonington facility will house several
profit and cost centers including a commercial scale shellfish
hatchery, a wet storage facility, a commercial scale nursery, up-
wellers. support space for near-shore longline and tray operations,
and mooring facilities for shellfish har\est and long-line tender
boats. The company has adapted several proven aquaculture tech-
nologies to create its own proprietary production systems.
Mohegan Aquaculture's production model utilizes a three spe-
cies portfolio approach — bottom seeding of quahog clams, scallop,
and oyster production in various types of floating midwater and
corral structures. The enterprise will also strive to augment its
production capabilities by working w ith third party contract grow-
ing partners. This hybrid production approach allows the enterprise
to focus on its core business competencies including production
techniques, management expertise, and shellfish-value adding
while utilizing available outside production capacity.
THE FIRST 18 MONTHS OF A COMMUNITY-BASED
SHELLFISH RESTORATION PROJECT FOR EASTERN
LONG ISLAND, NY. Marv F. Morgan. Kathleen K. Becker,
Marion Maino, and Kim Tetrault, Cornell Cooperative Exten-
sion of Suffolk County Marine Program, Marine Environmental
Learning Center. Southold. NY 1 1971.
Cornell Cooperative Extension of Suffolk County. New York is
in the second year of an expansion of its Marine Program to
include a community-based shellfish restoration model to foster
Milford AqLiaCLiltiire Seiniii;ir. Miltord. CoiuieL-licut
Ahsiniciy FebmuiA 2003 297
stewardship of the Peconic Estuary. Special Projects in Aquacul-
ture Training (SPAT) is based on the understanding that enhance-
ment of shellfish beds contribute greatly to the health iif estuarine
ecosystems, and that local communities can play a significant role
in stewardship and restoration. Bay scallops. Aii;opeclen iinulians
inacliuns. hard clams. Mercenaria inerccnaiia iiolata. and Eastern
oysters. Cnissostrea virginica are commercially, recreationally,
ecologically, and historically important species to the Peconic Es-
tuary, which currently supports only ]'/c of its historic stocks.
Nationally published data ha\e indicated that hands-on oppor-
tunities in the environment help people become good caretakers of
the environment. From the beginning, it has been a goal of the
project to involve community members in a long-Iemi effort both
to restore locally important marine resources and to develop a
stewardship ethic. While capturing the interest and dedication of
community members is a labor-intensive, year-round undertaking,
the project has motivated many members of our Long Island com-
munity. As of August 2002. 209 marine shellfish gardens are being
maintained by individuals or families totaling 284 individuals. The
community is involved in varying degrees in everything from tend-
ing their own aquaculture gardens, attending monthly seminars,
building and operating a community hatchery, developing an all-
volunteer creek water quality testing team, public education efforts
such as speaking at local civic groups, donating materials and
supplies, and to creating and selling a cookbook to raise funds for
the continuance of the project.
EFFECTS OF WEANING STRATEGIES ON GROWTH
AND SURVIVAL OF JUVENILE SUMMER FLOUNDER.
PARALICHTHYS DENTATUS. Jessica Musche and David A.
Benglson, Department of Fisheries. Animal and Veterinary Sci-
ence. University of Rhode Island, Kingston, RI 02881.
The transition from live feed to formulated diets, which aqua-
culturists call weaning, is a difficult period in the hatchery rearing
of some marine fish. Elevated mortality during this period is often
due to cannibalism as some fish adapt to the new diet and grow
while others do not adapt. Certain strategies have been developed
with other species to optimize the weaning process. We conducted
four experiments on the effects of weaning strategies on the
growth and survival of newly metamorphosed juvenile summer
flounder. Parol ichlhys dentalus. The goal of these experiments
was to reduce growth variability and increase survival.
Each experiment consisted of three treatment groups and one
control group, with three replicates (75-L aquaria) each. The con-
trol group in each experiment consisted of fish that were fed live
brine shrimp nauplii throughout the course of the experiment. In
each experiment, the actual weaning period lasted for two weeks,
followed by several weeks of feeding with the new diet. All
aquaria were on a fiow-through system, receiving Narragansetl
Bay water at 20°C.
In experiment I. we attempted to determine the optimum age at
which to wean the fish. Fish were weaned onto a dry commercial
pellet at 2, 4, or 6 wk post-metamorphosis. Fish that were weaned
at 6 wk post-metamorphosis had the smallest mean lengths, but
they had the least variability in growth and the highest sur\i\al.
We. therefore, conducted each of the remaining experiments be-
ginning at six weeks post-metamorphosis for that group of fish.
In experiment 11, we studied the timing of weaning diet pre-
sentation. Fish were given either a dry diet in the morning and
brine shrimp nauplii in the evening, both dry diet and brine shrimp
simultaneously in the morning and e\ening, or a dry diet and brine
shrimp on alternate days. There were no significant differences in
sur\ i\al or growth among the treatments; however, the fish given
brine shrimp and dry diet simultaneously had the lowest variability
in growth.
In experiment 111, we explored the use of intermediate weaning
diets. Fish were weaned directly to a dry pellet, weaned to frozen
adult brine shrimp and then a dry pellet, or weaned to a semi-moist
pellet and then a dry pellet. While there were no significant dif-
ferences in growth between the fish fed dry pellet only and those
fed frozen brine shrimp, the fish fed the semi-moist diet had a
significantly lower growth rate. The fish fed frozen brine shrimp
had the lowest variability in growth of the treatments. There were
no significant differences in survival among treatments.
In experiment IV, we attempted to use already-weaned fish to
teach unweaned fish to accept a pelleted diet. Aquaria in each
treatment were provided no already-weaned fish, one already-
weaned fish, or five already-weaned fish. Clear barriers that al-
lowed water to flow through were placed in the tanks to separate
already weaned from unweaned fish. At the end of the experiment
there were no significant differences in survival or growth among
treatments, and very little difference in growth variability. In each
experiment, the control groups had the highest survival. The con-
trol groups also had the lowest variability in size, with the excep-
tion of the first experiment in which those fish weaned at 6 weeks
post-metamorphosis had the lowest variability in size.
NATURAL SPAWNING OF BLACK SEA BASS. CENTRO-
PRISTIS STRIATA, AT THE NMFS MILFORD LABORA-
TORY AND THE UMASS DARTMOUTH LABORATORY
WITH OBSERVATIONS ON SPAWNING BEHAVIOR
David A. Nelson and Dean Perry. USDOC, NCAA, National
Marine Fisheries Service, Northeast Fisheries Science Center, Mil-
ford Laboratory, Milford, CT 06460; Edward Baker. 1.^6 Beech-
wood Hill Tr, Exeter, RI 02882.
The black sea bass, Cenlriiphstis striata, is an important sport
and commercial fishery along the United States Atlantic coast.
Black sea bass are managed under the Magnuson-Stevens Fishery
Conservation and Management Act and by the Atlantic States
Marine Fisheries Commission. Because the black sea bass is a
temperate reef species and is unavailable to bottom trawlers, cap-
ture is limited to anglers and pot fisheries. The demand for black
298 Abstracls. February 2003
Milford Aquuculture Seminar. Milt'ord. Connecticut
sea bass exceeds supply and the hiyh market \alue has prompted
researchers to evaluate its potential for commercial aquaculture.
Reproductively, black sea bass are protogynous hermaphro-
dites, developing first as females and later, at 3—4 y of age. trans-
forming into males. Early attempts at spawning black sea bass
centered around artificial spawning, collecting adult black sea bass
in spawning condition and hand-stripping both males and females.
Later attempts focused on inducing ovulation by intramuscular
injection of two hormones; human chorionic gonadotropin or
luteinizing hormone releasing hormone analog (LHRHa) and hand
stripping.
Milford Laboratory and UMASS Dailmouth Laboratory, have
used photothermal manipulation to induce spawning. Black sea
bass were placed in tanks of ambient, flowing seawater (10"C).
The day/night cycle was controlled by a timer that turned fluores-
cent lighting on and off. Lighting was adjusted every three days
to simulate the day/night cycle that was occurring in nature until
15 h of light and 9 h of darkness was reached. When ambient
temperature reached 1 8-20°C and the day /night cycle was 1 5 h of
light and 9 h of darkness the black sea bass spawned. Fish were
allowed to spawn in the tanks and embryos were collected on a
500- |xm screen or in a 800- ixm net. Fish were spawned under these
conditions from mid-April to the middle of July at the Dartmouth
Laboratory and from the end of May until the beginning of July at
the Milford Laboratory. Percent viable embryos ranged from 0%
(first eggs produced) to lOOVr (in the middle of the spawning
season).
We have also made observations on the spawning behavior of
black sea bass in the course of our conditioning procedures. One
dominant male (alpha) appeared to control spawning. The domi-
nant male segregated other males and females in the tank (10
females to 5 males at Milford and 9 females to 5 males at
UMASS). This one male prevented other males from mingling
with the females. When spawning occurred a female would swim
up to the alpha male and present herself. Both fish would move to
a separate portion of the tank where the female would release eggs
and the male would release milt. When spawning was complete the
female returned to the other females and the alpha male positioned
himself between the females and the other males.
GROWTH OF JUVENILE BLACK SEA BASS, CENTRO-
PRISTIS STRIATA, IN A RECIRCULATING SEAWATER
SYSTEM David A. Nelson, Dean M. Perry, and Robin Kater-
sky, USDOC. NOAA. National Marine Fisheries Service, North-
east Fisheries Science Center, Milford Laboratory, Milford, CT
06460; Stephen Metzler, End to End Technical Services Inc.,
Suite 102, 415 Port Centre Parkway, Portsmouth, VA 23704.
The black sea bass. Ceniropristis striata, is currently being
investigated as a potential aquaculture species. Work to date has
focused on spawning and larval development, conditions for cul-
ture of larvae, stocking densities of larvae and juveniles, feeding
trials of juveniles and sub-adults, cannibalism in juxeniles, habitat
preferences in juveniles, and the effect of water velocity on the
juveniles position and moxement. Many of the studies on juveniles
have been conducted with wild-caught black sea bass. Although
black sea bass show great potential for aquaculture. studies have
not demonstrated the time required to produce a market-size fish.
Our goal is to grow black sea bass from larvae to market size
adults (454-680 grams) in 24 months. Black sea bass are spawned
naturally by photothermal manipulation. Embryos are collected on
a 5()()-(j.m stainless steel screen. Viable embryos float and are
separated from dead embryos in an Imhoff cone. The embryos are
placed in 1,140 L cone-bottom tanks filled to 1,100 L with 20°C
seawater. These tanks are part of a closed, recirculating seawater
system with a biofilter and U.V. light. Embryos are allowed to
hatch (48 h) and grow into juveniles in these tanks. Fish remain in
this system for 3-4 mo and are culled by size before being trans-
ferred to two 1,067 L half-round tanks ( 120.5 cm diameter x 60.2
cm depth x 180.7 cm length). Filtered seawater ( IO-|xm) is recir-
culated in these tanks with 10% water replacement/day. These
tanks have biofilters and U.V. lights associated with them. Flow
rate is 113.6-151.4 L/min. Temperature in these tanks is main-
tained at 20 ± 1°C. Fish are weighed and measured on the day of
transfer and once every two weeks thereafter. After 477 days in
this recirculating seawater system, fish have grown from an initial
mean length of 91 mm and a mean weight of 15.6 g to mean
lengths of 232.3 mm and 197.7 mm and mean weights of 242.2 g
and 177.9 g in the two tanks. Juvenile black sea bass that were
produced from fish spawned in 2002 have grown from mean
lengths of 80 and 106.9 mm at transfer to 1 17.2 and 146.5 mm in
83 days. Mean weights have increased from 10.9 and 23.6 g to
32.8 and 65.5 g in 83 days. Fish spawned in 2001 had specific
growth rates of 0.499r and 0.56% per day. Black sea bass spawned
in 2002 had specific growth rates of 0.6% and 0.8% per day.
THE POTENTIAL OF POLYCHLORINATED BIPHENYLS
CONTAMINATION OF AQUACULTURE PRODUCTS
THROUGH FEED. Christopher Parkins. Bridgeport Regional
Vocational Aquaculture School, 60 Saint Stephens Road, Bridge-
port, CT 06605.
Polychlorinated Biphenyls (PCBs) are a group of industrial
organochlorine chemicals that are a major environmental concern.
They are used commercially because they are chemically inert
liquids, have low vapor pressures, are inexpensive to produce and
are excellent electrical insulators. Due to the fact that PCBs are
inert chemicals and soluble in fatty tissues. PCBs undergo bio-
magnification.
Most aquaculture products rely on commercially processed
feeds. These feeds are based on wild-stock fishmeal, which may be
contaminated with PCBs found in the natural environment.
Through the consumption of these aquaculture products PCBs
pose numerous health risks to humans. These include birth defects.
Milford Aquatulture Seminar. Milford. Connecticut
ALmraas. February 2003 299
carcinogenic potential and negative impacts to the immune system.
The feed types, which are being tested, include Zeigler Trout
Feed®. Silver Cup Floating and Sinking Trout Feed® and Hartz
Turtle Feed®. The PCBs are extracted from the feed samples using
a microwave extraction system following EPA method 3546. A
temperature programmable gas chromatograph with a dry electro-
lytic conductivity detector (DELCD) was used following EPA
method 8082 to determine the qualitative level of Aroclor® 1260
in the samples. Two trials were performed which showed the ab-
sence of Aroclor® 1260 in all tested samples.
® The use of trade names is to identify products and does not
imply endorsement by the National Marine Fisheries Service.
EFFECTS OF HIGH LEVELS OF AMMONIA. PH, AND
SALINITY IN ALGAL FEEDS ON THE MASS PRODUC-
TION OF ROTIFERS Dean M. Perry. David A. Nelson. Robin
Katersky. and Mark Dixon, USDOC, NOAA, National Marine
Fisheries Service, Northeast Fisheries Science Center. Milford
Laboratory, Milford, CT. 06460; Stephen Metzler. End to End
Technical Services Inc., Suite 102, 415 Port Centre Parkway,
Portsmouth. VA 23704.
The rotifer, Bmclnonus pikatilis. has been widely used as a
live food for feeding the larval stage of marine fishes. Successful
aquaculture of marine fish requires adequate and reliable produc-
tion of high-quality, nutritious rotifers. One method of culturing
rotifers is to feed them microalgal diets that promote rapid growth
and reproduction. The rotifers used in our aquaculture studies of
the tautog and black sea bass were fed the algal strain Tel rase I mis
sp. (PLY 429). This alga not only promotes rapid reproduction of
the rotifers, but also contains the n-3 and n-6 polyunsaturated fatty
acids that have been shown to promote growth and survival in
larval marine fish. Tetraselinis was cultured under semi-
continuous conditions in three large, open rectangular fiberglass
tanks that received constant lluorescent lighting. These tanks were
maintained between 2()0-3()(l L. Rotifers were fed Tetraselinis
from two of the three tanks on a rotating basis. Initially, for about
1 week, rotifers showed an increase from 4 to 16 million. After that
time, the rotifer population declined to five million and remained
at that level for 2 wk. During that time, and for the next 3 mo.
sporadic measurements of ammonia, salinity, and pH were taken in
each of the three algal tanks. High levels of unionized ammonia
(>1 mg/1). and abrupt changes in salinity (±5 ppt) and pH (±1 pH
unit) in the algal tanks coincided with decreases in the rotifer
population. Those measurements indicated that either individual
fluctuations in salinity. pH and ammonia, or a combination of two
or more of these factors adversely affected rotifer production. We
conclude that changes in salinity, pH. and ammonia levels, as well
as increased numbers of bacteria and ciliates in algal cultures can
be counterproductive to maintaining high rotifer populations. It is
recommended that algal tanks be inonitored daily during high ro-
tifer production times for salinity, pH, and ammonia levels. Also.
large open algal tanks should be monitored on a regular schedule
for bacteria {Vibrio) and ciliates. Some alternatives to using live
algae include concentrated algal pastes, baker's yeast and com-
mercial products.
EVALUATION FACTORS FOR AQUACULTURE GEAR
APPLICATIONS. Cori Rose, Senior Project Manager, United
States Army Corps of Engineers, New England District. Regula-
tory Division. 696 Virginia Rd., Concord. MA 01742: Peter Fran-
cis and Robin Bray, Connecticut Department of Environmental
Protection. Office of Long Island Sound Programs. 79 Elm Street.
Hartford. CT 06106; Tessa S. Getchis, Connecticut Sea Grant.
University of Connecticut. 1080 Shennecossett Road, Groton, CT
06340.
In response to the expansion of aquaculture activities and uti-
lization of developing rearing techniques, there is an increasing
need for review and evaluation of aquaculture proposals to ensure
adequate protection of the environment, wild populations and their
habitat, and the compatibility of such enterprises with existing
users of the public resource. Regulatory agencies (federal, state,
and local) are mandated to review applications for foreseeable
future impacts, which a grower may not consider or be aware of.
It is the charge of such agencies to achieve a balance between
sometimes competing interests while ensuring appropriate regula-
tion of the industry with due regard to the environment and its
many users. For example, it is some or all of these agencies'
responsibility to ensure that granting of a permit, lease or other
authorization will not adversely impact marine resources or pose
unacceptable disease, ecological, health, safety, or welfare risks to
persons, the environment, or aquatic resources. In addition, agency
determinations must also ensure that an authorized activity does
not conflict with or negatively impact any recreational, commer-
cial or other use of the proposed project area, or adversely impact
the \ alue or use of private property in and around the area.
The charge to an applicant proposing an aquaculture project,
especially for a project that entails innovative technologies not
currently used in a geographical area or for the culture of non-
indigenous stock, is to provide enough information for regulators
to make a reasoned decision. However, this can be a daunting task
and the various parties' differing expectations regarding the
amount and type of information needed may result in costly delays
or protracted regulatory reviews. The purpose of this talk is to
impart the type of information that should be submitted along with
an application for aquaculture in Connecticut in order to facilitate
the state/federal joint regulatory review process; and also to dis-
cuss regional guidance that currently exists to aid aquaculture ap-
plicants, convey expectations of the standard level, and provide the
quality of information that may be solicited from regulatory agen-
cies when seeking authorization of aquaculture projects.
300 Absiracts. February 2003
Milford AqiKicLilture Seminar. Milford. Connectieut
A COMPARISON OF MORTALITY IN THE AMERICAN
LOBSTER, HOMARUS AMERICANUS. USING TWO
METHODS OF TAGGING. Anthony Rossomando. Ryan Kil-
martin, and John Roy. The Sound Sehool. 60 South Water St.,
New Haven. CT 06519: Richard Cooper, UCONN. 1084 Shen-
necossett Road. Groton. CT 06340.
The American lobster. Hoinanis anieiicaiuis. has been the sub-
ject of tagging studies for the past several decades. The benthic life
cycle of the lobster and the ease with which they are trapped make
them a species that lends itself readily to recapture studies. Popu-
lation declines in southern New England during the past decade
have made investigations into the recruitment methods of this spe-
cies a priority for several studies. The means by which the species
propagate makes the female lobster the preferred sex for many
studies. A large percentage of the female animals that survive to
maturity will bear eggs annually. The impact of tagging female
lobsters in Southern New England, where the population is declin-
ing, warrants the investigation of the stress caused by the tagging
procedure.
Outcomes from catch and release studies that depend on the
capture of tagged animals to produce data are influenced by re-
capture percentages. While many factors influence the success of
the recapture rate, mortalities that result from the capture, tagging,
and subsequent release of aquatic animals adversely affect all
study outcomes. Investigators and scientific researchers have used
many methods of marking animals that have been taken in this
type of study. Students from the Sound School Regional Aquacul-
ture Center conducted a study to compare the effects of tagging
adult female lobsters with both Floy tags and Back tags.
The results from this study indicate that mortalities associated
with the stresses caused by tagging increased in tagged specimens.
Mortalities of 19.1% and 14.3% were recorded in Back and Floy
tagged lobsters respectively while the lobsters held as controls had
mortalities of 9.59r. The students at the Sound School have had
first hand experience with the dramatic declines in the lobster
populations in western Long Island Sound during the late 1990s.
We belie\e that it is becoming increasingly important to monitor
accurately the existing stocks of lobsters at all levels of the fishery.
However, it has become increasingly apparent through our studies
that tagging efforts, which employ either the Floy or Back tag to
study Hoiiuinis (iinericniuis. may be inflicting substantial mortali-
ties among the sampled portion of the population.
IT TAKES A COMMUNITY TO BUILD A HATCHERY.
Otto Schmid, Arniand DeLuca, and Kim Tetrault, Cornell Co-
operative Extension of Suffolk County Marine Program. Marine
Environmental Learning Center. Southold. NY 11971.
The program Special Projects in Aqiiaculture Training (SPAT)
at Cornell Cooperative Extension, in Southold. New York, has just
completed its second year of operation, having attracted over 200
families volunteering over 1 1 .000 h. Construction of a community
hatchery began in the fall of 2001 and was made operational in the
spring of 2002. SPAT members supplied all of the labor necessary
to do the carpentry, plumbing and electrical work. This became a
valuable learning experience for 12 core workers, augmented by
numerous additional SPAT members on an "as available" basis.
Utilizing many recycled materials, donations of supplies and
equipment and volunteer labor, the cost of the hatchery was mini-
mized. During the 2002 winter layover following its initial grow-
ing season, the layout and exterior were revised and improvements
made to make the hatchery more efficient, in addition to adding a
maintenance annex for tools and equipment. The hatchery at this
time houses six 400 L larval rearing conicals along with the nec-
essary aquaculture equipment needed to produce approximately
6-9 million larvae per spawn.
Selected species of bivalves are spawned in the hatchery and
the larvae are raised through metamorphosis, at which time they
are moved to downwellers in the Marine Center nursery. All coni-
cals are maintained three times weekly at which time the equip-
ment is cleaned and the larvae are culled and restocked to a desired
density. Larvae are fed a mixed diet of algae produced at the
Marine Center. Larvae are set using a variety of techniques.
The community hatchery is the product of the diverse talents of
many individuals. It serves as an invaluable tool for practicing the
concepts learned during the training initiatives of the program in a
hands-on and productive manner. As with many of the components
of the SPAT program, the hatchery is a work in progress and is
unique in many ways. The individuality and commitment of the
SPAT members have allowed the hatchet^ to perform effectively
in its first year of operation and is anticipated to greatly increase
production in the 2003-growing season. The emphasis will be
placed on the production of bay scallops (Argopecten irradians
irniJIaiis) with a target goal of 10-1.5 million post-set.
EFFECTS OF CONTAINER SIZE ON GROWTH AND
METAMORPHOSIS OF LARVAL SUMMER FLOUNDER.
PARALICHTHYS DENTATUS. Laurie Stafford, Jessica
Miische, and David A. Bengtson. Department of Fisheries. Ani-
mal and Veterinary Science. University of Rhode Island. Kingston.
Rl 02881.
Commercial aquaculture of the summer flounder, Paralichthys
dentatus. began in the 1990s. Although research on optimum con-
ditions and methods to rear the larvae has been conducted for
years, many factors remain to be studied. Growth rates of summer
flounder begin to vary greatly in the late larval period. Because
metamorphosis is size-dependent, the fastest growing fish settle
first and often cannibalize their slower-growing siblings who settle
later. Many variables may affect growth and metamorphosis. We
examined effects of container size by conducting an experiment of
49 days duration in which larvae were raised from age 12 days
(after hatch) through metamorphosis. Fish were obtained from
GreatBay Aquaculture in Portsmouth, NH and were the result of
Milford Aquaculture Seminar. Milford. CoihiclUlui
Ab<,lraci\. February 2003 301
their first pureh Fl male x Fl female crosses. The experiment
consisted of three treatments: 2-L, 20-L, and 150-L containers with
four replicates of each treatment and stocking densities in all con-
tainers of 10 fish/L. Three specific variables were examined: sur-
vival, growth (as measured by total length), and the rate of meta-
morphosis (as measured by settlement times of the fish; settled fish
were removed from each container every 3 days). Although there
were no significant differences in survival among the three treat-
ments, container size did affect growth and metamorphosis. The
length of the fish in the 20-L containers was significantly greater
than in the other two treatments until shortly before metamorpho-
sis, after which the fish in the 1 50-L containers surpas.sed the other
treatments (.^NOVA. P < 0.05). Analysis of the distributions of
settlement over time indicated that fish in the 20-L containers
metamorphosed earlier than fish in the 150-L containers (Kolmo-
gorov-Smimov test, P < 0.U5). but metamorphosis of fish in the
2-L containers was not significantly different from that of fish in
either 20-L or 150-L aquaria. Because commercial aquaculture of
summer flounder larvae is conducted in volumes of 1.000 L or
greater, our results may have more significance for the research
community than for the industry. Nevertheless, container size can
affect summer flounder larval growth and metamorphosis.
GENETIC STRATEGIES FOR CULTURE AND STOCK
ENHANCEMENT OF BIVALVES. Sheila Stiles. Joseph
Choronianski, and Dorothy Jeffress, USDOC, NCAA, National
Marine Fisheries Service. Northeast Fisheries Science Center. Mil-
ford Laboratory. Milford. CT 06460.
Genetic selection, which exploits the heritable component of
sanation through breeding, has enhanced significantly the effi-
ciency of livestock and crop production in agriculture. Aquacul-
tured species lag behind, but ha\e provided some successes. How-
ever, inadvertent selection and. therefore, narrowing of the gene
pool can occur with standard hatchery practices, such as spawning
small numbers of broodstock and screening or culling larvae and
juveniles for size. This inbreeding effect can be minimized by
developing appropriate strategies such as introducing new brood-
stock to increase genetic diversity. In addition, in some hatcheries,
there may be certain characteristics that are desired to improve or
increase production. Selecting animals for growth, disease resis-
tance or shell color could increase the frequency of these traits. For
example, some scallops have obvious shell markings, such as
stripes, which could be used in stock identification as nomta clams
are used in the clam industry, and oysters with disease resistance.
Selective breeding studies are underway employing scallops
with striped shells as markers for stock enhancement, as one major
limitation to previously conducted stock enhancement programs
has been a lack of identification of stocks. The objective of this
project is to investigate the feasibility of producing, through se-
lective breeding, increased numbers of bay scallops with distinc-
tive phenotypes for field identification. These naturally-occurring
scallops with distinctively \isible markers at low frequencies of
1-5% are being developed to determine the reproductive success
or genetic contributions of transplanted populations to stock en-
hancement efforts possibly in sanctuaries. Preliminary laboratory
results indicate a positive response to selective breeding with an
increased frequency of at least 50% of scallops with striped shells,
and favorable growth and survival.
Other components of a breeding program, which could include
marker-assisted selection (MAS) and quantitative trait loci
(QTLs). should consider the following points: the status of the
population, the goals to be attained (i.e.. harvesting, stock resto-
ration or enhancement), facility and personnel needs, mating
schemes, genetic monitoring (for genetic diversity), and periodic
assessments. Genetic monitoring methods include cytogenetics,
allozyme, mt and nuclear DNA analyses, and PCR technology.
Alternative biotechnological and supplemental approaches to
breeding encompass technology of gene transfer and chromosome
engineering, such as induced polyploidy (triploidy and tetra-
ploidy). Genetic applications additionally could involve DNA-
based probes and assays to detect disease agents as in MSX and
Deniio studies with oysters, as well as the generation of molecular
tags to identify stocks of shellfish. All of these diverse aspects are
applications of genetics to aquaculture and fisheries management
that should be considered in strategies to maximize production.
OYSTER TRIPLOIDY TRIALS ON MARTHA'S VINE-
YARD. Amandine Surier and Richard C. Karney, Martha's
Vineyard Shellfish Group. P. O. Box 1552, Oak Bluffs, MA
02557.
Triploidy is the condition of possessing three times the haploid
number of chromosomes in the cell nucleus. Because triploid bi-
valves are sterile, their meat quality remains constant throughout
the year and the energy usually used for reproduction is diverted
towards somatic growth and disease resistance. Because of those
unique qualities, their production has attracted worldwide attention
since the early 1980s. At this time triploidy has been successfully
applied to the economic benefit of the Pacific oyster industry on
the west coast of the US and also in France.
Under funding from the Sailors" Snug Harbor foundation of
Boston, triploidy was induced in the American oyster Crassostrea
viriiinica in an attempt to locally produce triploid strains of oysters
for the growers of the island of Martha's Vineyard. Triploidy was
induced with a low risk chemical. 6-DMAP that has been shown to
be slightly less efficient than Cylochalasin B but much safer to
handle and is water-soluble. The success of induction was mea-
sured by flow cytometry at the Virginia Institute of Marine Sci-
ence. By the third attempt, a 12-min treatment at a concentration of
400 (iM yielded 949r triploidy. After 9 days of development,
differential mortality led to a percentage triploidy of over 96% in
that same batch.
Although we were successful in producing the triploid oysters.
302 Abstracts. February 2003
Millord Aquaculture Seminar, Milford, Connecticut
the late production date coincided with deteriorating water quality
prevalent later in the summer. Due to a toxic algae bloom iPio-
roceiitriiin sp.), high bacteria {Pseiichvtioiuis sp.) and a minor oil
spill, only a couple of thousand triploid and diploid control sur-
vived and their growth was altered by the exposure. On October
2nd the surviving animals were transferred to one of the growers"
high flow, tidal upweller nurseries. However the oysters did not
grow and only time will tell if they were hardy enough to survive
overwintering.
RAZOR CLAM, ENSIS DIRECTUS, GROWTH RATES IN
NIANTIC RIVER, CONNECTICUT. John Wadsworth, Nian
tic Bay Shellfish, LLC. 15 First Street. Waterford, CT 06385,
USA; Tessa S. Gefchis and Nancy Balcom. Connecticut Sea
Grant, University of Connecticut. 1080 Shennecossetl Road.
Groton, CT 06340.
In 2001. Niantic Bay Shellfish. LLC partnered with the North-
eastern Regional Aquaculture Center as part of a regional project
to develop growout culture methods for the razor clam. £;?,v/,v
directus.
Approximately 10.000 seed (20 mm) were distributed evenly
(one clam per 6.45 square cm) into felt-lined wire cages
(0.6m:length x 0.6 nrwidth x 0.3 m:height) and were filled to a
height of 15 cm of sediment. The cages were set and buoyed on
leased ground in the Niantic Ri\er in Waterford. Connecticut.
MLW was 0.6-0.9 m (site-dependent) with a tidal height of 0.85
m. Monthly inventories to determine clam density and growth
(length and width to ± 0.01 mm) were performed beginning in
September 2001. The clams increased in length from 18.84 ± 2.22
mm to 74.25 ± 6.54 mm in the first 2 y of the project. Grow-out
trials have continued with limited success, as surviving clams are
slow growing and have been increasingly susceptible to predation
by green crabs.
THE LONG AND WINDING ROAD: TOWARDS SUSTAIN-
ABLE FISHERIES MANAGEMENT AND MEANINGFUL
SHELLFISH RESTORATION (WELLFLEET, MA). Bill
Walton. Wellfleet Shellfish Department. 300 Main Street. Well-
tleet. MA 02667.
Over the last year, the Town of Wellfleet (Cape Cod. MA.
USA) has begun the long and often contentious process of devel-
oping a long-term shellfish management plan. Here I describe the
evolution of this plan from a traditional fisheries management
approach (e.g.. gear limitations, increased fees, etc.) to a commu-
nity-driven document that relies on input from the shellfishing
community while promoting sustainability. Topics will include
spawnmg sanctuaries, cultching. predator control, disease manage-
ment, and monitoring efforts. In addition. I will review several
steps we have taken toward increasing the efficiency of local shell-
fish lestoration efforts.
MOVING TOWARDS COMMERCIALIZATION OF SOFT-
SHELL CLAM CULTURE ON MASSACHUSETTS"
NORTHSHORE. Scott Weston. Bonnie McAneney. Mark
Fregeau, and Joe Buttner, Northeastern Massachusetts Aquacul-
ture Center and Department of Biology. Salem State College, Sa-
lem, MA 01970.
To support community-initiated enhancement and aquaculture
efforts on Massachusetts' Northshore that target the softshell clam
(A/vo areiuiria). the Northeastern Massachusetts Aquaculture Cen-
ter (NEMAC) produced nearly 2 million juvenile clams in 2002.
Beyond serving as a regional hatchery and nursery, NEMAC ex-
panded outreach efforts that include technical assistance, educa-
tional activities and networking with shellfishers and regulators.
Survival of clams spawned by NEMAC personnel in 2002, to
2.0 mm, exceeded 80%. Resultant juvenile clams were distributed
in July to Massachusett"s sites: 650,000 (ave. In. = 2.5 mm) to
Rowley and 170,000 (ave. In. = 3.5 mm) to Martha's Vineyard.
Another 220,000 clams (ave. In. = 14.0 mm) over-wintered in
Smith Pool at NEMAC's Cat Cove Marine Laboratory (CCML)
were also transferred to Rowley. About 800.000 juvenile clams
were retained in spat bags placed in protective plastic cages and
floated in Smith Pool. By the end of the growing season, clams had
grown to 6.0 mm (ave. In.) with survival rates approaching 95%.
Clams are being over-wintered in submerged cages for release
onto approved flats in the spring/early summer 2003.
To facilitate and expand clam production, a dual use Dock/
Floating Upwelling System (FLUPSY) was acquired by the Town
of Rowley, through NEMAC's small grants program. Clams
(650.000) were cultured in the FLUPSY as a cooperative effort
involving Rowley shellfishers (maintenance and coordination), the
Boy Scouts (maintenance and data collection) and NEMAC (tech-
nical support, environmental monitoring and supplies). Sur\iving
clams were released onto the Rowley tidal tlats and covered with
predator exclusion netting (6.4 mm mesh). NEMAC also advanced
private seed collection and grow-out projects in Ipswich and
Gloucester. Massachusetts by providing materials and training. It
is anticipated that clams spawned at the CCML will attain market
size in 2003. the beginning of a sustainable, shellfish aquaculture
industry on Massachusetts" Northshore.
DEMAND FEEDING OF BAY SCALLOPS, ARGOPECTEN
IRRADIANS IRRADIANS USING AN AUTOMATED CON-
TROL SYSTEM. James C. Widman Jr. and David J. Veilleux.
USDOC. NOAA. National Marine Fisheries Service. Northeast
Fisheries Science Center. Milford Laboratory. Milford. CT 06460.
We have developed a system that allows juvenile bay scallops.
Aifiopecten imidiatis irradians. to be exposed to near-constant
concentrations of phytoplankton, even as scallops consume it.
Chlorophyll-a fluorescence levels are used to monitor phytoplank-
ton cell concentration in the juvenile scallop culture system. Sea-
water from the scallop culture is continuously pumped through a
Milford Aquaciilture Seniiiiai. Milford. Connecticul
Ahsumis. February 2003 303
WET® labs submersible nmiriuiieler using a Poiidinaster® mag-
netic drive pump. The fluorometer outputs an analog signal (volt-
age) proportional to the fluorescence. The analog signal is mea-
sured by an ADAC® model 5516 DMA data acquisition board
installed in a personal computer. An algorithm reads the voltage/
fluorescence and switches a relay on or off depending on the value.
When the fluorescence drops below a preset value, the relay turns
on and starts adding phytoplankton to the scallop culture with a
peristaltic pump. On reaching the desired fluorescence (algal cell
concentration), the algorithm switches the relay off which in turn
stops the addition of algae to the culture system. By continuously
monitoring the fluorescence level of the culture water, the algal
cell concentration can be maintained and scallops are fed on demand.
Scallops with an initial mean shell height of 7.2 mm grew to a
mean shell height of 18.4 mm in 78 days using our prototype
system. This growth was achieved while testing the mechanics and
logic of the system. Additional monitoring systems are being built
so we can analyze how algal cell concentration affects scallop
growth. Our goal is to maximize scallop growth while minimizing
phytoplankton consumption. This system would be amenable to
feeding oysters, clams, mussels, brine shrimp, rotifers, and other
phytoplankton grazers.
® The use of trade names is to identify products and does not
miply endorsement by the National Marine Fisheries Service.
A DECISION TREE FOR DESIGNING A PROCESS TO
PRODUCE MICROALGAL FEEDS FOR AQUACUL-
TURED ANIMALS. Gary H. Wikfors. Barry C. Smith, Shan-
non L. Meseck, Mark S. Dixon, and Jennifer H. Alix, USDOC,
NOAA, National Marine Fisheries Service, Northeast Fisheries
Science Center, Milford Laboratory. Milford, CT 06460.
Research, commercial, and publicly-funded mariculture facili-
ties generally have a need to produce microalgal cultures to feed
molluscan broodstock. larvae, and post-set, or to rear zooplankton
as live feed for larval finfish or crustaceans. Too often, facilities
and procedures for microalgal feed production are based on inap-
propriate, previously-existing examples, leading to production pro-
cesses that are under-scaled, expensive to operate, and undepend-
able. A production process based on quantitatixc and qualitative
needs would be preferable; a decision tree seems to be a useful tool
for designing a microalgal feed-production process in a new aqua-
culture operation or improving an existing facility.
Main considerations for process-design decisions are defined
by the nutritional and water-quality needs of the animals being
fed — What? How Much? And How Often'.' Choices of "What"
microalgae to grow will, to some extent, constrain the shapes and
configuration of containers, and culture management options (i.e..
batch or some form of continuous or semi-conlinuous culture man-
agement). In some applications, especially very intensive, recircu-
lating systems, microbiological, and chemical aspects of water-
quality become critical in defining feed-culture quality. Once ac-
ceptable qualitative food requirements arc identified, then the
quantitative characteristics of the process — "How Much and How
Often" — must be addres.sed. Daily harvest volumes can be calcu-
lated by dividing algal biomass quantities required to feed animals
by estimated (conservatively!) biomass densities per unit volume
of algal culture using the selected culture management. Options to
replicate small production units many times or to monitor and
manage several large cultures intensively can be considered in the
context of dependability of the process. Rationale for choices made
in Milford Laboratory microalgal feed-production processes — and
surprises and problems encountered during operation of these pro-
cesses— will be discussed.
INITIAL INVESTIGATION OF AN ANNUAL PROROCEN-
TRUM BLOOM IN LAGOON POND, MARTHA'S VINE-
YARD. William M. Wilcox, Marthas Vineyard Commission, PO
Box 1447, Oak Blutfs, MA 02557, USA; David W. Grunden,
Oak Bluffs Shellfish Department, PO Box 1327, Oak Bluffs, MA
02557.
For 5 out of the last 6 years blooms of Prorocemniin have been
observed in Lagoon Pond on Martha's Vineyard. This salt-water
embayment supports various uses including recreational boating
and shellfishing. It has two hatcheries located on its shores: the
Martha's Vineyard Shellfish Group's shellfish hatchery and the
Massachusetts Division of Marine Fisheries Lobster Hatchery.
This project is the first to investigate the possible causes of this
almost annual Proroccntnini bloom.
This report summarizes the lah and field data collected from
five sample locations in Lagoon Pond during the period from
mid-May through mid-September 2002. The data reported include
vertical profiles of temperature and dissolved oxygen; dissolved
and particulate nutrient analyses from both surface and deep
sample sites; and chlorophyll, bacterial and phytoplankton analy-
ses, and transparency.
July and August rainfall was 5.5 inches less than the historic
average for these two months. In addition, water table levels
throughout the outwash plain set new records for monthly low
stands as measured since 1991 (Wilcox 2003). This produced a
lower than usual amount of fresh water input from rainfall, ground-
water, runoff and fresh water surface inflow. As these sources are
major contributors of nitrogen, there was less input of this nutrient
to the system.
During the course of study, the Lagoon maintained good water
column transparency v\ith Secchi depth averaging 3 to 3.4 meters
and never falling below 2.1 meters. The dissolved oxygen satura-
tion was typically above 80 percent in the surface water but fell
briefly to a low of 12 percent at 5 to 7 meters depth at one station.
The pond is always limited by the availability of nitrogen but
cycles between times when silica is and is not limiting to the
growth of phytoplankton.
In July, the Shellfish Hatchery quahogs became heavily fouled
with vorticella, hydrozoans and bryozoans and showed symptoms
304 Abslmcts. February 2003
Milt'ord Aquaculture Seminar. Millord. Connecticut
of lack of food or poor quality food. There were some indications
of mild bacterial infection hut the usual die-off did not materialize
in July as it has typically in previous years.
A groundwater survey was conducted and. at the seeps
sampled, nitrogen was 20 to 100 times more concentrated than in
the pond. Silica was about an order of magnitude greater but
onho-phosphate was roughly equal to the in-pond concentration.
Groundwater is clearly a source of nitrogen and silica to the sys-
tem.
The Upper Lagoon Pond discharges through a herring ladder at
the Madeiras run. This freshwater pond experienced a severe algae
bloom with chlorophyll a concentrations rising from 8.2 micro-
grams per liter in mid-May to over 50 by mid-June. A second
bloom began in mid-August, peaked at 143.-5 txgfL on August 19
and continued through the last sample round on September 12.
The data collected hint at a complex cycle of phytoplankton
populations, grazers including jellyfish, water quality, and shell-
fish survival at the MVSG Hatchery. The jellyfish were numerous
this year and. it is suspected, their feeding on copepods and other
grazing organisms freed up primary phytoplankton growth, which
is influenced by both the availability of nitrogen and silica. Low
levels of nitrogen in the system probably subdued the phytoplank-
ton bloom that, as a result, was not as excessive as perhaps pre-
vious vears.
AN UPDATE ON BLUE MUSSEL CULTURE IN LONG IS-
LAND SOUND. Lawrence Williams, Jessie D.. Inc.. 68 Anchor-
age Drive, Milford, CT 06460; Tessa S. Getchis, Connecticut Sea
Grant, University of Connecticut, 1080 Shennecossett Road,
Groton, CT 06340-6048; Inke Sunila, Connecticut Department of
Agriculture, Bureau of Aquaculture. P.O. Box 96, Milford, CT
06460.
As fisherman and commercial shellfish harvesters continue to
struggle with fisheries disasters in Long Island Sound including
disease, drought, storms, invasive species, etc., they are able to
depend less and less on traditional fisheries. Maritime industry
members have partnered with state and federal agencies to inves-
tigate the possibility of new/alternative species for culture in Long
Island Sound.
In 2001. a pilot project was initiated to investigate long line
culture of blue mussels {Myliliis ediiUs) in Long Island Sound. A
natural set of seed mussels was collected on the long lines in the
area of Charles Island off the coast of Milford, Connecticut, USA.
Newly set mussels were observed on the long lines in the spring,
summer, and fall of 2001 and 2002. The mussels reached market
size of approximately 2.5 inches in less than 10 months.
An investigation into the feasibility of a commercial-scale op-
eration involving blue mussel culture in Long Island Sound has
been proposed.
Jininml of Shellfish Research. Vol. 22, No. 1, 305. 2003.
ABSTRACTS OF TECHNICAL PAPERS
Presented at The 95th Annual Meeting
NATIONAL SHELLFISHERIES ASSOCIATION
New Orleans. Louisiana
April 13-17. 2003
305
National Shellfisheries Association, New Orleans. Louisiana Abstracts. 20U3 Annual Meeting, April 13-17, 2003 307
CONTENTS
George R. Abbe. Candace A. Morrell, Carol B. McColloiigh and Christopher F. Diingan
Environmental effects on Pcrklnsus muriiiiis infection rates, growth and survival among dermo-disease-free juvenile
oysters in the Patuxent River, Maryland during drought conditions 317
Charles Adams, Effte Philippakos, Alan Hodges, David Mulkey. Dorothy Comer and Leslie Stunner
Economic impact of the cultured hard clam industry in Florida 317
Standish A. Allen Jr., A. J. Erskine, Elizabeth Walker, Ronald Zebal and Gregory A. DeBrosse
Production of tetraploid Suminoe oysters. C. unnkcnsis 317
Yvonne C. Allen, Charles A. Wilson. Harry Roberts, John Siipan and Ralph Pausina
Ground truthing hydroacoustic data with commercial ovster dredging 317
L. S. Andrews, B. Posadas, D. Barrage and Michael Jahncke
Oyster irradiation: Pathogenic Vibrio response and consiuiier difference testing 318
Linda S. Andrews and Susan De Blanc
Response of Vibrio vubujicits and V. parahafiuulyticiis 03:K6 318
William S. Arnold
Population collapse, depensation effects, and the time-scale of recovery of hard clam (Mercenaria
spp.) fisheries 318
William S. Arnold, Sarah L. Walters, Sarah C. Peters, Theresa M. Bert and Jon S. Fajans
Influence of congeneric aquaculture on hard clam [Mercenaria spp. ) population genetic structure 318
Corinne Audemard, Lisa M. Ragone Calvo, Kimberly S. Reece, Eugene M. Burreson and Kennedy T. Paynter
lit situ determination of Perkinsits marinus transmission dynamics 319
Jean-Christophe Avarre. Yannick Gueguen, Evelyne Bachere and Jean-Michel Escoubas
Functional genomics: A powerful approach to study the immune response of the Pacific oyster
Crassostrea gigas 319
Patrick D. Banks
Biological assessment of storm effects on the Louisiana public oyster resource: Tropical Storm Isidore and
Hurricane Lili 319
Carta D. Beals and Shirley Baker
Clearance rates and feeding selectivity of Crassostrea virginica and Mercenaria mercenaria; implications of increased
eutrophication in the Suwannee River Estuary 319
Donald L. Bishop
Engineering and economics as related to Oysters Grown in the Gulf of Mexico 320
Karine Bouilly. Helen McCombie, Alexandra Leitdo and Sylvie Lapegue
Persistence of atrazine impact on aneuploidy in the Pacit~ic oyster, Crassostrea gigas 320
Daniel Bourque, Thomas Landry, Jeff Davidson and Neil McNair
Impact of an invasive tunicate in Atlantic Canada: Recruitment and competition 320
V. Monica Bricelj, John Kraeuter, Eric N. Powell, John M. Klinck, Eileen E. Hofniann. Ray Grizzle
and Stuart Buckner
A simulation model of the population growth of hard clams {Mercenaria mercenaria). III. Effects of brown tide 320
Kenneth M. Brown. Gary Peterson, Mike McDonough, Patrick Banks and Brian Lezina
Deterrents to black drum predation on oyster leases 321
Nicole T. Brun, V. Monica Bricelj, Emmanuel E. Egbosimba. Thomas H. MacRae and Neil W. Ross
Stress responses in scallops and hard clams to heat and cold shock 321
Eugene M. Burreson. Kimberly S. Reece, Karen L. Hudson and Christopher F. Dungan
Perkinsns chesapeaki and Perkinsus amirensi are the same species 321
David Bushek, Donnia Richardson, Yvonne Boho, Loren Coen and Jennifer Cardinal
Evaluating shell quaiantine duration to limit the transfer of Perkinsus marinns when planting oyster cultch 321
Kevin R. Calci
High hydrostatic pressure inactiv alion of viruses 322
Lisa M. Ragone Calvo, Gene M. Burreson, Susan E. Ford. John N. Kraeuter, Dale F. Leavitt and Roxanna Smolowitz
Host genetic origin an important determinant of QPX disease 322
Mark D. Camara and Standish K. Allen Jr.
Experimental evaluation of crosses within and among five commercial strains of hard clams, Mercenaria mercenaria.
across a salinity gradient in Virginia waters 322
308 Ab.stnicts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana
Ruth H. Carmichael, Andrea C. Shriver, Erica T. Weiss and Ivan \ aliela
Growth of quahogs (Mercenaria mcnenaria) and softshell elams (Mya arenaria) in response to eutrophie-driven
changes in food supply and habitat 323
Ryan B. Carnegie. Mark D. Caniara. Lisa M. Ragone Calvo, Kimberly S. Reece and Patrick M. Gaffney
Development of a single nucleotide polymorphism (SNP) marker set for the hard clam. Merct'iuiriu mciccuaiia 323
Robert M. Cerrato, Amy E. Streck and Darcy J. Lonsdale
Trophic interaction between hard clams and natural assemblages of plankton 323
Maria del Refugio Castaneda Chavez, Erasino Orrantia B., Violeta Pardio Sedas and Fabiola Lango Reynoso
Presence of pathogenic bacteria in the lagoon systems La Mancha and Alvarado Veracruz. Mexico in water and
oyster ( Cnissoslrea virgiiiica) 323
Daniel P. Cheney. Andrew D. Suhrbier. Aimee E. Christy, Hector S. Beltran. Jonathan P. Davis. Kenneth M. Brooks
and Frank J. Smith
Mussel gi-owth and food utilization in relation to water column conditions on raft systems in Puget
Sound, Washington 324
Marnita M. Chintala and Karin A. Tamnii
Assessing the effect of habitat alteration on shellfish populations 324
Mary C. Christman. Cynthia J. Giffen. Jon H. Volstad and Lynn W. Fegley
Design and implementation of a survey of commercial blue crab effort in the Maryland portion of the
Chesapeake Bay 324
Fu-Lin E. Chu and Jean-Francois Samain
An integrated approach to bivalve domestication: introductory remarks 324
Loren D. Coen and Majbritt Bolton-Warberg
Evaluating the impacts of harvesting practices, boat wakes and associated shoreline erosion on intertidal creek
habitats in the southeastern US: Managers and restoration programs take note 325
David W. Cook
History of post-harvest treatment to reduce Vibrio spp. in shellfish 325
Hua Dan
Freshwater pearl culture and production in China 325
Richard L. Darden and Brian R. Kreiser
Population genetics of the blue crab ( Callinectes xapidtis) in the Gulf of Mexico 325
Patricia M. da Silva. Antonio Villalha and Jose Fuentes
Growth and mortalitv of different Osiica cdiilis stocks cultured in the Ri'a De Arousa (Galicia. NW Spain) 326
Patricia M. da Silva, Antonio Villalba. Maria J. Carballal and Jose Fuentes
Differences in disease susceptibility among Ostrea edidis stocks cultured in Galicia (NW Spain) 326
Joth Davis and Dennis Hedgecock
Crossbreeding in pacific oysters 326
Lewis E. Deacon
The effect of algal toxins on the isolated ventricle of the clam. Mercenaria incrccnavia 326
Lionel Degremont, Pierre Boudry. Patrick Soletchnick, Edouard Bedier. Michel Ropert. Arnaud Huvet, Jeanne Moal and
Jean Francois Samain
Genetic basis of summer mortality in juvenile cupped oysters 327
Maryse Delaporte. Philippe Soudant. Jeanne Moal and Christophe Lambert
Impact of environmental and nutritive conditions on defense mechanisms of oysters during an annual cycle 327
Leonard DiMichele. Stephan Towers and Donald Shepherd
Mucin secretions and nacre deposition in the formation of pearls 327
Angela K. Dukeman. Norman J. Blake and William S. Arnold
Reproduction in tfame scallops. Liiiui scahra scal'iv (born 1 778). from the lower Florida Keys 327
Christopher F. Dungan. Kimberly S. Reece and Karen L. Hudson
In vitro propagation of Peil<iiisii.\ sp. parasites from Japanese Manila clams. Riidiuipes phdippinarum 328
Vincent G. Encomio and Fu-Lin E. Chu
The role of heat shock proteins in tolerance to parasitic stress in the eastern oyster, Crassostrea virginica 328
National Shellfisheries Association. New Orleans, Louisiana Ahstnicrs. 2003 Annual Meeting, April 13-17. 2003 309
Martha Enriquez-Diaz. Stepliaiie I'ouvreaii. Caroline Fabioiix, Yvette Le Coguic, Jean Claude Cochard and
Marcel Le Pennec
Reproductive strategy: Variability of reproductive pattern in two populations genetically determined of
Crassostrea gigas 328
A. J. Erskine and Standish K. Allen. Jr.
Histological exaniinalion of ganietogenesis in genetic triploid Cnissostrea ar'uikensis in Chesapeake Bay 328
Ford Evans. Sean Malson. John Brake and Chris iMngdon
Effects of inbreeding on perfonnance traits in Pacific oysters ( Cnissostrea gigas) 329
Caroline Fahioiix. Arnaud Huvet. Frederic LeRoiix. Marcel LePennec and Jean-Claude Cochard
Oyster vasa-like gene: A specific marker ol the germ cell lineage in Crassusirca gigas 329
Jonathan S. Fajans and Patrick Baker
Tracking the spread of an in\asi\e mussel (Mytilidae: Penni viriilis) in Florida 329
Andrea Findiesen. Oded '/.mora. Moti Harel. Yonathan Zohar. Alicia Young-Williams and Anson H. Mines
Manipulation of environmental parameters for out-of-season egg and larval production in blue crab broodstock
( Calliiiccti's sapiJiis ) 329
Mark Finkbeiner. Bill Stevenson, Bill Anderson, Mike Yianopolous, Loren Coen, Ginger Martin and Karen Cullen
Managing and monitoring intertidal oyster reefs with remote sensing in coastal South Carolina 330
William S. Fisher
Is copper required for eastern oyster setting and metamoiphosis? 330
Pierre-Gildas. Fleury. Erwan Le Ber, Serge Claude, Florence Cornette, Florence d'Amico, Patrice Guilpain,
Hubert Palvadeau. Stephane Robert, Patrick Le Gall. Michel Ropert. Charlotte Simonne and Catherine Vercelli
Comparison of Pacific oyster {Crassostrea gigas) rearing results (survival, growth, quality) in French farming areas,
altera 10-year monitoring (1993-2002) by the IFREMER/REMORA network 330
George E. Flimlin. Jr.. Michael Celestino. John N. Kraeuter, Robert J. Macaluso and Michael Kennish
Evaluation of Raritan and Sandy Hook Bay hard clam. Mcrcenaria mercenaria. stocks for fishery management 330
Celine Garcia, Isabelle Arzul. Franck Berthe. Bruno Chollet, Jean-Pierre Joly, Nolwenn Kerdudou, Laurence Miossec,
Maeva Robert and Jean-Louis Nicolas
Potential pathogens associated with abnormal mortalities 331
Catherine M. Gatenby, Danielle A. Kreeger, Deborah Raksany and Richard J. Neves
Seasonal variation in the physiological status of three species of mussels in the Allegheny River. PA 331
Melanie Gay, Guenaelle iMUcelot, Bruno Chollet, Tristan Renault, Nathalie Cochennec, Franck Berthe,
Christophe iMmbert, Gwenaelle Choquet, Christine Paillard, Manolo Gouy, Frederique Le Roux and
Philippe Goulletquer
Characterization of Vibrio isolated from Pacific oysters" spat suffering form summer mortality outbreaks 331
Stephen P. Geiger and William S. Arnold
Restoration of bay scallops in highly modified and relatively pristine habitats on the west coast of
Florida. USA 33 1
Michael Goedken and Sylvain De Guise
Flow cytometry as a tool to quantify oyster phagocytosis, respiratory burst and apoplosis 332
Jon Grant. Marie Archambaull. Cedric Bacher and Peter Cranford
Integration of modeling and GIS in studies of carrying capacity for bivalve aquaculture 332
Jennifer Greene. Ray Grizzle and Jamie Adams
Mapping and characterizing eastern oyster {Crassostrea virginica) reefs using underwater videography and
quadrat sampling 332
Dianne L Greenfield. Darcy J. Lonsdale, Robert M. Cerrato and Glenn R. Lopez
The effects of background concentrations of the brown tide alga Aiireococciis annphagejferens on growth and feeding
in the bivalve Mercenaria mercenaria 332
Raymond E. Grizzle, Eileen E. Hofmann, John M. Klinck. Eric N. Powell. John N. Kraeuter, V. Monica Bricelj and
Stuart C. Buckner
A simulation model ol the population growth of hard clams {Mercenaria mercenaria). IV. Effects of
climate change 333
310 Abstracrs. 2003 Annual Meeting, April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana
Vincent Guillory, Harriet Perry and the Blue Crab Technical Taskforce
Status of blue crab populations in Lousiana based on fishery independent data collections (1967-2002) with
observations on relative abundance in other Gulf States 333
Ximing Giio, Susan Ford and Gregory DeBrosse
Breeding and evaluation of eastern oyster strains selected for MSX, dermo and JOD resistance 333
Terrill R. Hanson, Lisa O. House and Benedict C. Posadas
Marketing implications of consumer attitudes toward oysters 334
Matthew Hare, D. Merritt, K. Paynter, S. K. Allen, Jr., E. M. Burreson, M. D. Camara, Ryan Carnegie, M. Luckenbach
and K. S. Reece
How many larvae stay at home? Measuring patterns of local oyster recruitment using molecular markers 334
Leslie H. Haynes, Arielle Poulos, Lacey K. Smith, Aswani K. Volety and S. Gregory Tolley
Suitability of oyster clusters as habitat for reef-resident fishes and decapod crustaceans in the Caloosahatchee estuary 334
Helene Hegaret, Gary Wikfors. Philippe Soudant and Jean-Franfois Samain
Algal food quantity and quality affect immune function in oysters stressed by high temperature 334
Anson H. Hines, Jana L. D. Davis, Alicia Young-Williams, Yonathan Zoliar and Oiled Zmora
Assessing feasibility of stock enhancement for Chesapeake blue crabs ( Culliiiectcs sapicliis) 335
Eileen E. Hofmann, John M. Klinck, Eric N. Powell, John Kraeuter, Monica Bricelj, Ray Grizzle and Stuart Buckner
A simulation model of the population growth of hard clams (Henenaha mercenaria). 1. Model development
and implementation 335
Andrea C. Hsu, Roxanna M. Smolowitz, Andrei Y. Chistoserdov and Hemant M. Chikarmane
Comparison along the New England coast of epidemic shell disease in the American lobster.
Homarus americanus 335
Don Hubbs
Tennessee's pearl culture industry 336
Karen L. Hudson, Kimberly S. Reece, Christopher F. Dungan and Rosalee M. Hamilton
Prevalence and abundance of PcrkiiiMis mariiuis and Peikinsiis chesapeaki/andrewsi in Chesapeake Bay
oyster beds 336
Kristi L. Huels, Yolanda J. Brady, Mary A. Delaney and Joel A. Bader
Evidence of a cold shock response in Vibrio \ ulnificus, a human pathogen transmitted via raw eastern oysters,
Crassostrea virginica, from the Gulf of Mexico 336
Stephen J. Jordan and Jessica Vanisko
A fishery-oriented model of Maryland oyster populations 336
Stephen L. Kaattari and Christopher Earnhart
Development of biomarkers for Perkinsus marinus resistance in the eastern oyster {Crassostrea virginica) 337
Gregg Kenney, Andrew Kahnle, kathy Hatlala and Steven H. Jury
The blue crab fishery of the Hudson River Estuary 337
Marilyn B. Kilgen
Evaluation of commercial post harvest treatments for control of Vibrio viibiificus in oysters 337
Peter Kingsley-Smith
Polinices piilclielliis: The James Dean of gastropods: living fast, dying young 337
David M. Knott, Elizabeth L. Wenner and Susan L. Thornton
Observations on the unusual abundance of tropical CalUnectes species in the South Atlantic Bight in fall 2002. and
remarks on the non-indigenous Charyhdis liellerii 338
John Kraeuter, Eric N. Powell Eileen E. Hofmann, John M. Klinck, Ray Grizzle, V. Monica Bricelj and Stuart Buckner
A simulation model of the population growth of hard clams [Mercenaria mercenaria). II. Effects of fishing 338
Maureen K. Krause, John J. Dunn, Daniel van der Lelie and Sean McCorkle
Genomic signature tags: A novel method for genomic profiling with applicability to shellfisheries research 338
D. Kreeger, R. Thomas, H. Herder and D. Raksany
Spatial and temporal variation in oyster fitness in San Antonio Bay. Texas. 1998-2002 338
Cathy A. Laetz and Robert C. Cerrato
Reconstructing the growth of hard clams. Mercenaria mercenaria. under brown tide conditions 339
National Shellt'isheries Association, New Orleans, Louisiana Abstracts. 2003 Annual Meeting, April 13-17, 2003 31
Chrislophe Lamhert. Philippe SoudaiiU Gwenaelle Choqiiet, Christine Paillard. Stephane Fioiiel, Lionel Degremont,
Manse Delaporte. Jeanne Moat. Pierre Boiidry, Patrick Soletclinick, Michel Ropert. Edouard Bedier, Tristan Renault,
Beatrice Gagnieres, Arnaud Huvet and Jean-Francois Saniain
Ininiunological status of selected Crassostrea gigas families and descendants, reared in different
en\ ironmental conditions -^-^"
Panl iMng and Chris Langdon
Optimization of sperm cryopreservation for the Pacific oyster Crassostrea gigas: Evaluation of cooling rate 339
Chris iMngdon. Sean Matson, John Brake and Ford F^ans
Family-based selection improves yields of Pacific oysters Crassostrea gigas 339
J. David Lange, Jr., William D. DiiPaul and David B. Rudders
An evaluation of Sea Scallop closed area boundaries in the Mid- Atlantic 340
Amy A. Larson and Robert M. Cerrato
The role substrate characteristics have in altering the behavior, growth and survival of juvenile (postsettlement)
Mcrccuaria mcrcenaria 340
Gina Latendresse
One man's dream: American cultured pearls 340
Clare Lehane and John Davenport
Zooplankton ingestion by bi\ alves — more food for thought! 340
Susan J. Limbeck and Paul D. Rawson
Species-specific variation in thermal tolerance during larval development in blue mussels, Mytihis spp 340
Susan A. Little, Winsor H. Watson, III and Riidman Hall
Variations in the size structure of lobster [Hoiuuriis amcncantis) populations within the offshore fishery 341
Mark W. Luckenbach and Loren D. Coen
Oyster reef habitat restoration: A review of restoration approaches and an agenda for the future 34 1
Eric D. Lund, Fu-Lin E. Chu and Ellen Harvey
Progress in the development of chemotherapeutic protocol for eliminating/reducing dermo disease in
infected oysters 341
Richard A. Lutz, Timothy M. Shank and Daniel J. Foniari
Striking succession of mussels at newly formed deep-sea hydrothermal vents 341
M. Maille Lyons and J. Evan Ward
Suspension-feeding bivalves, marine aggregates and the accessibility of small particles 342
Sandra L. Macfarlane
Shellfish restoration: lt"s not just biology that matters 342
Scott MacQuarrie and V. Monica Bricelj
Evidence for natural selection for resistance to PSP toxins in eariy life history stages of the softshell clam,
Mxa arcnaria 34_
/ F. Mallet and iMndry
Optimizing oyster productivity in Caraquet Bay: Coordinating restoration and aquaculture 343
Aaron P. Maloy and Katherine J. Boettcher
Roseimarina crassostreae (gen. nov., sp. nov.) associated with JOD-signs in the absence of significant mortalities,
and first isolation from a New York epizootic 343
Roger Mann and Peter Kingsley-Smith
Finding the wheat in the chaff — oyster larval feeding in turbid, low salinity conditions 343
Michel Mathieu. Katherine Costil, Brice Dubois. Clothilde Heitde, Arnaud Huvet. Kristell Kellner
and Stephane Pouvreau
Characterization of summer mortalities of Crassostrea gigas oyster in relation to physiological parameters 343
Carol B. McCollough, Christopher F. Dungan, George R. Abbe and Candace A. Morrell
Perkiiisiis manniis infection rates in specitlc-pathogen-free juvenile oysters planted in the Patuxent River, Maryland 344
Ayana McCoy, Shirley Baker, Ruth Francis-Floyd and Anita Wright
Is Mcrcenaria mcrcenaria a host for Pcrkiiisiis species? 344
Earl J. Melancon, Jr., Dale Diaz and Badiollah Asrabadi
Recommendations to oyster harvesters on removing hooked mussels, Iscluulium rccurviim 344
312 Abstracts. 2003 Annual Meeting. April 13-17, 2003 National Shellfisheries Association. New Orleans. Louisiana
D. Mestey and G. E. Rodrick
A comparison of cryogenic freezing techniques and their usefulness in reduction of Vibrio vulnificus in
retail oysters 344
Coren A. Milhury and Patrick M. Gaffney
Using molecular genetic techniques to assess oyster restoration programs and projects 345
Thomas J. Minello and Lawrence P. Rozas
Creating salt marshes to enhance production of fishery species 345
Jeanne Moal, Edouard Bedier, Pierre Gildas Fleury, Aime Langlade, Y'vette LeCoguic, Lionel Degremont,
Pierre Boiidry, Jean Rene Le Coz. Stephane Pouvreau, Martha Enriquez-Diaz. Christophe Lambert, Philippe Soudant
and Jean Francois Saniain
Genetic xariability in reproduction and summer mortality in Crassoslrea gigas 345
James Moore, Thea Rohhins, Carolyn Friedman, iWeal Hooker, Thomas McCormick and Melissa Neuman
Preliminary pathological investigation of the white abalone. Haliotis sorenseni 345
Ken B. Moore
Utilization of post-harvest treatment as a strategy for reducing Vibrio vuhiificus illnesses 346
Brenda M. Morsey and Sylvain De Guise
Characterization of natural idller cell-like activity in the eastern oyster. Crassoslrea virginica 346
Jessica Munro and Carter Newell
Food availability in a mussel raft 346
Bruno Myrand, Lise Chevarie, Fabrice Pernet and Diego Mantovani
Comparing two Mya arenaria populations as potential candidates for seeding operations 346
Richard J. Neves, Jess W. Jones. William F. Henley and Rachel A. Mair
Propagation of freshwater mussels for freshwater pearl production 347
Carter Newell and John Richardson
An expert system for the optimization of shellfish raft culture 347
Roger L E. Newell, Christopher Gobler and Stephen T. Tettelbach
Linking hard clam (Mercenaria merccnaria) reproduction to phytoplankton community structure: II. Phytoplankton
community structure and food composition 347
David H. Nisbel
Commercial implementation of high pressure processing (HPP) for Pacific oysters 347
Melanie L. Parker, William S. Arnold and Dan C. Marelli
Optimal planting conditions for maximum reproductive output of cage-planted scallops, Argopecten irradians. in
Anclote, Florida 348
Landon D. Parr, Robert P. Romaire and W. Ray McClain
Water losses, seasonal mass loading, and best management practices for craw fish ponds 348
Susan E. Pate, Jeffrey J. Springer, Sandra E. Shumway and JoAnn M. Burkholder
Effects of Karcnia hrevis on shellfish: Does strain matter? 348
Wolf T. Pecker, Jose A. F. Robledo, Eric J. Schott and Gerardo R. Vasta
Assessment of the epizootiology of Perkinsus spp. on the Atlantic coast of USA using genus-, species-, and
strain-specific molecular probes 348
Harriet Perry, Kirsten Larsen, Bill Richardson and Traci Floyd
Ecological effects of fishing: Biological, physical, and sociological impacts of derelict and abandoned crab traps
in Mississippi 349
Esther C. Peters, Marilyn J. Wolfe and Jeffrey C. Wolf
The registry of tumors in lower animals: A resource for bivalve culture health studies 349
Bryan Piazza, John Plunket, John Supan and Megan LaPeyre
Using created oyster reefs as a sustainable coastal protection and restoration tool 349
Allen R. Place, Colin R. Steven and Xiaojun Feng
Blue crab ( Calliiwctes sapiihis) genetic structure and diversity 350
Allen R. Place, Andrea Findiesen and Nilli Zmora
Fiber digestion in the blue crab, Culinccles sapitlus 350
National Shciltisheries Association, New Orleans. Louisiana Ahsrraci.s. 2003 Annual Meeting. April 1.^-17, 2003 313
John I'hiiikcl and Megan Im Peyre
A comparison of finfish assemblages on subtidal oyster shell (cultched oyster lease) and mud bottom in Barataria
Bay. Louisiana 350
John Phinket, Gary Peterson. Bryan Piazza and Megan La Peyre
A comparison of nekton usage of mud bottom, created limestone, shell, and natural shell reef habitats in Terrebonne
Bay. Louisiana 350
Benedict C. Posadas and Linda S. Andrews
Consumer preferences and attitudes toward irradiated oysters 351
Martin H. Posey, Troy D. Alphin, Heather D. Harwell and Thomas J. Molesky
Form and function in oyster reefs: intluence of reef morphology on habitat function and oyster survival 351
Stephane Poiivreaii, Martha Lnriquez-Diaz. Pierrick Le Soiuhii, Jean Paul Connaii. Berlrand Le Roy,
Christian Mingant, Jeanne Moal, Maryse Delaporte, Jean Rene Le Coz and Jean Francois Saniain
Reproduction, bioenergetics and summer mortality of Cnissosireu t^ii^as: Experimental approach 351
Heidi Pye, Winsor H. Watson HL Christopher Rillahan, Rachel Hamilton and Jennifer Wishinski
A comparison and feasibUity study of tvvxi ddferent biomomtormg systems using the blue mussel. Mytihts cditlis. and
the American lobster. Hcniuiriis ainericamts 35 1
Paul D. Rawson
Lar\ al ecology: Molecular tools for the black box? 352
Sammy M. Ray and Thomas M. Soniat
•Status of Pcrkiiisiis nuiriiuis in Galveston Bay. Texas: Results of the Dermowatch Program 352
Deborah Raksany, Catherine M. Gatenby and Danielle A. Kreeger
Seasonal and temporal variability in condition index and tissue biochemistry of Elli/nin LuniphiiMla 352
Kimherly S. Reece
Nucleic acid-based aquatic pathogen molecular diagnostics for detection, research and environmental monitoring 352
P. W. Reno, Y-C. Su, M. Morrissey and D. Nisbet
Validation of post-harvest processing of Vihm parahemolyticus in oysters: Speed bumps on the road from the
research lab to the processing plant 353
John Richardson and Carter Newell
Computational flow modeling of aquaculture systems 353
Jose A. F. Rohledo and Gerardo R. Vasta
Characterization of the Crassostrea viriiinica SLC 1 1 A gene ( fomierly NRAMP) 353
Jose A. F. Robledo, Eric ./. Schott and Geraldo R. Vasta
Perkiiiiis iiianiuis cellular biologv using expression sequence tags (EST) 353
J. Flye Sainle Marie, S. E. Ford, E. Hofmann, F. Jean, J. Klinck, C. Paillard and E. Powell
Development of an individual, energy-balance based, growth model for the Manila clam
(Rudilapes phiUppinanim) 354
Eric J. Schott, Jose A. F. Robledo, Wolf T. Pecher. Florence Okafor and Gerardo R. Vasta
The antioxidant pathway of Pcrki)\sus inaiinus: Functional analysis and localization of two iron
superoxide disinutases 354
Donald Shepherd
Correlation of Hat pearl studies with pearl sac formation in a freshwater mussel (Cvrtoitaias tampicoeiisis) 354
P. Soletchnik, M. Ropert, A. Hiivet, J. Moal, L. Degremont, E. Bedier, J. F. Bouget, B. Dubois, J. L. Martin,
M. Enriquez Diaz, N. Faury, O. Le Moine, T. Renault, B. Gagnaire and J. F. Samain
Characterization of summer moralities of C. i^iaas oyster in France relation to environmental parameters 354
Laurie Carroll Sorabella and Mark W. Luckenbach
A comparison of two oyster [Crassostrea viriiiuica) slocks to determine suitability for use in oyster reef restoration
in Virginia 355
Melissa Southworth and Roger Mann
Decadal scale changes in seasonal patterns of oyster recruitment in the Virginia sub estuaries of the
Chesapeake Bay 355
Mary F. Stephenson, Sharon E. McGladdery, Michelle Maillet, Anne Veniot and Gary Meyer
First reported occurrence of MSX in Canada 355
314 Abstracts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association, New Orleans. Louisiana
Colin R. Steven, Kristen Hunter-Cevera, Allen R. Place, Mike Sheppard and Dick Lee
A quantitative, real-time PCR assay to detect the parasitic dinoflagellate HcimatnUnium sp. in blue crabs.
Calinectes sapidus ^^5
Colin R. Steven, Xiaojun Feng, Allen R. Place and Jeffrey L. Boore
The mitochondrial genome of the blue crab, Calinectes sapidus 356
Colin R. Steven, Johnatlmn Wilkes, Allen R. Place, Jessica Hill and Brian Masters
Developmenl of microsatellite markers in the blue crab. Calinectes sapidus 356
Bradley G. Stevens and Kathy Swiney
Settlement, survival, and predation of red king crabs on natural and artificial substrata 356
Bradley G. Stevens, J. Eric Miink and Peter A. Ciimniiskey
Use of log piling structures as artificial habitats for red king crabs Paralitlwdcs caiiitschaticiis 356
John E. Supan
Sustamable community development via an inshore molluscan aquaculture park: A concept for the Gulf
of Mexico -^ '
John Tesvich and Patrick Fahey
History of the development, commercialization and successful marketing of the first HACCP-based post-harvest
process for the remediation of Vibrio sp. in raw oysters — the AmeriPureProcess® 357
Stephen T. Tettelbach, Christopher F. Smith and Peter Wenczel
Selection of appropriate habitats/sites for bay scallop restoration 357
Stephen T. Tettelbach. Roger I. E. Newell and Christopher Gobler
Linking hard clam (Merccnaria mcrceuana) reproduction to phytoplankton community structure: L Clam growth and
reproductive cycles ^^'
S. Gregory Tolley, Aswani K. Volety, Mike Savarese and James T. Winstead
Influence of freshwater input on the habitat value of oyster reefs in three Southwest Florida estuaries 358
Stephan Towers, Leonard DiMichele and Donald Shepherd
Histological evaluation of early pearl-sac de\elopment in the Tampico pearly mussel iCyrtonaias tampicoensis) 358
Jessica Vanisko and Thomas Miller
Modeling individual eastern oyster (Ciassostrea virginica) growth in the Maryland portion of the
Chesapeake Bay 358
Itzel G. Villa, Fabiola L. Reynoso and Ma. del Refugio C. Chavez
Evaluation HACCP in the oyster acti\ ity in the lagoon system Alvarado. Veracruz. Mexico 358
Jeffrey S. Vincent, Dwayne E. Porter, Dave Biishek and Steve Schill
Remote sensing to map and assess inteilidal shellfish resources in the southeastern USA 359
Mike Voisin
History of commercial application of hydrostatic high pressure processing to molluscan shellfish 359
Aswani K. Volety, S. Gregory Tolley and James T. Winstead
Establishing minimum flows and levels of freshwater in the Caloosahtchee River. Florida, using responses
of oysters -^^^
Linda Walters, Paul Sacks, Lisa Wall, Jeffrey Grevert, Daniel Lejeune, Samantha Fischer and Andrew Simpson
Declining intertidal oyster reefs in Florida: direct and indirect impacts of boat wakes 359
Yongping Wang and Ximing Guo
Chromosomal mapping of ribosomal RNA genes and telomeric repeats in Zhikong and Bay scallops 360
J. Evan Ward, Kari B. Heinonen, Michael P. McKee, Bridget A. Holohan and Bruce A. MacDonald
Production of transplant exopolymer particles (TEP) by bivalves 360
Ami E. Wilbur
Estimating the impact of bay scallop restoration efforts using genetic data 360
Wan Xi Yang and Jun-Quan Zhu
Comparative spermatozoon ultrastructure of Arcidae bivalves Area olivacea and Scapluirea broughtoiii 361
Wan Xi Yang
Immunological studies on the origin of the lamellar complex (LCX) during spermiogenesis of Maerobraeluiiiii
nippunense (de Haan) 361
National Shellfisheries Association. New Orleans, Louisiana Ahslracts, 2003 Annual Meeting, April 13-17, 2003 315
Wan Xi Yang, Anion ina dos Santos, Luis Narciso, Ricardo Calado, Hong Zhou, Jian-Ping Lu, Nat-Cheng Jiang and
Xue-Piitg Ying
Microscopic observation of tegument and cement gland distribution of female pleopod in Chinese mitten crab,
Eriocheir sinensis 361
Gtiy M. Yianopoiilos, and William D. Anderson
Intertidal oyster restoration along an eroding shoreline: An assessment of substrate types for stabilization
and propagation 361
Xue-Ping Ying and Wan Xi Yang
The morphology and ultrastructure of spermatozoon of the gastropod Bitllacui exarata 362
Qian Zhang. Karen L. Hudson, Standish h. Allen Jr. and Kiittberly S. Reece
Population genetic structure of the Suminoe oyster as inferred from restriction fragment length polymorphism (RFLP)
and microsatellite markers 362
Jun-Quan Zhu and Wan Xi Yang
Fine structural analysis of spermatozoon of the bivalve Barhatia vircscens and its evolutionary characteristics 362
Nilli Zinora and John M. Trant
Characterization of key cDNAs of the endocrine axes regulating reproduction and nioltmg in the blue crab.
Callincites sapidiis 362
Yonalhan Zohar. Oded Zinora, Andrea Findiesen. Emily Lipinan, John Stttbblefield, Anson H. Hines
and J ana L. D. Davis
Hatchery mass production of blue crab ( Ccilliiiecles sapidiis) juveniles 363
Natioiuii Slielltislieries Association, New Orleans. Louisiana
Abstracts. 2(103 Annual Meeliny. April 13-17. 2003 317
ENVIRONMENTAL EFFECTS ON PERKINSUS MARINUS
INFECTION RATES, GROWTH AND SURVIVAL AMONG
DERMO-DISEASE-FREE JUVENILE OYSTERS IN THE
PATUXENT RIVER. MARYLAND DURING DR0U(;HT
CONDITIONS. George R. Abbe,* Candace A. Morrell. Acad-
emy of Natural Sciences Estuarine Research Center 10?45 Mack-
all Rd. St. Leonard. MD 20683; Carol B. McCollough and Chris-
topher F. Dungan. Sarbanes Cooperative Oxford Lab. Oxford.
MD216.S4.
In September 2000 specific pathogen-free (SPF) oysters were
transplanted to 3 sites in the Patuxent River, Maryland to investi-
gate environmental effects of Perkinsus mariiuis on infection rates,
growth and survival. During the first year, salinity at Holland Point
(upper river), Gatton (mid) and Town Creek (lower) averaged 1 1,
L^ and 14, respectively, but during the second year averaged 13.
16. and 17. Thirty oysters were collected from each site for assay
of P. mariniis infections by the whole body burden technique
allowing an estimate of time to initial infection and subsequent
progression of disease. An additional 30 oysters from the natural
bar at each site were checked by rectal tissue assay. Oysters at HP.
GAT and TC grew 23. 34 and 27 mm, respectively, and survival
was 95, 98 and 94% during the first year. During the second year,
growth was slightly better at HP (21 mm) than at GAT ( 16mm) or
TC (19nim). By August 2002, mortalities at HP, GAT and TC
were 60, 98 and 97%, respectively, and HP reached 97% 2 months
later.
PRODUCTION OF TETRAPLOID SUMINOE OYSTERS C.
ARIAKENSIS. Standish K. Allen Jr.*, A.J. Ersklne, Elizabeth
Walker, and Ronald Zebal, Aquaculture Genetics and Breeding
Technology Center, Virginia Institute of Marine Science, Glouc-
ester Point, VA 23062; Gregory A. DeBrosse, Haskin Shellfish
Research Lab, Rutgers University, Port Norris, NJ 08349.
Tetraploids have now been produced in three species Crassos-
trea. with the addition of the Suminoe oyster (C ahakensis) in
2002. Tetraploids are produced by a unique genetic manipulation
of eggs from triploids. This presupposes that there will be triploid
females with exceptional fecundity. We found 1-10% of 2- and
3-year old triploid C. ariakensis attained a level of fecundity suf-
ficient for accomplishing 21 tetraploid spawns in summer 2002
resulting in 12 that yielded spat. Number of spat obtained ranged
from about 30 to 4800: the proportion of tetraploids ranged from
0% (one case) to 90%, averaging about 65%. Fecundity of triploids
ranged from 1.25M to 75. 2M eggs. Average time in culture to first
eyed larvae was 15 days, ranging from 14 to 18 days. Eyed larvae
attained exceptional size before setting, averaging 424 |j,m, com-
pared with 340 |j.m in diploid C. ariakensis and 390 jxm in trip-
loids. We also experimented with decreased dosage of cytochala-
sin B. Half the working dose (0.25 jjig/ml) worked as well as our
typical working dose (0.50 (xg/nil) as indicated by increased initial
survival (6.5% and3.2%. respectively) and the same percentage
tetraploidy (74% and 76%, respectively).
ECONOMIC IMPACT OF THE CULTURED HARD CLAM
INDUSTRY IN FLORIDA. Charles Adams. Effie Philippakos,
Alan Hodges, David Mulkey, Dorothy Comer and Leslie Stur-
nier*, P.O. Box 89 Cedar Key. FL 32625 USA.
The hard clam aquaculture industry in Florida went from pro-
ducing 10 inillion clams in 1991 to 140 million clams, with farm
gate sales totaling $15 million, in 2001. This paper determines the
economic impact of this rapidly growing industry. Not only does
the industry contribute in terms of product sales and employment.
It produces a greater economic benefit because of the acti\ ity it
generates among the firms that provide inputs to the clam culture
firms. Further, employees within the industry generate economic
activity when they spend their income. Thus, the economic ben-
efits resulting from clam culture extend beyond the local area to
the general economy. For the cultured clam industry to thrive, it is
essential that water quality standards remain high, permitting and
regulatory measures continue to be favorable, and effective mar-
keting efforts are employed. Given the need for this support, the
state and local decision-makers must understand the value of clam
sales to their economy.
GROUND TRUTHING HYDROACOUSTIC DATA WITH
COMMERCIAL OYSTER DREDGING Yvonne C. Allen,
Charles A. Wilson, Harry Roberts, John Supan, Buddy Pau-
sina. Coastal Fisheries Institute Louisiana State University Baton
Rouge. LA 70803.
Traditional methods used to assess oyster reef distribution and
condition are only able to provide subjective point information,
which is often pooriy georeferenced. Maps of oyster habitat in
shallow waters are therefore typically extremely generalized, giv-
ing few details about the true distribution, character, extent and
dynamics of reefs. Sidescan sonar offers a significant advantage
for quick and accurate assessment of oyster reefs in the turbid
waters of coastal Louisiana.
We conducted four years of side scan surveys over the same
area of oyster reef in south Louisiana. We compared the resulting
imagery to the volume of shell Iroiii quadrant sampling and were
able to establish a strong quantitati\e relationship. In the summer
of 2002. we integrated dredge sampling into our ground truthing
efforts in the hope of making our results more relevant for the
oyster industry. We found a similar relationship between shell
present in dredge samples and pixel intensity. There is a very
promising future in using sidescan sonar and GIS to monitor a
producti\e oyster lease. This combination of tools will be very pow-
erful in helping the oyster grower to plant, harvest, monitor and track
changes to the lease - focusing efforts on productive areas.
318 Abstracts. 2003 Annual Meeting. April 13-17, 2003
National Shellfisheries Association, New Orleans, Louisiana
OYSTER IRRADIATION: PATHOGENIC VIBRIO RE-
SPONSE AND CONSUMER DIFFERENCE TESTING. L.S.
Andrews*. B. Posadas, D. Burrage, Michael Jahncke. Coastal
Research and Extension Center Mississippi Stale University 2710
Beach Blvd. Suite IE, Biloxi, MS 39531.
Pathogenic strains of Vibrio ( Vibrio viilitiflcKs and V. pani-
haemolyticus), although natural inhabitants of estuarine and ocean
environments, can cause serious illness and death in susceptible
persons when consumed along with raw half-shell oysters.
Objectives of this study were 1) establish the irradiation dose
needed to reduce pathogenic Vibrios to nondetectable levels and 2)
determine consumer's ability to differentiate between irradiated
and control oysters.
Live oysters, with naturally incuired and artificially inoculated
pathogenic Vibrios, were exposed to 0-3 kGy dose Cobalt-60
gamma radiation for microbial response. Consumer volunteers
were asked to determine differences between treated ( 1 kGy) and
untreated oysters by triangle difference testing.
Vibrio vulnificus (MO-624) was reduced from 106 cfu/g oyster
meat to nondetectable levels (<3 mpn/g oyster meat) at a dose of
0.75 kGy. Vibrio paraluieiiwlyticiis. 03:K6 (TX-2103), required
1.0 kGy for reduction to nondetectable levels. Sensory triangle
difference tests by 146 volunteers resulted in confirmation that
consumers, many of whom work in the seafood industry, could not
distinguish between control and iiradiated oysters (p <0.01).
RESPONSE OF VIBRIO VULNIFICUS AND V. PARA-
HAEMOLYTICUS 03:K6. Linda S. Andrews* and Susan De-
Blanc, Mississippi State University, Coastal Research and Exten-
sion Center. 2710 Beach Blvd., Suite IE. Biloxi. MS 39531.
Vibrio viiliiificiis and V. paraluivinolxticiis are natural inhabit-
ants of estuarine environments world wide. Pathogenic strains of
these bacteria can cause serious illness and death in susceptible
persons when consumed along with raw half-shell oysters.
Objectives of this study were to determine the time/temperature
parameters needed to reduce Vibrios in shell stock oysters to non-
detectable levels (<3 mpn/g oyster meat) using hot water pasteur-
ization followed by cold shock. Secondly, sensory evaluation stud-
ies were conducted to deterinine sensory changes associated with
the process.
Oysters containing naturally incurred Vibrio and artificially
contaminated pathogenic strains of V. vulnificus (M0624) and V.
parahacmolyticus 03:K6 (TX2071) (106 cfu/g oyster meat) were
pasteurized at 52o C for up to 22 minutes. Oysters, for .sen.sory
testing were harvested during the winter months and also pro-
cessed in 52o C circulating water bath for up to 22 minutes.
Pathogenic strains of V. v. and V.p.03:K6 proved to be more
process resistant than nonpathogenic environmental strains found
in Gulf of Mexico waters. High levels of Vibrio { 1 06 V. v. and V.p
03:K6 cfu/g oyster meat) were successfully reduced to nondetect-
able levels (<3 mpn) when internal oyster temperature achieved
>500C for 10 min. Processing at this T/T did not adversely affect
the sensory qualities consumers expect in raw half-shell oysters.
POPULATION COLLAPSE, DEPENSATION EFFECTS,
AND THE TIME-SCALE OF RECOVERY OF HARD CLAM
{MERCENARIA SPP.) FISHERIES. William S. Arnold*,
Florida Fish & Wildlife Conservation Commission Marine Re-
search Institute 100 8th Avenue SE St. Petersburg, FL 33701.
The commercial fishery for naturally occuixing hard clams has
a brief but eventful history in Florida waters. The first known
fishery, initiated in the early I900"s on the southwest coast of the
state, constituted one of the largest hard clam fisheries on record.
The population that supported that fishery collapsed in the 1940s
■ and has never recovered. Smaller fisheries developed in the early
I980"s and the early I990's in the Indian River on the Florida east
coast. Those fisheries also collap.sed, apparently in response to
freshwater inputs, and similarly have not recovered. The observed
lack of recovery of any of those populations may result from
depensation effects at low population density. Recovery may be
protracted even with intervention. An alternative management ap-
proach, taking into account the vagaries of hard clam recruitment
and population survival, is proposed for consideration.
INFLUENCE OF CONGENERIC AQUACULTURE ON
HARD CLAM {MERCENARIA SPP.) POPULATION GE-
NETIC STRUCTURE. William S. Arnold*, Sarah L. Walters,
Sarah C. Peters, Theresa M. Bert, Jon S. Fajans, Rorida Fish &
Wildlife Conservation Commission Marine Research Institute 100
Sth Avenue SE St. Petersburg FL 33701.
An aquaculture-based hard clam industry is developing on the
west coast of Florida. There, the species Mercenaria campechien-
sis predominates in the natural clam population whereas M. mer-
cenaria is the predominant species utilized by the industry. The
species hybridize extensively, and this study was conducted to
measure the genetic impact of M. mercenaria aquaculture on the
natural population of M. campechiensis near Cedar Key, Florida.
Clams (N = 257) were analyzed for genetic composition, age, and
the presence and stage of gonadal neoplasia. Results indicate that
the genetic composition of the clam population has changed since
the 1993 advent of aquaculture. Mercenaria mercenaria were non-
existent prior to the initiation of aquaculture but increased in abun-
dance post-aquaculture, as did hybrid clams. There was no differ-
ence in abundance of M. campechiensis pre- vs. post-aquaculture.
All taxa exhibited a high incidence (> S(Wc) of gonadal neoplasia,
but it is not clear if this high incidence results from the introduc-
tion of aquaculture or if neoplasia predates that introduction. These
results indicate that Mercetiaria culture can influence naturally
occurring congeneric populations in the vicinity of the culture
operation, although the long-term implications of that influence
remain to be seen.
National Shellfisheries Association, New Orleans. Louisiana
Ahstracrs. 2003 Annual Meeting, April 13-17, 2003 319
IN SITU DETERMINATION OF PERKINSUS MARINUS
TRANSMISSION DYNAMICS. Corinne Audeniard*. Lisa M.
Ragone Calvo. Kimberly S. Reece, Eugene M. Burreson.
Kennedy T. Paynter, Virginia Institute of Marine Science, The
College of William and Mary. Gloucester Point, Virginia 23062.
Dermo disease, caused by the protozoan parasite Perkinsus
marinus, is currently the most widespread and lethal infectious
disease of the oyster, Crassostrea virginica. During the last de-
cade, it has spread into low salinity areas raising que.stions about
parasite transmission dynamic. Our objective is to determine the
transmission dynamics of P. marinus in low to moderate salinity
areas. The functional relationship between disease-related mortal-
ity, ambient parasite abundance, and infection acquisition by naive
oysters was examined in three Chesapeake Bay tributaries-the Ma-
gothy. Patuxeiit. and James Rivers. From June through October
2002. water samples were collected weekly and parasite cell num-
bers were quantified using real-time PCR. Concurrently, on a
monthly basis, naive .sentinel oysters were deployed and monitored
for P. marinus acquisition and local oysters were monitored for
mortality and infection levels. The three studied rivers showed
very different P. marinus abundance: the high salinity site in the
James river showed up to 3000 cells/1, whereas the Patuxent site
showed less than 20 cells/1 during the whole study and. the Mag-
othy showed no parasite detection. These abundances and P. mari-
nus incidence in sentinel oysters were significantly correlated with
mortality of local oyster population and with salinity.
arrays were designed with cDNAs encoding proteins involved in
physiological functions such as immunity, wound healing, cell
proliferation or cell motility, in order to assess the effect of envi-
ronmental stresses on oyster health.
BIOLOGICAL ASSESSMENT OF STORM EFFECTS ON
THE LOUISIANA PUBLIC OYSTER RESOURCE: TROPI-
CAL STORM ISIDORE AND HURRICANE LILI. Patrick D.
Banks. P.O. Box 98000 Baton Rouge. LA 70898.
Effects of Tropical Storm Isidore and Hurricane Lili on Loui-
siana's public oyster resource were determined using a combina-
tion of square-meter and dredge sampling. Pre and post storm
samples were statistically analyzed for differences in percent mor-
tality and density of oysters (Crassostrea virginica). Results were
di.scussed in relation to environmental parameters such as salinity,
precipitation, and storm surge. Although percent mortality of oys-
ters in square meter samples significantly increa.sed on some pub-
lic oyster grounds following the storms, it was generally < 40%.
Oyster density data from square meter samples yielded mixed
results and dredge samples indicated a slight increase in percent
mortality of oysters in the Lake Pontchartrain basin following the
storms. Negative effects of Tropical Storm Isidore and Hurricane
Lili on the public oyster resource exhibited large spatial variation
(likely due. in part, to extensive spatial variation of Louisiana's
oyster habitat) with significant effects only occurring on some of
the public grounds sampled.
FUNCTIONAL GENOMICS: A POWERFUL APPROACH
TO STUDY THE IMMUNE RESPONSE OF THE PACIFIC
O'S STER CRASSOSTREA GIGAS. Jean-Christophe Avarre,
Yannick Gueguen, Evelyne Bachere and Jean-Michel Es-
coubas*. Defense and Resistance in Marin hnertebrates (DRIM)
UMR5098 (IFREMER. CNRS, UMII) Universite de Montpellier
112 place Eugene Bataillon. CC80. 3409.S Montpellier. FRANCE
Most of knowledge on oyster innate iminunity is based on
biological activities, and molecular features of immune effectors
remain largely unknown. To progress in oyster immune gene char-
acterization we generated expressed sequence tags (ESTs) from a
hemocyte cDNA library built from Crassostrea gigas subjected to
bacterial challenge. A total of 1 142 randomly selected clones were
single-pass sequenced. According to sequence similarities, a pu-
tative function could be assigned to 54V(r of the clones (for more
details, visit the database web site http://www.ifremer.fr/
GigasBase). Among them. 20 genes potentially involved in immu-
nity were identified. To investigate the expression pattern of these
genes. cDNA arrays were developed. Oysters were experimentally
injected with several Vilvio strains isolated from moribund ani-
mals during mortality outbreaks, and gene expression was com-
pared with unchallenged animals. First results showed that some of
these genes were over-expressed after bacterial challenge suggest-
ing their involvement in defense mechanisms. Likewise. cDNA
CLEARANCE RATES AND FEEDING SELECTIVITY OF
CRASSOSTREA VIRGINICA AND MERCENARIA MERCE-
NARIA; IMPLICATIONS OF INCREASED EUTROPHICA-
TION IN THE SUWANNEE RIVER ESTUARY. Carla D.
Beals* and Shirley Baker. Department of Fisheries and Aquatic
Sciences. University of Florida. 7922 NW 71st Street. Gainesville.
FL 326.'i3.
The objective of this study is to examine the potential effects of
increased eutrophication of the Suwannee River Estuary on the
feeding biology of clams and oysters. My hypotheses are that 1 1
the presence or absence of particular phytoplankton species will
affect bivalve clearance rates; and 2) bloom concentrations of
some phytoplankton species will reduce the particle selection and
clearance rates of bivalves. Oysters and clams collected from the
Estuary will be subjected to two concentrations of plankton (av-
erage and Suwannee bloom densities) and four types of plankton
assemblages: 1 ) natural phytoplankton. 2) monospecific cultures of
phytoplankton (not included in selectivity experiments). 3) labo-
ratory-manipulated phytoplankton assemblages, and 4) phy-
toplankton and niicro-zooplankton combinations. Changes in
clearance rate or particle selection ability will have implications
for the future productivity of clams and oysters in the Suwannee
River Estuary.
320 Abstracts, 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
ENGINEERING AND ECONOMICS AS RELATED TO
OYSTERS GROWN IN THE GULF OF MEXICO. Donald L.
Bishop. Bishop Aquatic Technologies Inc., P.O. Box 669. 1 lO-B
Bonnechere St. Eganville. Ontario. Canada. KOJ 1X0 / Engineer-
ing and Economics as Related to Oysters Grown in the Gulf of
Mexico.
Globally the demand for a quality, safe, consistently available
shellfish continues to outpace production. To deliver to the current
consumers as well as to yet to be developed markets will take the
focused involvement of biological, engineering and business plan-
ning aspects to further develop the industry. Currently there is little
correlation between the individuals that specialize in these areas,
yet solutions have been implemented and proven by a small mi-
nority of shellfish producers that understand how to match together
these dynamics. Husbandry technologies have been developed that
allow for the control of shell shape, appearance, size, meat yield
and even shelf life pre harvest. Unfortunately many within the
scientific community are unfamiliar with this area. These tech-
nologies also input control to bio fouling, and production manage-
ment further enhancing yield and profitability. Economics related
to new technologies with return on invested capital per acre per
year are significant. In the past economic models have been cre-
ated for the oyster industry based on past input, output and cost of
operation numbers. New husbandry technology and processes
change this processes significantly, a discussion relating these to-
gether with physical and biological aspects will be co\ered.
within and between generations and also support the genetic basis
previously found for this phenomenon.
IMPACT OF AN INVASIVE TUNICATE IN ATLANTIC
CANADA: RECRUITMENT AND COMPETITION. Daniel
Bourque*, Thomas Landry DEO. P.O. Box 5030. Moncton. New
Brunswick, EIC 9B6. Jeff Davidson. University of Prince Edward
Island, 550 University Avenue. Charlottetown. Prince Edward Is-
land, CIA 4P3; Neil McNair. PEI Department of FAE. P.O. Box
2000, Charlottetown, Prince Edward Island CIA 7N8.
The presence of the club tunicate. Sr\ela clava, was recently
noted in Eastern Prince Edward Island (PEI), Canada. This tuni-
cate presents a significant fouling problem for the blue mussel
{Mytibis ediilis) farms. S. clava has had a negative impact on
mussel culture, attaching in high densities to mussel socks and
equipment, competing for food resources and fouling equipment.
This tunicate is spreading rapidly in the waters of PEI and seems
to be mainly from anthropological mode as opposed to natural
mode. Recruitment, abundance and growth of S. clava were stud-
ied on a temporal scale. The impact of this new fouling organism
was investigated by evaluating its competition for food in relation
to the mussels. The eradication of this invasive tunicate from PEI
waters is considered impractical and therefore the development of
farm management strategies is considered as the only economi-
callv viable solution.
PERSISTENCE OF ATRAZINE IMPACT ON ANEUP-
LOIDV IN THE PACIFIC OYSTER. CRASSOSTREA GIGAS.
Karine Bouilly*. Helen McCombie. Alexandra Leitao, and
Sylvie Lapegue. IFREMER. Laboratoire de Genetique et Patholo-
gic. Avenue de Mus de Loup. 17390 La Tremblade. France.
Aneuploidy is the alteration of the normal diploid chromosome
number. In the Pacific oyster. Crassostrea gigas. hypodiploid
aiieiiploid cells have regularly been reported as has a negative
correlation between this phenomenon and growth and evidence for
a genetic basis. We previously demonstrated a positive relationship
between a pollutant, atrazine. and aneuploidy in Crassostrea gigas
adults and juveniles. To e\aluate the persistence of this impact, the
present study focused on a sample of the same juveniles exposed
to different atra/Jne treatments (0.01 nig/1 which represents a peak
value found m a polluted environment and 0.1 mg/1) for three and
a half months and evaluated them for aneuploidy after another two
and a half months in non polluted conditions. Their aneuploidy
level remained significantly different between the treatments ap-
plied. In addition, our study examined the offspring of the same
adult population previously treated and found that these offspring
exhibited significantly higher aneuploidy levels when the parents
had been exposed to atrazine. These results demonstrate the per-
sistence of the atrazine impact on Pacific oyster aneuploidy in time
A SIMULATION MODEL OF THE POPULATION
GROWTH OF HARD CLAMS [MERCENARIA MERCE-
NARIA). III. EFFECTS OF BROWN TIDE. V. M. Bricelj*,
J. N. Kraeuter, E. N. Powell, J. M. Klinck, E. E. Hofmann,
R. Grizzle, and S. Buckner. Institute for Marine Biosciences Na-
tional Research Council Canada 1411 Oxford Street Halifax. NS
B3H 3Z1.
Brown tides oi Aureococcus anophagejferens have occurred in
Great South Bay, NY, since the mid-1980's. Peak concentrations
usually occurs in June or July and have been attributed a role in the
decline of hard clam populations. Using a physiologically-based
model, simulations were run to examine the effect on clam popu-
lation growth by a) timing of blooms, b) Aureococcus concentra-
tion (105 to 2x106 cells ml-I). c) bloom duration, and d) food
supply. Brown tide effects were incorporated into the model by
assuming dose-dependent feeding inhibition of juveniles/adults. A
brown tide-induced, larval mortality function was generated based
on laboratory results obtained with bay scallop larvae. Sensitivity
of the model output lo variation in larval mortality was assessed.
Effects of a time-dependent, juvenile mortality function, based on
published data for 2 mm clams which experienced high moralities
after prolonged (4-wk) exposure to high brown tide levels (4x105
cells ml-1) were also tested. Preliminary results of a modeled
brown tide effects on individual scope for growth and egg produc-
National Shellfisheries Association. New Orleans. Louisiana
Abslracls. 2003 Annual Meeting. April 13-17. 2003 321
tion o\er a clam's lifespan for inUiNiduals varying in initial body
size and genotype will be presented.
DETERRENTS TO BLACK DRUM PREDATION ON OYS-
TER LEASES Kenneth M. Brown*, (iary Ptttrson, Mike Mc-
Donough, Patrick Banks, and Brian Lezina. Department of Bio-
logical .Sciences. Louisiana State University. Baton Rouge. Loui-
siana 70803.
Preliminary experiments indicated large black drum were ef-
fective predators, and small oysters were preferred, but that salin-
ity did not affect feeding. Further laboratory experiments indicated
scent of dead black drum (as hypothesized by lease holders) did
not lower fish feeding. Field experiments in Barataria Bay in 2
seasons indicated scent reduced feeding by 10-20 %. but only at
one site in one season. We conclude scent is not effective under
most conditions. Mortalities to all predators ranged from 60 % to
90 % within the four weeks on leases. The importance of black
drum and southern oyster drills varied among sites, as did temporal
patterns of mortality. Fish caused 74 % of mortality, and oy.ster
drills 23 %. and fish and oyster drill predation were inversely
related. Laboratory experiments with sound deterrents indicate
"drumming" by male black drum in the frequency range of 40-60
Hz does not deter predation. Sounds in the range of 10-30 Hz may
deter fish, but are impractical because they require considerable
power to broadcast over leases. We hope in future work to deter-
mine whether limited gill netting or trot line fishing can decrease
oyster mortality to black drum without impacting other fish popu-
lations through by-catch.
STRESS RESPONSES IN SCALLOPS AND HARD CLAMS
TO HEAT AND COLD SHOCK. Nicole T. Brun*' \ V.
Monica Bricelj", Emmanuel E. Egbosiniba", Thomas H. Mac-
Rae', and Neil W. Ross". 'Biology Department. Dalhousie Uni-
versity, Halifax NS B3J IZl, "National Research Council. Institute
for Marine Biosciences. Halifax NS B3H 3Z1.
In response to various stressors, such as temperature, organisms
increase production of stress proteins (SPs). Heat shock SP re-
sponses have been studied in mussels, but limited information is
available for other bivalves. Cold shock Stress Protein Response
(SPR) has not been previously investigated. Sea scallops Pla-
copecten magellaiiictis. a deeper water species, and the estuarine
bay scallop Argopecten irradians inadians differ in temperature
tolerance: the former is susceptible to high temperatures, whereas
the latter may be more vulnerable to low temperatures. Juvenile
hard clams Meneiiana mercenaha suffer heavy mortalities during
over wintering in Atlantic Canada and the mid-Atlantic US. The
SPR to acute heat shock, determined by SP-70. was ciimpared in
the two scallop species (10°C increase for 3h). No differences in
SP-70 expression were observed in sea scallops, except at 2 Id.
when levels were significantly lower than initial, control levels. In
contrast. SP-70 in bay scallops increased significantly during and
following heat shock, attaining a maximum by I2h, and exceeded
control levels after 8d-recovery. The SPR of bay scallops and hard
clams to acute cold shock (17°C decrease for 3h) was examined to
determine if this stressor also modulates SP-70. The latter in-
creased significantly in both bivalves, with levels still increasing
after 8d and 24h respectively. The same samples are being ana-
lyzed using SP-40. Characterization of the SPR to acute tempera-
ture shock may have application in acquired thermo tolerance of
bivalves transferred from hatchery to field growout sites.
PERKINSUS CHESAPEAKI AND PERKINSUS ANDREWSI
ARE THE SAME SPECIES. Eugene M. Burreson*. Kimberly
S. Reece, Karen L. Hudson. Christopher F. Dungan. Virginia
Institute of Marine Science Gloucester Point. VA 23062.
Perkinsiis chesapeaki was described from the soft-shell clam.
M\a arenaha. and Perkinsus andrewsi was described from the
Baltic macoma. Macoma halthica. both from Chesapeake Bay.
Sequence analysis of the internal transcribed spacer region (ITS),
the large subunit ribosomal RNA gene and actin genes from clonal
Perkinsus cultures derived from both hosts revealed that the two
species are synonymous. Multiple DNA clones of each region
were sequenced from each clonal isolate. Phylogenetic analyses
based on all three sequences placed isolates derived from the two
different hosts into a monophyletic group. Polymorphisms were
detected at each locus and sequence variation was observed within
clonal isolates at the multi-copy genes. ITS sequences from each
isolate were found in each of two monophyletic sister clades. One
clade included the GenBank deposited ITS sequence for Perkinsus
chesapeaki. and the sister clade included P. andrewsi ITS se-
quences. These results suggest variation observed among ITS se-
quences of these isolates is representative of polymorphisms
within a single parasite species from two different hosts. GenBank
deposited P. chesapeaki and P. andrewsi ITS sequences represent
sequence variants from a single Perkinsus species. The name P.
cliesapeaki has priority under Article 23 of the International Code
of Zoological Nomenclature.
EVALUATING SHELL QUARANTINE DURATION TO
LIMIT THE TRANSFER OF PERKINSUS MARINUS WHEN
PLANTING OYSTER CULTCH David Bushek*. Donnia
Richardson, Yvonne Bobo, Loren Coen and Jennifer Cardinal.
Baruch Marine Field Lab. University of South Carolina. PO Box
1630. Georgetown. SC 29442.
Freshly shucked oyster shell can carry harmful organisms such
as predators, non-natives or pathogens in remaining tissues. Thus,
planting fresh oyster cultch may spread harmful organisms. De-
composing Perkinsus marinus infected oyster tissue is a major
source of infective stages of P. nuirinus. Therefore, we examined
322 Absinicls. 2003 Annual Meeting, April 13-17. 2003
National Shellfisheries Association, New Orleans, Louisiana
changes in P. iiuiiiiuis abundance in tissues of shucked and whole
Gulf Coast oysters deployed in replicate shell piles between March
and July 2002 in Charleston, SC. Parasite abundance was deter-
mined by RFTM body burden assay, and parasite enlargement in
RFTM used to indicate viability. After 31 days, only 13% of
shucked oysters contained any tissue and total parasite abundance
had declined to 0.05%. No tissues remained in subsequent
samples. Tissues decayed much slower in whole oysters, but para-
site abundance still declined rapidly with just 1% remaining after
only 31 days. After 1 15 days, only two whole oysters contained
any observable tissue and total parasite abundance was a mere
0.005% of the original number. The impact of climate and shell
pile configuration should be more closely evaluated, but simply
quarantining oyster shell for one month or more on land can dra-
matically reduce the abundance of P. marinus. minimizing the
potential for transmission.
HIGH HYDROSTATIC PRESSURE INACTIVATION OF
VIRUSES. Kevin R. Caici*, US FDA PO Box 158 Dauphin
Island, AL 36528.
Viruses have been epidemically linked to the majority of the
illnesses associated with consumption of raw shellfish. The ma-
jority of the implicated shellfish were traced back to growing areas
in approved status which were thought to have become contami-
nated by illegal overboard discharges or failures of proximal
wastewater treatment facilities. High hydrostatic pressure (HHP)
processing is in use commercially to reduce Vibrio sp. in shellstock
oysters. Investigations are underway, to determine if HHP might
serve as a post-harvest treatment process to improve the safety of
oysters as related to viruses. Viruses under study include hepatitis
A virus and SM-17, a surrogate for Norwalk-Like virus. Oysters
(Crassostrea virginica) accumulated virus in a flow through sea-
water system. Shucked meats were packaged in plastic pouches
before subjecting to HHP processing. Results show HAV to be the
more pressure resistant requiring pressures > 400 MPa to achieve
a 3 log 10 reduction in 1 min. A similar reduction could be
achieved with SM-17 in 1 min at 275 MPa. The wide range of
pressures required to inactivate different viruses may make it dif-
ficult to select a pressure, that will be effective in destroying all
viral contaminates in oysters without damaging the quality of the
ovsters.
HOST GENETIC ORIGIN AN IMPORTANT DETERMI-
NANT OF QPX DISEASE Lisa M. Ragone Calvo*. Gene M.
Burreson, Susan E. Ford, John N. Kraeuter. Dale F. Leavitt,
Roxanna Smolowitz. Virginia Institute of Marine Science. Col-
lege of William and Mary, Gloucester Point, VA 23062.
Epizootics of QPX (Quahog Parasite Unknown) a protistan
pathogen of hard clams, Mercenaria mercenaria have occurred in
maritime Canada and Massachusetts, New York, New Jersey, and
Virginia, USA. Although it has been found in wild hard clam
populations, the parasite has most seriously affected cultured hard
clams, suggesting that aquaculture practices may promote or pre-
dispose clams to the disease. In this investigation we examined the
influence of host genetic origin and geographic location on QPX
su.sceptibility. Five clam strains, originating from Massachusetts,
New Jersey, Virginia, South Carolina, and Florida were produced
at a single hatchery and evaluated for growth, survival, and QPX
susceptibility at three QPX endemic sites (Massachusetts, New
Jersey and Virginia). Severe winter-associated clam losses oc-
curred at the Massachusetts site precluding completion of the study
at that location. At both the New Jersey and Virginia sites the
South Carolina and Florida clam stocks exhibited significantly
higher QPX prevalences and lower survival than the New Jersey
and Massachusetts clam stocks: while clams from Virginia had
QPX prevalences and survival rates that were intermediate to the
more '" northern" and " southern" clam stocks. These results sug-
gest that genotype-environment interactions are important deter-
minants of QPX disease.
EXPERIMENTAL EVALUATION OF CROSSES WITHIN
AND AMONG FIVE COMMERCIAL STRAINS OF HARD
CLAMS, MERCENARIA MERCENARIA, ACROSS A SA-
LINITY GRADIENT IN VIRGINIA WATERS. Marli D. Ca-
niara*, Standish K. .Yllen Jr. Aquaculture Genetics and Breeding
Technology Center Virginia Institute of Marine Science PO Box
1346 Gloucester Point, VA 23062.
Cultured Mercenaria mercenaria are a multi-million dollar in-
dustry in Virginia. Grow-out sites vary from ocean salinity outside
to mid-salinity estuarine sites inside the Chesapeake Bay. Pres-
ently, the industry uses essentially undomesticated genetic stocks,
and we know very little about the suitability of stocks to varying
environmental conditions. We evaluated the genetic influence of
hard clam strain selection on growth along a salinity gradient in
Virginia as well as the potential for enhancing production by out-
crossing available strains. We first created all fifteen possible com-
binations within and among five brood stock strains in the hatch-
ery. We subsequently raised the juveniles in common conditions
until they reached approximately 10 mm, at which point we split
the groups for planting at five sites encompassing the range of
salinities at which clams are grown. We measured them and com-
pared the growth of these groups in the hatchery, nursery, and
field, estimated the correlations among the performance measures
between life stages, compared the performance of within- and
among-strain crosses, and assessed site-specifity. We discuss the
results and their implications for strain selection, hatchery spawn-
ing procedures, and future efforts in selective breeding for superior
hard clam strains.
Niitional Shelirisheries Association, New Orleans, Louisiana
Ahsrracts. 2003 Annual Meeting. April 13-17, 2003 323
GROWTH OF QUAHOGS (MERCENARIA MERCENARIA)
AND SOFTSHELL CLAMS (M)A ARENARIA) IN RE-
SPONSE TO ELTROPHIC-DRIVEN CHANGES IN FOOD
SUPPLY AND HABITAT Ruth H. Carnikhael*. Andrea C.
Shriver. Erica T. Weiss, and Ivan \'aliela. Boston University
Marine Program, Marine Biological Laboratoiy. 7 MBL Street,
Woods Hole, MA 02543.
In recent years increased urbanization has increased nitrogen
loads to coastal estuaries, prompting eutrophication and changing
estuarine features. Increased N loads increase phytoplankton and
microphytobenthos concentrations, result in accumulation of or-
ganic matter from detritus of algae, reduce sediment and water
column oxygen content, and may change sediment composition.
These changes likely affect growth and survival of commercially
important bivalves like quahogs and soft-shell clams. To determine
how eutrophication-related changes affect these bivalves, we trans-
planted ju\eniles into estuaries of different land-deri\ed N loads,
measured changes in sediment and water column properties, and
recorded growth and survival of bivalves. We used N stable iso-
topes to link responses of bivalves to their food supply and land-
derived sources of N for management. We found growth rales of
quahogs and soft-shell clams increased as land-derived N loads
increased their food supply. Water column food sources had a
greater effect on growth than sediment sources, and low salinity
and high particulate organic matter may have limited growth in
some areas despite increased food supply. N stable isotope analysis
linked these growth responses to land-derived N primarily from
wastewater sources.
DEVELOPMENT OF A SINGLE NUCLEOTIDE POLY-
MORPHISM (SNP» MARKER SET FOR THE HARD
CLAM, MERCENARIA MERCENARIA. Ryan B. Carnegie*.
Mark D. Camara, Lisa M. Ragone Calvo, Kimberly S. Reece.
and Patrick M. Gaffney. Virginia Institute of Marine Science
P.O. Box 1346 Gloucester Point. VA 23062.
In aquaculture. molecular genetic markers can be used to e\ alu-
ate the diversity of wild shellfish stocks to be introduced into
hatchery breeding programs, to control pedigrees in hatchery lines,
and to track the performance of outplanted seed. While progress
has been made in developing molecular markers for Crassttstrea
spp.. the hard clam Meirenaria inercenaria. an extremely valuable
commercial species in eastern North America, has received rela-
tively little attention. Our objective was to develop a set of single
nucleotide polymorphism markers (SNPs) for M. inercenaria.
Hemocyte and mantle complementary DNA (cDNA) libraries
were created in plasmid vectors and then sequenced. Screening for
SNPs is being done using a panel of clams encompassing the
genetic diversity of VIMS hatchery stocks and reflecting the wide
geographic distribution of M. mercenaria. SNPs demonstrating
Mendelian inheritance will be immediately useful for evaluating
the relative performance of clams produced from Massachusetts,
New Jersey, Virginia, and South Carolina broodstock that are now
deployed at two QPX-enzootic and three QPX-free sites in Vir-
ginia. The markers will also be useful for characterizing wild M.
mercenaria germplasm diversity, and may begin to reveal allelic
variation underpinning the variable susceptibility of East Coast
clams to QPX.
TROPHIC INTERACTION BETWEEN HARD CLAMS
AND NATURAL ASSEMBLAGES OF PLANKTON Robert
M. Cerrato*. Amy E. Streck. and Darcy J. Lonsdale. Marine
Sciences Research Center Stony Brook University Stony Brook,
NY 11794-3000.
To examine whether intensive grazing by hard clams or cope-
pods shifts the composition of the plankton community toward
species of different nutritional quality, we conducted experiments
in 400-liter tanks at three locations in Great South Bay, NY. Treat-
ments were created by varying adult clam and copepod abun-
dances. After a 2-week acclimation period, several juvenile (2
mm) clams were added to each tank and allowed to grow for 4
weeks. In one location, where growth under ambient conditions
was high, juvenile growth declined by 57% in the treatment with
high adult clam grazing, suggesting that juveniles and adults were
competing. In the other two locations, where growth under ambi-
ent conditions was moderate to poor, juvenile growth improved by
60 to 200% in treatments with high adult clam grazing. Plankton
composition was altered in the high adult copepod treatments, but
no effect on juvenile hard clam growth was observed. Examination
of clearance and assimilation rates of naive clams exposed to treat-
ment water indicated that observed increases in juvenile clam
growth were related to food quality rather than quantity. Our re-
sults suggest that intense grazing by hard clams can have a positive
effect on the nutritional value of the plankton.
PRESENCE OF PATHOGENIC BACTERIA IN THE LA-
GOON SYSTEMS LA MANCHA AND ALVARADO VER-
ACRUZ, MEXICO IN WATER AND OYSTER (CRASSOS-
TREA VIRGINICA ). Maria del Refugio Castafieda Chavez*,
Erasmo Orrantia B., Violeta Pardio Sedas, Fabiola Lango Rey-
noso. Carr. Veracruz-Cordoba Km 12 C.P. 94290. Boca del. Ver.
Mexico.
Mexico maintains the 6th place in world-wide oyster produc-
tion, contributing the Gulf of Mexico with 76% of the total vol-
ume. In this coastal area, 30 and 36 sampling stations were estab-
Ikshed in the coastal lagoons of Alvarado. and La Mancha. Water
and oyster samples were taken during one annual cycle, and mi-
crobiological analysis were performed to determine according to
the Mexican Official Norm NOM-031-SSA 1-1993. Three stocks
of pathogenic vibrios were isolated from water samples of Lagoon
of Aharado. Vilvio alginolyticus. V. cliolerae (IN.ABA) and V.
choierae No-OI. besides Salmonellas and total coliforms. The V.
324 Abstnicls. 2003 Annual Meeting. April 13-17, 2003
National Shellfisheries Association. New Orleans, Louisiana
cholerae serotype INABA was reported in Alvarado during the
months of July. August and September. The V. alginolyticus was
reported in January. V. cholerae No-01 was reported in the La
Mancha during the rainy season exclusively. Analysis for V. chol-
erae no-01 from oyster samples of the Alvarado is not significantly
different to those reported from the oyster banks of La Mancha. It
was concluded that fecal discharges is the main cause of pollution
representing a health problem that must be considered due to the
possibility of survival of microorganisms when oysters are raw
consumed and not subjected to depuration.
efforts ha\'e been conducted to restore many of the altered areas
back to their original habitat value. What is not always clear is how
to define the value of a habitat to a particular species of interest.
This information is important to assess the impacts of habitat al-
teration on species that utilize those areas. The impacts of these
types of alterations to the critical life support functions of shellfish
populations will be reviewed. A characterization of the habitat
conditions that support the survival and continued viability of
shellfish populations is needed to properly assess habitat alteration
and e\aluate the success of restoration efforts.
MUSSEL GROWTH AND FOOD UTILIZATION IN RELA-
TION TO WATER COLUMN CONDITIONS ON RAFT
SYSTEMS IN PUGET SOUND. WASHINGTON Daniel P.
Cheney*. Andrew D. Suhrbier, .\iniee E. Christy. Hector S.
Beltran. Jonathan P. Davis, Kenneth M. Brooks, and Frank ,|.
Smith. 120 State Ave. NE #142 Olympia. WA 98501.
Suspended mussel and oyster culture on the U.S. west coast is
predicted to increase significantly in coming years. Description of
the changes associated with the culture of these crops is essential
for the siting and evaluation of new culture facilities and in im-
proving yield and production of existing facilities. This research
had three general objectives: 1 ) to assess at large-scale farm sites,
mussel growth and yield against a suite of measured physical,
chemical and biological variables; 2 ) to compare the same suite of
variables with measurements of mussel feeding and biodeposit
production; and 3) to utilize available nutrient and yield models to
estimate potential mussel carrying capacity in the farming area.
During a two year period (2001-03). multiple observations were
made of water currents, water chemistry, phytoplankton. mussel
growth, seston removal and absorption, fouling, and fish utilization
at commercial mussel raft culture sites in Totten Inlet and Penn
Cove. Washington. Although parameters, such as water currents
and phytoplankton abundance varied markedly inside and outside
the raft units and under different tidal regimes, these effects were
localized and did not correlate with mussel growth. This research
is supported by a Sea Grant National Marine Aquaculture Initiative
arant.
ASSESSING THE EFFECT OF HABITAT ALTERATION
ON SHELLFISH POPULATIONS. Marnita M. Chintala*.
U.S. EPA. NHEERL. Atlantic Ecology Division, Narragansett. Rl
02882; and Karin A. Tanimi. NOAA/R.l. Dept. of Environmental
Management. Narragansett, Rl 02882.
Habitat provides a variety of life support functions for many
species, such as providing shelter, substrate, food, and nursery
areas. Habitat alteration is one of the most important causes of
declines in ecological resources in North America, and habitats
essential to the well being of shellfish species are rapidly being
affected by many land-use activities. As a result, many restoration
DESIGN AND IMPLEMENTATION OF A SURVEY OF
COMMERCIAL BLUE CRAB EFFORT IN THE MARY-
LAND PORTION OF THE CHESAPEAKE BAY Mary C.
Christman,* Cynthia J. Giffen. Department of Animal and
.Avian Sciences. University of Maryland. College Park. MD
20742; Jon H. Volstad. Versar Inc.. 9200 Rumsey Rd., Columbia.
MD 21045; and Lynn W. Fegley. MD DNR. Tawes State Office
Building. 580 Taylor Ave.. Annapolis. MD 21401.
The Maryland Department of Natural Resources (MD DNR.
requires estimates of the fishing effort expended by commercial
crab fisheries in the Chesapeake Bay. We designed a three-prong
approach to obtaining instantaneous estimates of effort in the bay.
We collected field data on the commercial pot and trotline crab
fisheries, and telephone surveys for supplementary information.
Sampling included -160 stratified random transects for pots each
month to obtain estimates of spatially explicit pot densities. Survey
stations were modified transects; planar boards were used to de-
lineate the width of each transect. The trotline surveys were per-
fonned using both aerial flyovers and roving intercept surveys to
quantify the mean number of lines per boat and mean trotline
length. We describe methods for merging this information in ways
that can be used to estimate effort for similar fisheries.
AN INTEGRATED APPROACH TO BIVALVE DOMESTI-
CATION: INTRODUCTORY REMARKS. Fu-Lin E. Chu*,
Virginia Institute of Marine Science. College of William and
Mary. Gloucester Point. VA 23062; Jean-Francois Saniain*. If-
remer Centre de Brest, BP 70, 29820, Plouzane, France.
Environmental and disease stresses are worldwide problems
and have caused severe mortality in many cultivated and feral
bivalve populations. For years, scientists in France and US have
devoted time and effort in an attempt to improve the yields via
multi-disciplinary research. To coordinate activities of researchers
from various scientific disciplines in US and France, a US-France
Workshop on "Domestication of bivalve molluscan shellfish" was
held in La Tremblade, France, 2002. Via the meeting several short-
term US-France collaborative projects have been developed. To
accelerate information and technology exchange, ideas for future
technical workshops have been established. Currently a five-year
National Shellfisheries Association. New Orleans. Louisiana
Abxnucrs. 2003 Annual Meeting. April 13-17. 2003 325
multi-disciplinary research project on Crassostreci gifias summer
mortality is being conducted in France. Six disciplines are con-
tributing together to test the hypothesis of a complex interaction
between oyster, environment and opportunistic pathogens. The
study focuses on mortality dynamics in the field and determines
the relative role of different putative factors in contributing to the
mortality.
EVALUATING THE IMPACTS OF HARVESTING PRAC-
TICES, BOAT WAKES AND ASSOCIATED SHORELINE
EROSION ON INTERTIDAL CREEK HABITATS IN THE
SOUTHEASTERN U.S.: MANAGERS AND RESTORATION
PROGRAMS TAKE NOTE. Loren D. Coen* and Majbritt
Bolton-WarbiTji, Marine Resources Research Institute. SCDNR.
217 Fon Johnson Rd.. Charleston. SC 29412 and Graduate Marine
Biology Program. Grice Marine Lab. College of Charleston. 20,'i
Fort Johnson Rd.. Charleston. SC. 29412.
In areas where oysters are intertidal and fringe marsh-lined
creeks, they can act as shoreline "stabilizers". Recent work (FL.
SC. and NC) suggests that harvesting and boating, in addition to
natural phenomena, can significantly impact natural intertidal
habitats and restoration/enhancement efforts. We assessed oyster
populations prior to applied treatments, evaluating the direct im-
pacts of four common harvesting practices on oyster population
recovery at 12 sites, paired with controls. Concurrently, recruit-
ment, survival, and growth were also examined annually and popu-
lations reassessed -3 years later to evaluate "recovery". Simulated
boat wake experiments used shell treatments (with and without
mesh) to evaluate impacts of wakes on restoration efforts. Results
are discussed and current larger-scale study designs applying our
findings are summarized. Four study sites were established in 1999
to measure shoreline erosion. Over 2?-38mo. rates ranged from
~0-23cm/month; overall bank losses were from 69-1.54 cm. In
2001, we expanded sampling at nine additional sites using our
SCORE program. Erosion rates (4-16mo.) ranged from ~2-8cm/
month, with overall losses from 13- 104cm. These and other results
suggest that anthropogenic impacts may be having much greater
impacts on critical intertidal habitats than previously perceived.
HISTORY OF POST-HARVEST TREATMENT TO RE-
DUCE VIBRIO SP. IN SHELLFISH David W. Cook, Food &
Drug Administration Gulf Coast Seafood Lab P.O. Box 158 Dau-
phin Island, AL 36528.
Vibrio vidnificiis was first recognized as the cause of primary
septicemia in humans and its relationships to shellfish consump-
tion established in the early 1970's. K vulnificus is a naturally
occurring pathogen and it densities in shelltlsh at harvest are re-
lated to growing water temperature. To control illnesses caused by
this bacterial species, several approaches including time-
teniperaliire controls, harvest restrictions and consumer education
have been initiated. Research into post-harvest processing mitiga-
tion strategies to reduce Vibrio numbers without destroying the
raw characteristic of the shellfish was undertaken. In 1996, the first
commercial post-harvest treatment process, a mild heat treatment,
was recognized as capable of reducing V. vidnificus in shellfish to
a non-detectable level. Two other processes, freezing and high-
hydrostatic pressure processing, were validated in 2002 by com-
mercial processors. Other processes under study are depuration,
relaying of shellfish to waters free of V. vidnificus and irradiation.
Post-harvest processes for reducing V. parahaemolyticus in shell-
fish to non-detectable levels are also being validated.
FRESHWATER PEARL CULTURE AND PRODUCTION IN
CHINA. Hua Dan*. Freshwater Fisheries Research Center
(FFRC). Chinese Academy of Fisheries Sciences. Wuxi City
214081. Jiangsu Province. CHINA
Lustrous pearls have been called the queen of jewels, biil the
occurrence of quality pearls in wild mussels is rare. The technolo-
gies of freshwater pearl culture were developed in China some
2,000 years ago. However, commercial pearl culture dates back
only to the late 1960s. Gradual changes in technology and in the
type of mussel used {Hxriopsis cuiningii). resulted in the produc-
tion of greater quantities of larger and more lustrous round, near-
round, and baroque cultured pearls of various colors. Today, there
is a great demand for cultured freshwater pearls, and China pro-
duces 95*7^ of those pearls sold in the world market. China pro-
duces an estimated 800 to lOOO metric tons of freshwater cultured
pearls annually, of which roughly 400 to 500 metric tons are
exported to different continents and countries worldwide. Pearls 8
mm and larger represent a large percentage of those exported. This
presentation will re\ iew the techniques of freshwater pearl culture
in China, to include principles of pearl formation, mussel operation
procedures, and mussel culture post-implantation.
POPULATION GENETICS OF THE BLUE CRAB (CALLI-
NECTES SAPIDUS) IN THE GULF OF MEXICO. Richard L.
Darden* and Brian R. Kreiser, Department of Biological Sci-
ences. Universitv of Southern Mississippi. Hatliesburg. MS 39406.
Gene flow among populations of the blue crab {Callinectes
sapidus) is determined by larval dispersal and adult crab move-
ments. Assessment of population genetic structure allows infer-
ences about historic and contemporary patterns of gene flow. A
total of 1.920 crabs were collected from 26 locations around the
Gulf of Mexico coast between Naples. Florida and Brownsville.
Texas during 2001-02. A 650-base pair portion of the mitochon-
drial cytochrome oxidase I (COI) gene was amplified and se-
quenced for individuals from each location. Preliminary results
seem to indicate that Gulf of Mexico blue crab populations are not
326 Abslnicls. 2003 Annual Meeting, April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
genetically homogeneous. We will place these results into the between cumulative mortality and burden of pathological condi-
context of blue crab life history as well as prevailing theories tions was significant,
concerning blue crab dispersal and migration.
GROWTH AND MORTALITY OF DIFFERENT OSTREA
EDULIS STOCKS CULTURED IN THE RIA DE AROUSA
(GALICIA, NVV SPAIN). Patricia M. da Silva*. Antonio Vil-
lalba. and Jose Fuentes. Centre de Investigacions Marinas.
Aptdo. 13. 36620 Viianova de Arousa, Spain.
Nowadays. Bonamiosis is the most important constraint for the
Galician oyster industry. The development of a disease-resistant
stock by a selective breeding program seems a promising measure.
Oysters harvested from four genetically different populations were
used as broodstock to obtain 5 families from each stock in a
hatchery. Two of these stocks were obtained from two B. ostreae-
free areas in Ireland and Greece, and the other two from Coroso
and Ortigueira. two Galician areas where the parasite is present.
Spat of every family is being cultured in the Ria de Arousa since
Sept 2001. Growth and mortality data for one-year culture period
are analyzed in this presentation. Results show significant differ-
ences in growth and mortality, both among stocks and families. On
average, Galician and Greek stocks perform better (faster growth
and lower mortality) than the Irish one. However, the importance
of the differences detected among families in both variables di-
minishes the relevance of those among stocks.
CROSSBREEDING IN PACIFIC OYSTERS. Joth Davi.s*.
Taylor Shellfish Farms, Quilcene, WA 98376; Dennis Hedgecock.
Bodega Marine Laboratory. Bodega Bay, CA.
Intraspecific hybrid lines of Pacific oysters {Crassastrea giaas)
were made in 2001 by crossing inbred oysters in a full factorial
mating design at the Taylor Shellfish Farms bivalve breeding fa-
cility in Quilcene, WA. Two cohorts of hybrid oysters were reared
from inbred lines produced by the Molluscan Broodstock Program.
Families generated from individual pair-matings were reared and
set using standard techniques. Seed from individual families was
reared in the field in a 2 month replicated experiment to test for
differences in yield among hybrid families. Oysters were subse-
quenlly redeployed in replicate cages for a 1 2-month yield trial.
Final yield measurements (total count and biomass) made in Au-
gust 2002 demonstrated a positive correlation between yield at the
seed stage and yield in harvest-ready oysters. Inbred lines and
hybrid combinations that generated superior yield at both the seed
and harvest stages were identified. Stock improvement via cross-
breeding emphasizes yield testing at the seed stage to help predict
tlnal yield in oyster production, and offers some advantages over
the cost and effort associated with traditional selection and breed-
ing programs.
DIFFERENCES IN DISEASE SUSCEPTIBILITY AMONG
OSTREA EDULIS STOCKS CULTURED IN GALICIA (NW
SPAIN). Patricia M. da Silva*. Antonio Villalba, Maria J. Car-
ballal. and Jose Fuentes. Centro de Investigacions Marinas.
Aptdo. 13, 36620 Viianova de Arousa, Spain.
Bonamiosis is the bottleneck for Galician oyster industry. A
program to develop a Bonamia ostreae resistant strain is being
performed. Oysters from different populations were selected as
broodstock: Ireland and Greece bonamiosis-free areas, and two
Galician areas. Ortigueira (bonamiosis is epizootic), and Coroso
(low bonamiosis pressure). Five families per stock were trans-
ferred to a raft in the Rfa de Arousa on September 2001 . Mortality
is estimated monthly and samples of each family are taken and
historically processed. The most prevalent pathological conditions
detected until October 2002 were intranuclear inclusions, suggest-
ing viral infection, and disseminated neoplasia. RLO in digestive
epithelia and Haplosporidium-like plasmodia were rare.
Haemocytic infiltration, granulocytomas and necrosis were also
observed. B. ostreae was detected in September and October 2002
with very low prevalence, although increment is expected in the
second year. Significant differences in the burden of pathologic
conditions were detected anions stocks and families. Correlation
THE EFFECT OF ALGAL TOXINS ON THE ISOLATED
VENTRICLE OF THE HARD CLAM, MERCENARIA MER-
CENARIA. Lewis E. Deaton. Biology Department. University of
Louisiana at Lafayette. Lafayette. LA 70504.
While many species of algae have been associated with mass
mortalities of shellfish, relatively little is known about the specific
effects of algal toxins on the organ systems of mollusks. Isolated
ventricles in aerated seawater were exposed to varying concentra-
tions of saxitoxin, brevetoxin 2 and brevetoxin 9. Saxitoxin had no
effect, even at a concentration of Ix 10-6 M. Brevetoxin 2 caused
a prolonged negative inotropy in the ventricles; the threshold is
about 1 X 10-9M and the effect is dose-dependent. Brevetoxin 9 (I
X 10-9 M) caused a decrease in the amplitude and increase in the
diastolic tone; these effects were transitory. Higher doses (10-8.
10-7 M) of brevetoxin 9 did not increase the inhibitory effect. The
hearts of bivalves are myogenic, and are not affected by the neural
Na-i- channel blocker, tetrodotoxin. The lack of any effect of sax-
itoxin is therefore unexceptional. Brevetoxins open Na-i- channels;
whether this is the mechanism of their inhibition of the Mercenaria
ventricle will require further study.
National Slielltisheries Association. New Orleans. Louisiana
Ahstnict.s. 2003 Annual Meeting. April LVI7. 2003 327
GENETIC BASIS OF SUMMER MORTALITY IN JUVE-
NILE CUPPED OYSTERS. Lionel Degremont*, Pierre
Boudry and Patrick Soletchnick. LGP-LCPC. F- 1 7390 La Trem-
hlade; Edouard Bedier. LCB. F-.'i647() La Trinite; Michel Rop-
ert. LCN. F- 1 4520 Port-en-Bessin; Arnaud Huvet, .Jeanne Moal
and Jean Francois Samain, LPL F-2y2S0 Plouzane.
The French project "Merest", coordinated by IFREMER, aims
to understand the causes of the simimer mortalities in Crassostrea
gii^tis. In 2001, three sets of families were bred following a nested
half-sib mating design. 1 7 halt-sib families (HSF) were obtained in
this first generation (Gl) and reared in 3 sites. Significant differ-
ences in survival were observed among HSF. and some HSF
showed high levels of mortality in all sites, clearly indicating a
genetic basis for survival. In 2002. a second generation (02).
including divergent selection and inbred lines, was constituted.
Monitoring of survival and growth of G2s were the same as in
2001. Significant differences in survival were found between the
offspring of the "high" and "low" selected groups and between
inbred lines. The high realized heritability for survival indicates
that selective breeding programs could efficiently improve sur-
vival of juvenile oysters.
MUCIN SECRETIONS AND NACRE DEPOSITION IN THE
FORMATION OF PEARLS. Leonard DiMichele* and
Stephan Towers. Department of Wildlife and Fisheries Sciences.
Texas A&M University, College Station, TX 77843; Donald
Shepherd. Professional Pathology Laboratories. Ltd, P.O. Box
326. Tow. TX 78672
Cultured pearls originate within a pearl-sac formed by the in-
sertion of a nucleus and graft tissue into a surgically created pouch.
Within the pouch, the host animal initiates a classical wound heal-
ing response and then nacre-secreting cells from the graft prolif-
erate, lining the lumen of the pouch. Maturing pearl-sac epithelia
from a freshwater mussel (LInionidae: Cyrloiniias tampicacnsis)
were examined. Mucopolysaccharide secretions gradually in-
creased after 30 days of development. By day 43. all mucins were
actively secreted by host epithelia. Although the pearl-sacs were
morphologically mature, there was no evidence of calcium secre-
tion. However, natural pearl-sacs in the same mussels exhibited
calcium secretions. The various proteins and calcium secretions
formed an aragonite - protein laminate (nacre). Using atomic force
microscopy and acid extractions, we characterized natural pearls
and shell nacre of Cyrtomiias tampicoensis. Our results were simi-
lar to those reported from several salt water species and were
consistent with evidence from Asian freshwater mussels.
IMPACT OF ENVIRONMENTAL AND NUTRITIVE CON-
DITIONS ON DEFENCE MECHANISMS OF OYSTERS
DURING AN ANNUAL CYCLE. Maryse Delaporte*. Philippe
Soudant. Jeanne Moal and Christophe Lambert, Maryse De-
laporte Laboratoire de Physiologic des Invertebres Centre Ifremer
de Brest, BP 70 29280 Plou/ane (France).
In the frame of MOREST project, a common biological mate-
rial, resulting of a mixture of different families produced in ex-
perimental hatchery, was reared in two different environmental
fields: Normandy and Charente. Concomitantly, a pool was con-
ditioned at the Ifremer Argenton hatchery with three different al-
gae levels: 4%, S'/r and 12% of algal dry weight per oyster dry
weight. During the experiments, five immune parameters were
studied in parallel with survival rate and reproductive status
(stages and intensity).
Site location, seasonal variations and experimental diet level
clearly intluenced oyster immune parameters. Hemocyte counts
were higher for oysters reared in Normandy than those reared in
Charente and ni hatchery. Granulocyte percentage was drastically
reduced in hatchery conditions compared to in situ conditions.
Moreover, hemocyte activities were also affected by the in situ
conditions and dietary treatments in relation to reproductive cycle
and mortality events. In example, in vitro haemocyte adhesion
capacities were more affected by pathogenic Vibrio when oysters
suffered mortality.
REPRODUCTION IN FLAME SCALLOPS, LIMA SCABRA
SCABRA (BORN 1778), FROM THE LOWER FLORIDA
KEYS. Angela K. Dukeman*. 100 8th Avenue SE St. Petersburg,
FL 33701. Norman J. Blake and William S. Arnold.
Sex ratio, gonadal characteristics, and the reproductive cycle of
the flame scallop. Lima scabra scabra (Bom 1778), collected from
Boca Chica Key. FL were investigated over a 21-month period
from January 1998 through September 1999. Gametogenic cycles
were examined using qualitative and quantitative methods and the
results were analyzed within the context of environmental varia-
tion. Gamete development was initiated in winter and coincided
with cooler water temperature and moderate food concentration.
Maximum gamete ripeness and size occurred in late summer, when
water temperatures were at maximum values (33 C), and food
quantities were increasing (>0.2 ug/l). Both quantitative and quali-
tative results indicated a clearly defined spawning event that oc-
curred in autumn in association with decreased female gonad size,
increased presence of partially spawned, spent, and early gameto-
genic gonads, rapidly decreased water temperature (~7 degrees),
and maximum measured chlorophyll-a concentrations (1 ug/I).
Less defined periods of spawning activity occurred in February
and June but could not be related to specific changes in environ-
mental conditions. The presence of ripe and partially spawned
flame scallops and adequate chlorophyll-a concentrations through-
out the year suggests a continuous spawning reproductive strategy,
common in tropical marine invertebrates.
328 Abslnicts. 2003 Aiiiuial Meeting. April 13-17, 2003
National Shellt'isheries Association, New Orleans, Louisiana
IN VITRO PROPAGATION OF PERKINSUS SP. PARA-
SITES FROM JAPANESE MANILA CLAMS. RUDITAPES
PHILIPPINARUM. Christopher F. Dungan*, Maryland DNR.
Cooperative Oxford Laboratory. Oxford. MD 21654; Kimberly S.
Reece. and Karen L. Hudson. Virginia Institute of Marine Sci-
ence. Gloucester Point. VA 23062
Perkinsus sp. is destructive parasites of Manila clams. Rudi-
tapes philippinarum from Korea. Japan, and Spain, but parasite
isolates are not reported from this host. Gills of Japanese Manila
clams collected in Gokasho Bay, Mie prefecture were infected by
Perkinsus sp. parasites at 97% prevalence and moderate infection
intensities. Parasite cells in gill and gonad tissue samples were
enlarged for 48h at 28C in Ray's fluid thioglycollate medium: then
inoculated into DME: Ham's F-12 Perkinsus sp. culture medium.
Enlarged parasite cells zoosporulated to produce hundreds of mo-
tile zoospores, which subsequently gave rise to schizogonic in
vitro cell lines that zoosporulated intermittently at low frequency.
Four Perkinsus sp. isolates were propagated, cryopreserved, and
cloned. In vitro cell morphologies and cell cycles of these isolates
differed from those reported for other Perkinsus sp.. and DNA
sequences suggest that at least one of our isolates is genetically
distinct from described Perkinsus species.
REPRODUCTIVE STRATEGY: VARIABILITY OF RE-
PRODUCTIVE PATTERN IN TWO POPULATIONS GE-
NETICALLY DETERMINED OF CR.ASSOSTREA GIGAS.
Martha F^nriquez-Diaz*. Stephane Pouvreau, Caroline Fabi-
oux, Yvette Le Coguic. Jean Claude Cochard, Marcel Le Pen-
nec; UMR PE2M. IFREMER; BP70. 29280 Plouzane. France.
In the literature, the reproductive cycle of C. giiicis has been
well described and is generally characterized in three steps: ( 1 )
energy storage: (2) gamete development and (3) spawning. But the
genetic intra-variability of this cycle has been scarcely investigated
in C. gigas. During the French MOREST program, a genetic se-
lection based on the survival criteria allowed to obtain a resistant
stock (named "' R") and a susceptible stock (named " S"). The
gametogenic activity of these two stocks was characterized in field
(South Brittany. France) on the basis of quantitative histology of
the gonad (gonad volume, number and egg size) and by the ex-
pression of the vasa gene, specific marker of the germinal cell.
Results showed that the reproductive strategy, especially the re-
production effort and the spawning intensity, was strongly differ-
ent between the two groups and these results suggest that a genetic
triggering mechanism might exist for the onset and flexibility of
gametogenesis.
THE ROLE OF HEAT SHOCK PROTEINS IN TOLER-
ANCE TO PARASITIC STRESS IN THE EASTERN OYS-
TER. CRASSOSTREA VIRGINICA. Vincent G. Encomio*; Fu-
Lin E. Chu. Virginia Institute of Marine Science. College of Wil-
liam and Mary. Gloucester Point, VA 23062.
Thermal stress could affect disease resistance mechanisms by
depressing immune defense and physiological fitness . We are
investigating the relationship between heat tolerance and P. mari-
nus resistance among Dermo "resistant" and "susceptible" oyster
stocks and the role of heat shock proteins (hsps) in protection of
oysters from thermal and disease stress. Results revealed that
Chesapeake stocks had higher thermal tolerance than Louisiana
stocks. Levels of hsp 70 did not vary between these two stocks and
only increased slightly as water temperatures increased. No con-
sistent differences in thermal tolerance were found among Chesa-
peake resistant and susceptible stocks, and a resistant hatchery
strain. Exposure of oysters to a sublethal heat shock improved their
survivorship when subsequently exposed to a lethal temperature.
We are presently examining how induced thermo tolerance and
hsps mediate interactions between parasitic and thermal stress in
uninfected and P. marinus challenged oysters This study is sup-
ported by ODRP. Sea Grant. NOAA (Award*: 1 14926-GL100I4.
Project* VA-OD-01-05).
HISTOLOGICAL EXAMINATION OF GAMETOGENESIS
IN GENETIC TRIPLOID CRASSOSTREA ARIAKENSIS IN
CHESAPEAKE BAY A.J. Erskine* and Standish K. Allen, Jr.,
College of William and Mary. Virginia Institute of Marine Sci-
ence. P.O. Box 1346, Gloucester Point. VA 23062.
Combating the loss of the oyster resource in Chesapeake Bay
has been ongoing for decades. Recently, focus has turned to the
non-native Suminoe oyster, Crassostrea ariakeusis and the possi-
bility of its introduction as reproducing diploid or a triploid for
aquaculture only. In field tests, triploid C. ariakensis has exhibited
high survival, growth, and disease tolerance in Chesapeake Bay.
As reported for several other shellfish species, triploidy often re-
sults in abnormal or arrested gametogenesis. Documenting the
extent of gamete development in triploid C. ariakensis is an im-
portant biological variable addressing the risk associated with non-
native introduction. Nine diploid females and one tetraploid male
were used as parents for this triploid spawn. These genetic triploids
were deployed at six sites along Chesapeake Bay ranging from low
salinity (~13%c) to high salinity (-35%r). Diploid native controls
were sampled at each site to track the "normal" cycle of gameto-
genesis. Paraffin histology of triploids revealed abnormal gamete
production typical of triploid. However, a few sites produced un-
usually mature ova and spermatozoa for triploids. Samples late in
the season indicated spawning had occurred in both diploid and
triploid males and females.
National Sliellfisheries Association. New Orleans. Louisiana
Ahstimls. 2(103 Annual Meeting, April 1.V17, 2003 329
EFFECTS OF INBREEDING ON PERFORMANCE TRAITS
IN PACIFIC OYSTERS (CRASSOSIREA GIGAS). Ford
Evans*, Sean Matson, John Brake, and Chris Langdon. Hat-
field Marine Science Center, Oregon State University. Newport.
OR, 97365.
Understanding the elTects of iiihrccding ls critical lo the long-
term viability of shellfish breeding programs, hibreeding depres-
sion in shellfish is well documented among the offspring of self
fed individuals and full-sib crosses. This study was conducted to
determine if crossing more distantly related parents would result in
measurable inbreeding depression of performance traits in aduh
raised in a commercial inter-tidal growing environment. Families
were created with average estimated inbreeding coefficients (F) of
0. 1/16, and 1/5. Average family yield, individual growth rate, and
survival were recorded after the first and second growing seasons.
After two growing seasons, significant inbreeding depression of
yield and individual growth rate was observed in families with
F= 1/16 and F= 1/5. Significant depression of survival at harvest
was observed only in families with F= 1/5. These results empha-
size the importance of maintaining pedigree records in breeding
programs to help avoid the dcleteriiius effects of inbreeding de-
pression, even among crosses of distantly related parents.
TRACKING THE SPREAD OF AN INVASIVE MUSSEL
(MYTILIDAE: PHRNA VIRWIS) IN FLORIDA Jonathan S.
Fajans*, Patrick Baker. Department of Fisheries and Aquatic
Sciences, University of Florida, 7922 NW 7r' ST, Gainesville, PL
32653
The green mussel Feiiia virulis was introduced to Tampa Bay,
Florida in 1998. Since April 2002. we have been conducting sur-
veys to chart the population growth of the mussel and monitor its
spread. Three sites within the Bay were chosen as representatives
of estuarine. introdtiction epicenter, and oceanic environments.
Monthly collections were made for population density estimates.
Densities within the Bay have reached 4033. 3675. and 41 17 per
square meter, respectively. Coastal sites throughout Florida were
visited annually to determine presence or absence of P. viriclis. As
of January 2003 the range of pt)pulations has been extended to Fort
Myers Beach to the south of Tampa Bay and Indian Rocks Beach
to the north. Additionally, a new population has been found south
of St. Augustine extending to Ponce Inlet on Florida's east coast,
and several specimens have been reported from Pensacola in the
panhandle.
OYSTER VASA-LIKE GENE: A SPECIFIC MARKER OF
THE GERM CELL LINEAGE IN CRASSOSTREA GIGAS.
Caroline Fabioux*. .Arnaud Huvet. Frederic LeRoux, Marcel
LePennec, Jean-Claude Cochard. UMR PE2M. Itiemer, BP70
29280 Plou/ane, France.
Identification of physiological mechanisms implied in repro-
duction of Crassiistrea i>igii.s is essential to improve control re-
production in hatchery. Origin and developmental pattern of first
germ cells in oyster are steel unclear underlying the need of mark-
ers for gametogenesis initiation. The vasa gene, isolated from sev-
eral organisms such as Drosophila, Caenorhabditis. Xenopus or
Zebrafish are specifically expressed in germ cells and are essential
for gonad differentiation. We isolated and characterized an homo-
logue of the vasa gene in C. .i;/,?ai by RT-PCR. The spatio-
temporal expression pattern of vasa gene was established by In
Situ Hybridization or real-time PCR. Results showed that vasa is
only expressed in germ cells and not in somatic cells. Moreover,
vasa appeared differentially expressed during gametogenesis: from
high expression in oogonia and spermatogonia to zero in gametes.
Oyster Vasa-like gene appeared to be a relevant marker of germ
cells for further studies such as the analysis of environmental
effect on the kinetic of gametogenesis and reproductive effort of C.
gigas.
MANIPULATION OF ENVIRONMENTAL PARAMETERS
FOR OUT-OF-SEASON EGG AND LARVAL PRODUC-
TION IN BLUE CRAB BROODSTOCK {CALLINECTES
SAPWUS). Andrea Findiesen*, Oded Zniora. Moti Harel, and
Yonathan Zohar, Center of Marine Biotechnology, University of
Maryland Biotechnology Institute. Baltimore. MD; Alicia Young-
Williams and An.son H. Hines. Smithsonian Environmental Re-
search Center, Edgewater. MD.
Blue crab production techniques are being developed at the
Center of Marine Biotechnology (COMB) to evaluate the possi-
bility of restocking the diminished Chesapeake Bay blue crab
population. Mature mated females were introduced into 2 m3 tanks
with phase-shifted environmental conditions. By manipulating
photoperiod, temperature and salinity, we have successfully in-
duced females to ovulate, produce egg masses (sponges) and pro-
vide viable larvae all year-round. We also have been able to pro-
duce up to four successive sponges per female. Sponge production
seems to be affected by a combination of photoperiod and tem-
perature: long photoperiod ( 14 hours light: 10 hours dark) and high
temperature (23oC) generated the most sponges. Our data indicates
that high temperatures, though optimal for sponge production, in-
crease susceptibility to disease when exposed over long durations
of time. Sand is necessary for sponges lo adhere properly to a
female's abdomen. There doesn't seem to be any difference be-
tween inaintaining the females at 25 or 30 ppt. Future work may
include hormonal manipulation of broodstock to provide more
predictable ovulation and larval production.
330 Absrnicis. 2003 Annual Meeting, April 13-17, 2003
National Shellfisheries Association. New Orleans, Louisiana
MANAGING AND MONITORING INTERTIDAL OYSTER
REEFS WITH REMOTE SENSING IN COASTAL SOUTH
CAROLINA. Mark Finkbeiner*. Bill Stevenson. Bill Ander-
son. Mike Yianopolous, Loren Coen. Ginger Martin, and
Karen Cullen. NCAA Coastal Services Center 2234 South Hob-
son Ave, Charleston. SC 29405.
Intertidal oyster reefs are a keystone species in South Caroli-
na's estuaries and a major commercial and recreational resource.
The South Carolina Dept. of Natural Resources (SCDNR) is re-
sponsible for conserving oyster reefs and regulating their harvest.
The current oyster reef database for South Carolina was developed
by field assessment in the 1980s and an update is needed to assess
resource status and trends across the coastal zone. Coastal devel-
opment and associated waterway usage are suspected of altering
the extent and density of the state's oyster resources. The NCAA
Coastal Services Center is working with SCDNR to develop meth-
ods for using high-resolution remote sensing data to assess inter-
tidal oyster reefs along the South Carolina coast.
The objective of the project is to provide SCDNR with a new
methodology for assessing intertidal oyster resources. The project
examined digital and analog aerial photography in two pilot areas
located in Charleston and Beaufort Counties. A variety of image
processing and photogrammetric methods were evaluated includ-
ing manual delineation, spectral clustering, and digital texture
analysis. The.se methods focused on determining the perimeter and
spatial characteristics of oyster reefs. Results of this study will
support future efforts to update the entire state database.
IS COPPER REQUIRED FOR EASTERN OYSTER SET-
TING AND METAMORPHOSIS? William S. Fisher, US. En
vironmental Protection Agency National Health and Environmen-
tal effects Research Laboratory Gulf Ecology Division Gulf
Breeze, FL 32561.
Recent field research with eastern oysters demonstrated higher
defense activities, including hemocyte numbers, locomotion and
bactericidal ability, associated with locations exhibiting relatively
high chemical contamination. Copper and zinc, found in high con-
centrations in tissues of oysters collected from these sites, are
known to accumulate almost exclusively in amebocytes. These
data have led to a re-evaluation of potential roles for copper and
zinc in oyster physiology. A role for copper in setting and meta-
morphosis of oysters was previously proposed by Herbert F.
Prytherch (1934), who found that larval oysters would not set or
metamorphose without 0.05 to 0.6 mg L- 1 copper for at least short
durations in the surrounding water. High concentrations were not
toxic for these short durations, and setting was stimulated within
minutes of copper addition. Salinity altered the amount of time
required for larvae to fix to the substrate but was not ultimately
critical to setting. Consequently, oyster setting near river mouths
may be due to incoming copper rather than the variable salinity to
which it is sometimes attributed. If true, our understanding of
oyster distributions and larval setting success would be greatly
altered. Yet. by all appearances, these observations have never
been validated.
COMPARISON OF PACIFIC OYSTER (CRASSOSTREA GI-
GAS) REARING RESULTS (SURVIVAL. GROWTH. QUAL-
ITY) IN FRENCH FARMING AREAS, AFTER A 10-YEARS
MONITORING (1993-2002) BY THE IFREMER/REMORA
NETWORK. Pierre-Gildas Fleury*. Erwan Le Ber. Serge
Claude, Florence Cornette, Florence d'Aniico. Patrice Guil-
pain. Hubert Palvadeau. Stephane Robert, Patrick Le Gall,
Michel Ropert. Charlotte Simonne. Catherine Vercelli.
IFREMER. F-56470 La Trinite-sur-mer. France.
Since 1993 the network IFREMER /REMORA has carried out
annual standard monitorings of survival, growth and quality crite-
ria of the Pacific oyster ^Crassostrea gigas) among the main
French farming areas. The network provides data series for each
site, mean values (references) and allows multifactorial analysis of
oyster rearing results. It must be pointed out that no general cor-
relation was found between growth and mortality. A large range of
results was exhibited both between years and between sites. How-
ever, unusual mortalities, annual variations of growth, or increas-
ing infestation by the worm Polydora could be focused and quan-
tified. Moreover, local trends may be of interest for collective
oyster management. At last. REMORA data may support various
types of studies, such on oyster quality, biological indicators for
coastal waters or explanatory models of the oyster-farming eco-
systems.
EVALUATION OF RARITAN AND SANDY HOOK BAY
HARD CLAM, MERCENARIA MERCENARIA. STOCKS
FOR FISHERY MANAGEMENT. George E. Flimlin, Jr.*,
Michael Celestino, John N. Kraeuter, Robert J. Macaluso,
Michael Kennish. Rutgers Cooperative extension 1623 Whites-
ville Rd. Toms River. NJ 08755.
The hard clam fishery in the Raritan and Sandy Hook Bays
took a twenty year hiatus started by a hepatitis outbreak in the
early 1960's. The use of a clam relay and depuration allowed
clammers to re-enter the fishery in 1983. Since then the fishery has
grown steadily to about 200 full and part-time participants sup-
plying clams to two depuration plants with others relaying their
catch to approved beds in another county for purging.
A stock assessment was done the State in 1 983 with no further
evaluation until 2000 when the Bureau of Shellfisheries covered
the same area again. Simultaneously, studies were done examining
the age and growth of the shellfish as well as a natural mortality
study. Armed with this information, the industry and the state can
better work together to manage the harvest pressure and the par-
ticipation in the area. Analysis of the data indicates that the stocks
are at higher levels than when harvest restarted in 1983, possibly
allowing for further exploitation.
National Shellt'ishenes Association. New Orleans, Louisiana
Abstracts. 2003 Annual Meeting. April 13-17. 2003 331
POTENTIAL PATHOGENS ASSOCIATED WITH ABNOR-
MAL MORTALITIES. Celine Garcia,* Isabelle Arzul. Francl<
Bcrthe. Bruno Chollet, .Jean-Pierre Joly, Nolwenn Kerdudou,
Laurence Miossec. Maeva Robert and Jean-Louis Nicolas. Ge-
netic-Pathology and Aquaculture Shellfish Research Laboratory.
IFREMER 17300 La Tremblade. France.
In France abnormal mortalities of mollusks affect many species
of bivalves. They occur mainly in summer and concern all the
French coastline. For Crassostrea gigas. they affect all life"s
stages but more particularly spat. A pathology monitoring net-
work. REPAMO. was created at the beginning of the nineties in
France in order to answer European requirements as regards mol-
lusk pathology. REPAMO observes whether there is abnormal
mortality and keeps track of the health situation of mollusk stocks
including the presence of pathogens notifiable to the European
L'nion and OIE.
When mortalities occur, the network REPAMO. samples the
populations and performs different types of analysis (histology.
bacteriology, viral detection) in order to detect potential patho-
gens. In France, different agents have been sometimes associated
with abnormal mortalities of bivalves such as herpes-virus in Cnis-
sostreci gigas. Bacterial agents can be also involved. Indeed
hemolymph of moribund oysters from open sea and from hatchery
are often invaded by one Vibrio species belonging to V. spleiuUdus
group or V. aestiiarianus. These observations suggest that Vilirio
could induce or aggravate mortality in oysters weakened by envi-
ronmental or physiological (maturation) factors.
SEASONAL VARIATION IN THE PHYSIOLOGICAL STA-
TUS OF THREE SPECIES OF MUSSELS IN THE ALLE-
GHENY RIVER, PA. Catherine M. Gatenby*, The Academy of
Natural Sciences 1900 Benjamin Franklin Parkway Philadelphia.
PA 19103; Danielle A. Kreeger, Deborah Raksany, and Rich-
ard J. Neves.
A necessary precursor to identifying suitable feeding regimes
for maintaining endangered freshwater inussels in captivity is de-
fining their nutritional requirements. Similarly, a better under-
standing of their physiological status and use of food resources is
needed to assess their role in natural systems and develop man-
agement plans that protect existing populations from further de-
cline. We quantified the seasonal and interspecific variation in
condition index and tissue biochemistry of representative unionid
families from a large bed in the Allegheny River. Condition
[jeaked in July and was similar between November and May for all
species. Protein content peaked in November and May for EUiptio
(Ulatata and Lasiiiigoini costaia (>40'7r). but did not differ season-
ally in Actinoiiaias ligaineiitinu (>307r). Lipid content was high in
November and May for A. ligamentina and E. dilatata ( >29'7c ). but
peaked in July in L. costata. (23%). Carbohydrate content was
similar among species and times. The overall physiological status
and specific demands for protein and lipid varied considerably
among seasons and species. Hence, the formulation of diets for
maintaining captive mussels should target these changing de-
mands. As well, efforts to assess the ecological importance of
mussels should anticipate variation in physiological rate functions.
CHARACTERIZATION OF VIBRIO ISOLATED FROM PA-
CIFIC OYSTERS* SPAT SUFFERING FROM SUMMER
MORTALITY OUTBREAKS Melanie Gay*, Laboratoire de
Genetique et Pathologic IFREMER 1 7390 La Tremblade France.
Guenaelle Lancelot, Bruno Chollet, Tristan Renault, Nathalie
Cochennec. Franck Berthe, Christophe Lambert, Gwenaelle
Choquet, Christine Paillard, Manolo Gouy, Frederique Le
Roux, Philippe Goulletquer.
The pathogens related to summer mortality outbreaks are a
heipes virus and two bacterial strains one belonging to Vibrio
splemlidiis biovar II and the other to Vibrio splendidus spp. How-
ever, the feature pathogen/opportunist of these strains is still un-
known. Several strains belonging to the genus Vibrio have been
identified as pathogen for different mollusk species.
In the context of the French program Morest. experiments of
cohabitation have been used to demonstrate the potential presence
of a transmissible infectious agent in batches of oysters suffering
from summer mortality outbreaks. More than one hundred Vibrio
strains have been isolated from these experiments. These strains
have been phenotypically and genotypically characterized. Their
virulence has been evaluated by infection trials.
Two Vibrio lentus strains have been selected. The mortality rate
induced by them injected together is always higher than the mor-
tality rate induced by each strain injected individually. A histo-
logical examination of injected animals showed damaged
hemocytes and muscle. However, bacteria have only been ob-
served in the tissue surrounding the muscle and in the kidney. We
have shown that physiological and genetic factors had an effect on
the sensitivity of Crassostrea gigas to the experimental model of
bacterial infections.
RESTORATION OF BAY SCALLOPS IN HIGHLY MODI-
FIED AND RELATIVELY PRISTINE HABITATS ON THE
WEST COAST OF FLORIDA, USA. Stephen P. Geiger* and
William S. Arnold. Florida Fish and Wildlife Conservation Com-
mission Manne Research Institute 100 8th Avenue S.E. St. Peters-
burg. FL 33701 USA.
The density of scallops in many populations within Florida has
declined greatly while other populations have remained healthy
enough to allow recreational harvest. We have been attempting to
restore four of the populations that experienced declines. Two of
these populations exist in coastal areas with expansive seagrass
meadows and low i?iipact from de\elopment. Two populations
exist in embayments which have been modified by anthropogenic
impacts such as hardened shorelines, filled wetlands, channeliza-
tion, and construction of causeways. In one coastal population.
332 Abstmcts. 2003 Annual Meeting. April 13-17, 2003
National Shellfisheries Association. New Orleans. Louisiana
adult density has returned to har\estable levels. Good management
practices, natural variability, and restoration efforts may have all
played a role. The density in the second coastal community has
also increased but not to harvestable levels. Neither population in
embayments has recovered despite restoration efforts. Evidence
from surveys of adult scallops and recruitment of spat in these four
populations as well as three additional populations where no res-
toration efforts occurred suggest that habitat alteration may am-
plify negative variations in the population. One example is the rate
of recovery from declines related to harmful algal blooms. Con-
tinued de\elopment in northwest Florida may exacerbate the popu-
lation declines, especially if those regions serve as a source for
recruits in other areas.
CIS to examine the effects of culture density and location on
seston depletion in Tracadie Bay, an important site in the PEI
mussel industry. Models have been constructed at several levels
including box models of the estuary, and fully coupled physical-
biological models set up on a detailed hydrodynamic grid. In the
latter case, maps of seston depletion and biodeposition are gener-
ated as a function of culture density and distribution. Model results
are integrated as data layers in the GIS, and calculations are made
w ithin grid cells using spatially explicit conditions to predict mus-
sel growth and bioenergetics. A comprehensive field program in-
cluding moorings with current meters, particle sensors, sediment
traps, and surveys with a towed vehicle was used to provide
boundary conditions as well as groundtruthing of model results.
FLOW CYTOMETRY AS A TOOL TO QUANTIFY OYS-
TER PHAGOCYTOSIS. RESPIRATORY BURST AND AP-
OPTOSIS. Michael Goedken*. and Sylvain De Guise. Depart-
ment of Pathobiology and Veterinary Science. University of Con-
necticut. 61 N Eagleville Road. U-89. Storrs. CT 06269.
The parasites Perkiiisus iiuiriiiiis and Haplosporidium nelsoiii
have generated losses in the hundreds of millions of dollars. The
relationship between parasites and oyster defense mechanisms is
unclear. A better understanding of the iinmunopathologic associa-
tion may reduce these economic losses. Defense mechanisms of
the Eastern Oyster {CrassDstii'a virginica) were quantified at the
single cell le\el utilizing flow cytometry. Phagocytosis was mea-
sured using fluorescent beads. Respiratory burst activity was quan-
tified as the increase in dichlorofluorescein-associated fluores-
cence upon stimulation. Apoptosis was evaluated with TUNEL
assay. Three sub-populations of heniocytes (granulocytes. h>ali-
nocytes and intermediate cells) were identified with unique func-
tional characteristics. Granulocytes were most acti\e at phagocy-
tosis and peroxide production while hyalinocytes were relatively
inactive. TUNEL assay application allowed quantification of
hemocyte apoptosis. which was more frequent in dividing cells.
Flow cytometry can rapidly, accurately and directly quantify the
morphology and function of a large number of individual cells, and
will lead to a better understanding of the bivalve immune system
and susceptibility to disease.
INTEGRATION OF MODELING AND GIS IN STUDIES OF
CARRYING CAPACITY FOR BIVALVE AQUACULTURE
Jon Grant. Marie .Archambault *, Cedric Bacher. and Peter
Cranford. Dpt Oceanograph> Dalhousie University Halifax. NS
B3H4J1 Canada.
Estimation of carrying capacity for bivalve culture is important
in predicting the effect of the environment on culture yield, as well
as the effect of culture on the environment. Areas of Prince Ed-
ward Island (Canadian Maritimes) appear saturated with respect to
mussel farms, and there is a requirement for estimation of culture
density relative to sustainability for growth rates and ecosystem
health. We have combined field studies, biophysical modeling, and
MAPPING AND CHARACTERIZING EASTERN OYSTER
iCRASSOSTREA VIRGl.MCA) REEFS USING UNDERWA-
TER VIDEOGRAPHY AND QUADRAT SAMPLING Jenni-
fer Greene*. Ray Grizzle and Jamie Adams. Jackson Estuarine
Laboratory University of New Hampshire iS.'i Adams Point Rd.
Durham. NH 03824.
This project attempts to develop an economical technique to
map oyster ( Cra.v.vo.sfrra virginica) reef boundaries as well as char-
acterize the general health of oyster populations using videography
and quadrant sampling. In New Hampshire, oyster monitoring by
resource managers has been impeded by lack of an effective meth-
odology for determining distribution and abundance. Videography
was conducted in Great Bay, NH by systematically imaging mul-
tiple sampling cells in a grid covering two study reefs. In each cell.
a 5-10 s digital video image was recorded (0.25 m2 area) with
location determined by DGPS. A representative still image was
selected for each cell and combined into a photomontage overiaid
onto a geo-referenced base map using ArcView GIS. Quadrat
samples (0.25 m2) were collected from 8-10 of the imaged areas
on each reef and all live oysters were counted, measured and
returned to the reef. Initial results suggest that systematic videog-
raph\ can accurately delimit reef boundaries, yield quantitative
data on shell densities, and provide information on reef characteristics
and structure. Additional reefs will be sampled in 2003 using a com-
bination of continuous video transects with peri(xiic camera drops in
an attempt to pro\ ide finer scale determination of reef boundaries.
THE EFFECTS OF BACKGROUND CONCENTRATIONS
OF THE BROWN TIDE ALGA AUREOCOCCUS ANOPH-
AGEFFERENS ON GROWTH AND FEEDING IN THE BI-
VALVE MERCENARIA MERCENARIA. Dianne I. Green-
field*. Darcy J. Lonsdale. Robert M. Cerrato, and Glenn R.
Lopez. Marine Sciences Research Center. Stony Brook University.
Stony Brook NY 11794-5000.
This study examined the extent to which background levels,
defined as concentrations too low for toxicity to inhibit feeding, of
Aureococciis anopliagefferens (brown tide) influenced the growth
National Shellfisheries Associalioii. New Orleans. Louisiana
Abstracts. 2003 Annual Meeting. April 13-17. 2003 333
and feeding physiology of hard clanis. Mcnenaria iiicireiuiria. in
the laboratory compared to other phytoplankton common to Long
Island. NY. waters. Juvenile clams were fed either unialgal cul-
tures or diets mixed with background levels of brown tide. Ab-
sorption efficiency (AE) was determined using the 14C:51Cr dual-
tracer method and growth was determined by biomass change.
Results showed that unialgal diets resulting in the highest AE.
specifically Isocliiysis galhana and Thalassiosira pseudonana. re-
sulted in rapid M. mercenaria growth. A unialgal diet of Nitzschiu
closteriiim resulted in a comparatively low AE and loss in clam
biomass. Diets mixed with brown tide resulted in a significantly
lower AE than the corresponding unialgal diet for all phytoplank-
ton species except N. closteriiim. Additionally, mixed diets re-
sulted in slightly less clam growth than unialgal diets. This sug-
gests that when brown tide occurs in the field at background levels,
clams may suffer subtle, chronic effects. Moreover, the responses
of M. mercenaria to each diet have implications for understanding
how phytoplankton community composition influences bixalve
growth in the field.
A SIMULATION MODEL OF THE GROWTH OF HARD
CLAMS {MERCENARIA MERCENARIA), IV. EFFECTS OF
CLIMATE CHANGE. Raymond E. Grizzle*, Eileen E. Hof-
mann. .|ohn M. Klincli. Eric N. Powell. John N. Kraeuter, V.
Monica Bricelj. and Stuart C. Buekner. Jackson Estuarine Labo-
ratory University of New Hampshire 83 Adams Point Rd. Durham.
NH 03824.
A phvsiologically-based model that simulates indi\idual
growth of the hard clam. Mercenaria mercenaria. in response to
changes in environmental conditions has been developed. We are
applying this base model to esaluate the effects of possible climate
change scenarios. Thus far. our climate change modeling has fo-
cused on water temperature and the timing of spring and fall phy-
toplankton blooms because these are major factors that control
growth of hard clams. Actual water temperature data sets from
Great South Bay. NY as well as sites in Chesapeake Bay and North
Inlet. SC were used to simulate the long-term warming trend pre-
dicted by all major climate change inodels. Each data set was used
in combination with different spring and fall phytoplankton bloom
scenarios. When bloom times were held constant, long-term warm-
ing resulted in increased growth and the predicted rates matched
published values for clams from each area from which water tem-
perature data were used. The timing of blooms had a dramatic
effect on growth, suggesting that year-to-year variations may be
more important than overall temperature trends as climate change
ensues. Details on the modeling results will be presented and dis-
cussed.
STATUS OF BLUE CRAB POPULATIONS IN LOUSIANA
BASED ON FISHERY INDEPENDENT DATA COLLEC-
TIONS (1967-2002) WITH OBSERVATIONS ON RELA-
TIVE ABUNDANCE IN OTHER GULF STATES. Vincent
Guillory*. Louisiana Department of Wildlife and Fisheries. P.O.
Box 1 89. Bourg, Louisiana 70343; Harriet Perry, Center for Fish-
eries Research and Development. Gulf Coast Research Laboratory,
College of Marine Sciences, the LIniversity of Southern Missis-
sippi. P.O. Box 7000. Ocean Springs. Mississippi 39566-7000; and
tlie Blue Crab Technical Taskforce, Gulf States Marine Fish-
eries Commission.
The 33-year ( 1967-2000) database from the Louisiana Depart-
ment of Wildlife and Fisheries bottomfish/shrimp monitoring pro-
gram is the most extensive and continuous fishery independent
blue crab data set in the Gulf of Mexico. Long term and recent
trends in recruit (<4() mm CW), juvenile (40-99 mm CW). sub-
legal (100-124 mm CW). and legal 0123 mm CW) blue crabs
were examined, as well as overall catch per unit of effort sub-legal
crabs did not significantly change over the long term, although
overall CPLIE for these size groups showed a significant increase
from 1967-1989 with a significant decrease in recent years ( 1990-
2002). Catch per unit of effort of recruits significantly increased
over the long term with a downward trend noted in cuirent data.
Trends in relative abundance are discussed in relation to habitat
changes in northern Gulf of Mexico estuaries and to biological
factors such as predation.
BREEDING AND EVALUATION OF EASTERN OYSTER
STRAINS SELECTED FOR MSX, DERMO AND JOD RE-
SISTANCE. Ximing Guo*, Susan Ford, and Gregory De-
Bros.se. Haskin Shellfish Research Laboratory. Rutgers Uni\er-
sity. 6939 Miller Avenue. Port Norris. NJ 08349; Roxanna
Smolowitz. Marine Biological Laboratory. 7 MBL Street. Woods
Hole. MA 02543; Inka Sunila. Bureau of Aquaculture and Labo-
ratory. Milford. CT ()6460.
Rutgers University has been breeding oysters for disease-
resistance since the early 1960s and produced strains showing
strong resistance to MSX and some resistance to Dermo. Breeding
at the F.M. Flower Oyster Company has produced a strain (FMF)
showing superior growth and JOD-resistance. We undertook a
project to evaluate the Rutgers NEH strain, the FMF strain and
their hybrids (HYB) along with a global susceptible control (ME)
and local controls that were normally cultured at each of the four
deplovment sites. Oysters were produced in June 2000. deployed
in Jul\ 2000 and evaluated for 27 months. Dermo exposure was
heavy at most sites, while MSX and JOD infections were low or
absent. At Cape Shore (NJ) where infection was the heaviest. NEH
and HYB had the lowest cumulative mortality. 43.5% and 43.6%
respectively, compared with 82.3% for FMF. 99.4% for ME and
81.1% for the local control (Delaware Bay wild). In growth. HYB
was the same as FMF and faster than NEH. while ME and the local
334 Absmicls, 2003 Annual Meeting, April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
controls grew the slowest. The hybrid offered the highest yield by
surviving as well as the NEH strain and growing as fast as the FMF
strain.
MARKETING IMPLICATIONS OF CONSUMER ATTI-
TUDES TOWARD OYSTERS Terrill R. Hanson*, Lisa O.
House. Benedict C. Posadas. Dept. of Agricultural Economics
P.O. Box 5187 Mississippi State. MS 39762.
As US consumption of oysters declined during the 1990's an
understanding of why consumers purchase and consume oysters is
important to marketing oysters effectively and reversing this trend.
In 2000-2001. a survey was administered to U.S. residents on the
topic of seafood consumption. Results and findings of this survey
are useful for sellers to use in targeting consumers likely to in-
crease their oyster consumption and for processors to use in de-
signing programs likely to improve food safety considerations.
Reasons for eating oysters included enjoyment of the flavor
{SOVc of consumers) and addition of variety to their diet (37%).
Oyster consumer's identified price (38%), product safety (29%),
and lack of availability of fresh product (20%) as the main reasons
for not consuming oysters more often. Forty-three percent of oys-
ter consumers and 54% of those concerned about product safety
indicated their consumption of oysters would increase if depura-
tion methods were used to 'guarantee' oyster safety. Sixty-one
percent stated a willingness to pay of $0.34/oyster over the raw-
oyster price for a 'guaranteed' safe product. This option may be
profitable if depuration costs do not exceed the increases in con-
sumer's willing to pay.
HOW MANY LARVAE STAY AT HOME? MEASURING
PATTERNS OF LOCAL OYSTER RECRUITMENT USING
MOLECULAR MARKERS. Mattliew Hare*. D. Merritt and
K. Paynter. Biology Department University of Maryland College
Park. MD 20742; S.K. Allen. ,Ir.. E.M. Burreson. M.D. Camara.
Ryan Carnegie. M. Lucixenbach and K.S. Reece.
Recruitment enhancement is one of the primary objectives of
oyster restoration in the Chesapeake Bay. Given the tremendous
spatial and temporal variability in recruitment patterns that have
been documented in Chesapeake Bay oysters, correlations between
broodstock plantings and spat set provide only a partial and po-
tentially misleading index of recruitment strength from enhance-
ment efforts. We are using the unique genetic signature of disease-
tolerant selected strains to measure the geographic scale of recruit-
ment provided by restored reefs. With the highly variable DNA
polymorphisms that we have developed, every newly settled oyster
is "tagged" with a genotype that links it to its parents and its source
population. In cooperation with several agencies and organizations
we planted CROSBreed selected strains in the Great Wicomico
River in 'Virginia and the Little Choptank River in Maryland dur-
ing the summer of 2002. Genetic markers provide better than 95%
accuracy for assigning indi\ idual oysters to their source, selected
strain or natural broodstock. CROSBreed oysters planted in 'Vir-
ginia averaged 6 cm. large enough to potentially breed during
2002. We will present analyses of 2002 Virginia spat indicating the
proportion of recruitment derived from planted oysters versus
natural broodstock.
SUITABILITY OF OYSTER CLUSTERS AS HABITAT FOR
REEF-RESIDENT FISHES AND DECAPOD CRUSTA-
CEANS IN THE CALOOSAHATCHEE ESTUARY Leslie H.
Haynes.* Arielle Poulos. Lacey K. Smith. Aswani K. Volety and
S. Gregory Tolley. 1227 S.W. 25th St. Cape Coral. FL 33914.
The habitat suitability of oyster clusters for reef-resident fishes
and decapod crustaceans was examined in the Caloosahatchee Es-
tuary. Lift nets representing three habitat treatments were deployed
during three seasonally wet and three seasonally dry months on an
oyster reef located in the lower estuary. Nets contained either no
oyster shell (control), dead, articulated clusters, or live oyster clus-
ters. Based on the results of Hester-Dendy sampling conducted at
the same site, nets were deployed for a period of 1 month to ensure
full recruitment. Analysis of variance indicated that articulated
oyster shell (dead or living clusters) exhibited greater species rich-
ness, biomass. and dominance than did the controls. Furthermore,
organism abundance was higher in living oyster clusters compared
to dead, articulated clusters; both treatments with oyster shell ex-
hibited significantly greater abundances than the control. In addi-
tion, biomass of all treatments was significantly greater during the
dry season than during the wet season. The results of this study
suggest that the habitat value of oyster clusters to reef-resident
fishes and decapod crustaceans lies primarily in the three-
dimensional structure created; dead, articulated oyster shell exhib-
ited levels of as.sociated biomass and species richness similar to
that of clusters containing living oysters.
ALGAL FOOD QUANTITY AND QUALITY AFFECT IM-
MUNE FUNCTION IN OYSTERS STRESSED BY HIGH
TEMPERATURE. Helene Hegaret*. Gary Wilvfors. NCAA
Fisheries. Milford. CT. USA. Philippe Soudant. LEMAR, lUEM-
UBO, Plouzane. France, and Jean-Fran^-ois Saniain. Laboratoire
de Physiologic des Invertebres. IFREMER. Brest. France.
Oyster seed from a hatchery must resist environmental stresses
when planted in the sea. We conducted an experiment to analyze
the influence of nutrition on oyster. Crassostrea virigiiiica's. im-
mune capability. Cultured microalgal diets were varied factorially
in quantity ( 10 and 50% dw/dw microalgae/oyster soft tissue per
day) and quality (Skeletonema. Tetraselmis, and a 50/50 mix),
with unfed controls. Oysters were fed five weeks at 20°C and then
temperature-stressed for one week at 28°C. Before and after heat
stress, we used flow-cytometry and multivariate statistics to ana-
lyze the following hemocyte functions: viability, aggregation.
National Shell! ishcries Association. New Orleans, Louisiana
Ahsiracly 2003 Annual Meeting, Apiil 1.^-17, 200.^ 335
phagocytosis, and respiratory burst. Discriminant Analysis showed
significant effects of food quantity and quality on hemocyte func-
tion. Principal Components Analysis revealed the main effects of
heat stress to be increased respiratory burst and decreased phago-
cytosis; this decoupling of the two steps in pathogen defense was
more severe in starved or poorly-fed oysters.
ASSESSING FEASIBILITY OF STOCK ENHANCEMENT
FOR CHESAPEAKE BLUE CRABS (CALLINECTES SAPI-
DUS). Anson H. Hines*. Jana L.D. Davis, Alicia Young-
Williams. Smithsonian Environmental Research Center, Edgcwa-
ter. Maryland 2 1 037, USA; Yonathan Zohar. Oded Zmora, Uni-
versity of Maryland Biotechnology histitute, Baltimore. Maryland
21202, USA.
In overexploited. recruitment-limited fisheries, enhancement
with hatchery-produced juveniles, coupled with traditional man-
agement techniques and habitat restoration, may be required for
effective stock management. Enhancement, used most frequently
for finfish stocks, has rarely been attempted with crustaceans. The
Chesapeake blue crab stock exhibits key characteristics as an ap-
propriate candidate for enhancement: SIVr decline in biomass over
the past decade, recruitment limitation, and extensive habitat with
reduced juvenile mortality and densities below carrying capacity.
The goals of this work were ( I ) to determine the enhancement
potential of blue crab subpopulations by releasing hatchery-reared
crabs (25.000 juveniles <23mmCW) on spatial scales of 10-15 ha,
and (2) to identify factors intluencing survivorship of hatchery
crabs in the wild. In four separate cohorts (3,700-9,500 juveniles)
that were sampled over S-16 weeks, released tagged hatchery crabs
successfully enhanced local subpopulations, growing rapidly and
surviving to contribute to the spawning stock. Hatchery and wild
crabs were similar in most respects, but differed initially in burial
rate, carapace morphology, and susceptibility to predation. How-
ever, differences disappeared within days in the field, suggesting
ways to improve success of future released crabs. These initial
results contribute to determining whether enhancement on a larger
scale is possible.
A SIMULATION MODEL OF THE POPULATION
GROWTH OF HARD CLAMS (MERCENARIA MERCE-
NARIA). I. MODEL DEVELOPMENT AND IMPLEMENTA-
TION. Eileen E. Hofmann*, ,|ohn M. Klinck, Eric N. Powell,
John Kraeuter, Monica Bricelj. Ray Grizzle, Stuart Buckner.
CCPO, Crittenton Hall Old Dominion University Norfolk, VA
23529,
A physiologically-based model that simulates the population
growth of hard clams, Mercenaria incrcenaria. in response to tem-
perature, salinity and food supply has been developed and applied
in Great South Bay. The model structure model allows indepen-
dent simulation of shell and tissue growth, which permits calcu-
lation of animal conditi()n as a diagnostic. Also, length and age are
independently tracked, thereby allowing specification of size-
frequency and age-frequency distributions to describe population
structure, and more importantly to define age dependent, as well as
size-dependent, processes. The model structure includes a genetic
component that permits simulation of a range of genotypes, which
are combined into cohorts to construct a population. The simulated
hard clam growth obtained using environmental conditions char-
acteristic of Great South Bay match weight and length values that
are ob.served for populations in this region. The extension of the
simulations of individual clams to cohort and populations scales
shows the imponance of assimilation rate and the apportionment
between reproductive and somatic growth in determining inter-
cohort variability and overall long-term population characteristics.
The results of these simulations as well as those that examine
model sensitivity to assumptions made for key model processes
and parameteri/ations will be presented.
COMPARISON ALONG THE NEW ENGLAND COAST OF
EPIDEMIC SHELL DISEASE IN THE AMERICAN LOB-
STER, HOMARUS AMERICANVS. Andrea C. Hsu*, Boston
University Marine Program Marine Biological Laboratory Woods
Hole. MA 02543; Roxanna M. Smolowitz, Marine Resources
Center Marine Biological Laboratory Woods Hole. MA 02543:
Andrei Y. Chistoserdov. Department of Biology University of
Louisiana at Lafayette Lafayette. LA 70504; and Hemant M.
Chikarmane. Marine Resources Center Marine Biological Labo-
ratory Woods Hole, MA 02543.
During the last six years, shell disease has been found at high
levels in wild lobsters along the New England coast. This study
utilizes a combination of scanning electron microscopy (SEM).
denaturing gradient gel electrophoresis (DGGE). and histological
analysis to describe and define bacterial cells on the infected cara-
pace of wild-caught lobsters. Diseased lobsters used in this study
were collected starting from Eastern Long Island Sound. New
York, up toward Cape Cod Bay. Massachusetts, with control ani-
mals from Maine.
SEM analysis revealed and statistical tests verified five sepa-
rate morphological types of bacteria present in shell lesions. Halo-
like holes surrounding all bacterial types suggest boring as their
causative mechanism for degrading the lobster carapace. Prelimi-
nary DGGE data indicated up to fourteen independent phylotypes
of bacteria were present in lobster lesions. At least two of them
were found in all diseased lobsters used in this studv. Histopath-
ologically. the carapace matrix was usually absent or lattice-like
cuticular remnants were found attached to underlying less de-
graded cuticle. Bacteria were the predominate organisms found at
the leading edge of erosions. Combined results from SEM, DGGE,
and histological analyses present evidence that an assemblage of
bacteria may be the cause of New England epidemic shell disease.
336 Absinicls. 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
TENNESSEE'S PEARL CULTURE INDUSTRY. Don Hubbs.
Mussel Program Coordinator. Tennessee Wildlife Resources
Agency. P.O.B. 70. Camden. TN 38320.
The Tennessee Wildlife Resources Agency (TWRA) regulates
freshwater pearl culture in Tennessee. Administrative rules, proc-
lamations and contracts are employed to regulate the industry, and
protect and manage its utilization of the native mussel resource.
Although experiments in pearl culturing began in the 1960's, gov-
erning regulations were not developed until 1988. A panel com-
posed of TWRA fisheries personnel and industry representatives
drafted the first regulations. The washboard, Megalonaias iien-o.sa
(Rafinesque, 1820). is the primary freshwater mussel species used
by the pearl culture industry. Because washboards command the
highest price in the commercial shell market, and legal-sized in-
dividuals can be scarce, industry experts convinced the TWRA to
permit the use of sub-legal sized washboards for economic rea-
sons. Contracts, seasons, and quotas were established to control the
harvest of wild washboard mussels for the pearl culture industry.
Permission for use of sub-legal sized washboards for pearl culture
proved unpopular with many commercial shell harvesters and
wholesale shell dealers.
EVIDENCE OF A COLD SHOCK RESPONSE IN VIBRIO
VULNIFICUS, A HUMAN PATHOGEN TRANSMITTED
VIA RAW EASTERN OYSTERS, CRASSOSTREA VIR-
GINICA, FROM THE GULF OF MEXICO. Kristi L. Huels*,
203 Swingle Hall Auburn University. Auburn. AL 36049-5419.
Yolanda J. Brady, Mary A. Delaney, Joel A. Bader.
This study examined the response of Vibrio vulnificus to incu-
bation at 13 and 4o C. It focused on changes in protein expression
using one and two dimensional gel electrophoresis. Although dif-
ferent proteins were expressed following cooler temperature ex-
posure no major cold shock protein was identified. As hypoth-
esized, longer incubation times at l3o C resulted in increased
variations. Proteins expressed at the cooler temperature were only
transiently expressed, classical of stress responses. These prelimi-
nary results suggest there is a cold shock response active in V.
vulnificus that requires further investigation in order to properly
evaluate and alter the general management practices for collection
and processing of the Eastern Oyster, Crassostrea viri^inica. from
the Gulf of Mexico.
PREVALENCE AND ABUNDANCE OF PERKINSUS MARI-
NUS AND PERKINSUS CHESAPEAhl/ANDREWSl IN
CHESAPEAKE BAY OYSTER BEDS Karen L. Hudson*,
Kimberly S. Reece, Christopher F. Dungan, and Rosalee M.
Hamilton. Virginia Institute of Marine .Science. Gloucester Point.
VA 23062.
Three described species oi Perkinsus have been reported in the
Chesapeake Bay region of the United States. Perkinsus inarinus is
a well known pathogen of the eastern oyster. Crassostrea vir-
ginica. Perkinsus chesapeaki and Perkinsus undrewsi are more
recently described species from the soft-shell clam. Mya arenaria
and the Baltic clam. Maconia Ixdthicu. respectively. Recent mo-
lecular studies, however, suggest that these two species are syn-
onymous (Dungan et al. 2002). In 2001. Coss et al. reported P.
aruirewsi infections in oysters. The routine test used to diagnose P.
niariinis infections from oysters. Ray's fluid thioglycollate me-
dium (RFTM) however, is not species-specific. The objective of
this study was to survey oyster beds in the Chesapeake Buy area
located adjacent to a variety of clam species in order to assess
prevalence and abundance of Perkinsus species in oyster and clam
hosts. Prevalence was assessed by standard PCR using two spe-
cies-specific PCR primers: one P. nnn-inns-^pecifii: and the other
P. chesapeaki I andrewsi- specific. Abundance was accomplished
using quantitative PCR using the same species-specific primers.
Two species-specitlc in situ hybridization probes were developed
and tested. Results of the assay development and en\ironmental
screening will be presented.
A FISHERY-ORIENTED MODEL OF MARYLAND OYS-
TER POPULATIONS Stephen J. Jordan* and Jessica Vani-
sko. USEPA. Gulf Ecology Division 1 Sabine Island Drive Gulf
Breeze, PL 32561.
We used time-series data to calibrate a model of oyster popu-
lation dynamics for Maryland's Chesapeake Bay. Model param-
eters were fishing mortality, natural mortality, recruitment, and
carrying capacity. We calibrated for the Maryland bay as a whole
and separately for 3 salinity zones. Simulations indicated that a
long-term declining trend in the Maryland bay-wide stock of har-
vestable oysters could be reversed by controlling fishing mortality
and enhancing recruitment. For example, an exponential increase
in stock size was predicted by simulating a 40% reduction in
fishing mortality; initial losses to the fishery were more than com-
pensated by large gains after a few years. In the low salinity zone,
where the harvestable stock has been maintained largely by relay-
ing seed oysters, recruitment rates are too low to support a sig-
nificant population increase, but stocks in the medium and high
salinity zones appear to have potential for recovery within 10-20
years. The model is sensitive to mortality and recruitment rates,
but not to carrying capacity, which is much larger than current
stock sizes. Measures of uncertainty for model predictions include
(1) confidence limits for mean predicted trends, and (2) percent-
ages of iterative simulations that satisfy specified criteria.
NutiDiial Shellfisheries Assoeialion. New Orleans. Louisiana
Ahslmcts. 2(103 Annual Meeting. April 13-17. 2003 337
DEVELOPMENT OF BIOMARKERS FOR PERKINSUS
MARINUS RESISTANCE IN THE EASTERN OYSTER
(CRASSOSTREA VIRGIMCA). Stephen L. Kaattari and
Christopher Earnhart. Department ol Environmental and
Aquatic Animal Health. Virginia Institute of Marine Science, Col-
lege of William and Mary. Gloucester Ponit. VA 23062.
The development of biomarkers for the determination of Per-
kin.'iiis mariiuis resistance in the eastern oyster would be of great
utility to the oyster industry and would also serve as an important
tool in the study of disease pathogenesis. To achieve such a goal
we have capitalized on the observation of the ability o't P. inaiinus
cells to respond in a specific manner to extracts of susceptible
oyster tissue. Generally co-incubation of P. inaniuis with host
tissue extracts can elicit a variety of effects including altered cel-
lular differentiation, protease expression, growth rates, infectivity.
and parasite lethality. The application of this analysis to stock
assessment and deployment decisions, as well as their use in the
selection of future oyster broodstock could provide a needed com-
petitive edge to the American oyster industry. Further, investiga-
tion in this arena should yield useful models for the analysis of the
developmental process of oyster protozoan parasites.
EVALUATION OF COMMERCIAL POST HARVEST
TREATMENTS FOR CONTROL OF VIBRIO VULNIFICUS
IN OYSTERS. Marilyn B. Kilgen*. Department of Biological
Sciences, Nicholls State University, Thibodaux, LA 70310.
Post harvest treatments of freezing, low dose ionizing irradia-
tion, and hydrostatic high pressure (HHP) were commercially
tested in collaboration with oyster industry members from the gulf
and east coasts. Six vinegar-based oyster marinades were also
developed in collaboration with the NSU Chef John Folse Culinary
Instiiiiie. All reduced Vihric vKliiificits levels from 240,000 MPN/g
to non-detectable levels (<3MPN/g) after 24 hours of marinating at
35F. and one received the highest sensory score from 1,116 tasters
(80%) at the Louisiana Boat Show. Commercial cryogenic (liquid
C02) freezing of half-shell oysters reduced V. viilnificii.s levels
from 460,0(10 MPN/g to 0.74 MPN/g by 6 weeks post freezing.
Commercial ionizing irradiation with Co60 reduced levels of
Vihiid Yubuficus in live shellstock oysters from 46().0()() MPN/g to
<0.3 MPN/g at 1.0 kilogray (KGy). In recent studies, live oysters
were treated with hydrostatic high pressure for the first time. In
commercial applications, 35,000 psi for 3 minutes at 70F was
determined to be most economically feasible and was validated to
reduce V. vulnificus from > 100.000 to <3 MPN/g. It was also
initially discovered in these studies that oyster adductor muscle
protems were denatured at the shell attachment resulting in me-
chanical shucking of the oyster.
THE BLUE CRAB FISHERY OF THE HUDSON RIVER ES-
TUARY. Gregg Kenney*. Andrew Kahnle, Kathy Hattala, and
Steven H. Jury. 21 South Putt Corners Road New Paltz, NY
12561.
Despite its economic and recreational importance, there has
been relatively little systematic inquiry into blue crab {Callinectes
sapidus) abundance, distribution, and habitat utilization in the
Hudson River Estuary. Blue crab abundance is generally consid-
ered to have increased in this system as indicated by expanding
recreational and commercial fisheries. The New York State De-
partment of Environmental Conservation has implemented a pro-
gram to investigate the extent of the commercial fishery and sea-
sonal movements of blue crabs in the estuary. The commercial
fishery catch was monitored during the 2000. 2001 and 2002 crab-
bing seasons and fishery independent sampling was conducted
weekly throughout the 2002 season. Catch per unit effort was
found to fluctuate temporally and spatially in a manner similar to
that found in other temperate estuaries. The relationship between
blue crab abundance and changes in temperature and salinity is
presently being analyzed. This project will provide data that can be
used to monitor changes in relative abundance and distribution of
blue crabs in the Hudson River to fulfill the goal of maintaining a
sustainable fishery.
POLINICES PULCHELLUS: THE JAMES DEAN OF GAS-
TROPODS: LIVING FAST, DYING YOUNG Peter Kingsley-
Smith*. VIMS P.O. Box 1346 Gloucester Point. VA 23062.
The gastropod. Poliniccs puklwllus. is patchily distributed on
subtidal muddy sand in Red Wharf Bay. Wales, LIK. Competent
pediveligers metamorphosed in response to sediment collected
from the adult habitat, such that the adult distribution may be
explained by preferential larval settlement. Polinices pulcheltus
densities were significantly higher in summer than in winter,
which it is proposed arose from mating aggregations. Small indi-
viduals (4-5 mm) were present throughout the year indicating an
extended period of low-level recruitment, which was reflected in
the year-round production of egg collars in the laboratory. Larger
females had higher fecundities than smaller females, however,
smaller females continued to lay egg collars later in the year. Small
females (4-5.9 mm) grew rapidly during the warm, summer
months (April to August), attained sexual maturity at 8-9.9 mm
and began laying egg collars in mid-Septeinber. The relationship
between shell length and statolith diameter was determined for
newly hatched larvae through to fully-grown adults ( 16 mm). Es-
timates of mean shell length at the formation of the first and
second prominent rings supported the conclusion that prominent
statolith rings are formed annually. Polinices putchellus reaches its
maximum si/c in 2-3 years.
338 Abstracis. 2003 Annual Meeting. April 13-17, 2003
National Shellfisheries Association. New Orleans. Louisiana
OBSERVATIONS ON THE UNUSUAL ABUNDANCE OF
TROPICAL CALLINECTES SPECIES IN THE SOUTH AT-
LANTIC BIGHT IN FALL 2002. AND REMARKS ON THE
NON-INDIGENOUS CHARYBDIS HELLERII. David M.
Knott*, Elizabeth L. Wenner. and Susan L. Thornton, South-
eastern Regional Taxonomic Center at the Marine Resources Re-
search Institute, South Carolina Department of Natural Resources.
Charleston. SC 29412.
Tropical species of Callinecles typically appear in the South
Atlantic Bight only occasionally, and then usually only in isolated
occurrences. In fall 2002. however, commercial fishermen near
Charleston reported the capture of mature C. exasperatus and C.
bocourti in abundances great enough to warrant inquiry about their
identity and the legality of selling them. Although quantitative
estimates are not available (landings reports do not include species
composition), we believe that these species were fairly common
and widespread in the vicinity, beginning in mid-October. A single
specimen of C. lan'atus was also collected by SCDNR staff in
mid-November, at about the same time that the last C. bocourti
was seen. The more recent collection of moribund C. exasperatus
on December 10. after water temperatures had dropped below
1 l°C, indicates that these species may be unable to survive typical
winter condition in the area. Possible explanations for this unusual
event will be discussed. Additional remarks will address the inva-
sive portunid Charybdis hellerii in the SAB. and the original report
of its occurrence in the western Atlantic will be updated.
A SIMULATION MODEL OF POPULATION GROWTH OF
HARD CLAMS (MERCENARIA MERCENARIA). II. EF-
FECTS OF FISHING. Kraeuter*, Haskin Shellfish Research
Laboratory Rutgers University 6959 Miller Axenue Port Norris.
NJ 08349; Powell. Hofmann, Klinck. Grizzle, Bricelj and Buck-
ner.
A physiologically-based model that simulates hard clam, Mer-
cenaria mercenaria. growth in response to environmental condi-
tions of temperature, salinity and food supply has been developed.
We are applying this base model to evaluate the effects of various
fishing scenarios on Great South Bay. New York clam popula-
tions. Comparison of fishery-independent samples with landings
suggests the population was heavily over fished in the late 1970
until at least the mid I980's. Base simulations illustrate the effect
of changing salinity and food environments. The spawner/recruit
relationship is based on limited data so the effects of variation in
this parameter have been evaluated. Fishing simulations evaluate
the effects of proportional fishing (all marketable sizes of clams
are removed in proportion to their abundance) at various percent-
ages of removal. In addition, exclusive removal of various per-
centages of commercial size categories; littleneck, topneck. cher-
rystone or chowder is explored. Finally, population recovery rates
are evaluated under scenarios of a total fishing ban or limited
percentage removal. In general, simulations indicate recovery time
is on the order of a decade or more, and fishing proportionally or
on littlenecks at greater than 25 to 35% of adult standing stock will
decrease fishing yields and clam populations.
GENOMIC SIGNATURE TAGS: A NOVEL METHOD FOR
GENOMIC PROFILING WITH APPLICABILITY TO
SHELLFISHERIES RESEARCH. Maureen K. Krause*. Dept.
of Biology. Hofstra University. Hempstead. NY 11549; John J.
Dunn. Daniel van der Leiie, Sean McCorkle. Biology Dept..
Brookhaven National Laboratory. Upton. NY 1 1973.
Genomic Signature Tags (GSTs) are the products of a new
high-throughput, direct sequence-based approach for characteriz-
ing genomes that does not rely on a priori knowledge of the
genome. Our technique produces large numbers of positionally
defined tag sequences that can. in principle, provide limited rep-
resentation of all the DNA molecules in a sample. A GST analysis
of the 4.7 Mb Yersinia pestis EV766 genome validates that the
GST technique provides a route to obtaining numerous sequence
tags that can be used to identify the DNA source. Additionally, our
data show that the presence or absence of particular tags can be
used to characterize intraspecific genetic variability. One exciting
extension of GST analysis, ribosomal GSTs. shows tremendous
potential for analyzing microbial communities, including those
that may be associated with shellfish disease. Overall knowledge
of microbial cotnmunities associated with diseased versus non-
diseased shellfish remains poor due to constraints of culturability
and microscopy. Ribosomal GST profiles have the potential to not
only determine what microbial species are present, but their rela-
tive abundance, as well. The power of our approach is that it is
both qualitative and quantitative, and can directly provide se-
quence information without electrophoretic isolation of amplicons.
SPATIAL AND TEMPORAL VARIATION IN OYSTER FIT-
NESS IN SAN ANTONIO BAY, TEXAS, 1998-2002. D. Kree-
ger*. R. Thomas, H. Hertler. and D. Raksany. Patrick Center for
Environmental Research. Academy of Natural Sciences. 1 900 Ben
Franklin Parkway. Philadelphia. PA 19103.
Adult oysters {Crassostrea virginica) were sampled eleven
times between October 1998 and May 2002 from four locations in
San Antonio Bay to quantify spatial and temporal variability in
body size, condition index, and tissue biochemical composition.
All measures of oyster fitness varied spatially, seasonally and
among years. Seasonal differences were consistent with expected
norms for healthy adults that undergo an annual reproductive cycle
(fall/winter conditioning: winter/spring gametogenesis and spawn-
ing). This pattern was not observed every year at every location,
however, and spatial and inter-annual variability interacted
strongly. Spatial variation was greatest along an axis extending
from the upper to lower bay, and inter-annual differences were
greatest at upper bay locations. Seaward oysters were consistently
Natioiuil Shellfisheries Association. New Orleans. Louisiana
Absimcis. 2003 Annual Meeting. April 13-17. 2003 339
fit. hut ovster fitness in the upper hay varied widels hetvseen drv
and wet years. Follov\ing major floods, upper bay oysters had a
smaller size (or were inorhid) and demonstrated a subdued sea-
sonal conditioning cycle compared to seaward oysters: whereas, in
drier years upper bay oysters were largest and attained the highest
condition of all locations. The stable oyster beds in the lower bay
appear to serve as critical broodstock that provide larvae to re-
plenish upper bay stocks follov\ing major disturbance events.
RECONSTRUCTING THE GROWTH OF HARD CLAMS.
MERCENARIA MERCENARIA, UNDER BROWN TIDE
CONDITIONS. Cathy A. Laetz* and Robert C. Cerrato. 4.^01
Greenwood Ave N Apt # 102 Seattle WA 98103.
Hard clams have been an important resource in Great South
Bay. New York for decades despite severe population declines.
One suspected cause of declines in recent years is brown tides, or
blooms of the phytoplankton Aureococcus anophagefferens which
have been found to cause a slowing or cessation of feeding activity
in various shellfish. Growth in hard clams planted in Great South
Bay for one year during a brown tide bloom v\ as similar to growth
in clams measured in years prior to brown tides. Similarly, ar-
chived shells from the Town of [slip's annual shellfish surveys
showed comparable growth rates between brown tide and pre-
brown tide years. Rapid shell growth was observed in the spring
and fall, whereas no growth occurred when water temperatures fell
below 6"C. Although there was no relationship between brown
tide concentration and clam growth, a strong relationship was ob-
served with temperature, which accounted for65'A of the variation
in shell growth rate. In contrast to other shellfish, brown tide does
not appear to have as great a negative effect on the growth of hard
clams in Great South Bay. possibly due to acclimation, growth
compensation, or population selection over time.
"bad"). Their immunological status was compared after four
months in the three sites. Another comparison was performed after
13. 15. and 17 months but only in one site. Concomitantly, im-
munological parameters of triploTdes and diploide oysters were
followed during summer in Charentes. Significant differences
were measured between good and bad families but were less
marked during the year 2. Triploi'des and diploides presented clear
differences. To discuss the possible genetic transmission of im-
mune parameters, status of 8 divergent families from crossed good
or bad families was studied.
OPTIMIZATION OF SPERM CRYOPRESERVATION FOR
THE PACIFIC OYSTER CRASSOSTREA GIGAS: EVALUA-
TION OF COOLING RATE Paul Lang* and Chris Langdon,
Coastal Oregon Marine E.xperiment Station. Hatfield Marine Sci-
ence Center. Oregon State University. Newport. Oregon. 97365.
Sperm suspension with a concentration of 109^ dimethyl sulf-
oxide was prepared from calcium-free Hanks' balanced salt solu-
tion (-800 mOsmol/kg) and sperm of five Pacific oysters (Cras-
sostrea giges). Plastic straws (2.5-mL) were filled with 2 mL
suspension, placed in a chamber previously cooled to either -30 °C
or -70 °C. and plunged into liquid nitrogen (-196 °C) when inter-
nal straw temperature fell within ~2 C of the chamber temperature
(7 min at -30°C. 4 min at -70"Cl. Straws were thawed in a water
bath (70 "C) for 30 sec. Eggs of females (n = 5) were fertilized
using thawed or fresh (control) sperm at equal sperm-to-egg ratios
(20:1 ). and incubated in lO-mL tubes. Fertilization (the percentage
of eggs to have reached the 4-cell stage) was 22% ± 9% for eggs
fertilized with sperm cooled to -30°C. 51'7r ± SVc for -70 °C. and
57 ± 49c for fresh sperm. Larval development (the percentage of
initially fertilized eggs to have reached D-stage) was 29% ± 10%
for eggs fertilized with sperm cooled to -30°C. 62% + 13% for
-70"C. and 72% ± 5% for fresh sperm.
IMMUNOLOGICAL STATUS OF SELECTED CRASSOS-
TREA GIGAS FAMILIES AND DESCENDANTS, REARED
IN DIFFERENT ENVIRONMENTAL CONDITIONS. Chris-
tophe Lambert*. Laboratoire des sciences de fenvironnement
marin (LEMAR) Institut Universitaire Europeen de la Mer
(lUEM) Universite de Bretagne Occidentale (UBO) Place Coper-
nic. technopole Brest Iroise 29280 Plouzane. FRANCE: PhHippe
Soudant. Gwenaelle Choquet, Christine Paillard. Stephane
Frouel. Lionel Degremont. Maryse Delaporte. Jeanne .Moal.
Pierre Boudry. Patrick Soletchnick. Michel Ropert. F^douard
Bedier, Tristan Renault. Beatrice Gagnieres Arnaud Huvet
and Jean-Francois Saniain.
Defense mechanisms variability in Crassostrea gigas is sus-
pected to result from genetic factors. In the French program MO-
REST, bi-parental families, obtained from a nested half-sib cross-
ing design, were reared four months in three culture sites. Six
families were selected on their survival performance ("good" and
FAMILY-BASED SELECTION IMPROVES YIELDS OF
PACIFIC OYSTERS. CRASSOSTREA GIGAS. Chris Lang-
don*. Sean Matson, John Brake. F'ord Evans. Coastal Oregon
Marine Experiment Station and Dept. Fisheries and Wildlife. Or-
egon State University. Newport. Oregon 97365.
The Molluscan Broodstock Program (MBP) was established in
1 995 to improve yields of Pacific oysters on the West coast, U.S.,
through family-based selection. Parental families (PI ) in three co-
horts of about 60 families each were selected based on superior
live weight and meat yields at harvest. Live weight yields of prog-
eny (Fl) from crossing PI selected families were significantly
greater than those of non-selected control families in four out of
seven trials (ANOVA, p<0.001). resulting in an average gain of
9.5% after one generation of selection. ANOVA indicated a sig-
nificant (P<0.01) genotype by environment interaction effect on
yields for families planted at both inter-tidal and sub-tidal sites.
340 Abstracts. 2003 Annual Meeting. April 13-17, 2003
National Shcllt'isheries Association, New Orleans. Louisiana
Nonetheless, it was possible to identify four to six "generalist"
families that were among the top ten families at both sites. Further
evaluation is needed to determine if the best strategy to improve
oyster yields will be to select "generalist" families or whether it
will be more effective to develop site-specific lines instead.
M. inerceiniriii abundance differed and was nonadditive. Sediment
and faunal effects in shell hash were not different, although there
was some indication the sediment effect may be greater. In sand
and large shell pieces, alternative prey availability may be more
important for small M. ineirenaiia survival than physical refuge
from predation.
AN EVALUATION OF SEA SCALLOP CLOSED AREA
BOUNDARIES IN THE MID-ATLANTIC J. David Lange.
Jr., William D. DuPaul*. and David B. Rudders. VIMS PC
Bo.\ 1346 Gloucester Point, VA 23062-1346.
A formal area management strategy for the U.S. sea scallop
fishery is being developed under Amendment 10; Sea Scallop
Fishery Management Plan. Area closures impacting the sea scallop
fishery occurred in 1994 on Georges Bank to protect groundfish
resources. Also, in 1998, area closures in the mid-Atlantic (Hudson
Canyon and Virginia Beach) were enacted to protect concentra-
tions of pre-recruit scallops. This study determined if scallop abun-
dance was reflective of closed area boundaries designated by co-
ordinates on navigational charts.
Data was collected among two post-closure stock abundance
surveys. A total of 329 standard survey tows were conducted both
inside and outside the closed areas. Survey data were evaluated
and results indicate that the use of electronic vessel monitoring
systems to track fishing activity can be an effective tool in the
enforcement of area management strategies. The effective bound-
ary is described as the location at which the scallop population
differed as a result of an absence of fishing mortality due to the
protection provided by the area closures.
THE ROLE SUBSTRATE CHARACTERISTICS HAVE IN
ALTERING THE BEHAVIOR, GROWTH AND SURVIVAL
OF JUVENILE (POST-SETTLEMENT) MERCENARIA
MERCENARIA. Amy A. Larson*, and Robert M. Cerrato, De-
partment of Biology San Diego State University 5300 Campanile
Drive San Diego, California 92182-4614.
Indirect effects can be the primary structuring mechanism in
soft-sediment communities, but can be overlooked in experiments
that do not test for effects at appropriate levels of habitat com-
plexity. Interactions between physical factors (azoic sediments)
and biotic factors (faunal communities from the different sub-
strates) on growth and predation of small Mercenaria mercenaiia
were tested in different habitat types: sand, shell hash, large pieces
of shell and a control with no substrate. In the sand, competition
between small M. mercenaria and infauna reduced growth of M.
mercenaria. Shell hash and the associated fauna had no effect on
growth. On large pieces of shell, both competition and effects of
the substrate were important, and the combined effect of the two
was additive, resulting in the slowest growth rate overall. Preda-
tion rates were approximately equivalent in the different habitat
types, but the relative importance of physical and biotic factors on
ONE MAN'S DREAM: AMERICAN CULTURED PEARLS
GIna Latendresse*, American Pearl Company. 807 Watts Lane
#B. Nashville, TN 37209.
The late John Latendresse was the visionary behind pearl cul-
ture in the United States. His forty-five year journey from local
fisherman, entrepreneur, shell exporter, pearl importer and finally
to the originator of the American cultured pearl walks us through
the many facts of his life and details of his business success and
failures. His venture into pearl culture started with a challenge
from a Japanese colleague. Later he would be known as an evan-
gelist for pearl culture in the United States, To accompany this
presentation will be a display of exceptional American natural
pearls and jewelry designs with American cultured pearls, to in-
clude coin. bar. triangle, loaf, cabochon, teardrop .ind round.
ZOOPLANKTON INGESTION BY BIVALVES— MORE
FOOD FOR THOUGHT. Clare Lehane* and John Davenport.
Dept. of Zoology & Animal Ecology, University College Cork,
Lee Makings. Prospect Row. Cork. Ireland.
Bi\al\es have generally been thought of as herbivorous, gain-
ing nutrition from phytoplankton. However, since the 19th century
researchers ha\e reported finding zooplankton species in the stom-
achs and excreta of bivalves. A study was carried out to determine
if four species of bivalves, namely blue mussels, common cockles,
queen scallops and horse mussels could ingest zooplankton.
Though a series of suspended cage experiments and sampling bi-
valves from their natural habitats, it was determined that all four
species ingested zooplankton representative of that found in the
water column on the days of experiment. A second experiment
dealt with determining if a fabricated bed of blue mussels could
deplete zooplankton in overflowing water. It was found that zoo-
plankton numbers were lowest in the middle of the bed. signifying
that mussels may have the ability to affect zooplankton popula-
tions to some degree.
SPECIES-SPECIFIC VARIATION IN THERMAL TOLER-
ANCE DURING LARVAL DEVELOPMENT IN BLUE MUS-
SELS. MYTILUS SPP. Susan J. Limbeck*, and Paul D. Raw-
son, School of Marine Sciences, University of Maine. Orono. ME
04469.
Two species of blue mussel, Mytihis eclidis and Mytilits tros-
siihis. are sympatric throughout much of the Canadian Maritime
Provinces and into the Gulf of Maine. While the distribution of A/.
National Shellfisheries Association. New Orleans. Louisiana
Ahslrmls. 2003 Annual Meeting. April 13-17. 2003 341
eihilis extends south to the Mid-Atlantic, that of M. trasuilKs ends
abruptly in the Gull of Maine. We hypothesized that species-
specific variation in larval thermal tolerance influences differences
in distribution. Previously, we found that M. lro\siihi\ experienced
higher mortality than M. cJiilis when larvae were held at tempera-
tures above 1 5 C throughout development. Our current experiment
examines whether species-specific sensitivity to elevated tempera-
tures is dependent upon larval age. Growth and mortality were
monitored for larvae exposed to three experimental temperatures at
three time points during development. Preliminary analysis sug-
gests that M. trosMiliis larvae experience higher mortality at 1 S^C
and 22°C but the effect is dependent on larval age. The importance
of these findings with respect to patterns of larval dispersal and
coastal water temperatures in the Gulf of Maine will be discussed.
VARIATIONS IN THE SIZE STRUCTURE OF LOBSTER
(HOMARUS AMEKICANUS) POPULATIONS WITHIN THE
OFFSHORE FISHERY Susan A. Little \ Winsor H. Watson.
HI. and Rudman Hall. Dept of Zoology University of New
Hampshire Durham. NH 03824.
The offshore lobster fishery is cunently managed as one unit
(Area 3), although it extends from New Jersey to Maine. To de-
termine if there were differences in the size structure of lobster
populations within Area 3. we examined 36,815 lobsters from
three regions: North: Georges Bank and offshore Gulf of Maine;
Middle; offshore Massachusetts to south of Cape Cod. and; South:
offshore Rhode Island to New Jersey. Each region included mid-
shelf areas (30-40 fathoms) out to continental shelf canyons (120-
150 fathoms). In the North 2% of the catch was sub-legal, com-
pared to 409r in the Middle and 29Vc in the South. This pattern was
reflected in the average size in each region: North = 110mm CL
(carapace length); Middle = 89mm and; South = 91 mm. There
were also more lobsters > 100mm CL in the North (37%) than in
the Middle (4%) and South (3%). and. conversely, more lobsters
<65mm CL in the Middle (3%) and South (5%). than in the North
(<I%). Despite similarities in depth and bottom temperature, the
populations differ between the three regions examined, which may
have important management implications. These patterns may re-
sult from regional differences in growth rates, migratory patterns
or fishing pressure.
tiated within the past few- years. New urgency is given to evalu-
ating the success of these efforts by the serious consideration cur-
rently being given to the introduction of an exotic oyster species.
We will summarize these projects and assess what is known about
their success. Patterns emerging from this review indicate some
unifying themes, but also point to the importance of site specific-
ity. Several studies reveal the importance of reef architecture (size,
shape and spatial configuration) and substrate composition, but
questions remain about how to optimize the placement of material.
Hatchery-produced and wild oysters have been transplanted onto
reefs in a variety of locations, but the efficacy of this has only
occasionally been tested. Altered hydrographic regimes and chang-
ing water quality conditions in many estuaries increase the com-
plexity of restoring these habitats. The importance of improving
our understanding of the genetic implications of restoration strat-
egies, larval dispersal patterns, factors affecting early post-
settlement survival, disease dynamics and landscape-level patterns
in restoring oyster reefs is emphasized.
PROGRESS IN THE DEVELOPMENT OF A CHEMO-
THERAPEUTIC PROTOCOL FOR ELIMINATING/
REDUCING DERMO DISEASE IN INFECTED OYSTERS.
Eric D. Lund*, Fu-Lin E. Chu and Ellen Harvey. Virginia In-
stitute of Marine Science, College of William and Mary, Glouc-
ester Point, VA 23062.
There is a need for protocols to eliminate/reduce Perkinsiis
marinus infection of oysters for hatchery, aquaculture operations
and basic research on the disease, yet no practical method for
treating infected oysters currently exists. Studies on the antimicro-
bial drug triclosan have shown that the drug is a .specific inhibitor
of Fab 1 . an enzyme in the type II class of fatty acid synthetases
which are found in bacteria, plants and some protozoans, but not
animals. The effects of triclosan on 7. 15 and 48-day-old P. mari-
nus cultures were tested. Results revealed that triclosan (5-10 mi-
cromolar) added to the medium inhibits fatty acid synthesis and
stops the proliferation of P. marinus meronts in vitro. The inhibi-
tory effects of triclosan were highest in the 7-day cultures and
somewhat attenuated in older cultures. These results suggest that
triclosan may be a useful chemotherapeutic agent for treating oys-
ters infected with P. marinus. This study has been supported by
NSF award #0131 108.
OYSTER REEF HABITAT RESTORATION: A REVIEW
OF RESTORATION APPROACHES AND AN AGENDA
FOR THE FUTURE Mark W. Luckenbach* and Loren D.
Coen. Virginia Institute of Marine Science P.O. Box 350
Wachapreague, VA 23480.
Reefs produced by the Eastern oyster have been degraded by
many factors, including overfishing, disease, sedimentation, pol-
lution, hydrographic alterations and boat wakes. Numerous oyster
reef restoration projects are currently underway or have been ini-
STRIKING SUCCESSION OF MUSSELS AT NEWLY
FORMED DEEP-SEA HYDROTHERMAL VENTS Richard
A. Lutz*. Timothy M. Shank, and Daniel J. Fornari. Institute of
Marine and Coastal Sciences. Rutgers University. New Brunswick,
NJ 08901.
In April, 1991 a volcanic eruption obliterated existing biologi-
cal communities within extensive regions along the crest of the
East Pacific Rise between 9°45'N and 9°52'N (depth 2520 m). The
342 Ahsinuls. 2003 Annual Meeting, April 13-17, 2003
National Shellfisheries Association. New Orleans. Louisiana
initiation of hydrothermal venting during the eruptive process af-
forded the unique opportunity to follow, over a 10+ year period,
temporal changes in biological community structure from the
"birth" of numerous hydrothermal vents. Vestimentiferan tube-
worms, amphipods. copepods. octopods. and galatheid and
brachyruan crabs gradually colonized the vents during the first 2
years. Noticeably absent during this time was any evidence of the
mussel Bathymodioliis thermophilus. Small mussels (<l cm shell
length) were first observed on basaltic substrates 3 1/2 years after
the eruption, but were noticeably absent on the tubes of the domi-
nating vestimentiferan Riftia pachyptila during this period. By 4
1/2 years after the eruption, mussels with shell lengths >5 cm were
common in the region and had begun to colonize the tubes of Riftia
pachyptila. concomitant with declining concentrations of H2S in
the venting diffuse flow fluids. Over the next 5-6 years, the abun-
dance of mussels increased dramatically until most of the existing
communities previously dominated (in biomass) by tubeworms
were now dominated by extensive populations of mussels.
SUSPENSION-FEEDING BIVALVES. MARINE AGGRE-
GATES AND THE ACCESSIBILITY OF SMALL PAR-
TICLES. M. Maille Lyons*, J. Evan Ward, Department of Ma-
rine Sciences. University of Connecticut. Groton CT 06340.
Marine aggregates (i.e., marine snow, organic aggregates.
floes, and detritus) are common in coastal waters where large
populations of bivalves dominate benthic communities. Suspen-
sion-feeding bivalves actively pump seawater through their pallial
cavities and extract particles for food. Retention efficiencies of
small, freely suspended, particles (~1 p,m) are generally low. Small
particles are often concentrated within aggregates. The retention
efficiency of the larger, amorphous aggregates (>10 |j,m) should be
100%. However, the percentage of aggregates ingested, compared
to the percent rejected as pseudofeces, is not known. The focus of
this study is to determine the fate of the particles embedded within
aggregates. Determining the ingestion rate of aggregates is an
important step in assessing the role aggregates play in trophic
interactions between water column microbiota and benthic bi-
valves. To test the hypothesis that the presence of marine aggre-
gates increases the accessibility of small, otherwise poorly re-
tained, particles, experiments were designed using artificial aggre-
gates generated on a rolling table. Fluorescent beads ( I |xm and 10
|xm) were incorporated into aggregates in order to track the fate of
embedded particles. The percentage of beads in the bivalves gut.
evaluated by direct counts, was compared to controls (fluorescent
beads not incorporated into aggregates). Preliminary results indi-
cate species specific differences, increasing the accessibility of
small particles for the sea scallop (Placopecten inagellanicus), but
showing no significant increase in accessibility for the marsh mus-
sel (Geukensia demissa).
SHELLFISH RESTORATION: IT'S NOT JUST BIOLOGY
THAT MATTERS. Sandra L. Macfarlane. Coastal Resource
Specialists P O Box 1 164 Orleans. MA 02653.
Shellfish restoration projects have been practiced in most
coastal states for years. But as stocks decline, water quality de-
grades and population pressures increase throughout the coastal
zone, restoration projects have become more urgent in many sec-
tors. While biological factors such as predator/prey relationships
and physical factors such as current and sedimentation are impor-
tant for the success of a restoration program, other less tangible
factors can be equally important. Increasingly, the success of a
project may depend on a holistic approach of embayment man-
agement that often includes land use issues, topics such as storm-
water runoff, nutrient loading, proliferation of docks or erosion
control structures and other human use impacts, issues that may
not considered when planning a restoration effort. As coastal area
population increases, land use and human marine use issues may
have greater influence on the success of the restoration effort than
traditional biological and physical factors. And yet. as land uses
continue to degrade water quality, shellfish restoration projects are
being considered as a counter measure, using the natural filtering
capacity of shellfish to boost water quality. This paper discusses
these issues as challenges to shellfish restoration efforts.
EVIDENCE FOR NATURAL SELECTION FOR RESIS-
TANCE TO PSP TOXINS IN EARLY LIFE HISTORY
STAGES OF THE SOFTSHELL CLAM. MYA ARENARIA.
S. MacQuarrie*. V. Monica Bricelj. 1411 Oxford St. Halifax.
Nova Scotia. Canada. B3H 3Z1.
Our prior research has demonstrated that sensitivity to paralytic
shellfish poisoning (PSP) toxins, measured by behavioral and
physiological indices, varies significantly among Mya arenaria
populations with differing toxin exposure histories. Populations
from PSP-affected areas are predominantly resistant whereas naive
populations are dominated by sensitive individuals. An extensive
survey of M. arenaria populations supports this correlation over a
wide geographical range. This study identifies the life history
.stages susceptible to selective pressures and demonstrates the po-
tential for strong selection. Effects of toxin exposure were deter-
mined for larvae and spat from a population previously character-
ized as sensitive. Veliger larvae exposed to bloom levels of a
highly toxic Alexandrium tamarense strain (PR18b) in a mixture
with non-toxic algae showed no increased mortality relative to
controls. However, spat (3.5mm) exposed to a monospecific sus-
pension of PR 1 8b suffered 95% mortality after 1-week exposure,
resulting in a population dominated by resistant clams. Video ob-
servations suggest that anoxia of the pallial cavity may be respon-
sible for mortalities. Ingestion of toxic cells is necessary to induce
mortality and a single bloom of sufficient toxicity is capable of
selecting for resistance at these stages. Results will be discussed in
context of ecological relevance and fitness consequences.
National Shellt'isheries Association, New Orleans, Louisiana
Abstiuas. 2003 Annual Meeting. April 13-17. 2003 343
OPTIMIZING OYSTER PRODUCTIVITY IN CARAQUET
BAY: COORDINATING RESTORATION AND AQUACUL-
TURE. J. F. Mallet , and T. Landry. DFO. P.O. Box 5030.
Moncton NB. EIC 9B6.
Caraquet Bay represents the most northern location with a sus-
tainable oyster (Crassostrea virginica) population. In recent years,
a decrease in the productivity of oysters from the natural beds has
generated interest in restoration projects. To determine the benefits
of restoration activities, information on the distribution, abundance
and population structure of oysters was collected in 1999. Over
60% of the oysters found were pre-recruits (35-75mm). These
oysters were mainly found in the northern portion of the bed,
which is locally renowned for its "stunted oysters". In 2001,
"stunted" oysters along with control oysters were transfened to
four stations and monitored for growth. Results to date show that
growth oysters are associated with site and bottom conditions. In
2002, samples of "stunted" oysters were placed at three stations in
various holding situations to evaluate the effects of vertical posi-
tioning in the water column and tidal position. The results from
this experiment revealed that oyster productivity varies in relation
to their geographical location. They will provide key information
for the oyster fishers and aquaculturists to develop management
strateaies.
ROSEIMARINA CRASSOSTREAE (GEN. NOV.. SP. NOV.)
ASSOCIATED WITH JOD-SIGNS IN THE ABSENCE OF
SIGNIFICANT MORTALITIES, AND FIRST ISOLATION
FROM A NEW YORK EPIZOOTIC Aaron P. Maloy*. 5735
Hitchner Hall University of Maine Orono ME. 04469: and
Katherine J. Boettcher.
The alpha-proleobacterium Roseiiiiariiio crassostreae (gen.
nov., sp. nov.) has. to date, been isolated exclusively from episodes
of juvenile oyster disease (JOD) in .Maine. With few exceptions,
isolates have been recovered from animals that probably would
have died from the disease. Mortalities have been reproduced by
experimental exposure to the bacterium, although without typical
JOD-signs (e.g. conchiolin). Herein we describe induction of con-
chiolin in oysters challenged with R. crassostreae. Further, we
report a 907f correlation between conchiolin and colonization by
Roseimarina in a natural (but unusual) occurtence of JOD. The
affected animals were from Maine's Damariscotta River where
cumulative mortalities were < 5% in 2002 (down from 50% in
2001 ). Thus, these bacteria were isolated in the absence of signifi-
cant mortalities. In fact, most of the 9% of animals with conchiolin
appeared otherwise healthy. Finally, we document the first isola-
tion of R. crassostreae from JOD outside of Maine. Analyses of
the 16S-23S rDNA internal transcribed spacer region revealed that
isolates from a 2002 New York epizootic were the same genotype
(GTl) as those from Maine epizootics in 1997 and 1998. For
unknown reasons, a slightly different genotype (GT2) appeared in
Maine in 2000. and thereafter replaced GTl as the etiological
acent here.
FINDING THE WHEAT IN THE CHAFF— OYSTER LAR-
VAL FEEDING IN TURBID. LOW SALINITY CONDI-
TIONS. Roger Mann and Peter Kingsley-Sniith. VIMS P.O.
Box 1346 Gloucester Point, VA 23062.
Oyster restoration efforts in the Middle Atlantic States focus on
a combination of benthic habitat refurbishment and brood stock
supplementation, predominantly in low salinity sanctuaries from
endemic disease. Central to this approach is the assumption that
efforts increase local recruitment, yet we are ignorant of the in-
fluence of low salinity, elevated turbidity, and limited food avail-
ability on the survival and growth of oyster larvae. We suggest that
in high turbidity regions available food is essentially diluted by
indigestible inorganic material, and larvae may be food limited
despite an apparently adequate absolute concentration of food be-
cause the relative food concentration is low. If this is the case then
watershed management practices emphasizing nutrient reduction
policies in excess of concomitant sediment load reduction may
serve to reduce larval survival in receiving water bodies, and com-
promise restoration efforts. We offer numerical estimates of the
impact of elevated turbidities on oyster restoration through de-
creased lar\al survival. We then investigate larval feeding behav-
ior, as a proxy for overall larval viability, under both controlled
salinity-turbidity conditions in the laboratory, and along a salinity-
turbidity cline in the York-Mattaponi river systems of the Chesa-
peake Bay.
CHARACTERIZATION OF SUMMER MORTALITIES OF
CRASSOSTREA GIGAS OYSTER IN RELATION TO
PHYSIOLOGICAL PARAMETERS. M. Mathieu*. B. Dubois,
K. Costil. C. Heude. A. Huvet, K. Kellner, S. Pouvreau. Physi-
ologic Ecophysiologie des MoUusques Marins IFREMER Univer-
site de Caen. 14032 Caen Cedex, France.
Pacific oysters are characterized by high fecundity, and follow
a seasonal breeding pattern beginning in autumn with gamete
maturation in spring and early summer. Summer mortalities occur
during spawning period, but according to the environmental con-
ditions gametes are spaw ned or reabsorbed. In Normandy, which is
the northern more oyster cultivation area in France, spawning is
often partial or absent. Mortalities were observed in both situations
but more often when gamete release is delayed. The implication of
hemocytes in gamete resorption as in storage tissue restoration was
observed. The level of fecundity varies with trophic conditions.
Storage material is accumulated in specific cells mainly in autumn
and winter, and then mobilized to support gametogenesis. Resorp-
tion of gametes contributes to storage tissue development. Incor-
poration of metabolites in storage cells is performed by diffusion
through cell membrane and by two transport systems sensitive to
344 Abstnicts. 2003 Aniuuil Meeting. April 13-17. 2003
National Shellfisheries Association. New Orieans. Louisiana
internal regulation. The storage tissue follows a seasonal cycle
regulated by internal factors, with reversion of its metabolism in
summer.
PERKINSUS MARINUS INFECTION RATES IN SPECIFIC-
PATHOGEN-FREE JUVENILE OYSTERS PLANTED IN
THE PATUXENT RIVER, MARYLAND. Carol B. MeCol-
lough* and Christopher F. Dungan. Sarbanes Cooperative Ox-
ford Laboratory. Oxford. MD 2l6.'i4; George R. Abbe and Can-
dace A. Morrell. Academy of Natural Sciences Estuarine Re-
search Center, St. Leonard, MD 20685.
Specific-pathogen-free (SPF) oysters were set and reared in
artificial seawater and transferred to four sites in the Patuxent
River along a salinity gradient. Three sites were adjacent to natural
oyster bars and one, Sandgates, was remote from existing oyster
populations. Samples of 30 oysters were assayed at 2 and 4 weeks
post-deployment for infection by P. mariuus using an enhanced
RFTM whole body burden technique. Assays continued at 4-week
intervals. Deployments were made in May and September 2002.
From the May deployment. SPF oysters placed at Sandgates, re-
mote from existing populations, acquired infections by day 27
(13%' prevalence), as did juveniles deployed at TC (7%) and HP
(3%). At all sites oysters acquired infections within 62 days, with
prevalences of 10%, 63%, 43%, and 37%^ (TC - HP). By 91days
post-deployment all sites, with the exception of TC, had infection
prevalences greater than 90%, and these elevated prevalences con-
tinued through 127 days. At 9 1 days TC prevalence remained low
at 10%, but by 127 days it also increased, to 53%. In October
prevalences declined at TC. GA. and SG (154 days), and all re-
mained above zero into mid-November. SPF juveniles deployed in
late September acquired P. mariuus infections by 27 days at all
sites, however prevalences were low and declined at 55 days, with
infections detected then only at HP.
IS MERCENARIA MERCENARIA A HOST FOR PERKIN-
SUS SPECIES? Ayana McCoy*, Shirley Baker, Ruth Francis-
Floyd, and Anita Wright, University of Florida Department of
Fisheries and Aquatic Science 7922 NW 71st St Gainesville. Fl
32653.
Perkinsus marimts is an endoparasitic protistan that infects the
Eastern oyster. Crassostrea virginica. This parasite has caused
mass mortalities of oysters along the Atlantic and Gulf coasts. The
commercially important Mercenaria mercenaria is cultured in ar-
eas naturally populated by C virginica. Whether the hard clam, M.
mercenaria, is susceptible to Perkinsus infection or serves as an
intermediate host has not been well studied. Therefore, the objec-
tives for this study were ( 1 ) to examine the diversity of Perkinsus
species associated with A7. mcrccnarici and C. virginica in the
environment, and (2) to experimentally test the susceptibility of
hard clams to P. marinus and P. andrewsi infections. M. merce-
naria and C. virginica were collected from the Cedar Key area on
the Gulf Coast of Florida. Both species-specific PCR assays and
standard Ray's Fluid Thioglycollate Media assays were u.sed to
identify associated parasites and determine levels of infection.
Laboratory studies are in progress to determine virulence of Per-
kinsus species in M. mercenaria. This project should help to pro-
\ ide an indication of virulence potential of Perkinsus species for
the hard clams on Florida's Gulf Coast and the possible threat of
these parasites to the rapidly growing aquaculture industry in the
region.
RECOMMENDATIONS TO OYSTER HARVESTERS ON
REMOVING HOOKED MUSSELS, ISCHADIUAf RECUR-
VUM. Earl J. Melancon. Jr.*. Biology Department, Nicholls
State University. Thibodaux, La 70310; Dale Diaz, Mississippi
Department of Marine Resources. Biloxi. Miss. 39530; Badiollah
Asrabadi. Math Department, Nicholls State University.
Our results indicate that high salinity as a physiological factor
to kill mussels is of minimal value to removal success. Predation,
often by the blue crab, is the driving force on the removal of
mussels from oysters. In addition, the physical process of harvest-
ing with a dredge, using water cannons to move oysters on deck
and then again to plant overboard can resulted in as much as a
33-38% direct mussel mortality; in turn, the dead and dying mus-
sels attract predators. Planting to down-bay (high salinity) habitats
will remove mussels within a short period of time if predators are
present; perhaps in as little as week in summer water temperatures
(25-30°C). Use of water cannon to move oysters on deck sup-
presses temperatures within the pile and allows relaying during
summer months without harming oysters. The spray from the hose,
the dripping from the stacked oysters and evaporative cooling all
work together to keep air temperatures well below the heat toler-
ance of mussels and oysters. Observations also suggest that culti-
vation by breaking up oyster clusters may reduce mussel fouling in
mid-bay and down-bay sites, but not necessarily at up-bay (low-
salinity) sites.
A COMPARISON OF CRYOGENIC FREEZING TECH-
NIQUES AND THEIR USEFULNESS IN REDUCTION OF
VIBRIO VULNIFICUS IN RETAIL OYSTERS D. Mestey and
G.E. Rodrick*, University of Florida Dept. Food Science and
Human Nutrition, Gainesville, Fl. 3261 1.
Freezing the oysters and storing them at freezing temperatures
suppress the number of recoverable V. vulnificus from the oyster
meat. There are various methods that can be used to achieve a
frozen product. In this study the effectiveness of carbon dioxide
and nitrogen was analyzed. A comparison of freezing whole oys-
ters versus half shell oysters with this two freezing methods was
also studied.
The oysters were processed using the commercial practices at
each of the three seafood-processing plants. The samples were
analyzed using the guidelines provided by the FDA Bacterial Ana-
National Shellt'isheries Association. New Orleans, Louisiana
Absrracls. 2003 Annual Meeting. April 13-17. 2003 345
lytical Manual. An initial sample of fresh unfrozen oysters was
analyzed to determine the initial \'ihriii viiliiifkus load followed by
analysis of frozen samples at 1.7. 14 and 21 days after storage at
-10°C.
The study demonstrates that there is lower number of recover-
able V. viilnifkiis when C02 is used for freezing than when nitro-
gen is used, but the overall decrease in V. vulnificus load in the
fresh to frozen product is by 200,000 organisms per gram of oyster
meat. There are few organisms recovered by 21 days regardless of
the freezina method.
USING MOLECULAR GENETIC TECHNIQUES TO AS-
SESS OYSTER RESTORATION PROGRAMS AND PRO-
JECTS. Coren A. Milbury* and Patrick M. Gaffney. College of
Marine Studies. 700 Pilottown Road. Lewes. De 19958.
Restoration efforts are becoming essential in managing many
of our ecological resources. Equally important are the assessment
and monitoring of restoration programs. Recent advances in ge-
netic techniques allow for the use of high-throughput and cost
effective methods in restoration assessment and monitoring. We
have used molecular methods to assess a restoration project by the
Maryland Oyster Recovery Partnership and the University of
Maryland. Four million Louisiana oysters were planted in the
Choptank Ri\er. Maryland. Crassostrea virginica exhibits region-
ally diagnostic mitochondrial haplotypes. which provide a means
to genetically differentiate Gulf Coast oysters from native oysters.
Detection of newly recruited spat possessing the Gulf Coast hap-
lotype in the Choptank River confirms the survival and propaga-
tion of the outplanted oysters and the contribution of new progeny.
A high-throughput, synthesis-by-sequencing technique (Pyrose-
quencing) was used to determine the mitochondrial hap-
lotypes of spat collected in the Choptank River. Of 4,566 spat
analyzed. 94.2% possessed the North Atlantic haplotype. 5.39^ had
the South Atlantic haplotype. and 0.1% possessed the Gulf Coast
haplotype. The results demonstrate the contribution of the out-
planted Louisiana oysters to the resident Choptank River popula-
tion, and show that effective monitoring of stock enhancement
projects can be achieved with high-throughput molecular genotyp-
ing techniques.
CREATING SALT MARSHES TO ENHANCE PRODUC-
TION OF FISHERY SPECIES Thomas J. Minello* and
Lawrence P. Rozas, National Marine Fisheries Service. Southeast
Fisheries Science Center, Galveston Laboratory, 4700 Avenue U.
Galveston. TX 77551. U.S.A.
Salt marshes in the northern Gulf of Mexico are valuable nurs-
ery habitats for fishery species such as penaeid shrimps and blue
crabs. Extensive marsh loss has led to numerous restoration pro-
jects in the region, but little design information has been available
for optimizing fishery productivity from these created wetlands.
We have sampled the small-scale spatial distributions of shrimps
and blue crabs in natural and created marsh systems and developed
models to 1) estimate populations of these fishery species in
marshes of different land- water configurations and 2) simulate
population changes in created marshes with different land-water
patterns. The amount of vegetation-water interface or edge in salt
marshes is an important characteristic that can determine the fish-
ery value of these habitats. Marsh creation projects that maximize
edge are likely to be most productive for commercially important
decapod crustaceans. Terracing and the formation of small marsh
islands are two restoration techniques that produce a great amount
of marsh edge and should provide productive habitats for penaeid
shrimps and blue crabs.
GENETIC VARIABILITY IN REPRODUCTION AND SIM-
MER MORTALITY IN CRASSOSTREA GIGAS. Jeanne
Moal*. Edouard Bedier, Pierre Gildas Fleury, Ainie Langlade,
■^'vette LeCoguic. Lionel Degreniont. Pierre Boudry. Jean
Rene Le Coz, Stephane Pouvreau. .Martha Enriquez-Diaz,
Christophe Lambert. Philippe Soudant, Jean Francois Sa-
main. Ifremer. centre de Brest BP 70 29280 Plouzane. France.
Bi-parental families were produced in hatchery and tested in
the field in 2001. Two sets of 5 families were constituted, selected
on their high (R) and low (S) survival. These two sets were reared
in Brittany from March to November 2002. Samplings were per-
formed twice a month to obtain data on biometry, survival, repro-
ductive cycle, biochemical composition, adenylate energy charge,
hemolymph parameters (ions and defense system) and muscle
strength.
"R" and ■"S" oysters exhibited different reproductive effort and
spawning strategy. "R" oysters allocated less energy in gonad than
"S" ones and presented a complete spawning at the end of August
contrary to the "S" which spawns partially. Mortality started in
July when the seawater temperature reached 19"C and affected
mainly "S" oysters. Concentrations of Na and CI ions in
hemolymph were different for "S" and "R" from May to June. A
bacterial increase in hemolymph (R and S) was observed during
the same period. The adenylate energy charge was more lowered
for "R" than for "S" oysters, just before the spawning event How-
ever, other physiological and immunological parameters were
similar between the two sets during the phases of maturation and
mortality but discriminated groups after the spawning peak.
PRELIMINARY PATHOLOGICAL INVESTIGATION OF
THE WHITE ABALONE. HALIOTIS SORENSENI. James
Moore*. Thea Robbins. Carolyn Friedman. Neal Hooker.
Thomas McCormick. Melissa Neunian. Bodega Marine Labo-
ratory P. O. Box 247 Bodega Bay CA 94923 USA.
Populations of white abalone HalUnis sorenseni. deep water
inhabitants, were severely exploited in the 1970s following serial
depletion of several other species found in shallower water. This
346 Abslracls. 2003 Annual Meeting, April 13-17. 2003
National Shellt'isheries Association. New Orleans, Louisiana
species appears to be nearing extinction and in 2000 became the
first marine invertebrate to be listed under the federal Endangered
Species Act. Acquiring health information is critical for planning
recovery of this species. White abalone broodstock were collected
in 1999-2000 prior to federal listing. Deaths of eleven of these
animals appeared to be related to collection injuries or water qual-
ity problems rather than infectious disease. The etiological agent of
withering syndrome (WS-RLP, withering syndrome associated
Rickettsiales-like prokaryote), was not detected in any of the dead
animals by histology or PCR. Juveniles produced from broodstock
were held at 12. 15 and ISC and were exposed to the WS-RLP.
Marked losses of and pedal atrophy in animals with severe WS-
RLP infections demonstrated that white abalone are susceptible to
withering syndrome. As in other abalone species, cool water pro-
vided some refuge from WS-RLP pathogenicity. No other signifi-
cant pathogens were observed. The susceptibility of white abalone
to WS must be considered in the formulation of recovery plans.
UTILIZATION OF POST-HARVEST TREATMENT AS A
STRATEGY FOR REDUCING VIBRIO VULNIFICUS ILL-
NESSES. Ken B. Moore. ISSC 209-2 Dawson Road Columbia
SC 29223.
Illnesses and deaths associated with the consumption of raw
molluscan shellfish continue to be a significant public health con-
cern for the Interstate Shellfish Sanitation Conference (ISSC). In
1995, the ISSC highlighted three main approaches for reducing V.
vulnificus-Telated illnesses and deaths involving high-risk consum-
ers. These included education of "high-risk" groups to avoid raw
shellfish, more rapid post-harvest refrigeration of shellfish to pre-
vent increases in numbers of the pathogen, and encouraging and
promoting shellfish post-harvest treatments to reduce Vibrio
vKlnificits to non-detectable levels. The role of post-harvest treat-
ment as a strategy to control Vibrio vulnificus has become more
significant since the passage of the mandatory Vibrio vubtificus
Illness Reduction Plan by the ISSC Voting Delegates in 2000. The
establishment of collective illness reduction goals for core states
has created a proactive approach for addressing Vibrio vubiificus-
related illnesses and deaths. The ISSC remains committed to
Vibrio vulnificus illness reduction and is continuing efforts to iden-
tify additional effective strateaies.
CHARACTERIZATION OF NATURAL KILLER CELL-
LIKE ACTIVITY IN THE EASTERN OYSTER, CRASSOS-
TREA VIRGINICA. Brenda M. Morsey* and Sylvain De Guise,
Department of Pathobiology and Veterinary Science, University of
Connecticut, 61 N Eagleville Road, U-89. Storrs, CT 06269, USA.
Natural killer (NK) cells are an important part of the innate
immune system of mammals. However, little is known about NK-
like cell activity in the Eastern Oyster, Crassostrea virginica. NK-
like cell activity of oyster hemolymph cells was measured by a
flow cytometric assay in which oyster hemocytes were incubated
with DiO-labeled K-562 target cells, and propidium iodide to label
dead target cells. For every individual oyster tested, higher effec-
tor-to-target cell ratios resulted in higher levels of target cell death.
Moreover, NK-like activity of individual oysters was further en-
hanced by recombinant human interleukin-2. Enhancement of NK-
like cell activity by interleukin-2 was more pronounced in pooled
oyster hemolymph compared to individual oyster hemolymph
samples. Our data demonstrate for the first time the presence of
NK-like cell activity in a marine invertebrate. This activity can be
enhanced by physiologically relevant concentrations of mamma-
lian interleukin-2 which further suggest that some structural and
functional homologues of the mammalian innate immune func-
tions are conserved in invertebrates such as the oyster. The im-
portance of oyster NK-like activity in protection against disease
and pathogen control will be assessed.
FOOD AVAILABILITY IN A MUSSEL RAFT Jessica
Munro* and Carter Newell. Great Eastern Mussel Farms, P.O.
Box 141. Tenants Harbor, Maine. 04860.
Current speed, phytoplankton concentration, detritus concen-
tration, mussel biomass and mussel density are important deter-
mining factors in the growth rate of raft cultivated mussels. Peri-
odic measurements of flow and food with depth inside and outside
mussel rafts are used to determine seasonal and site specific food
availability and mussel raft consumption. Field data is collected
with Seabird CTD and current meter casts, water sampling, and
weighing mussel lines with a crane scale. Seasonal stratification
causes vertical variation of food availability to mussel rafts in
Maine waters. The depletion of available food is a function of the
biomass of a mussel raft and mussel raft hydrodynamics.
COMPARING TWO MYA ARENARIA POPULATIONS AS
POTENTIAL CANDIDATES FOR SEEDING OPERA-
TIONS. Bruno Myrand*, Station Technologique Maricole des
Iles-de-la-Madeleine, Cap-aux-Meules, Canada, GOB I BO, Rejean
Tremblay, Societe de Developpement de ITndustrie Maricole.
Gaspe, Canada, G5X 1T5; Lise Chevarie. Societe de Developpe-
ment de ITndustrie Maricole, Cap-aux-Meules, Canada, GOB IBO;
Fabrice Pernet, Universite du Quebec a Rimouski-Centre Aqua-
cole Marin de Grande-Riviere, Grande-Riviere, Quebec, GOC
IVO; and Diego Mantovani, Institut des biomateriaux du Quebec.
Universite Laval, GIK 7P4.
It is important to identify a source of clams for seeding in
lles-de-la-Madeleine. Two populations were examined: Havre-
aux-Basques (HB) and Dune-du-Nord (DN). No neoplasia were
found. Both populations belong to the same stock and have a low
multilocus heterozygosity. Growth was better at DN site for both
populations and better for the DN clams at both sites. The HB
clams had a very limited growth. These results will be interpreted
according to scope for growth measurements. The fragility of the
National Shcllfislieries Association. New Orleans. Louisiana
Abstracts. 2003 Annual Meeting, April 13-17, 2003 347
shell was higher tor HB elanis. Therefore, the HB clams appear
unsuitable tor seedine.
PROPAGATION OF FRESHWATER MUSSELS FOR
FRESHWATER PEARL PRODUCTION. Richard J. Neves*.
Jess W. Jones, William F. Henley, and Rachel A. Main. Fresh-
water Mollusk Conservation Center, Virginia Cooperative Fish
and Wildlife Research L'nit, Virginia Tech. Blacksburg, VA
24061.
The commercial harvest of mussel species suitable for pearl
production could provide an incentive to replace wild-caught
adults with laboratory-reared juveniles to sustain populations. The
Freshwater Mollusk Conservation Center at Virginia Tech was the
first facility in the LInited States to begin an annual propagation
and release program focused on endangered mussel species. Initial
research to identify host fishes, develop production and culture
methods, and test culture technology required nearly 10 years of
experimentation. Endangered juvenile mussels were released first
in 1997. and subsequent annual releases total nearly 370.000 ju-
veniles of 10 species. A new facility dedicated to propagation was
completed in 2002, with capacity to address commercially har-
vested species, as well as those under federal protection. Should
the harvest of particular species such as those with colored nacres
increase, then culture techniques are now available to replace har-
vested specimens with progeny produced from the parental popu-
lation.
AN EXPERT SYSTEM FOR THE OPTIMIZATION OF
SHELLFISH RAFT CULTURE. Carter Newell* and John Ri-
chardson. Great Eastern Mussel Farms P.O. Box 141 Tenants
Harbor. Maine.
An expert system combining computer-based methodologies
for determining tidally driven flows, wave heights, flow through
shellfish raft systems, and consumption of food by the shellfish
with specially designed data collection techniques is being used to
improve shellfish production on mussel rafts in Maine. Elements
of the expert system are being incorporated into a single computer
that operates in a "point and click" manner. A large scale flow
model develops tidal flow boundary conditions for the three di-
mensional computational fluid dynamics (CFD) raft model, and
predicts wave conditions relative to mooring specifications and
site risk assessment. The detailed CFD raft model predicts cunent
speed and chl a consumption relative to ambient flow speed and
direction, shellfish biomass, and density distribution. Field data
collection involves flow profiles, wave gauges, CTD casts, sedi-
ment traps and feeding chambers. Mussel biomass on culture ropes
is monitored using a crane scale. Optimization of production
cycles on shellfish rafts involves careful consideration of raft hy-
drodynamics, seasonal changes in food availability, and stocking
densities.
LINKING HARD CLAM {MERCENARIA MERCENARIA)
REPRODUCTION TO PHYTOPLANKTON COMMUNITY
STRUCTURE: II. PHYTOPLANKTON COMMUNITY
STRICTURE AND FOOD COMPOSITION Roger I.E. New-
ell*. Horn Point Laboratory UMCES Cambridge. MD
21613. Christopher Gobler. and Stephen T. Tettelbach.
Hard clam. Mercenaria mercenar/a. recruitment has declined
in some southern bays of Long Island, NY and we hypothesized
that this was associated with changes in the phytoplankton com-
munity structure and overall patterns of primary production. We
collected hard clams over an annual cycle for analysis of repro-
ductive condition from five south shore bays of Long Island. Con-
currently, ambient water was filtered for analyses of organic car-
bon and nitrogen, total and size-fractionated chlorophyll, and mi-
croscopic counts for the harmful brown tide picoplankter.
Aiireococciis anophagejferens. We found appreciable differences
in seston composition that related to the observed differences in
hard clam reproductive and tissue condition. Bay Shore and
Patchogue had the highest total Chl a levels and organic carbon
nitrogen and carbon of any bay. Paradoxically, clams from this
location had the lowest condition index and reproductive effort.
The size fractionated Chl a data, however, showed that the high
levels of organic material at these two locations was mainly con-
tributed by cells < 2 \i.m which are too small to be efficiently
retained by adult hard clams and hence have no nutritional value.
In addition, both Bay Shore and Patchogue had brown tide blooms
at cell concentrations that inhibit adult hard clam feeding. We
conciude that changes in the floristic composition of the phy-
toplankton community in at least some of the Long Island south
shore bays is translating into appreciable differences in hard clam
condition and ultimately into reducing tcital reproductive effort.
COMMERCIAL IMPLEMENTATION OF HIGH PRES-
SURE PROCESSING (HPP) FOR PACIFIC OYSTERS.
David H. Nisbet*, Nisbet Oyster Co., P.O. Box 338 Bay Center,
WA 78527.
High Pressure Processing (HPP) was first used commercially
on Pacific oysters, Crassostrea gigas by Nisbet Oyster Co.. Inc. a
cultivator, processor and packer of Pacific oysters on Willapa Bay
in Washington State. Initial pilot scale experimentation was cen-
tered on the oyster shucking protocol for pressure and dwell time
regimes. Physical material flow proved a major obstacle to resolve
in the feed and outfeed of the equipment. .\n engineering study
was commissioned to determine real-time throughput capabilities
of commercially available HHP equipment. Building design, prod-
uct flow and ergonomics were also researched as the company
expanded its processing facility. When the commercial high-
pressure equipment installation was completed, studies were un-
dertaken in collaboration with the Oregon State University Sea-
food Laboratory and Seafood Consumer Center. Extended sensory
analysis and Vibrio control studies were considered most impor-
348 Ahsnacts. 2003 Aiinuul Meeting. April 13-17, 2003
National Sliellfisheries Association, New Orleans, Louisiana
tant, as well as the development of other possible \alue added
product candidates. The commercial considerations tor high pres-
sure processing included specific end product related studies and
building design features including product flow, throughput analy-
sis, ergonomics, equipment maintenance, and cleanup. Addition-
ally, the physical size of Pacific oysters relative to available hy-
drostatic chamber size capabilities constitutes special consider-
ations for HPP commercial installations.
OPTIMAL PLANTING CONDITIONS FOR MAXIMUM
REPRODUCTIVE OUTPUT OF CAGE-PLANTED SCAL-
LOPS. ARGOPECTEN IRRADIANS. IN ANCLOTE.
FLORIDA. Melanie L. Parker*. William S. Arnold and Dan C.
Marelli, Florida Marine Research Institute 100 Eighth A\enue SE
St. Petersburg. FL 33701.
As part of an ongoing effort to restore bay scallop populations
on the west coast of Florida, we compared the growth, survivor-
ship and gonadal development of bay scallops planted in cages at
\arious densities and planting conditions in the Anclote estuary.
To test density effects, scallops were planted in 0.6-in L x 0.6-m
W cages, constructed from 12.7-mm-mesh. within a seagrass bed.
Densities of 50, 150 and 300 scallops per cage were tested in
triplicate. Growth, survivorship, and gonadal development were
monitored every six weeks between July 1999 and July 2000.
Planting at 150 scallops per cage resulted in the most live scallops
available for fall spawning. To test the effect of planting condition.
50 scallops per cage were planted in triplicate in each of four
treatment combinations including within and outside a seagrass
bed and either directly on the substrate or raised 20 cm above the
substrate. Growth and survivorship were monitored every si.x
weeks between September 1999 and April 2000. Results indicate
that growth and survivorship were significantly lower in the cages
planted directly on the substrate within the seagrass bed. but no
significant difference was detected among the remaining treat-
ments.
WATER LOSSES. SEASONAL MASS LOADING. AND
BEST MANAGEMENT PRACTICES FOR CRAWFISH
PONDS. Landon D. Parr*, Robert P. Romaire. and W. Ray
McClain. Louisiana State University AgCenter. Aquaculture Re-
search Station. 2410 Ben Hur Road. Baton Rouge. Louisiana
70820.
Some crawfish (Procamhanis clarkii and P. zoiuingiili(s) ponds
discharge into impaired water bodies in Louisiana. The objectives
of this research were to develop water discharge models, determine
seasonal mass loading of solids and nutrients, assess effluent qual-
ity during final drawdown (May through June), and identify best
manageinent practices for crawfish ponds. Average crawfish pond
water loss during a production cycle was 228 cm and was parti-
tioned among evapotranspiration (68%). precipitation overflow
(13%). final drawdown (13%). and infiltration (6%). Modeling
indicated that 15-cm of water storage capacity reduced precipita-
tion oserflow by 28% in high precipitation years. 61% in average
precipitation years, and 100% in low precipitation years. Predicted
mass loading was greatest in the winter (precipitation overflow)
and late spring through early summer (final drawdown). During
final drawdown, total suspended solids (TSS) were high in the first
5% and last 20% of water discharged. During final drawdown,
deep vegetated ditches provided the best TSS reduction compared
to narrow , shallow, non-vegetated ditches. Slow draining from the
water surface and avoiding drainage of the final 20% of the pond
\olume are recommended best management practices. The final
20% of the pond volume can be treated in deep vegetated ditches,
setthng basins, or constructed wetlands.
EFFECTS OF KARENIA BREVIS ON SHELLFISH: DOES
STRAIN MATTER? Susan E. Pate.* Jeffrey J. Springer, and
JoAnn M. Burkholder. Center for Applied Aquatic Ecology,
North Carolina State University, Raleigh, NC 27606; Sandra E.
Shuniway, Department of Marine Sciences, University of Con-
necticut, Groton, CT 06340,
Red tides are found in the Gulf of Mexico and the coast of
Florida and consist primarily of the toxic dinoflagellate, Karenia
hrevis (Davis). Previous studies show lipid-soluble polyether tox-
ins (bre\'etoxins, PbTx) can accumulate by .several species of shell-
fish exposed to A', hrevis. Bloom characteristics, shellfish grazing
rates, and biotransfoniiati\'e processes influence shellfish toxin
le\'els. Little is known regarding interactions between shellfish and
varying strains of K. hrevis.
Experiments were conducted involving three bivalve species
(Argopeeteii irradians. Crassostreu yiri>iniea, Mereenaria inerce-
naria). The three K. hrevis strains represent low, moderate, and
high levels of brevetoxin production and were introduced at cel-
lular concentratiims during a bloom event. Behavioral response
and grazing rates were determined for each species versus each K.
hrevis strain. In addition, we microscopically examined fecal ma-
terial to determine whether cells remained intact and viable after
passage through the digestive tract. Preliminary results indicate
that some cells pass through the shellfish digestive tract intact.
ASSESSMENT OF THE EPIZOOTIOLOGY OF PERKIN-
SUS SPP. ON THE ATLANTIC COAST OF USA USING
GENUS-. SPECIES-, AND STRAIN-SPECIFIC MOLECU-
LAR PROBES. Wolf T. Pecher*. Jose A. F. Robledo. Eric J.
Schott. and Gerardo R. Vasta. Center of Marine Biotechnology
701 East Pratt Street Baltimore. MD 21202.
P. marinus represents a major cause of mortality of the eastern
oyster {Cnissostrea viriiiiiica) along the Gulf of Mexico and At-
lantic coasts of the USA. Based the fluid thioglycolate medium
(FTM) assay. Perkinsiis infections attributed to P. marinus have
been reported as far north as Maine but although infection preva-
National Shellfisheries Association. New Orleans. Louisiana
Ahsinicls. 2003 Annual Meeting. April 1.^-17. 2003 349
lence in Northeast regions may be high, it may not correlate w ith
oyster mortality. In addition to the influence of environmental
factors, the presence of other PcrkiiisLi.s species/strains that evhibit
reduced pathogenicity for C. viri;ii}iLa may explain the.se observa-
tions. Two recently described species. P. clwsupeaki and P. tm-
drewsi that test positive by the FTM assay, can also be present in
clams and oysters, but their \irulence remains unknown. Thus, the
accurate pre\ alenee assessment of Perkinsiis v/7/j is needed for the
detailed understanding of epizootic events. To discriminate be-
tween P. iiniriiins. P. aiidrewsi and other Perkinsiis species our
laboratory has developed species-specific PCR-based assays. We
are applying these molecular probes to investigate the epizootiol-
ogy of Perkinsiis species and strains in oysters, hard clams, and
other shellfish along the East Coast (from ME to VA). [Supported
by ODRP. NOAA award NAO6RG01U1-5. through the MD Sea
Grant College, to GRV).
ECOLOGICAL EFFECTS OF FISHING: BIOLOGICAL,
PHYSICAL, AND SOCIOLOGICAL IMPACTS OF DER-
ELICT AND ABANDONED CRAB TRAPS IN MISSISSIPPI.
Harriet Perry*. Kirsten Larsen, Center for Fisheries Research
and Development. Gulf Coast Research Laboratory. College of
Marine Sciences. The University of Southern Mississippi. P.O.
Cox 7000. Ocean Springs. Mississippi 39566-7000; Bill Richard-
son and Traci Floyd. Mississippi Department of Marine Re-
sources. 1141 Bayview Avenue. Suite !01. Biloxi. Mississippi
39530.
The wire crab trap dramatically changed the Gulf of Mexico
Blue crab (Cailinectes sapidiis Rathbun) fishery. Crab traps were
introduced in Louisiana and Texas as early as 1948 and by the
mid-1950s were widely accepted throughout the Gulf. While adop-
tion of the crab trap had a positive impact on fishing efficiency and
harvest, proliferation of traps has resulted in an increase in the
problems associated with lost or discarded traps. Derelict traps
contribute to the mortality of blue crabs and other bycatch, exac-
erbate user group conflicts, create visual pollution, and may cause
damage to sensitive habitats. Derelict traps result form abandon-
ment of fishable traps by fishermen and the inadvertent loss of
actively fished traps from: I ) weather/hydrological factors, 2) de-
terioration of buoys, lines, or knots, 3) negligence in assembling
and maintaining gear, 4) use of plastic jugs/bottles as floats, 5)
clipping of float lines by vessel propellers, and 6) intentional cut-
ting of buoy lines by vandals. Conservative estimates of trap loss
for the Gulf of Mexico approach 250.000 traps per year. Hundreds
of traps litter coastal waters in eastern and western Mississippi
Sound. Concern over the magnitude of the problem and the po-
tential impacts to the blue crab resource prompted Mississippi to
develop a program to remove these traps form near shore waters.
THE REGISTRY OF TUMORS IN LOWER ANIMALS:
A RESOURCE FOR BIVALVE CULTURE HEALTH STUD-
IES. Esther C. Peters*. Tetra Tech, Inc., Fairfax. VA 22030;
Marilyn J. Wolfe and Jeffrey C. Wolf. Experimental Pathology
Laboratories, Inc., Herndon, VA 20172-0474.
Neoplastic diseases have been recognized in several orders of
bivalves. Of particular concern for culture efforts are hemopoietic
neoplasms of mussels and soft-shell clams and gonadal neoplasms
of quahogs. The etiologies of these diseases are unknown but
studies suggest that factors which could be manipulated in culture,
such as diet, genetics (hybridization or breeding for disease resis-
tance and faster growth rate to market size), and environmental
conditions (water quality, crowding) could influence the develop-
ment of these and other cellular proliferative disorders. The Reg-
istry of Tumors in Lower Animals (RTLA) has been moved to
Experimental Pathology Laboratories. Inc.. under contract to the
National Cancer Institute, and will continue to provide a global
resource for investigators interested in bivalve diseases. The col-
lection of contributed specimens and reprints will be expanded and
Internet access to a searchable and illustrated database provided.
The RTLA welcomes visitors (by appointment) and will offer
diagnosis of bivalve diseases contributed for archi\ ing and training
in comparative histopathology.
USING CREATED OYSTER REEFS AS A SUSTAINABLE
COASTAL PROTECTION AND RESTORATION TOOL.
Bryan Piazza*, John Plunket, John Supan and Megan La
Peyre. U.S.G.S. Louisiana Fish and Wildlife Cooperative Re-
search Unit. School of Renewable Natural Resources. Louisiana
State University Agricultural Center, Baton Rouge, LA 70803.
Protection and restoration of coastal shorelines remains a pri-
ority worldwide. This study tested the viability of creating sus-
tainable oyster reefs for use as a coastal protection and restoration
tool in Caillou (Sister) Lake. Louisiana. Six oyster shell reefs
(approximately 25 m x 2 m x 0.75 m) were created along the
shoreline during June 2002 in two areas representing typical low
and high-energy environments. Reefs were located approximately
3-5 m from shore (60 - 90 cm deep). Marsh vegetation was
dominated by Spartina allerniflora. Jiincus roemerianus. and Dis-
tichlis spicata. The value of reefs for protecting shorelines was
determined by tracking shoreline position and adjacent marsh
health (vegetation biomass, redox, sediment accretion) at paired
cultched and non-cultched sites. Reef sustainability was deter-
mined by measuring recruitment and survival of oyster spat. Fish-
eries value of the reef was quantified by sampling nekton. Recruit-
ment and survival of oyster spat increased throughout the spring
and summer. Fish community usage of cultched and non-cultched
sites was similar and dominated by Anchoa mitchilli. Shoreline
retreat appears to be slightly higher in high energy, non-cultched
sites. Minimal movement and reworking of shell through two
tropical storm events showed that reefs were stable.
350 Abstracts. 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
BLUE CRAB {CALLINECTES SAPIDUS) GENETIC
STRUCTURE AND DIVERSITY. Allen R. Place*. Colin R.
Steven, and Xiaojun Feng. Center of Marine Biotechnology Suite
236 701 E. Pratt Street Baltimore. MD 21202.
A responsible approach to marine stock enhancement requires
that potential negative impacts upon the gene pools of wild popu-
lations be mitigated through the use of genetically sound breeding
and release protocols. Studies over the past decade of patterns of
genetic variation and divergence in a variety of pelagic marine
organisms have demonstrated that high dispersal potential at any
of several life-history stages does not necessarily indicate high
levels of actual gene flow and uniformity in population structure.
Three published studies describing the population genetics of
Calliiiectes sapidiis all indicate substantial gene flow, with values
sufficiently high to infer panmixia between all blue crab popula-
tions from New York to Texas. Despite this high level of gene
flow, two striking patterns of temporal and geographic differen-
tiation occurred: genetic patchiness and clinal variation. These
studies were done with protein polymorphisms (allozymes) which
are less diagnostic of population substructure than the more vari-
able genetic markers found in mitochondrial and nuclear DNA. To
help distinguish hatchery-raised crabs from wild cohorts we have
characterized the genetic variability in both the mitochondrial ge-
nome and nuclear genomes of Calliiiectes sapidus. The implica-
tions of these findings to the overall genetic structure of Calli-
nectes sapidus will be addressed.
A COMPARISON OF FINFISH ASSEMBLAGES ON SUB-
TIDAL OYSTER SHELL (CULTCHED OYSTER LEASE)
AND MUD BOTTOM IN BARATARIA BAY. LOUISIANA
John Plunket*. Megan La Peyre. U.S.G.S. Louisiana Fish and
Wildlife Cooperative Research Linit. School of Renewable Natural
Resources. Louisiana State University. Baton Rouge. LA 70803.
Recent research suggests that oyster reefs provide unique three-
dimensional habitat for many tlsh species. Along the northern
shore of the Gulf of Mexico, oyster shell bottoms are predomi-
nantly flat, sublidal and cultched. providing a very different habi-
tat. In this study, we compared finfish assemblages and gut con-
tents at subtidal oyster shell (cultched oyster lease) and mud bot-
tiims in Barataria Bay. Louisiana. Three mud and three shell sites
were sampled from October 2001 to October 2002. using gill nets
with mesh ranging from 25.4-63.5 cm. and 60 x 50 cm substrate
trays. Data from the gill nets were used to compare fish assem-
blages, and to document diets through gut content analysis. Data
from the substrate trays were used to document benthic fish and
invertebrate communities associated with the subtidal cultched
oyster shell habitat. Finfish abundance was greater at shell
(N = 223) versus mud (N= 170) bottoms, with higher numbers of
sciaenid fishes over shell. Substrate trays collected a variety of
benthic fish and invertebrates, primarily naked gobies (Gohiosoma
base), skilletfish (Gobiesox stnimosis). toadfish (Opsaiuis beta)
and xanthid crabs. These results support the contention that shell
bottoms support unique communities of tlsh. as compared to mud
bottom habitats.
FIBER DIGESTION IN THE BLUE CRAB. CALLINECTES
SAPIDUS. Allen R. Place*. Andrea Findiesen. and Nilli Zmora.
Center of Marine Biotechnology 701 East Pratt St. Baltimore. MD
21202.
A wide range of digestive enzymes have been reported in Crus-
tacea indicative of the diverse dietary preferences of the different
species. Two of the most important carbon containing compounds
in the blue crab diet are chitin (an unbranched homopolymer of b
1-4 linked N-acetyl-D-glucosamine residues, NAG) and cellulose
(an unbranched homopolymer of b 1-4 -D-glucose residues. Glc).
The traditionally held view of chitin and cellulose digestion in
higher animals and invertebrates has been that gut microbes confer
the ability to degrade these two polymers. However, recently the
genes for chitinase and cellulase have been detected in the ge-
nomes of Crustacea. Accordingly, using degenerate primers de-
signed from aligned sequences of chitinases and cellulases, we
have begun screening a heptopancreas cDNA library of the blue
crab. Currently, we have isolated a 479 bp fragment that is highly
homologous to the vertebrate and insect chitinase and just starting
to probe for crab cellulases. Given that these two polymers are the
two most abundant and renewable energy resource on earth, ef-
fective utilization of these fibers especially in diets for aquaculture
rearing will be an important key to improving production and feed
conversion efficiency in the future.
A COMPARISON OF NEKTON USAGE OF MUD BOT-
TOM, CREATED LIMESTONE. SHELL. AND NATURAL
SHELL REEF HABITATS IN TERREBONNE BAY. LOUI-
SIANA. John Plunket*. Gary Peterson. Bryan Piazza and
Megan La Peyre. U.S.G.S. Louisiana Cooperative Fish and Wild-
life Research Unit. School of Renewable Natural Resources, Loui-
siana State University Agricultural Center, Baton Rouge, LA
70803.
Restoration of coastal environments increasingly involves habi-
tat creation for fisheries species. The creation of artificial reefs is
based on the assumption that estuarine hard-bottom habitats sup-
port more diverse, complex communities than soft bottom habitats.
In Louisiana, the creation of artificial reefs has recently become a
focus of activity among recreational fisherman and coastal man-
agers. In 2002, we compared finfish abundance on a natural shell
reef, a created clam shell reef, a created limestone rubble reef, and
a mud bottom site in lower Lake Pelto, Louisiana. The four sites
were sampled over one year using 200' experimental gill nets, an
8' otter trawl and fish traps. On average, species diversity was two
times higher on natural and created reefs (N= 15), as compared to
mud bottom (N = 7). The created limestone and natural reef con-
sistently supported the more diverse, as well as the more even
(Pielou's J) communities throughout the year. Sorenson's commu-
National Shellfisheries Association. New Orleans. Louisiana
Abstracts. 2003 .-Xnnual Meeting. April 13-17. 2003 3.SI
nity similarity index indicates large dissimilarities between the
created reefs and the mud bottom (S<0.23). and more similar com-
munities between both created and natural reefs (S>0.5). The two
artificial reefs support communities of greater diversity and even-
ness than mud bottom habitat, and are comparable to natural reefs
in diversity, but vary in species composition.
CONSUMER PREFERENCES AND ATTITUDES TOWARD
IRRADIATED OYSTERS. Benedict C. Posadas* and Linda S.
Andrews. Mississippi State University. Coastal Research and Ex-
tension Center 2710 Beach Blvd. Ste. 1-E. Biloxi, MS 39531.
Consumer attitudes and preferences toward raw oysters in gen-
eral, and irradiated oysters, in particular, were evaluated from
results of consumer surveys conducted through personal and tele-
phone interviews. Seventy five interviews were conducted at the
MSU-Coastal Aquaculture Unit Open House in Gulfport. Missis-
sippi on December 6. 2001. Another survey was conducted at the
MSU-Coastal Research and Extension Center booth among 140
participants of the 2002 International Boston Seafood Show in
Boston. Massachusetts on March 12-14. 2002. Telephone inter-
views with a simple random sample of adults living in the Balti-
more and Houston MSAs in households w ith telepht)nes were done
by the Survey Research Unit (Social Science Research Center at
Mississippi State University) in June of 2002. Households were
selected using random digit dialing procedures. Of the eligible
respondents contacted in the Baltimore Metropolitan Statistical
Area (MSA). 610 completed the interview and S5 refused to par-
ticipate. Of the eligible respondents contacted in the Houston
MSA. 606 completed the interview and 67 refused to participate.
FORM AND FUNCTION IN OYSTER REEFS: INFLUENCE
OF REEF MORPHOLOGY ON HABITAT FUNCTION AND
OYSTER SURVIVAL Martin H. Posey*, Troy D. Alphln,
Heather D. Harwell and Thomas J. Molesky. Center for Marine
Science. UNC-Wilmington 5600 Marvin K. Moss Lane Wilming-
ton. N.C. 28409.
With the decline in natural oyster's reefs there is increasing
interest in restoration of reef habitat for fishery and ecosystem
functions. Oyster reefs provide important structural habitat and
have significant ecosystem impacts. However, the function of oys-
ter reefs varies with reef morphology, especially venical complex-
ity that may affect 3-diniensional characteristics of the reef sur-
face, edge convolution that may affect encounter surfaces for in-
tertidal reefs and reef fragmentation. We have begun a multi-year
study examining the influence of vertical complexity, edge con-
volution and fragmentation on faunal use. ecosystem function, and
oyster settlement and survival on intertidal created oyster reefs in
southeastern North Carolina. Reefs have been established with
blocked high and low vertical coinplexity and circular versus con-
voluted edge as well as small and larce frasment reefs. We are
assessing sediment nutrient fluxes, benthic microalgae. infauna.
epifauna. and nekton use of these reefs through a variety of sam-
pling approaches to examine community responses to variations in
landscape factors. Reefs were established in 2002 and initial re-
sults indicate strong effects of vertical complexity and fragmenta-
tion and weaker effects for edge characteristics. Efforts to restore
oyster reefs should consider the potential influence of reef design
on ultimate habitat function.
REPRODUCTION, BIOENERGETIC AND SUMMER
MORTALITY OF CRASSOSTREA GIGAS: EXPERIMEN-
TAL .APPROACH. Stephane Pouvreau*. Martha Enriquez-
Diaz, Pierrick Le Souchu, .Jean Paul Connan, Bertrand Le
Roy, Christian Mingant. .Jeanne Moal, Maryse Delaporte, Jean
Rene Le Coz, and Jean Francois Saniain, *UMR PE2M
WVPhysiologie et Ecophysiologie des Mollusques MarinsWV". Sta-
tion Experimentale dWVArgenton. 1 1 presqu\\\"ile du vivier. 29840
Argenton (FRANCE).
As a part of the French MOREST program, we examine ex-
perimentally, in 2002. the relationships between food level, repro-
ductive processes, bio-energetic status and mortality on 3 batches
of the same hatchery oyster population, produced in 2001 . Each lot
underwent a same annual temperature cycle (from 8 to 20 "C). a
same food composition (4 algae), but a different food level: low
(-30 cell.jjLl-1 ). medium (-60 cell.|jil-l) and high (-100 cell.|jLl-l).
Each month, several parameters were followed: (I) somatic
growth, storage, gametogenesis using quantitative histology: (2)
clearance rate, absorption efficiency, oxygen consumption and
scope for growth (SFG). (3) biochemical composition. Results
demonstrated that oysters under high food availability showed an
accelerated gametogenesis and the highest reproductive effort. At
the maximum of gametogenesis development (i.e. July), these oys-
ters exhibited also the highest oxygen consumption and conse-
quently the lowest SFG values. E.xperimental infection (by Vibrio
lentils as infectious agent) confirmed this relative weakness in
relation with the reproductive effort.
As a conclusion, it appears that food level that controls the
reproductive effort can generate a bioenergetic imbalance at high
trophic conditions. Thus, summer mortalities in eutrophic areas
could be partly explained by these processes.
A COMPARISION AND FEASIBILITY STUDY OF TWO
DIFFERENT BIOMONITORING SYSTEMS USING THE
BLUE MUSSEL, MYTILUS EDULIS. AND THE AMERICAN
LOBSTER, HOMARUS AMERICANVS. Heidi Pye*, Winsor
H. Watson III, Christopher Rillahan, Rachel Hamilton, and
Jennifer Wishinski. 46 College Rd Zoology Department-UNH
Durham. NH 03820.
The advantages of biomonitoring in accordance with traditional
techniques include: 1. behavioral and physiological responses are
more sensitive indicators of contaminant-induced stress. 2. While
352 Abstracts. 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
traditional instrumentation measures specific substances, organ-
isms integrate all stressors to provide an indicator of overall water
quality. 3. and if utilizing keystone species the information will
help assess impact of the contamination at population and com-
munity levels. To effectively use a bioindicator it is necessary to
characterize its response and sensitivity (detection threshold) to
contaminants. Our goal was to compare the response and sensi-
tivities of the American lobster. Homanis americanus and the blue
mussel. Mytihis echilis. to four different heavy metals (CuCl.
CrC13. PbCI2, CdC12) common in the Great Bay Estuary. In gen-
eral, detection levels were lower for mussels (O.Sppm CuCl.
<lppm PbC12. >30ppm CdC12) than lobsters (Ippm CuCl. 50ppm
CrC13. > 50ppm PbC12. CdC12). Clear responsiveness was limited
to CuCl which occurred close to lethal levels (for H. americanus
Ippm response. 2ppni LD50). Given these results we would rec-
ommend using mussels, due to their higher sensitivity and ease of
use. The only drawback is that mussels are sensitive to a variety of
other environmental perturbations that can make responses to
heavy metals difficult to elucidate.
LARVAL ECOLOGY: MOLECULAR TOOLS FOR THE
BLACK BOX? Paul D. Rawson. School of Marine Sciences
5751 Murray Hall. University of Maine Orono, ME 04469-5751.
Many marine invertebrates, including ecologically and com-
mercially valuable shellfish, have biphasic life histories with a
relatively long-lived and highly dispersive larval stage. Ecologists
have recognized the role that larval supply and settlement play in
population and community dynamics while geneticists have fo-
cused on the impact that larval dispersal has on the distribution of
genetic variation. Dispersal and settlement, in turn, are dependent
on the local abundance of larvae, which can be extremely variable
in space and time. Traditional methods for identifying and enu-
merating larvae can be time consuining. and because of the mor-
phological similarity between larvae of many species, requires
specialized training. Thus, our understanding of the links between
planktonic processes that generate larval patchiness and larval
settlement can perhaps be represented by a black box. Molecular
methodologies, in particular PCR-based methodologies, provide
tools for peering into this black box by allowing the rapid, and
perhaps quantitative, analysis of larval abundance. We will discuss
the development of some of these methodologies, the advantages
and pitfalls associated with them, as well as providing examples of
their application from work currently being conducted in our lab.
STATUS OF PERKINSUS MARINVS IN GALVESTON BAY.
TEXAS: RESULTS OF THE DERMOWATCH PROGRAM
Sammy M. Ray.* Department of Marine Biology. Texas A&M
University at Galveston. Galveston. TX 77553; Thomas M. So-
nlat. Department of Biology. Nicholls State University.
DermoWatch is a web site (www.blueblee.com/dermo). a
monitoring program and an online community for the management
of the oyster parasite, Pcrkinsus inariniis. The web site contains an
embedded model, which calculates a time to critical level of dis-
ease from an initial weighted incidence of disease and water tem-
perature and salinity. Six public reefs and three private leases in
Galveston Bay have been sampled monthly since December 1998.
The web site displays the most recent data from each site on the
home page and archives all data, such that an historical record is
maintained. Historical data show high levels of disease during the
drought years of 1999 and 2000. With the cessation of the drought
in 2001 and heavy rains associated with tropical storm Allison in
June of 2001, disease levels throughout the Bay have been de-
pressed.
SEASONAL AND TEMPORAL VARIABILHTY IN CONDI-
TION INDEX AND TISSUE BIOCHEMISTRY OF ELLIP-
TIO COMPLANATA. Deborah Raksany*. Catherine M.
Gatenby and Danielle A. Kreeger. The Academy of Natural Sci-
ences 1900 Ben Franklin Pkwy Philadelphia. PA 19103:
Due to diminishing biodiversity and habitat, it is imperative
that we better understand the biology and the ecological function-
ing of our existing freshwater mussel populations. Temporal vari-
ability in the condition and physiological status of marine shellfish
has been well studied, but there remains a dearth of knowledge
with respect to these trends in freshwater mussels. Our goal was to
quantify variability in physiological condition of Elliptic) coinpla-
iiata. a common freshwater mussel in the Atlantic drainage. Con-
dition index and proximate tissue biochemistry (protein, lipid, and
carbohydrate) were monitored in adults collected from a healthy
population over a three-year period. Both parameters varied sea-
sonally and among years for similar seasons. For example, condi-
tion index peaked in August of 2000. but reached its peak in
October of the following year. These results reflect the reproduc-
tive and seasonal conditioning processes of these animals, which
may be responsive to environmental cues. By understanding tem-
poral shifts in the physiological status of these animals in nature,
we will be better equipped to gauge their functional roles in fresh-
water ecosystems and formulate appropriate diets to sustain them
in captivity.
NUCLEIC ACID-BASED AQUATIC PATHOGEN MO-
LECULAR DIAGNOSTICS FOR DETECTION, RE-
SEARCH AND ENVIRONMENTAL MONITORING Kim-
berly S. Reece. Virginia Institute of Marine Science. Gloucester
Point. VA 23062.
Advances in molecular genetic technology have facilitated
progress on many fronts of aquatic disease research including
pathogen identification, detection, and studies examining transmis-
sion dynamics, epizootiology. virulence mechanisms and host/
parasite/environment interactions. Probes for in situ hybridizations
and primers for use in PCR are now available for many pathogens
found in the aquatic environment. These nucleic acid-based mo-
National Shelltisheries Associatiiin. New Orleans, Louisiana
Ahsimay 2003 Annual Meeting. April 13-17. 2003 353
leciilardeteetion methods can improve sensitivity and efficiency of
disease diagnoses and detection of organisms in environmental
samples, especially where it is difficult and/or time-consuming to
isolate and identify pathogens. Rapid and accurate molecular de-
tection assays have been developed to facilitate both field moni-
toring programs and studies to examine the effects of various
environmental parameters on growth and distribution of patho-
gens. Studies that employ different molecular detection techniques
will be presented including those where real-time PCR assays are
being used to quantify the number of pathogen cells in water
samples for environmental monitoring programs and disease trans-
mission studies. In situ hybridization assays ha\'e been developed
for confirming the identity of parasites in host tissues and for
detecting pathogenic organisms in the gut contents of bivalves that,
because of their filter-feeding behavior, are natural integrators of
the water column.
VALIDATION OF POST-HARVEST PROCESSING OF VI-
BRO PARAHEMOLYTICUS IN OYSTERS: SPEED BUMPS
ON THE ROAD FROM THE RESEARCH LAB TO THE
PROCESSING PLANT. P. VV. Reno*. V-C. Su. M. Morrissey,
and D. Nisbet. HMSC 2030 S. Marine Science Dr. Newport, OR
97365-52%.
Small scale laboratory experiments were canied out to deter-
mine the efficacy of high pressure processing in inactivating
Vibrio paralu'inolyticus (VP), particularly serotype 03;K6, with in
Pacific oysteis. Oysters were held at HMSC isolation facility with
u\ -irradiated, sand-filtered seawater that had no detectible VP in
the incoming water or in oysters. The oysters were exposed \ ia
bath for 3 h in static seawater with between 103 and 106 cfu/mL
of VP. Bacterial counts per gram of oyster meat approximated the
VP count per niL water. Bacterial counts remained stable in oys-
ters for at least 10 h at IOC. The results of these tests indicated
>l05/g reduction in colony counts was achieved at 3IOmP;i/2min
in a 1.5-L pressure unit. Transfer of the technology from the small
scale (1.5 L capacity) research laboratory to a pressure unit oper-
ating under commercial processing conditions was undertaken to
validate the process to accede to anticipated FDA requirements.
Using a commercial pressure unit of 42 L volume, a series of
time/pressure combinations are currently under way to determine
the efficiency of killing under commercial conditions on oysters
exposed by the techniques used in the research laboratory. The
process is still ongoing, but results appear promising.
COMPUTATIONAL FLOW MODELING OF AQUACUL-
TURE SYSTEMS. John Richardson*, Alden Research Labora-
tory, 30 Shrewsbury St., Holden. MA 01520-1843; Carter Newell.
Great Eastern Mussel Farms, P.O. Box 141. Tenants Harbor.
Maine 04860.
The successful design of floating raft-culture systems requires
knowledge of how water circulates through the raft-culture struc-
tures. In this research advanced Computational Fluid Dynamics
(CFD) Techniques were used to model fiow through ratl-culture
systems used to grow oysters and Blue Mussels. The basic mod-
eling techniques are general, and they can also be used to model
the flow through other types of aquaculture systems (marine or
teiTestrial). The analysis techniques used for this study are capable
of accurately simulating the 3-dimensional flow of water through
raft-culture structures located in areas with complex bathymetries.
The analysis scheme can. additionally, be used to simulate the
transport of nutrients and wastes through the floating rafts.
CHARACTERIZATION OF THE CRASSOSTREA VIR-
GINICA SLCllA GENE (FORMERLY NRAMP). Jose A. F.
Robledo* and Gerardo R. Vasta, COMB, UMBI. University of
Maryland. Baltimore. MD 21202, USA.
Pt'ikinsits imiriniis has been associated to extensive damage to
oyster populations, with catastrophic consequences for shellfish-
eries. Although, selective breeding approaches for development of
disease-resistant oyster stocks are promising, the identification of
genes that are directly linked to disease-resistance/susceptibility
represents an attractive alternative. The Slclla (former Nramp:
natural resistance-associated macrophage protein) is a divalent cat-
ion transporter, demonstrated to be a determinant of resistance/
susceptibility to intracellular pathogens. Most parasites have de-
veloped efficient mechanisms for iron acquisition from their hosts.
Reciprocally, most hosts have developed mechanisms to prevent
pathogens from acquiring iron. Iron sequestration from the patho-
gen is also a non-specific host response to infection (nutritional
immunity), and SIcl la is a critical component in this response. We
have already characterized the P. inariinis SIcl 1 (PmSlcl la) and
obtain partial sequence of the C. viriiiuica Slclla (CvSlclla).
Sequence information was used for screening a C virginica ge-
nomic library resulting in several clones" positives for CvSIcl la.
The characterization of CvSlclla gene in both host and parasite
will provide insight into their competition for iron, and yield in-
formation on the mechanisms underlying disease susceptibility
(Grant No NA06RG010I-5 from ODRP. NOAA, through the
Maryland Sea Grant College to GRVj.
PERKINSUS MARINUS CELLULAR BIOLOGY USING EX-
PRESSION SEQUENCE TAGS (EST). Jose A. F. Robledo*.
Eric J. Schott. and Gerardo R. Vasta COMB, UMBI, University
of Maryland, Baltimore, MD 21202, USA.
During the last five years virtually all fields of biology have
benefited from the tremendous volume of information generated
by genomic approaches. Embedded within genomic sequence data
is information needed for identifying targets for drug development
and for dissecting the biological aspects that may constitute the
basis for infectivity and pathogenicity. Perkinsus marimis has been
associated with mass mortalities of the eastern oyster, Crassoslrea
viriiinica. for more than 50 years and although substantial progress
354 Abstracts. 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
in understanding the disease has been accomplished, effective pre-
vention or treatment methods are still lacking. Previously, we pre-
sented a dataset consisting of 300 ESTs generated from two P.
monniis cDNA libraries constructed using P. inariniis propagated
in standard culture medium and in medium supplemented with C.
virginica serum. We now present the analysis of a more extensive
EST dataset corresponding to both libraries, focusing on those P.
mahniis genes or metabolic pathways that may be unique to this
parasite, or which have been targeted for intervention in other
parasite species. Based on our increased knowledge of P. inariiiiis
genomics/biology. possible strategies to enhance anti-parasite re-
sponses in the oyster will be discussed [ODRP. NOAA award
NA06RG0101-5. through the MD Sea Grant College, to GRV].
DEVELOPMENT OF AN INDIVIDUAL. ENERGY-
BALANCE BASED. GROWTH MODEL FOR THE MA-
NILA CLAM (RUDITAPES PHILIPPINARUM). J. Five
Sainte Marie*, S. E. Ford, E. Hofmann. F. Jean. J. Klinck. C.
Paillard. E. Powell. LEMAR. Univ. Bretagne Occidentale Inst.
Univ. Europeen de la Mer. Place Copernic F-29280 PLOUZANE.
To study Brown Ring Disease in the Manila clam. Ritditapes
pliiUppiiiaiiim. caused by the bacterial pathogen Vibrio tapetis. an
environment-host-pathogen interaction model is being developed.
As a base upon which to build a population model, an individual
growth model, which does not include the pathogen, was first
developed. The aim of the present study was to calibrate, to vali-
date and to do a sensitivity analysis on this model. The model
simulates the length and weight increase of an average individual
under forcing of two environmental variables: temperature and
food. Model simulations approximate the scope for growth and
spawning events observed in nature. On the other hand, the simu-
lations showed that chlorophyll a concentrations are not an ad-
equate substitute for food availability for this infaunal bivalve.
Although additional data are needed to develop a relationship be-
tween growth and food availability in the field, .sensitivity analysis
showed that this model is responsive to the parameters that deter-
mine scope for growth.
sure to superoxide and hydrogen peroxide (H202). but not hypo-
chlorite. These findings are consistent with two observations: ( I )
Viable trophozoites are able to destroy hydrogen peroxide in vitro;
(2) extracts of P. imiriiuis contain abundant iron-type superoxide
dismutase (FeSOD) activity, as well as ascorbate dependent per-
oxidase (APX) activity. We previously described the cloning and
characterization of two P. imiriiiKs FeSODs that have the potential
to convert superoxide to H202 in vivo. Recombinant PmSODl
and PmSOD2 proteins have been crystallized for structural analy-
ses, and used to raise specific antisera for immunolocalizations.
The APX activity appears to be a 35 kD protein. Continuing analy-
sis of P. marinus SOD and APX functions will be presented. The
unique characteristics of the P. marinus antioxidant system may
provide the basis for disease prevention or therapy strategies | Sup-
ported by ODRP. NOAA award NA06RG0I0I-5. through the MD
Sea Grant College, to GRV].
CORRELATION OF FLAT PEARL STUDIES WITH
PEARL SAC FORMATION IN A FRESHWATER MUSSEL
(CYRTONAIAS TAMPICOENSIS). Donald Shepherd*. Profes-
sional Pathology Laboratories. Ltd. P.O. Box 326, Tow. TX
78672.
Flat pearl studies can illustrate the process of biominerali/atiiin
of molluscan shell, by placement of a flat material, between the
mantle and inner shell of the mussel. Protein and calcium carbon-
ate crystals can be evaluated by specific stains and light and po-
larization microscopy. The initial stage is secretion of a protein
layer of glycoproteins on the insert as the nucleating protein sheet.
After several days, secretion of calcium carbonate crystals begins
trom the epithelial cells of the mantle. These crystals are calcite.
which form rhomboid crystals by 15 to 17 days. A .second crystal
forms on the calcite crystals; it is an isoform of calcium carbonate
- aragonite. The switch from calcite to aragonite is accomplished
by a change in acid protein secretion (Lustrin A). The aragonite
forms small bricks as in a wall to form the mother-of-pearl nacre.
Photos of natural pearls from Tampico pearly mussels will be
presented to illustrate natural pearl formation.
THE ANTIOXIDANT PATHWAY OF PERKINSUS MARI-
NUS: FUNCTIONAL ANALYSIS AND LOCALIZATION OF
TWO IRON SUPEROXIDE DISMUTASES. Eric J. Schott*.
Jose A. F. Robledo. Wolf T. Pecher. Florence Okafor. and
Gerardo R. Vasta, Center of Marine Biology 701 E. Pratt St
Baltimore. MD 21202.
The economic and environmental impacts of Pcrkinsiis mari-
nus epizootics make imperative the understanding of this parasite's
virulence mechanisms. It has been proposed that viable P. marinus
trophozoites rapidly suppress or detoxify reactive the oxygen burst
characteristic of oyster hemocytes. We now report that cultured P.
marinus trophozoites are remarkably insensitive to transient expo-
CHARACTERISATION OF SUMMER MORTALITIES OF
C.GIGAS OYSTER IN FRANCE IN RELATION TO ENVI-
RONMENTAL PARAMETERS. P. Soletchnik. M. Ropert.
A. Huvet. J. Moal. L. Dcgremont. E. Bedier, J.F. Bouget. B.
Dubois, JL. Martin. M. Enriquez Diaz. N. P'aury. O. Le Moine,
T. Renault. B. (lagnaire and J.F. Samain. Ifremer 17390 La
Tremblade, France.
Field characterization of summer mortality was performed in
France in the frame of the Morest project. Natural and hatchery
spat were compared between three oyster production areas in
France. Regardless of the natural or hatchery origin, oysters died
during the reproduction period after temperature reaches 19°C.
National Shelltishcries Association, New Orleans, Louisiana
Absiivcrs. 2003 Annual Meeting, April 13-17. 2003 355
Thus, in southern ureas, temperature accelerated gametogenesis of
small spat ( 10mm) as well as adults, and mortality appeared for the
two age classes. In contrast, sexual maturation proceeded more
slowly in northern where spat mortality was lower compared to 18
months old oysters. Hov\e\er, critical gametogenesis and tempera-
ture were not sufficient to induce mortalities, as observed in e.x-
aniples w ith stable environment. Alternatively sediment proximity
m addition to oyster manipulations increased mortality during
spring and summer, suggesting that some additional environmental
stresses were necessary to reproduce the phenomena. These inter-
action processes will be detailed in the other Morest contributions.
course of recent human history - a decadal time scale. Analysis of
long term trends in oyster settlement periodicity since 1960 in
three major sub estuaries (James, Piankatank and Great Wicomico
Rivers) of the Chesapeake Bay show marked changes in this pe-
riodicity within the 40 year time frame with the 50th percentile of
cumulative recruitment occun'ing between day 194 and 250 of the
year depending on year and location. Significant coherence in
interannual variation is observed across a wide range of sites.
These are discussed in relation to pre- and post-disease (both MSX
and Pfrkinsus) events, periods characterized by high and low river
flow, varying harvest pressure, and trends arguably associated with
alobal warming.
A COMPARISON OF TWO OYSTER {CRASSOSTREA VIR-
GINICA) STOCKS TO DETERMINE SUITABILnV FOR
USE IN OYSTER REEF RESTORATION IN VIRGINIA
Laurie Carroll Sorabella* and Mark W. Luckenbach, Virginia
Institute of Marine Science P.O. Box 1346 Gloucester Point. VA
23062.
Restoration efforts for eastern oysters (Crassostrea virginica)
in Virginia have focused on constructing sanctuary reefs that are
intended to serve as spawner sanctuaries. Frequently, these reefs
are stocked with hatchery-produced oysters to enhance regional
recruitment rates. An important unresolved issue is the suitability
of specific oyster stocks to achieve maximal reproductive output
on sanctuary reefs. The efficacy of using stocks selected for aqua-
culture verses wild stocks for oyster reef restoration is not well
established. We compared the performance of two hatchery-reared
oyster stocks, the CROSBreed selected stock and a wild-caught
oyster stock (Lynnhaven), after deployment onto reefs in the
Lafayette River (Chesapeake Bay). Performance was evaluated
based on growth, survival, female fecundity, sex ratio, disease
status and cumulative egg production. Results indicate that repro-
ductive performance of the two stocks varied depending on which
disease predominated. Where MSX disease pressure was high, the
CROSBreed stock outperformed the Lynnhaven stock for cumu-
lative egg production; where dermo disease pressure was high, the
Lynnhaven stock outperformed the CROSBreed stock. This work
suggests that to maximi/'e reproductive output, broodstocks used in
reef restoration should be selected based on knowledge of disease
pressure in the region.
DECADAL SCALE CHANGES IN SEASONAL PATTERNS
OF OYSTER RECRUITMENT IN THE VIRGINIA SUB ES-
TUARIES OF THE CHESAPEAKE BAY. Melissa South-
worlh* and Roger Mann. Virginia Institute of Marine Science
P.O. Box 1346 Gloucester Point, VA 23062.
Reproductive periodicity of sessile estuarine invertebrates re-
flects local seasonality of both environmental (temperature, salin-
ity) and biological (food) parameters. Estuaries are ephemeral fea-
tures in geological time, but considered somewhat constant in the
FIRST REPORTED OCCURRENCE OF MSX IN CANADA.
Mary F. Stephenson*, Sharon E. McGladdery, Michelle Mail-
let and Anne Veniot. Gulf Fisheries Centre. Department of Fish-
eries and Oceans. P.O. Box 5030. Moncton. New Brunswick.
Canada EIC 9B6: Gary Meyer, Pacific Biological Station. De-
partment of Fisheries and Oceans, 3190 Hammond Bay Road,
Nanaimo. British Columbia. Canada V9T 6N7.
The first reported occurrence of MS.X (HupUispdiidian iiclsdni)
in American oysters (Cnissostrea virf>iiiic(i) was observed on the
Atlantic Coast of Canada in October 2002 associated with mor-
talities of >80% in adult oysters from St. Patrick's Channel. Bras
d"Or Lakes, Nova Scotia. Histological examination revealed the
plasmodial stage of MSX with confirmation using DNA probes
received from the Office International des Epizooties (OIE) Ref-
erence Laboratory for Haplosporidiosis at the Virginia Institute for
Marine Science. In collaboration with the Provinces. Industry, and
First Nations, an extensive disease survey was conducted from
October to December 2002 while affected areas were closed to the
harvest of oysters. Heavy infections, adult oysters with plasmodia
or spores, were contained within Bras d'or Lakes while light back-
ground levels were described from other areas. Stakeholders con-
tinue to work collaboratively on the development of MSX control
strategies within Atlantic Canada.
A QUANTITATIVE. REAL-TIME PCR ASSAY TO DE-
TECT THE PARASITIC DINOFLAGELLATE HEMATOD-
INWM SP. IN BLUE CRABS, CALINECTES SAPIDUS. Colin
R. Steven*, Kristen Hunter-Cevera, Allen R. Place, Mike
Sheppard, and Dick Lee, Center of Marine Biotechnology Suite
236 Baltimore, MD 21202.
Hematodinium sp. is a parasitic dinotlagellate that infects and
kills several species of commercially valuable crustaceans, includ-
ing the blue crab. This dinoflagellate is found in several different
morphologies in the hemolymph and tissues of blue crabs. Hema-
todinium infections in the Chesapeake Bay show strong salinity
and temperature dependencies during their seasonal fluctuations.
We present our work towards the development of an ultra sensi-
356 Ahstnicts. 2003 Annual Meeting. April 13-17. 2003
National Sliellfisheries Association. New Orleans, Louisiana
tive. real-time, fluorescence-based. PCR assay for the detection
and quantification of Hciuntddiniiiiii infection. This assay builds
on a previously developed PCR-based diagnostic that relies on
specific oligonucleotide primers designed against a section of the
Hematodiniiim 18S rRNA gene (AF421184). Our quantitative,
real-time assay incorporates a fluorescently-labeled. gene-specific
probe as well as two gene-specific primers which allow us to
accurately detect approximately 1.4 Heniatiicliniiiin cells/ml
hemolymph. This new diagnostic tool will allow investigators to
quickly and easily monitor the extent and severity of Heniatod-
iiuiim infections in blue crabs, and ensure that infected crabs are
not released from hatcheries.
THE MITOCHONDRIAL GENOME OF THE BLUE CRAB,
CALLINECTES SAPIDUS. Colin R. Steven*. Xiaojun Feng,
Allen R. Place, and Jeffrey L. Boore. Center of Marine Biotech-
nology Suite 236 701 E. Pratt Street Baltimore. MD 21202.
In animals. mtDNA is generally a small (15-20 kB) genome
containing 37 genes that is maternally inherited. There is generally
a single large non-coding region which, for a few animals, is
known to contain controlling elements for replication and tran-
scription. Animal mtDNA displays extensive intraspecific poly-
morphism {often in the non-coding cimtrol region) and often
evolves faster than typical single-copy nuclear DNA. Most
mtDNA variants involve nucleotide substitutions or small length
changes; gene order is highly stable over short evolutionary time.
No published studies using blue crab mitochondrial polymor-
phisms exist and the only crustacean mitochondrial genome de-
posited in GENBaiik is that for Artemia. Recently the DOE Joint
Genome Institute has begun a Mitochinidrial genomics program.
We ha\e initiated a collaborative project to sequence the entire
Callinectes sapidits mitochondrial genome which w ill allow us to
find variable regions for distinguishing the mothers of hatchery
derived juveniles from those in the wild. Depending of the vari-
ability observed, these same markers would assist in defining the
genetic substructure of blue crab in the Chesapeake Bay.
Investigators use microsatellites to distinguish genetic subpopula-
tions as well as individuals at the genetic level with a very high
degree of certainty. We have isolated approximately two dozen
dinucleotide and tetranucleotide microsatellite loci, and are in the
process of screening these loci to determine their usefulness. Once
validated, these microsatellite loci will be used to examine the
genetic structure of the Chesapeake Bay blue crab fishery, and to
determine the impact that restocking efforts would have on the
natural fishery.
SETTLEMENT, SURVIVAL. AND PREDATION OF RED
KING CRABS ON NATURAL AND ARTIFICIAL SUB-
STRATA. Bradley G. Stevens*. NMFS/NOAA Kodiak Fisheries
Research Center 301 Research Ct.. Kodiak. AK 99615; and Kathy
Swiney.
In tests with structurally complex live substrata, postlarxal
(glaucothoe) and juvenile red king crabs ParaUthodcs ccinitsclniti-
iiis prefen'ed hydroids and algae, over sand or worm colonies.
Survival to stage CI was highest for controls, least on sand, and
intermediate on other substrata. Predators (larger crab) caused in-
creased mortality of glaucothoe, but neither shelter presence or
type, or predator size had any effect. Survival of juvenile crabs was
significantly decreased by shelter absence, predator presence, and
predator size. Density of juvenile crabs on shelters was higher than
that o( glaucothoe. and increased in the presence of larger preda-
tors, whereas that of glaucothoe did not. Despite active selection
for complex substrata by settling glaucothoe. significant predation
occurs there, and behavior of glaucothoe is not compensatory. In
contrast. juNcnile crabs modify their behavior to achieve higher
densities in sheltered habitats, which dampens the effect of preda-
tion. These survisal strategies have probably evolved to compen-
sate for the much greater risk of predation in open habitats. Bio-
genic oases are important to settling larvae, and should be pro-
tected from disturbance by fishing activities. Knowledge of
settlement behavior is essential prior to considering the potential of
king crabs for stock enhancement or aquaculture.
DEVELOPMENT OF MICROSATELLITE MARKERS IN
THE BLUE CRAB, CALLINECTES SAPIDUS. Colin R.
Steven, Johnathan Wilkes, Allen R. Place. Jessica Hill, and
Brian Masters. Center of Marine Biotechnology Suite 236 701 E.
Pratt Street Baltimore. MD 21202.
Current tagging methods for blue crabs, which include, fluo-
rescent elastomers and coded wire tags can be expensive, labor-
intensive and/or relatively short-lived. We have initiated the iden-
tification and characterization of genetic markers, or microsatel-
lites, to augment current tagging methods. Microsatellites. or
simple sequence repeats (SSRs), are tandemly repeated units of
two to six nucleotides, located randomly throughout the genome of
all organisms. The high variability among these loci has become a
powerful and popular tool for ecology and population genetics.
USE OF LOG PILING STRUCTURES AS ARTIFICIAL
HABITATS FOR RED KING CRABS PARALITHODES
CAMTSCHATICUS. Bradley G. Stevens*. NMFS/NOAA Ko-
diak Fisheries Research Center 301 Research Ct.; J. Eric Munk,
and Peter A. Cummiskey.
Juvenile king crabs use wooden dock pilings as habitats. We
studied whether pilings could be used to mitigate for natural habi-
tat lost during construction of a breakwater. Scuba divers counted
organisms on six piling structures and adjacent seatloor areas at
quarterly intervals. Site, sea.son, and their interaction had signifi-
cant effects on abundance. Abundance of juvenile (age 0 tol-i-)
king crabs increased steadily from July 1997 through March, then
declined in June I99S. Crab abundance was significantly higher on
pilings than on the adjacent substratum, and at more exposed sites
National Shclltisheries Associatiim. New Orleans. Louisiana
Abslmcls. 2003 Annual Meeting. April I. VI 7. 200.^ 357
than at sheltered sites. Red king erabs were assoeiated with the
presenee of green urehins. deeorator crabs, leather stars, and
sculpins. Each site could be discriminated by their unique com-
munity of inhabitants. Why juvenile king crabs are attracted to
pilings is unknown. Pilings are inefficient habitats that are not
structurally complex, do not persist in the environment, and may
not be the best structure for habitat enhancement. For these rea-
sons, and because there is no evidence that RKC are habitat-
limited, we do not recommend the use of pilings as artificial habi-
tats to mitigate for the loss of natural habitat.
SUSTAINABLE COMMUNITY DEVELOPMENT VIA AN
INSHORE MOLLUSCAN AQUACULTURE PARK: A CON-
CEPT FOR THE GULF OF MEXICO John E. Supan \ La
Sea Grant College Program. LSU. Baton Rouge. LA 70803.
Industrial parks are areas permitted and/or zoned for the op-
eration of prescribed businesses without the need for individual
permitting. Such community programming is commonly used in
the economic development of inner cities and rural areas across the
nation. This same concept can be applied to coastal waters delin-
eated and permitted for certain farming activities for economic
development of coastal regions.
The concept of state aquaculture parks was proposed in March
\9H9 by the National Research Council's Committee on Assess-
ment of Technology and Opportunities for Marine Aquaculture in
the U.S as a means of fostering entrepreneurship through technol-
ogy transfer and commerciali/.ation. A well-planned and adminis-
tered aquaculture park can circumvent user conflicts, navigation,
security, and liability issues that may otherwise hinder such use of
coastal waters. A public entity could be the authority that selects
the site, obtains public input, necessary permits. Coast Guard ap-
proval, and administers park operations, such as leasing areas to
farmers, providing security.
The Gulf region's semitropical climate provides ideal condi-
tions for sea farming. Oyster genetics research has created superior
stocks, which can be coupled with technically ad\anced grow-out
methods in a park setting to achieve their full economic potential.
HISTORY OF THE DEVELOPMENT, COMMERCIALIZA-
TION AND SUCCESSFUL MARKETING OF THE FIRST
HACCP-BASED P0ST-HARVF:ST PROCESS FOR THE RV-
MEDIATION OF VIBRIO SP. IN RAW OYSTERS— THE
AMERIPIREPROCESS". .lohn Tesvich* and Patrick Fahey.
AmeriPure Processing Company. Inc.. Franklin. LA.
The development of a hot water/cold shock treatment to reme-
diate Vibrio sp. In raw oysters without removing the oyster from its
natural shell was initiated in response to growing public health
concerns and marketplace reaction to raw oyster-related Vibrio sp.
Illnesses. This led to the patenting and commercialization of the
first raw. yet dead (as a result of the PHT) in-shell oyster product,
as well as the first HACCP-based PHT process to remediate Vibrio
sp. Ill raw oysters. The marketing of a value-added raw oyster with
reduced risk of infection and excellent shelf life opened markets
previously closed to raw oysters, particularly from the Gulf of
Mexico. It akso paved the way for other HACCP-based PHT pro-
cesses and has sparked considerable interest among other oyster
processors to license the AmeriPure Process*. The process is de-
pendable, simple and economical. It is also adaptable to large and
small operations with equipment that is easy to maintain and avail-
able from numerous manufacturers/fabricators.
SELECTION OF APPROPRIATE HABITATS/SITES FOR
BAY SCALLOP RESTORATION. Stephen T. Tettelbach*.
Christopher V. Smith, Peter Wentzel. Natural Science Division
Southampton College of Long Island University. Southampton,
NY 11968.
Strategies for restoration of bay scallop, Argopectcn irradums
irrculiciiis. stocks include collection of setting larvae in spat bags,
direct seeding of juveniles or adults on the bottom ("free-
planting"), or placement of broodstock in protective enclosures.
Aggregations of the latter type of enclosures are often refen'ed to
as spawner sanctuaries. Larval collection is often attempted adja-
cent to spawner sanctuaries or. when data are available on tidal
circulation patterns, in other areas where larvae are likely to be
entrained. In Long Island. New York waters, we have evaluated
potential sites for free-planting of scallops on the basis of several
criteria, including: historical scallop productivity, anticipated lar-
val dispersion, predator abundance, bottom characteristics (includ-
ing sediment type and presence of SAV's). degree of exposure to
prevailing NW winter winds (which can cause stranding of scal-
lops on adjacent beaches), and the potential for scallop burial (in
winter) by shifting sediments. Placement of net enclosures has
been based on most of the above criteria, but additional factors of
particular importance include water depth, potential hazard to
navigation, and suitability for securing appropriate permits. The
choice of appropriate strategies and habitats/sites must be consid-
ered simultaneouslv.
LINKING HARD CLAM (MERCENARIA MERCENARIA)
REPRODUCTION TO PHYTOPLANKTON COMMUNITY
STRUCTURE: I. CLAM GROWTH AND REPRODUCTIVE
CYCLF^S. Stephen T. Tettelbach*, Natural Science Division,
Southampton College of Long Island, Roger I.E. Newell, Chris-
topher Gobler.
Hard clam. Merccnariu mcrccnaria. populations and fisheries
have declined dramatically in the south shore bays of Long Island,
New York since the mid- 1 970s. We hypothesized that this decline
in recruitment was associated with variation in either the timing of
gametogenic development or synchronicity of reproduction w ithin
the population due to changes in cnerall patterns of primary pro-
duction. Quantitative histological techniques were used to assess
the reproductive cycles of adult (>4() mm) female hard clams, from
358 Abstracts. 2003 Aniuuil Meeting, April 13-17. 2003
National Sliellfisheries Association, New Orleans. Louisiana
October 2000 - October 2001. at tlve sites in south shore bays of
Long Island. For comparison, we also sampled tv\ o sites in Raritan
Bay, New Jersey where regular hard clam recruitment supports a
large fishery. Timing of peak reproduction was nearly identical at
the 5 south shore bay sites and was 1 to 2 weeks later in Raritan
Bay. There were appreciable differences in reproductive effort
between locations, with female clams from Bayshore and
Patchogue showing the lowest and the two Raritan sites having the
highest Gamete Volume fraction. Clam growth and condition in-
dex differed even more dramatically between sites, with poorest
growth and condition also being exhibited at the Bayshore and
Patchogue sites. University Southampton, NY 11968.
INFLUENCE OF FRESHWATER INPUT ON THE HABI-
TAT VALUE OF OYSTER REEFS IN THREE SOUTH-
WEST FLORIDA ESTUARIES S. Gregory Tolley*, Aswani
K. Volety, Mike Savarese and James T. Winstead, Florida Gulf
Coast University, 10501 FGCU Blvd S, Fort Myers. FL 33965.
In order to examine the influence of freshwater input on the
habitat value of oyster reefs, a spatiotemporal comparison of reef-
resident fishes and decapod crustaceans was conducted during
three seasonally dry and three seasonally wet months in three
Southwest Florida estuaries: the Caloosahatchee and Estero rivers,
and the Faka-Union Canal. Lift nets containing 5 liters of oyster
clusters were deployed monthly at three sites along the salinity
gradient of each system. Salinities within each system varied both
spatially and seasonally, with mean salinities being significantly
higher downstream and significantly lower during wet months.
Analysis of variance also indicated significant spatial and seasonal
differences in the community metrics examined. Overall results
suggested that abundance, biomass, and species richness of reef-
resident organisms increased downstream where salinities were
higher. Diversity (H") and richness were also greatest downstream
in the Caloosahatchee. but diversity in the Faka-Union was highest
upstream. In general, both biomass and diversity exhibited a sig-
nificant positive correlation with salinity. Our results suggest that
freshwater input (salinity) plays a significant role in structuring
oyster-reef communities in southwest Florida estuaries. These re-
sults can be used to inform water management practices as well as
efforts at oyster-reef restoration.
HISTOLOGICAL EVALUATION OF EARLY PEARL-SAC
DEVELOPMENT IN THE TAMPICO PEARLY MUSSEL
{CYRTONAIAS TAMPICOENSIS). Stephan Towers*, and Le-
onard DiMichele. Department of Wildlife and Fisheries Sciences,
Texas A&M University. College Station. TX 77843: and Donald
Shepherd, Professional Pathology Laboratories, Ltd. P.O. Box
326. Tow, TX 78672.
Pearl-sac development in the Tampico pearlymussel was evalu-
ated histologically. Hemocytes massed at the wound entrance,
sealing it off and staunching blood loss. Hemocytes also lined the
incision track. Mucopolysaccharides formed an extracellular ma-
trix important in wound healing, restructuring of blood sinuses,
and development of a basal membrane. Epithelial cells originating
from the graft began to proliferate onto the newly formed basal
membrane. The peari-sac was formed, and tall columnar cells be-
gan active secretions by day 30. Our results indicate that pearl-sac
development is remarkably consistent across taxa and among im-
plantation sites. The primary role of the host is seemingly to seal
the wound, reconstruct blood sinuses, and provide a basal mem-
brane. The role of the donor tissue is to provide epithelial seed
cells. Both epithelia (lateral and medial) of the graft may prolif-
erate, but only those from the lateral surface of the mantle appear
to be involved with pearl formation.
MODELING INDIVIDUAL EASTERN OYSTER {CRASSOS-
TREA VIRGINICA) GROWTH IN THE MARYLAND POR-
TION OF THE CHESAPEAKE BAY. Jessica Vanisko*. Co
operative Oxford Laboratory. MDDNR. Oxford. MD 21654: and
Thomas Miller. Chesapeake Biological Laboratory. UMCES, So-
lomons MD 20688.
Eastern oyster populations have declined dramatically in the
Chesapeake Bay during the last century. A clear and quantitative
description of oyster population dynamics is essential for the
implementation of effective restoration efforts. Growth remains an
important, but poorly understood component of these dynamics
providing the link between spat (young-of-year oysters) and the
reproductive and fishable stocks. Catch-at-length data collected at
55 sites from fishery-independent surveys were used in a length-
based analysis of growth through modal decomposition, allow ing
the mean growth of individuals within a cohort to be followed
through time over a maximum of 6 years. Initial sizes of spat were
highly variable both temporally and spatially (mean = 24.03.
CV = 32.81%). Maximum and minimum observed growth were
1.02-46.22 mm/yr. Growth rates declined with age class. Growth
rates were also highly variable among sites due to site-specific
differences such as salinity. These data were used to develop re-
gion-specific age-length relationships for oysters.
EVALUATION HACCP IN THE OYSTER ACTIVITY IN
THE LAGOON SYSTEM ALVARADO, VERACRUZ:
MEXICO. Itzel G. Villa*. Fabiola L. Reynoso. and Ma. del
Refugio C. Chavez, km 1 2 carr. Veracruz-cordoba. boca del no.
Veracruz. Mexico cp 94290.
In the last 30 years the oyster production in the national envi-
ronment has not been stable, presenting this in the State of Ver-
acruz a descending behavior, diminishing of 40,569.4 t obtained in
1988 to 9,653.8 t for 1994, that which demonstrates that the oyster
veracruzana's activity faces limitations, caused by the over exploi-
tation, the contamination, the accumulation of sludge in the coastal
lagoons, the climatological interferences, the ecological changes
National Shellfisheries Association. New Orleans, Louisiana
Absiimls. 2003 Annual Meeting, April 13-17, 2003 359
and the sanitary problems: in some cases these alterations ha\e
caused serious problems of public health and even the exhaustion
of the banks of oyster. Considering that the concept HACCP in-
volves all the potential dangers of security of the foods (biological,
chemical and physical), either that they happen in natural form, for
en\'ironmental changes or that was generated by failure in the
production process. The present project carried out a diagnose of
the lagoon system of Alvarado using the HACCP v\ith the aim to
propose a handling plan for the exploitation of the American oyster
[Crassosirea virginiai). and this way to guarantee its sanitary
quality as food for human consumption and to fulfill the regula-
tions sanitary to product exportation.
REMOTE SENSING TO MAP AND ASSESS INTERTIDAL
SHELLFISH RESOURCES IN THE SOUTHEASTERN USA.
Jeffrey S. Vincent. USC Dept. of Geography; Dwayne E. Porter,
use Baruch Inst, and School of Public Health. Loren Coen,
SCDNR Marine Resource Research Inst.; Dave Bushek*, USC
Baruch Inst.; and Steve Schill. GeoMetrics. Inc., Baruch Marine
Field Laboratory /USC. PO Box 1630. Georgetown, SC 29442.
Oyster resources in the southeastern USA are predominantU
intertidal. Water clarity and tidal stage limit the use of passive
remote sensing systems while shallow water limits the ability of
sonar to accurately map beds and reefs. Oysters can be observed
directly during low tide exposure, but inaccessibility and other
problems make mapping these intertidal oyster resources difficult
and tedious with questionable accuracy. Currently, maps are pro-
duced via a lengthy process of ground surveys and manual inter-
pretation of aerial photographs, both of which are time-consuming
and prone to human error. This project is developing a library of
hyperspectral imagery to identify spectral end members of shell-
fish from in situ and remotely sensed (HyMAP) imagery. Prelimi-
nary results indicate separation in hyperspectral characteristics of
oyster resources compared to surrounding habitats. Furthermore.
HyMAP spectral end members show reasonable separation and
similarity with in-situspectral end members. We will use these
spectral characteristics to classify and map the distribution and
condition of intertidal shellfish resources. If successful, we will
develop an automated mapping technique in a GIS environment
that can be used by resource managers to obtain more timely
information on the changing condition of oyster resources and
better direct enhancement/restoration efforts.
HISTORY OF THE COMMERCIAL APPLICATION OF
HYDROSTATIC HIGH PRESSURE PROCESSING TO
MOLLUSCAN SHELLFISH Mike Voisin. P.O. Box 3916
Houma, La. 70361-3916.
The history of the commercial application of Hydrostatic High
Pressure to molluscan shellfish will be discussed by the CEO of
the firm that developed the application. The challenges and op-
portunities during the development of this revolutionary process
will be discussed. The process reduces certain {Vibrio) bacteria to
non-detectable levels and shows potential to inactivate viruses in
shellfish, at the same time the shellfish's inuscle releases from the
shell creating an easily processed product with reduced labor cost
and increased yields.
ESTABLISHING MINIMUM FLOWS AND LEVELS OF
FRESHWATER IN THE CALOOSAHATCHEE RIVER,
FLORIDA. USING RESPONSES OF OYSTERS. Aswani K.
Volety*. S. Gregory Tolley and James T. Winstead, Florida Gulf
Coast University, 10501 FGCU Blvd, Fort Myers, FL 33965.
Alterations in freshwater intlow resulting from watershed de-
velopment and water management practices have impacted salinity
and water quality and led to declines in oyster populations within
southwest Florida estuaries. In the Caloosahatchee Estuary,
Florida watershed management is typified by large freshwater re-
leases during wet summer months and little or no releases during
dry winter months. Effects of watershed management on oysters
were investigated to provide guidelines for establishing minimum
How s and levels of freshwater in the Caloosahatchee Estuary, Re-
productive patterns, Pfrkiijsiis marinus disease, spat recruitment,
and juvenile oyster growth, were investigated. Oysters in the Ca-
loosahatchee Estuary spawn continuously from April-October. Up-
stream, sub-tidal locations exhibited good spat recruitment, low
disease intensity, and higher juvenile growth rates compared to
downstream, intertidal sites. High freshwater flows during summer
flush out oyster larvae and spat from areas with suitable cultch
and/or reduce salinities to unfavorable levels for spat settlement
and survival. Limited freshwater releases during winter coupled
with decreased releases in summer will result in suitable condi-
tions for survival and enhancement of oyster reefs. Water quality
targets that should sustain, enhance and restore oyster reefs have been
both identified and communicated to water resource managers.
DECLINING INTERTIDAL OYSTER REEFS IN FLORIDA:
DIRECT AND INDIRECT IMPACTS OF BOAT WAKES
Linda Walters*. Paul Sacks. Lisa Wall. Jeffrey Grevert, Daniel
Lejeune, Samantha Fischer, and Andrew Simpson, Department
of Biology University of Central Florida Orlando, FL 32816.
Numerous intertidal reefs of the eastern oyster Crassostrea
viifiinica have dramatically declined over the past 50 years along
the east coast of central Florida. Many reefs are significantly
smaller than in the past and have large dead margins on their
seaward edges. It is hypothesized that these differences are due to
increased recreational boating activity. To better understand the
impact of boating on intertidal oyster reefs, we have begun to run
replicated field trials in Mosquito Lagoon that include a motorboat
passing a reef at one of three speeds (5, 10, 20 mph), one of three
distances from shore (15, 30, 45 ml and one of two propeller
360 Ahstnicts. 2003 Annual Meetmg. April 13-17. 2003
National Sliellfisheries Association. New Orleans. Louisiana
angles (45 and 90 degrees). On shore, observers have recorded
dislodgment of shells, flow rates, w ake height. v\ ind speed, propa-
gation time, and turbidity. With the present configuration, all 3
variables had a significant impact on the oyster reef.
CHROMOSOMAL MAPPING OF RIBOSOMAL RNA
GENES AND TELOMERIC REPEATS IN ZHIKONG AND
BAY SCALLOPS. Yongping Wang,*' - and Ximing Guo.'
'Haskin Shellfish Research Laboratory, Rutgers University. 6959
Miller Avenue. Port Norris. NJ 08349. USA: -Experimental Ma-
rine Biology Laboratory. Institute of Oceanology CAS. Qingdao.
Shandong 266071. PRC.
Chromosomal localization of major (18-5.8-28S) and minor
(5S) ribosomal RNA genes, and the \ertebrate telomeric repeat
(TTAGGG)n were studied in two scallop species, zhikong scallop
Clikimys faneri and bay scallop Argopecten irradians. using fluo-
rescence in situ hybridization (FISH). Probes were made by PCR
amplification, labeled with digoexigenin-1 1-dUTP and detected
with fluorescein-tagged anti-digoxigenin antibodies. In zhikong
scallop, the major and minor genes were mapped to two different
regions of Cliromosome 5. The major rRNA genes were located at
the telomeric region of the short arm. while the 5S rRNA gene w as
located at an interstitial site on the long arm. In bay scallop, the
major rRNA genes had two loci one on Chromosome 4 and the
other on Chromosome 8. both at telomeric regions of the short
amis. The 5S rRNA was found at an interstitial site of an acro-
centric chromosome (Chromosome 10). In both species, the ver-
tebrate telomeric repeat hybridized to telomeres of all chromo-
somes, and no interstitial sites were observed. The finding of major
differences in the distribution of the rRNA genes between the tv\ o
species suggests that chromosomal rearrangements may have
played an important role in the esolution of scallops.
PRODUCTION OF TRANSPARENT EXOPOLYMER PAR-
TICLES (TEP) BY BIVALVES J. Evan Ward . Kari B. Hei-
nonen, Michael P. McKee, Bridget A. Holohan. Department of
Marine Sciences. University of Connecticut. Groton. CT 06340;
Bruce .\. MacDonald. Department of Biology. University of New
Brunsw ick. Saint John. N.B.. Canada, E2L 4L5.
In the marine environment, dissolved polysaccharide-rich or-
ganic matter coalesces to form transparent exopolymer particles
(TEP). In turn. TEP has substantial impact on the flocculation of
phytoplankton and other particles into aggregates (marine snow)
which increase deposition of organic matter to the benthos. Pre-
vious studies have demonstrated that exudates and lysates from
phytoplankton and bacteria contribute to the production of TEP.
Little is known, however, about other sources of TEP precursors,
especially in near-shore environments. The purpose of this study
was to investigate production of TEP by several species of bi-
\al\es iMytiliis cJiilis. Argopecten irradians. Crassostrea vir-
ginica ).
In laboratory studies, several individuals of one bivalve species
were isolated in static or recirculating seawater chambers and al-
lowed to feed for up to 9 hr. In the field, groups of oysters were
isolated in flow-through, benthic chambers and allowed to feed for
1 to 2 hr. Water samples were taken periodically and analyzed for
TEP. dissolved organic carbon (DOC), and bacterial numbers. TEP
cimcentration was determined using an Alcian Blue staining tech-
nique and quantified using a spectrophotometer. Results indicated
that TEP concentration in chambers with actively feeding bivalves
w as significantly higher than in control chambers without bivalves.
No significant differences in bacterial numbers were found be-
tween control and experimental chambers suggesting that the ef-
fects of bacteria were similar in all treatments. Mixed results were
obtained for DOC concentration. Our results indicate that bivalves
do produce TEP. probably during feeding when large volumes of
water pass o\'er mucus-coated feeding structures. We suggest that
bivalves may be an important source of TEP in near-shore waters.
ESTIMATING THE IMPACT OF BAY SCALLOP RESTO-
RATION EFFORTS USING GENETIC DATA. Ami E. Wil-
bur. Biological Sciences/CMS University of North Carolina-
Wilmington 5600 Mar\in K. Moss Lane Wilmington. NC 28409.
Shellfish populations in many areas are being augmented with
hatchery-produced animals in an effort to counteract the effects of
overfishing, habitat degradation and disease. While such efforts
have the immediate effect of increasing local abundance, it is the
expectation that the restorations will have a more dramatic effect
on subsequent generations. Until recently, it has been difficult to
evaluate the contribution made by such restorations because the
offspring of hatchery-produced animals are not readily distinguish-
able from wild conspeciflcs. The constraints of hatchery method-
ologies, however, prevent the production of stocks that mimic
natural populations with respect to genetic variation. These inevi-
table genetic differences between hatchery-produced and wild
stocks can be used to differentiate individuals in the cohort fol-
lowing restoration. Recent efforts to assess the contribution of
hatchery-produced bay scallops based on sequence analysis of mi-
tochondrial DNA markers serve as a field test of this approach.
Assessment of restoration efforts in Florida provided no genetic
evidence of a contribution from the hatchery stock despite sub-
stantial increases in abundance following the restoration. In con-
trast, a substantial contribution from hatchery-produced scallops
deployed in Chincoleague Bay was suggested by mtDNA analysis,
indicating that the restoration effort was in part responsible for the
increase in abundance.
National Shellfisheries Association. New Orleans. Louisiana
Ahsinicls. 2003 Annual Meetnig. April 13-17. 2003 361
COMPARATIVE SPERMATOZOON ULTRASTRUCTURE
OF ARCIDAE BIVALVES ARCA OLIVACEA AND
SCAPHARCA BROUGHTONI. VVan-Xi Vang*, School of Lite
Sciences, Zhejiang University, Hangzhou 310012, China; Jun-
Quan Zhu. Department of Marine and Fisheries, Ningbo Univer-
sity. Ningbo 31521 I.China.
The uitrastructure of mature spermato/oon of two Arcidae fam-
ily species Ana olivticea and Scapliana briiughtoni was com-
pared using transmission electron microscopy for the first time.
The mature spermatozoon of both species consists of a head which
is composed of a cone-shaped acrosome and a round nucleus and
a tail region. Spermatoaoon of both species has a round solid
nucleus, which exhibits a triangular posterior invagination, hous-
ing the centriolar complex and proximal portion of the axoneme.
The acrosome of Scapliana bidiiglitoni is fat while that of Area
olivacea is very thin. In Scapliana hroKi^htoiii. the subacrosomal
space contains an axial rod and a basal plate, while in Ana oli-
vacea. no such structures were obserxed. Within the middle piece,
the spermatozoon of Scapliana hnmf>hhmi has five spherical mi-
tochondria, and in contrast, only four mitochondria were observed
in Area olivacea. Both species has long whip-like end portion,
which is composed of an axoneme w ith the typical 9+2 structure.
MICROSCOPIC OBSERVATION OF TEGUMENT AND
CEMENT GLAND DISTRIBUTION OF FEMALE PLEO-
POD IN CHINESE MITTEN CRAB. ERIOCHEIR SINENSIS.
Wan-Xi Vang*, College of Life Science. Zhejiang University,
Hangzhou 310012. China; Antonina dos Santos, Inst. Nac. In\.
Agraria e das Pescas IPIMAR. A\. de Brasilia, s/n 1449-006 Lis-
boa, Portugal; Luis Narciso and Ricardu Calado. Laboratorio
Maritimo da Guia-Faculdade de Ciencias da Universidade de Lis-
boa. Estrada do Guincho, 2750-642 Cascais, Portugal; Hong
Zhou, Jian-Ping Lu and Nai-Cheng Jiang. College of Life Sci-
ence. Zhejiang University. Hang/hou 310012. China; Xue-Ping
Ving. Department of Biological and En\ ironmental Science. Wen-
zhou Normal College. Wenzhou 325027. China.
Eriocheir .sineii.sis is a vitally important economic species of
China. In recent years, its production falling down partially be-
cause of egg-loss during larval aquaculture. To reveal possible
causes of egg loss, we primarily studied the pleopod tegument
structure and its cement gland distribution. The pleopod tegument
consists of exoskeleton (subdivided into epicuticle. exocuticle and
endocuticle) and epithelial cell layer, while the cement glands lie
closely to the epithelial cells, with fine gland tubules come across
the exoskeleton. We primarily consider that cement glands in the
pleopod function in the egg attachment in Eriocheir sinensis.
IMMUNOLOGICAL STUDIES ON THE ORIGIN OF THE
LAMELLAR COMPLEX (LCX) DURING THE SPERMIO-
GENESIS OF MACROBRACHIUM NIPPONENSE (DE
HAAN). Wan-Xi Vang. School of Life Science. Zhejiang Uni-
versity. Hang/hou 310(112. China.
Lamellar complex (LCX) is a transient organelle, which is
believed to be deri\ed from Golgi apparatus and lysosome during
spermiogenesis of caridean shrimp Macrohrachiuni nipponensc
(de Haan). Conventional electron microscopical evidence shows
that, in the round spermatid, no LCX observed surrounds the
nucleus while saccules of Golgi apparatus begin to separate and
move to the nucleus along with the condensation of cytoplasm.
Typical LCX can be seen when nucleus of spemiatid begins the
sickle-shaping process, and it locates on the convex side of the
nucleus. One important feature is that lysosomes merge into the
Golgi saccules while the saccules open a cut or cuts. Most part of
the LCX conies from Golgi apparatus. To prove this, we use
GM130 monoclonal antibody to localize the Golgi apparatus. Im-
munofluorescence data show that GMI30 exists mostly in the
LCX. and immunocytochemistry results show that gold particles
(representing GM130) distribute mainly on the LCX. All these
evidence support that the idea that LCX originates mostly from
Golgi apparatus.
INTERTIDAL OVSTER RESTORATION ALONG AN
ERODING SHORELINE: AN ASSESSMENT OF SUB-
STRATE TVPES FOR STABILIZATION AND PROPAGA-
TION. Guy M. Vianopoulos. and William D. Anderson*, Ma-
rine Resources Division. South Carolina Department of Natural
Resources, Charleston. South Carolina 29422.
Gulf coast Crassostrea virginica shell. South Carolina inter-
tidal oyster shell, whelk shell {Biisycon spp.) and intertidal seed
oysters were established as cultch material along an eroding inter-
tidal shoreline ( 1.83m mean tidal range) to compare the efficacy of
substrate types for propagation to three-dimensional oyster popu-
lations. Four treatment areas were asses.sed for matrix accumula-
tion, growth and recruitment over a three-year period. Shell treat-
ments were covered with polypropylene netting (Cinlotlex *)
mesh size of 3. 1 75cm x 3.8 1 cm to provide stabilization. Recycled
South Carolina intertidal oyster shell and whelk shell demonstrated
the best matrix propagation, with whelk shell accumulating the
most spat. Gulf coast shell recruited lov\er numbers, but grew
larger spat. Transplanted intertidal seed oysters suffered mortali-
ties during the three-year study but continued to recruit significant
numbers of spat.
362 Abstracts. 2003 Annual Meeting. April 13-17. 2003
National Shellfisheries Association. New Orleans. Louisiana
THE MORPHOLOGY AND ULTRASTRUCTURE OF
SPERMATOZOON OF THE GASTROPOD BULLACTA EX-
ARATA. Xue-Ping Ying. Department of Biological and Environ-
mental Science. Wenzhou Normal College. Wenzhou 325027.
China; Wan-Xi Yang*. College of Life Science. Zhejiang Uni-
versity. Hangzhou 310012. China.
The morphology and ultrastructure of spermatozoon of mud
snail Biillacta exafcita are first described. It is composed of a head
in which a simple cap-shaped acrosomal complex and a elongated
nucleus are included and a tail containing middle piece, principle
piece and end piece. The nucleus is cylindrical, tapering gradually
towards the anterior tip. A posterior nuclear fossa is observed
clearly. In the Middle piece, there is a ring consisting of 5 occa-
sionally 6 mitochondria, which closely contacted the posterior por-
tion of the head. The proximal centriole lies in posterior nucleus
fossa and the distal one is in the center of the mitochondrial ring.
The principal piece with 9-1-2 structure consists of axoneme and
lateral fins. The end piece is short with relatively simple structure.
FINE STRUCTURAL ANALYSIS OF SPERMATOZOON
OF THE BIVALVE BARBATIA VIRESCENS AND ITS EVO-
LUTIONARY CHARACTERISTICS. Jun-Quan Zhu. Depart
ment of Marine and Fisheries, Ningbo University. Ningbo 315211.
China; Wan-Xi Yang*. School of Life Sciences. Zhejiang Uni-
versity. Hang/hou 310012. China.
The ultrastructure of mature spermatozoon of Baihatia vire-
sceiis was observed using transmission electron microscopy and its
evolutionary significance was analyzed. The mature spermatozoon
consists of a head and a tail. The head is composed of an apical,
umbrella-shaped acrosome and cylindrical nucleus. In the longi-
tudinal sections, striations can be seen clearly, which come across
outer acrosomal membrane. The nipple-shaped subacrosomal
space contains small granules. The nucleus has a Ll-shaped anterior
invagination and an inserted V-shaped posterior one. The nucleus
is highly condensed. The tail of the spermatozoon includes a
middle piece sunounded by five or occasionally six spherical mi-
tochondria and a long whip-like end piece with an axoneme with
the typical 9-h2 structure. A phylogenetic path can be traced by
comparative study of sperm ultrastructure in the Family Anidae.
The spermatozoon of B. viresceiis has a very important role in the
reproductive evolution of the Family Arcidae.
POPULATION GENETIC STRUCTURE OF THE SUMI-
NOE OYSTER AS INFERRED FROM RESTRICTION
FRAGMENT LENGTH POLYMORPHISM (RFLP) AND
MICROSATELLITE MARKERS. Qian Zhang*. Karen L.
Hudson, Standish K. Allen Jr. and Kimberly S. Reece. Virginia
Institute of Marine Science. College of William and Mary. Glouc-
ester Point. VA 23062.
The Suminoe oyster. Crassostrea ariakeitsis. is being evaluated
and considered as a non-endemic aquaculture species for Chesa-
peake Bay. To date, published reports on the taxonomic status and
genetic characterization of this species have focused on inter-
specific relationships within the genus Crassostrea. and little is
known about the population genetic structure of C ariakensis in its
native range. In this study, we used restriction fragment length
polymorphism (RFLP) markers based on the mitochondrial cyto-
chrome oxidase I (COll gene and the first internal transcribed
spacer (ITS- 1 ) region of the nuclear ribosomal RNA gene region to
examine the genetic variation within and among five geographi-
cally separated samples of C. ariakensis and hatchery stocks.
RFLP data using nuclear and mitochondrial loci showed that the
samples shared common haplotypes. but significant frequency dif-
ferences were observed between the samples in the northern group
(Northern China and Japan) and southern group (Southern China)
indicative of population structure (P= 0.000). These results sup-
port previous phylogenetic analy.ses based on ITS 1 and CGI DNA
sequences of 5-10 individuals from each sample. Microsatellite
markers are currently being employed to further examine the popu-
lation structure and to determine whether a bottleneck effect has
occurred in hatchery stocks.
CHARACTERIZATION OF KEY CDNAS OF THE ENDO-
CRINE AXES REGULATING REPRODUCTION AND
MOLTING IN THE BLUE CRAB. CALLINECTES SAPIDUS.
Nilli Zmora* and John M. Trant. 701 E. Pratt St. Baltimore. MD
21202.
For the first time in brachyurans. a number of cDNAs encoding
key hormones, enzymes and receptors of the reproductive/molting
endocrine axes were isolated from the blue crab. Using 5' and 3"
RACE. O-methyltransferase. the major regulatory enzyme for me-
thylfamesoate (MF) production, was isolated from the mandibular
organ. The deduced amino acid (AA) sequences are 74% identical
to the Metapeiiaeits ensis enzyme. The enzyme that activates ec-
dysone, 20-hydroxylase (CYP4), was isolated from the Y-organ
and is 59% identical to the Cherax quadricarinatiis enzyme at the
AA level. The ecdysone receptor (ECR) and vitellogenin (Vg)
cDNAs were isolated from ovary. The AA sequence of ECR shares
-96'7(- identity with the Fiddler crab and many insect ECRs. A 2 kb
fragment of the 5' -terminus of a putative Vg transcript was iso-
lated, however there is a low degree of homology when compared
to crayfish and penaeid Vg sequences. Our attempts to isolate the
mandibular-organ-inhibiting-hormone (MOIH) from the X-organ
were unsuccessful. The above cDNAs, together with the published
molt inhibiting hormone (MIH) sequence, will be used to develop
the molecular assays for the investigation of the endocrine regu-
lation of reproduction, molting and growth.
Natiiinal ShcllCisheries Association. New Orleans. Louisiana
AhstniLls. 2003 Annual Meeting, April 13-17. 2003 363
HATCHERY MASS PRODLCTION OF BLUE CRAB
iCALUNECTES SAPIDUS) JUVENH^ES Yonathan Zohar*.
Oded Zmora, Andrea Findiesen, Emily Lipnian. John Stubble-
field. Anson H. Hines and Jana L.D. Davis. Center of Marine
Biotechnology, University of Maryland Biotechnology Institute
701 E. Pratt St. Baltimore. Maryland 21202. USA.
Responding to the rapidly declining abundance and harvests
of the blue crab in the Chesapeake Bay. a multidisciplinary re-
search program was established to study the blue crab basic biol-
ogy, develop hatchery technologies for its mass production and
examine the feasibility of its stock enhancement. This presenta-
tion will address the hatchery work. Exposing wild-caught, mated
blue crab females to phase-shifted photo-thermal conditions re-
sulted in out-of-season hatchinc of millions of zoeae I. Larval
rearing to the zoea 8/megalopa stage was conducted at densities of
40-1 10 individuals per liter based on a diet comprised of microal-
gae. rotifers and Anemia naiipUi. Zoeae 8/megalopae were
produced in an average 22 days, and survival rates of 41.5%.
Maximal survival was 74%. Secondary growth of zoeae 8/mega-
lopae to 20 mm juvenile crabs was conducted at lower densities of
2-40 individuals per liter. To reduce cannibalism, ample shelter
structure was introduced and the crabs were graded by size. Diet
was comprised of adult Artemia, shredded squid and artificial pel-
lets. In large-scale conditions, 20 mm juvenile crabs were pro-
duced in 64 days at a survival rate of 46%. During summer/spring
2002. we produced 40,000 juvenile crabs, of which 25,000 were
individually tagged and experimentally released to the Chesapeake
Bay.
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Watts. R. J.. M. S. Johnson & R. Black. 1990. Effects of re-
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Book:
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Meegan E. Vandepeer and Robert J. Van Barneveld
A comparison of the digestive capacity of blacklip {Haliotis nihni) eind grecnlip (Haliotis laevii^aia) abalone 171
W. Gregory Cope, Teresa J. Newton and Catherine M. Gatenby
Review of techniques to prevent introduction of /cbru mussels {Drcissciin jxilxnidiplui) during native mussel
(Unionoidea) conservation activities 177
W. L. Marshall, S. M. Bower and G. R. Meyer
A comparison of the parasite and symbiont fauna of cohabiting native if'ioiolluuci .siaiiiincii) and introduced
( Venerupis philippinarum and Nuttalia obscurata) clams in British Columbia 1 85
D. E. Morgan, M. Keser, J. T. Swenarton and J. F. Foertch
Population dynamics of the Asiatic clam. Corhicula flmninea iMiiller) in the lower Connecticut River: Establishing a
foothold in New England 193
Robert S. Anderson, Brenda S. Kraus, Sharon McGladdery and Roxanna Smolowitz
QPX. a pathogen of quahogs (hard clams), employs mucoid secretions lo resist host antimicrobial agents 205
Bruce A. Macdonald and Lisa M. Nodwell
A portable and practical method to monitor bi\alve feeding activity in the field using time-lapse video technology 209
Vera L. Trainer, Bich-Thuy I^ Eberhart, John C. W'ekell. Nicolaiis G. Adams. Linda Hanson, Frank Cox and Judy Dowell
Paralytic shellfish toxins in Puget .Sound, Washington state 213
Matthew M. Nelson, Bradley J. Crear, Peter D. Nichols and David A. Ritz
Feeding southern rock lobster, Jasus edwardsii Hutton. 1875. phyllo.somata in culture: Recent progress with
lipid-enriched Arlcniia 225
R. J. B. Musgrove and P. J. Babidge
The relationship between haemolymph chemistry and moult increment for the southern rock lobster.
Jasu.s cdwurdsii Hutton 235
Juan C. Chaves and David B. Eggleston
Blue crab mortality in the North Carolina soft-shell industry: Biological and operational effects 241
Pablo D. Ribeiro, Carolina G. Luchetti and Oscar O. Iribarne
Sex-specific response to disburbance in a fiddler crab 251
Dominique Audet, Derek S. Davis, Gilles Miron, Mikio Moriyasu, Khadra Benhalima and Robert Campbell
Geographical expansion of a nonindigenous crab. Caiciniis maoias (L.). along the Nova Scotian shore into the
soulheastem Gulf of St. Lawrence. Canada 255
William J. McGraw and John Scarpa
Minimum environmental potassium for survival of Pacific white shrimp Litopmuwiis vwwamei (Boone)
in freshwater 263
Leticia Arena, Gerard Cuzon, Cristina Pascual, Gabriela Gaxiola, Claud Soyez, Alain van Wormhoudt and Carlos Rosas
Physiological and genetic variations in domesticated and wild populations of Litopenaeus vannamci fed with different
carbohydrate levels 269
Lucia Ocampo, Carlos Rosas and Humberto Villarreal
Effect of temperature on post-prandial metabolism of brown shrimp Faifaiuepenaeus ralifonuensis 281
Abstracts of technical papers presented at the 23rd Annual Milford Aquaculture Seminar. Mi"'ird, Connecticut, February
24-26. 2003 285
Abstracts of technical papers presented a( the 95th Annual Meeting of the National Shellfisheries Association. New Orleans.
Louisiana, April 13-17. 2003 305
COVER PHOTO: Mussels. Mylilus cdtdis. Photo: S. E. Shumway.
The Journal of Shellfish Research Is indexed In the following: Science Citation Index*. Sci Search®. Research Alert®. Current
Contents*/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current
Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography,
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.
JOURNAL OF SHELLFISH RESEARCH
Vol. 22, No. 1 June 2003
CONTENTS
Mingfang Zhou and Standish K. Allen, Jr.
A review ot published work on Crassoslrea ariakensis 1
Jonathan H. Grabowski, Sean P. Powers, Charles H. Peterson, Monica J. Powers and David P. Green
Consumer ratings of non-native [Crassostrea gigas and Crassosueti iiriakcnsis) vs. native (Crassostrea
virginica) oysters 21
Ziniu I'm, Xiaoyu Kong, Liusuo Zhang, Ximing Guo and Jianhai Xiang
Taxonomic status of four Crassostrea oysters from China as infened from mitochondrial DNA sequences 31
John N. Kraeuter, Susan Ford and Walter Canzonier
Increased biomass yield from Delaware Bay oysters (Crassostrea virginica) by alternation of planting season 39
Lisa House, Terrill R. Hanson and S. Sureshwaran
U.S. consumers: Examining the decision to consume oysters and the decision of how frequently to
consume oysters 51
John N. Kraeuter, Michael J. Kennish, Joseph Dobarro, Stephen R. Fegley and G. E. Flimlin, Jr.
Rehabilitation of the northern quahog (hard clam) [Merceiiaria iiieneiuiria) habitats by shelling — 1 1 years in
Bamegat Bay. New Jersey 61
Jorge L. Gutierrez and Oscar O. Iribarne
Spatial variation in the body mass of the stout razor clam, Tagelus plelwiiis: Does the density of burrowing crabs,
Cluismagnalliiis graniilata. matter? 69
William R. Congleton, Jr., Bryan R. Pearce. Matthew R. Parker and Robert C. Causey
Mariculture siting — Tidal currents and growth of Mva arcnaria 75
A. Campbell and M. D. Ming
Maturity and growth of the Pacific geoduck clam, Panopea ahriipla. in southern British Columbia, Canada 85
M. A. Delaney, Y. J. Brady, S. D. Worley and K. L Huels
The effectiveness of N-halamine disinfectant compounds on Perkinsus marinus. a parasite of the Eastern oyster
Crassostrea virginica 91
A. Louro, J. P. De la Roche, M. J. Campos and G. Roman
Hatchery rearing of the black scallop, Chlaniw varia (L.) 95
Lorelei A. Grecian, G. Jay Parsons, Patrick Dabinett and Cyr Couturier
Effect of deployment date and environmental conditions on growth rate and retrieval of hatchery-reared sea scallops,
Placopecten nuigellanicus (Gmelin, 1791 ), al a sea-based nursery 101
Seifu Seyoum, Theresa M. Bert, Ami Wilbur, William S. Arnold and Charles Crawford
Development, evaluation, and application of a mitochondrial DNA genetic tag for the bay scallop.
Argopecten irraJians Ill
A. P. Maloy, B. J. Barber and P. D. Rawson
Gametogenesis in a sympatric population of blue mussels, Mytilus edulis and Mytilus trossidus, from Cobscook
Bay (USA) 119
F. M. Suplicy, J. F. Schmitt, N. A. Moltschaniwskyj and J. F. Ferreira
Modeling of tllter-feeding behavior in the brown mussel, Perna perna (L.), exposed to natural variations of seston
availability in Santa Catarina, Brazil 125
Jorge Cdceres-Marti'nez, Miguel A. Del Rio-Portilla, Sergio Curiel-Ramirez Gutierrez and Ignacio Mendez Gomez Humardn
Phenotypes of the California mussel, Mxtihis californiainis. Conrad (1837) 135
G. Darrigran, C. Damborenea, P. Penchaszadeh and C. Taraborelli
Adjustments of Limnoperna fortunei (Bivalvia: Mytilidae) after ten years of invasion in the Americas 141
Wolfgang B. Stotz, Sergio A. Gonzalez, Luis Caillaux and Jaime Aburto
Quantitative evaluation of the diet and feeding behavior of the carnivorous gastropod, Concliolepas concholepas
(Bruguiere, 1789) (Muricidae) in subtidal habitats in the southeastern Pacific upwelling system 147
D. A. Lopez, M. L. Gonzalez and M. C. Perez
Feeding and growth in the keyhole limpet, Fissurella picla (Gmelin, 1791) 165
CONTENTS CONTINUED ON INSIDE BACK COVER
JOURNAL OF SHELLFISH RESEARCH
VOLUME 22, NUMBER 2
SEPTEMBER 2003
The Journal of Shellfish Research
(formerly Proceedings of the National Shellfisheries Association)
is the official publication of the National Shellfisheries Association
Editor
Sandra E. Shumway
Department of Marine Sciences
University of Connecticut
Groton, CT 06340
Standish K. Allen. Jr. (2004)
Aquaculture Genetics and Breeding
Technology Center
Virginia Institute of Marine Science
College of William and Mary
P.O. Box 1346
Gloucester Point, Virginia 23062
Shirley Baker (2004)
University of Florida
Department of Fisheries and Aquatic Sciences
7922 NW 7r' Street
Gainesville, Florida 32653-3071
Bruce Barber (2005)
School of Marine Science
University of Maine
5735 Hitchner Hall
Orono, Maine 04469
Brian Beal (2004)
University of Maine
9 O'Brien Avenue
Machias, Maine 04654
Neil Bourne (2003)
Fisheries and Oceans
Pacific Biological Stadon
Nanaimo, British Columbia
Canada V9T 6N7
Andrew R. Brand (2003)
University of Liverpool
Port Erin Marine Laboratory
Port Erin, Isle of Man IM9 6JA
United Kingdom
Eugene Burreson (2003)
Virginia Institute of Marine Science
P.O. Box 1346
Rt. 1 208 Create Road
College of William and Mary
Gloucester Point, Virginia 23062
Wnrme Biological Laboratory '
Woodi Hole Oceanographic Institution
Library
OCT 2 7 2003
Woo'Js I >oifc, Ma
543
EDITORIAL BOARD
Peter Cook (2004)
Austral Marine Services
Lot 34 Rocky Crossing Road
Warrenup
Albany, W.A. 6330. Australia
Simon Cragg (2004)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom
Leroy Creswell (2003)
University of Florida/Sea Grant
8400 Picos Road, Suite 101
Fort Pierce, Florida 34945-3045
Lou D'Abramo (2004)
Mississippi State University
Department of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762
Christopher V. Davis (2004)
Pemaquid Oyster Company, Inc.
P.O. Box 302
1957 Friendship Road
Waldoboro, Maine 04572
Ralph Elston (2003)
Aqua Technics/Pacific Shellfish Institute
455 West Bell Street
Sequim, Washington 98382
Susan E. Ford (2004)
Rutgers University
Haskin Shellfish Research Laboratory
6959 Miller Avenue
Port Norris, New Jersey 08349
Raymond Grizzle (2003)
Jackson Estuarine Laboratory
Durham, New Hampshire 03824
Karolyn Mueller Hansen (2004)
1524 Barley Circle
Knoxville, Tennessee 37922
Journal of Shellfish Research
Volume 22, Number 2
ISSN: 0730-8000
September 2003
www.shellfish.org/pubs/jsr.htm
Mark Luckenbach (2003)
Virginia Institute of Marine Science
Eastern Shore Lab
P.O. Box 350
Wachapreague, Virginia 23480
Bruce MacDonald (2004)
Department of Biology
University of New Brunswick
Saint John, New Brunswick
Canada E2L 4L5
Roger Mann (2004)
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
Islay D. Marsden (2004)
Department of Zoology
Canterbury University
Christchurch, New Zealand
Jay Parsons (2005)
Memorial University
Marine Institute
Box 4920
St. John's, Newfoundland
Canada AlC 5R3
Tom Soniat (2004)
Biology Department
Nicholls State University
Thibodaux, Louisiana 70310
J. Evan Ward (2004)
Department of Marine Sciences
University of Connecticut
1080 Shennecossett Road
Groton, Connecticut 06340-6097
Gary Wikfors (2004)
NOAA/NMFS
Rogers Avenue
Milford, Connecticut 06460
Joiirihil oj Slifllfish Research, Vol. 22, No. 2, 365-375, 2003.
BIOCHEMICAL INDICATOR OF SEA SCALLOP {PLACOPECTEN MAGELLANICUS) QUALITY
BASED ON LIPID CLASS COMPOSITION. PART I: BROODSTOCK CONDITIONING AND
YOUNG LARVAL PERFORMANCE
FABRICE PERNET,' * REJEAN TREMBLAY." AND EDWIN BOURGET"
^GIROQ. Pavilion Vachon. Uuiversite Laval. Cite Universitaire, Quebec, Qc. Canada. GIK 7P4;
'Univer.site du Quebec a Rinwuski — Centre .Aqiuicole Marin. 6 Rue du Pare. Centre Aqiuteole Marin
MAPAQ. 6 Rue du Pare CP. 340. Grande-Riviere. Qc, Canada. GOC IVO: and ''Vice-reetorat a la
Recherche. Pavilion Central, Univer.site de Sherhrooke. Sherbrooke, Qc. Canada. .1 IR 2R1
ABSTRACT The aim of this study was to test the validity of a lipid based indicator of larval quality of sea scallop Placopeclen
ma,uell(iiiici(S. Objectives were 2-fold: ( 1 ) to determine the link between lipid class content and reproductive state of adults in the field
and in the laboratory and (2) to follow lipid class content, growth, and survival during embryonic and early larval development. Adult
scallops were periodically sampled during gametogenesis for lipid class and histological analysis of the gonads in the field at two
locations and in the laboratory after feeding three different diets. Females were induced to spawn and lipid class content, larval growth,
and survival of five batches of eggs were followed for 8 days after fertilization. Site, diet, and time had significant effects on lipid class
composition of male and female gonads and gametogenesis of females. Triacyglycerol accumulation during vitellogenesis was
characteristic of female gonads and explained respectively 56.4% and 71.3%' of the variability in maturity and egg size. When spawning
was induced, no major effect of location or diet on lipid composition of gonad and subsequent eggs was detected. Nevertheless, the
mean number of eggs produced by females increased with atresia level in gonad, suggesting that egg quantity was incompatible with
egg quality. Lipid class composition during embryogenesis and young larval development showed a high demand for triacyglycerol.
KEY WORDS: broodstock nutrition, gametogenesis. hatching, larval growth, lipids, scallop, Placopeclen
INTRODUCTION
The expansion of aquuculttire has increased the demand for
juveniles of a wide range of bivalve species. As a consequence,
hatcheries need to produce large quantity of eggs and larvae of
good quality. Larval production has to be considered in two
phases: broodstock conditioning and larval rearing. Under northern
temperate conditions, young larvae of most bivalves rely on en-
dogenous sources of energy during embryogenesis before the tran-
sition to exogenous sources. The diet provided to adults can affect
the biochemical composition of their gonads and their resulting
eggs and larvae. For instance, the essential fatty acid composition
of the microalgae fed to adult scallop Peeten maximiis was re-
flected in the composition of gonads, eggs and larvae until 5 days
after fertilization (Delaunay et al. 1992, Samain et al, 1992). Then,
maturity and hatching success of eggs from adult P. maximus were
improved with a diet based on Isoehrysis sp. rich in essential fatty
acids (Soudant et al. 1996a, Soudant et al. 1996b). Given the
above, the performance of young larvae is directly linked to ma-
ternal nutrition.
Studies focusing on gametogenesis and early larval develop-
ment highlight the primordial role of lipids and the relative im-
portance of triacylglycerol (TAG), For example, gametogenesis in
the scallop Argopecten purpuratus was associated with an increase
in the ovary lipid content (Barber & Blake 1981 ). The sea scallop
Placopeclen inai;ellaiucii.s stores large quantities of TAG in the
gonads before spawning (Napolitano & Acknian 1992). Then, lipid
reserves accumulated in the eggs of the scallop Crassadoma gi-
gantea cover 47,6% of energetic needs during embryogenesis
(Whyte et al. 1991). TAG were preferentially cataboli/.ed during
egg development of the clam Mercenaria inercenaria and the oys-
*Corresponding author. National Research Council 1411 O.xford Street
Halifax, Nova Scotia, Canada, B3H 3Z1. Telephone: 902-426-8289;
Fax: (902) 426-9413; E-mail: fabrice.pemet@nrc.ca
ter Crassostrea virginica (Gallager et al. 1986). Finally, the mass
of lipid in the eggs is correlated with the hatching success of
P. maximus larvae (Dorange 1989, Devauchelle & Mingant 1991).
The reproductive cycle of the sea scallop, like other marine
bivalves, includes five distinct periods: vegetative, cytoplasmic
growth, vitellogenesis. spawning and. finally, resorption of non-
released gametes (see Barber & Blake 1991, Eckman 1996),
Spawning is synchronous among individuals at a particular site,
and most populations display a single annual spawning period
extending over 1 or 2 mo between July and October, depending on
latitude. In other scallop species, site-specific variation in game-
togenic cycles has been observed (e.g.. Bricelj et al. 1987).
Histological preparation of gonadal tissue provides the means
to assigned numerical values to the developmental stages. For
instance, mean egg diameter is indicative of the stage of the ga-
metogenic cycle. Eggs gradually increase in size during gameto-
genesis, reaching a maxitnum size prior to spawning and decrease
sharply after spawning as mature eggs are released (Barber &
Blake 1981, Barber et al. 1988, Paulet & Boucher 1991). Histology
allows quantification of the fraction of the gonad occupied by
developing, mature, and resorbing (atresic) gametes (Beninger
1987, MacDonald & Bourne 1987).
The aim of this .study was to test the validity of lipid class
composition of gonad, egg, and larvae as a predictor of quality of
sea scallop P. magellanicus. In the present article, "quality" refers
to a set of physiological variables (lipid composition) that could
explain the variability of reproductive state of adult (maturity,
atresia and egg size) and larval performance (survival and growth).
For example, the mass of lipid in bivalve larvae is a good indicator
of quality because it has been positively correlated with growth
and survival (Gallager et al. 1986). To our knowledge, gonad
quality has never been assessed using lipid composition. We de-
signed our study with two objectives in mind: 1 ) to examine the
link between lipid class variation in the gonads and reproductive
state of adults, in the field and in the laboratory- and 2) to examine
365
366
Fernet et al.
the link between lipid class composition of gonads and eggs with
performance during early development (number of eggs released,
growth, and survival),
MATERIAL AND METHODS
Animal Maintenance
This study was conducted at the experimental hatchery of Min-
istere de 1' Agriculture, des Pecheries et de TAlimentation du
Quebec at Grande-Riviere (Gaspe coast, Quebec, Canada). Male
and female adult scallops of comparable size (110 cm ± 10 cm)
were harvested by SCUBA diving at the end of May 2001 at two
sites on Gaspe coast: Perce (Si) at a depth of 30 m and Pointe
Saint-Pierre (S2) at a depth of 20 m (48 °30'N; 65 °15'0). Labo-
ratory held animals were maintained in a flow-through sea water
system (28 ppt) and fed continuously with living microalgae. The
latter were produced semicontinuously in the f/2 nutrient mixture
(Guillard 1975). Temperature was maintained at 8 °C from the
beginning of the experiment until July 9th and temperature fluc-
tuated between 10 and 13 "C until spawning. Photoperiod was set
at a constant cycle 16:8 (light/dark). Spawning was induced by
thermal rise to 16 °C and mechanical shock using air-lift systems.
Fertilization was made with a mixture of sperm at ca. 10 sperm to
each oocyte. The fertilized eggs were left undisturbed in 1000 L
Xactic® tanks (one tank per treatment) at 12-14 °C. Swimming
embryos were then siphoned 24 h later into another tank, where
they were maintained in suspension with a light bubbling. Four
days after fertilization, as D-veligers emerged, water was poured
through a 20-|xm pore mesh, and the tank was cleaned. Larvae
were reared until day 8 at a density of 1.5 larvae per niL.
Experimental Design
Adult Conditioning and Field Sampling
Adult scallops collected in the bay of Perce were fed from the
beginning of June until mid-July 2001 with three artificial diets
June
V
July
(Fig. 1 ). The daily dry mass of algal ration was adjusted to 4%
of scallop dry mass for each adult diet. Based on preliminary
measurements, mean dry mass was assumed to be ca. 25 pg celP'
for Isochiysis sp., Chaetoceros inuelleri, and Pavlova lutheri and
150 pg cell"' for Skeletonema costatum. Adult diet A was a mix of
hochi-ysis sp. (clone T-iso), P. lutheri. S. costatum (40/40/20
cells), adult diet B consisted of the standard mix of Isochiysis sp.,
P. lutheri. S. costatum. and C. muelleri (25/25/25/25 cells) and,
finally, adult diet C was made of Isochiysis sp. and C. muelleri
(25/75 cells). The adult scallops harvested at Pointe Saint-Pierre
were conditioned from mid-July to mid-August with the standard
diet B (treatment S2B, Fig. 1 ). Microalgae were harvested every
3 to 4 days for lipid class (n = 15) and fatty acid (« = 5) analyses
during the entire feeding period. Adults maintained in the labora-
tory on the three artificial diets were periodically sampled in trip-
licate and their gonads analyzed from the beginning of the experi-
ment (early June, t^) during vitellogenesis (early July, t,) until
spawning (mid-July, t;). Adults living in the wild at the two sites
were sampled at t,,, t,. and t, but also later during the reproductive
cycle (mid-August, t, and mid-September, ly. Fig. 1 ). Three pieces
of ca. 100 mg of gonad were collected to perform lipid class (both
sex) and histological analyses (females only).
Spawning Induction and Larval Rearing
Females from SI -fed diets A, B, C, and females from S2 were
induced to spawn in separate buckets in mid-July and females from
treatment S2B were induced to spawn in mid-August (Fig. 1).
Wild animals were induced to spawn right after arrival in the
laboratory. Number of spawning females varied from two to five
per treatment. Eggs of each female were counted and sampled
separately for lipid class analysis and size measurement. Egg fer-
tilization was conducted with a mixture of spermatozoa from three
to five males per treatment. Individual spawnings were pooled in
one tank per treatment for fecundation and embryonic develop-
Aug.
h
Sept.
LAB
3 diets
FIELD
2 sites
SI-
82-
FIELEH-LAB S2B
1 site+l diet
0
■D
t
Gametogenesis
D
Spawning
Larval rearing
i
Sampling period
f
Larval sampling (day) : 0 4
Figure 1. Experimental design.
\
Biochemical Indicator of Sea Scallop Broodstock Quality
367
ment fno treatment replication). Each group of 4-day-old larvae
was separated among three tanks of ca. 200 to 400 L and fed
different diets: a) /.mchiysis sp. and Pavlova lutheri (50/50 cells).
b) Isochnsis sp. and Chaetoceros imielleri (50/50 cells), and
c) Isochnsis sp. with C. muelteri grown under silicate deprivation
to enhance TAG accumulation (50/50 cells). Larvae were sampled
for lipid analysis, density and growth measurements on day 4 and
8 after fertilization.
Laboratory Analysis
Sample Collection
Samples of 10 mL microalgal culture, 10,000 eggs, and 5000
larvae were filtered on prebaked GF/C filters at 450 °C and stored
in 1 mL of dichloromelhane in amber glass vials with Teflon liner
caps under nitrogen at -20 C until lipid extraction. Gonad
samples for lipid analysis (ca. 100 mg) were directly stored in
dichloromelhane. Gonad samples for histologic analyses were
stored at room temperature in Helly's fixative. Finally, samples of
eggs and larvae used for size measurements were stored in 10'7f
formaldehyde.
Lipid Extraction
Lipids were extracted after a 4-day to 1-mo storage period.
Microalgae. egg, and larvae samples were first sonicated three
times in 1.5 mL of CHXL-MeOH (2:1; v/v) in an ice bath to
remove the organisms from the filter. Gonads were ground in 6 mL
of CH,CK-MeOH (2:1: v/v). KCl (0.88'7f) was added to the pre-
vious solution to obtain CHXL-MeOH-KCl (2: 1 :0.6; v/v/v; Folch
et al. 1957). The homogenates were mixed and centrifuged at 4000
rpm for 2 min to obtain a biphasic system. The lipid fraction (lower
phase) was removed and transferred to a clean tube. The solvent
was evaporated under a nitrogen fiow and lipids suspended in 0.05,
0.1. or 1 niL CH-,CU for eggs and larvae, microalgae, or gonads,
respectively. Lipid extracts of microalgae were fractionated in two
aliquots to analyze lipid classes and fatty acid. Manipulations were
carried out on ice and under nitrogen whenever possible.
Lipid Class Composition
Lipids (0.5% to 10% of total extraction depending on sample
tissue) were spotted onto the S-lII Chromarods (latron Laborato-
ries Inc., Tokyo, Japan) using a Hamilton syringe. Four different
solvent systems were used to obtain three chromatograms per rod
according to Parrish (1987). This method separates aliphatic hy-
drocarbons (HCs), ketones (KETs), TAGs, free fatty acids (FFAs),
free fatty alcohol (ALCs), free sterols (STs), diglycerides (DCs),
acetone mobile polar lipids (AMPLs), and phospholipids (PL).
Between each development, Chromarods were scanned by the
flame ionization detection system of the analyser latroscan
Mark-V (latron Laboratories Inc., Tokyo, Japan). Lipid classes
were identified and quantified with the use of standard calibration
curves obtained for each lipid class. The load applied to the rod
ranged from 0.05 to 5.9 |xg. Within each set of rods, one was used
for the lipid standard and another one for extraction blank.
Fatty Acid Composition
Lipid extract of microalgae was analyzed by gas chromatogra-
phy. Fatty acid methyl ester (FAME) were prepared from about
0.2 mg of the total lipids following the method of the American Oil
Chemists" Society using BF,/CH,OH (12%; AOCS, 1989). FAME
were suspended in 40 (xL of hexane, and a 2-p,L aliquot was
injected with a 1:37 split in a Perkin Elmer Sigma 300 capillary
chromatograph, equipped with a Supelco Omegawax™ 320 fused-
silica capillary column 30 m x 0.32 mm x 0.25 ixm ID. The
following chromatographic conditions were used: 190 °C for 20
min, followed by an increase of 4 °C min"' to 210 °C for 25 min,
followed by an increase of 5 °C min"' to 240 °C for 5 min. Helium
was the carrier gas at a flow rate of 2 mL min"'. The gas chro-
matograph was equipped with flame ionization detectors and the
integrator software Varian Star Chromatography Workstation
5.51. FAME were identified by their retention times compared
with standard (Supelco 37 component FAME Mix, Menhaden Fish
Oil and PUFA-3, Supelco Bellefonte, PA) and quantified with
tricosanoic acid (c23:0) as an internal standard. Notation used in
fatty acid identification is L:BnX where L is the chain length, B is
the number of double bonds and nX is the position of the double
bond closest to the terminal methyl group.
Histology
Female gonads were dissected and stored in Helly's fixative.
After rinsing, the samples were dehydrated through an ascending
alcohol series, cleaned in toluene and embedded in paraffin. Speci-
mens were sectioned (6 |j.m m thickness) and stained with Harris
hematein and eosin. Examination of gonad sections was made
using a compound microscope at a magnification of 40x with an
image capture kit CoolSNAP-Pro cf Digital Kit™ 4.1. Percentage
area of gonads occupied by mature and atresic eggs and size dis-
tribution of eggs for each sampling date were measured with Im-
age-pro plus® 4. 1 .0 package software. Eggs were considered ma-
ture when stalked or ripe. Eggs with a much deformed appearance
(jigsaw-puzzle shapes) were considered as atresic (Dorange 1989).
Three counts were made for each tissue section and one tissue
section was examined per individual.
Growth Measurements
Shell size was calculated as the average of the length (anterior-
posterior distance) and height (dorsal-ventral distance) of larvae.
Larvae were measured using a compound microscope (magnifica-
tion of 40x) with an image capture kit CoolSNAP-Pro cf Disital
Kit"! 4.1.
Data Analysis
Diet composition, in terms of lipid class and fatty acids, was
submitted to one-way multiple analysis of variance (MANOVA).
Fatty acids were grouped as saturated (SEA), monounsaturated
(MUFA), and polyunsaturated fatty acids (PUFA). Among PUFA,
20:5n3 (eicosapentaenoic acid. EPA), 22:6n3 (docosahexaenoic
acid, DMA), total n3 and n6 and finally n3-n6 ratios were distin-
guished. As the /; value was smaller than the number of fatty acids
(independent variables), there were not enough degrees of freedom
to apply MANOVA without data grouping.
Lipid class composition and histology of gonads depending on
treatment (diets A, B, and C and sites SI, S2) and time (from early
June, t„ to mid-July, t,), were investigated using two-way
MANOVA by sex. The total sum of squares was partitioned be-
cause of the asymmetric experimental design (Underwood 1997).
At t(„ there were only two treatments (SI, in which A, B, and C
were confounded and S2) whereas at t, and t^, there were five
distinct treatments. Consequently, contrasts were carried out be-
tween control and experimental treatments (t^, vs. t,) and then a
368
Pernet et al.
two-way MANOVA was run among experimental treatments at t,
and t,. Site-specific effects on the independent variables (lipid
class composition and histology of gonads) from t, to tj were
investigated using a two-way MANOVA by sex. It was not pos-
sible to perform an overall analysis due to the fact that animals in
the laboratory were not sampled from t^ to tj. then leading to an
asymmetric design (Fig. 1 ).
One-way MANOVA was used to investigate treatment effects
on lipid class content and histological data of ovaries prior to
spawning. Each individual was considered as an experimental unit
(n = 3 per treatment).
One-way MANOVA was used to investigate treatment effects
on lipid class content, size and quantity of eggs produced by
adults. Each batch of eggs produced per female was considered as
an experimental unit (n = 2 to 5 per treatment). The number of
replicates among adult feeding regimes differed.
Age effects on lipid class content and growth from early em-
bryogenesis until day 8 were analyzed using one-way MANOVA.
As previously mentioned, individual spawnings were pooled in
one tank for fecundation and embryonic development. The pooled
eggs and larvae from each treatment were considered as experi-
mental units {II = 5). Values of lipid class content and size of
the pooled eggs used for fecundation were obtained by weighing
the contribution of each female to the total number of eggs in the
group (individual fecundity). This is a means to assess the initial
composition of the pooled eggs, allowing their inclusion in the data
analysis.
Finally, lipid class composition, quality, size and survival of
8-day-old larvae were subjected to one-way MANOVA to deter-
mine larval diet effects (/; = 3).
When overall differences were detected. Least-square means
multiple comparison tests (LSMean. SAS Institute Inc. 1999-
2000. Gary, NO were used to determine which means were sig-
nificantly different. Probability levels were divided by the number
of degrees of freedom of the tested factor (Bonferroni correction ).
Homoscedasticity was tested using Levene's test and was con-
firmed by graphical examination of the residuals (Sherrer 1984).
A stepwise multiple regression was used to examine the rela-
tionships between histological data as response variable [% area of
gonads occupied by both mature and atresic eggs and egg diam-
eter) and lipid class content as explanatory variable (/! = 49).
Another model was used to assess the relation between spawning
performance as response variable (number and size of eggs at
release and hatching success) and gonad histological data (egg
maturity, atresia and size) and lipid composition as explanatory
variable. Finally, a stepwise multiple regression was also used to
examine the relationship between larval performance as response
variable (growth and survival) and egg lipid composition as ex-
planatory variable (n = 5). A significant threshold of 0.05 was
adopted for all statistical tests. All statistical analyses were carried
out using SAS 8.01 (SAS institute Inc. 1999-2000).
RESULTS
Lipid Class and Fatty Acid Composition of Diet
Diets A. B. and C fed to giant scallop contained respectively
88.31. 74.67, and 129.88 mg of lipid per g of algal dry mass
{P < 0.001). Lipid class composition of diets A and B differed
from diet C particularly in TAG {P < 0.001, Fig. 2). TAG content
of diet C was 100 and 10 times higher than that of diets A and B
respectively. Diet C contained significantly higher level of MUFA
O < O
5 u. J
P u. ^
Lipid class
Figure 2. Mass of each lipid class (±SD, n = 14) expressed In mg per
g dry mass in diet A (D). B (D ), and C (■) (For compositions of diet,
see text). Lipid classes detected were wax estser (WE), ketone (KET),
triacylglycerol (T.\G). free fatty acid (FF.\), fatty alcohol (.\LC).
cholesterol (ST), acetone mobile polar lipid (.\MPL), and phospho-
lipid (PL).
and PUFA than diets A and B. and differences of PUFA were
mainly attributed to n6 fatty acid (Table I ). Values of 20:5n3 and
22:6n3 did not differ among diets (P = 0.061 and P = 0.082,
respectively).
Gonad Lipid Class Content and Maturation
General Pattern
We examined the influence of three diets in the laboratory and
two sites in the field on ovarian lipids and histology from vitello-
genesis until the resorption period. Based on the percentage of
mature eggs and egg diameter measurements, the first major
spawning probably occurred at the beginning of July (t, ) and lasted
until the beginning of August (t,. Fig. 3). Egg size increased sig-
nificantly from t(, to t| and reached a maximum in mid-July (t,).
Thereafter, egg maturity and size gradually decreased in gonads
until end of September (tj). Adults reared in the laboratory dis-
played partial spawning at t, and were induced to spawn at t, (July
II. 12, and 15 for diets B, A. and C, respectively).
Total lipid level (TL) of female gonad varied from 6-16% dry
mass during the experiinental period (Fig. 4f). The highest level of
TL was observed at the end of vitellogenesis (t, ), followed imme-
diately by a sharp decrease during spawning (t, to t,, P = 0.008,
Fig. 4f). TL remained low until the end of the experiment. In male
gonad, TL increased from tg to t, (Fig. 41). The gonad lipid fraction
varied between 4.5 to 7.5% dry mass. This suggests that, during
gametogenesis. lipids were highly solicited in females but only
moderately in males. TAG explained 84% of the variability of TL
in female gonads, whereas PL explained 65% of the variability of
TL in male gonad during the course of experiment.
Time, Treatment, and Site-Specific Effects on Ovaries
Mature eggs in the female gonads varied in time depending on
the diet. There was a highly significant time effect from t,, (end of
May) to t| (beginning July, P = 0.003) and a significant interac-
tion of treatment and time [from t, to t, (mid-July)J (P < 0.001,
Biochemical Indicator of Sea Scallop Broodstock Quality
369
TABLE 1.
Fatty acid mass (nig g ' dry mass) and % (relative to the sum of
fatty acid mass) in adult diets A ilsochrysis sp., Pavlova liilheri, and
Skeletonema costaliim 40/40/20), B [Isochrysis sp., P. lutheri.
S. coslatiim, and Chaeloceros muelleri 25/25/25/25), and
C. ilsochrysis sp. and C. muelleri 25/15). ii = S.
Diet A
Diet B
Diet C
Fatty Acid Mass
%
Mass
%
Mass
%
12:0
14:0
14:ln5
l.'5:0
16:0
I6:ln7
16:2n4
16:3n4
18:0
lS:ln9
IS:ln7
18:2n6
18:3n6
I8:3n3
I8:4n3
20:0
20:ln9
21:0
20:4n6
20:4n3
20:5n3
22:0
22:ln9
21:5n3
22:5n3
24:0
22:6n3
0.05
5.63
0.19
0.16
3.22
3.22
0.91
3.68
0.24
2.30
0.73
3.13
0.23
1.67
2.66
0.02
0.00
0.00
0.21
0.01
6.80
0.06
0.00
0.00
0.00
0.00
3.99
0.12
14.40
0.49
0.40
8.22
8.24
2.34
9.40
0.62
5.89
1.85
8.00
0.58
4.27
6.80
0.06
0.00
0.00
0.54
0.02
17.40
0.15
0.00
0.00
0.00
0.00
10.20
0.09
4.66
0.18
0.17
3.10
5.21
1.14
4.56
0.31
1 .55
0.59
2.02
0.26
1.03
1.75
0.00
0.00
0.00
0.46
0.01
7.13
0.07
0.00
0.00
0.02
0.00
2.86
0.23
12..54
0.48
0.47
HM
14.01
3.(J6
12.27
0.84
4.17
1.60
5.43
(171
2.77
4.70
0.00
0.00
0.00
1.23
0.02
19.18
0.18
().()()
0.00
0.06
0.00
7.70
0.05
10.80
0.38
0.44
8.01
15.36
2.21
7.40
0.67
3.11
1.39
4.03
0.8!
2.01
3.05
0.08
0.07
0.00
1.73
0.02
12.65
0.07
0.02
0.00
0.05
0.01
4.27
0.06
13.73
0.48
0.56
10.18
19.52
2.80
9.41
0.86
3.95
1.76
5.13
1.03
2.56
3.88
0.10
0.08
0.00
2.19
0.03
16.07
0.09
0.02
0.00
0.06
0.02
5.43
ISFA
IMUFA
IPUFA
Xn3
Vn6
In3/In6
9.38
6.44
23.29
15.13
3.57
4.91
23.98
16.47
59.55
38.68
9.12
8.40
7.53
21.24
12.80
2.74
5.27
22.61
20.26
57.14
34.43
7.37
20.14
20.31
38.22
22.05
6.56
^.^3
25.60
25.82
48.58
28.03
8.35
Fig. 3). At t|. animals fed diet C showed a higher maturity than
those fed diets A and B. Scallops in the field showed maturity
levels between those fed diets A and B. At t,, the maturity of field
scallops and scallops fed diet B dropped to values observed at t,,
the maturity of those fed diet C remained high whereas that of
scallops fed diet A increased significantly to reach the le\el of
those fed diet B at t,. Thus, scallops maintained in the laboratory
initiated vitellogenesis faster than those in the field, and those fed
diet C matured more quickly and completely than those fed diets
A and B. Thus, scallops fed diet B and adults maintained in the
field started to spawn at t,. earlier than adults fed diets A or C with
lower maturity levels. Levels of atresia in female gonads showed
significant time and treatment effects. In fact, percentage area
of gonads occupied by atresic eggs increased from t^, to t, {P =
0.019) and decreased markedly between t, and t, (P = 0.023),
except for scallops from Pointe Saint-Pierre (S2). The lowest level
of atresia was observed in scallops fed diet C. There was no site
effect on maturity and atresia levels in female gonads. Scallops
3
(0
E
O)
LU
E
■Jo
O)
tu -
— • —
-S1
— ■ —
-S2
s?
30-
- - -a- '
-A
Q
- - -A -
-B
(A
9
20 -
a-A---r
. - O- -
-C
L.
,-5
/ \
■--,
■ - * -
-S2B
<
10 J
• ' ' J
^' V
L
T ' ' y^
.^ A\.
n J
^'-l^^^^ ^--^^^-^
i^~
May 9 June 8 July 8 Aug 7 Sept 6 Oct 6
Figure 3. Female giant scallop maturity, egg size (mean diameter), and
atresia as a function of time and treatment (±SD, n = 3). Broodstock in
the field (normal line) at Perce (SI, #), Pointe Saint-Pierre (S2. ■), and
in the laboratory (dashed line) fed with diet A (D), B (open triangle),
and C (O) and Pointe Saint-Pierre-fed diet B since mid-July (S2B, ■).
from Perce (SI) had smaller eggs than those from Pointe Saint-
Pierre (S2; Fig. 3).
In ovaries, TAG levels sharply increased at the end of vitello-
genesis. between t,, to t, (P = 0.005). and dropped sharply during
the spawning period, from t, to t, {P = 0.016. Fig. 4a). TAG
levels gradually decreased from t, to tj {P < 0.001 ). Levels of TAG
were highly variable depending on treatment and time. From t, to
t,, scallops from Perce (SI), and those fed diets A and C had
significantly higher TAG content than those fed diet B. scallops
from Pointe Saint-Pierre (S2) exhibiting intermediate levels. Fi-
nally, ovarian TAG levels were influenced by location since there
were significant interactions of site x time (P = 0.03). In fact.
370
Pernet et al.
Ovary
(A
(fl
19
E
■D
a
May 9 June 8 July 8 Aug 7 Sept 6 Oct 6 May 9 June 8 July 8 Aug 7 Sept 6 Oct 6
— S1
-82
Time
B
S2B
Figure 4. Mass of each lipid class expressed in mg per 100 mg dry mass ( % ) in ovary (left) and testis (right) as a function of time and treatment
(±SD, n = 3). Broodstock in the field (normal line) at Perce (SI, #1, Pointe Saint-Pierre (S2. ■). and in the laboratory (dashed line) fed with diet
A (D), B (open triangle), and C (C) and Pointe Saint-Pierre-fed diet B since mid-July (S2B. ■). Lipid classes detected were TAG, FFA, ST,
AMPL, and PL. TL were obtained by summation of each lipid class.
scallops from Pointe Saint-Pierre (S2) did not exhibit a significant
gonadal TAG increment as observed in scallops from Perce (SI ) or
those reared in the laboratory. TAG maxima occurred at different
times depending on site (t, for SI and t, for S2).
FFAs were a minor lipid class in female gonads accounting for
ca. 6% of total lipid (Fig. 4b). In fact, there was no accumulation
or depletion of FFAs from tg to t, but FFAs markedly increased
from t, to ti in all the treatments. Thereafter, FFA levels gradually
decreased toward initial values until t4 except for scallops from S 1 .
ST content decreased from t„ to t, {P = 0.003), reaching the
lowest value at t, (Fig. 4c). Values of ST measured at t, and tj
were intermediate between t, and ty, suggesting a slow recovery
after spawning. ST were affected by treatment in such a way that
scallops fed diet B exhibited the highest level, those fed diets A
and C and from Perce (SI) were intermediate and scallops from
Pointe Saint-Pierre (S2) were the lowest. Moreover, a site effect
was detected on ST because levels in scallop gonads from 81 were
lower than those from 82 (P = 0.012).
Biochemical Indicator of Sea Scallop Broodstock Quality
371
AMPL. mainly glycolipids. pigments and remaining neutral
lipids, gradually increased during the period of study (P < O.OOl.
Fig. 4d|. There was no significant effect of treatment but a sig-
nificant interaction of time x site (P = 0.008). PL content of
gonads gradually decreased during the study as a function of treat-
ment and site (Fig. 4e). Particularly, scallops from Pointe Saint-
Pierre (S2) showed a pronounced drop from t, to t, (P = 0.014).
TL decreased throughout the spawning period, trom t, to tj
(Fig. 4f).
Time, Treatment, and Site-Specific Effects on Testes
Lipid class composition of male gonad consisted mainly of
structural lipids, such as ST and PL (ca. 88% of TL). TAG were
consistently low (Fig. 4g). FFA were a minor lipid class in testes
and accounted for ca. 2% of total lipids (Fig. 4h). There was a
significant interaction of time and site on FFA content ( P < 0.00 1 ).
but no clear pattern emerged. ST content increased from t„ to t,
(P = 0.028) but subsequently was not affected by time, diet, or
site (Fig. 4i). AMPL gradually increased all over the period of
study with no significant effect of diet or site (Fig. 4j). The AMPL
pattern observed in testes was similar to that in ovaries (student
f-test, P = 0.487). PL increased before spawning from to to t,
(P < 0.001 ) and decreased markedly from t, to t^ (Fig. 4k). There
was no significant difference in PL in the male and female gonads
(student t test, P = 0.198) except that PL content in testes in-
creased from t(, to t,. Finally, TL content in testes increased from
t|, to t, (P < 0.001 ) and gradually decreased until t4 as for PL (Fig.
41). TL was also infiuenced by site since values in scallops from
Perce (SI) were lower than those from Pointe Saint-Pierre (S2,
P = 0.041).
Lipid Class Composition and Histological Analyses
A stepwise multiple regression was conducted to examine the
relationship between lipid class and female maturity (Table 2). The
model, including TAG and FFA. explained 61.7% of the variabil-
ity of maturity. Based on this model, increasing ovarian TAG
increases maturity level. TAG" only explained 16.7% of the vari-
ability of maturity, but it indicates that TAG values above a certain
threshold are linked with lower maturity. TAG alone explained
56.4% of the variability of maturity. Variability of atresia in fe-
male gonads was weakly linked with ST (r^ = 0.12). The model
suggests that atresia increases as ST decreases. Finally, egg size
was related to TAG and ST. The model suggests that egg size
increases with TAG until a threshold (quadratic effect) and also
increases as ST decreases. However, the influence of ST (partial r
= 0.03) was negligible compared with that of TAG (partial
r- = 0.68).
Lipid Class Content, Growth, and Survival During Embryonic and
Early Larval Development
Females fed diets A. B. C. and originating from Pointe Saint-
Pierre (S2) were induced to spawn in mid-July (t,) and those from
S2B in mid- August (t,). Lipid composition and maturity of gonads
collected prior to spawning differed markedly in relation to treat-
ment. Significant effects were detected in FFA (P < 0.001). ST
(j)=0.Q\5). and PL (P = 0.035). Individual fed diet C showed the
highest maturity, followed by those fed diet A and finally those
from S2. S2B and B (P < 0.001). It seemed that broodstock con-
ditioning just after the first major spawning (S2B) maintained but
did not increase maturity (Fig. 3).
TABLE 2.
Stepwise multiple regression analysis using lipid class composition of
female gonads as explanatory variables and maturity, atresia, and
size of eggs in gonad as response variables, n = 49.
Maturity
Regression Equation
\ = 4.4.1 + 7.89 X
X FFA, r- = 0.617, f
TAG - 0.47 X
< 0.000
TAG- - 8.04
Regression
Step No.
Source
of Variance
Partial r^
P Value
1
2
TAG
TAG^
FFA
0.396
0.167
0.052
<.000
<.000
0.015
Atresia
Regression Equation
Y = 18..39 - 27.08
X ST, r= =
0.1
20. P = 0.014
Regression
Step No.
Source of
Variance
Partial r'
P Value
1
ST
0.120
0.014
Egg Size
Regression Equation
Y = 31.70 + 4.87 X
xST, r- = 0.713, f
TAG - 0..
< 0.000
19 X
TAG' - 10.34
Regression
Step No.
Source of
Variance
Partial r
P Value
1
2
3
TAG
TAG'
ST
0.473
0.210
0.030
<.000
<.000
0.034
There was no effect of the treatment on lipid composition of
eggs (except in FFA. P< 0.001, Fig. 5) and on the number of eggs
released (Table 3. P= 0.366). In contrast, egg size varied according
to the treatment (P = 0.004) and was correlated with size of
8-day-old larvae (r = 0.82, P = 0.035). Eggs released by fe-
O) 6
O)
<u
O)
(A 4
(A
(0
E
•o
■q. 2
i
^£i
DA
DB
■ C
aS2
■ S2B
TAG FFA ST AMPL PL
Lipid class
TL
Figure 5. Lipid class profile of eggs as a function of treatment (±SD,
/I = 2 to 5). Broodstock were liarvcsted in the field at Pointe Saint-
Pierre (S2) and in the laboratory fed with diets A, B, C and Pointe
Saint-Pierre-fed diet B since mid-July (S2B). Lipid classes detected
were TAG, FFA. ST, AMPL, and PL, TL were obtained by summation
of the lipid classes.
372
Pernet et al.
TABLE i.
Effect of broodstock treatment (diet A, B, C, site S2 and site S2 fed diet B since mid-July [S2B]) on number of eggs and survival
(% determined from day 0 to 4 and 4 to 8) and size of egg and larvae (±SD when replicated).
Treatment
A
B
C
S2
S2B
No. of female spawning
Number of eggs { 10")"
4
6.42 ± 4.39
5
2.78 ± 1.94
1
3.28 ± 3.65
1
19.3 ± 11.35
3
15.05 ±20.8
Survival (%)
4-day old''
8-day old'
2.44
39.95
1.58
91.67
5.94
67.09
1 1 .50
61.60
2.09
90.57
Size (|xm)
Egg"
4-day old*
8-day old^
79.0 ± 1.16
93.63
106.08
75.0 ± 1.31
91.81
103.16
79.0 ± 0.46
97.05
111.71
85.2 ± 2.63
103.38
118.14
82.2 ± 2.55
100.57
111.07
' The average value; female were individually induced to spawn by thermal shock.
* Calculated on the pooled eggs; for each treatment, individual spawnings were pooled in one tank for fecundation and embryonic development.
"The average value; each group of four day old larvae was placed in three tanks fed a different diet.
males originating from S2 were significantly larger than those
from other treatments.
A stepwise multiple regression was conducted to examine the
relation between gonad histology prior to spawning and spawning
performance (number and size of eggs at release and hatching
success). The model showed that number and size of eggs released
increased with atresia (Fig. 6). Hatching success was not correlated
with any variable.
From the time of spawning to 8 days of age, TAG levels de-
creased whereas FFA. ST, AMPL. and PL content increased sig-
0<o
I. O
Z 0)
50
40
30 -
20-
10
0
y = 0.89X + 2.03
r2=0.31,p=0.030
5 10 15
Atresia (%)
90
85 H
0)
N ^^
•5) E
O) i
S^ 75
80
70
y = 0.39X + 76.56
r =^ = 0.68, p= 0.002
10
Atresia (%)
15
20
Figure 6. Significant regression obtained after the stepwise procedure
using ovarian histology before spawning as explanatory variables (egg
maturity, astresia. and size) and number and size of eggs released and
hatching success as responsible variables. The number and size of eggs
were considered per female spawning in = 16) whereas hatching suc-
cess was measured on the group of eggs (/; = 5).
nitlcantly (Fig. 7). No effect of larval age on TL could be detected
(P = 0.256).
In the larval feeding experiment, diet-specific effects were ob-
served in TAG content. Larvae fed diets B and C exhibited ca.
twice as much TAG as larvae fed diet A (Table 4). However, such
differences were apparently not sufficient to produce a significant
effect on larval size and survival.
DISCUSSION
Gonad Lipid Class Content and Maturation
Gametogenesis of female giant scallop relies on the accumu-
lation of neutral lipid in the gonad, particularly TAGs (Fig. 4a). In
contrast, male gonads did not accumulate lipids during the experi-
ment, and slight variations in TL were partly explained by varia-
tions of structural lipid content. The lipid composition of male and
20 _
■o
(/)
(A
ra
E
■o
a
□ Egg
B4 d
■ 8d
TAG FFA ST AMPL PL TL
Lipid class
Figure 7. Lipid class profile of eggs and young larvae as a function of
treatment (±SD, h = 5). Broodstock were harvested in the field at
Pointe Saint-Pierre (S2) and in the laboratory fed with diets A, B, C
and Pointe Saint-Pierre-fed diet B since mid-July (S2B). Lipid classes
detected were TAC. FFA, ST, AMPL, and PL. TL were obtained by
summation of the lipid classes. Groups with different letters are sig-
nificantly different {P < 0.05).
Biochemical Indicator of Sea Scallop Broodstock Quality
373
TABLE 4.
(a) Results of lipid class composition (ng larvae"'), size (finil, and
survival C/r determined from day 4 to 8) of larvae aged 8 days
depending on diet (a, b, and c): (b) summary of MANOVA; and
Ic) multiple comparisons.
Lipid Class
Source
of
Variance TAG FFA ST AMPL PL TL
Others
Size Survival
(a)
(b)
a
0.21
0.S2
0.1 fi
1.07
2.59
4.55
110.89
65.37
b
0.47
0.71
0.18
1.17
3.67
6.19
109.92
65.20
c
0.42
0.64
0.15
1.56
3.55
6.31
109.28
79.96
(1.002 0.514 0.770 0.295 0.127 0.08 1 0.917 0.590
(c) A C B NS NS NS NS NS NS
NS
Results are arranged in increasing order of estimated means from left to
right. Groups underlined are not significantly different. Significant prob-
abilities are in bold {P < 0.05).
NS, nonsignificant.
female gonads of giant scallop P. magelUmicus was consistent
with previous results for this species (Napolitano & Ackman 1992,
Napohtano at al. 1993) and for the Chilean scallop Argopecien
purpuratus (Caers at al. 1999). showing that the lipid level in testis
was significantly lower than in ovaries. In fact, eggs accumulate
large amounts of TAG during gametogenic development presum-
ably to provide energy during embryoganasis. Spermatozoa con-
tain little lipid, except PL and ST in cell membrane (Soudant et al.
1996a). Consequently, ovarian lipid metabolism during gametoge-
nesis principally involved deposition of lipid reserves, whereas
testis lipid metabolism was mainly governed by structural lipid
dynamics.
TAG content of female gonad increased from the beginning of
the experiment to reach a peak al the beginning of the spawning
period and decreased with spawning (Fig. 4a). The decrease of
total lipid in female gonad was previously attributed to the loss of
eggs rich in TAGs during spawning in the scallop Pecten maximus
(Pazos et al. 1997) and two clam species Tapes decussatiis and
T. philippinanim (Beninger 1984). Variations of ovarian maturity
and egg size during gametogenesis were linked with TAG levels.
A positive correlation between total lipid level and female gonad
index (r^ = 0.779) and mean egg diameter (r- = 0.418) has been
reported in the literature (Pazos et al., 1997). Thus, TAG mass
appears to be a good indicator of ovarian maturity.
Our study revealed that FFA levels sharply increased after the
first major spawning (Fig. 4b). These molecules in a nonesterified
form are usually observed in minor amounts in marine organisms.
The presence of large quantities of FFAs in animal tissue is an
indication of lipid degradation (Parrish 1999). FFA might result
from the spawning, which leads to tissue breakdown and produces
dead cells. Testis did not accumulate FFA during the spawning
suggesting that the pattetTi of FFA is sex specific and closely
related to TAG metabolism.
PLs were also a major lipid class constituent of ovaries and
exhibited a slight continuous decline over the study (Fig. 4e).
Maxima of PL in two clam species were observed during active
gametogenesis. while the subsequent decrease coincided with ga-
mete release (Beninger 1984). In our study. PL declined at t,,,
before spawning. PL might be catabolized before spawning to
sustain energetic needs to complete gametogenesis or to synthesize
TAG. Thus, PL may constitute an energy reserve catabolized be-
fore spawning in ovaries. In testis, PL increased before spawning
and then sharply decreased. This pattern is consistent with previ-
ous reports on clam species (Beninger 1984).
The effect of feeding regimen during broodstock conditioning
has been extensively studied (see Lifting & Millican 1998). The
originality of our study is that the three diets tested in this experi-
ment differed in terms of lipid class and fatty acid profiles. The
lipid rich diet C {hochrysis sp. and C. muelleri. 25:75) promoted
a greater and faster maturity and lower atresia than diets A and B
(Fig. 3). In a previous study. Isochiysis giilhaiui (Clone T-ISO)
enhanced the rate of vitellogenesis and lowered atresia more than
the other two diets during conditioning of Pecten maximus
(Soudant et al. 1996a). This result was related to the high level of
essential fatty acid 22:6n3 in T-ISO. In contrast with this result,
diet C in our experiment had the same level of 22:6n3 as diets A
and B. The higher and fasfet maturity and the lower atresia ob-
tained with diet C could be attributable to the high KETs, TAGs,
AMPL, or PL content or total MUFA or PUFA levels. It was not
possible to attribute the observed differences in gonad lipid com-
position and histology to a specific lipid class or fatty acid because
several components varied simultaneously.
Lipid Class Content, Growth, and Survival During Embryonic and
Early Larval Development
Our study showed a positive relation between the number of
eggs released and the level of atresia in the female gonad (Fig. 6).
Similarly, a negative correlation between fecundity of P. maximus
and the number of D-larvae was found and was attributed to the
atresia of ovaries (Le Pennec et al., 1998). In spite of the lack of
correlation between atresia in gonad and subsequent hatching suc-
cess of eggs produced, our results suggest that the released of a
high number of eggs is incompatible with high egg qualify.
Lipid composition of gonad and egg failed in predicting spawn-
ing (number and size of eggs released) and larval (survival and
growth) performances. In contrast, previous studies showed that
hatching success of P. maximus is a function of egg lipid reserves
(Dorange 1989, Devauchelle & Mingant 1991). The absence of
relationship in our study might be due to the similarity of lipid
composition between egg groups.
Lipid class composition during embryonic and larval develop-
ment showed a strong TAG depletion (Fig. 7). In fact, 52% of egg
TAG reserves were consumed during embryogenesis (egg to
4 days) and 32% during early larval development (4 to 8 days).
Consequently, despite the fact that larvae feed on exogenous phy-
toplancton at day 4, TAGs continued to decrease until day 8.
Similarly, scallop larvae of Patinopecten yeoenssis consumed 54%
of the initial reserves of neutral lipids during embryogenesis
(Whyte et al. 1991) and scallop larvae of P. maximus started lipid
accumulation 4 days after the first feeding (Delaunay et al. 1992).
Our study confirms the high TAG demand during embryogenesis
and the effect on larval lipid content.
Concomitantly, increases of STs and PLs during embryonic and
larval development have been observed (Fig. 7). This underlines
the changes from eggs, full of energy reserves, to young larvae,
rich in structure. Thus, structural lipids, and particularly PL seem
to be synthesized at the expense of TAG. It has been suggested that
TAG play a double role in oyster Osirea edulis larvae by storing
large amounts of saturated fatty acids for energy purposes while
acting as temporary reservoir of PUFA transferred to the structural
374
Pernet et al.
lipids (Napolitano et al. 1988). However. PL decrease during tlie
early development of P. maximiis (Delaunay et al. 1992) and fish
larvae (Tocher et al. 1985. Fraser et al. 1988), suggesting an en-
ergetic role of PL. A more detailed experimental design is needed
to interpret interactions between egg origin and age of larvae on
lipid dynamic.
A significant increase of FFA from trace amounts in eggs to ca.
11% of TL in 8-day-old larvae was observed (Fig. 7). This result
is in agreement with previous studies (Napolilano et al. 1988). As
mentioned earlier. FFA is generally considered a degradation prod-
uct and may be attributed to the increasing number of moribund
larvae in samples. However. Napolitano et al. ( 1988) discussed the
possible physiologic role of FFA and concluded that FFA probably
remain adsorbed to specific proteins rather than being freely cir-
culating, toxic lipids. Thus. FFA may not necessarily reflect inad-
equate storage or extraction conditions.
AMPL content increased during larval development from day 4
to 8 (Fig. 7). AMPL containing pigments (Parrish 1987) likely
reflected ingestion of microalgae by larvae. This would also ex-
plain the lack of an increase of AMPL during embryogenesis when
larvae are not able to feed from exogenous sources.
Surprisingly, our experiment did not show a significant effect
of development on TL content (Fig. 7). In contrast, other studies
have reported a marked decline of TL during embryogenesis of
scallop larvae (Gallager et al. 1986. Whyte et al. 1991, Lu et al.
1999). Graphical examination of TL pattern during embryogenesis
suggests a specific egg group effect (Fig. 6). hence this interpre-
tation need to be taken with caution.
ACKNOWLEDGMENTS
The authors thank E-J. Arsenault and S. Bourget for their as-
sistance in broodstock conditioning and microalgae and all the
staff of CAMGR (Centre Aquacole Marin de Grande-Riviere) of
MAPAQ (Ministere de 1' Agriculture des Peches et de
TAIimentation du Quebec). Thanks are also addressed to S. Belvin
for the histologic work and E. Demers from CTPA (Centre de
Transformation des Produits Aquatiques) of MAPAQ for their
help with GC analyses and fatty acid identification. Funding for
this research was provided by CORPAQ (Conseil des Recherches
en Peche et en Agro-alimentaire du Quebec), MAPAQ. Techno-
pole maritime and GIROQ (Groupe Interuniversitaire de Recher-
ches Oceanographiques du Quebec). We are grateful to Dr. L.
Fortier for the use of his latroscan. Thanks are also addressed to
G. Daigle, Departement de mathematiques et statistique. Univer-
site Laval, for validating statistical analyses, and V. Moreau.
M. Cusson and L. Lapointe for their constructive and critical
discussions.
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BIOCHEMICAL INDICATOR OF SEA SCALLOP {PLACOPECTEN MAGELLANICUS) QUALITY
BASED ON LIPID CLASS COMPOSITION. PART II : LARVAL GROWTH, COMPETENCY
AND SETTLEMENT
FABRICE FERNET,' * REJEAN TREMBLAY," AND EDWIN BOURGEX"
^GIROQ. Pavilion Vachon. Universite Laval, Cite universitaire. Quebec; Qc, Canada. GIK 7P4:
'Universile dii Quebec a Rimouski — Centre Aquacole Marin. 6 Rue du Pare, Centre Aquacole Marin
MAPAQ. 6 Rue du Pare C.P. 340. Grande-Riviere. Qc. Canada. GOC IVO: and ^Vice-rectorat a la
Recherche. Pavilion Central. Universite de Sherhrooke. Sherbrooke. Qc. Canada. JIR 2RI
ABSTRACT The purpose of this study was to examine the lipid class content of larval .stages of the sea scallop Piacopecten
magellaiiicus during development and to examine the potential effects of varying feeding regimes on larval lipid content, growth,
survival, settlement behavior, and survival of postlarval stage. The potential of lipid class ratios to forecast larval growth, survival,
settlement behavior, and success was examined. At the start of exogenous feeding (day 4) three diets, which differed in triacylglycerol
(TAG) content, were applied. Diet A consisted oilsochrysis sp. and Pavlova tutheri. diet B was a mix of Isochnsis sp. and Chaeloceros
muelleri. and diet C consisted of the same two species, but C. muelleri was grown under silicate deprivation to enhance TAG
accumulation. Larvae were periodically sampled for lipid class analysis, growth measurement and survival assessment. Behavior of
pediveliger larvae for each diet was recorded using an endoscopic camera during settlement. Experiments were replicated twice and
repeated 1 mo later. Our study shows that TAG level in larval food was positively correlated with growth rate, larval TAG content and,
as a consequence, larval "quality," as measured by TAG-sterol (ST) or TAG-phospholipid (PL) ratios, prior to settlement. A positive
relation between number of competent lar\ ae produced and larval quality at day 8 was found, suggesting that survival at competency
was partly explained by the recovery efficiency of energetic reserves as TAG after embryogenesis. Higher growth rates obtained with
the diet enriched with TAG reflect its high caloric content and the presence of sufficient essential fatty acids. TAG-ST ratio of
competent larvae was negatively correlated with settlement success (day 40). High quality larvae explore the same period of time
whatever their age, whereas low quality larvae decrease exploration time with age. Consequently, the low settlement success observed
in our experiments with high quality larvae might reflect delayed metamorphosis in response to poor environmental conditions.
KEY WORDS:
behavior; larval nutrition; lipid; microalgae; Piacopecten magcllanicus: silicate deprivation, scallop
INTRODUCTION
Success of bivalve larval culture depends to a large e.xtent on
larval energy reserves to support embryogenesis and metamorpho-
sis. Energy reserves depend on the nutritional value of microalgal
diets supplied to the larvae (Whyte et al. 1989). Webb and Chu
(1983) reviewed the role of chemical constituents in phytoplankton
and concluded that lipids were the most important constituent of
the algal diet for larval rearing, particularly polyunsaturated fatty
acids (PUFAs). Since then, attention has been paid to understand-
ing the nutritional role of these essential components for bivalve
larvae (Whyte et al. 1989, Delaunay et al. 1993, Soudant et al.
1996).
To optimize larval quality, here referring to physiological char-
acteristics that could explain variability of growth, survival, and
success of metamorphosis, total lipid or lipid composition would
appear most appropriate. In fact, total lipid is a good indicator of
larval quality because it has been correlated with growth and vi-
ability of bivalve larvae. When larvae are able to feed from exog-
enous sources, excess energy is stored mainly as triacylglycerol
(TAG), the major storage lipid (Gallager et al. 1986). During em-
bryogenesis. the larvae of scallop Patinopecten yessoen.^is depend
on endogenous reserves, and lipid accounts for 47.6% of their
energetic needs (Whyte et al. 1991). During metamorphosis, neu-
tral lipids, particulariy TAG. are the primary energy reserve of the
oyster Oslrea cdiilis (Holland 1978. Gallager et al. 1986) proteins
being used later. Thus, lipids and proteins account for approxi-
mately 95% of total energy requirements of oyster larvae (Holland
& Spencer 1973).
*Corre,sponding author. Telephone; 902-426-8289; Fax: (902) 426-9413;
E-mail: fabrice.pemeKa'nrc.ca
Given the above, quality assessment has used ratios of lipid
classes, TAG content can be related to larval quality, but as TAG
content is directly dependent on larval size, it must be normalized.
The use of TAG-sterol (ST) ratio takes into account the size de-
pendency of TAG content because there is a positive correlation
between ST content and larval mass (Fraser 1989). In a study of
stressed larvae of marine bivalves. Crustacea and fish. TAG-ST
ratio reflects the quality of the larvae (Fraser 1989). This ratio has
been applied to fishery experiments in the field (Hakanson 1989.
Hakanson et al. 1994, Lochmann et al. 1995. Ouellet et al. 1995)
and in the laboratory (Delaunay et al. 1992, Ouellet & Taggart
1992. Miron et al. 1999. Miron et al. 2000). These studies showed
that TAG-based ratios are linked with larval growth, survival, and
habitat selection during settlement.
The larval cycle of sea scallop, Piacopecten magellanicus is
typical of bivalves. Once gametes are released in the water column
and fertilization has occurred, embryogenesis proceeds toward de-
velopment of veliger larvae, a process completed after ca. 4 days.
Then, swimming larvae spend 4 wk or more feeding in the water
column until they reach a length of ca. 220 (xm (day 28), where
they become pediveligers, with eyespots and a foot. At this stage,
larvae explore the bottom to find a suitable substratum to settle and
undergo metamorphosis (Culliney 1974).
In this study, growth, survival, settlement, and lipid class con-
tent of sea scallop larvae reared under a variety of feeding regimes
are reported to verify the validity of a TAG based indicator of
larval quality. We designed this experiment with several objec-
tives: ( 1 ) to tnonitor the lipid class content of larvae over the entire
larval cycle. (2) to verify the effect of feeding regimen on lar\ al
lipid content, growth, survival and settlement, and (3) to examine
the effect of larval quality, as determined by a TAG-based ratio, on
growth, survival, and settlement.
377
378
Pernet et al.
MATERIALS AND METHODS
Rearing Procedures
This study was conducted at the experimental hatchery of Min-
istere de TAgricuhure. des Pecheries et de TAIimentation du
Quebec at Grande-Riviere. Male and female adult scallops were
harvested by SCUBA diving at Pointe Saint-Pierre at a depth of 20
ni on July 15, 2001. Spawning induction, fertilization and rearing
of young larvae was as previously described in Pernet et al. (2003).
Briefly, five females were induced to spawn separately right after
arrival in the laboratory. Individual spawnings were pooled in one
tank for fecundation and embryos development. Fertilization oc-
curred with a mix of spermatozoa provided by five males. Larvae
were reared in 500-L tanks with aeration, at 13°C, at an initial
density of 1.5 individual per niL. They were fed at ca. 15 000 algal
cells mL"'. Microalgae were produced by a semicontinuous
method, grown in the f/2 nutrient mixture (Guillard 1975), and
harvested every 3 to 4 days for diet characterization (lipid class
and fatty acid composition) during the experimental period (40
days). Water renewal in the settling tanks followed the method of
Bourne etal. (1989).
Experimental Design
Larvae were split into six batches to apply three diets in du-
plicate 4 days after fertilization, as D-veliger emerged. Diet A
consisted a standard mixture of Isochiysis sp. and Pavlova lutheri
(50/50 cells), diet B was a mixture of Isochrysis sp. and Chaeto-
ceros imielleri (50/50 cells), and diet C was the same as diet B, but
the diatom C. imielleri was grown under silicate deprivation. This
process slow cell division and enhances TAG accumulation (Lom-
bard! & Wangersky 1991), whereby the energy normally allocated
to silicate uptake and deposition is diverted to lipid production
(Coombs et al. 1967). At day 28, collectors were added to culture
tanks since larvae reached competency (>507f larvae had devel-
oped visible eyes). Larvae were allowed to settle until day 40.
Swimming larvae were harvested at day 4. 8. 12. 20. 28. 32. 36.
and 40 after fertilization for growth, survival, and lipid class analy-
sis whereas settled larvae were sampled at the end of the experi-
ment (day 40). Behavior of individual pediveliger larvae on day 36
and 40 for each diet treatment and replicate culture was recorded
on videotape using an endoscopic camera (see below). Endoscopic
recorded behavior were categorized as follows: ( 1) larvae actively
exploring or (2) not moving, remaining attached to the screen, (3)
actively swimming, or (4) passive in the water column. Based on
these observations, a time budget (relative time spent by each
larvae exhibiting a specific behavior) was determined. Exploration
distance and exploration rate (distance. time"') were measured for
each larvae as well.
The complete experiment was repeated from mid-August to
September, without the behavioral aspects. However, the second
experiment was conducted without replicates because of the poor
Laboratory Analysis
Shell size was average of length (anterior-posterior distance)
and height (dorsal-ventral distance) of at least 30 larvae. Larvae
were measured using a compound microscope at a magnification
of 40x with image capture kit CoolSNAP-Pro cf Digital Kit ' ^' 4. 1 .
Lipid analysis was conducted as previously described (Pernet et
al. 2003). Briefly, 5000 veliger larvae or 500 pediveligers were
filtered on prebaked GF/C filters and stored in dichloromethane at
-20°C until lipid extraction. Samples were sonicated and lipids
were extracted according to Folch et al. (1957). Then, lipids were
spotted onto the S-III Chromarods (latron Laboratories Inc., To-
kyo, Japan) and separated according to Parrish (1987). Lipid
classes were quantified with the analyser latroscan Mark-V (latron
Laboratories Inc., Tokyo, Japan). Fatty acid composition of mi-
croalgae was analyzed by gas chromatography. Fatty acid methyl
ester (FAME) were prepared following the method of the Ameri-
can Oil Chemists' Society (AOCS 1989) and injected in a Perkin
Elmer Sigma 300 capillary chromatograph, equipped with a Su-
pelco Omegawax^''' 320 fused-silica capillary column. FAMEs
o>
E
in
(0
rs
EXP I
130
70
10
i
aab
aaa aaa
aoi
J
EXP
aaa
aaa
^
aaa
^
i
aab
1
I
J aaa
M
Q.
2
-1
Q.
ID
h-
lU
<
Lipid class
<
O
-J
<
CO
a.
<
Figure 1. Mass of each lipid class (±SD, n = 9 and h = 6 for experiments I and II, respectively ) expressed in mg per g dry mass in diet A {Isochrysis
sp. + Pavlova lutheri. □), B (Isochrysis sp. + Chaetoceros muelleri. D ). and C (Isochrysis sp. + Chaetoceros muelleri with silicate deprivation, ■)
during experiment I and 11. Lipid classes detected were wax ester (WE), ketone (KET), triacylglycerol (TAG), free fatty acid (FFA), fatty alcohol
(ALC), cholesterol (ST), acetone mobile polar lipid (.\.MPL), and phospholipid (PL).
Biochemical Indicator of Sea Scallop Larvae
379
were identified by their retention times compared with standard
(Supelco 37 component FAME Mix. Menhaden Fish Oil and
PUFA-3, Supelco, Bellefonte, PA| and quanlilied with tricosanoic
acid (c23:0) as an internal standard.
The methodology used to examine e.\pk)ratory behavior of
competent larvae was previously described (Walters et al. 1999).
The equipment used was an Olympus K 17- 1 8-90 endoscope (1.7
mm diameter. 186 mm length) attached to a video camera fixed to
a micromanipulator arm, allowing two-dimensional movement.
Video output was sent to a video 8 mm integrated to a monitor
(Optiscan lUS-l ). An Olympus 250 W high-intensity xenon light
source (model ILV-2) provided cold light to the extremity of the
endoscope. Behavioral observations were performed on swimming
larvae sampled in settling tank during water renewal on day 36 and
40 for diets B and C. These larvae were carefully transferred to
circular tank type "Plankton Kreisel." A two-dimensional (23 x
16.5 cm) polypropylene collector (200-(xm pore mesh) was placed
perpendicular to the tlow (ca. 1 cm s '). Observation of larval
behavior in each trial lasted until the larvae had stopped movement
for 5 min or was out of the field covered by the camera (5.5 x 3.5
cm). Between each recording, collector was shaken and rinsed to
detach larvae and new larvae were injected into the system. Be-
havioral observations lasted for average 6.3 min, at least 1 min to
a maximum of 33 min.
Data Analysis
The lipid class (n = 15) and fatty acid compositions (/i = 5)
of the diets were compared by one-way multiple analysis of vari-
ance (MANOVA). Fatty acid were grouped in saturated (SFA),
monounsaturated (MUFA). and polyunsaturated fatty acids
(PUFAs). Among PUFAs, 20:5n3 (eicosapentaenoic acid, EPA),
22:6n3 (docosahexaenoic acid, DHA), total n3 and n6 and finally
n3-n6 ratio were distinguished (see Pemet et al. 2003).
Shell size, lipid class composition, and quality of swimming
larvae depending on diet and age were investigated by two sepa-
rate two-way MANOVA. Groups of larvae fed diet A were lost at
day 28, leading to an unbalanced design. Then, the first analyzes
included data from day 4 to 28 whereas the second analyses in-
cluded data from day 28 to 40. Where differences were detected.
Least Square Mean multiple comparison tests (LSMean) were used
to determine which means were significantly different with prob-
ability levels divided by the number of degrees of freedom of the
tested factor (Bonferroni correction). Shell size, lipid class com-
position, and quality of larvae depending on diet, age and behavior
were investigated by one-way MANOVA. Treatments consisted of
different combinations of factors: diets B or C at days 28 and 40
and. at day 40, larvae settled or swimming. Contrasts were per-
formed to verify a posteriori particular effect.
Exploration rate and exploration distance of larvae depending
on diet and age were submitted to two-way analysis of variance.
Homoscedasticity was tested by running Levene's test and was
confirmed by graphical examination of the residuals (Sherrer
1984).
Finally, to compare survivorship according to diet from day 4
to 28, Life Test procedures were used. When differences were
detected, the x" comparison tests were applied to determine which
treatments differed significantly. A significant threshold of 0.05
was adopted for all statistical tests. All statistical analyses were run
in SAS 8.01 (SAS Institute Inc., 1999-2000, Gary, NG).
RESULTS
Lipid Class and Fatly Acid Composition of Ixinal Diet
As expected, lipid class content differed among larval diets
(P = 0.0221 ). TAG levels were highly different among diets (Fig.
1 ). In experiment I. diet C had higher TAG content than diets A
(/> < 0.0001 ) and B (P < 0.0001 ) and diets A and B also differed
{P = 0.0333). In fact, diets A. B and C contained 2.68, 8.92 and
65.11 mg.g"' algal dry mass of TAG which represent 2.32, 8.31,
and 31.19% of lipid class composition respectively. Free fatty
acids (FFA), cholesterol (ST), and total lipids (TL) levels were
also different among diets (Fig. 1 ). During the course of experi-
ment II, microalgae lipid class composition revealed nearly the
same differences of TAG and total lipid content among diets.
However. FFA and ST contents were the same whereas ketone
levels were different (P = 0.0001 ). In short, the lipid composition
of the diatom Chaetocerus mitelleri was altered by varying the
silicate level in the culture medium. The TAG content of cells
TABLE L
Fatty acid mass (mg g"' dry mass) and % (relative to the sum of
fatty acid mass) in larval diet X {Isochrysis sp. + favlora lutheri), B
ihochrysis sp. + Chaetocerus mitelleri), and C {Isochrysis sp. +
Chaetoceros muelleri with silicate deprivation) during experiment I
and II (H =5).
Fatty
Acid
A
B
C
Mass
%
Mass
%
Mass
%
12:0
0,07
0.09
0.07
0.08
0.20
0.09
14:0
13.29
17.24
12.01
14.80
28.80
13.16
14:ln5
0.58
0.75
0.52
0.64
0.53
0.24
15:0
0.23
0..10
0.40
0.49
1.79
0.82
16:0
7.56
9.81
9.15
11.27
53.73
24.55
16:1 117
6.06
7.87
11.18
13.77
48.37
22.10
16:2n4
0.86
1.12
1.28
1.57
2.46
1.13
16:3n4
0.42
0.55
4.44
5.46
4.42
2.02
18:0
0.24
0.32
0.98
1.21
3.67
1.68
18:ln9
4.55
5.91
4.32
5.32
6.32
2.89
18:ln7
1.20
1.56
1.38
1.70
1.77
0.81
18:2n6
3.50
4.,S4
4.76
5.87
5.45
2.49
18:3n6
0.72
0.94
0.81
0.99
6.55
2.99
18:3n3
4.33
5.61
3.44
4.24
3.48
1.59
18:4n3
1 1 .56
15.00
8.33
10.26
13.48
6.16
20:0
0.02
0.02
0.09
0.11
0.09
0.04
20:ln9
0.23
0..W
0.29
0.36
0.09
0.04
21:0
0.02
0.02
0.00
0.00
0.56
0.25
20:4n6
0.28
0.36
0.90
1.11
7.58
3.46
20:4n3
0.07
0.09
1.43
1.76
0.65
0.30
20:5n3
1043
13.53
8.26
10.17
19.47
8.89
22:0
0.06
0.08
0.16
0.19
0.38
0.17
22:ln9
0.04
0.06
0.07
0.09
0.06
0.03
21:5n3
0.00
0.00
0.00
0.00
0.00
0.00
22:5n3
0.02
0.02
0.03
0.03
0.27
0.12
24:0
0.02
0.03
0.02
0.02
0.00
0.00
22:6ii3
10.71
13.89
6.88
8.47
8.68
3.97
SSFA
21.51
27.90
22.87
28.17
89.21
40.76
IMUFA
12.68
16.45
17.77
21.88
57.14
26.11
IPUFA
42.90
55.65
40.55
49.95
72.49
33.13
In3
-17.11
48.14
28.37
34.94
46.03
21.03
In6
4.51
5.84
6.47
7.97
19.58
8.95
In3/In6
8.24
5.18
2.42
380
Pernet et al.
300
♦
4 8 12 16 20 24 28 32 36 40 4 8 12 16 20 24 28 32 36 40
Day
Figure 2. Growth of larvae fed diet A (■), B ( ♦ ), and C (•) depending on larval stage and experiment 1 1 or III. Distinction between settled (open
symbol) and plantonic (filled symbol) larvae was made at day 40 (±SD. ;i = 2 and n = 1 for experiments I and II. respectively).
cultured in the silicate deprived medium was higher than those in
the complete medium.
Fatty acid analyses performed of diets A, B, and C during
experiment I and II revealed that diets A and B did not differ
(Table 1 ). However, silicate deprivation enhanced fatty acid accu-
mulation in diet C with no distinction among SFAs. MUFAs, or
PUFAs. Relative content of PUFAs from diets A and B (ca. 53%)
differed from that of diet C (33%). As a consequence, diet C may
be qualified as a high TAG-rich SFA and MUFA diet. The three
diets had all essential fatty acid requirements, each of them con-
taining eicosapentaenoic (EPA. 20:.'in3) and docosahe.xaenoic acid
(DHA. 22:6n3). Diet C was the richest in total n3 and 20:5n3
whereas diet A exhibited the highest level of 22:6n3. DHA-EPA
ratios of diets A. B and C were respectively 1.03. 0.83 and 0.45.
Larval Growth and Sunival
Diet and time interacted in their effects upon larval growth
(P = 0.032). The difference of larval size among diets first ap-
peared when larvae had reached competency at day 28 (Fig. 2). In
fact, larvae fed diet C exhibited a higher mean size (220.09 |j.m)
than larvae fed diet B (204.56 (xm. P = 0.0011) and diet A
(200.74 p.m. P < 0.0001). There was no size difference between
larvae fed diets A and B (P = 0.3960). Consequently, diet C
increased larval growth by ca. 20% compared with diets A and B.
However, these results were not maintained at day 40 because
larvae fed diet B reached the same size as those fed diet C (re-
spectively 257.16 and 248.96 |xm. P = 0.339). Furthermore,
settled larvae were larger than swimming ones (253.9 vs. 23 1 jjim.
P = 0.004). Larvae in experiment II showed the same tendency as
in experiment I. but dietary effects seemed more pronounced (Fig.
2). In fact, mean size of larvae fed diets A, B and C at day 28 were
respectively 199.29. 210.65 and 230.90 jj-m and discrepancies
among size related to diets were maintained after settlement.
Larvae fed diet A suffered high mortality on day 28 in both
experiments (Fig. 3). In experiment I. survivorship patterns were
similar between larvae fed diets B and C but larvae fed diet B
seemed to have a better settlement success than those fed diet C at
day 40. In experiment II. larvae fed diet C seemed to promote
higher survival than those reared on diets B and A at competency.
100
>
3
(0
EXP
o
n r-
4 8 12 16 20 24 28 32 36 40
4 8 12 16 20 24 28 32 36 40
Day
Figure 3. Survival of larvae fed diet A (■). B ( ♦ ), and C (•) depending on larval stage and experiment (I or II). The distinction between settled
(open symbol) and planktonic (filled symbol) larvae was made at day 40 (±SD, h = 2 and ;i = 1 for experiments I and II, respectively).
Biochemical Indicator of Sea Scallop Larvae
381
and a higher settlement success than larvae fed diet B trespectively
4.6% vs. 2.7%. Fig. 3).
Lipid Class Composition During Larval Cycle
From D-Veligers to Competency
Age and diet interacted in their effects up(in lipid class com-
position of sea scallop larvae [P = 0.0001 ). particularly. TAG, ST.
PL and. as a consequence, total lipid TL (Figs. 4 and 5). FFA and
acetone mobile polar lipids (AMPL) were accumulated during lar-
val development without any diet effect.
From the beginning of the experiment to day 20. TAG content
of larvae was constant with no difference attributable to diet. TAG
content of 28 days old larvae fed diets B and C rose by factors of
15 and 70 respectively, while it remained constant for larvae fed
diet A (Figs. 4 and 5). Consequently, the high TAG level in diet C
was correlated with a better accumulation of TAG in larvae prior
to metamorphosis compared with larvae fed diet A (P < 0.0001)
and B (P = 0.012). Moreover, diet B allowed a greater accumu-
lation of TAG than diet A (P = 0.0007).
The two structural lipids. .ST and PL followed the same trend
(Fig. 4 and 5). From the beginning of larval development until day
20. ST and PL were gently rising, independently of diet. After day
20. these lipids were accumulated at different rates according to
diet. PL and ST contents of larvae fed diets B and C showed a
marked rise, whereas they remained low for larvae fed diet A.
25
20 -I
15
10 -I
AMPL
0)
t
c
(0
(0
flj
60
50
40
30
20
10
0
PL
-T ! r-
4
3 -I
2
1
ST
— , 1 1 1 1 — ~T 1 1 1—
4 8 12 16 20 24 28 32 36 40
8 12 16 20 24 28 32 36 40
Day
Figure 4. Lipid class profile of larvae of experiment I fed diet A (■), B ( ^ ), and C (#) depending on larval stage. Distinction between settled
(open symbol! and planktonic (filled syniboll larvae was made at day 40. Lipid classes detected were TAG. FFA, ST, AMPL, and PL. TL was
obtained by summation of eacb lipid class (±SU, n = 2).
382
Pernet et al.
60
50
40
30
20
10
5.0
2.5
0.0
AMPL
^
12
0)
ra
t
y
(9
C
6
(0
(A
3
$
1 1 r-
o -
ST
•-
2
/" o
/ o
1 -
^.^
0 -
, --t--^^^'^-^
4 8 12 16 20 24 28 32 36 40
Day
Figure 5. Lipid class profile of larvae of experiment II fed dies A (■), B ( ♦ ), and C (•) depending on larval stage (no replication). Distinction
between settled (open symbol) and planktonic (filled symbol) larvae was made at day 40. lipid classes detected were TAG. FFA, ST, AMPL, and
PL. TL was obtained by summation of each lipid class.
FFA displayed distinct patterns compared with the other lipid
class. Despite a significant time effect on FFA content. FFA were
not accumulated during larval development in experiment I (Fig.
4). In fact, the level of FFA in young larvae at day 8 was the same
as that of pre-competent larvae at day 28 (P = 0.16). Average
FFA content was 9.01%. High levels of FFA in sample may be
attributed to the presence of moribund larvae. The presence of
large amounts of FFA in animal tissues (more than 10%) is usually
an indication of lipid degradation and decreases in the amounts of
TAG and PL.
Finally, AMPL content of larvae increased gradually from day
4 to competency without any measurable effect of the feeding
regimen (P = 0.543, Figs. 4 and 5). AMPL consist principally of
pigments and may reflect ingestion of microalgae.
From Competency to Settlement
In experiment 1. TL level in larvae fed with diet B increased
significantly from day 28 to 40 (P = 0.041). This effect was
mainly attributable to the augmentation of PL level during this
period (P = 0.009, Fig. 4). This pattern was not observed in larvae
fed diet C. Levels of TAG in larvae fed diet C were higher than
those fed diet B (P = 0.003). Settled larvae had higher TAG and
TL content than planktimic ones (P = 0.018 and P = 0.015
Biochemical Indicator of Sea Scallop Larvae
383
respectively). However, data of experiment II showed inverse re-
sult since settled larvae ted diet C had a lower TAG and TL
contents than planktonic larvae (Fig. 5).
Larval Quality
ST and PL were highly correlated with larval size, confirming
the adequacy of these structural lipids as weighting factors for the
size dependency of TAG levels (Fig. 6). Thus, TAG-ST and TAG-
PL ratios were used as indicators of larval quality.
From D-Veligers to Competency
Age and diet interacted in their effects upon TAG-ST and
TAG-PL ratios (P < 0.001, Fig. 7). Between day 4 and day 20.
there was no difference in either TAG-ST or TAG-PL ratios. At
day 20, larvae fed diet C showed higher ratios of TAG-ST and
TAG-PL than those fed diets A and B. From day 20 to day 28, just
before settlement, a sharp rise of both ratiiis was observed for
larvae fed diets B and C in both experiments. High TAG level in
diet C suggests higher larval quality prior to metamorphosis com-
pared with diets A and B.
Correlation analysis showed a significant positive relation be-
tween size of 28-day-old larvae and their TAG-ST and TAG-PL
ratios at day 20 (Table 2). There was also a positive relation
between survival of 28-day-old larvae and TAG-ST and TAG-PL
ratios at day 8. The strength of this relationship gradually de-
creased with age of larvae (Table 2).
From Competency to Settlement
TAG-ST and TAG-PL ratios of planktonic larvae did not vary
significantly during settlement [P = 0.258 and P = 0.359 respec-
tively. Fig. 8). Planktonic larvae fed diet B showed lower levels of
TAG-ST and TAG-PL ratios (P = 0.001 and P < 0.001, respec-
tively).
The level of ratios of settled larvae remained constant com-
pared with those observed at competency (Fig. 7). However, there
was a significant effect of diet on TAG-ST and TAG-PL ratios of
settled larvae. Larvae fed diet C maintained higher TAG-ST and
TAG-PL ratios than larvae fed diet B. Settled and swimming lar-
vae at day 40 had the same ratio values, despite different TAG
levels.
A negative correlation between larval quality (TAG-ST ratio)
of 28-day-old individuals and settlement rate at day 40 was ap-
parent (Fig. 9). The higher the TAG-ST ratio, the lower was the
settlement rate. However, this relation was not significant for
TAG-PL ratio.
Larval Behavior During Seltlement
Larval behavior time budgets (relative time spent by each lar-
vae exhibiting a particular behavior) at day 36 showed no evident
effect of feeding regimen or larval quality. Larvae fed diets B (low
TAG-ST ratio) and C (high TAG-ST ratio) spent most of their time
swimming in the water column. Active exploration of the collector
consisted in ca. \6'7c of observation time whereas immobility on
(0
t
(A
JS
O
a
(A
(A
r^ = 0.88
EXP II
PL
ST
100
150
200 250
300
Larval size (|im)
Figure 6. Relation between structural lipid a.s cholesterol (STI and phospholipids (PI, I, and larval size in experiments I and II (/; = 39 and n =
19, respectively).
384
40 n
0)
5
0
O
a:
3.0
2.5
2.0
1.5
1.0
0.5
0.0
TAG/PL
-1 1 1 1 1 1 1 r-
4 8 12 16 20 24 28 32 36 40
Day
Figure 7. Triacvlglycerol (TAG)-cholesterol (ST) or TAG-phospholipid (PI-1 ratios of larvae-fed diet A (■), B (♦), and C (•! depending on
larval stage in experiment I and II. Distinction between settled (open symbol! and planktonic (filled symbol) larvae was made at day 40 (±SD,
H = 2 and n = 1 for experiments 1 and II, respectively).
the screen accounted for ca. 34% of observation time of each
larvae (Fig. 10). However, larvae fed diet B or C exhibited differ-
ent time budgets at the end of the experiment (day 40). In fact,
larvae fed diet C showed active exploration of the collector for 9'7c
of the observation time whereas larvae fed diet B exhibited little
exploration behavior {<2'7f ). The relative time spent immobile on
the screen by larvae was higher at day 40 compared with day 36,
and. at day 40, it was higher for larvae fed diet B than those fed
diet C. Exploration rate of 40 day old larvae seemed to be higher
than observed for 36-day-old larvae fed diet B (P < 0.05, Fig.
1 lA). Finally, exploration distance was similar for larvae fed both
diets (Fig. IIB).
TABLE 2.
Matrix of Pearson correlation coefficients between size or survival
of 12-, 20-, and 28-day-old larvae with TAG-ST and TAG-PL ratios
of 8-. 12-. and 20-dav-old larvae.
■Age
TAGAST
TAG/PL
Variable
(d)
8
12
2(1
8
12
20
Shell Mze
12
-0.03 1
0.327
(jjini)
20
-0.070
-0.061
-0.023
-0.062
28
0.519
0.667
0.739
0.070
-0.056
0.734
Survival
12
0.289
-(J.0I5
(%r
20
0.449
0.427
0.4.59
0.210
28
0.706
0.543
0.313
0.760
0.476
0.309
" Based upon initial number of 4-day-old larvae.
Data of experiment I and II were pooled {n = 9 larval cultures
Significant probabilities are in bold {P < 0.05).
DISCUSSION
Lipid Composition of Larvae
During the first 20 days, lipid reserves of the larvae of the sea
scallop Phicopecten magelkmicus remained low (Figs. 4 and 5). A
similar pattern for total lipids occurs in Japanese scallop Pati-
nopecten yessoensis (Whyte et al. 1987). From late embryogenesis
to 20 days, energy from food intake might be insufficient to sustain
sitiiultaneously larval growth and lipid accumulation. In contrast, a
continuous increase of TAG from day 3 is normal in developing
larvae of the great scallop Pecten maximus and suggests that food
was efficiently assimilated (Delaunay et al. 1992). Thus, the ob-
served pattern in sea scallop larvae might reflect a lag in the
metabolic and digestive processes of food assimilation and storage.
From day 20 to 28, TAG and structural lipids accumulated as
the larvae reached pre-metamorphic condition. During this period,
dietary sources of energy were directed toward growth, develop-
>
Biochemical Indicator of Sea Scallop Larvae
EXP I
385
TAG/PL
28
32
36
40 28
32
36
40
Day
Figure 8. Triacjlglyccrol (TAG)-cholesterol (ST) and TAG-phospholipid (PL) ratios of competent planlvtonic larvae fed diet B (0) and C (O)
depending on age in experiments I and II (±SD. ;i = 2 and h = I for experiments I and II, respectively).
nient iif primary gill filaments and foot, as well as storage to meet
the energy demand for metamorphosis (Whyte et al.. 1987).
Finally, during settlement, from day 28 to 40, lipid levels re-
mained stable. These results contrast with the low lipid levels
following metamorphosis of oysters (Holland and Spencer, 1973;
Labarta et al., 1999), scallops (Whyte et al. 1992) and barnacles
(Lucas et al. 1979). Three reasons might be evoked to explain this
pattern. Firstly, a low level of lipid reserves following metamor-
phosis of marine invertebrates is still debated. For example, lipids
provided 59.3'Jf of energy needs during metamorphosis of Ostreci
echilis (Holland and Spencer. 1973) whereas another study showed
that metamorphosis of the same species was fuelled mainly by
50
0
r^0.66,p =0.049
£^
40 J
♦
**
c
•
oi
\^
E
30 -
^N^
a>
\s^
S
N.
20 -
10 -
♦ X,^^
r^=0.30,p =0.259
10
20
30
40
0
1
TAG/ST TAG/PL
Figure 9. Triacylglycerol (TAO-cholesterol (ST) and TAG-pholpholipid (PL) ratios of larvae fed diet B ( ♦ ) and C (•) at competency (day 28)
as a function of ttie settlement success. Settlement success is tile percentage of settled larvae at day 40 based of the number of competent larvae
at day 28. Data of experiments I and II were pooled (h = 6 larval culture).
386
Pernet et al.
Diets
DietC
Day 36
TAG/ST= 9.69
TAG/PL= 0.79
N=2S
36%
16%
44%
TAG/ST= 16.67
TAG/PL=1.63
N=19
32%
15%
47%
Day 40
TAG/ST= 8.04
T.AG/PL=0,54
N=8
TAG/ST= 23.43
TAG/PL= 1.55
N=27
53%l
11%
83%
34%
Figure 10. Time budgets for sea scallop larvae according to diet and
time. Recorded behavior were larvae actively crawling (D) or remain-
ing fixed on the screen <■) swimming (■ ) or passive in the water
column {[J )■ Each pie chart represents the proportion of observation time
spent by each larva exhibiting a specific behavior. (n:2 replicate culture)
proteins, lipids accounting for only 16.8% (Rodriguez et al. 1990).
Secondly, low TAG levels have been observed in newly settled
larvae (Holland & Spencer 1973). In our experiments, larvae may
not have been as newly settled as necessary to observe a TAG
depletion. Lipid analysis were performed on sample of ca. 5000
larvae containing settled larvae of different ages. Thus, some of
them may have started recovering energy reserves and increasing
the mean lipid level of the cohort. For instance, post-metamorphic
larvae of O. ediilis had recovered pre-metamorphic neutral lipid
mass 4 to 11 days after settlement (Holland & Spencer 1973).
Finally, sea scallop larvae may continue to feed on microalgae
during settlement and counterbalance the lipid utilization. In fact,
the oyster Crassostrea rirginica has the ability to feed during
settlement and metamorphosis (Baker & Mann 1994). The feeding
hypothesis agrees with the maintenance of dietary differences in
TAG levels during settlement.
Effects of Dietary Triglyceride Enrichment
The diet enriched in TAG promoted higher larval growth (Fig.
2). Chaetoceros muelleri grown under silicate limited conditions
leads to the highest growth rate of juvenile oysters at low feeding
rations whereas at higher feeding rations, the silicate limited cul-
ture was a poorer diet than the control culture (Enright et al. 1986).
The high level of calorie-rich SFAs and MUFAs in the silicate-
limited diet was evoked by the authors to explain higher growth
rates of the oysters at the lowest feeding ration. At the higher
feeding ration, the fatty acid composition, and particularly the
relatively low content of 22:6n3 became a limiting factor and
might explain the lower growth rate of oyster larvae fed with the
silicate limited cells. In our experiments, high growth rates were
obtained with a diet including silicate limited culture of C. muelleri
(diet C) probably because SFA and MUFA were 4 times higher
and PUFA were also twice higher than in diets A and B.
Abnormally high mortality was observed for larvae fed diet A
in both experiments and was correlated with extremely low TAG
and low total lipid levels in diets and larvae. This agrees with
previous studies showing a positive relation between larval lipid
content and survival (Gallager et al. 1986, Delaunay et al. 1992,
Ouellet & Taggart 1992). It seems that low lipid or TAG level in
the feeding regimen might have deleterious effect on survival,
whereas there was no difference in survival for the other regimes.
TAG levels in competent larvae were correlated with TAG
levels in diet (Figs. 4 and 5). A possible explanation is that the
metabolic cost involved in TAG synthesis and storage could be
lower when feeding upon microalgae rich in SFA and MUFA.
TAG, or more generally, total lipid rather than with other bio-
chemical sources such as protein or carbohydrate. However, con-
clusions based on these observations must be tempered by the
possibility of effects as a result of variations of other unmeasured
variables such as digestibility or palatability due to diatom cell
wall properties or biochemical compounds such as amino-acids
and vitamins (see Robert & Trintignac 1997. for review). In short,
we cannot argue a direct causal relationship between diet and
larval lipid composition.
Liinal Quality
We used two indicators to assess larval quality: TAG-ST and
TAG-PL ratios. During larval development prior to competency,
both ratios have led to the same results and conclusions. As PL
have an energetic role during early larval development or starva-
tion period (Tocher et al. 1985. Fraser el al. 1988, Fraser 1989,
Delaunay et al. 1992) the use of the more conservative TAG-ST
ratio should be more appropriate to estimate larval quality. How-
ever, PL depletion was not observed in our study of sea scallop
larvae, as reported for larvae of Patinopecten yessoeiisis (Whyte
1987). This allows us to use both indicators without distinction.
Based on these indicators, a positive relation between size of
28-day larvae and larval quality at day 20 was found (Table 2). It
has previously been reported that poor growth was related to a low
TAG-organic matter (OM) ratio in D-larvae. while higher levels of
ratio were not necessarily reflected by growth rate (Delaunay et al.
1992). Consequently, it seemed that ST was a better denominator
than OM. This might be due to the fact that OM values include
both structural and storage molecules, thus decreasing accuracy of
a storage-structure ratio.
A positive relationship between number of competent larvae
produced and 8 d larval quality indices was found (Table 2). This
is in accordance with results of previous studies. Indeed, risks of
mortality of the shrimp Pandalus borealis were well correlated
with the condition indices of the larval group as measured by the
proportion of larvae exhibiting a TAG-wet mass ratio <0.2 (Ouel-
let & Taggart 1992). Moreover, high mortality of larvae of scallop
P. maxiiHHs could be related to an abnormally low initial TAG-OM
ratio (Delaunay et al. 1992). Our study suggests that survival at
competency was partly explained by the efficiency of recovery of
lipids after embryogenesis and. as discussed by Delaunay et al.
(1992), reflects the difficulties of the transition to the D-veliger
stage.
Finally, a negative correlation between the quality (TAG-ST
ratio) of competent larvae (28-day-old) and settlement success (at
40 days) was found (Fig. 9). The better the quality, the lower the
settlement success. In the barnacle Semibalanus balanoides, it ap-
pears that cyprids of high physiological condition, as measured by
Biochemical Indicator of Sea Scallop Larvae
387
B36
C36 B40 C40
J^
B36
C36
840 C40
Figure 11. Exploration rate (Al and distance (B) as a I'unction of diet (B or C) and age of larvae (36 or 40 days old). Exploration rate is the
distance traveled by exploring larvae (h varied from 2 to 14 as a function of treatment combination) per minute. Exploration distance is the
average value of the distance traveled by all the observed larvae (;i varied from 8 to 28). Vertical bars represent standard errors between replicate
cultures.
a TAG-ST ratio, settle in the best quality habitats compared with
those in low condition (Miron et al. 1999). In fact, TAG-ST ratio
of cypiids was highest at low intertidal level, the preferred attach-
ment location site. This suggests that larvae fed with rich TAG diet
C and having high TAG-ST ratio might delay metamorphosis to
encounter better quality settlement sites, whereas larvae fed with
poor TAG diet B and having low TAG-ST ratio would not have
enough reserves to make to a meticulous selection of settling site
and therefore would settle more rapidly. According to this sce-
nario, larvae in good condition would delay metamorphosis until
reaching a critical physiologic threshold where settlement would
become urgent. In support of this hypothesis, behavioral observa-
tions showed that larvae fed diet C (high TAG-ST) spent 12-15%
of observation time exploring the substratum whatever the age,
whereas larvae fed diet B (low TAG-ST) did not explore the sub-
stratum at day 40 (Fig. 10). Thus, it seemed that larvae fed diet B
lost selectivity with age as observed with the barnacle Balaniis
amphitrilc (Rittschof et al. 1984. Pechenik et al. 199.^. Qian &
Pechenik 1998. Miron et al. 1999). Moreover, in our study, larvae
spent 15% to 47% of observation time swimming in the water
column. Such swimming activity in competent larvae has previ-
ously been reported for the bryozoan Bugula ncritiua (25% to 45%
of observation time), which was attributed to the stillness of the
water (Walters et al. 1999). Similarly, substrates became attractive
to the cyprids of Balamis amphitrite in flow, whereas no explora-
tion was observed in individuals from the same cohort placed in
still water (Miron et al. 2000). Larvae of other invertebrate species
also seem to avoid settlina in low flow conditions (Mullineaux &
Butman 1991. Pawlik et al. 1991). For example, larvae of the
polychaete Phragmatopoma califoniica tumbled along the bottom
in the presence of fast flow, whereas they were swimming in the
water column in slow flow (Pawlik et al. 1991). Thus, the low
settlement success observed in larvae of high quality might be the
effect of delayed metamorphosis because of inappropriate hydro-
dynamic conditions in our experiment. If this interpretation was
correct, larval quality need to be considered jointly with hydrody-
namics to fully understand the decision process of settling larvae.
ACKNOWLEDGMENTS
The authors thank E.-J. Arsenault and S. Bourget for their
assistance in the culture of larvae and microalgae and all the staff
of Centre Aquacole Marin de Grande-Riviere of Ministere de
FAgriculture des Peches et de TAlimentation du Quebec for help
in hatchery and laboratory. Thanks are also addressed to E. Dem-
ers from Centre de Transformation des Produits Aquatiques
(CTPA) for teaching GC analyses and fatty acid identification.
Funding for this research has been provided by CORPAQ (Conseil
des Recherches en Peche et en Agro-alimentaire du Quebec).
MAPAQ and GIROQ (Groupe Interuniversitaire de Recherches
Oceanographiques du Quebec). We are grateful to Dr. L. Fortier
from Universite Laval, who kindly let us use the latroscan. and Dr.
B. A. MacDonald from University of New Brunswick for his en-
doscope camera. Thanks are also addressed to G. Daigle, Depar-
tement de mathematique et statistique. Universite Laval, Quebec
for validating the statistical analysis, and V. Moreau. M. Cusson
and L. Lapointe for their constructive and critical discussions.
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Jounud oj Shellfish Research. Vol. 22, No. 2. 389-.W1. 2()().V
A RAPID TEST FOR THE DETERMINATION OF THE SPAWNING STATUS OF THE BAY
SCALLOP, ARGOPECTEN IRRADIANS (LAMARCK, 1819)
STEPHEN L. ESTABROOKS*
Nantucket Marine Laboratory, 0 Eastern Street. Nantucket. Massachusetts 02554
ABSTRACT The bay scallop. Argopecten iiraduiiis irnidians (Lamarck. 1819). is a generally semelparous. commercially imponant
marine bivalve found along the shores of the Northeast Atlantic from Cape Cod, Massachusetts to New Jersey. It can be found in areas
of varying conditions, including current flow, nutrient levels, salinities, siltation levels, all factors that can affect its size when it
becomes legally harvestable. A harvestable scallop is defined as having a visible growth ring signifying that it has completed its
reproductive cycle. However, there are areas on the island of Nantucket, MA. that produce scallops that lack this classic growth ring,
giving rise to disagreements between scallop fishermen and regulatory agencies concerning the legality of harvesting them or returning
them to the water. A rapid. 10-min test has been developed to quickly determine whether scallops in a particular area have spawned.
It was determined that bay scallops, at least those found in Nantucket waters, retain mature spermatozoa in their gonads throughout
the scallop harvest season, which in Massachusetts, runs from October through March of the next year. Detection of their presence
could be useful in determining their spawning status. A small piece of male gonad is removed, homogenized bnetly. and stained to
detect the presence of these residual spermatozoa. It is hoped that the implementation of this rapid test will help to settle some of these
local disputes, which should help ensure the protection of seed scallops.
KEY WORDS: scallops, Argopecten. spawning, seed
INTRODUCTION
The bay scallop, Argopecten irradians irradians (Lainarck.
1819), found in shallow bays along the Northeastern United States
coast from Cape Cod to New Jersey, is a hermaphroditic, generally
semelparous bivalve that is sexually mature at the age of I yr
(Belding 1910). The reproductive period inay last from mid-.lune
into September in the waters surrounding Cape Cod. depending on
local conditions, after which time scallops may be harvested be-
cause most will not survive to complete a second reproductive
season (Belding 1910, Marshall 1960, Taylor & Capuzzo 1983.
Tettelbach et al. 1999).
The waters surrounding the island of Nantucket. Massachu-
setts, have yielded steadily declining scallop harvests from a high
of 117,000 bushels in 1980 to a low of 6.800 bushels in 1998
(Curley 2002). It has been long recognized that seed scallops must
be protected because they are the primary source of the next year's
harvest (Belding 1910). Scallops resulting from late spawning tend
to be much smaller the next year, although many may catch up in
size with scallops spawned earlier in the year, again, depending
on local conditions (Auster & Stewart 1984). However, many of
these late-spawned scallops may lack the distinctive growth ring,
generating confusion among fishermen and regulatory agencies as
to whether these are seed scallops and should not be harvested
(MacFarland 1991).
Massachusetts laws governing the taking of mature bay scal-
lops require the presence of a ""well defined raised annual growth
ring" (MGL.cl30,s.70). However, in some areas of Nantucket,
varying conditions, such as water temperature, food supply, cur-
rent flow, and heavy siltation. among others, can lead to scallops
lacking this distinctive ring. In addition, several investigators have
documented late spawning events that give rise to smaller scallops
with very small growth rings very near the hinge line (McFarland
1991, Tettelbach et al. 1999, Tettelbach et al. 2001). Scallops
*Correspondence. Tel.: 1-843-546-4047; E-mail; estabrooks(a'sccc.tv
spawned near the beginning of the spawning season, generally
beginning around the middle of June when water temperatures
reach 15°-16°C in Nantucket waters, in an area of good current
flow and sufficient food, can often yield seed scallops that are as
large or larger than the average adult by the time harvest season
arrives (Kelley & Ceely 1980). During one very productive year,
1990, in Pleasant Bay on Cape Cod, MacFariand (1991), found
that if scallops were harvested based on size alone, as was recom-
mended by local fishermen, 66'7f of those harvested would have
been large seed.
In 1999. one area. Madaket Harbor, was closed to scalloping
because of the presence of a large number of seed, and the fol-
lowing year saw the initiation of much stricter enforcement of
taking only scallops with the distinctive growth ring. This has led
to controversy because Madaket Harbor has generally poorer
growing conditions and often yields scallops without the classic
annual ring. Fishermen point to a fine line, usually within a cen-
timeter of the hinge, as the annual growth ring, giving rise to the
local term "nub" scallop, i.e., a scallop that has spawned (1-t- yr)
but that demonstrates no normal growth ring. Others state that
this is a first-year scallop that has not spawned (O-i- yr) and there-
fore should be returned to the water. Heretofore, confirmation has
relied on preserving scallops in formalin and sending the speci-
mens to a laboratory to have histologic slides prepared and read,
with the results often obtained weeks later. To help eliminate this
confusion, a rapid yet definitive test was developed to aid local
regulatory agencies in differentiating seed scallops from those that
have spawned. This test is based on the observation by the author
over several years that bay scallops, at least those found in Nan-
tucket waters, retain residual mature sperm in their gonads into
March and April of the following year, whereas residual eggs
are generally resorbed quickly, most likely because of their high
energy content and the scallop's need to store energy for the up-
coming winter. The purpose of this investigation was 2-fold: to
determine whether this sperm retention was a sporadic event or
was generally found throughout the scallop population and sec-
ondarily, to develop a rapid test to detect the presence of the
residual sperm.
389
390
ESTABROOKS
METHODS
Fifty bay scallops that displayed the classic annual growth ring
(1+ yr) were collected each month from Nantucket Harbor from
October 1998 through March 1999. Scallops were obtained from
three sources, SCUBA diving, from commercial scallopers. and from
upwellers and lantern nets inaintained at Nantucket Marine Labora-
tory. Because the purpose of this study was to see whether scallops
that had spawned retained sperm throughout the harvest season, only
those scallops that had clearly spawned were used. In addition to the
presence of the growth ring, the gonad was large and flaccid, the
upper shell was generally encrusted with flora and fauna, and the
bottom valve was distinctly of a greater curvature than the upper shell.
Also, 25 known seed scallops (0+ yr) that had been obtained in July
and August of 1998 from sets onto onion bags and subsequently
maintained in upwellers and lantern nets were also tested each month.
Residual sperm in bay scallops were examined by clipping a
small piece (1-2 mm" is sufficient) from any part of the male
gonad (see Fig. 1) and placed in a disposable 1.5-mL plastic coni-
cal tube containing approximately 0.5 mL of \0'7c formalin (this
amount is not critical).
The tissue was ground for 15-30 sec using a pellet pestle, and
a drop of this mixture was placed onto a glass slide and spread.
Once air-dried, the slide was dipped in methanol to fix the tissue,
and stained for 10-30 sec in a Safranin: Wright-Giemza: Water
(1:2:10) stain.
The slide was rinsed briefly in running tap water, air-dried and
read at lOOOx magnification under oil immersion. Results can be
hastened by drying the slide at each step on a slide warmer or hot
plate. This procedure also lends itself to sampling by needle biopsy
if sacrificing of the scallop needs to be avoided (Schneider et al.
1997).
RESULTS
Sperm with characteristic bullet-shaped heads (S) were stained
grayish-blue with Wright-Giem/a, and the tails (T) were stained
i
Figure L Anatomy of the bay scallop, Argopecten irradians (L.) seen
with the upper or left valve removed: male gonad (MG); female gonad
(FCl; adductor muscle (.AMI; gill (CL); digestive gland (DG); heart
(HTl; mantle (MT).
• -.
u.
i
4
Figure 2. Residual sperm ( KKlOx) seen in a representative bay scallop
harvested in February 2001 confirming that it has previously spawned
and is legally harvestable. Sperm heads (S); tails (Tl; hemocytes (H).
pink by the safranin (Fig. 2). These can be readily distinguished
from the larger nucleated hemocytes (H) that tend to become more
pervasive in the gonad as the season progresses, ostensibly phago-
cytizing the remaining sperm. Late in the scallop season, i.e.,
February and March, some scallops may demonstrate mostly tails
(Fig. 2).
Results for 100% of the seed scallops (O-t- yr) tested negative
for the presence of spermatozoa. In the post spawning scallops
( 1-1- yr), of 300 individuals tested from October through March,
241 displayed residual sperm, 55 demonstrated only tails, and the
remaining 4 tested negative (these were from the scallops collected
in March).
DISCUSSION
Because one of the primary concerns in maintaining a semelpa-
rous and commercially important species, such as Argopecten
irradians irradians. is ensuring the survival of the seed scallops, it
is important that they not be taken before their contributing to the
following year's population.
Traditional methods of detemiining whether bay scallops have
spawned, in addition to the presence of a distinct annual growth
ring, include a larger gonad, grayish in color or with a whitish line
or band as compared with a much smaller gonad, shiny black in
color, as seen in reproductively immature scallops. Also, a dis-
tinctly curved lower or right shell as compared with the upper shell
and a generally rougher appearance due to a greater longevity in
the water with a concomitant accumulation of flora and fauna upon
its upper shell help to separate the older scallop from its younger
cohort (Belding 1910).
However, these differences are often subjective and can lead to
controversy between fisherman and local regulatory agencies. In
addition, the spawning season in Massachusetts waters, as de-
scribed by Belding (1910) and Sastry (1963). generally has run
from the middle of June through mid-August but now seems to be
Spawning Status of the Bay Scallop
391
extended to include September (Kelley & Sisson 1981. McFarland
19<:)1). Tettelbach (1999) found similar results for some scallop
populations in New York state.
In Nantucket, these late-spawned or nub scallops lack the dis-
tinctive growth ring seen in earlier-spawned scallops. These may
spawn the next year, but in one experiment. 807f of these nub
scallops held in cages survived yet an additional year {2+ yr) and
50% of those spawned (Conant. K. Assistant Town Biologist. Nan-
tucket. MA, personal communication, 2002). However, many fish-
ermen see a line, usually falling within 10 mm of the hinge, as the
growth ring, and harvest these as adults. McFarland ( 1991 ) found
that 9% of the scallop population had growth rings between 4 and
8 mm from the hinge line, of which 50% spawned the next year
and the remaining 50% spawned the following year. It remains
unclear as to the significance of the contribution of this small
portion of the population, but it inay play a role in the persistence
of some scallop populations (Tettelbach et al. 1999). Although the
significance of this secondary spawning remains unclear, it is ab-
solutely clear as to the significance of taking scallops that have yet
to spawn. It is hoped that the development of a rapid yet simple test
to determine v\'hether scallops in an local area have spawned or not
will be useful in reassuring both regulatory agencies and scallop
fishermen that only adult animals are being harvested.
ACKNOWLEDGMENTS
This research was supported by grants from the PADI Foun-
dation. The Nantucket Land Council, and the Nantucket .Shellfish
and Harbor Advisorv Board.
LITERATURE CITED
Auster, P. J. & L. L. Stewart. 1984. Compensatory growth in the bay
scallop. Argopeclen irradians (L.). J. Northwest Atlantic Fish. Sci.
5:103-104.
Belding. D. L. 1910. A repon upon the scallop fishery of Massachusetts.
Boston: The Commonwealth of Massachusetts. 150 pp.
Curley. T. 2002. Shellfish Catch Reports. Nantucket. MA: Nantucket An-
nual Town Report. Shellfish and Marme Department.
Kelley. K. & M. Ceely. 1980. Studies of bay scallops, Argopecleu irradi-
ans. on Nantucket 1979-1980 season. Nantucket. MA: Shellfish and
Marine Department, pp. 16-34.
Kelley. K. M. & J. D. Sisson. (1981). Seed sizes and their use in deter-
mining spawning and setting times of bay scallops on Nantucket. In:
K.M. Kelley. editor. The Nantucket Bay scallop fishery: the resource
and its management. Nintucket. MA: Shellfish and Manne Depart-
ment, pp. 43—19.
MacFarlane, S. L. 1991. Managing scallops .4/;i;()/)fcrc)i irradians irradi-
ans (LamiU'ck) in Pleasant Bay. Massachusetts; large is not always
legal. In: S. E. Shumway and A. P. Sandifer. editors. IntT Compen-
dium of Scallop Biology and Culture. World Aquaculture Soc. 264-
272.
Marshall. N. I960, Studies on the Niantic River. Connecticut with special
reference to the bay scallop. Aequipecten irradians. Limnol. Oceanogr.
5:86-105.
Massachusetts General Laws. Chapter 1 30. Section 70.
Sastry, A. N. 1963. Reproduction of the bay scallop, Aequipecten irradians
Lamarck. Influence of temperature on maturation and spawning. Biol.
Bull. 125:146-153.
Schneider. P.. R. Smolowitz, C. Smith. J. Degiorgis & M. McCafferty.
1997. Comparison of three techniques for evaluating seasonal game-
togenesis in Spisula solidissima. Biol. Ball. 193:233-234.
Taylor. R. E. & J. M. Capuzzo. 1983. The reproductive cycle of the bay
scallop. Argopecten irradians irradians (Lamarck), in a small coastal
embayment on Cape Cod. Massachusetts. Estuaries 6:431-435.
Tettelbach. S. T. C. F. Smith. R. Smolowitz. K. Tetrault & S. Dumais.
1999. Evidence for fall spawning of Northern Bay scallops Argopecten
irradians irradians (Lamarck, 1819) in New York. / Shellfish Res.
18:47-58.
Tettelbach, S. T., P. Wenczel & S. W. T. Hughes. 2001. Size variabihty
of Juvenile (O-t- Yr) Bay Scallops Argopecten irradians irradians
(Lamarck, 1819) at Eight Sites in Eastern Long Island. New York. The
Veliger 44(4):389-397.
Jomnal of Shellthh Research. Vol. 22, No. 2. 393--W9, 2003.
OPTIMIZATION OF SETTLEMENT OF LARVAL ARGOPECTEN PURPURATUS USING
NATURAL DIATOM BIOFILMS
RUBEN AVENDANO-HERRERA,' CARLOS RIQUELMES,'* FERNANDO SILVA,'
MIGUEL AVENDANOD,- AND RUTE IRGANG'
' Liihoratorio de Ecologia Mkiohiami. Departameuto de Acuicidtuni, Uuiversidad de Antofagasta
Cusilla 170, Antofagasta; 'Departameuto de Aciiicidtiira. Uuiversidad de Antofagasta, Casilla 170,
Antofagasta
ABSTRACT Larval settlement is a critleul stage in the artificial production of Argopeclcii iniipurauis. The study investigated the
feasibility of improving post-larval settlement of this .species using a substrate (cultchi that was pre-treated with a biofilm of native
diatoms. Four species of diatoms were isolated from the surface of collectors that had high numbers of juvenile scallops (spat). These
four species were able to attach themselves and grow on a polystyrene substrate. Scallop post-larval settlement was evaluated
experimentally in two ways: (I) laboratory experiments in lO-L buckets; and (2) under natural condition by in situ experiments at the
Marine Reserve "La Rinconada" (Antofagasta. Chile). Effects of biofilm treatments were examined using collectors that were coated
with diatoms and collectors handled using normal culture methods (new netlon held in filtered seawater that did not have a biofilm).
Results of the laboratory experiments showed a higher percentage oi A. purimmhis post-larval settlement on collectors coated with
Fwgiliaropsis pseudomma compared with control collectors (P > 0.03). Results comparing biofilms of the diatoms F. pseudonaiia and
Navicida venela showed higher settlement on collectors pretreated with N. venehi ( 1.136 ± 172 spat per collector "' ). Statistical analysis
showed that the addition of diatom biofilms enhanced spatfall and always produced larger settlement compared with untreated
collectors. These results indicate that addition of cultured diatom biofilms improves scallop larval settlement.
KEY WORDS: Argopecten purpuratus. diatoms, biofilms, post-larval settlement
INTRODUCTION
The northern Chilean scallop, Argopecten piirpiiraliis (Lama-
rck 1819), is the most important commercial bivalve species in
Chile. Production in 1999 was 20.668 I. valued at $13 million (US)
and the industry provided 3.600 direct jobs (Lozano 2000). The
aquaculture production, however, is not sufficient to satisfy the
international demand for this species. A major reason is the large
variation in natural seed production (Navarro et al. 1991, Disalvo
1991. Avila et al. 1994. Riquelme et al. 1995. Avendano et al.
2001) that supplies about 30% of the annual Chilean production
(Farias et al. 1998).
A major problem with seed supply occurs at metamorphosis
when larvae settle on a substrate (Keough & Downes 1982). Fol-
lowing attachment larvae undergo considerable morphologic and
physiologic changes as they metamorphose from a pelagic to a
benthie existence (lUanes 1990). There are generally a large num-
ber of larval mortalities (Tremblay 1988. Bourne et al. 1989.
Castagna 1975). Ambrose et al. (1992) reported that there was
little information concerning factors that influence scallop larval
settlement and this led to numerous studies on the subject in the
mid 1990s. A goal of these investigations was to increase settle-
ment by improving substrates for settling larvae including color of
the substrate, size, monofilament density and composition of the
collector (Miron et al. 1995. Pouliot et al. 1995. Pearce & Bourget
1996). Further, the mechanism by which scallop larvae detect and
settle on a particular substrate is still not understood (Harvey et al.
1997). Many studies showed that biologic, chemical and physical
factors could induce larval settlement of marine invertebrates
(Weiner et al. 1989; Bonar et al. 1986. Christensen 1989, Maki et
al. 1990, Chevolot et al. 1991). Many of these studies showed that
bacterial films were important for triggering larval settlement
(Meadows & Campbell 1972, Kirchman et al. 1982. Weiner et al.
Corresponding author. E-mail; criquelmeCs'uantof.cl
1989, Maki et al. 1990, Pearce & Bourget 1996). Bacterial com-
munities were found associated with other microorganisms such as
diatoms forming a multi-specific biofilm that was firmly attached
to a substrate. These multi-specific biofilms emitted several types
of signals, including; ( 1 ) peptic (Zimmer-Faust & Tamburri 1994)
or associated fatty acids (Pawlik 1986) and (2) polysaccharides
and glycoproteins (structure of a biolfilm) (Hadtleld 1986) that
would stimulate marine invertebrate larvae to settle (Pawlik 1992.
Keough & Raimondi 1995).
Harvey et al. (1995). using electron microscopy showed that
biofilms were not only composed of bacteria but microalgae and
detritus as well. The various organisms may have different effects
on settlement of different species of scallops. Benthie diatoms that
colonize substrates might not only be a source of nutrition for more
advanced post-larval stages of marine invertebrates (Takami et al.
1997) but also may be necessary for the settlement of mollusc
larvae, as shown in abalone culture (Seki 1980, Hahn 1989).
The purpose of this study was to isolate native diatoms from
scallop collectors that had high levels of settled spat and evaluate
the feasibility of improving post-larval scallop settlement by using
of biofilms composed of specific diatom species. Results of labo-
ratory and in situ field experimental work are reported here.
MATERIALS AND METHODS
The study was perfomied in three stages; ( I ) isolation of native
diatom species: (2) laboratory experiments undertaken at the
hatchery of the Facultad de Recursos del Mar de la Uuiversidad de
Antofagasta (FAREMAR, Faculty of Marine Resources at Anto-
fagasta University); and (3) in situ field experiments undertaken in
"San Jorge" bay at the Marine Reserve "La Rinconada"
(27°03'24"S-70°5r30"W).
Isolation of Diatoms
Diatoms isolation was undertaken at the Cultivos Marinos In-
ternacionales hatchery in "Inglesa" bay, Chile (27°03'24"S-
393
394
Avendano-Herrera et al.
70°5r30"W). Netlon collectors with high levels of scallop-spat
settlement were selected (more than 2.500 spat per collector"').
Seventy pieces of netlon mesh were cut into 100 cm" sections.
They were washed several times with a marine saline solution
(SSM) (Austin 1988), and placed in Schott bottles with 50 ml of
seawater filtered to 0.2 |jim. Diatoms were removed from the mesh
with an Ultrasonic Homogenizer (Cole-Parmer) for 60 sec. The
resulting solution was diluted in test tubes with 9 mL of F/2 com-
mercial Fritz Chemica Inc. supplemented with sodium metasilicate
(F/2M, Guillard & Ryther 1962). These tubes were incubated for
7 d at 20 ± rC. photoperiod 12:12 and a light intensity of 100
(jimol m"~s~'. Dominant diatom species were isolated using the
microfishing technique described by Hoshaw and Rosowski
( 1979). Four species were identified after the method of Rodriguez
(1998) as: Navicula veneta (Kutzing), Navicida ciyptocephala
(Hustedt). Navicula menisciihis (Schuman) and Fragilariopsis
psciicloiuma {}i-ds]e). The species were then purified by exposure to
a wide range of antibiotics (Hoshaw & Rosowski 1979).
Adherence of Diatoms to Polystyrene Substrates
Adherence assays to attach diatoms to the experimental sub-
strate were performed using the method of Gawne et al. (1998).
Polystyrene petri dishes with a diameter of 3 cm were filled with
5 mL of filtered seawater (0.2 |xm) autoclaved for 15 min at 12rC
and inoculated with either species of N. veneta (Nv). N. crylo-
cephala (Nc), N. menisculus (Nm), and F. pseudonana (Fp) at a
concentration of 5 x 10' cells x ml"' (3.5 x 10*^ cells x cm"-) in
the pre-stationary phase. Three replicates of each culture were
incubated in a controlled environment room at 20 ± 1 °C and a
photoperiod of 12:12 for 48 h. Each petri dish was washed 5 times
with 0.2 (im filtered seawater to insure that only those diatoms
adhering to the bottom of the dishes were retained, non-adhering
diatoms were thus eliminated. Diatom adherence at incubation
times of 1,6. 12, 24, and 48 h were recorded by direct count with
an inverted microscope Olympus 1X50 at a magnification of x 1 00
(Guillard 1973) The percentage of diatom adherence was calcu-
lated by comparing the concentration of inoculated diatoms with
those observed on the bottom of the petri dish.
Growth of Diatoms on Polystyrene Substrates
Polystyrene petri dishes with a diameter of 3 cm were filled
with 4 mL of F/2M solution autoclaved for 15 min at 121°C. Each
dish was inoculated with one of the four species of diatoms from
the pre-stationary phase at a concentration of 5 x 10"* cells x ml"'
(2.8 X 10^ cells X cnr). To assess the growth of diatoms, microal-
gal counts were performed every 48 h for a period of 144 h under
identical conditions to those in the adherence experiments.
Stage U-Laboratory Experiments on Settlement of
Post-Larval Scallops
The effect of native diatoms on the settlement of A. purpwatits
post-larvae, was evaluated by conforming settlement among
coated with biofilm of the four species of diatoms.
(a) Determination of post-larvae settlement substrate pre-
treated with diatoms (according to the criteria of diatom adherence
on substrate).
Bioassays were performed in buckets containing 10 L of 1 |j.m
filtered seawater and no aeration. Each bucket was inoculated with
strains of diatoms in the stationary phase (Fox 1983) at a concen-
tration of 5 X 10' cells X ml"'. After inoculation of the diatoms, a
piece of netlon collector was placed in each bucket (length x width
= 30 X 60 cm ) and incubated for 48 h. A set of collectors that were
placed and kept in 10 |j.m-filtered seawater was pre-treated ac-
cording to procedures done by commercial companies (natural).
The control was new netlon that did not have a biofilm (Ct s/b).
At the end of the incubation period, "eyed" scallop larvae
(>220 p.m) were added to each bucket at a density of 1 larva x
mP', and maintained for a 7-day-period. During this time, the
water in each bucket was changed daily, larvae were filtered on a
120 |j.m screen, washed on 205 (jlhi screens, and returned to their
respective buckets. Larvae were fed daily with a mixed diet of
7,500 cells X ml"' of Chaetoceros calcitrans and 10.000 cells x
ml"' of C gracilis. After seven days the netlon collectors were
removed, cleaned with horsehair brush and the spat collected on a
205-|jLm screen. The number of attached spat was determined using
an Olympus BH2 stereoscopic microscope. Results were expressed
as "percent settlement" calculated by comparing the number of
attached spat on collectors to the number of "eyed" larvae added to
each bucket (Avendaiio-Herrera et al. 2002).
Settlement i
Number of attached post-larvae x 1 00%
I X lO"* "eyed" larvae
(b) Determination of post-larvae settlement on substrate pre-
treated with diatom (according to the criteria of diatom growth on
substrate).
Bioassays to assess diatom growth were performed in buckets
containing 10 L of 1 p,m filtered seawater using a constant 24-h
photoperiod with a light intensity of 50 ixmol m"" s"' and aeration.
Buckets were inoculated with diatoms at concentrations similar to
the polystyrene substrate growth experiments. To stimulate growth
during the incubation period, treatments and controls were en-
riched with the addition of F/2M. Netlon spat collectors measuring
30 X 60 cm that are typically used by commercial companies were
placed in each bucket and incubated for a 96 h period.
The bioassays with larvae were carried out as previously de-
scribed.
Stage Itl-in Situ Field Experiments of A. Purpuratus Attachment to
Collectors Treated with Diatoms
When the effect of the four diatom species on settlement of
scallop larvae was known from the laboratory experiments, strains
of F. pseudonana and N. veneta were selected for further testing in
the natural environment. Buckets with 20 L of l-|j.m filtered sea-
water were inoculated with diatoms in the stationary phase at a
concentration of 5 x lO"* cells x ml"' and incubated with aeration
and constant 24 h photoperiods at a light intensity of 50-|jimol m'
s"'. A biofilm was established on one set of collectors using the
method commonly used by commercial companies (Natural) and
as control was used new the control used new netlon without
biofilm (Ct s/b). Treatment and control buckets were enriched with
the addition of F/2M and incubated for 10 days after inoculation of
the diatom six netlon spat collector (30 x 60 cm) and placed in
each bucket.
A collector from each experiment was sampled to determine
the density of diatoms attached to the surface of the collectors at
the end of the incubation period. Three pieces of netlon were cut
into 25 cnr pieces, washed repeatedly with SSM and placed in
50-ml Schott bottles, and the diatoms attached to pieces of netlon
were removed using an Ultrasonics Homogenizer for 60 sec. The
number of diatoms attached to the monofilaments of each piece of
Settlement of Larval A. pukpuratus and Diatom Biofilms
395
netlon was determined by direct counting using a Neubauer cham-
ber and an Olympus BH-2 microscope. Results were extrapolated
for the complete area of the collectors (1.800 cnr).
The five remaining collectors from each treatment and control
were placed in 1 x 1 mm "onion" bags, labeled, and placed in the
ocean at a depth of 16 m at the Marine Reserve Area for 38 days
(January \5 to February 22. 2002). Prior to placing the collectors
in the water, plankton-sampling method was used to assess A.
piiipiiiatus spatfall. Water temperature was recorded to evaluate
larval and spat growth during the 38-d period (17°C ± T'C).
After 38 days the collectors were removed from the ocean
following the method of Wallace (1982) and taken to the Labora-
torio de Ecologia Microbiana de la Universidad de Antofagasta
(Microbial Ecology Laboratory of the University of Antofagasta)
to assess spatfall. The effect of diatom biofilms on settlement of /\.
piirpiiraliis was determined by counting the juveniles (spat) that
were firmly attached to the monofilament of the treated collectors.
Results were not affected by those spat that fell off collectors
during transport because the interest was on spat that were firmly
attached to the collectors. Each collector was removed from the
onion bag. washed with circulating water for 5 min. and the at-
tached material collected was deposited on a 205-|xm-mesh screen.
To avoid loss of spat, each collector was cleaned with a horsehair
brush and the spat were preserved in (70%) ethanol for counting
with a stereoscopic Olympus microscope.
Statistkal Analysis
The growth rate of diatoms was calculated using Guillard's
equation (Stein 1979), which describes mean microalgal duplica-
tion velocity:
K = [3.322/(t- -t')] X (log N-/N')
Where K is the mean microalgal duplication velocity of the
microalgal biomass. N' is the cellular density of the beginning of
the experiment, N" is the cellular density at the end of the experi-
ment, t' is the time at the beginning of the experiment and t" the
time at the end. Results were tested by ANOVA to compare
growth rates and maximum density values (Sokal & Rohlf 1980).
To evaluate the effect of diatoms on settlement of larvae in
laboratory experiments, the results were tested by ANOVA with
the statistical significance criteria (alfa = 0.05) and Multiple LSD
Comparison Test (Sokal & Rohlf 1980). The influence of selected
diatoms on settlement of larvae in the "in situ" field experiments
was realized counting the collector naturally pre-lreated (Natural)
as one treatment. Results were submitted to the Dunnet test com-
paring results from the various treatments to those of the control
(Zar 1984).
RESULTS
Isolalion of Diatoms
Four species of diatoms were isolated from the microflora that
was attached to the surface of scallop collectors. Only four species
could be purified to an axenic condition using a mixture of che-
motherapeutics and these were; Navicula veneta (Kutzing). Na-
vicula ciyptocephala (Hustedt), Navicula menisculus (Schumann),
and Fragilariopsis pseudonaim (Hasle).
Adherence and Growth of Diatoms on Polystyrene Substrates
N. veneta rapidly colonized plastic substrates without the ad-
dition of nutrients and 100% adherence was observed 48 h after
inoculation (Fig. 1). A similar situation was observed with N.
ci-\ptocepliala and N. menisculus.
Two growth patterns were observed for the four diatom species
when F/2M was added to the cultures, an accelerated growth for N.
veneta and N. menisculus and slower growth for N. cryptocephala
and F. pseiitlonana. Figure 2 illustrates that the four species were
in the exponential phase of growth after 96 h of culture and a
maximum cell production was observed after 144 h. When dupli-
cation velocity was compared, the rate for rapid growing species
was (K = 1.57 ± 1 duplication x days"') and the slow growing
species was (K = 1.38 ± 1 duplications x day"'), the difference
was significant (P < 0.05).
Stage II-Effect of Diatoms on Attachment of Scallop Larvae in
Laboratory Studies
Results of experimental laboratory studies showed a higher
percentage of post-larvae attached to collectors incubated with
diatoms for 96 h in 1 (xm filtered seawater enriched with the
addition of F/2M (criteria of diatom growth on substrate) com-
pared with collectors incubated with diatoms for 48 h in 1 p,m
filtered seawater (criteria of diatom adherence on substrate) (Fig.
3). Collectors treated with F. pseudonana had a larger number of
spat attached to the collectors. The breakdown was 2.567 ± 205
and 7,727 ± 107 post larvae x collector"' under the criteria of
diatom adherence and diatom growth on substrate, respectively.
Collectors without biofilms had lower numbers of spat on them.
Statistical analysis of settlement of larvae between collectors with
and without diatoms films after 48 h incubation showed a signifi-
cant difference between treatments with different diatom species
and the control that had no film {P < 0.05). There was no statistical
difference between settlement on the control and cultch that had
been treated with 10 p. in filtered seawater.
The only significant difference in settlement on cultch treated
with different species of diatoms was cultch treated with F. pseu-
donana (P < 0.05), incubated with diatoms for 96 h in I (xm
filtered seawater enriched with the addition of F/2M, and where
settlement reached 77.27% of the total available post-larvae. Un-
der laboratory conditions, collectors treated with 10 |xm filtered
seawater showed a significant increase in the number of settled
larvae (33.84 ± 7.13%). This number was close to the average of
those treated with the five species of diatoms (31.91 ± 2.21%).
125 1
too -
r 75
50 -
Figure 1. Percent attachment of the axenic diatoms Navicula veneta
Navicula (Nv), cryptocephala (Nc). Navicula menisculus (Nm), and
Fragilariopsis pseudonana (Fp) incubated for 48 h on polystyrene sub-
strate,s. Vertical lines show standard deviation.
396
Avendano-Herrera et al.
240 1
.-/!i
■^E
_«_
Nv -»-Nc -*-Nm -»«-Fp
>;
■fo
" __...z:....
o
2 '^°
1
g
^
Z-
^
■55 -^
S 60
o
o
U
^^
0 ^
0 48
Time (hours)
95
144
Figure 2. Growth curves of the axenic diatoms Navicula veneta (Nv),
Navicula cryptocephala (Nc), Navicula menisciiliis (Nm) and Fragilari-
opsis pseudonana (Fp) grown with the addition of culture medium
marine phytoplaniiton F/2M. \ ertical lines show standard deviation
Stage Ul-in Situ Field Experiments of Settlement of A. Purpuratus
Larvae on Collectors Treated with Diatoms
The best results for stimulating settlement in scallop lar\ae
were found with the diatoms F. pseiuloiuma and N. veneta and
these species were used in field experiments. After 10 days of
incubation the concentration of these two diatoms on cultch was 73
± 3.5 X lO'* and 47 ± 3.7 x 10'* cells x cm"", respectively. Plankton
sampling showed a concentration of 8,197 larvae x m""* with a
mean size of 181.4 ixm in water column in the Reserve. Recorded
seawater temperatures during the 38 days of the experiment in the
area of "Rinconada" did not show drastic changes.
Results of settlement on collectors after 38 days in the natural
environment are shown in Figure 4. Collectors treated with the
diatom N. veneta had a higher number of spat (1,156 ± 172 spat x
collector"') compared with the controls and other treatments. Sta-
tistical analysis showed that spat settlement on collectors with
diatom biofilms was always higher than controls (P < 0.05).
DISCUSSION
The presence of diatoms in the microflora of biofilms on sub-
strates is a natural phenomenon and formation of such a micro-
environment on a clean surface is inevitable (Cooksey & Wiggles-
worth-Cooksey 1995). Harvey et al. (1955) showed that the sec-
ondary surface colonizers after bacteria were a diverse species of
100 1
80
60
40
20
■JJJri
Cts/b
natural Nv Nc
Treatments
Nm
Fp
D Adherence ■ Growth
Figure 3. Percentage of A. purpuratus spat that settled on collectors
pre-treated with native diatoms according to diatom adherence crite-
rion (White rectangle = 48 hi and diatom growth criterion (Black
rectangle = 96 h). Vertical lines show standard deviation.
cts^
Treatments
Figure 4. Number of A. purpuratus spat on collectors coated with
different diatoms species in field experiments. Vertical lines show stan-
dard deviation.
benlhic diatoms and these have been traditionally used as a settle-
ment surface for abalone (Seki 1980, Hahn 1989). In this study, the
diatoms N. veneta and N. menisculus adhered better and grew
faster on the plastic substrate compared with iV. cryptocephala and
F. pseudonana. The diatoms N. veneta and N. menisculus are
probably more adapted to adherence and formation of a primary
biofilm on such substrate compared with the other two species
(Characklis & Bryers 1990). Numerous investigators have stated
(hat adherence and development of a biofilm are associated with
the physical and chemical properties of the substrate (Wiggles-
worth-Cooksey & Cooksey 1992. Callow & Fletcher 1994). Struc-
ture of the diatoms has an important role in facilitating adherence
as well as the production of extracellular polymers that interact
with the substrate and may affect diatom adherence positively or
negatively (Wetherbee et al. 1998).
Results of larval settlement in laboratory experiments showed
variable rates of settlement for the four species of diatoms. The
presence of spat was always greater when diatoms were present
compared with clean substrates. Studies of the biology and culture
of marine invertebrates indicate that before settling on a substrate,
the larvae require biofilm capable of emitting to the environment
chemical signals that stimulate their settlement (Kavouras & Maki
2000.).
Studies of the effect of microbial biofilms on attachment of
pectinid larvae have shown variable results. In laboratory experi-
ments, Hodgson and Bourne (1988) showed higher attachment of
Chlamxs hastata on surfaces that had biofilms compared with
surfaces without biofilms and Parsons et al. (1993) reported similar
results for Placopecten magellanicus. In this study, the higher
percentage of spat attached to collectors that were exposed to 10
|jLm filtered seawater under controlled growth conditions (3,384
spat X collector"'), may have resulted from stimulation by live
organisms in the biofilm. Microbenthic components are considered
to be among the principal diatom components. Cyanoficeas epi-
benthic and their associated bacteria (Meadows & Anderson 1968)
in the presence of nutrients would increase their density, coloniz-
ing a higher percentage of the substrate that are used for adherence,
favoring the settlement of scallop larvae. Because they colonize
the substrate and cause higher spatfall. characteristics of biofilm
growth or production of some unidentified substance may cause
this higher spatfall. The composition of natural diatom biofilms
and associated microfiora that colonized the netlon are unknown
but they could be variable and produce changes in composition and
Settlement of Larval A. purfuratus and Diatom Biofilms
397
structure of the community that could produce irregular settlement
(Suzuki et al. 1987).
Butman et al. ( 1988) suggested that stimulation of invertebrate
larval settlement is commonly enhanced by substances that enrich
the substrate. In metamorphosis of scallops, it has been shown that
if no stimulation is present then no settlement will occur thereby
suggesting that specific stimuli may be necessary for different
species (Padilla 1979).
There were significantly more spat on collectors incubated for
96 h with F. pseudonana compared with collectors with N. veneta,
N. cnptocei'hala and N. inenisciiliis that may indicate a selectivity
of scallop spat for a specific species of diatoms. Studies of the
ingestion of A. piirpiiratus larvae exposed to probiotic bacteria (II,
77 y C33) showed larvae selected two of the strains (Riquelme et
al. 2000). Reasons for selection of biofilm surfaces for settlement
are unknown but various theories exist. Bourne and Hodgson
( 1991 ) proposed that selection was due to differences in nutrition
that occurred during transition among the ciliate velum of the
planktonic phase and the filamentous gill of the young benthonic.
Bivalve larvae may respond to colonized substrates of biofilms
that serve as a bridge between planktonic feeding and tillering
feeding, by using the foot for pedal feeding. Observations made
under microscopy have allowed visualizing the gradual detach-
ment caused by the movement of the food and the ingestion (Un-
published MS). Studies of settlement of abalone larvae showed
that the success of settlement and density of juveniles on cultch
depended on diatom species (Daume et al. 1999). Initial studies of
attachment of five species of diatoms on polyethylene showed
minor colonization of the F. pseudonana strain after 48 h of in-
cubation but none by the other four species (Unpublished MS).
Probably the majority of spat in the F. pseudonana treatment
settled because of the formation of a primary biotllm that was
favored by the incorporation of nutrients and also physical char-
acteristics of the substrate surface (Characklis & Marshall 1990).
The diatoms are not only a nutrient source for marine invertebrate
larval stage, but they also have the capacity to liberate chemical
stimuli or extracellular component into the environment (Welher-
bee et al. 1998). This extracellular component could be absorbed
by pectinid larvae, improving the larval survival in the substrate
(Pearce & Bourget 1996, Kavouras & Maki 2000). It is also pos-
sible that the gradual biofilm detachment could be used as food
(Unpublished MS). Some investigators report that the microbial
biofilm gradually detached from the substrate, and the detachment
of cells from the biofilms is a nature process in the biofilm devel-
opment (Stoodley et al. 2001). This detachment phenomenon
shows that the gradual cell detachment from the biofilm could be
used as food for the pectinid larvae. Studies on settlement of
abalone larvae to biotllms with 18 species of diatoms showed
better attachment of the larvae to biofilm with high density of
diatom (Kawamura & Kikuchi 1992). Increase in density of colo-
nizing diatoms on the substrate, static conditions in the experi-
ment, and relative confinement of larvae probably facilitated de-
tection of diatom biofilms by larvae.
Results of adherence of diatoms F. pseudonana and N, veneta
in field experiments showed similar values (lO*" cells x cm"").
Collectors incubated with N. veneta had the majority of spat and
showed that pre-conditioning the surfaces with a diatom biofilm is
a preferred substrate for scallop larvae. Pearce and Bourget (1996)
proposed that larvae of the sea scallop. Pkicopecten magellanUus.
were able to choo.se between different substrates for settlement,
favoring monofilaments with a biofilm. Harvey et al. (1997) found
a significant effect of natural biofilms on bivalve settlement (66%)
and of Pecten magellanicus (35%) compared with cultch without
a biofilm. Hence there is a preference of settlement substrates by
some pectinid larvae, one criterion being determined by nutrition
(Bourne & Hodgson 1991 ). The stimulus for settlement can be due
to various factors including pectinid species, diatom composition,
and density of the biotllm. Diatoms may be effective because of a
particular microcosm with extracellular material that enhances
settlement. Further, the constant supply of artificial substrates with
specific and adecuated biofilms is the key to produce higher
growth and survival (Hahn 1989, Takami et al. 1997).
In conclusion, diatom biofilms enhanced settlement of A. pur-
pwatus larvae in laboratory and field experiments, spatfall reach-
ing higher values than on collectors without biofilms or using
traditional biofilms. This suggests that native diatom biofilms may
be used to increase production of spat of other bivalves, including
northern Chilean scallop Argopecten purpuratus.
ACKNOWLEDGMENTS
The authors thank Professor Ismael Kong for the revision and
commentary. Dr. Neil Bourne for the invaluable critical reading
and improvement to the manuscript. Professor Marcela Cantillanez
for her collaboration during the "in situ" stage, and Professor Luis
Rodriguez for the identification of native diatoms. This study was
financed by the project FONDEF N° DOOI1I68.
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Jounml of Shellfish Research. Vol. 22. No. 2. 40 1 -+02, 2003.
ADHESIVES TO ATTACH JUVENILE BAY SCALLOPS TO PLASTIC NETTING
IN AQUACULTURE
ENID K. SICHEL AND RICHARD C. KARNEY
The Woods Hole Oceanographic Institution. Woods Hole, Massachusclts 02543; The Martha 's Vineyard
Shellfish Group. Inc.. Oak Bluff's. Mas.sachiisctts 02557
ABSTRACT Fanning Ihe hay scallop. Argopeclen irradians inadiims. is a labor-intensive effort, primarily due to biofouling control
on the netting of culture cages. We tested commercially available adhesives for possible application in a cageless scallop aquaculture
methodology: attaching juvenile hay scallops via adhesives to polyethylene netting. The new culture method holds promise to minimize
the culture structure surface area subject to biofoulmg and to facilitate harvesting. We present results for five adhesives.
KEY WORDS: hay scallop, Aif^opecteii imulians. aquaculture
INTRODUCTION
Traditionally, bay scallops have been reared in floating cages or
lantern nets in the United States. Biofotiling of cage netting and a
subsequent decrease in water flow and food availability is a major
obstacle for growers of filter feeding shellfish. Physical removal of
fouling organisms by brushing and power washing represents a
labor-intensive expense. Cageless culture methods, the topic of
this article ehminate Ihe labor and expense of cleaning fouled
netting. Methods used to attach seed shellfish to floating structures
include, piercing the "ears" of scallops to attach a line or using
adhesives to bond the shell to netting. A student group has tested
adhesives for tagging marine mammals (private communication).
Ear hanging experiments with bay scallops -10 mm diameter at
the Martha's Vineyard Shellfish Group (MVSGl cii. 1990 were
performed by piercing the ears with a Dremel® tool and stringing
them on monofilament line. Although the shell surfaces were heav-
ily covered with fouling organisms, the scallops grew at remark-
able rates. Over the course of the growing season, the drilled ear
failed to grow and eventually the weight of the growing scallops
caused the ears to break.
In an attempt to replace ear hanging with an alternative cage-
less method, we report our recent study to find suitable adhesives
and specialty cements to affix juvenile scallops to hanging struc-
tures in the water column. The ideal adhesive must be strong,
adhere to damp surfaces, set up quickly, cure under water and not
break down in seawater. Further, it must not injure the shellfish,
interfere with their growth, or leave any toxic residue in the tissue.
To be useful in aquaculture, the adhesive must be cost-effective
both in the cost of the labor to affix the shellfish to netting and the
cost of the adhesive.
METHODS
We attached bay scallops to high-density polyethylene netting
(ADPl Enterprises, Inc. Durethene BOP-2L, mesh size 2.25-inch
by 2.2.'>-inch). The animals were quickly blotted dry and the ad-
hesive was applied in air. After a cure time of about 15 min, the
scallop was immersed in seawater, either in a tank or off a dock at
the MVSG facility. The adhesives are listed in Table I .
Testing
Tests of adhesives on live scallops were "pass or fail" tests. If
the adhesive held the .scallop to the netting, it was graded pass. If
the adhesive failed to hold and the scallop dropped off, the test was
graded fail. We discovered that it was important to engulf the
polyethylene netting in adhesive to form a good bond. Shells were
about 3 cm in height.
Scallop shells were cleaned free of algal fouling. Live animals
were scrubbed with a brush in buckets of seawater to remove
algae, tubeworms, barnacles, and other fouling. Shells were blotted
dry with paper towels. In addition to removal of biofouling, some
tests were peiformed by touching each shell top with anhydrous
ethanol or blowing dry with compressed air. No significant im-
provement in results was noted with these drying techniques, prob-
ably because of the high ambient humidity. When bonding shells
to netting, it is easiest to place the animal on top of the netting with
the adhesive sandwiched between the animal and the netting. How-
ever, the animal "drools", which keeps the adhesive wet, and
"claps", which disturbs the bond as it is setting up. We tried both
configurations (shells under netting and shells on top of netting)
and found no significant differences in bonding.
Our results are shown in Table 2. The last column (#bonds) is
the number of animals bonded to netting at the beginning of the
study. The time that the animals were out of water is noted in the
second column. In three cases, anhydrous alcohol flowed past the
shell edge and the adhesive bonds were intact but several animals
died. The number of bonds to empty shells (dead animals) is noted
in parentheses.
TABLE 1.
List of adhesives
Adhesive
Manufacturer
Adhesive Type
Ceramicrete
Bone & dental cement .SI 458
PSI-326 (Smart Glue)
Fastcure epoxy 0.51 135-08107
Prism 454
Dr. Arun Wagh, Argonne Nat. Lab.
Stoelting co.
Polymeric systems Inc
3M Company
Loctite Co.
Ceramic cement +5% phosphoric acid
Cement/methacrylic acid ester/amine
Two-pan epoxy
Two-part epoxy
Single component
401
402
SiCHEL AND KARNEY
TABLE 2.
Results on bay scallops.
Adhesive
Air Time
Life Test Conditions
Results
# Bonds
PSI-326 (Smart Glue)
15 niin
Seawater; 19 wk
3 out of 12 intact (2 dead)
12
PSI-326 (Smart Glue)
15 min
Seawater: 14 wk
5 out of 24 intact
24
PSI-326 (Smart Glue)
15 min
Alcohol dry: seawater;
15 wk
4 out of 12 intact (3 dead)
12
Fastcure epoxy 051 135-08107
15 min
Seawater; 4 mo
7 out of 25 intact
25
Fastcure epoxy 051 135-08107
15 min
Seawater; 14 wk
8 out of 24 intact
24
Fastcure epoxy 051 135-08107
15 min
Alcohol dry; seawater.
14 wk
4 out of 12 intact (1 dead)
12
Stoelting bone cement 51458
20 min
Seawater; 17 wk
0 out of 4 intact
4
Stoelting bone cement 51458
20 min
Seawater; 16 wk
6 out of 12 intact
12
Stoelting bone cement 51458
15 min
Alcohol dry; seawater;
14 wk
9 out of 12 intact (5 dead)
12
Ceramicrete
20 min
Seawater; 20 wk
2 out of 5 intact (1 dead)
5
Ceramicrete
30 min
Seawater; 19 wk
6 out of 9 intact
9
Prism 454
15 min
Seawater, 5 wk
25 out of 25 intact (1 dead)
25
CONCLUSIONS
The most promising adhesives are Fastcure epoxy 051135-
08107 (3M Company) and Ceramicrete (developed by Dr. Arun
Wagh, Argonne National Lab.). The Ceramicrete powder was
mixed with phosphoric acid (diluted to 5% by weight in water) to
speed the setting time. Dr. Arun Wagh has recommended another
additive (magnesium oxide) to further speed setting. Initial tests of
"quick set" Ceramicrete with magnesium oxide additive were dis-
appointing. Stoelting bone cement also proved to be a good adhe-
sive for this application but may be too expensive; additionally, it
sets up too fast to use with large numbers of animals at a time.
Initial tests of Prism 454 (Henkel Loclite, Rocky Hill, CT) were
promising but the cost of this one-part adhesive is high. Equally
important for all adhesives are tests for toxicity, which remain to
be done. Proper curing of epoxies requires that the ambient tem-
perature be sufficiently high for the thermal energy to support
molecular motion so that the chemical reaction of resin and hard-
ener can go to completion. A good rule of thumb is that the
reaction should occur at temperatures above the glass transition
temperature, T„. (The glass transition temperature is the tempera-
ture at which a polymer changes from a glassy to a rubbeiy state.
Above Tg, portions of the polymer molecules are mobile.) There-
fore, application of adhesives in winter poses additional chal-
lenges. Resins that are liquid at 0 °C and materials with T^, near
0°C would be useful for cold weather curing.
ACKNOWLEDGMENTS
This project was supported in part by NSF grant DUE-0I0I632
and by the Southeastern Massachusetts Aquaculture Center
(SEMAC). We benefited from many helpful suggestions from Dr.
A. Pocius, 3M Company. Polymeric Systems, Inc. and 3M Com-
pany generously provided free samples of adhesives. Assistance
was provided by student technician, Ann Bodio.
LITERATURE CITED
A high school student research project, "Upward Bound" in Ohio in
2002, evaluated adhesives to attach tags to whales. Marine Quest 1492,
University of Akron, Goodyear Polymer Center, Akron, OH.
Gary Harp was the graduate student advisor (private communica-
tion).
Hamada, T., N. Yamashita, T. Watanabe & S. Natsume. 2001. Drilling
position of the ear affects growth and mortality of scallop {Palino-
pecten yessoensis. Jay) in ear-hanging culture. Aquaculture 193:
249-256.
Harold Hudson, J. 1972. Marking scallops with quick-setting cement. Proc.
Nm. Shellfish Assoc. 62:59-61.
Lemarie, D. P., D. R. Smith, R. F. Villella & D. A. Weller . 1996. Evalu-
ation of tag types and adhesives for marking freshwater mussels. (Ab-
stract only). J. Shellfish Res. 15:528.
Mallet, A. 2000. Reports on oysters attached with masonry cement to lines
for aquaculture. Presentation at the Northeast Aquaculture Conference
and Expo in Portland, Maine. December 7-9.
Wagh, A. 2002. Perfect Patch? Adrian Cho (ed). Science 295:619.
Joiinuil of Shellfish Research. Vol. 22, No. 2, 403-4U8, 200.^.
EVIDENCE FOR THE INVOLVEMENT OF CYCLIC AMP IN THE METAMORPHOSIS OF
BAY SCALLOP, ARGOPECTEN IRRADIANS (LAMARCK) LARVAE
TAO ZHANG, HONGSHENG YANG, HUAYONG QUE,* GUOFAN ZHANG, SHILIN LIU,
YICHAO HE, AND FUSUI ZHANG
Institute of Ocecmology, Chinese Acadciity of Sciences. 7 Ncinhai Road Qint^duo.
Shandong 266071. China
ABSTRACT The putative involvemenl of cyclic AMP (cAMPi in the metamorphosis of bay scallop /I /xo/w/ch irradians larvae has
been investigated on three integrated aspects. First, we conducted experiments on response of competent larvae to selective inhibitors
of phosphodiesterase (PDE), theophylline, and caffeine, which presumably lead to elevated concentration of intracellular cAMP by
preventing the degradation of cAMP to 5'-AMP. Second, the endogenous levels of cAMP were determined during larval development.
Third, monitoring the variation of cAMP content in larvae when exposed to neuroactive compounds tested (i.-DOPA and epinephrine)
and to elevated concentrations of potassium ion. was carried out to examine the possible role of cAMP as a second messenger in
metamorphic pathway stimulating artificially. Consistent results have been obtained in all the three experiments. The two putative PDE
inhibitors that were tested stimulated metamorphosis in A. irradians larvae significantly above control level in a dosage-dependent
manner. The inductive effects did not vary significantly with exposure time. At the optimum concentration of 1.0 mM. percent
metamorphosis increased by 33% and 36.0 1'/r when subjected to theophylline and caffeine respectively. The endogenous level of
cAMP varied dramatically over larval development. In particular, significant increase in cAMP content from 2129 pmoL/(mg protein!
for eyed larvae (Day 13 post-fenilization, PF) to 15,195 pmol/(mg protein) for spats (Day 17 PF) occurred dunng the metamorphic
process. This finding indicates that metamorphic pathway involves cAMP in appreciable quantities. Furthermore, the endogenous
cAMP content increased significantly in competent larvae exposed to excess potassium ion, epinephrine, or l-DOPA, suggesting cAMP
plays an important role in metamorphic signal transduction pathway triggered by the three chemical cues. Evidences presented here
show that cAMP becomes involved in the metamorphic pathway oi A. irradians larvae.
KEY WORDS: cAMP, metamorphosis, Argopecten irradians. catecholamines, L-DOPA, PDE inhibitors
INTRODUCTION
Larval metamorphosis is a crucial process in the development
of most murine invertebrates. Evidence indicates that this process
is triggered by specific endogenous and exogenous chemical cues
(Burke 1983, Baloun & Morse 1984, Coon & Bonar 1986, Yool et
al. 1986, Bonar et al. 1990, Inestrosa et al. 1993a, Leise & Had-
field 2000. Pires et al. 2000, Zhang et al. 2002a, Zhang et al.
2002b). Recent evidence suggests that neurotransmitters (norepi-
nephrine, dopamine, and 5-hydro.\ytryptamine) play an important
role in regulating metamorphosis of mollusk larvae (Coon & Bo-
nar 1986. Pires et al. 2000, Zhang et al. 2002a). The cAMP/PKA
(protein kinase A) pathway is one of the most important signal
transduction pathways involved in the neurotransmitter regulation.
Previous studies revealed or inferred that the cAMP, as an
important mediator of cellular metabolism and cell-to-cell signal-
ing, was possibly involved in the metamorphosis of certain species
of marine invertebrates, such as the polychaete Pluigmatopoma
califoniica (Jensen & Morse 1990), the barnacle Balanus amphi-
trite amphitrite (Clare et al. 1995), the red abalone Haliotis rufe-
scens (Baxter & Morse 1987). It remains unclear as to whether
cAMP is involved in the metamorphosis of some marine inverte-
brate species such as of Crassostrea gigas (Coon & Bonar 1987.
Bonar et al. 1990, Coon et al. 1990) and Hydroides elegans (Holm
et al. 1998). The signal transduction pathway involving cAMP,
however, is incompletely understood, in the majority of previous
studies, because there is no direct proof of variation in endogenous
larval cAMP level during the metamorphic process.
It is still unknown whether cAMP is involved in the metamor-
phosis of the bay xaXXo^ Argopecten irradians (Lamarck). In this
study, we investigated the potential role of cAMP and sought the
*Corresponding author. E-mail; hqueCs'ms.qdio.ac.cn
direct evidence on the involvement of cAMP in the metamorphosis
of A. irradians larvae. We designed three experiments to test the
putative involvement of cAMP in the metamorphosis of A. irra-
dians. The first experiment investigates larval response to phos-
phodiesterase (PDE) inhibitors. PDE is known to function in
stimulating the hydrolysis of cAMP to 5'-AMP. Response of lar-
vae exposed to PDE inhibitors (e.g., theophylline and caffeine),
which is assumed to increase endogenous cAMP level, would
provide proof revealing function relationship between cAMP and
the metamorphic pathway. The second experiment is designed to
manifest potential role of cAMP in the natural metamorphic pro-
cess by monitoring endogenous levels of cAMP over larval devel-
opment. The third experiment is designed to elucidate the possible
relationship between the inductive activities of the commonly
adopted exogenous chemical cues and cAMP. Results from the
three experiments are expected to provide fuller understanding of
the signal transduction pathway that involves cAMP in marine
mollusks.
MATERIALS AND METHODS
Collection of Larvae
Larvae of the bay scallop. Argopecten irradians (T^amarck).
were obtained from Xujia Maidao Hatchery, Institute of Oceanol-
ogy Chinese Academy of Sciences. Larvae collected with Nitex
screen were cultured with the methods as described by Zhang et al.
(1986, 1991).
Test of Chemical Cues
All experiments were conducted in 6-well plastic tissue culture
plates using l-p-m filtered natural seawater at 23°C, 32 ppt. The
selective chemical cues, including the two PDE inhibitors (the-
ophylline and caffeine), l-DOPA, epinephrine (Fluka), KCI, pre-
403
404
Zhang et al.
pared as 10 stock solutions in distilled water prior to experiments,
were kept under 4°C. All chemicals were purchased from Sigma
Company unless denoted.
For experiments, the stock solutions of chemicals tested were
allowed to be equilibrated to the desired temperature and then were
diluted to the appropriate concentration with seawater containing
A. inadiaiis larvae. Approximately 50-100 larvae in 10 niL of
filtered seawater were placed in each well of the plastic tissue
culture plates. Seawater in controls was diluted with distilled water
to match that in experimental groups. Test solutions of PDE in-
hibitors were applied at concentrations of lO""*. 10"~. 10"', 1, and
10 mM in seawater. Exposure time of PDE inhibitors varied from
1 h to 24 h. For the assessment of endogenous cAMP, competent
larvae were exposed to l-DOPA or epinephrine at a concentration
of 10 |xM for 8 h, or to 13.42 mM KCl for 24 h.
On completion of the treatment, larvae were rinsed and re-
placed in fresh filtered seawater to be ready for other procedures.
Larvae were cultured for an additional 72 h before they were fixed
with iodine and observed under a dissecting microscope to deter-
mine the percentage of larvae that had metamorphosed and the
mortality rate. Metamorphosed larvae were verified by the com-
plete formation of dissoconch, the newly grown adult shell. Three
replicates were conducted for each experiment with 50-100 larvae
per replicate using different batches of larvae.
Analysis of Endogenous cAMP Content
Samples of different developmental stages of A. irradians lar-
vae for cAMP assay were taken as follows: D-stage larvae (Day 3
post-fertilization, PF), umbo-stage larvae (Day 7 PF), 10% eyed
larvae (Day 10 PF), 100% eyed larvae (Day 12 PF). 100% eyed
larvae (Day 13 PF) and spats (Day 17 PF). Larvae following
exposure to elevated concentration of K*. l-DOPA, or epinephrine
were also sampled for cAMP assay.
For the measurement of cAMP content -100-200 larvae were
used in each sample. The extraction of cAMP was carried out by
homogenizing in 5% trichloroacetic acid and centrifuging at 3,000
rpm for 30 min. The supernatant was washed with saturation ether
to remove trichloroacetic acid and then dried on 70-75°C water
bath. The residue was redissolved in TE buffer for cAMP assay as
described by Oilman (1970). The P-E 240 Elementary Analyzer
(Perkin Elmer, USA) was used for protein assay. The amount of
larvae was counted prior to the measurement of cAMP content in
larvae. The cAMP content is finally expressed as follows, with its
unit of pmol cAMPAmg protein): Content of cAMP = (cAMP
content per larva)/(protein content per larva) Where the protein
content per larva was calculated using the following: Protein con-
tent per larva = (absolute content of nitrogen x 6.25)/(larval
amount).
Data Analysis
Percentage of response of larvae to chemical cues was com-
pared by two-way analysis of variance (ANOVA). All analyses
were conducted usmg Microsoft Excel program.
RESULTS
Influence of Theophylline on Larval Metamorphosis and Mortality
Theophylline exhibited high and consistent inductive activity
on the metamorphosis of A. irradians larvae. The percentage of
metamorphosed larvae increased by over 14% at concentrations of
0.001-10 mM for 1-24 h of exposure compared to controls. The-
ophylline induced larvae to metamorphose in a concentration de-
pendent manner (P < 0. 1 ). Increased concentration of theophylline
led to an increase in percentage of larvae that had metamorphosed.
At theophylline concentration of 1.0 niM, there is an average
maximum increment of 33% over control levels. The mean per-
centage of larvae metamorphosing increased by 23.15% and
21.97% in response to 0.1 mM and 10 mM theophylline respec-
tively (Table 1 ). On the other hand, the effect of exposure duration
on the metamorphosis of A. irradians larvae was not significant (P
> 0. 1 ). The average metamorphosis increment varied from 1 9.07%
to 26.1% for the exposure duration of 1-24 h at various concen-
trations of theophylline (Table 1 ).
Theophylline treatment at concentrations lower than 1 niM for
brief periods of time did not cause obvious mortality to the larvae
of A. irradians. In 1 1 of 20 cases, larvae in theophylline-treated
groups showed higher survival rates than that in the control groups
(Table 2). Lethal effect emerged, however, when high concentra-
tions of theophylline or prolonged exposure time were applied.
The larval mortality increa.sed by 24.69 ± 3.56% when treated with
10 mM theophylline for 16 h compared with that of the control
group. It seemed that prolonged exposure time had more impact on
larval survival, as suggested by the increase in larvae mortality, by
17.64 ± 3.56%, 15.48 ± 3.45%, and 39.23 ± 4.36% for 24 h
exposure at the concentration of 0.1, 1.0, and 10 mM, respectively.
Overall, exposure to theophylline had no significant effect on the
TABLE L
Effects of theophylline concentration and exposure time on metamorphosis of A. irradians.
Increment of Percent Metamorphosed 1
Larvae Above Controls Level
1
(mM)
1 h
8h
16 h
24 h
Average
0.001
22.43 ± 3.45
12.97 ±2.56
17.50 ±4.23
20.91 ±2.56
18.45
0.010
18.74 ±2.76
17.29 ±1.29
8.40 ±1.73
13.84 ±2.10
14.57
0.100
20.79 ± 2.25
28.64 ± 3.65
23.94 ± 4.72
19.23 ± 2.89
23.15
1.000
21.33 ±4.67
39.49 ± 6.45
33.37 ± 4.30
37.81 ±2.57
33.00
10.00
29.54 + 5.12
32.13 ±2.64
12.16±1.26
14.06 ± 1.14
21.97
Average
22.57
26.10
19.07
21.17
Competent larvae were exposed to theophylline as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater
that had been diluted to match thai in experimenlal group correspondingly. Three replicates were made with 50-100 larvae per replicate. Metamorphosis
was defined as complete formation of dissoconch.
Data are expressed as mean percentage and standard deviation.
Cyclic AMP in Metamorphosis of the Bay Scallop
405
TABLE 2.
Effects of theophylline concentration and exposure time on mortality of A. irradians.
Concentration
(mM)
Increment of Larval Mortality Relative to Controls Level
1 h
8 h
16 h
24 h
Average
0.0(11
2.73 ± 0.45
-2.84 ± 0.23
-2.61 ±0.78
-8.03 ± 0.89
-2.69
0.010
10.96 ± 2.32
-7.53 ±1.23
0.26 ± 0.09
0.05 ± 0.02
0.94
0.100
-4.62 ± 0.98
-21.16 + 2.45
-8.99 + 2.11
17.64 ±3.56
-4.28
1 .000
-13.65 + 2.34
-4.00 ±1.34
-0.91 ±0.08
15.48 ±3,45
-0.77
10.00
-4.26 ± 0.89
3.56 ± 0.78
24.69 ± 3.56
39.23 ±4.36
15.81
Average
-1.77
-6.39
2.49
12.87
Competent larvae were exposed to theophylline as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater
that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate.
Data are expressed as mean percentage and standard deviation.
mortality of A. irradians larvae in coinparison with larvae in con-
trols (P> 0.1).
Influence of Caffeine on Larval Metamorphosis and Mortality
Caffeine like theophylline stimulated the inetainorphosis of A.
irradians larvae remarkably. The metamorphosis increment aver-
aged over 10% for the treatment of caffeine at varying concentra-
tions for 1-24 h. The action of caffeine on the metamorphosis was
dose-dependent. Concentration of caffeine had significant effect
on the metamorphosis induction (P < 0. 1). Optimum inducing of
metamorphosis was achieved at a concentration of 1 .0 niM caf-
feine, with mean percentage of metamorphosed larvae increasing
by 36.01% among the four exposure durations (Table 3). The next
effective concentration for caffeine was 10 mM. averaging incre-
ment of 26.43% metamorphosis. Caffeine exposure time does not
appear to significantly influence the percentage of metatnorphosed
larvae. Among various durations of exposure time, metamorphosis
increased by 19.65-22.02% above the control levels (Table 3). The
correlation between treatment duration and efficiency of larval
metamorphosis inducing was not significant (P > 0. 1 ).
Statistic analysis showed that the caffeine treatment had sig-
nificant effect on larvae mortality of A. irradians (P < 0.01). In
particular, larvae mortality occurred more than control levels when
the exposure time was prolonged to 24 h or when caffeine con-
centration reached 10 mM. Increment of larvae mortality rose up
to 21.29 ± 0.95% when larvae were treated with 10 mM caffeine
for 24 h compared to the control groups. Exposure of A. irradians
larvae to caffeine below 24 h or 10 mM, however, resulted in the
increase in larval survival (Table 4).
Variation of Endogenous Levels of c AMP During Larval Development
The endogenous cAMP level increased over the time-course of
the development in A. irradians larvae (Fig. I). The larval cAMP
content underwent a gradual increase from 10% eyed larvae to
100% eyed larvae and eventually a sharp climb in the cAMP level
following the completion of metamorphosis. The content of cAMP
increased by 6.1 times from eyed larvae (100%, Day 13 PF) to
spats (Day 17 PF). This result showed that cAMP played an active
role in the metamorphic pathway that naturally occurs in A. irra-
dians.
Variation of Endogenous cAMP Level Following Excess K* Treatment
Larvae of A. irradians underwent an increase in the cAMP
level significantly above the control groups" level when exposed to
excess potassium ion (Fig. 2). The content of cAMP in larvae
treated with 13.42 mM KCI for 24 h was elevated to 7.8 times
higher than that in the control groups, that was, from 2129 pmol/
(mg protein) to 18.656 pmol/(mg protein).
Variation of Endogenous cAMP Level Following Treatment of
Epinephrine or 1.-DOPA
The content of cAMP in A. irradians larvae increased follow-
ing the treatment of IO-|jlM epinephrine or l-DOPA for 8 h (Fig.
TABLE 3.
Effects of caffeine concentration and exposure time on metamorphosis of A. irradians.
Concentration
ImMl
Increment of Percent Metamorphosed
Larvae Above Controls Level
1
1 h
8h
16 h
24 h
Average
0.001
12.55 ± 1.34
21.52 ±2.45
16.00 ±3.13
12.46 ±2.56
15.63
0.010
7.78 + 0.99
8.39 ± 1.12
8.40 ±1.45
24.98 ± 3.56
12.39
0.100
19.10±2.12
12.08 ± 1.56
9.42 ±1.68
13.70 ±1.98
13.58
1.000
23.92 ±2.53
35.73 ± 2.97
46.43 + 3.76
37.94 ± 3.98
36.01
10.00
34.89 ±4. 13
32.37 ± 2.38
20.00 ± 1.99
18.44 ±2.01
26.43
Average
19.65
22.02
20.05
21.50
Competent larvae were exposed to caffeine as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater
that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate. Metamorphosis
was defined as complete formation of dissoconch.
Data are expressed as mean percentage and standard deviation.
406
Zhang et al.
TABLE 4.
Effects of caffeine concentration and exposure time on mortality of A. irradians
Concentration
(mM)
Increment of Larval Mortality Relative to Controls Level
1 h
8h
16 h
24 h
Average
0.001
-4.50 + 0.14
-5.60 ±0.14
-6.43 ± 0.45
5.53 ± 0.97
-2.75
0.010
-8.60 ±0.87
-9.47 ± 0.87
-6.60 ± 0.76
6.07 ± 0.74
-4.65
0.100
-4.98 ± 0.57
-2.88 + 0.30
-9.29 ± 0.89
5.10 + 0.78
-3.01
1.000
-4.07 ± 0.49
-6.59 ±0.71
-5.73 ±0.76
9.75 ± 0.89
-1.66
10,00
-3.49 ± 0.37
5.64 ±0.45
1.07 ±0.02
21.29 ±0.95
6.13
Average
-5.13
-3.78
-5.40
9.55
Competent larvae were exposed to caffeine as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater
that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate.
Data are expressed as mean and standard deviation.
3). Larval cAMP content in treated groups increased by 1.5 and
10.7 titnes than that ot the control groups, (i.e., from 4007 pmol/
[mg protein] in the control groups to 9882 pniol/[nig protein] and
46,824 pniol/[mg protein]) for epinephrine and l-DOPA treatment
groups respectively.
DISCUSSION
It is generally believed that the pathway taking cAMP as sec-
ond messenger is an important signal-transduction pathway in in-
vertebrate tissues. Involvement of cAMP in larval metamorphosis
varies with the species of marine invertebrates and there is no
evidence of involvement of cAMP in metamorphosis of Pacific
oysters Crassostrea gigas. The alpha- 1 adrenergic receptor served
as the receptor of norepinephrine to regulate metamorphosis of C.
gigas larvae (i.e., norepinephrine was through intracellular mes-
sengers DG [diglyceride] and IP, [1,4,5-trisphosphoinositidel) not
cAMP, to regulate metamorphosis of C. gigas larvae (Coon &
Bonar 1987, Bonar et al. 1990, Coon et al. 1990). One report
revealed that cAMP level in the gastropod Concholepas conchole-
pas larvae reduced by 20 times during metamorphosis (Inestrosa et
al. 1993b). An earlier report, dealing with the metamorphosis of
this species, revealed that the degree of larval internal protein
phosphorylation increased during the metamorphosis process
18000
16000 •
■i 14000
2
^12000
E
10000
r 8000 -
< 6000
4000
2000
0
15195
1)96
1402
1770
m.
2129
3 7 10 12 13 17
Developing days (PF)
Figure 1. The variation of endogenous level of cAMP in A. irradians
larvae during consecutive different developing stages as follows: D-
stage larvae [Day 3 post-fertilization, (PF)|, umho-stage larvae (Day 7
PR), 10% eyed larvae (Day 17 PF). Data on the top of the bars rep-
resents cAMP content. Data are averages of three duplicates with
standard deviation indicated as vertical bars.
(Campos et al. 1991 ). Therefore, Inestrosa et al. (1993b) concluded
that the phosphorylation of protein following metamoiphosis had
no relation with PKA but presumably was triggered by other kinds
of kinase, including PKC. The investigators, however, did not
clarify whether or not cAMP is involved in the metamorphosis of
C. coiuholepas larvae.
On the other hand, several studies revealed that cAMP is in-
volved in the process of settlement and metamorphosis in certain
invertebrate species (Jensen & Morse 1990, Clare et al. 1995). It
has been shown that cAMP is involved in the morphogenetic path-
way in the larvae of the red abalone. Haliotis rufescens (Baxter &
Morse 1987). Cholera toxin has been found to be effective in
inducing metamorphosis in Cassiopea andromeda larvae, whereas
db-cAMP is not effective in initiating the settlement and metainor-
phosis of the same species (FitI et al. 1987). Furthermore, endog-
enous cAMP level in C. andromeda larvae did not undergo sharp
variation as did in mammal species. Based on earlier observation,
the authors concluded that cAMP is involved in the metamorpho-
sis, but not in initiating the settlement and metamorphosis process.
The drug induction method has been used for the research in
signal-transduction pathway in the process of marine invertebrate
larvae settlement and metamorphosis, i.e., the signal transduction
pathway could be inferred from the response of larvae to the spe-
cific drug that induces larval settlement and metamorphosis. This
method is simple and practical and much progress has been made
through this method. Because of the complexity of the biochemical
reaction involved in the metamorphic process, there is still linii-
25000 r
m.
Figure 2, The variation of cAMP content in A. irradians larvae ex-
posed to elevated K*, Larvae were treated with 13.42 mM KCl for 24 h.
Data on the top of the bars represent cAMP content. Data are averages of
three duplicates with standard deviation indicated as vertical bars.
Cyclic AMP in Metamorphosis of the Bay Scallop
407
60000
50000
g^40000
ail
E
o 30000
£
S 20000
<
10000
9882
m
Control Epinephrine L-DOPA
Figure 3. The variation of cAMP content in A. irradians larvae ex-
posed to epinephrine and 1,-DOPA. Larvae were treated with 10 jjM of
the testing neuroactive drug for 8 h. Data on the top of the bars
represent cAMP content. Data are averages of three duplicates with
standard deviation indicted as vertical bars.
tation in using this method because of deviation to some extent.
Therefore, we combined both drug induction method and direct
assay of endogenous level of cAMP in larvae for veiification of
involvement of cAMP in the process of metamorphosis in A. ir-
radians. As for drug induction, theophylline and caffeine, which
could affect the intracellular level of cAMP. were used to test the
mechanism of A. irradians metamorphosis. Results presented here
show that both theophylline and caffeine are effective in promot-
ing metamorphosis in A. irradians larvae. On the other hand.
cAMP level is found to vary different larval developmental stages,
especially with significant increase from eyed larvae to spats. Fur-
thermore, the internal cAMP level in larvae increases significantly
following exposure to excess K*. epinephrine or l-DOPA that are
known as the common inductive agents for settlement and meta-
morphosis in larval marine invertebrates. All these results suggest
that cAMP is involved in the metamorphosis of A. irradians lar-
vae. The drastic increase of cAMP, however, occurs after meta-
morphosis not before. Therefore, we believed that the process of
metamorphosis of A. irradians larvae was not triggered by cAMP.
although cAMP is involved in this process. The triggering process
might be through other pathways.
Of particular interest is that mortality of metamorphosing lar-
vae exposed to either theophylline or caffeine in most of the cases
was much lower than that in controls. The increase in larval sur-
vival was possibly due to the metamorphosis promotion of com-
petent larvae in treated groups, which shortens the time elapsed in
the metamorphic process. We have found that the delay of meta-
morphosis resulted in increasing loss of competent larvae in A.
irradians. This finding indicates that theophylline or caffeine is
potentially useful for promoting yield of metamorphosed spats in
A. irradians. which is essential for the efficiency of seed produc-
tion in commercial hatcheries, and their use as exogenous meta-
morphosis inducers by hatcheries engaging in seed production of
bay scallops in China will result in promising and cost efficient
commerciali/ation of bay scallop aquacullure.
Ill the settlement and metamorphosis model of C. gigas, L-
DOPA, as the precursor of neurotransmitter, is absorbed by the
larvae and transformed into dopamine, which initiates the settle-
ment of C. gigas larvae followed by the secretion of neurotrans-
mitters, such as norepinephrine. This process causes metamorpho-
sis in the larvae through the a_, adrenergic receptor (Bonar et al.
1990. Coon et al. 1990). In this study, the intracellular level of
cAMP increased significantly following exposure of larvae to l-
DOPA and epinephrine, which suggests that P-adrenergic receptor
is involved in the metamorphosis of A. irradians larvae (i.e., epi-
nephrine regulates the metamorphosis of A. irradians through
3-adrenergic receptor). It seems that the mechanism of A. irradi-
ans metamorphosed was obviously different from that of C. gigas.
In this study, endogenous level of cAMP increased with expo-
sure of A. irradians larvae to excess potassium ion. It is generally
believed that K* induces larval metamorphosis through directly
depolarizing excitable cells involved in the larval perception of
inductive stimuli (Yool et al. 1986. Baloun & Morse 1984). It,
however, remained unknown as to how depolarization causes lar-
val metamorphosis. Based on our results, we propose that as a
result of cell membrane depolarization resulting from excess po-
tassium ion exposure, nerve impulse occurs and then increases
intracellular level of c AMP through certain pathways, which trig-
gers phosphorylation of PKA. and eventually regulates metamor-
phosis of larvae of bay scallops.
In summary, this study shows that the second messenger cAMP
is involved in the regulation of metamorphosis in A. irradians
larvae. Since cAMP functions by activating PKA, it means that
PKA is possibly involved in the metamorphosis of the bay scallop
larvae. However, further proof of PKA involvement in metamor-
phosis of this species has to be found in future studies.
ACKNOWLEDGMENT
We thank Mr. Jianghu Ma at Maidao Hatchery for providing
larvae for experiments and to numerous scholars at lOCAS who
extended their help to this study. This study was supported by
China Natural Science Foundation Grant No. ,^9970.'i88 and No.
30200214.
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Journal of Shellfish Rf.morh. Vol. 22. No. 2, 409-414. 2003.
DEPURATION CONDITIONS FOR GREAT SCALLOPS {PECTEN MAXIMUS)
WILLIAM J. DORR,* JENNIFER FARTHING, AND IAN LAING
Centre for Eiiviriiiiineii! Fisheries and Aqiuieiilture Seieuee. Weymouth Laboratory. Barraek Road.
Weymouth. Dorset. United Kingdom
ABSTRACT Trials were undertaken to deternilne appropriate conditions for depurating hand-gathered great scallops {Ptrteii maxi-
mu.f). Scallops were contaminated with Escherichia coli to levels consistent with those requiring depuration hy relaying in sewage
impacted waters for a minimum of 2 weeks. These scallops were then purified for 42-48 h in both laboratory and small-scale
commercial depuration systems under varying conditions. Levels of E. coli were monitored before and after depuration to assess the
effect of temperature, salinity, shellfish-loading arrangements, and the use of artificial seawater on the depuration process. Self-righting
trials were used to assess the amount of stress imposed on the scallops caused by transport, handling, and the depuration procedures.
Results to date demonstrate that the use of artificial seawater cannot be recommended. During depuration, natural seawater should be
maintained at a salinity ^SO'^r and at a temperature >10°C. Our results demonstrate that scallops could be depurated in a double layer
within trays at a nominal density of 250 scallops m"" with a shellfish-to-water ratio of 1:12 (kg:L).
KEY WORDS: great scallops, depuration conditions, purification
INTRODUCTION
Sewage-containinated bivalve mollu.scan shellfish can present a
significant health risk if consumed raw or lightly cooked (Rippey
1994. Cliver 1997). To minimize these health risks, most countries
operate legislative controls on the harvesting and placing on the
market of live bivalve shellfish (Lees 2000). Such controls gener-
ally rely on the use of Escherichia coli as an indicator of fecal
pollution in these shellfish. European Community (EC) Directive
91/492 (Anon 1991) stipulates such controls for the EC and re-
quires classification of shellfish harvested areas depending on the
degree of fecal pollution, as judged from monitoring for E. coli
contamination of bivalve tlesh. This classification determines
whether bivalve shellfish can be sold direct for consumption or
must be treated before sale. There are four classification categories
(Table I ). Bivalves from category B areas require short-term self-
purification in tanks of clean seawater by a process termed depu-
ration (Richards 1988). All bivalves sold for consumption whether
treated or not must comply with an end-product standard of <230
E. coli 100 g^'.
There is increasing interest in farming great scallops (Pecten
maximus) in Europe (Chataigner 1996, Dao et al. 1998) and sev-
eral studies have examined the environmental requirements for
cultivation of this species (e.g., Brynjelsen & Strand 1996, Fleury
et al. 1996, Chauvaud et al. 1998, Laing 2000, 2002). However,
scallops held in inshore areas have been shown to be as capable of
accumulating equal amounts of sewage-derived micro-organism as
other commercially cultivated bivalve shellfish (Silk 2000). The
availability of pristine (category A) waters for scallop cultivation
is limited in the United Kingdom. Most (64%) of the 249 recog-
nized shellfish-harvesting areas in England and Wales are cur-
rently classified as category B. About 69% of over 120 Scottish
shellfish sites are category B for all or part (seasonal classification)
of the year. At least two of the three present scallop farms in
Northern Ireland are likely to hold a B classification (Heath &
Pyke 2002). The market for scallops is predominantly for a live
product. Where bivalves are sold as live product the treatment
process most commonly used is depuration, which represents a
major control point during the production of bivalve molluscs
*Corresponding author. E-mail: w.j.doreCScefas. co.uk
world wide (Richards 1998). Depuration has not been applied to
scallops landed in the United Kingdom because they are tradition-
ally considered to be fished in offshore locations deemed to be
microbiologically secure and so are exempt from classification
requirements (Anon 1991 ). To realize the full aquaculture potential
of great scallops in the United Kingdom, there is a need to apply
successful treatment processes that will remove microbiological
contaminants. Depuration is likely to be the preferred option.
Depuration relies on bivalves continuing filter-feeding activity
when placed in tanks of clean seawater and purging themselves of
sewage contatninants. To ensure this is achieved, suitable condi-
tions must be met. Criteria for some of these conditions are com-
mon to all species, such as adequate water quality, shellfish con-
dition, and system design. However, some conditions, such as
temperature, salinity, and loading arrangements, vary depending
on the species depurated. These conditions have been carefully
determined in the United Kingdom for a variety of bivalve mol-
luscan species, including oysters (Ostrea edulis, Crassostrea gi-
gas), mussels (Mytilus edulis). cockles (Cardium edule), and clams
(Ensis spp., Mercenaria inercenaria. Tapes philippinarwn. T. de-
ciissatus. and Spisula .solida). Some preliminary investigations
have been conducted to determine the effect of a number of con-
ditions for scallop depuration (Heath & Pyke 2002). However, it
was determined that further work would be required to define these
and other conditions more closely before regulatory authorities
could sanction the use of depuration as a treatment process for
scallops.
This study investigated the effect of temperature, salinity, and
emersion time before depuration and shellfish-loading arrange-
ments on scallop purification, principally using E. coli elimination
as a measure of depuration efficiency. The aim of the study was to
produce sufficient information that would allow minimum depu-
ration criteria for scallops to be determined.
MATERIALS AND METHODS
Experimental Animals and Environmental Contamination
Market-size scallops were obtained from a commercial culti-
vation site and were distributed into lantern nets at field sites that
were impacted by sewage contamination. The nets were filled with
six to seven scallops in each of the 12 compartments and sus-
pended from floating pontoons with the top of the net at least 1 m
409
410
DORE ET AL.
TABLE 1.
Criteria for classifying bivalve molluscan slielirish harvesting areas
(EU Shellfish Hygiene Directive 91/492/EEC).
Classification
E. coli
Category
100 g-' Flesh
Comment
A
Less than 230
Suitable for consumption. Can
be marketed.
B
Less than 4,600
Depuration needed (or relaying
in category A area or cooking
by an approved method)
C
Less than 46.000
Relaying (minimum of 2 mo) m
category A or B area needed
(or cooking by an approved
method)
Prohibited
Above 46,000
Cannot be taken for placing on
the market
from the seawater surface. The nets were deployed for at least 2
weeks to allow microbiological contamination of the scallops. Two
field sites were used throughout the study and both had previously
been identified as areas where the scallops would reliably accu-
mulate E. coli to a level at which they would require depuration.
After contamination, scallops were collected from the field site as
required for depuration experiments. They were transported in
groups of 60-70 animals in 40-L rectangular plastic bins covered
with a dampened hessian sack to maintain a high level of humidity.
For all experiments scallops were transported to the laboratory in
less than 3 h.
Depuration Tanks
Experiments were conducted in two types of depuration tanks
both using UV sterilization. Laboratory scale systems had dimen-
sions of 1050 mm (length) by 300 mm (width) by 450 mm (depth)
with a working volume of 200 L. Seawater was recirculated
lengthways through the tank at a rate of 400 L h~' and sterilized by
irradiation in a 15 W UV sterilizer (type l5/3p: UVAQ Ltd., Sud-
bury, UK). Temperature was inaintained by placing the whole tank
in a controlled temperature room. Dissolved oxygen levels were
maintained by the use of a spray bar for recirculated water. Shell-
fish were depurated in plastic mesh baskets (no. 41042; Sommer
Alibert [UK] Ltd.. Droitwich. UK) raised off the base of the tank
to avoid recontamination by voided fecal material.
Standard design small-scale commercial systems (SFIA 1995)
had dimensions 1 140 mm (length) x 950 mm (width) x 600 mm
(depth) with a working volume of 550 L. Seawater was recircu-
lated through the tank at a rate of 900 1 h"' and sterilized by
irradiation in a 15 W UV sterilizer (type 15/3p; UVAQ Ltd.. Sud-
bury. UK). Scallops were loaded into six mesh baskets (no. 41042;
Sommer Alibert [UK] Ltd.. Droitwich. UK) stacked three high in
two columns. Temperature was maintained by the use of an
aquarium heater (Tronic 100 watt; Hagen [UK] Ltd.. Castleford.
UK) or chiller units (model RA680; Teco Ltd.. Ravenna. Italy).
Dissolved oxygen levels were maintained by the use of a spray bar
for recirculated water. Baskets in the bottom layer were raised off
the base of the tank by 50 mm to avoid recontamination by voided
fecal material.
Depuration
Natural or artificial seawater was circulated through the depu-
ration system and UV irradiated for at least 24 h before each
experiment. Artificial seawater was made using a standard salt mix
widely used in the UK for shellfish depuration from a commercial
supplier (Seainix; Peacocks Ltd. Glasgow. UK). Contaminated
scallops were thoroughly washed and damaged or gaping shellfish
discarded. Prior to depuration an initial sample of 20 or 30 scallops
was removed and analyzed as duplicate or triplicate samples of ten
animals. Scallops were loaded into mesh baskets with cupped shell
down generally in a single layer except in loading configuration
experiments. For all experiments, except a trial investigating the
effect of length of emersion, depuration commenced within 4 h of
shellfish collection. After an initial trial using what was believed to
be optimal conditions, trial parameters were changed to investigate
the effect of artificial seawater. salinity levels, temperature, emer-
sion time before depuration, and loading arrangements. Details of
the parameters investigated are discussed further in the relevant
part of the results section. A control treatment where the parameter
under investigation was not varied was included for each experi-
ment.
All depuration experiments were run for between 42 and 48 h,
after which time duplicate samples of 10 scallops for each treat-
ment were removed for E. coli analysis. Levels of dissolved oxy-
gen, temperature, ammonia, and pH were recorded periodically
throughout the depuration period.
E. coli Analysis
Scallops were thoroughly washed and scrubbed under running
potable water. Dead and open scallops not responding to percus-
sion were discarded. Ten scallops were aseptically opened using a
flame-sterilized shucking knife to sever the adductor muscle and
meats and intravalvular fluid removed. These were diluted and
homogenized as described previously for oysters (Dore & Lees
1995).
Diluted homogenates were assayed for E. coli using a standard
most-probable number (MPN) method used for shellfish analysis
(Donovan et al.l998). Briefly, this is a five-tube, three-dilution
procedure involving inoculation of tubes containing minerals
modified glutamate broth (CM607; Oxoid Basingstoke UK) fol-
lowed by incubation at 37''C for up to 48 h. Tubes displaying acid
production were confirmed as containing E. coli by subculture on
to Tryptone Bile Glucuronide Agar and incubation at 44°C for 24
h. After incubation, the number of tubes that were positive for
(3-glucuronidase activity after subculture was recorded. The MPN
was then calculated by reference to standard tables (Donovan et al.
1998). The nominal limit of sensitivity for the assay is 20 MPN
100 g~'. All results are expressed as an average for the duplicate
samples.
Self-Righting Experiments
Self-righting experiments were performed on some surplus
scallops as a simple assessment of pre- and postdepuration stress
levels in the animals (Minchin et al. 2000). For these trials. 10-20
scallops were placed upside down (flat shell down) in 25 cm depth
of sea water in a 3()0-L rectangular tank supplied with a continuous
flow of aerated unfiltered sea water at ambient temperature and
salinity (>30%f ). The number of scallops self-righting after 1 h
was recorded and the result compared with that for control ani-
mals. Repeated observations were made with the same scallops
every 3-5 days for up to 15 days or until at least 50% of the
scallops in both control and treatment groups righted within 1 h.
Mortality of the scallops in the tanks was recorded. After each
Depuration Conditions for Great Scallops (Pecten maximus)
411
experiment the scallops that self-righted were marked with a small
spot of permanent ink and all scallops were returned to the normal
(cupped side down) position until the next observation in the ex-
periment.
Scallops collected from the cultivation site and delivered di-
rectly to the laboratory provided the control for predepuration
self-righting trials to ensure that the results from the depuration
experiments were not compromised by stress caused by the effects
of holding, transporting, and handling of the animals during the
contamination phase.
Control scallops for assessing the effect of depuration (in the
control depuration treatment) were taken from surplus animals
collected from the field site. Comparisons were also made of post-
depuration scallops from individual treatments compared with
scallops from the control depuration treatment.
RESULTS
Initial Depuration Experiment
An initial experiment was conducted under what was expected
be acceptable conditions for scallop depuration based on require-
ments for oysters. Conditions for the experiment were salinity
levels of 36%o, temperature of 15°C ± 1°C, with a scallop to water
ratio of approximately 1:50 (1:1 ratio being equivalent to 1 kg to
I L of seawater). Dissolved oxygen levels were maintained above
90% saturation throughout the experiment. E. coli levels of 805
MPN 100 g"' (consistent with a category B classification) were
reduced to nondetectable levels indicating that it was possible to
purify category B scallops under these conditions. Further inves-
tigations varied one parameter at a time.
Artificial Seawater and the Effect of Salinity Concentration
Experiments were conducted to investigate the effect of salin-
ity. Initial trials used fresh tap water to make artificial seawater
from standard salt mixes. However these trials produced high mor-
tality rates and poor levels of E. coli elimination were observed
(Table 2).
These results apparently indicate that decreasing salinity causes
an increase in mortality. However the fact that 20% mortality
occurred in the control treatment (35'^f) compared with no mor-
talities in the initial trial using natural seawater at a siinilar salinity
described above, indicates that salinity alone was not responsible
for the mortalities.
A further trial comparing artificial seawater made up to a final
concentration of 30%t with natural seawater diluted with freshwa-
ter to also give a final concentration of 30%f demonstrated 100%
mortality in the tank using artificial seawater compared with
TABLE 2.
Percentage mortality of scallops during depuration under varying
salinity ranges.
Trial Date
Salinitv (%)
Mortality (%)
14/2/01
27/2/01
14/2/01
27/2/01
25
30
35
35
100
55
20
20
no mortality using diluted natural seawater. E. coli levels in shell-
fish in the natural seawater tank were reduced from 265 MPN 100
g"' to 20 MPN 100 g"' indicating successful depuration.
To determine whether the problem associated with using arti-
ficial seawater was because of the salt mix used or the fresh water
in which it was diluted, an experiment comparing artificial seawa-
ter (30 '^(f ) prepared by adding potable water and using freshwater
that had been treated by passing through an activated charcoal
filter. Scallops depurated in seawater made up in untreated water
had a 20% mortality level compared with no mortalities in scallops
depurated in filtered water. E. coli reductions in the scallops during
depuration also differed; initial levels of 2.300 MPN 100 g~' were
reduced to <20 MPN 100 g"' in the treated water tank compared
with 300 MPN 100 g"' in the untreated water tank. Chlorine and
ammonia levels recorded during these experiment were low in
both tanks (<0.06 mg mL~' chlorine and <0.02 mg mL"' for
ammonia). It therefore appears that there was some unknown con-
stituent in the untreated water salt solution that was causing the
mortality in scallops, which was removed by treatment with acti-
vated carbon filtration.
Further salinity trials were performed using natural seawater
that was diluted in fresh water treated by passing through an ac-
tivated carbon filter. Other parameters during these experiments
were maintained at optimal conditions. Dissolved oxygen levels
were maintained above 80% saturation and temperature at 15°C ±
1°C. Scallop to water ratios were maintained at approximately
1:50. Results are shown in Table 3.
A salinity concentration of 28%c or higher appeared to allow
successful elimination of E. coli although caution should be used
in interpreting some of this data given the relatively low initial E.
coli levels observed in some of the trials. In all further experiments
investigating the effect of other physiologic parameters, full .saline
natural seawater (range 35 to 38%c) was used.
Temperature Trials
Results from the experiments to investigate the effect of tem-
perature on depuration efficiency are shown in Table 4. Depuration
at 10. 16. and 20" C was shown to be effective at reducing £. coli
to end product levels (<230 E. coli MPN 100 g"' ) even from levels
consistent with a category C classification (>4600 E. coli MPN
100 g"'). In contrast a minimal reduction (10%) was observed
when depuration was carried out at 7°C.
TABLE 3.
E. coli levels in scallops before and after depuration under varying
salinity ranges.
E. coli MPN 100 g-'
Post
Percent
Trial Date
Salinity (%)
Predepuration
Depuration
Reduction
13/3/0 1
25
465
210
55.2
24/4/01
28
330
<20
>94
13/3/01
30
465
<20
>96
24/4/01
30
330
<20
>94
2/5/01
30
2.300
<20
>99
Artificial seawater was made using standard salt water mixes dissolved In
potable standard water.
All values are averages of duplicate samples. Artificial seawater was made
using standard salt water mixes dissolved in water treated with an activated
charcoal filter.
412
DORE ET AL.
TABLE 4.
E. coli levels in scallops before and after depuration under varying
temperature ranges.
Temperature
E. coli MPN 100 g-'
Post
Percent
Trial Date
CCl
Predepuration
Depuration
Reduction
31/7/01
10
2200
<20
>99.1
31/7/01
16
2200
<20
>99.1
21/8/01
10
9750
220
95.5
21/8/01
20
9750
<20
>99.8
4/9/01
7
600(1
5400
10
All values are averages of duplicate samples.
Emersion Time Before Depuration
One trial was performed to assess the effect of the length of
time scallops were emersed before depuration had on the treatment
process. Scallops were held out of water at 15°C ± TC for a total
of 6. 10. and 22 h before being placed in depuration tanks at 14°C
± 1°C for 42 h. Initial E. coli levels of 1200 MPN 100 g"' were
reduced to 30. 30. and 145 MPN 100 g'' for 6, 10. and 20 h
emersion treatment respectively. All scallops were successfully
reduced from a category B level to end product standard, although
it appears that 20 h emersion may have a detrimental effect on the
efficiency of depuration compared with a lO-h immersion period.
Loading Arrangements
An initial trial was conducted with 60 scallops loaded in two
layers, cup side down, into one basket in a laboratory scale depu-
ration tank under optimal conditions. The scallops moved substan-
tially and several scallops escaped from the basket and on to the
base of the tank amongst fecal strands that had settled there. Scal-
lops did not escape from the basket in the control tank where only
20 animals were placed in one basket. After depuration, samples
were taken randomly from the top and bottom layers of the treat-
ment basket. Although reductions of E. coli were similar between
the control and treatment (96% and 98%. respectively), scallops
that had escaped the basket and were sitting on the base of the tank
were not tested.
Further trials placed mesh nets over the baskets so that the
scallops could not escape. A space was left between the scallops
and the net so that it did not impinge on the ability of the scallops
to open and filter.
A trial was conducted to confirm that scallops could be depu-
rated in two layers. Sixty scallops were loaded into one basket in
two layers and 20 scallops into another basket in the same system
to act as a control. Initial E. coli levels of 3500 MPN 100 g~' were
reduced to 30 and 20 MPN 100 g"' in the top and bottom layer of
the treatment basket, respectively. E. coli levels in the control were
reduced to 30 MPN 100 g"'.
Three trials were conducted in the commercial scale depuration
system fully loaded with scallops on the basis of a double layer of
50-60 scallops in each of six baskets. This gave a scallop to water
ratio of about 1:12. Trials were conducted at 1 5 ± 1 °C and salinity
of 36%f'. Control tanks containing just 20 scallops were also used
(scallop to water ratio in excess of 1 :50). In all cases E. coli levels
were reduced to below 230 MPN 100 g"' (Table 5). Dissolved
oxygen decreased in the treatment tanks in all three trials, but
remamed above 70% saturation at all times. Total ammonia in the
three treatment tanks increased to a level between 2.5 and 5 mg
L~' during the three trials compared with maximum levels 0.5 mg
L"' in the control tank but did not appear to have a detrimental
effect.
Self-Righting Experiments
The percentage number of scallops self-righting in the control
groups was variable between experiments, from 40-80%, but was
generally consistent for each batch of scallops for every repeated
observation within experiments. It was often the same (marked)
animals that righted on each occasion.
It was shown that holding scallops at the field site and trans-
porting them to and from the site did not apparently impose any
stress. In eight righting trials these scallops performed similariy to
control scallops delivered directly to the laboratory from the cul-
tivation site. The mean percentage righting responses, for the first
observations only, were 64.4% for control scallops and 55.6% for
treatment scallops. A paired r-test showed that the difference was
not significant (t = 1.08. P = 0.314. for 7 df).
The self-righting response of scallops following depuration in
the control treatment was similar to that for scallops from the same
batch collected from the field site at the same time but not depu-
rated. Mean righting response was 63.8% and 60.8% respectively
(paired t = 0.515. P = 0.634. for 4 df).
Scallops from the artificial seawater treatment, which was
found to be not suitable for depuration, did not show any self-
righting above 10% over the 14 days for which this experiment
was continued, by which time there was 40% mortality.
With the salinity experiments, scallops depurated at 25%o took
20 days to recover to a level of self-righting response of only
28.6%, although there was no mortality in this group. Scallops in
the 289ft treatment showed no difference to the control scallops
immediately following depuration at this salinity, with the same
number of scallops self-righting in both groups.
At low temperatures, scallops from the 7°C depuration treat-
ment did not show any recovery above 10-20% self-righting over
15 days, by which time there was 70% mortality. In three obser-
vations, between 50-70% of the scallops depurated at 10°C self-
righted in 1 h. a similar result to those from the control treatment.
It was also shown that scallops from an ambient temperature of
18°C that were held at 10°C for 42 h in a simulated depuration
experiment showed no sign of stress as measured by these experi-
ments. A similar number of control (untreated) scallops and scal-
lops from this treatment self-righted.
In four separate self-righting trials using scallops from the high
density (double layer) depuration experiments, including the three
carried out in the commercial scale systems, there were no differ-
ences in righting response between high and low stocking densities
in the depuration tanks. The mean righting responses, for the first
observations only, were 64.8% (control, 20 scallops in tray) and
61.1% (double layer, 50-60 scallops in tray; paired t = 0.502.
P = 0.65. for 3 df).
DISCUSSION
The predepuration self-righting experiments showed that the
conditions used for holding and handling the experimental ani-
mals, including transportation to and from the field site and the
laboratory, did not cause stress to the scallops. Most of the other
infomiation available on transporting scallops is in respect of mov-
Depuration Conditions for Great Scallops (Pecten maximus)
413
TABLE 5.
E. coli levels before and after depuration in scallops taken from various positions throughout small-scale commercial depuration systems.
Sample Position
E. coli MPN 100 g-
Predepuration
Post Depuration
% Reduction
99.8
99.7
98.2
99.2
99.6
88.1
92.4
97.9
92.4
92.4
97.9
97.9
97.9
95.7
97.9
97.9
98.9
97.8
98.9
97.8
97.8
4/12/01
Control
Top basket top layer
Top basket bottom layer
Bottom basket top layer
Bottom basket bottom layer
17/12/01
Control
Left top basket top layer
Left top basket bottom layer
Right top basket top layer
Right basket bottom layer
Left mid. basket top layer
Left mid. basket bottom layer
Right mid. basket lop layer
Right mid. basket bottom layer
Left bottom tray
Right bottom tray
26/2/02
Control
Left top basket
Left bottom basket
Right top basket
Right bottom basket
9,100
925
10,000
20
30
165
20
40
115
70
20
70
70
20
20
20
40
20
20
110
220
110
220
220
Scallops were loaded in plastic mesh trays in double layers at a density of approximately 50-60 scallops per tray.
ing juveniles from a hatchery or collector site to a cultivation site.
This has shown that periods of up to 12 h out of water have no
observable effect, provided the scallops are maintained in a humid
atmosphere (Maguire et al. 1999. Christophersen 2000. Minchin et
al. 2000). One of the current depuration experiments showed that
the process could be run effectively with scallops that had been
immersed for this amount of time. Longer periods of emersion may
compromise the ability of the scallops to depurate, and there was
some indication of this from the results of this study.
Initial trials had indicated that it was possible to successfully
depurate category B level scallops using standard procedures used
in the United Kingdom without any detrimental effects on the
product quality. Subsequently this study concentrated on finding
the minimum acceptable requirements for parameters, such as tem-
perature, salinity, etc., when depurating scallops.
The use of artificial seawater during depuration is common
practice for a wide range of species. However, results during this
study indicated that the use of artificial seawater is unacceptable
under the conditions applied here. This finding does not concur
with previous work (SFIA 1996). which demonstrated that it was
possible to use artificial seawater in tanks for degritting scallops
without any reduction of scallop activity or increased mortality.
However, no details of what was used to dilute the artificial salt
mix were given for that study. The scallop depuration trials con-
ducted by Heath and Pyke {2001 ) used natural seawater only. The
results obtained here indicated that an unidentified constituent of
the tap water used to make the artificial seawater was responsible
for the mortalities observed. Artificial seawater has been used on
numerous occasions at the CEFAS Weymouth laboratory to un-
dertake depuration trials with other shellfish species without any
effect on shellfish quality, and this result is unique to these scallop
trials. Because it was not possible to identify the constituent re-
sponsible for the problem, it remains unclear whether this situation
is unique to the water used at this laboratory or would be a wide-
spread problem if used in the field. However, until further work is
done to investigate the use of artificial seawater during scallop
depuration the use of artificial seawater in this role cannot be
recommended.
Salinity was found to have a critical effect on efficiency of
depuration and levels of 25%o had a detrimental effect on the rate
of E. coli clearance. The next lowest concentration of salinity that
was investigated was 28%t. and E. coli levels were successfully
eliminated at this salinity. However only one trial was conducted
at this salinity, and the initial level of E. coli in this trial was only
330 MPN 100 g"'. It is questionable whether this can be consid-
ered a suitable challenge to test this condition. Given this it is
recommended that in the absence of further work a minimum
salinity concentration of 30%c for scallop depuration should be
maintained during depuration. This is a relatively high salinity
compared with minimum concentrations set for other species. This
is not surprising given that scallops are an open seawater species
that will normally be exposed to full-salinity seawater. It should be
noted that the consistent availability of natural seawater at a sa-
linity in excess of 30%c might present a constraint in some com-
mercial settings. This requirement should be carefully considered
by operators at the outset of any plans to depurate scallops.
Temperature was also found to have a significant effect on E.
coli reduction. Minimal reductions were observed at 7°C, whereas
E. coli levels were successfully depurated at 10°C. These results
do not agree with those from previous studies (Heath & Pyke
414
DORE ET AL.
2001), which concluded that temperatures as low as 6.6°C could
effectively reduce levels of E. coli. They are, however, in agree-
ment with the results of McNamara (SFIA 1996), who recom-
mended a temperature range of 10-1 8°C for scallop degritting
based on measurements of shellfish activity. In the absence of any
further work, a minimum temperature of IO°C is recommended for
use during scallop depuration. No experiments were conducted to
define upper temperature limits for depuration. This is because
higher temperatures do not usually compromi.se the depuration
process, although they may affect product quality. In the one ex-
periment (Table 4) in these trials in which scallops were depurated
at 20°C the scallops depurated effectively, but there was some post
depuration mortality. Heath and Pike (2001) recommended an up-
per temperature limit of 16°C for depuration and McNamara
(SFIA 1996) a limit of I8°C for degritting.
Although scallops depurated to below the end product standard
in all treatments during the single experiment investigating emer-
sion time, there was some evidence that animals immersed for 22
h reduced E. coli levels less successfully compared with those
immersed for 10 h. However this single result must be considered
inconclusive, although it does concur with previous work (SFIA
1996). That study concluded that scallop activity was reduced
during degritting in scallops that were immersed for 24 h before
processing. A general conclusion from various studies on the effect
of emersion time on viability of great scallops is that they should
not be kept out of water for longer than 12 h (Maguire et al. 1999,
Christophersen 2000. Minchin et al. 2000).
It was possible to depurate successfully in a double layer with
nominal capacity of 230 scallops m'~. Scallops loaded at this
density showed a considerable tendency to move and, if left un-
confined, escaped from the basket and deposit themselves on the
base of the tank. This is considered unacceptable during depuration
as much of the fecal material excreted by the scallops during the
depuration cycle will settle on the base of the tank. Movement of
the scallops in this area will resuspend this material, which may be
reingested and recontaminate the scallops with microbiological
organisms present in the sediment. During this trial, plastic mesh
was placed over the baskets to prevent the scallops escaping. It is
critical for the depuration process that scallops should be contained
within the basket. Any procedure for doing this must not interfere
with the ability of the scallops to open and filter. The lowest
scallop to shellfish water ratio investigated in this trial was 1:12
because this was the maximum that could be achieved using the
double layer arrangement. This is a higher ratio than may be found
in some of the high-intensity systems that may be used to depurate
other species. These may have shellfish-to-water ratios of as low as
1 :3 when fully loaded. However it is considered unlikely that
high-density depuration of scallops is likely to be required in the
near future. Given this and results from density trials conducted
elsewhere (Heath & Pyke 2001 ), it is recommended that scallop to
water ratios should not fall below 1:12.
In general, results from the post depuration self-righting (stress)
experiments gave good agreement with the results from the depu-
ration experiments. That is, scallops from conditions that sup-
ported effective depuration showed no difference in stress to con-
trol scallops, whereas scallops from conditions in which they did
not depurate showed high levels of stress, sometimes accompanied
by subsequent high mortality. Also, the lower temperature and
salinity limits, below which the scallops will not depurate effec-
tively, are similar to the lower limits for optiinuni growth perfor-
mance of great scallops (Laing 2000. 2002).
ACKNOWLEDGMENTS
This research was funded by the UK Department for Environ-
ment, Fisheries and Rural Affairs.
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Joiirmil of Shellfish Research, Vol. 22, No. I, 41S-42I, 2003.
CIRCADIAN METABOLIC RATE AND SHORT-TERM RESPONSE OF JUVENILE GREEN
ABALONE (HALIOTIS FULGENS PHILIPPI) TO THREE ANESTHETICS
OSCAR CHACON,' MARIA TERESA VIANA,' ANA FARIAS," CARLOS VAZQUEZ,' AND
ZAUL GARCIA-ESQUIVEL' *
^Instititto de Investigaciones Oceanologicas. Universidad Autonoma de Baja Ccdifomia. Apdo. Postal
453, 22 800 Enseiiada. B.C. Mexico: 'Instituto de Aciiicultura, Universidad Austral de Chile, Campus
Puerto Montt. Puerto Monti. Chile: and ^Universidad Autonoma de Mexico, Facultad de Ciencias
Veterinarias. Ciudad Universitaria. Mexico. DF
ABSTRACT Time-course experiments were performed on juvenile green abalone {Hiilmlis fiili;fii.^\ to assess the degree of stress
caused by the anesthetics magnesium sulfate (MS), benzocaine (BZ). and phenoxyethanol (PE). Metabolic rate (VO,) of abalone was
reduced by 65. 35. and 18% during short-term (10 or 20 mini exposure to MS. BZ. and PE. respectively. Abalones significantly
increased their VO. above control values (1.5-fold) after removal of PE from metabolic chambers, whereas those treated with MS or
BZ recovered their VO, to preanesthesia values. Visual criteria of recovery generally coincided with those of metabolic measurements
(i.e., 80% of abalone regained "normal" activity after 35 min postanesthesia), yet metabolic measurements showed that "fast"
recovering abalone treated with PE maintained high VO, values during 3-h postanesthesia. Abalone treated and nontreated with
anesthetics exhibited a circadian metabolic rhythm, with 20-35% higher rates observed during dark than light hours. Despite the
short-term metabolic alterations with MS. BZ. and PE, the present study suggests that all three anesthetics may be safely used in
abalone. However, detailed evaluations are still needed to assess the effect of anesthesia on other physiological variables. The results
obtained in this study highlight the importance of physiological evaluations when different chemical substances are used in aquatic
invertebrates.
KEY WORDS: abalone, anesthetics, circadian rhythm, metabolism, Haliotis fidgens
INTRODUCTION
Current culture methods for abalone involve vaiious steps in
which organisms need to be dislodged from their rearing substra-
tum for the purpose of size grading, adjusting densities, tagging,
and/or transfening from indoor to outdoor culture facilities (Hahn
1979, Juefeng & Shiuan 1996). Because of the natural ability of
abalone to strongly adhere on most surfaces, forced removal not
only results in excess mucus production with the consequent en-
ergy losses (Peck et al. 1987, Davies & Williams 1995, McBride
et al. 2001 ), but it can also result in injuries in the .soft tissues that
may eventually result in death (White et al. 1996). It is thought that
the lack of clotting mechanisms in abalone facilitate prolonged
bleeding and/or the appearance of bacterial infections in wounded
tissues (Armstrong et al. 1971 ), thus increasing the probabilities of
death (Juefeng & Shiuan 1996).
Farms have produced several solutions to remove abalone from
the substrate without producing injuries. All of them are aimed to
relax the soft tissues or decrease the degree of awareness in aba-
lone and include thermal shock and desiccation (Hahn 1989) in
addition to the use of anesthetic substances, such as CO,, urethane,
chloral hydrate, barbitol, diethyl carbonate, benzocaine, ethyl al-
cohol, propylene phenoxytol, potassium chloride, procaine hydro-
chloride, MS-22, pentobarbital, magnesium sulfate, and phenoxy-
ethanol (Hahn 1989. Juefeng & Shiuan 1996, White et al. 1996,
Aquilina & Roberts 2000). The last three substances have been
reported as effective and nonlethal anesthetics for abalone because
organisms usually recover within the first few hours of application
(Hahn 1989, White et al. 1996. Aquilina & Roberts 2000).
Excess magnesium sulfate interfere with neuromuscular trans-
mission signals in mammals because magnesium ions block the
*Con-esponding author. Tel. -1-52-646-174-4601; Fax -I-52-646- 1 74-5303;
E-mail; sgarcia(a'uabc.mx
release of the neurotransmitter acetylcholine from motor nerve
endings, by competitively binding to N-methyl-d-aspartate
(NMDA). a glutamic acid receptor (Iwatsu et al. 2002). The overall
effect of this blockade is a sedative effect of the neuromuscular
system, followed by muscle paralysis, respiratory depression,
coma, and death (Swain & Kaplan-Machlis, 1999). It is thought
that phenoxyethanol also binds competitively to NMDA receptors
(Mushoff et al. 1999) and causes depression of the central nervous
system and hypoxia when delivered in excess (American Veteri-
nary Medical Association 2001 ). The mechanism of action of local
anesthetics, such as benzocaine (BZ), is a blockade of the voltage-
activated sodium channel at the neuronal cell membrane, which
prevents the generation and conduction of the nerve impulse (Cat-
terall & Mackie 1996). Excess BZ in mammals may result in
prolonged sedation, cardiac arrhythmias, respiratory depression,
tremors, and death (Catterall & Mackie 1996).
The effectiveness of anesthetics in marine molluscs has been
largely evaluated on the basis of visual observations, such as the
degree of gaping respon.se after tactile stimuli in bivalves (Culloty
& Mulcahy 1992, Heasman et al. 1995, Mills et al. 1997), degree
of muscle relaxation, and coloration in squids (Garcia-Franco
1992), and degree of adhesion, muscle relaxation, and mortality in
abalone (Hahn 1989, White el al. 1996, Aquilina & Roberts 2000).
In most cases, short-, medium-, or long-term effects of anesthetics
have not been studied in detail, even though the magnitude of
stress during and shortly after the application of anesthetics is well
documented with visual observations. Therefore, detailed under-
standing of the effect of anesthetics on abalone is still needed at the
physiologic level, especially because they represent a potential tool
for research and management.
Several physiological parameters have been reported to in-
crease at night in abalone, including motor (Donovan & Carefoot
1998), feeding activities (Barkai & Griffiths 1987), and metabolic
rate (Uki & Kikuchi 1975). The latter is known as a highly sen-
415
416
Chacon et al.
sitive parameter in molluscs because it readily changes in response
to stress factors, such as temperature (Newell 1973, Paul & Paul
1998). pH (HaiTis et al. 1999), nitrite (Harris et al. 1997) and
ammonia concentrations (Harris et al. 1998), and starvation (Gar-
cia-Esquivel et al. 2002). Respiration is controlled by the central
nervous system, and therefore it is not surprising that metabolic
rate of abalone is affected by substances such as magnesium sul-
fate (Edwards et al. 2000). In the present study, time-course mea-
surements of metabolic rate were performed in juvenile green aba-
lone (Haliotis fulgens) in the presence and absence of anesthetics
to assess the magnitude and duration of metabolic stress produced
by magnesium sulfate, phenoxyethanol and benzocaine.
METHODS
Experimental Conditions
One and a half year-old juvenile abalone (Haliotis fulgens) with
shell lengths ranging from 25 to 35 mm, originally obtained from
BC Abalone farm in Erendira B.C., Mexico, and maintained at the
laboratory facilities at the University of Baja California, were used
for the different experiments. Abalone was kept in a shallow water
tray (180 x 90 x 20 cm, length x width x height) under flow-
through (ca. 300 mL min"'), aerated seawater conditions. Seawater
temperature was maintained at 23 ± 1°C with a digitally controlled
heater (CLEPCO. 1000 watts) located in a reservoir. Inert food
was offered at night on a regimen of 12 h per day, with a diet
(Table 1 ) made in the laboratory as recommended by Viana et al.
(1996). Light intensity was kept at ca. 2 x 10"*^ p-E/s/cm" with
several layers of a plastic mesh (70% shed) placed around the
system. A photoperiod of 12:12 (Light: Dark) was maintained
throughout the study.
Experimental Design
Experiments were of two types: (1) Time series to identify
circadian changes in the metabolism of H. fulgens. and (2) Time
series to assess the short-term (3 h) and medium-term (2 days)
effects of magnesium sulfate (MS), phenoxyethanol (PE), and BZ
on the metabolic rate.
Circadian Changes
Twenty-four abalone were randomly selected from the main-
tenance tray and distributed among eight respiration chambers
(three organisms per chamber) with a volume of 1.8 L each. Four
additional chambers were also used as controls (without organ-
isms). Chambers were maintained with open flow and without
food during 24 h for the abalone to acclimate to the system. Feces
and any remaining particles were siphoned out from each chamber
before beginning the first measurement of oxygen consumption.
Respiration was measured using closed-cell respirometry. Incuba-
tions of ca. 1- to 1 .5-h duration were conducted every hour during
a total period of 48 h. At the end of this period, the live weight and
total length of the experimental abalone was recorded.
Anesthetics
Two experiments were carried out with using the anesthetics
magnesium sulfate (MS. Sigma M-75()6) at a final concentration of
4% w/v (Hahn 1989); phenoxyethanol (PE, Sigma P-1 126) at 0.1%
v/v (Edwards et al. 2000) and benzocaine (BZ, Sigma E-1501) at
0.01% v/v (Hahn 1989). The latter was dissolved in 95% ethanol
(10% w/v) before use. All final solutions were prepared in 5-p,m
filtered seawater just before application.
TABLE L
Percent composition (dry weight basis) of the balanced diet used
offered to juvenile green abalone Haliotis fulgens.
Ingredients
(g/100 g diet)
Balanced Diet
Fish meal*
Corn starch
Kelp meal''
Corn tlour''
Gelatin (50 blooms)
Soybean meal'
Cellulose''
Modified starch*^
Mineral mixture'
Vitamin mixture^
Fish silage''
Stay-C
Choline chloride
Sodium benzoate
BHT'
Composition (%)
Protein
Ash
Nitrogen-free extract
30.00
14.66
10.00
10.00
10.00
8.00
5.00
5.00
4.00
1.50
1.40
0.20
0.10
O.iO
0.04
30.8 ± 0.7
12.6 ±0.1
6.3 ±0.1
50.3 ± 04
* 64% protein.
'' Made from Macrocyslis pyrifera.
'' Corn flour (Maseca).
" 39Vf protein, 2V7c lipid.
'' «-cellulose (Alphacel).
■■■ Modified com starch (Clearjel®).
' ICN vitamin diet fortification.
* ICN salt mixture #5 Brigges.
'' Acid fish silage from tuna viscera.
' Ascorbyl polyphosphate (kindly donated by Roche).
' Butylated hydroxy toluene.
In the first experiment, a step-wise approach was used for
quantifying the short-term response of juvenile abalone exposed to
anesthetics. Metabolic rate was measured before, during, and after
the application of anesthetics. Each anesthetic was evaluated on
different days using four replicate chambers per anesthetic (three
organisms per chamber), three control chambers (abalone without
anesthetic), and three reagent controls (seawater with anesthetics,
but no abalone). Experimental organisms were transferred to the
chambers 24 h before the treatment, as described in the previous
section. At the end of this period, chambers were cleaned and
respiration rate of abalone was measured during 1-1.5 h. The
water was completely renewed ( 100% oxygen saturation) and an-
esthetics were added directly into the incubation chambers, while
recordings of oxygen consumption continued. Abalones were in
contact with anesthetics for a fixed period of 10 min (BZ and PE)
or 20 min (MS). These treatment periods were based on prelimi-
nary visual observations of the organism's response to these an-
esthetics. Chambers were flushed with fresh seawater (ca. 6 vol-
ume changes) after the exposure period to eliminate anesthetics. It
was assumed that anesthetics got rid off the chambers during flush-
ing, as the reagent control and experimental chambers regained a
constant oxygen baseline afterwards. Incubations continued every
1 or 1.5 h during the following 3 h to measure the metabolic
response postanesthesia on the same organisms.
CiRCADiAN Rhythm and Anesthetics in H. fulgens
417
The second experiment consisted of a time series of 48 li v\'ith
simultaneous measurement of VO, in abalone treated with all three
anesthetics (MS, PE, BZ) to assess the duration of metabolic stress.
Experimental abalone were removed from the maintenance tray
with the help of a spatula and transferred to plastic buckets con-
taining I L of seawater. When all organisms had adhered to the
walls, all three anesthetics (MS, PE, BZ) were added separately
into the buckets and the anesthetized organisms were transferred to
metabolic chambers (3 abalone per chamber), where respiration
measurements began 3h later. Incubations lasted between 1 .3 and
2 h, with a measurement frequency of ca. 8h, and a total elapsed
time of 47 h. At the end of each trial, oxygen consumption rate was
measured in the same experimental chambers without abalone, to
correct for the oxygen consumed by sources other than abalone
(electrodes, microorganisms). A total of three replicates per treat-
ment (anesthetics) and two control replicates (abalone without an-
esthetics) were used for this trial.
Measurements
Metabolic Rate
Oxygen consumption by H. fulgens was recorded every 30 sec
with two computer-controlled polarographic oxygen sensors
(Strathkelvin Instruments Ltd.. Ireland). Each oxygen meter had
six channels, such that 12 chambers could be monitored simulta-
neously. Aerated seawater was used for calibration to 100%, and
sodium sulfite was used for 0% calibration. A magnetic stir bar
( lO-mm diameter x 8-mm length) was used for mixing the water in
each incubation chamber under a perforated acrylic sieve (4-mm
mesh) to prevent a direct contact between the stir bar and organ-
isms. After incubations all abalone from the chambers were blot
dried with a piece of cloth, measured with digital calipers (MAX-
CAL, ± 0.03 mm) on their longest dimension, and weighed in a
portable scale (AND SV-200, ± O.Olg). Oxygen consumption rate
(metabolic rate, VO,l was estimated from the corrected slope of
the oxygen evolution curve (abalone minus non-abalone cham-
bers), after transforming the VcO-, saturation to |j.mol of dissolved
O, in seawater. from known values of oxygen solubility (Green
and Cairitt. 1967). The following equation was used for calculat-
ing metabolic rate:
VO,, = (Cs*m*60)/(100%* Wwt) (I)
where VO,^. = metabolic rate of the experimental organism (|j.L
O, /g/hT
Cs = total amount of O, in the incubation chamber at 100%
saturation (jjiL O,).
m = slope of the O^ evolution curve (9'rO-,/min)
60 = factor used to transform from minutes to hours
Wwt = live weight (g) of organisms in the incubation chamber.
Visual Assessment of Anesthesia and Recovery
Direct observations of abalone behavior during and after ap-
plication of each anesthetic were conducted on 36 organisms dis-
tributed in 12 buckets (three organisms per bucket). The time taken
from the application of anesthetics to the moment an abalone fell
off the walls of the bucket was considered as the period needed for
induction to anesthesia. Similarly, the time taken for an anesthe-
tized abalone to regain an upright position (ventral side firmly
attached to the container's walls) was considered a visual criterion
for recovery postanesthesia (White et al. 1996). Mortality was evalu-
ated on anesthetized organisms after 2 or 4 weeks postanesthesia.
Statistics
In all cases, time-dependent changes of metabolic rate were
statistically tested using an analysis of variance (ANOVA) with
repeated measures. When significant differences were found, least
squares pre-planed comparisons of means were used to identify
specific differences. These tests were carried out using a general
linear model procedure (GLMi included in the statistical package
SAS. version 6.08 (SAS, 1998).
RESULTS
Short-Term Effect of Anesthetics
Juvenile abalone treated with all three anesthetics exhibited
time-dependent differences in their metabolic rate (VO,), as this
was significantly reduced (Table 2) by 65% (MS), 35% (BZ), and
18% (PE) of initial values during the exposure period (Fig. la-c).
TABI^E 2.
Results of repeated analysis of variance for comparison of short-term (5 h) and long-term |48 h) changes of metabolic rate of juvenile
abalone, Haliotis fulgens, after exposure to three anesthetics (Anest).
Source of
Variation
Mean Squares
F
DF
MS
PE
BZ
MS
PE
BZ
Short term
Replicates
3
28.0
60.9
46.9
0.9 NS
3.8*
1.0 NS
Between Subjects (Anest)
1
390.5
1137.8
30.7
12.8**
70.3**
0.7 NS
Within Subjects (lime)
3
824.5
431.3
234.5
27.0**
26.6**
5.1**
Time x Anest
3
842.0
439.6
378.3
27.6**
27.1**
8.2**
Error
17
30.5
16.2
46.2
Long term
Replicates
2
51.8
3.1 NS
Between Subjects (Anest)
3
141.2
8.4**
Withm Subjects (time)
6
115.22
68.8**
Time x Anesth
18
46.9
2.8**
Error
47
16.7
Short-term trials were conducted independently for each anesthetic and its control whereas the long-term trial was conducted simultaneously for the
anesthetics magnesium sulfate (MS), phenoxyethanol (PE). henzocaine (BZ) and a control without anesthetics (C).
* P < 0.05; **P < 0.01 ; NS = not significant at P > 0.05.
418
Chacon et al.
D)
CM
o
80 -
(b)
r<^-^^^
3.
S
60 ■
-<
^J^^^-^
(T
o
^i
o
3
40 J
u
Elapsed Time (h)
Figure 1. Short-term changes of metabolic rate in juvenile green aba-
lone {Haliotis fiilgens) before, during, and after exposure to magne-
sium sulfate (a), phcoxyethanol (bt, and benzocaine (cl. Arrows indi-
cate the moment when anesthetics were added i and removed T from
the respiration chambers.
Abalone from the MS and BZ treatments re-established their VO,
after flushing away the anesthetic from the respiration chambers,
and remained similar to control values thereafter (P > 0.05). In
contrast, abalone treated with PE significantly increased their VO.
by a factor of 1 .5 above control values (Table 2) and remained high
for the ne.xt 2 h posttreatment. with a trend to decrease thereafter
(Fig. lb). Oxygen consumption of reagent control chambers (an-
esthetics, without abalone) also increased in the presence of these
chemicals. It accounted for lO^r (MS). 13% (PE), and 54.3% (BZ)
of the total O. consumed by experimental abalone during the ex-
posure period, yet no significant O, consumption was observed in
the reagent chambers (P > 0.05) after anesthetics were flushed
away (data not shown).
Diel Changes of Metabolic Rale
A circadian rhythm was observed in juvenile abalone in the
absence of anesthetics (Fig. 2a). with significantly higher meta-
bolic rate observed during dark than light hours (P < 0.01 ). Meta-
bolic rate (VO-,) decreased and remained relatively constant (42-
32 jjiL OMg Wwt) during the period of 10;00 to 16:00 h (light
conditions), and significantly increased (P < 0.01) by ca. 20%
during the period of 22:00 to 4:00 h (dark conditions). The tran-
sition period (light switched on or off. at 8:00 and 20:00 h. re-
spectively) was characterized by rapid changes of metabolic rate,
such that abalone exhibited most of the time a dark- or a light-
adapted metabolic rate (Fig. 2a). The circadian VO, pattern was
maintained throughout the 48-h measuring period, even though the
absolute values showed a trend to decrease in the second day of the
trial (P < 0.05). Abalone exposed to anesthetics exhibited signifi-
cant anesthetic and time effects (Table 2), and the same circadian
rhythm identified above. VOj values obtained at 8:00 or 20:00 h
(Fig. 2b) also corresponded to the transition period. Higher values
(65 to 70 [jLLO,/h/g Wwt) were observed during dark hours and
lower values during daylight hours (Fig. 2b). Time vs anesthetic
interaction was also significant (Table 2). PE-treated abalone ex-
hibited significantly higher VO, values than control organisms (P
< 0.05) during the first measurement (3 h postanesthesia), yet these
differences were not statistically significant thereafter (P > 0.05).
Visual Criteria
Based on motor activity, it was observed that SM acted slowly
and asynchronously on juvenile abalone. These organisms showed
a complete relaxation of the mantle and became narcotized some-
DAY
NIGHT
DAY
NIGHT
I
3.
B
o
75
60
45
30
90
(a)
75 -
60
45
30
16
24
32
40
48
Elapsed Time (h)
Figure 2. Two-day recordings of metabolic rate exhibited by juvenile
green abalone (Haliolis fulgens) without anesthetic treatment (a) and
after a lO-min exposure (b) to the anesthetics magnesium sulfate (MS),
phenoxyethanol (PK), and benzocaine (BZ). A control treatment (aba-
lone, no anesthetics) is also shown. Time 0 = 10:00 AM.
CiRCADiAN Rhythm and Anesthetics in H. fuluens
419
TABLE 3.
Visual assessment of minimal (min) and maximal (max) exposure period (minutes) necessary to anesthetize juvenile abalone. Haliotis Julgeiis,
by magnesium sulfate (MS), phenoxyethanol (PE) and benzocaine (BZ).
MS
PE
BZ
ID
Min
Max
Min
Max
Min
Max
1
2.4
19.0
2.3
4.2
4.5
4.5
2
3.2
5.1
1.1
4.2
3.7
6.5
3
2.3
25.0
1.9
2.7
2.7
4.4
4
2.7
18.6
2.3
5.7
2.6
4.2
5
4.0
20.0
2.3
4.2
3.0
9.6
6
2.1
23.0
2.1
5.6
2.5
4.4
7
2.4
34.0
2.8
3.0
2.4
9.2
8
4.0
19.0
3.2
4.3
4.5
4.8
9
1.5
23.0
2.6
3.2
2.9
2.9
10
14.0
23.0
2.4
3.1
6.5
7.1
11
12.0
26.0
3.0
4.7
2.4
3.1
12
2.4
18.0
2.6
3.3
3.7
6.1
Mean+ SE
4.4+ 1.2
21.1 + 1.9
2.4 + 0.2
4.0 + 0.3
3.4 + 0.3
5.6 + 0.6
Data based on evaluations of 12 independent chambers per each anesthetic (15 abalone/chamber).
time between 2 and 21 min after exposure. In contrast, organisms
expose(i to PE anii BZ became generally narcotized within the first
2 to .^ min (Table 3) and were characterized by their rigidity.
Recovery postanesthesia varied among anesthetics. About 80% of
abalone regained their normal upright position after 18 min (PE)
25 min (BZ) and 35 min (MS) post-anesthesia, and nearly 100%
had been recovered after 1 h in all treatments (Fig. 3).
DISCUSSION
Short-Term Effect of Anesthetics
The induction/recovery periods visually determined for H. fiil-
gens in this study were similar to those reported for other mollus-
can species, including the abalone H. gigantea and H. midcie (Hahn
1989, White et al. 1996), the scallop Pecten fumatus (Heasman et
al. 1995) and the pearl oysters Pinaata albina and P. margu-
ritifeni (Norton et al. 1996). The heterogeneous anesthetizing ef-
fects of MS contrasted with the homogeneous, rapid action of PE
and BZ assessed visually. Similar observations have been previ-
ously reported for H. midae (Hahn 1989, White et al. 1996) and
may be related to the degree of access of these anesthetics to the
site of action. It is known that topical anesthetics like BZ readily
and locally interact with any nerve cell receptor (American Vet-
erinary Medical Association 2001). whereas MS affects the
smooth muscle or the central nervous system of vertebrates (Swain
& Kaplan-Machlis 1999) by blocking the release of the neurotrans-
mitter acetylcholine. The relaxation symptoms of abalone tissues
observed in this and other studies (White et al. 1996) are consistent
with the symptoms described for the neuromuscular system of
humans (i.e.. Swain & Kaplan-Machlis 1999) and other vertebrates
(American Veterinary Medical Association 2001).
To our knowledge, this is the first study that documents in
detail a time-course response of metabolism in marine inverte-
brates exposed to anesthetics, including the observation of rapid
depression of the respiratory system and concomitant recovery
following the elimination of anesthetics from the chambers (Fig.
1). White et al. ( 1996) recorded an inhibitory response of the tarsal
muscle of H. midae when exposed to MS. PE, and procaine, but
their study was more focused at demonstrating the effectiveness of
these substances as anesthetics, rather than documenting time-
dependent physiologic effects on abalone. The short-term (i.e., <3
h postanesthesia) metabolic response of abalone was only partially
coincidental with visual criteria of recovery. In this regard, the
VOt exhibited by H. fulgens immediately after MS and BZ were
flushed away from the incubation chambers were similar to the
controls, whereas organisms treated with PE maintained a high
VO2 even after 3 h postanesthesia (Fig. lb). Conversely, visual
observations suggested that organisms exposed to PE and BZ re-
covered faster and more uniformly than those exposed to MS (Fig.
I ). Although no other physiological variables were measured in
this study, it has been reported that the trout OncDihyiulinx iiiykiss
experienced an increase in blood pressure after being exposed to
PE (Fredricks et al. 1993). Therefore, the actual physiologic state
of abalone (this study) was more likely reflected in the metabolic
response curve, as this variable is highly sensitive to exogenous
0)
>
o
o
(1>
a.
0)
>
3
B
3
o
. -^::jC^Z-^^-<^-
'~Z..---
PE
BZ
■ MS
^-^
,.•■■
n = 36
75
/ '' ■'
50 ■
/
/J
25
■J !
t
n -
}y
Time Post-anesthesia (min)
Figure 3. Cumulative percent recovery from anesthesia of juvenile
abaicme, Haliotis fulgens), based on visual criteria. Magnesium sulfate
(MS), phenoxyethanol (PE), and benzocaine (BZ).
420
Chacon et al.
and endogenous perturbations. The reason for the post-anesthesia
increase of VO, in the presence of PE is not known and need
further and detailed studies in abalone.
Diel Changes
The consistent circadian rhythm exhibited by control and an-
esthetic-treated H. fidgens suggests that these chemicals did not
significantly affect the functioning of the central nervous system
after a few hours of application. Furthermore, the observed rhythm
is consistent with previous reports of night-accelerated metabolism
in the Japanese abalone H. discus hannai (Uki & Kikuchi 197.'i).
Other physiologic variables, such as food intake (Barkai & Grif-
fiths 1987) and motor activity (Donovan & Carefoot 1998), typi-
cally increased at night in abalone. Circadian physiologic rhythms
have also been observed in other molluscan species. For example,
Watanabe ( 198.^) reported that the repair and growth of shell in the
bivalves P. murtensii and Helisoma duryi was highest under con-
tinuous darkness. Taken together, these results suggest that the
endogenous clock of these species may be cued by light. It has
been demonstrated that changes in light intensity/photoperiod are
among the major environmental cues responsible for the activation
of clock genes in organisms ranging from fruit flies to mammals
(Schibler & Lavery 1999). In addition, it has been shown that the
ocular circadian rhythm of the marine snail Bulla i;inildiana is a
complex process regulated at the level of transcription, translation,
and phosphorylation and involves the presence of a cyclin-
dependent protein kinase, whose activity coincides with a circa-
dian clock (Krucher et al. 1997). Earlier reports suggest that the
hypothesis of a light-controlled circadian rhythm may still be con-
troversial haliotids. In this regard. Jan et al. (1981) found that
Haliotis diversicolor supertexta exhibited a circadian metabolic
rhythm when exposed to a natural diurnal cycle, but not under
continuous light. Accordingly. Peck el al. (1987) did not find
significant differences in the metabolic rate of H. iLdtercidata be-
tween day and night (12:12 L:D photoperiod), and suggested that
such behavior resulted from an excess in the available food. The
contradictory results found among haliotids may either suggest
that there are species-specific differences in the response to envi-
ronmental cues and/or there are subtle methodological differences
that may explain the observed results. In this regard, it was shown
that the gastropod A. californica exhibited a circadian feeding
rhythm under conditions of 12:12 L:D photoperiod, with shorter
feeding response associated with light hours. Such a pattern con-
tinued when organisms were shifted to a 0:24 (L:D) photoperiod
(Kohn 1983). but the whole experimental period lasted only three
days, and therefore no conclusive results could be drawn as to the
role of light on cueing the observed rhythm. Therefore, detailed
and long-term experiments are needed to test whether the circadian
rhythm observed in this and other studies can be generalized in
haliotids. Despite these controversies, the results of the present
study may have implications for growth/production protocols in H.
fidgens because the total energy drain (i.e., respiration) of H. fid-
gens is highest at night. The trend of decreasing metabolic rate
observed during the second day of measurement (Fig. 2a) was
likely due to a higher amount of food remaining in the abalone's
gut (i.e., SDA component of VO.) in the first day of measurement.
It has been shown that a complete gut evacuation can take between
18 h to 7 days in abalone (Wee et al. 1992, Maguire et al. 1993,
Britz et al. 1996, Mai et al. 1998).
Overall, the results of this study highlight the importance of
physiologic evaluations when different chemical substances are
used in aquatic invertebrates. The combined visual and metabolic
evaluations confirmed that all three anesthetics might be poten-
tially used for handling abalone, since all of them effectively in-
duced anesthesia, rapid post-anesthesia recovery and no mortality.
Nevertheless, careful evaluations are still needed to assess the
long-term effects of anesthesia on other physiologic variables such
as growth, food intake and activity of abalone. In this regard,
Edwards et al. (2000) found that the abalone H. laevigata and H.
rubra exhibited a significantly lower growth rate than control or-
ganisms after 6 weeks of exposure to PE and BZ.
ACKNOWLEDGMENTS
Financial support was partially obtained through a grant
CONACYT (G281I9B) awarded to MTV and a grant (SINVE
002-DE) awarded to ZGE by Gobiemodel Estado de, Baja, Cali-
fornia. The authors thank Marco A. Gonzalez, Roberto Escobar,
and Laura Gomez for their valuable help during most of the trials.
Thanks to two anonymous reviewers who helped to improve the
article.
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Journal of Shellfish Research, Vol. 22, No. 2, 423-+30, 2003.
LABORATORY HYBRIDIZATION OF THE MUSSELS, MYTILUS TROSSULUS AND
M. GALLOPROVINCIALIS: LARVAL GROWTH, SURVIVAL AND EARLY DEVELOPMENT
SEAN E. MATSON,'* JONATHAN P. DAVIS," AND KENNETH K. CHEW'
^Oregon State Universit}: Hatfield Marine Science Center. 2030 SE. Marine Science Dr.. Newport.
Oregon 97365: 'Taylor Resources, Inc.. Quilcene. Washington 98376: and ^University of Washington
School of Fisheries and Aquatic Sciences. Seattle. Washington 98 J 95
ABSTRACT Expenmenis were performed to determine whether hybrid larvae of Mytihis trossiilus (Baltic mussel) and Mytilus
galloprovincialis (Mediterranean mussel) could be produced in a shellfish hatchery environment and whether early development,
survival, or growth differences existed between the two species and their reciprocal hybrids at full and reduced salinity. Hybrids of
these two species are uncommon in Puget Sound. Washington and on the northern west coast of North America. Broodstock were
screened morphologically and positively identified at two nuclear DNA loci using polymerase chain reaction and restriction fragment
length polymorphism techniques. Hybrid larvae were produced in both reciprocal combinations, and were successfully reared through
metamorphosis. There was no apparent hybrid vigor because hybrids did not grow consistently larger (or survive better) than the
parental crosses, nor did one reciprocal cross grow consistently larger than the other. Both reciprocal hybrid crosses and the parental
cross, M. rro.ssuliis. grew faster than the other parental cross, M. galloprovincialis. at low salinity (20 ppt). These results concur with
the two species' physiologic and ecological characteristics. Mytilus trossiilus grows well in areas of low and variable salinity (much
of Puget Sound) and M. galloprovincialis grows well in areas of stable, full salinity, and recruits pooriy in Puget Sound. Hybrids
showed generally lower fertilization rates and slower early development than parental crosses, although they were sufficient to produce
larval cultures and postlarvae. The successful fertilization, growth, and survival of these hybrids suggests that some factor other than
genetic incompatibility is likely responsible for the rarity of these hybrids in Puget Sound. One such factor could be the limited overlap
of the spawning periods of the two species in this region. A differentia! species growth-response to salinity was observed in this study.
KEY WORDS: hybrid, mussel, Mytihis irossuliis. galloprovincia
INTRODUCTION
Two species of mainne mussels found in Puget Sound, Wash-
ington are Mytilus trossiilus (Baltic mussel) and M. galloprovin-
cialis (Mediterranean mussel). The two species occur both sym-
patrically and allopatrically in Puget Sound. Both species possess
distinct ecological and physiologic characteristics (Johnson 1978,
Kautsky 1987, Brooks 1991,Margus 1991. Sarver & Loudenslager
1991, Sarver & Foltz 1993, Geller et al. 1994, Hoffman & Somero
1995, 1996). Mytilus tros.ndus thrives in areas of widely fluctuat-
ing salinity, as is the character of much of Puget Sound, whereas
M. iialloprovincialis occurs in bays with more stable, high salinity
(Brooks 1991).
These two species hybridize naturally in isolated populations in
Puget Sound (Brooks 1991, Suchanek et al. 1996). Their hybrids
also occur in other bays in Washington, Oregon, and California.
Although these two species often co-occur throughout their distri-
bution within Puget Sound, the overall frequency of hybrids has
remained very low (Brooks 1991, Suchanek et al. 1996). One
reason this laboratory attempt at hybridizing these two species was
performed was to help elucidate what sort of barrier to hybridiza-
tion may be responsible for this lack of hybrids in the wild. Bar-
riers to hybridization can be rooted in genetic incompatibility or
physiologic and ecological differences, such as disease, salinity
tolerance, or the tiining of spawning events.
Both mussel species are commercially important within the
United States and throughout the world. Their physiologic and
ecological differences pose challenges to tho.se who culture them.
Mytilus trossulus suffers high mortalities before the end of its
second year of life becau,se of high summer water temperatures
and the disease hemic neoplasia (Brooks 1991). Up to 75% of aM.
trossiilus mussel crop in Penn Cove, Puget Sound often dies before
*Corresponding author. E-mail: sean.matsonCa'oregon.state.edu
lis
it is old enough to be harvested (Brooks 1991). Mytilus gallopro-
vincialis typically grows to a larger size than M. trossulus and is
resistant to hernic neoplasia (Brooks 1991). Mytilus galloprovin-
cialis has been observed suffering significant mortalities when
salinities have dropped to 20 parts per thousand (ppt), and 100%
mortality below 10 ppt (Kautsky 1987, Margus 1991). Low salin-
ity conditions have repeatedly coincided with substantial mortali-
ties of M. galloprovincialis in Holmes Harbor. Puget Sound.
Washington (Kurt Johnson. Taylor Resources, personal commu-
nication), eventually leading to the closure of the mussel farm
there. The financial implications of the aforementioned mortalities
are severe enough to wanant examining biologic alternatives that
might ameliorate lost farm revenues and even closures as a result
of salinity- and disease-related crop loss. One such alternative
worth examining is hybridization of the two mussel species. Myti-
lus trossulus and M. galloprovincialis possess characteristics that
if expressed in a hybrid (variable salinity tolerance of M. trossulus
and the disease resistance of M. galloprovincialis) might result in
increased mussel production for industry. Mytilus galloprovincia-
lis X M. edulis hybrids carry some of M. galloprovincialis' disease
resistance to a trematode parasite (Coustau 1991). Sturgeon hy-
brids have been found to be more resistant to thermal and salinity
shock than either parental species (Chikhachev 1979). Both inter-
specific and intraspecific hybrid vigor have been documented in
bivalve mollusks (Loosanoff 1954. Hedgecock et al. 1996. Bayne
et al. 1999) and it could occur in hybrids of these two Mytilus
species. Hybrid vigor is defined here as an increase in growth or
survival of hybrid crosses over pure-species crosses.
This investigation was performed to determine whether hybrid
larvae of two locally occurring species of marine mussels (A/.
trossulus and M. galloprovincialis) could be produced in a shell-
fish hatchery environment, and whether survival and growth dif-
ferences existed between the two species and their reciprocal hy-
brids at full and reduced salinity. This was the necessary first phase
of evaluating the culture potential of hybrid mussels.
423
424
Matson et al.
METHODS
Broodstock
Broodstock mussels were collected from areas known to have
essentially monospecific populations. After preliminary Brood-
stock selection was made based on morphology, molecular meth-
ods were used to positively identify all of the Broodstock in this
study. Penn Cove was chosen for collection of M. trossuhis based
on Brooks 1991. 1996 and 1997. Only mussels several years old
that tit the typical morphology for its species were collected. Hy-
brids often have a shell morphology intermediate to that of the
parent species (Lubet 1984). Large mussels (1.5 inches or longer)
of either species are easier to tell apart than very young ones, so
only larger mussels were collected.
These two species have different morphologies (Brooks 1991.
McDonald & Koehn 1991). The valves of Mytilus trossuhis are
typically narrow and long. The ventral shell margin is usually
concave or straight. The anterior end (the umbo) is gradually bent;
sometimes referred to as "beaked." The periostracum of M. tros-
sulus is typically thin and rubs off near the umbo. In sagittal
section, the ventral shell margin is straight. M. galloprovincialis
has a very broad valve. The ventral margin is often convex. The
umbo appears sharply hooked. M. galloprovincialis ' periostracum
is typically thick and black. In sagittal section, the ventral shell
margin is rolled inward at the joining of the two valves. Mussels
that fit these criteria were chosen for Broodstock. Those that ap-
peared intermediate to these morphotypes were not collected to
avoid hybrid Broodstock.
Molecular Identification
Broodstock mussels were identified using two different types
of nuclear DNA markers (Heath et al. 1995. Rawson et al. 1996).
These diagnostic molecular markers enabled positive discrimina-
tion between the three members and hybrids of the Mytilus com-
plex. Both of these nDNA markers were based on the polymerase
chain reaction and one used restriction fragment length polymor-
phism analysis. The first marker is based on the Glu gene, which
encodes the mussel polyphenolic adhesive protein (Rawson et al.
1996). That protein is key in mussel attachment to the substrate.
The Glu-5' marker enables differentiation between all three spe-
cies in the Mytilus complex; Mytilus trossuhis. M. galloprovincia-
lis. and M. ediilis.
In this study, tissue samples were digested using CTAB isola-
tion buffer (IB) and proteinase k ( 10 mg/niL). CTAB was used to
remove mucopolysaccharides in the bivalve tissue that could co-
extract with the DNA and negatively affect later polymerase chain
reaction (PCR). The CTAB IB (2% w/v CTAB, 1.4 M NaCl, 0.2%
w/v 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris/HCl, pH 7.5)
was preheated at 50''C. Mantle edge tissue was chopped up with a
razor blade and put into 1 .5-inL polypropylene centrifuge tubes
with 10 (J.L of proteinase k and an equal volume of CTAB IB. The
mixture was incubated at 55"C for 3 h in a Rossi agitating incu-
bator, vortexed for 10 s each, and then held at 50°C in a water bath
overnight. In the morning, DNA was extracted from the tissue
digestion with an equal volume of 24:1 chloroform:isoaniyl alco-
hol mixture. The mixture was centrifuged at I 1,500 g in a mi-
crofuge for 10 min. It was necessary to repeat the extraction two
or three times to get a clear supernatant. Two volumes of 100%
ethanol were added and the mixture was held in a -20°C freezer
over night to allow precipitation of the DNA. The next day, the
extraction was centrifuged for 30 min at 1 1 ,500 g. The alcohol was
removed and the pellet was rinsed with 0.5 mL of 70% ethanol.
The pellets were dried in a centrifugal evaporator and then dis-
solved in 100 p.L of TE ( 10 mM Tris/HCl, 1 mM EDTA, pH 7.6).
A "Gene Quant" spectrophotometer (Pharmacia) was used to
quantify DNA stock solutions. The stock solutions were diluted to
make a 100 ng/jji.L working solution for use in PCR and were
stored in refrigerator at 4°C. The stock solutions were frozen at
-20°C for long term storage.
The sequences of the primers used for Glu-5' (Rawson et al.
1996) in this study were: 5'-GTAGGAACAAAGCATGAACCA-
3' (forward) and 5'-GGGGGGATAAGTTTTCTTAGG-3' (re-
verse) The PCR recipe of Rawson et al. (1996) and their thermal
cycler protocol were adapted. The end concentrations of chemicals
in the PCR were 0.8x TBE buffer (20x TBE buffer solution: 121
g/L Tris base, 61.7 g/L boric acid, 7.44 g/L Na2EDTA*2H20),
0.32 dNTPs, 1.5 mM MgCU, 4 |j.M forward primer, 4 |jiM reverse
primer, 4 ng/p-L of DNA template, and 0.04 U/|xL of Taq DNA
polymerase. The total reaction volume was 12.5 p.L, and samples
were amplified in a Techne thermal cycler using a hot-start pro-
tocol. The thermal cycler protocol used for this marker was one
cycle of 94° for 3 min and then 24 cycles of 94° for 20 sec, 53° for
20 sec, and 72° for 45 sec. After PCR. the products were size-
fractionated on 3% agarose TBE gels and stained with SYBR
green (Molecular Probes) for approximately 1 h. They were visu-
alized using a Molecular Dynamics 575 Fluorlmager. The banding
pattern observed for Glu-5' in M. galloprovincialis was one band
of 300 base pairs (bp) and one 500 bp band or just one 300 bp
band. One 240 bp band was observed for M. trossuhis.
The second DNA species marker used was also PCR-based but
was followed by restriction fragment length polymorphism analy-
sis (Heath et al. 1995). This codominant marker was based on
internal transcribed spacer (ITS) regions between the 18S and 28S
nuclear rDNA coding regions. Heath et al. (19951 showed that it
worked very well in distinguishing M. trossuhis from M. gallo-
provincialis or Mytilus edulis. This marker cannot distinguish be-
tween M. galloprovincialis and M. edulis. but because M. edulis is
not yet known to occur in any of the Broodstock collection sites,
or anywhere else in Puget Sound. It was reasonable to use this
marker in conjunction with the Glu-5' marker and morphologic
screening. The sequences of the primers used (Heath et al. 1995)
in this study for the ITS marker were 5'-GTTTCCGTAGGT-
GAACCTG-3' (forward) and S'-CTCGTCTGATCTGAGGTCG-
3' (reverse). The Heath et al. (1995) PCR recipe and thermal cycler
protocol were both optimized for the facility where the work was
performed. The end concentrations of chemicals in the PCR were
Ix buffer, 0.8 mM dNTPs, 1.5 mM MgCU, 0.3 p,M forward
primer, 0.3 p.M reverse primer, 0.5 ng/(xL of DNA template, and
0.05 U/|xL of Taq DNA polymerase. The thermal cycler protocol
used was 94° for 3 min and then 30 cycles of 94° for 20 sec, 50°
for 20 sec, and 72° for 45 sec. The total reaction volume was I6p,l
and a Techne thermal cycler was used. The PCR was hot-started.
After PCR. 1 jjiL of each product was electrophoresed on a 1.5%
agarose gel to check for amplification success. The products were
then cut with Hluil restriction endonuclease overnight. Conditions
for one digestion reaction was 0.04 p,L Hhal enzyme, 1.0 p-L lOx
NEB #4 buffer (50 mM potassium acetate, 20 mM Tris acetate, 10
mM magnesium acetate, 1 mM DTT, pH 7.9 @ 25°C), 0.1 \x.L of
lOOx bovine serum albumin, and 3.5 p.L of sterile double distilled
water, and 5 |jiL of template DNA in TE (100 ng/p-L). The frag-
Laboratory Hybridization of M. trossulus and M. galloprovincialis
425
ments were separated on a S'/r agarose gel. stained with SYBR
green for approximately 1 h and visualized using a Fluorlmager.
Heath et a), reported that in M. ciliilis and M. i^iilloprovincialis.
the 1250-bp PCR product was cut into two 45()-bp fragments and
two 180-bp fragments. In Mytilus trossulus. the 1250-bp product
was cut into two 280-bp fragments, two 1 80-bp fragments, and a
few fragments smaller than 100 bp. In this study, the PCR product
was closer to 1050 bp long in both species. The two species mark-
ers, Glu-5' and ITS both worked well at distinguishing M. trossu-
lus from M. galloprovincialis and from hybrids. All Broodstock
individuals were positively identified using the Glu-5' marker. All
but three males and two females were identified at the ITS locus.
Those few were most likely not identifiable because of sample
degradation. Both banding patterns were seen when equal portions
of DNA from each species were mixed together and amplified
(with either marker). This shows that one species' DNA was not
preferentially amplified over the other's. Hybrid mussels would
show the banding patterns of both of their parents (Rawson et al.
1994). The use of two loci increased the power of detection of
hybrids.
Larval Rearing
Mussels were spawned and reared in 12-L plastic bags at the
Taylor Shellfish Hatchery on Dabob Bay. Washington. It was nec-
essary to condition M. trossulus Broodstock for a few weeks be-
fore spawning was attempted. M. trossulus normally spawn in
March through May in Puget Sound (Johnson 1978). The mussels
were held in tanks at ambient Dabob Bay temperature (10 to I2°C)
and were fed large amounts of algae to encourage the necessary
development of the gonad. Brenko and Calabrese (1969) found
that food was the primary controlling factor for gonad develop-
ment in Baltic mussels, and that rise in water temperature was the
triggering factor for spawning in the natural environment. It was
not necessary to condition the fully ripe M. galloprovincialis
Broodstock. as they were already in spawning condition. They
could not be held in water overnight because they spawned out in
the holding tank before morning when this was attempted. M.
galloprovincialis are typically in spawning condition December
through March in Puget Sound. Up to 100 mussels of each type
were used in spawning attempts for the first experiment to ensure
that enough mussels actually spawned to make the crosses. Only
approximately one fourth of tho.se mussels that were induced,
spawned enough gametes to produce a culture. Approximately .^00
mussels of each species were induced in the spawning attempts
that led to the second experiment. The mussels were induced to
spawn by agitation, followed by heat shock (Loosanoff & Davis
196.^). The mussels were taken from ambient IT or 14°C water,
shaken in buckets for approximately two minutes, and then placed
into spawning trays with flowing seawater. The water temperature
in the trays was changed from as high as 24° to as low as 11 °C
repeatedly. Dense, live algal food was also added occasionally for
periods of about 20 min to encourage spawning.
Once a mussel began spawning, it was immediately removed
from the tray, its sex was identified, the mussel's interior and
exterior was rinsed with seawater, it was placed in a separate clean
dish, and was allowed to spawn further. After that, its interior and
exterior and its dish were rinsed a second time. Then it was al-
lowed to spawn the gametes that would be used in the crosses.
When all of the individuals had spawned, the mussels were re-
moved from the dishes and the gametes were screened. All screens
were cleaned and soaked with hot fresh water between batches of
gametes. Spawning mussels, each in its own dish, were kept sepa-
rated in different areas on different tables by both sex and by
species. Every batch of eggs and sperm were carefully examined
under the microscope for contamination by other gametes. A batch
of eggs or sperm was only used in a cross if it was observed to
have zero signs of development in it, and any contaminated ga-
metes were discarded. Fertilization was confirmed in each culture
under the microscope. After fertilization, the embryos were
screened and rinsed to remove excess sperm. All screens were
cleaned and soaked with hot fresh water between batches of em-
bryos. Two samples of one ml each were taken after fertilization
for early development analyses and later growth measurements.
Sixty-four different mussels were used in all, to produce the 32
pair matings used in the experiment. Eight individuals of each sex
were used to establish each cross (16 parents for each cross). Each
replicate represented one single-pair mating. No replicates shared
either a sire or dam. The four crosses made were M. trossulus x
trossulus (TT). galloprovincialis sperm x trossulus egg (GT). tros-
sulus sperm x galloprovincialis egg (TG). and galloprovincialis x
galloprovincialis (GG). The low-salinity treatment was 20 ppt and
the high salinity was 30 ppt. The embryos were then placed in 32
separate 12-L culture bags, with four replicate bags per cross by
salinity treatment. (See Fig. I for a graphical description of the
experimental design.) The culture bags were hung in a water bath
with a thermostat-controlled immersion heater and circulating
pumps. Culture temperatures were maintained at 18°C. The cul-
tures were covered with shade cloth to prevent algal growth.
The larval density and the algal density of each culture were
both standardized (regulariy made equal between cultures). Larval
density was equalized twice per week (at each water change) to
prevent density-dependent growth or survival. This was performed
by counting the larvae in each culture, and then decreasing the
water volume in all bags until they had the same larval density as
the culture with the highest survival (10 larvae/ml initially, de-
creasing to 3 larvae/mL by day 14). The algal density was equal-
ized once per day, by counting the algae in each culture, and then
feeding a different amount to each culture to maintain the desired
algal density (20,000 cells/mL initially, gradually increased to
80.000 cells/mL at pediveliger).
Larvae were initially fed 20.000 cells/mL of naked flagellates
Mytilus trossulus speim X Mytilus
trossulus egg (TT)
Higli salinity
Low salinity
QQQQ QQQQ
Mytilus trossLilus speim x Mytilus
gaUopimincialis egg (TG)
High salinity
Low salinity
SQQQ 0QQQ
Mytilus galloprovincialis speim X
Mytilus galloprmincialis egg (GG)
High salinity
Low salinity
QQ00 QQQQ
MytHus galloprovincialis sperm X
Mytilus trossulus egg (GT)
High salinity
Low salinity
QQQQ QQQQ
Figure 1. Two-factor experimental design used in examining survival
and growth in larvae of M. trossulus, M. galloprovincialis. and their
reciprocal hybrids at high and low salinities (.'2 ppt and 2(1 ppl. re-
spectively). The i2 larval cultures were produced by 32 separate-pair
matings. Eight cultures were used in each of four crosses, and four
cultures were used lor each salinity level within each cross. For ex-
ample, the M. trossulus sperm by M. galloprovincialis egg cross in-
cluded four cultures at high salinity and four at low salinity.
426
Matson et al.
(Isochrysis sp. Tahitian isolate). Algal cell concentration was de-
termined using a Coulter Counter model ZBI. On the day follow-
ing fertilization, the cultures were fed a mixture of flagellates and
diatoms {.Tahitian Isochnsis. Chaetoceros calcitrans. Thalassio-
sira pseudonana [University of Washington 3H clone], and Ske-
letonema [species unidentified]). A mixture of two algal species
supported faster growth than one alone, according to Bayne
(1965), when he fed Isochiysis galhana and Monochiysis liitheri
together. The amount of food given increased incrementally to a
maximum of 80,000 cells/mL at the pediveliger stage. The density
of larvae and algae was kept equal between cultures to reduce the
possible influence on larval growth rate due to crowding. Sampling
for survival was done twice per week at each water change. Each
culture was condensed to 100 mL and one count was taken. The
variability between counts was kept below 5% (tested beforehand)
by condensing the culture and using a paddle stirrer. Cultures were
resuspended in one liter between counting and bag refilling. Bags
were cleaned with bleach, sodium thiosulfate. and rinsed with hot
water at each water change. Bag water volumes were then adjusted
to equalize larval density and larvae were resuspended in their
bags. Two samples of 1 mL each were taken after fertilization for
early development analyses and later growth measurements. Esti-
mates of the proportions of larvae at each developmental stage
present in the cultures were made from those samples as well. A
total of 200 larvae were counted from each sample, and the number
of larvae at each developmental stage was noted. Fifty larvae were
chosen randomly and the distance from umbo to lip (shell length)
of each was measured in microns using a compound light micro-
scope and ocular micrometer.
Figure 2. Early development of hybrid and pure specie.s Mytilus lar-
vae. Larvae of the TT cross developed significantly faster than those of
the GT hybrid cross iP = 0.007). TT. M. trossulus; GT. M. gallopro-
vincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. gal-
loprovincialis egg; GG, M. galloprovincialis. Bars represent the mean
transformed proportion of the larvae that were at or beyond the
blastula stage after 12 h at 16 C at a salinity of 30 ppt.
growth in the low salinity treatment than in the high salinity treat-
ment, while the GG cross did not. It had a slightly higher mean
growth in the high salinity treatment than in the low salinity treat-
ment.
Survival
RESULTS
Early Development
Early development was measured as the proportion of embryos
that had developed to the blastula stage or beyond, at 12 h post-
fertilization. This proportion was arcsine transformed to conform
to the normality and homogeneity of variance assumptions of the
analysis of covariance. It was also adjusted for egg density by
using egg density as a covariate. The regressions for the covariate
were significant for developmental success (proportion of blastu-
las; P < 0.0001 ). The slopes of the lines for the different crosses
were equal for developmental success (NS cross by egg density
interaction). Cross (P = 0.045) was a significant factor. The mean
development of the TT cross was significantly higher than that of
the TG cross" mean (P = 0.007. Fig. 2). No other differences were
significant.
Growth
At day 3. the mean length of the GG cross was significantly
higher than those GT and TT crosses (P = 0.026 and < 0.001.
respectively). The TG cross's mean length was significantly higher
than the TT cross's mean length also (P = 0.001. Fig. 3). These
results are similar to an earlier experiment performed with the
same crosses (Matson 2000). The low salinity treatment was ap-
plied at day 3. No significant differences exi.sted in growth be-
tween crosses or salinities froiu day 3 to 7.
Salinity was a highly significant factor affecting growth be-
tween day 3 and 14 (P < 0.001, Fig. 4, Table 1). Cross was not a
significant factor from day 3 to 14 (P = 0.256). The three crosses
with a M. trossulus component (TT. GT, and TG) had higher mean
Cross was a significant factor {P = 0.011) affecting day 3
survival. The TT cross's mean survival was significantly higher
than those of the GT, TG, and GG cross's mean survival (P =
0.042. 0.026. and 0.020 respectively. Fig. 5). No significant dif-
ferences existed in survival between crosses or salinities from day
3 to 7. or from day 3 to 14, though there was an interesting pattern
in the means. Each hybrid cross survived most like its sire (TG
cross survived better at low salinity, GT cross survived better at
high salinity).
1
UJO 1
"5
■1?
=3
sn -
fin -
T
i
It
-tn -
A
.-.t.
E^•
c
*7
3n -
■ '-I
??
n
1_J
TT GT TG GG
Cross
Figure 3. Shell length of hybrid and pure species Mytilus larvae at day
3. At day 3. the mean length of the GG cross was significantly greater
than those GT and TT crosses (P = 0.026 and <0.001, respectively). TT,
M. trossulus; GT, M. galloprovincialis sperm x M. trossulus egg; TG,
M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis.
Cultures were maintained at 18 C in 30 ppt seawater.
Laboratory- Hybridization of M. trossulus and M. galloprovinciaus
All
GLov?(^2Dppti
■ Kigb(30ppt.i
Figure 4. Change in shell length of hybrid and pure species Mytilus
larvae from day 3 lo day 14 at two salinities (20 ppt and 30 ppt).
Salinity was a significant factor affecting growth of the larvae (P <
O.OOI ). Larvae of the TT. (JT, and TG crosses grew more than at low
salinity than at high salinity. Cultures were maintained at 18°C. TT,
M. trossulus; GT, M. galloprovincialis sperm x M. trossulus egg; TG,
M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis.
Figure 5. Survival of hybrid and pure species Mytilus larvae at day 3.
The mean survial of the TT cross was significantly higher than the
mean survival of the GT, TG, and GG crosses {P = 0.042, 0.025, and
0.020, respectively). TT, M. trossulus: GT, M. galloprovincialis sperm x
A/, trossulus egg; TG, M. trossulus sperm x A/, galloprovincialis egg;
GG, A/, galloprovincialis. Cultures were maintained at 18°C in 30 ppt
seawater.
DISCUSSION
Barriers to Hybridization
Hybrid larvae were produced in both species-egg combinations
and larvae were successfully reared through settlement. The suc-
cessful fertilization, growth, and survival of these hybrids suggest
that some factor other than genetic incompatibility is responsible
for the rarity of these hybrids in Puget Sound. One such factor
could be the limited overlap of the two species' spawning periods
in Puget Sound. This would be an example of a partial temporal
barrier to hybridization. Both M. trossulus and M. galloprovincia-
lis. have one peak or mass-spawning time per year and one or more
periods when a much smaller proportion of each species spawns
(trickle-spawning). Mass spawning of M. galloprovincialis occurs
in the late-winter through early spring in Totten Inlet (Dr. Jonathan
Davis, personal communication). Brooks (1991) found ripe M.
galloprovincialis during November and December of 1988 though
1990 in Puget Sound. M. trossulus that were examined at the same
time had gonads that were still in the resting stage with little
gamete formation. M. trossulus typically mass-spawn in March or
April in Holmes Harbor in Puget Sound (Johnson 1978). These
observations support M. galloprovincialis being primarily a win-
ter-spawner and M. trossulus being a primarily spring-spawner in
Puget Sound, and thus also support the theory of a partial temporal
barrier to hybridization. The comparatively low abundance of M.
galloprovincialis in the region (Suchanek et al. 1996) may interact
with or exacerbate the effects of a temporal barrier. There would
likely be fewer opportunities for hybrids to be formed if the two
species spawning times are different and if one of the species was
present in much lower numbers than the other. Mytilus gallopro-
vincialis, in this case, occurs in Puget Sound at much lower abun-
dance than M. trossulus (Suchanek et al. 1996, Brooks 1991),
probably due to M. galloprovincialis' preference for high, stable
TABLE 1.
Two-factor analysis of variable table for growth (change in shell length from day 3 to 14) of the four crosses of Mytilus pure-species and
hybrid larvae at high and low salinities.
Dependent
Variable
LENGTH Difference in
Microns Day
3 to 14
Type III Sum
Mean
Eta
Noncent.
Observed
of Squares
df
Square
F
Sig.
Squared
Parameter
Power
Source corrected model
14626.461
7
2089.494
4.539
0.003
0.58
31.771
0.965
Intercept
284579.457
1
284579.5
618.1
0
0.964
618.145
1
Cross
1993.008
3
664.336
1.443
0.256
0.158
4.329
0.33
Salinity
8030.93
1
8030.93
17.44
0
0.431
17.444
0.979
Cross* salinity
3901.449
3
1300.483
2.825
0.061
0.269
8.474
0.6
Error
10588.661
23
460.377
Total
315391.818
31
Corrected total
25215.122
30
TT, M. trossulus: GT. M. galloprovincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis.
Salinity was a significant factor affecting growth of the larvae (P < 0.001).
Computed using alpha = 0.05
R squared = 0.580 (Adjusted R squared = 0.452)
428
Matson et al.
salinity bays and M. trossiiliis' preference for low temperature,
variable salinity bays (Margus 1991. Sarver & Foltz 1993, Hilbish
1994. Geller 1994. Hoffman & Somero 1995. 1996).
There are many examples of barriers to hybridization among
mollusk and echinoderm species, and some of these are temporal
in nature. A temporal barrier to hybridization is believed re-
sponsible for the lack of natural hybridization of the sea stars
Leptasterias polaris and Asterias vulgaris (Hamel & Mercier
1994). The two sympatric species were shown to hybridize readily
in the laboratory, although they do not in the wild, due to their
distinct breeding seasons. Another example of a temporal barrier
to hybridization is found in Montastraea corals. Szmant (1997)
found that the coral, Montastraea faveolata consistently spawned
I to 1.5 h before M. franksi and M. annularis in experi-
ments. These species have very specific spawning periods that
are both seasonal and closely related to lunar cycles. Szmant dis-
cussed this as a potential temporal barrier to fertilization and hy-
bridization in these species because there was no inherent pre-
zygotic barrier to cross-fertilization among three species he stud-
ied.
Other examples of barriers to hybridization include genetic,
ecological, geographic, and physiologic barriers. In the hard clam,
Mercenaria mercenaria x M. campechiensis hybrids were shown
to have an excessive susceptibility to gonadal neoplasia, relative
to either parental species (Bert et al. 1993). This cellular disease
acts as a barrier to hybridization by decreasing fitness of the hy-
brids relative to the parental species. Genetic barriers to hybrid-
ization have been shown in oyster species. Allen et al. (1993)
attempted hybridization between Crassostrea gigas and C. vir-
ginica, as well as between C. rivularis and C virginica. They
found that larvae survived only 8 to 10 d and grew little; therefore,
the hybrids were considered genetically inviable. Allen and
Gaffney (1993) found that C. gigas and C. rivularis yielded viable
hybrids when crossed in the laboratory. Crassostrea gigas and C.
sikamea have also been hybridized, although they were only suc-
cessful in one direction (Dr. Anja Robinson, pers. comm.).
An unusual physiologic barrier to hybridization is seen in the
interaction of the urchins, A. vulgaris and S. droebachiensis ga-
metes. Although shown to be physiologically able to hybridize
with L. polaris (which has a different spawning period), the eggs
of A. vulgaris were observed to disable heterospecific sperm from
S. droebachiensis (believed to have an overlapping spawning pe-
riod) within 12 sec (Hamel & Mercier 1994). It is thought that a
very diffusible substance involved in the phenomenon is secreted
only from mature eggs that appears to disable sperm in the direct
vicinity of an egg.
Salinity, Growth, and Sunival
A differential species response to salinity was observed in this
study. From day three to 14, the TT, GT, and TG crosses all
grew much faster at low salinity, and the GG cross grew slightly
faster at high salinity (Fig. 3). These differences agree with the
two species' physiologic and ecological characteristics (Kautsky
1987, Margus 1991, Hoffman & Somero 1995, 1996, Sarver
& Foltz 1993, Geller etal. 1994, Johnson 1978, Brooks 1991), and
are likely to be inherited genetic differences. Mytilus tros-
sulus has been shown to be a low, variable salinity mussel
and M. galloprovincialis has been shown to be a high, constant
salinity mussel (Brenko et al. 1977, His 1989, Margus 1991,
Sarver & Foltz 1993, Hilbish 1994, Hoffman & Somero
1996). These data suggest that the hybrids inherited the ability
to grow well in low salinity water from their M trossulus pa-
rent.
Significant cross-dependent differences in survival existed
only during the first week, where the TT cross survived better
than the GT, TG, and GG crosses (Fig. 5). There were no sig-
nificant between-cross differences in larval survival after the
first week. These data are in agreement with a previous experiment
by Matson (2000). Hybrid larvae of both reciprocal crosses
survived through settlement and as juveniles. No significant
survival differences existed between crosses or salinities from
day 3 to 7, or from day 3 to 14, though there was an interesting
pattern in the means by day 14. Each hybrid cross survived most
like its sire (TG and TT crosses survived better at low salinity,
GT and GG crosses survived better at high salinity). It may be
worth examining this more closely, perhaps with greater replica-
tion (more than 4x per cross at each salinity, more than /; = 32
total) to see if salinity tolerance/preference may be paternally in-
herited.
Hybrid Vigor
Although significant cross-dependent differences were found
in early growth, survival, and eariy development, most of them
seem to have been due to factors other than the phenomenon
of hybrid vigor. Hybrid vigor was defined here as an increase
in fitness of the hybrids over either of the parental crosses, exhib-
ited in either growth or survival. Hybrids were not consistently
larger than parental crosses, nor did one reciprocal consistently
grow faster than the other. These findings concur with a previous
between-cross experiment (Matson 2000). The hybrid cross using
M. galloprovincialis eggs generally grew larger than its recipro-
cal, which used M. trossulus eggs, during the first week. This
may have been because of maternally dependent conditioning ef-
fects, or species-specific temperature effects that were also
maternally dependent. Early cross-dependent larval growth was
probably also influenced by maternal effects (Lannan et al.
1980). These maternal effects may have been the result of
species- or population-dependent differences in egg nutrition
(Bayne 1978), or differences in egg condition (Lannan et al. 1980),
reflected by the different peak spawning times of each species
in Puget Sound (Johnson 1978, Brooks 1991). When spawned,
the M. galloprovincialis mussels were at the end of their spawning
season, and the M. trossulus mussels were almost at their peak.
This observation concurs with previous seasonal examinations
of these two species gonadal condition (Johnson 1978, Brooks
1991).
These results are similar to those of Beaumont et al. 1993 (in
terms of lack of hybrid vigor), who hybridized M. galloprovincia-
lis with M. edulis. Beaumont et al. (1993) found that after initially
higher mortality, veliger larvae of both reciprocal hybrid crosses
grew as fast (Trial Three) or significantly faster than (Trial One)
M. galloprovincialis larvae in one trial, but not the other. Hybrid
crosses didn't grow consistently faster than parental crosses. Lubet
( 1984), who created hybrid M. galloprovincialis x M. edulis mus-
sels and examined their juveniles and adults in the field, concluded
that those two species are closely related, and exhibit minimal
barriers to hybridization as well as minimal fitness differences
between hybrids and parentals.
Laboratory Hybridization of M. tkossulus and M. gallofrovincialis
429
ACKNOWLEDGMENTS
Thanks to Paul Bentzen, Ginger Arbrust. Patrick, and Pam
Jensen from the University ot Washington (UW) Marine Molecu-
lar Biotechnology Laboratory; thanks to Hal Beattie and Amily
Caffe at the Washington Department of Fish and Wildlife. Pt.
Whitney Shellfish Laboratory, and thanks to William Hershberger
from UW. Special thanks to everyone at the Taylor Shellfish
Hatchery. Quilcene. WA. This project was funded by the Victor
and Tamara Loosanoff Endowed Fellowship and the Research and
Scholarship Committee at the University of Washington School of
Fisheries.
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RIBOSOMAL RNA CHARACTERIZATION OF NON-TRANSCRIBED SPACER AND TWO
INTERNAL TRANSCRIBED SPACERS WITH 5.8S RIBOSOMAL RNA OF PERKINSUS SP.
FOUND IN UNDULATED SURF CLAMS (PAPHIA UNDULATA) FROM THAILAND
SUFANNEE LEETHOCHAVALIT,'* E. SUCHART UFATHAM," KWANG-SIK CHOI,'
FICHAN SAWANGWONG/ KASHANE CHALERMWAT/ MALEEYA KRUATRACHUE^
'institute of Marine Science. Bunipha University. Bangsaen. Clwnhiiri 20131. Tliailcmd: 'Faculty of
Science. Department of Biology. Burapha University. Bangsaen. Chonburi. 201 3 f Tliailanil: ^Faculty of
Applied Marine Science, College of Ocean Science. Cheju National University. 1 Ara 1-Dong Jeju City
Jeju-Do 690-76-56. S. Korea: ^Faculty of Science. Department of Aquatic Science. Burapha University.
Bangsaen. Chonburi 20131. Thailand: ^Faculty of Science. Department of Biology. Mahidol University,
Rama 6 road. Payathai. Bangkok. 10400. Thailand
ABSTRACT The genetic divergence of Perkinsiis sp. loLind in the undulated surf clam i.Puphiu iimliilala) from the Gulf of Thailand
and other known Perkiiisiis species was exammed using the non-transcribed spacer and two internal transcribed spacers with 5.S S
rRNA gene. The sequences of non-transcnbed spacer (NTS) and internal transcribed spacer region (ITS) that includes the 5.8S rRNA
gene flanked by ITS I and ITS2 (ITSI-5.8S-ITS2) were cloned and sequenced. The sequences were compared with those of Perkin.sus
olseni from Australia. P. allimiiciis from Korea. P. marimis and P. aiidrewsi from the United States and P. qugwadi from Canada. The
lengths of the obtained nucleotide sequences of NTS. ITS-1 5.85 rRNA and ITS-2 were 1,167, 183, 159, and 371 bp, respectively. The
nucleotide sequences of NTS and ITS-5.8S rRNA of Thai Perkiiuus and P. olseni showed 98.69% and 99.85% identity, respectively.
When compared with P. allanticus identities were 96.27 and 99.71%, in P. marinus 75.38 and 94.88% and in P. andrewsi 46.55 and
86.23%. The nucleotide sequences of ITS-5.8S rRNA between Thai Perkinsiis and P. chesapeaki showed an identity of 87.05%. This
is the first report of the occurrence of Perkinsiis sp in the Gulf of Thailand.
KEY WORDS: Perkinsiis sp.. Pupliia iindutata. nucleotide sequence, non-transcribed spacer, internal transcribed spacerl, internal
transcribed spacer 2
INTRODUCTION
The pathogenic protozoans. Perkinsiis spp. causes Perkinsosis
disease in marine bivalves (Andrews 1988). According to Perkins
(1976) and Levine (1978). they were classified as an apicom-
plexan. However, recent molecular phylogenetic analyses by Sid-
dall et al. (2001) and Recce et al. (1997) have placed these para-
sites within the Dinoflagellata. Traditionally, diagnosis of Perkin-
sus infection depends on the fluid thioglycollate medium (FTM)
assay for identification and Choi's 2 M NaOH digestion technique
on FTM cultivated tissues for quantification (Choi et al. 1989,
Almeida et al. 1999). However, the FTM assay does not discrimi-
nate different Perkinsiis species and has a potential to introduce
misleading positive results between Perkinsiis and other di-
noflagellate species (Almeida et al. 1999). A more precise appli-
cation for detection, identification, and numeration of these para-
sites is based on molecular characterization. The internal tran-
scribed spacers (ITS), 5.8 S regions of the ribosomal RNA (rRNA)
and non-transcribed spacer gene (NTS) can be used to discriminate
among the Perkinsiis species because these regions are largely
non-coding with high evolutionary rate, and have been used to
identify Perkinsiis species isolated from different hosts and geo-
graphical regions (Kotob et al. 1999. Robledo ct al. 1999, Robledo
et al. 2000). These NTS and ITS regions have also been used to
distinguish between strains and species of other protozoa (Cai et al.
1992, Goggin 1994, Cunningham 1997). We have identified Per-
kinsiis in the undulated surf clam, Paphia lunliihila, a major com-
mercial species from the Gulf of Thailand using FTM assay. In this
study, we have characterized the Thai Perkinsiis ribosomal RNA,
the nucleotide sequences of ITS-5.8S rRNA, and NTS and com-
*Corresponding author. E-mail: sp02l7@yahoo.com; Fax: -1-66-38-391674
pared the sequences with rRNA sequences that have been reported
for other known Perkinsiis species.
MATERIALS AND METHODS
Isolation of Prezoosporangia
Live specimens of the undulated surf clam {Paphia iindidata)
were obtained from food markets in Chonburi Province. Thailand.
The infected gills of clams were cultured in fluid thioglycollate
medium supplemented with streptomycin (500 |j.g/ml) and peni-
cillin G potassium (500 unit/ml) at 27°C in the dark for 3 days. The
tissue was then digested by trypsin (0.25% in sterilized seawater)
at rootn temperature for .3-4 h. and the obtained prezoosporangia
were then isolated by filtration through a silk net. The resulting
pellets were finally washed 3 times using sterilized seawater and at
each washing the pellets were cenlrifuged at x490,i? for 8 min.
DNA Isolation
Genomic DNA was extracted from prezoosporangia using a
DNA trap kit, according to details provided by the manufacturer
(Tissue Protocols for DNA isolation, DNA TEC, Thailand).
PCR Amplification
The complete region of ITS1-5.8S-ITS2 and NTS genes were
amplified from genomic DNA using a forward primer of the small
subunit (SSU) 5'AGGAAGGAGAAGTCGTAACAAGG 3'
(Hamaguchi et al. 1998) and a reverse primer of the large subunit
(LSU) 5'ACCCGCTGAATTTAAGCATA 3' (Goggin 1994). The
NTS region was amplified by using a forward primer 5' AAGTC-
CTTAGGGTGCTGCTGGCT 3' and reverse primer 5' CTACTG-
GCAGGATCAACCAGGT 3' (Park et al. 2002). The polymera.se
431
432
Leethochavalit et al.
chain reactions were performed in a final volume of 20 |xL reac-
tion mixture containing 2 jxL of 10 x reaction buffer (50 mM KCL;
10 mM Tris-HCL, pH 8.3: 1.5 mM MgCK). 2 jxL of 1 mM de-
oxyribonucleoside-5'-triphosphate (dNTPs)(Proniega Corp.,
Madison, WI), 1 (jlI of 5 |jlM upstream primer, and 1 (jlL of 5 jjlM
downstream primer, 0.8 p.L of 5 units Taq DNA polymerase
(Promega Corp.) and 4 |xL of 5 ng/fil of DNA template. The final
volume was adjusted with sterilized distilled water to 20 (xL. The
mixture was amplified in a PCR Thermal Cycler (GeneAmp®PCR
system9700, Albertville, MN) for 35 cycles with initial denatur-
ation at 94°C for 3 min. The process was followed by 35 cycles of
denaturation at 94°C for 30 sec and annealing at 55°C for 30 sec.
The primer extension was performed at 72°C for 1 min followed
by a final extension at 72°C for 5 min. The PCR products were
analyzed utilizing 1% agarose gel electrophoresis and visualized
for DNA bands under UV light and photographed using digital
photography (Delidow et al. 1993, Hamaguchi et al. 1998, Carne-
gie et al. 2000).
DNA Cloning and Sequencing
The PCR products of the ITSI-5.8S rRNA- ITS2 and NTS
fragments were excised from the agarose gel and purified by a
cleaning reagent consisting of exonuclease I (Exo I) and shrimp
alkaline phosphatase (SAP). The NTS and ITS-5.8S fragments
were cloned into pGEM™-T Easy vector (Promega Corp.) and
isolated. At least one clone of each NTS and ITS-5.8S fragment
was sequenced following standard procedures in an automatic
DNA sequencer, ABI PRISM model 377 (Dicker et al. 1993,
Hamaguchi et al. 1998). The nucleotide sequences of NTS were
analyzed for nucleotide similarities with P. olseni (GenBank ac-
cession number AF466527), P. atlanticus (AF438150), P. marimts
(AF497479), P. andrewsi (AF102171). The nucleotide sequences
of ITS1-5.8S-ITS2 were analyzed for nucleotide similarities with
P. mariniis (AF497479), P. andrewsi (AFI02171), P. atlanticus
(AF473840), P. olseni (U07701), P. qiigwadi (AFI51528), and P.
chesapeaki (AF09154I) by BLAST and CLUSTALW programs
provided by GenBank and the European Bioinformatics Institute.
RESULTS
From this study, the sequences of the NTS. ITS-1, ITS-2, and
5.8S rRNA fragments PCR amplified from prezoosporangias of
Thai Perkinsiis found in P. imdulata were 1 167,183, 371, and 159
bp in length, respectively. These sequences were submitted to
GenBank and given an accession number (AF522321 ). The nucle-
otide sequences of NTS from Thai Perkinsiis were compared with
the completed sequences of P. olseni isolate P 01 (Murrell el al.
unpublished data). P. atlanticus (Park et al. 2002), P. mahnus
isolate TXsc (Robledo et al. 1999), and P. andrewsi (Coss et al.
2001). The sequence similarity between the NTS region of Thai
Perkinsus and P. olseni. P. atlanticus. P. marimis. and P. andrewsi
were 98.69%, 96.27%, 75,38%, and 46.55%, respectively (Table I ).
To determine the ITS with 5.8S rRNA sequence similarities of
the Thai Perkinsus. we compared the complete sequences of this
species with completed sequences of P. marimts isolate TXsc
(Robledo et al. 1999), P. andrewsi (Coss et al. 2001), P. atlanticus
(Park et al., in press), P. olseni (Goggin 1994), P. qugwadi (Hervio
et al.. unpubl. data), and P. chesapeaki (Kotob et al. 1999). The
ITS-5.8S rRNA sequences of Thai Perkinsus was 94.887r similar
to P. marinus isolate TXsc, 88.34% similar to P. andrewsi, 99.71%
similar to P. atlanticus. 99.85% similar to P. olseni, 68.02% simi-
lar to P. qugwadi, and 87.05% similar to P. chesapeaki (Table I ).
The sequence of 5.8S rRNA of Thai Perkinsus showed 100%
similarity to P. olseni, P. atlanticus, and P. marinus.
DISCUSSION
Several species of Perkinsus have been reported from different
locations in the worid including Australia (Goggin 1994). China
(Liang et al. 2001 ). Japan (Blackbourn et al. 1998, Hamaguchi et
al. 1998, Choi et al. 2002). Korea (Park & Choi 2001), New
Zealand (Goggin 1994), Portugal (Azevedo 1989) and USA
(Mackin et al. 1950). There has been no report of Perkinsus sp. in
any species of shellfish in Thailand and no species of shellfish in
Thailand has been reported to exhibit symptoms of Perkinsosis
diseases. However, additional research in this area may reveal
otherwise.
In our study, we targeted and analyzed the NTS. ITS-1, ITS-2,
and 5.8S rRNA genes for species-specificity of Thai Perkinsus
found in P. undulata. The results showed that the NTS region of
Thai Perkinsus is slightly different from that of P. olseni (1.31%)
and P. atlanticus (3.73%) but highly different to P. marimis
(24.62%) and P. andrewsi (53.45%). As proposed by Coss et al.
(2001 ). this implies that the NTS region of P. andrewsi is dramati-
cally different in both length and sequence from those of P. mari-
nus and P. atlanticus. Robledo et al. (1999) concluded that the
NTS region can accumulate a high degree of sequence variability
between closely related species. The sequence of 5.8S rRNA of
Thai Perkinsus showed 100% similarity to P. olseni. P. atlanticus.
TABLE 1.
The length and sequence similarity ( % ) of non-transcribed spacer, internal transcribed spacerl, 5.8S ribosomal RNA and internal
transcribed spacer2 of Thai and other Perkinsus species.
Length
NTS
Length
ITS-1
Length
5.8S rRNA
Length
ITS-2
Accession
Organism
(bp)
(^similarity)
(bp)
(% similarity)
(bp)
(% similarity)
(bp)
(% similarity)
No.
Thai Perkinsus
1.167
100
183
lUO
159
100
371
100
AF522321
P. marinus
KL-ig
75.38
197
85.27
161
100
372
93.27
AF497479
P. andrewsi
1,551
46.55
185
79.45
159
98.74
368
82.60
AF102171
P. atlanticus
—
—
183
99.45
159
100
371
99.73
AF473840
P. atlanticus
1.146
96.27
—
—
—
—
—
—
AF438150
P. olseni
—
—
183
99.45
159
100
371
100
U07701
P. olseni
1,153
98,69
—
—
—
—
—
—
AF466527
P. qugwadi
—
—
204
47.05
158
93.63
363
63.08
AF102171
P. chesapeaki
—
—
18S
87.76
159
96.22
379
82.45
AF091541
Characterization of Spacers in Thai Perkinsus
433
and P. marinus. As reported by Goggin (1994). the 5.8S rRNA
sequence regions from P. olseni. P. atlanticiis. P. marinus. and
unidentified Perkinsus (from/4, trapezia and C. pacificus) were ail
identical. However, the 5.8S rRNA sequence of Thai Perkinsus
differs at 2 positions when compared with P. andrewsi and 10
positions when compared with P. ijugwudi. Coss et al. (2001 )
reported that 5.8S rRNA of P. andrewsi differed from 5 isolates of
Perkinsus spp. reported by Goggin (1994) in 2 positions but dif-
fered from P. qujiwadi in 14 positions. Murrell et al. (2002) re-
cently updated the phylogenetic position of the genus Perkinsus
and considers P. olseni and P. aikinticus to be synonyms.
Our results show high levels of sequence homology in 1TS-5.8S
rRNA region among Thai Perkinsus. P. olseni. and P. allanticus.
The nucleotide sequences of ITS-5.8S rRNA in Thai Perkinsus
were highly similar to P. olseni (99.85%) and P. atlanticus
(99.7 IVr). In this region. Thai Perkinsus differs from P. olseni at
1 position in ITS-1 but differs from P. atlanticus at 2 positions in
both ITS-1 and ITS-2. Goggin (1994) found that P. olseni from
Australia and P. atlanticus from Portugal had an identical se-
quence for ITS I but differed in ITS-2 at .^ positions by substitution
of one nucleotide and he suggested that these two species belong
to a single species. From our study the Thai Perkinsus is most
closely related to P. olseni and P. atlanticus. At the same time, the
Thai Perkinsus showed genetic divergence at the ITS-5.8S rRNA
region from P. marinus. P. andrewsi. and P. cjui^wadi. The nucle-
otide sequence of Thai Perkinsus ITS-5.8S rRNA showed 94.88%
homology to P. marinus. 86.23% homology to P. andrewsi, and
68.02% homology to P. qugwadi. At this fragment, the sequences
of Thai Perkinsus versus those of P. marinus and Thai Perkinsus
versus P. andrewsi were more different in ITS-1 (14.73% and
20.55%) than ITS-2 (6.73% and 17.40%). Goggin (1994) also
reported that the sequences of ITS-1 and ITS-2 of P. marinus from
American oysters differed significantly from P. olseni. P. atlanti-
cus. and an unidentified Perkinsus from Anadara trapezia and
Chama pacificus. Furthermore, he found that the variation among
4 isolates of Perkinsus and P. marinus was greater in the ITS- 1
(23%) than the ITS-2 (7-8%) region. Goggin (1994) concluded that
12% differences of nucleotide deletions were most common in the
ITS-1. Our study shows that the sequences of lTS-1 and lTS-2 in
Thai Perkinsus and P. qugwadi were substantially different. Coss
et al. (2001) also found genetic divergence from ITS-1 and ITS-2
regions, between P. andrewsi and P. qugwadi and suggested that
P. qugwadi is not closely related to the other Perkinsus species.
In conclusion, molecular evidence of the ribosomal RNA from
Perkinsus found in Paphia undulata from the Gulf of Thailand
shows that it is distinctly different from P. marinus. P. andrewsi.
and P. qugwadi. Although homology of NTS, ITS-1, 5.8S rRNA,
and ITS-2 sequence in Thai Perkinsus with P. olseni and P. at-
lanticus are high, the level of homology required to discriminate
between species of Perkinsus have not been determined (Goggin
1994). Therefore, we do not specify a species-specific name for
Perkinsus sp. found in Paphia undulata from the Gulf of Thailand
at this point in time.
ACKNOWLEDGMENTS
The authors thank the Institute of Marine Science for use of
facilities and laboratory space. Partial research funding was pro-
vided by the Graduate Program in Biological Science, Graduate
School and Faculty of Science, Burapha University. The Shellfish
Aquaculture and Research Laboratory, Faculty of Applied Marine
Science. College of Ocean Science. Cheju National University
provided funds for travel and research in Korea. We also thank Dr
Wansuk Senanan for reading and commenting on the manuscript.
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Journal of Shellfish Research, Vol. 22, No. 2, 435^M1, 2003.
A STUDY OF GONADAL DEVELOPMENT IN RUDITAPES DECUSSATUS (L.) (MOLLUSC A,
BIVALVIA), USING IMAGE ANALYSIS TECHNIQUES: INFLUENCE OF FOOD RATION AND
ENERGY BALANCE
M. DELGADO AND A. PEREZ CAMACHO*
Instituto Espauol cle Occauografia. Miiellc de Animas.
s/n. E- 15001 A Conma. Spain
ABSTRACT This study evaluated the inlluence of food availability on sexual maturation ui RiiclinqH-x ilecussaUis (L.) in conditions
of positive (daily rations of 0.10, 0.24, 0.42. and 0.96<;f). zero (O.OS'/f ration), and negative energy balance (0.025% ration). The
percentages correspond to the organic weight of the phytoplankton supplied as a proportion of the live weight of the clams. The gonadal
occupation index (GOI) and the percentage of ripe oocytes in the gonad, calculated using image analysis techniques, were taken as
indicators of the degree of sexual maturity. Gonadal development in R. decussauis occurred under all food rations and energy balance
conditions, even when the organic weight of the clams decreased during the period of sexual development. All conditions registered
a gradual increase in GOI and the percentage of ripe oocytes throughout the experimental period. Maximum values for GOI varied
between 30% and 40% in females and between 55% and 75% in males, according to the amount of food available. Similarly, mature
sexual cells were observed under all experimental conditions, with maximum percentages in females of between 30% and 40%. The
extent of gonadal development is directly related to the amount of food available, which in turn has a direct bearing on the rate of
gonadal development, with smaller rations leading to a lower rate of increase in the gonadal occupation index and the percentage of
ripe oocytes.
KEY WORDS: food ax'ailability. gonadal development, image analysis. Rudilapes decitssams
INTRODUCTION
The majority of studies of the reproductive cycle of R. decus-
satus in its natural habitat (Perez-Camacho I9S0. Beninger 1982,
Shaffee & Daouidi 1993. Villalba et al. 1993). are based either on
indirect indicators of gonadal development (condition index, go-
nadosomatic index, flesh weight), the discharge of gametes, smear
techniques (Berthou et al. 1980), or histologic studies of the gonad
that describe the various stages of gametogenesis (Holland &
Chew 1974).
A more objective determination of the degree of maturity is
provided by methods that measure the area occupied by sexual
cells and the frequency distribution of oocyte sizes. As a result, it
has been possible to produce more accurate inter- and intraspecies
comparative analyses of several bivalve species (Navarro et al.
1989, Xie & Burnell 1994, Lai^elle et al. 1994, Rodri'guez-
Moscoso & Amaiz 1998). However, the data on bivalve reproduc-
tive histology provided by image analysis are more accurate and
precise than that obtained by the traditional stereological method
in which an ocular graticule is used (Lowe et al. 1982).
Temperature is one of the main factors influencing the game-
togenic cycle in bivalves (Sastry 1975, Mann 1979). It would
appear to define both the starting point and the rate of gonadal
development, whereas diet appears to have a direct effect on the
duration of gametogenesis (Lubet 1980-1981). The above-
mentioned studies, however, tend to support the involvement of
several environmental parameters on sexual activity in bivalves.
The reproductive phenomenon is studied in the natural habitat.
making it difficult to separate the particular effect of one factor
from those of the others. In fact, there are very few studies of the
individual infiuence of each environmental variable on the repro-
ductive process under controlled conditions (Sastry 1966. Gima-
zane 1972, Bayne et al. 1975, 1978. Pipe 1985). The use of image-
analysis techniques to determine the effects of a single environ-
mental variable, in this case food availability, on gonadal
development in R. decussanis is the main aim of this study.
MATERIALS AND METHODS
Breeding Stock
*Corresponding author. E-mail: alejandro.perez@co.ieo.es
The experiments were performed in two years running, using
clams of two sizes. In the first experiment specimens of R. decus-
sanis with a length of 20.8 ±0.15 mm (mean plus standard devia-
tion) and a live weight of 1.60 ±0.31 g were used. In the second
experiment, average clam length was 36 ± 0.19 mm and live
weight 9.97 ± 1.53 g.
Experimental Design and Conditions
The experiments were performed in a flow-through system
containing seawater filtered through a p.m cartridge and main-
tained at a constant temperature (18°C) and salinity (33%o). As a
consequence of the large number of individuals in each experi-
ments (400 and 420) and long duration of the surveys (46 and 70
days), clams were maintained within large groups, in plastic tanks
of 12 1. In this way, food concentration is more stable and equal for
all clams at each experimental conditions are closer to natural
ones. Food consisting in different rations of the microalga Isoch-
rysis galbana was added to the circulating water on a continuous
basis by means of a variable flow peristaltic pump. The different
rations were obtained by maintaining food concentration constant
and varying both the flow of water into the tanks and the number
of clams per tank. Through-flow in the vessels was reduced after
each sampling, to adjust it to the number of clams remaining.
Experiment I
The following daily food rations, with percentages correspond-
ing to the organic weight (ask free dray weight) of food supplied
as a proportion of the live weight of the clams, were assayed in this
experiment: 0.24% (Al). 0.48% (A2), and 0.96% (A3).
The initial number of specimens was 140 for ration, and the
number of clams for tank 1 40. 70. and 35 for the rations A 1 , A2,
and A3 respectively. The experimental period lasted 46 days, with
samples being taken on days 12, 26, 35, and 46. On each occasion
10 specimens from each diet were used to determine soft tissue dry
435
436
Delgado and Perez Camacho
weight, with a further 10 specimens used for histologic studies.
Where necessary, the number of specimens per sample was in-
creased to obtain a minimum of four specimens of each sex.
Experiment 2
The rations used in this experiment were 0.025% (BI), 0.05%
(B2). and 0.10% (83). The initial number of specimens was 200
for ration Bl, and 100 for rations 82 and 83. and the number of
clams for tank 200. 100, and 50, for the rations Bl. B2. and 83,
respectively. The experimental period lasted 70 days and samples
were taken on days 25, 41, and 70, with 10 specimens being used
to determine soft tissue dry weight and a further 10 specimens used
for histologic studies. Where necessary, the number of specimens
per sample was increased to obtain a minimum of four specimens
of each sex.
Soft Tissue Growth: Total, Somatic and Gonadal
The anatomic features of the gonad in this species make it
difficult to separate from the rest of the organism, so indirect
methods are usually used to determine the changes that take place
(Perez Camacho 1979). In our case, total clam flesh growth (FG)
corresponds to the difference between initial and final dry weight
(DW). DW was obtained by freeze-drying the total amount of soft
tissue.
When there was an increase in weight during the experimental
period, gonadal growth (GG) was calculated from the difference
between the DW of the initial sample (when gonadal development
was nil, or very little) and that of the final sample (when the gonad
was well developed). To discount any growth of the organism
during the experimental period, initial DW was calculated for a
standard clam of the same length as the mean length of the final
sample, using the length-DW equation of the initial sample. So-
matic growth (SG) was taken as the difference between the in-
crease in total DW and sonadal srowth (FG-GG).
stage of vitellogenesis, or ripe, when their maximum diameter
exceeded 50 |jim (Vilela 1950).
Males
Colorimetrics was used to analyze images of the male clams,
with each different part of the soft tissue being color-coded. This
division of soft tissue corresponded to gametes (deep purple stain),
muscle tissue and reserves (deep and pale pink stain), and empty
zones (white). The area occupied by each color in the image being
studied was measured, and the previously mentioned expression
(GOD was calculated, the area occupied by gametes corresponding
to that occupied by spermatozoids, spermatids, sperinatocytes and
spermatogonia. Each specimen was assigned a mean value for GOI
and a percentage of ripe oocytes present in the gonad, obtained
from the nine images analyzed in each case.
Statistical Methods
Comparisons between the different rations for flesh dry weight,
conditioning index, gonadal occupation index and oocyte diameter
were established by analysis of variance (ANOVA) for a signifi-
cance level of 95%, and by analysis of covariance (ANCOVA) to
compare slopes of the regression lines of those equations having
the greatest determination coefficient. Cochran's test was used to
guarantee the homogeneity of the variances. When there was a
direct relationship between the mean and the standard deviation,
logarithmic Iransfomiation was used to homogenize the variances.
Parameters expressed as percentages were modified, prior analysis
using angular transformation (arcsineV%). Multiple comparisons
between experimental conditions were performed with the mul-
tiple rank test using the least significant difference (LSD) method.
All the statistical analyses were performed with Statgraphics plus
3.0 software, according to the methods described by Snedecor and
Cochran (1980) and Zar (1974).
RESULTS
Histology and Image Analysis
A conventional histology protocol was followed. The soft tis-
sues were fixed with Bouin's fixative, sealed in paraffin, and 4-|jLm
slices were taken. Harris" hematoxylin and eosin stain was used
(Bancroft and Stevens 1996). For each specimen, nine fields of
vision of the gonad were chosen at random, corresponding to three
different depths in the body of the clam. Microimage software
(Olympus) was used to process and analyses the images obtained.
Females
Because sexual maturation in venerids is characterized by an
increase in size of the gonadal follicles and their progressive oc-
cupation by ripe gametes, which then separate from the follicle
walls, it was decided to focus on the area of the gonad occupied by
oocytes. The area of each of the oocytes visualized was obtained
automatically (Microimage software). On average, measurements
of more than 500 oocytes were obtained for each specimen.
The gonadal occupation index was defined as follows:
GOI: (area occupied by gametes/area of the field analyzed) x 100.
Gametogenic development in females is also characterized by a
considerable increase in oocyte size, and maximum diameters were
therefore measured. Oocytes were considered to be in the final
Total. Somatic, and Gonadal Growth
The clams in experiment 1 were fed daily rations of 0.24, 0.42,
and 0.96%. All three diets produced a positive energy balance,
leading to a considerable increase in flesh dry weight (DW) that
was directly proportional to the amount of food available (Fig. la).
The total increase in DW, expressed as a percentage of initial DW,
was 35.8% for ration Al, 48.9% for A2, and 80.4% for A3. The
differences between the increases in DW recorded for each of
these diets were statistically significant (ANOVA, P < 0.001 ; mul-
tiple rank test (LSD), P < 0.05).
In experiment 2, diet 83 (0.10%) produced a positive energy
balance leading to an increase of 18.6% in DW over the initial
value. For diet 82 (0.05%) DW stayed approximately constant
during the experimental period, indicating a zero energy balance,
as corresponds to a maintenance diet. Diet Bl (0.025%) led to a
negative energy balance and a loss of 20% DW by the end of the
experimental period (Fig. lb). The differences between the varia-
tions in DW of clams fed with these diets were statistically sig-
nificant (ANOVA, P < 0.05; multiple rank test (LSD), P < 0.05).
Most of the energy acquired by the clams in positive energy
balance conditions in our experiments was expended on gonadal
development. Accordingly, as can be seen in Figures la and lb,
gonadal growth accounts for 90% of the total increase in DW for
the highest diets (experiment 1). and 98% for diet 83 in expert-
Gonadal Development in R. decussatus
437
80
70
ei
60
£
SO
%
40
o
I.
Ul
o
20
10
0
P
y
Al
a:
Diets
Aj
□ GG B SG
100
80
60
'm
40
E
71)
j=
0
S
-20
£
-40
o
-60
-80
-100
-120
W
r
^^fl^K'
\y
/
y^
i\ B2
Diets
B3
□ GG B SG □ FG
Figure I. Total flesh growth (FG), somatic growth (SG), and gonadal
growth (GG) during the experimental period, (a) Experiment I (46
days): diets Al (0.24% ), A2 (0.48% ), and A3 (0.96% ). (b) Experiment
2 (70 days): diets Bl (0.025%), B2 (0.50%), and B3 (0.10%).
ment 2. Gonadal development in diets Bl and B2 occurred at the
expense of previously stored reserves, and cannot therefore be
quantified by the same method.
GOI
GOI increased throughout the experimental period in both
males and females for all diets. Although there was clear evidence
of gonadal development in all cases, there were obvious differ-
ences, attributable to the different rations. Statistical comparisons
were based only on data from samples taken up to days 26 (ex-
periment 1) and 41 (experiment 2). Partial spawning observed in
the experimental tanks after these dates would have affected the
interpretation of the data corresponding to later samples.
Experiment I
Females
The two highest rations in experiment 1 (A3 and A2) both
produced a rapid increase in the GOI to approximately 35'7r by day
12, after which it remained constant (Fig. 2a). The rate of increase
for ration Al was slower, and although maximum GOI was simi-
lar to those for diets A2 and A3 (Fig. 2al this did not occur until
day 35.
GOI was related to time by means of a potential equation
(Table 1). A comparison of the slopes of these equations after
applying logarithmic transformation reveals statistically signifi-
cant differences between the lowest ration (Al ) and the two high-
Females
50 -
40 .
5 30 -
I
i
o
o
20
10
0
b
Males
r
10 20 30 40 50
Conditioning period (days)
o A3 ^ A2 A Al
O
X
15
10
20
30
40
50
Conditioning period (days)
A3
A2
Al
Figure 2. Gonadal occupation index (GOI) during experiment 1 with
diets Al (0.24% l, A2 (0.48% ), and A3 (0.96% ). (al Females, (b) Males.
Average data (± SD).
est (A2 and A3). No significant differences were observed between
the latter two rations (P > 0.05). The amount of food available did
not lead to any significant difference in the maximum GOI for any
of these diets (ANOVA. P > 0.05).
Males
The GOI was much higher in males than in females. Maximum
values of between 60 and 75% were obtained, according to the
amount of food available. This factor, together with the energy
balance, has a more noticeable effect on variations in the male
GOI: there is a constant increase throughout the experimental pe-
riod, with the highest diets showing the greatest rate of increase
(Fig. 2b). There was a marked decrease in the GOI of clams fed on
ration A3 after day 26, once maximum GOI (75%) had been
reached. This coincided with the partial spawning observed in the
experimental tanks.
The best fit between GOI and time (Fig. 2b) is given by a linear
equation (v = a + bx). Comparison of pairs of regression lines
(Table 1) shows significant differences between the slopes of these
equations {P < 0.05). The ANOVA performed between the maxi-
mum values of the GOI for each ration shows significant differ-
ences (P < 0.05) between the lowest diet (A I) and the two highest
(A2 and A3). No statistically significant differences were observed
between the latter two rations.
438
Delgado and Perez Camacho
TABLE 1.
Parameters of the regression lines between the gonadal occupation index (%, y) and time (days, x).
Diets
Comparison
of Slopes
Females
Males
Females
Males
Al (1)
17. IS
0.57
0.74
0.0040
12
A1-A2
0.0100
A2(l)
18.65
0.65
0.75
0.0001
13
A1-A3
NS
A3(1)
18.51
0.59
0.69
0.0005
13
A2-A3
0.0200
Al (2)
33.61
6.64
0.63
0.0036
11
A1-A2
0.0040
A2 (2)
33.10
11.15
0.70
0.0007
12
A1-A3
0.0020
A3 (2)
27.87
15.55
0.88
0.0000
11
A2-A3
0.1040
Bl (3)
6.45
18.63
0.85
0.0001
10
B1-B2
0.0010
B2(3)
6.78
26.26
0.91
0.0000
13
B1-B3
0.0001
B3(3)
5.69
31.59
0.97
0.0000
9
B2-B3
0.0500
Bl (1)
8.41
14.32
0.78
0.0001
13
B1-B2
NS
B2(!)
9.38
13.25
0.80
0.0004
10
B1-B3
NS
B3(l)
13.24
13.35
0.66
0.0001
16
B2-B3
NS
(1) Potential model (y
'"); (2) Linear model (y = a + bx); (3) Logarithmic model (y
bliix). NS. not significant; /;. number of observations.
Experiment 2
Females
Ma.ximum GOI (39.81'7c) was reached after 41 days (Fig. 3a)
for those clams fed on a ration that produced a positive energy
balance (B3). Clams fed on ration B2. the maintenance ration, did
not reach the same GOI until the end of the experimental period
(day 71). Diet Bl, with a clearly negative energy balance, gave
both a slower rate of increase in the GOI and a lower maximum
value (35.5%). The differences in maximum GOI values between
rations were not, however, statistically significant (ANOVA, P >
0.05).
Although maximum gonadal occupation is similar for all the
diets in this experiment, the rate of gonadal development is deter-
mined by the amount of food available, and a comparison of the
GOI time regression slopes (Table 2) shows statistically significant
differences (ANCOVA, P < 0.05).
Males
Variations in the GOI of males in experiment 2 were similar for
all rations, with maximum values of around 60% (Fig. 3b). Com-
parisons between the slopes of pairs of regression lines (Table 2)
show no significant differences between any of them (ANOVA.
P > 0.05), and neither were there any significant differences be-
tween the maximum values obtained for each ration (ANOVA,
P > 0.05).
Percentage of Ripe Oocytes
Experiment 1
The percentage of ripe oocytes (i.e.. with diameters of over 50
(xm) in the clams in experiment I increased rapidly during the first
two weeks of the experimental period to approximately 25%. This
rate of increase then diminished, and by day 26 average values of
26.3, 32.3, and 34.8% were recorded for rations Al, A2. and A3,
respectively. After this date partial spawning was observed in the
tanks containing clams fed on the two highest rations (A2 and A3),
this being reflected in a decrease in the percentage of ripe oocytes,
followed by a subsequent recovery (Fig. 4a).
Although the maximum percentage of ripe oocytes is similar
for all three rations at close to 40% (ANOVA, P > 0.05), the rate
of increase of this percentage is directly related to the amount of
food available, and the increase of the slopes of the regression lines
between the percentage of ripe oocytes and time coincides with an
increase in food (Table 2). The corresponding ANCOVA shows
significant differences between the slopes of rations Al and A3, at
a 95% confidence level. No statisticallv significant differences
a
Females
O
o
20 40 60
Conditioning period (days)
X B3 ^. 82 ^
Bl
20 40 60
Conditioning period (days)
80
B3
82
Bl
Figure 3. Evolution of the gonadal occupation index (GOI) during
experiment 2 with diets Bl (0.025% ), B2 (0.05% I, and B3 (0.10% ). (a)
Females, (b) Males, .\verage data (±D).
Gonadal Development in R. decussaws
439
TABLE 2.
Parameters of the regression lines between the proportion of ripe
oocytes {%, v) and time (days. \).
Diets
a
b
r
P
/I
Al
9.92
10.29
0.71
0.0003
A2
10.39
12.96
0.70
0.0004
A3
8.72
13.6
0.76
0.0000
Bl
-7.41
7.47
0.73
0.0320
B2
-11.66
12.87
0.87
0.0000
B3
-11.33
12.74
0.92
0.0001
9
Linear model (y = a + b.x). P. probability level; n. number of observations.
periment 1 (Fig. 4b). The lower percentages recorded at this point
for clams fed with ration B3 coincide with partial spawning ob-
served in the experimental tanks.
As in experiment 1, the increase in the percentage of ripe
oocytes was directly related to the amount of food available, al-
though the differences between maximum percentages of ripe oo-
cytes for each ration were not statistically significant (ANOVA.
P > 0.05). Accordingly, comparison of the slopes of the regression
lines between the percentage of ripe oocytes and time (Table 2)
shows statistically significant differences (ANCOVA. P < 0.05)
between the lowest ration (Bl: 0.()25'7f ) and the two highest (82:
0.057f. B3: 0.10%). No statistically significant differences were
observed between the latter two rations.
were observed between the slopes of rations A2 and A3, or Al
and A2.
Experiment 2
If the amount of available food is lower, as in experiment 2.
where daily rations of 0.025. 0.050. and 0.10% were used (rations
B 1 . B2. and B3. respectively), the percentage of ripe oocytes in the
gonad increases at a lower rate than for the higher diets in experi-
ment 1 (daily rations of between 0.26% and 0.96%). Under these
conditions, after 25 days the average percentages of oocytes with
a diameter greater than 50 ixni were 12.8. 14.6. and 23.5%. for
rations Bl. B2. and B3. respectively, and 70 days were needed to
reach values similar to those of the third sample (day 25) in ex-
a 60
50
fc 40 i
30
a 20^
10
0 .^
b
60
50
1
-i-
,_,
40
??;
1
4>
30
^
20
K
in
cd
0
J,
ml
12 26 35 46
Conditioning period (days)
BA3aA2oAl
[t
-10
1
25
41
70
Conditioning period (days)
a B3 B 82 Q Bl
Figure 4. Percentage of ripe oocytes during the experimental period.
(ai Experiment 1: diets Al (0.24% ), A2 (0.48% ). and A3 (0.96% ). (bl
Experiment 2: Bl (0.025%), B2 (0.05%), and B3 (0.10%). Average
data (±SD).
DISCUSSION
Image analysis has been used by several authors to compare the
reproductive cycles of different species, using methods based on
the frequency of different sizes of oocyte or the proportion of
gonadal tissue occupied by oocytes. For example. Laruelle et al.
(1994) and Xie and Bumell (1994) detected differences in the
extent and intensity of reproductive activity in species, such as R.
decussatus and Ruditapes philippinarum (Adams and Reeve). The
parameters used in the present study, i.e.. the percentage of ripe
oocytes and the gonadal occupation index, would also seem to be
good indicators of the degree of gonadal maturity in R. decussatus.
although they give no indication of the total amount of gonadal
tissue.
Navarro et al. (1989) associated interannual differences in the
reproductive cycle of Ceiastodenna edute (L.) with fluctuations in
the nutrient storage cycle caused by variations in food availability.
The amount of food available in the environment is a determining
factor of the amount of energy incorporated by the animal, and
must therefore affect processes such as somatic and reproductive
growth, as our experiments clearly show.
Accordingly, when the daily amount of available food (ex-
pressed as a percentage of clam live weight) is equal or greater
than 0.10%. as in rations 83. Al. A2. and A3, a positive energy
balance ensues. In these situations, there is a corresponding in-
crease in the amount of clam soft tissue, which under the tempera-
ture conditions prevailing in our experiments, corresponds princi-
pally to an increase in reproductive tissue.
Ration B2 produces a zero energy balance, in which energy
acquisition and expenditure by the organism were equal. When
there is a negative balance, as in the case of ration B 1 . the energy
obtained from food is insufficient to meet the energy demands of
the organism, resulting in a considerable loss of body weight.
Gonadal development took place in both situations, possibly as a
result of the high temperature at which the experiments were per-
fonned. but in this case at the expense of previously stored re-
serves.
Our results show that the amount of available food influences
both the extent of gonadal development and the rate of gonadal
maturation, with the higher rations producing a faster rate. Simi-
lariy, Buchanan et al. (1998) detected differences in the gameto-
genic development and the conditioning index of Crassostrea vir-
ginica (Gmelin). which he associated with nutritional and tempera-
ture differences between laboratory conditions and the natural
medium that produce a faster rate of gonadal development in
specimens conditioned at a higher temperature and optimal nutri-
tional conditions.
440
Delgado and Perez Camacho
There are noticeable differences in the GOI of males and fe-
males, with maximum values ranging from 55-75% for the former
and 35 and 40% for the latter (Figs. 2 and 3). Spontaneous release
of gametes can occur when these values are reached. A similar
phenomenon is observed regarding the proportion of ripe oocytes.
with spawning taking place when percentages reach between 30
and 40% (Fig. 4).
These are not total spawnings because the variations in DW.
GOI, and the percentage of ripe oocytes are only moderate, and in
the case of the last-mentioned parameter they are followed by a
rapid recovery. In this respect our results coincide with the period
of continued spawning described by Laruelle et al. (1994) and
Rodri'guez-Moscoso (2000) for R. decussatus, characterized by
partial but continued release of gametes once a certain level of
gonadal occupation has been reached. This reproductive strategy
regulates the continued and progressive process of follicular oc-
cupation, which does not appear to be compensated by an adequate
degree of reabsorption of gametes in this venerid. The spawning
period starts earlier under favorable nutritional conditions, since
the first partial spawnings correspond to the diets with the greatest
abundance of food. These partial discharges of gametes may. on
the other hand, be responsible for the reduced synchronization
between specimens, and for the high degree of variation in the data
from the final stages of the experiment. Toba et al. (1993) also
describe a greater synchronization between specimens in the early
stages of gonadal maturation in R. philippinaniin in Tokyo Bay.
which decreases considerably in the later stages of maturity.
Bayne (1975), however, in contrast with the findings of our
study, discovered a certain increase in the rate of gametogenic
development in Mytihts ediilis (L.) under conditions of nutritional
stress during the initial stages of gametogenesis, although in this
species this process is completed by the reabsorption of gametes.
In a later study on the effects of thennal and nutritional stress on
the eggs of M. edulis, Bayne et al. ( 1978) establish a relationship
between decreases in the volumetric fraction of gametes and
spawning periods when temperatures are high and food abundant.
When food is scarce, these decreases correspond to reabsorption
processes or a low level of gametogenesis. In our case, and has
already been mentioned, decreases in GOI for the higher diets are
associated with spontaneous spawnings, but we have seen no sig-
nificant decreases associated with nutritional deficiency in either
zero or negative energy balance situations.
Based on the relationship between gonadal development and
the accumulation and use of nutrients, species can be classified as
being either conservative or opportunist (Bayne. 1976). In the
former category, gametogenesis takes place at the expense of pre-
viously accumulated reserves (Zandee et al. 1980, Bayne et al.
1982). In the latter, gametogenesis occurs when there is an abun-
dance of food in the environment, and sexual maturation parallels
the accumulation of nutrients.
Our results show that the behavior of R. decussatus varies
according to the amount of food available. When there is an abun-
dance of food it adopts an opportunist behavior, developing the
gonad at the expense of ingested food, but when food is scarce it
behaves like a conservative species, with gametogenesis taking
place at the expense of accumulated reserves.
ACKNOWLEDGMENTS
We are grateful to P. Espineira. G. Rico. H. Regueiro. C. Pena,
and P. Mallo for their technical assistance. This study was financed
by the project PGIDT - 99MAR60401. M. Delgado was supported
by a research personnel training grant from the European Social
Fund - Spanish Oceanographic Institute (1998-19991 and by a
grant from the Consello Regulador do Me.xillon de Galicia (Board
of Control of the Galician Mussel) (2000-2001 ) while working on
this study.
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ABSORPTION OF BIOCHEMICAL COMPONENTS AND FEEDING BEHAVIOR WITH
NATURAL AND CARBOHYDRATE-RICH DIETS IN RUDITAPES DECUSSATUS AND
VENERUPIS PULLASTRA CLAMS
M. ALBENTOSA'*, M. J. FERNANDEZ-REIRIZ", U. LABARTA", AND A. PEREZ-CAMACHO'
Institiito Espanol de Oceaiwgrafi'a. Centra Oceaiwgrdfico de A Corima. Miielle de Animas, s/n. 15001
A Corumi. Spain and 'Consejo Superior de Investigaciones Marinas. Institutn de Investigaciones
Marinas. Eduardo Cahello. 6, 36208 Vigo. Spain.
Abstract The feeding behavior and the efficiency of the absorption of biochemical component.s in the diet of specimens of two species
of clams. Rudiuipes deciissatiis and Venenipis piillastra fed on natural and carbohydrate-rich diets were studied. Both the natural diet,
which consisted of the microalga Isochrysis ajf. galhana. clone T-ISO and ashed sediment, and the carbohydrate-rich diet, which
consisted of microalgae and corn starch as organic ingredients, and ashed sediment as the inorganic component, were assayed at a
concentration of total paniculate matter close to I mg TPM L"'. and a concentration of particulate organic matter of approximately
0.6 mg POM L"'. which are similar conditions to those found in the Galician Rias. The feeding behavior of both species for each diet
is described with reference to the clearance and ingestion rates, whereas the absorption of the biochemical components of the two diets
was determined by biochemical analysis of the diet and the resulting feces. Both mgestion and absorption rates were higher for V.
/yulkistni when the clams were fed on a natural diet. Enriching the diet with carbohydrates led to a notable increase in the ingestion
and absorption rates in both species, although this increase was greater in R. decussatus than in V. piillastra, and in consequence the
energy absorbed from the carbohydrate-rich diet was greater in the case of R. decussatus. The energy absorbed by R. decussatus fed
on this latter diet was three times greater than that absorbed on the natural diet, allowing it to maintain similar rates of protein
absorption for both diets. However, in the case of V. pullastra. the amount of total energy absorbed that denved from proteins is 50%
lower in the carbohydrate-rich diet than in the natural diet. The energy absorbed from carbohydrates in the carbohydrate-rich diet was
greater for R. decussatus than for V. pullastra. The contribution ot lipids to the total energy absorbed was found to be almost double
in R. decussatus fed on the carbohydrate-rich diet, in comparison with the natural diet, although in V. pullastra this contribution was
lower. Thus, the effect of diet on the feeding behavior of both species, i.e.. the increase in the ingestion rate and the corresponding
increase in the absorption rate, allows R. decussatus to compensate for the nutritional deficiencies of the carbohydrate-rich diet,
whereas in the case of V. pullastra it does not appear to be sufficient for the clams to maintain the same protein absorption rate as on
the natural diet. These results are discussed in relation to the possible existence of major differences in the metabolism of the two
species of clams, differences which would be connected to the habitats in which they live.
Keywords: absorption, biochemical components, clams, diets, feeding behavior. Ruditapes
INTRODUCTION
Difference.s in the characteristics of the habitat occupied by a
given species, particularly food availability and quality, give rise
to functional adjustments in individual members of the species to
allow them to maintain adequate levels of energy acquisition.
These adjustments can take place at different levels, e.g., filtration
activity, production of pseudo-feces, ingestion rate, digestive ca-
pacity, transfer of food to the digestive gland, and enzyme pro-
duction. The efficiency with which the food is absorbed after
ingestion, i.e., absorption efficiency, is one of the most decisive
parameters in establishing the amount of energy available to a
specimen for growth and reproduction.
Although the absorption processes of bivalves, in terms of total
organic matter, have been the subject of extensive study (Thomp-
son & Bayne 1972, Widdows 1978. Griffiths & King 1979, Na-
varro & Winter 1982, Bayne & Newell 1983; Bayne at al. 1989,
Beiras et al. 1993, Navarro & Thompson 1996, Perez-Camacho et
al. 1997. amongst others), there are few references in the bibliog-
raphy on the efficiency with which each individual biochemical
component in the diet is absorbed (Langdon 1989, Bayne et al.
1993: Kreeger & Langdon 1994, Ibarrola et al. 1996, 1998), it
having been observed that the quality of the diet affects the effi-
*Corresponding author.
Tel.: -1-34-8 1-205362; Fax: ■i-34-S 1-229077: e-mail: marina.albentosa@co.
ieo.es
ciency with which its different components are absorbed, this be-
ing closely related to the digestive processes.
Studies of the absorption efficiencies of specific elements of
the diet, such as carbon or nitrogen, are to be found in greater
number (Hawkins and Bayne 198.*), Cranford 1995, Iglesias et al.
1996, Urrutia et al. 1996). and from these it is possible to predict
efficiencies for proteins in relation to carbohydrates and lipids.
Another approach to establishing the nature of the mechanisms by
which different components of the diet are used is based on the
oxygen consumption : nitrogen excretion (0;N) ratio, which is an
indirect indicator of the relative use of protein ( Kreeger & Lang-
don 1993).
As a result of the work of our group in recent years on the two
species of clams included in the present study. Ruditapes decus-
satus and Venerupis pullastra, we have established the existence of
major differences between these two species in terms of both nu-
tritional requirements and physiological parameters, as a result of
the different ecological niche they each occupy (Labarta et al.
1997). The purpose of the present work has been to study the
absorption of the biochemical components of the diet and the
feeding behavior of the two species of clam when fed on a natural
diet and on a carbohydrate-rich diet.
MATERIAL AND METHODS
Acclimatization
Specimens of the clams, R. decussatus and V. pullastra, of
approximately 40 mm in length were collected in the surrounding
443
444
Albentosa et al.
area and transferred to the Centro Oceanogratlco de A Coruna,
where they were acclimatized to laboratory conditions over a mini-
mum of 7 days. Throughout the whole of the acclimatization pro-
cess, clams were kept in an open-flow system with a flow rate of
approximately 2 L ind"' h"' of seawater filtered to 1 |j.m and
enriched with the microalga Isocliiysis aff. galbana. clone T-ISO.
The organic weight of microalgal cells was calculated by filtration
of a volume of the algal cultures through Whatman GF/C glass
fibre filters that had previously been ashed and then rinsed with a
0.5-M ammonium formate solution. Filters were dried to constant
weight at 100°C and ashed at 450°C in a mufie furnace. The
concentrations of the microalgal cultures were determined using a
Multisizer Coulter Counter. The daily food ration during the ac-
climatized period, approximately 3%, expressed as a percentage of
organic matter in the diet in relation to total tlesh dry weight, was
supplied at a concentration of approximately 0.5 mg MO L"''.
these being similar conditions as those applying during the experi-
mental period. Water temperature was maintained at 19 ± 1°C.
Experimental Conditions
Similar-sized specimens (« = 10) of each species were chosen
from the stock of acclimatized clams and placed in individual
vessels connected to an open-flow system by multichannel peri-
staltic pumps. Each vessel was fitted with an inlet-tube at the base
and an outlet-tube near the surface, the latter being covered with a
nylon mesh to prevent loss of feces. Each pump was also con-
nected to two vessels containing no clams to obtain samples of the
diet supplied. The tlow-rate was 2 L ind~' h"' and the temperature
was maintained at 19 ± l°C in a controlled environment.
Experimental Diets
The natural diet was designed so as to reproduce the annual
average values of total particulate matter (TPM; mgL"'), hence
particulate organic matter (POM; mgL~'), and percent organic
matter observed in the Galician Rias. The diet comprises two
particulate components: Isochiysys aff. galbana. clone T-ISO,
cells, and sediments from underneath the bottom that had been
ashed and freeze-dried.
The carbohydrate-rich diet consisted of a mixture of microalgae
and com flour starch (commercial corn starch MAIZENA from
Bestfoods Espana, S.A.) as its organic components and ashed sedi-
ment as the inorganic component. The stability of the diet over a
24-h period, in both quantitative and qualitative terms, was moni-
tored from samples obtained from the outlet tubes of the clam-free
control vessels. The daily ration of com flour starch and sediment
was resuspended in seawater, using an electrical stirrer and sieved
at 60 (xm before adding to the system. Size of the com starch
particles used ranged from 4 to 30 (xm, being the mean particle size
15 |jim.
Both diets (Table 1 ) were assayed at a concentration of total
particulate matter of approximately 1 mg TPM L"', and a concen-
tration of particulate organic matter of around 0.6 mg POM L ',
these being similar to the conditions prevailing in the Galician Ri'as
(Babarro et al. 2000). The concentration of organic matter in the
carbohydrate-rich diet was increased to 0.77 mg POM L"', so that
when expressed in units of energy (Table 1 ) this concentration
would be equivalent to that assayed in the natural diet, given the
lower energy content of corn flour starch in comparison with mi-
croalgae. Both experiments were conducted in summer, being the
water temperature for both experiments around 19°C.
Samples (2 L) were taken daily from the outlet-tubes of the
clam-free vessels directly on to Whatman GF/C fiberglass filters
that had previously been washed, ashed, and weighed. After fil-
tration, these filters were rinsed with a 0.5 M ammonium formate
solution. Samples were taken in triplicate over a 24-h period to
determine both particulate matter, whether total (after oven-drying
to constant weight at 100°C) or organic (after ashing in a muffle
furnace to constant weight at 450°C) and biochemical components.
The filters used for biochemical analysis were freeze-dried and
stored at -30°C until the analyses were performed.
Physiological Parameters
The physiological rates were established from the total amount
of feces produced over a specific period of time by means of the
biodeposition method (Iglesias et al. 1998). The clams were main-
tained on the experimental diet for 24 h. after which they were
cleansed of feces and the period of accumulation of total feces
commenced, these being collected after 24 h. The total feces pro-
duced were collected on Whatman GF/C filters that had been
treated as described above. A proportion of the feces were used to
establish their inorganic content and thus detemiine ingestion rates
and absorption efficiency. The remainder were collected on filters,
which were freeze-dried, weighed to obtain the total ingestion rate,
and then stored at -30°C until biochemical analyses were per-
fomied.
The sum of the weight of the feces distributed among the dif-
ferent filters (total egestion rate), together with their inorganic
content (inorganic and organic egestion rate) and the inorganic
content of the diet allows us to calculate the clearance rate, which
when multiplied by the concentration of organic matter in the diet
gives us the organic ingestion rate. Absorption efficiency was
obtained from the organic content of the feces (e) and the diet (f),
according to the formula established by Conover (1966):
AE = {f-e)l((l -e)*f).
TABLE 1.
Characteristics of the diets used: natural diet: Isochrysis galbana. clone T-ISO, and ashed sediment: carbohydrate-rich diet: /. galbana. clone
T-ISO, corn flour starch, and ashed sediment.
TPM POM
Energy
JL '
POMH-PM
Protein
Carbohydrate
% total POM
IJpids
Diet
mgL'
Natural
Carbohydrate-rich
0.77 ± 0.07 0.56 + 0.07
0.95 ±0.10 0.77 ±0.06
14.1
15.1
0.73
0.82
40.5 + 6.5
7.0 ±0.1
17.1 ±2.5
79.7 ± 0.2
42.4 ±4.8
13.3+0.3
TPM, total particulate matter; POM, particulate organic matter. Average values ± standard deviations are shown (n
6).
Absorption of Diet Biochemical Components in Clams
445
Clearance rates were standardized for both species for a specimen
of I g flesh dry weight using the expression:
where CR^ is the standardized clearance rate. W^. is ihc ficsji dry
weight of each specimen, and CR^. is the observed clearance rate of
the same specimen. The exponent applied. /). was 0.68. which
relates clearance rate to the size of the specimen, expressed in
terms of weight, for clams (Delgado 2002).
Absorption of Biochemical Components
The biochemical composition of the diet and the feces pro-
duced was ascertained by analyzing the contents of the filters of
food and feces, according to the following methodology. Proteins
were calculated using the method described by Lowry et al. (1951 )
after alkaline hydrolysis with NaOH 0.5N/30"C. Carbohydrates
were quantified as glucose by the phenol-sulphur method (Strick-
land & Parsons 196S). Lipids were extracted according to a modi-
fied Bligh and Dyer ( 1959) method (Feniandez-Reiriz et al. 1989).
Total lipids were determined by the Marsh and Weinstein method
(1966), with tripalmiline used as a standard. Based on the results
of the biochemical analyses of the contents of the food and feces
filters, ingestion rates for the different biochemical components
were calculated from the product of the organic ingestion rate and
the proportion of each biochemical component in the diet. The
absorption efficiencies of the various components (AE^.^^pi AEp.
AEf.. and AEj ) were obtained by applying the following formula
(IbaiTolaet al. 1998):
^Eci.mp= ifoiupi^ ~ aiiiipf, ( 1-/\E))/ camp,-,
in which coiiipf, (/>,„ Cp and Lp) and ciwip,, {P^. C,, and Lp) are
the contents of each component in the feces (F) and the diet (D),
respectively. The absorption rates of the different biochemical
components were obtained from the product of the ingestion rate
of the biochemical component in question and its absoi-ption effi-
ciency. Component absorption rates were transformed to energetic
units using the following energy equivalents: 18.0 Kj (g protein)"'.
17.2 Kj (g carbohydrate)"', and 35.2 Kj (g lipid)"' (Beukema & de
Bruin 1979).
Statistical Analysis
The differences observed in the different physiological param-
eters between the experimental diets used and between the two
species studied in this experiment were submitted to statistical
analysis of variance ( ANOVA, P < 0.05; Zar 1 984). Angular trans-
formation (arc sin V(AE/100)) was used to transform the results for
absorption efficiency in order to guarantee standardisation of the
data. The Bartlett test was used to check homogeneity of the vari-
ances. In the case of non-homogenous variances, logarithmic or
reciprocal transformation was used to transform the data, after
which their homogeneity was once again checked.
RESULTS
Characteristics of the Diets
Table 1 shows the characteristics of the diets used. The main
components of the organic fraction in the natural diets were pro-
teins and lipids, each accounting for approximately 40*. whereas
the proportion of carbohydrates is much lower at 17.1%. In the
carbohydrate-rich diet, however, the relative percentages of pro-
teins and lipids are much lower, with values of 7.0 and 13.3%,
respectively, the main component being carbohydrates, which ac-
count for 79.7%.
Both diets were assayed at concentrations similar to those ob-
served in their natural environment (Navarro et al. 1991, Babarro
et al. 2000). The ratio of the concentration of organic matter to
total particulate matter was 0.73 for the natural diet and 0.82 for
the carbohydrate-rich diet. Food concentrations, expressed as en-
ergy equivalents, were similar for both diets, being 14.1 and 15.1
J L"' for the natural and carbohydrate-rich diets, respectively.
Physiological Parameters
Average clearance rates (CR). organic ingestion rates (//?„),
organic absorption efficiencies {AEj and organic absorption rates
(ARj together with their standard deviations for a specimen of I
g flesh dry weight for each species of clam and for both diets are
shown in Table 2. Organic ingestion rates of natural diet were
significantly higher in V. piillastra than in R. clecussalits {P < 0.05,
ANOVA test). When clams were fed on the carbohydrate-rich diet
organic ingestion rates were significantly higher than those regis-
tered for the natural diet. This increase in the ingestion rate was
much more noticeable in R. clecussalus than in V. pullastni. thus
leading to higher rates in R. ilecussatiis
The absorption efficiencies of total organic material were simi-
lar for both species fed on the natural diet (ANOVA; P > 0.05),
with a value of close to 70%. However, when the clams were fed
on the carbohydrate-rich diet, absorption efficiencies decreases in
both species at around 37%. Thus, the increase in the proportion of
carbohydrates in the diet leads to an increase in the ingestion rate,
and this in turn supposes a decrease in the efficiency with which
the ingested food is absorbed. The relation between the ingestion
rate and the absorption efficiency is given by a model that fits the
equation ,4£= a*L/?^ in which a = 6.37 (±0.575) and b = -0.404
(±0.095) (r = -0.9493, R- = 90.13%. P = 0.0507).
The organic absorption rate (/1R„) behaves in a similar manner
to the //?^, in natural-diet fed clams: the AR^^ was significantly
higher (ANOVA. P < 0.05) in V. piillastra than in R. deciixsaliis.
When the carbohydrate diet was used, organic absorption rate was
three times greater than that for the natural diet in the case of R.
deciissaliis. but only 50% higher in comparison with the natural
diet in the case of V. piillastra.
Absorption of Biochemical Components
The difference in biochemical composition between the two
diets determines the ingestion rates of each biochemical compo-
nents of the diet. In V. piillastra, although the total ingestion rate
of the carbohydrate-rich diet is three times greater than that of the
natural diet, the quantity of protein ingested in the former is only
half that in the latter (Table 2). The value of lipids ingested is
similar in both diets in this species, whereas the quantity of car-
bohydrates ingested is much greater in the carbohydrate-rich diet.
In the case of R. deciissalns. however, the protein ingestion rate is
the same for both diets whereas lipid ingestion doubles with the
carbohydrate-rich diet, in which the quantity of carbohydrates in-
gested increases considerably.
Although total organic absorption efficiency is the same for
both species when fed on the same diet, the efficiency with which
proteins are absorbed by V. piillastra on the carbohydrate-rich diet
is less than that of R. deciissalns. and this, together with the smaller
amount of proteins ingested by V. piillastra, as described above.
446
Albentosa et al.
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gives us a protein absorption rate whicin is 60% lower than that
observed for the natural diet. Lipid absorption efficiencies are
simihir between the two species, and between diets for each spe-
cies, at around 56%. thus producing the same difference in lipid
absorption rates as have previously been described for ingestion
rates, i.e., R. deciissarus doubles its lipid absorption rate, whereas
that of V. piiUasira remains the same when the diet changes. Car-
bohydrate absorption efficiencies decrease for both species with
the carbohydrate-rich diet, but because carbohydrate ingestion
rates are very high on this diet, the absorption rates for this com-
ponent are nevertheless much higher than those observed for the
natural diet.
Whereas the highest absorption rate was given by proteins in
both species when fed on the natural diet, the main component
absorbed were carbohydrates when clams were fed on the carbo-
hydrate-rich diet, due to the fact that they comprise the highest
quantity of the organic matter ingested. In R. deciissuiiis. protein
absorption rates maintain similar values (53.5 ixg ind~' h~ ) to
those described for the natural diet (33.8 (jig ind"' h"'). in spite of
the noticeable decrease of the protein content of the diet. V. pul-
liisini. however, is unable to maintain the same level of protein
absorption as with the natural diet (84.5 |xg ind"' h"' ). dropping to
35.3 p.g ind^' h"'. a lower rate than any of those obtained for R.
deciissatiis on either of the diets. The increase in total ingestion for
both species fed on the carbohydrate-rich diet leads to a consid-
erable increase in the carbohydrate absorption rate when compared
to that obtained with a natural diet, although this increase is much
greater in R. Jecii.ssaius. with an AR^ of 244.4 as opposed to 178.2
|jig of carbohydrates absorbed in V. pullastra. With regard to lipid
absorption, this is maintained at a similar level to the natural diet
(A/?, . 66.4 (xg ind^' h"') by V. pullastra when fed on a carbohy-
drate-rich diet (A/?L. 55.4 ^g ind"' h"': ANOVA. P = 0.0838)
whereas in the case oi R. decussatus it doubles (75.0 p.g ind"' h~' )
its value with respect to the figure obtained for the natural diet
(40.2 (jLg ind"' h"').
Absorption Levels Expressed in Units of Energy
Figure la and b shows the absorption rates of the biochemical
components of the diet e.xpressed in their energy equivalents. In
both species the greatest amount of energy absorbed comes from
lipids, when they are fed on a natural diet (54% of total energy
absorbed), but from carbohydrates when they are fed on a carbo-
hydrate-rich diet. On a natural diet, the energy absorbed by V.
pullastra is 70% higher than that absorbed by R. decussatus, but on
a carbohydrate-rich diet R. decussatus absorbs 40% more energy
than V. pullastra. When fed on a carbohydrate-rich diet. R. decus-
satus absorbs three times the energy than it does when fed on a
natural diet and is thus able to maintain protein absorption, in
energy terms, at similar levels for both types of diet. However, V.
pullastra reduces the contribution of energy supplied from proteins
to the total of absorbed energy by 50% when fed on a carbohy-
drate-rich diet in comparison with a natural diet. The energy ab-
sorbed from carbohydrates when both species were fed on a car-
bohydrate-rich diet was greater in R. decussatus than in V. pullas-
tra. In the case of lipids, the amount of energy deriving from this
component in R. decussatus when fed on a carbohydrate-rich diet
is almost double that obtained on a natural diet, whereas in V.
pullastra this figure decreases.
Absorption of Diet Biochemical Components in Clams
447
Natural Diet
□ Proteins
H Carbohydrates
□ Lipids
D Total
R decussatus
V- pullastra
Carbohydrates-rich Diet
□ Proteins
H Carbohydrates
□ Lipids
□ Total
I
B
R. decussatus
V. pullastra
Figure 1. Absorption rates (ARl of biochemical components, ex-
pressed in energy equivalents, in two clam species, Ruditapes decussa-
tus and Venerupis pullastra when fed on a natural diet (a) and a car-
bohvdrate-rich diet (b).
DISCUSSION
Physiological Parameters
The most important difference observed between the feeding
physiology of the two species of clam when fed on a natural diet
are caused by the ingestion rates. According to the results of our
study, organic ingestion rates in V. pullastra are SOVr higher than
in R. decussatus (Table 2; V. pullastra/R. decussatus index, Vp/Rd
= 1 .50), which when taken together with the slightly higher food
absorption efficiency in V. pullastra gives a total organic absorp-
tion rate for this species that is almost 60% higher than that of R.
decussatus {Vp/Rd = 1.59). This difference in energy absorbed is
in consonance with the findings of other authors (Perez-Camacho
1980, Beiras et al. 1993, Albentosa et al. 1996. Laing et al. 1987,
Laing & Child 1996). who in their studies note that both growth
and food consumption rates in R. decussatus are lower than those
observed in other venerids such as V. pullastra or Ruditapes phil-
ippinarum.
When the clams are fed on a carbohydrate-rich diet, important
differences can also be observed in the feeding behavior of the two
species, although of an opposite nature to those described for the
natural diet. In these circumstances, the ingestion rate for R. de-
cussatus when fed on a carbohydrate-rich diet is higher than that
observed for V. pullastra. giving us in this case an index of Vp/Rd
= 0.74. An increase in ingestion is observed in both species when
fed on the carbohydrate-rich diet, this increase being of the order
of 6 and 3 times greater in R. decussatus and V. pullastra. respec-
tively. It is therefore true to say that the effect of the diet is the
same in both species, i.e., an increase in ingestion when compared
with the natural diet, although quantitatively much greater in the
case of R. decussatus. If we bear in mind that the food concentra-
tion, expressed as total particulate matter, is only 1 .4 times higher
in the carbohydrate-rich diet than in the natural diet, these quan-
titative differences would not account for the increase in ingestion
observed. Furthermore, when expressed in terms of energy, the
food content of both diets was similar (Table 1 ). Navarro et al.
(2000) describe a feeding behavior similar to the one observed in
our study, in Argopecien purpuratus. These authors describe an
increase in ingestion of up to 6 times, just as is the case with R.
decussatus in our study, when the microalgal diet is supplemented
with carbohydrates obtained from potato starch. They also note a
similar behavior when the diet is supplemented with lipids, but this
time the ingestion rate increases by a factor of 8 in comparison
with that obtained on a pure microalgal diet. They suggest the
existence of chemical receptors on the gills or labial palps that are
capable of detecting specific nutritional components of the diet and
which would stimulate an increase in the clearance rate and hence
the ingestion rate. The com flour starch used in the present study
consists of particles of a much greater density than the microalgal
cells, or if expressed in terms of unit volume, the organic content
of com flour starch particles are some 4 times greater than that of
microalgae cells (unpublished data). If we consider that bivalves
are continuous filter-feeders, i.e.. their digestive system is continu-
ously occupied by food, then we can assume that the digestive
capacity of both species, expressed in terms of the amount of
organic matter that can be contained inside the digestive tract, must
be much greater when the clams are fed on a carbohydrate-rich diet
than when fed on a natural diet, because of the above-mentioned
difference in particle density between the two diets. Given the
great similarity of food concentration at which both diets were
assayed (0.6-0.8 mg POM L"'), the total occupation for an equal
volume of the digestive system would be obtained by the existence
of higher clearance rates for the carbohydrate-rich diet, which
would account for the differences found between the ingestion
rates for the two diets.
Total organic matter absorption efficiency is reduced by half in
both species when they are fed on a carbohydrate-rich diet, owing
to the considerable increase in the ingestion rate. The relation
between food ingestion rate and absorption efficiency has been
much studied in bivalves (Foster-Smith 1975, Navarro & Winter
1982, Bayne & Newell 1983, Beiras et al. 1993, Albentosa et al.
1996. Ibarrola et al. 1998) with a similar behavior being described
in all instances, i.e.. a decrease in absorption efficiency as the
ingestion rate increases, principally because of the reduced transit
time through the digestive tract and hence the reduced length of
time during which food is exposed to the digestive enzymes. Na-
varro et al. (2000) also describe a decrease in absorption efficiency
when the microalgal diet is supplemented with either carbohy-
drates or lipids, although to a lesser extent than that observed in the
present study.
Total organic matter absorption efficiency within each diet was
the same for both species, so the differences observed between
species in absorption rates ( Vp/Rd = 1 .59 for the natural diet and
Vp/Rd = 0.72 for the carbohydrate-rich diet) reflect the differ-
ences observed in the ingestion rate. Although there is a consid-
erable decrease in the efficiency with which ingested food is ab-
sorbed when the clams are fed on a carbohydrate-rich diet, the total
organic matter absorption rates are higher, even more so in the case
of/?, decussatus (Vp//?d = 0.72). The absorption of total organic
matter was three times higher in the carbohydrate-rich diet than in
448
Albentosa et al.
the natural diet for R. decussaius. whereas in the case of V. pul-
lastra this increase was only 1.5 times greater.
Absorption of Biochemical Components
There are few references in the literature to the process of
absorption of the various biochemical components of the diet in
bivalves (Kreeger & Langdon 1994, Ibarrola et al 1996. 1998).
particularly when the biochemical composition of the diets differs
as much as it does in the present study. Ibarrola et al. (1998). in
studies of specimens of Cenistoderma editle fed on diets consist-
ing of microalgae and sediment in varying proportions (some of
which are comparable with the natural diet assayed in our study),
show that the most efficiently absorbed biochemical component in
high quality diets (i.e.. diets with the highest proportion of organic
matter) are carbohydrates, whereas in low quality diets lipids are
the most efficiently absorbed component. The authors attribute this
high rate of carbohydrate absorption in high quality diets to an
increase in the activity of certain carbohydrases to be found in the
digestive gland. Protein absorption efficiency, however, remains
unaffected by the quality of the diet. In our study, on the other
hand, the biochemical component that is most efficiently absorbed
by both species is protein, regardless of diet. This discrepancy may
be due to interspecies differences between enzyme production in
the digestive systems of cockles and clams, or also to the different
biochemical composition of the microalgae used in the two studies.
The high protein content (63.9*7^) of the microalgal portion of the
diets assayed by Ibarrola et al. (1998) when compared to the pro-
tein content of the two diets used in the present study (40.5% for
the natural diet and 7.0% for the carbohydrate-rich diet) may well
account for the differences in protein absorption efficiency regis-
tered between the two studies.
The effect of diet on the feeding behavior of the two species in
our study, i.e.. the increase in ingestion and the resulting increa.se
in absorption, allows R. decussaius to compensate for the nutri-
tional deficiencies of the carbohydrate-rich diet, whereas V. pul-
lastni seems unable to compensate fully for these deficiencies
because it does not maintain the same level of protein absorption
as observed in the natural diet. This latter level of absorption can
be taken to be sufficient for this species, because it is a reflection
of the conditions found in its natural habitat. If we consider that
protein absorption is of fundamental importance for all organisms,
because proteins are the source of necessary essential amino acids
in the biosynthetic routes in the metabolism, this leads us to sup-
pose that V. pullastra has a lesser capacity to respond to diets with
a high carbohydrate content than does R. decussatus, which may
be an indication of the existence of different metabolic routes in
the two species.
Studies that have been performed by our research group (re-
viewed by Labarta et al. 1997) in connection with feeding behav-
ior, the biochemical composition of body tissues, and the nutri-
tional requirements of the two species of venerids studied in the
present work suggest that lipid demand is higher in V. pullastra
than in R. decussatus. whereas carbohydrate demand is higher in
the latter than in the former, provided that there is sufficient pro-
tein in the diet. This may be related to the mechanism by which
each species adapts to its specific habitat: R. decussatus. which
characteristically inhabits the inter-tidal zone and is subject to
periods of emmersion as a result of the tidal cycle, would possess
an anaerobic metabolism in which carbohydrates are a more ap-
propriate source of energy than lipids. V. pullastra, on the other
hand, a species that is permanently submerged because of its sub-
tidal habitat, would not have these same nutritional requirements,
which are more appropriate to an anaerobic metabolism, and
would instead find lipids to be a more relevant source of energy,
since they are the appropriate fuel for the aerobic routes of the
metabolism. In our experiment both species were exposed to a
completely unbalanced diet that contained a very high proportion
of carbohydrates at the expense of protein and lipids. Both species
responded in a similar manner, in qualitative terms, showing a
considerable increase in ingestion which allowed them to counter
the low protein content (proteins being an essential component of
the diet) of the unbalanced diet. In quantitative terms, however,
there are major differences between the two species: R. decussatus
is able to maintain protein absorption levels, and even manages to
double the quantity of lipids absorbed, whereas V. pullastra is
unable to keep protein absorption at the same level, registering a
60% decrease, and is barely able to maintain lipid absorption.
These results would appear to reinforce the previously mentioned
hypothesis regarding metabolic differences between the two spe-
cies.
ACKNOWLEDGMENTS
Funding for this research was provided by Comision Intermin-
isterial de Ciencia y Tecnologia. Spain, project MAR99-0240-
C02. The authors thank P. Espineira from the Centro Ocean-
ografico de A Corufia (lEO) for her helpful technical assistance in
the physiological measurements and L. Nieto and B. Gonzalez
from the Instituto de Investigaciones Marinas (CSIC) for their
technical assistance in the biochemical assistance. This study was
conducted in accordance with the legal and ethical standards of the
countries involved.
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Jounuil of Shellfish Ri'.minh. Vol. 22. No. 2, 451-464. 2003.
THE PERSISTENCE OF NEW JERSEY'S OYSTER SEEDBEDS IN THE PRESENCE OF OYSTER
DISEASE AND HARVEST: THE ROLE OF MANAGEMENT
STEPHEN R. FEGLEY,'* SUSAN E. FORD," JOHN N. KRAEUTER," AND
HAROLD H. HASKIN- t
Comi?ii; School of Ocean Studies. Maine Maritime Academy. Castine. Maine 04420; and ~Haskin
Shellfish Research Laboratory. Rutgers University. 6959 Miller Avenue. Port Norrls. New Jersey 08349
ABSTRACT New Jersey'^ Delaware Bay oyster fishery developed along a pathway common to many fisheries. Perennially large
harvests led to depletion of the oyster resource, which led to increasing, but ineffective, harvest restrictions and cumbersome
nianagemeni. In the 1950s, two events altered the management structure. In the beginning of the decade, a university researcher
dedicated himself to having oystermen and the state regulatory agency use information from research and monitoring programs directly
in their decision making. He achieved limited success until a previously unknown oyster disease, eventually called MSX, occurred that
threatened to drive the oyster fishery to extinction. The presence of MSX led oyster harvesters to become dependent on the information
provided by the university. In addition, the regulatory agency and its regulations had to be responsive to shon-tenn changes in the
intensity and prevalence of disease. A tripartite management structure developed in which: I ) the oystermen. researchers, and state
regulatory agency acted cooperatively and 2l flexible guidelines were developed that could respond to annual variation in oyster
abundance and disease. Several aspects of this management arrangement could prove useful in other fisheries.
KEY WORDS: oyster, management, fishery
INTRODUCTION
Over the past decade, an increasing sense of urgency to develop
effective, nontraditional approaches to fisheries management has
developed. Too frequently, government-directed management has
had problems sustaining fisheries resources at hai^estable levels
while providing economic and social stability for the fishery par-
ticipants (McGoodwin 1990, Hannesson 1996). Alternative man-
agement models that have been suggested include adaptive man-
agement (Waiters 1986), ecosystem management (Schramm &
Hubert 1996), and responsible management (FAO 1995). All al-
ternative management models suggested to date involve greater
participation by fishery participants in the management decision
processes, a management structure generally referred to as co-
management. Many observers of and participants in fisheries are
wary of including the principal users of the resource: they doubt
that those who would gain immediate benefit from using a re-
source would sacrifice current profit for future sustainability
(Jentoft et al. 1998). In contrast, Jentoft et al. ( 1998) have argued
that there are numerous social and institutional elements that allow
a more positive expectation of the outcome of co-management
models.
Co-management has developed in some fisheries without a de-
liberate effort to develop a nontraditional management program
(Jentoft & McCay 1995). Contingent needs can lead all partici-
pants in a fishery to search for an operating environment to solve
certain problems. Such is the case with New Jersey's Delaware
Bay oyster fishery. A detailed examination of the ontogeny and
structure of this particular fishery provides several benefits. First.
it allows those who are considering developing co-management
programs to learn from the successes and failures of those who
have already incorporated co-management. Co-management pro-
grams are emerging. For example, in the state of Maine, co-
management has been legislated recently for the lobster fishery
*Corresponding author. E-mail: sfegley@iTima.edu
tDeceased.
(Acheson et al. 2000). Other fisheries in the region are expected to
follow the same path. Second, the contingent need that had to be
solved in New Jersey's Delaware oyster fishery was the presence
of diseases that affected the resource. Apparently several popula-
tions of marine species have an increasing incidence of disease-
induced mortality (Harvell et al. 1999). Managing in the presence
of disease may be a more common feature of fisheries in the future.
Accordingly, we present the following case study.
Historically low abundances of the eastern oyster, Crassostrea
virginica. presently occur throughout much of the middle Atlantic
US coast. Many factors have contributed to the decline of the large
oyster populations that existed in Chesapeake and Delaware Bays,
including management that failed to prevent overharvesting (Ha-
ven et al. 1978, Kennedy & Breisch 1983). A major factor con-
tributing to recent declines of midcoast oyster populations and
frustrating restoration efforts is the presence of one or more oyster
diseases. Disease-induced mortalities have been so intense that in
some areas oysters are rare and local oyster fisheries have become
extinct (Bosch & Shabman 1989).
In Delaware Bay, the principal oyster disease organism for
most of the past four decades has been the MSX parasite Hap-
lospohdium nelsoni. Since 1990, a southern oyster parasite, Per-
kinsiis marinus, which causes Dermo disease, has invaded the Bay
becoming the principal disease agent affecting oysters. Epizootics
produced by both parasites have caused extensive oyster mortali-
ties in Delaware Bay; however, large numbers of oysters persist.
Continued high abundances of oysters in Delaware Bay have been
possible because many natural oyster beds occur in a spatial refuge
from disease in the upper regions of the Delaware estuary. Salini-
ties in this area frequently fall below levels necessary to sustain
MSX infections. The Dermo parasite survives in these reduced
salinities but does not produce lethal infections. The natural beds
have been a primary source of seed oysters for the industry since
the mid 1800s. Until recently, direct marketing from the beds had
been prohibited. All seed oysters had to be transplanted to private
leases in the lower bay where their growth and meat quality would
be greatly enhanced before marketing. With the advent of Denno
451
452
Fegley et al.
disease, movement of oysters into the lower Bay became uneco-
nomical, and limited direct marketing from the beds began (Ford
1997).
Because the natural beds are located in the upper estuary, the
seed resource would have survived the depredations of oyster dis-
ease without human intervention. However, a management scheme
that developed shortly before the 1957-1959 MSX outbreak sta-
bilized postepizoolic yields from seed oysters. Oysters still had to
be transplanted onto leased grounds where enhanced growth and
fattening was now countered by higher disease pressure that in-
creased mortality. After the outbreak of Dermo disease, an entirely
new strategy had to be developed to sustain the industry in the face
of a disease with very different characteristics. We believe that a
description of New Jersey's management structure provides in-
sights for those desiring an effective management structure for
many fisheries. Below we describe the physical and biologic con-
text of the seed oyster fishery in Delaware Bay. Next, we provide
a brief history of the fishery and describe the development of the
present management structure and how it functions. We then sum-
marize important aspects of the role of oyster diseases and how the
management scheme responded to challenges from the diseases.
We conclude by highlighting the unique elements of the manage-
ment structure that we feel led to its success. It is important to note
that the authors were participants in many events described below
and may be burdened with preconceptions as to the value and
importance of different aspects of the management structure. How-
ever, our direct and extensive knowledge of the inner workings of
the management structure allows us to place events and circum-
stances in a context that would not be available to an outsider.
Physical Description of Delaware Bay
Detailed descriptions of the physical and bathymetric charac-
teristics of Delaware Bay are available elsewhere (Shuster 1939,
Maurer & Watling 1973, Galperin & Mellor 1990a, 1990b). Dela-
ware Bay is bounded on the north and east by New Jersey and on
the south and west by Delaware (Fig. 1 ). The bay extends 75.2 km
from its southeastern-facing mouth between Cape May and Cape
Henlopen to the entrance of the Delaware River in its northwestern
comer. The average depth is ca. 10 m with the greatest depths
occurring near the central long axis of the bay. The eastern side of
the bay has extensive tidal flats. The bottom consists largely of
soft-substrates (sands and muds) with hard substrate limited to
spatially discrete oyster beds and cobble aggregates.
Delaware Bay experiences predominately semi-diurnal tides
with a 1-1.25 m tidal range near its mouth. Around 12% of the
annual freshwater input enters the bay from the Delaware and
Schuylkill Rivers. Salinity near the mouth ranges from 30-3 1 ppt
and decreases with distance in a roughly uniform fashion up the
bay to 0-4 ppt near Wilimington, DE. Water temperatures range
from -1.8 to 29.0°C annually.
Oysters in Delaware Bay
Historically, natural oyster beds existed throughout Delaware
Bay (Ford 1997). Before the mid- 1800s, however, harvest prac-
tices and the distribution of oyster predators (primarily oyster
drills, Urosalpinx cinerea and Euplcuni caudata) eliminated beds
in the lower bay. The geographic location of extant natural (seed-
oyster) beds has remained fairly constant and predates the appear-
ance of MSX in Delaware Bay (Engle 1953, Maurer et al. 1971 ).
These oyster beds occur in several small rivers entering the bay
NEW JERSEY
DELAWARE
Figure 1. Location of the seedbeds (shaded areas) and the planting
(leased) grounds (areas inside the broken lines) in Delaware Bay. The
double line extending down the center of the bay represents the ship-
ping channel. It separates the New Jersey and Delaw are portions of the
bay. .Abbreviations for the seedbeds are the same as in Table 1. The
labels of five small beds, located inland of EIS and NWB-STR, are
indicated by letters (a— NPT. b— HGS, c— HWN, d— VEX, and e—
BDN ). DPW is the Deepw ater site.
(Broadkill. Leipsic, Mispillion. Murderkill, and St. Jones Rivers in
Delaware; Back, Cedar, and Nantuxent Creeks and Cohansey and
Maurice Rivers in New Jersey) and in the bay itself between Egg
Island Point in the south and Arnold's Point in the north (Fig. 1 ).
Most of the beds are in the eastern or New Jersey half of the bay.
Oyster beds vary in size from those that are a few m" in area
("lumps") to some that exceed 6 x 10'^ nr. The density of oysters
per unit area is highly variable within and between beds.
Salinity of the water immediately over the oyster beds varies
with distance from the mouth of the bay (Engle 1953. Maurer &
Watling 1973. Fegley et al. 1994). Over the lowermost beds (those
closest to the mouth of the bay) bottom salinity typically ranges
from 16.0-20.0 ppt. The uppermost beds generally experience a
bottom salinity ranging from 7.0-15.0 ppt. Oysters experience
reduced predation rates on all but the lowermost beds because the
most abundant and effective oyster predators in Delaware Bay, the
oyster drills, are inhibited by salinities less than 15 ppt (Engle
1953). In contrast to survival, oyster growth rates decline along the
decreasing salinity gradient in Delaware Bay. Oysters transplanted
from the lowermost beds have generally required a single growing
season to reach marketable size whereas most oysters moved from
the uppermost beds have needed to remain on the planting grounds
at least 2 years before they could be landed.
Adult oysters spawn throughout the summer with most repro-
Management of New Jersey's Oyster Seedbeds
453
ductive activity occurring from mid June to mid July. The larvae
remain in the water column trom 10 to 20 d. depending on water
temperature. Oyster spat set over most of the bay. The densest sets
generally occur in the eastern portion of the bay south of Egg
Island Point, where no beds exist and where oysters rarely survive
to adulthood because of high predation. disease, and winter ice
mortalities (Engle 195,^, Ford & Haskin 1988).
Oyster Fisheries in Delaware Bay
Several detailed accounts of the history of Delaware Bay oyster
fisheries exist (Miller 1962, Maurer et al. 1971, Ford 1997). The
following description, based on these histories, concentrates on the
New Jersey portion of the fishery.
In colonial times, natural oyster beds occurred throughout the
bay although then, as now, most beds were located in the eastern
(New Jersey) portion. Oysters were harvested directly from the
beds and most were taken directly by ship to markets in Philadel-
phia. The concept of planting small "seed" oysters onto private
leases for growth and fattening before taking them to market was
introduced to Delaware Bay in the mid 1 800s. Leases were estab-
lished in the lower bay because the market quality of oysters was
better there and because the local, natural beds had been largely
destroyed by that time. Transplanted oysters were usually left on
these relatively high-salinity leased grounds for 1-3 y before they
were marketed. The seed oysters came primarily from the extant
natural "seed" beds in the upper bay and in the creeks where low
salinity protected small-sized oysters from predation. These beds
remained a "public" resource. The practice of planting oysters was
codified independently by laws in the States of Delaware and New
Jersey. Until recently, planting seed oysters was the principal
means of producing oysters in Delaware Bay. As planting became
more widespread, the oyster fishery became dominated by com-
panies that owned large schooners and used dredges to harvest
oysters; hand tongers oystering from small boats have remained a
marginal component of the fishery since that time (Fig. 2).
From 1900 to 1930. Delaware Bay oyster landings produced
between one million and two million bushels annually (Ford
1997). After 1930 and until the mid 1950s, the productivity of the
industry declined slightly and annual landings remained at or just
below one million bushels (-40 million L, Fig. 3). Landings of this
magnitude, although supplemented by planting of seed oysters
collected from outside of the estuary (primarily Chesapeake Bay
and Long Island Sound), removed tremendous numbers of oysters
from the natural seeds. By the early 1900s, seedbeds near the
planting grounds were reported to be out of production. Subse-
quent harvest practices (e.g., failure to return oyster shell to the
seedbeds and the introduction of engines into the sailing schooners
used to dredge seed oysters) and physical-biologic interactions
(e.g., persistent droughts that increased the range and abundance of
oyster drills) led to further degradation of the seedbeds. Finally,
several years of poor recruitment onto the seedbeds and some
unexplained mortalities of adult oysters in the 1940s and 1950s left
oyster abundances on the seedbeds at historical lows.
Development of Oyster Seed Fishery Management
Legislation enacted by the States of New Jersey and Delaware
during the 19th century attempted to regulate oyster fisheries in
both states (Ford 1997). The overall goal was to preserve the oyster
resource. Specific laws introduced culling (returning oyster shells
to the bottom), restricted taking oysters from public seedbeds to a
>
Q
CO
Z
o
BOATS IN NEW JERSEY'S
DELAWARE BAY OYSTER FISHERY
C T vessels > 5 gross metric tons
^^M vessels < 5 gross metric tons
o vessels dredging seed beds
IVISX Denno
1900 1910 1920 1930
1940 1950
YEAR
1960 1970 1980 1990
Figure 2. The number of vessels registered annually in New Jersey's
Delaware Ba> fisher). Open bars represent vessels greater than 5 gross
metric tons in size. Shaded bars represent vessels less than 5 gross
metric tons in size. From 1958 to 1991, the number of vessels partici-
pating in seedbed dredging each year is indicated by the diamonds. In
the last 3 yr (1995 to 1997) the diamonds indicate the number of boats
harvesting the seedbeds in the spring and fall for direct marketing of
the oyster seed. The broken vertical lines indicate the first year the
respective diseases were observed in the bay. No data were available in
vears when the beds were closed.
specific season, allowed the first private leasing of grounds, and
created a variety of organizations to monitor and enforce the leg-
islation. Enforcement was a perennial problem and. at the request
of many oystermen. the State of New Jersey took control of both
the public and private grounds in 1899 (the State of Delaware had
done so in 1 873, just two years after private grounds were devel-
oped there).
The principal regulation affecting the New Jersey seedbeds
limited the period for oyster dredging to May and June. During this
period, known as bay season, licen.sed vessels were permitted to
take as many oysters as they could dredge and carry from the
seedbeds for transplanting onto private leased grounds. Beyond
limiting the length of bay season, there were no attempts to restrict
the numbers of oysters taken from the beds. Prior to the 1950s, the
seedbeds were closed to harvest only once, in 1928, to protect a
large set of spat (newly settled oysters up to one year of age;
Nelson 1929). During this time, information on year-to-year
changes in oyster abundance on the seedbeds was not gathered.
Few data were available to provide a basis for decisions by man-
agement.
Management of New Jersey's oyster resource can be traced to
1888. In that year Julius Nelson, a member of Rutgers University's
New Jersey Agricultural Experimental Station, convinced the
school to create the Department of Oyster Culture. Julius Nelson,
and later his son, Thurlow. became leaders in the field of oyster
biology and established a tradition of using scientific methods to
produce information useful to the oyster industry (Nelson 1913,
1928, 1947). In the early 1950s, when oyster abundances on the
Delaware Bay natural seedbeds reached historical lows, the De-
partment of Oyster Culture, then under the direction of Harold
Haskin. began studying the factors limiting oyster abundance on
the seedbeds and gathering data that would suggest management
strategies to rehabilitate the beds. The collection of data on oyster
life-history in Delaware Bay in a regular and consistent manner
454
Fegley et al.
O
LU
_l
O
>
to
o
100 -I
80
60
40 -I
20
0
NJ Seed bed harvest
Tf
MSX
Dermo
Ml|l|l|l^
1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990
80
^ 60
NJ market landings
from Delaware Bay
in
o
0
30
25
20
15 -,
10
1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990
Market value of
Delaware Bay oysters
' I ' ' ' ' I ' ' ' ' I ' '
1880 1890 1900 19101920 1930 1940 1950 1960 1970 1980 1990
YEAR
Figure 3. Historical changes in seed bed harvest, market landings, and
market value in New Jersey's Delaware Bay fishery. The broken ver-
tical lines indicate the first year that the respective diseases were ob-
served in the bay. The absence of bars prior to 1957 indicate that no
data are available. The absence of bars after 1957 indicate 0 values for
the respective measures. Harvests are mostly for direct marketing and
not planting starting in 1996.
has continued for 45 y and has provided the basis for what we
believe has been an effective management scheme.
At its inception the seedbed rehabihtation program consisted of
two key elements: gathering quantitative data on oysters (Research
Component) and advocating the use of these data in making man-
agement decisions (Applied Component). The research component
consisted of several studies conducted yearly including: ( 1 ) deter-
mining the temporal and spatial abundance patterns of oyster lar-
vae. (2) determining the temporal and spatial patterns of oyster
spat settlement and fouling organisms (invertebrates that compete
with spat for space) onto artificial collectors, (3) detecting annual
changes in the abundances of spat, yearlings, and older oysters on
the seedbeds, and (4) estimating the volume of seed oysters trans-
planted from the beds. Much of the funding for monitoring in the
early years derived from University sources, a condition that is
uncommon in our experience. The applied component entailed a
determined effort on the part of the Director of the Department of
Oyster Culture (Haskin) to convince the state management agency
and, more importantly, the oystermen of the need for additional
restrictions on seed transplants and of the usefulness of scientifi-
cally collected data in decision-making. The use of scientifically
collected data is now an accepted element. Both components have
had continued importance in the overall management of the re-
source.
Research Component
Of the several studies in the research component, two have
been consistently of greatest use to management of the resource:
collecting dredge samples from seedbeds and estimating the
amount of oyster seed transplanted during bay season. Since the
onset of Dermo disease, data on infection levels and oyster mor-
tality rates have also been used on a regular basis in making
management decisions.
For dredge sampling, several grids, each consisting of contigu-
ous 275-m X 370-m rectangles (approximately 0.2 min of longi-
tude by 0.2 min of latitude, respectively), were created for each of
the 25 spatially largest seedbeds that had historically contributed
the bulk of oyster production. Each year, generally between No-
vember and March, approximately 10% of the grids were chosen
from each bed using a stratified random sampling design. Samples
were taken from the middle of each grid. In the grid an oyster
dredge (with a 71-cm tooth bar and a bag capacity of -80 L) was
towed on the bottom for one minute at constant boat speed (i.e.
approximately constant effort) three separate times. Approxi-
mately 13-14 L of the contents of each of the three hauls were
retained, pooled, and returned to the laboratory as a single sample.
First, the volumes of live oysters (adults, yearlings, and spat),
cultch (oyster shell with no live oysters attached), and debris
(sponges, algae, wood, etc.) of each sample were estimated. Then
the following quantitative attributes were determined by direct
examination: ( 1 ) the number of oysters older than I y, (2) the
number of "yearlings"" (oysters that were about 1 y old), (3) the
number of spat, (4) the number of ""boxes"" (articulated but empty
oyster valves), (5) the number of "gapers"" (recently or nearly dead
oysters that do not fully close their valves when handled), and (6)
the number of dead spat and, if any distinctive drill or crab valve
damage was apparent, the source of spat mortality.
Estimates of seedbed yields were made by research crews every
day that dredging occurred on the seedbeds from 1956 to 1991.
How many and which boats dredged, which beds the boats
dredged, and estiinates of the volume of oysters moved to the
planting grounds at the end of the day were obtained by direct
inspection. Estimating the volume of oysters harvested was done
by noting the size of the pile on the deck and the position of the
water line on the oyster boat. In several years research crew esti-
mates were compared with estimates of seed oyster volume made
by the boat captain and by direct measurements. Remote observer
estimates were generally within W7c of the captain"s estimates and
of direct measures.
Estimates of the percent composition of commercial dredge
samples were also made during bay season. On Thursday (usually)
of each week of seed planting season uncalled 40 L samples of
oysters and shell were taken directly from the decks of oyster
boats. Boats were selected on the basis of which beds they
dredged. The beds of interest were those that had experienced the
greatest amount of dredging activity during the week or that had
begun the week with relatively low percentage (by volume) of
oysters. On shore a committee composed of industry members,
managers, and laboratory personnel sorted the samples into oyster
(live adults, yearlings, and spat) and shell (anything without an
oyster attached) and estimated the relative volumes of the two
portions. This information was then used to decide whether to
Management of New Jersey's Oyster Seedbeds
455
close some of the beds or to end the seed tninsplant early. If the
average percent of oysters by volume was less than 40*^ for a bed
the committee gave serious consideration to closure.
The 40% value was a "rule of thumb" benchmark that was
never supported by statute or regulation. It was not supported by
scientific evidence. When the seedbed rehabilitation program be-
gan the approximate percent oyster on many beds was around 40'7f
and many felt that it should not go lower. The industry members
understood this measure (as opposed to more complex statistical
indices) that required simple math and that they could derive on
their own via examination of dredge hauls. Also, when percent
oyster did drop much below 407c harvesting oysters became pro-
hibitively expensive for boats using manual culling. Use of the
40% rule was flexible. Depending on other factors (abundance of
oysters elsewhere, number of spat in the sample, perceived eco-
nomic needs of the oystermen) a bed could be closed before the
percent oyster measure reached 40% or at a considerably lower
percentage (as low as 20% in a few cases).
Applied Component
A shellfish council, officially consisting of industry members
appointed by the Governor, had long been in place to advise the
state agency in charge of the seedbeds (the council also supervised
the private leases approving transfers, vacancies, boat licenses,
etc.). In the mid 1950s incorporating research results into the coun-
cifs decision-making proved difficult. The concept of managing
oyster beds with recently collected data was foreign to both the
state agency (NJ Bureau of Shellfisheries) and the oystermen.
However, the greatly depleted condition of the beds indicated that
restrictions on seed transplants would be austere for some time to
come. The patent threat to the fishery by the condition of the
seedbeds and the persistent efforts of the Director of the Depart-
ment of Oyster Culture advocating the utility of research results
led to the development of a tripartite management scheme. An
independent source of information. Rutgers University, was added,
in an informal advisory role, to the shellfish council and state
regulators (Fig. 4). This system remains in effect today.
In late winter, several months prior to the beginning of bay
season, data collected from the seedbeds by the university re-
searchers are presented to the shellfish council and representatives
of the state management agency. The primary concerns are the
Shellfish Council
* INDUSTRY
INDEPENDENT
RESEARCH AND
MONITORING
STATE MANAGEMENT
AGENCY
RESOURCE
Oyster seed beds
Figure 4. Diagram illustrating the relationships among the compo-
nents of the New Jersey Delaware Bay oyster fishery. .Solid lines indi-
cate formal informational pathways. Broken lines indicate informal
informational pathways. The X represents the control of industry ac-
cess to the seedbeds via state management.
relative compositions of dredge samples taken from the seedbeds
(percent oyster) and the seedbed spat abundances. An oral presen-
tation of these data (usually supplemented with a written sum-
mary) is made to the shellfish council members who use this
information and, in some years, their own direct observations of
the beds, to decide ( I ) whether there will be a bay season. (2) how
long the season will be, and (3) whether any beds will be excluded
from fishing. The council's recommendations are then submitted
to the state management agency (specifically the Commissioner of
New Jersey Department of Environmental Protection who directs
the Bureau of Shellfisheries), where they have generally been ap-
proved.
Onset of MSX Disease
In the spring of 1957, widespread mortalities of oysters planted
the previous year occurred on the New Jersey leased grounds.
Within two years the epizootic had killed over 90% of the oysters
on the planted grounds and almost half of those on the seedbeds
(Haskin et al. 1966). The causative agent, H. netsoni (popularly
referred to as MSX), was identified in 1958 and has remained
enzootic in the estuary (Ford 1997). Since 1957, dockside landings
of oysters from Delaware Bay have remained well below a half a
million bushels (-20 million L) of oysters annually (Fig. 3), al-
though significant undeireporting of these landings may be occur-
ring (Haskin & Ford 1983).
Uninfected oysters residing in salinities greater than 15 ppt can
become infected with H. nelsoni from June to early November.
The disease progresses to a lethal stage within several weeks in
susceptible oysters. Mortalities are delayed in Delaware Bay native
stock; it has developed a degree of resistance to the disease (Ford
& Haskin 1987). Some oyster deaths occur in late summer or fall
of the first year of planting, but these are usually tolerably low
(Ford & Haskin 1982). Mortalities are cumulative, however, and
become unacceptably high if oysters are not marketed within a
year. The large oyster mortalities produced by MSX on the planted
grounds altered the practices of the Delaware Bay oyster fishery.
First, importation of oyster seed from other regions ended. Second,
only relatively large oyster seed could be transplanted from the
seedbeds to the planting grounds because only a single growing
season was likely to be available to growers. It was no longer
possible for small oysters to survive in the lower bay for the two
to three years necessary to reach market size. Planters could not
stockpile oysters on their leases anymore. Third, oystermen con-
centrated their planted oysters in a relatively small area of the bay
less prone to disease. Leased bottom was made available that
encroached onto the lower seedbeds in an attempt to provide less
saline and less disease-ridden planting grounds. Fourth, oyster
boats decreased operating costs by using automatic culling ma-
chines instead of manual labor to separate oysters from cultch.
Fifth, regulations were changed to permit marketing oysters earlier
in the year. This allowed planters to land oysters as soon as they
reached market size instead of waiting until 1 September as they
had previously. Sixth, a limited fishery based on boat size was
established in 1981 to prevent a large influx of participants during
good times who had no commitment to preservation of the re-
source.
The onset of MSX disease initiated a long-term monitoring
program that followed the spatial and temporal patterns of the
disease in the bay and consequent oyster mortality (Ford and
Haskin, 1982). Results garnered from this effort helped interpre-
456
Fegley et al.
tation ot data acquired from the annual seedbed sampling program.
At approximately one month intervals, oysters were dredged, using
the same device described above, from the larger seedbeds and
several locations on the planting grounds. The dredge samples
were taken only from the most productive grids on the seedbeds.
In contrast to the fall/winter seedbed sampling procedure, several
successive dredge hauls were conducted until a bushel (-40.7 L)
containing only live oysters, gapers, and bo.xes was obtained. All
gapers and boxes were examined for evidence of shell damage that
could be attributed to crabs, drills, or dredging. Gapers and boxes
with undamaged valves were assigned to the nonpredation mor-
tality category. The interiors of the boxes were further inspected to
determine whether any fouling organisms had recruited onto the
inner surfaces of the valves. Boxes with no fouling on the inner
valve surfaces were considered "recently dead." Spatial and tem-
poral variation in the rates of valve fouling were estimated by
placing clean valves in the field at regular intervals and examining
them at subsequent intervals for the presence of fouling organisms.
Seasonal "fouling intervals" ranged from 2 to 3 wk in the summer
and up to 10 wk in the winter (Ford & Haskin 1982). Estimation
of the annual mortality from predation and nonpredation sources
were made by accumulating mortalities determined over short in-
tervals.
MSX disease prevalence and intensity was determined via his-
tologic procedures in live oysters and gapers collected during the
mortality sampling. After sectioning and staining, the abundance
and location of MSX parasites in the tissues were determined via
microscopic examination. In local infections (nonlethal at the time
of collection) the parasites occur only in the gills. In systemic
infections parasites are distributed through all oyster tissues. Sys-
temic infections are found in 90'7f of oysters that die of MSX
disease (Ford & Haskin 1982).
Onset of Derino disease
Infections by the southern oyster parasite, P. mariniis, causative
agent of Dermo disease, had been historically of little consequence
in Delaware Bay. During the mid 1950s light infections were
found in planted oysters after parasitized seed was imported from
Virginia where the disease was endemic. Dermo infections became
rare in the bay after importation of seed ended. The water tempera-
tures in Delaware Bay were generally believed to be too cold for
Dermo disease to persist (Ford & Haskin 1982) and sampling spe-
cifically for Dermo disease ended in 1963. In the late summer of
1990, oyster mortalities that did not fit the pattern associated with
MSX disease were documented in several locations in Delaware Bay
(Ford 1996). The causative agent was quickly identified as P. inari-
nus. Since 1990. Dermo infections have been persistent, wide-
spread, and responsible for continuing oyster mortality in the bay.
In contrast to the pattern of MSX distribution, Dermo infections
have extended onto the seedbeds and caused substantial monalities
of seed oysters. P. mariiuis is much more tolerant of low salinity
than H. nelsoni. It survives on most of the seedbeds, even though
it does not cause many lethal infections on the uppermost beds.
Parasites proliferate rapidly in oysters transplanted to the planting
grounds in spring, stimulated by both high temperature and high
salinity. Under these conditions, transplanted oysters typically die
before the fall market season. The consequences of a mortality
pattern quite different from the delayed mortalities induced by
MSX disease was forcefully demonstrated to the planters shortly
after the onset of the Dermo epizootic. Planters were advised of the
presence of Dermo disease in Delaware Bay immediately after it
was identified in the summer of 1990. During the remainder of the
summer and fall, the disease spread to all planting areas and to the
lower seedbeds, but caused relatively little mortality and yields
from planted oysters were the best in many seasons. The following
year, the abundance of oysters on the seedbeds was the best since
the early 1980s and nearly 300,000 bushels (1.2 x 10^ L) were
moved to the planted grounds. A large majority of these oysters
were already infected with P. manniis, which quickly proliferated.
Despite advisories about relatively high infection levels by re-
searchers (including warnings by oyster disease researchers from
institutions other than Rutgers University expressed in a special
public meeting), most planters, remembering the profitable results
of the previous year, chose to leave their oysters on their leases
rather than to harvest early. Mortalities, when they began, were
severe and only about a quarter of the oysters survived to the fall
market season.
The MSX surveillance program was severely diminished after
the mid 1980s because of funding limitations and an expressed hesi-
tation by university administrators to commit to long-term monitoring
programs. Tlie advent of Dermo disease, however, raised enough
concern within the industry that limited monitoring was resumed. It
centered primarily on disease diagnosis in oysters collected during the
fall seedbed survey, which provided information on the spatial dis-
tribution and intensity of Dermo disease on the natural beds at a time
of peak prevalence and intensity (Ford & Tripp 1996). Because it is
likely that oysters rarely, if ever, completely rid themselves of P.
mariini.s. even under the low temperature and low salinity conditions
that are unfavorable to the parasite (Ragone-Calvo & Burreson 1994,
Ford et al. 1999). the fall sampling provided a good estimate of what
percentage of oysters are infected on each bed. Subsequent sam-
pling in the spring before bay season, provided additional infor-
mation on infection intensity, which typically decreases over the
winter in proportion to temperature and fresh-water influx. Infec-
tion intensity in oysters likely to be transplanted provided a rough
measure of whether infections would progress to the lethal stage
relatively sooner or later after planting.
The results from the Dermo disease surveillance program and
from the earlier MSX program were presented to the shellfish
council and to individual planters. In recent years, mailings to all
lease holders describing the most recent levels of oyster mortality
and disease prevalence were made.
Delaware Bay Oyster Fishery Activity. 1953 to 1991
Seed dredging has occurred in most years since the first MSX
epizootic (Fig. 5). Generally all of the beds were open, but oys-
termen concentrated their efforts in just a few beds. The 1960s
harvests were relatively small and came primarily from the upper-
most beds. By the end of the 1 960s most oyster seed came from the
beds in the middle of the seedbed region. Four beds, Cohansey,
Shell Rock, Bennies, and New Beds, produced 68.2'7f of the oyster
seed from 1958 to 1991. These are among the largest beds and
perennially have relatively high abundances of moderately large
oysters (Table I).
As would be expected, samples collected for the weekly esti-
mation of relative oyster volume during bay season were taken
from where most of the harvest activity occurred. During the 1960s
and early to mid 1980s the relative volumes of oysters in the
samples were generally less than 40% (Table 2). Only a quarter of
these samples ( 1 1 of 42 instances) was less than 30% and in only
Management of New Jersey's Oyster Seedbeds
457
LU
_l
o
>
LU
<
C/5
LU
O
cr
LU
0.
ANNUAL MEAN PERCENT VOLUME OF OYSTERS
80
60
40 -
20
S
n > ^
I
X
o
o
■n !2 i
■o 0) ■¥ £
0) </) > "^
T-
T
I
I"
T
II
li
T-
I
ill
T
T
T
T
-r ^ '
III
T
1955
A
I960
1965
1970
MSX
T
1975
YEAR
T
t:
T
(1)
o
— to
^ </l ^ CO
S J ^ ^
~-- "^ "O in
u) "^ c J£
^ ^ CO 5
> -D o: 00
CO
T
T
CM (^
TT
I
ll
1980
1985
1990
Dermo
1995
Figure 5. Average (±1 SE) perteni volume of oysters in dredge samples for each year. The average is calculated across all beds; ;i ranges from
60 to 120 for any given year. The arrows indicate the first year the respective diseases were observed in the bay. The horizontal broken line
indicates a percent volume of 40%. Text above each bar describes the length of the subsequent bay season. Abbreviations for seedbeds are the
same as in Table 1.
three instances were the proportions less than 209c. During the mid
to late 1970s the relative oyster volume frequently exceeded 40%.
but these data were never used to extend a seedbed harvest season
beyond the length that had been agreed upon earlier in the year.
Individual seedbeds were closed before the end of bay season only
four times (Shell Rock Bed, 1961: Cohansey Bed, 1967; Shell
Rock Bed. 1972; and Bennies Bed. 1974). Low percent oyster was
the reason in half of these closures, while protection of spat led to
the other early closures.
The fishery benefited from very successful recruitment in 1972
although relative abundances of oysters on the beds were increas-
ing before this year (Fegley et al. 19941. The large 1972 set pro-
vided oysters until the early 1980s. The persistence t)f harvestable
oyster seed for almost a decade after the 1972 set was aided by the
management and harvest practices of the fishery participants. For
instance, despite large abundance of oysters in 1974 the length of
bay season was not extended to take immediate advantage of this
bounty either that year or in any successive year. Within years, the
efficiency of seed harvest (actual harvest/potential harvest x
100%) remained near 60% throughout the period (the potential
harvest was based on estimates of the total abundance of oysters
present on the seed bed large enough to be suitable for transplant).
The observed efficiency was most likely a function of boat harvest
limitations rather than conscious efforts of the harvesters. Oyster
recruitment onto the seedbeds was relatively low in the years after
1972; another "large" set (only a third the si/e of the 1972 set) did
not occur until 1 986. Restrained harvesting of the large 1 972 set by
the fishery, combined with average or above-average annual Dela-
ware River flow into the bay. remains the most likely explanation
for the continued presence of oysters on the seedbeds into the late
1970s and early 1980s.
In the mid 1980s seedbed harvests began to decline. During this
time there were increased prevalences and intensities of MSX
disease throughout the bay (Fig. 6); widespread mortality of oys-
ters followed. This was the first time since the mid 1960s that the
seedbeds exhibited such high levels of disease and predation. The
mid 1980s were also the first time since the mid 1960s that the
annual mean Delaware River flow remained below the long-term
average for several successive years (Fegley et al. 1994). No seed
dredging occurred for 3 yr (1987-1989). During this protracted
closure of the fishery there were modest increases in the abun-
dances of oysters on the beds and seed transplants began again in
1990. Unfortunately that was also the first year of a Dermo disease
epizootic.
The effects of Dermo di.sease upon the New Jersey oyster fish-
ery have been substantial. Data provided by university researchers
informed oystermen that most of the oysters they would plant were
infected with the Dermo parasite and would not survive long after
planting. Based on this monitoring information, the shellfish coun-
cil voted to close the seedbeds in 1992. 1993. and 1994. By 1995.
after 3 yr without a planting season, it was obvious that the tradi-
tional transplant scheme would work no longer.
In 1995. a new strategy was agreed upon and tried for the first
time that allowed direct marketing from the seedbeds. Up to this
time, all oysters removed from public seedbeds had to be trans-
planted onto private grounds before they could be marketed. In the
new scheme, which was developed by the Bureau of Shellfusheries
and agreed to by the Shellfish Council, each licensed vessel re-
458
Fegley et al.
TABLE 1.
Some characteristics of the seed beds related to seed harvest. The area of the seed bed includes nonproductive bottom. Mean percent oyster
is based on dredge samples taken in the random sampling program (1953-1991). Mean individual size is estimated by dividing the volume of
a dredge sample consisting of oysters by the number of oysters present. The harvest data are the total volume of seed removed from each
bed between 1958 and 1991. The five largest >alues in each category appear in boldface. The names of the beds, which are listed from those
uppermost in the bay to those that are lowermost, are given below.
Area
% Oyster
Indiv.
Harvest
Bed*
(Hectare)
Rank
(±1 SD)
Rank
Size (niL)
Rank
L X 10'
Rank
RIS
162
13
73.6(13.5)
2
37
18
3,066
13
UAR
121
15
74.1(14.3)
1
51
16
787
17
ARN
232
9
70.7(18.4)
3
49
17
8.249
10
UMD
20
18
49.9(27.7)
13
63
15
1.S55
15
MID
374
7
64.9(17.2)
5
70
13
17,617
5
COH
545
3
62.2(17.5)
6
78
8
43,735
2
SHJ
454
4
66.5(18.5)
4
74
10
16,2.^1
6
SHR
404
5
61.7(20.6)
7
85
6
42,784
3
BNS
101
17
59.9 (22.3)
8
91
5
4.335
8
BEN
636
2
48.0(25.2)
14
98
4
40,941
4
NPT
212
11
53.0(22.8)
11
69
14
2.476
14
HGS
111
16
46.8(23.8)
15
76
9
11.270
7
NWB-STR
829
1
53.4(26.9)
10
85
7
62,496
1
HKN
202
12
55.0(19.4)
9
71
12
1,121
16
BDN
293
8
43.4 (25.9)
17
135
2
203
18
VEX
162
14
52.8(20.1)
12
74
11
4.051
11
EIS
394
6
43.6(25.4)
16
112
3
8.945
9
LDG
TT)
10
33.0 (23.0)
18
146
1
3,479
12
* RIS, Round Island; UAR. Upper Arnold's; ARN. Arnold's; UMD, Upper Middle; MID. Middle; COH. Cohansey; SHJ, Ship John; SHR. Shell Rock;
BNS, Bennies' Sand; BEN, Bennies; NPT. Nantu.xent Point; HGS, Hog Shoal; NWB, New; STR. Strawberry; HKN. Hawk's Nest; BDN, Beadon's;
VEX. Vexton; EIS. Egg Island; LDG. Ledge.
ceived a quota (of equal size, regardless of boat size). Vessel
owners were required to buy a tag costing $1.25 per bushel for
each bushel they expected to harvest up to their quota. A time
period was set in which the quota was to be used. After this period
the status of the resource, markets and other factors were evalu-
ated, and another quota decision was made. In most cases the quota
per boat was increased. This activity has generated a considerable
amount of revenue (Table 3). Purchase of tags alone totaled
$374,615 (through the fall of 1998). This money was deposited
into an "Oyster Resource Development Account" that is used for
shell planting and moving oysters from upper to mid bay beds.
Direct marketing from public beds goes against a trend towards
privatization, which is generally considered more efficient than
public fisheries (Haven et al. 1978). Because direct marketing does
not require maintaining leased private grounds nor capital invest-
ment in moving oysters from the public to the private grounds
there is a possibility that new participants could more easily enter
the fishery. Yet, direct marketing from the New Jersey Beds has
been the only obvious use of the resource under prevailing disease
conditions. For instance, in 1991 and 1995 (the beds were closed
from 1992 through 1994). a total of 390,000 bushels was taken
from the seedbeds and transplanted to the leased grounds, but
because of high subsequent mortality, only 63,000 bushels (2.6 x
10* L) were landed, producing a total return of $1,189,190. For
each bushel removed from the seedbeds, direct marketing has re-
turned nearly seven times more in dockside value compared with
typical planting returns during periods of high Dermo disease
(Table 4).
The presence of Dermo disease has increased the reliance of
oystermen and state officials on the results of university research
and monitoring. In the past, information about MSX prevalence
was of secondary importance to the shellfish council when they
were deciding whether to have a bay season (MSX was generally
uncommon on the seedbeds). In contrast, the high prevalences of
Dermo disease in oysters on the seedbeds raised concerns about
transplanting infected oysters, which could result in rapid prolif-
eration of the disease and high oyster mortalities before they could
be marketed. Data on Dermo disease prevalence has been the
primary information leading to limited seed transplanting in the
past few years. A 4-week bay season was agreed to in 1995, but the
shellfish council closed the beds after two weeks. Shellfish council
deliberations cover a range of issues when the council makes de-
cisions on closures (Appendix I ).
DISCUSSION
Aspects of the Delaware Bay Management Structure
Management of the Delaware Bay New Jersey oyster fishery
has the elements common to many fishery management structures.
It consists of a management agency, an industry, a means of data
collection and evaluation, an industry council, and a set of statutes
and regulations. The difference between this system and other
fishery management structures is the way these entities relate to
each other. Although these special relationships cannot, by them-
selves, be credited with the continued persistence of harvestable
oyster populations in Delaware Bay, we believe their implemen-
tation has developed an atypical management program.
There are at least six basic differences — some obvious, others
subtle — between the Delaware Bay New Jersey management
scheme and many others. First, as for several estuarine shellfish-
Management of New Jersey's Oyster Seedbeds
459
TABLE 2.
Weekly estimations of average percent oyster volume during seed bed harvest season. Values belovt' 40% are shaded. Bed designations are
the same as in Table I. ND = no data.
Year
RIS
ARN
MID
COH
SH,I
SHR
BNS
BEN
OB
NPT
HGS
NWB
VEX
LDG
EIS
AVG
1958
33.5
36.8
33.4
30.7
34
33.7
1%1
1962
1964
1966
1968
1969
1970
1971
1972
1973
1974
1975
1976
1977
1978
1979
1980
1981
1982
1983
1984
1985
1986
1990
ALL SEED BEDS CLOSED TO HARVEST (1959-1960)
35.6 40.3 37.1 24.1
32 29.8 31.7 30.3
ALL SEED BEDS CLOSED TO HARVEST (1963)
17.3 28.2 15.9
ALL SEED BEDS CLOSED TO HARVEST (1965)
74.3 38.6 33.5 23.7
ALL SEED BEDS CLOSED TO HARVEST (1967)
64.4
50.8
58.6
56
55.3
85.5
71
754
69.9
79
81.9
74
71
85.8
66.4
51.7
54.9
64
60.7
36.3
36
36.2
49.7
30.7
69.7
ALL SEED BEDS CLOSED TO HARVEST (1987-1989)
52.4 52
63
78.1
68.2
52.6
39.5
49.4
32
37.4
46.8
35
48. 7
45.5
47
55
45.5
36.6
28.3
32.2
63.5
58.7
52.2
59
54.8
30.8
19.2
25.9
23.6
34.3
30.9
20.4
43
ND
ND
ND
45.5
56.3
57.2
57.1
54.8
66.5
52.5
69.1
78
75.5
64
78.5
65
69.9
57
71
64.3 39
56.8
43.8
53.9
52.5
43.5
44.2
61.3
48.7
ND
31.3
37.3
38.6
44
35
37.1
31.8
27
43.7
31.8
35
42.3
25
30.9
52.2
eries, there is no formally written and passed management plan,
nor has there ever been one. Only a number of very basic provi-
sions are encoded in State statute and regulations. Second, the
fishery has been closed to new entries since 1981. Third, all of the
major participants are housed in close proximity to each other and
have been for nearly a century (the Haskin Shellfish Research
Laboratory is located at the home port of the New Jersey oyster
fleet and the State Bureau of Shellfisheries has an office in the
Laboratory building). Fourth, these three groups have worked, and
continue to work, together closely in various combinations. Fifth,
a tripartite relationship exists in which each entity has a specific
role: the industry is represented by the Delaware Bay Shellfish
Council, the State Bureau of Shellfisheries provides the adminis-
trative support for the Shellfish Council and the Commissioner of
the Department of Environmental Protection makes final decisions
based on Council recommendations, and an independent group (in
this case the Haskin Shellfish Research Laboratory) collects and
provides data to the other two. Sixth, formal, informal, and per-
sonal information exchange between all three parties takes place
on a regular basis. The actual importance of these six differences
in the development, evolution, and execution of the management
strategy is not easily evaluated; however, the salient features of
each are described below.
I. The lack of a written plan provides flexibility. The process
required to make changes can be adapted to the situation at
hand and, with the exception of those portions that are en-
coded in law, most issues are settled in Council meetings.
All decisions are made openly (regularly scheduled shellfish
council meetings are advertised in the paper, anyone may
attend the meetings and express their opinions to the gath-
ering, minutes are taken and distributed at the next meeting,
newspaper journalists generally attend and publish articles
on decisions within one to two days, and special unsched-
uled meetings are held after all industry members have re-
ceived notification by direct mailings.). The decision pro-
cess however is not burdened by regulatory needs for formal
hearings, published notices, comment periods, etc. If all
three parties (industry. State, and the Laboratory) agree,
even major changes can be accomplished relatively rapidly.
The change to harvest practices brought about by Dermo
disease provides an example of this flexibility. This disease
caused such high losses in oysters that by 1995 it was ob-
vious that the traditional movement of oysters from the
seedbeds to the planted grounds in spring was neither com-
mercially viable nor biologically desirable. Discussion be-
gan in the fall Council meetings about harvesting directly
from the seedbeds. This was in direct opposition to over 100
y of practice and the proposal generated a great deal of
heated debate. In general, the older members of the fishery
were opposed and the younger members thought that the
new approach should be tried. At the end of March, after
five to six meetings and an industry evaluation of the seed-
460
Fegley et al.
XXXX X xxxxxxxxxxxxxxxxxx
1960 1965 1970 1975 1980 1985 1990
YEAR
Figure 6. Percentages of live oysters infected with either MSX or
Dermo disease on several seedbeds and one site located in the planting
grounds (DPW. Deepwater). The absence of a symbol indicates no
samples were taken from that location in that year. The sites are
arrayed from the least saline (ARNi to the most saline (DPW). The
abbreviations for the seedbeds are the same as in Table 1.
beds in mid-March, general agreement was reached that a
direct harvest should take place beginning in mid-April,
with each boat limited to 1000 bushels instead of the normal
unlimited transplantation. This change in statute was intro-
duced into the New Jersey State legislature with three pro-
visions: 1 ) a limited direct market program should be at-
tempted, 2) if oysters were to be harvested from the beds, a
per-bushel fee should be imposed with the proceeds used for
bed rehabilitation, and 3) the harvest would have to be ac-
tively managed to be successful; recommendations for open-
ing and closing the season should be in the hands of the
Council, with final authorization being given by the Com-
missioner of the Department of Environmental Protection
(Appendix 2). Once these aspects were "agreed upon", leg-
islation was drafted, introduced into the NJ Legislature in
May, passed and signed by the Governor by the first week
in September. Fall direct harvest from the seedbeds began
one week later.
The closed nature of the fishery means that all participants
are known and readily contacted for regular or special meet-
ings. Mailings of informational bulletins are easily accom-
plished. Contact is uncomplicated even though out-of-state
interests control a significant part of the industry because
they have local representatives who act in much the same
fashion as other local fishery participants.
The importance of all groups having significant on site rep-
resentation cannot be overemphasized in fostering the flow
of information and appreciation of differing outlooks. The
close proximity permits daily contact among the parties, but
more importantly, nurtures a sense of community. It allows
each individual and group to become aware of the other's
point of view and to understand their biases. This does not
mean all groups agree on every issue, but it does allow
interested participants to evaluate what is being said in a
context broader than that of a formal meeting.
Working together in various capacities is partly an out-
growth of the close proximity of the different parties and
adds to their overall ability to understand and communicate
with each other. For instance, since 1989 the industry has
donated a boat and captain for the Laboratory's annual sur-
vey of the seedbeds. Without this donation continuation of
the annual shellbed survey would have not occurred given
the existing University resources during that time. The State
often collects samples for the Laboratory, has collected
samples of interest to the industry, and often allows industry
members to sample the beds "out of season." Laboratory
representatives regularly attend Shellfish Council meetings
where they present results of ongoing projects or simply
answer questions on issues of immediate interest.
The tripartite scheme, with a party independent of the man-
agement authority collecting basic data, holds in check the
belief common to many fishermen that data obtained by
manageiTient agencies are biased, or that the interpretation
of those data is biased. In the current scheme, both the
management agency and the industry are free to criticize
data collection and/or evaluation in any way they see fit.
This provides a check and balance, somewhat equivalent to
"peer review" on the data collection and presentation pro-
cess. In addition, a research organization can use funds from
competitive funding sources to support research that does
not have an immediate interest to management or the indus-
try. However, these "pure" research projects can occasion-
ally provide new information to the attention of the industry
and the management agency that they would not have oth-
erwise.
The formal, informal, and personal relationships, as with the
close physical proximity, allows communication and infor-
mation exchange to take place on many different levels.
What is said in private conversations is often not represen-
tative of the positions presented in public meetings. This is
because each group has personal views that may not be
appropriate for expression in a formal meeting. For instance,
the formal role of the researchers is to present the facts and
to elucidate potential biologic risks. Their opinion on man-
agement alternatives is frequently sought, and they may en-
dorse certain options, but they generally refrain from advo-
cating a specific action. These scientists may have views on
whether the industry is making optimal economic use of the
resource, but this would be not be expressed in a formal
presentation of the data on the status of the resource. Simi-
larly, an individual in the industry may think the resource is
being exploited too heavily, but because of social relation-
ships in a small community, not wish to express this view in
Management of New Jersey's Oyster Seedbeds
461
TABLE 3.
Direct murkt'liii); of oysters from Delaware Bay, New Jersey Seed Oyster Beds.
Time Period
Numher of Bushels
Landed (vol. in L)
Approximate \ alue
of Bushels Landed
Value of Tags Sold
Spring 1996 (10 weeks)
Fall 1996 (7 weeks)
Spnng 1997 (10 weeks)
Totals
17.828 (7..^ X 10')
42.570 ( 1.7 X 10'')
27.479(1.1 X 10")
S7.S77(.^.6x lO*")
$,^20,904
$89.^,970
$.'i77.0.'i9
$1,791,933
$22.28.5
$52,213
$34,349
$108,847
Time period includes the length of the dredging season. We present the harvest in the fishery's traditional bushels but we also convert those volumes into L.
a formal meeting. The Industry iiieiiibers kuik to the Labo-
ratory or the State to present this view in the formal context.
Two obvious characteristics of management for the New Jersey
seed oyster fishery has been the high degree of cooperation and
mutual respect among the oystermen. State officials and university
researchers. In formal Council discussions each entity generally
honors each other's expertise and role. The relationship has been
uneasy, particularly when the resource was scarce. In recent years,
however, the restrictions on the fishery imposed by severe oyster
disease have been important in maintaining a mutual dependency
of the three parties. Scientific data have become recognized as
being more significant than ever in the management process. Co-
operation of all parties has been crucial in implementing and test-
ing new practices. The persistence of disease and its potential to
kill oysters has forced the industry to proceed cautiously and to
husband the oyster resource thoughtfully. The industry may have
acted in an equally prudent way in the absence of the existing
management structure, although pre- 1950s fishery practices sug-
gest otherwise. We believe that the interactive management struc-
ture, described above, has fostered effective decisions about the
use of the oyster resource in the presence of disease.
Scientific Data: Formal Use
Critical to the management structure has been the availability
of current population data, collected in a consistent manner over a
prolonged period. Although the data are clearly used, the manner
of use has varied, depending on the status of the resource and the
industry at the time. Below, we provide instances where the bio-
logic data can be shown to have influenced Council decisions,
others where more informal uses of the data are evident, and still
others where the data were generally ignored.
Prior to 1991 (when the Dermo disease epizootic became a
decisive factor) the abundance of oysters and spat, and to a lesser
degree MSX disease prevalence, were considered when decisions
TABLE 4.
Comparison of returns per bushel of oysters removed from the
seedbeds by planting (in 1991 and 1995) and by direct marketing
(1996-1997) during periods of high Dermo disease.
Seedbed Oysters
Fate
Bushels (vol. in L)
Average
Return per
Total Sales Bushel
Leased grounds
Direct marketing
-m).(JOO ( 1 .6 X 10')
87,877 (3.6 X 10")
$1,189,190
$1,791,933
$3.05
$20.39
We present the harvest in the fishery's traditional bushels but we also
convert those volumes into L (vol. in L).
were made about the length of seedbed season. A general, direct
relationship of these measures and the resultant occurrence or
length of the season is apparent (short or no season when percent
oyster <40%, longer seasons when percent oyster >40%; Fig. 5).
On specific occasions, the data clearly influenced decisions. In
1972 Bennies Bed was closed to dredging. At that time the relative
abundance of oysters was over 40% and the proportion of oysters
infected with MSX in the preceding two springs was low; how-
ever, oysters were available on other beds and the opportunity to
allow previous good sets on Bennies Bed to mature undisturbed by
dredging was realized. The usefulness of this decision was never
formally tested because in 1972 that bed and the remainder of the
bay experienced another, even larger, recruitment event that
proved to be an important source of oysters for years to come.
Data use has been amply illustrated since 1991 when it was
recognized that planting oysters infected with the Dermo parasite
would likely result in unacceptably high losses of planted oysters
and loss of shell from the seedbeds. This realization clo.sed the
seed fishery for 3 consecutive years despite lost income to the
fishery and the opposition by some industry members. The desire
of these members to continue to plant as usual was muted because
most participants in the fishery shared beliefs that restrained the
degree of risk that the fishery as a whole would take. The shared
beliefs included the following: I ) that the "disease problems"
would eventually lessen (as they did with MSX). making preser-
vation of the resource until that time an important and common
goal; 2) that data gathered and presented by the "third-party" re-
searchers were accurate and unbiased (although conclusions about
the data were not always widely shared); and 3 ) that the experience
of oystermen concerning when and where to plant, and when to
harvest, were important in making decisions about the advisability
of dredging seed oysters.
Scientific Data: Informal Use
There is no clear correlation in the long-term data between
MSX prevalence and oyster mortality on pi'ivate leases. A major
reason is because the total mortality on a particular ground is only
partly a function of disease levels. It is also influenced by decisions
of the lease holders who transplanted oysters. Oystermen fre-
quently solicited information about MSX prevalence and intensity
from the Laboratory. If MSX prevalence and intensity seemed to
be increasing on the leased grounds, .some planters would equip
extra boats to harvest oysters to insure they retrieved all market-
able individuals before they died (L. Jeffries, pers. comm., 199.5).
Not all lease owners availed themselves of the data or. if they did,
acted on them. Oystermen were free to ignore the monitoring data
and gamble that the disease would be less destructive than ex-
pected.
462
Fegley et al.
Little or No Data Use
The "40% rule" was ignored on several occasions during the
1960s and late 1980s (2 and Fig. 5). The industry was still reeling
from the financial losses caused by the initial MSX epizootic in the
1960s and was severely stressed again in the 1980s because of a
drought that stimulated renewed MSX activity. Economic pres-
sures clearly predominated over the biologic data; however, the
data were not entirely ignored because the length of bay season
was restricted to only 2 wk in most of these years.
Economic and Financial Pressures
Economic considerations continually threatened this manage-
ment strategy. Oystemien had to maintain cash flow during pro-
longed periods when oyster harvests were small or impossible;
most responded by diversifying their activities. Boat owners who
also owned shucking houses kept the houses active by shucking
oysters from other locations (primarily Connecticut, but also from
the Gulf Coast) or by processing surt' [Spisiila solidissima) and
mahogany clams {Arctica iskmdka). Some oystermen moved
boats into the Atlantic surf clam fishery or used them to harvest
finfish. blue crabs (CalUuectes sapichis). whelks {Busycon spp.). or
horseshoe crabs {Liimilus polyphemus) in Delaware Bay. Others
diversified economically by direct marketing of multiple seafood
products or managing marinas. Some of the older oystermen pos-
sessed sufficient cash reserves to temporarily retire. Many younger
participants left the industry; the on-again off- again nature of the
fishery restricted their ability to reenter. The large costs of prepar-
ing a boat to work in the fishery when economic return was so
uncertain resulted in a de facto limited entry fishery prior to the
establishment of a regulatory limited fishery. Only those who
could risk substantial financial losses could continue to participate.
CONCLUSION
New Jersey's management of the Delaware Bay oyster seed
fishery demonstrated an ability to respond relatively quickly to
both threatening and promising changes in the dynamics of oyster
populations and oyster mortality sources. Despite this flexibility
and the fact that management is largely in the hands of the industry
itself, the resource has been generally well conserved. In fact, the
impact of seed dredging on the oyster population cannot be sta-
tistically measured (Fegley et al. 1994). We suggest that the pri-
mary reasons for the persistence of the resource include (1 ) the
high degree of communication among the three parties involved in
the management strategy. (2) the presence within the industry of a
few individuals who took a long-term and relatively conservative
management view and who were generally respected by others in
the industry; and (3) the perception of a shared risk among industry
members, which also constrained their activities.
Not all aspects of the Delaware Bay management system may
apply to other fisheries. For example, the fishery has relatively few
participants who operate in a geographically constrained area. Part
of the resource lies within an area where diseases and predators are
absent or reduced by prevailing environmental conditions. Both of
these conditions reduced the scale of management complexity in
the present case. However, several characteristics of this fishery
and its management structure could be exported to other locations.
We argue they include the following: Human harvest activities on
some parts of the resource need lo be limited. Long-term, reliable,
third-party monitoring of the resource, disea.ses, and harvest ac-
tivities should be integrated as a consistent part of the decision
processes of the management structure. Continued personal con-
tact through meetings, discussions and working together is essen-
tial in transmitting information. Last and most importantly, the
participants in the fishery should agree on the basic goals of the
program and all must play a role in the management of the bed and
its dependent fishery. Participating groups must agree on their
respective formal roles, restrain themselves from "stepping be-
yond"" their areas of expertise, and respect the role and viewpoints
of the other participants.
ACKNOWLEDGMENTS
A large number of individuals contributed to the projects de-
scribed in this paper. Three contributed more than the rest: Labo-
ratory biologist, Donald Kunkle, and the two boat captains over the
period from 1953 to 1990, William Richards and Clyde Phillips. In
recent years, the supportive efforts of J. Dobarto and R. Reed of
the New Jersey Bureau of Shellfisheries have been substantial.
Financial support was received for much of this research from the
State of New Jersey and from Public Law 88-309 funds. The
authors thank Walt Canzonier for his comments and insight. This
is New Jersey Agricultural Experiment Station Publication No.
D-32403-1-03 and contribution no. 2003-19 from the Institute of
Marine and Coastal Sciences, Rutgers University.
APPENDICES
/. An Example of Shellfish Council Deliberations Before the Advent
of Large-Scale, Direct Marketing from the Seedbeds
The following account describes deliberations by the Delaware
Bay Shellfish Council during bay season of 1995. Oyster planters,
representatives of the New Jersey State management agency, Rut-
gers University personnel, and shellfish council members partici-
pated in what was often a chaotic discussion. However, a consen-
sus was reached. A cursory description is presented here to provide
an example of the issues considered when making decisions and of
how biologic information provided by the University was inte-
grated with economic realities faced by planters.
Bay season had begun on 10 April and was scheduled to last for
a minimum of two weeks. A decision on the closing date was to be
made near the end of the second week. On 20 April 1995, the
shellfish council met to examine dredge samples that had been
collected from the beds that day and to consider extending bay
season. By that date approximately 3000 bu of oysters had been
marketed directly from the beds at approximately $15-$ 17 per
bushel. A little more than 20 boats harvested (seed for planting
plus direct market) a total of about 100,000 bushels. Most of the
harvest was from New, Bennies, and Bennies Sand Beds. Some
harvest was from Ledge Bed. Sampling to determine percent oys-
ter on the beds was conducted on 13 April and 20 April from
Bennies Sand and New Beds, and from New Beds on 20 April.
Mean percent oyster was high on both dates (Bennies Sand = 61%
and New Beds = 63% on the 1 3th and New Beds = 62% on the
20"^).
Although there was general agreement that plenty of oysters
remained on all of the beds, several other concerns were discussed.
First, prices for oysters marketed directly from the beds were low
and only 3' oysters were acceptable. This meant that a good deal
of costly on-board sorting was required to produce a marketable
product. Second, as nearly all seed was infected with P. marinus,
any oysters planted on the leased grounds would have to be mar-
keted before July to avoid mortality. Third, the season had been
Management of New Jersey's Oyster Seedbeds
463
good so far. Transplanting more oysters to the planting grounds
would likely lead to decreases in profit because summer prices are
usually low and the cost of moving oysters might not be recovered
if subsequent mortality was high. Fourth, if oysters were not
moved and they died on the seedbeds, at least the shells would
remain as cultch. Fifth, if the beds remained open, everyone would
keep fishing in spite of the economic risk. After listening to all
these issues the shellfish council opted for a conservative strategy
and decided to close the seedbeds for the season.
//. An Example of Shellfish Couneil Deliberations After the Advent of
Direct Marketing from the Seedbeds
Direct marketing of oysters from the seedbeds has had mi.xed
results. This process provided $4.3 million in revenues to the in-
dustry for harvests in 1996. 1997. and the spnng of 1998, and
allowed the industry to maintain a presence in the markets and
maintain boats. Tag fees provided for an enhanced shelling effort.
The down side to this form of landing was that the industry was
restricted to the time period agreed to and could not stockpile
oysters on the planted grounds to satisfy markets at other times.
Because the oysters were harvested from lower salinity waters, the
meat quality was not as good as in oysters from farther down bay
and the price received for the product was not as high as it might
have been. Chiefly because of these latter conditions, some indus-
try members wished to plant oysters.
The State achieved direct revenue ($1.25/bu) from oysters re-
moved from the seedbeds for market, but would only receive pay-
ment on planted oysters once they were landed. Thus in the former
case the State (and directly the oyster industry accounts) received
payment up front, while in the latter case the State took on the
majority of the risk. If the oysters died on the planted grounds the
resource would not be paid for. the shell would no longer be on the
seedbeds, and no funds would have been generated to replace it.
In 1997 the State and industry agreed to a spring direct harvest
followed by an evaluation of the seedbeds to determine if a plant-
ing season could be allowed in the summer of 1998. The chief
reason for the planting would be to allow meat quality to improve
during the late summer and fall. The chief worry was the level of
the oyster disease Dernio. University researchers sampled for
Dermo and reported to the council in an open meeting. Samples
were removed in July from the five beds deemed by the industry
to have the greatest probability of being harvested. The samples
revealed that oysters on all beds were heavily infected with Dermo.
The summer had been hot and dry and the forecast was for a
continuation of these conditions.
The discussion in the August 1998 council meeting was heated
because some segments of the industry wished to move oysters
anyhow, while others were reluctant to risk the resource. The latter
group said that the resource would remain for later harvest if it was
not moved. The group finally agreed to wait and monitor condi-
tions further. Laboratory researchers took samples in August. Con-
ditions had not improved and the Council deferred a seed move
and decided to allow direct market harvest to begin (1500 bu/
license! beginning on 17 August. The council requested a Septem-
ber sample of disease prevalence: it remained high. The council
decided to have a 5-d transplant in an 8-d period beginning 7
October. To participate each boat would have to participate in a
one day intermediate transplant (.5 and 6 October) in which oysters
from up bay would be moved to an intermediate bed. Direct market
harvest would cease when the transplant began. A meeting was
scheduled for 1 October to make final adjustments to this plan. In
October the direct market program allocation was increased by
1000 bu/license. otherwise the transplant program was to occur as
decided earlier.
As of the November council meeting the direct market program
had landed approximately 73.000 bu: 10.000 bu were moved in the
intermediate transplant and 58.800 bu were transplanted to the
leases.
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Jotinuil ofShelljhh Rcscanh. Vol. 22. No. 2, 465-474, 20U3.
INFLUENCE OF TIMING OF BROODSTOCK COLLECTION ON CONDITIONING, OOCYTE
PRODUCTION, AND LARVAL REARING OF THE OYSTER, CRASSOSTREA GIGAS
(THUNBERG), AT SIX PRODUCTION SITES IN FRANCE
JORGE CHAVEZ-VILLALBA,'* JEAN BARRET." CHRISTIAN MINGANT,- JEAN-
CLAUDE COCHARD,- AND MARCEL LE PENNEC'
'U.M.R. C.N.R.S. 6539. Institut Univcrsitaire Eitropeen de la Met: 29280 Plouzane. France:
-IFREMER, Centre de Brest, Udnmitoire de Physiologie des Invertebres. BP 70. 29280 Plouzane.
France
ABSTRACT Ganietogenic development and response to conditioning procedures of six samples of oysters Crassoslrea gigas
(Thunberg) collected in the Bassin d'Arcachon. each cultivated at a different production site along the Atlantic coast of France were
compared simultaneously from December 1998 to July 1999. Oysters were conditioned with and without food (fed oysters and unfed
oysters, respectively). Samples at northern production sites (Bale des Veys, Aber Benoit, and Baden) initiated gonadal development
and spawning about one month earlier than those at southern production sites (Bouin. La Tremblade, and Arcachon). Three condi-
tioning expenments (December 1998 to February 1999. February to April 1999. and April to June 1999) favored Bale des Veys and
Aber Benoit oysters, because these resulted in higher body component indices and higher proportions of mature oocytes in the three
conditionings that produced more gametes than the other samples in all expenments. Unfed oysters from Bale des Veys and Aber
Benoit produced viable gametes and larvae in all the experiments. No significant difference was observed in larval culture (growth and
mortality I among samples, of both fed and unfed animals. Differences in the timing of gametogenesis and response to conditioning
among northern and southern samples seem adaptive and non-genetic in nature, since all oysters were collected from the same
population in the bay at Arcachon. Nutrient recycling seems to have been an important regulating factor for gametogenesis in the
northern samples. The occurrence of oysters in different locales having differences in the timing of gametogenesis and response to
conditioning has implications for spat production in hatcheries.
KEY WORDS: conditioning. Crassoslrea gigas. gametogenesis, larvae, oocytes
INTRODUCTION
The Pacific oyster Crassostrea gigas (Thunberg) was intro-
duced to France to replace the Portuguese oyster C. angulata that
was decimated by a virus in the 1970s (Heral 1989). The Pacific
oyster exhibited high survival rates and adequate growth. Since
1982. spat importation from Japan and progenitors from British
Columbia were no longer needed because the collection of juve-
niles in French bays and lagoons became sufficient to sustain
oyster cultures (Mann 1983). The oyster industry expanded exten-
sively throughout this time and France became the fourth largest
oyster producer in the world in 1994. At the present time, there is
great inter-annual variability of spat setting at collection sites
caused by collector overcrowding and hydroclimate variations
(Robert & Gerard 1999). Moreover, the demand for spat increased
continually over time, but hatcheries produced only 10% to 15% of
the juveniles required by oyster farmers. Under these circum-
stances, national management programs are directed at studying
reproductive factors affecting this species under hatchery condi-
tions to improve current spat production procedures.
The culture of C. gigas in France is conducted in four stages;
(I) spat collection; (2) intermediate culture; (3) culture; and (4)
fattening (Cochard 1990). Juveniles (12 to 18 mo) are transferred
from collection areas at Marennes-Oleron embayment and Arca-
chon lagoon to production sites, where they continue development
until reaching commercial size (24 to 36 mo later, depending on
the region). In France, C. gigas reaches first sexual maturity in 12
*Corresponding author. Present address: Centro de Investigaciones Bi-
ologicas del Noroeste, Guaymas Unit. (CIBNOR), A. P. 349. Guaymas,
Sonera 85465. Mexico. E-mail: jechavez@cibnor.mx Fax: -1-52-622-221-
2238.
to 18 mo (Soletchnik et al. 1997). This means that oysters can
complete two reproduction cycles before the end of the harvest
period at production sites. Since oysters are exposed to fluctuations
in temperature, photoperiod. and quality and quantity of suspended
fine particulate matter (seston) that affect their physiology and
growth (Barille et al. 1994. Goulletquer et al. 1996). we would
expect important geographic variations of gametogenesis along the
French coast.
Under laboratory conditions, many factors including those af-
fecting gametogenesis and broodstock conditioning influence lar-
val development in both early and late juvenile stages (Martinez et
al. 2000). Le Pennec et al. (1998) pointed out that pectinid egg
development and consequent larval production are extremely vari-
able in hatcheries and that results are not reproducible from one
year to the next. For C. gigas. Lannan et al. (1980) demonstrated
that this variation is related to gonadal development of parental
oysters and that this involved environmental and heritable com-
ponents. Seasonal studies have shown that environmental factors,
such as temperature and food availability, are closely related to
reproductive performance in bivalves (Ruiz et al. 1992). For in-
stance, the quantity of phytoplankton in temperate and high-
latitude seas varies seasonally, producing cyclical changes in avail-
ability of nutrients (Gabbott 197.'i. Abad et al. 1995). Regulatory
substances with gonadotrophic action vary periodically, and they
play important roles in spawning and in maturation of oocytes and
adults (Deridovich & Reunova 1993).
To obtain gametes in hatcheries in an optimum state of devel-
opment, it is essential to know the gonadal stage of the parents at
the time of conditioning, as well as the rate of gametogenesis
during the conditioning intervals (Lannan et al. 1980). Moreover,
during the conditioning of broodstock from different localities,
seasonal variations in gonadal development must be identified
465
466
Chavez-Villalba et al.
(Chavez-Villalba et al. 2002). The objective of this study is to
discover the response to artificial conditioning procedures of oys-
ters originating in Arcachon, but cultivated in six different geo-
graphic regions of France. This was accomplished by simulta-
neously comparing conditioning with and without food, oocyte
production, and larval rearing during three consecutive periods of
oyster cultivation from Bale des Veys (BV), Aber Benoit (AB).
Baden (BA). Bouin (BO), La Tremblade (LT). and Arcachon
(ARJ.
MATERIAL AND METHODS
Experimental Conditions
At the end of November 1998, 150 oyster samples were taken
from each of six production areas along the Atlantic coast of
France, where they had been cultured in plastic bags on tables for
almost two years. These oysters had been collected in the Bassin
d' Arcachon, grown at Baden until they were 18 mo old. and dis-
tributed in March 1 997 to the various culture sites. Two sites are
situated along the English Channel: Bale des Veys (BV) in Nor-
mandy and Aber Benoit (AB) in Brittany. Four are located along
the Bay of Biscay on the Atlantic coast: Baden (BA), Bouin (BO).
La Tremblade (LT) and Arcachon (AR) (Fig. 1). Specimens were
obtained at the same time from all sites and transported immedi-
ately to the IFREMER center at Brest, where they were placed in
a tlow-through seawater system for one week. Temperatures in the
tanks were maintained in close proximity to those found at the
production sites at the time of collection; 10°C in December, 1 TC
in February and 12°C in April. The procedure was conducted three
times: December 98 (first conditioning), February 99 (second con-
ditioning), and April 99 (third conditioning). Additionally, oysters
were collected in June and July 99, but they were not conditioned
because they were mature (Lango-Reynoso et al. 2000).
After acclimation, each sample was divided into two groups for
conditioning and transferred to seawater maturation tanks, where
the temperature was increased 1°C per day until 19°C (heating
period) and the photoperiod was adjusted to 16 h of daylight.
Oysters were subjected to two conditions: with and without food.
The fed groups had a diet used commonly for conditioning in
experimental hatcheries: a mixture of two microalga species (10*^
cells of each species/day /animal) from monospecific cultures of
Isochrysis aff. galbana Green (Clone T-iso: Tahitian Isochrysis)
and Chaetoceros calcilrans Takano. The samples recovered in
June were tested for histology only, and the samples collected in
July were tested for histology and stripped immediately (see larval
culture).
Sampling
Upon arrival at the laboratory, 20 oysters were chosen ran-
domly from each sample for biometrical measurements. Weights
of whole animals, empty shells, and soft tissue were determined to
within ±1 mg using a digital balance. The soft tissues of 10 oysters
were freeze-dried during 48 h, and dry weights were measured.
The body component index of Walne and Mann (1975) was cal-
culated:
WMI--
DSTW* 1000
' DSW
where WMI is the Walne-Mann index, DSTW is the dry soft tissue
weight in grams, and DSW is the dry shell weight in grams.
Only fed oysters were considered for histologic study. Two
samples of 10 oysters each were taken from each group during the
conditioning experiments. The first sample was obtained before
the heating period, and the second sample was taken at the end of
the conditioning period. For individuals collected in June and July,
the histologic samples were taken immediately after biometric
measurements.
Semi-quantitative Histologic Analysis
Oysters used for histology were opened, and a section of ap-
proximately 1 cm"* visceral mass was taken from above the peri-
cardial area, and fixed in Bouin's solution for at least 48 h.
Samples were dehydrated with a series of ethanol treatments of
English Channel" "T^
Sale des^ys
o\
»
r— 1 .
Normandy
Aber BenoTt^^'^'*^ VO^S«^-^
_ ^ Brittany
BadeiA^-n
ATLANTIC Boul.j>
OCEAN A
~ Bassin de Marennes-Oleron^
>
La Tremblade
^
n Spat collection Bassin d'Arcachon
5
# Production sites 1
Figure L Location of the six Crassostrea gigas production sites studied. Spat collection and production sites are also marked.
Influence of Timing of Broodstock Collection
467
increasing concentration, cleared in toluene, and embedded in par-
affin following a standard procedure. Sections (5 (jini) were cut.
mounted on glass slides, and stained with Groat's hematoxylin and
eosin Y solution (Martoja & Martoja-Pierson 1967). The histology
slides were examined under a microscope connected to a video
camera to determin oocyte size and frequency, and gametogenic
activity. Recorded images were processed by digital image analy-
sis.
Oocytes were measured and histology classified following the
description by Lango-Reynoso et al. (2000). These operations
were conducted on 100 randomly chosen oocytes per oyster, and
measurements followed a standard bias reduction procedure for
selecting measurement fields. Transects of gonad preparations
were oriented to maximize coverage of the larger vertical or hori-
zontal oocyte field axis. All oocytes with a well-defined germinal
vesicle in a field were measured, and every oocyte measured was
assigned to a reproductive stage based on diameter and histologic
characteristics of the gonad (Table 1).
Larval Rearing (larval yield estimation)
Both fed and unfed oyster groups were considered for larval
rearing. Following seven weeks of conditioning, oysters were
opened and their sex was determined by observing a fresh smear
sample from the gonad under a microscope. After this procedure,
females and males were separated and gametes from both sexes
were recovered using the scarification technique described by
Allen and Bushek (1992). Gonads of all oysters were scarified by
a light incision of the gonadal tegument. Oocytes were collected in
beakers by rinsing the gonad with filtered seawater. Then the
oocytes were passed through a 60-(a.m sieve to eliminate undesir-
able material. Mature oocytes were retained in a 20-(xm sieve.
These were rinsed several times and placed in 2 or 5-L beakers. To
determine oocyte production, three 50-|jlL samples per group were
examined, and counted under a profile projector. Males underwent
the same procedure, but spermatozoa suspensions were examined
under a microscope for mobility. Batches of spermatozoa of low
mobility were discarded. A minimum of three batches was mixed
together and diluted 10- to 20-ml P' for fertilization. Oocytes were
fertilized in 3-L beakers, and checked for normal progress 0.5 to
I h later (Robert & Gerard 1999).
After fertilization, an equal number of embryos from all oysters
of each group were pooled together and placed one group per tank
in 150-L experimental tanks at concentration .^3 embryos per ml.
After 48 h the tanks were emptied and the larvae recovered by
sieving. Three 50-[xL larvae samples from each tank were taken
for larval yield estimation: number of D larvae after 48 h of cul-
ture/initial number of embryos.
Standard methods were used during larval rearing. Tank sea-
water temperature was maintained at 20°C throughout the experi-
ment, and larval diet consisted of a mixture of three microalga
species: 40% Chaetoceros pumilum. 40% l.soclirysis aff. gall'iiiui
Green (Clone T-iso. Tahiti Isochiysix). and 20% Pavlova hiilicri.
Feeding increased from an initial concentration of 80,000 cells/ml
to a final concentration of 150,000 cells/ml. Seawater in the tanks
was renewed every two days, and larvae were recovered by siev-
ing. Larvae were measured during the second, ninth, and sixteenth
days of culture by sampling I or 2 mL of seawater containing
larvae from each experimental tank after sieving. Larval samples
were placed on microplates and fixed with formaldehyde (5%).
Two or three pictures of each sample were taken using a Scioncorp
frame grabber and processed by image analysis for size evaluation
(Scion Image for Windows). Larvae length was deemed equivalent
to that of the major axis of the best-fitting ellipse.
Data Analyses
The oocyte proportion corresponding to each reproductive
stage was calculated according to Lango-Reynoso et al. (2000).
and arcsine transformed (Snedecor & Cochran 1972) for each oys-
ter. The logarithms of oocyte production data were calculated. The
transformed proportions and logarithms were compared using the
Kruskal-Wallis test. A two-way ANOVA test was used to examine
the effect of conditioning and sample on ( 1 ) early, growing, and
mature oocyte categories; (2) the Walne-Mann index; (3) oocyte
production; and (4) the D larval yield. A three-way ANOVA test
was run to analyze the effect of conditioning, days of culture, and
origin (sample) on larval culture. Statistics were analyzed at sig-
nificance level a = 0.05.
RESULTS
Gametogenesis
The results of oocyte evolution from December 98 until July
1999 are presented here without regard to conditioning experi-
ments. The mean oocyte size for each sample is shown in Fig-
ure 2. Oysters from the six samples had the same oocyte diameter
distribution except the AB sample that had degenerating oocytes in
December 1998. In this sample we detected that the proportion of
oocytes in the early gametogenesis stage increased significantly
(36.4-92.4%) at the same time as that of degenerating oocytes
decreased significantly (44.8-0.0%) from December to February.
TABLE L
Reproductive scale for Crassostrea gigas proposed by Lango-Reyno.so et al. (2000). Each reproductive stage is based on an oocyte diameter
(fim) interval. Cytological characteristics corresponding to each stage are included.
Stage
Interval
(Mm)
Histologic Description
Early gametogenesis
Growing
Mature
Degenerating
3.0-12.0 Follicles are elongated and often isolated in the abundant connective tissue, with walls consisting of primary
oocytes of homogeneous size.
12.1-30.0 Start of oocyte growth. A large range in oocyte size at all gametogenic stages can be observed, including some
free oocytes. Interfollicular connective tissue disappears.
30.1^1.0 Follicles of homogeneous size completely filled with mature oocytes with distinct nucleus.
41.1-60.0 Follicles containing degenerating oocytes, often elongated in shape, sometimes broken. Obvious redevelopment
indicated by increased number of primary oocytes.
468
Chavez-Villalba et al.
50
40
30
20
10
BV
a
o
AB
I I I
O 40
£
ra 30
0)
20
BA
>» 10-
o
o
O
50
40
30
20
10
— T- T I I I I I
LT
^
I I I
BO
T I I I I I I 1
s
AR
T
t
1
F=H=«=
DJFMAMJJA DJFMAMJJA
Time (1998 -1999)
Figure 2. Evolution of oocyte diameters (mean ± SD) over time in Crassoslrea gif;as specimens collected at six production sites on the Atlantic
coast of France: BV (Bale des Vevs), AB (Aber Benoit). BA (Baden), BO (Bouin), l,T (l.a Tremblade), and AR (Arcachnn).
In the other samples, oocytes at this stage did not change signifi-
cantly during the same period. In February, growing oocytes were
observed only in the oysters from BV. AB. and BA (4.0. 8.0. and
3.0% respectively). In all samples, oocytes in growing and mature
stages increased significantly from February to April, and from
April to June, respectively. The BV. AB. and BA samples, which
contained growing oocytes in February, were the same samples in
which mature oocytes decreased significantly from June to July.
The oocytes in the early gametogenesis and growing stages in-
creased significantly in the BV (2.4-28% and 18.6^7.6% respec-
tively) and AB (0.0-3.'^. 27r and 1.3^9.0% respectively) samples
during the same period. In the other samples (BA. BO. LT and
AR). no significant change was detected from June to July.
Conditioning
The results of the three conditioning procedures performed in
this study are presented in Figure 3. In the BV sample, the pro-
portion of growing oocytes increased significantly in the first (3.0-
18.8%) and second (3.7-30.7%) conditionings, and in the last ex-
periment the proportion of oocytes in this category decreased sig-
nificantly (57.0-7.0%). The proportion of mature oocytes
increased significantly in this sample in all the experiments. By the
end of the first conditioning, degenerating oocytes were no longer
observed in the AB sample, and there was no significant change in
growing oocytes, but the proportion of mature oocytes increased
significantly in the three conditionings. For the BA and BO
samples, the same pattern as for the AB sample was observed,
except during the second conditioning experiment when the pro-
portion of growing oocytes increased significantly (2.9-18.0% in
BA. and 0.0-27.2% in BO). The proportion of growing oocytes
also increased significantly in the second conditioning for the LT
sample (0.0-37.2%), but that of mature oocytes increased signifi-
cantly only in the second (0.0-41.5%) and third (0.0-89.7%) ex-
periments. Finally, the proportion of AR sample growing oocytes
increased significantly during the first (0.0-39.5%) and the second
(0.0-21.8%) conditionings but the proportion of mature oocytes
increased significantly only in the last experiment (7.0-88.2%).
The two-way ANOVA test on early gametogenesis, growing,
and mature oocytes showed that the effect of the sample was not
significant in any conditioning experiment, but the effect of con-
ditioning was evident on the proportion of early gametogenesis
stage oocytes, which increased significantly in the first and the
second conditionings. No significant conditioning effect was de-
tected on oocytes at the growing stage during the three experi-
ments. Finally, conditioning increased the proportion of mature
oocytes significantly during the second experiment.
Index
Values of the Walne-Mann index (WMI) are presented in Fig-
ure 4. The WMI increased significantly in BO (3rd experiment),
LT (2nd and 3rd experiments) and AR (1st and 3rd experiments)
by the end of conditioning. In the other samples no significant
difference was detected between the start and end of conditioning.
The two-way ANOVA test concerning the effect of condifioning
showed that WMI values in the third experiment were significantly
higher than those in the two previous conditionings, but these
Influench of Timing of Broodstock Collection
469
120
80
120
>> 80
u
C 40
0)
3 0
CT '20
0)
^ 80
First conditioning
S E
A^..^^^
Second conditioning
S E
.^^
-^^
A l/L-.^
k i/k
l\ |/ Wo>^
Third conditioning
S E
.^^'^^^
BV
--^^
AB
BA
.-J\
BO
LT
/^es=^
AR
40 $0 0
40 60
40 60 0 20 40 60
Oocyte diameter {\im)
Figure 3. Temporal variation in oocyte diameter of specimens conditioned with food, from six sites of Crassostrea gigas production durin;-
December 1998 to February 1999 (tlrst conditioning); February to April 1999 (second conditioning); and April to June 1999 (third conditioning).
Each line represents one individual. S (start of conditioning). E (end of conditioning), BV (Bale des Veys), AB (Aber Benoit), BA (Baden), BO
(Bouin). LT (La Tremblade), and AR (Arcachon).
values were significantly lower than those of oysters collected in
July. The WMI values of oysters from the AB sample were sig-
nificantly higher than those from the other samples, except BV.
Gamete Production
The highest gamete production was observed in oysters col-
lected in July except for those of BO, conditioned in April to June
(Fig. 5). The two-way ANOVA test showed that there were sig-
nificant conditioning and sample effects on gamete production. We
observed that gamete production increased significantly from the
first to the second experiment, and also from the second to the third
conditioning. No difference between the last conditioning and that
of oysters collected in July was observed. The AB sample pro-
duced significantly more gametes than that of BO.
We observed that only the unfed groups of AB and BV pro-
duced gametes during the first conditioning, and that the quantity
was significantly higher in AB oysters. In the second conditioning,
four groups produced gametes, and are ranked by gamete quality
as follows: AB, BV. BA. and BO. The AB and BV groups pro-
duced significantly more oocytes than the BA and BO groups. In
the third conditioning, two-way ANOVA revealed significant ef-
fects of conditioning and sample on gamete production. All groups
produced significantly more gametes except LT. which produced
no oocytes during the three conditioning procedures. AB oysters
produced significantly more gametes than any other group (Fig. 5).
D Larval Yield
The two-way ANOVA test showed no significant effect of
conditioning or sample on D larval yield of either fed or unfed
oysters. D larval yield for fed and unfed oysters during the three
conditioning experiments, as well as that for animals collected in
July are presented in Figure 5. During the first conditioning, the
highest larval yield (80%) was observed for fed oysters from BV,
while the lowest corresponded to those of AB and BA (28 and
22%. respectively). In the second conditioning, the BO group had
the highest percentage (90%), while the lowest (51%) was ob-
served for LT oysters. In the third conditioning, and for oysters
collected in July, we found that larval yield was homogeneous
(=60%) for all groups. We had technical problems with unfed
oysters during the second experiment, so the D larval yield was not
measured and consequently, no larvae were reared. Nevertheless,
we observed no significant difference in larvae yields between fed
and unfed oysters during the first and third conditionings.
Larval Growth
Three-way ANOVA demonstrated significant effects of condi-
tioning and time on size of larvae produced by both fed and unfed
oysters (Fig. 6). Larvae from fed oysters, were significantly larger
in the third conditioning than in the tlrst or second conditionings.
The first conditioning of unfed animals in groups BV and AB were
larger also. There was no significant sample effect on larval size of
fed or unfed animals. We compared the size of larvae from fed and
unfed oysters, and those of oysters collected in July on the last day
of culture (16th day), and found no significant difference.
DISCUSSION
The gametogenic development of Crassostrea gigas in this
study is similar to that reported by Lango-Reynoso et al. (2000)
for two populations in Brittany and one in Marennes-Oleron. We
observed that the gametogenic cycle (December 1998 to July
470
Chavez-Villalba et al.
120 1
100
80-
X
0)
•a
c
0)
c
5
60-
40-
20
Start of conditioning
I
I
1
Irt
1
jA^
xu
x
IJ
120-
100-
80-
60
40 -f
20
0
End of conditioning
123N 123N 123N 123N 123N 123N
BV
AB
BA
BO
LT
AR
Oyster samples
Figure 4. Walne-Mann indices (WMI I of specimens conditioned « ith food, troni six sites of Crassostrea gigas production during December 1998
to February 1999 ( 1 1, February to April 1999 (2|. April to June 1999 (3). and July 1999 (M. BV (Bale des Veysl, AB ( Aber Benoit), BA (Baden),
BO iBouinl. LT (La Tremblade) and AR (Arcachonl. An asterisk (*) represents a significant difference between WMIs at the start and end of
conditionings in a Kruskal-Wallis test [P < 0.05).
1999) of all samples examined in this study followed the same
pattern. Primary oocytes were evident from December to February.
In the Aber Benoit sample, a large proportion of degenerating
oocytes (457^) were detected in December, but not in February.
Degenerating oocytes occur because the oysters at this site have
partial spawnings from September to January, and gametes in the
gonad are reabsorbed very slowly (Chavez-Villalba et al. 2001 ).
Histologic observations show that only northern samples BV, AB,
and BA had growing oocytes in February. Oocytes grew in all
groups from February until maturity in June, and there were al-
ways more than 75% mature oocytes. Histologic observations
show that northern oysters spawned only partially between June
and July, and that the proportions of mature oocytes were 25%.
18%. and 52%, respectively. Moreover, we detected primary and
growing oocytes in the gonads of these samples during the same
period, indicating the development of a new oocyte generation. In
contrast, the proportion of mature oocytes of southern samples BO.
LT. and AR continued above 80%. Lango-Reynoso et al. (2000)
found that oysters at northern sites initiated gonad growth,
achieved maximal gonad development, and began spawning about
one month earlier than oysters from Marennes-Oleron. The results
of this study and those of previous experiments in our laboratory
(Chavez-Villalba 2001 ) were consistent with the observations of
Lango-Reynoso et al. (2000). Differences between northern and
southern oysters in the timing of gametogenesis during the condi-
tioning experiments revealed that northern samples perfomied best
in laboratory conditions. These oysters in the three conditionings
presented higher Walne-Mann index values and higher propor-
tions of mature oocytes and produced more oocytes than other
samples in all experiments. Moreover, unfed BV and AB oysters
produced viable gametes and larvae in all experiments. This dem-
onstrates that differences between northern and southern sites in
environmental influences regulate the initiation or completion of
gametogenesis.
Differences between populations in the term and extent of go-
nad growth, apan from genetic differences, suggest the existence
of environmental factors regulating gonad development (Barber et
al. 1991). The differences found in this study should not be con-
sidered genetic since all juvenile oysters were collected in the
Bassin d'Arcachon. Thus, we believe that there is intraspecific
variation in gametogenesis of C. gigas in France that is an adap-
tation to different local environmental factors. Dinamani (1987)
stated that the pacific oyster shows flexible reproductive behavior
that includes changes in timing and length of gametogenesis de-
pending on the environment in various regions of the world. It is
known that water temperature is a principal environmental factor
affecting gonad development in marine bivalves (Loosanoff &
Davis 1963). Goulletquer and Heral (1997) pointed out that the
temperate climate in France is affected by the Gulf Stream, with a
geographic barrier around Brittany, limiting the distribution of
marine species between the coldest regions in the north and warm-
est in the south. The fact that oysters from northern locations
Influence of Timing of Broodstock Collection
471
Gamete production x 10®
D larval yield (%)
80
70
60
SO
40
30
20
10
0
80
70
60
50
40
30
20
10
0
80
70
80
50
40
30
20
10
0
80
70
60
50
40
30
20
10
0
First conditioning
100
^ fl^ rl,
gl- ^^ ^
-^
1
1
a
CL
£D_
X
1
i
w wo w wo
BV AB
w wo w wo
BA BO
Oyster samples
Figure 5. Gamete production and D larval yield of specimens collected at the end of each conditioning with IW) and without (WO) food, at six
sites of Crassostrea gigas production during December 1998 to February 1999 (first conditioning), February to April 1999 (second conditioning),
April to June 1999 (third conditioning), and July 1999. BV (Baie des Veys), AB (Aber Benoit), BA (Baden), BO (Bouin), LT (La Tremblade),
and AR (Arcachon).
acclimated to lower temperatures than those from the south and
began gonad growth earlier, eliminates temperature as the single
regulator of gonad development in C. gigas. Goulletquer and Hera!
(1997) indicated that another difference between northern and
southern locations is variations in trophic conditions caused by
tidal effects. Tidal cycles can vary markedly in the quality and
amount of suspended particulate matter (Pastoureaud et al. 1996).
We believe that differences detected in this study result from varia-
tion in stored reserves that depend on food availability (Thompson
et al. 1996). This view is supported by MacDonald and Thompson
( 1988). who reported site-specific variation in the gonad develop-
ment of Placopecten inagellanicus. due to adaptation to local
variations in environmental factors, most notably food availability.
There is evidence that periods of reserve accumulation and
gamete production are temporally separated in temperate species
(Emmett et al. 1987, Thompson & MacDonald 1990). Berthelin et
al. (2000) found that re.serves in C. gigas are constituted during the
autumn and the winter, and that these reserves are used later in
472
Chavez-Villalba et al.
N
'55
(0
250
200
150
100
50
250
200
150
100
50
Fed oysters Unfed oysters
Bale des Veys
La Tremblade
Arcachon
16 2
Time (days)
16
Figure 6. Change in larval size until day 16 of culture of specimens conditioned with and without food, from six sites of Crassoslrea gigas
production during December 1998 to February 1999 (1). February to April 1999 (2), April to June 1999 (3), and July 1999 (N).
gametogenesis. This suggests greater food accessibility that fa-
vored nutrient accumulation in oysters from northern locations.
Some considerations for assuming this are for example, that the
region of Baie des Veys is a high carrying-capacity ecosystem
(Goulletquer et al., 1996) and that the national production program
of C. gigas in France gets the highest meat yield per year from the
oysters of Aber Benoit (Goyard 1997). In contrast. Bouin oysters
have poor growth rates and low biologic yields compared with the
rest of French oyster production (Gerard, 1995). Heral et al. ( 1986)
found evidence of biologic overload in the Marennes-Oleron basin
(La Tremblade) produced by a huge oyster biomass (95,000 tons),
and Pastoureaud et al. ( 1996) indicated low seston quality encoun-
tered by oysters in this bay. Barber and Blake (1983) suggested
that potential food supply for the scallop Argopeaen iiradians
decreases with latitude and that metabolic rate increases with tem-
perature. The metabolic rate in the Japanese oyster increases with
temperature (Bougrier et al. 1995). Previous observations suggest
that the metabolic rate of C. gigas increases with decreasing lati-
Influence of Timing of Broodstock Collection
473
tude. bill there is less food that results in less energy for repro-
duction.
It was significant that the best laboratory performance coin-
cided with partial spawning in nature. Ropert (1999) reported that
ovsters in Baie des Veys have a partial spawning during their
reproductive cycle, and Cha\ez-Villalba et al. (2001) found that
oocytes left from the incomplete spawning of Aber Benoit oysters
are slowly reabsorbed from September to January. This led us to
think that apart from the advantage of ambient food at northern
sites, it is possible that nutrient recycling from reabsorption of
unreleased gametes within the gonad is a regulating factor in the
timing of gametogenesis. Post-spawning reabsorption has been
observed in C. gigas. in which gametes remaining after spawning
are reabsorbed (Steele. 1998). Beninger and Le Pennec (1991)
suggest that reabsorption of residual gametes leads to nutrient
rec>cling in scallops. Le Pennec et al. (1991 1 found evidence for
lipid catabolism during reabsorption of unreleased gametes in
Pecten maximus and they suggested that the products of these
catabolic activities could be stored as glycogen. This was con-
firmed by several investigators, including Berthelin et al. (2000).
and could indicate that nutrients in northern oysters could be re-
cycled from residual gametes during the autumn-winter period and
then used for gametogenesis. It would be interesting to compare
the conditioning response of northern oysters maintained in natural
conditions throughout the year with those returned to natural con-
ditions in the autumn or winter after artificial spawn in July or
August.
We observed greater gamete production in fed oysters than
unfed. Robinson ( 1992) found comparable results when comparing
gamete production of C. gigas oysters maintained with and without
food. However, in our study the D larva yield of animals condi-
tioned with and without food was close, in particular for northern
oysters. Moreover, we found that there is no difference in larval
growth without considering broodstock culture conditions. It
seems that these oysters maintain oocyte quality by reducing their
number when there is not enough food.
Gametogenic cycles in bivalves are strongly tied to glycogen
stocking cycles and to ultimate synthesis, de novo, of lipids during
spring \itellosenesis. which depends on stocked glycogen (Gab-
bott 1975). Interruption of these cycles, due to artitlcial condition-
ing at high temperature, might force oocyte development before
sufficient glycogen has been accumulated for lipid synthesis. The
consequence might be production of few gametes with low bio-
chemical quality (Gallager & Mann 1986). Our observations sug-
gest that the stocking reserve in unfed oysters allows production of
fewer gametes of high quality. It seems that viability and survival
of reared larvae are directly related to the initial quantity of lipids
during gamete emission (Holland & Spencer 1973, Gallager &
Mann 1986). Apparently, unfed BV and AB oysters maintain their
lipid stock during conditioning, probably due to large glycogen
reserves, which assures not only lipid synthesis but also gamete
development.
When comparing lar\al development in the three conditioning
experiments, we observed that larval growth of fed and unfed
oysters is significantly inferior during the first two experiments.
Although Lannan et al. (1980) showed the importance of season in
the timing of broodstock collection for artificial conditioning, they
had no explanation concerning mechanisms that govern egg qual-
itv and the variability of survival during larval rearing. However,
Gallager and Mann (1986) noticed that growth and survival of
Mercenaria mercenaria and C. virginica larvae were associated
directly with the initiation and duration of conditioning. Berthelin
(2000) found that glycogen stores in the gonads of C. gigas during
autumn and the beginning of winter remained low in spring, while
proteins and lipids increase significantly from March to April,
coincident with the first phytoplankton blooms. Results of condi-
tioning during December to February suggest that reserves used
for larval growth in fed and unfed oysters were accumulated in
autumn and winter, and reserve allocation during spring increased
fecundity and larva growth but not necessarily D larval yields.
Knowledge of the general condition of animals before exposure
to experimental conditions is important to obtain gametes in the
optimum state of development. This study shows that the stored
reserves of northern oysters allow them to perform better during
conditioning than southern oysters. The existence of oysters hav-
ing distinct gametogenic development and therefore distinct re-
sponses to conditioning has implications for oyster spat production
in hatcheries. Broodstock from northern locations can be condi-
tioned starting in December because they mature after six weeks of
exposure at elevated temperatures (19°C). According to Chavez-
Villalba et al. (2002) this occurs because 60% of oocytes in the
gonad are mature after conditioning. The response of these oysters
to artificial conditions can be maintained throughout gametogenic
development, whereas the oysters from southern locations mature
only after commencing conditioning in April. These conditioning
experiments suggest that using oysters from northern locations in
hatchery operations should result in substantially increased hatch-
ery production.
ACKNOWLEDGMENTS
The authors thank CONACYT (Mexico) for a scholarship grant
to Jorge Chavez-Villalba for PhD studies at Universite de
Bretagne Occidentale, France. This work was supported by the
project IFREMER/Contrat Uni\ersitaire LTBO, No. 98/2521426.
Editing staff at CIBNOR reviewed and improved the English text.
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APPEARANCE AND PATHOGENICITY OF OVARIAN PARASITE MARTEILIOIDES
CHUNGMUENSIS IN THE FARMED PACIFIC OYSTERS, CRASSOSTREA GIGAS, IN KOREA
MI SEON PARK,* CHANG-KEUN KANG, DONG-LIM CHOI. AND BO-YOUNG JEE
National Fisheries Research & Development Institute. Sirani;-ri, Gijang-Guu. 619-902 Biisan, Republic
of Korea
ABSTRACT The ovarian parasite Marlcilioidcs chuui;mucnsis that ijilects the ovaries of Pacific oyster Crassostrea gigas ha.s
increased in frequency in farmed oysters on the southern coast of Korean peninsula since the early 1990.S. The appearance and
pathogenicity of the ovarian parasite in the farmed oyster in Jinhae Bay, Korea, were investigated in 1996 and 1997. Infection by M.
chiingiiiucnsis was highest during spawning (from June to August) and gonadal regenerating .season of the oysters (from September
to October), with prevalences ranging from 1,1. ,1 to 57.1% in 1996 and from 28.6 to 6l.59f in 1997, respectively. The surveyed oysters
showed signs of recovery from the infection after October. Glycogen levels were considerably lower in M. chiingiiuieiisis-\nfected
oysters those that of uninfected oysters. A rapid accumulation of glycogen was observed in uninfected oysters together with the gonadal
regeneration after the summer spawning. By contrast, no increase in glycogen content was found in infected oysters until the end of
the investigation. Lipid levels were slightly higher in the infected oysters than in the uninfected oysters. Serum protein concentrations
were significantly lower in the infected oysters than in the uninfected oysters. Also, the increase of serum protein concentration after
the summer spawning was apparent in the uninfected oysters but not in the infected oy.sters. These results indicate that the infections
by M. chungimiensis may have an adverse impact on metabolic recovery after spawning of the oysters.
A'£)' WORDS: Pacific oyster, Crassoslrea gigas. ovarian parasite, Marteilioides chungmiiensis
INTRODUCTION
The ovarian parasite Marreilloiiles chiingiiuien.^i.'^ is found in
the ovaries of the Pacific oyster, Crassostrea ,?(,?fl.s. The parasite is
a Protozoan belonging to the Phylum Ascetospora Sprague
(Comps et al. 1986, Park & Chun 1989). It is found in the cyto-
plasm of the ovum and measures 3-3,5 |jim in diameter (Comps et
al. 1986).
The effects of the ovarian parasite on the growth of oysters
have been studied for the last 10 years. Park et al. ( 1999) reported
that the parasite could induce ovary necrosis and prohibit normal
growth of fertilized eggs. Oysters infected by this parasite show
grossly visible ovary deformations, with lump-like hypertrophy
that renders them unmarketable. Therefore, the parasite is consid-
ered one of the most serious problems for oyster production in
Korea, The parasite has been found in oysters from almost all
oyster culture areas of Korea (Chun 1970, 1979. Park & Chun
1989, Park et al. 1999), although prevalence and intensity of in-
fection vary with region and season. Infection levels along the
southern coast have increased since 1990 and the oyster industry in
this area is facing increasing production challenges as a result of
poor seed collection.
Studies of M. cliiiii^mia'nsis have concentrated on histopathol-
ogy, infection dynamics, and transmission pathways, but little
work on the effects of the parasite on the physiology of the oyster
has been conducted. To better understand the effects of this ovar-
ian parasite on oyster aquaculture, this study examined the rela-
tionship between M. cluiiii>niuensis infection on the oysters and
gonad regeneration and its related physiologic parameters,
MATERIALS AND METHODS
Collection of Oyster Broodstock
Oysters were collected from the culturing sites around Chil-
cheon Island in Jinhae Bay of Korea (Fig. I). Although many
*Corresponding author. E-mail: parkms@nfrdi.re.kr
oyster beds still operate in this bay, sulTicient seed for stocking
purposes produced no longer in this formerly productive seed col-
lection area since 1 990. For broodstock sampling, one oyster string
was divided into three sections (upper, middle, and bottom), and
30 individuals were collected monthly from each section.
Examination of M. chungmuensis Infection Levels
Sampled broodstock were washed with clean seawater and
shucked by hand. A 3-mm thick dorsoventral cross section through
the anterior thiid of the soft tissues was fi,\ed in Davidson solution
and processed for light microscopy. Paraffin-embedded sections (4
jxm) were stained with Harris' hematoxylin-eosin for microscopic
examination. Because early stages of infection are difficult to de-
tect by light microscope, additional tissue smears were made from
ovarian tissues and stained with eosin-methylene blue (Fig, 2).
Evaluation of Infection Levels and Oyster Condition Factors
The oyster samples were divided into two groups, infected and
uninfected, based on gross evidence of M, chungmuensis infec-
tions, to evaluate infection effects on specific oyster condition
parameters. To have an accurate gross indication of infection, oys-
ters were collected from May to September when infections are
most obvious to the naked eye (Fig. 3). Infection levels were
divided into two levels by observation of slides that were smeared
with reproductive tissues and stained with eosin-methylene blue
(H group: heavy infection of >50'yf prevalence; M group: moderate
infection of <50'7r prevalence). Of condition parameters, glycogen
content (excluding ovarian tissues) was measured using the
method of Whyte and Englar (1982); biochemical composition of
the meat using the AOAC method (1990); and serum protein con-
centrations using the Lowry method (1951).
RESULTS
M. chungmuensis Infection Levels
Prevalences of infection from 1996 to 1997 ranged between 0.0
to 61.5% (Fig. 4), Infection levels of M, chungmuensis were high-
475
476
Park et al.
K ORE A
A ^^
(^
■n\
.o.
128°10'
128° 30-
Figure 1. Map showing sampling site in Jinhae Bay, Korea.
128°:50-E
34°55'N
34° 50-
=J34°45'
est in September 1996 (57.1%) and in August 1997 (61.5%). No
infections were detected from January to April 1996. In 1997, the
parasite was detected all the year round, with highest prevalences
in August.
Correlation Condition Factor to Levels of M. cliungmuensis Infection
Glycogen
Monthly mean glycogen levels ranged from 2.0 to 14.8%, from
2.1 to 16.0%, and from 4.0 to 20.2% in H group, M group, and
uninfected oysters, respectively (Fig. 5). Glycogen levels in all the
three groups were maxima in May and minima in August. Glyco-
gen levels in the infected oysters were similar between H and M
groups but consistently lower than in uninfected oysters (analysis
of variance. P < 0.01 for all the sampling months. Table 1). The
glycogen level increased abruptly in September when the gonadal
tissues were regenerated (Fig. 5). However, recovery of glycogen
level after summer spawning was observed in the infected oysters.
Biochemical Analysis of Oyster Tissue
Lipid levels fluctuated in the narrow ranges from 11.5 to
14.0%, from 10.0 to 13.0%. and from 5.0 to 9.0% in H group.
"juT^IidFtMBWGW'^AOW '^^■■H Figure 3. The external view of an oyster with an advanced M. cliun-
Figurc 2. I'hotdmicrograph of a smear preparation from a heavily gmHfn.vis infection associated with ovarian hypertrophy, rendering the
infected ovary. Pa, parasite. Eosin-methylene blue (x200). meat 'iumpy" in appearance.
Appearance and Pathogenicity of Ovarian Parasite M. chungmuensis
All
Figure 4. Monthly variations of the % prevalence of the ovarian
paraste M. chungmuensis of the Pacific oyster, Crassostrea gigas.
i
■ Heavy infection
■ Moderate infection
nUninfection
^
i\
jH
m
Figure 5. Monthly variations of levels (% of dry tissue weight ± 1 SD)
in the infected and uninfected oysters from May to September.
M group, and uninfected oysters, respectively (Fig. 6). The lipid
contents in the infected oysters were similar between H and M
groups (paired /-test. P = 0.174) and showed slightly greater
levels than those in the uninfected oysters (paired t test. P = 0.06
for both infected groups). Protein levels ranged from 56.4 to
62.09^, from 55.1 to 58.9% and from 53.2 to 55.8% in H group. M
group, and the uninfected oysters, respectively. No apparent dif-
ferences were found between infected and uninfected oyster
(paired r-test, P = 0.111 between H and M groups, P = 0.562
between H-group and the uninfected oysters, and P = 0.673 be-
tween M group and the uninfected oysters). Carbohydrate levels
ranged from 4.2 to 17.0%>, from 9.1 to 20.1%. and from 8.7 to
25.6% in H group. M group, and the uninfected oysters, respec-
tively. Because glycogen levels accounted for most of total car-
bohydrate levels, temporal variations of carbohydrate levels par-
alleled those of glycogen with maxima in May and minima in
August (Fig. 6). Ash levels ranged from 1 1.5 to 21.1%. from 16.0
to 25.1%, and from 10.7 to 25.6 in H group, M group, and the
uninfected oysters, respectively, with maxima in August and
minima in May. No pronounced correlation to infection was found
for ash content (paired /-test. P = 0.930. P = 0.384, and P =
0.085. respectively, for the same paired variables as the statistical
treatment of protein content).
Serum Protein
Mean serum protein concentration ranged from 3.1 to 4.0 p,g/
(jlL, from 3.7 to 6.2 |jig/|jiL, and from 5.1 to 11.4 (jig/(jiL in H
group, M group, and the uninfected oysters, respectively (Fig. 7).
Serum protein concentrations in the infected oysters were signifi-
cantly higher in H-group than in M-group (analysis of variance, P
< 0.001 for each sampling month except September). Serum pro-
tein concentrations were then significantly lower in infected oys-
ters then in uninfected oysters (analysis of variance, P < 0.001 for
all the sampling months. Serum protein concentration in unin-
fected broodstock was highest in May and lowest in August, with
an obvious increase after the summer spawning of the oysters.
However, in H-group oysters, the low concentration of mean s 4.0
|jLg/fj.L remained constant from May to September. In M group
oysters, the serum protein concentration was highest in May and
lowest in August-September. Finally, no increase in the serum
protein concentration after the summer spawning was observed in
both groups of the infected oysters.
TABLE 1.
Results of ANOVA and Tukey post-hoc test (a = 0.05) for absolute values of tissue glycogen and serum protein in each sampling month.
Uninfected
Parameters
Month
H Group
M Group
Oysters
P
Tissue glycogen (% of dry tissue)
May
14.8 ±2.5
=
16.0 ± 1.8
<
20.2 ± 2.6
<0.001
June
11.9 + 2.2
=
13.5 ±2.0
<
16.8 ±0.5
<0.001
July
3.8 ± 0.5
=
4.7 ±1.2
<
5.7 ± 0.9
<0.001
August
1.8 + 0.6
=
2.2 ± 0.5
<
4.0 ± 0.5
<0.001
September
2.0 + 0.7
=
2.1 ±0.7
<
6.8 ± 0.6
<0.001
Serum protein ((jLg/|j.L)
May
4.0 ± 0.7
<
6.2 ±0.7
<
11.4 ±0.5
<0.001
June
3.5 ±0.4
<
6.2+1.3
<
8.3 ± 0.4
<0.001
July
3.7 ± 0.5
<
4.8 ± 0.3
<
7.2 ±0.5
<0.00l
August
3.1 ±0.5
<
4.1 +0.5
<
5.1 ±0.8
<0.001
September
3.1 ±0.7
=
3.7 ± 0.7
<
6.5 ± 1.0
<0.001
Data represent mean ± 1 SD.
478
Park
ET AL.
Heavy infection
100%
■ ■ ■ ■
"■1
100%
80%
'II
1
80%
s
60%
60%
g
m
40%
llll
1
40%
20%
1 1 1 1
1
20%
0%
■ ■ ■ ■
JU
0%
Moderate infection
I ■ ■ ■ ■
Uninfection
CO
1 00%
80%
60%
40%
20%
0%
■ Ash
■ Carbohydrates
D Total Lipids
■ Proteins
M J J A S
Figure 6. Monthly variations in the biochemical composition of infected and uninfected oysters.
DISCUSSION
Park and Chun (1989) reported M. chungmuensis infection
prevalences of 5.3-6.7% between 1986 and 1987 in the oyster
growing area of Hansan-Geoje Bay in the southern coast of Korea.
i
■ Heavy infection
■ Moderate infection
D Uninfection
i
JL
1
Figure 7. Monthly variations of serum protein concentrations [\i?J\il^
± 1 SD) in the infected and uninfected oysters from May to September.
The parasite was detected from June to October. Park et al. ( 1999)
reported prevalences of 15.0-18.6% in oysters from Goseong Bay
and Geoje Bay in 1993, with infections detected from May to
September. During a subsequent survey in 1997. the parasite was
detected all the year round, with an average prevalence of 26.6%.
Therefore, the prevalences of M. chimginiiensis have been in-
creased annually during last decade in Korea.
M. chungmuensis only infects oyster ovarian tissues, inducing
necrosis of the ova and massive hypertrophy of the gonad. The
parasite impedes development of the fertilized eggs (Matsuzato et
al. 1997, Matsuzato & Masumura 1988, Park & Chun 1989). Park
et al. (1999) compared infected eggs with uninfected eggs and
concluded that the infected eggs did not undergo fertilization. And
also uninfected eggs isolated from infected oysters could be fer-
tilized but their growth pattern was abnormal. More than 80% of
the fertilized eggs which were from infected oysters showed ab-
normal shapes and died during the early umbo stage. To date, there
is no clear evidence that the parasite M. chungmuensis induces
mortality of the mature oyster.
Glycogen reserves have been considered to be the main energy
reserves both for the formation of gametes of marine bivalves,
especially under conditions of nutrient stress and also for the main-
tenance during nutritional stress (Beninger & Lucas 1984, Enco-
mio & Chu 2000). It is accumulated in storage tissues of the
digestive gland, gonad, and mantle (Berthelin et al. 2000). Glyco-
gen reserved in the gonad and mantle is used for gamete matura-
Appearance and Pathogenicity of Ovarian Parasite M. chungmuensis
479
tion. Lower content of glycogen in infected hroodstock indicates
that the parasite may directly reproductive success. Serum protein
increased after the summer spawning in the uninfected oysters, but
decreased in infected oysters. Reduced serum protein as well as
reduced glycogen content may exacerbate morbidity, but there is
no clear evidence to date that this has a direct correlation to mor-
tality of oyster hroodstock. Protein may not be used for gameto-
genesis, but is considered to be an essential metabolic requirement
(Berthelin et al. 2000).
Increased serum protein after spawning is normal; however,
this did not occur in infected oysters examined. Thus, as with
tissue protein, the effects of infection may have a significant meta-
bolic impact, whereby the drop in prevalence of infection in Oc-
tober was caused the death of heavily infected oysters, rather than
recovery from infections.
The prolistan parasite Perkiiisus nuiiiinis has been responsible
for high mortality of eastern oyster Crassostrea virginica in the
United States. The physiologic effects of P. marinus infection are
most apparent as a reduction in growth rate as well as reproductive
capacity (Barber and Mann 1994, Paynter 1996, Dittman et al.
2001). The physiologic effects of M. chungmuensis infection on
Crassostrea gigas may reduce reproductive capacity of oyster
population in Korea.
ACKNOWLEDGMENTS
The authors thank Dr. Sharon E. McGladdery at Department of
Fisheries and Oceans Canada, Gulf Fisheries Centre, Centre des
Peches du Golfe DFO Headquarters, Moncton, Ottawa, Canada for
her critical corimients and suggestions on the article. This work
was supported by the Ministry of Maritime Affairs and Fisheries-
Special Grants for Fisheries Research and Development Project in
Korea.
LITERATURE CITED
AOAC. 1990. OtTicial Method.^ of Analysis of the Association of OtTicial
Analytical Chemists. 15th Ed. Arlington. Virginia: Association of Of-
ficial Analytical Chemists Inc.
Barber, B. J. & R. Mann. 1994. Growth and mortality of eastern oyster,
Crassostrea virginica (Gmelin, 1791 ). and Pacific oysters, Crassostrea
gigas (Thunberg. 1793) under challenge from the parasite. Perkinsus
marinus J. Shellfish Res. 13:109-114.
Beninger, P. G. & A. Lucas. 1984. Seasonal variations in condition, re-
productive activity and gross biochemical composition of two species
of adult clam reared in a common habitat: Tapes ileciissatiis L.. (Jef-
freys) and Tapes philiphiiiariim (Adams and Reeve). J. Exp. Mar. Biol.
Ecol. 79:19-37.
Berthelin. C. K. Kellner & M. Mathieu. 2000. Storage metabolism in
Pacific oyster [Crassostrea gigas) in relation to summer monalities and
reproductive cycle (west coast of France). Comp. Biochem. Physiol.
125:359-369.
Chun, S.-K. 1970. Diseases of oyster. I. Pathological study. Bull. Korean
Fish. Sac. 5:1-7.
Chun. S.-K. 1979. Amoeba infection on oyster. Bull. Korean Fish. Soc.
12:281-285.
Comps, M., M. S. Park & I. Desportes. 1986. Etude ultrastrurale de Mar-
teilioides chungmuensis n.g., n. sp. parasite des ovocytes de I'huitres
Crassostrea gigas) TH. Prolistologica 22:279-285.
Dittman, D. E., S. E. Ford & D. K. Padilla. 2001. Effects of Perkinsus
nmriiuis on reproduction and condition of eastern oyster, Crassostrea
virginica. depending on timing. J. Shellfish Res. 20:1025-1034.
Encomio, V. & F.-L. E. Chu. 2000. The effect of PCBs on glycogen
reserves in the eastern oyster Crassostrea virginica. Mar. Environ. Res.
50:45-49.
Lowry. O. H.. N. J. Rosebrough, A. L. Farr & R. J. Randall. 1951. Protein
measurement with the Folin phenol reagent. J. Biol. Chem 193:265-
275.
Matsuzato. T. & K. Masumura. 1988. Abnormal enlargement of the ovary
of oyster. Crassostrea gigas (Thunberg) by an unidentified parasite.
Inter. J. Aqua. Fish. Tech 9:3-7.
Matsuzato, T., T. Hoshina, K. Y. Arakawa & K. Masumura. 1977. Studies
on the so-called abnormal egg-mass of Japanese oyster, Crassostrea
gigas (Thunberg). Distribution of the oyster collected in the coast of
Hiroshima Pref., and parasite in the egg-cell. Bull. Hiroshima Fish.
Exp. St. 8:9-25.
Park. M. S. & S. K. Chun. 1989. Study on Marteilioides chungmuensis
Comps et al., 1986 parasite of the Pacific oyster, Crassostrea gigas
Thunberg. / Fish Pathol. 2:53-70.
Park. M. S.. H. Y. Lyu & T. S. Lee. 1999. Investigation on the cause of bad
natural seed collection of the Pacific oyster. Crassostrea gigas: rela-
tionships between the conditions of mother shell and the viability of the
released eggs and larvae based on the pathological and embryological
survey. J. Korean Fish. Soc 32:62-67.
Paynter, K. T. 1996. The effects of Perkinsus marinus infection on physi-
ological processes in the eastern oyster. Crassostrea virginica. J. Shell-
fish Res. 15:119-125.
Whyte. J. N. C. & J. R. Englar. 1982. Seasonal variation in the chemical
composition and condition indices of Pacific oyster, Crassostrea gigas.
growing in trays and on the sea bed. Can. J. Fish. Aquat. Sci. 39: 1084—
1094.
Joumcit ofShetlfish Research, Vol. 22, No. 1. 481-185. 2003.
MOLECULAR PHYLOGENETICS OF FIVE CORBICULA SPECIES DETERMINED BY PARTIAL
28S RIBOSOMAL RNA GENE SEQUENCES
GAB-MAN PARK'*AND EE-YUNG CHUNG"
^Dcpcirtmeii! o) Parasitology. Kwamhmg University College of Medicine. Gangnimg. Gangivon-Jo
210-701. Korea; 'Department of Marine Living Re.wurces. College of Ocean Science anil Technology,
Knnsan National University. Knnsan 573-701. Korea
ABSTRACT Partial 28S rihosomal RNA (rRNA) gene sequences of five species (C fliiminea. C. papyracea. and C leana from
Korea. C. japonica from Japan, C. larifillifrii from China) in the genus Corbkuhi were investigated for their genetic divergence.
Neighbor-joining analysis on the alignment of 412 base pairs of C. flmninea. C. largillerti. C. papyracea. C. leana and C. japonica
(with Polymesoda maritima. P. caroliniana and Sphaerhtm comeiim chosen as an outgroup) provides a robust molecular phylogeny
for the genus; (C. japonica, C. papyracea. C. largillieni. C. leana. C. fluminea. P. maritime. P. caroliniana. and S. corn
results of this study provide potential use of 28S rRNA gene sequence for phylogenies in the family Corbiculidae.
KEY WORDS: Corbiculoidea. Corbiciila spp.. 28S rRNA. phylogeny, China. Korea. Japan
, The
INTRODUCTION
Corhicula is conservative, possessing few moiphologic char-
acters useful for species discrimination and displaying a broad
range of subtle variability, especially with respect to shell form and
color. The genus Corbicula is present in freshwater, brackish wa-
ter, and estuaries in southeastern Asia, Africa, the Indian subcon-
tinent, the Pacific Islands, and South America, where it is an
important component of benthic communities in both lentic and
lotic environments (Leveque 1973, Britton & Morton 1979). In
Korea, six species, C. fluminea, C. leana, C. fenouilliana, C. pa-
pyracea. C. colorata and C. portentosa, are recognized based on
shell form {Kwon et al. 1993).
Corhicula species can be categorized as 3 major groups based
on reproductive characters and ecology (Miyazaki 1936); the spe-
cies belonging to Group 1 are monoecious, viviparous, and incu-
batory. They have nonswimming planktonic veliger larvae and live
in freshwater; the species belonging to group 2 are dioecious,
oviparous, nonincubatory, and also live in freshwater regions; the
species belonging to group 3 are dioecious and oviparous. They do
not incubate the young, has free-swimming planktotrophic larvae
and live in brackish waters. The phylogenic relationship among
these three groups cannot fully be clarified with these taxonomic
characters alone. Recently, the chromosome numbers and the de-
grees of genetic differentiation from a Hmited number of species of
the genus Corbicula have been investigated (Okaiiioto & Arimoto
1986, Lee & Kim 1997, Park et al. 2000).
Within the tandemly repeated rRNA gene complex, coding
sequences for small (18S) and large (5.8S + 2SS) subunit rRNA
components are flanked by nontranscribed and internal transcribed
spacer regions. As a result of functional constraints within the
ribosome. coding regions are in general more conserved than the
spacer regions (Raue et al. 1990, Mulvey et al. 1998). The 28S
rRNA gene contains "conserved" core regions interspersed with
more variable "expansion segments" or domains, designated D 1 to
D18 (Raue et al. 1988). Hillis and Dixon (1991) reported that, if
chosen carefully, many divergent domains in the gene coding for
large subunit ribosomal RNA are useful for reconstructing recent
events. Sequence data from the 28S rRNA gene have been suc-
*Corresponding author: Tel: -1-82-33-649-7480; Fax: -1-82-33-641-1074;
E-mail: gmpark@kwandong.ac.kr
cessfuUy used for intergeneric resolution within the Corbiculoidea
(Park & Ofoighil 2000). This study is base on analysis of se-
quences from 5' end 28S rRNA gene five common Corbicula
species.
MATERIALS AND METHODS
Sample Collection and DMA Extraction
Five Corbicula species C. fluminea (Cheorwon. Gangwon
Province); C. papyracea (Yeongwol, Gangwon Province); C.
leana (Wanju, Chunbuk Province) from Korea, C. japonica
(Iwaki, Fukushima Province) from Japan and C. largillieni
(Tung-ting lake, Hunan Province) from China were analyzed and
nucleotide sequences were applied to five specimens in each spe-
cies (Fig. I). Polymesoda maritima (Corbiculidae, GBDB Acces-
sion no. AF1310I0), P. caroliniana (Corbiculidae. GBDB
AF131011) and Sphaerium cornium (Sphaeriidae), GBDB
API 3 101 3) were also analyzed as an out group. Voucher speci-
mens of the Corbicula species used in this study have been placed
in the Department of Parasitology, Kwandong University College
of Medicine. Korea.
Genomic DNA was isolated from fresh tissues using DNeasy
Tissue Kit (Qiagen #69504) following manufacturers instructions.
The 28S gene regions were amplified by the polymerase chain
reaction (PCR) from 20 to 40 ng of genomic DNA. For the 28S,
primers used were forward 5'-GATTACCCGCTGAACTTAAG-
CATAT-3' and 5'-GCTGCATTCACAAACACCCCGACTC-3'
reverse and DIF and D6R were used (Park & Ofoighil 2000). PCR
amplification was conducted over 40 cycles using the following
conditions; 1 min at 95'"C. 1 min at 54°C, and 1 .5 min at 72°C with
a final extension of 7 min at 72°C. The PCR products were purified
A B C D E
Figure 1. Shells of Corbicula species. A, Corbicula fluminea; B, C.
leana; C, C. papyracea; D, C. japonica; E, C. largillierti.
481
482 Park and Chung
1. C. fluminea AACCAGGATTCCCCCAGTAACGGCGAGTGAAGCGGG-AAGAGCCCAGCACCGAATCTCCC
2. C. largillierti -
3 . C . papyra cea G • ■ • •
4 . C. japonica - . . . .
5. C. leana T G----
6. P. maritima •••A TT ---A-
7. P. cariliniana •••A T -....
8. S. corneum •••A TT - CT-
1. GGCCTGACGGGCGGCGAGAAATGTGGTGTATAGGCGGCCGATTGTTGCCGGGTCCGGCGCTCAA-GTCCTCCTGATCGTG
2 . CG A -
3. CG-
4. CG-
6 . ATG CC TT •
7 . - • • • ATG C CG •
8 . • A - • ATG ■ • • C - - • • AG C ■ • • AA GC - • AGTC •-G-T-C---G--A
1. GCCTTGCCCAGAGCGGGTGTCAGGCCCGT GGCGGCGCTGGAGACGGCGGCTTCGAGCCTCCTTGGAGTCGGGTTGTT
2. --- A
3. --- A
4. A
5. ---
6. T AT---T--T---
7. T G---TG---T
8. ---A-A G-ATC- - • -CT-GAC-CGG- AA ■
1 . TGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCACCTAAGGCTAAATACTGGCACGAGTCCGATAGCGGACAAGTACCGT
2. A
3.
4.
5.
6. A
by gel extraction (Qiagen Co.) and ligated into a T cloning vector (IPTG) and X-gal. DNA from positive recombinants was purified
(Novagen Co.). Clones were generated by transforming Escheri- using the QIAprep spin plasmid kit (Qiagen Co.). DNA sequenc-
chia coli NovaBlue competent cells provided in the T cloning ing was performed using the dideoxy chain termination method
vector kit, according to the protocol of the supplier. The recom- and an automated DNA sequencer (Applied Biosystems, Model
binant plasmid was screened using isoprophy-p-thiogalactoside 373A. Perkin Elmer). At least two clones were sequenced per
Molecular Phylogenetics of the Genus Corbicula
483
1. AGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGTACGTGAAACCGCATAGAGCCAAACGGGTGGATCCGCAG
2. G
3.
4. T
5.
6. T G
7. T G
8. A GT
1.
2.
3.
4.
5.
6.
7.
AGTCGACCCGGGGAATTCAGCCCGGCGGGTGCC
GA • • T
GA- -C
CAG- • ■ -G- • -T
Figure 2. Aligned of 5' 28S rRNA gene sequences of Corbiculoidea. Dashes represent gaps in the alignment.
isolate, and additional clones were sequenced as necessary to re-
solve ambiguous sites.
Sequences Analyses
Nucleotide sequences were aligned using Clustal X (Thompson
et al. 1997). Phylogenetic analyses were performed by a distance
method, using Kimura 2-parameters distance, to obtain a neighbor-
joining tree (Saitou & Nei 1987) and using the MEGA vl.OI
program. (Kumar et al. 199.3). Gaps were considered as an addi-
tional character state in pairwise comparisons. The statistical con-
fidence of a particular cluster of sequences was evaluated by the
bootstrap procedure (1000 replicates).
RESULTS
The alignment of the partial 28S rRNA gene sequences of C.
fluminea. C. papyracea. C. leana. C.japonica, C. largillierti. Poly-
mesoda maritima, P. caroliniana, and Sphaerium corneum is
shown in Figure 2. Nucleotide sequence data reported in this study
are available in the GenBank database under the accession num-
bers; C. fluminea (AY052553), C. largillierti (AY052534). C. pa-
pyracea (AY052555), C. japonica (AY052556) and C. leana
(AY052557). The 28S sequence was 412 base pairs, which in-
cluded gaps in length. Nucleotide sequence differences for the
various pairs of Corbiculoidea are presented in Table 1 . For this
gene segment, interspecies differences from recognized species
within any species where clones from the different sequenced
isolates (C. fluminea. C. papyracea. and C. leana). Interspecies
variation within the genus Corbicula was detected at a low level of
0.73 to 1.70% (from 4-22 nucleotides). Among the genus, how-
ever, Corbicula, Polymesoda, and Sphaerium exhibit more varia-
TABLE 1.
Nucleotide sequence differences between pairs of Corbicula taxa for
28S rRNA region.
Species (Origin)
2
3
4
S
6
7
8
L
Corbicula fluminea (Korea)
6
6
4
3
IT
18
56
1
C. largillierii (China)
5
4
7
18
16
57
3.
C. papyracea (Korea)
4
6
Tl
20
57
4.
C. japonica (Japan)
6
19
17
57
5.
C. leana (Korea)
22
18
56
6.
Pohmesoda maritima (USA)
12
62
7.
P. caroliniana (USA)
56
8.
Sphaerium corneum (Germany)
484
Park and Chung
79 1 Corticula papymcea
*6 H— c japonica
87
791 '-""
6Ji-C
79
C largilliaflj
C tluminaa
C leans
92
PoiymasoCa mantima
-P carolimana
Sphaenum comeum — Sphaeriidae
Corblculidae
Figure 3. Tree dipicting relationships among genus Corbicula inferred
from 28S rRNA gene sequence data using P. maritina. P. caroliniana.
and S. corneiim as an outgroup. A distance matrix was calculated using
the Kimura-2-paramater model and the tree constructed using the
Neighbor-Joining method.
tions (1.70-15.1%, from 22-62 nucleotides). The distances be-
tween the genus Polymesoda and Sphaerium and the various Cor-
bicula species are significantly greater than some interspecies
distances with the Corbicula genus. The phylogenetic tree
shows relationships among the interspecies based on the 28S
sequences (Fig. 3). Analyses using P. maritima. P. caroriniana
and S. comeum as outgroups supported the monophyly of genus
Corbicula. Also, in the neighbor-joining tree, monophyly was
strongly supported for both the families Corblculidae and Sphaeri-
idae.
DISCUSSION
In the family Corblculidae, there are three genera: Corbicula.
Batissa and Polymesoda. Of these, only Corbicula has a signifi-
cant number of freshwater and brackish-water species. The other
genera are dominantly brackish-water clams and characteristically
have reducing sediments in tropical mangrove swamps. There are
marked ecologic and reproductive differences between interspe-
cies of Corbiculoidea (Table 2). Geographic variation in physiol-
ogy, sex determination, and reproduction are undefined. There are
references in the literature to a single species (C fluminea) pos-
sessing different sexual strategies (e.g., protandry, protogyny,
separate sexes) in different parts of its range (Morton 1982). Re-
production in these species must be by parthenogenesis, but mature
sperm are found in the gonads.
The earliest corbiculid (mid-Jurassic) and dreissenid (Eocene)
fossils were clearly marine (Keen & Casey 1969, Nutall 1990)
and all dreissenid and some corbiculid species retain an indirect
mode of development involving broadcast spawning and a pelagic
veliger larval stage (Morton 1985, Morton 1989, de Severeyn
et al. 1994). A planktonic veliger larva is considered to be
nonadaptive in riverine freshwater environments because it lives
in colonies at upstream habitats (McMahon 1991). Some fresh-
water corbicuid species have evolved parental care of young
in association with a greatly reduced (C. fluminea ) (King et al.
1986) or completely absent (Neocorbicula limosa ) (Ituarte 1994)
pelagic larval ontogeny. From the comparisons of the chromo-
some numbers and karyotypes in three species, Okamoto
and Arimoto (1986) assumed that the ancestral species of the
hermaphroditic species including C. leana originated from the
ancestral species of C. sandai that had originated from the an-
cestral species of C. japonica. Lee and Kim (1997) reported
that the genetic similarity coefficient of C. fluminea. C. leana,
and C. colorata in freshwater was very closed (Rogers S <0.970).
whereas C japonica in brackish-waters was genetically distant
(S = 0.873) from them. In this study, despite widespread geo-
graphic origins of the Corblculidae, their percentage sequence
variation in the 28S rRNA was low, <5.3%. Phylogenetic tree
calculated using neighbor-joining method is shown in Figure 3.
Instead of a single monophyletic Corbicula lineage, C papyracea.
C japonica. and C larf>illierti are members of a separate clade
distinct from that shared by C. fluminea and C leana: (i.e., within
the Corbiculinae there are two sister groups) both of which contain
species currently assigned to Corbicula. Based on the 288 rRNA
data, the genus Corbicula is indistinguishable by biologic habitat
features; C. japonica live in brackish-water, while other species
live in freshwater. Partial 28S rRNA gene sequences provide use-
ful data for resolving phylogenies within the Corbicula species
groups.
TABLE 2.
Habitats, reproduction, and chromosome numbers in 12 species of the superfamily Corbiculoidea.
Habitats
Reproduction
Chromosomes
Species
2n
References
Corbiculidae
C. jhiminea
Freshwater
Hermaphrodite
54
Park et al., 2000
C. papyracea
Freshwater
Hermaphrodite
54
Park et al., 2000
C. leana
Freshwater
Hermaphrodite
54
Okamoto & Arimoto.
1986
C. colorata
Freshwater
Dioecious
38
Park et al., 2000
C. japonica
Brackish-water
Dioecious
38
Okamoto & Arimoto,
1986
C. sandai
Freshwater
Dioecious
36
Okamoto & Arimoto,
1986
Sphaeriidae
Pisidium coreanum
Freshwater
Hermaphrodite
190
Park et al., 2002
P. ca.'^ertantnu
Freshwater
Hermaphrodite
ca. 150, 180
ca. 190
Barsiene et al., 1996
Burch et al., 1998
Sphaerium comeum
Freshwater
Hermaphrodite
36
Keyl, 1956
S. occidenlale
Freshwater
Hermaphrodite
ca. 209
Burch et al., 1998
S. striatinufii
Freshwater
Hermaphrodite
ca. 68-98
ca. 152
Woods. 1931
Lee, 1999
Musculium secure-^
Freshwater
Hermaphrodite
ca. 247
Burch et al., 1998
Molecular Phylogenetics of the Genus Corbicul\
485
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Joitmal ,\f Shellfish Research. Vol, 22. No. 2. 487-4').1. 200.^.
DOMINANCE OF THE ASIATIC CLAM, CORBICULA FLUMINEA (MULLER), IN THE
BENTHIC COMMUNITY OF A RESERVOIR
ALEXANDER Y. KARATAYEV,'* LYUBOV E. BURLAKOVA,' THOMAS KESTERSON,' AND
DIANNA K. PADILLA-
^ Department of Biology. Stephen F. Austin State University. P.O. Box 13003. SFA Station, Nacogdoches,
Texas, 75962-3003 and 'Department of Ecology and Evolution. State University of New York at Stonv
Brook, Stony Brook. New York 11794-5245
ABSTRACT Corbuula fluminea dominated the benthic community of Latce Nacogdoclies, East Te,\as, composing 97% of the total
biomass of benthic invertebrates. C. fluminea appears to be restricted to the httoral zone. Lower depths have lower oxygen, especially
during the stratified period, which may restrict the distribution of C. fluminea. C. fluminea was found only down to a depth of 4 m and
had and extremely patchy distribution. The greatest density within a patch was found at 1 m depth (35.8 ± 13.8 m"-) and the greatest
biomass withm a single patch was at 2 m ( 137.17 ± 69.21 g • m""). C. fluminea density differed significantly among substrate types. The
maximum density (43 ± 14 xvC') was found in sediments with dead C. fluminea shells and course detritus, and the lowest density (3.6
± 3.6 m"') was found in silt. The spatial distributions of C. fluminea and three species of unionids were similar both in depth and across
substrates in the reservoir. We found no correlation between the densities of C fluminea and other benthic invertebrates. Finally, we
contrasted the effect of C. fluminea on benthic coninuinities to what is known about the impacts of another invasive bivalve, the zebra
mussel.
KEY WORDS: Corhicula fluminea. benthic comnuinity. Hydrilla. invasive species
INTRODUCTION
Asiatic clams [Corbicula fluminea (Muller)] are native to
Southeast Asia and have been successfully invading North Ameri-
can water bodies since the beginning of the 20th century. They
currently occur in 36 states in the United States and northern and
central Mexico; however, they are not found in Canada (McMahon
1982, McMahon 1999, McMahon & Began 2001). C. fluminea
invaded Texas in the 1960s and has now spread statewide (How-
ells 1992). C. fluminea is a simultaneous hermaphrodite that is
ovoviviparous. Fertilized eggs are brooded in the interlamellar
spaces of the gills through the trochophore and veliger stage and
released at the nonswimming pedi veliger stage (McMahon 1999).
Because of a high reproductive potential (<68,000 pediveligers
adult"' y"' ), C. fluminea can rapidly increase in population density
within a short period of time (Aldridge & McMahon 1978, Mc-
Mahon 1991, McMahon 1999, McMahon & Bogan 2001). C. flu-
minea is infaunal, usually burrowing in soft sediments. Adults can
grow to 50-70 mm in size and can live for 3—4 y (reviewed in
McMahon 1999). One of the reasons for its success may be the
ability of C. fluminea to feed both from the water column (using
siphons; Cohen et al. 1984, Boltovskoy et al. 1995), and from the
sediments (using the foot to pedal-feed; Reid et al. 1992, Haken-
kamp et al. 2001)
Carried into raw water systems on intake flows, C. fluminea
nonswimming pediveligers and juveniles may settle in places with
water currents below 1.2-1.5 m sec"' and form adult populations
>20,000 m"- (McMahon 1999). The total damage caused by C.
fluminea for US industries in 1986 was estimated at $1 billion
(Isom 1986). C. fluminea can also play an iinportant role in at|uatic
ecosystems as a benthic-pelagic coupler (Lauristen 1986, Haken-
kamp & Palmer 1999). C. fluminea can reduce phytoplankton lev-
els (Cohen et al. 1984), seston concentration (Leef et al. 1990),
particulate phosphates (Greer & Zeibell 1972) and chlorophyll a
levels (Beaver et al. 1991). Water clarification by clam filtering
*Corresponding author. E-mail: akaratayev@sfasu.edu
favors the growth of rooted macrophytes, shifting primary produc-
tion from planktonic to benthic communities (Phelps 1994. Mc-
Mahon 1999). As a consequence, C. flu)uinea is becoming a major
component of benthic communities in freshwater environments
across North America (McMahon 1983, Counts 1986, Poff et al.
1993. McMahon 1999).
C. fluminea may also influence bottom fauna as a result of
pedal-feeding via bioturbation of sediments or consuming benthic
fauna directly (Hakenkamp & Palmer 1999, McMahon 1999, Hak-
enkamp et al. 2001). Although there are some reports that the
Asiatic clam can compete with native unionid bivalves (Kraemer
1979, Leef et al. 1990, Howells 1992), there are no data about the
impact of this invasive bivalve on biodiversity and functioning of
the macroinvertebrate community or productivity and food web
interactions. Hakenkamp et al. (2001) found that an increasing
abundance of C. fluminea was negatively associated with the abun-
dance of benthic bacteria and flagellates but had no apparent effect
on other benthic protists or meiofauna. This contrasts with studies
of another invading bivalve, the zebra mussel, Dreissena polymor-
plia (Pallas) (reviewed in Karatayev et al. 1997, Karatayev et al.
2002).
We determined the abundance and distribution of C. fluminea
along depth gradients and among substrate types and their role in
the benthic community, especially possible impacts on native
fauna including unionid bivalves. We also compared patterns of
the distribution of C. fluminea and its impact on bottom inverte-
brates with those found for zebra mussels.
METHODS
Study Area
Studies were conducted at Lake Nacogdoches, a monomictic
reservoir in East Texas (31' 37'N, 94"49'W), Lake Nacogdoches is
the municipal water supply reservoir for the city of Nacogdoches,
Texas. The dam-forming Lake Nacogdoches was completed in
July 1976. The reservoir has a surface area of 8.94 km", maximum
487
488
Karatayev et al.
storage capacity of 49.7 million nr. maximum depth of 13 m, and
an average depth of 5.6 m.
The upper shallow (<5 m depth) and more eutrophic part of the
reservoir is situated north of an island in the lake and constitutes
approximately 40% of the water body (Fig. 1). Bottom sediments
in this shallow part are mainly silt and a mixture of silt and clay.
The lower part of the reservoir is less eutrophic. deep ( up to 13m).
and has a variety of substrates, including sand, gravel, clay, shells,
course detritus, and silt, as well as various combinations of these.
The drainage area of the reservoir is 231 km", and Loco Bayou is
the primary tributary (Prater 1991). During December to March,
there is a long period of homeothermy. In spring, summer, and fall
the water column of the reservoir is stratified. A lack of mixing and
high productivity in the reservoir cause complete oxygen depletion
below the thermocline by late spring. As a result, the oxygen
content at depths greater than 6 m never exceeds 1 mg L"' from
May to August (Taylor 1980).
In the early 1980s, Hydhlla verticillata (l.f.) Royal was acci-
dentally introduced into the Lake Nacogdoches and by 1989 cov-
ered approximately 45'7r of the reservoir (Prater 1991). H. verti-
cillata spread mainly in the upper shallow part of the water body,
where it completely covered the reservoir. In contrast, m the lower
part less than 3% of the reservoir is covered with H. verticillata
(Fig. 1).
Sampling Protocol
To determine the distribution of C. fluminea and its effect on
the benthic community of Lake Nacogdoches, a total 96 bottom
NACOGDOCHES
RESERVOIR
ELEVATION 279' (65 M)
Figure 1. Location of transects sampled in Lake Nacogdoches. Shaded
area represents the extent of the reservoir covered by Hydrilla verti-
cillata.
samples were taken in September (transect 1) and October
(transects 2-6) 2001 (Fig. 1). For each transect, samples were
collected from 1, 2. 3. 4. 6. and 8 m. except for transects 5 and 6.
where samples were collected at depths of 1. 2, 3, and 4 m. These
last two transects were situated at the upper shallow part of the
reservoir with a maximum depth less than 5 m. In addition, the
deep (profundal) part of the lake was sampled separately (6 and 10
m depth). Three or more replicate samples were taken at each
depth with an Eknian grab (sampling area = 0.0233 m") and
washed through a 550-(xm mesh. At each sampling point, water
transparency, bottom temperature, pH, oxygen, and conductivity
were recorded (Table 1). After sampling, all macroinveilebrates
were transferred to containers with W7c neutral-buffered formalin
and labeled. All macroinvertebrates were identified to the genus or
species level, counted, and weighted to the nearest 0.0001 g after
being blotted dry on absorbent paper (wet mass). For oligochaetes,
only Braiuhiiira sowerbyi Beddard and Stylaria laciislris (Lin-
naeus) were identified to species level. All C. fluminea and union-
ids were cut open with a scalpel to remove water from the mantle
cavity, measured, weighed (wet mass), and identified to species.
The average mass of individual C. fluminea in a sample was cal-
culated by dividing the total mass by the number of clams in the
sample. Because several samples contained no C. fluminea (den-
sity = 0), we used nonparametric Kruskal-Wallis test to analyze
the data. When multiple statistical tests were conducted on the
same data, we used a Bonferroni correction to determine the criti-
cal alpha for significance.
RESULTS
Corbicula fluminea Distribution
During our September sampling, the reservoir was still well
stratified for temperature and oxygen to around 6 m depth (Table
1 ). In October, the lake was well mixed and both temperature and
oxygen did not vary appreciably with depth. Oxygen content was
low only at the deepest sampled site (10 m depth). Water pH and
conductivity did not show sharp changes across the thermocline
(Kruskal-Wallis test, P = 0.20).
C. fluminea was found only in the lower part of Lake Nacog-
doches (transects 1-4; Fig. 1). We did not find any live C. flu-
minea, or even their dead shells, in the upper part of the reservoir,
which was covered with H. verticillata (transects 5 and 6).
We found a significant difference in some chemical parameters
between regions of the lake with C. fluminea (transects 2-4, 1-4 m)
and the area of the lake with H. verticillata. where we did not find
clams (transects 5-6, 1-4 m). The pH was slightly higher in the
upper region (7.96 ± 0.009. /; = 12) vs. 7.86 ± 0.011 [n = 1):
Kruskal-Wallis test, P = 0.0005). Dissolved oxygen was slightly
lower in area covered with H. verticillata (9.26 ± 0.06 (n = 7) vs.
9.76 ± 0.15 mg L-' (n = 12); Kruskal-Wallis test, P = 0.016),
but this difference was only marginally significant (critical alpha
with the Bonferroni Correction = 0.012). Conductivity was lower
in the upper part of the reservoir (93.1 1 ± 0.28 (/( = 7) vs. 95.66
± 0.53 m Siemens cm"' (n = 12); Kruskal-Wallis test, P =
0.002). Transect 1 was not included in these analyses as it was
sampled 20 days earlier.
The average (± SE) C fluminea density and biomass in the
lower portion of the reservoir (transects 1^, depths 1-8 m) was
15.6 ± 5.3 m~' and 71.9 ± 18.8 g m"", respectively. There were no
significant differences in density or biomass of C. fluminea be-
tween the four transects (Kruskal-Wallis test, P > 0.44). in addi-
Dominance of Corbicula in Benthos
489
TABLE 1.
Oxygen concentration, temperature, conductivity and pH in Lake Nacogdoches.
Depth (m)
Parameter
1
2
3
4
6
8
10
Transect 1
Oxygen, mg • L"'
9,72(1)
9,.'i7 (1)
9,40(1)
9,26(1)
3,03(1)
0,45 ( 1 )
—
Temperature. °C
27.8(1)
27,7 (1)
27,6(1)
27,6(1)
26,3(1)
24,3(1)
—
Conductivity. mSiemens
cm-'
96,9 ( 1 )
95,3(1)
94,3 ( 1 )
94,2 (1)
97.7 (1)
116,7 (1)
—
pH
7,87(1)
7,90(1)
7,90(1)
7,87(1)
7,76(1)
7,60 ( 1 )
—
Transects 2-4
Oxygen, nig ■ L '
10,24 ±0,36 (3)
9,62 ±0,39 (3)
9,64 + 0,30(3)
9,52 ±0,25 (3)
7,83 ±0,40 (3)
6.25 ±2,15 (2)
—
Temperature, C
23,10 ±0,17 (3)
22,69±0..30(3)
22,83 ±0,12 (3)
22,82 ±0,12
(3)
22.45 ±0,05 (3)
22,30 ±0(2)
—
Conductivity. mSiemens
cm-'
96,27 ± 1,77(3)
95,2 ±0,95 (3)
95,2 ±0,68 (3)
95,97 ± 1,06(3)
95,70 ±0,20 (3)
97,65 ±2.25 (2)
—
pH
7,87 ±0,03 (3)
7,86 ±0,02 (3)
7,86 ±0,01 (3)
7,84 ± 0,02
(3)
7,84 ±0,03 (3)
7,80 ±0,02 (2)
—
Transects 5-6
Oxygen, mg ■ L"'
9,30 ± 0,20 (2)
9,20 ±0(2)
9,33 ±0,12 (2)
9,15 (1)
—
—
—
Temperature. °C
22,30 ± 0,20 (2)
22,31 ±0,16(2)
22,41 ±0,27(2)
22,7(1)
—
—
—
Conductivity, mSiemens
cm"'
92.60 + 0(2)
93,5 + 0.50(2)
93,55 ±0,75 (2)
92,50(1)
—
—
—
pH
7,96 ±0,03 (2)
7,95 ±0,04 (2)
7,96 ±0,005 (2)
7.97(1)
—
—
—
Profunda]
Oxygen, mg ■ L"'
—
—
—
—
8,45 (1)
—
0,85(1)
Temperature, "C
—
—
—
—
23,4(1)
—
21,3(1)
Conductivity, mSiemens
cm"'
—
—
—
—
93,6 ( 1 )
—
123,0(1)
pH
—
—
—
—
7,14(1)
—
7,80(1)
Transect 1 was sampled in September; all other transects were sampled in October. Transects 5 and 6 were in an area of the lake covered with Hydrilla
venicillala and no Corbicula flmninea. Transects 2-4 had C fliiminea and no H. verticiUata. In addition, we sampled two sites deep in the lake (profunda!.
6 and 10 m). Average values + standard errors of mean are given, sample sizes are in parentheses, — = no data.
tion. density and wet mass did not differ significantly with depth
down to 4 m (Kruskal-Wallis test: density: P = 0.29; bioniass; P
= 0.40; Figs. 2 and 3). This lack of significance is probably
caused by the high degree of patchiness (Index of Dispersion Test:
I = 59.7; X- = I * (n - 1) = 59.7*(48 - I) = 2805. x',,025. 47
= 67.8, P < 0.001). Many samples had no C, fluminea, which
resulted in an increase in the variance in observed means. The
1 150
I 100
50
12
12
12
fla^
I o I Live mussels
■i Dead shells
Meant SE
12
m
12 3 4 5 6
Depth (m)
Figure 2. Density of Hve and dead shells of Corbicula fluminea at
different depths in Lake Nacogdoches, Averages, standard errors of
mean, and sample sizes are given.
mean density o'i C. fluminea was greatest at 1 m (35,8 ± 13,8 m""),
and the maximum biomass was at 2 m (137,17 ± 69,21 g m""). The
maximum density of C. fluminea in a single sample was 172 m""
(transect 4, I m) and maximum wet mass (soft body + shells) in a
single sample was 770 g m"" (transect 2, 2 m).
The average individual mass for C. fluminea (total sample
mass/density) differed significantly with depth (ANOVA,
Depth (m)
Figure 3. Total wet mass (left axis) and average individual wet mass
(total wet ma.ss divided by the number of clams in the sample, right
axis) of Corbicula fluminea at different depths in Lake Nacogdoches.
Averages, standard errors of mean, and sample sizes are given.
490
Karatayev et al.
16
.■& 8
12
12
rol Unionid Density
H Unionid Wet Mass
Mean ± SE
12
12
12
T
12 12
150
50
Depth (m)
Figure 4. Density (left axis) and total net mass (right axis) of unionids
at different depths in Lake Nacogdoches. Averages, standard errors of
mean, and sample sizes are given.
P < 0.029; Fig. 3). The smallest average individual mass (2.95 ±
1.02 g) was at 1 m. and the largest average individual mass (7.33
± 0.86 g) was at 2 m.
C. fluminea shells were found down to 6 m (Fig. 2). The dis-
tribution of shells with depth was not uniform (Kruskal-Wallis
test, P = 0.0002) and differed from the distribution of live C.
fluminea (Kolmogorov-Smirnov test, P < 0.001).
C. fluminea density differed significantly among substrate
types (Kruskal-Wallis test, P = 0.04). Dead C. fluminea shells
and course detritus had the highest density (43 ± 14 m"-). and the
lowest density (3.6 ± 3.6 m~") was found in silt (Table 2). Dead C.
fluminea shells were not found uniformly among substrate types.
and were most abundant in clay with stones (272 ± 56 m"";
Kruskal-Wallis test, P = 0.0008).
Distribution of Benthic Animals
We found 38 taxa (species, genera or higher taxa), including 17
chironomids (identified to the species or genus level). The average
density of benthic animals, excluding bivalves, over the entire
reservoir was 901 ± 91 m^~ with a biomass of 2.76 ± 0.29 g m"".
The most abundant insect larvae were the chironomid Coelotanx-
pus tricolor (Lowe) (185 ± 25 m"*, total number of samples =
96), Cliironomus sp. (69 ± 17 m""), and the phantom midge Clia-
oborus punctipennis (Say) (137 ± 35 m""). Among oligochaetes,
Brandnura sowerhyi Beddard was dominant (71 ± 12 m~"). Only
a single species of amphipod, Hyallelu azteca (Saussure), was
found (24 ± II m"-).
Corhicula fluminea dominated the benthic biomass in the lit-
toral zone from 1— t m in the lower part of the reservoir (transects
1—4) and was responsible for more than 97% of the total wet mass
of the benthic community. At depths s 6 m. Cliironomus sp.,
C. punctipennis. and B. sonerbyi were responsible for 43%, 17%,
and 26% of the total benthic biomass, respectively.
In the upper region of the reservoir (transects 5 and 6) the
average density (1165 ± 216 m"~) and average biomass (3.57 ±
0.54 g m~") of benthic animals were marginally higher (Kruskal-
Wallis test, density: P = 0.073; biomass: P = 0.061) than in the
lower part (excluding C. fluminea and unionids density 843 ± 104
m^", biomass 2.60 ± 0.35 g m""). However, because of the pres-
ence of C. fluminea in the lower region of the reservoir, the total
macrobenthos (including C. fluminea) biomass (74.5 ± 18.9 g m~")
was 20 times greater than in the upper part.
There were three species of unionids in the lake, Pyganodon
grandis (Say), Ligumia subrostrata (Say), and Toxolasma texas-
ensis (Lea). Two P. grandis. one L. subrostrata. and six T. texas-
ensis were found on transects 2, 3, and 4 on depths of 1—4 m (Fig.
4). These unionids completely overlapped with the distribution of
C. fluminea (Kolmogorov-Smirnov test, P > 0.10).
For tran.sects 1—1. the maximum density and biomass of union-
ids was in clay (17.9 ± 8.3 m"-, 97.5 ± 42.8 g • m"", total number
of samples n = 12) and in course detritus with C. fluminea shells
(10.8 ± 5.6 m"-, 98.4± 71.1 g ■ m'-.n = 12). The lowest density
and biomass of unionids was in silt (3.6 ± 3.6 m"", 14.9 ± 14.9
g-m~", n = 12). There were no significant correlations between the
C. fluminea density or the density of C. fluminea shells and any
invertebrate taxon.
DISCUSSION
C. fluminea Distribution
The exotic plant Hydrilla verlicillata covers approximately
45% of Lake Nacogdoches and is the dominant macrophyte spe-
cies in this community (Prater 1991). Another exotic species. C.
fluminea. dominated the benthic community of this reservoir.
However, the spatial distribution of these two nuisance species did
not overlap. During our study, neither live C. fluminea nor dead
shells were found in the upper part of the reservoir, which is
covered with H. verticillata. Prater ( 1991 ) sampled the benthos of
Lake Nacogdoches monthly over 12 mo in 1989-1990 and never
found C. fluminea in the H. verticillata region of the reservoir as
well. Two factors may contribute to the absence of C. fluminea in
the upper part of the reservoir. First, dense H. verticillata mats may
deplete the oxygen in the water to levels below those critical for C.
fluminea survival. We found a significant decrease in oxygen in
TABLE 2.
Abundance of live Corhicula fluminea and shells in various substrata in Lake Nacogdoches in 2U01,
Substrate Type
Density, Ind. m
Biomass, g ■ m
C. fluminea Shells m
C. fluminea shells and course detritus
Clay and stones
Clay
Sand
Silt
43.0 ± 14.0(12)
19.1 ± 14.5(9)
25.1 ±8.3(12)
14.3 ± 14.3(3)
3.6 + 3.6(12)
162.68 ±56.01 (12)
95.36 ± 63.75 (9)
149.93 ±66.26 (12)
45.87 ±45.87 (3)
17.77 ± 17.77(12)
71.7 ±25.6 (12)
272.3 ±55.5 (9)
100.3 ±21.4 (12)
28.7 ±28.7 (3)
112.9 ±28.6 (24)
The abundance of live C. fluminea was estimated from transects 1—4, from 1—4 m. The abundance of dead shells was estimated from transects
1-6 m. Average values ± standard errors of the mean are given. Sample size in parentheses.
■ and
Dominance of Corbicula in Benthos
491
the portions of the lake covered with H. verlicillata. but this dif-
ference was relatively small. Second, bottom substrates in the up-
per part of the reservoir are predominantly silty clay, which may be
unfavorable for C. fliiiiiiiieci.
C. fluminea was found in all four transects in the lower part of
Lake Nacogdoches at depths up to 4 m. and C. fluminea dead
shells were found up to 6 m depth. Deeper in the reservoir. C.
fluminea was probably limited by low oxygen, especially during
the summer, when the water column is stratified and the oxygen
content deeper than 6 m never exceeds 1 mg L"' (Taylor 1980). C.
fluminea is known to be intolerant of even moderate hypoxia (Mc-
Mahon 1991. McMahon & Bogan 2001 ). and low oxygen is con-
sidered to be one of the main sources of mortality for C. fluminea
(Sieckel 1986). Although live C. fluminea were most dense at 1 m
depth and had highest total biomass at 2 m, their dead shells were
most abundant at 3 and 4 m (Fig. 2). which suggested that the
depth of maximum C. fluminea abundance may vary with time or
that dead shells were transported to deeper water by water motion.
C. fluminea density and biomass also varied among substrate
types. Clams were most abundant in sediments formed by shells
and course detritus and least abundant on silt. The mean population
density and biomass of C. fluminea we found were very similar to
those found 10 years earlier by Prater (1991). He found that clam
density in 1989-1990 varied from 0 to 60 m"- (average 24.4 ± 5.5
m"'). This suggests that the population density of C. fluminea in
Lake Nacogdoches is rather stable. C. fluminea can occur in dense
aggregations, exceeding 2000 m'" (Gardner et al. 1976. Phelps
1992). These densities are much higher densities than those that
have been recorded in Lake Nacogdoches. However, these higher
densities were reported for a limited period of lime, shortly after
initial invasion (Phelps 1994) or from a local spot in a water body
(Eng 1979). For example, after the initial invasion in the Potomac
River in 1977, C. fluminea reached a maximum density in 1986
(722 g m~" wet weight, including shell) but then sharply declined,
and in 1992 was at 24.87^ of 1986 levels (Phelps 1994). In the
sediment bars of the Delta-Mendota Canal, the maximum density
of C. fluminea at one site was 1 3 1 ,200 m""; however, the average
density was much smaller (Eng 1979). In another Texas lake. Lake
Arlington, the mean density of C. fluminea in 1975 was very
similar to the densities we found in Lake Nacogdoches (32.1 ±
16.5 nr-. Aldridge & McMahon 1978).
Dominance in Benthos
C. fluminea appears to dominate the benthic community of
water bodies it invades (McMahon 1983, Counts 1986, McMahon
1991, Poff et al. 1993, McMahon 1999). We found that in littoral
zone of Lake Nacogdoches C. fluminea comprises more than 97%
of the total wet mass of the macrobenthic community. We found
no correlations between C. fluminea density and biomass and other
nonmolluscan invertebrates.
Impact on I'nionids
Whether C. fluminea and native bivalves compete is controver-
sial (McMahon 1999, Strayer 1999). According to some authors,
C. fluminea may out compete native unionids (Kraemer 1979,
Belanger et al. 1985, Leef et al. 1990, Howells 1992). The com-
petitive advantage of C. fluminea over native bivalves has been
suggested because it has a much higher filtering rate than native
species (Mattice 1979, Lauristen 1986). In addition, by being able
to use both filter and pedal feeding, C. fluminea may have an
advantage over native bivalves that are only able to filter feed
(Hakenkamp & Palmer 1999). However, most of the evidence for
the competitive impacts of C. fluminea on native bivalves is based
on an analysis of their spatial distributions, and much of these data
are anecdotal and qualitative rather than quantitative (Strayer
1999). According to many authors (reviewed in Strayer 1999), C.
fluminea and native bivalves have nonoverlapping spatial distri-
butions, implying that C. fluminea can out compete other bivalves.
However, we found that in Lake Nacogdoches unionids and C.
fluminea are both abundant and occupied the same areas. The
depth distribution of C. fluminea and unionids was completely
overlapping. In addition, both unionids and C. fluminea were abun-
dant in the same type of substrate (course detritus with C. fluminea
shells and clay). The lowest numbers and biomass of both C.
fluminea and unionids were in silt.
Several other authors have found that unionids and C. fluminea
coexist (Clarke 1988. Beaver et al. 1991. Miller & Payne 1994).
These data may suggest that the impact of C. fluminea on native
unionids is not as strong as the impact of zebra mussels, which can
cause mass mortality of unionids (reviewed in Karatayev et al.
1997).
Impacts of Corbicula fluminea versus Dreissena polymorpha
In Lake Nacogdoches C. fluminea was never found in areas
over grown by H. verlecillata. In contrast, zebra mussels are often
found at their highest densities on submerged macrophytes, which
they use as sites for attachment (Lewandowski 1982, Lyakhnovich
et al. 1994, Karatayev et al. 1998). During our study. C. fluminea
was most abundant on shelly sediment. This sediment is also one
of the best substrates for the zebra mussel (Lyakhnovich et al.
1994. Karatayev et al. 1998). Silt is the poorest substrate for both
C. fluminea (Duarte & Diefenbach 1994) and D. polymorpha (Zha-
din 1946, Draulans & Wouters 1988, Karatayev & Burlakova
1995) and often limits their distributions. Belanger et al. (1985)
found in their field and laboratory studies that C. fluminea pre-
ferred the following sediments in decreasing order: fine sand, or-
ganically enriched fine sand, and coarse sand. C. fluminea, a bur-
rowing animal, preferred tine sediments: however, the zebra mus-
sel, which attaches to hard substrate, forms especially high
densities on rocks (Lyakhnovich et al. 1994, Burlakova 1998).
Low oxygen may be another important limiting factor for both C.
fluminea (McMahon 1991, McMahon & Bogan 2001) and the
zebra mussel (Mikheev 1961. Spiridonov 1972. Shkorbatov et al.
1994).
Both C. fluminea (McMahon 1983, Counts 1986, McMahon
1991, Poff et al. 1993, McMahon 1999) and D. polymorpha
(Sokolova et al. 1980, Karatayev et al. 1994) dominate benthic
communities and are responsible for more than 95Vf of the bio-
mass in lakes where they occur. C. fluminea live in soft sediment,
crawl through sediment with a foot, and feed both as a filter feeder
from the water column (Cohen et al. 1984, Boltovskoy et al. 1995),
and from the sediments as a pedal feeder (Reid et al. 1992, Hak-
enkamp et al. 2001) and thus may negatively impact burrowing
detritivores (McMahon 1999). Zebra mussels, in contrast, can live
only on the surface of the sediments, where they attach to hard
substrates and each other with proleinacious byssal threads creat-
ing complex three-dimensional structures (Karatayev et al. 2002).
D. polymorpha constantly filter the water for both feeding and
respiration. Filtered particles are either consumed or bound in
mucus, preventing immediate re-suspension. This zebra mussel
492
Karatayev et al.
activity builds a direct connection between the planktonic portion
of water body and the benthos (benthic-pelagic coupling) and
greatly enhances the rates of deposition of both organic and inor-
ganic material on the bottom. D. polymorpha provide food and
shelter for many benthic invertebrates, which have increased den-
sity and biomass in zebra mussel beds. Simultaneously other spe-
cies (mainly filter feeders) may decrease or disappear from the
community (Karatayev & Burlakova 1992. Stewart et al. 1998.
Stewart et al. 1999). This well-documented effect of zebra mussel
on benthic communities contrasts with the unknown impact of C.
fluminea on composition, structure and densities of native inver-
tebrates. In a recent study Hakenkamp et al. (2001) found that
when they experimentally increased C. fliiiiiiuea density in the
field, there was no apparent impact on the abundance or taxonomic
composition of the meiofauna.
In some circumstances C. fluminea may compete with native
bivalves for food or substrate. In contrast, the negative impact of
D. pohmorpha on native unionids is more diverse. Besides re-
source competition, zebra mussels also show direct interference
competition through overgrowth of unionids. By attaching to
unionids, zebra mussels can make it more difficult for them to
burrow and move through the sediment. They can weight down
their host unionid. resulting in burial in very soft sediments, can
increase drag and the likelihood of dislodgement by water motion
for species living near shore, prevent opening valves for respira-
tion, feeding and reproduction, or preventing the closing valves
(reviewed in Karatayev et al. 1997, Burlakova et al. 2000).
Mass mortalities of unionids caused by D. polymorpha over-
growth are most common during the initial stages of colonization,
when mussel populations are growing rapidly. After initial peaks
in zebra mussel abundance, D. polymorpha can coexist with
unionid bivalves (Nichols & Amberg 1999, Buriakova et al. 2000).
Similarly, we hypothesize that the strength of competition between
C. fluminea and native bivalves may depend on various factors
including unionid species, C. fluminea density, and time since C.
fluminea invasion.
ACKNOWLEDGMENTS
We would like to thank Dmitry and Vadim Karatayev for as-
sistance in the field and with sample processing. DKP acknowl-
edges the support of the Distinguished Research Fellow program.
Bodge Marine Laboratory, University of California. Davis (BML
contribution 2184).
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Russian). Saratov: Saratov University Press, pp. 15-21.
Stewart, T. W., J. C. Gafford, J. G. Miner & R. L. Lowe. 1999. Dreissena-
shell habitat and antipredator behavior: combined effects on survivor-
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Stewart, T. W., J. G. Miner & R. L. Lowe. 1998. Quantifying mechanism
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Strayer, D. L, 1999. Effects of alien species on freshwater mollusks m
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ervoir, Nacogdoches County, Texas. Master of Science Thesis, Stephen
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Zhadin. V. 1. 1946. The traveling shellfish Dreissena. Priroda. 5:29-37.
Joimuil of Shellfish Research. Vol. 22, Nii. 1. 493-500, 2003.
PATTERNS OF EMERGENCE AND SURVIVAL OF CONCHOPHTHIRVS ACUMINATUS
(CILIOPHORA: CONCHOPHTHIRIDAE) FROM DREISSENA POLYMORPHA
(BIVALVIA: DREISSENIDAE)
ALEXANDP:R v. KARATAYEV,' * SERGEY E. MASTITSKY,- DANIEL P. MOLLOY,' AND
LYUBOV E. BURLAKOVA'
^Department of Biology. Stephen F. Austin State University; Nacogdoches. Tews 75962-3003; 'General
Ecology Department. Belanisslun State University. 4 Skorynu Ave.. Minsk. 220050 Belarus; and
Division of Research & Collections. New York State Museum. Albany. New York 12230
ABSTRACT Thi.s is the first study to quantify the penodic emergence of a Cdihluiplnhinis sp. from its bivalve host. Emergence rates
of C. acuininanis from Dreissena polymorpha over the entire 24-day experiment appeared to be directly correlated with infection
intensity. The rate of ciliate emergence from individual mussels varied considerably throughout the experiment at both I4'C and 210.
It was not uncommon to have a sampling period in which no emergence was observed immediately followed by a period of high
emergence, e.g., at I4°C from 0 to 25 ciliates and at 21°C from 0 to 720 ciliates. The total mean number of ciliates that were observed
to have emerged from each mussel during the 24-day experiment was significantly higher at 21°C (207 ciliates/mu.ssel) than at 14°C
(29 ciliates/mussel). Our experiments suggested that C. acuininanis have a short survival period outside their host. Although we
observed a maximum survival period of 144 hr (6 days), most ciliates died within 48 h.
KEY WORDS:
Conchiipliiliini.y ucuminulus. ciliate, commensal, host, bivalve, Dreissena polxiiiorpha. /.ebra mussel, mantle cavity
INTRODUCTION
The ciliate Conchophthirus ucwninatus (Claparede & Lach-
mann) (Scuticocillatida: Conchophthiridae) is the most common of
34 endosynibionts associated with zebra tnussels {Dreissena poly-
morpha (Pallas)) (Molloy et al. 1997). Although tiot known from
North America, this ciliate is very common in European zebra
mussel populations, including in Bulgaria (Raabe 1934). Denmark
(Fenchel 1965), Hungary (Raabe 1950). Macedonia (Raabe 1966).
Poland (Dobrzanska 1958). and Switzerland (Claparede & Lach-
mann 1858). Its widespread distribution was recently confirined by
its presence in all 21 zebra mussel populations surveyed in Belarus
(Burlakova et al. 1998, Karatayev et al, 2000a). Among all zebra
mussel protozoan symbionts, this ciliate typically has the highest
prevalence (i,e,. percentage of mussels with ciliates) and intensity
of infection (i.e., number of ciliates per infected mussel) (Molloy
et al. 1997, Burlakova et al. 1998. Karatayev et al. 2000a).
Conchophthirus acmninatus appears to be very specific to Dreis-
sena and has never been reported from any other host. Raabe
(1950) never observed it in unionid mussels, even though they
were sometimes completely covered by C acwninatus-'miecXeA
zebra mussels. Although its feeding on the sperm cells of D. poly-
morpha has been documented (Laruelle et al. 1999), C acuminatus
is likely a commensal organism which ingests a variety of organic
particles present on Dreissena'^ mantle epithelial suifaces (Molloy
et al. 1997). C. acwniiuitiis is typically found on the epithelial
surfaces of the mantle, gills, visceral mass, and labial palps, and
within gill water tubes and suprabranchial cavities (Laruelle et al.
1999).
As with other Conchophthirus spp.. C acuminatus appears to
have an obligate association with its bivalve host, with the only
free-living phase of its life cycle occurring during its transfer to
new hosts. The longer these ciliates can live in open water, the
greater their success in reaching new hosts, particularly distant
zebra mussel populations. An investigation of this free-living
phase in the C acuminatus life cycle was the focus of this study.
*Corresponding author. E-mail: akaratayev@sfasu.edu
In a series of laboratory experiments, we quantified the frequency
that these ciliates emerged from zebra mussels and measured their
survival rate in open water. The results presented herein are part of
an extensive investigation that we. as members of the International
Research Consortium on Molluscan Symbionts (Molloy 2003), are
conducting to characterize the systematics. biology, ecology, and
distribution of Di-eissena\ endosymbionts (Molloy et al. 1996.
Molloy et al. 1997, Molloy et al. 2001. Burlakova et al. 1998,
Laruelle et al. 1999. Karatayev et al. 2000a. Karatayev et al.
2000b, Karatayev et al. 2002, Laruelle et al. 2002, Fokin et al,
2003). This current study, in particular, will hopefully contribute to
a better understanding of the emergence patterns and subsequent
free-living phase of C. acuminatus and will thereby provide in-
sights into the life cycle of a commensal — a type of symbiont
which, relative to parasites and mutualists, has received little re-
search attention.
MATERIALS AND METHODS
Laboratory experiments were conducted during 1998-2002 in
the Republic of Belarus using zebra mussels collected at a ca. 1 .5
m depth from the Dnieper-Bug Canal (52°06'N. 26°00'E) and the
Svisloch River (53°55'N. 27°32'E).
Emergence of C. acuminatus /row D. polymorpha
To determine the frequency of emergence of C. acuminatus
from zebra mussels, an experiment was conducted in April 1998 in
which 48 mussels from the Dnieper-Bug Canal were placed indi-
vidually in 20-mL Petri dishes containing a suspension of the alga
Sceneilesinus acuminatus (LagerheimI in 10 mL of unchlorinated
tap water. For 24 days, half of these dishes were held at 14 (±1 )°C
and half at 21 (±1)°C. Mean mussel lengths in the I4°C and 21°C
dishes were, respectively, 13.8 mm and 14.3 mm (Tables 1 and 2).
Every 2 to 3 days, the water in each dish was transferred to a
plankton counting chamber and fresh, unchlorinated tap water and
algae were added to each dish. Water in the counting chamber was
examined for C. acuminatus using a stereomicroscope (20x), with
ciliates counted and discarded.
495
496
Karatayev et al.
TABLE 1.
Pattern of emergence of C. acuminatus from D. polymorpha at the 14 (±1)°C.
\Iussel
Number of Ciliates Collected Outside Their Host
Infection
Mussel
Length
Total During
Intensity
No.
(mm)
Day 3
Days
Day 7
Day 10
Day 12
Day 14
Day 17
Day 19
Day 21
Day 24
Experiment
on Day 24
1
13,4
6
0
0
1
4
1
1
0
11
7
31
34
2
13.2
1
0
0
1
0
6
1
0
4
6
19
74
3
15.5
1
0
1
2
5
3
0
2
17
24
55
313
4
14.0
3
0
0
1
0
0
1
-)
17
6
30
19
5
14.6
~i
1
T
3
3
->
-)
0
25
28
68
125
6
13.1
1
0
0
1
0
—
1
I
6
1
12
1
7
13.2
0
0
T
1
~i
0
2
1
14
5
27
117
8
13.6
0
0
0
0
0
5
1
1
4
4
15
3
9
13.0
0
0
1
0
0
-)
0
■>
8
24
37
41
10
13.5
1
0
0
3
2
3
")
4
11
7
33
24
11
14.0
1
0
0
2
1
0
1
0
8
8
21
13
12
14.1
4
6
26
19
0
8
13
4
3
12
95
0
13
14.0
1
0
0
3
0
1
0
1
4
6
16
5
14
14.0
0
0
0
0
-)
0
T
T
5
29
40
89
15
14.2
1
0
3
3
1
0
1
3
5
1
19
0
16
13.9
0
0
0
1
0
0
0
1
13
6
21
89
17
14.1
0
0
3
0
0
6
9
5
5
6
34
10
18
13.6
3
0
2
1
1
0
0
0
2
10
19
4
19
13.6
0
1
1
1
0
2
0
0
6
3
14
69
20
13.9
1
3
1
1
T
0
—
1
5
7
21
16
21
13.2
0
2
4
2
I
1
0
4
6
18
38
20
22
13.4
1
0
0
1
0
-)
0
0
4
17
25
77
23
13.6
1
0
1
2
1
0
0
0
0
1
6
11
24
13.6
0
1
0
0
-)
0
1
0
0
5
9
10
Mean
13.8
1.2
0.6
2.0
2.0
1.1
1.8
1.7
1.4
7.6
10.0
29.4
48.5
SE
0.02
0.06
0.06
0.22
0.16
0.06
0.10
0.13
0.06
0.25
0.36
0.83
2.85
To determine infection prevalence and intensity at the begin-
ning of the experiment, we dissected 13 14-mm long mussels from
the above-mentioned Dnieper-Bug Canal sample. Infection preva-
lence and intensity were also calculated at the end of the experi-
ment by dissecting the 48 mussels used in the Petri dishes. During
dissection, mussel mantle cavities were repeatedly flushed with
unchlorinated tap water using a pipette to remove all ciliates from
exposed epithelial surfaces. Because C. acuminatus were also
present within gill water tubes and suprabranchial cavities, gills
were lacerated with forceps and then flushed by pipette. The num-
ber of C. acuminatus in all rinse water was determined in a plank-
ton counting chamber using a stereomicroscope (20x).
Survival of C. acuminatus Outside D. polymorpha
Three laboratory experiments were conducted to determine
how long C. acuminatus survive outside their host in open water.
In all experiments, C. acuminatus were transferred with a pipette
into dishes containing water. Dishes were then covered with lids to
prevent evaporation and half of them were held at 14 (±1 )°C and
the other half at 21 (±1 )°C. Using a stereomicroscope (20x), dishes
were inspected until all ciliates had died.
Experiment 1
In November 1998, mussels were collected from the Svisloch
River and dissected. Ciliates were held in groups of 10 in each of
six lO-mL Petri dishes containing 2 mL of unchlorinated tap water
and were inspected daily.
Experiment 2
In January 2000. 40 C. acuminatus obtained by dissection from
zebra mussels collected in Dnieper-Bug Canal were held individu-
ally in lO-mL Petri dishes containing 3 mL of unchlorinated tap
water. Mortality was scored at 6, 21, 70, and 90 h.
Experiment 3
In July 2002. 20 C. acuminatus obtained by dissection from
zebra mussels collected from the Svisloch River were held at 14
(±I)°C and 23 (±1)°C in 40 individual 4-mL plastic dishes con-
taining 2 mL of filtered ( lOO-jjim mesh net) Svisloch River water.
Mortality was scored at 6, 24. 30, 48, and 54 h. During each dish
inspection. I mL of water in each dish was replaced with fresh
filtered water. Since ciliates may be more sensitive to environmen-
tal changes than their hosts (Beers 1959), we followed Beers'
suggestion to collect mussels as needed and to use the ciliates at
once. Therefore, in experiment 3 we repeated the same exact pro-
cedure three times, starting on three consecutive days using ciliates
from freshly collected mussels.
Data Analysis
The Box-Cox procedure (Krebs 1999) indicated that the best
transformation to achieve a normal distribution was X' = {X+ \f~.
Emergence and Survival C. acuminatus
497
tablp: 2.
Dynamics of the emergence of C. acuminatus from D. polymorpha at the 21 (±1)°C
\Iu$$el
Num
ber of CiUates Collected Outside Their
Host
Infprtinn
Mussel
Length
Total During
■ iiitri. iiuii
Intensity
No.
(mm)
Day 3
Day 5
Day 7
Day 10
Day 12
Day 14 Day 17
Day 19
Day 21
Day 24
Kxperiment
on Day 24
1
13.1
-)
1)
0
3
(1
0 70
176
15
26
292
0
2
13.1
1
0
17
99
29
9 22
15
16
1
210
0
3
14.0
2
9
2
2
5
3 2
85
171
35
316
7
4
15.5
0
720
186
45
14
5 4
0
12
1
987
63
5
14.0
1
3
6
4
(1
3 31
20
12
5
85
1
6
15.5
9
0
6
120
159
87 156
32
24
47
640
143
7
15.8
1
0
1
-)
0
0 1
5
5
32
47
49
8
15.2
43
9
22
6
1
4 14
7
4
4
114
129
9
14.6
20
-}
0
0
3
9 105
24
37
16
216
114
10
13.0
0
2
0
28
15
9 34
6
9
3
106
168
11
14.3
3
72
3
1
4
11 39
14
24
41
212
732
12
13.5
0
3
10
")
1
2 0
1
1
5
25
262
13
14.5
1
0
0
0
1
3 66
45
15
7
138
331
14
14.5
1
2
1
T
0
5 8
3
6
11
39
158
15
15.2
9
76
1
1
5
0 6
29
9
6
142
1035
16
13.9
5
0
2
0
4
13 4
4
1
1
34
0
17
15.8
2
55
1
3
6
33 61
150
34
61
406
93
18
14.0
0
7
0
1
2
15 200
29
52
6
312
175
19
14.1
1
0
0
0
0
3 3
3
6
11
27
264
20
13.4
0
1
4
7
11
7 26
8
11
3
78
39
21
13.7
0
5
0
0
3
0 1
6
5
5
25
21
22
13.2
0
3
I
0
1
3 9
39
25
133
215
82
23
13.6
5
1
3
11
10
6 20
72
12
0
140
27
24
15.4
0
8
0
19
24
14 81
10
4
3
163
351
Mean
14.3
4.4
40.8
11.1
14.8
12.5
10.2 40.1
32.6
21.3
19.3
207.0
176.8
SE
0.04
0.39
6.10
1.57
1.30
1.34
0.74 2.16
1.91
1.43
1.23
9.17
10.23
To compare transformed data, we used Welch's approximate t test
(or t test if variances were homogeneous) in Statistica software
(Windows Release 6.0, StatSoft. Inc.). Effects were considered
stati.stically significant at P < 0.05.
RESULTS
Emergence of(S. acuminatus /rom D. polymorpha
The rate of ciliate emergence from individual mussels varied
considerably throughout the experiment at both 14°C and 21°C. It
was not uncommon to have a sampling period in which no emer-
gence was observed, immediately followed by a period of high
emergence, e.g., at I4°C from 0 to 25 ciliates (Table I: mussel 5.
day 19 vs. day 21) and at 2I°C from 0 to 720 ciliates (Table 2:
mussel 4, day 3 vs. day 5).
At I4°C. typically s3 ciliates were observed outside a host
mussel each sampling day. but this pattern was typically inter-
rupted by periods of higher emergence, particularly toward the end
of the experiment (Table I ). The mean number of ciliates that were
observed outside of the 24 mussels at I4°C ranged from 0.6 to 10.0
ciliates/mussel (Table 1). During the first 19 days of the experi-
ment, a mean of 1.5 ciliates was observed outside the 24 mussels
at I4°C (Table I ). Dissection data indicated that infection intensity
in the 14°C mussels during the experiment remained constant at
about 48 ciliates/mussel (day 0 and day 24 intensities of, respec-
tively 47.3 and 48.5 ciliates/mussel. Table 1). This indicated that
during the first 19 days of the experiment on average ca. 3% (i.e..
1.5/49.5) of ciliates were outside their hosts on a sampling day.
Emergence rates increased toward the end of the I4°C experiment
with a mean emergence of 10.0 ciliates/mussel at the termination
of the experiment on day 24 (Table I). Because the 24 mussels
dissected at the end of the I4°C experiment had a mean infection
intensity of 48.5 ciliates/mussel (Table I), this indicated that ca.
17% (i.e., 10.0/58.5) of all ciliates present within the 24 dishes
were outside their hosts on day 24.
A similar irregular pattern of ciliate emergence was ob.served at
21°C (Table 2). Typically <I5 ciliates were observed outside a
host mussel at 2 1 °C, but the majority of mussels also had at least
one sampling period during which very high numbers (e.g., 72-
720 ciliates) were observed to have emerged. The total mean num-
ber of ciliates that were observed to have emerged from each
mussel during the 24-day experiment was significantly higher at
2I°C than at 14°C (Welch's t test: t = 6.35, P < 0.001) and was,
respectively, 207.0 and 29.4 ciliates/mussel (Tables 1 and 2). The
higher number of emerged ciliates in the 2I°C dishes was almost
certainly related to the significantly higher infection intensity that
had developed in mussels at this warmer temperature. Mean in-
fection intensity in the I4°C mussels at the end of the 24-day
experiment was 48.5 ciliates/mussel (Table I ) and was not signifi-
cantly different it test: t = 0.06, P = 0.95) from the infection
intensity at the beginning of the experiment, i.e., 47.3 ciliates/
mussel. In contrast, mean infection intensity in mussels held at
2!°C increased to 176.8 ciliates/mussel by the end of the experi-
ment (Table 2) and differed significantly from the initial infection
498
Karatayev et al.
intensity (Welcii's t test: t = 2.32, P = 0.026) and the infection
intensity in mussels held at 14°C (f test: r = 2.43, P = 0.019). In
contrast to the 14°C data, emergence rates at 21°C were not higher
toward the end of the experiment. At the termination of the 2 1 °C
experiment on day 24, a mean of 19.3 emerged ciliates were ob-
served (Table 2). Because dissections revealed that these 24 mus-
sels had a mean infection intensity of 176.8 ciliates/mussel (Table
2), this indicated that ca. 10% (19.3/196.1) of all the ciliates in the
24 dishes were outside their host on day 24.
Survival of C. acuminatus outside D. polymorpha
In experiment 1 , C. acuminatus exhibited mortality during first
24 h, but 20% were still alive after 96 h at 2 TC and after 144 h at
14°C (Fig. 1). During experiment 2, there was a shorter survival
period, and all ciliates died by 21 h at 2I°C and by 90 h at 14°C
(Fig. 2). In experiment 3 ciliates began to die during first 6 h at
both temperatures (Fig. 3), and as in previous experiments, ciliates
tended to perish faster at higher temperature.
DISCUSSION
Emergence of C acuminatus /rom D. polymorpha
This is the first study to quantify the periodic emergence of a
Conchophthirus sp. from its bivalve host. Emergence rates of C.
acuminatus over the entire 24-day experiment appeared to be cor-
related with infection intensity. Higher infection intensities led to
a higher emergence of ciliates possibly because of higher ciliate
reproduction at 21°C compared with 14"C. We hypothesize that
these results can explain the seasonal change in zebra mussels
infection intensity with C. acuminatus that we have observed in the
field, i.e., higher intensity in summer and lower in winter
(Karatayev et al. 2000b ).
The data at both 14°C and 21°C suggested that C. acuminatus
emergence from an individual zebra mussel does not occur at a
constant periodic rate, but is rather an irregular pattern marked
occasionally with sudden fluctuations. When data on ciliate emer-
gence was pooled for the entire test group of 24 mussels at either
temperature, however, the day-to-day fluctuations in emergence
rates were considerably reduced. This suggested that in nature the
total number of C. acuminatus emerging from a single mussel
might fluctuate markedly from day to day, but at the same time the
100
100
24
48 72 96 120
Duration of experiment (hours)
168
Figure
its host
(±1)°C
1. Experiment 1. Mean (±SE) survival of C. acuminatus outside
zehra mussel at 14 (±1) C (solid line, filled squares) and at 21
(dashed line, open circles).
10 20
30 40 50 60 70
Duration of experiment (hours)
100
Figure 2. Experiment 2. Survival of C. acuminatus outside the host
zebra mussel at 14 (±1) C (solid line, filled squares) and at 21 (±1) C
(dashed line, open circles).
total number emerging from the entire zebra mussel populations
would vary far less. Pooling data from all 24 dishes at each tem-
perature provided rough estimates (e.g., 3%, 10%. 17%) of the
total C. acuminatus outside their hosts, suggesting that a consid-
18 24 30 36
Duration of experiment (hours)
Figure 3. Experiment .^. Survival of C. acuminatus outside the host
zebra mussel in three consecutive tests (A, B, and C) at 14 (±1) C (solid
line, filled squares) and at 2^ (±1) C (dashed line, open circles).
Emergence and Survival C. acuminatus
499
enable portion of the C. acuminalus population might be in open
water in search of new hosts. This, in addition to the commensal
nature of this symbiont. is likely a key factor explaining why
prevalence of this ciliate is typically near 100% in almost all
European zebra mussel populations (Molloy et al. 1997. Karatayev
et al. 2000a).
Fenchel (1965) observed that Cuiulinplitliinis spp. in non-
dreissenid bivalves quickly emerged in large numbers from their
damaged or dying hosts. Burlakova et al. (1998) confirmed this
same pattern in laboratory trials in which they recorded rapid and
massive emergence of C. acuDiinaliis from dying zebra mussels.
Our present experiment supplements these latter studies by pro-
viding information on emergence patterns from live zebra mussels.
Because prevalence of C. acuminatus in zebra mus.sel hosts is
frequently 100%, it was surprising, therefore, to observe in our
experiment that some infected mussels (i.e.. ciliates emerged from
them during the experiment) were completely uninfected by the
end of the experiment (Table I. mussels 12 and 15; Table 2.
mussels I, 2, and 16). This suggests that C. acuminatus infection
can be temporary. In nature, however, mussels are likely infected
periodically by C. acuminatus from other infected zebra mussels,
whereas in our experiment mussels were individually isolated,
with transinfection prevented.
Hopefully this experiment has provided some insight into the
frequency to which C. acuminatus emerge from their host zebra
mussels. Future trials, however, may want to expand on its design
as follows:
1. More frequent obseiTcitions. We likely underestimated the
numbers of ciliates that emerged. Since C. acuminatus is a
relatively small organism (L x W s 100 x 50 |jim), it was
extremely difficult to see dead/decomposing individuals us-
ing the stereomicroscope. Thus, our counts were almost ex-
clusively based on observation of live ciliates exhibiting
movement (i.e., swimming, cilia beating, etc.). and ciliates
that emerged and died between the 2- to 3-day sampling
periods were likely overlooked. A higher frequency of ob-
servations, possibly every 3 h. would be required to address
this problem.
2. Dishes with more than one mussel. Host density may affect
ciliate emergence rates, and this was not accounted for in
our experimental design. The possibility exists C. acumina-
tus may be stimulated to emerge from their hosts when
chemical cues indicate the presence of other nearby poten-
tial host zebra mussels, particularly uninfected juvenile
mussels. Our study measured emergence only from isolated
zebra mussels (i.e.. one mussel per dish).
3. Mussel siphoning ohsenations. Is ciliate emergence active
(i.e.. do they swim out of the mussel) and/or passive (i.e.,
ejected from the mussel)' Do ciliates emerge through the
mussel's inhalant siphon and/or exhalant siphon? Do some
ciliates reenter their hosts, and if so, through which siphon?
Direct observation, including video recording, would be
helpful to shed light on these questions.
Siinival of C. acuminatus Outside the Host
Our experiments suggest that C. acumiiuitus have a short sur-
vival period outside their host. Although we observed a maximum
survival period of 144 h (6 days), most ciliates died within 48 h.
These results are similar to those of Beers (1959). who studied the
survival of Ctmchophthinis mytili DeMorgan (syn. Peniculistomu
mytili (DeMorgan)) inhabiting the mantle cavity of marine bivalve
Mytilits edulis Linneaus. He found that a period of 84 h in open
water was fatal for the ciliate at 14°C but ciliates died faster at
22°C (48 h) and 30°C (10 h). Fenchel (1965) found that 50% of
Peiuculistoma mytili survived outside their bivalve host for 100 h.
Ancistrum mytili (Quennerstedt) for about 100 h. and Ancistro-
cotna myae (Kofoid and Busch), Ancistrum caudatum Fenchel. and
Thiginophrya saxicavae Fenchel for ca. 50-100 h. Kidder (1934)
studied Conchophthirus ;,pp. from nondreissenid bivalves and
found them to live not longer than 24 h.
Just because a ciliate is alive does not mean it is capable of
reproduction — an essential requirement for establishing a popula-
tion in a new host. Thus, future trials examining C. acununatus
survival should investigate the relationship between duration of
time outside a host and the ability of such surviving ciliates to
successfully reproduce following entry into a new zebra mussel
host.
We set up our survival experiments with ciliates obtained by
dissection of zebra mussels. Future trials may want to measure the
survival of ciliates that have emerged naturally from their hosts. It
is possible that this latter group ciliates may contain greater food
reserves and thus may have great longevity in open water.
How does C. acuminatus maintain its infection in expanding
zebra mussel populations? Dreissena spp. often spread to other
waterbodies by the downstream dispersal of their planktonic lar-
vae, sometimes being carried hundreds of kilometers from their
origin (Stoeckel et al. 1997). These planktonic larvae lack a mantle
cavity and are too small to contain C. acuminatus. Yet C. acumi-
natus is virtually ubiquitous in all freshwater European zebra mus-
sel populations. Since zebra mussel larvae can stay suspended in
downstream currents for more than a week (Hillbricht-Ilkowska &
Stanczykowska 1969. Skalskaya 1976). it would appear from our
experimental data that C. acuminatus would not be able to survive
for as long a duration as the zebra mussel larvae that are at the
leading edge of the dispersing population. Over time, however. C.
acuminatus would likely establish itself throughout the entire ex-
panded population by smaller incremental steps of dispersion.
ACKNOWLEDGMENTS
In the Republic of Belarus, the research was supported by grant
288/73 from the Ministry of Natural Resources and Environmental
Protection Republic of Belarus and grant number 892/51 from
Belarussian State University (A.Y.K.). We gratefully acknowledge
Lyudmila K. Volkova and Vladimir V. Volosyuk for their techni-
cal assistance.
LITERATURE CITED
Beers, C. D. 1959. Some observations on the autecology of the ciliate
Conchophthirus mytili. J. Elislui Mitchell Sci. 75:3-10.
Burlakova. L. E., A. Y. Karatayev & D. P. Molloy. 1998. Field and labo-
ratory studies of zebra mussel {Dreissena polymorpha) infection by the
ciliate Conchophthirus acuminalus in the Republic of Belarus. J. In-
verlebr Pathol. 71:251-257.
Claparede. E. & J. Lachmann. 1858. Etudes sur les Intusoires et les
Rhizopodes. Geneva. Switzedand; Messman.
500
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Dobrzanska. J. 1958. Sphenophryu dreissemie sp. n. (Ciliata. Holotncha.
Thigmotrichida) living on the gill epithelium of Dreissena polymorplui
Pall.. 1754. Bull. Acad. Pol. Sci. Sen Sci. Biol. 6:173-178 (+ Fig. 6-10
on unnumbered pages).
Fenchel, T. 1965. Ciliates from Scandinavian molluscs. Ophelia 2:71-174.
Fokin, S. 1.. L. Giambenni. D. P. Molloy & A. bij de Vaate. 2003. Bacterial
endocytobionts within endosymbiotic ciliates in Dreissena polymorplui
(Lamellibranchia, Mollusca). Ada Parasitol. Pol. in press.
Hillbricht-Ilkowska. A. & A. Stanczykowska. 1969. The production and
standing crop of planktonic larvae of Dreissena polymorpha (Pall.) in
two Mazurian lakes. Pol. Arch. Hydrobiol. 16:193-203.
Karatayev, A. Y.. L. E. Burlakova, D. P. Molloy & L. K. Volkova. 2000a.
Endosymbionts oi Dreissena polymorpha (Pallas) in Belarus. Int. Rev.
Hydrobiol. 85:539-555.
Karatayev. A. Y.. L. E. Burlakova. D. P. Molloy. L. K. Volkova & V. V.
Volosyuk. 2002. Field and laboratory studies of Ophiyoglena sp. (Cili-
ata: Ophryoglenidae) infection in zebra mussels. Dreissena polymor-
pha (Bivalvia: Dreissenidae). J. Invertebr. Pathol. 79:80-85.
Karatayev, A. Y., D. P. Molloy & L. E. Burlakova. 2000b. Seasonal dy-
namics of Conchophlhirus acuminatus (Ciliophora: Conchophthindae)
infection in Dreissena polymorpha and D. bngensis (Bivahia: Dreis-
senidae). Eur. J. Prolistol. 36:394-404.
Kidder. G. W. 1934. Studies on the ciliates from fresh water mussels. 1.
The structure and neuromotor system of Conchophthirus anodontae
Stein. C. curtiis Engl., and C. magna sp. nov. Biol. Bull. 66:69-90.
Krebs, C. J. 1999. Box -Cox Transformation. Ecological Methodology, 2nd
ed. Addison Wesley Educational Publishers, Inc.. Menlo Park, CA.
pp. 561-564.
Laruelle. F.. D. P. Molloy. 1. Fokin & M. A. Ovcharenko. 1999. Histologi-
cal analysis of mantle-cavity ciliates in Dreissena polymorpha: their
location, symbiotic relationship, and distinguishing morphological
characteristics. / Shellfish Res. 18:251-257.
Laruelle. F., D. P. Molloy & V. A. Roitman. 2002. Histological analysis of
trematodes in Dreissena polymorpha: their location, pathogenicity, and
distinguishing morphological characteristics. J. Parasitol. 88:856-863.
Molloy. D. P. 2003. International Research Consortium on Molluscan
Symbionts: A Research Network Organized by the New York Slate
Museum. Available at: (http/Zwww. nysm.nysed.gov/biology/ircoms/
bioJrcoms.html)
Molloy. D. P.. L. Giamberini. J. F. Morado. S. I. Fokin & F. Laruelle.
2001. Characterization of intracytoplasmic prokaryote infections in
Dreissena sp. (Bivalvia: Dreissenidae). Dis. Aquat. Org. 44:203-216.
Molloy, D. P., A. Y. Karatayev, L. E. Burlakova. D. P. Kurandina & F.
Laruelle. 1997. Natural enemies of zebra mussels: predators, parasites
and ecological competitors. Rev. Fish. Sci. 5:27-97.
Molloy. D. P.. V. A. Roitman & J. D. Shields. 1996. Survey of the parasites
of zebra mussels (Bivalvia: Dreissenidae) in northwestern Russia, with
comments on records of parasitism in Europe and North America. J.
Helminthol. Soc. Wash. 63:251-256.
Raabe. Z. 1934. Weitere Untersuchungen an einigen Arten des Genus
Conchophthirus Stein. Mem. Acad. Pol. Sci. Lettr. Ser. B. Sci. Nat.
1934:221-235.
Raabe. Z. 1950. Recherches sur les cilies Thigmotriches (Thigmotricha Ch.
Lw.). V. Cilies Thigmotriches du lac Balaton (Hongrie). Ann. Univ.
Mariae Curie-Skadowska Sect. C Biol. 5:197-215.
Raabe. Z. 1966. The parasitic ciliates of Dreissena polymorpha and other
Bivalvia in the Ohrid Lake. Acta Protozoal. 4:1-14.
Skalskaya. I. A. 1976. Colonization of new substrates in Gorkovskoe Res-
ervoir by Dreissena polymorpha Pallas. Biol. Vnutr. Vod. Inf. Byull.
31:30-34 (in Russian).
Stoeckel, J. A., D. W. Schneider, L. A. Soeken. K. D. Blodgett & R. E.
Sparks. 1997. Larval dynamics of a riverine metapopulation: implica-
tions for zebra mussel recruitment, dispersal, and control in a large-
river system. J. N. Am. Benthol. Soc. 16:586-601.
Jouniul of Shellfish Research. Vol. 22. No. 2, 501-50.^ 2003.
A NOVEL METHOD FOR LOCATING TAGGED INFAUNAL BIVALVES:
SUBMERSIBLE PULSE TECHNOLOGY METAL DETECTORS
RONALD B. TOLL.' ROBERT S. PREZANT," AND HAROLD B. ROLLINS'
^Department of Biology. University of Central Arkan.sas, Conway, Arkan.fas 72035: "Department of
Biology. Montclair State Unirer.<;ity. Upper Montclair. New Jersey 07043: Department of Geology and
Planetary Science. University of Pittsburgh, Pittsburgh, Pennsylvania J 5260
ABSTRACT Harddams. Meixenaria mercenaria (Linne. 1758), tagged with brass washers attached to the outer shell surface and
replanted into their natural habitat, were located remotely through the use of a commercially available, fully submersible, pulse
technology metal detector. The ability to remotely locate tagged, replanted clams can increase the speed and efficiency of field
operations associated with studies of clam population dynamics. Also, this methodology can reduce localized disturbances to the habitat
that routinely accompany extensive hand probing to relocate experimental clams in traditional tag and recapture based studies.
KEY WORDS: Mercenaria. tagging, infaunal, recapture, metal detector
INTRODUCTION
Studies on the population dynamics of infaunal bivalves rou-
tinely involve the mark and recapture of measured, marked indi-
viduals. Ma.\inii/ation of recovery rates of experimental clams
returned to their natural habitats is essential to support robust
analyses of various parameters related to population biology. Vari-
ous studies have used fenced enclosures to retain experimental
organisms within predescribed plots and minimize losses from the
population under study. However, enclosures can alter various
biotic and abiotic parameters of the microhabitat under study, in-
duce localized erosion, call unwanted attention to the experimental
plot from passers-by. and damage fragile habitats.
In cases where clam motility is considered to be minimal, en-
closures can be deemed unnecessary and simple marking of the
boundaries of the study plot followed by intensive hand probing
of the substratum may be sufficient to recapture a statistically
significant sample of marked individuals. However, complete re-
connaissance of the study plot by hand probing can result in
significant damage to the substratum and associated infaunal
and epifaunal animals, and rooted or attached plants and algae.
Also, even minimal lateral translocations of clams, by passive
or active means, across the boundary of the study site can lead to
loss of these organisms from the study population because of
the impracticalities of hand probing extensive areas beyond the
experimental plot limits. While some loss is expected through
predation. erosive exhumation associated with stochastic high-
energy events, etc.. these losses could be significant in terms of
previously unrecognized micro- to meta-displacements that have
dispersal consequences when viewed cumulatively over time
scales ranging from months to years (Prezant et al. 1990. Prezant
et al, 1994).
To facilitate our ongoing studies of the population dynamics of
Mercenaria mercenaria at St. Catherine's Island, Georgia, a typi-
cal barrier island ecosystem located near the apex of the Georgia
Bight, a novel technique for locating marked, replanted individuals
was developed. This new method for remote location of replanted
infaunal clams has several distinct advantages to future studies of
population dynamics of various clam species including maximiz-
ing recovery rates of tagged individuals from both within and
outside of the study plot as well as limiting habitat disturbances
during recovery operations.
MATERIALS AND METHODS
Inexpensive, common brass washers were obtained in several
sizes ranging from approximately 10 to 30 mm in diameter from
local building supply centers. Approximately 125 clams of varying
sizes ranging from about 25 to 110 mm total length were hand
collected from a variety of habitats on St. Catherine's Island. Geor-
gia. The posterior portion of one or both valves was cleaned using
a synthetic, abrasive scouring pad and fine sandpaper as necessary
to prepare a clean surface for bonding of the washer, A variety of
adhesives and glues were tried including two part epoxies and
exterior grade construction adhesives.
Clams were held in in vivo positions (posterior end uppermost)
by placing them into a shallow tray filled with beach sand. Adhe-
sive was applied to the previously prepared and dried shell surface
with care to not glue the two valves together. The washer was
pressed into the adhesive, which was then allowed to dry or cure
as per manufacturer's instructions. Some clams received two
washers, one on each valve (see Fig. 1 ). In some cases, masking
tape was used to hold the washer in place until the adhesive set up
and the washer was secured firmly in place.
Clams were replanted in a variety of habitats including barrier
beaches (quartz sand) and ebb-dominated point bars (richly or-
ganic detritus) and placed in in vivo orientation and substratum
depths, A hand-held Fisher (Fisher Research Laboratory. Los
Banos, CA) "Impulse" (fully marine submersible, pulse technol-
ogy) metal detector was used to locate the tagged clams from 2 to
5 days after replanting. The metal detector was used according to
the manufacturer's instructions with the search coil moved parallel
to and just above the surface of the substratum in overlapping
sweeping arcs.
RESULTS AND DISCUSSION
In a series of trials involving 15-25 brass washer-tagged Mer-
cenaria mercenaria, nearly 100% of all clams were located using
the submersible, pulse technology detector. Individual washers
placed by hand could be located to depths of up to 20-25 cm,
depending on substratum type and prevailing local conditions. In
actual use as markers on clams, there was no discernible difference
in the ability to locate clams with a single washer versus those with
two washers under the conditions encountered in this study. Under
501
502
Toll et al.
^^t^U^
Figure 1. Posterior view of Mercenaria mercenaria with two brass
washers (diameter = 22 mm) attached as targets for pulse technology
metal detection.
other conditions (e.g.. deeper burial) two washers should provide
a greater potential target. The ability to ground balance the detector
to eliminate the background signal from the naturally highly min-
eralized mud substrata, found commonly in the tidal tributary sys-
tems of St. Catherine's Island, was essential to discriminate the
brass targets.
Because of the highly corrosive nature of marine sediments,
many anthropogenic targets, especially those composed of ferrous
compounds (nails, fishhooks, etc.) are rapidly oxidized and elimi-
nated from the habitat. These trash items, if present, could result in
false positive returns as they do in more traditional metal detecting
scenarios. Pre-screening of clam relocation study sites for such
unwanted targets with the detector could allow for study site op-
timization.
To maximize the discrimination capabilities of the detector, the
brass washers were bonded to the exterior of the shell in an ori-
entation that would cause them to be parallel to the surface of the
substratum when the clam was in a normal living position. In doing
so, the brass washer targets create low yet conspicuous surface
irregularities on the shell. However, it has been our experience
following direct observation of thousands of hardclams that con-
siderable epibiont growth, particularly oysters and barnacles, is
known to occur on clams from native populations around the study
sites at St. Catherine's Island, particularly those recovered from
tidal creeks within well established salt marshes. Upon careful
examination, even clams with heavy epibiont loads appear to be
healthy and have growth rates similar to non-epibiont carrying
clams (Walker & Tenore 1984, Walker 1985, Walker 1987).
Therefore, it is highly unlikely that the presence of the brass
washer has any direct deleterious effect on the health and viability
of the tagged clam.
Detachment of targets from the valve surface occurred in a
small percentage of the clams (<10%) resulting in retrieval of only
the brass washer. Target loss can be minimized by careful surface
preparation and adhesive choice to ensure solid bonding. While the
two part (resin and hardener) products had excellent bonding char-
acteristics, their use is more time consuming due to the need to mix
small quantities at a time. The exterior grade construction adhe-
sives, available in large tubes and extruded with the use of a
standard caulking gun, were cheaper, easier to use, more time
efficient, and had nearly the same efficacy as more expensive
epoxy products. As shell surface characteristics can vary from
habitat to habitat even within the same species, experimentation
trials with different adhesives are recommended before large-scale
deployment of tagged clams (Walker & Tenore 1984).
With the excellent sensitivity and discrimination capabilities of
the submersible, impulse technology metal detector, tagged clams
could be precisely located within a lateral distance of 5-20 cm
depending on the size of the target (brass washer) and the depth of
the target. Therefore, disturbance to the habitat by hand probing is
minimized substantially as compared with hand probing of the
entire study plot.
Detection of metal targets depends on a variety of factors.
including target size and depth of burial. Burrowing depth of Mer-
cenaria mercenaria is known to be positively correlated with clam
size (Walker 1985. Walker 1987). Also, smaller clams are known
to exhibit increased vertical motility (Walker 1985. Walker 1987)
within the substratum. As such, small brass washers placed on
small clams should have similar chance of detection as large wash-
ers placed on large clams. In theory, the impact of the washers on
the clam, if any. would then be similar across the various size
classes.
The use of pulse technology, submersible metal detectors for
the location of tagged bivalves represents a simple and extremely
cost effective methodology potentially applicable to a wide range
of freshwater and marine clam species. Continuing improvements
in metal detection technology should increase the practicality and
efficacy of the use of metal detectors for studies of infaunal or-
ganisms. For example, metal detectors with target recognition ca-
pabilities could provide expanded opportunities to remotely rec-
ognize tagged clam cohorts by size without the need to recover
individual clams.
While all detector use in our trials was performed at low tide
with the substrata either fully exposed or covered by less than 15
cm of seawater, the fully submersible operation of this detector
would allow for it to be used over the side of a small boat, pref-
erably one with a fiberglass hull, or handheld by a person using
snorkel or scuba gear.
ACKNOWLEDGMENTS
Special thanks to Fisher Research Laboratory, Los Banos, Cali-
fornia, for technical assistance and the initial loan of the metal
Metal Detector Locates Tagged Infaunal Bivalves 503
detector used in this study. Amy Daniels. Janet Fallon. Alexandria and generously offered his vast knowledge of the island itself
Toll. Micah Toll, and Danielle Toll assisted with the lab and field- Important financial support for this research was provided by
work. Mr. Royce Hayes, superintendent of St. Catherine's Island. grants from the St. Catherine's Island Foundation. Inc. adminis-
provided logistical support and assistance on St. Catherine's Island tered by the American Museum of Natural History. New York.
LITERATURE CITED
Prezanl. R. S.. H. B. Rollins & R. B. Toll. 1990. Dispersal of adult hard resources in coastal Georgia. Georgia Marine Science Center. Techni-
clams as an adjunct to larval recruitment. Am. Zool. 30:89A. cal Report Series 85-1:164.
Walker. R. L. 1987. Hard clam Mercenaria mercenaria (Linne) popula-
tions of coastal Georgia. Georgia Marine Science Center. Technical
Report Series 87-1:73.
Walker. R. L. & K. R. Tenore. 1984. The distribution and production of the
hard clam. Mercenaria mercenaria, in Wassaw Sound. Georgia. E.siii-
Walker, R. L. I98.'i. Subtidal hard clam, Mercenaria mercenaria (Linne), aries 7:19-27.
Prezant. R. S.. H. B. Rollins. R. B. Toll & S. Y. Skoog. 1994. Population
dynamics of hard clams on point bars of St. Catherine's Island, Geor-
gia. Abstr. National Association of Biological Scientists Conference.
ll(l):138.
Joiirmil oj Shellfish Rfseanh. Vol. 22, No. 1, 505-519, 2003.
EFFECTS OF STARCH TYPE, MACROALGAL MEAL SOURCE, AND p-CAROTENE ON
GONAD YIELD AND QUALITY OF THE GREEN SEA URCHIN, STRONGYLOCENTROTUS
DROEBACHIENSIS (MULLER), FED PREPARED DIETS
CHRISTOPHER M. PEARCE,'* TARA L. DAGGETT,' AND SHAWN M. C. ROBINSON"
^Ross Island Salmon Ltd.. P.O. Bo.x 1304. Grand Maiian. New Biunswkk. Canada E5G 4M9; 'Applied
Aqitaciiltiire Section, St. Andrews Biological Station. Fisheries and Oceans Canada. 531 Brandy Cove
Road. St. Andrews. New Brunswick. Canada E5B 2L9
ABSTRACT Adult green sea urchins {Srrongylocentmius droebachien.sis) were collected from the wild, placed in land-based tanks,
and fed one of 12 prepared feeds or a control diet of kelp {Liimimiria tongicruris or L. digilata) for a period of 12 wk (April 8 to July
I, 1999). The prepared diets were formulated to examine three experimental factors: (I) starch type (corn, potato, or tapioca); (2)
macroalgal meal source |kelp (L. Unigururis) or rockweed {Ascoplixllmn nodosum) meal]; and O) P-carotene concentration (0 or 200
mg kg"' dry weight of feed). The experiment was a 3 x 2 x 2 completely crossed design. A number of gonad attributes were quantified
during the 12-wk experiment including percent yield, percent water, color, texture, firmness, and taste. Color was assessed subjectively
by eye and objectively with a reflected-light, fiber-optic spectrophotometer to generate CIE L*, a*, and b* values. Results from sea
urchins fed prepared feeds were compared and contrasted with those of wild specimens collected from the source population at weeks
0 and 12 of the experiment. After 12 wk, sea urchins fed prepared diets had significantly higher percent gonad yields (range of means:
19.2-24.3%) than sea urchins given kelp (mean ± SE: 14.5 ± 3.9%) or those collected from the wild at the end of the experiment (2.8
± 0.5%). Percent gonad yield of sea urchins fed prepared feeds increased significantly over time, but was not significantly affected by
starch type or macroalgal meal source. By the end of the experiment, feeds containing 3-carotene had produced significantly lower
percent gonad yield than feeds without the pigment. Increase in gonad yield was not a result of the addition of water because percent
gonad water decreased significantly over time in sea urchins fed prepared diets. Gonad color of sea urchins fed prepared diets or kelp
was, generally, pale yellow/orange to yellow-brown/orange-brown at the end of the experiment and did not differ significantly among
any of the feeding treatments. Gonad color did improve significantly over time as evidenced by color ratings done by eye and
spectrophotometric data. Gonad color was not significantly affected by starch type or macroalgal meal source, but was influenced by
P-carotene concentration-feeds with pigment generally giving better gonad color than feeds without it. At the end of the experiment,
sea urchins fed prepared diets had gonads that were typically smooth to very smooth with distinct gonad segment halves (texture
rating), firm (firmness rating), and ranging from satisfactory to good in flavor (taste rating). Gonad texture and firmness of sea urchins
fed prepared diets was as good or better than kelp-fed sea urchins or wild controls. Gonad taste, however, was significantly belter in
kelp-fed or wild individuals than in sea urchins given the prepared feeds. Gonad texture, firmness, and taste were generally uninfiu-
enced by starch type, macroalgal meal .source, p-carotene concentration, or time. Results indicate that gonad enhancement diets for sea
urchins should incorporate pigment for optimum coloration, but thai the type of starch or macroalgal meal used (at least of those tested)
may be less critical for optimizing gonad quantity or quality.
KEY WORDS: P-carotene, gonad, macroalgal meal, prepared feed, roe quality, sea urchin, starch, Strongvlocentrolus droebacluensis
INTRODUCTION decreasing the necessary ainount of stock required to generate the
Sea urchins are harvested worldwide with the majority destined ^^'"'^ ''^^^' °*^ '"come.
for the Japanese fresh Osh markets. Japan is the world's largest ^''"''"' ''^'^ "^^J"'' 'P*^^*" »* echinoids of economic interest
consumer of sea urchin gonads, termed "roe" or "uni" in the com- ^°' P"'^"""' ^qu^^-ult^re development-Dwffe,m, setosum. Echi-
mercial trade, importing approximately 6100 tonnes of sea urchin
nils esculenliis. Loxechinus albiis. Lytechinits variegatus. Paraceu-
product worth USD million $251 in 1994 (Sonu 1995). Whereas "'°'"' '"'"'"'• P^^m'^echimis miUaris. Strongylocentrotus droe-
market demand remains steady, recent years have seen declines in ''"f'"^"^"' S- franciscaniis. S. intennedius. S. nudus. S. purpura-
catch statistics in many of the world's major sea urchtn fish.nt' ""■ Tripneiistes gratillci. T. ventricosus (Lawrence & Bazhin
countries (Keesing & Hall 1998, Andrew et al, 2001). It is gener^- '998)-are predominantly macroalgal grazers (see review by
ally acknowledged that the only way that future market demand Lawrence 1975), the use of macrophytes tor feeding in large-scale
can be reliably tiiet is through aquaculture production. Aquaculture enhancement or grow-out operations will be problematic due to the
production could involve spawning adult brood-stock and rearinu '^^P^"'^ ^"'^ '°g''"" '"^°'^^'' '" '^e collection and storage of the
larvae/juvemles through to market size or using enhancement tech"- '"^'''^^ quantities ot algae required for such an undertaking. In
niques where commercial-size sea urchins of low gonad yield are ^^'''"''"- n^-'^'rophyes can vary in nutritional quality with season
captured from the wild, held in captivity, and fed natural or pre- ""'^ '''^'^""" ""'^ ""^ '^""'"'" ^ ^1^°'^ ^""^ «* *""''"§ organisms,
pared feeds to increase their percent yield. The latter method, '^^^ evolution of a successful sea urchin culture industry will un-
while still reliant on natural stock, could help wild-capture fishery doubtedly require the development of suitable, low-cost prepared
sustainability by increasing gonad yield and quality and, hence, '^'^'' "'^' ^'^ '^^^''^ ''°'''^^ ""'^ nutritionally reproducible.
A number ot scientific studies have shown that enhancement ot
gonad yield in captive sea urchins can be readily achieved by
♦Corresponding author. E-mail: pearcec@pac.dfo-mpo,gc.ca feeding them prepared diets (Lawrence et al. 1992. Lawrence et al.
Present address: Pacific Biological Station, Fisheries and Oceans Canada, 1997, de Jong-Westman et al. 1995a, Klinger et al, 1997, McBride
3l90HammondBayRoad.Nanaimo, British Columbia, Canada V9T6N7. et al, 1997. McBride et al. 1999, Barker et al, 1998, Fernandez &
505
506
Pearce et al.
Boudouresque 1998, Fernandez & Boudouresque 2000, Goebel &
Barker 1998, Walker & Lesser 1998, Spirlet et al. 2000. Olave et
al. 2001. Pearce et al. 2002a. Pearce et al. 2002b. Pearce et al.
2002c. Robinson et al. 2002). Surprisingly few investigations.
however, have focused on how prepared diets affect gonad quali-
ties such as color, texture, firmness, or taste (see Motnikar et al.
1997, Goebel & Barker 1998. Watts et al. 1998, McLaughlin &
Kelly 2001, Pearce et al. 2002a. Pearce et al. 2002b, Pearce et al.
2002c, Robinson et al. 2002) — factors at least as important as
gonad yield in the commercial roe industry.
Diets that have been developed specifically for gonad enhance-
ment generally contain five major components: ( 1 ) feed binders
(e,g,, agar, gelatin, ligno-sulfonate, sodium alginate, starch); (2)
protein sources (e.g., macroalgae, com meal, fish meal, soybean
meal, wheat meal); (3) lipid sources (e.g. com oil, canola oil, fish
oil); (4) vitamins and minerals; and (5) pigments (e.g.. (J-carotene.
lutein, zeaxanthin. capsanthin). Whereas corn, potato, and wheat
starches have been used individually as binders or fillers in a
number of experimental diets (Lawrence et al. 1989. Klinger et al.
1994. de Jong-Westman et al. 1995a, de Jong-Westman et al.
1995b. Motnikar et al. 1997. Robinson & Colbome 1997. Barker
et al. 1998, Goebel & Barker 1998, McBride et al. 1998, Pearce et
al. 2002a, Pearce et al. 2002b, Pearce et al. 2002c, Robinson et al.
2002), no study has examined the potential effect of various starch
types in prepared feeds on gonad quantity or quality in sea urchins.
Starches may, indeed, be able to affect gonad quality. Previously,
it was shown that prepared diets bound solely with unmodified
com starch produced significantly better-colored gonads than diets
made with other binders such as gelatin, guar gum, or sodium
alginate (Pearce et al. 2002a). The authors hypothesized that com
starch may have led to greater production of storage glycogen and,
thus, a lighter or whiter gonad background and a brighter gonad
color (Pearce et al. 2002a).
Several studies utilizing prepared diets for feeding sea urchins
have incorporated kelp plants or kelp meal in the diets (Levin &
Naidenko 1987, Klinger et al. 1997. Lawrence et al. 1997,
McBride et al. 1998. Watts et al. 1998. Havardsson et al. 1999.
Pantazis et al. 2000. McLaughlin & Kelly 2001 . Olave et al. 2001 ).
Considerably fewer studies have used rockweed meal, derived
from Ascophyllum nodosum, in prepared feeds (Pearce et al.
2002a, Pearce et al. 2002b, Pearce et al. 2002c). Of the common
species of macroalgae available to S. droebachiensis in its natural
environment, kelps (such as Alaria, Lawinaria. Macrocysris.
Nereocystis) are usually preferred, with fucoid algae {Fiicits spp.
and A. nodosum) generally eliciting intermediate to low preference
(Vadas 1977, Larson et al. 1980. Himmelman 1984, Himmelman
& Nedelec 1990). Green sea urchin populations in the Bay of
Fundy may be an exception to this generalization, however, as they
express a preference in feeding trials for A. nodosum (Mackay
1976). the most abundant alga present in the ecosystem. No study
has tested the efficacy of kelp meal versus rockweed meal in
prepared diets on the gonad yield or quality of sea urchins.
The development of suitable color in gonads of sea urchins fed
prepared feeds can be problematic if appropriate levels of certain
dietary pigment sources are not included in the diet (Barker et al.
1998. Grosjean et al. 1998. Watts et al. 1998. Pearce et al. 2002a.
Pearce et al. 2002b). de Jong-Westman et al. (1995a) found that
inclusion of p-carotene (60 mg kg"' dry weight of feed) in pre-
pared feeds significantly increa.sed gonad growth in S. droe-
bachiensis as opposed to feeds without the pigment, but did not
examine the gonad quality of sea urchins given feeds containing
P-carotene. Research by Robinson et al. (2002) with S. droe-
bachiensis has shown that p-carotene is an effective pigment
source for producing the bright yellow/orange coloration sought
after by the commercial roe market and that the most effective
concentration is 200-250 mg kg"' dry weight of feed. No studies
have examined the effect of pigmentation source or concentration
on gonad quality factors, such as texture, firmness, or taste. The
objective of this study was to conduct a multi-factor experiment to
examine the combined effects of (I) starch type (com, potato, or
tapioca); (2) macroalgal meal source (kelp or rockweed meal); and
(3) p-carotene concentration (0 or 200 mg kg"' dry weight of feed)
on the gonad yield and quality of adult green sea urchins, S. droe-
hacliiensis, held in a land-based culture facility.
MATERIALS AND METHODS
Sea Urchin Collection and Maintenance
Adult green sea urchins. 5. droebachiensis. were collected by
SCUBA divers on March 24-27. 1999 off Bancroft Point. Grand
Manan Island. Bay of Fundy. Canada (44°43'N, 66"44'W) on a
rocky, cobble bottom at a depth of -10 m (high tide). Mean test
diameter and wet weight of a sub-sample of these sea urchins were
61.2 ± 4.7 mm and 100.5 ± 20.4 g. respectively (mean ± SD. n =
30). Sea urchins were placed in plastic tote boxes with ambient
seawater and transported to the laboratory within 3 h of collection.
They were then put in white plastic tanks (L x W x H: 50 x 50 x
28 cm) that were supplied with flow-through, ambient seawater at
a flow rate of -5 1 min"'. These tanks were equipped with double
standpipes (ID of outer pipe: 35 mm; ID of inner pipe: 18 mm) and
were designed so that seawater entering into the tanks at the top
exited at the bottom. Initial stocking density was 100 urchins
tank"' or -168 kg m"' of water volume or -14 kg m"' of tank
surface area. Sea urchins were starved for 12-15 d prior to experi-
mentation to standardize relative hunger levels. Any individuals
that died during that period were removed and replaced.
Seawater temperature was automatically recorded in a header
tank every 15 min during the experiment by a temperature data
logger. The temperature gradually increased during the experimen-
tal period (min: 3.6°C, max: I3.6°C. mean ± SD: 7.4 ± 2.0°C. n =
7839). Lighting for the experiment was provided by overhead
fluorescent lights (34 W Sylvania "Cool White") set to a constant
photoperiod of 15.5 h light and 8.5 h dark (i.e.. photoperiod cor-
responding to early July in the Bay of Fundy).
Diet Preparation
Ingredients used in diets are shown in Table 1 . Kelp meal was
produced by collecting fronds of Laminaria longicruris. drying it
(either solar drying or in a convection dryer at -20°C), and grind-
ing it into small flakes using a hammer mill. Rockweed meal,
derived from AscophyUum nodosum, was produced by Tidal Or-
ganics Inc. (Lower East Pubnico. Nova Scotia. Canada) and pur-
chased from Shur-Gain. Maple Leaf Foods Inc. (Truro. Nova
Scotia, Canada). Dulse powder. Pabnaria pabnata. was produced
by and purchased from Roland's Sea Vegetables (Grand Manan,
New Brunswick. Canada). Com starch, corn oil, and molasses
were purchased from a local supermarket. Potato starch was pro-
duced by World Flower (Gemiany) and purchased from East Coast
Scale Company Ltd. (Dartmouth, Nova Scotia, Canada). Tapioca
starch was produced by the National Starch and Chemical Com-
pany (Bridge water, NJ) and purchased from Kennedy Distribution
Gonad Enhancement of Stronuylucentrotus druebachiensis
507
TABLE 1.
Ingredients used in diets
("Kelp" = kelp meal. "Rock" = rockweed meal), and p-carotene
concentration ("0" = 0 mg kg . "200"
200 mg ks"'). For
Ingredient
Dry Weight ( % )
Starch (corn, potato, or tapioca)
Macroalgal meal (kelp' or rockweed'')
Rovimix p-carotene 0.2%'^^
Soybean meal
Dulse powder''
Molasses
Gelatin (pork)
Canola oil
Dicalcium phosphate
Lecithin
Ethoxyquin
Vitamin pre-mix"^
Mineral pre-mix'
Vilamni C (Stay C)
TOTAL
24.0 or 14.0
22.8
0.0 or 10.0
27.9
10.0
5.0
5.0
2.0
L8
LO
0.2
O.I
0.1
0.1
100 0
'^Laminaria longicniris
^Ascophyllum nodosum
'Contains 98% wheat middlings, 1.8% starch-coated matrix of gelatin and
carbohydrates, and 0.2% (J-carotene. Obtained from Shur-Gain. Maple
Leaf Foods Inc.
''Palmaria pahnata
'Contains ground wheat, vitamin E. vitamin C (Stay C). inositol, ethox-
yquin. vitamin D,, niacin, calcium pantothenate, vitamin K. soybean oil.
vitamin B,,, biotin, riboflavin, pyridoxine, thiamine, vitamin A, and folic
acid. Levels are proprietary information. Obtained from Shur-Gain, Maple
Leaf Foods Inc.
'Contains ground wheat, manganese sulphate, iron sulphate, zinc sulphate,
soybean oil, calcium iodate. selenium selenale. and copper chloride. Levels
are proprietary information. Obtained from Shur-Gain. Maple Leaf Foods Inc.
Inc. (Moncton, New Brunswick, Canada), Pork gelatin, with a
bloom factor of 175, was obtained from CSP Foods Inc. (Moncton.
New Brunswick, Canada). This was found to be the best of a
number of different binders tested in terms of maintaining pellet
stability (Pearce et al. 2002al. Rovimix (J-carotene 0.2% was pro-
duced by Hoffmann-La Roche Ltd. (Cambridge. Ontario, Canada)
and purchased from Shur-Gain. This product contained 0.2%
P-carotene. 1.8% starch-coated matrix of gelatin and carbohy-
drates, and 98% wheat middlings. Feeds with P-carotene added
had a concomitant reduction in starch level while all other dietary
ingredients remained at the same percentage (Table 1 ). All other
ingredients were purchased from Shur-Gain.
A small Hobart mixer/grinder (Hobart Corporation, Troy, OH)
was used to mix the dry ingredients with hot freshwater (~100"C)
and to extrude a moist pellet (diameter; 5/16" or 7.9 mm). These
pellets were then air dried at ~20°C in a forced-air drying oven and
later stored at 3-5°C in covered plastic boxes until used in ihc
experiment. Twelve different prepared diets were formulated to
examine three experimental factors: ( 1 ) starch type (com. potato.
or tapioca); (2) macroalgal meal source [kelp (Laniinaria longi-
cruris) or rockweed (Ascaphyllum nodosum) meal]; and (3)
P-carotene concentration (0 or 200 mg kg"' dry weight of feed).
Each level of one factor was present in combination with each
level of the other two factors in a totally crossed experimental
design. For brevity and clarity, treatment names have been abbre-
viated using the starch type ("Com" = com starch, "Pot" =
potato starch, "Tap" = tapioca starch), macroalgal meal source
example, a feed containing corn starch, rockweed meal, and 200
mg kg"' of P-carotene would be abbreviated "Com Rock 200,"
Experimental I'roloeoh
The experiment was conducted for 12 wk (April 8 to July I.
1999). In addition to the 12 prepared diet treatments (see "Diet
Preparation"), there was a control treatment of kelp (fronds of
Laniinaria liini;icniris and/or L. digitaui. predominantly the
former). Three replicate tanks were established for each of the 13
treatments, each replicate having 100 sea urchins at the beginning
of the experiment and being placed in a separate group in a com-
pletely randomized block design. Sea urchins were fed twice a
week (generally Monday and Friday) at a rate of 3.0% body weight
d"' of kelp or 0.5% body weight d"' of prepared feed (n.b., total
food amounts took into consideration all days in between feedings
including the day of feeding). Further research — examining gonad
production of sea urchins fed the Corn Rock 0 diet at ration levels
of 0.25. 0.50, and 1.00% body weight d"' — has shown that gonad
yield is inaximized at 0.50% body weight d"' (Pearce et al. 2002c).
Sea urchins were hand fed and attempts made to ensure that all
individuals had equal access to feed. Tanks were cleaned before
feeding by removing the standpipes. allowing the tanks to drain,
and washing the uneaten feed and fecal material out of the tanks
with ambient seawater. While feeding rates were not measured
directly, generally there was little uneaten food remaining in the
tanks at the time of cleaning. Dead individuals were removed from
the tanks as soon as they were observed, but not replaced.
A random sample of 30 sea urchins, chosen from extra indi-
viduals that were not part of the study, was taken at the beginning
of the experiment to assess initial gonad yield, color, texture, and
firmness. Yield and color were quantified every second week for
the 12-wk duration of the experiment while texture and firmness
were assessed on weeks 6 and 12 of the experiment. This was done
by randomly sampling 10 sea urchins from each replicate tank.
Gonad taste was as.sessed at weeks 6 and 1 2 of the experiment by
randomly sampling three sea urchins from each replicate tank;
each individual being sampled by two independent tasters, both
unaware of treatment designation. Feeding rates were adjusted for
sampled individuals but not dead ones, since percent mortality
during the experiment was low [<10.3% per 12 wk in all treat-
ments (see Results)]. Thirty sea urchins were randomly sampled
from the wild source population at the end of the experiment for
assessment of yield and quality.
Sampled sea urchins were vigorously shaken to remove excess
external water and their test diameter and mass measured using
digital calipers and a digital balance, respectively. Urchins were
then cracked open, thoroughly drained of internal fluid, and re-
weighed. Gonads were scooped out of the tests, rinsed in seawater,
and gently shaken using forceps to remove as much water as
possible, but not blotted dry. The gonads were then placed in
pre-weighed aluminum pans, weighed, assessed for quality, dried
to a constant weight in a 70°C oven for a minimum of 48 h, and
then re-weighed. Color was assessed using 79 different paint card
samples (Home Hardware. Beauti-Tone) that were later converted
to a rating of 1—4 (see scale later). Gonad color was always as-
sessed under standardized light conditions [i.e., 50 cm away from
a single-point, artificial light source (20 W Sylvania Cool White
fluorescent light) with no natural lighting]. At weeks 6 and 12 of
508
Pearce et al.
the experiment, gonad color was also subjectively rated without
the use of the paint samples using the 10 randomly sampled indi-
viduals from each replicate tank. Gonad characteristics were quan-
tified as follows:
Gonad Yield (%) = (wet gonad weight/wliole urchin weight) x 100
Gonad Water {%) = |(wet gonad weight -
dry gonad weight)/wet gonad weight] x 100
Gonad Color— Subjectively by Eye With or Without Paint Samples
(Rating 1^)
1 = bright yellow or orange (equivalent to Grade A in com-
mercial roe industry)
2 = paler yellow or orange, mustard (Grade A or Grade B)
3 = yellow-brown, orange-brown, red-brown, cream (Grade B
or Grade C)
4 = any other color (e.g., dark brown, grey) (Grade C)
Gonad Texture — Subjectively by Eye (Rating 1-4)
1 = two distinct gonad segment halves, very smooth
2 = two distinct gonad segment halves, smooth (distinction
and smoothness <1 )
3 = distinction of gonad segment halves possible but <2,
rough/granular
4 = distinction of gonad segment halves not possible, rough/
granular
Gonad Firmness — Subjectively by Eye (Rating 1-4)
1 = very firm
2 = firm
3 = soft
4 = very soft
Gonad Taste — Subjectively by Two Independent Tasters
(Rating 1-6)
1 = excellent (very sweet)
2 = very good (very sweet, but <1)
3 = good (sweet)
4 = satisfactory (bland: not sweet, not bitter)
5 = poor (bitter)
6 = very poor (very bitter)
Gonad color was also objectively quantified at weeks 0. 6,
and 1 2 using a retlected-light, fiber-optic spectrophotometer taking
three replicate measurements of L* (intensity or lightness), a* (hue
or redness), and b* (chroma or yellowness) from each of 30 sea
urchins at week 0 and three replicate measurements from each of
five randomly sampled sea urchins from each replicate tank in
weeks 6 and 12. For a full description of the spectrophotometric
system see Robinson et al. (2002).
Statistical Analyses
For percent gonad yield, percent gonad water, gonad color done
by eye, gonad texture, and gonad firmness of experimental ani-
mals, a mean was calculated using the 10 sea urchins sampled from
each replicate tank (for the wild urchins sampled at weeks 0 and
12. thirty individuals were randomly placed in three groups often
to obtain three mean values). This mean "tank" value was then
used in subsequent statistical analyses (/( = 3). For gonad taste, a
mean value was calculated for each sea urchin using the two in-
dependent observations and this value was then used in the calcu-
lation of mean tank values. These tank values were then used in
statistical analyses (/i = 3). For gonad color assessed with the
spectrophotometer, mean values of L*, a*, and b* were calculated
for each sea urchin using the three measurements taken. These
individual means were then used to calculate a tank mean from the
five sampled sea urchins taken from each tank. Tank means were
then used in subsequent statistical analyses (n = 3).
For the various gonad characteristics, two-way ANOVAs
(completely randomized block design with tank as the blocking
factor) were used to determine the significance of the differences
among all treatments at the end of the experiment, including the
kelp-fed sea urchins and wild samples taken at the beginning and
end of the experiment. To examine more closely the combined
effects of starch type, macroalgal meal source, and ^-carotene
concentration, four-way ANOVAs (starch type, macroalgal meal
source, p-carotene concentration, and tank as a blocking factor)
were conducted on the various gonad characteristics at the end of
the experiment. To assess the effects of starch type, macroalgal
meal source, and (B-carotene concentration on percent gonad yield,
percent gonad water, and gonad color over time, four-way repeated
measures ANOVAs were used. In these analyses the blocking
factor was left out so as not to produce a cumbersome tlve-way
ANOVA. In multi-way ANOVAs with significant interaction
terms, the effect of one main factor was examined within each
level of the other main factors with two-way ANOVAs (treat-
ment and block). Where significant P-values were generated in
ANOVAs, Fisher's LSD post-hoc comparison tests were used to
evaluate differences among pair-wise means {P < 0.05). Probabil-
ity plots were used to confirm that data were normally distributed
and Cochran's tests (P < 0.01) used to verify that variances were
homogeneous.
RESULTS
Mortality
At the end of the 12-wk experiment, cumulative percent mor-
tality in the various feeding treatments ranged from a low of 1 .7 ±
0.7% (mean ± SE) for Com Kelp 200 and Tap Kelp 200 to a high
of 10.3 ± 5.9% for Pot Rock 0 with most treatments having a mean
cumulative percent mortality under 5% (Fig. lA). There were no
significant differences in cumulative percent mortality among any
of the treatments including the kelp control (2.3 ± 0.7%) (Table 2).
Gonad Yield
Mean percent gonad yield increased from 15.1% at the begin-
ning of the experiment to at least 19.2% in all prepared diet treat-
ments (Fig. IB) with percent gonad yield increases per week rang-
ing from a low of 0.3% for Tap Kelp 200 to a high of 0.8% for Tap
Kelp 0. Mean percent gonad yields for the feeding treatments at the
end of the 12-wk experimental period and the wild samples col-
lected at the beginning and end of the experiment differed signifi-
cantly (Table 2). All prepared feed treatments had significantly
higher percent gonad yields (range: 19.2-24.3%) than the kelp
control (14.5 ± 3.9%) or wild sample collected at the end of the
experiment (2.8 ± 0.5%) (Fig. IB). All prepared feed treatments
except Tap Kelp 200 (19.2 ± 1.1%) and Tap Rock 200 (19.6 ±
2.1%) had significantly higher percent gonad yields than the wild
sample taken at the beginning of the experiment (15.1 ± 1.0%)
(Fig. IB). There was only one significant pair-wise comparison
among the prepared feeds at the end of the experiment: Tap Kelp
0 (24.3 ± 1.3%) had a higher percent gonad yield than Tap Kelp
Gonad Enhancement of Strongylocentrotus droebachiensis
509
A
s
^f^hk
t\
Li"
O 10
■*■ *" <e ^ -t ,^'' # jf -k- i"^ •J-*jf
^ ^ ^ ^jf -^ ^ «^if
n
At) r
I ABAB^B
No Oisljnct Halves
Rough/Granular
Less Dislincl Halves ^
Rough/Granular -^
Dislina Halves E
Smoolh ij_ 2
Dislincl Halves
a" Q° 'fi aV ■*
1 0(&O^OJj.OJj'i^O^^^
Very Firm
3 81
5 80
Ma
iXhX.j
o^<5>,°„o°
^'/^/ -^'
.^.^
u
A AB
1 i
Very Poo(
Very Bitlei
Very Good
Very Sweel(<1)
Excellenl
Very Sweet
<f</
Figure 1. Mean cumulative percent mortality (A), mean percent gonad yield (B), mean percent gonad water (C), mean gonad texture rating (D),
mean gonad firmness rating (E). and mean gonad taste rating i¥) lor all experimental treatments and wild controls at the end of the experiment.
See text for full explanation of gonad quality ratings. Error bars are SE and n = i. Letters above bars indicate the results of Fisher's LSD multiple
comparison post-hoc tests showing significant pair-wise differences among experimental treatments and wild controls within each graph. "NS"
denotes no significant differences among treatment means.
200 (Fig. IB). The kelp control and wild sample collected at the
beginning of the experiment had significantly greater percent go-
nad yield than the wild sample taken at the end of the experiment
(Fig. IB).
A 4-way ANOVA examining the effects of starch type, mac-
roalgal meal source, p-carotene concentration, and block on per-
cent gonad yield at the end of the experiment revealed a significant
effect of P-carotene concentration, but no other significant main or
interaction effecfs (Table 3). Feeds with 200 mg kg"' of p-carotene
produced significantly lower percent gonad yield than those with-
TABLE 2.
Results of separate 2-way ANOVAs on cumulative percent mortality, percent gonad yield, percent gonad water, gonad color rating. L*, a*
b*, gonad texture rating, gonad firmness rating, and gonad taste rating at the end of the 12-wk experiment. Sources of variation are
treatment, block, and error.
Source
SS
DF f-Ratio
P-Value
SS
DF f-Ratio
/"-Value
SS
DF f-Ratio
P-Value
Mortality
Yield (%)
Water {%)
Treatment
228.97
1 2 0.94
>0.5
1253.83
14 11.37
<0.001
58.14
14 4.04
<0.001
Block
161.08
2 3.98
<0.05
15.93
2 1.01
>0.1
0.56
2 0.27
>0.5
Errcir
486.26
24
220.63
28
28.81
28
Color
Rating (with paint samples)
Color Rating (without paint samples)
L* (brightness)
Treatment
1.58
14 0.77
>0.5
1.13
14 1.^5
>0.1
419.60
14 1.54
>0.l
Block
0.41
2 1 .40
>0.1
0.29
2 2.42
>0.1
8.46
2 0.22
>0.5
Error
4.10
28
a* (redness)
1.68
28
b* (yellowness)
543.46
28
Texture Rating
Treatment
85.56
14 1.63
>0.1
122.22
14 3.71
<0.005
3.98
14 2.89
<0.01
Block
3.72
2 0.50
>0.5
5.36
2 1.14
>0.1
0..30
2 1.51
>0.1
Error
104.74
28
Firmness Ratmg
65.89
28
Taste Rating
2.76
28
Treatment
1.5.^
14 0.89
>0.5
13.26
1 3 3.65
<0.005
Block
0.14
2 0.56
>0.5
0.19
2 0.33
>0.5
Error
,^.4.^
28
7.27
26
510
Pearce et al.
TABLE 3.
Results of separate 4-way ANOVAs on percent gonad yield, percent gonad water, gonad color rating, L*, a*, b*, gonad texture rating,
gonad firmness rating, and gonad taste rating at the end of the 12-wk experiment. Sources of variation are starch type (S), macroalgal meal
source (M), (5-carotene concentration (B), block, and error.
Source
SS
DF
f-Ratio
P-Value
SS
DF
f"-Ratio
P-Value
SS
DF
F-Ratio
f-Value
Yield (%)
Water (%)
Color
Rating
(with paint ^
>amples)
S
6.17
-)
0.52
>0.5
3.40
2
2.13
>0,1
0.02
1
0.10
>0.5
M
5.18
I
0.88
>0.1
0.34
1
0.42
>0.5
0.32
1
3.77
>0.05
B
44.98
1
7.64
<0.05
0.07
1
0.09
>0.5
0.19
1
2.20
>0,l
SxM
8.84
-)
0.75
>0.1
0.38
2
0.24
>0.5
0.01
1
0.01
>0,5
SxB
24.18
T
2.05
>0.1
1.60
1
1.00
>0.1
0.12
1
0.71
>0.5
MxB
0.41
1
0.07
>0.5
0.22
1
0.28
>0.5
0.01
1
0.01
>0,5
SxMxB
1.44
-)
0.12
>0.5
1.32
1
0.83
>0,1
0.05
1
0.30
>0.5
Block
7.72
->
0.66
>0.5
1 .05
1
0.66
>0.5
0.61
-)
3.58
<0.05
Error
129.53
n
17.53
->2
1.88
22
Color
Rating (without paint
samples)
L*
(brightness)
a*
(redness)
S
0.08
2
0.81
>0.1
8.41
T
0.23
>0.5
1.87
T
0.26
>0,5
M
0.20
1
4.08
>0.05
51.39
1
2.86
>0.l
1.22
1
0.33
>0.5
B
0.23
1
4.70
<0.05
45.70
1
2,54
>0.1
17.78
1
4.87
<0.05
SxM
0.01
1
0.07
>0.5
100.08
1
2,78
>0.05
12.86
T
1.76
>0.1
SxB
0.02
2
0.16
>0.5
27.82
1
0.77
>0.1
6.73
">
0.92
>0.1
MxB
0.03
1
0.68
>0.1
15.87
1
0.88
>0.1
12.91
1
3.53
>0.05
SxMxB
0.01
1
0.02
>0.5
33.70
2
0.94
>0.1
2.43
1
0.33
>0.5
Block
0.18
1
1.81
>0.1
2.34
2
0.07
>0.5
0.78
2
0.11
>0.5
Error
1.09
T>
396.04
11
80.41
22
b*
(yellowness)
Texture Rating
Firmness Rating
S
13.58
-i
3.20
>0.05
0.07
2
0.39
>0.5
0.17
1
0.68
>0.5
M
1.83
1
0.86
>0.1
0.32
1
3.53
>0,05
0.01
1
0.08
>0,5
B
4.93
1
2.32
>0.1
0.07
1
0,78
>0,1
0.28
1
2.21
>0,1
SxM
4(1.25
2
9.49
<0.001
0.31
2
1,69
>0.1
0.14
1
0.53
>0.5
SxB
18.14
2
4.28
<0.05
0.24
2
1,32
>0.1
0.03
1
0.11
>0,5
MxB
24.73
1
11.66
<0.005
0.01
1
0.05
>0.5
0.01
1
0.08
>0,5
SxMxB
1.54
1
0.36
>0.5
0.07
2
0.41
>0.5
0.08
1
0.32
>0,5
Block
1.50
2
0.36
>0.5
0.06
2
0.33
>0.5
0.09
1
0.37
>0.5
Error
46.65
11
2.00
22
2.83
22
Taste Rating
S
0.07
2
0.12
>0.5
M
0.54
1
1.84
>0.1
B
0,92
1
3.13
>0.05
SxM
0.59
2
1.00
>0.1
SxB
0.08
1
0.13
>0.5
MxB
0.29
1
1.00
>0.1
SxMxB
0.07
1
0.11
>0.5
Block
0.50
1
0.85
>0.1
Error
6.46
11
out the pigment (Fig, 2A). A 4-way repeated ANOVA examining
the effects of time, starch type, macroalgal meal source, and
P-carotene concentration on percent gonad yield showed a signifi-
cant effect of time, but no other significant main or interaction
effects (Table 4). Percent gonad yield increased over time with all
pair-wise comparisons among weeks being significantly different
except for comparisons between weeks 0 and 4. weeks 0 and 6, and
weeks 4 and 6 (Fig. 2B).
Gonad Water
Mean percent gonad water decreased during the 12-wk experi-
ment from 84.2 ± 0.3% at the beginning of the experiment to less
than 81.0% in all feeding treatments at the end of 12 wk (Fig. IC).
A 2-way ANOVA analyzing the effects of treatment and block on
percent gonad water at the end of the experiment showed a sig-
nificant effect of treatment (Table 2). All prepared feed treatments
(range: 79.5-81.0%). as well as the kelp control (80,9 ± 1.0%) and
wild samples taken at the end of the experiment (81.9 ± 0.8%), had
significantly lower percent gonad water than the initial sample
(84,2 ± 0.3%) (Fig. IC). There were no significant pair-wise dif-
ferences in percent gonad water among any of the prepared feeds
or kelp control (Fig. IC).
A 4-way ANOVA examining the effects of starch type, mac-
roalgal meal source, P-carotene concentration, and block on per-
cent gonad water at the end of the experiment revealed no signifi-
cant main or interaction effects (Table 3). A 4-way repeated
ANOVA examining the effects of time, starch type, macroalgal
meal source, and p-carotene concentration on percent gonad water
showed significant starch type and tiine main effects and a sig-
nificant starch type x (B-carotene concentration interaction (Table
4). For feeds with 0 mg kg"' of p-carotene, those containing potato
Gonad Enhancement of Strongylocentrotus droebachiensis
511
2
>-
■a
to
c
o
O
30
A
2b
A
-l-
1
B
-r
20
15
10
_L
-
5
0 200
[ll-Carotene] (mg kg"')
30
25
°^ 20
0)
>-
CD
c
o
O
15
10
B
B
A
-
D
E
D
-t
D
C
10
12
Time (week)
Figure 2. (A) Mean percent gonud yield at week 12 In feeding treatments with and without (i-carotene. Error bars are SE and n = 18. Letters
above bars indicate the results of an ANOVA showing significant difference between treatment means. (B) Mean percent gonad yield of all
feeding treatments at each sampling interval. Error bars are SE and ;i = 36. Letters above bars indicate the results of a Fisher's LSD multiple
comparison post-hoc test showing signiTicant pair-wise differences among weeks.
Starch produced gonads with significantly higher percent water
than those containing tapioca starch (Fig. 3A). There were no
significant differences, however, among starch types for feeds con-
taining 200 mg kg"' of P-carotene (Fig. 3A). There were no sig-
nificant differences between the two p-carotene concentrations at
any of the starch type levels. Percent gonad water decreased over
time with all pair-wise comparisons among weeks being signifi-
cantly different except for comparisons between weeks 0 and 2.
weeks 0 and 4, and weeks 2 and 4 (Fig. 3B).
Gonad Color
At the end of the experiment, mean color ratings of sea urchin
gonads from prepared diet treatments varied between 2.5 ± 0.3
(Tap Kelp 200) and 3.0 ± 0. 1 (Com Rock 0) for ratings done with
paint samples and between 2.3 ± 0.2 (Corn Kelp 200) and 2.8 ± 0. 1
(Tap Rock 0) for ratings done without paint samples (Fig. 4A. B).
There were no significant differences at the end of the experiment
among any of the prepared feeds, kelp control, or wild controls in
either color rating data set (Table 2, Fig. 4A, B).
Four-way ANOVAs examining the effects of starch type, mac-
roalgal meal source, p-carotene concentration, and block on gonad
color ratings at the end of the experiment revealed a significant
effect of p-carotene concentration for ratings done without paint
samples, but not for ratings done with paint samples (Table 3). In
both data sets, feeds with P-carotene produced better gonad color
than those without the pigment (Fig. 5A). There were no other
significant main or interaction effects in either color rating data set
(Table 3).
A 4-way repeated ANOVA examining the effects of time,
starch type, macroalgal meal source, and P-carotene concentration
on gonad color ratings done with paint samples showed a signifi-
cant effect of time, but no other significant main or interaction
effects (Table 4). Gonad color improved over time; sea urchins
sampled in weeks 6. 8, 10. 12 had significantly better gonad color
than those sampled in weeks 0 and 4 while those measured in
weeks 10 and 12 had significantly better gonad color than those
measured in weeks 0. 2. and 4 (Fig. 5B). Time also significantly
affected gonad color ratings done without paint samples, although
the effect of time was dependent on the interaction with macroal-
gal meal source (Table 4). For sea urchins fed kelp meal diets,
gonad color ratings significantly improved at each subsequent
sampling date; week 12 was significantly better than week 6,
which was significantly better than week 0 (Fig. 5C). For sea
urchins fed rockweed meal diets, gonad color ratings in weeks 6
and 12 were significantly improved from the beginning of the
experiment, but there was no significant difference between weeks
6 and 12 (Fig. 5C). There was no significant difference in gonad
color ratings between kelp and rockweed meal diets at weeks 0 or
6, but feeds containing kelp meal produced significantly better
gonad color ratings than feeds containing rockweed meal by week
12(Fig. 5D).
At the end of the experiment, mean values of L* of sea urchin
gonads from prepared diet treatments varied between 45.4 ± 2.3
for Com Rock 0 and 55.1 ± 0.4 for Corn Kelp 0 (Fig. 4C). There
were no significant differences at the end of the experiment among
any of the prepared feeds, kelp control (52.0 ± 1.3), or wild con-
trols (0 wk: 49.5 ± 2.8, 12 wk: 44.3 ± 4.0) (Table 2, Fig. 4C). There
were no significant main or interaction effects in the 4-way
ANOVA conducted on week 12 L* data (Table 3) and only the
effect of time was significant in the 4-way repeated ANOVA
(Table 4). The mean value of L* was higher in week 12 than in
weeks 0 or 6 of the experiment, but only the comparison between
weeks 6 and 12 was significantly different (Fig. 6).
Mean values of a* (hue or redness) of sea urchin gonads from
prepared diet treatments in week 12 varied between 19.2 ± 0.7 for
Com Kelp 0 and 23.0 ± 1.5 for Com Kelp 200 (Fig. 4D). There
were no significant differences at the end of the experiment among
any of the prepared feeds, kelp control (20.3 ± 0.9), or wild con-
trols (0 wk: 18.9 ± 1.3, 12 wk: 23.1 ± \A) (Table 2, Fig. 4D).
There was a significant effect of P-carotene concentration on a*
values at the end of the experiment, but no other significant main
or interaction effects (Table 3). Feeds with 200 mg kg"' of P-caro-
tene produced significantly higher a* values than feeds with 0 mg
kg"' of pigment (Fig. 7A). Only the effect of time was significant
in the 4-way repeated ANOVA (Table 4). Values of a* were
512
Pearce et al.
significantly higher in week 12 than in weeks 0 or 6 of the ex-
periment (Fig. 7B).
At the end of the experiment, mean values of b* (chroma or
yellowness) of sea urchin gonads from prepared diet treatments
varied between 16.2 ± 0.2 for Com Kelp 0 and 21 .5 ± 1 .4 for Corn
Rock 0 (Fig. 4E). Mean values of b* for the feeding treatments at
the end of the 1 2-wk experimental period and the wild samples
collected at the beginning and end of the experiment differed sig-
TABLE 4.
Results of separate 4-Hay repeated ANOV.\s on percent gonad yield, percent gonad water, gonad color rating, L*, a*, b*, gonad texture
rating, gonad firmness rating, and gonad taste rating. Sources of \ariation are starch type (S), macroalgal meal source (M), (i-carotene
concentration (B), time (Tl, and error.
Source
SS
DF
F-Ratio P-Value
SS
DF f-Ratio P-Value
SS
DF F-Ratio P-Value
S
M
B
SxM
SxB
MxB
SxMxB
Error
T
TxS
TxM
TxB
TxSxM
TxSxB
TxMx B
TxSxMxB
Error
S
M
B
SxM
SxB
MxB
S xMxB
Error
T
TxS
TxM
TxB
TxSxM
TxS xB
TxMxB
TxSxMxB
Error
S
M
B
SxM
SxB
MxB
SxMx B
Error
T
TxS
TxM
TxB
TxSxM
TxSxB
TxMxB
TxSxMxB
Error
55.48
23.30
126.68
20.99
40.66
7.78
25.93
799.39
2121.78
55.36
19.80
34.00
34.39
34.50
22.60
60.27
515.00
1
-)
24
6
12
6
6
12
12
6
12
144
Yield (9r)
2 0.83
1 0.70
1 3.80
2 0.32
2 0.61
0.23
0.39
98.88
1.29
0.92
1.59
0.80
0.80
1.05
1.40
>0.1
>0.1
>0.05
>0.5
>0.5
>0.5
>0.5
<0.001
>0.1
>0.1
>0.1
>0.5
>0.5
>0.1
>0.1
Color Rating (without paint samples)
0.10
0.01
0.11
0.01
0.03
0.01
0.04
2.06
1.81
0.09
0.44
0.13
0.01
0.04
0.03
0.06
2.28
9.42
3.20
0.41
19.25
49.47
0.50
1.48
78.89
878.57
53.62
18.44
5.76
36.46
34.72
62.60
0.81
180.88
24
0.59
0.01
1.25
0.02
0.18
0.11
0.23
19.07
0.47
4.58
1.40
0.02
0.22
0.26
0.29
4
48
b* (yellowness)
2 1.43
1
1
2
2
1
")
24
4
4
2
4
48
0.97
0.12
2.93
7.53
0.15
0.23
116.57
3.56
2.45
0.77
2.42
2.30
8.31
0.05
>0.5
>0.5
>0.1
>0.5
>0.5
>0.5
>0.5
<0.001
>0.5
<0.05
>0.1
>0.5
>0.5
>0.5
>0.5
>0.1
>0.1
>0.5
>0.05
<0.005
>0.5
>0.5
<0.001
<0.05
>0.05
>0.1
>0.05
>0.05
<0.001
>0.5
8.29
0.10
1.72
1.69
8.12
0.09
1.24
24.95
577.15
10.26
2.99
1.24
5.12
10.06
2.53
7.80
80.79
5.11
0.63
13.91
9.69
28.36
21.54
7.22
285.29
145.14
24.40
84.40
31.88
114.50
32.00
10.77
48.34
940.94
0.18
0.14
0.03
0.18
0.13
0.01
0.06
2.24
0.31
0.13
0.19
0.05
0.28
0.43
0.01
0.30
4.00
Water ( ':f )
2 3.99
1 0.09
1 1.66
2 0.8 1
2 3.91
1 0.09
: 0.60
Color Rating {with paint samples)
24
6
12
6
6
12
12
6
12
144
L* (brightness)
2 0.22
171.46
1.52
0.89
0.37
0.76
1.50
0,75
1.16
1
1
2
24
0.05
1.17
0.41
1.19
1.81
0.30
3.70
0.31
2.15
0.81
1.46
0.41
0.28
0.62
4
48
Texture Rating
2 0.96
24
4
4
2
4
48
1.51
0.29
0.96
0.67
0.03
0.31
1.87
0.39
1.12
0.27
0.85
1.28
0.01
0.90
<0.05
>0.5
>0.1
>0.1
<0.05
>0.5
>0.5
<0.001
>0.1
>0.5
>0.5
>0.5
>0.1
>0.5
>0.1
>0.5
>0.5
>0.1
>0.5
>0.1
>0.1
>0.5
<0.05
>0.5
>0.1
>0.1
>0.1
>0.5
>0.5
>0.5
>0.1
>0.1
>0.5
>0.1
>0.5
>0.5
>0.5
>0.1
>0.5
>0.1
>0.5
>0.1
>0.1
>0.5
>0.1
0.02
0.14
0.35
0.21
0.07
0.06
0.03
3.91
6.25
0.62
0.88
0.22
0.67
0.59
0.23
0.98
15.62
6.43
0.08
10.73
4.98
1.12
6.23
2.05
87.53
129.05
24.17
3.68
9.18
8.27
11.61
7.21
2.40
224.40
0.12
0.01
0.23
0.06
0.03
0.01
0.02
2.86
1.61
0.09
0.01
0.14
0.57
0.04
0.03
0.07
6.82
24
6
12
6
6
12
12
6
12
144
a*
">
1
1
1
24
2
4
2
2
4
4
0.06
0.83
2.16
0.65
0.21
0.35
0.10
9.59
0.48
1.35
0.34
0.52
0.46
0.36
0.75
(redness)
0.88
0.02
2.94
0.68
0.15
1.71
0.28
13.80
1.29
0.39
0.98
0.44
0.62
0.77
0.13
4
48
Firmness Rating
0.51
2
1
1
2
2
1
2
24
4
48
0.11
1.94
0.26
0.14
0.01
0.10
5.66
0.15
0.02
0.50
1.01
0.07
0.10
0.12
>0.5
>0.1
>0.1
>0.5
>0.5
>0.5
>0.5
<0.001
>0.5
>0.1
>0.5
>0.5
>0.5
>0.5
>0.5
>0.1
>0.5
>0.05
>0.5
>0.5
>0.1
>0.5
<0.001
>0.1
>0.5
>0.I
>0.5
>0.5
>0.1
>0.5
>0.5
>0.5
>0.1
>0.5
>0.5
>0.5
>0.5
<0.01
>0.5
>0.5
>0.5
>0.1
>0.5
>0.5
>0.5
continued on next page
Gonad Enhancement of Strongylocentrotus droebachiensis
513
TABI.F 4.
Continufd
Source
SS
DF
f-Ratio
P-Value
Tasie Rating
S
0.05
2
0.04
>0.5
M
1.23
1
1.95
>0.1
B
0.01
1
0.01
>0.5
SxM
0.24
T
0.19
>0.5
SxB
0.16
2
0.12
>0.5
MxB
0.07
1
0.11
>0.5
SxM>
B
1.10
1
0.87
>0.1
Error
15.14
24
T
0.11
1
0.29
>0.5
TxS
0.07
2
0.09
>0.5
TxM
0.01
1
O.OI
>0.5
TxB
2.08
1
5.75
<0.05
T X S X
M
2.28
2
3.14
>0.05
T X S X
B
0.02
2
0.03
>0.5
TxM>
B
1.06
1
2.94
>0.1
T X S X
MxB
1.69
T
2.34
>0.1
Error
8.7(1
24
nitlcantly (Table 2). Gonads from wild sea urchins generally had
lower b* values than those from sea urchins fed prepared feeds or
kelp, although only Com Rock 0. Com Rock 200 (19.5 ± 0.5). and
Tap Kelp 200 (20.9 ± 0.4) had significantly higher b* values than
wild samples (Fig. 4E). In the 4-way repeated ANOVA, there was
one significant main effect (time) and three significant interactions
(starch type x p-carotene concentration, time x starch type, and
time x macroalgal meal source x (J-carotene concentration) (Table
4). Comparing the two concentrations of (J-carotene at each inter-
action level revealed three out of 12 (week 0 data not tested)
pair-wise comparisons to be significant; feeds with 0 mg kg"'
P-carotene had significantly higher b* values than feeds with 200
mg kg"' P-carotene for Com Kelp and Pot Kelp at week 6 whereas
the reverse was true for Tap Kelp at week 12 (Fig. 8). Comparing
the three weeks at each interaction level revealed sicnificant dif-
ferences for all treatments except Com Kelp 0. Pot Kelp 0. Pot
Rock 200. Tap Kelp 0. and Tap Rock 200; weeks 0 and 12 gen-
erally had significantly higher b* values than week 6 (Fig. 8).
Gonad Texliirc, Firmness, and Taste
At the end of the experiment, mean gonad texture ratings of sea
urchins fed prepared diets varied between 1 .3 ± 0.1 for Com Kelp
0 and 1 .9 ± 0. 1 for Com Rock 0. Pot Rock 0. and Tap Kelp 0 (Fig.
ID). Mean texture ratings for the feeding treatments at the end of
the 12-wk experimental period and the wild samples collected at
the beginning and end of the experiment differed significantly
(Table 2). All prepared feeds, kelp control (1.7 ± 0.2). and wild
0-wk control (1.8 ± 0.2) had significantly lower texture ratings
than the wild 12-wk control (2.8 ± 0.3) (Fig. ID). Among the
prepared diets. Com Kelp 0 had significantly better gonad texture
than Com Rock 0. Com Rock 200 ( 1 .9 ± 0.2), Pot Rock 0, Tap
Kelp 0. and Tap Rock 0 ( 1.9 ± 0.2); there were no other signifi-
cant pair- wise differences among prepared feeds (Fig. ID). There
were no significant main or interaction effects on gonad texture in
either the 4-way ANOVA (Table 3) or 4-way repeated ANOVA
(Table 4).
Mean gonad firmness ratings of sea urchins fed prepared diets
varied between 1.9 ± 0.3 for Pot Kelp 200 and 2.5 ± 0.3 for Tap
Kelp 0 (Fig. IE). There were no significant differences among any
of the prepared feeds, kelp control (1.8 ± 0.2), wild 0-wk control
(2.1 ±0.3). or wild 12-wk control ( 1.8 ± 0.1 ) (Table 2. Fig. IE). A
4-way ANOVA examining the effects of starch type, macroalgal
meal source, p-carotene concentration, and block on gonad firm-
ness ratings at the end of the experiment revealed no significant
main or interaction effects (Table 3). Time was the only significant
effect in the 4-way repeated ANOVA (Table 4). Gonads were
significantly fimier at week 6 than at weeks 0 or 12. but there was
no significant difference in gonad firmness between weeks 0 and
12 (Fig. 9).
Mean gonad taste ratings of sea urchins fed prepared diets
varied between 3.3 ± 0.2 for Pot Rock 0 and Tap Rock 0 and 4.2
± 0.1 for Pot Kelp 0 (Fig. IF). A 2- way ANOVA revealed sig-
JO
to
c
o
O
yo
" H Corn
a Pot
■ Tap
8b
80
AB
jr.
A
B
rh
NS
0 200
[R>-carotene] (mg kg '
B
TO
TO
c
o
CD
90
85
80
75
B
A
A
A
B
C
D
E
-
10 12
Time (week)
Figure 3. (A) Mean percent gonad water o>tr the 12-wk experiment in feeding treatments with and without (J-carotene with the three difTerent
starches. Error bars are SE and n = 42. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing
significant pair-wise differences among starch types at each (5-carotene level. "N.S" denotes no significant difference among treatment means. (B)
Mean percent gonad water of all feeding treatments at each sampling interval. Error bars are SE and n = .^6. Letters above bars indicate the
results of a Fisher's LSD multiple comparison post-hoc test showing significant pair-wise differences among weeks.
514
Pearce et al.
AU
Hini
.•^ ,^ "^ ^ .^ ^ .^
Dark Brown
Grey/Black
Yellow-Brown
Orange-Brown
Pale Yellow
Pale Orange
Bnghl Yellow
Bnght Orange
E
03
o
o
o
i, A
Q ^ O ^ _Q, ^ Q ^ O ^ .^ c?" J^
Dark Brown
Grey/Black
Yellow-Brown
Orange-Brown
Pale Yellow
Pale Orange
Bnght Yellow
;n sfN Bnghf Orange
y^^ -r /^ o^ />o -c _5- <
^'-°</</
*^*
1^ ^ ^h
i
o°/o°>
O^ O^ O^ <5cO Q^ o^a^^-^
o
BCD
CD 1
CD I ■^
'^ f O^ tt ct° ^ j^ ^
Figure 4. Mean color rating with paint samples (A), mean color rating without paint samples (B), mean CIE lightness (Cl. mean CIE hue ID),
and mean CIE chroma (E) for all experimental treatments and wild controls at the end of the experiment. See text for full explanation of gonad
quality ratings. Error bars are SE and n = 3. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing
significant pair-wise differences among experimental treatments and wild controls within each graph. "NS" denotes no significant differences
among treatment means.
nificant differences among treatment means at the end of the ex-
periment (Table 2). All prepared feeds produced significantly
worse tasting gonads than the kelp control (2.3 ± 0.3) while the
wild sample collected at the end of (he experiment (2.5 ± 0. 1 1 had
significantly better tasting gonads than all prepared feeds except
Pot Rock 0 (3.3 ± 0.2) and Tap Rock 0 (3.3 ± 0.2) (Fig. IF). There
were no significant pair-wise differences among any of the pre-
pared feeds in terms of gonad taste ratings (Fig. IF). A 4-way
ANOVA examining the effects of starch type, macroalgal meal
source, P-carotene concentration, and block on gonad taste ratings
at the end of the experiment revealed no significant main or in-
teraction effects (Table 3 ). A four- way repeated ANOVA on gonad
taste ratings over time revealed no significant main effects and
only one significant interaction — time x p-carotene concentration
(Table 4). There was no significant difference between weeks 6
and 12 in gonad taste of sea urchins fed prepared feeds without
P-carotene. but gonad taste was significantly better at week 6 than
at week 12 for sea urchins given feeds with p-carotene (Fig. IDA).
There was no significant difference in gonad taste of sea urchins
fed feeds with or without pigment at either week 6 or week 12 (Fig.
lOB).
DISCUSSION
The experiment was begun during the natural spawning season
of the green sea urchin. Gonad yields of individuals sampled at the
beginning of the experiment were high (15.1 ± 1.0%), but natural
populations had completely spawned out by the end of the experi-
ment in July (gonad yield = 2.8 ±0.5'7f ). In contrast, experimental
sea urchins maintained in the laboratory under ambient tempera-
ture and fixed photoperiod and fed prepared diets did not undergo
complete spawning. Partial spawning occurred in some individuals
early in the experiment as evidenced by a slight drop in gonad
yield during the second week of the experiment. This was also
observed macroscopically as a number of individuals were leaking
gametes early in the experiment. After the second week, however,
there was a steady increase in gonad yield at each subsequent
sampling period. This increase in gonad yield was not due to
increasing water, however, since percent gonad water decreased
during the experiment. These results are interesting from a com-
mercial culture perspective since it shows that gonads of sea ur-
chins fed prepared feeds can be enhanced even during periods of
the year when natural populations are spawning. While sea urchins
given prepared feeds showed an increase in percent gonad yields
over the 12-wk experimental period, individuals fed kelp actually
showed a decrease, albeit not significant, over the same time in-
terval. Previous experiments have also shown that kelp can be an
inferior feed in relation to prepared diets in terms of optimizing
gonad yield (Pearce et al. 2002a, Pearce et al. 2002b, Pearce et al.
2002c). While kelp was inferior to prepared feeds in terms of
increasing gonad yield, it did produce significantly better tasting
gonads than the prepared diets.
Gonad Enhancement of Strongylocentrotus droebachiensis
515
"to
Qi
k_
o
o
O
o Without Samples
s Witti Samples
NS
r^
[+1
0 200 0 200
[n>-carotene] (mg kg ')
2 4 6 8 10 12
Time (week)
Dark Brown
Grey/Black
Yellow-Brown
Orange-Brown
Pale Yellow
Pale Orange
Bright Yellow
Bright Orange
Kelp Rock
CO
a.
E
to
to
'3
o
= 2
CD
_o
O
O 1
D
2 Kelp
□ Rock
N*^
-in
ph
A
B
-
Dark Brown
Grey/Black
Yellow-Brown
Orange-Brown
Pale Yellow
Pale Orange
Bright Yellow
12 Bright Orange
Time (week)
Figure 5. (A) Mean color rating («ith and without paint samples) at week 12 in feeding treatments with and without (J-carotene. Error bars arc
SE and n = 18. Letters above bars indicate the results of ANOVAs showing significant difi'erence between treatment means. "NS" denotes no
significant difference between treatment means. (B) Mean color rating (with paint samples! of all feeding treatments at each sampling interval.
Error bars are SE and ii = id. Letters above bars indicate the results of a Fisher's LSD multiple comparison post-hoc test showing significant
pair-v\ise differences among weeks. (C) Mean color rating (without paint samples) in feeding treatments with kelp or rockweed meal at the three
sampling intervals. Error bars are SE and n = 18. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests
showing significant pair-wise differences among weeks at each level of macroalgal meal source. (D) Mean color rating (without paint samples)
at the three sampling intervals for the kelp or rockweed meal treatments. Error bars are SE and ;i = 18. Letters above bars indicate the results
of ANOVAs showing significant difference between treatment means. "NS" denotes no significant difference between treatment means.
Whereas all prepared feed treatments experienced gonad
growth, the rate of increase (range: 0.3-0.8% wk~' ) was somewhat
slower than pubhshed rates of increase in previous studies that
have used prepared feeds to enhance 5. droebachiensis ( Klinger et
al. (1997): 1.4% wk"': Motnikar et al. (1997): 1.2-2.6% wk"';
Havardsson et al. (1999): 0.7-0.8% wk"': Pearce et al. (2002a):
1.2-1.4% wk"'; Pearce etal. (2002b): 0.9-1.3% wk"'; Robinson et
al. (2002): 1.6-2.2% wk"'). Differences in rate of gonad increase
may be attributed to variations in dietary components, especially
protein concentration (de Jong-Westman et al. 1995a) and/or pro-
tein source ratio (Pearce et al. 2002b). Time of year may also affect
rate of increase in percent gonad yield. In this study, while experi-
mental sea urchins maintained in the laboratory did not spawn
completely out, gonad yield dropped slightly in the second week of
the experiment suggesting partial spawning in some individuals.
Partial spawning was also evidenced by the presence of gonads
leaking gametes in many individuals early in the experiment. This
partial spawning event decreased the overall rate of gonad yield
increase.
Gonad yield was unaffected by starch type or macroalgal meal
source. This result was not surprising given that: ( 1 ) protein ap-
65
60
CO
c
55
.^ 50
LU
O
45
40
AB
-i-
0 6 12
Time (week)
Figure 6. Mean CIE lightness (L''') of all feeding treatments at each
sampling interval. Error bars are SE and ;; = 36. Letters above bars
indicate the results of a Fisher's LSD multiple comparison post-hoc
test showing significant pair-wise differences among weeks.
516
Pearce et al.
0 200
[fl-carotene] (mg kg^)
26
24
TO, 22
<D
^ 20
LU
o ''
16
14
B
A _
B B ■ -
n I
0 6 12
Time (week)
Figure 7. (A) Mean CIE hue or redness (a*l at week 12 in feeding treatments with and without p-carotene. Error bars are SE and n = 18. Letters
above bars indicate the results of an ANOVA showing significant difference between treatment means. (B) Mean CIE hue or redness (a*) of all
feeding treatments at each sampling interval. Error bars are SE and ii = 36. Letters above bars indicate the results of a Fisher's LSD multiple
comparison post-hoc test showing significant pair-wise differences among weeks.
pears to be the dietary component that may be predominantly
responsible for gonad growth (de Jong-Westman et al. 1995a) and
(2) the three starches contain minimum quantities of protein
(<0.3%, manufacturer's specifications) while the two macroalgal
meals have similar protein concentrations. The rockweed meal
used in the experiment had a protein concentration of -6% (Jeff
Whitman, Intervest Trading, pers. comm.). Protein concentration
of kelp can vary substantially with year, season, species, geo-
graphic location, and plant part, but Black (1950) reported that
fronds of L. saccharina. collected between April and July of two
successive years, had a range of 5.5-13.3% crude protein (% dry
weight), depending on month of collection.
It is unclear why feeds with p-carotene (or more specifically
Rovimix) supported significantly lower gonad growth than feeds
without the pigment. Robinson et al. (2002) had similar results in
one of their experiments, but this finding is contradictory to the
results of de Jong-Westman et al. (1995a) who reported that the
inclusion of p-carotene in prepared diets significantly increased
gonad growth in green sea urchins. More research on prepared
feeds with P-carotene is required to fully understand the mecha-
nisms at work.
The gonad color of sea urchins fed prepared diets improved
over time. This was shown subjectively with both color rating
schemes and objectively with L*. a*, and b* readings. These re-
sults indicate that formulated diets can be used to significantly
improve sea urchin gonad color. Feeds containing p-carotene pro-
duced significantly better gonad color by the end of the experiment
than feeds without the pigment. Similarly, Robinson et al. (2002)
reported that they could significantly improve gonad color of 5.
droehachiensis by incorporating a spray-dried form of microalga
25
*
CO
E
o
20
15
O 10
UJ
O 5
NS NS
ill
□ 0 mg kg '
n 200 mg kg '
NS
NS n
NS NS NS NS
\K
NS
M
NS
^
NS
NS
M
NS
NS
NS
ik
Kelp Rock
Kelp Rock
Kelp Rock
Kelp Rock
Kelp Rock
Kelp Rock
Kelp Rock Kelp Rock Kelp Rock
Corn
Pot
OWeek
Tap
Corn
Pot
6 Week
Tap
Corn Pot Tap
12 Week
Figure 8. Mean CIE chroma or yellowness lb*) for each feeding treatment at each sampling interval. Error bars are SE and n = }. Letters above
bars indicate the results of ANOVAs showing significant pair-wise differences among (J-carotene levels at each time/starch/macroalga level.
Numbers at bottom of bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing significant pair-wise differences
among time intervals within each starch/macroalga/p-carotene level.
Gonad Enhancement of Strongylocentkotus droebachiensis
517
a:
(/)
CO
CD
c
B
r^
A
r^
Very Soft
Soft
0 6 12
Time (week)
Firm
Very Firm
Figure 9. Mean firmness rating of all feeding treatments at each sam-
pling interval. Error bars are SE and ;/ = 36. Letters above bars
indicate the results of a Fisher's LSD multiple comparison post-hoc
test showing significant pair-wise differences among weeks.
{Diinaliella salina). rich in p-carotene. into prepared diets. They
tested prepared feeds containing a range of P-carotene concentra-
tion (50. 100, 250. 500 mg kg"' dry weight of feed) and found that
250 mg kg"' was the most effective level at producing suitable
gonad color (Robinson et al. 2002). Similarly, McLaughlin and
Kelly (2001 ) reported that prepared diets containing the microalga
Phaeodacnliim tricomiiliiiii (having as its major carotenoid pig-
ments fucoxanthin and diadinoxanthin and small amounts of
P-carotene) significantly improved gonad color of the sea urchin
Psammechiniis miliuris over control diets without microalgae
added. In contrast. Goebel and Barker (1998) reported that pre-
pared feeds containing synthetic P-carotene did not significantly
affect gonad color of the sea urchin Evechinus chloroticus. They
used a much higher concentration (i.e., 60 parts per thousand) than
recommended by Robinson et al. (2002) for optimizing color en-
hancement. Prepared diets containing astaxanthin or canthaxan-
thin — higher oxidation-state carotenoid pigments — do not signifi-
cantly improve gonad color of P. miliaris (Cook et al. 1998, Kelly
et al. 1998) or S. droebachiensis (Havardsson et al. 1999, Pearce et
al. 2002a). This suggests that sea urchins cannot use higher oxi-
dation state carotenoids, such as astaxanthin, as metabolic precur-
sors for p-carotene or echinenone. p-carotene is a precursor of
echinenone (Griffiths & Perrott 1976, Tsushima & Matsuno 1990,
Tsushima et al. 1993). both pigments being major common carot-
enoids found in the gonads of a number of echinoid species in-
cluding 5. droehachiensis (Griffiths & Perrott 1976, Matsuno &
Tsushima 2001).
Gonad yield, color, texture, firmness, and taste were not sig-
nificantly affected by varying the starch type or macmalgal meal
st)urce indicating that gonad quantity and quality are independent
of these two factors, at least with the starch and algal sources
tested. This suggests that economic considerations or product
availability, rather than biological factors, may influence the
choice of starch or macroalgal meal sources for inclusion in sea
urchin feeds. On the east coast of Canada for instance, rockweed
meal is readily available commercially and considerably cheaper
than kelp meal (non-bulk pricing: $0.80 CAD kg"' and $30.80
CAD kg"', respectively). Algal meal made from Ascophyllum no-
dosum is often sold and packaged erroneously as "kelp meal".
End-users of kelp meal should confimi with producers or suppliers
to ensure they are receiving a product inade from kelp.
ACKNOWLEDGMENTS
Project funding was provided by the National Research Council
of Canada Industrial Research Assistance Program (NRC-IRAP),
the Atlantic Canada Opportunities Agency (ACOA), and Ross
Island Salmon Ltd. (RISL). Many thanks are expressed to Phillip
Reece (NRC-IRAP), Andrew Woyewoda (NRC-IRAP). and
Nancy Williston (ACOA) who were all instrumental in securing
funding and reviewing project progress. Christopher Pearce was
partially supported by an Industrial Research Fellowship from the
Natural Sciences and Engineering Research Council of Canada.
Ken Brown, president of RISL, is greatly acknowledged for help-
ing to initiate and fund this research. We are indebted to the
technicians who helped run this experiment, Annise Brown and
Blaine Brown. Special gratitude is extended to Robert Young and
Joel Foster for assisting in the collection of sea urchins and to
Wade Blanchard (Statistical Consulting Service of Dalhousie Uni-
versity) for statistical consultation.
CD
a:
w
NS
■in
D Week 6
D Week 12
B
Arh
^
0 200
[R>-carotene] (mg kg ')
en
c
00 3
B
NS
■h
D 0 mg kg '
D 200 mg kg '
Very Poor
Very Bitter
Poor
Bitter
Satisfactory
Bland
Good
Sweet
Very Good
Very Sweet (<1)
Excellent
Very Sweet
6 12
Time (week)
Figure II). (A, B) Mean gonad taste rating of feeds with and without pigment (0 and 200 mg kg ' |3-carotene) at weeks 6 and 12. Error bars are
SE and n = 18. Letters above bars indicate the results of ANOVAs showing significant pair-wise differences between pigment concentrations.
"NS" denotes no significant difference between treatment means.
518
Pearce et al.
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Journal «f Shellfish Research. Vol. 22, No. I, 521-.'i25. 20U3.
PRODUCTION OF RED SWAMP CRAWFISH {PROCAMBARUS CLARKII) IN EARTHEN PONDS
WITHOUT PLANTED FORAGE: ESTABLISHMENT, MAINTENANCE AND HARVEST
OF POPULATIONS
LOUIS R. D'ABRAMO* AND CORTNEY L. OHS
Department of Wildlife and Fisheries. Mississippi State University. Box 9690. Mississippi
State. Mississippi ^9762
ABSTRACT Two separate studies were conducted in successive years to evaluate the effect of initial stocking densities and
restocking versus natural recruitment during successive years on the production of red swamp crawfish (crayfish). Proeambanis clarkii.
in earthen ponds without planted forage. In the first study, permanently flooded ponds (mean depth = 1.05 m) that contained no
crawfish populations were stocked with adult populations of red swamp crawfish (assumed male:female ratio of 1:1 ) at initial biomass
densities of 168.8, 225; and 281.8 kg/ha. Production was compared with unstocked ponds with recruitment populations arising from
animals that remained after harvest of the previous season (initial stocking density of 225 kg/ha). Crawfish populations were fed a
commercially available 25'7r crude protein crawfish feed, twice daily and trap harvested. The mean yield and individual harvest weight
of natural recruitment ponds ( 1 584 kg/ha) was not significantly different from that of ponds that were initially stocked at the increasing
densities (1623, 1755, and 1816 kg/lia, respectively). In the second study, restocking (112 kg/ha) versus natural recruitment and
crawfish versus catfish feed were evaluated. Crawfish were harvested by both trap and seine. The mean yield and harvest weight of
crawfish harvested from natural recruitment ponds (2374 kg/ha, 19. Ig) was not significantly different from that of crawfish harvested
from ponds that were restocked with broodstock (2160 kg/ha, 18.7g). Mean individual weight of harvested crawfish decrea.sed from
28.5 g in October to 18.4 g in July. There were no significant differences relative to the different feeds used. The results suggest that
after initial stocking, good management practices should result in sufficient recruitment to obviate restocking while still achieving
consistent annual yields.
KEY WORDS:
crawfish aquaculture in earthen ponds, Proeambanis elarkii. management
INTRODUCTION
Louisiana provides 90% of the US supply of crawfish (cray-
fish), approximately 47,240 mt per year (mean of 1991-1995)
from a combination of forage-based culture fisheries and capture
fisheries of P. clarkii. the red swamp crawfish (Huner 1997). Tra-
ditional culture of red swamp crawfish is based upon a flood and
drain management of shallow ponds in association with a planted
forage that is the basis for stimulation of the detrital food chain
(Huner etal. 1994). The practice of double-cropping crawfish with
a forage-feed, such as rice, may not always be possible or preferred
in states outside of Louisiana. As commonly practiced in Louisi-
ana, double-cropping with a forage limits realization of the true
potential of crawfish growth and production because this manage-
ment practice actually limits the time when ponds can be flooded
during the growing and harvest season. In addition, the presence of
forage restricts the mode of harvest to trap (Huner et al. 1994). In
addition to Louisiana, commercial culture of the red swamp craw-
fish, Proeambanis clarkii is conducted in Mississippi, Texas,
Delaware, North Carolina, Arizona, and many other states in the
United States (Huner et al. 1994).
Two of the major management problems associated with craw-
fish culture in systems with planted forage are depletion of suffi-
cient amounts of food prior to the end of the growing and harvest
season, and chronically low levels of dissolved oxygen. Production
ponds are actually shallow flooded fields where rice is planted. In
these systems, crawfish consume food derived directly or indi-
rectly from the decomposition of the forage during the fall. How-
ever, by spring, this food resource is often depleted and a large
population of resident crawfish that has yet to be harvested is left
without sufficient food resources. As a result, growth during the
*Corresponding author. E-mail: Ldabramocacfr.msstate.edu
rising spring temperatures cannot be fully realized (Avaull &
Brunson 1990). Warm-water temperatures combined with the
characteristic decomposition of plant material in this system are
conducive to low levels of dissolved oxygen. Levels of dissolved
oxygen less than 3 ppm are considered unsuitable for crawfish
(Huner et al. 1994). Frequent incidence of low levels of dissolved
oxygen induces stress in the crawfish, and feed efficiencies cor-
respondingly decrease (Avault & Brunson 1990). Under these ad-
verse conditions crawfish may even seek to respire atmospheric
oxygen, and even emigrate from the pond.
A management approach that eliininates the use of planted
forage in crawfish farming is attractive because permanently
flooded, deeper ponds permit better control of the quality of water
and food within the production system (D'Abramo & Niquette
1991, McClain & Romaire 199,5). Permanently flooded, deeper
ponds (D'Abramo & Niquette 1991) will result in water tempera-
tures that are cooler and not so susceptible to wide daily fluctua-
tion. Transition from an extensive to semi-intensive production
system offers resident crawfish populations a potentially more
suitable environment, as described by McClaine and Romaire
(1995).
In an effort to move toward semi-intensive management and
more reliable production from year to year in crawfish farming,
D'Abramo and Niquette (1991) evaluated the feasibility of seine
harvest and feeding of formulated pelleted diets as alternatives to
exclusive trap harvest and the use of planted or volunteer forage as
a food resource. Studies were designed to determine the best man-
agement practices for the production of red swamp crawfish in
earthen ponds without planted forage. They observed that two
different initial stocking rates of broodstock did not appear to
affect mean harvest weight, suggesting a high degree of plasticity
and unpredictability associated with production systems based on
natural recruitment.
521
522
D"Abramo and Ohs
MATERIALS AND METHODS
Common Pond Management Practices
Levels of dissolved oxygen and temperature for all experimen-
tal ponds were recorded daily for the entire harvest season to guide
the implementation of different management practices. When the
levels of dissolved oxygen were anticipated to decrease below 5
mg/L, pond water was aerated using a 0.5 hp aerator (Air-O-Lator
Corporation, Kansas City, MO). Emergency aeration was provided
by PTO driven paddlewheels when levels of dissolved oxygen
decreased below 3 mg/L. Every third day during May through
August. pH was recorded during the late afternoon. If pH values
exceeded 9.2, all ponds received an application of cracked com
(56.3 kg/ha) to increase the production of CO, and thereby lower pH.
Study 1
Before stocking, 1 2 earthen ponds ranging in water surface area
from 0.040 to 0.053 ha were treated with a chemical insecticide
(Ambush, ICI Agricultural Products) at a concentration of 50 ppb.
After 24 h the ponds were drained, filled half way, and again
drained to remove any residual insecticide. This procedure effec-
tively eliminated any resident crawfish from the designated ex-
perimental ponds. Ponds were then filled (mean depth of 1.05 m)
and initially stocked with adults (assumed male:female ratio of
1:1) at biomass densities of 168.75. 225, and 281.75 kg/ha into
ponds. The latter stocking densities exceeded those recommended
for conventional forage based ponds (56-84 kg/ha) in an attempt to
determine whether feeding could increase survival of young and
total annual harvest. An additional treatment consisted of four
ponds that already contained populations compo.sed of animals
remaining from the harvest of the previous season (initial first year
stocking density of 225 kg/ha) and natural recruitment.
Crawfish populations were fed a commercially available 15%
crude protein crawfish feed (Arcadiana Choice "25") twice daily.
Feeding rates (Table 1 ) were based on an estimated pond biomass
and water temperature. Total feed provided per treatment was in
proportion to the initial stocking biomass.
Crawfish were harvested using pyramid style traps constructed
of L91-cm diameter hexagon mesh with 61 -cm extended necks
TABLE \.
Monthly feeding rates by percent of total and kg/ha/yr.
Percent of Total
Month
Study
1 Study 2
January
0
0
February
0
0
March
15
8
April
15
15
May
25
20
June
20
20
Julv
5
8
August
0
0
September
0
0
October
5
11
November
10
10
December
5
8
Total feed fed (kj
;/ha/year)
*
,^700
'■ 2400 = low density; 3200 = mid density; 4000 = high density.
and three funnel openings between 3.8 and 4.4 cm. Traps were
baited with approximately 150 g of a commercially available bait
(Acadiana Choice - Medium), on Monday and Wednesday and
harvested on Tuesday, Wednesday. Thursday, and Monday. Craw-
fish were harvested a total of 97 days during periods of November
21, 1995 to December 7, 1995 and March 3, 1996 to August 1.
1996. On one day during each week, up to 50 individuals harvested
from each pond were randomly selected and individual weight and
sex were recorded. Males were classified as sexually active (form
I) or sexually inactive (form II) (Hobbs 1974).
All ponds were harvested once per month from May to August
with a 1.91-cm mesh, knotted nylon seine, 1.5 m in height, that
was modified through the attachment of a heavy nylon mud line to
the existing lead line. Fifty individual crawfish were randomly
selected from each seine harvest of each pond, and individual
weight and sex were recorded.
Study 2
The relative value of an annual management practice of re-
stocking (1 12 kg/ha) versus natural recruitment was investigated.
In addition, two feeds, a 25% crude protein formulated crawfish
feed (Acadiana Choice 25) and a sinking pelleted catfish feed
(2%% crude protein), were evaluated. Eight ponds, previously
stocked in the first study, were randomly assigned to each of the
two treatments, four ponds per treatment. An additional eight
ponds from the previous year were restocked with crawfish (112
kg/ha) at an assumed male:female ratio of 1:1. Four ponds were
randomly assigned to each feed thereby providing a 2 x 2 factorial
arrangement of treatments. The annual rate of application of the
commercially manufactured pelleted feeds was 3,700 kg/ha, pro-
portionally distributed over eight months based upon pond water
temperature and estimated resident biomass (Table 1).
Harvest was by trap and seine. Harvest by trap occurred on 99
days extending over 9 mo, from October 15, 1996 through De-
cember 13 and from March 5 to August 1, 1997. All ponds were
seine harvested once in May, June, and August. The same traps.
trap density, and seine described in study I were used. Trap har-
vest occurred on Monday. Tuesday. Thursday, and Friday. After
harvest of unbaited traps on Mondays and Thursdays, traps were
baited with approximately 150 g of a commercially bait (Acadiana
Choice - Jumbo) and harvested on the following Tuesday and
Friday, respectively. Compared with study 1, this management
protocol used one less day of baiting and modified the two 72 h
unbaited soak periods of study 1 to one 48 h and one 72 h. Data
were collated to examine the relationship of yield to water tem-
perature based upon whether the traps were baited.
Once weekly, up to 50 crawfish from each pond representing
the different treatments were randomly selected. In addition. 50
individual crawfish were randomly selected from each pond that
was seine harvested. In both instances, individual weight, sex. and
reproductive form of the males (Hobbs 1974) were recorded.
These data were collected to determine the composition of the
crawfish population at different times of the year and identify any
possible bias in harvest method relative to sex or male reproduc-
tive condition.
Statistical Methods
For study 1 . an analysis of variance ( ANOVA) using SAS (SAS
Institute 1988) was used to determine whether significant differ-
ences in mean individual weight, total production, and survival
Establishment of Red Swamp Crawfish in Earthen Ponds
523
existed among treatments. For study 2. a two-way analysis of
variance (ANOVA). using the general linear model procedure
(PROC GLM) of SAS (SAS Institute 1988), was used to determine
whether significant differences in mean individual harvest weight,
total production (kg/hal. and survival (%) existed between treat-
ments and whether a significant interaction between factors (feed
and stocking procedure) existed. An ANOVA was also conducted
to determine whether the proportions of each sex and male devel-
opmental stage harvested were significantly different for the treat-
ments in each study. All differences were considered significant at
P < 0.05. The relationship between trap yields and water tem-
perature for traps with and without bait was examined via linear
regression and the best fitting lines were then calculated.
1996- 1997
RESULTS
Sliidv ]
The mean yield of natural recniitmenl ponds ( 1589 kg/ha) was
not significantly different from that of ponds that were initially
stocked at densities of 168.75. 225. and 281.75 kg/ha ( 162.^, 1755.
and 1816 kg/ha. respectively). Mean individual weight of crawfish
harvested from natural recruitment ponds (17.3) was also not sig-
nificantly different from that of ponds that were initially stocked at
densities of 168.75. 225. and 281.75 kg/ha (17.9. 19.1. and 16.9 g.
respectively).
Yields from seine harvests, individual harvest weights. M/F
ratios, and 7r of form I males are presented in Table 2. Seine
harvest comprised 13-359^ of the total wet weight harvest per
month. The mean percent of form 1 males that were trap harvested
increased from 47c in April to 96% in August and declined to 12%
by December. When seine harvest was conducted, the proportion
of form 1 males was significantly lower than that obtained by trap
harvest for the same week (Fig. 1 ).
Study 2
The interaction between quality of feed and stocking proce-
dures was not significant (P = 0.45). The mean yield of natural
recruitment ponds (2374 kg/ha) was not significantly different
TABLE 2.
Mean yields from individual seine harvests relative to stocking
density treatments (Study 1).
1995-1996
May 8
June 5
July 1
July 30
Kg/ha
High
40.2
101.9
136.4
71.5
Medium
76.5
108.4
165.6
55.0
Low
58.8
71.9
126.6
73.8
Average wt.
(g)
High
12.0
13.7
14.8
18.8
Medium
13.0
14.6
15.7
18.3
Low
13.5
15.0
16.4
19.7
M/F ratio
High
1.08
0.91
1.04
0.91
Medium
1 .05
0.8
1.02
0.85
Low
1.15
1.46
0.9X
0.94
Form 1 male (%)
High
39
42
72
47
Medium
39
37
71
64
Low
42
47
69
51
100
S 40
Week
1997- 1998
100
80
60
40
20
u
□ Trap
■ Seine
22
28
Week
Figure 1. The relative percentages of form I males harvested by trap
and seine during the same week.
from that of ponds that were restocked with broodstock (2160
kg/ha). Mean individual weight of crawfish harvested from natural
recruitment ponds ( 19. 1 g) was not significantly different from that
of restocked ponds (18.7 g) and decreased in all ponds from 28.5
g in October to 18.4 g in July. There were no significant differ-
ences in production based upon feeds used.
Yields, individual weights. M/F ratios, and % of form I males
from seine harvests are presented in Table 3. Seine harvest com-
prised 10-40% of the total wet weight harvest per month. The
mean percent of form I males in all ponds increased froin 5% in
March to 95% in August. The proportion of form II males in seine
harvests indicates that they were not harvested in the same pro-
portion by trap (Fig. I ).
For crawfish harvested by trap, the relationship between mean
harvest/trap/day and temperature, with and without the use of for-
mulated bait, is presented (Fig. 2). The best fitting lines derived
from harvest data obtained from baited and unbaited traps were
detemiined. The intersection of the best fitting lines for unbaited
and baited traps is 15C. Above this water temperature, the use of
formulated bait becomes increasingly more effective than the use
of no bail as the divergence of lines indicates. Soak time for baited
traps was approximately 24 h. whereas the soak time for un-baited
traps was either 48 or 72 h.
DISCUSSION
Populations of crawfish initially stocked into earthen ponds
without planted forage appear to be self-sustaining for successive
annual harvests when sufficient food is provided, good water qual-
ity is maintained, and the harvest practices outlined in studies 1
524
D"Abramo and Ohs
TABLE 3.
Mean yields, maleifemale ratios, and percent form I males from
individual seine harvests relative to treatments representing
different management practices (Study 2).
1996-1997
May 6
June 20
Augl
Kg/ha
Nat. Rec./crawfish
82
126.2
84
Nat. Rec/catfish
27.8
85.8
54.7
Restockycrawfisli
118.2
144.1
96.2
Restock/catfish
41.2
93.6
105.8
Average wt. (g)
Nat. Rec./crawfish
15.6
15.5
17.3
Nat. Rec./catfish
13.5
16.1
14.8
Restock/crawtlsh
15.3
15.1
16
Restock/catfish
14.4
15.3
16.3
M/F ratio
Nat. Rec./crawfish
l.ll
0.99
Nat. Rec./catfish
0.88
0.69
Restock/crawfish
1.0
0.9
Restock/catfish
0.97
.77
Form I males (%)
Nat. Rec./crawfi.sh
48.9
68.4
Nat. Rec./catfish
68.6
66.4
Restock/crawfish
55.7
63.5
Restock/catfish
57.4
66.6
and 2 are conducted. Increases of 33.3% and 66.7% in stocking
biomass did not affect total annual production, even with corre-
sponding increases in feeding rates. This suggests that a carrying
capacity controlled by a variety of abiotic and biotic factors is
reached and maintained as crawfish are harvested, i.e. removed
from the pond. When ponds are well managed, restocking is un-
necessary unless an annual harvest is unexpectedly low. suggesting
that insufficient broodstock would be present for satisfactory re-
cruitment for the following year.
Active harvest by seine revealed that information about popu-
lation characteristics collected through passive trap harvest is not
accurate. Data obtained from siene harvest show that the propor-
tion of form II males in the pond population during May. June, and
July is higher than that suggested by data obtained from trap har-
vest. Form II males are not caught by trap in the same proportion
as they are present in the male population. This observation may be
a manifestation of a behavioral hierarchy that exists among differ-
ent males due to differential attraction to bait as a source of food.
Another behavioral-based explanation is that form II males are
more likely to leave the trap after the bait has dissolved as sug-
gested by Romaire (1995).
At water temperatures below 15°C. use of the commercial for-
mulated bait in traps was not effective in this study. Above 15°C.
baited traps are more effective, daily harvest exceeding that of
unbailed traps by approximately 0.01 kg/trap for every degree
Celsius increase. As a result, a difference of 0.06 kg harvested/
trap/day is realized by 20°C and 0.13 kg harvested/trap/day by
30°C. When bait is used, the higher daily yields/trap begin to be
consistently realized at a water temperature of s 1 7C and collec-
tively contribute to an overall increase in annual yield. The tem-
perature-dependent effectiveness of the bait used in this study may
be related to its ingredient composition. Effective baits for trap
harvest at temperatures <17 C may be achieved through modifi-
cation of the ingredient composition. Further research needs to be
conducted to evaluate different harvesting efforts, strategies, and
formulated baits within specific temperature ranges.
The lack of significance of many of the management practices
designed to improve production and evaluated in our studies may
simply be due to the lack of sufficient control. Production from
year to year is based upon a level of recruitment that cannot be
totally controlled. However, a management strategy that does not
incorporate an annual drain down followed by the planting of
forage seems to offer a greater chance for consistent production
from year to year. Nonetheless, the draining of conventional ponds
for the planting of forage does offer two indirect benefits, control.
0.7
0.6
0.5 H
:| 0.4
0.2
0.1
0.0
• Unbaited
o
Unbaited Predicted
o Baited
o
-
Baiteid Predicted
•
o
o
88
^
"
• o
o
•^-^
o
o
o
*-
-^
o 8
o*
o
o
•
o 8
- — • to
•
o
•
o
o
• •
•
•
W"
.-*-*^^S^
• • ••
• o
/^ o
•
o 8*
Q ^00
10
15
30
35
20 25
Temperature (C)
Figure 2. Linear regression displaying predicted mean yields (kg/trap/day) for harvest at different water temperatures with (;i = 50) and without
(H = 51) the use of formulated bait.
Establishment of Red Swamp Crawfish in Earthen Ponds
525
if needed, of predaceous fish that unintenlionally become establish
in the ponds and the oxidation of anaerobic sediments that may
have resuhed. Under the management practices described in this
investigation, an annual production of approximately 1700 to 1800
kg/ha can be expected. Higher yields could be realized with the
implementation of different harvest strategies. However, these ap-
proaches need to be weighed against the labor required. Practices
that are designed to increase production may be inherently limited
because of density-dependent factors, including cannibalism, for
which management has little control. Some increase in production
could be realized by methods to reduce the incidence of cannibal-
ism. In addition, changes in the distribution of production over an
annual cycle may be possible, but the increases may not be realized
because only a particular range of production can be realized given
the restrictions imposed by the nature of the management system.
The quality of food provided can have an effect but once a certain
level of quality of feed is attained, similar levels of production will
be achieved, because the feeds primarily serve as an indirect
source of nutrients. Most of the food provided appears to stimulate
secondary productivity within a pond similar to the detrital food
chain that is stimulated by the decomposition of planted forage in
traditional ponds. The level of secondary productivity is limited
and thereby controls the production of crawfish that can be real-
ized. Certainly, routine removal by harvest does contribute to
higher production. The red swamp crawfish is a species that has
many characteristics that make it appealing as an aquaculture spe-
cies. However, some management practices are probably not suc-
cessful simply because of the restrictions imposed by the nature of
the production system. In addition, the innate level of variation
among production ponds may preclude identification of significant
treatment-related differences that would only be observed with a
inordinate number of replicates. Any management practices that do
succeed must still remain within the confines of realizing a posi-
tive net return. Those management practices that are ultimately
identified as being most efficient and cost-effective must be trans-
ferred to larger (at least 0.5 ha) ponds to verify applicability.
ACKNOWLEDGMENTS
The authors thank the staff of the Eastern Unit of the National
Warmwater Aquaculture Center, Mississippi State University, par-
ticularly Ms. Beth Peterman. Mr. Bubba Groves, and Mr. Angus
Irvine, for their assistance in the management of the water quality
of the ponds, the feeding of the crawfish, and the harvesting. We
also thank Mr. Mack Fondren. Manager of the Eastern Unit, for his
cooperation and interest as well as Dr. Patrick Gerard, Department
of Agricultural Information Science and Education. Mississippi
State University for his guidance in the experimental design and
statistical analysis. The research was funded through a special
aquaculture grant from the United States Department of Agricul-
ture. Mississippi Agricultural and Forestry Experiment Stafion
Publication Number J 10230.
LITERATURE CITED
Avault, J. W. & M. W. Brunson. 1990. Crawfish forage and feeding sys-
tems. Rev. Aqualic Sci. .^:1-10.
D'Abramo, L. R. & D. J. Niquette. 1991. Seine harvesting and feeding of
formulated feeds as new management practices for pond culture of red
swamp crawfish. Procambarus clarkii (Girard. 1852). and white river
crawfish. / Shellfish Res. 10:169-177.
Hobbs. H. H., Jr. 1974. A checklist of the North and Middle Amencan
crayfishes (Decapoda: Astacidae and Cambaridae). Smirhsnnian Conli:
Zool. 166:1-153.
Huner. J. V.. M. Moody & R. Thune. 1994. Cultivation of freshwater
crawfishes in North America. In: J. V. Huner. editor. Freshwater craw-
fish aquaculture in North America, Europe, and Australia. Families
Astacidae. Cambaridae. and Parastacidae. Binghaniton. NY: Haworth.
pp. 5-1-V5.
Huner. J. V. 1997. The capture and culture fisheries of North American
crawfish. World Aquaculture 28:44-50.
McClain. W. R. & R. P. Roniaire. 1995. Management considerations for
the production of large Procamband crawfish. J. Shellfish Res. 14:553-
560.
Romaire. R. P. 1995. Harvesting methods and strategies used in commer-
cial Procambarid crawfish aquaculture. / Shellfish Re.s. 14:545-551.
SAS Institute. 1988. SAS/STAT Users Guide, release 6.03. SAS Institute,
Gary, NC. 1056 pp.
Jounud u] Shellfish Rcsuaich. Vol. 22, No. 1, 527-531, 2003.
PRODUCTION OF RED SWAMP CRAWFISH (PROCAMBARUS CLARKII) IN EARTHEN PONDS
WITHOUT PLANTED FORAGE: EVALUATION OF TRAP AND SEINE HARVEST STRATEGIES
LOUIS R. D'ABRAMO, *CORTNEY L. OHS, AND KATHLEEN C. ELGARICO
Deparliiicnt of Wildlife ciiul Fisheries. Mississippi State University. Box 9690, Mississippi State,
Mississippi 39762
ABSTRACT The et'lect of trap density and trap versus seine harvest on the production of red swamp crawfish were evaluated in
earthen ponds without planted forage. Crawfish were harvested from traps at densities of either 81 or 1 2 I/ha. 4x/week. from October
through July of the followmg year. In another treatment crawfish were harvested from traps at a density of 12 I/ha when water
temperatures were >I9'C and seine harvested when water teinperatures were between 15 and I9°C. Mean annual production ranged
from 2173 to 2606 kg/ha, and mean harvest weight ranged from 16.6 to 17.8 g. Total production and catch per unit effon for seine
and trap harvests at water temperatures between 15 and 19°C were not significantly different. Mean individual weight of seine
harvested crawfish was significantly less (1 1.4 g) than that of trap-harvested crawfish (21.6 and 17.6 g). In a second .study, the effects
of different harvesting strategies and two formulated feeds were evaluated. Crawfish were fed either a 32% crude protein, extruded,
slow-sinking formulated diet or a 32% crude protein, pelleted sinking diet, and harvested from traps either 3x (81 traps/ha) or 2x ( 121
traps/ha) per week. Trap harvest at 81 traps/ha, 3.x/week and 121 traps/ha, 2x/week produced 2447 and 1884 kg/ha, and a mean
individual harvest weight of 18.7 and 19.8 g. respectively. A significantly lower individual weight (16.2 g) was associated with the
pelleted sinking feed relative to the extruded, slow-sinking feed. However, mean total production was not significantly different
between treatments. Over 90% of the annual yield was harvested from April through October when water temperatures were >19°C.
KEY WORDS: crawfish aquaculture in earthen ponds, Procumbanis cUirkii, seine and trap harvest
INTRODUCTION
The goal of harvest strategies is to enhance efficiency of the
labor expended while maximizing production. In traditional for-
age-based fanning of the red swamp crawfish (crayfish), labor
accounts for 30-70"^ of the total direct operational expenses and is
primarily attributed to harvesting. Therefore, efficient harvest
based upon conditions of when, how, how often and at what level
of effort to harvest, is critical to the economic success of crawfish
farming systems. The ainount of labor is affected by trap density,
number of harvest days/week, number of trap sets/day (the number
of times a trap is prepared, with or without bait, to harvest crawfish
daily), and current market price of crawfish (Romaire 1995).
The size of harvested crawfish also must be considered because
quality of the product relative to market demand should not be
compromised in exchange for a reduction in labor. In forage-based
ponds for culture of crawfish, when soak time increases, larger but
fewer crawfish are harvested from traps (Romaire 1995). There-
fore, a harvesting strategy also must consider the number of craw-
fish that are harvested at one set. Inverting traps to prevent access
when bait is not provided might result in an increase in mean size
at harvest and yield because crawfish would be allowed to feed,
reproduce, and molt in the pond for longer periods of time during
the harvest season.
A decrease in catch per unit effort for trap harvest is encoun-
tered commonly for consecutive harvest days in production ponds
with (McClain et al. 1998) and without (D'Abramo & Ohs 2003)
planted forage. Proper management of the harvest schedule could
lead to a more consistent yield with a corresponding decrease in
labor. Trap density is also a component of an optimal harvest
strategy, whereby a reduction in cost per unit effort can be real-
ized.
Water temperature is also a major factor that infiuences the
efficiency of harvest. Temperature-related differences in harvest
*Corresponding author: E-mail: Ldabramo@cfr.msstate
strategy can also optimize trapping effort. Significant increases in
catch per unit effort (CPUE) were achieved with the u.se of com-
mercially manufactured baits when water temperature is equal to
or exceeds 19 C (D"Abramo & Ohs 2003). A similar relationship
between temperature and CPUE using formulated bait was ob-
served in production ponds with planted forage (Romaire 1995).
Efficient, cost-effective harvest at water temperatures less than
19°C requires a different approach that may include bait, soak
time, or method of harvest.
A preliminary investigation of the utility of seine harvest of
crawfish in production ponds without planted forage was con-
ducted by D'Abramo and Niquette (1991). However, consistent
yields were not achieved and mean individual weight of harvested
crawfish declined because as the number of harvested crawfish
accumulated in the seine, the ability of small crawfish to escape
through the mesh decreased. D'Abramo and Ohs (2003) used pe-
riodic seine harvesfing at pond water temperatures >19°C in an
atteinpt to reduce biomass density and density-dependent growth
reduction, with the intent to increa.se total annual yield. However,
seine harvest may be most effective at water temperatures <I9°C
when the effectiveness of a passive trap harvest in conjunction
with a formulated diet decreases.
In the absence of planted forage, formulated diets are needed to
serve as both a direct and an indirect source of protein and other
nutrients for the growth of crawfish. Uneaten food serves as both
an inorganic and organic fertilizer of the pond, thereby contribut-
ing to an increase in natural food of the crawfish through stimu-
lation of the delrital food chain. Diets that are more water stable
will presumably increase the possibility of crawfish using the feed
as a direct source of nutrients. An extruded feed is generally more
water stable than a pelleted feed due to the heating process used in
manufacture (De Silva & Anderson 1995). Buoyancy may also
play an important role because a slow-sinking pellet contains air
pockets that contribute to a lower rate of sinking and greater po-
tential for distribution throughout the pond. This study evaluated
trap density (81/ha versus 121/ha), and the effecfiveness of seine
versus trap harvest when pond water temperatures ranged between
15 and 19 C.
527
528
D"Abramo et al.
MATERIALS AND METHODS
Study 1
Twelve earthen ponds with estabhshed crawfish populations
from initial stocking either 1 or 2 y previously and, ranging from
0.045 to 0.069 ha in surface area, were used in this study. The
duration of the study period was September through July of the
following year. There were three treatments, four ponds/treatment.
For the first treatment, eight seine harvests were conducted be-
tween October 31 and March 17 when water temperatures were
between 15 and 19°C. When water temperatures consistently ex-
ceeded 19°C, crawfish were then trap harvested exclusively at a
trap density of 81 /ha. For the second treatment, crawfish were
harvested exclusively by trap at a density of 121/ha for the entire
study period. For the third treatment, the harvest schedule was the
same as the second treatment except trap density was 81 traps/ha.
An additional three seine harvests were performed when water
temperatures were >19°C, once each in May, June, and July. Pyra-
mid traps used in this study were constructed with 1.91 -cm wire
mesh (Gulf Coast Wire Products, Kaplan, LA). The traps had three
funnel entryways, elongated necks that extended above the water
surface, and polyvinyl chloride-retaining rings at the top. Traps
were harvested four days per week (Monday, Tuesday, Thursday,
and Friday) at temperatures >19°C. Harvests on Tuesday and Fri-
day occurred after baiting on the previous days (24 h soak). Har-
vests on Thursday and Monday occun'ed with no bait after 48 and
72 h soak times, respectively. Traps were baited with approxi-
mately 150 g of a commercially available bait (Gros Rouge,
Cargill, Minneapolis, MN). Four ponds were randomly assigned to
each treatment stocked previously ( 1 or 2 y). No harvest was
conducted when pond water temperatures were below 15°C. Craw-
fish were fed a 28% protein extruded, slow-sinking formulated
feed for nine months (Table 1 ).
Traps were harvested a total of 105 days extending over 1 1 nio.
from September 16 through July 31. The seine used for harvest
was nylon, 1 .5 m in height, and consisting of 1 .9 cm mesh and was
modified through the attachment of a heavy nylon mud line to the
existing lead line. Once per week, after harvest, up to 50 crawfish
from each pond were randomly selected and individual weight and
TABLE \.
Monthly feeding rates (percent of total) and total amount of feed fed
annually for studies 1 and 2.
Percent of Total
Month
Study 1
Study 2
January
0
7.25
February
0
6.5
March
10
7.0
April
17
11.5
May
15
14.25
June
14
12.5
July
10
7.0
August
0
4.0
September
8
7.75
October
10
8.0
November
10
7.0
December
6
7.25
Total feed fed (kg/ha/y)
4400
5635
sex were recorded. From each seine harvest, fifty individual craw-
fish were also randomly selected and individual weight and sex
were recorded.
Levels of dissolved oxygen and water temperature for each of
the experimental ponds were recorded daily for the entire year. If
dissolved oxygen was anticipated to decline below 5 mg/L, surface
aeration was provided by a 0.5-hp Aquarian aerolators (Air-O-
Later Corp.. Kansas City, MO). Additionally, tractor powered
paddlewheels were used when the concentration of dissolved oxy-
gen was anticipated to decline below 3 mg/L. From May to Au-
gust, pH was measured every third day from water samples col-
lected from each pond. In June, all ponds were treated with gyp-
sum at approximately 182 kg/ha to control the sporadic and rapid
increase in pH. The value of different harvesting methods and
strategies was compared through calculation of CPUE. The calcu-
lations were based upon the assumptions that one worker (laborer)
with a boat can harvest 150-300 crawfish traps/h (Romaire 1995),
and a crew of three laborers, with the proper equipment, can seine
harvest a I ha pond in 1 h. Seine harvest requires the removal of
traps. However, no additional investment of labor is necessary if
the traps are removed at the same time they are last harvested. The
different labor investments required for the different strategies of
harvest during water temperatures between 15 and I9°C, were
standardized by assuming a 1 ha production pond. CPUE (kg/ha/
laborer/h) was calculated by dividing the total harvest (kg/ha) for
the entire period when water temperatures between 1 5 and 1 9^C by
the number of harvest days, and then dividing by the number of
hours required to complete harvest.
Study 2
Twelve earthen ponds were used in the evaluation of the effects
of an extruded feed and an increased trap density. There were three
treatments, four replicates (ponds) per treatment. Nine ponds had
been in continuous production for either 2 or 3 y as part of pre-
vious investigations. The remaining three ponds were stocked with
a 1:1 ratio of males to females at 1 12.5 kg/ha during July 1998.
One of these ponds was randomly assigned to each of the three
treatments. The management practices represented by the first
treatment were the feeding of a 32% crude protein, pelleted, for-
mulated diet, and a trap density of 81 /ha. Traps were harvested 3
days/week, 2 consecutive days, followed by 24 h of soak. Traps
were then inverted one day, baited the following day, and then
harvested after a 24 h soak time. The second treatment was the
same as the first treatment except a 32% crude protein, extruded,
slow-sinking, formulated diet was fed. The final treatment con-
sisted of the feeding of extruded, slow-sinking formulated diet, a
trap density of 121/ha, and 2 consecutive harvest days/week with
a 24 h soak time. When traps did not contain bait, they were
inverted.
Harvest was conducted with the pyramid traps described in
study I. Trapping with bait occurred at water temperatures >19°C
using a lOO-g piece of formulated bait (Gros Rouge. Cargill, Min-
neapolis, MN). When water temperatures were between 15 and
I9°C, traps were not baited and remained soaked. Under these
conditions, a higher mean harvest weight would be expected be-
cause smaller crawfish would have more time to exit out of the
trap. Dissolved oxygen concentrations and pH were measured and
managed as described in study I .
Some management constraints were imposed on trap harvest to
maximize return on trapping effort. If the weekly harvest yielded
Production of Red Swamp Crawfish in Earthen Ponds
529
<15 kg/ha/treatment, or mean individual liarvest weight of the
crawfish was <I5 g/treatmenl/week, trap harvest was suspended
for the next week. Also, if the average water temperature for all
ponds decreased to <15°C. then harvest was suspended and re-
sumed when water temperatures returned to IS^C.
From September 8 until November 3. crawfish in all ponds,
representing ail three treatments were fed a 32% crude protein,
sinking, formulated feed manufactured by pelletization (Producers
Feed Company. Isola. MS). Thereafter, the diets that were part of
the previously described three treatments were fed. The results of
the proximate analysis of each of the two different feeds used as
part of the investigation are presented in Table 2.
CPUE (kg/ha/laborer/h) was calculated as described for the
first study. To evaluate the differences between trap densities, a
theoretical 1 ha pond was used and it was also assumed that 130
traps can be harvested per hour by one laborer with a boat. Total
weight harvested (kg/ha) was determined for each pond day when
trap harvest was conducted. Yields were either combined or sepa-
rated to reflect harvest yields at water temperatures of either >19C
or between 15 and 19 C. The cumulative harvest weights were
divided by the number of harvest days, and then divided by the
number of laborer hours required for trap harvest.
Statistical Analysis
A one-way analysis of variance using the general linear model
of SAS (Statistical Analysis System, version 8.1, Cary, NC) was
used to determine whether differences existed among treatments
for mean yields (kg/ha and number/ha), mean individual weights
and mean CPUE overall and relative to harvest temperature of the
pond water. Significant differences were identified at the P < 0.05
level.
RESULTS
Sltidy 1
The mean total production (kg/ha), and mean individual harvest
weight (g) of crawfish harvested from ponds with a trap density of
81 /ha were not significantly different from those ponds with a trap
density of 121/ha (Table 3).
Total yield (kg/ha) at water temperatures between 1 5 and 1 9"C
was not significantly different among treatments (Table 4). How-
ever, the mean individual weight (g) of the crawfish harvested by
seine (11.4 g) was significantly less than those harvested from
traps at densities of 81/ha (21.6 g) and 121/ha (17.6 g).
Seine harvest required a greater amount of labor than trap har-
vest and contributed to the lowest CPUE (10.1 kg/ha/laborer hour).
TABLE 2.
Results of the proximate analysis for extruded, slow -sinking, and
pelleted diets fed to crawfish in ponds without planted forage.
Component (% Dry Weight)
Extruded
Slow-Sinking Diet
Pelleted
Diet
Ash
8.4
7.1
Crude protein
37.8
39.1
Crude lipid (acid hydrolysis)
5.4
4.3
Crude fiber
6.7
6.2
Nitrogen-free extract (carbohydrate).
by difference
41.7
43.3
TABLE 3.
Mean annual production (kg/ha) ± SE and mean individual weight
(g) ± SE of harvested crawfish (Study I ).
Treatment
Total Production
(kg/ha)
.Mean Individual
Weight (g)
Kl traps/ha 2606 ± 400
1 2 1 traps/ha. trap < 1 9°C 23 1 8 ± 2 1 1
121 traps/ha. seine < 1 9"C 2I73±239
I7.S + 0.4
16.7 + 0.6
16.6+ I.O
CPUE was highest for trap harvest at 121 traps/ha (13.9 kg/ha/
laborer hour) during water temperatures between 15 and 19 C
(Table 4).
Each of the three seine harvests conducted during the summer
months when water temperatures exceeded 19 C in ponds that
contained 81 traps/ha yielded between 50 and 100 kg/ha. Greater
yield was achieved from a cumulative four day trap harvest than
one seine harvest during the same week two of three times. The
individual harvest weight of seine harvested crawfish was less than
that of trap harvested crawfish collected during the first seine
harvest in May. Under an equal number of trap days, the yields for
the two different trap densities were similar. Further evaluation of
trap density and trapping effort is warranted.
Study 2
The mean total production, mean individual weight, and mean
number/ha did not differ significantly among treatments (Table 5).
A 33% decrease in the number of harvest days for ponds contain-
ing 121 traps/ha resulted in a 23% decrease in annual production
(kg/ha) and a 27% decrease in total number of crawfish harvested
per hectare. CPUE (kg/ha/laborer hourj for the trap harvest con-
ducted twice per week was 58% greater than that conducted 3x/
week.
At water temperatures between 15 and 19°C mean total pro-
duction (kg/ha), mean individual weight, and mean number/ha
were not significantly different among treatments (Table 6). To
evaluate the feasibility of trap harvest at water temperatures less
than I9°C. CPUE was calculated and multiplied by a market price
of $2.75 US/kg. Maximum return for one hour of labor to harvest
81 traps/ha three times a week would be 37.8% less than the return
realized from harvest of 109 traps/ha twice a week.
TABLE 4.
Total production (kg/ha), CPUE ( kg/ha/laborer hour), and mean
individual weight (g) of crawfish harvested when water
temperatures were between 15 and 19 C (trap harvest was
conducted for 15 separate days and seine harvest occurred eight
different times, study 1 ).
Treatment
Total
Production
(kg/ha)
CPUE
(kg/ha/laborer
hour*)
Mean
Individual
Weight (g)
81 traps/ha. trap
1 2 1 traps/ha. trap
121 trap.s/ha. seine
293
284
298
10.5
13.9
10.1
21.6
17.6
11.4
* Assumed labor required: one laborer can harvest 1 50 traps per hour, three
laborers can seine harvest a I ha pond in 1 hour. Values are based upon the
mean of each treatment.
530
D'Abramo et al.
TABLE 5.
Mean total annual production (kg/ha) ± SE and mean individual weight (g) ± SE of harvested crawHsh (Study 2).
Treatment
Mean Total Production
(kg/ha)
CPUE
(kg/ha/laborer hour*)
Mean Individual
Weight (g)
Number/ha
81 traps/ha 3x. ESS
8 1 traps/ha, 3x, PS
121 traps/ha. 2x. ESS
2447 ± 169
2294 ± 309
1884 ±260
13.2
12.4
20.8
18.7 + 0.9
16.2 ±0.5
19.8 ± 1.5
130.764 ±3.781
141.233+ 18.416
95.928 ± 18.878
At trap densities of 81/ha and 121/ha. the total number of harvest days was 100 and 67 days, respectively. Either a e.xtruded slow-sinking (ESS) or a
pelleted sinking (PS) diet was fed.
* Assumed labor required: one laborer can harvest 150 traps her hour. Values are based upon the mean of each treatment.
For trap harvest when water temperature exceeds 19''C. mean
total production (kg/ha), mean individual weight, and mean num-
ber/ha were not significantly different. However, with a mean
increase of 500 kg/ha and 32.000 crawfish/ha. the potential eco-
nomic impact is obvious (Table 7). A mean weight increase of 1 g
with the decreased trapping effort was not statistically significant.
DISCUSSION
Large inherent variation of production parameters of ponds
within the same treatment does present some problems in the iden-
tification of the relative value of different management strategies.
This condition is characteristic of the system under investigation,
that is, production from one year to the next cannot be directly
controlled and is principally determined by recruitment success.
Nonetheless, some recommendations can emerge and future areas
of investigation can be defined.
Results indicate either an extruded, slow-sinking diet or a pel-
leted 32-35% crude protein, sinking diet can be fed. and selection
should be determined by cost and availability. A sinking catfish
diet that is not particularly water stable works as well as a formu-
lated diet. These results suggest that stimulation of the detrital food
chain may be the best way to serve the nutritional needs of the
crawfish as long as a selective harvest schedule is sufficiently
intense to remove a satisfactory amount of biomass through time.
Further investigation into the use of other alternative feedstuff's is
warranted because the cost of feed represents a large proportion of
the total operational costs. The results of the two studies suggest
that trap density is sufficient at 81/ha and that trap harvest is a
better strategy when water temperature is <19°C. Although the
catch per unit effort is greater al a density of 121/ha because of less
labor for harvest, this apparent benefit must be weighed against the
cost of additional traps and the higher production that can be
achieved for the entire harvest season when traps are harvested
three times/week. The comparatively poor performance of seine
versus trap harvest is probably caused by a less-than-efficient de-
sign for harvest. A design specific to the harvest of pond raised
crustaceans may result in an attractive option. Other potential ap-
proaches to enhance yield from seine harvest would be provision
of food (bait) just prior to a scheduled harvest, and/or harvest soon
after dusk when foraging activity is believed to be highest.
An alternative management strategy that needs investigation is
a modification in the proportion of trap days per month when trap
harvest is conducted at water temperatures >19°C. This procedure
would consist of a decrease in trapping effort from March through
May. followed by a corresponding increase in effort from June
through October. The ultimate goal of this management strategy
would be maintenance of equivalent annual production but with
the amount of production being proportionately greater when tra-
ditional capture and culture fisheries can no longer provide the
product. Those management practices that are ultimately identified
as being most efficient and cost-effective must be transferred to
larger (at least 0.5 ha) ponds to verify applicability.
ACKNOWLEDGMENTS
The authors thank the staff of the Eastern Unit of the National
Warmwater Aquaculture Unit for their assistance in the manage-
ment of the water quality of the experimental ponds, the distribu-
tion of feed to the ponds, and the harvest of crawfish. We also
thank Dr. Patrick Gerard of the Department of Agricultural Infor-
mation Science and Education, Mississippi State University for his
assistance in the establishment of an experimental design and guid-
ance in performance of the appropriate statistical analysis. The
research was supported by the U.S. Department of Agriculture
through a special grant for aquaculture research. Mississippi Ag-
ricultural and Forestry Experiment Station Publication Number
J 10249.
TABLE 6.
Total production (kg/ha), CPUE (kg/ha/laborer hour), and mean individual weight (g) of crawfish harvested when water temperatures were
between 15 and 19 C (Study 2).
Treatment
Total Production
(kg/ha)
CPUE
(kg/ha/man hour*)
Mean Individual
Weight (g)
Number/ha
81 traps/ha, 3x. ESS
81 traps/ha. 3x. PS
121 traps/ha. 2x. ESS
200
126
136
4.5
2.8
6.2
20.7
19.8
20.7
9679
7721
7082
Trap harvest was conducted a total of 24 and 16 days at trap densities of 81/ha and 121/ha, respectively. Either an extruded slow-sinking (ESS) or a
pelleted sinking (PS) diet was fed.
* Assumed labor required: one laborer can harvest 150 traps per hour.
Production of Red Swamp Crawfish in Earthen Ponds 531
TABLE 7.
Total annual production (l<g/ha), CPUE (kg/ha/laborcr hour), and mean individual weight (g) of crawfish harvested when water temperature
was >I9 C iStudv 2).
Total Produc
tion
CPUE
Mean Individual
Treatment
(kg/ha)
(kg/ha/man hour*)
Weight (g)
Number/ha
81 Iraps/ha, 3x. ESS
2246
1(1.0
18.6
121.08."^
81 trap.s/ha, 3x, PS
2168
15.4
16.0
133,512
121 traps/lia. 2x. ESS
1748
25.5
19.7
88.846
Trap harvest was conducted a total of 76 and 50 days at trap densities of 81/ha and 121/lia. respectively. Either an e.xtruded slow-sinking (ESS) or a
pelleted sinking (PS) diet was fed.
* Assumed labor required: one laborer can harvest 150 traps per hour.
LITERATURE CITED
D'Abramo. L. R. & D. J. Niquette. IWl. Seine harvesting and feeding of De Silva. S. S. & T. A. Anderson. 1995. Fish nutrition in aquaculture.
formulated feeds as new management practices for pond culture of red London: Chapman & Hall. 340 pp.
swamp crawfish, Procambarus cUiikii (Girard, 1852). and white river McClain, W. R., J. J. Sonnier & D. T. Miller. 1998. Effects of vertical
crawfish. / Shellfish Res. 10:169-177. substrate on crawfish growth and survival. 90th Annual Research Re-
D'Abramo, L. R. & C. L. Ohs. 2002. Production of red swamp crawfish port. Rice Research Station, Louisiana Agricultural Experiment Sta-
(Procambarus clarkii) in earthen ponds without planted forage: estab- tion, Louisiana State University Agricultural Center, pp. 481^84.
lishment, maintenance, and harvest of populations. J. Shellfish Res. Romaire, R. P. 1995. Harvesting methods and strategies used in commer-
22:340 p. cial Procambarid crawfish aquaculture. J. Shellfish Res. 14:545-553.
Journal of Shellfish Research. Vol. 22. No. I. S33-S4{). 2(){)3.
DISTRIBUTION, SHELTER FIDELITY, AND MOVEMENTS OF SUBADULT SPINY LOBSTERS
iPANULIRUS ARGUS) IN AREAS WITH ARTIFICIAL SHELTERS (CASITAS)
ENRIQUE LOZANO-ALVAREZ, PATRICIA BRIONES-FOURZAN, AND
MARIA EUGENIA RAMOS-AGUILAR
Instituto de Ciencias del Mar y Limiwlogi'a. Uiiidad Acadcniica Puerto Morelos, Universidad Nucionul
Auu'moma de Mexico. Ap. Posted 1 152. Caiuiiu. Q. R. 77500 Mexico
ABSTRACT In Balu'a de la Ascension, a large bay on the Caribbean coast of Mexico, artificial shelters (casitas) have been used in
the fishery for spiny lobsters (Panulinis argiis) for several decades. We selected two bay sites that differed in their ecological
characteristics: site I was a protected inner-bay site, rich in benthic vegetation (settlement and postsettlement habitat) and site 2 was
a more exposed, outer-bay site, closer to the coral reef tract, with less vegetation and more open hard bottoms. In each site, we explored
the size distribution, population density, and patterns of aggregation of lobsters in casitas. as well as the site and shelter fidelity and
the short-term movement ranges of individually tagged subadults (mean ± SD carapace length: 68.1 ± 10.9 mm). We expected that,
owing to its lush vegetation, site I would have a higher density of lobsters of a smaller mean size than site 2. but that because of the
occurrence of casitas in both sites, site and shelter t~idelity and the movement ranges of subadull lobsters would be similar in both sites.
As expected, site 1 had significantly more lobsters encompassing a wider size range, but with a smaller mean size, than site 2. Lobsters
were also more aggregated beneath casitas in site 1 than in site 2. Subadull lobsters exhibited similar site fidelity and short-term
movement ranges in both sites, but a marginally higher shelter fidelity in site 2. However, shelter fidelity in both sites was lower than
expected based on studies conducted by other workers in areas with natural shelters only. Although not conclusive, our results suggest
that, because casitas might all afford a similar shelter quality to lobsters, lobsters in areas with casitas exhibit lower shelter fidelity and
wider movement ranges than lobsters in areas with natural shelters only.
KEY WORDS: Pamiliriis argiis. artificial shelters, casitas. site fidelity, shelter fidelity, movements
INTRODUCTION
The spiny lobster Pamdirus argus (Latreille, 1804), a major
fishing resource throughout the Caribbean area, has several onto-
genetic shifts in habitat and sociality during its benthic life. After
a protracted, oceanic larval phase, the postlarvae of P. tirf^iis settle
in shallow, vegetated habitats, where the ensuing algal-phase ju-
veniles (6 to 15-20 mm carapace length, CL) remain widely dis-
persed, displaying asocial behavior. The postalgal juveniles (15-
20 to approx. 45 mm CL) remain close to the settletnenl habitats
but occupy crevice-type shelters and become socially gregarious.
The subadults (45-80 mm CL) are more nomadic and may aggre-
gate in large shelters but tend to migrate towards nearby coral reef
tracts as they approach the adult phase (>80 mm CL). Adults dwell
in caves and crevices in coral reefs and rocky bottoms on wide
expanses of continental shelf and undergo massive, organized sea-
sonal migrations (reviews in Herrnkind 1980, Butler & Hermkind
1997).
Shelter availability plays an important role in the survival of
spiny lobsters (Smith & Herrnkind 1992. Mintz et al. 1994. Bri-
ones-Fourzan & Lozano-Alvarez 2001 ) and much of the individual
and social behavior of spiny lobsters revolves around the shelter
(Childress & Herrnkind 1996, 2001). Spiny lobsters must balance
their need to remain in a shelter to avoid predation with the op-
posite need of leaving that shelter to forage (Sih 1992. Vannini &
Cannicci 1995) but have the ability to relocate known shelters
(Hermkind et al. 1975, Cobb 1981, Nevitt et al. 2000, Lozano-
Alvarez et al. 2002). On the other hand, spiny lobsters prefer
shelters that allow cohabitation (Spanier & Zimmer-Faust 1988.
Eggleston et al. 1990, MacDiarmid 1994), and individuals of P.
argus may use conspecifics as cues both to locate and to assess the
quality of a shelter (Ratchford 1999, Nevitt et al. 2000, Childress
& Henmkind 2001).
Vegetated and hard-bottom habitats have a fractal structure.
which decreases the amount of shelter for large animals compared
with small animals (Morse et al. 1985. Caddy 1986). Paucity of
shelter may affect the movements and residence time of spiny
lobsters in different ways. In areas poor in shelter, juveniles may
exhibit either high rates of nomadism (Hermkind 1980), which
increases their risk of predation, or restricted foraging movements,
which precludes them from exploiting available food resources
(review in Lipcius & Eggleston 2000) and may result in a poor
nutritional condition (Briones-Fourzan et al. 2003). Also, shelter
scarcity would increase shelter fidelity in spiny lobsters, i.e.. the
propensity of lobsters to return to a previously used shelter (Herm-
kind et al. 1975. Ratchford 1999).
Casitas. or artificial shelters for spiny lobsters, have been em-
pirically used for a number of decades in the fishery for P. argus
in Bahi'a de la Ascension, a large, shallow bay on the Caribbean
coast of Mexico (Briones-Fourzan et al. 2000; Fig. I ). Casitas may
increase lobster abundance and biomass in areas with limited natu-
ral shelter (Briones-Fourzan & Lozano-Alvarez 2001) by increas-
ing protection from predators (Eggleston et al. 1990, Mintz et al.
1994). Casitas used in Bahi'a de la Ascension are scaled to accom-
modate mostly subadults and adults (i.e.. lobsters >45 mm CL). but
because of their gregarious behavior lobsters that occupy casitas
are 10-145 inm CL (Lozano-Alvarez et al. 1991). However, mean
size of lobsters is generally larger in "outer-bay" sites (sites be-
tween the mouth of the bay and the reef tract, see Fig. 1 ) than in
"inner-bay" sites (elsewhere in the bay; Eggleston et al. 1990,
Lozano-Alvarez et al. 1991).
The area of the bay suitable for using casitas has been divided
in parcels (called campos) allotted to the members of the local
fishing cooperative. Fishers decide how many casitas and where to
deploy them within their campos. Favored substrates are vegetated
habitats and hard bottoms. Unvegetated soft bottoms are generally
avoided because on these substrates casitas tend to sink or their
sheltering space becomes obstructed by sediment (Briones-
Fourzan et al. 2000). Therefore, although Camarena-Luhrs et al.
(1996) estimated an average of 3.3 casitas ha"' in some bay areas.
533
534
Lozano-Alvarez et al.
19°40'-
19°30'N-
Figure 1. Location of the two study sites: an inner-bay site (site 1) and
an outer-bay site (site 2) in Bahi'a de la Ascension (Caribbean coast of
Mexico). Blacli areas represent the coral reef tract.
the distribution of casitas throughout the fishing areas in the bay is
highly heterogeneous.
Lozano-Alvarez (1993) hypothesized that, in addition to de-
creasing predation risk of lobsters, the occurrence of numerous
casitas over large expanses could allow spiny lobsters to exploit
food resources over more extensive areas, because after their noc-
turnal foraging excursions lobsters could retreat into any casita
available in their vicinity. Moreover, lobsters foraging close to a
casita may be attracted by chemical cues emanating from other
lobsters already sheltered in that casita (Ratchford & Eggleston
1998. Nevitt et al. 2000). This hypothesis implies a low shelter
fidelity among lobsters occurring in areas with casitas.
We explored the lobster density and the pattern of lobster ag-
gregation in casitas in two sites in Bahi'a de la Ascension that
differed in their environmental characteristics: an inner-bay site
(site 1) and an outer-bay site (site 2). Based on previous studies
(e.g., Lozano-Alvarez et al. 1991. 1994). we expected 1 ) a smaller
mean size and a higher abundance of lobsters in site 1 than in site
2. and 2) larger aggregations of lobsters in casitas in site 1 than in
site 2. We also explored the site and shelter fidelity and the short-
term movements among casitas of lobsters >45 mm CL (i.e., sub-
adults and young adults). Despite the environmental differences
between both sites, we hypothesized that, owing to the presence of
casitas. ( 1 ) site and shelter fidelity of lobsters would be similar in
both sites, and (2) short-term movements of these lobsters would
be similar between both sites but greater than those reported for
areas with natural shelters only.
MATERIALS AND METHODS
Study Sites
Site 1 was located west of Punta Hualastok, an inner-bay area
highly protected from wave surge (Fig. I). The water in this site
was very calm and reddish in color as a result of the thick man-
grove forests bordering the nearby coasts to the east and south of
the site. Depth was 3^ m. The bottom in site I was mostly fine
calcareous sand and mud. extensively covered with dense mead-
ows of macrophytes that included mixed seagrass {Thalassia
testudinum and Syringodium filifonne) and abundant macroalgae,
such as Laurencia intricata. Dictyota divaricata. Jania adiuierens,
Caulerpa sp.. Halimeda iiicrassota, H. monile. Batophom oersie-
dii. and Ripocephalus phoenix.
Site 2 was located in an outer-bay zone, leeward of the coral
reef tract (Fig. I ) and was more exposed to wave surge than site 1 .
Water in site 2 was generally very clear, and depth was 3-3.5 m.
The bottom in site 2 was coarse calcareous sand with a few small
coral heads and patches of exposed calcareous pavement. The
bottom type changed gradually towards the coast, where a few
patches of dense vegetation were interspersed with vast expanses
of sparse vegetation and open sand. The macrophytes consisted of
mixed seagrass with interspersed macroalgae. especially Halimeda
spp., Laurencia scoponia, Penicillus diimetosus. Udotea flavelhun.
U. conghainata and U. spinidosa.
Lobster Sampling
In Bahi'a de la Ascension, casitas harbor more lobsters towards
the end of the closed season (1 March-30 June), which is reflected
in significantly higher catches during the first month (July) than
during the rest of the fishing season (August-February) (Lozano-
Alvarez et al. 1991 ). Therefore, to avoid bias in our results caused
by fishing activities, our study was conducted in June 1990 and
May through June 1991.
Two divers towed by a boat in a systematic pattern surveyed
each of the two sites for casitas. When a casita was found, it was
marked with an individually numbered buoy. Casitas were more
widely dispersed in site 1 than in site 2. We marked 22 casitas in
site 1 and 25 in site 2. The size and shape of the 47 casitas were
similar (-1.8 m long x 1.2 m wide x 6-8 cm high) and all were
constructed with the same materials (a palm-trunk frame and a
ferrocement roof). We delimited the area enclosing the marked
casitas in each site with additional buoys, measured the distance
between adjacent buoys, and estimated the surface area of each
site. This was approximately 25 ha in site 1 and 12 ha in site 2. The
delimited areas were surveyed again, but no further casitas were
found.
Divers censused the lobsters beneath the 22 casitas in site I on
six occasions between 15 and 23 June 1990. On each of the first 4
days, all the lobsters sheltering beneath a randomly chosen casita
were prodded into the cod-end of a seine net (Lozano-Alvarez et
al. 1991. Lipcius et al. 1998). The cod-end was kept underwater at
the side of the boat to maintain the lobsters submerged and pro-
tected from direct sunlight. Lobsters were then extracted from the
net one at a time to determine their sex and to measure CL with
calipers (±0.1 mm. between the rostral horns and the posterior
margin of the cephalothorax). Subadults (individuals 2:45.0 mm
CL) were then tagged and returned to their original casita. Tags
consisted of a color-coded flag of adhesive tape held by a rubber
band around the carapace between the fourth and fifth pair of
pereiopods that allowed for identification of both the individual
and the casita from where it was extracted. Over subsequent sur-
veys, we recorded the data of resighted lobsters and of the casitas
where they sheltered.
Lobsters beneath the 25 casitas in site 2 were censused on 15
occasions between May 7 and June 9, 1991. On eight dates be-
tween May 7 and 24. all the lobsters from one casita were mea-
sured and the subadults tagged. In addition to the color-coded tag.
SuBADULT Spiny Lobsters in Areas with Casitas
535
o
in
CM
o
CO
in
CO
o
un o
■>d- Ln
in
o
CD
ID
CD
O
in
o
00
in
c»
o
CJ>
in
Carapace length (mm)
I Site 1 (N = 161) HSite2(N = 122)
Figure 2. Panulirus argiis. Size distribution of lobsters captured from beneath casitas in an inner-bay site (site 1 ) and an outer-bay site (site 2)
in Bahi'a de la Ascension. The arrow indicates the class size from which individuals were tagged.
which aicieii in the rapid icientification of the original casita. we
also applied to these lobsters individually numbered Australian
"spaghetti" tags (Chittleborough 1974), modified for small lobsters
(Lozano-Alvarez 1992. Negrete-Soto et al. 2002), on the dorsolat-
eral muscle between the cephalothorax and abdomen. With these
tags, Lozano-Alvare/ (1992) estimated a tag-related mortality of
-5% after three months in individuals of P. argiis over the same
size range as in our study.
Lobster Density and Patterns of Aggregation in Casitas
In each site, lobster population size, losses (death -i- emigration)
and immigration were estimated using the Fisher-Ford model
(Fisher & Ford 1947), which relies on several tagging and recap-
ture dates as well as on multiple recaptures of individuals. When
capture-recapture data are scarce, the Fisher-Ford model tends to
yield more reliable results than other models based on multiple-
recapture data (Bishop & Sheppard 1973, Begon 1979, Lozano et
al. 1982, Negrete-Soto et al. 2002). The Fisher-Ford model as-
sumes a constant survival rate ((()) but provides a method to test for
this assumption (Begon 1979). Because the number and frequency
of sampling dates varied between sites, we used only the data from
censuses conducted over consecutive days (four dates in site 1 and
six dates on site 2) to estimate lobster abundance. This would also
increase the probability of a constant survival rate over such short
periods. We then obtained the density of lobsters in each site by
dividing the number of lobsters estimated by the model over the
site area (Begon 1979). We also estimated the density of the por-
tion of the lobster population sheltering in casitas in each site by
dividing the daily number of lobsters censused beneath casitas
over the site area. The propensity of lobsters to aggregate in casitas
was analyzed in each site by plotting the number of lobsters in
each casita vs. the number of casita surveys over the sampling
period (Briones-Fourzan et al. 2000) and fitting the data to a ran-
dom distribution.
Site and Shelter Fidelity Among Lobsters
The percent of tagged lobsters that were resighted at least once
in each site was considered as a measure of site fidelity (Butler &
Herrnkind 1997). Because consecutive censuses were conducted 1
to 6 days apart, we used two measures of shelter fidelity 1 ) the
percent of occasions a tagged lobster returned to the shelter it used
the previous day (Ratchford 1999). and 2) the percent of occasions
a tagged lobster returned to the shelter it occupied on the previous
census date throughout the study periods. We compared site and
shelter fidelity of lobsters between sites using contingency table
analyses (Zar 1999).
Movements of Lobsters
Although lobsters may forage following complex, circuitous
routes (Jemakoff 1987), we considered as the minimum daily dis-
tance moved by a tagged lobster the distance measured on a
straight-line between casitas occupied by that lobster on consecu-
tive days (Acosta & Butler 1997. Ratchford 1999). We used con-
tingency table analyses (Zar 1999) to compare between sites the
median daily distance moved by those lobsters that shifted casitas
on the first post-tagging day and during the first post-tagging week
(Jemakoff et al. 1987). We also measured the angle between ca-
sitas occupied by tagged lobsters on consecutive dates with an
underwater compass, and analyzed the circular distribution of the
angles with a Rayleigh test (Zar 1999) to determine whether lob-
sters showed directional or random movements. To assess the
movements of lobsters over periods longer than those encom-
pas.sed by our study, fishermen were requested to report the cap-
ture of tagged lobsters and their location of capture after the open-
ing of the fishing season on July the first of each year.
RESULTS
Size Distribution of Lobsters
The number of lobsters extracted from four randomly chosen
casitas in site 1 was 161, over a size range of 22.3-99.5 mm CL
(mean + SD: 58.7 ± 17.5 mm CL). In site 2, 122 lobsters were
extracted from eight casitas. These lobsters were 34.8-96.9 mm
CL (mean ± SD: 66.9 ± 10.9 mm CL) (Fig. 2). Mean size of
lobsters was significantly different between sites (Student's ?-test
with log-transformed data to homogenize variances, t = 5.024.
536
Lozano-Alvarez et al.
df = 272, P < 0.0001 ). The difference was the result of the greater
occurrence of small, postalgal juveniles (i.e.. juveniles <45 mm
CL) in site 1 (Fig. 2). Postalgal juveniles made up 31.7% of lob-
sters sampled in site 1 but only 4.5% of lobsters sampled in site 2.
When postalgal juveniles were excluded from the comparison, the
mean size of subadults was similar in both sites (site 1: 68.15 ±
12.4 mm CL; site 2: 68.25 ± 9.3 mm CL: t = 0.008, df = 215, P
> 0.50). Sex ratio was around 1;1 in both sites.
Lobster Density and Patterns of Aggregation in C'asitas
We tagged and returned to their original casitas 136 subadults
and young adults (45.1-97.5 mm CL) in site 1, and 1 17 (45.1-96.9
mm CL) in site 2. Of these, 67 (49.3%) were resighted at least once
in site 1 and 71 (60.7%) in site 2. Table I shows the statistics
derived from the Fisher-Ford model for each site. As expected,
population size, losses and immigrations were higher, but more
variable, in site 1 than in site 2. Survival rate ((})) was also higher
in site 1 (4) = 0.865) than in site 2 ((}> = 0.745). Based on the
estimates of population size, mean ± SD lobster density was esti-
mated as 47.7 ± 9.7 lobsters ha"' in site 1 and 25.8 ± 3.8 lobsters
ha"' in site 2 (Table 1). These mean densities were significantly
different (t = 4.673. df = 6. P = 0.0034).
The daily number of lobsters beneath the 22 casitas in site 1
ranged from 295 to 467, yielding a density of 1 1 .8-1 8.7 lobsters in
casitas ha"' (mean + SD: 16.6 ± 2.7). In site 2. the daily number
of lobsters in the 25 casitas fluctuated between 1 1 1 and 174, yield-
ing a density of 9.3- 14.5 lobsters in casitas ha"' (mean ± SD: 13. 1
± 1.6), significantly different from that of site 1 (t = 3.653. df =
19, P = 0.0017). When considering only those dates included in
the Fisher-Ford model, the mean number of lobsters beneath ca-
sitas accounted for 34.4% and 52.1%, respectively, of the mean
number of lobsters estimated throughout sites 1 and 2.
The distribution of lobsters in casitas departed significantly
from a random distribution in both sites (site 1: x" = 156.865; P
< 0.001: site 2: x" = 40.493; P < 0.001 ). However, lobsters tended
to be more aggregated in site 1 than in site 2 (Fig. 3). In site 1 , 52%
casitas harbored over 20 lobsters and the maximum number of
lobsters sheltering beneath a casita was 60, whereas in site 2 these
figures were, respectively, 3% and 40. In both sites, some casitas
harbored no lobsters (Fig. 3), but casitas with no lobsters on a
given date had lobsters on the following date and vice versa.
Site and Shelter Fidelity Among Lobsters
Table 2 summarizes the results on site and shelter fidelity of
subadult lobsters in both sites. Some lobsters that were not re-
sighted on the first few post-tagging days were seen again later,
whereas others were never seen again. Site fidelity was higher, but
not significantly different, in site 2 than in site 1 . Mean shelter
fidelity A (percent of occasions a tagged lobster returned to the
same casita it used the day before) did not differ significantly
between sites, whereas the difference in mean shelter fidelity B
(percent of occasions a tagged lobster returned to the casita it used
on the previous census date throughout the study period) was mar-
ginally significant. Tlie P values may indicate that the power of the
tests was low, but the overall results suggest that lobsters in site 2
exhibited slighdy higher site and shelter fidelity than lobsters in site 1 .
Movements of Lobsters
On the first post-tagging day, lobsters that shifted casitas
moved 58^16 m overnight in site 1 (median distance = 165 m)
and 25-290 m in site 2 (median = 108 m). The medians were not
significantly different (x-= 2.110: df = \: P = 0.220). During
the first post-tagging week the movements remained similar, both
within (median of site 1: 133 m; of site 2: 1 10 m) and between sites
(x' = 1.100; df = I, P = 0.431). Therefore, lob.sters from both
sites exhibited similar movement ranges during the first post-
tagging week. Lobsters that used more than two casitas moved
1 55—400 m among casitas over the study periods. The movements
of lobsters within site 1 (mean angle ± angular deviation: 154.6° ±
TABLE 1.
Panulirus argus: statistics of the Fisher-Ford model for spiny lobsters in (a) an inner-bay site (site 1) and (b) an outer-bay site (site 2) in
Bahia de la Ascension, Mexico.
Sampling
Date
Spiny Lobsters
Population Estimates
Captured
Tagged
Size (N)
Losses
Emigration
Density
(Lobsters ha"')
Inner-bay (site 1 )
15 June
16 June
17 June
18 June
Mean ± SD
Outer-bay (site 2)
07 May
08 May
09 May
10 May
11 May
12 May
Mean ± SD
293
410
467
466
175
185
131
146
160
169
37
46
17
0
40
19
9
14
0
0
1196
1435
948
1196 + 246
346
320
355
273
251
320 ± 45
161
194
128
88
82
91
70
64
401
47.8
■294
57.4
—
37.9
47.7 ±9.7
62
28.8
117
26.7
9
29.6
47
22.8
—
20.9
25.8 ± 3.8
All lobsters were captured from beneath artificial shelters (casitas). Losses are deaths -i- emigration. Density of lobsters was estimated by dividing the
population size over the surface area of each site (25 ha in site 1. 12 ha in site 2).
SuBADULT Spiny Lobsters in Areas with Casitas
537
(A
"55
(0
o
c
o
Q.
O N ^ -b ^ b fe A % <^ sO sN ^^ ^^ ^^ ^^ K^ <\ ^% ^<i nO n> r/> ri> r> T^ nfo X> n% r« 4i 45
Number of lobsters per casita
I Site 1 (N = 128) SSite 2 (N = 374)
Figure 3. Panulirus argus. Distribution of lobsters beneath casitas in an inner-bay site (site 1) and an outer-bay site (site 2) in Bahia de la
Ascension. N is the number of casita surveys conducted throughout the study period in each site.
71.6°) were non-directional (Rayleigh's test: z = 2.162, n = 45,
P > 0.10). In contrast, movements of lobsters within site 2 (mean
angle ± angular deviation: 82.6° ± 69.1°) were not uniformly dis-
tributed around the circle (z = 3.1, « = 42, P < 0.05). These
lobsters showed a tendency to move towards the coral reef, which
lies at 80° from site 2 (V-test, u = 2.493, n = 42, P < 0.01).
Fishermen recaptured 33 lobsters tagged in site 1 during July
1990. 4-8 wk after being tagged. Of these. 17 remained within site
1, but 16 were recaptured 2,000-14,600 m away from this site. In
contrast, fishermen recaptured 20 lobsters tagged in site 2 during
July 1991 (8-13 wk after being tagged), of which 19 remained
within site 2 and only one was caught outside this site (distance not
recorded). Lobsters recaptured by fishermen (67.5-84.2 mm CL)
had increased 4.3-20.2 mm in 6-13 wk.
DISCUSSION
As expected, the inner-bay site (site 1 ) had significantly more
lobsters encompassing a wider size range, but with a smaller mean
size, than the outer-bay site (site 2). Although we sampled site 2
one year later than site 1 , our results are consistent with previous
findings. In Bahi'a de la Ascension, larger lobsters occur in many
bay areas but are more common in the outer-bay, whereas smaller
lobsters commonly occur at higher densities in more protected
inner-bay areas, rich in settlement and post-settlement habitats
(Eggleston et al. 1990; Lozano-Alvarez et al. 1991, 1994). Similar
results have been obtained in shallow areas of northern Quintana
Roo (Arce et al. 1997. Sosa-Cordero et al. 1998) and Belize
(Acosta 1999).
TABLE 2.
Panulirus argus: comparisons of site and shelter fidelity of subadult spiny lobsters tagged in an inner-bay site (site 1) and an outer-bay site
(site 2) in Bahia de la .Ascension. Mexico.
Number of Lobsters
Site
Tagged
Resighted
Site
Fidelity (%)
Shelter Fidelity {%)
B
Inner-bay (site 1)
Outer-bay (site 2)
X' value
P value
136
117
67
71
49.3
60.7
3.31
0.069
18.4
30.3
3.17
0.074
31.6
47.4
3.94
0.047
All lobsters were captured from beneath artificial shelters (casitas). Site fidelity is the percent of tagged lobsters resighted at least once within the
respective site. Two measures of shelter fidelity were considered: (A) the percent of occasions a tagged lobster returned to the same casita it used the
day before, and (B) the percent of occasions a tagged lobster returned to the same casita it used on the previous census date. Census dates were 1 to 6
days apart. Degrees of freedom = I in all comparisons.
538
Lozano-Alvarez et al.
Density of lobsters beneath casitas was also higher and lobsters
were more aggregated in site 1 than in site 2. Lobsters tend to
aggregate more beneath large artificial shelters deployed over veg-
etated habitats, where juvenile density is higher, than over hard
bottoms (Lozano-Alvarez et al. 1994, Mintz et al. 1994, Arce et al.
1997, Sosa-Cordero et al. 1998. Briones-Fourzan et al. 2000. Bri-
ones-Fourzan & Lozano-Alvarez 2001 ). This pattern of aggrega-
tion may indicate a "guide-effect."' which is a consequence of
conspecific attraction related to lobster density (Childress & Herm-
kind 2001).
Short-term movements (and hence site fidelity) of lobsters
could be affected by disturbance caused by capture and tagging
(Hermkind 1980). However, initial capture had only short-term
effects, and tagging had no additional effect on the movement of
individual Jasiis edwardsii (MacDiarmid et al. 1991); capture and
handling had no short-term effects on movements of individually
tagged P. cygiuis (Jernakoff et al. 1987). and disturbance of lob-
sters had no apparent effect on the selection of shelter by other
lobsters (Ratchford 1999). Disturbance probably had little effect
on our tagged lobsters because there were no significant differ-
ences in movement ranges and site fidelity between our study sites,
and tag-related mortality was unlikely in either site. Therefore,
lobsters that were not resighted may have been predated, moved
beyond the boundaries of the sites, or occupied unsurveyed natural
shelters throughout the sites.
We did not survey the potential natural shelters occurring in
each of our sites; this would have been a formidable task given
their large surface area. However, it has been shown that benthic
vegetation, in addition to providing settlement habitats and feeding
areas, may also provide shelter to juvenile P. argiis. In Bahi'a de la
Ascension, Lipcius et al. (1998) plotted algal biomass vs. survival
of tethered juvenile P. argits (30-75 mm CL) and obtained a
hyperbolic habitat-survival function. Their results indicate that
even a modest increase of algal biomass. which increases the ar-
chitectural complexity of the habitat, significantly enhances the
survival of juvenile P. argus. In Belize, greater numbers of juve-
nile P. argus moved into and from habitats surrounded by seagrass
than those surrounded by rubble, which suggests that vegetated
substrates may function as movement corridors for juvenile lob-
sters, facilitating their dispersal to areas containing new resources
(Acosta 1999). This would explain the greater variations in popu-
lation estimates of juveniles in our site 1 compared with site 2.
Moreover, Acosta and Butler (1997) found that large juveniles of
P. argus have similar survival when sheltering among mangrove
prop roots and in coral crevices. Our inner-bay site, in addition to
having more benthic vegetation, was close to thick mangrove for-
ests; therefore, the higher survival rate estimated for lobsters in site
1 may reflect the additional protection provided by these vegetated
substrates. Also, the lesser habitat complexity in site 2. where
vegetation was scarce, could underlie the slightly higher shelter
fidelity exhibited by lobsters in site 2 compared with site 1 .
Herrnkind (1980) devised a conceptual model postulating that
lobsters in areas of abundant food and shelter will tend to be
residential, whereas lobsters in areas of scarce shelter and disperse
food supply will tend to be more nomadic owing to intraspecific
competition for shelter. But evidences for a relationship between
site and shelter fidelity, lobster size, and shelter abundance remain
equivocal (Hermkind et al. 1975, Hermkind 1980, MacDiarmid et
al. 1991, Acosta & Butler 1997, Butler & Hermkind 1997, Bri-
ones-Fourzan & Lozano-Alvarez 2001). Some studies report that
smaller lobsters display stronger shelter fidelity than larger lob-
sters, whereas others report that subadults and young adults are
more transient and nomadic (which implies a low shelter fidelity)
than old adults. However, these evidences have been obtained in
areas with natural shelter only. For example, in the case of P.
argus. average shelter fidelity A of tagged subadult and young
adult individuals was estimated at 38% (range: 15-88%) by Ratch-
ford ( 1999), similar to the 42% reported for old adults by Herm-
kind et al. (1975). and Acosta and Butler ( 1997) found average den
residence times for postalgal P. argus of 2.0 to 4.38 days over five
consecutive days (equivalent to a shelter fidelity A of 40-87%).
Compared with these values, the average shelter fidelity A of our
subadult P. argus (18.4% in site 1; range: 20-60%, and 30.3% in
site 2; range: 20-75%) was rather low.
The occurrence of casitas could partially explain these results,
as proposed by Lozano-Alvarez (1995), because casitas presum-
ably reduce competition for shelter by allowing cohabitation of
large numbers of individuals. However, based on laboratory ex-
periments, Ratchford (1999) suggested that the longer a lobster
resides in an area and becomes more familiar with the shelters in
that area, the lower its shelter fidelity will appear. This could also
explain the overall low shelter fidelity A of our lobsters as well as
the marginal difference in shelter fidelity B among lobsters be-
tween our sites 1 and 2. The large number of postalgal juveniles
cohabiting in casitas with subadults in the inner-bay site 1, rich in
settlement and post-settlement habitats, suggests that these sub-
adults had probably remained in that area since settlement. But this
inner-bay area may cease to be an appropriate habitat once sub-
adult lobsters reach a critical size. These subadults would then
immigrate to other outer-bay habitats (Cmz et al. 1986, Lozano-
Alvarez et al. 1991), thus explaining the distant locations where
lobsters tagged in site 1 were recaptured by fishermen a few weeks
later. In contrast, individuals beneath casitas in site 2 were mostly
subadults, which had probably immigrated recently to this site
from other, more vegetated areas. The proximity of the coral reef,
the habitat preferred by subadults and adults, could also underlie
the more directional movements of subadults towards this habitat
in site 2.
Some species of spiny lobsters are highly mobile [e.g. PanuU-
rus cygnus (Jernakoff 1987. Jemakoff et al. 1987) and P. argus
(Hermkind et al. 1975, Ratchford 1999)] and others are more
sedentary [e.g. Jasus edwardsii (MacDiarmid et al. 1991) and P.
guttatus (Negrete-Soto et al. 2002, Lozano-Alvarez et al. 2002)].
However, even in highly mobile species, the extent of the daily
movement range appears to depend on the occurrence of suitable
structured shelter. Previous studies estimating the daily move-
ments of tagged postalgal juveniles and adults of P. argus have
been conducted in areas with natural shelters only. Hermkind et al.
( 1975) used sonic tags to individually track 27 large, adult P. argus
(average size approx. 1 10 mm CL) in a coral reef habitat over five
consecutive nights. These lobsters typically moved 30-90 m over-
night and used three or four dens within 140 m, with a maximum
den shift just under 500 m (Hermkind 1980). In shallow coastal
areas, postalgal juveniles (average size approx. 37 mm CL) moved
5.4 to 24.5 m overnight when shifting shelters (Acosta & Butler
1997). whereas lobsters 70.6-134.0 mm CL moved 10-185 m
ovemight when shifting shelters and up to 270 m among shelters
over a period of four weeks (Ratchford 1999). Our subadult P.
argus (mean size: 68 mm CL) moved 25—116 m overnight when
shifting casitas, and 155^00 m among casitas over the study
periods. These movements are greater than those reported by
Acosta and Butler (1997) for postalgal juveniles and Ratchford
SuBADULT Spiny Lobsters in Areas with Casitas
539
( 1999) for subadults and adults in areas with natural shelters only,
suggesting that the occurrence of casitas does increase the move-
ment range of subadult P. argtis.
Areas with few natural, appropriate shelters would favor be-
havior that allows lobsters to efficiently relocate previously used
shelters (Ratchford 1999). Because of their physical properties,
casitas allow cohabitation of many individuals over a wide size
range and afford — at least in theory — a similar quality of shelter,
although the latter may vary somewhat depending on the type of
substrate around individual casitas (Meiners-Mandujano 2002).
Therefore, areas with numerous casitas would allow lobsters to
forage over greater areas by reducing their need to relocate a
previously used casita. Moreover, a lobster could be attracted to
any nearby casita at the end of its foraging excursion by cues
emanating from lobsters already sheltered in that casita (Nevitt et
al. 2000, Ratchford & Eggleston 2000, Childress & Herrnkind
2001). This would be reflected in low values of shelter fidelity A
and wide short-term movement ranges, as suggested by our results.
These results are, however, inconclusive, because to fully test
this hypothesis it would have been necessary to compare shelter
fidelity and movement ranges of subadult lobsters in areas of the
bay with and without casitas. This was unfeasible because an
estimated 20,000 casitas occur throughout the lobster habitats in
Bahi'a de la Ascension (Briones-Fourzan et al. 2000). However,
preliminary results of a controlled field experiment recently con-
ducted in the reef lagoon at Puerto Niorelos, Mexico indicate a
significant increase in the daily movements of postalgal juvenile P.
argus after the introduction of casitas scaled to their size (Meiners-
Mandujano 2002, Lozano-Alvarez et al., unpublished data).
ACKNOWLEDGMENTS
The authors thank F. Negrete-Soto for his invaluable help in the
fieldwork. Much appreciated logistic support was provided by the
crew of the boat "Fipesco 207." Capt. Daniel Duran, Pedro Men-
dez, and Michel Moreno, and the local lobster fisher Manuel
Cahuich. DGAPA (Direccion General de Asuntos del Personal
Academico, UNAM) provided a scholarship forMERA. This work
was partially funded by World Wildlife Fund-U.K. through Aso-
ciacion de Amigos de Sian Ka'an, A.C., and Universidad Nacional
Autonoma de Mexico (UNAM).
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Arce, A. M., W. Aguilar-Davila, E. Sosa-Cordero & J. F. Caddy. 1997.
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Begon, M. 1979. Investigating Animal Abundance: Capture-Recapture for
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Briones-Fourzan, P. & E. Lozano-Alvarez. 2001. Effects of artificial shel-
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Prog. Ser. 221:221-231.
Briones-Fourzan, P., E. Lozano-Alvarez & D. B. Eggleston. 2000. The use
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Jo:iimil of Slu'lljlyh Ri'scairh. Veil. 22. No. 2. .'i41-.S4.S, 200.V
TETRAPLOID INDUCTION BY HEAT SHOCKS IN CHINESE SHRIMP,
FENNEROPENAEUS CHINENSIS
FUHUA LI, JIANHAI XIANG,* XIAOJUN ZHANG. CHANGGONG WU,
CHENGSONG ZHANG, LINGHUA ZHOU, AND KUIJIE YU
lustiliitc (if Occciiu)loi>y. Cliiiwsc Academy of Sciences. 7 Nanhai Road. Qiiiiidao 26607 f
People '.V Republic of China
ABSTRACT Tetraploid induction in the Chinese shrimp Fenneropenueus chinensis was studied. Tetraploid larvae were successfully
produced through mitosis I inhibition hy heat shock in this species. Proper tiine window for tetraploid induction was optinii/ed. and
the highest induction level was inore than 909^ as measured by How cytometry. At spawning temperature of Ih C. the best starting
time for heat shocks was 98 to 1 10 min po.stfertili/ation. Tetraploid embryos had less viability compared with diploids. The highest
tetraploid level detected at nauplius stage was .IS'/t. Further work is needed to increase the viability of tetraploid larvae.
KEY WORDS: tetraploid. heat shocks, flow cytoinetry. Fenncnipfiuwiis chinensis
INTRODUCTION
Chinese shrimp Feuiicrnpenacits cliiiieusis is one of the most
important aquaculture species in China. Because of its meal quality
and cold resistance, the Chinese shrimp is one of the best species
for shrimp culture. In recent years, the prevalence of vinjs disease
has devastated shrimp culture worldwide. Genetic improvetnent is
being used to enhance growth rate or disease resistance in culture
fish and shellfish.
It was reported that triploid shellfish were useful for aquacul-
ture because of their sterility, superior growth and improved tneat
quality, and increased disease resistance (Allen et al. 1989. Hand
et al. 1998. Quo 1999). In fish, triploids were produced to improve
growth (Flajshans et al. 1993, Pandian & Koteeswaran 1998),
control reproduction, or reduce contamination for transgenic spe-
cies (Devlin & Dotialdson 1992, Pandian & Marian 1994). Since
triploid induction was rarely 100% effective, the best way to pro-
duce triploids is using telraploids to hybridize with diploids (Arai
et al. 1993, Guo et al. 1996). Tetraploid production however, is
challenging according to the reports to date because of the low
viability of tetraploids. Until now, tetraploid production has been
successful only in a few species of fish and shellfish (Guo & Allen
1994, Pandian & Koteeswaran 1998, Yang et al. 2000). Theoret-
ically, tetraploid induction can be achieved by inhibiting mitosis of
fertilized eggs. Through this method, production of tetraploids has
been reported in a few fish species (Thorgaard et al. 1981), but
there is no report on successful tetraploid production through in-
hibiting mitosis I in shellfish. Tetraploid embryos have been pro-
duced in the Pacific oyster by heat shock induced mitosis I inhi-
bition, but the larvae did not survive beyond tnetamorphosis (Guo
et al. 1994). Tetraploids could be produced by inhibiting the first
polar body of the eggs from triploids (Guo & Allen 1994, Eudeline
et al. 2000, He et al. 2000) or by inhibiting meiosis I of diploid
zygotes (Yang et al. 2000, Zhang et al. 2000). Studies on chro-
mosome manipulation for cultured shrimp have progressed slowly.
Successful triploid production for shrimp was reported in a few
species (Xiang et al. 1998, 2001 ; Li et al. 1999, 2002. 2003; Norris
et al. 2001). Because of difficulties with artificial fertilization in
shrimp, there is to date no way to induce triploids on large scale.
The only way to induce triploids in shrimp is to treat fertilized eggs
from one shrimp at a time. The need for tetraploids seetBS tnore
'Corresponding author. Fax: -i- 1-532-289-8578; E-mail: jhxiang(9'ms
qdio.ac.cn
urgent in shrimp than in other species. To our knowledge, there has
been only one report about tetraploid induction in shrimp (Xiang et
al. 1993). In this study, tetraploid induction was performed and
optimal treatment conditions for tetraploid induction were deter-
mined in Chinese shrimp.
MATERIALS AND METHODS
Source of Gravid Shrimp
Gravid shrimp were collected from the wild from the Yellow
Sea or from an over-wintered population from a hatchery nearby
Qingdao. The gravid shrimp were brought into the aquarium of our
institute and put into 4 m' tanks. Twenty individuals were put in
each tank, where the seawater temperature was set at 12-13°C. At
that time, ovaries of the gravid shrimp were at stage IV. The
shrimp were kept at I2-I3°C for 3—4 days to acclimate them to the
conditions of our laboratory. Then water temperature was raised
gradually (0.5°C/day) to the ptoper spawning temperature (16-
18°C). Meanwhile, the normal light cycle for these tanks was
reversed according to the method that was developed in our labo-
ratory to make shrimp spawn at daytime (Xiang et al. 1993).
Gravid shrimp were fed with polychates and fresh clam meat.
Collection of Fertilized Eggs
Shrimp with good ovary development that would spawn in 1 or
2 days were put into 300-L tanks with controlled temperature and
light cycle. According to their behavioral changes, shrimp that
would spawn immediately were taken out and put into 20-L con-
tainers. Usually the spawning process for gravid shrimp lasts about
10 min. After the spawning, they were placed in larger tanks to be
cultured until re-maturation again. Spawned eggs were collected
and concentrated for tetraploid treatment.
Treatment of Fertilized Eggs
Experiments were designed to compare tetraploid induction
level under treatments of different starting time, different intensity,
and different treatment duration. Heat-shocks were used to inhibit
the first mitosis of fertilized eggs. Proper window for starting
treatments was determined according to the tetraploid induction
efficiency from a 3^ min heat shock of 33-34°C applied at dif-
ferent starting times from 90-1 14 min. Tetraploid induction effi-
ciency was determined at embryo stage using flow cytometry then
induction efficiency under different treatment intensity, including
heat-shock temperature and duration time, was compared. For each
treatment, about 700-800 fertilized eccs taken out from the con-
541
542
Ll ET AL.
700 »0 300 ISO 400 4fi0 R.I SOO
2n
60 100 160 TOO 260 300 380 400 4B0 FL1 GOO
2n
j 4n
^1
300 ■
260 -
2n
f
200 -
1
IBO
100 '
i
4n
W
L
MBbtMWHUw
260 MO ]eO 400 460 FLt BOO
100 ISO 100 tSO 300 ISO 400 4B0 FL1 SOO
Figure 1. Flow cytometry analysis of embryos after heat shock treatment starting at different times after fertilization: (a) 90 min, (bl 96 min,
(c) 102 min, (d) 106 min, (e) 108 min, and (f) 114 min.
centrated eggs were put into 1000 ml beakers containing about 600
mL hot seawater with the desired temperature. The beakers that
were used to treat fertilized eggs were put into a water bath with
temperature that stabilizes the treatment condition. When the treat-
ment was almost finished, the beakers with fertilized eggs were
removed from the water bath and most of the hot water in the
beakers was removed. Seawater with natural temperature (15-
120
100
80
60
40
20
0
1 8°C) was added to the beakers to change the water temperature to
1 8-20°C. The treated eggs were then incubated at ~20°C. For each
treatment, fertilized eggs without any treatment were used as the
control group. Usually for each group of experiments, all fertilized
eggs for the treatment were from the same gravid shrimp to ex-
clude variation in egg quality. After -24 h of incubation, 70-80
embryos were taken out for ploidy detection using flow cytometry.
120
100
MM
■ 2 min
■ 4 min
D6 min
32. 5 33 33. 5
90 92 94 96 98 100 102 104 106 108 110 112
starting time (minutes)
Figure 2. Tetraploid level detected at the embryo stage when a 3—1 under different beat-shock temperatures and dilTerent duration of
min heat shock of 33-34 C was applied at different starting times. treatment.
treatment temperature C C)
Figure 3. Comparison of tetraploid level detected at embryo stage
Tetraploid Induction in Chinese Shrimp
543
300 -
coum
»0
b
200 •
160
4n
lOO ■
50
0
*t>^
4G0FLI 600
460rLI 600
Figure 4. Flow cytometry analysis of embryos from telraphiicl Induction treatment under optimized conditions in Cliinese shrimp Fennerope-
naeus chinensis. (a) control and (b) tetraploid embryos.
and the remaining embryos were kept until they hatched into nau-
plii. about 20-30 of which were used for final ploidy analysis.
Ploidy Detection
Tetraploidy were detected using flow cytometry. For ploidy
detection at embryo stage, 70-SO embryos were put together and
triturated in 0.2 niL preparation buffer consisting of 2% citrate acid
and 0.5% Tween 20 in distilled water. For nauplius stage. 20-30
larvae from each treatment were triturated in preparation buffer.
Tissue debris was removed using nylon screen, and 0.7 mL 2 mg/L
DAPl solution was added to stain the nuclei. Embryos or larvae
from the control groups were treated in the same way and used as
diploid controls. Percentages of triploids and tetraploids in the
sample were determined by comparing areas of different peaks.
Hatching Success
For every treatment, percentages of nauplii hatched in the
treated and control groups were recorded to determine the rela-
tionship between tetraploid levels and hatching levels.
Statistical Analysis
To compare the effects of different factors such as starting time,
treatment intensity, and duration on tetraploid induction. F-test
was used to analyze the effect of different factors on the efficiency
of tetraploid induction.
RESULTS
(2-6 min) were tested and compared (Fig. 3). Tetraploid frequency
detected at embryo stage rose apparently with extension of treat-
ment duration from 2 to 6 min at 32-33. 5°C. There was no sig-
nificant difference in tetraploid levels between different treatment
temperatures for the same treatment duration in the range of 32-
33.5°C. The data indicated that 32-33.5°C temperature could ef-
fectively inhibit the first mitosis of fertilized eggs when treatment
duration was 4-6 min. Tetraploid level detected under 34°C was
much higher than that at 32-33. 5°C when the treatment lasted for
4 min. There was no difference in tetraploid level between 4 and
6 min treatment at 34°C. It showed that at certain range of treat-
ment temperature, proper duration of the treatment was a key
factor for tetraploid induction.
Evaluation of Effects of Different Factors on Tetraploid Level
Starting time, treatment duration and treatment temperature are
major factors affecting tetraploid induction. Totally, 5 levels of
starting time (79, 81, 85, 91, 96, 100 min), 3 levels of treatment
duration (2, 4, 6 min), and 5 levels of treatment temperature (32,
32.5, 33, 33.5, 34°C) were tested. F-test showed that tetraploid
level detected at different starting time for the treatment among
different treatments and treatment duration (2, 4, 6 min) had sig-
nificantly different effects among groups; and that different treat-
ment temperature had no significant effects. At low treatment tem-
peratures, longer treatment duration increased tetraploid induction
Effect of Starting Time on Tetraploid Inducing Rate
To determine the optimal window for tetraploid induction, dif-
ferent starting times for treatment were tested. Flow cytometry
analysis of embryos from treatment starting at different times is
shown in Figure 1. Usually two peaks, diploid (2n) and tetraploid
(4n or G2 phase of 2n) peaks, were present in each sample. With
changes in starting time, the relative 4n area changed greatly. After
numerical repeats for each treatment, the optimal starting time for
tetraploid induction was determined based on data in Figure 2. The
proper starting time for tetraploid induction was 98-1 10 min under
a spawning temperature of 16'C. When starting time was at 1 12
min, tetraploid level dropped sharply. The window for tetraploid
induction was only about 10 min. Out of this range, the treatment
could not effectively inhibit mitosis I.
Effect of Different Treatment Duration and Different Temperature on
Tetraploid Rate
Using the optimized time window for the treatments, different
treatment intensities (32-34°C) for different treatiTient duration
0{control)
70-100
30-40 41-50 51-60
tetraploid rate (%)
Figure 5. Relationship between tetraploid level and hatching success
in Chinese shrimp Fenneropenaeus chinensis.
544
Li et al.
efficiency. When the spawning temperature of gravid shrimp was
16''C, the proper starting time for treatment should be 102-1 10
min after fertilization. If the spawning temperature was lower or
higher, then the starting time for treatment should be later or
earlier. After the induction condition was optimized, tetraploid
level detected at embryo stage reached almost 100% (Fig. 4).
Relationship Between Tetraploid Rate at Embryo Stages and
Hatching Rate
Higher treatment temperatures led to more tetraploids. they also
led to reduced survival of the treated embryos. There was strong
negative correlation between tetraploid induction efficiency and
larval survival (Fig. 5).
Production of Tetraploid Larvae
Although tetraploid levels detected at embryo stages were high,
tetraploid embryos experienced problems in hatching. The hatch-
ing success rate of tetraploids was low. In our experiments, the
highest tetraploidy rate detected at nauplius stage was about 38%
(Fig, 6a) while tetraploid levels detected at embryo stage was 55%
(Fig, 6b), This result was obtained under spawning temperature of
15.7°C. with a 3-min heat shock at 34°C starting 110 min after
fertilization. During the hatching process of tetraploid embryos,
some live embryos in membrane were observed, but their mor-
phology was abnormal. And when the.se abnormal embryos were
selected for detection of ploidy, it was found that most of these
embryos were tetraploids (data not shown).
DISCUSSION
Available data showed that the Chinese shrimp Fennerope-
naens chinensis. tetraploids could be produced by inhibiting the
first mitosis. Reported methods for producing tetraploids in aquatic
animals include inhibiting first mitosis (Thorgaard et al. 1981,
Varadi et al. 1999), inhibiting the first meiosis of diploid fertilized
eggs (Yang et al. 2000, Zhang et al. 2000), or inhibiting polar body
I in eggs from triploids (Guo & Allen. 1994, He et al. 2000). In
shellfish, there is no successful production of viable tetraploids by
inhibiting mitosis I (Guo et al. 1994), although tetraploid embryos
can be produced. In our experiments on shrimp, no tetraploids
were produced through inhibiting meiosis. This study showed that
tetraploid embryos could be produced at high rate in shrimp. How
to make more embryos hatch into nauplii, however, remains a
problem that must be solved. The challenge is to improve the
treatment conditions so that they lead to high-level production of
tetraploids without causing serious damage to treated embryos. It
is also possible that tetraploid embryos have limited viability or
ability to hatch, and they can be obtained by improving hatching
conditions. During tetraploid induction, the exact time of thermal
or pressure shock applied to inhibit mitosis I is important. Inhib-
iting different processes will lead to different viability according to
an analysis of tetraploid induction in fish (Pandian & Koteeswaran
1998).
This study showed that heat-shock is effective in inhibiting
mitosis I in the Chinese shrimp Fenneropenaeus cliinensis. This
shrimp is a temperate species, so it is more sensitive to heat than
tropical species. Heat shock has an advantage over chemical treat-
ments, in that there is no pollution to the environment. For sub-
tropical or tropical species, cold shocks may be more helpful. To
our knowledge. Fenneropenaeus chinensis is the only shrimp spe-
cies where tetraploid induction has been reported. Until now, there
is only one report that tetraploid was produced in this species
(Xiang et al. 1993). In earlier reports in this study, tetraploids were
induced by cytochalasin B (CB) and ploidy was detected through
chromosome counting. In this work, heat shock was used and
optimal treatment was identified. The use of flow cytometry as a
method for detecting tetraploidy was a key factor in our successful
evaluation of heat shock treatment. Compared with chromosome
counting, flow cytometry analysis allows rapid and accurate ploidy
determination of many experiment groups. The application of flow
cytometry techniques has greatly advanced polyploidy research in
shrimp (Zhou et al. 1999).
Although heat-shocks can effectively inhibit mitosis 1 in
shrimp, optimal conditions for the hatching of fertilized eggs need
further investigation. Extending treatment duration might increase
the tetraploid rate, but reduce hatching success. The proper strat-
egy to induce triploids should be to achieve certain high levels of
tetraploid and hatching rates. Hatching rates varied from brooder
to brooder when the tetraploid rate was the same because of dif-
ferent egg quality. There is the common tendency however, that
hatching success decreases when tetraploid level increases. In Fen-
neropenaeus chinensis. 40-60% tetraploid rate is preferred to ob-
tain viable larvae. Although no viable tetraploid post-larvae were
obtained, this study showed that high percentages of tetraploids
could be produced by heat shock. The optimization of heat shock
treatments is an important first step in successful tetraploid induc-
tion. Further work is needed to improve the survival of tetraploids
so that viable tetraploid shrimp can be eventually obtained.
ACKNOWLEDGMENTS
The authors thank Dr. Ximing Guo from Rutgers University,
USA for his kind instructive comments and revision of this manu-
script and Dr. Xiaolin Liu for his help in statistical analysis of data.
2n
^^ifmnt/^t^tu^ti^ t*^ t ^ s
2n
4n
iiMi|Uii<HiW
rSO 300 3U
M*-
«ra 460 FLI EDO 0 SO 100 ISO 1 00 NO 300 3 GO 4t» 460 FL1 BOO
Figure 6. Flow cytometry analysis at nauplius (a) and embryo (b) stages of one group treated tor tetraploid induction in Chinese shrimp
Fenneropenaeus chinensis.
Tetraploid Induction in Chinese Shrimp
545
This research was funded by International Foundation for Sciences
(A2027-2), National Key Fundamental Research Programme
0199901 2009 and Knowledge Creative Programme of the Chinese
Academy of Sciences (ZKCX 2-211).
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Jtnimal of Slwlljhh Kesearch. Vol. 22. No. 1. 547-553, 2003.
SELECTION AND USE OF DIFFERENT DIETS IN A STUDY ON CHINESE SHRIMP,
FENNEROPENAEUS CHINENSIS
GUOQIANG HUANG, SHUANGLIN DONG,* FANG WANG, AND SHEN MA
Murkuhure research laboratory. Fisheries College. Ocean University of China. Qingdao. 266003.
People's Republic of China
ABSTRACT A 30-day feeding e,\periment was conducted to investigate the dietary selectivity in Chinese shiinip. Fenneropenaeus
chinensis. Si.x groups of shrimp with initial body weight of 1.530 ± 0.047 g (mean ± SD. n = 6) were used, in which the first five
groups were fed to satiation with single diets of FF, llesh of fish (Sardinella zunasi); SF, tlesh of shrimp (Trachypenaeus curvirostris);
CF. foot of clam (Ruditapes vangata); PW, polychaette worm (Neanthes japonica) FD. a commercial formulated diet; and the last group
received MD. mi.xed diet. The feeding tfials were conducted simultaneously and shrimp were fed to satiation. The specific growth rate
(SGR), food intake (FI), food conversion efficiency (FCE), and apparent dige.stive ratio (ADR) were determined. The results showed
thai specific growth rates of dry weight, protein, and energy (SGR^. SGR^. and SGR,.) were highest in the MD fed group, food
conversion efficiencies (FCEj, FCEp. and FCR,.) were highest at PW fed group. Food ingestion in terms of dry weight, protein, and
energy were significantly higher in CF and MD fed groups than others. The highest ADR was observed in CF fed group. In mixed diet
feeding group, percentages of the five ingested diets to the total ingested amount based on dry material, protein, and energy were: FF.
13.07%; SF, 9.60%: CF. 46.45%; PW, 30.88%; and FD. 0%; FF. 15.56%; SF, 11.44%; CF. 45.09%: PW. 27.91%; and FD. 0%; FF.
13.66%; SF, 10.48%; CF, 44.00%; PW, 31,86%; and FD, 0%; respectively. This indicates that Chinese shrimp possess the ability to
discriminate different diets. The optimal foraging strategy of Chinese shrimp in this experiment was to gain energy as much as possible
to meet energy needs of variable physiologic activities under the premise of maximizing growth. Additionally, the protein sparing effect
of dietary E/P ratio and lipid content was also observed in this experiment.
KEY WORDS: dietary selectivity. Chinese shrimp. Fenneropenaeus chinensis
INTRODUCTION
Because of their economic significance to fisheries and impor-
tant function in aquatic ecosystems, several studies were con-
ducted to investigate feeding habits of shrimp and crab. The most
commonly used direct method was to analyze the stomach content
or foregut of the animal, from the wild or under culture condition,
and evaluate its feeding habits from the composition (Chong &
Sasekumar 1981. Phil & Rosenberg 1984, Cockcroft & Mclachlan
1986, Prakash & Agarwal 1989, Nunes et al. 1997, Roy & Singh
1997, Kulkami et al. 1999. Minami 2000, Schwambom & Griales
2000). The activity of different digestive enzymes in the animals
was also used to judge their feeding habits (Biesiot & Capuzzo,
1990). In recent years, stable isotope analysis method was also
applied in analyzing of the feeding habits of the animals (Newell
et al. 1995. Nunes et al. 1997. Schwambom & Griales 2000). Ivlev
(1961 ) proposed a selection index to describe the dietary selectiv-
ity of fish. Pinn et al. ( 1998) used Strauss" Linear Selection Index
to describe in their study the dietary selectivity of two mud-shrimp.
Nevertheless, the selectivity of a diet item is affected by such
factors as energy content, difficulty of foraging and handling, and
so on (Sunaga 1971, Griffiths 1975, Manghagen & Wiederhohn
1982. Mikheev 1984, Buskey et al. 1991, Alam et al. 1996, Meh-
ner et al. 1998). The theory of optimal foraging is based on the
evolutionary premise that individuals within a population that for-
age most efficiently and maximize their net rate of energy intake
will possess greater fitness and contribute more genes to future
generations (Calow & Townsend 1981 ). It has been found that the
dietary selectivity of animals is partly or completely subjected to
the law (Kislalioglu & Gibson 1976. Elner & Hughes 1978).
The dietary condition of shrimp is variable in wild and exten-
sive or semi-intensive cultural waters, and the abundance and com-
position of diet vary greatly in different waters and time periods
*Corresponding author. E-mail: dongsKs'mail.ouc.edu.cn
(Marte 1980, Luna-Marte 1982). In most cases, abundance of its
preferred diet is likely to decrease to a low level because of the
natural fluctuation or high feeding pressure. Hence, the shrimp
cannot select the diet species in accordance with its actual prefer-
ence. It is probable that shrimp might ingest the diet species that is
not preferred, to release the pressure of starvation or innutrition
and satisfy its growth or development. Therefore the methods men-
tioned previously, to analyze its feeding habit, are quite difficult to
reflect or define its dietary selection or preference. Quantitative
study of the preference of some diet items of animals can be really
conducted in only controlled conditions when different diet items
(include items differing in nutritional composition, origin, size,
and so on) are provided.
In the wild environment, crustaceans, polychaette worms, and
juvenile bivalves are major diet items of Chinese shrimp (Fen-
neropenaeus chinensis) (Wang, 1997). In this study Chinese
shrimp, widely cultured and distributes in China, were used and
five diet items were provided equally in excessive amounts to
study the feeding preference of the shrimp and the strategy of its
diet selectivity.
MATERIALS AND METHODS
Rearing Conditions
Chinese shrimp were kept in glass aquaria (45 x 30 x 30 cm^.
water volume of 35 dm'), and each rearing unit was stocked with
four shrimp. The room temperature was controlled by an air con-
ditioner, and water temperature was 25 ± 0.5°C. Aeration was
provided continuously and 0.50-0.67 of water was exchanged ev-
ery other day. Seawater used in the experiment was filtered by
composite sand filter. During the experiment, dissolved oxygen of
water was maintained above 5.5 mg/L, pH was about 8.0, the water
salinity was between 30-33%c, photoperiod of 14 h of light: 10 h
of darkness was used.
547
548
Huang et al.
Diets Preparation
The five diets used in the experiment were: fish flesh (FF) — the
flesh of sardine iScinlinella ziinasi) without head, scales, fins, bow-
els, and bones: shrimp flesh (SF) — small shrimp (Trachypenaeus
cur\nrostris) without head and shell; clam foot (CF) — from {Rii-
ditapes varigata): PW, polychaette worm — Neanthes japonica:
FD formulated diet — a commercial sold shrimp diet (Sea-Horse
Brand. Fujian Mawei Unite Feed Ltd. China) comprised of bean
powder, fish powder, shrimp powder, compound vitamins, and
compound minerals; MD, mixed diet — equal combination of the
five diets. Shrimp were fed diets to satiation. Each diet item was
cut into almost the same size as the formulated diet (about 4 mm
in length and diameter of 2 mm) before feeding. Biochemical
composition of the diets is listed in Table 1.
Source and Acclimation of Shrimp
The experiment was carried out at the Mariculture Research
Laboratory, Ocean University of Qingdao, People's Republic of
China. The shrimp used in the experiment were collected from the
Tianheng Shrimp Farm, Qingdao. Prior to the experiment, the
shrimp were transferred into aquaria and underwent a 6-day accli-
matization period during which they were fed with formulated diet
(FD) at satiation level twice a day (at about 6:00 and 18:00).
Experiment Design
After 24 h starvation. ^6 shrimp with an initial wet weight of
1.530 ± 0.047 g (mean ± SD) were selected from acclimated ani-
mals and placed in 24 aquaria to form 6 experimental groups fed
with different diets of FF, SF, CF, PW. FD, and MD. A complete
randomized block design was used to arrange the 24 aquaria of 6
groups.
Sample Collection and Analysis
Three groups (eight shrimp each) were sampled from the ac-
climated shrimp simultaneously while experimental shrimp were
selected to determine the initial body composition of the experi-
mental shrimp. After the 30-d experiment, the shrimp of all groups
were starved for 24 h and then sampled. The shrimp from the
individual aquaria were pooled as a sample and there were 24
samples of final shrimp.
During the course of the experiment the daily food supply was
recorded and uneaten food was collected 3 h after feeding. Feces
were collected promptly. Shrimp and food were weighed to the
nearest 0.001 g using an electronic balance after carefully blotted
with paper towel to remove excess moisture.
After the weight was obtained all samples of shrimp, feces, and
food were dried in an oven at 70°C to constant weight, homog-
enized with a glass mortar, and stored at -20°C. Before chemical
compositions were analyzed, the samples were re-dried at 70"C to
constant weight.
The N content was measured using the Micro-Kjeldahl meth-
ods and the crud protein content was calculated by multiplying
Kjeldahl N content by 6.25(AOAC, 1984). Crude lipid was deter-
mined by the Soxthlet method (AOAC, 1984). ash was determined
by combusting dry samples in a muffle furnace at 550°C for 1 2 h
(AOAC. 1984). and the gross energy content of dry samples was
determined by PARR 1281 calorimeter (PARR Instrument Com-
pany. USA). An analysis of each sample was conducted in tripli-
cate (three sub-samples for each sample).
Calculation of Data
Specific growth rate (SGR), food ingestion (FD. apparent di-
gestive ratio (ADR), food conversion efficiency (FCE), and Ivlev's
index of dietary selectivity (I,) were calculated as follows:
SGRw(%/day) = 100(In W-In W„)/T (Ricker 1979)
Fl^C^r body weight/day) =
lOOC/IT (W, + W„)/2] (Wu et al. 2()()())
ADR (%) = 100(C-F)/C (Smith 1971)
FCEw{ •7r) = 100(W,-W„)/C (Matty & Smith 1978)
Where W, and W„ were the finial and initial wet weight of the
shrimp. T was the duration of growth period in days. F was the dry
weight of feces, and C was the dry weight of consumed food.
SGR. Fl. ADR. and FCE in terms of dry matter (SGRj, FIj,
ADRj, and FCE^). protein (SGRp. FIp, ADRp, and FCEp), and
energy content (SGR^, FI^, ADR^. and FCE^) were calculated
similariy.
I, = (r,-p,)Ar, + p,) (Ivlev 1961)
Where r, was the portion of one diet in the total ingested diet, and
TABLE 1.
Biochemical composition and energy content of experimental diets (Mean ± SE).'
Diets
Composition
FF
SF
CF
PW
FD
MD"
Moisture (%)
77.23 ± 0.38
80.33 ± 1.27
79.35 ±3.15
74.18 ±0.80
7.70 + 0.15
76.57 + 0.10
Protein (%)
83.98+1.12
84.13 ±0.65
68.49 ± 0.59
63.73 + 0.44
42.57 ± 0.50
71.I4±0.18
Lipid (%)
5.18 ±0.01
5.00 ± 0.01
5.96 ±0.01
16.32 ±0.03
9.93 ± 0.02
8.96 ± 0.02
Ash (%)
6.41 ± 0.02
3.2 ±0.01
5.38 ±0.02
6.89 ± 0.02
10.75 + 0.03
5.77 ± 0.02
Energy
22.15 ±0.24
22.95 ± 0.04
19.89 ±0.05
21.66 + 0.09
19.23 ±0.09
21.02 ±0.03
E/P
26.38 ±0.14
27.28 ±0.1 7
29.17 + 0.20
33.99 ±0.17
45.20 ± 0.70
29.54 ±0.11
L/P
0.061 ±0.001
0.059 ±0.001
0.087 ±0.001
0.256 ± 0.002
0.233 ± 0.003
0.126 ±0.001
' Moisture is percentage content of wet sample, moisture = 100 x (WW - DS)AVW. WW: wet weight. DW: dry weight: Protein. Lipid and Ash are
percentage content of dry sample; Unit for energy content is KJ.g"' in dry sample; E/P: energy/protein ratio, unit for E/P is KJ.g''; L/P: Lipid/protem
ratio, unit for E/P is g.g"'.
•^ Composition of mixed diet was calculated after the experiment basing on the ingested dry weight of the first five diets, it is a weighted value according
to the portions of every diet in the total ingested weight in mixed diet fed group.
Selective Diets of Fenneropenaeus Chinensis
549
Pj was the portion of one diet in tiie total provided diet r, and p,
were calculated in terms of dry matter.
Statistical Analysis
Statistics were performed using SPSS 10.0 statistical software
with possible differences among diet treatment being tested by
one-way ANOVA. Tukey's-b multiple range tests was used to test
differences between treatment groups. Differences were consid-
ered significant at a probability level of 0.05.
RESULTS
Food Consumption and Feces
Table 2 lists the food consumption and feces for the six diet
treatments. Shrimp fed with CF and MD consumed significantly
more food than the others did. The largest amount of feces in terms
of dry matter emerged in MD but it was not significantly larger
than FD. However, the largest amount of feces was observed in FD
in terms of protein and energy.
Growth
At the end of the experiment no significant difference existed
among CF-, PW-, and MD-fed shrimp in terms of WW. DW, P,
and E, and all were significantly higher (df = 5, P < 0.05) than the
other three groups (Table 3). FF and SF were the lowest of the six
groups in all terms of WW, DW. P. and E. and no significant
difference existed between them (Table 3).
No signitlcant difference was observed among CF. PW and
MD in SGR,,. SGRj. SGR^, and SGR^. respectively, and all were
significantly higher than the other three groups. Except for the high
SGR„ (2.76 ± 0.06) observed in PW, the highest SGRj (2.99 ±
0.07), SGRp (2.91 ± 0.07), and SGR^. (3.15 ± 0.07) all appeared in
MD { Fig. 1 ). FD was significantly higher than SF and FF in SGRj,
SGRp, and SGR^. Every parameter of FF and SF was lower than
other groups (Fig. I ).
Food Conversion Efficiencies
Figure 2 illustrates that the FCE in terms of DW, P, and E. PW
was significantly higher than other groups in FCEj, FCEp, and
FCE,, and it had the highest values of 22.86 ± 1.63, 22.87 ± 1.65,
and 21.39 ± 1.49, respectively. FD was significantly higher than
other groups except PW in FCEp, and it was not significantly
different from CF and MD though it was significantly higher than
FF and SM had the lowest FCE
(only about 20-25% of PW). MD had significantly lower FCE than
PW when four diets were ingested in different portions.
Food Ingestion of Six Diet Treatments
Fl„, FI(j, FIp, and Fl^ in CF and MD fed groups were 5.58 ±
0.24. 22.89 ± 0.84, 23.62 ± 0.87, 23.59 ± 0.86, and 5.94 ±0.14,
23.28 ± 0.45, 24.97 + 0..50, 24.98 ± 0.48, respectively (Fig. 3). CF
and MD were significantly higher than other groups in Fl for all
the four terms, and no significant difference between CF and MD
was observed (Fig. 3). FI in FD was significantly lower than the
others because of its lowest protein content. FI of FF and SF fed
groups were the lowest in all measures except protein (Fig. 3).
Percent Composition of Ingested Diets and Indexes of Selectivity of
Five Provided Diets in MD
When five diets were provided simultaneously and in excess,
Chinese shrimp ingested four diets of FF, SF, CF, and PW, and no
FD was ingested (Fig. 4). Percentages of dry weight and energy
consumed by shrimp of FF and SF fed groups were not signifi-
cantly different from each other, but the percentage of protein
consumed in SF was significantly lower than FF. Among the four
ingested diets, CF had significantly higher percentage of dry
weight (46.45 ± 1.63). protein (45.09 ± 1.49) and energy (44.00 ±
1.60) than the other three. Percentages of dry weight, protein, and
energy of consumed PW were 30.88 ± 2.06, 27.91 ± 1.93, and
31.86 ± 2.08, respectively, and significantly higher than FF and
SF.
Indexes of selectivity of five provided diets (based on dry mat-
ter) in MD treatment were FF, -0.210 ± 0.017: SF, -0.352 ± 0.016;
CF, 0.397 ± 0.016; PW, 0.210 ± 0.030; and FD, -I ±0 respec-
tively. It indicated that Chinese shrimp performed positive selec-
tivity on CF and PW. Negative selectivity on FF and SF was
observed, and FD was excluded under experimental conditions.
Apparent Digestive Ratio of Diets
The highest ADR in terms of dry weight, protein, and energy
was in CF (92.97 ± 0.35, 98.05 ± 0.10, and 99.10 ± 0.04, respec-
tively). ADR,, ADRp, and ADR, of FD were 77.97 ± 1.92, 86,15
± 2.06, and 91.10 ± 1.55, respectively, which are significantly
lower than other groups (Fig. 5).
DISCUSSION
Feeding behaviors of shrimp and crab have been studied by
methods of analyzing stomach contents or foregut, activity of di-
gestive enzymes, and by stable isotope technique (Chong & Sase-
TABLE 2.
The dry weight (g, DW), energy (KJ, E), and protein (g, P) content of the food consumed and feces for the six diet treatments.
Treatments
(mean ± SE)
FF
SF
CF
PW
FD
MD
Food consumption
DW
1.383 + 0.0-18''
1.378 ±0.163"
3.897 ±0.185"
2.215 ±0.074"
2.020 ±0.1 74"
4.3.M ± o.oes-"
P
1.183 ±0.033"
1. 169 ±0.138"
2.681 ±0.127'
1.425 ±0.048"
0.868 ± 0.075"
3.084 ± 0.048''
E
30.352 ± 0.838"
31.610 ±3.729"
77.509 ±3.67f
47.990 ± 1.610"
38.841 ± 3.342"
91.014 ± 1.295"
Feces
DW
O.I 23 ±0.020"
0.234 ±0.018"
0.275 ± 0.023"
0.307 ± 0.030"
0.4-30 ± 0.03 r
0.436 ±0.018'
P
0.066 ±0.001'
0.041 ±0.001"
0.052 ±0.001"
0.074 ±0.001''
O.I 14 ±0.003'
0.090 ±0.001'=
E
0.473 ± 0.078"
0.742 ± 0.057"
0.704 ± 0.059"
1.350 ±0.133"
3.336 ± 0.243"
2.040 ± 0.084'
Values with different letters in the same line were significantly different (df = 5, P < 0.05) from each other.
550
Huang et al.
TABLE i.
Initial and final shrimp wet weight (g. WW), dry weight (g, DW), protein Ig. P) cuntent and energy (KJ. E) for the six diet treatments.
Treatments
(mean
±SE)
FF
SF
CF
PW
FD
MD
Initial shrimp
WW
1.536 ±0.010
1.520 ±0.040
1 .525 ± 0.030
1 .497 ± 0.026
1.519 ±0.021
1.535 ±0.008
DW
0.360 ± 0.002
0.356 ± 0.009
0.357 ± 0.007
0.351 ±0.006
0.356 ± 0.005
0.360 ± 0.002
P
0.243 ± 0.002
0.240 ± 0.006
0.241 ±0.005
0.237 ± 0.004
0.240 ± 0.003
0.243 ±0.001
E
6.802 ± 0.048
6.722 ±0.1 70
6.755 ±0.1 34
6.627 ±0.1 17
6.726 ± 0.090
6.802 ± 0.036
Final shrimp
WW
2.057 ±0.108"
2.204 + 0.151-
3.133 ±0.152''
3.418 ±0.139"
2.371 ±0.136"
3.335 ± 0.096"
DW
0.423 ± 0.016"
0.448 ± 0.040"
0.780 ± 0.050'
0.855 ± 0.044'-
0.591 ±0.045"
0.882 ±0.014'
P
0.288 ±0.011"
0.292 ± 0.026"
0.517 ±0.033'
0.561 ±0.029'
0.378 ± 0.029"
0.581 ±0.009'
E
7.753 ±0.305"
7.777 ±0.687"
15.193 ±0.981'
16.864 ±0.862'
11.229 ±0.862"
17.501 ±0.286'
Values with different letters in the same line were significantly different from each other (df = 5. P < 0.05 ).
kumar 1981. Phil & Rosenberg 1984. Cockcroft & Mclachlan
1986, Prakash & Agarwal 1989. Biesiot & Capuzzo 1990. Newell
et al. 1995, Nunes et al. 1997, Roy & Singh 1997, Minami 2000.
Schwamborn & Griales 2000). Because of the variation of food
abundance and composition in natural and extensive or semi-
extensive cultural water, these results cannot reflect the real food
preference of the animals to some dietary organisms. Although
Finn et al. (1998) analyzed the selectivity on dietary organisms in
two mud crabs by utilizing the Strauss" Linear Index, it was af-
fected by such factors as diet density, difficulty of searching, dif-
ficulty of handling, and soon (Sunaga 1971, Griffiths 1975. Mang-
hagen & Wiederholm 1982, Mikheev 1984, Buskey et al. 1991.
Alam et al. 1996, Mehner et al. 1998). The dietary selectivity of
Chinese shrimp on five diets with same availability was observed
in these studies, and indicates that the Chinese shrimp possess the
ability to discriminate different diets. The shrimp selected CF and
PW. but ingested almost no FD (Fig. 4).
The dietary selectivity of animals are affected by many factors
such as dietary energy content, abundance, and difficulty of
searching, handling, digesting, and so on. In foraging, animals gain
energy by ingesting prey (food) and expand energy in searching
and handling prey (food). Composition of diets of some fish ac-
corded with the prediction by the theory of optimal foraging in
many experimental studies (Kislalioglu & Gibson, 1976; Tytler &
Calow, 1984). Feeding behaviors of some crabs on snails and bi-
valves also agreed with the Optimal Foraging Theory on the whole
(Finer & Hughes 1978. Boynton 1979. Hughes & Finer 1979.
Kennedy et al. 1983, Lawton & Hughes 1985. Seed & Hughes
1997). Diets with almost the same encounter probability, size, and
difficulty of handling were provided in this study. It was observed
that the Chinese shrimp did not exhibit behaviors that maximized
the net energy gain in the selection of diets under this experimental
condition, and a great deal of CF. which was lower in energy
content, was ingested. The correlation coefficient between in-
gested percent quantity and per unit energy content of diet was
only 0.131. which indicated that no significant correlation existed
(/? = 20. P = 0.589). Poor relativity between Ivlev's index of
selectivity and per unit energy content of diet was also observed
(R = 0.207. /I = 20. P = 0.380).
In this experiment, every diet provided to the shrimp was in
excess but equally divided. Major factors that affect the dietary
selectivity of shrimp are diet nutrition, difficulty of digesting, ufi-
lizing rate, attraction to diets, and so on. Shrimp of group MD
showed distinct selectivity of different natural diets provided in the
experiment. ADR of four natural diets were significantly higher
than FD. The highest portion (based on dry weight) of 46.45% was
observed in CF (Fig. 4), and the portion of PW (30.88%) was also
comparatively higher than those of FF (13.07%) and SF (9.59%).
It could be concluded from the results the shrimp preferred diets
that met their high growth requirement (such as CF and PW), then
they selected diets based on the digestibility (indicated by ADR),
namely, they ingested more CF, which had relative higher ADR,
Figure 1. Special Growth Rati (SdRl of all diet treatments (Different
letters above the bars denote significant differences (P < 0.05) among
columns in the same cluster). Where FF = Fish Flesh, SF = Shrimp
Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = Formulated
Diet, and MD = Mixed Diet: S(;R„, SGR,,, StiRp, and SGR.. are Spe-
cial Growth Rates in terms of wet weight, dry weight, protein, and
energy of shrimp body respectively; Verical bar = SE (n = 4).
Figure 2. Food Conversion Efficiencies (FCE) in diet treatments (Dif-
ferent letters above the bars denote significant differences (P < 0.05)
among columns in the same cluster). Where FF = Fish Flesh, SF =
Shrimp Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = For-
mulated Diet, and MD = Mixed Diet: FCE^. FCEp, and FCEe are Food
Conversion Efficiencies in terms of dry matter, protein, and energy
respectively; Vertical bar = SE (n = 4).
Selective Diets of Fenneropenaeus Chinensis
551
IFF
@SF
ICF
IPW
^FD
Figure 3. Food Ingestion (FI) of all groups (Different letters above the
bars denote significant differences (P < 0.05) among columns in the
same cluster I. Where FF = Fish Flesh. SF = Shrimp Flesh. CF = Clam
Foot, PW = Polychaette Worm, FI) = Formulated Diet, and MI) =
Mixed Diet; FI„, FIj. Fl^, and FI^. are Food Ingestion in terms of wet
weight, dry matter, protein, and energy respectively; Vertical bar = SE
(n = 4).
than PW. The optimal foraging strategy of Chinese shrimp in this
study was to gain as much energy as possible to meet the needs of
variable physiologic activities, under the premise of ensuring fast
growth, not to select diets to gain the highest FCE. In Group MD.
the shrimp ingested a large amount of CF. which was easily di-
gested (Fig. 5). so that they were able to ingest more diet continu-
ously during the period to ma.ximize the dietary energy ingestion.
More studies on the effect of feeding attractants of these diets on
dietary selectivity are still needed.
It was found in a few of studies that the decisive factors af-
fecting the utilization, expressed in protein efficiency ratio (PER)
and food conversion efficiency (FCE), of diets, were other ingre-
dients when dietary protein content was above a reasonable level.
These phenomena occurred in fish (Degani & Viola 1987, Viola &
Lahav 1991. Erfanullah & Jafri 1995. Company et al. 1999. Morais
et al. 2001, Shalaby et al. 2001. Das 1991). and also existed in
shrimp and crab (Andrews et al. 1972. Colvin 1976. Sedgwick
1979. Xu & Li 1988). Xu and Li (1988) found, a protein sparing
effect of lipid, in a study on the optimal protein, carbohydrate,
fibrin, and lipid contents for Chinese shrimp diet that the increase
of lipid content significantly promotes PER at all of the three
protein contents of 36%, 40%, and 44%. Dietary protein contents
of all the diets provided in this study exceeded 40% (Table 1) and
could satisfy the demands for protein of Chinese shrimp. The
IFF
^SF
iCF
IPW ■FD(a)
Dry Weight Frotein hri.MRv
Figure 4. Percent compostion of ingested diets in mixed diet fed group
(Different letters above the bars denote significant differences (P <
0.05) among columns in the same cluster). Where FF = Fish Flesh, .SF
= Shrimp Flesh, CF = Clam Foot, PW = Polvchaette Worm, FI) =
Formulated Diet, and MD = Mixed Diet. Vertical bar = SF (n = 4).
Note: FD = 0(a) in all cluster.
Figure 5. Apparent Digestive Ratio (.XDR) of every diet group (Dif-
ferent letters above the bars denote significant differences (P < 0.05)
among columns in the same cluster). Where FF = Fish Flesh, SF =
Shrimp Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = For-
mulated Diet, and MD = Mixed Diet; ADR^, ADR^, and ADR,, are
Apparent Digestive Ratios in terms of dry matter, protein, and energy
respectively; Vertical bar = SE (n = 4),
energy to protein ratio (E/P) and lipid content, however, varied
greatly in different diets. The lipid contents of PW and FD were
16. .32% and 9.93% respectively, and they were higher than other
provided diets (Table 1 ). Although the lipid content in shrimp diet
should not exceed 10% (Xu & Li 1988, Li 1990), it was found in
this study that high lipid content had positive effect on FCE in
Chinese shrimp, and the highest FCE was observed in PW, which
was the highest in lipid content (16.32%). This result indicates that
the shrimp could use more lipid than indicated in other reports, and
diet for shrimp should be higher than 10% while protein content
was high. E/P of these two diets are 33.99 KJ.g"'and 45.20 KJ.g"'
respectively, which are also higher than other diets. It was prob-
able that the protein sparing effect of these two parameters sig-
nificantly improved the FCEp of PW and FD (reaching 22.87% and
15.46%, respectively). Although shrimp of CF treatment ingested
a larger amount diet in terms of DW. P, and E than PW treatment
(Fig. 3), their SGR, FCE, and final body weight were not signifi-
cantly higher than PW due to the lower lipid content and lower E/P
ratio.
Munoz and San Feliu (1984) found in an experiment that Japa-
nese shrimp, Penaeus japanicits fed on natural diets grew faster
than those fed on formulated diet. In this study, the Chinese shrimp
fed on FD grew significantly faster than those fed on FM and SM,
and slower than those fed on CF and PW. Because the shrimp
ingested no FD when it was fed simultaneously with natural diets,
it was necessary that natural diets and the formulated diet should
not be fed simultaneously in practice to avoid the wasting of for-
mulated diet. Because the Chinese shrimp ingested no FD in the
MD group, there was no significant difference among FD, FF, SF,
and PW groups in FI^, (Fig. 3). Furthermore, FIj of FD was sig-
nificantly higher than that of FF, SF, and CF, and the FCE^ of FD
was significantly higher than that of other diets except PW (Fig. 2).
This result indicates that FD was le.ss contaminating than FF, SF,
and CF because of less nitrogen and organic matter loss in the
water when these diets were ingested. It was found that fish culture
used trash fish as feed, which results in heavier contamination than
dry and artificial feeds (Wu, 1995). Considering the heavy con-
tamination that can be caused by feeding the shrimp with natural
diets in pond culture practice and the diets resource limitation (Wu
1995, Dong et al. 2000), it is reasonable to propose a high quality
formulated diet to be used in culture practice.
552
Huang et al.
ACKNOWLEDGMENTS
This work was supported by funds from the Chinese National
Science Foundation for Talent Youths (Grant no. 39725023). the
Project under the Major State Basic Research of China (Grant no.
G1999012011) and the National Tenth five-year Scientific and
Technological Key Project (Grant no. 2001BA505B-04).
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Juuiiial of Shelljisli Rcseiirdi. Vol. 22, No. 2, 555-559. 2003.
EFFECT OF SALINITY ON SURVIVAL, GROWTH, AND OXYGEN CONSUMPTION OF THE
PINK SHRIMP FARFANTEPENAEVS PAULENSIS (PEREZ-FARFANTE 1967)
MONICA Y. TSUZUKI,'* RONALDO O. CAVALLl,' AND ADALTO BIANCHINI-
'Liihoraldrio cle Mariciiltiira. Dcpiirtanwiito Je Oceanogrcifia. Fuuda<;M> Universidude Federal do Rio
Grande. Caixa Postal 474, 96201-900. Rio Grande, RS. Brazil: 'Lxiboratorio de Zoofisiologia,
Departamento de Ciencias Fisiologicas. Fiiiidd^cio Universidade Federal do Rio Grande, Caixa Postal
474. 96201-900, Rio Grande. RS. Brazil
ABSTR.ACT Survival, growth, and o.xygen consumption rates of Farfantepenueus pauleiisis postlarvae (PL) were examined at
different salinities. Initially, PL 15 maintained at 30%c salinity were gradually acclimated to 2. 5. 10. 20, and 30%c over 5 days.
Afterwards, survival, growth, and oxygen consumption rates of shrimp reared at these salinities were determined over a 42-day
experimental period. Lower wet weight and cephalotorax length, and higher mortality rates were observed in shrimp reared at 2%o
salinity, especially when compared with those reared at lO'At salinity (P < 0.05). In the range of S'/tr to 30%f salinity, growth was
optimized at Iff/n salmity. although this response was not significant. Salinity affected the oxygen consumption rates of F. paiilensis
postlarvae. At the beginning of the growth trial, oxygen consumption rate was markedly lower at 27ii salinity than at 10%c or 30%o
salinity (P < 0.05). This response was probably associated with a metabolic depression that preceded the shrimp death. Thereafter,
oxygen consumption at 2%t salinity showed a nonsignificant increase due to a higher variability of measurements probably associated
with a better performance of surviving shrimp, which were tolerant to low salinity levels. At the intermediate salinities (5%c-20'^t).
oxygen consumption was higher at 10%f salinity. At the end of the experiment, oxygen consumption reached similar and low levels
irrespective of the salinity level. Oxygen consumption rate of shrimp reared at 30^. salinity was constant and close to 5-(iL mg dry
weight"' hr"' throughout the experiment.
KEY WORDS: shrimp. Faifantepenaeus paiilensis. growth, oxygen consumption, salinity
INTRODUCTION
FaifaiUepciuiciis paiilensis (Perez-Farfante 1967) is a cold tol-
erant shrimp naturally occurring between Mar del Plata. Argentina,
and Ilheus, Brazil (D'Incao 1995). It is an important fishery re-
source, especially in Southern Brazil, where catches by artisanal
fisheries have averaged around 3500 metric tons/yr in the last 40
years. However, unpredictable fluctuations in capture caused by
climatic and oceanographic factors (Castello & Moller 1978,
D'Incao 1995) usually result in a severe socio-economical prob-
lem. Some studies have examined the viability of cultivation and
restocking programs with this species (Olivera et al. 1993.
Wasielesky et al. 1995, Peixoto et al. 2002). The release and
growth of F. paulensis in pen enclosures is routinely carried out at
the estuary of the Patos Lagoon, Southern Brazil (Wasielesky
2000). which is characterized by abrupt and wide variations in
salinity (Baptista 1984).
Salinity is one of the most important environmental factors
affecting growth and survival of penaeids as it influences food
consumption, conversion efficiency, and metabolic responses
(Venkataramiah et al. 1972. Castille & Lawrence 1981. Dalla Via
1986, Staples & Heales 1991. Clark 1992, Brito et al. 2000). The
knowledge of the species tolerance limits and optimum salinity
levels is necessary to evaluate the viability of F. paulensis culti-
vation at variable environmental conditions. Furthermore, it is im-
portant to understand the effects of salinity when shrimp is reared
in nursery grounds characterized by sudden salinity tluctuations
and extreme environmental conditions. Salinity might have an in-
direct influence on the survival and growth of postlarvae when
they penetrate estuarine areas, and also on the migration of juve-
niles back to the ocean. For example. Staples ( 1980) observed that
reductions in salinity caused the migration of Fenneropeiiaeiis
merguiensis juveniles from nursery grounds to oceanic waters.
Salinity tolerance limits and the effects of acclimation to sa-
linity on the survival of F. paulensis have already been evaluated
(Tsuzuki et al. 2000). However, as the optima! salinity range for
growth is narrower than for survival, growth occurs when the
metabolic demands for maintenance and feeding activity are sat-
isfied. Several studies have analyzed the metabolism and activity
in crustacean decapods through oxygen consumption measure-
ments (Kutty et al. 1971, Venkataramiah et al. 1974, Venkat-
aramiah et al. 1975, Gaudy & Sloane 1981, Du Preez et al. 1992,
Villarreal & Rivera 1993). Since the rate of oxygen consumption
is modified by changes in the energetic demand for biologic ac-
tivities, it is expected that salinity variations would lead to changes
in oxygen consumption of shrimp, as demonstrated by Kutty et al.
(1971). It is also expected that changes in metabolic rates induced
by salinity can affect shrimp growth and production, as pointed out
by Dalla Via (1986).
In light of discussion earlier, the objective of this study is to
investigate the effects of salinity on survival, growth, and oxygen
consumption of F. paulensis postlarvae.
MATERIAL AND METHODS
General Rearing Conditions
This study was conducted at the Marine Aquaculture Station
"Prof Marcos A. Marchiori" of the Funda^'ao Universidade Fed-
eral do Rio Grande (Southern Brazil). Postlarvae (PL) oi Faifante-
penaeus paulensis were reared at 22-25°C, 30Sff salinity, and
natural photoperiod. In the initial stages of development, PL were
fed with newly-hatched Artemia nauplii, and afterwards with Ar-
temia nauplii and finely chopped meat of white clam (Mesodesnui
maelroides), tlsh (various fresh fish) and squid (llle.x sp). Water of
different salinities was obtained by mixing dechlorinated tap water
with natural seawater. Salinity was measured with an optical re-
fractometer ( I .O'/n precision. Atago Co., Tokyo, Japan).
Survival, Growth, and Oxygen Consumption
Fifteen-day-old PL were reared in the conditions described ear-
lier and were gradually acclimated from 30 to 2. 5, 10, and 20%o
555
556
TSUZUKI ET AL.
salinity over a 5-day period, by daily reductions of 6, 5, 4. and 2%c
salinity, respectively (Tsuzuki et al. 2000). Postlarvae maintained
at 30%o salinity were used as control. After the salinity acclimation
period, PL survival and growth in each salinity were examined
over 6 wk by stocking 80 PL in a 100-L plastic tank. Shrimp were
fed ad lihinim twice a day with a commercial diet containing 45%
crude protein (Tetra DoraMarin. Pfizer Co., USA). Every day. pH
and temperature were monitored, and organic residuals were si-
phoned out from the bottom of the tanks when at least 10% of the
water was renewed. Every two weeks, 20% of the animals in each
tank were counted and individually weighed to the nearest 0.1 mg
(wet weight). Cephalotorax length and dry weight (60°C for 48 h)
were measured to the nearest 0.01 mm and 0.1 mg. respectively, at
the beginning (» = 48) and at the end of the trial. At the end of the
experiment (week 6). all living shrimp were weighed (wet and dry
weights) and measured {n = 26-16.^). Cephalotorax length was
measured using a stereoscopic microscopy (Nikon. Japan).
At the end of the salinity acclimation period, and every 2 wk
during the growth trial, oxygen consumption was measured using
a Barcroft-Warburg respirometer (Oser 1965). Values were ex-
pressed as (xL of O, per mg of dry weight per hr.
Statistical Analysis
Each treatment was done in triplicate. However, no significant
difference was detected between replicates and results were then
pooled for further analysis. Differences between replicates and
treatments were analyzed by one-way analysis of variance
(ANOVA) followed by the Tukey's test. The significance level
adopted was 95% (P < 0.05).
RESULTS
Water temperature throughout the experiment was 24.9 ± 0.1 °C
(mean ± SB), while mean values of pH and salinity were 7.5, 7.6,
7.6, 7.8, and 7.9 at 2. 5. 10, 20. and 30%c salinity, respectively.
After the five-day acclimation period to different salinities,
mean weights (wet and dry weights) and cephalotorax length of
20-day-old PL did not change with the acclimation salinity (P >
0.05). Therefore, all values were pooled and only one mean was
calculated. Mean (± SE) wet and dry weight and cephalotorax
length was 9.2 ± 0.2 mg. 2.0 ± 0.1 mg, and 2.1 ± 0.0 mm. respec-
tively. Survival rates of these PL were higher than 95% and there
were also no significant difference between treatments (P < 0.05)
(data not shown). However, after two weeks of experiment, sig-
nificantly lower survival rates (28.1%) were observed at 2%o sa-
linity (results not shown). At this salinity, only 15.8% survival was
observed at the end of the growth period (Table 1 ).
Figure 1 shows the PL growth as wet weight at different sa-
linities throughout the experimental period. From the second to the
fourth week of experiment, a higher mean wet weight was ob-
served in PL reared at 10%p salinity, especially when compared
with those reared at 2%i, 59cc. and 20%^ salinity (P < 0.05). After
six weeks of experiment, wet weight of shrimp reared at 2%c
salinity was significantly lower than those reared at 10%r salinity
(P < 0.05). For salinities between 5%c and SO^ft, PL wet weight
was higher at 10%c salinity although this difference was not sta-
tistically significant (Fig.l, Table 1). At 2%< salinity, PL dry
weight tended to be lower at the end of the growth period, but no
significant changes were detected. Cephalotorax length was sig-
nificantly smaller in PL reared at 2%c salinity than in those reared
at 5%c. or lOVcc salinity (P < 0.05) (Table I ).
After the salinity acclimation period, oxygen consumption of
PL acclimated to 2%c salinity was lower than that observed in PL
acclimated to 10%fi or 30%c salinity. At 2%f' salinity, oxygen con-
sumption increased after the second week and reached a maximum
value at the fourth week of experiment. Afterwards, a marked drop
in oxygen consumption rate occurred. At the intermediate salinities
(from 5%c to 20%c), oxygen consumption was higher at 10%c sa-
linity until the second week of the experiment although not statis-
tically different (P > 0.05). At the end of the growth period, oxy-
gen consumption reached similar and low levels (around 4 p-L Oo
mg dry weight"' hr~') irrespective of the salinity level tested.
Oxygen consumption of shrimp at 30%c salinity was constant and
close to 5 |jiL O2 mg dry weight ' hr~' throughout the experiment
(Table 2, Fig. 1).
DISCUSSION
In this study, survival of Faifantepenaeus paiilensis postlarvae
(PL) was extremely low ( 15.8% ) after a 6-wk growth period at 2%c
salinity. A similar result was verified by Cawthome et al. (1983)
when only 34% of Pemieus monodon juveniles survived at that
salinity for two weeks. Although Tsuzuki et al. (2000) verified an
increase in salinity tolerance of F. paulensis postlarvae with aging
(from PL 15 to 30) when PL were directly transferred from 30%o
to 27co or 5%o salinity, the low survival rate observed at 2%p salinity
in this study indicates that 20-day-old PL were not able to cope
with low salinity levels for a long period of time (6 wk). Also, the
lower shrimp growth rates observed at 2%c salinity confirms the
physiologic disturbance induced by low salinity in F. paidensis
PL. Dalla Via (1986) suggested that reductions in shrimp growth
at low salinities can be related to a higher energetic expenditure to
keep the osmotic equilibrium at these saline conditions. The same
author showed that exposure to 10%o salinity for five months re-
sulted in reduction (up to 33%) of the ash-free organic content.
Therefore, in low salinity environments a significant reduction in
shrimp production might be expected. However, this hypothesis
can only be considered if one assumes that the food assimilation
TABLE 1.
Survival, wet and dry weights, and cephalotorax length of Farfantepenaeus paulensis postlarvae reared at different salinities for 6 weeks.
Salinity (%r)
Survival (%)
Wet Weight (mg)
Dry Weight (mg)
Cephalotorax Length (mm)
5
10
20
30
15.8 ±4.7 (a)
81.3 ±5.2 (b)
88.3 ± 0.6(b)
82.9 ± 9.0(b)
70.0 ± 14.3(b)
102.4:
135.2:
147.2:
140.3 :
140.9:
11.3(u)
5.1 (ab)
4.6 (b)
7.6 (ab)
10.0 (ab)
27.2 ±3.7 (a)
30.8 ± 1.2(a)
33.2 ±1.1 (a)
33.2 ± 1.9(a)
34.2 ± 2.6 (a)
4.8 ±0.4 (a)
5.7 ± 0.2(b)
5.7 ±0.1 (b)
5.1 ±0.2(ab)
5. 1 ± 0.2 (ab)
Data are means ± SE (;i = 26-163). Same letters indicate absence of significant difference between salinities (P > 0.05).
Growth and Ox-igen Consumption of Farfantepenaeus paulensis
557
160
150 -
140 -
130
120 -
110
100
3 90 -
E 80
I 70
S 60
50
40
30
20
10
Time (weeks)
Figure 1. Wet weight of Farfantepenaeus paulensis postlarvae reared
at different salinities. Data are means ± SE (;i = 26-1631. Different
letters indicate sij^niflcant differences between salinities at the same
time of cultivation {P < (1.(15). Salinities (%c): O = 2; D = 5; A = 10; V
= 20; 0 = 30.
rate is not dependent on salinity. Marques & Andreatta (1998)
found significant differences in dry matter consumption of F. pau-
lensis reared in low salinity levels while Wasielesky et al. (2002)
reported that food consumption in this species was not affected by
salinity. Therefore, further investigation is needed to clarify this
question.
It has been demonstrated for several penaeids that higher
growth rates usually are observed at salinities ranging from 57(i to
35%c, depending on the species and the ontogenetic phase consid-
ered. In Liiopenaeus vannamei PL. higher growth rates occurred at
20^r salinity when compared with those observed al 5'^r and 45"^?
salinity (Huang 1983). Bray et al. ( 1994) reported that juveniles of
the same species reared at 5%t and \57cc salinity achieved higher
increment in wet weight than those reared at 25%c, 35%f. and 49'ycc
salinity. Venkataramiah et al. (1974) verified that F. aztecus
growth was enhanced at '^.S'/n and 17"y?f salinity. Henning & Le-
mos ( 1994) verified that L. schmilli growth was similar at 5'/tf and
30%c salinity, but higher at 10%c salinity. In this study, a better
growth rate was observed at 10%f salinity, being significantly dif-
ferent from that observed at 2%c salinity throughout the experi-
ment. Venkataramiah et al. ( 1975) observed in estuaries where F.
aztecus is naturally found, that a higher abundance occurs in sa-
linities that are close to the optimum level estimated under labo-
ratory conditions. The same fact is observed for F. paulensis dis-
tribution in the Patos Lagoon estuary, where a higher abundance is
observed in areas with salinities below lO'^f salinity, although
shrimp can be found in salinities between 0%6 and 'iVic (DTncao
1991).
Before growth occurs, the metabolic demand for maintenance
and feeding activity must be satisfied. The knowledge of such
demands under different environmental conditions is necessary
(Brett 1970). Several studies have used the oxygen consumption
measurement to analyze metabolisin and activity in crustacean
decapods (Kutty et al. 1971, Venkataramiah et al. 1974, Gaudy &
Sloane 1981; Villarreal & Rivera 1993). Since the oxygen con-
sumption alters with changes in the energetic demand for biologic
activities, it would be expected that salinity variations could lead
to changes in oxygen consumption (Kutty et al. 1971).
At the beginning of the experiment, the oxygen consumption of
20-day-old PL reared at 27i< salinity was markedly lower com-
pared with PL reared at the other salinities tested. This fact prob-
ably indicates that PL could be in a metabolic depression stage that
would precede death. In fact, a high mortality rate (71.9%) was
observed in the first two weeks of the experiment. In Penaeus
semisukatus. Clark (1992) also observed a decrease in the respi-
ration rate after a salinity reduction from 409ft to \%9ii salinity. He
also noticed that shrimp were moribund and died 12 hours after
being exposed to the salinity shock. Chen & Fang (1986) consid-
ered that the respiratory depression observed in Metapenaeus ensis
after a salinity change was caused by a reduction of the w aler flow
through the gills to resist the salinity shock, leading to a reduction
of the oxygen consumption. In this study, a low oxygen consump-
tion was observed throughout the experiment at 2%c salinity, ex-
cept after four weeks when a non-significant increase in oxygen
consumption was observed. In this case, mortality rates did not
significantly change after two weeks of experiment. Therefore, the
increase in oxygen consumption observed after four weeks of ex-
periment at 2%c salinity could be attributed to a higher variability
of the oxygen consumption measurements due to a better perfor-
mance of surviving animals, which probably were more resistant to
lower salinity levels.
Until the second week of the experiment, higher oxygen con-
sumption rates were observed in shrimp maintained at \Wcc salin-
ity. Concomitantly, higher growth rates expressed as wet weight
and cephalotorax length were generally observed for shrimp kept
at this salinity. Yagi & Ceccaldi (1984) verified in Palaemon
seiratus larvae that the oxygen consumption was maximum in
salinities ranging from 25'^f to 30%f. which could be explained by
a higher physiologic activity related to larvae food utilization.
Moreover, between 25%c and 30%(: salinity the energetic demand
for osmoregulation seems to be lower and growth higher. It is
important to emphasize that an attempt to correlate energetic ex-
penditures for ionic and osmotic regulation with oxygen consump-
tion rates is speculative, once the subject is still controversial.
Some investigators point out that the energy expended to osmo-
regulation can be evaluated by oxygen consumption measurements
in aquatic invertebrates (Lofts 1956. Rao 1968). In this case, oxy-
gen consumption can be expected to increase for osmoregulators
TABLE 2.
Oxygen consumption rates (jtL {), mg dry weight' hr ') of
Farfantepenaeus paulensis postlarvae reared at different salinities
for 6 weeks.
Salinity
{■7<,)
Time of Cultivation (Weeks)
-)
3.7 ± 0.6 (a)
.'i.3±().y(ah)
5
7.9 ± 2.3 (ab)
8.2 ±1.4 (a)
10
1 3.0 ± 2.8(b)
12.9 ± 3.3 (ab)
2(J
9.1 ±2.6(ab)
8.1 ± 1.3 (ab)
30
6.3 ± 0.4(b)
4.3 ± 0.5(b)
12.3±3..S(a) .^.0±0.8(a)
3.9 ± 0.7(b) 3.6 ±0.5 (a)
5.6±0.9(ab) 4.3 ±0.5 (a)
4.6 ± 0.5 (ab) 4.3 ± 0.5 (a)
4.4±0.y(ab) 4.1 ±0.7 (a)
Data are means ± SE (n = 3-6). Same letters indicate absence of signifi-
cant difference between salinities (P > 0.05).
558
TSUZUKI ET AL.
when the osmotic difference between the hemolymph and the en-
vironment increases, resulting in an increase in the metabohc de-
mand to keep constant the hemolymphatic concentration. Never-
theless, changes in metabolic rates related to salinity are, in most
cases, too big to be attributed only to the energetic cost with ionic
and osmotic regulation. In this case, it would be difficult to relate
oxygen consumption rates exclusively to energetic requirements
for osmoregulation (Potts & Parry 1964). Therefore, not only the
anisosmotic regulation of extracellular tluids. that seems less likely
the general cause of metabolic changes of the organism (Duncan
1966, Kinne 1971 ) should be taken into consideration, but also the
isosmotic regulation of the intracellular fluids involving the mo-
bilization of organic substances and changes in the energetic needs
for ionoregulation (Wheatly 1988). Additionally, the interference
of the locomotion activity should be considered (Beamish &
Mookherji 1964).
The comparatively low and stable oxygen consumption rates of
shrimp reared at 30^c salinity, and the low oxygen consumption at
the end of the experiment irrespective of salinity levels, indicate
more economical respiration rates at salinities where animals are
genetically adapted or acclimated for a longer period (Kinne
1971).
ACKNOWLEDGMENTS
The authors thank Alvaro Montenegro Neto for his technical
assistance. This study was supported by the Brazilian CNPq. R.
Cavalli and A. Bianchini are research fellows of this agency (Proc.
n^ 300131/01-1 and 300536/90-9, respectively).
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Journal of Shellfish Rcmirch. Veil. 22, No. 2, 561-56S, 2003.
ANATOMICAL DAMAGE TO HUMPBACK SHRIMP, PANDALVS HYPSINOTUS (BRANDT 1851)
CAUGHT BY TRAWLING AND TRAPPING
P. M. TROFFE, S. ONG, C. D. LEVINGS,* AND T. F. SUTHERLAND
Di'partnu'iir of Fisheries and Oceans, Wesr Vancuuver Laboratory 4160 Marine Drive, West Vancouver,
V7V-1N6. Canada
ABSTRACT We compared the anatomical damage, individual size, total catch, and bycatch when humpback shrimp. Pandaliis
hypsiiuniis (Brandt 1851). were harvested using otter trawls, beam trawls, and traps in Simoom Sound. British Columbia. Regional
body damage (RBD) and total body damage (TBD) to humpback shrimp were assessed for four major regions of the shrimp body
(rostrum, carapace, abdomen, and tailfan). TBD was higher for otter and beam trawling compared with traps, with a significant
difference observed between the otter trawling and half-day trapping. After standardizing trawl data by fishing effort (area swept and
fishing time). TBD was significantly higher for beam trawl. RBD was significantly different across fishing methods and there were also
significant differences among the various body parts. Trawl caught humpback shrimp showed the highest ratio of damaged/total
individuals relative to those caught by traps. In general, the carapace and rostrum body regions were more damaged relative to the
abdomen and tail fan. The survival of humpback shrimp released after trawling or trapping will depend on the extent of the body
region-specific anatomical damage that has occurred and its functional importance.
KEY WORDS: damage, otter trawling, beam trawling, trapping, shnmp, fishing gear, bycatch, andaliis hypsinonis
INTRODUCTION
Studies exploring the use of selective fishing gear are ongoing
and past studies have focused on the size and shape of net meshes
as well as the use of extruders in trawl nets to separate the target
and bycatch species (e.g., DeAheris & Reifsteck 1993, Suuronen
et al. 1996. Richard 1999). Most studies comparing fishing gear
bycatch have focused on the volume of bycatch and only a few
more recent studies have focused on damage to the catch and
subsequent survivability of organisms, (e.g., Mensink et al. 2000,
Stevens et al. 2000. Bergmann & Moore 2001 ). This study, how-
ever, turns a lens to the damage to humpback shrimp. Pandahts
hypsinotus, harvested in an inshore ecosystem in Pacific Canada
with three different fishing gear types: beam trawl, otter trawl, and
traps. Humpback shrimp are caught in directed trap and trawl
fisheries in British Colunibiu and are also commonly found as
incidental catch in shrimp trawl {Pandahts spp.) and spot prawn
{Pandalus platyceros) trap fisheries (Boutillier & Nguyen 1999).
No information has been published about fishing gear-related ana-
tomic damage caused by these harvesting methods in Pacific
Canada. In this study we focused on three objectives: 1 ) the rela-
tive total damage to humpback shrimp among fishing methods
(total body damage [TBD]); 2) susceptibility to gear-related dam-
age among major anatomic regions of shrimp (regional body dam-
age |RBD|); and 3) comparison of catches of target and nontarget
species among gear types.
This study was part of a larger project designed to determine
whether trawling or trapping would be a preferable method of
harvesting humpback shrimp, as a representative crustacean spe-
cies, in an ecosystem-based management system (e.g.. Jamieson &
O'Boyle 2001 ). One of the aspects of such a management system
would be to avoid "bykill" or unwanted fishing mortality of un-
dersized shrimp or nontarget shrimp species by minimizing the
practice of discarding bycatch if there were high levels of collat-
eral damage during harvest. Previous studies have showed that
shrimp trawling can result in damage to benthic habitats (e.g..
♦Corresponding author. Tel: 604-666-7915: fax: 604-666-3497: E-mail:
levingsc@pac.dfo-mpo.gc.ca
Hansson et al. 2000). However, data to compare damage by trawl-
ing relative to other gear types are not available, and the interaction
between, gear type, bycatch. and collateral damage have not been
presented to date.
MATERIALS AND METHODS
Experimental Trawling and Trapping
Simoom Sound, an inlet off Fife Sound on the central coast of
British Columbia was chosen as the study location (Fig. 1 ). Bottom
salinity and temperature ranged between 31.5 to 33.5 psu and 7.5
to 8.7^C, respectively, using a Sea-Bird CTD (model SBE-
911 plus) deployed in October 2001. The surface sediment in Si-
moom Sound consists of approximately 90% silt and 21% organic
content. Beam trawling, otter trawling, and trapping were used to
catch humpback shrimp in Simoom Sound during November 2000
(otter trawl, trap), and February 2001 (beam trawl). Each gear type
was deployed in a separate "block" of the seafloor (approximately
700 m by 400 m) characterized by relatively uniform depth and
sediment type. The gear used was representative of that used in the
commercial fishery and complete details on methods, vessels, and
gear dimensions are given eLsewhere (Ong et al. 2002). The shrimp
trawl industry in British Columbia has voluntarily adopted a 100%
implementation of bycatch reduction devises (BRDs) in their nets
since 2000 and all trawl nets used in this study were fixed with
rigid type bycatch reduction grids (Department of Fisheries and
Oceans 2002).
Otter Trawling
Six otter trawls were conducted on three transects on Novem-
ber 14. 2001 (Fig. 1). with two trawls performed on each of the
transects. The water depths ranged between 55 and 60 m.
Transects lengths were between 643 and 677 m and each trawl was
10-13 min in duration, not including the time required for net
haul-back. The otter trawl net measured 36.8 m long with a head-
rope and footrope (without a tickler chain) of 23.8 m and 30.5 m.
Codend mesh size was 38 mm. Catches were sorted and counted
by species and weighed to the nearest 0.1 kg. Humpback shrimp
specimens used for the otter trawl damage assessment (/; = 106)
561
562
Troffe et al.
v-\
, British >
^Columbia>
Figure 1. Map of Simoom Sound. BC, witli approximate locations of
beam trawl, other trawl, and traplines used in this study.
were collected from the catch after the codend contents had been
placed onto a sorting table. Samples were frozen in labeled freezer
bags for later analysis in the laboratory. After collection, care was
taken to keep specimens flat to minimize damage because of han-
dling.
Beam Trawling
Beam trawls were completed on three transects, west of the
otter trawl lines on February 22, 2001 (Fig. 1), with two trawls
conducted on each of the three trawl lines. The trawl duration,
length, and depths ranged among 15-17 min, 313 and 660 m, and
46 and 55 m. respectively. The beam trawl net measured 26.6 m in
total length, with a headrope and footrope length of 14.0 m and
16.5 m. Codend mesh size was 44 mm. Beam trawl catches were
sorted by species, then counted and weighed to the nearest 0.1 kg.
Humpback shrimp specimens used in the beam trawl damage as-
sessment {n = 132) were collected and frozen using the same
techniques described for otter trawling.
Trapping
Traps were set out twice, east of the otter trawl lines, during
November 15-16, 2000, on three transects (62-75 m deep; Fig. 1 ).
Two time periods were used. The first set of traps remained sub-
merged for approximately 6 h (half-day traps), during the day, and
the second set of traps was submerged for 17 h over night (over-
night traps). Approximately 40 traps were set on each trap line
with spacing of about 1 5 m between each trap. The three transects
measured from 558 to 660 m long. Most sets included traps outside
the defined block of sea tloor because the groundline used was
longer than the predetlned transect length of 500 m. Traps were
baited with salmon fish feed pellets, cut-up Pacific herring. Chipea
harengus pallasi. and shiner perch. Cymatogaster aggregaui. col-
lected on site as bycatch from the trawling experiments. The traps
were conical and measured 76.2 x 30.5 x 71.1 cm. with a stretch
mesh size averaging 45 mm. Each trap weighed approximately 1.4
kg. On one overnight set. humpback shrimp were only collected
from traps that fished the predetermined line. All the other hump-
back shrimp were from at least 10 traps within the predetennined
line and from a few of the traps extending outside of it. The traps
were emptied into a plastic tote and the catches from each trap
were then identified to species and counted. Catches of all shrimp
species were weighed to the nearest 0. 1 kg. A subsample of the
humpback shrimp catch was frozen using the same techniques
described for otter and beam trawling. 139 humpback shrimp were
collected from the half-day traps and 145 from the overnight traps.
Sample Processing
Humpback shrimp were thawed in the laboratory for 1-2 h.
Weight, length, and sex were recorded for each individual. Lengths
were recorded to the nearest millimeter using manual or electronic
Vernier calipers. Carapace length was measured from the poste-
rior-most part of the orbit to the posterior middorsal margin, and
total length, from the tip of the rostrum to the tip of the telson. As
several humpback shrimp from the beam trawl were in a transi-
tional stage (from male to female phases), their total lengths were
calculated based on the relationship between male carapace length
and total length. Sexing was accomplished by noting the presence
of eggs in the head or abdomen, and by examination of the endo-
pods of the second pleopods (Butler 1980).
Damage Assessment
A table was constructed to delineate the principal body parts in
the four major regions (rostrum, carapace, abdomen and tailfan) of
the shrimp body, as shown in Butler ( 1980). The body parts chosen
for analysis were those that would be required for the survival of
a humpback shrimp if it were to be released. Each body part within
each of the four major body regions was given a score from 0 to
1.0. with zero being a missing body part, and 1.0 representing a
fully intact body part, with intermediate scores representing vary-
ing levels of damage, that is. the rostrum is composed of nine body
parts and a summed score of 9.0 reflects zero percent damage
whereas a score of 6.0 represents. (1 - [6.0/9.0] 1 00). or 33.3%
damage. Table 1 describes the codes and damage scores used for
each of the body regions and Table 2 depicts the particular body
parts and their accompanying functions. Humpback shrimp from
each of the fishing methods were assessed for damage using this
scheme (Table 1) resulting in data on RBD and TBD. TBD was
assessed by summing the damage scores from all body regions and
dividing the score by the total number of body parts assessed from
each gear transect and expressing the resultant as a percentage.
Because of time constraints, some of the trap-caught humpback
shrimp were assessed using a low-resolution scheme wherein dam-
age to the abdomen was not assessed (Ong et al. 2002). Only data
from the high-resolution scheme are presented herein.
Statistical Analysis
Statistical analysis was performed on the humpback shrimp
catch data using Systat'"' v. 10 statistical software. A Bartlett's test
for homogeneity of variances was performed on the data set prior
to statistical analysis and for parametric analyses, proportional data
was arcsine transformed (Zar 1984). A single factor analysis of
variance (ANOVA) and Tukey HSD multiple comparison tests
were used to test for significant differences in RBD and TBD. In
cases where were parametric assumptions were not met. a single
factor Kruskal-Wallis ANOVA by ranks was performed. Untrans-
p. HYPSiNOTUs Damage by Trawling and Trapping
563
Region
Code
TABLE 1.
List oF humpback slirinip body regions assessed for damage.
Body Part
Damage Score
Rostrum
Rl
Rostrum
0 =
R2
Eye stalk and cornea (R)
0 =
R3
Eye stalk and cornea (L)
0 =
R4
Antennae 1 (L)
0 =
R5
Antennae 1 (R)
0 =
R6
Antennae 2 (L)
0 =
R7
Antennae 2 (R)
0 =
R8
Antennal scale (L)
0 =
R9
Antennal scale (R)
0 =
Carapace
CI
Third ma.xilliped (L)
0 =
C2
Third maxilliped (R)
0 =
C3
Pereiopod I (L)
The
C4
Pereiopod I (R)
0 =
C5
Pereiopod II (L)
0.2
C6
Pereiopod II (R)
0.5
C7
Pereiopod III (L)
0.7
C8
Pereiopod III IR)
0.9
C9
Pereiopod IV (L)
0.9
CIO
Pereiopod IV (R)
0.9
CU
Pereiopod V (L)
0.9
C12
Pereiopod V (R)
0.9
C13
Carapace itself
0.5
Abdomen
Al
Somites I-VI
0.5
A2
Pleurons I-V
0.5
A3
Pleopods I(L and R)
0.5
A4
Pleopods II (L and R)
0.5
A5
Pleopods III (L and R)
0.5
A6
Pleopods IV (L and R)
0.5
A7
Pleopods V (L and R)
0.5
Tail Fan
Tl
Tel son
0 =
T2
Uropods (L)
0 =
T3
Uropods (R)
0 =
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage. I
completely broken off. 0.5 = some damage. I
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage, I
completely broken off, 0.5 = some damage. I
completely broken off, 0.5 = some damage. I
completely broken off, 0.5 = some damage, 1
following scores apply to all pereiopods:
broken off below (bob) coxa O.I = bob basis
= bob ischium 0.3 = bob merus, 0.4 = bob c;
= bob propodus, 0.6 = damaged chela
= broken off chela, 0.8 = broken off exopod
= broken off epipod, 1 .0 = intact
= broken off epipod, 1 .0 = intact
= broken off epipod, 1 .0 = intact
= broken off epipod, 1 .0 = intact
= broken off epipod, 1 .0 = intact
= some damage, 1 .0 = intact
= some damage, 1 .0 = intact
= some damage, 1 .0 = intact
= some damage, I.O = intact
= some damage, 1 .0 = intact
= some damage, 1 .0 = intact
= some damage, 1 .0 = intact
= some damage, 1.0 = intact
completely broken off, 0.5 = some damage. I
both broken off. 0.5 = some damage. 1 .0 = i
both broken off. 0.5 = some damage, 1 .0
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.0
=
intact
.irpus
.0 =
ntact
ntacl
Scores for each body region are assessed based on the total score assigned to each body part in the region (e.g.. Rostrum has nine body parts, minmium
score = 0, maximum score = 9).
formed data were used because arcsine transformation did not
change the ranks of the parameters. A parametric ANOVA was
al.so used to compare catch weights of humpback and pink shrimp,
Pandalus eous (P. borealis), among hairest methods. Four major
hypotheses were tested: 1 ) gear-related damage to humpback shrimp
was not equal among fishing methods, 2) proportional damage to the
four major shrimp body regions differed with fishing methods, 3)
total numbers and biomass of humpback shrimp in the catches
TABLE 2.
List of humpback slirimp body parts according to function.
Region
Bodv Part
Function
Rostrum Rostrum
Antennae I (antennules)
Antennae 2
Antennal scales
Carapace 3rd Maxilliped
Pereiopod I
Pereiopod II
Pereiopods III to V
Abdomen Abdomen (somites and pleurons)
Pleopods
Tail Fan Tel son
Uropods
The head spine that helps deter small predators
Detect waterborne smells
For touch and to detect approaching predators
Provide stability while swimming
Holds food while pieces are pulled off with claws; used when sparring uith other shrimps
If chelate, it is used to catch small prey
Chelate leg with articulated carpus for grooming and retrieving scraps of food
Walking legs; pereiopod V may have brushes used for grooming and cleaning eggs
With tail fan, the strong muscles are used for fast backward swimming (in escape response)
For forward swimming, and to brood eggs
Bears the anus; involved in backward swimming
Involved in backward swimmins
564
Troffe et al.
differed among fishing methods, and 4) individual weight of
humpback shrimp was different among fishing methods tested.
RESULTS
Raw data on damage scores, lengths, and weights for all indi-
vidual humpback shrimp together with catch data from each fish-
ing gear are presented elsewhere (Ong et al. 2002). Specific analy-
ses are summarized below.
TBD to Humpback Shrimp Among Fishing Methods
TBD tended to be higher in trawls than traps and the otter trawl
caught humpback shrimp were significantly more damaged than
those from half-day traps (9.9 ± 5.0% vs. 2.0 ±1.1%: P = 0.023).
Other comparisons were not statistically significant (P > 0.0: Table
3). A comparison of standardized TBD data between otter and
beam trawl methods resulted in a significant difference (5.7 ±
4.1% vs. 23.6 ± 8.6%; P < 0.001). TBD to trap-caught humpback
shrimp was standardized by soak time. There was no significant
difference (P > 0.05) in total percent damage per hour between
traps set out for 6 h in daytime compared with 17 h overnight
(Table 3). There were also marked differences in the proportion of
individual humpback shrimp that received any damage to the 32
body parts observed. Trawl caught humpback shrimp received the
highest ratio of damaged/total individuals (otter 89.9%; beam
78.8%; overnight traps 45.0%; half-day trap 37.4%).
RBD to Humpback Shrimp Among Fishing Methods
Considering all three fishing methods, the carapace was the
most damaged (Table 3) and showed the greatest variability of the
four body parts, however, there were differences between gear
types, as explained below carapace damage was represented by
disfigurement, depression, partial tear-off and detachment from the
thorax. There was very weak negative correlation (r^ = 0.049)
between the carapace length of humpback shrimp and percent
carapace damage across all tlshing methods.
There were significant differences (P = 0.047) in carapace
damage between fishing methods with otter trawls (16.4% ± 10.0)
and beam trawls (10.3% ± 0.4) resulting in higher proportions of
carapace damage than humpback shrimp harvested by overnight
trap (4.5% ± 1.8) or half-day traps (2.9% ± 1.9). However, there
were no pair-wise significant differences (f > 0.05) assessed with
Tukey tests (Table 3).
Damage to the rostrum differed among fishing gear {P =
0.003; Table 3). Damage to the rostrum of humpback shrimp har-
vested by otter trawl was significantly greater (12.0% ± 2.6) com-
pared with both overnight traps (5.6% ± 2.4; P = 0.02) and half-
day traps (2.6% ± 1.2; f = 0.002). The rank of the proportional
damage to the rostrum of humpback shrimp was the same as re-
ported for the carapace, with otter trawl (12.0% ± 2.6) incurring
the most damage followed by beam trawls (7.2% ± 0.9), overnight
traps (5.6% ± 2.4). and half-day traps (2.6% ± 1.2), respectively
(Table 3).
Regardless of fishing method, the abdomen and tailfan of
humpback shrimp were less damaged than the carapace and ros-
trum (Table 3). There was a significant difference {P = 0.028) in
the damage to the abdomen between gear types, with the beam
trawl causing at least five times more damage than any other
fishing method, and significantly more than the overnight traps
(Tukey test, 0.01 < P < 0.025; Table 3). There were also significant
differences in the amount of damage to the tailfan among fishing
methods (P = 0.034). Tailfan damage from the otter trawl catch
was the highest (3.0% + 1.9), with the beam trawl (1.8% ± 0.9),
half-day traps (0.2% ± 0.2). and overnight traps (0.2% ± 0.2)
following, respectively (Table 3). As with carapace data, there
were no pair- wise significant differences (P > 0.05) when assessed
with Tukey tests (Table 3).
RBD to Humpback Shrimp by Each Fishing Method
Damage to specific humpback shrimp body parts differed
within otter trawl activity {P = 0.002; Table 4). The carapace
received the highest damage scores and was significant-
ly more damaged than the abdomen (Tukey test, P = 0.023)
(Table 4).
The damage assessments for humpback shrimp caught by beam
trawl were similar to those revealed in the otter trawl catch (Table
4). There were significant differences (P < 0.001) in the damage to
various shrimp body parts (Table 4). The carapace of the beam
trawl caught humpback shrimp had significantly more damage
TABLE 3.
RBD and TBD data
TUKEY
Otter
Beam
Traps
Traps
ANOVA
Gear Type,
Body Region
Trawl
Trawl
Overnight
Half-Day
Gear Type
P Values
RBD: Carapace
16.4 ±10
10.3 ±0.4
4..5± 1.8
2.9 ±1.9
H =
7.95. P = 0.047*
NS
RBD: Rostrum
12.0 ±2.6
7.2 ±0.9
5.6 ± 2.4
2.6+1.2
F =
11.14. P = 0.003*
O vs HT. 0.002
0 vs OT, 0.02
RBD: Abdomen
0.2 ± 0.2
1.7± 1.7
0.0 ± 0.0
0.3 ± 0.0
H =
9.13, P = 0.028*
B vs OT, 0.01 < P < 0.025
RBD: Tailfan
3.00 ±1.9
1.8 ±0.9
0.2 ± 0.2
0.2 ±0.2
H =
8.76. P = 0.034*
NS
TBD
9.9 ±5.0
7.4 ±0.7
3.4 ± 1.4
2.0± 1.1
F =
5.58, P = 0.023*
O vs HT, 0.027
TBD standardized hy total catch
(kg) by area swept (km") per hour
.'i.7±4.l
23.6 ± 8.6
ND
ND
F =
21.2, P = 0.001*
—
TBD standardized by trap gear soak
time (hr)
ND
ND
0.2 ± 0.08
0.3 ± 0.2
NS
—
Mean percent ± SD; » = 3 for humpback shrimp compared by gear types with ANOVA and Tukey P values; Kruskal-Wallis test applied to nonparameCric
comparisons, a = 0.05.
* Statistically significant: only statistically significant comparisons given for Tukey tests.
O, otter trawl; B, beam trawl; HT, half-day traps, OT, overnight traps.
p. HYPSiNOTus Damage by Trawling and Trapping
565
TABLE 4.
RBD (mean percent + SD) (h = 3) for humpback shrimp caught by
different gear types compared with \NO\ A and Tukey /' values.
anova
Tl'KEY
Gear Type
Bod) Region
Body Region, P \ alues
Otter trawl
H
= 14.46. P = 0.002*
C vs A. 0.02.^
Beam trawl
F
= 40.08. P< 0.001*
C vs R. 0.03 1
C vs A. <0.001
C vs T. <0.001
R vs T. <0.001
R vs A. <0.001
Overnight traps
H
= 9.24. P = 0.026*
R vs A, 0.05
Half-day traps
H
= 8.69. P = 0.0.34*
NS
Kruskal-Wallis test applied lo non-parametric comparisons, a = 0.05.
* Statistically significant; only statistically significant comparisons are
shown for Tukey tests.
C. carapace; R. rostrum; A. abdomen; T. tail fan.
than the rostrum, abdomen, and tailfan (P = 0.0.31; P < 0.001;
P < 0.001. respectively) and the rostrum had significantly more
damage than the abdomen and tailfan (P < 0.001. P < 0.001.
respectively. Table 4).
Trapping caused less damage to humpback shrimp than trawl-
ing; however, there were still significant differences among shrimp
body parts (Table 4). A Kruskal-Wallis ANOVA with data from
the half-day traps suggested there was a significant difference
(P = 0.034) in the amount of damage assessed between body
parts, but a ranked Tukey HSD test failed to reveal any significant
differences {P > 0.05: Table 4). Damage to various humpback
shrimp body parts from overnight traps, like the half-day traps.
were significantly different (P = 0.026; Table 4). The greatest
amount of damage in overnight traps was to the rostrum followed
by the carapace tailfan and abdomen. The rostrum and abdomen
were significantly different ^P = 0.05) when tested with a ranked
Tukey HSD test (Table 4).
Assessmenis of Humpback Shrimp Catch and Bycalch
On average humpack shrimp catches were significantly higher
(P < 0.001; numbers, biomass) on the trap lines relative to the
beam and otter trawl transects (Table 5). Highest catches were on
the overnight traplines (582, 7.9 kg). The average weight of indi-
vidual humpback shrimp was higher for trap-caught animals. (P <
0.0001). Overnight traps collected the largest humpback shrimp
(13.7 ± 0.5 g) followed by half-day traps (13.0 ± 0.3 g). otter trawls
(8.3 ±0.5 g), and beam trawls (7.1 ± 1.5 g). respectively (Table 5).
There were significant differences in the individual weights of
humpback shrimp caught by both otter and beam trawl when com-
pared with both half-day (P < 0.001) and overnight traps (P <
0.001; Table 5).
In addition to humpback shrimp, several other fish and inver-
tebrate species were caught. Beam trawl and otter trawl bycatch
was dominated by demersal fish, roundfish, and other shrimp spe-
cies. Bycatch from the traps included decapod crustaceans, echi-
noderms, roundfish and smaller shrimp species (Table 6). The
average bycatch of finfish was 4 ± 3 per trapline. 5 1 ± 1 8 per beam
trawl, and 376 ± 218 per otter trawl. Pink shrimp were present in
the catch of all harvest methods. However, the abundance of this
species was significantly higher in the otter trawl catches (P <
0.0001; Table 6).
DISCUSSION
Effects on Sunival
The type and extent of damage to individual shrimp will likely
affect survivorship if humpback shrimp are released following
their capture, as found for other Crustacea. Stevens (1990) inves-
tigated the survival of trawl-caught king crab (Paralithodes
camtschaticus) and tanner crdh {Chionocceres bainii and C. opilio)
in the Bering Sea. After injuries to both body and legs of the crabs,
survival rates in experimental tanks were about 507^ and 75%,
respectively, for the two crabs. Lancaster and Frid (2002) docu-
mented survival of undersize brown shrimp, Crangon crangon.
from an UK beam trawl fishery. They reported low mortality and
only the occasional loss of a telson or antennae after the catch was
brought aboard and sorted by mechanical riddle. However. Berg-
mann and Moore (2001 ) suggested that post-trawling mortality of
discarded decapod crustaceans have been underestimated, and
showed that damaged decapods had a significantly lower longer-
term survival {30% ) than controls (72-83%). Mensink et al. (2000)
showed that only 40% of common whelks, Bucciniim iiiniatiiiu.
TABLE 5.
Mean biomass and counts (±SD) per transect for humpback shrimp catches arranged by gear types (n = 3).
Gear Type
Otter
Trawl
Beam
Trawl
Traps
Overnight
Traps
Half-Day
ANOVA
Gear Type
Mean catch weight (kg) 0.3 + 0.2 3.2+1.6 7.9 ±1.5 6.6 ±1.4
Mean catch count (no.) 41 ±22 458 ± 226 582 ±131 508 ± 99
Mean individual shrimp weight (g) 8.3 + 0.5 7.1 + 1.5 13.7±0.5 13.0±0.3
20.4. />< 0.001*
8.9, P = 0.006*
F = 44.0. P< 0.0001^
TUKEY
Gear Type, P Values
B vsOT. 0.01
O vs HT. 0.002
O vs OT. <0.0001
B vs O, 0.027
O vs HT. 0.015
O vs OT, 0.007
B vs HT, <0.001
B vs OT, <0.001
O vs HT, 0.001
O vs OT. <0.0001
Biomass and counts of various harvests compared separately among gear types with ANOVA and Tukey tests, a = 0.05.
* Statistically significant; only statistically significant comparisons are shown for Tukey tests.
O. otter trawl; B. beam trawl; OT. overnight traps; HT. half-day traps.
566
Troffe et al.
TABLE 6.
Mean number of animals harvested from beam trawl, otter trawl, individual trap and trapline catches in Simoom Sound in = 3).
Species
Beam Trawl
Otter Trawl
Individual
Trap HD
Individual
Trap ON
Trapline HD
Trapline ON
Common Name
Scientific Name
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Mean ± SD
Shrimp
Humpback
Pandalus hypsinotus
441 + 339
41 ±23
13 ±2
15 ±3
508 ± 99.2
582 ± 151
Prawn
Puiukdus platyceros
—
—
0.08 ±0.1
0.4 ± 0.3
3 ±4
17 ± 13
Spiny pink
Pciiulalus eous (P. borealis)
42 ± 63
6123+ 1059
0.2 ±0.1
0.2 ±0.1
8.3 + 7
10 ±6
Two-spined crangon
Crangon communis
31 ± 10
1 ±0
—
—
Short-scaled eualid
Eiiahis suckleyi
0.2 ± 0.3
—
0.2 ±0.2
7±3
8±8
267 ± 140
Flatfish
English sole
Pleuroneaes vetulus
0.3 ± 0.3
—
—
—
—
—
Flathead sole
Hippoglossoides elnssodon
26 ± 12
0.7 ± 0.6
—
—
—
—
Rock sole
Pleuronectes hdineatus
0.5 ± 0.5
—
—
—
—
—
Arrowtooth flounder
Atheresdies stoniias
0.2 ± 0.3
—
—
—
—
—
Selachii
Spiny dogfish
Squalus acanthias
0.2 ± 0.3
—
—
—
—
—
Spotted ratfish
Hydrolagus colliei
9±3
—
—
—
—
—
Longnose skate
Raja rhina
0.2 ± 0.3
—
—
—
—
—
Roundflsh
Pacific tomcod
Microgadus proxinnis
—
0.7 ±0.6
—
—
—
—
Pacific herring
Cliipea harengus pallasi
—
229 ± 29
—
—
—
—
Blackbelly eelpout
Lxcodopsis pacifica
0.8 ±0.3
—
—
—
—
—
Lingcod
Ophiodon elongarus
0.2 ± 0.3
—
—
—
—
—
Black cod
Anoplopoma fimbriii
—
—
0.03 + 0.01
0.008 ±0.01
1.0 ±0.0
0.3 ± 0.6
Walleye Pollock
Theragra chalcogramma
1.2± 1.6
1.2 ± 1
—
—
—
—
Sandlance
Ammodytes hexaplerus
—
1.5 ±2.6
—
—
—
—
Dwarf wrymouth
Lyconectes ateutensis
—
—
0.008 ±0.01
0.05 ± 0.02
0.3 ± 0.6
2.0 ± 1.0
Shiner perch
Cymatogaster aggregata
14 ± 13
144.5 ±96
0.05 ± 0.03
0.008 ±0.01
2.3 ± 1.5
0.3 ± 0.6
Showy snailtlsh
Liparis pulchellus
0.2 ± 0.3
—
—
—
—
—
Staghom sculpin
Leptocottus annatus
—
—
0.008 ± 0.01
0.008 + 0.01
0.3 + 0.6
0.3 + 0.6
Prickleback
Stichaeidae (Family)
—
0.2 ± 0.3
—
—
—
—
Capelin
Mallolus villosiis
0.2 ±0.3
—
—
—
—
—
Invertebrates
Clams
Bivalvia (class)
0.5 ± 0.5
—
—
—
—
—
Smbby squid
Rossia pacifica
0.2 + 0.3
—
—
—
—
—
Squid
Lidigo opalescens
—
0.3 ± 0.6
—
—
—
—
Flatworm
Turbellaria (class)
—
0.3 ± 0.6
—
—
—
—
Dungeness crab
Cancer niagister
—
—
0.02 ± 0.03
—
0.7 ±1.2
—
Decorator crab
Majidae (family)
—
—
0.008 ±0.01
—
0.36 ± 0.6
—
Spider crab
Majidae (family)
—
—
0.008 + 0.01
—
0.3 ±0.6
Tanner crab
Chionoecetes bairdi
—
—
0.008 ±0.01
0.008 ±0.01
0.3 ± 0.6
0.3 + 0.6
Sunflower star
Pycnopodia helianlhoides
—
—
0.03 ±0.1
0.05 ± 0
1.3 + 0.6
2.0 + 0.0
Trapline data calculated for 40 traps. ON. overnight traplines sets of approx. 17 h; HD. half-day traplines sets of approx. 6 h; — indicates no catch.
harvested by a beam trawl survived after a 6-week experimental
period. Over 95% of whelks harvested in baited traps survived,
suggesting that trapping was relatively benign compared with
trawling. Other models assessing collateral mortalities to benthic
megafauna have suggested that 5-39% of annual mortalities in
fisheries on the Dutch continental shelf can be attributed to trawl
discards, with half of the species observed showing values of
greater than 20% annual mortality (Bergmann and van Santbrink
2000). Future studies should calibrate damage assessment with
survivorship to determine the level of resolution required to create
meaningful damage indices.
Possible Causes of Damage
It is likely that the relatively severe damage to humpback
shrimp from otter trawling was caused by a combination of factors.
Otter trawl gear was towed at higher speeds for a relatively long
distance (643-677 in), had a larger net opening, a finer cod-end
mesh and caught more non-target species than trapping or beam
trawling. The otter trawl yielded the smallest catch of humpback
shrimp of the three gears, but exhibited the largest bycatch of
finfish and pink shrimp, which likely resulted in higher internal net
pressures, more scouring by other spiny crustaceans, and ulti-
mately more damage to the humpback shrimp catch. However,
when total damage to humpback shrimp caught by trawl gear was
standardized by total catch weight (kg) by area (km") fished per
time (h) TBD to humpback shrimp caught by beam trawl was
higher compared with otter trawl caught animals. It is important to
standardize damage data in a format that will allow for cross-
comparisons to take place between investigations. We have chosen
to standardize the data according to catch, fished area, and fishing
p. HYPsiNOTUs Damage by Trawling and Trapping
567
duration to incorporate effects imposed by factors such as catch
size, duration of fishing activity, and boat speed.
BRDs reduction devices were shown to reduce head, carapace,
and tail damage to trawl caught penaeid prawns (Salini et al.
2000). These authors also mentioned that prawn damage was
higher when large animals were present in the trawls, possibly
because of their trashing effect in the codend. In our study, it is
possible that the trawls we used might have intlicled more damage
to humpback shrimp if BRDs had not been used. Further data are
needed to confirm this.
Haulup water pressure and bycatch may have also caused dam-
age to humpback shrimps in traps. However, as the traps were
hauled through the water over a short distance (62-75 m, verti-
cally) at a relatively slow rate (about 15 m/minute) and caught
fewer bycatch species these factors were lessened relative to trawl-
ing. The rostrum and carapace of humpback shrimp from the over-
night traps were more damaged than half-day traps possibly be-
cause the 17 h soak allowed for more time for the humpback
shrimp to collide with each other in efforts to escape. This may
have been particularly important at night when shrimp are more
active and may have even been attempting to swim off the bottom.
Catches were also higher in the overnight sets, leading to more
crowding in the traps. However, the differences in damage to the
rostrum and carapace for half day and overnight traps were not
evident when standardized by soak time.
Additional Damage to Humpback Shrimp from Fishing Gear
In addition to the fragmenting and crushing injuries we ob-
served on the exoskeleton of humpback shrimp, other important
but subtle anatomic damage likely occurred during or after capture
by both traw I and trap. Examples would be the loss of integumen-
tal scales which function as distance receptors (Mauchline et al.
1977) and sensilla (20-500 [jtm in length) found on the antennae,
carapace, walking legs, abdomen, telson, and uropods (Heinisch &
Wiese 1987). As observed with our methods, the tailfan and ab-
dominal region of humpback shrimp were the most resistant to
damage (<2% damage), and the tailfan received more damage than
the abdomen. Tailfan damage differed among gear types, with the
beam trawl resulting in the most damage. The telson, which is
borne on the tailfan, was reported to carry two pairs of tuft organs
used as chemosensors (Mauchline et al. 1977). In our study, the
highest damage scores were assigned to the carapace in all cases
except overnight trapping, where the rostrum received the highest
damage. The carapace of humpback shrimp also received the high-
est cumulative damage scores of any body region surveyed and is
likely the most critical part of the shrimp's anatomy for survival as
it houses the cardiac, gastric and branchial organs. The visceral or
gastric region of lobsters {Homanis americanus) were also iden-
tified by Ganz (1980) as particularly vulnerable to damage from
otter trawling, especially during the molting phase. Because our
work only assessed damage to humpback shrimp when they are not
molting, the estimates presented herein are minima. Further work
at different seasons would be required to investigate effects on
humpback shrimp at different life stages.
Humpback Shrimp Catch and Biomass
There were considerable differences in the biomass of hump-
back shrimp harvested in Simoom Sound among fishing methods.
Although they fished over approximately the same distance along
the bottom of Simoom Sound (500-700 m) humpback shrimp
catches in traps were higher than trawl catches. A similar pattern
can be seen in the commercial fishery, with trap vessels usually
landing more humpback shrimp compared with trawlers (average
20 metric tonnes (t) vs 10 t per year; Boutillier & Nguyen 1999).
Standardization of catches by trawling and trapping vessels may be
possible using fuel consumption, but data on specific vessels
would be required. The mean individual weights of humpback
shrimp caught by trap were significantly higher than those from
beam trawl or otter trawl, suggesting that traps were selecting for
larger individuals. Wright and Panek (2000) http://www.orst.edu/
Dept/IIFET/2000/papers/wright2.pdf have suggested that there is
an inverse relationship between the trap soak-time and the weight
of prawns {Pandahis platyceros) harvested. However, we found no
significant differences between the individual size of humpback
shrimp caught in half-day versus overnight traps.
ACKNOWLEDGMENTS
Thanks are owed to the Masters of the fishing vessels for their
cooperation and assistance during this study. Victor Keong, Beth
Piercey, Shane Petersen, and Hugh McLean provided great help in
the fieldwork and laboratory analyses. Jim Helfield, Tamara Ro-
manuk, Laurie Convey, and Sung-Yun Hong kindly provided com-
ments on the manuscript. Dario Stucchi provided the oceano-
graphic data for Simoom Sound. Funding for this study was pro-
vided by the DFO Environmental Sciences Strategic Research
Fund and Science Branch. Pacific Resion.
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Journal of Shellfish Research. Vol. 22, No. 2, 569-579, 2003.
INTRASPECIFIC AGGREGATION STRUCTURE OF A SHOAL OF A WESTERN
MEDITERRANEAN (CATALAN COAST) DEEP-SEA SHRIMP, ARISTEUS ANTENNATUS (RISSO,
1816), DURING THE REPRODUCTIVE PERIOD
FRANCESC SARDA,'* JOAN B. COMPANY,' AND ARTURO CASTELLON"
^Institut de Ciencies del Mar (CMIMA-CSIC) and 'Unidad de Teaiologia Marina (CMIMA-CSIC).
Passeig Maritim de la Barceloneta 37-49. 0,H003 Barcelona. Spain
ABSTRACT The deep-sea rose shrimp, Arisleus amennalus. constitute an important fishery resource in the Western Mediterranean
Sea. The spatio-temporal behavioral pattern of A. antennatiis is well-known, with the species forming seasonal aggregations on the
middle slope at depths between 400 and 900 m. These aggregations form between late winter and early summer. The object of the
present study is to determine the internal structure of shoals of the western Mediterranean (Catalan coa.st) rose shrimp along the slope
on the grounds where the species is tlshed (from 400 to 10(X) m) at the tmie of peak density during the reproductive period. Interactions
between fishing and research vessel have been used to sample synchromcally and bathymetrically the shoals of the deep-sea shrimp
to determine intra and interspecific shoal structures. The results of this study on A. uineiiiiatiis have specifically shown that ( 1 ) The
pattern shrimp shoal distribution is such that density rises rapidly in the portion located in the shallower distribution range of this
species and then gradually decreases at greater depths; (2) the distribution of this resource straddles both sides of the ecological
boundary located at 900 m, though with changes in the sex-ratio and individual size: (3) species coexisting with this shrimp species
are concentrated at depths other than the depths of peak shrimp density; (4) commercial trawlers deploy according to the abundance
pattern of the resource; and (5) the reproductive portion of the stock is heavily exploited.
KEY WORDS:
shoals
Arisleus aiueimauis.
Mediterranean Sea. population structure, aggregation, sex ratio, size frequencies, fisheries.
INTRODUCTION
The deep-sea rose shrimp, Arisleus anteniuitiis (Risso, 1816)
(Crustacea. Decapoda, Dendrobranchiata, Aristeidae), represents
an important fishery in the Western Mediterranean Sea (Sarda &
Martin 1986, Demestre & Lleonart 1993, Bianchini & Ragonese
1994, Carbonell et al. 1999). This species is a characteristic com-
ponent of the demersal muddy bottom community on the middle
slope at depths between 400 and 1,200 m (Cartes & Sarda 1993),
where Cartes & Sarda ( 1992) and Maynou & Cartes (2000) have
defined it as a nektobenthic species of moderate-to-high swimming
mobility. However, the distribution of this species is also fished
frequently between 400 and 800 m in other Mediterranean areas
(Bianchini & Regonese 1994, Carbonell et al. 1999, Papaconstan-
tinou & Kapiris 2001, Cau et al. 2002). The distribution of this
species is nonetheless considerably broader, reaching at least to
depths of 2250 m (Sarda & Cartes 1992, 1993), indicating that the
species is eurybathic with a distribution considerably broader than
that of other decapod crustacean species.
The spatiotemporal behavioral pattern of /I. antennanis is well
known, with the species forming seasonal aggregations on the
middle slope at depths between 400 and 900 m. These aggrega-
tions form between late winter and early summer (Tobar & Sarda
1987, Demestre & Martin 1993, Sarda et al. 1994). Towards the
end of summer, the shrimp shoals tend to break up and move inside
submarine canyons, with the shrimp being fished at shallower
depths (400-700 m) along the margins of the canyons, locations
that are less accessible to trawlers (Sarda 1993, Sarda et al. 1994,
Sarda et al. 1997).
During the period in which this species forms aggregations
(late winter to early summer), shoals consist of reproductive adult
females. Copulation takes place at the start of the aggregation stage
(Relini Orsi, 1980, Sarda & Demestre 1987) with a percentage of
*Corresponding author. E-mail; siscu@icm.csic.es
males in the population of less than 20% (Sarda & Cartes 1992,
Demestre & Martin 1993, Sarda et al. 1994). Tursi et al. (1996)
reported that during copulation in late winter, males can be 50% of
the population in Ionian Sea. Studies conducted on the catchability
of shoals of this species (Sarda & Maynou 1998) have suggested
that the shoals take on an elongate shape parallel to the coast. It is
exactly at this time when the shrimp stock bears the brunt of
fishing effort (Tudela et al. 2003), because shoal formation is at its
peak on the part of the slope most readily accessible to trawlers
and females attain maximum size, that is, biomass concentration is
also at its peak. In addition, marketability of this species is also
highest at this time.
Studies on schooling in pelagic species (Swartzman et al. 1994.
Nonacs et al. 1994, Nottestad et al. 1996) particularly using echo-
sounding, and in species in captivity (Pitcher 1983, Pitcher et al.
1985), have been common, but there have been very few such
studies on benthic or benthopelagic species. Gordoa & Duarte
(1991) considered some Merliiccius species and reported size-
dependent schooling behavior. Macpherson & Duarte ( 1991 ) also
related size and depth for different fish species and discussed the
possible existence of a general size-depth relationship. On the
whole, studies on schooling and shoaling behavior have been quite
diverse in terms of methodology used, and they have also dealt
with a range of different aspects. Furthermore, although shoaling
of coastal prawns and migratory displacements relating to their life
cycles are well known (Garcia & Le Reste 1987), our literature
review has not disclosed any similar studies on shoaling patterns
for species dwelling at depths below the margin of the continental
shelf
Accordingly, the object of the present study was to determine
the depth structure of shoals of the Catalan coast rose shrimp,
Arisleus antennanis (Risso, 1816), along the slope on the grounds
where the species is fished (from 700 to 1000 m) at the time of
peak density (aggregation). Bearing in mind, however, that the
depth distribution for this species extends across several commu-
nity boundaries (down to at least 3000 in in depth; Sarda 2001 ). the
569
570
Sarda et al.
role of the noncommercially exploited portion ot the population on
the population as a whole has also been discussed. Shoaling struc-
ture has been considered in terms of both intraspecific aspects,
such as density, size range, and sex ratio, and interspecific aspects,
i.e.. density relationships between the rose shrimp and other fish
and crustacean species dwelling in the same faunal assemblage, on
the basis of depth. Our goal has been to underscore the importance
of understanding the intra and interspecific structure of aggrega-
tions of marine species as a significant factor in establishing the
actual level of vulnerability to exploitation by fisheries. In addi-
tion, over and above a simple discussion of the results presented
here, this article aspires to be an example of studies of this kind
and thus also includes a consideration of ecological and fisheries
aspects in the discussion, relating them to the specialized literature.
MATERIAL AND METHODS
A study was conducted jointly by the RN Garcia del Cid and
commercial trawlers on 21 to 23 June 2000 on the "Serola" fishing
grounds located off Barcelona (Northwest Mediterranean Sea).
where mature females of the deep-sea rose shrimp typically ag-
gregate at that time of year (Fig. 1 ).
To be able to obtain an instantaneous view of the aggregation
structure of a shoal of this deep-water species, operations must be
completed in the shortest possible time to avoid variations in re-
sponse to sudden environmental changes affecting community
structure. The weather was sunny and good over the 48 h in which
sampling was performed and remained stable over the course of
the survey.
The fishing vessels operating in this area are trawlers from the
port of Barcelona specialized in the shrimp fishery, with engine
power ratings ranging between 800 and 1100 horsepower and
lengths between 17 and 21 m. Five fishing trawlers conducted
fishing operations during their normal operating hours at depths
41.4-
41.3-
41.2-
41.1
50 m
100 m
200 m
400 m
700 m
900 m
^«
,^v^^^
.<iii
SS''
ijiSs
c,e»
1000 m
1100 m
1200 m
2.2
2.3
2.4
2.5 E
Figure 1. Study area showing the transects on which hauls were per-
formed by the research vessel (dotted lines) and the trawlers (solid
lines).
between 780 and 850 ni. Haul duration was typical for the fishery,
namely, two hauls daily lasting about 3.5 h each. Landings by
these trawlers were recorded on June 22. 2000. by a surveyor at the
wharf. Trawler headings and locations were monitored continu-
ously using the Automatic Radar Plotting Aid (ARPA) radar sys-
tem on board the research vessel, which made it possible to follow
the courses of their hauls from start to finish (Fig. 1).
The RA' Garcia del Cid is 38 m in length with an engine power
rating of 1 100 horsepower. It operated concurrently with the trawl-
ers in the same area, but over a broader depth range, between 700
and I 200 m (Fig. 1). A total of 11 daytime and nighttime hauls
were conducted on June 21 to 23. 2000. at least two hauls in each
of the 700-. 800-. 900-. 1000-. and 1200-m depth intervals. Depths,
towing speed, starting time, and ending time were recorded for
each haul (Table 1 ). The horizontal mouth opening of the gear
between the wings (13.5 m) was also recorded using remotely
operated Scanmar sensors.
Haul duration was 1 h to ensure that the sampling data would
be discrete and suitable for use in discriminatory analysis. Biologic
data collected consisted of the number and individual weight of all
specimens caught. Standard carapace length (CL in mm), indi-
vidual weight in g, sex. and maturity stage were recorded for all
rose shrimp specimens. The data from fishing trawlers and the data
from the research vessels are not directly comparable and only
relative comparisons were undertaken. These data were presented
in different graphics and with different units. Only biologic data
and size frequencies obtained on board the research vessel were
used in data treatment to avoid potential deviations. Only those
females in an advanced gonad maturity stage (maturity stages IV
and V according to the gonad coloration scale published by Relini
& Relini ( 1979) as expanded by Demestre & Fortufio ( 1992) were
classified as mature). Percentage size frequency values were com-
pared using multivariate analysis to evaluate similarity of the sur-
face areas of the bars and using the Kolmogorov-Smimov test
(P < 0.05) to compare the cumulative frequency values.
The sampling protocol used, using short hauls yielding data that
were highly discrete in terms of time, provided a "snapshot" of the
resource. This strategy has furnished good results when used in
studies of the density and spatial distribution of benthic (Gonzalez-
Gurriaran et al. 1993. Maynou et al. 1996) and benthopelagic
(Carter et al. 1993) crustaceans. Samples of longer duration would
have entailed the risk of introducing new variables, principally in
relation to changes in weather, which would definitely be a po-
tential source of noise in the data analysis. On the basis of the
results of previous work conducted in this same area published by
Tobar & Sarda (1987). Demestre & Martin. (1993). Sarda et al.
(1994). and Sarda et al. (1998), shoals of A. antennatus are con-
tinuously present at the sampling depths from late winter to early
summer.
Biomass in number and individual weight standardized to km",
on the basis of the area swept by each haul, have been graphically
represented using the sampling data collected by the research ves-
sel. Measurements were effected individually and overall and on
the total of fish and other shrimp species. In the case of the com-
mercial trawlers, which were not equipped with remotely con-
trolled monitoring systems, shrimp landings (kgh"') were
weighted on the basis of the length of the working day (7 h-d"').
Specialized personnel was on board of each commercial vessel
weighting the shrimp caught. Also bills of sales in auction was
collected to compare data on board. Figure 1 includes trend lines
fit visually to facilitate interpretation and discussion of the data.
Intrasphcific Aggregation of the Shrimp A. antennatus
571
Haul
Code
TABLE 1.
List oC hauls etYected.
Depth (m) D/N
Local Starting
Time (h)
Local KndinK
Time (hi
Starting Position
Ending Position
Swept Area
(km-)
HI
700
D
15:03
16:05
41° 09' 01" N 02 IS' 04" E
41' 08' 42" N 02 22' 01" E
0.06290
H2
800
D
17:52
18:47
41° 08' 08" N 02° 20' 32" E
41° 08' 27" N02° 16' 49" E
0.06806
H3
900
N
21:43
22:56
41= 07' 44"N02° 21' 37" E
41° 07' 43"N02" 25' 43" E
0.08001
H4
1000
N
3:53
5:20
41° 06' 07" N02° 23' 06" E
41° 08' 16"N02° 29' 13" E
0.09779
H5
1200
D
8:03
9:16
41° 05' 53" N02° 28' 56" E
41° 04' 06"N02° 25' 42" E
0.06826
H6
750
D
12:17
13:18
41° 08' 30" N 02° 21' 21" E
41° 08' 40"N02° 17' 50" E
0.06645
H7
1000
D
15:32
16:33
41° 07' 08"N02° 24' 34" E
41° 08' 15"N02° 28' 51" E
0.09005
H8
1200
N
19:33
21:09
41° 04' 23"N02° 27' 05" E
41° 05' 04"N02° 21' 55" E
0.08116
H9
750
N
0:18
1:42
41° 08' 38"N02° 21' 01" E
41° 08' 5r'N02° 16' 09" E
0.07038
HIO
800
N
3:44
4:40
41° 08' 18"N02° 20' 36" E
41° 08' 14"N02° 23' 58" E
0.07006
Hll
900
D
6:52
7:54
41= 07' 34" N 02° 24' 44" E
41° 08' 40" N 02° 27' 59" E
0.06714
D/N, day-night hauls
A matrix consisting of species (columns) and hauls (rows) was
constructed for community analysis. Species that occurred only in
a single haul and species occasionally represented by only a single
individual in some hauls have not been included. The data were log
transformed In (x+1 ) and used in multivariate cluster analysis. The
linear correlatio)! value was used as the similarity index and
UPGMA as the aggregation algorithm.
The abundance ratios for rose shrimp to other crustacean and
fish species were calculated by dividing the number of rose shrimp
individuals by the total number of individuals of all species in the
other two groups, crustaceans and fishes, respectively. Diversity
was calculated using Simpson's index, a good discriminator for
indicating dominance by a given species or group of species (May.
1975), which is the case of the rose shrimp here, the predominant
species in the present study. This diversity index has been recom-
mended for use in comparisons of marine communities (Lambs-
head et al.l983).
RESULTS
Abundance and Distribution
Shrimp abundance on the basis of the samples collected by the
research and commercial vessels have been depicted in biomass
(Fig. 2a and b) and number of individuals (Fig. 3). These figures
show that the lowest catches, with densities of about 20 ind. km"",
were made at around 700 m in depth, whereas the highest catch
densities, of around 1700 ind. -km"", were made at 800 m. Indi-
vidual density levels then tapered off progressively with increasing
depth. These results clearly define a specific structure across the
shoal with depth, with rose shrimp density augmenting sharply in
the shallowest portion of the shoal and then gradually falling off
towards the deepest portion.
The yields obtained by the trawler that effected tows at a depth
of around 800 m (Fig. 2b) were 2-fold those of the three trawlers
operating at greater depths and 5-fold those of the trawler operat-
ing at a shallower depth. Trawler deployment thus mirrored the
distribution of the shrimp resource being fished: one vessel oper-
ating at 700 m. where shrimp density was lowest: one vessel
operating at 800 m, where shrimp density was highest: and three
vessels operating at more than 800 m, where biomass began to
taper off This spatial deployment of the fishing trawlers was dic-
tated by the amount of space that had to be left between them to
ensure proper maneuverability. The first vessel to reach the fishing
grounds begins to work at the depth the skipper deems best to
achieve the highest yields. Vessels arriving later will then take up
a position next to the vessels already present, though always on the
deeper side, where rose shrimp density will tend to be lower still
profitable.
Day-Night Shoal Structure
Figures 2a and 3 depict the hauls conducted in the daytime
(hollow circles) and nighttime (solid circles). The distribution pat-
Research vessel
a
600 700 800 900 1000 1100 1200 1300
7-|
Fishing
vessels
6-
5 -
e
A
b
4 ■
\
3
/ '\
2-
I \
1 -
t
0 -
600 700 800 900 1000 1100 1200 1300
Depth (m)
Figure 2, Catches in weight by depth taken by the research vessel (a)
and the trawlers (b). Hollow symbols, day samples. Solid symbols,
night samples. Grey points, different fishing vessels.
572
Sarda et al.
600 700 800 900 1000 1100 1200 1300
Depth (m)
Figure 3. Densities by depth made by the research vessel. Hollow
symbols, day samples. Solid symbols, night samples.
tem can be observed to differ according to depth. In the depth
range between 750 and 900 m catches were higher at night than in
the daytime, which suggests that part of the population migrate to
the upper portion of the slope at night. The low individual densities
at 700 m were insufficient to allow any reliable inferences con-
cerning daytime-nighttime movements. Differences between day-
time and nighttime catches appeared to decrease with depth; how-
ever, it should be noted that commercial day-night catch data were
unavailable tor comparison with the experimental catch data, be-
cause commercial trawlers are not allowed to operate at night. This
observations coincides with the migrations of decapods suggested
by Cartes et al. (1993).
Size Frequencies
Figure 4 shows the size frequencies for females at the different
sampling depths. The bell-shaped size frequency curves tended to
flatten out and have wider tails with depth both in the daytime and
at night. At depths around 800 m the population tended to consist
of females with a modal mean size of 40 mm CL, ranging from a
minimum of 20 mm CL to a maximum of 5 1 mm CL. A similar
structure was observed at 1000 m. However, from 1000 m. there
was a change in the size frequency distribution, with the propor-
tions of both the smallest sizes and the largest sizes increasing.
This trend was quite distinct at 1200 m. despite the low number of
individuals, however due to the low occurrence of A. antenimtus in
this depth, the number of individuals caught was considered suf-
ficient for a good size spectrum on this depth. Because of the small
number of individuals caught at 700 m. it was not possible to
construct a sufficiently representative size structure for that depth.
The similarity analysis for the size frequencies (Table 2) indi-
cated significant differences (P < O.O.'i) between the size frequen-
20
18
IS
14
12
10
8-
S-
2
0
DAY
■ I ■ .111!
n = 108
800 m
ll..l ..
20 1
NIGHT
18
16
14
12
10
8
11 = 62
800 m
4
2
n
.1 lllllllll
,1,1, ,.,.,,,.
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20.1
is-
le
14
12
10
S-
6
4
2
0
Jr^^
Al
n = 51
900 m
n = 116
900m
xJiJi
I. Ill ,i,i,i, ,1
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20
18
16
14
12-
ID-
S'
6
4
2
0
■I III lllillli
n = 63
1000 m
llll,l..ll
20
18
16
14
12
10
8
6
4 -
2 ■
0
■ ■.■■■III Hill
n = 96
1000m
III !■!!■ ■
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
20
IS
16
14
12-
10
n = 17
1200 m
111 11 I
E
n = 26
1200 m
m
20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50 52 54 56 58 60
CL (mm)
Figure 4. Size frequencies for females by depth and by daytime-nighttime. CL, carapace length.
Intraspecific Aggregation of the Shrimp A. antennatus
573
TABLE 2.
Similarity between frequency values.
H3
900 ni
N
0.724
H4
1000 m
N
0.717
0.728
H?
1200 m
D
0.306*
0.373*
0.378*
H7
lOOOni
D
0.653
0.677
0.697
0.349
H8
1 200 m
N
0.380*
0.365
0.460
0.469
0.426
HIO
800 m
N
0.694
0.736
0.649
0.305*
0.657
0.310*
Hll
900 m
D
0.683
0.723
0.670
0.333*
0.638
0.386*
0.711
H2
800 m
N
H3
900 m
N
H4
1000 m
N
H5
1200 m
D
H7
1000 m
D
HS
1 200 m
N
HIO
800 m
N
H. haul code; D. day; N. night.
* Significanl differences (f<0.05) Kolmogorov-Sminiov test.
cies for the 800-900 m and 1000-1200 in depth intervals but not
between daytime and nighttime. There was a tendency towards
greater spread of the sizes at deeper depths, with higher propor-
tions of both juvenile and larger individuals. It is interesting to note
that the mo.st relevant changes in the size structure of the A. an-
tennatus population were linked to the community boundary (be-
low 900 m) for the species, as will be discussed in the following
section. Males exhibited a trend similar to that of the females, with
greater proportions of the extreme sizes (small and large individu-
als) at depths greater than 900-1000 m (Fig. 5). However, because
of the low occurrence of males in the population at the time of year
when the study was carried out. no reliable analysis of the level of
significance was possible.
Sex Ratio
The sex ratio (Fig. 6) was characterized by the low presence of
males at depths of 800 m «20%) and 900 m (<5'7f .). The propor-
tion of males increased progressively from 1000 m, gradually ris-
ing to nearly 40%. Virtually 100% of the adult females were
mature, and all bore a spermatophore on the telycum. which con-
firms that the shoal was at the spawning stage, as demonstrated in
various earlier papers (Sarda & Demestre 1987, Demestre & For-
tuiio 1992, Sarda etal. 1994).
Assemblages Differences
The increase in biomass with depth (Figs. 7 and 8) was caused
principally by the presence of very large species, e.g.. Alepoceph-
atits rostratus. Mora mow. and Lepidion lepidion, which are typi-
cal of the community below 900 m, and their occurrence also
raised abundance levels (Table 3). According to the results of the
cluster analysis, the main discriminating factor was the presence of
deep-water fish and crustacean species in the depth interval con-
sidered in this study, such as Bathypterois mediteiraneus. Mora
mora. Nezuinia aeqiialis. Acanthephyra eximia, Geryon longipes.
Miinida teninmana. Paromola cuvieri. and Sergestes arcticus. as
opposed to species that are characteristic of shallower depths, such
as Hymenocephalus italicus, Phycis hiennoides, Trachyrhynchus
scabnis. Scliyliorliinus canicula. Aristeus antennatus, Pasiphaea
multidentata. Pasiphaea sivado, and Polycheles typhlops (Fig. 9),
Trawls nos. 1 , 2, 9, 6, and 10 made up a group comprising the 700-
and 800-m depth intervals. Trawls nos. 3. 5. 4. 7. II. and 8 made
up a group comprising the depth intervals between 900 and 1200
40
35
30
25
20
15
10
5 -
0
40
35
30
25
20
15
10
5 -
0
40
35
30
25
20
15
10
5
0
40
35 ■
30 ■
25
20
15 •
10
5
0
n = 29
800 m
■ I III ■■II III
15 17 19 21 23 25 27 29 31 33 35 37 39
n = 6
900 m
17 19 21 23 25 27 29 31 33 35 37 39
n = 33
1000 m
oLl
u
15 17 19 21 23 25 27 29 31 33 35 37 39
n= 17
1200 m
I ■ I .l-LI.
15 17 19 21 23 25 27 29 31 33 35 37 39
CL(mm)
Figure 5. Size frequencies for males by depth. CL, carapace length.
574
Sarda et al.
50
40
30
20
10
800
1200
900 1000
Depth (mm)
Figure 6. Sex ratio by depth expressed as tlie percentage of males.
m. It is important to bear in mind that, unlike most other species,
distribution of the deep-water rose shrimp is virtually continuous
from 700-800 m to more than 1200 m (Fig. 2). Accordingly, this
species is represented and attains high abundance levels in both of
the assemblages revealed by the cluster analysis.
Density-Dependent Exclusion
The density of other species was lowest between 800 and 1000
m, where shrimp shoal density was highest. As A. aniennatus
density decreased with depth, the density of other species in-
creased progressively (Fig. 10), which suggests a high degree of
density-dependent exclusion between the shoal of shrimps, the
dominant species, and the distribution of other species at the same
depth. This was also reflected by diversity, which displayed little
variation between trawls, with Simpson index values between 0.09
and 0.19 (Fig. 11). High index values are indicative of lower
diversity due to dominance by one or just a few species. Index
values were highest (reflecting single species abundance and low
diversity) and displayed less dispersion for the depth intervals
between 800 and 1000 m. as a consequence of the greater occur-
rence of shrimps in those intervals. Diversity index values were
most variable for the extreme depth intervals sampled (700 and
1200 m), bearing out the preceding results.
DISCUSSION
A number of workers have described the composition of the
community supporting the deep-sea rose shrimp, Aristeus anten-
natiis. fishery in the Western Mediterranean Sea separately for
crustaceans and for fish (Abello et al. 1988, Stefanescu et al. 1992,
Cartes & Sarda 1993, Stefanescu et al. 1994). This community,
dwelling over muddy bottoms on the middle slope, is composed
25000
20000
E
•*: 15000
■o
c
■| 10000
3
^ 5000
0
♦
♦
♦
600 700 800 900 1000 1100 1200 1300
Depth (m)
Figure 7. Individual abundance by depth.
E
1600
1400
1200
1000
800
600
400
200
0
♦
♦
600 700 800 900 1000 1100 1200 1300
Depth (m)
Figure 8. Biomass by depth.
principally of the target species. Aristeus antennatus. along with
other species of no commercial interest, e.g., Genoii loni^ipes,
Polycheles typhlops, Lepidioii lepidion. Alepocliephaliis rostratus,
and Trachyrhynchus scabrus. In any case, A. antennatus is an
interesting species as compared with the other species dwelling in
the community because of certain specific biologic characteristics,
namely, (1 ) its broad depth distribution, making it a highly eury-
bathic species, and (2) even though fishing pressure has been
extremely high over the past 40 y, the population seems to be in a
healthy state of exploitation. Cartes & Sarda (1993) defined three
main zonations for the deep-sea decapod fauna in the Western
Mediterranean: the upper middle slope above 670 ni; the lower
middle slope between 850 and 1200 m; and, below this last-
mentioned depth, a transition zone to the lower slope community
(down to 2000 m). However, sampling between 650 and 900 m in
that study was inadequate, and the depth limit between the upper
middle slope and lower middle slope assemblages could not be
accurately determined. Based on the samples collected in the
present study, the boundary between the upper middle slope and
the lower middle slope would appear to be more exactly situated at
around 900 m related exclusively for A. antennatus. The above-
mentioned boundaries represent genuine barriers to distribution for
different decapod crustacean and fish species, but not for A. an-
tennatus. which enjoys a continuous distribution from 550 and at
least 3000 m (Sarda et al. 1993, Sarda 2001), However, we must
consider here that the definition of boundary is a controversial
question, often depending on the sampling adequacy and data used
in the analysis. Haedrix & Merret ( 1990) and Koslow (1993) and
Stefanescu et al. (1993) and Moranta et al. 1998, provided respec-
tively different results investigating in the same areas, however
only fishes are considered in these studies. In this paper we present
clusters including crustacean and fishes, reaching similar results as
Morales-Nin et al. (2003) with a first boundary around 800 m
depth. However, as has been observed in the present study,
changes in the internal population structure of this species are
apparent, linked to a community boundary existing at around 900
m. Similarly, the findings presented here have demonstrated that
the main stock, in terms of fishable biomass, is distributed chiefly
between 700 and 1,000 ni during the period of gonadal maturation
from late winter to early summer. This portion of the /\. antennatus
population consists primarily of females, with low proportions of
males (<10%) and medium-sized individuals. The highest fishing
effort is expended during the reproductive period of females. All
these aspects would appear to suggest that this species can be
expected to quickly become overexploited. but this does not seem
Intraspecific Aggregation of the Shrimp A. antennatus
575
TABLE 3.
Species abundances (number individuals km"').
Haul Code
1
9
6
*)
10
11
3
4
7
5
8
Species Depth (m)
700
700
750
800
800
900
900
1000
1000
1200
1200
Pisces
Alepocephuliis loslralus
0
0
60
(1
0
3098
462
2516
2254
689
4731
Antonogadus megulokynodun
97
71
0
15
14
0
0
0
0
0
0
Bathypterois mediterraneus
0
0
0
0
0
0
0
20
11
0
123
Benthocomeles rohustus
0
0
0
0
300
0
0
0
11
0
12
Coelorhynchus coelorhynchus
0
782
301
44
414
0
50
0
0
234
554
Chauliodus sloani
16
14
0
0
29
0
0
0
0
0
0
Epigonus lelescopus
48
14
0
15
0
(1
0
0
0
0
0
Hymenocepluiliis ilalicus
403
85
45
29
0
0
0
0
0
0
0
Lampanycms crocodilits
48
71
15
73
29
119
37
10
111
73
0
Lepidion lepidion
113
384
150
191
271
1162
1850
1391
1199
1143
1799
Myclophidae
16
57
15
0
43
0
0
C)
11
0
0
Mora mom
0
171
120
59
157
149
75
61
477
308
308
Nettastonia melwninim
0
0
0
0
0
0
25
61
0
29
25
Nezumia aequalis
16
28
0
88
29
447
612
654
644
293
357
Nolacamlms honapunei
64
355
75
59
71
60
137
1023
167
15
74
Phycis blennoides
225
57
120
59
29
149
137
20
44
15
0
Symphiirus nigrescens
48
14
30
0
14
0
0
0
0
0
0
Trachyrhynchus scabrus
1208
298
316
720
571
1 549
2125
1166
655
469
789
Selaceans
Etmoplenis spinax
0
0
0
0
0
30
0
0
11
59
74
Galeus melanoswmus
113
242
241
309
200
387
525
123
144
322
308
Scyliorhinus canicula
48
28
0
15
0
0
0
0
0
0
0
Crustaceans
Acaiitephyra eximia
0
14
15
0
0
119
62
757
155
483
838
Aristeus amennatiis
32
568
30
1028
1856
819
1350
1135
888
308
456
Geryon loiigipes
113
43
45
88
143
194
187
317
122
132
271
Monodaeus coiichi
0
0
0
0
14
0
0
0
0
0
271
Mttnida tenitimana
16
227
0
59
200
89
37
61
33
293
961
Paromola ciivieri
16
43
0
15
57
209
50
51
67
88
99
Pasiphaeu mtdlidentata
129
28
150
59
29
30
87
20
0
29
49
Pasiphaea sivado
0
0
15
0
0
0
175
0
0
15
0
Plesionika acanthonotus
0
57
0
0
14
119
0
20
67
0
25
Plesionika mania
741
28
211
59
29
30
50
0
0
0
0
Polycheles thyphlops
290
227
75
176
428
179
137
102
144
44
25
Ponlophilus iwrvegicus
0
0
0
0
57
89
87
41
144
59
25
Sergesles arcticus
0
0
0
0
14
30
0
0
56
147
0
Sergio rohiisiu
16
0
0
0
0
89
0
0
11
59
0
to be the actual condition of the stock (Demestre & Lleonart 1993).
Accordingly, perhaps the biology and internal population structure
of this species may somehow include the necessary features to
avert potential overexploitation.
The community boundary at around 900 m described here is
mainly the result of the upper limit to the depth distribution range
for such species as Alepocephalus rostratus, Lepidion lepidion,
Nezumia aequalis. Acanthephira eximia, and Geryon longipes,
species with high abundance and biomass levels. Also, the ARPA
log system results indicate that this same depth is the maximum
fishing depth at which commercial trawlers operate following the
A. antennatus shoals. Therefore, this community boundary could
be a direct effect of the high fishing pressure down to the said
depth of 900 m. On the other hand, at the present time no technical
constraints preventing fishing operations at deeper depths exist, yet
fishermen seem to be aware that there is a community boundary at
that level and thus do not operate at deeper depths, in the knowl-
edge that yields of A. antennatus there will be insufficient.
The deep-sea rose shrimp presents a well-defined distribution
pattern across this boundary at 900 m. The number of individuals
making up the shoal rose sharply from 750 to 800 m. that is. over
a depth interval of around 500 m. spatially equivalent to about one
mile, given the bottom configuration at the study location. From
900 m shrimp abundance fell off gradually over a distance of about
5 or 6 miles down to a depth of around 1200 m. though shrimp
distribution continues over a distance of several dozen miles out to
the bathyal zone (Sarda et al. 1993, Maynou & Cartes 2000).
Shoals were tongue-shaped situated parallel to the depth profile,
with peak abundance in the shallower portion (Sarda & Maynou
1998). Small daily or weekly variations in shoal location caused
the fishing trawlers to relocate operations over the depth of peak
shrimp abundance (Sarda & Maynou 1998). To date trawlers have
not undertaken shrimp fishing operations at deeper depths for tech-
nical reasons: insufficiently large winch size, distance to the fish-
ing grounds offshore, unfaniiliarity w ith the bottoms, etc.; even so,
in recent years trawlers have been observed to expand their fishing
depth gradually down to 1000 m. Nevertheless, shrimp specimens
caught experimentally at depths below 1000 m have been shown to
576
0.32 _
•rH
M
a
•l-H
1.00 J
Sarda
ET AL.
0.4
0.35
0.3
.2
0.25
0.2
0.15
0.1
0.05
0
O
^ -" 00
-NlOO-^'-gODCD-»--^-^CD-»-
OOOOOOMOOONJ
ooooooooooo
o o o o
Figure 9. Cluster illustrating the similarity between hauls carried out
at different depths.
be. on average, smaller in size (Sarda et ai. 1994). The size fre-
quencies set out herein bear out that observation. Furthermore, the
proportion of males increases (Sarda et al. 1993), with males being
smaller than females as a consequence of this species' sexual
dimorphism, as illustrated in Figure 5. These features lower the
commercial attractiveness of the deeper shoals and cause the fish-
ing trawlers to stay within the more commercially profitable depth
range. Trawlers have never been recorded on the deeper portion of
these fishing grounds (Sarda et al. 1998), suggesting that the shoal
structure described here remains unchanged at this time of year.
The literature contains no discussion of the role of this unexploited
or pristine portion of the stock (below 1000 m) in relation to the
exploited portion of the stock at shallower depths. Furthermore,
stock assessment studies (Demestre & Lleonart 1993, Martinez-
Baiios 1997, Garcia-Rodriguez & Esteban 1999) have suggested
that despite the high level of fishing pressure to which they are
subjected as a target species, shrimp stocks are not overexploited.
The stability of the population in the face of fishing pressure would
seem to suppoit the assumption that the stocks are replenished by
500 600 700 800 900 1000 1100 1200 1300
Depth (m)
Figure 10. Ratios for the number of shrimp individuals in relationship
to other species. Hollow circles, fishes (solid line); solid circles, crus-
taceans (dotted line).
an influx of individuals from the pristine populations located in
deeper waters.
Based on the size frequency data, modal size at t~irst maturity
for males would appear to be in the neighborhood of 2 1 mm CL
(Sarda & Demestre 1987, Sarda & Cartes 1997). Mean size of
males appeared to be located mainly in the 800 m depth interval,
where they coexist with sexually mature females. The implication
is that mating takes place principally between adult females and
two-year-old males around the time of first maturity (Demestre &
Fortuiio 1992), with smaller and larger males being less aggregated
and mainly present at deeper depths, down to 1200 m (Sarda &
Cartes 1997). To date there is no further evidence to suggest that
these depths are recruitment zones or are subject to lower preda-
tion and thus are more conducive to juvenile development, as
postulated for deep-water species by Gage & Tyler ( 1990).
There is little information on daytime and nighttime variations
in catches, the only data available being provisional data for the
Gulf of Taranto (Maiorano et al. 1999 and unpublished data), the
area off Sardinia (Sabatini et al. 1999 and unpublished data), and
the area off Algeria in North Africa (A. Campillo and A. Nouar,
0.20-
0.15
I—
0)
■5 0,10-
Q
0.05^
0.00 J
i ▲
▲ A
A
A
700
1200
800 900 1000
Depth (m)
Figure 11. Plot of Simpson's diversity index by depth.
Intraspecific Aggregation of the Shrimp A. antennatus
577
personal communications). The literature reviewed contains no
further studies on this topic. According to the researchers just
mentioned above, the shrimp population carries out nocturnal mi-
grations up the depth profile and can thus be caught at shallower
depths at night. A sunilar pattern was observed over the depth
interval considered in the experiment reported here, with nighttime
catches being somewhat higher than daytime catches and the dif-
ference peaking in shallower waters al around .SOO m. The differ-
ence decreased with depth, suggesting the possible involvement of
an effect related to light levels (directly or indirectly), with the
population located at deeper depths thus being less affected by
decreases in luminosity or by the effect of light on migratory
mesopelagic organisms dwelling in the higher layers in the water
column. Studies of stomach contents would be needed to be able
to establish relationships between the vertical migrations of me-
sopelagic species and food availability at the different depths and
in the water column. The present results corroborate the hypoth-
eses described in Cartes et al. ( 1993). These authors observed that
certain nektobenthonic species seem to undergo migrations along
Ihe bottom to shallower areas of the slope at night. Moreover
vertical migrations into the water column above would seem to be
an unlike explanation, in view of the small share of planktonic prey
items in the nocturnal diet of A. antennatus (Cartes 1991).
The main question requiring elucidation is why shrimp shoals
present the characteristic structure described, in terms of both
shoal morphology and size and sex composition. The presence of
a lower proportion of larger males and of females of different sizes
with increasing depth might be attributable to the adaptation of the
metabolisms of large individuals to deeper habitats, which are
more oligotrophic and less favorable to high biomass levels com-
posed mainly of reproductive females. An alternative hypothesis
could be that adult females in advanced stages of gonadal maturity
have certain nutritional requirements that are best filled at depths
between 800 and 1000 ni, where there may be some sort of envi-
ronmental features at certain times of year that trigger the popu-
lation structure observed. Puig et al. (2001 ) have proved this hy-
pothesis for shrimps of the genus Plesionika. They observed char-
acteristic distributions of berried and juvenile females at certain
depths, associated with the presence of nepheloid layers in the
same area in spring and fall. However, no such relationship has yet
been demonstrated for A. antennatus.
Cartes & Sarda (1989) and Maynou & Cartes (1997, 1998)
consider this species to occupy one of the lower positions in the
benthopelagic food chain but to be atypical among deep-sea de-
capod crustaceans in that it exhibits a relatively high proponion of
full stomachs as compared with other deep-sea decapod crusta-
ceans. The high metabolic and growth rate demonstrated for this
species by Company & Sarda (1998. 2000) is likewise indicative
of this. Furthermore, more mobile species tend to have higher
metabolic rates, that is. they have higher energy requirements.
which translates into a higher daily ration (Koslow 1996). Given
the reduction in food sources in deep-sea habitats, causing dietary
overlap and competition for food (Gage & Tyler 1990). it seems
reasonable to suppose that A. antennatus will have specific nutri-
tional requirements during spawning and will therefore tend to
adopt a distribution at optimum depths to fulfill those require-
ments. This could be one of the main reasons for the high level of
dominance found for this species in the depth interval studied. In
the Catalan Sea total consumption by balhyal decapod crustacean
assemblages is higher on the upper middle slope (400-900 m) than
on the lower middle slope (900-1200 m). The generally lower food
consumption by decapod crustaceans with depth is consistent with
the commonly accepted notion that food availability also declines
with depth, which holds both for the suprabenthos (one of the main
sources of food for benthic decapod crustaceans) and for mesope-
lagic decapods and euphausiid crustaceans and other crustacean
taxa (Carpine 1970. Cartes 1998. Cartes & Maynou 1998. Mura et
al. 1998). The reduction in food resources takes place around the
zonation boundary located at 900 m. with deep-water rose shrimp
shoals being located above that depth.
Temperature did not appear to be a determining factor in these
processes, in that temperature in the Mediterranean is constant at
around 13 ± 0.5 'C below 200 m (Hopkins 1985), hence the popu-
lation structure and behavior of A. antennatus can be considered
temperature-independent. In the deep-water habitat that concerns
us here, food availability in the deep-sea food web would seem to
be the principal limiting factor (Gage & Tyler 1990).
In conclusion, the results of this study on the Catalan Sea, have
specifically shown that the shoals of A. antennatus during the
reproductive period has the following structure:
(1) The pattern shrimp shoal distribution is such that density
rises rapidly in the portion located in the shallower portion
and then gradually decreases with greater depth;
(2) The distribution of this resource straddles both sides of the
ecological boundary located at 900 m, although with
changes in the sex-ratio and individual size;
(3) Species coexisting with this shrimp species are concen-
trated at depths other than the depths of peak shrimp den-
sity;
(4) Commercial trawlers deploy according to the abundance
pattern of the resource;
(5) The reproductive portion of the stock is heavily exploited;
(6) There is substantial evidence that ecological aspects need
to be taken into account when evaluating the dynamics of
exploited populations with a view to sustainable manage-
ment.
On the whole, the following salient aspects would appear to
merit consideration: Fishing on the shrimp stock takes place
mainly during the season of aggregation and maturation of repro-
ductive females, which heightens the population's vulnerability to
fishing activity. This factor needs to be taken into account for
purposes of assessment and management. Nevertheless, studies
published by Demestre & Lleonart (1993), Martinez-Bafios
( 1997), and Tursi et al. (1996) have reported the status of exploi-
tation of this species to be near the maximum sustainable yield
(MSY) in different parts of the Mediterranean Sea. This is where
the unexploited portion of the stock inhabiting the lower-middle
slope (from 900 m to at least 2200 m) comes into play. This
portion of the stock may act as a reserve, contributing additional
biomass to the exploited portion of the stock and thereby prevent-
ing overexploitation. However, it should be noted that this hypoth-
esis has not yet been demonstrated and that studies focusing di-
rectly on this aspect are needed.
ACKNOWLEDGMENTS
Funding for this research was provided by the CICYT of Spain
(Project MAR97/()640 C02-01 ) co-ordinated by Dr. J. Lleonart. In
particular, the authors thank the crew of the RA' Garcia del Cul.
578
Sarda et al.
and Mr. M.A. Estevez of the CSIC's Unidadde Gestion de Buques
Oceanograficos [Oceanographic Vessel Operating Division] for
their assistance. Thanl^s are also due to the colleagues who par-
ticipated in the sampling. S. Tudela. G. Rotllant. J. Aguzzi. J. A.
Garcia. B. Molf. J. Ri'os, and 1. Catalan, and to Mr. LI. Llado,
skipper of the fishing trawler "Bona Mar."
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Jminuil of Shellfish Reseanh. Vol. 22, No. 2, 58I-5SS. 2UUJ.
SEAFOOD DEALERS' SHRIMP-PURCHASING BEHAVIOR AND PREFERENCES WITH
IMPLICATIONS FOR UNITED STATES SHRIMP FARMERS
FERDINAND F. WIRTH* AND KATHY J. DAVIS
Food and Rcsoiiicc Economics Department. Indian River Researcli and Editcali<m Center, University of
Florida. IFAS. 2199 South Rocli Road. Fort Pierce. Florida .U945-M 38
ABSTRACT The US shrimp-farming industry has been expandmg in the southern United States in response to the strong market
demand tor shnmp. However. US farmers have difficulty competing on price with imports in fro/en shrimp commodity markets. This
study identified the shrimp-purchasing behavior and preferences of seafood wholesalers and retailers in nine southeastern US states to
provide shrimp farmers the market information needed to develop successful marketing strategies. Results of a mail survey of the
seafood dealers, including a conjoint analysis experiment, are presented and discussed. There appears to be a potential market for fresh,
farm-raised shnmp in a vanety of sizes, but there is considerable dealer resistance to the whole or live head-on shrimp form. Shrimp
farmers interested in successfully marketing to seafood dealers may be required to process their product to offer shrimp tails, rather
than whole shrimp.
KEY WORDS: shrimp, buyer preferences, marketing, conjoint analysis
INTRODUCTION
According to the "Top Ten Seafoods" summary prepared by
the National Fisheries Institute, shrimp was the leading seafood
consumed in the United States in 2001, surpassing tuna for the first
time in a decade (National Fisheries Institute 2001). Per capita
consumption of shrimp was 3.4 pounds/person in 2001. or 23% of
total US seafood consumption. Demand for seafood in the United
States far exceeds the amount produced by US commercial fish-
ermen and aquaculture producers. In 2001, 882.6 million pounds
of shrimp, about 85% of the total supply, were imported into the
United States, primarily from Southeast Asia. These imports were
valued at $3.6 billion and accounted for 37% of the value of total
edible fishery product imports (National Marine Fisheries Service
2001). Domestic farmed shrimp production accounts for less than
5% of the total US supply (Harvey 2002).
Interest in the shrimp farming industry has been growing rap-
idly in Florida and other southern states in response to the excess
domestic market demand for shrimp. The most viable candidate
shrimp species for large-scale culture in Florida appears to be the
Pacific white shrimp. Liiopenaeiis vainniiiiei. because of its market
popularity, fast growth, adaptability to diverse salinities, and abil-
ity to be cultured at high densities.
In the past, expansion of marine shrimp culture in Florida has
been constrained by high coastal land prices, competing uses of
coastal land, and concerns over potential environmental damage to
sensitive coastal ecosystems. However, aquaculture researchers in
I'lorida have successfully acclimated the marine shrimp L van-
iHiinei to hard freshwater at the age of 3 weeks (12-15 days post-
larvae). The freshwater found in much of central and south Florida
and other southern states contains the mineral balance to support
this species. Farmers with hard freshwater wells are now able to
demonstrate the technical feasibility of raising shrimp from post-
larvae to commercial market size in inland locations. Attempts to
demonstrate economic and market feasibility are ongoing.
US shrimp farmers, including those in Florida, wish to harvest
and market their shrimp as quickly as possible. However, US
farm-raised shrimp cannot compete effectively on price with im-
ports in fresh-frozen shrimp commodity markets for the most
♦Corresponding author. E-mail: ffwirth@ifas.un.edu
popular forms and sizes. Furthermore, although some domestic
farms will undoubtedly develop processing capability, the equip-
ment, packaging, and marketing required to assure the success of
value-added products and to satisfy food safety requirements
(HACCP) are beyond the capability or interest of many sinall
farmers. Thus, shrimp farmers in Florida and other southern states
are particularly interested in the potential for marketing their prod-
uct to seafood dealers (retailers and wholesalers) as live shrimp or
fresh, head-on shrimp.
This research is part of a more comprehensive study designed
to identify and characterize the most attractive direct markets for
fresh, farm-raised shrimp. The specific objectives of this research
were 1) to identify the shrimp-purchasing behavior, preferences,
and attitudes of seafood dealers (wholesale and retail) in the south-
eastern United States and 2) to characterize marketing challenges
and opportunities associated with the seafood dealer market.
LITERATURE REVIEW
Relatively little published information specific to domestically
cultured shrimp is available relating to preferences and purchase
behavior of seafood dealers. Most recent research has been focused
on wild-caught and farmed finfish. This paucity further empha-
sizes the need for reliable market research information for farm-
raised shrimp.
Dore (2000) provided an overview of the shrimp distribution
system in the United States. The retail food business in the United
States is dominated by superinarkets. Specialty retail seafood mar-
kets are typically located on the coasts or in large cities, and tnany
of these combine retail sales with a wholesale or restaurant busi-
ness. Similarly located, specialty seafood wholesalers primarily
supply restaurants. Although some retail food stores do buy
through wholesale grocers, most supermarkets are supplied
through their own purchasing departments, with smaller chains
more likely to buy direct.
The wholesale, retail, and food service sectors of the seafood
industry create significant economic activity within many non-
coastal areas of the country: this is becoming even more pro-
nounced given the rapid development of inland aquaculture (Ad-
ams 1998). Market analyses for several aquaculturally produced
finfish have demonstrated a strong retailer and wholesaler prefer-
ence for highly processed product (fish fillets), consistent with a
581
582
WiRTH AND Davis
noted consumer preference for convenience and ease of prepara-
tion (Gok & Nelson 1999, Wirth et al. 1990).
Shrimp are sold in a variety of fresh or frozen product forms,
including whole (head-on) or tails, shell-on or peeled, and round or
split and deveined. Growth in US consumption of shrimp is pri-
marily in the form of raw headless, raw peeled, or cooked peeled
shrimp (National Marine Fisheries Service 1996). Head-on shrimp
is increasingly important in Europe, but the US market for this
product is still very small and consists primarily of oriental res-
taurants (Dore 2000). Shrimp prices vary according to a wide
variety of factors, including size, supply, quality, origin, species,
and color (Yokoyama et al. 1989). Many species of shrimp are
consumed in the United States, but white shrimp are generally
preferred ( Keithly et al. 1993). Sales and shipments are reported by
size categories of shell-on shrimp tails, defined by count per
pound. Customary commercial size classifications in the United
States are U/15 (under 15 shrimp/lb), 16/20, 21/25, 26/30, 31/35,
and so on.
General information concerning retailer and wholesaler shrimp
purchase behavior was extracted from three small studies con-
ducted in Hawaii and Florida. These were the only readily avail-
able reports containing information specifically relating to shrimp
dealers. Shang (1990) interviewed 63 fish distributors in Hawaii
and found that the shrimp dealers sold shrimp in six forms: frozen
head-off, frozen peeled and deveined, breaded, canned, dried, and
fresh. Frozen head-off was the most important category, account-
ing for about 70% of the total volume sold; fresh shrimp accounted
for only 1 % of the total volume. Dealers preferred large shrimp for
frozen tails and frozen peeled and deveined shrimp. Firms that
indicated foreign imports as their major supply source most often
cited "best price" as their reason, firms that relied on US supply
sources did so for "best quality" or "steady supply."
Schumann (2000) surveyed Florida shrimp broker/distributors
regarding their willingness to purchase live shrimp. Only about
10% of respondents reported that they currently purchase live
shrimp but about 35% indicated that they would probably purchase
live shrimp in the future. Shrimp buyers indicated a willingness to
pay $3.50-$4.80/lb for farmed shrimp in 1999 and an interest in
marketing full shrimp farm production capacity.
The Florida Department of Agriculture and Consumer Services
(2001 ) interviewed US seafood wholesalers at the Boston Seafood
Show concerning their shrimp purchase behavior. About one half
(49%) of the dealers indicated they purchase only frozen shrimp
and 62% purchase only head-off shrimp forms. Almost one half
(46%) of the dealers currently purchase farm-raised shrimp, and
74% indicated a willingness to purchase white shrimp farmed do-
mestically in fresh water and to purchase shrimp directly from a
shrimp farmer. At the time of the interview, these dealers also had
the opportunity to evaluate Pacific White shrimp farmed in fresh-
water; 83% of the dealers rated the shrimp as good or excellent
overall.
Although published information specific to domestic buyer
preference for shrimp is very limited, a strong demand appears
to exist for high-quality, rea.sonably priced shrimp. The shrimp
farmer is ideally situated to provide a consistent supply of
fresh shrimp and can adapt production to meet buyer demands for
size, quality, quantity, and timeliness. However, head-off forms
seem to dominate shrimp sales and consumption, so shrimp farm-
ers may encounter some resistance to direct marketing of whole
shrimp.
METHODS AND MATERIALS
This study was designed to characterize preferences within the
domestic seafood dealer market and identify opportunities and
challenges associated with marketing to seafood dealers. A ques-
tionnaire was developed and administered by mail to 3038 seafood
dealers, identified by Standard Industrial Classification code, in
nine southeastern U.S. states (AL, AR, FL, GA, LA, MS, NC, SC,
and TN). The mailing list included the entire population of seafood
wholesalers, retailers, and processors in the nine states, as identi-
fied by American Business Information, a commercial provider of
business directories. The questionnaire solicited information con-
cerning the location and size of the seafood business, business
sales structure, shrimp-buying practices, and preferences for vari-
ous shrimp product forms and attributes. A conjoint analysis was
conducted to quantify the utility value and relative importance of
key shrimp product attributes that are within the control of shrimp
farmers; size, refrigeration state, form, and price. A thank-you/
reminder postcard was mailed to each dealer approximately 4 days
after the survey mailing.
Conjoint Analysis
Conjoint analysis has become a popular marketing research
tool for designing new products. A conjoint analysis allows for a
buyer's overall preferences for a product to be disaggregated
among the complement of that product's features. This requires
knowledge of the overall evaluations of a set of alternative prod-
ucts that are prespecified in terms of levels of different features
(Green & Srinivasan 1978). Using conjoint analysis, a researcher
can analyze a heterogeneous product market and obtain results that
can be highly disaggregated to homogeneous groups of buyers.
Alternatively, aggregating results for buyers who have similar
preference or utility functions can be useful in modifying current
products or services and in designing new ones for selected market
segments (Green & Wind 1975).
The features and feature levels that define the conjoint design
must be carefully selected. The features correspond to product
characteristics that have been demonstrated or are hypothesized to
influence purchase behavior. The feature levels are sample values
for each of the selected factors, and the levels should span the
realistic range of each feature. Table 1 summarizes the four fea-
tures (size, refrigeration state, product form, and price) and feature
levels selected for the conjoint analysis experiment in this study.
The levels for price were selected based on published retail prices
TABLE L
Shrimp features and feature levels for conjoint analysis.
Feature
Feature Levels
Size (tail count/lb)
Extra large (16-2.S/lb)
Large (26-35/lb)
Medium (36-50/lb)
State
Fresh (never frozen)
Frozen
Form
Whole (head-on)
Shell-on tails
Peeled & deveined tails (P & d)
Price
$3.(K)/lb
$5.50/lb
$8.00/lh
Seafood Dealers" Shrimp-Purchasing Behavior
583
for fresh, shell-on shrimp tails over the range of sizes included in
the conjoint analysis. These prices were then adjusted to compen-
sate both for retail mark-up and for the conversion from shell-on
tail weight to round weight, to arrive at realistic wholesale prices
for whole (head-on) fresh shrimp.
The conjoint experiment uses a full-profile approach in which
respondents rate a set of hypothetical products defined by a speci-
fied level for each feature. In a full-factorial design, in which every
possible combination of feature levels is rated, the number of
products to be rated quickly becomes very large and the task
becomes unrealistic for survey participants. A fractional factorial
design is generally used instead, in which an orthogonal subset of
feature level combinations is selected. The orthogonality permits
estimation of all single-factor, or main, effects, although informa-
tion concerning feature interactions is lost (Green 1974). Every
level of each feature occurs with every level of every other feature,
thus unrealistic combinations of feature levels may occur in the
design.
The orthogonal design was developed using CONJOINT
DESIGNER, a software package developed by Brelton-Clark.
Only nine hypothetical products were required to represent the
orthogonal design described in Table 1, as opposed to 54 for a
full-factorial design. In addition, the experiment included one
"holdout" product defined to closely resemble realistically mar-
ketable farm-raised shrimp. Holdout products are used to validate
results as well as to gather data on particular products of interest
(Herman 1988). The coefficients of the conjoint model are esti-
mated using only the products that determine the orthogonal de-
sign, without use of any holdout products. The actual ratings of the
holdout products can then be compared with those predicted by the
conjoint model as an indication of the predictive validity of the
model. The 10 shrimp products presented to the survey participants
are described in Table 2.
Several important product characteristics, such as farm-raised
vs. wild-caught, raw vs. cooked, and domestic vs. imported, were
deliberately omitted from the conjoint experiment to limit the
number of tasks required of the survey respondents. Seafood deal-
ers were asked to rate each of the products shown in Table 2 on a
scale of 0-10, where 0 was the least desirable combination of
product attribute levels, and 10 was the most desirable combina-
tion of product attribute levels.
Model Specification
A conjoint preference model is used to estimate the influence of
various product features on preferences indicated by the respon-
TABLE 2.
Hypothetical products rated by seafood dealers for conjoint analysis.
Product
No.
Size
State
Form
Price
1
2
3
4
5
6
7
8
9
10 ("holdout"
Medium
Medium
Large
Extra large
Medium
Extra large
Large
Large
Extra large
Large
Frozen
Fresh
Fresh
Frozen
Frozen
Frozen
Frozen
Frozen
Fresh
Fresh
P&d
Tails
Whole
Tails
Whole
Whole
Tails
P&d
P&d
Whole
$s.on/ib
S.^.OO/lb
$8.00/lb
ss.no/ib
S5.5()/lb
$.^.00/lb
S-S.SO/lb
$.\00/lb
$5.5(J/lb
S5.50/lb
dents. The specification of the conjoint preference model as de-
scribed by Wirth et al. (1990) involves two steps. First, the func-
tional form for each product feature must be specified. Next, the
functional forms for each feature are combined into a conjoint
preference model for estimation.
There are three ways to model a buyer's utility (unction for
each product feature: a part-worth or dummy variable function
model, a linear vector model, and a quadratic or ideal-point model.
Green and Srinivasan (1978) provide a detailed theoretical discus-
sion of the three functional forms. The most general and most
commonly used utility model is the part-worth model, which is
especially appropriate for qualitative variables. The part-worth
model requires separate estimates of the contribution or part-worth
of each level of a feature. Quantitative features with two or three
feature levels, such as price, can be modeled using the part-worth
model, the vector model, or the ideal-point model. For this study,
the part-worth function model was used to model all four shrimp
product features: size, state, form, and price. The part-worth model
provides the greatest flexibility in the shape of the utility function
for each of the product features. However, this model also requires
estimation of the greatest number of parameters (perhaps reducing
the reliability of the estimates).
In conjoint analysis, a buyer's utility for a particular product, as
represented by the preference rating, is modeled as the sum of the
buyer's utilities for each product feature. The part-worth function
model posits that for a set of t features, where Vjp denotes the level
of the p"" feature for the j"" product, the preference Sj is given by
the following:
2./p<y.ip)
p=i
(I)
where /p is the function denoting the part-worth of different levels
of Vjp. In practice, /p(yjp) is estimated only for the selected set of
feature levels, with values for intermediate levels obtained by lin-
ear interpolation (Green & Srinivasan 1978). The general model
for this study can be expressed as follows:
Rating =/(Size, State, Form, Price)
(2)
where
rating = preference rating given to the hypothetical shrimp prod-
ucts by survey respondents,
size = shrimp size (extra large, large, or medium),
state = refrigeration state (fresh or frozen),
form = shrimp product form (whole, shell-on tails, or peeled and
deveined tails), and
price = product price ($.^.()0/lb. $5.5()/lb. S8.00/lb).
This study used mean deviation coding for the dummy variable
specification and the coefficients were estimated using ordinary
linear regression. This dummy variable coding technique is math-
ematically equivalent to traditional dummy variable coding, but
the coefficient for the base level is easily calculated as the negative
sum of the coefficients for the other k- 1 levels. The intercept is the
overall mean preference rating, and dummy variable coefficients
measure deviation from the mean rating (Harrison et al. 1998).
RESULTS AND DISCUSSION
Mail Survey
A four-page questionnaire was mailed to 3,038 seafood dealers
in the nine states comprising the southeastern United States. A
584
WiRTH AND Davis
total of 253 (8.3%) surveys were returned as undeliverable. Two
hundred and fifty (250) of the remaining 2.785 surveys were com-
pleted and returned. gi\ing an effective response rate of 9.0'^f. The
survey included questions concerning the location and size of the
seafood business, business sales structure, shrimp buying prac-
tices, and the conjoint experiment described in the Methods and
Materials section.
Almost half (4691^) of the responding dealers were located in
Florida, followed by Louisiana (169^), Georgia (11%), and North
Carolina (10%). The businesses were fairly evenly distributed be-
tween rural, suburban, and urban locations (22-35%). with fewer
in resort areas. Most (87% ) of the seafood dealers can be classified
as small businesses, with 25 or fewer employees.
Dealers were asked to describe their business in terms of the
percentage of their total sales in each of four specified categories:
wholesale to wholesale, wholesale to retail, retail, and other. For
this report, dealers were classified as "wholesalers"" if they indi-
cated that more than 50% of their total sales were wholesale to
wholesale and/or wholesale to retail. Similarly, dealers were clas-
sified as "retailers" if they indicated that more than 50% of their
total sales were retail. All other dealers were classified in a "com-
bination/other'" category. Respondents were fairly evenly split be-
tween the "wholesaler"" and "retailer" designations, but approxi-
mately 70% of responding dealers reported some retail sales, sug-
gesting that many seafood dealers are diverse, selling in multiple
markets.
Dealers were then asked several questions about their current
shrimp-buying practices. Of those responding, 85% indicated that
they currently purchase shrimp and reported their total annual
shrimp purchases. About two thirds of dealers who buy shrimp
purchase 50,000 pounds or less annually. .Mmost 10% buy more
than one million pounds annually.
These dealers were also asked to list the percentage of their
total shrimp purchases in each of several specified sizes and prod-
uct forms. Figure 1 shows the percent of responding shrimp buyers
who indicated they currently purchase any shrimp in the specified
sizes and forms. The results indicate that shrimp dealers carry the
full range of shrimp sizes from 16/20 count to shrimp smaller than
41/50 count. Figure 2 shows the shrimp product forms currently
being purchased by responding shrimp dealers. The majority of
shrimp dealers (85%) carry shrimp tails, but 50% of shrimp dealers
purchase some whole, head-on shrimp. More than 25% of shrimp
dealers also purchase peeled & deveined (p&d) tails and/or peeled
& undeveined (pud) tails.
The dealers were asked several questions specific to US farm-
raised shrimp. Of the dealers responding, 73% were familiar with
aquaculture and 54% indicated they currently buy faiin-raised
shrimp, although the country of origin was not identified. Seventy-
five percent would offer domestic farm-raised shrimp if it were
readily available and 72% would be willing to purchase shrimp
directly from a farmer. Only 38% of dealers were familiar with
Pacific White shrimp raised in fresh water, but 55% would be
willing to purchase these shrimp.
Figure 3 shows the percent of dealers in each sales category
that indicated willingness to buy shrimp directly from a US shrimp
farmer. About 1 8% of dealers classified as "wholesalers" for this
study specifically stated that they were not willing to buy directly
from shrimp farmers, whereas only 7% of "retailers"" were unwill-
ing to buy direct. Because of survey length constraints, dealers
were not specifically asked about their willingness to buy whole
shrimp directly from farmers.
Willingness to buy directly from farmers does not appear to be
directly correlated with any of the other basic dealer characteristics
recorded in this survey, including business location (state, or rural
vs. urban), company size, and shrimp volume. However, willing-
ness to buy directly from a shrimp fanner is likely contingent on
many factors not measured in this study due to survey length
constraints, such as the dealer's proximity to the farm, product
quality and quantity available, and the level of services (e.g.. pack-
ing, grading, delisery) provided by the farmer.
These results do suggest that US shrimp farmers should find a
ready dealer market for their product, especially with seafood re-
tailers. Farmers who are willing and able to perform the process-
ing, storage, transportation, and other marketing functions nor-
mally performed by seafood wholesalers may receive prices higher
than normal farm-gate prices and capture a share of the farm-retail
price spread.
Finally, dealers were asked to rate various shrimp product fea-
tures from 0-10. with 10 indicating the feature is ""most important""
in their shrimp purchase decisions. Table 3 shows the mean rating
100
90 -
80
70
a 60
S- 50
40
30
20
10
largerltian 16/20 21/25 26/30 31/35 36/40 41/50 smallerthan
16/20
41/50
shrimp size
Figure 1. Percent of shrimp dealers currently buying any shrimp in specified sizes.
Seafood Dealers" Shrimp-Purchasing Behavior
585
whole
tails
p&d tails pud tails butterfly
shrimp product form
Figure 2. Percent of shrimp dealers currently buying any shrimp in specified forms.
other
and ranking of each product feature for all dealers combined and
for those identified as wholesalers or retailers. Ratings were con-
sistent among wholesalers and retailers. Quality, freshness, and
smell were the three most important shrimp product features to the
responding dealers, each with mean rating greater than 8.5. Un-
fortunately, from the perspective of US shrimp fanners, production
source (impoiled vs. wild-caught vs. farm-raised) and country of
origin appear to be relatively unimportant to dealers. Dealers also
do not consider the whole (head-on) shnmp form, or fresh (never
frozen) state to be very important.
Cimjoint Analysis
The seafood dealers were asked to rate the 10 hypothetical
shrimp products shown in Table 2 on a scale of 0-10, with 0
indicating least preferred and 10 indicating most preferred. These
products were designed to permit quantification of seafood dealer
preferences for four shrimp product features that are within the
control of shrimp farmers: size, refrigeration state (product), form,
and price. The first nine products comprised the orthogonal frac-
tional factorial design for the analysis. The tenth "holdout" product
was selected to represent the most feasible whole shrimp product
for shrimp farmers to market directly, without processing.
The conjoint model parameters were estimated using ordinary
least squares regression: results are shown in Table 4. Coefficients
were estimated for the entire sample of dealers, and for subgroups
of dealers who attributed more than 50% of their total sales to
wholesale (wholesale-to- wholesale and wholesale-lo-retail com-
bined) or to retail. The coefficients for all dealers combined were
statistically significant al P = 0.05, except for the coefficients for
state = fresh, and for price = $5.50/lb (significance varies for
dealers in each sales category). The regression constant was esti-
mated at 3.83 for all dealers, and is interpreted as the mean pref-
erence rating, with feature level coefficients measuring deviation
from that rating in response to a particular product attribute. The
adjusted R-Square value computed for this model, interpreted as
the proportion of the variability in the dependent variable (rating)
that can be explained by the variability in the independent vari-
100
90
80
70
60
50
40
30
20
10
0
W^%^
hl^
■
1
■ 1
■ I
no I uncertain
all
yes
no 1 uncertain j
wholesalers
yes no uncertain
retailers
yes
no 1 uncertain
combo/other
yes
willingness to buy directly from shrimp farmer
Figure 3. Dealer willingness to buy directly from shrimp farmers, within each sales category.
586
WiRTH AND Davis
TABLE 3.
Mean rating and ranking of shrimp features in dealer
purchase decisions.
TABLE 5.
Utility of shrimp product feature levels to seafood dealers.
Product
Feature
Mean Rating (Ranking)
All
Wholesale
Retail
Combo
Quality
Freshness
Smell
Price
Color
Size
Consistent size
Taste
Consistent taste
Tails
Raw
Frozen
Fresh
Whole
Country of origin
P&d
Wild caught
Nutritional value
Farm raised
Imported
Cooked
9.51(1)
8.82(2)
8.75 (3)
7.73 (4)
7.61 (5)
7.51 (6)
7.37(71
7.17(8)
6.93 (9)
6.49(10)
5.88(11)
5.82(12)
4.64(13)
4.23(14)
4.19(15)
3.44(16)
3.40(17)
3.14(18)
3.00(19)
2.73 (20)
1.50(21)
9.57(1)
8.88(2)
8.66(3)
8.04(5)
8.04(4)
7.80(6)
7.70(7)
7.19(81
6.97 (9)
6.42(11)
5.86(12)
6.63(10)
4.22(15)
4.29(14)
4.32(13)
3.53(18)
3.73(17)
4.11 (16)
3.36(19)
3.26(20)
2.04(21)
9.61 (1)
9.03 (3)
9.12(2)
7.78(4)
7.44(5)
7.27(7)
7.10(8)
7.38(6)
6.94(9)
6.79(10)
6.08 (11)
5.79(12)
5.00(13)
4.32(14)
4.14(15)
3.23(16)
3.16(17)
2.74(19)
3.01 (18)
2.65 (20)
1.25(21)
9.38 ( 1 )
8.44(5)
8.31 (6)
7.06(10)
7.44(8)
8.13(7)
8.94(3)
8.56(4)
9.31(2)
7.13(9)
5.33(12)
4.27(15)
4.33(14)
4.19(16)
5.44(11)
4.56(13)
4.06(17)
2.38(19)
2.31 (20)
2.93(18)
0.93(21)
ables (size, state, form, and price) is 0.096. The low value is due
in part to the highly cross-sectional nature of the data. Aggregating
responses across individuals introduces additional variation due to
differences in each respondent's subjective rating for the same
product (Harrison et al. 1998). The F-statistics indicate that all
models were statistically significant at the a = 0.05 level.
The regression coefficients provide a direct measure of utility
for the levels specified for each feature. The effects coding tech-
nique used in this study constrains the utility of the levels of each
feature to sum to 0, so the utility of the base level for each attribute
is easily calculated (Table 5). The relative importance of each
attribute is then the range of utility over all levels of that attribute,
expressed as a percentage of the sum of the utility ranges for all
attributes. Only ratings of the nine products included in the frac-
Utilitv
Feature
Level
All
Wholesale
Retail
Combo
Size
State
Form
Price
Extra large
Large
Medium
Fresh
Frozen
Whole
Tails
P&d
$3.00/lb
$5.50/lb
S8.00/lb
0.49
0.28
-0.76*
0.06
-0.06*
-0.92
1.51
-0.59*
0.66
0.23
-0.90*
0.66
0.34
-1.00*
-0.14
0.14*
-0.87
1.36
-0.49*
0.99
0.19
-1.19*
0.26
0.90
0.34
0.07
■0.59*
-0.97'
0.14
0.07
■0.14*
-0.07^
■1.03
-0.87
1.66
1.40
-0.63*
-0.54'
0.54
1.09
0.20
0.65
-0.74*
-1.74'
* Calculated.
tional factorial design were used to determine the utility and rela-
tive importance of each attribute.
In the conjoint model, each level of each feature is considered
an independent variable contributing to the overall rating of the
product described by those feature levels. Of course, many com-
binations of these variables are logically impossible (for example,
it is not possible for a shrimp product to be simultaneously large
and extra large), hence the variables are not independent in reality.
This confounds interpretation of the significance of the coeffi-
cients for computation of feature relative importance. It is unlikely
that any of the specified features or levels genuinely have no
importance at all in buyer decisions. It is customary to calculate the
relative importance of the features, shown in Table 6, based on the
calculated coefficients of all feature levels without regard to their
individual levels of significance (Wirth et al. 1990, Harrison et al.
1998). and that practice is observed here. The calculated relative
importance of each attribute is generally affected very little by this
convention.
Product form was the most important shrimp feature for all
dealers, contributing almost 50% to the preference rating. Tails
were strongly preferred, and contributed more to the product utility
value than any other feature or feature level. Price contributed
almost 30% to the rating and was slightly more important than
TABLE 4.
Shrimp dealer conjoint model coefficients, estimated by linear regression.
All
Wholesale
Retail
Combo
Coeff.
SE
P
Coeff.
SE
P
Coeff.
SE
P
Coeff.
SE
P
Constant
3.83
0.10
0.00
3.75
0.17
0.00
3.91
0.14
0.00
4.41
0.39
0.00
Size extra large
0.49
0.14
(1.0(1
0.66
0.23
0.00
0.26
0.19
0,17
0.90
0.52
0.09
Size large
0,28
0.14
0.04
0.34
0.23
0.14
0.34
0.19
0.07
0.07
0,52
0.89
State fresh
0.06
0.10
0.55
-0.14
0.17
0.42
0.14
0.14
0.31
0.07
0.39
0.86
Form whole
-0.92
0.14
0.00
-0.87
0.23
0.00
-1.03
0.19
0.00
-0.87
0.52
0.10
Form tails
1.51
0.14
0.00
1.36
0.23
0.00
1.66
0.19
0.00
1.40
0.52
0.01
Price $3.00/Ih
0.66
0.14
0.00
0.99
0.23
0.00
0.54
0.19
0.00
1.09
0.52
0.04
Price $5. 50/1 h
0.23
0.14
0.09
0.19
0.23
0.40
0.20
0.19
0.28
0.65
0.52
0.21
F (model)
29.51
0.00
12.46
0.00
15.34
0.00
3.31
0.00
Adj. R-Square
0.10
0.11
0.09
0.10
Seafood Dealers' Shrimp-Purchasing Behavior
587
TABLE 6.
Relative importance of shrimp product features to seafood dealers.
Relative Importance (%)*
Attribute
All
Wholesale
Retail
Combo
Size
23.3
26.1
17.9
26.4
State
2.2
4.4
5.5
1.9
Form
45.3
35.2
52.0
31.9
Price
29.1
34.3
24.7
39.8
* Relative importance does not sum to 100% because of rounding.
size. As expected, the highest preference was for the lowest price
and the largest size. Refrigeration state had no significant effect on
Ihe product rating, suggesting that dealers are completely indiffer-
ent to the shrimp refrigeration state in their shrimp-purchasing
decisions. Results were fairiy consistent between all dealers com-
bined and the wholesaler and retailer groups, except that form was
more important and size was less important to retailers.
The model can be validated by comparing the actual mean
dealer ratings with the ratings predicted by the model for the
"holdout" product #10 (large, fresh, whole shrimp for $5.50/lb).
The buyer utility for the product is the base utility level plus the
sum of the utility values for each selected product feature. The
predicted utility for the "holdout" product #10 was calculated as
3.48. The actual dealer mean rating for product #10 was 3.08 with
a standard deviation of 4.08. Thus the model's predicted rating is
quite accurate.
CONCLUSIONS
The demand for seafood in the United States far exceeds the
amount produced by US commercial fishermen and aquaculture
producers. The US shrimp farming industry has been expanding
rapidly in the southern United States in response to the excess
market demand for shrimp. Shrimp farmers wish to harvest and
market their products as quickly as possible, at the lowest possible
costs, so the shrimp product form leaving the farm is likely to be
live shrimp or fresh, head-on shrimp. One marketing alternative,
especially during the early stages of industry development, is for
shrimp farmers to market their products directly to seafood dealers.
This research was designed to identify and characterize the
shrimp-purchasing behavior of seafood dealers (wholesale and re-
tail) in Ihe southeastern United States and to identify challenges
and opportunities associated with the seafood dealer market.
The results of the seafood dealer survey and conjoint analysis
of dealer product ratings suggest that the shrimp dealer market is
not an especially good candidate for direct sales of whole, farm-
raised shrimp. The large majority of dealers are willing to buy
farm-raised shrimp direct from the farmer but dealers revealed a
strong preference for shrimp tails, rather than whole shrimp. The
small percentage of dealers willing to purchase whole shrimp
would only be able to support a small volume of shrimp products
in a niche market.
This research also reveals other potential barriers to the dealer
market. Price is extremely important to dealers, contributing 30%
to the shrimp purchase decision. Shrimp dealers attach little im-
portance to farm-raised vs. wild-caught or to country of origin, so
dealers may be unwilling to pay higher prices for domestic farm-
raised shrimp, compared with shrimp from other sources. Dealers
are also completely indifferent to the shrimp refrigeration state
(fresh vs. frozen) in their shrimp-purchasing decision, suggesting
that domestic shrimp farmers cannot obtain any competitive ad-
vantage or product differentiation by selling fresh, never frozen,
shrimp, which is the farmers' preferred refrigeration state for mar-
keting purposes.
Overall, the results of this study indicate a potential dealer
market for fresh, farm-raised shrimp in a variety of sizes, but there
is considerable resistance to the whole or live, head-on shrimp
form. The mail survey and conjoint experiment results suggest that
shrimp farmers interested in successfully marketing to seafood
dealers may be required to process their product to offer shrimp
tails, rather than whole shrimp. Each shrimp farmer will have to
compare his own costs versus returns for both whole shrimp and
shrimp tails before choosing the product form and outlet that yields
the highest profit margin.
ACKNOWLEDGMENTS
This research was supported by the Florida Agricultural Ex-
periment Station, and approved for publication as Journal Series
No. R-09329. The authors thank Tara Minton for formatting this
document and Brian Boman, Elizabeth Lamb, Zhenli He, and an
anonymous reviewer for their helpful comments.
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pp. 81-103. http://www.st.nmfs.gov/stl/econ/oleo/chap6.pdf
National Marine Fisheries Service. 2001. Fisheries of the United States,
2001. http://www.st.nmfs.gov/stl/fus/fus01/
Schumann. D. 2000. President. Indian River Aquaculture. Vero Beach,
Florida. Personal Communication.
Shang. Y. C. 1990. Advances in world aquaculture. Volume 2. Aquaculture
economic analysis: an introduction. Baton Rouge, LA: The World
Aquaculture Society.
Wirth, F. F.. C. K. Halbrendt & G. F. Vaughn. 1990. Conjoint analysis of
the mid-atlantic food-fish market for farm-raised hybrid striped bass.
Experiment Station Bulletin No. 488, College of Agricultural Sciences.
University of Delaware.
Yokoyama, K. M., S. T. Nakamoto & K. Wanitprapha. 1989. Shrimp
economic fact sheet. Department of Agncultural and Resource Eco-
nomics. University of Hawaii.
JoKiiuil oj Shclljish Kescarch, Vol. 22, No. 2, 589-595, 2003.
OLFACTORY DETERRENTS TO BLACK DRUM PREDATION ON OYSTER LEASES
KENNETH M. BROWN,' GARY W. PETERSON," PATRICK D. BANKS,^ BRIAN LEZINA,^
CHARLES RAMCHARAN,' AND MICHAEL MCDONOUGH'
^Department of Biologic Sciences: 'Coastal Ecology Institute, Louisiana State University. Baton Rouge.
Louisiana 70803: Louisiana Department of Wildlife and Fisheries Baton Rouge. Louisiana 70898:
Departnwnt of Coastal Sciences Gulf Coast Research Lahoratoiy Ocean Springs. Mississippi 39564
ABSTRACT Black drum [Pogonias croiiiis) predation is a serious tlireat to oyster production on Louisiana leases, and leaseholders
hypothesize that black drum carcasses suspended above leases deter predation. We conducted experiments under laboratory and field
conditions to test whether the scent of dead con-specifics deterred black drum predation. Preliminary experiments indicated that fish
>70 cm total length were effective predators, and oysters <70 g wet total weight were preferred, and we used these sizes in subsequent
experiments. Salinity did not affect feeding rates. Experiments in 30.000 L raceways indicated that scent did not significantly lower
feeding rates. Parametric analyses of factorial experiments on oyster leases at two sites in Barataria Bay. Louisiana, during the fall and
spring (periods of the year when fish feeding is most intense), indicated that scent reduced feeding rates by 10% to 20%. but only at
one site in one season. Nonparametric analyses corroborated seasonal differences indicated by parametric analyses, but not the scent
effect. We therefore conclude that scent froin dead con-specifics is not an effective control strategy under most conditions. Dredge
hauls during experiments suggested mortalities to all predators ranging from 63.1% to 92.5% within the first 4 weeks after seeding.
The relative mortalities to black drum, southern oyster drills [Snamcmita haeinastoimi) or possibly PerkiiLsiis marinus infections varied
among sites, as did temporal patterns of mortality within and among seasons.
KEY WORDS: black drum, oysters, deten-ents. olfactory
INTRODUCTION
Oyster reefs (Crassostreu virginica) are important components
of Gulf of Mexico coastal ecosystems, providing shelter for several
economically important invertebrates and larval fish, improving
water quality, and stabilizing shorelines (Bahr & Lanier 1981,
Zimmerman et al. 1989). Oyster production in the northern Gulf of
Mexico is greatest in an "optimal" salinity band ranging frotii 5-15
psu in coastal marshes. Survivorship is physiologically constrained
at lower salinities, while predation losses are too high to sustain
populations at higher salinities (Melancon et al. 1998), except at
interlidal sites where oysters have a refuge from predation (Roeg-
ner & Mann 1995, O'Beirn et al. 1996. Brown & Stickle 2002).
Oysters spawn in the northern Gulf of Mexico as water tem-
peratures rise above 25°C in the spring, and then resume spawning
as temperatures drop below that level in late summer and fall
(Supan 198.3, Banks & Brown 2002). Warmer temperatures in
summer months are also associated with increased prevalence of
the parasitic protozoan Perkinsus marinus C'Demio'"), which can
result in mortality as high as 50% in oyster populations, especially
at higher salinities (La Peyre et al. 2003).
The Gulf of Mexico oyster industry was developed in the mid-
eighteen hundreds and was further spurred by the development of
the oyster dredge in the early nineteen hundreds. Oysters are har-
vested from public areas (mostly reserved as "seed grounds" where
leaseholders can collect small oysters to plant on their leases) and
private leases. Seed oysters are planted in the fall, and typically
harvested in their second spring when they reach market size.
Louisiana currently has approximately 8700 leases covering
419.000 acres under cultivation. Louisiana's oyster production av-
erages almost one third of the national production, and the Gulf of
Mexico region produces 60% of the national production, worth
almost 50 million dollars annually.
Coastal zones producing maximal oyster yields are however
This research was supported through the Gulf Oyster Industry Program of
the National Sea Grant College.
changing, as coastal erosion results in saltwater intrusion, shrink-
ing the optimal salinity band (Melancon et al. 1998). As salinities
increase, predation by black drum (Pogonias cromis) and southern
oyster drills {Stramonita haemastoma) increases, as does preva-
lence of Dermo. The iinportance of black drum as predators was
clearly indicated by a survey of Louisiana oyster leaseholders
(Louisiana Department of Wildlife and Fisheries [LDWF] 1999)
indicating significant loss in 55% of the leases. Seed oysters may
be stressed during transport from seed areas to oyster leases, and
they produce scents that attract black drum. Almost 80% of lease-
holders who had recently seeded oysters reported significant losses
to black drum.
Black drum inhabit near-shore and estuarine waters in the Gulf
of Mexico (Simmons &. Breuer 1962), mature at the end of their
second year, and spawn in coastal passes from February through
March. The larvae are transported into estuaries where the juve-
niles mature. The largest black drum in the Gulf of Mexico are
over 40 years old, and reach 105 cm in length and 29 kg in weight
(Sutter et al. 1986, Beckman et al. 1990). Black drum consume
over 30 oysters per night (Sutter et al. 1986), with small "seed"
oysters planted in leases preferred over natural reefs (Cave 1978,
Cave & Cake 1980, Dugas 1986). Oysters are consumed by fish
greater than 40 cm in total length, and oyster sizes consumed
increase with length. Juvenile black drum feed on a variety of
invertebrates (Pearson 1929, Gunter 1945, Darnell 1958). Produc-
tion losses may be as high as 1500 sacks per lease in some parishes
(LDWF 1999). Most oysters are lost in March, when groups offish
return to coastal leases to feed after spawning, or in October,
immediately after small seed oysters are bedded.
Our long-term goal is to develop deterrents to black drum pre-
dation on oyster leases. We first performed preliminary experi-
ments to understand basic aspects of the predator-prey interaction,
such as the vulnerability of different size classes of oysters, the
role that drum body size plays in determining feeding rates and
prey-size selection, and how the predator-prey interaction is af-
fected by salinity. Based on prior experimental work (Cave 1978),
or analyses of diet (Dugas 1986, Luquet 1992), our hypothesis wa.s
589
590
Brown et al.
that larger fish would be more effective predators, and small oys-
ters most at risk. We also expected reduced feeding rates with
lower salinity, because leases in coastal areas experience higher
mortality (LDWF 1999). These experiments also determined
which sizes of predators and prey and salinities were used in the
laboratory olfactory cue experiments discussed in the next para-
graph.
Second, we compared feeding rates in the laboratory, with or
without a black drum carcass; our hypothesis being that alarm
substances deter predation, as leaseholders report that carcasses
suspended above leases reduce losses (P. Vujnovich Jr., Pers.
Comm.). Alarm substances have been shown in other cases to
cause avoidance behavior in fish (reviewed in Smith et al. 1994,
Mathis et al. 1995, Chivers & Smith 1998). If scent cues reduce
feeding rates, scent, or components of scent, could be added above
the lease as a deterrent.
Third, to determine if olfactory cues were practical deterrents
under field conditions, we conducted experiments on commercial
leases in Barataria Bay, Louisiana. With the help of a leaseholder,
we planted leases with seed oysters with or without drum car-
casses. Comparison of predation rates between control and experi-
mental plots determined whether scent was an effective deterrent.
We placed oysters in trays and recorded their survival at 2 dis-
tances from scent sources in plots to determine how effective
scents were at a distance, and the role of current in displacing
scents. We also made hauls with a dredge to independently esti-
mate losses to predation. This design was replicated on two leases,
and in both the fall and spring.
MATERIAL AND METHODS
Preliminary Experiment
Predation rates and size preference of black drum were mea-
sured in experiments conducted from December 1999 to March
2000 at the LDWF Lyle St. Amant Marine Laboratory on Grand
Terre Island, Louisiana. Black drum captured by hook and line or
trot lines were held for 5 days at ambient salinities with oyster prey
for acclimation, and were then starved for 2 days before experi-
ments to standardize hunger levels. Experiments were conducted
with a single fish for 5 days in 2000-L tanks with biologic filters.
Barataria Bay water was mixed with fresh water to achieve aver-
age salinities of 13 (±0.4, standard error of the mean) and 36 (±0.3)
psu; water temperature averaged I5°C (±1.0). Oysters were pro-
vided in three sizes (10 <50 g total wet mass, 10 between 51-150
g, and 7 >151 g) and were replenished daily. We used two size
classes of black drum (30-70 cm (average = 51.4 ± 0.7] and >70
cm [90.6 ± 0.9] total length). Because prey sizes were presented in
unison to the fish, they were not independent treatments. We there-
fore used a multivariate analysis of variance (Peterson & Reynaud
1989). Effects of predator and prey size, and salinity were evalu-
ated in the factorial arrangement of treatments (2 predator x 2 prey
sizes X 2 salinities).
Laboratory Experiment
These experiments were conducted during October to April of
1999 to 2000 (to avoid low dis.solved oxygen concentrations) at the
Grand Terre Laboratory in large outdoor raceways (concrete block
tanks measuring 10 x 3 x 1 m deep) holding 30,000 L of water.
Tanks initially received filtered seawater (ambient salinity aver-
aging 27.9 ± 0.7 psu, and temperature averaging 22.4 ± 0.7°C)
from Barataria Bay, and water was re-circulated through oyster
chip filters during experiments. Two black drums, greater than 70
cm total length, were held in each tank. Fish were measured,
tagged, and acclimated in the tank for 1 week before starting
experiments. Temperature, dissolved oxygen, and salinity were
measured daily. Seventy-five small and 25 medium oysters were
added initially. Broken shells or missing animals were counted
daily and oysters replenished.
For each 5 day long experiment, two tanks were randomly
selected as experimental tanks, and two as controls. Experimental
tanks had a single black drum carcass suspended in a buriap bag at
one end of the tank. After each experiment, fish were removed; the
tank was drained and scrubbed, and refilled with filtered water
from Barataria Bay. Fish used in experimental treatments were
used next in controls (or the reverse) and were either then released
or sacrificed and used for the scent deterrents. Numbers of oysters
consumed were compared between the two treatments with a one-
way analysis of variance.
Field Experiment
Whereas laboratory experiments aid in understanding predator
behavior under controlled conditions, field experiments determine
if olfactory cues deter predation under natural conditions and are
feasible for industry use. We therefore made arrangements with a
leaseholder to conduct experiments on two leases in Barataria Bay
with a history of predation problems. The leases were in Lake
Grand Ecaille in southeast Barataria Bay (29°35'06"N,
89°33'47"W) with historically high predation levels (Mr. Peter
Vujnovich, Jr., pers. comm.), and in Creole Bay in west Barataria
Bay (29°35'73"N, 89°34'10"W), a site with intermediate historical
levels of black drum predation (Fig. 1 ). The field experiments were
replicated twice, once in October 2000 and again in March 2001.
At each site, four 60-m diameter, circular plots were seeded by
the leaseholder with oysters at densities typical for leases. Each
plot was randomly assigned as a control or treatment, and plots
were located 100-m apart to minimize infiuence from other treat-
ments. Immediately after seeding, two black drum carcasses were
enclosed in burlap bags and suspended from a PVC pole in the
center of the experimental plots, and plastic "oyster grow out"
trays (60 x 50 x 10 cm) each with a minimum of 100 oysters were
placed in all four plots to assess predation rates. Three trays were
near the center, and one tray was set at the end of three equidistant
rays (Fig. 2). Trays were inspected for predation. oysters replen-
ished, and the deterrent sources renewed at 1-week intervals, for 4
weeks. The number of oysters gaping (a sign either of oyster drill
predation. Brown & Richardson 1987, or possible mortality to
Dermo), or missing (presumably either consumed or at least
handled and removed from trays by black drum) were recorded at
each date. Separate trays (2 per plot) enclosed with 3 cm Vexar and
retrieved at the end of the experiment, indicated that natural oyster
mortality, or mortality caused by handling, averaged only 2%, and
so we have assumed that gaping oysters were mostly the result of
predation by oyster drills. Oyster drills were quite common on
trays when they were retrieved, averaging from 2.7/tray at the
coastal site to 8.5/tray at the estuarine site.
Each plot was also sampled with a dredge (0.3 x 0.6 m open-
ing) to as.sess oyster densities. Three 30-m long dredge hauls (par-
allel to each axis on which trays were .set) were taken in each plot
at the start, after 2 weeks, and at the end of the experiment (after
4 weeks), and all oysters were pooled for each plot and date.
Data were analyzed in repeated measures factorial analyses of
Olfactory Deterrents to Black Drum
591
Figure 1. Map of Barataria Bay, with botli sites where experiments were conducted on commercial oyster leases indicated.
variance. Data from trays for each date, or the three dredge hauls,
were the repeated variables in the two way design (presence or
absence of scent versus two seasons) conducted separately for each
site. Prehminary statistical analyses at all sites indicated no sig-
nificant effect of distance (e.g., center versus edge of plot. P > 0.08
in all cases) so all trays in a plot were considered replicates.
Dependent variables were percent of oysters surviving (n = 12
trays for both plots in each treatment at each site at each date),
percent mortality because of black drum, percent of oysters gap-
ing, and numbers of oysters retrieved from dredge hauls (n = 2
plots per treatment per site per date). In several cases, data were
not nomially distributed, even after log transformation. We there-
fore performed a nonparametric factorial test, a 2-way (season x
scent treatment) Sheirer-Ray-Hare extension of the Kruskal Wallis
test (p. 446. Sokal & Rohlf, 1995). This test is essentially a two-
way analysis of variance performed on the ranked data that pro-
vides H statistics that test the treatment and interaction effects.
Each of the 4 weeks was considered replicates in this analysis.
RESULTS
Preliminary Experiment
Black drum size had a highly significant effect on feeding rate
(Table I, Wilk's \ = 0.81, F = 7.9, P = 0.008), with larger fish
consuming on average 3.8 oysters and smaller fish only 0.9 oysters
per week. Oyster size was also important (Wilks's \ = 0.64, F =
9.4, P = 0.0006). with a Tukey's a posteriori test indicating that
the 5.2 small oysters consumed on average was significantly
greater than the 1.7 medium oysters consumed. No large oysters
were consuined by the fish. In contrast, salinity neither had an
effect on feeding rates (Wilk's \ = 0.99, F = 0.43. P = 0.52),
nor were any of the interactions between the main treatment effects
significant. Based on these experiments, we only used fish larger
than 70 cm total length in later experiments, and small or medium
sized oysters as prey. Because salinity had no consistent effect, we
used ambient salinity water for the laboratory scent experiments.
592
Brown et al.
TRAYS WITH
SEED OYSTERS
LOCATION OF SCENT
DETERRENT
CONTROL PLOT 1 EXPERIMENTAL PLOT 1
EXPERIMENTAL PLOT 2 CONTROL PLOT 2
Figure 2. Layout of the four circular plots at each of the two sites. Two
plots were controls, and two plots had centrally-located black drum
carcasses renewed weekly during the 4-week long experiments. Loca-
tions of trays containing seed oysters are also indicated. Distances
among plots are not to scale.
Laboratory Experiment
Under laboratory conditions, the presence of the scent of dead
con-specifics depressed feeding rates (Fig. 3) but not significantly
(F, ,T = 0.9. P = 0.37). Feeding rates were quite variable among
replicates, and overwhelmed differences between the two treat-
ments.
Field Experiment
At Creole Bay (Table 2), the repeated measures analysis of
variance indicated a strong difference in oyster survival among
weeks, and a significant interaction between time and season. The
general pattern was for survival rates to increase with time (Fig. 4),
although the shape of the curves differed between the fall and
spring experiment. Evidently black drum were quickly attracted to
the seeded leases, but moved away later (especially in the fall) as
seed oysters were depleted on the lease. The significant season
TABLE L
Average feeding rates |x ± SE, N in parentheses) for 2 size classes of
black drum feeding at 2 salinities on 3 size classes of oysters.
Fish
Salinity
Small
Medium
Large
<70cm
\n, (5)
1.8 + 0.6
(1
■if^c (4)
3.5 ± 1.2
0
>70 cm
\yi, (6)
10.0 ±4.5
4.0 ±2.6
36%t (3)
5.7 ±4.7
2.7 ±2.7
T3
E
V)
c
o
O
(0
<0
CO
>
O
Si
E
3
Z
40
30
20
10
Control Scent
Treatment
Figure 3. Number of oysters consumed (x ± SE) in the control and
scent treatments in the laboratory experiment. There were 6 replicates
in each treatment.
main effect still however indicates that mortality rates were much
higher overall in the spring than in the fall. Comparison of Tukey's
a posteriori tests indicated that survival rates differed among sea-
sons for each of the 4 weekly samples. In contrast, the scent
treatment was not significant, nor were any of the interactions of
scent and other treatments significant. The nonparametric test also
indicated a strong difference between seasons (H = 8.5. P< 0.01),
but an insignificant treatment (H = 0.3, P > 0.05) and interaction
(H = O.I, P>0.Q5) effect.
At Lake Grand Ecaille, there were also differences among
weeks in oyster survival (Table 2), with a significant interaction
between time and season. In the fall, survival rates were high
initially, but dropped considerably, probably caused by movement
of black drum onto the lease (Fig. 5). The significant season main
effect again suggested different mortality rates among seasons, and
survival was essentially zero in most of the weeks during the
spring, probably caused by high predation rates by black drum
present at the site from the initiation of the experiment (Fig. 5).
Comparison of Tukey's a posteriori tests indicated significant dif-
ferences in survival between seasons, for each of the weeks. There
was also a significant treatment effect at this site however, al-
though the increased survival in scent treatment plots averaged
only 10-20%, and occurred only in the fall, when mortalities were
TABLE 2.
F values from two-way repeated measures analyses of variance of
oyster survival at two sites in Barataria Bay, Louisiana, in two
seasons. The repeated measures are four samples through time at
each site and season.
Source of Variation
Creole Bay
Lake Grand Ecaille
Time
Time x Season
Time x Scent
3 way interaction
Season
Scent
Scent x Season
80.8**
22 9**
0.4
0.1
117.9**
1.0
0.03
92.4**
94.9**
3.2*
1009.0**
13.7**
5.4*
* Significant at P < 0.05
** Significant at P < 0.01.
Olfactory Deterrents to Black Drum
593
100
c
^>
'>
3
CO
to
I.
0)
>
O
Week
Figure 4. Percent survival of oysters I x ± SE, pooled over both scent
treatments, n = 24) in samples collected during 4 weeks at Creole Bay
in l«o seasons.
TABLE 3.
F values from t\»o-way repeated measures analyses of variance of
oysters collected in dredge hauls at tv\o sites in Barataria Bay,
Louisiana, in two seasons. The repeated measures (= time) are three
dredge hauls through time at each site and season.
Source of Variation
Creole Bay
Time
L3.1
Time x Season
2.7
Time x Scent
1.2
3 way interaction
L3
Season
7.4
Scent
0.3
Scent X Season
2.9
* Significant <H P < 0.05.
** Signitlcam at /> < O.OI.
Lake Grand Ecaille
32.7**
8.0
1.8
6.6
7.0*
0.1
Lfi
much lower than in the spring (Fig. 5). However, a posteriori tests
indicated treatment plots differed from controls only for the last
week of the experiment in the fall. The nonparanietric test again
indicated a significant season effect (H = 15.1, P < 0.01 ). but not
a significant treatment (H = 0.4. P > 0.05) nor interaction effect
(H = 0.\. P> 0.05).
Oysters collected in dredge hauls also declined dramatically
through time (Table 3, Fig. 6) corroborating the high mortality
rates suggested by the data from the trays deployed in plots. Oyster
densities also differed between the two seasons, but there were
neither scent treatment effects nor interactions. Oyster survival
(estimated by dividing final mean densities by initial densities)
varied from 7.5'7f in the fall at Creole Bay to 8.9'7r in the spring.
At Lake Grand Ecaille, 36.9% of the oysters survived in the fall,
but only 9.7% in the spring. Thus these data also suggest, as did the
data from trays, that mortality rates were higher in the spring at the
coastal site. Nonparanietric tests were not necessary here as data
were normally distributed (SAS, Inc. 1988, procedure Univariate).
Mortality caused by black drum (again as estimated by the
fraction of shells missing from trays) varied with time and season
(Table 4. Fig. 7). At Creole Bay in the fall, mortality due to drum
peaked during week 2, and was always at least 50%. In the spring,
mortality caused by fish steadily declined. At Lake Grand Ecaille,
mortality due to black drum steadily increased through the experi-
ment in the fall, and was constant and near 100% in the spring.
Conclusions from nonparanietric tests were again similar: no sig-
nificant treatment effects occurred at Creole Bay, but a significant
seasonal effect (H = 43, P < 0.01 ) occurred at Lake Grand Ecaille.
The percentage of oysters gaping in trays also varied with time,
and was dependent on season at one of the sites as well (Table 4).
At Creole Bay, percentage of oysters gaping was fairly consistent
through time in the fall, but increased with time in the spring. At
Lake Grand Ecaille. percent of shells gaping was high initially in
the fall, but declined through time. Gaping shells were essentially
absent in all but the final week in the spring, explaining the strong
seasonal effect at this site. The nonparanietric tests re-enforced
these conclusions: none of the treatment contrasts were significant
{P > 0.05) at Creole Bay, but there was a significant seasonal effect
(H = 15.1, P < 0.01) at Lake Grand Ecaille.
DISCUSSION
Predator Prey liiteraetioii
Our data indicate that large black drum are much more signifi-
cant predators of oysters than smaller fish, and that smaller oysters
are much more at risk. These findings corroborate earlier labora-
tory feeding data (Cave 1978), studies of the diet of field caught
fish (Pearson 1929, Gunter 1945, Darnell 1958, Dugas 1986), and
results of surveys of oyster leaseholders (LDWF 1999). Oyster
100
O)
c
80
>
>
3
60
(/)
(0
40
(1)
*^
(0
>
O
20
0
12 3 4
Week
Figure 5. Percent survival of oysters (x ± SE, n = 12) in samples
collected in 2 scent treatments during 4 weeks at Lake Grand Ecaille
in two seasons.
3
CO
X
CO
k_
CO
>.
O
1 60
♦ CB Fall
— ^- CB Spr
120
i S.— '— — — . T — H - GE Spr
\ ■,,
'V \
80
V. V
■•• X » V
X * ^ N
T \ -Q. X T
40
?-.. \ " ^
"^--^^^^:^^:^^r:~::3!
1 2 3
Date
Figure 6. Number of oysters collected in hauls (x ± SP-, « = 4 plots,
pooled over both scent treatments) collected at three dates during field
experiments at both sites in two seasons.
594
Brown et al.
TABLE 4.
F values from two-way repeated measures analyses of variance of
oyster percent gaping and mortality caused by black drum at 2 sites
in Barataria Bay, Louisiana, in two seasons. The repeated measures
(= time) are four samples through time at each site and season.
% by
Site
Source
% Gaping
Black Drum
Creole Bay
Time
Time x Season
36.7**
7.9**
50.7**
6.3**
Time x Scent
0.1
0.1
3 way interaction
Season
0.5
3.2
0.8
0.9
Scent
0.1
0
Scent X Season
1.1
1.5
Lake Grand Ecaille
Time
Time x Season
21.5**
17.6**
50.5**
51.8**
Time x Scent
4.1*
3.2*
3 way interaction
Season
1.4
48.7**
LI
128.3**
Scent
0.4
0.1
Scent X Season
0.7
0.4
' Significant at P < 0.05.
■* Sianificant A P < 0.01.
leaseholders reported much higher predation rates on leases seeded
with oysters the previous fall in comparison to inactive leases, or
active leases that were not seeded the previous fall. Two mecha-
nisms could explain this higher loss. First, our experiments suggest
these small, individual oysters are easily consumed by fish. In
comparison, naturally occurring oyster reefs along the Louisiana
coast occur inter-tidally, where predation pressure by fish, stone
crabs and oyster drills is reduced because of greater aerial expo-
sure, and where oysters grow in aggregations that are hard for fish
to feed on (Brown 1997. Brown & Stickle 2002). Second, oysters
that are transported on boat decks from state-maintained seed areas
on the eastern side of the Mississippi River to commercial leases
that are often 40-80 Km away undoubtedly experience stress, and
injured oysters produce scents that attract black drum (LDWF
1999).
Surprisingly, we found no evidence that reduced salinities al-
tered feeding rates. Perhaps salinities below the 13 ppt used in
O
c
o
Q.
100
80
60
40
20
0
B^-__ a
-_J.r_7_r_m-,^
"^?'<§v .
y
y^ ^'X^"
\X \^
tr JF \ \
i A V
\T 1
/
./ ■ CB Fall
/
\t 1
/ "©" CB Spr
I -,
L- _«... QE Fall
J^ - B - GE Spr
2 3
Week
Figure 7. Percent mortality (x ± SE. n - 12) caused by black drum at
two sites in two seasons in the field experiments on leases.
these experiments reduce predation. This salinity occurs at the
so-called "conch line" in Lousiana estuaries, north of which oyster
drills are not considered serious predators (Bahr c& Lanier 1981,
Butler 1985). We also found evidence from the field experiments
that losses to oyster drills were higher in the fall particularly at
Creole Bay, whereas fish predation rates were higher in the spring
at Lake Grand Ecaille. Black drum are apparently replacing nutri-
ent reserves lost during spawning in the previous winter, and by
voraciously feeding in groups, far outweigh any advantages of
oyster growth during the winter months.
Our field experiments certainly corroborate that predation can
be a considerable mortality source for oysters planted in leases.
Losses to drum occurted at high rates in both seasons, but were
particulariy high at Lake Grand Ecaille in the spring, where es-
sentially all oysters were handled and consumed in most of the
weeks. There was also a tendency for mortality to be high initially
at Creole Bay. and for the reverse pattern to occur at Lake Grand
Ecaille in the fall. These different temporal patterns are probably
explained by black drum either being resident on the site at the
start of the experiment (for example at Creole Bay in the spring).
or being attracted to the site after the leases were seeded (for
example at Lake Grand Ecaille in the fall).
Other mortality sources for oysters include predation by south-
em oyster drills, and mortality to Dermo infections. We hypoth-
esize that Dermo infections in these experiments caused minimal
mortalities for four reasons: ( 1 ) southern oyster drills were recov-
ered in high numbers on trays, especially at the estuarine site in the
fall; (2) oysters in covered cages survived well (average of 98%)
in all experiments; (3) temperatures were not extreme, as experi-
ments were conducted in the spring and fall, not summer months
when prevalences are higher in Louisiana oyster reefs (Cook et al.
1998. La Peyre et al. 2003), and (4) Dermo prevalences were low
(weighted incidence of 0.2-0.9 on Mackin scale) in seed grounds
where oysters were collected to seed our experimental plots (P.
Banks, LDWF, Pers. Comm.). Assuming gaping shells were the
result of southern oyster drill predation. mortality to these inver-
tebrate predators appeared to be greatest in the fall, not spring
months, and occurred more frequently at the more estuarine
site.
Importance of Scent
Laboratory experiments did indicate some reduction in feeding
in the presence of scent of a dead conspecific. but the effect was
overwhelmed by differences in the feeding rates of individual fish
in each of the independent experimental replicates. Observations
indicated that fish were not fully acclimated to the laboratory
settings even after a week. Although raceways were large, fish
repeatedly rubbed against walls, and the concrete surface produced
scrapes on the fish skin. Cave (1978) kept fish for long periods in
aquaria similar to ones in our preliminary experiments, undoubt-
edly resulting in better acclimation, and perhaps producing feeding
rates that are more comparable to field conditions.
Our field experiments did not indicate that scent of dead black
drum was a practical feeding detertent. Increases in oyster survival
occuned in scent plots at Lake Grand Ecaille. but only during the
fall, when mortality rates were overall lower, and survival was
increased only by 20% at the most. Black drum feed in groups, and
a scent stimulus may have to be maximal to deter these voracious
predators, especially when they feed on leases in the spring to
Olfactory Deterrents to Black Drum
595
renew energy reserves. We deployed two careasses at the center of
a 700-nr circular plot, and it could be argued that deploying larger
numbers of carcasses would be more effective. However, this is a
small area (selected as the smallest area where an oyster boat could
circle and wash off seed oysters onto the sediment). Furthermore,
if greater numbers of black drum are fished off leases and de-
ployed, the question arises as to whether the improved survival of
oysters is caused by the scent stimulus or simply reduced fish
abundance and thus reduced predation pressure.
ACKNOWLEDGMENTS
This research was funded by the Gulf Oyster Industry Prograin
of the National Sea Grant College. The authors thank the Louisiana
Department of Wildlife and Fisheries Lyle St. Amant Laboratory
for providing lodging and a logistical base for the research, also
Dr. Frank Truesdale, and Pete Vujnovich Jr. who was instruinental
in providing oysters for use in experiments, and in seeding experi-
mental leases in Barataria Bay.
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semblages: the importance of taxal differences and spatial and tidal
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Beckman, D. W., A. L. Stanley. J. H. Render & C. A. Wilson. 1990. Age
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Brown. K. M. & T. D. Richardson. 1987. Foraging ecology of the southern
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Brown. K. M. & W. B. Stickle. 2002. Physical constraints on the foraging
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Butler. P. A. 1985. Synoptic review of the literature on the southern oyster
dnll. Thais haemastoma. NOAA Tech. Rep. NMFS No. 35:9.
Cave. R. N. 1978. Predator-prey relationships involving the American
oyster. Cras.wslrea virginica (Gmelin). and the black drum. Pogonias
cromis (Linnaeus), in Mississippi Sound. MSc Thesis. Hammond. LA:
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Chivers, D. P. & R. J. F. Smith. 1998. Chemical alarm signalling in aquatic
predator-prey systems: A review and prospectus. Ecoscience 5:338-
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Cook. T.. M. Folli. J. Klinck. S. Ford & J. Miller. 1998. The relationship
between increasing sea-surface temperature and the northward spread
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La Peyre. M. K.. A. D. Nickens. A. K. Volety. G. S. Tolley & J. F. La
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Joimial of Shellfish Research. Vol. 22, No. 2, 597. 2003.
ABSTRACTS OF TECHNICAL PAPERS
Presented at the 56th Annual Meeting
NATIONAL SHELLFISHERIES ASSOCIATION
(Pacific Coast Section)
&
PACIFIC COAST SHELLFISH GROWERS ASSOCIATION
Newport, Oregon
September 27-30. 2002
597
NSA & PCSGA. Newport. Oregon Abstracts, September 27-30. 2002 599
CONTENTS
Dan L. Ayres and Ervin J. Schumacker
Assessing populations of Pacific razor clams {Silii/iiii panda) along the Pacific coast of Washington State 601
Colleen A. Burge, Yuiclii Eugene Saito and Carolyn S. Friedman
Relationships between summer mortality and immune responses in (he Pacific oyster. Crassostrea gigas 601
Melinda D. Chambers, C.S. Friedman, L. Hauser and Glenn R. Vanblaricom
Population structure and recovery dynamics of black abalone (Halinlis cnwherodii) at San Nicholas
Island. California 601
Aimee E. Christy
2002 monitoring of harmful algae in South Puget Sound and Willapa Bay - species of concern and
future considerations 601
Aimee E. Christy and Stuart D. Glasoe
Literature review - impacts of urbanization on water quality in shellfish growing areas in Puget Sound, Washington 602
Marion Dumont
The history and development of the Puget Sound commercial geoduck industry 602
Ford Evans, Sean Matson, John Brake and Chris Langdon
Relative importance of survival and growth rate in determining yields of Pacific oysters. Crassostrea gigas 602
Carl A. Finley, Tliea T. Robbing and Carolyn S. Friedman
Life history of an exotic sabellid polychaete Terehrasabella lieteroiincinata: fertilization strategy and influence of
temperature on reproduction 602
C.S. Friedman, C.A. Burge, D.P. Cheney, R.A. Elston, A.D. Suhrbier, G.N. Cherr, F.J. Griffin, A. Hamdoun
and C.J. Langdon
Summer mortality of the Pacific oyster. Crassostrea gigas, along the West Coast of the U.S.: performance of family
lines and environmental parameters 603
Carolyn S. Friedman, James D. Moore, Thea T. Rabbins, Beverly A. Braid, Carl A. Finley, Ronald P. Hedrick,
Dolores V. Baxa, Karl B. Andree, Eric Rosenblum, Mark R. Vianl, Ronald S. Tjeerdema, Peter L. Haaker,
Mia J. Tegner and Luis 1. Vilchis
Withering syndrome of abalone in California 603
Graham E. Gillespie, Randy Webb and Todd Johansson
.Assessment and management of intertidal clam resources in British Columbia 603
Blaine Griff en, Chris Langdon and Ted DeWitt
Feeding rates of the mud shrimp Upogehia piigettensis and implications for estuarine phytoplunkton abundance 604
K. Holsman, P. Sean McDonald. D. Armstrong and J. Ruesink
Patterns in intertidal habitat use by subadult Dungeness crab (Cancer magister) 604
Geoff Hosack, David Armstrong, Brett Dumbauld. Brice Semmens and Jennifer Ruesink
Seasonal utili-^ation of intertidal habitats by fish in a Washington State ceiastal estuary 604
R. Russ Jones, Carl Schawrz, Bart DeFrietas and Lynn Lee
Biomass surveys and active management of intertidal razor clams (Silic/iia patula) at beaches near Massett, Haida
Gwaii. Canada 60.5
Matthew J. Krachey and Steven C. Hackett
Economics of California's Dungeness crab (Cancer magister) fishery, preliminary results 605
Chris Langdon, Sean Matson, John Brake and Ford Evans
The Molluscan Broodstock Program: family-based selection improves yields of Pacific oysters. Crassostrea gigas 605
Heather M. Macrellis, Jennifer L. Ruesink and Brett Dumbauld
The role of culture practices in structuring interactions between cultured oysters and native eelgrass 606
Sean E. Matson and Chris Langdon
A specific pathogen free culture system for Crassostrea gigas larvae and spat 606
P. Sean McDonald, Gregory C. Jensen and David A. Armstrong
Biotic resistance to European green crab. Carcinus maenas, by native analogs in the Northeastern Pacific 606
C. Pearce, T. Daggett, T. Chopin, K. MacKeigan, V. Zitko and S. Robinson
Effect of diet on somatic growth of juvenile green sea urchins (Sirongylocentrotiis droebachiensis) 607
Don P. Rothaus, R.E. Sizemore, M.J. Ulrich and Carolyn S. Friedman
Trends in pinto abalone (Hatiolis kamtschatkana) abundance at ten sites in the San Juan Islands and management of
the species in Washington State 607
600 Abstracts. September 27-30. 2002 NSA & PCSGA, Newport. Oregon
Steven S. Rumrill and Victoria K. Poulton
Ecological role and potential impacts of molluscan shellfish culture in the estuarine environment of Humboldt
Bay. CA 607
B.C. Smith, C.E. Gnie, N.P. Kohn and J.P. Davis
The effects of the herbicide Rodeo® on Pacific oyster gametogenesis and tissue accumulation 608
Andrew D. Suhrbier, .Aimee E. Christy, Hector S. Beltran, Daniel P. Cheney, Jonathan P. Davis, Kenneth M. Brooks
and Frank J. Smith
Mussel growth and food utilization in relation to water quality on a raft system in Puget Sound, Washington 608
Vera L. Trainer, Barbara M. Hickey and Ervin J. Schumacker
Results from the Olympic Region Harmful Algal Bloom (ORHAB) Project on the Washington State coast: the value
of a collaborative project 608
B. Vadopalas, L.L. LeClair and P. Bentzen
Genetic differentiation amongst geoduck clam (Panopea ahnipta) populations revealed by allozyme and
microsatellite analyses 609
B. Vadopalas and Don P. Rothaus
Trial use of the U.S. Navy Remotely Operated Vehicle (ROV) SORD IV for sampling deep water geoduck clams
( Panopea ahnipta) 609
Donald E. Velasquez, S.F. Burton, D.A. Sterritt and B. McLaughlin
Shell condition testing of Dungeness crab in Puget Sound. Washington 609
NSA & PCSGA, Newport, Oregon
Abslmcis. September 27-30, 2002 601
ASSESSING POPULATIONS OF PACIFIC RAZOR CLAMS
(SIUQUA PATULA) ALONG THE PACIFIC COAST OF
WASHINGTON STATE. Dan L. Ayres, Washington Depart-
ment of Fish and WildMfe. 48 Devonshire Road. Montesano, WA
98563; and Ervin J. Schumacker, Quinault Department of Natu-
ral Resources. PC Box 189. Taholah, WA 98587.
Perfect habitat for the Pacific razor clam (Silii/tia panda) is
found along the Pacific Ocean beaches in Washington State. To
detennine total abundance of razor clams, the newly designed
Pumped Area Method recently became the method of choice. This
method requires water to be pumped from the surf or a nearby
lagoon. This water, as it is directed through a handheld PVC wand,
is used to liquefy the sand within an aluminum ring ( '/: square
meter in area). The razor clams found float to the surface and are
removed, measured and returned. This process is repeated along a
randomly selected transect with 6 rings completed every 50 feet.
Each transect requires one turn of the tide (5 hours). For each mile
of razor clam habitat determined to be on management beach, one
transect is completed. The data collected is used to calculate the
average number of razor clams per square meter. Using an estimate
of the number of square meters of razor clam habitat, the total
number of razor clams can be determined. The State of Washing-
ton and the Quinault Indian Nation use this jointly determined
abundance estimate to co-manage the harvest of razor clams at the
Copalis, Mocrocks and Kalaloch manageinent beaches.
RELATIONSHIPS BETWEEN SUMMER MORTALITY
AND IMMUNE RESPONSES IN THE PACIFIC OYSTER.
CRASSOSTREA GIGAS. Colleen A. Burge, Yuichi Eugene
Saito and Carolyn S. Friedman, School of Aquatic and Fishery
Sciences, University of Washington, Seattle, WA 98195.
The Pacific oyster, Crassostrea gigas. has experienced summer
mortality events in the Pacific Northwest and Japan since the mid
1950's and in California starting in 1993. Summer mortality events
have been linked to multiple stressors associated with planting
times and height including extreme dissolved oxygen and tempera-
ture fluctuations. Hemocytes are integral in many important physi-
ological processes such as nutrient digestion and transport, excre-
tion, wound repair and pathogen defense. Cellular defense is the
primary immune mechanism of marine invertebrates. Hemocytes
found in hemolymph and interstitial spaces function in host de-
fense via inflammation, wound repair, encapsulation, and phago-
cytosis. Hemocyte performance may contribute to observed dif-
ferences in mortality between selected family lines of C. gigas
from the Molloscan Broodstock Program (MBP) of Oregon State
University. To better understand the differences in oyster perfor-
mance, we examined the immune response of oysters from two
different MBP families grown at a site with low mortality (Totten
Inlet). These families were selected based on mortality rates: high
mortality (MBP family 10-116) vs. low mortality (MBP family
10-115). The ability of hemocytes to phagocytose or engulf foreign
particles, move towards a chemical stimulus (chemotaxis). and kill
Vibrio paraluu'inolylicits was examined. Differences and similari-
ties in immune responses between the two groups of oysters will
be described.
POPULATION STRUCTURE AND RECOVERY DYNAM-
ICS OF BLACK ABALONES {HALIOTIS CRACHERODII)
AT SAN NICOLAS ISLAND, CALIFORNIA. Melinda D.
Chambers, C. S. Friedman, L. Hauser, School of Aquatic and
Fisheries Sciences, University of Washington, Seattle, WA 98105;
and Glenn R. Vanblaricom, Washington Cooperative Fish and
Wildlife Research Unit. School of Aquatic and Fisheries Sciences.
University of Washington, Seattle, WA 98105.
Populations of black abalone have experienced declines of 85-
99% since the emergence of the disease Withering Syndrome
(WS) in 1985. Black abalone populations in the California Channel
Islands formerly harbored unprecedented densities. Since 1981 we
have collected data that documents the change in abundance on
San Nicolas Island. Recent data indicate the first recruitment event
since the onset of WS with observations of individuals sized <50
mm. A drift card study conducted in August 2002 on San Nicolas
Island, indicated that dispersal was largely localized. Subsequent
genetic studies will be conducted throughout 2002 and 2003 to
confirm speculation that genetic differentiation corresponds with
geographic distance. Tissue samples will be collected from each of
the California Channel Islands that support black abalone popula-
tions of high density and genetic analysis will be conducted using
allozymes and mtDNA. Additionally, further drift card studies will
be conducted to improve our understanding of circulation patterns
in the Channel Islands and temperature data will be monitored
using TidbiT stowaway devices, as elevated sea surface tempera-
tures are tightly associated with WS symptoms in black abalone.
2002 MONITORING OF HARMFUL ALGAE IN SOUTH
PUGET SOUND AND WILLAPA BAY— SPECIES OF CON-
CERN AND FUTURE CONSIDERATIONS. Aimee E.
Christy, The Evergreen State College, Olympia, WA 98505.
Harmful Algal Blooms (HABs) are natural phenomena and
have occurred worldwide for hundreds of years. The impacts of
these blooms include both economic and health concerns for shell-
fish growers, consumers and the local economies dependent on
shellfish resources. Pacific Shellfish Institute (PSI) is a member of
the Olympic Region Harmful Algal Bloom (ORHAB) Partnership
working to understand HABs and reduce HAB impacts on humans
and the environment. In addition to monitoring toxic algae in
Willapa Bay, PS! independently monitors plankton communities at
several locations in south Puget Sound. Monitoring efforts have
detected numerous species of plankton that form HABs and are of
special concern to the shellfish industry. Evidence exists that the
incidences of problems associated with toxic algae are rising. Pos-
602 Abstracts. September 27-30. 2002
NSA & PCSGA. Newport, Oregon
sible explanations for the increased frequency and intensity of
blooms include natural dispersal of plankton via currents, climatic
changes, nutrient enrichment and the transport of new species in
ballast water. Understanding the factors that contribute to HABs.
studying life cycles of local species, diligent monitoring and re-
sponse efforts, innovative methods for quick and economic toxin
detection and the ability to predict HAB occurrences are necessary
steps in the protection of human health and shellfish resources.
LITERATURE REVIEW— IMPACTS OF URBANIZATION
ON WATER QUALITY IN SHELLFISH GROWING AREAS
IN PUGET SOUND, WASHINGTON. Aimee E. Christy, The
Evergreen State College, Olympia, WA 98505; and Stuart D.
Glasoe, Puget Sound Water Quality Action Team, Olympia, WA
98504.
In response to population growth, urbanization and worsening
bacterial contamination trends throughout the region, the Puget
Sound Water Quality Action Team is undertaking a study to better
understand the impacts of urbanization on water quality in shell-
fish growing areas. A literature review was conducted to assemble
available information to determine the cunent understanding of the
relationship between urbanization and bacterial contamination in
the nearshore environment. Results of the literature search indicate
distinct differences in bacteria sources and transport pathways be-
tween rural and urban watersheds. The concentration and rapid
transport of urban pollutants into receiving waters caused by the
conversion of native vegetation to impervious surfaces and drain-
age networks is well documented. A number of indicators (imper-
vious surface coverage, developed land, population, housing den-
sity) are being examined and some appear more significant than
others in correlating development and bacterial contamination.
Findings encourage protecting natural filtration areas, preserving
buffers and native vegetation, disrupting connectivity between im-
pervious surfaces and receiving waters, educating the public, and
using innovative planning and low-impact development techniques
to mimic and preserve natural hydrologic functions. Understanding
the relationship between urbanization and water quality will pro-
vide the tools necessary to develop in ways that support future
growth, natural resources, public health and clean water.
THE HISTORY AND DEVELOPMENT OF THE PUGET
SOUND COMMERCIAL GEODUCK INDUSTRY. Marion
Dumont, Univ. of Washington, Tacoma, Washington.
The commercial geoduck industry had its official beginnings in
Washington State in 1970, the onset of a decade defined by con-
flict, transition and change. The men and women, politicians, har-
vesters, leaseholders and government agents were fiercely com-
petitive and adventurous, given to quarreling and trouble making.
They proved a driving force for an innovative and booming in-
dustry.
RELATIVE IMPORTANCE OF SURVIVAL AND
GROWTH RATE IN DETERMINING YIELDS OF PACIFIC
OYSTERS, CRASSOSTREA GIGAS. Ford Evans, Sean Mat-
son. John Brake, and Chris Langdon. Coastal Oregon Marine
Experiment Station and Dept. Fisheries and Wildlife, Oregon State
University, Newport, OR 97365.
Data were collected on three cohorts (C-6, C-7, C-9) of unse-
lected full-sib Pacific oyster (Crassostrea gigas) families. The
roles which individual growth rate and survival play in determin-
ing average family yield were investigated. C-6 families were
planted subtidally in Yaquina Bay. OR. C-7 families were planted
intertidally in Tomales Bay, CA, and subtidally in Yaquina Bay,
OR. C-9 families were planted intertidally in Totten Inlet, WA.
Once market size, oysters were harvested and yield, average indi-
vidual growth rate and survival recorded for each family. Pheno-
typic correlation coefficients (rp) between performance characters
were estimated within each cohort. Correlation coefficients of per-
formance characters between sites were estimated for C-7. Sur-
vival was significantly correlated with yield within all cohorts
(r = 0.66 to 0.98, p<0.05). Average individual growth rate was
significantly correlated with yield in all cases (r^ = 0.65 to 0.93,
p<0.05 ) except for C-7 in Yaquina Bay, OR (rp = 0.52. p = 0.20).
Correlations between average individual growth rate and survival
tended to be higher in cohorts planted intertidally (r^ = 0.70 to
0.71 ) than in cohorts planted subfidally (rp = -0.130 to 0.32). C-7
yield was significantly correlated between Tomales Bay, CA. and
Yaquina Bay, OR (rp = 0.88, p<0.01J. Yield stability appeared to
be driven primarily by the significant correlation of survival be-
tween sites (rp = 0.89. p<0.01). Individual growth was not corre-
lated between sites (rp = 0.32, p = 0.51 ). These results indicate
the relative importance of survival and growth rate in contributing
to yields of Pacific oysters varies between sites and cohorts. The
implication of these results on breeding schemes targeting oyster
yield will be discussed.
LIFE HISTORY OF AN EXOTIC SABELLID POLY-
CHAETE, TEREBRASABELLA HETEROUNCINATA: FER-
TILIZATION STRATEGY AND INFLUENCE OF TEM-
PERATURE ON REPRODUCTION. Carl A. Finley, Thea T.
Robbins, California Department of Fish and Game. Bodega
Marine Laboratory. P.O. Box 247, Bodega Bay, CA 94923; and
Carolyn S. Friedman, School of Aquatic and Fishery Sciences,
University of Washington. Box 355020. Seattle. WA 98195.
Abalone culture facilities have been devastated by an exotic
sabellid, Terehrasabella heterowuinata. following its introduction
from South Africa in the late 1980s. Infestations are associated
with shell deformities, increased mortality and financial losses. In
addition, the potential introduction and establishment of this exotic
pest into the natural environment was unknown. The development
of an effective management strategy is dependent upon under-
standing the life history of this sabellid, including its fertilization
NSA & PCSGA, Newport, Oregon
Ahstracls. September 27-30, 2002 603
strategy and generation time. In the present study, red abalone,
Haliotis rufescens. with single sabellid infestations were isolated
in containers al 18°C. This first, parental generation was held in
isolation until individuals produced F, larvae, which were subse-
quently isolated until indniduals produced a second, F,, genera-
tion. In a separate study, uninfesled abalones were exposed to
infested abalone at three temperatures typically encountered in
California. Transmitted larvae were observed as they developed to
specific life stages; initiation of feeding, sexual maturation and
production of motile, infestive, larvae. This research demonstrated
that isolated individuals are functional hermaphrodites and do pose
the threat of producing fully functional offspring and that the gen-
eration time of T. heieroiincinata is significantly temperature de-
pendent. The aquaculture industry. UC Santa Barbara researchers
and Department of Fish and Game (DFG) initiated an aggressive
eradication program in 1996, and DFG policy was established in
1997 to prevent further spread of the sabellid. Culling of infested
stocks and strict hygiene protocols including freshwater treatment
of tanks proved effective in curbing new infestations. Results of
recent eradication efforts will be described.
SUMMER MORTALITY OF THE PACIFIC OYSTER,
CRASSOSTREA GIGAS, ALONG THE WEST COAST OF
THE U.S.: PERFORMANCE OF FAMILY LINES AND EN-
VIRONMENTAL PARAMETERS. C. S. Friedman, C. A.
Burge, University of Washington. Seattle, WA 98195; D.P.
Cheney, R. A. Elston, A. D. Suhrbier, Pacific Shellfish Institute.
Olympia, WA 98501; G. N. Cherr, F.J. Grimn, A. Hamdoun,
Bodega Marine Lab, UC Davis. Bodega Bay, CA 94923: and C. J.
Langdon, Hatfield Marine Science Center, OSU, Newport, OR
97365.
Mortality of the Pacific oyster, Crassostrea gigas, has occurred
in the U.S. west coast and Japan since the mid !950's. Multiple
stressors have been implicated as contributing to these mortality
events. In an attempt to alleviate the >50'7f annual oyster mortality
observed in California and variable losses in Washington state, we
examined the interaction between survivorship, growth and stress
response of family lines from the Molluscan Broodstock Program
(MBP) of Oregon State University, and planting time and height,
and selected environmental parameters. To examine differential
performance between family lines and planting period three oyster
families were each outplanted during Fall 1999, 2000, 2001 and
Spring 2000, 2001, 2002 at 2-3 sites in California, 3 sites in
Washington (Spring only), and 1 site in Oregon (Spring 2002
only). Fall plants survived significantly more than did oysters
planted in the spring (p<0.05) in California. In addition, two fami-
lies (one commercial strain and MBP family 10-115) outper-
formed MBP family 10-1 16 (p<O.OOI ) at all locations. During the
study period inter-annual variation in phytoplankton was more
pronounced than spatial variation. While suspected hannful algal
species were present throughout the study period, phytoplankton
did not appear to be directly involved in oyster mortalities. How-
ever, in California a 2000 mortality coincided with a Gyinnodiniuin
sangninewn bloom, while 2001 and 2002 mortalities were not
associated with any phytoplankton bloom. Extreme temperature
and dissolved oxygen fluctuations were repeatedly associated with
oyster mortalities at the Washington and California study sites.
WITHERING SYNDROME OF ABALONE IN CALIFOR-
NIA. Carolyn S. Friedman. School of Aquatic and Fishery Sci-
ences, University of Washington, Box 355020, Seattle, WA
98195: James D. Moore, Thea T. Robbins, Beverly A. Braid,
Carl A. Finley, California Department of Fish and Game, Bodega
Marine Laboratory, P.O. Box 247, Bodega Bay, CA 94923;
Ronald P. Hedrick, Dolores V. Baxa, Karl B. Andree, Depart-
ment of Medicine and Epidemiology, UC Davis. CA 95616; Eric
Rosenblum, Mark R. Viant, Ronald S. Tjeerdema, Department
of Environmental Toxicology, UC Davis, CA 95616: Peter L.
Haaker, California Department of Fish and Game, Los Alimitos.
CA 90720; Mia J. Tegner and Luis I. Vilchis, Scripps Institution
of Oceanography, La Jolla, CA 92093.
Catastrophic declines in many abalone species in California,
both wild and cultured, have been attributed to the bacterial dis-
ease, Withering Syndrome (WS). The etiological agent was re-
cently described as a novel rickettsial bacterium, "Candidatus Xe-
nohaliolis adifomiensis" which infects gastroepithelial cells of
abalone and results in morphologic changes in the digestive gland
(degeneration and metaplasia) and foot muscle (atrophy). Diges-
tive gland metaplasia appears pathognomonic for WS. Differences
in susceptibility and tissue changes were noted between species
with black abalone. Haliotis cracherodii. being more susceptible
to WS than red abalone, H. rufescens. Climatic variation associ-
ated with ENSO events has been demonstrated to result in devel-
opment of WS in red abalone, and exacerbate disease development
in black abalone. Survivors appear relatively resistant to WS and
are being considered as captive broodstock in species restoration
programs. Molecular tools and therapeutants have been developed
and will play a key role in the abalone culture industry and captive
broodstock programs, particularly for the endangered white aba-
lone, H. sorenseni. which is being cultured in a WS endemic
region.
ASSESSMENT AND MANAGEMENT OF INTERTIDAL
CLAM RESOURCES IN BRITISH COLUMBIA. Graham E.
Gillespie, Fisheries and Oceans Canada, Pacific Biological Sta-
tion, Nanaimo, BC Canada V9T 6N7; Randy Webb. Fisheries and
Oceans Canada, 457 East Stanford Avenue, Parksville. BC,
Canada V9P IV7: and Todd Johansson, Fisheries and Oceans
Canada, PO Box 2159, Unit 10, 9250 Trustee, Port Hardy, BC
VON 2P0.
Intertidal clams continue to be an important resource in British
Columbia, and are utilized by commercial, recreational and Ab-
original harvesters as well as the aquaculture industry. The most
604 Abstracts, September 27-30. 2002
NSA & PCSGA, Newport. Oregon
important species is the Manila clam, or Japanese littlenect;. al-
though native littleneck. razor and butter clams are also landed
commercially. The basis for assessment of clam resources is a
statistically rigorous survey, with clearly defined protocols for
design, field procedures, data management and analyses. These
protocols are used by government programs, co-management pro-
grams with First Nations or Industry, and by contract biologists.
How assessment data are used varies between fisheries and de-
pends upon capacity for gathering assessment data and allocation
policies. The regular commercial fishery is managed using historic
expectations of production and landings that are monitored for
indications of depleted legal size stocks. The depuration fishery
and First Nations pilot program allocate individual beaches to
harvest groups. These groups must undertake stock assessments
before quotas can be determined. Beaches were initially managed
experimentally using harvest rates of 25 or 50% of estimated legal
biomass. Review of stock responses to harvest has led to devel-
opment of a sliding scale of harvest rates determined by abundance
thresholds. In the Area 7 fishery, assessment surveys are done
annually on index beaches, which are used to determine trends in
biomass indices for subareas that are fished. A simple feedback
gain model is used to set harvest thresholds for each subarea.
FEEDING RATES OF THE MUD SHRIMP UPOGEBIA
PUGETTENSIS AND IMPLICATIONS FOR ESTUARINE
PHYTOPLANKTON ABUNDANCE. Blaine Griffen. Chris
Langdon, Coastal Oregon Marine Experiment Station and Dept.
Fisheries and Wildlife. Oregon State University, Newport, OR
97365: and Ted Dewitt, US-EPA - Pacific Coastal Ecology
Branch, Newport. OR 97365.
The burrowing shrimp Upogebia pitgettemis is an abundant
inhabitant of Pacific Northwest bays and estuaries where it lives
commensally with the clam Ciyptomya californica. Suspension-
feeding activities of the shrimp and its commensal clam, as well as
particle settlement within the burrow, represent three potential
causes of phytoplankton reduction within shrimp habitats. These
three components together comprise what we call the "shrimp-
burrow complex". Laboratory measurements of particle filtration
rates indicated that shrimp were responsible for filtering the ma-
jority of phytoplankton removed by the shrimp-burrow complex;
however, particle settlement in burrows and adhesion to burrow
walls could also be responsible for removal of significant propor-
tions of phytoplankton. Particle filtration efficiencies of shrimp -i-
burrows and clams were similar to those of Pacific oysters, Cras-
sostrea gigas. for particles 2 to 10 microns in diameter, indicating
a potential for food competition among these species under food-
limiting conditions. A population filtration model, based on field
measurements of shrimp filtration rates together with data on phy-
toplankton concentrations and shrimp populations in the Yaquina
estuary, Oregon, predicted that shrimp-burrow complexes in this
estuary were capable of filtering the entire body of overlying water
between one and two times daily.
PATTERNS IN INTERTIDAL HABITAT USE BY SUB-
ADULT DUNGENESS CRAB [CANCER MAGISTER).
K. Holsman, P. Sean McDonald, D. Armstrong, and J. Ruesink,
Department of Zoology, University of Washington, Box 351800,
Seattle, WA 98195.
Complex intertidal habitats characteristic of northeastern Pa-
cific coastal estuaries provide critical nursery environments for
young-of-the-year Dungeness crabs. Cancer magister. yet their
role in supporting subsequent year classes remains unclear. As
with other brachyuran species that undertake diel intertidal migra-
tions, subadult C. magister (40 -I30mni; l-i- and >l-t- year classes),
which reach densities as high as 4,300 crabs ha ' in subtidal chan-
nels during low tides, may migrate during flood tides from subtidal
refuges into intertidal habitats to forage. Results of a bioenergetic
model for crabs in Willapa Bay, Washington, indicate that inter-
tidal foraging may contribute significantly to the energy budget of
subadult C. magister and may facilitate the high abundance of
crabs observed in large coastal estuaries. We conducted bay wide
trapping surveys in intertidal oystershell. eelgrass, and bare mud
habitats in order to elucidate patterns of habitat use by subadult
crabs, and underwater video was used to observe tidal migrations
in these habitats. Significant differences in crab abundance and the
magnitude of migrations were observed across habitats, with low-
est densities of C. magister occurring in older shell beds concur-
rent with high densities of red rock crabs (C productus). Obser-
vations of tidal migrations using underwater video suggest that the
physical structure of plants may hinder crab movement in eelgrass
beds since the number and size (carapace width, CW) of crabs
migrating was smallest in this habitat. Although the density of prey
species may be less in open mud or sand habitats, the lack of
structural hindrance and interspecific competition may render open
mud the most valuable intertidal habitat to subadult crabs. The
importance of intertidal habitats to subadult crabs has direct im-
plications in coastal estuaries of the Northeastern Pacific where
anthropogenic and biotie modification of intertidal areas threaten
the productivity of intertidal habitats and may adversely impact
estuarine populations of C. magister.
SEASONAL UTILIZATION OF INTERTIDAL HABITATS
BY FISH IN A WASHINGTON STATE COASTAL ESTU-
ARY. Geoff Hosack, David Armstrong, School of Aquatic and
Fishery Sciences, Box 355020. University of Washington, Seattle.
WA 98195; Brett Dumbauld. Washington State Department of
Fish and Wildlife. Willapa Bay Field Station, P.O. Box 190, Ocean
Park, WA 98640; Brice Semmens and Jennifer Ruesink, Dept. of
Zoology, University of Washington, Seattle, WA 98195.
Estuaries are regarded as important nursery areas for juvenile
marine and anadromous fish. Estuaries also support fisheries and
aquaculture and the effects of these activities on fish habitat are
NSA & PCSGA, Newport, Oregon
Abstracts. September 27-30, 2002 605
becoming increasingly scrutinized under tlie Endaui-ered Species
Act and Magnusou-Stevens Act . We are conducting a study to
evaluate the importance of the intertidal environment tor juvenile
fish within Willapa Bay, Washington with respect to aquaculture.
Our objectives are to compare commercially cultivated and uncul-
tivated habitats in order to; (1) elucidate potential habitat prefer-
ences among juvenile fishes. (2) establish possible mechanisms for
habitat preferences, and (3) evaluate the function of intertidal habi-
tats for fish foraging, predator avoidance, and mobility behaviors.
One-meter high hoop nets were deployed over three habitats (oys-
ter culture, eelgrass and unvegetated open mud/sand) to determine
habitat preference at three locations in 2001. They were also de-
ployed monthly to determine seasonal presence/absence of fish
species and at three different tidal elevations in 2002. Preliminary
results show that intertidal use by the majority of species exhibits
a pronounced increase during late spring and early summer. Few
significant differences in habitat use were found, but a prototype
two-boat surface trawl was designed and tested in 2002 to further
investigate potential differences in utilization of these low inter-
tidal habitats and adjacent subtidal channel by juvenile Chinook
salmon (Oncorhyncus tshawytscha). Finally, Chinook salmon and
shiner perch (Cymatogaster aggregata) have been marked with
acoustic tags and held in a large enclosure to observe fine-scale
movement over a suite of intertidal habitats.
BIOMASS SURVEYS AND ACTIVE MANAGEMENT OF
INTERTIDAL RAZOR CLAMS [SILIQUA PATULA) AT
BEACHES NEAR MASSETT. HAIDA GWAII, CANADA.
R. Russ Jones, Haida Fisheries Program. P.O. Box 98. Skidegate,
Haida Gwaii VOX ISO; Carl Schwarz, Department of Mathemat-
ics and Statistics. Simon Fraser University, Bumaby, BC V5A
1S6; Bart DeFreitas, Haida Fisheries Program, P.O. Box 87, Mas-
sett, Haida Gwaii VOT IMO; Lynn Lee, Marine Toad Enterprises.
P.O. Box 74, TIell. Haida Gwaii VOT lYO.
Intertidal razor clam populations and biomass were estimated
for commercial clam beaches near Massett, Haida Gwaii for the
period 1994 to 2000 using a three stage sampling design. Clams
were collected by fluidizing the substrate in a 0.5 ni" sampling
cylinder. Population was estimated for three size fractions (Shell
Length (SL) >4 mm, >20 mm and >90 mm, the latter being the
commercial size limit) at three beach sections on 18.8 km of beach
accessible to the commercial fishery. Calculations varied consid-
erably in some years depending on assumptions about transect
length and beach area. There was a record catch of 237 t in the
fishery in 2000 that led to concerns by managers about possible
overfishing. However surveys indicated that the biomass of clams
>90 mm at the start of 2000 was 1876 t (SE 157 t). Biomass was
shown to have been at a historic high in 2000 with large numbers
of two year old clams in the population, most of which were
expected to recruit to the fishery in 2001. The fishery had been
passively managed using size limits for many years. However an
examination of razor clam gonads showed that only 50% were
mature at 87 mm. Beginning in 2001. in addition to the size limit,
an annual quota was introduced in the fishery based on the annual
bioinass survey and a harvest rate of 12.3% (2/3 of a 1994 estimate
of Fmsy)-
ECONOMICS OF CALIFORNIA'S DUNGENESS CRAB
(CANCER MAGISTER) INDUSTRY. PRELIMINARY RE-
SULTS. Matthew J. Kraehey, Department of Fisheries Biology.
Humboldt State University, Areata, CA 95521 and Steven C.
Hackett, School of Business and Economics, Humboldt State Uni-
versity. Areata. CA 95521.
The cunent management regime for California's Dungeness
crab fishery has led to a derby, with the vast majority of the catch
occurring with the first six weeks of the six-month long season.
Questions have been raised about the impacts of the derby on
industry structure, prices, and product quality. One thrust of our
work is to identify baseline economic characteristics of the indus-
try under current management. These include value added, product
mix, employment, and capital investment in the processing sector,
as well as value added by fishermen. Our preliminary findings
indicate that, unlike former derby fisheries for tlnfish, the product
forms that economically dominate are not suppressed by derby
conditions. Moreover the derby fishery promotes large-scale pro-
cessing facilities that create important jobs and processing capa-
bility for other fish species in economically less robust coastal
communities. Ongoing research will focus on fishery participant's
opinions on management alternatives, number of traps deployed,
and marginal fishing cost.
THE MOLLUSCAN BROODSTOCK PROGRAM: FAMILY-
BASED SELECTION IMPROVES YIELDS OF PACIFIC
OYSTERS, CRASSOSTREA GIGAS. Chris Langdon, Sean
Matson, John Brake and Ford Evans, Coastal Oregon Marine
Experiment Station and Dept. Fisheries and Wildlife, Oregon State
University, Newport. OR 97365.
The Molluscan Broodstock Program (MBP) was established in
1995 to improve yields of Pacific oysters on the West coast, U.S.,
by family-based genetic selection. Parental families (PI) in three
cohorts of about 60 families each were selected based on superior
live weight and meat yields at harvest. Live weight yields of prog-
eny (Fl) from crossing PI selected families were significantly
greater than those of non-selected control families in four out of
seven trials (ANOVA, p<0.001). resulting in an average gain of
9.5% after one generation of selection. The response to selection
was greatest if Fl families were tested at the same site as that used
for their parents' selection rather than at a different site. There
were weak (p = 0.06; p = 0.04) positive correlations between the
yields of families planted at both inter-tidal and sub-tidal sites,
indicating strong genotype by environment interaction effects on
606 Ahslnicts. September 27-30. 2002
NSA & PCSGA. Newport. Oregon
yield. Nonetheless, it was possible to identify four to six "gener-
alist" families that were among the top ten families at both sites.
Further evaluation of families across a wider range of environ-
ments is needed to determine if the best strategy to improve oyster
yields will be to select "generalist" families that perform well
along the whole Pacific coast, or whether it will be more effective
to develop site-specific lines instead.
THE ROLE OF CULTURE PRACTICES IN STRUCTUR-
ING INTERACTIONS BETWEEN CULTURED OYSTERS
AND NATIVE EELGRASS. Heather M. Macrellis, Jennifer L.
Rue.sink, Zoology Department. Box 351800. University of Wash-
ington. Seattle. WA 98195; and Brett Dumbauld, Washington
State Department of Fish and Wildlife. Willapa Bay Field Station,
P.O. Box 190. Ocean Park. WA 98640.
The potential for positive interactions between aquaculture spe-
cies and native eelgrass {Zostera marina) is the subject of growing
interest in the Pacific Northwest. We conducted surveys of cul-
tured oysters {Cnissostrea gigas) and Z. marina density to deter-
mine the nature of the relationship between these two species, and
to determine whether this relationship changes under different cul-
ture practices used in Willapa Bay. Washington. Culture practices
assessed included ground culture harvested by dredgmg. ground
culture harvested by hand, and off-bottom line culture. The role of
planting density was also assessed in two separate experiments
where small plots were planted with several densities of oyster
seed and two year old oysters respectively. Eelgrass production
and density were measured throughout the experiments. Results of
the survey and experiments will be discussed.
A SPECIFIC PATHOGEN FREE CULTURE SYSTEM FOR
C. GIGAS LARVAE AND SPAT. Sean E. Matson and Chris-
topher Langdon. Hatfield Marine Science Center. Oregon State
University. Newport. OR 97365.
The Molluscan Broodstock Program (MBP). a selective breed-
ing program for the Pacific oyster. Crassosirea gigas. uses a Spe-
cific Pathogen Free culture system for all production and mainte-
nance of larvae, spat, broodstock and microalgae. This system is
necessary to exclude infectious agents of Haplosporidian costale
(Seaside Organism. SSO). which has been found in Pacific oysters
grown in Yaquina Bay. Oregon, for the safe outplanting of MBP
spat in field test sites along the West coast (USA). All seawater
entering MBP facilities is filtered through sand, diatomaceous
earth, and 20, 5. and l|jLm cartridge filters. Seawater to mass algal
cultures and the nursery is also irradiated with UV-light at >30.000
micro- Watts-sec/cni" (MWS) as a back-up precaution. Since the
system's inception, no MBP spat have been identified as being
contaminated with SSO. or any other infectious agent. A series of
laboratory experiments was performed to assess the effects of UV
water on larval growth and survival, spat growth and survival, and
microalgal culture density. Experiments with oyster larvae indi-
cated that both the micro-filtration system and UV water treatment
had a significant negative effect on larval growth (p = 0.0001 ). A
significant reduction in growth was evident at UV intensities as
low as 10.000 MWS (p<0.05). Methods that have significantly
improved larval growth, survival, speed to metamorphosis and spat
growth within the SPF culture system include substituting a 0.2|jim
filter and charcoal for a UV filter when rearing larvae, and the
addition of calcium bentonite (2nig/ml/day) or calcium montmo-
rillonite (5mg/ml/day) to larvae and spat cultures (p<0.05).
BIOTIC RESISTANCE TO EUROPEAN GREEN CRAB,
CARCINVS MAENAS, BY NATIVE ANALOGS IN THE
NORTHEASTERN PACIFIC. P. Sean McDonald, Gregory C.
Jen.sen and David A. Armstrong, School of Aquatic and Fishery
Sciences. University of Washington. Seattle. WA. 98195.
The notion of "biotic resistance", which holds that characteris-
tics of native biota act to prevent establishment and persistence of
nonindigenous species, remains a dominant component of invasion
biology theory. Yet. studies of nonindigenous marine species have
often focused on impacts to the recipient community while ignor-
ing effects of the latter on the former. The case of the European
green crab. Carcinus maenas. provides one such example; the
species has successfully colonized temperate coastal embayments
throughout the world and its attendant adverse consequences to
nafive biotic communities have been well-documented. However,
the distribution and habitat use of C. maenas in the northeastern
Pacific is more limited than would be expected based on Atlantic
populations, and peak abundances occur only in isolated, back-
marsh or high intertidal locations. We conducted a limited survey
of crab populations in Bodega Bay Harbor (BBH), California, in
1998. and subsequent intensive sampling was undertaken in 2001
in BBH and other central California estuaries. Results from snorkel
surveys and trapping data suggest that C. maenas are largely ab-
sent from areas occupied by native ecological analogs (Cancer
spp.). Incidence of limb autotomy in C. maenas is significantly
higher at BBH sites shared with Cancer spp. than in isolated areas
uninhabited by the latter or in Atlantic populations. A series of
tethering experiments similarly supports the assertion that preda-
tion/aggression by Cancer spp. affects the distribution and habitat
utilization of C. maenas. The significance of these interactions to
the eventual distribution of C. maenas in the northeastern Pacific
is discussed, as well as implications for monitoring and control
efforts.
NSA & PCSGA, Newport, Oregon
Abstracts, September 27-30, 2002 607
EFFECT OF DIET ON SOMATIC GROWTH OF JUVE-
NILE GREEN SEA URCHINS (STRONGYLOCENTROTVS
DROEBACHIENSIS). C. Pearce, Fisheries and Oceans Canada.
Pacific Biological Station. 31 W Hammond Bay Road, Nanaimo,
BC V9T 6N7: T. Daggett, Ross Island Salmon Ltd.. P.O. Box
1 304. Grand Manan. NB E5G 4M9; T. Chopin. Centre for Coastal
Studies and Aquaculture. Centre for Environmental and Molecular
Algal Research, Department of Biology, University of New Bruns-
wick Saint John. P.O. Box .5050. Saint John. NB E2L 4L5;
K. MacKeigan. V. Zltkos and S. Robinson, Fisheries and Oceans
Canada. St. Andrews Biological Station. 53 1 Brandy Cove Road.
St. Andrews. NB E5B 2L9.
Populations of sea urchins, harvested for their gonads, are in
decline worldwide and so research is now focusing on full life-
cycle grow out. The objective of this study was to compare the
somatic growth rates of juvenile green sea urchins (Strongylocen-
trotiis droebachiensis) fed one of seven diets. Sea urchins (test
diameter: 4.5 - 10.7 mm) were collected from the wild, held in
laboratory tanks supplied with flow-through seawater, and fed ad
libitum one of seven diets: (Da prepared diet. (2) Poipliyra pur-
purea. (3) Pabnaha pabnata. (4) Enteromorpha linza. (5) a mix-
ture of Ulvaria obscura and Ulva lactuca. (6) Lamiiuiria longi-
cruris collected from an Atlantic salmon culture site, and (7) Laini-
naria longicruris collected from a site uninfluenced by salmon
culture. Each diet was randomly assigned to three separate tanks
with each tank containing 19 individually housed urchins. Test
diameter and whole wet weight measurements from each urchin
were initially taken at the start of the experiment and then again
once per month for a period of 12 months. Feed type significantly
affected growth rate in terms of both diameter and wet weight.
Porpbyra purpurea and the prepared diet supported the best
growth while Laminaria longicruris collected from a site uninflu-
enced by salmon culture was the least effective diet.
TRENDS IN PINTO ABALONE (HALIOTIS KAMTSCHAT-
KANA) ABUNDANCE AT TEN SITES IN THE SAN JUAN
ISLANDS AND MANAGEMENT OF THE SPECIES IN
WASHINGTON STATE Don P. Rothaus, R. E. Sizemore,
M.J. Ulrich. Washington Department of Fish and Wildlife. Fish
Management Program, Central Shellfish Unit, Olympia WA
98501-1091; and Carolyn S. Friedman. University of Washing-
ton. School of Aquatic and Fishery Sciences. Seattle WA 98195-
5680.
As a result of concerns regarding the stability of pinto abalone
(Haliotis kamtschatkana) populations in Washington and the clo-
sure of the abalone fishery in neighboring British Columbia,
Canada, the Washington Department of Fish and Wildlife
(WDFW) established index stations at ten sites in the San Juan
Islands. These stations varied in size from 50 m" to 380 m", av-
eraging about 220 m~. WDFW divers systematically surveyed each
of these stations in 1992, 1994, and 1996. A decrease in total
abalone abundance at these ten index stations from 1992 to 1994
(n = 351 to n = 288). along with anecdotal information of popu-
lation decline by University of Washington (UW) researchers and
WDFW Enforcement personnel, resulted in the closure of the
Washington pinto abalone fishery in 1994. Following the closure,
a 1996 survey by WDI^W resulted in a combined n = 297. Re-
search in other regions indicate that sedentary invertebrates, such
as abalone, must be within 1.0-2.0 m of one another
(ds0.337ndash;0.15 abalone/nr) for successful fertilization. The
average abalone density (d) from one half of the sites surveyed in
1996 contained ds0.15 abalone/m".
Based on survey data, and information from abalone fisheries
around the world, it is clear that additional stock assessment is
needed to analyze the trend in Washington abalone stocks. Addi-
tional index sites, early juvenile life history, population genetics,
and the potential for enhancement have been propo.sed for study.
ECOLOGICAL ROLE AND POTENTIAL IMPACTS OF
MOLLUSCAN SHELLFISH CULTURE IN THE ESTUA-
RINE ENVIRONMENT OF HUMBOLDT BAY, CA. Steven
S. Rumrill and Victoria K. Poulton. Estuarine and Coastal Sci-
ences Laboratory. South Slough National Estuarine Research Re-
serve. Charieston, OR 97420.
The intertidal mudflats of Humboldt Bay. CA. provide habitat
for eelgrass {Zostera marina), invertebrates, shellfish, tlnfish, and
birds. Humboldt Bay is also the leading producer of Pacific oysters
(Crassostrea gigas) in California. We have completed the first
year of a 3-year project to identify and quantify the effects of
commercial oyster mariculture in tidetlat habitats, eelgrass beds,
and invertebrate communities. Experimental oyster long-line spac-
ing plots were established for comparison to a ground culture site
and 6 reference sites (no oysters). We sampled study plots quar-
terly between Aug 2001 -Aug 2002 for presence of eelgrass, oys-
ters, and other cover types. We collected infaunal cores, deployed
fish traps, and measured water quality, sedimentation, light inten-
sity, and oyster growth characteristics. Eelgrass shoot density and
percent cover were consistently highest in an eelgrass bed control
site, lowest at the 1.5-ft. long-line spacing plot, and most variable
at the ground culture site. Eelgrass metrics in the other long-line
spacing plots were generally lower but within the range of varia-
tion exhibited by the reference sites. Preliminary analysis of in-
vertebrate cores has produced a species list of over 70 taxa, many
of which are known prey items for estuarine fish. Sedimentation
measurements showed no consistent patterns among experimental
long-line plots. Oyster growth measurements did not differ sub-
stantially between long-line plots; oysters grew 20-35 mm in
length and 16-22 mm in width between May and Aug 2002. Light
intensity was lower beneath oyster long-lines, but did not differ
substantially between the 1 .5 and 5 ft. spacing plots.
608 Abstracts. September 27-30. 2002
NSA & PCSGA. Newport, Oregon
THE EFFECTS OF THE HERBICIDE RODEO* ON PA-
CIFIC OYSTER GAMETOGENESIS AND TISSUE ACCU-
MULATION. B. C. Smith, C. E. Grue, University of Washing-
ton, School of Aquatic and Fishery Sciences, Seattle. WA: N. P.
Kohn, Battelle Marine Sciences Laboratory. Sequim. WA; and
J. P. Davis, Taylor Shellfish Company, Quilcene. WA.
In Willapa Bay. WA, Rodeo® (Monsanto Agricultural Co., St.
Louis. MO) is being used to control Spartina (Spartina alterni-
flora). an invasive cordgrass native to the Atlantic Coast. Spartina
alters the tideland habitat by trapping sediment and raising the
elevation of the mudflats, thus reducing the available habitat for
oyster culture. Rodeo tank mixes include a surfactant to reduce the
surface tension of the spray. R-I I is currently the surfactant used
in the Bav. R-I I belongs to a class of non-ionic surfactants com-
prised of alkylphenol ethoxylates (APEO). Breakdown products of
APEOs have been implicated as endocrine disruptors in fish and
observed to cause delays in development of oyster veligers. The
objectives of our study were to assess whether applications of
Rodeo tank mixes 1 ) result in tissue concentrations of glyphosate
in oysters that exceed the established tolerance of 3 ppm wet
weight edible tissue and 2) impair oyster gametogenesis. To de-
termine this. Pacific oysters (Crassostrea gigas) were subjected to
five treatments; Rodeo; Rodeo tank mix (with R-11 surfactant);
two concentrations of R-1 1; and a control. The oysters were ex-
posed for 12 h once a week for 4 wks. Tissue samples were
collected for residue analysis of glyphosate, AMPA and APEO and
cross-sections of gonadal tissue were collected for histological
examination. Initial results indicate that exposure to Rodeo without
the surfactant results in concentrations of glyphosate below the
established human health criteria. Tissue resides of APEO and an
assessment of treatment effects on gametogenesis will be deter-
mined this fall.
MUSSEL GROWTH AND FOOD UTILIZATION IN RELA-
TION TO WATER QUALITY ON A RAFT SYSTEM IN
PUGET SOUND, WASHINGTON. Andrew D. Suhrbier,
Aimee E. Christy, Hector S. Beltran, Daniel P. Cheney, Pacific
Shellfish Institute, Olympia, WA 98501; Jonathan P. Davis, Tay-
lor Shellfish Farms, Shelton. WA 98584: Kenneth M. Brooks,
Aquatic Environmental Science Lab, Port Townsend, WA 98368;
and Frank J. Smith, Northwest Research Associates. Inc., Belle-
vue, WA 98007.
With an annual production of approximately 3 million pounds
live weight on the U.S. west coast, suspended mussel and oyster
culture is predicted to increase significantly in coming years. De-
scription of the changes associated with the culture of these crops
is essential for the siting and evaluation of new culture facilities
and in improving yield and production of existing facilities. This
research has three general objectives; ( I ) to assess mussel shell
growth and meat yield against measured physical, chemical and
biological variables; (2) to compare a suite of variables with mea-
surements of mussel feeding and biodeposit production; and (3) to
collaborate with an on-going nutrient modeling study to estimate
potential mussel carrying capacity in an entire farming area. Dur-
ing the first year (2001-02) multiple observations were made of
water currents, water chemistry, phytoplankton, mussel growth,
seston removal and absorption, fouling, and fish utilization at a
commercial mussel raft culture site in Totten Inlet, Washington.
Certain parameters, such as phytoplankton abundance varied
markedly inside and outside the raft units and under differing tidal
conditions, although these preliminary data suggest feeding effects
on phytoplankton are highly localized and largely contained in the
immediate raft system. The second project year (2002-03) will
continue the Totten Inlet experiments and add a study site at a
commercial mussel farm in Penn Cove. Washington. This research
is supported by the Sea Grant Program Office National Marine
Aquaculture Initiative grant no. NAI6RG159I.
RESULTS FROM THE OLYMPIC REGION HARMFUL
ALGAL BLOOM (ORHAB) PROJECT ON THE WASHING-
TON STATE COAST. THE VALUE OF A COLLABORA-
TIVE PROJECT. Vera L. Trainer, NMFS, Northwest Fisheries
Science Center, 2725 Montlake Blvd. E., Seattle WA 981 12; Bar-
bara M. Hickey, University of Washington. School of Oceanog-
raphy. Seattle WA 98195-7940; Ervin J. Schumacker, Qumault
Department of Natural Resources. PO Box 189. Taholah. WA
98587.
Harmful Algal Blooms (HABs) became a serious problem to
the coast of Washington state in 1991 when blooms of pennate
diatoms of the genus Pseudo-nitzschia produced the potent neu-
rotoxin, domoic acid. Pacific razor clams, (Siliqua patula). and
Dungeness crab, {Cancer magisler). bio-accumulated toxic levels
of domoic acid and recreational and commercial fisheries were
shut down in many areas. Since 1991 Pseudo-nitzschia blooms
have recurred many times along the Washington coast causing
suspensions of fisheries with associated economic and cultural
losses for coastal residents. Federal funding for HAB monitoring
projects since 1991 have been contingent on collaborative efforts
that include local stakeholders. The Olympic Region Harmful Al-
gal Bloom (ORHAB) project secured federal funding in year 2000
to investigate and monitor HABs along the Olympic peninsula.
Participants include state and federal agencies, the University of
Washington, non-profit research institutions, commercial shellfish
growers, coastal tribes and shellfish managers. The ORHAB proj-
ect has made significant findings regarding the physical and
chemical processes which create and transport HABs to the Wash-
ington coast. An initiation site for Pseudo-nitzschia blooms has
been found in the Juan de Fuca eddy region adjacent to Washing-
ton state and Vancouver island. Blooms from this area may be
transported by storm events to the coast where they are ingested by
shellfish. Monitoring and the use of new technologies by ORHAB
participants have better protected the public health and paved the
NSA & PCSGA, Newport. Oregon
Abslracls. September 27-30. 2002 609
way for better understanding of west coast HABs and more etTi-
cient means of early detection and monitoring.
GENETIC DIFFERENTIATION AMONG GEODUCK
CLAM iPANOPEA /lB/ft/Pr.4) POPULATIONS REVEALED
BY ALLOZYME AND MICROSATELLITE ANALYSES.
B. Vadopalas, Scliool of Aquatic and Fishery Sciences. University
of Washington. Seattle. WA 98195; L. L. LeClair, Washington
Department of Fish and Wildlife. Olympia. WA 98504; and
P. Bentzen, Department of Biology. Dalhousie University. Hali-
fax, NS B3H4J1.
The genetic population structure of geoduck clams (Panopeci
ahnipiii) in inland waters of Washington may affect fishery man-
agement and aquacultural practices involving this species. To in-
vestigate genetic differentiation in geoduck clams, samples were
collected from 16 Washington State sites located in the five Puget
Sound subbasins. southern Georgia Strait, and the Strait of Juan de
Fuca. A collection from Clarence Strait in SE Alaska was included
as an outgroup. Individuals were genotyped at 1 1 allozyme and 7
microsatellite loci. To investigate the level of isolation by distance,
we analyzed correlations between pairwise geographic distances
and multilocus Fst values. The Freshwater Bay collection in the
Strait of Juan de Fuca was differentiated from others at both mi-
crosatellite and allozyme loci. For both marker classes, there was
no evidence of significant correlation between genetic and geo-
graphic distance measures. In contrast to the microsatellite loci, the
allozyme loci were in Hardy-Weinburg Equilibrium (HWE). De-
viations firom HWE expectations at microsatellite loci were inter-
preted as being primarily due to primer site sequence variation
rather than population level processes such as inbreeding. These
results may be due to stochastic variation in reproductive success
and recruitment, and warrant further investigation into temporal
genetic differentiation.
TRIAL USE OF THE US NAVY REMOTELY OPERATED
VEHICLE (ROV) SORD IV FOR SAMPLING DEEP
WATER GEODUCK CLAMS (PANOPEA ABRUPTA).
B. Vadopalas, School of Aquatic and Fishery Sciences. University
of Washington. Seattle WA 98195; and Don P. Rothaus. Wash-
ington Department of Fish and Wildlife, Fish Management Pro-
gram, Central Shellfish Unh. Olympia WA 98501-1091.
The existence of geoduck clams (Panopea abrupta) below the
legally fishable depth of 21 m in Puget Sound, Washington has
been surmised from video camera drops in one embayment, but the
study of population dynamics and genetic relationships has been
hampered by the lack of practical deep water sampling methodol-
ogy for macrobenthic infauna. We initiated a deepwater geoduck
sampling trial using the U.S. Navy ROV SORD IV (Submerged
Ordnance Recovery Device). The ROV suction dredge and video
system were modified to enhance geoduck excavation and re-
trieval. The trial was conducted along a depth gradient seaward of
a commercial geoduck bed in central Hood Canal. In four hours of
ROV bottom time between 35 and 80 m depth we positively iden-
tified and attempted sampling of three geoducks. We obtained two
specimens approximately 15 meters apart that share many charac-
teristics, including small size, thin valves, and poor viscerosomatic
condition. The two animals were of the same age. a result unlikely
to arise by chance based on age frequencies in a proximate shallow
collection (p<0.01). Genetic analyses indicated that the two clams
are most likely full siblings (p<0.001). These findings suggest
large variation in year class strength and bias in reproductive suc-
cess among spawners. and underscore the need for further inves-
tigation into population dynamics and recruitment processes in
deep water geoduck.
SHELL CONDITION TESTING OF DUNGENESS CRAB IN
PUGET SOUND, WASHINGTON. Donald E. Velasquez. S.F.
Burton, Washington Department of Fish and Wildlife. 16018 Mill
Creek Blvd.. Mill Creek. WA 98012-1296; D. A. Sterritt and
B. McLaughlin, Washington Department of Fish and Wildlife.
1000 Point Whitney Road, Brinnon, WA 98320-9899.
Since 1997 the State of Washington and the Treaty Tribes have
been conducting cooperative shell condition testing of Dungeness
crab over a large portion of Puget Sound. The purpose of testing
has been to determine when it is best to conduct fisheries during
the year and limit the problems associated with handling softshell
crab.
A number of conclusions have been made regarding the data
collected since the program began. The peak molting season for
legal male crab differs between subareas within Puget Sound. A
pattern where legal-sized crabs finish molting earlier in the year in
Central Puget Sound and later in the year for areas adjacent to the
Canadian border is apparent. Data also indicate the schedule for
the peak softshell period in any given subarea can differ somewhat
from year to year. In a few subareas, it is difficult to assign a single
softshell period because either the softshell crab are not easily
detected or multiple softshell events appear to occur. Possible
explanations for the variation of the peak softshell period within
Puget Sound will be discussed. Additional observations of syn-
chrony and asynchrony in the life cycle of Dungeness crab were
made during shell condition sampling and will be covered.
THE NATIONAL SHELLFISHERIES ASSOCIATION
The National Shellfisheries Association (NSA) is an international organization of scientists, manage-
ment officials and members of industry that is deeply concerned and dedicated to the formulation of
ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man-
agement of shellfish resources. The Association has a membership of more than 1000 from all parts of
the USA. Canada and 18 other nations: the Association strongly encourages graduate students" mem-
bership and participation.
WHAT DOES IT DO?
— Sponsors an annual scientific conference.
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— Produces a Quarterly Newsletter.
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WHAT CAN IT DO FOR YOU?
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— You will get peer review through presentation of papers at the annual meeting.
— If you are young, you will benefit from the experience of your elders.
— If you are an elder, you will be rejuvenated by the fresh ideas of youth.
— If you are a student, you will make useful contacts for your job search.
— If you are a potential employer, you will meet promising young people.
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book review section, information on other societies and their meetings, a job placement section, etc.
HOW TO JOIN
— Fill out and mail a copy of the application blank below. The dues are 65 US $ per year ($35 for students)
and that includes the Journal and the Newsletter!
NATIONAL SHELLFISHERIES ASSOCIATION— APPLICATION FOR MEMBERSHIP
(NEW MEMBERS ONLY)
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Student members only — advisor's signature REQUIRED:
Make checks (MUST be drawn on a US bank), international postal money orders or VISA for $65 ($35 for
students with advisor's signature) payable to the National Shellfisheries Association and send to Nancy Lewis,
Bookkeeper, PO Box 350, V.I.M.S. Eastern Shore Lab, Wachapreague. VA 23480, USA.
INFORMATION FOR CONTRIBUTORS TO THE
JOURNAL OF SHELLFISH RESEARCH
Original articles dealing with all aspects of shellfish re-
search will be considered for publication. Manuscripts will be
judged by the editors or other competent reviewers, or both, on
the basis of originality, content, merit, clarity of presentation.
and interpretations. Each article should be carefully prepared in
the style followed in prior issues of the Journal of Shellfisli
Research before submission to the Editor. Papers published or
to be published in other journals are not acceptable.
Title, Short Title, Key Words, Abstract: The title of the
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beginning of the article. No separate summary should be in-
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Text: Manuscripts must be typed double-spaced throughout
on one side of the paper, leaving ample margins, with the pages
numbered consecutively. Scientific names of species should be
underlined or in italics and. when first mentioned in the text,
should be followed by the authority. Common and scientific
names of organisms should he in accordance with American
Fisheries Society Special Publications 16 and 17; Common and
Scientific Names of Aquatic Invertebrates from tlie United
States and Canada: Molhtsks and CSNAIUSC: Decapod Crus-
taceans, or relevant publications for other geographic regions.
Abbreviations, Style, Numbers: Authors should follow the
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Literature Cited: References should be listed alphabeti-
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should be those recommended in the American Standard for
Periodical Title Abbreviations, available through the American
National Standard Institute. 1430 Broadway, New York, NY
10018. For appropriate citation format, see examples below;
Journal:
Watts, R. J.. M. S. Johnson & R. Black. 1990. Effects of re-
cruitment on genetic patchiness in the urchin Echinometra
mathaei in Western Australia. Mar. Biol. 105;145-151.
Book:
Claudi. R. & G. L. Mackie. 1994. Practical manual for Zebra
Mussel monitoring and control. Boca Raton, FL; CRC Press.
227 pp.
Chapter in Edited Book:
Davio, S. R., J. F. Hewetson & J. E. Beheler. 1985. Progress
toward the development of monoclonal antibodies to saxitoxin;
antigen preparation and antibody detection. In: D. M. Ander-
son. A. W. White & D. G. Baden, editors. Toxic dinoflagel-
lates. Amsterdam: Elsevier, pp. 343-348.
Page Charges: Authors or their institutions will be charged
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for all manuscripts accepted for publication.
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Shellfish Research. Black and white photographs and color
illustrations will be considered.
Corresponding: An original and two copies and electronic
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ation should be sent to the Editor, Dr. Sandra E. Shumway.
Department of Marine Sciences, University of Connecticut,
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shumway@uconn.edu or sandrashumway@hotinail.com
Membership information may be obtained from the Editor
or the Treasurer using the form in the Journal. Institutional
subscribers should send requests to: Journal of Shellfish Re-
search. P.O. Box 465. Hanover, PA 17331.
Alexander Y. Karatayev, Sergey E. Mastitsky, Daniel P. Molloy and Lyubov E. Burlakova
Patterns of emergence and survival of Conchophthirus acuminatus (Ciliophora: Conchophthiridae) from Dreissena
polymorpha (Bivalvia: Dreissenidae) 495
Ronald B. Toll. Robert S. Prezanl and Harold B. Rollins
A novel method for locating tagged infaunal bivalves: Submersible pulse technology metal detectors 501
Christopher M. Pearce, Tara L. Daggett and Shawn M. C. Robinson
Effects of starch type, macroalgal meal source, and (i-carolene on gonad yield and quality of the green sea urchin
Slnmgylocenlmtiis droebachiensis (Miiller) fed prepared diets 505
Louis R. D'Abramo and Cortney L. Ohs
Production of red swamp crawfish (Procamharus clurkii) in earthen ponds without planted forage: Establishment,
maintenance and harvest of populations 521
Lxjuis R. D 'Abramo, Cortney L. Ohs and Kathleen C. Elgarico
Production of red swamp crawfish {Procamharus clarkii) in earthen ponds without planted forage: Evaluation of trap
and seine harvest strategies 527
Enrique Lozano-Alvarez, Patricia Briones-Fourzdn and Maria Eugenia Ramos-Aguilar
Distribution, shelter fidelity, and movements of subadult spiny lobsters [Pamdirus urgiis) in areas with artificial
shelters (Casitas) 533
Fuhua Li, Jianhai Xiang, Xiaojun Zhang, Changgong Wu, Chengsong Zhang, Linghua Zhou and Kuijie Yu
Tetraploid induction by heat shocks in Chinese shrimp Feniieropeiiaeus chinensis 541
Guoqiang Huang. Shuanglin Dong, Fang Wang and Shen Ma
Selection and use of different diets in a study of Chinese shrimp. Fenneropenaeus chinensis 547
Monica Y. Tsuzuki, Ronald O. Cavalli and Adalto Bianchini
Effect of salinity on survival, growth, and oxygen consumption of the pink-shrimp Farfantepenaeus paulensis
(Perez-Farfante 1967) 555
P. M. Troffe, S. Ong, C. D. Levings and T. F. Sutherland
Anatomical damage to humpback shrimp, Pandalus hypsinotus (Brandt 1851) caught by trawling and trapping 561
Francesc Sardd, Joan B. Company and Arturo Castellan
Intraspecific aggregation structure of a shoal of a western Mediterranean (Catalan coast) deep-sea shrimp, Aristeus
antennalits (Risso, 1816), during the reproductive period 569
Ferdinand F. Wirth and Kathy J. Davis
Seafood dealers" .shrimp-purchasing behavior and preferences with implications for United States shrimp farmers 581
Kenneth M. Brown. Gary W. Peterson. Patrick D. Banks. Brian Lezina, Charles Ramcharan and Michael McDonough
Olfactory deterrents to black drum predation on oyster leases 589
Abstracts of technical papers presented at the 56th Annual Meeting of the Pacific Coast Section. National Shellfisheries
Association, Newport, Oregon, September 27-30, 2002 597
COVER PHOTO: Sea urchins (Strongylocentroliis droebachiensis, S. frunciscanus. and 5. purpuralus) being used in
a gonad enhancement experiment at the Pacific Biological Station (Fisheries and Oceans Canada, Nanaimo, British
Columbia, Canada) to test the efficacy of various prepared feeds to produce suitable gonad color, taste, firmness, and
texture. Photo: Chris Pearce.
The Journal of Shellfish Research is indexed in the following: Science Citation Index®. Sci Search®, Research Alert®, Current
Contents*/Agriculture, Biology and Environmental Sciences, Biological Abstracts, Chemical Abstracts, Nutrition Abstracts, Current
Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography,
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.
JOURNAL OF SHELLFISH RESEARCH
Vol. 22, No. 2 September 2003
CONTENTS
Fabrice Fernet, Rejean Tremblay and Edwin Bourget
Biochemical indicator of sea scallop (Placopecten magellaniciis) quality based on lipid class composition. Part I:
Broodstock conditioning and young larvae performance 365
Fabrice Fernet, Rejean Tremblay and Edwin Bourget
Biochemical indicator of sea scallop iPliuapecteii magellaniciis) quality based on lipid class composition. Part II:
Larval growth, competency and settlement 377
Stephen L. Estabrooks
A rapid test for the determination of the spawning status of the bay scallop. Argopecten inadians
(Lamarck, 1819) 389
Ruben Avendano-Herrera. Carlos Riquelmes, Fernando Silva. Miguel Avendanod and Rate Irgang
Optimization of settlement of larval Argopecten purpuraius usnig natural diatom biofiliiis 393
Enid K. Sichel and Richard C. Karney
Adhesives to attach juvenile bay scallops to plastic netting in aquaculture 401
Tao Zhang, Hongsheng Yang, Huayong Que, Guofan Zhang, Shilin Liu, Yichao He and Fusui Zhang
Evidence for the involvement of cyclic AMP in the metamorphosis of the bay scallop. Argopecten uiadians
(Lamarck ) larvae 403
William J. Dore, Jennifer Farthing and Ian Laing
Depuration conditions for great scallops (Pecten niaximus) 409
Oscar Chacon, Maria Teresa Viana, Ana Farias, Carlos Vazquez and Zaul Garcia-Esquivel
Circadian metabolic rate and short-term response of juvenile green abalone [Halioiis Jiilgens Philippi) to
three anesthetics 415
Sean E. Matson, Jonathan P. Davis and Kenneth K. Chew
Laboratory hybridization of the mussels. Mylilus Irossulus and M. galtoprovincialis: Larval growth, survival, and
early development 423
Supannee Leethochavalit, E. Suchart Upatham, Kang-Sik Choi, Pichan Sawangwong, Kashane Chalermwat
and Maleeya Kruatrachue
Ribosomal RNA characterization of non-transcribed spacer and two internal transcribed spacers with 5.8S ribosomal
RNA or Perkinsiis sp. found in undulated surf clams (Paphiii uitdiihila) from Thailand 43 1
M. Delgado and A Perez Camacho
A study of gonadal development in Ruditapes deciissateu (L.| (Mollusca. Bivalvia), using image analysis techniques:
Influence of food ration and energy balance 435
M. Albentosa, M. J. Ferndndez-Reiriz, U. Labarta and A. Perez-Camacho
Absorption of biochemical components and feeding behavior with natural and carbohydrate-rich diets in Ruditapes
decussatus and Venenipis pidla.sira clams 443
Stephen R. Fegley, Susan E. Ford, John N. Kraeuter and Harold H. Haskin
The persistence of New Jersey's oyster seedbeds in the presence of oyster disease and harvest: The role
of management 45 1
Jorge Chavez- Villalba, Jean Barret, Christian Mingant, Jean-Claude Cochard and Marcel Le Pennec
Influence of timing of broodstock collection on conditioning, oocyte production, and larval rearing of the oyster,
Crassdsirea giga\ (Thunberg). at six production sites in France 465
Mi Seon Park, Chang-Keun Rang, Dong-Lim Choi and Bo-Young Jee
Appearance and pathogenicity of ovarian parasite Marteitioides clwngmuensis in the farmed Pacific oysters,
Crassoslrea gigas. in Korea 475
Gab-Man Park and Ee-Yung Chung
Molecular phylogenetics of five Corbicula species determined by partial 28S ribosomal RNA gene sequences 481
Alexander Y. Karatayev, Lyubov E. Burlakova, Thomas Kesterson and Dianna K. Padilla
Dominance of the Asiatic clam, Corhiciila Jlumtnea (MUller), in the benthic community of a reservoir 487
CONTENTS CONTINUED ON INSIDE BACK COVER
JOURNAL OF SHELLFISH RESEARCH
VOLUME 22, NUMBER 3
DECEMBER 2003
The Journal of Shellfish Research
(formerly Proceedings of the National Shellfisheries Association)
is the official publication of the National Shellfisheries Association
Editor
Sandra E. Shumway
Department of Marine Sciences
University of Connecticut
Groton, CT 06340
EDITORIAL BOARD
Peter Cook (2004)
Austral Marine Services
Lot 34 Rocky Crossing Road
Warrenup
Albany. W.A. 6330, Australia
Simon Cragg (2004)
Institute of Marine Sciences
University of Portsmouth
Ferry Road
Portsmouth P04 9LY
United Kingdom
Leroy Creswell (2005)
University of Florida/Sea Grant
8400 Picos Road, Suite 101
Fort Pierce, Florida 34945-3045
Lou D'Abramo (2004)
Mississippi State University
Department of Wildlife and Fisheries
Box 9690
Mississippi State, Mississippi 39762
Christopher V. Davis (2004)
Pemaquid Oyster Company, Inc.
P.O. Box 302
1957 Friendship Road
Waldoboro. Maine 04572
Ralph Elston (2005)
Aqua Technics/Pacific Shellfish Institute
455 West Bell Street
Sequim, Washington 98382
Susan E. Ford (2004)
Rutgers University
Haskin Shellfish Research Laboratory
6959 Miller Avenue
Port Norris, New Jersey 08349
Raymond Grizzle (2005)
Jackson Estuarine Laboratory
Durham, New Hampshire 03824
Karolyn Mueller Hansen (2004)
1524 Bariey Circle
Knoxville, Tennessee 37922
Journal of Shellfish Research
Volume 22, Number 3
ISSN: 0730-8000
December 2003
www.shellfish.org/pubs/jsr.htm
Standish K. Allen, Jr. (2004)
Aquaculture Genetics and Breeding
Technology Center
Virginia Institute of Marine Science
College of William and Mary
P.O. Box 1346
Gloucester Point, Virginia 23062
Shiriey Baker (2004)
University of Florida
Department of Fisheries and Aquatic Sciences
7922 NW 71" Street
Gainesville, Florida 32653-3071
Bruce Barber (2005)
School of Marine Science
University of Maine
5735 Hitchner Hall
Orono, Maine 04469
Brian Beal (2004)
University of Maine
9 O'Brien Avenue
Machias, Maine 04654
Neil Bourne (2005)
Fisheries and Oceans
Pacific Biological Station
Nanaimo, British Columbia
Canada V9T 6N7
Andrew R. Brand (2005)
University of Liverpool
Port Erin Marine Laboratory
Port Erin. Isle of Man IM9 6JA
United Kingdom
Eugene Burreson (2005)
Virginia Institute of Marine Science
P.O. Box 1346
Rt. 1208 Create Road
College of William and Mary
Gloucester Point, Virginia 23062
Mark Luckenbach (2005)
Virginia Institute of Marine Science
Eastern Shore Lab
P.O. Box 350
Wachapreague, Virginia 23480
Bruce MacDonald (2004)
Department of Biology
University of New Brunswick
Saint John, New Brunswick
Canada E2L 4L5
Roger Mann (2004)
Virginia Institute of Marine Science
Gloucester Point, Virginia 23062
Islay D. Marsden (2004)
Department of Zoology
Canterbury University
Christchurch. New Zealand
Jay Parsons (2005)
Memorial University
Marine Institute
Box 4920
St. John's, Newfoundland
Canada AlC 5R3
Tom Soniat (2004)
Biology Department
Nicholls State University
Thibodaux, Louisiana 70310
J. Evan Ward (2004)
Department of Marine Sciences
University of Connecticut
1080 Shennecossett Road
Groton, Connecticut 06340-6097
Gary Wikfors (2004)
NOAA/NMFS
Rogers Avenue
Milford, Connecticut 06460
Joiimul oj Shcllftsli Rcscunh. Vol. 22, No. 3. 611-613, 2003.
FEB 3 2004
V.'CT:.':
Melbourne Romaine Carriker
Honored Life Member
Melbourne Cuniker, or "Mer" as he is known ti) his many students, colleagues, and friends is a world recognized student of
Malacology, and an authority on marine subjects as diverse as functional morphology, biominerah/.ation. larval ecology, and predator-
prey interactions. Mel's interest in shellfisheries extends from his intense interest in molluscs, their ecology, biology, and morphology.
Scientist, scholar, husband, father, and friend — his career and his life have been punctuated by transition and achievement.
Mel's fascinating story began on February 2.Sth, 1915 when he was born in Santa Marta. Colombia. For the first twelve years of his
life. Mel li\ed on a coffee plantation (called Vista Nieve) with his American parents. His parents. Myrtle Carmela Carriker de Flye and
Melbourne Armstrong Carriker. Jr.. developed and managed the coffee plantation in the Siena Nevada de Santa Marta Mountains.
During his early years, Mel lived in an agrarian community among crops of coffee and sugarcane, hnmersed in rugged surroundings,
he and his siblings happily lived on the edge of a tropical paradise. When he was ten. Mel began accompanying his father, an
accomplished amateur naturalist and ornithologist, on short field trips to collect birds, birds' eggs, and small mammals. Undouhtably,
these experiences sparked his interest in the natural world and the seemingly secret lives that animals lead.
In 1927, Mel's parents sold the coffee plantation and moved the family to southern New Jersey, taking up residence in Beachwood.
His father took a position at the Academy of Natural Sciences of Philadelphia as Associate Curator of Ornithology, and Mel was enrolled
in Toms River grade school. After struggling through the depression years with his family, Mel graduated from Toms River High School
in 1934. Immediately after graduation, he accompanied his father on an ornithological expedition into Bolivia, South America. This
"enviable, exhilarating experience" (p. 273, Carriker 2()()()), reinforced Mel's desire to further his education in the field of zoology, in
particular ornithology. In the fall of 1935 Mel entered Rutgers University, New Jersey, majoring in agricultural research and minoring
in zoology. During several summers, he worked as the director of aquatic and recreation programs at the Boy Scout Camp, Burton-at-
Allaire in southern New Jersey to earn money for college. It was at Rutgers that Mel met Thurlow C. Nelson, his undergraduate adviser
and mentor, who offered him an opportunity that shaped his scientific career. Through Nelson's urging. Mel began working on Rutgers'
College of Agriculture's houseboat in Barnegat Bay. New Jersey, in the summer of 1938. studying the life history of oyster larvae. In
subsequent summers of 1939 to 1941. he continued this pursuit on the "Cynthia." broadening his studies to include the general biology
and ecology of oysters.
In the fall of 1939. Mel traveled to the University of Wisconsin where he began graduate work with Lowell E. Noland. For his
graduate work, he studied the biology of the pond snail, Lynmaeu sU)i>iuilis. a host of the trematode worm that causes swimmer's itch.
It was here that Mel began honing his skills as a scientist, studying invertebrate anatomy and physiology, and prepared his first paper
on the boring mechanisms of the oyster drill snail. Wisconsin also introduced Mel to one other love, his future wife Scottie McAllister.
In 1942. he participated in his first NSA annual meeting, presenting his first scientific paper on oyster-drill boring mechanisms! Mel
graduated (in June of 1943) with a doctoral degree in invertebrate zoology and physiological chemistry, and with the rank of ensign in
the U.S. Naval Reserve.
Immediately after graduating from the University of Wisconsin, he entered the Naval Training School. Harvard University, where he
was trained in naval communications. During World War II. he served on a PC 780 ship in the Aleutian and Hawaiian Islands as
communications officer. Although naval duty interrupted Mel's career, his love for malacology continued; rumor has it that during his
time off Mel would explore the coast around Adak (Aleutian Islands) collecting marine molluscs and their hemolymph to mail to Rutgers
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612 Ward
University for ongoing systematic studies. After the War. he returned to the east coast and accepted a position as instructor in the
Department of Zoology at Rutgers in 1946.
Mel worked at Rutgers for eight years, being promoted to Assistant Professor before leaving in 1954. During his time as a faculty
member at Rutgers, he developed courses (e.g.. estuarine ecology graduate course), taught, and. during the summers, worked with T.C.
Nelson and Harold Haskin (see Kraeuter and Ford 1999), investigating the biology of the quahog. In the summers of 1947 to 1949. he
returned to the houseboat "Cynthia" in Little Egg Harbor. New Jersey, establishing a research program in shellfish biology that would
span his career. From a small laboratory in the stem of the houseboat, he studied quahog ecology and continued researching the
shell-boring mechanisms of predatory gastropods. This research was the foundation for several classic published works including.
"Critical Review of Biology and Control of Oyster Drills Urosalpinx and Euplewa" (Carriker 1955). and "Interrelation of Functional
Morphology, Behavior, and Autecology in Early Stages of the Bivalve Mercenaria mercenarid" (Carriker 1961 ). Mel lived on the boat
with his wife Scottie and two children, Eric and Bruce; a happy but nonetheless crowded existence.
In 1954. Mel was offered, and accepted, a position as Associate Professor at the University of North Carolina (UNC). Chapel Hill.
He taught marine ecology and conducted marine-related research in the Department of Zoology. During the summers of 1953 to 1955
he also conducted research on pond culture of oysters and clams on Gardiner's Island. New York. This work was sponsored by the J.
& J.W. Elsworth Oyster Company and the U.S. Fish & Wildlife Service. Mel's work on Gardiner's Island was productive and brought
him in contact with shellfish biologist Victor Loosanoff. In 1956. his research on clam larvae was shifted to the UNC Institute of
Fisheries Research in Morehead City. Over the next five years Mel interacted with scientists at the Institute and at Duke University
Marine Laboratory a few miles away, focusing his research on larval biology and the predatory drilling snails of oysters. In 1961. due
to unfriendly politics that can be encountered in academia. Mel left UNC and took a position with the U.S. Bureau of Commercial
Fisheries Biological Laboratory. Oxford, Maryland. At the Oxford Laboratory he began working on an emerging disease of oysters
known as MSX. and this research consumed all of his time. The move to Oxford, however, was to be short lived. In 1962. Mel was
enticed by an offer to head a new systematics and ecology program at the Marine Biological Laboratory in Woods Hole. Massachusetts.
The Carriker family moved to Falmouth, Massachusetts, in the fall of 1962. where Mel assumed the position as Director of the
Systematics-Ecology Program. The long-term goal of this program was to spearhead research and training in marine systematics and
ecology, and enhance the scientific knowledge of organisms in the Cape Cod region. This Program turned out to be "highly successful
and functioned productively for ten years" (p. 281. Carriker 2000). One of the most recognized accomplishments of the Program was
the publication of a set of keys and check lists of the common invertebrates of, essentially, the waters of southeastern New England. First
published in 1964. the "Keys to Marine Invertebrates of the Woods Hole Region" (edited by Ralph I. Smith) provided nonsystematists
a useful guide for the identification of many common invertebrates in the region, and were invaluable to students and scientists alike.
The first complete revision of these keys in 35 y began in 1999. and the first revised sections can be viewed on the Marine Biological
Laboratory's web site. Unfortunately, due to a shortage of funds, the Program was closed in 1972. By then. Mel's reputation as an
outstanding marine scientist proceeded him. and he was offered a full professorship at the new College of Marine Studies (CMS).
University of Delaware, in Lewes.
In the fall of 1972. Mel and his wife Scottie moved to Delaware where he taught, conducted research, and helped shape the CMS
graduate program for thirteen years. During this time he studied oyster shell ullrastructure and chemistry as related to shell penetration
by oyster borers, taught a course in malacology, and supervised the research efforts of many graduate students (including some from
Central and South America). Mel officially retired in February 1985 at the age of 70. receiving the title of Professor Emeritus. After
retiring, he served as president of the Delaware-Panama Partners of the Americas: he continues his scholarly contributions through his
writings about his family and the science he loves. In 2000. Mel published a book concerning the fascinating history of his family and
their coffee plantation titled "Vista Nieve." from which much of this biography has been gleaned.
Mel is an accomplished scientist, publishing over 45 abstracts and 160 scientific papers and reports, and coining well-known
malacological terms such as the "accessory boring organ" (ABO) of muricids, and the "pediveliger" stage of bivalve molluscs. He has
presented technical papers at meetings and chaired scientific session over 255 times. From 1965 to 1977. Mel served as editor for the
manuals on the Marine Flora and Fauna series produced by the National Marine Fisheries Service. His dedication to the scientific
community is evidenced by the many positions he has held including chairman of the Division of Invertebrate Zoology. American
Society of Zoology ( now the Society of Integrative and Comparative Biology): vice-president of the Association of Marine Laboratories
of the Caribbean: and president of the Institute of Malacology, the American Malacological Society, and the Atlantic Estuarine Research
Society.
For almost a 50 y period. Mel has served NSA in various capacities, including: Secretary-Treasurer from 1953 to 1954. Vice President
between 1955 to 1957, President from 1957 to 1959, and as a source of trusted advice for many an Executive Committee ever since. As
Secretary-Treasurer, he was instrumental in formalizing the regular publication of the Association's meeting notes as the "Proceedings
of the National Shellfisheries Association (PNSA)," serving as its first Editor from 1954 to 1957. Mel also served several times on the
Publications Committee, including during 1979 to 1980 when the name of the NSA publication was changed from the PNSA to the
Journal of Shellfish Research. In 1978, Mel was presented with the Honored Life Member award by NSA. and in 1998 was recognized
for his years of dedication and scientific achievement in shellfish research when the first NSA student research award was named in his
honor. Presently. Mel serves as Historian of the Association, recently completing an historical account of NSA as it emerged from earlier
oyster meetings and groups, titled "Taming of the Oyster" (in press).
Throughout his career Mel has been a teacher, researcher, editor, and mentor. He has supervised 35 graduate students ( 17 Ph.D., 18
M.S.) and has served on numerous graduate student committees. Those of us who have had the pleasure of being a student of Mel's know
his objective, quiet approach to seemingly unsurmountable problems, and his deft ability to hone a piece of writing — with comments
neatly scripted in pencil on just about every page of many a proposal or paper (often to the immediate displeasure of his students) — so
Honored Life Member M. R. Carriker 613
that it was clear and concise. Mel is a source of knowledge and encouragement, and continues to mentor, albeit informally, young
students, former graduate students, and colleagues at yearly scientific meetings and e\ents. The scientific fields of malacology, shellfish
biology, and marine ecology have prospered from his life's work, and all of us who have had the pleasure of interacting with him have
benefitted by Mefs wisdom, poise, and grace.
J. Evan Ward
Groton. Connecticut
REFERENCES
Carriker. M. R. 2000. Viski Merc. Blue Mantle Press. Rio Hundu. Texas. 313 pp.
Kraeuter. J. & S. Ford. 1494. Harold Haley Haskin. Honored Life Member. J. Shellfish Res. 18:337-339.
Joiiniul of Shellfish Research. Vol. 22. Nci. ?. (il?-6l7. 2003.
Michael Castagna
Honored Life Member
Michael Castagna. known to almost everyone in NSA as Mike, was horn in Janesvijie. Wisconsin on October 21. 1927. His parents
immigrated to this country from Sicily; his father worked in a General Motors factory in Janesville and his mother worked in the home
and for a time in a woolen mill. Following graduation from Janesville High School in 1945. Mike joined the Navy, received his initial
training in the Great Lakes, and first viewed the ocean when he shipped out for the Pacific. Mike was stationed in Honolulu where he
served as a Pharmacist Mate 2nd Class from 1945 until 1949.
After leaving active duty in the Navy, he enrolled at Florida State University as an undergraduate where he participated in
intercollegiate sports, swimming on the all-Navy swim team. In 1951. with only one semester of study remaining at FSU. Mike was
recalled to active duty for the Korean conflict as a Hospital Corpsman 2nd Class. Mike's swimming talents were quickly put to use as
he became one of the first Navy divers to use SCUBA, taking part in many of the initial dives that led to the development of the now
familiar dive tables. When his tour of duty was over in 1953. he returned to FSU to complete work on his Bachelor of Science degree.
While enrolled in school. Mike supported himself by working in the Women's Department of Physical Education. After receiving his
B.S. degree in 1953. he was admitted to the graduate program at FSU where he worked on a Master's degree. He completed this degree
in 1955 with a study of the distribution and ecology of the hogchoker (Trinecles nniciilatiis) in the Wakulla River under the guidance
of Dr. Ralph Yerger.
In his first job out of graduate school, many of Mike's talents — swimming, fisheries, biology, and a keen love of the ocean — were
used as an Assistant Curator at Marine Studios of Marineland. located just south of St. Augustine. FL. He literally swam with dolphins
and was in charge of caring for and treating any of the animals that became ill. At this time Mike and his wife of 48 y. Mary Sperry.
got married. Mary worked for many years as a nurse and she and Mike have four children.
In 1956. Mike was hired by the Bureau of Commercial Fisheries (BCF) in Boothbay Harbor. ME. to work on the herring investi-
gations under Les Scattergood. This job put him back out on the ocean with frequent sampling trips offshore. During the two years Mike
spent in Boothbay Harbor, he served in the Naval Reserves and on several occasions was sent to Key West. FL. for Underwater
Demolition Training. There, as the oldest member of the team at nearly 30. he was called "Grandpa" by the younger team members, but
he went on to graduate with highest honors.
In 1958 he left Boothbay Harbor and began work at a small BCF laboratory in Franklin City. VA. This laboratory was placed at the
end of a long causeway on a former railroad spur, which extended into Chincoteague Bay. The sheet metal building was built next to
the former railroad pier. It was a perfect place for Mike who has both the ability to develop new techniques and a hands-on work ethic.
Mike has always had a firm commitment to understanding the fundamental ecology of the area where he was working. This included
what was present, where it could be found, general observations on abundance, and life history biology. To that end. he helped to design
and fabricate the gear needed to investigate the marine life of the bay.
After a short time in Franklin City, he was asked by a fellow Florida State graduate. Bill Hargis. to become the Scientist-in-Charge
of the Virginia Institute of Marine Science (VIMS). College of William and Mary laboratory in Wachapreague, VA (At the time it was
only known as the "Eastern Shore Laboratory", and it was not until much later that the formal connection to William and Mary was
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established). This new position included moving into a newly constructed, single floor building housing offices, wet and dry laboratories.
and two dormitory rooms. This was an inspired choice, because it allowed Mike"s skills, of leadership, mentoring, innovation, and hard
work to flourish. Mike began this position in 1962. remained at the Eastern Shore Laboratory moving up the ranks at VIMS until he
retired as a Professor and Division Director in 1992. He continues to work at the laboratory as a Professor Emeritus.
Mike instituted a prograin to gather basic biological information on the flora and fauna of the local area. He became intimately
involved in seeking information froin, and giving information to. the local fishing community and encouraged others to take field trips
to the Wachapreague area.
In addition to the basic science efforts. Mike coordinated the Eastern Shore components of many oyster trials that were being
conducted at VIMS in Gloucester Point. VA. Efforts to control oyster drills with the pesticides Polystream and Sevin. numerous studies
on oyster disease and the effectiveness of disease resistant stocks in the higher salinity waters were among the research
projects that kept Mike and many others busy with field work.
Although his diving skills were not used heavily at Wachapreague. he often helped members of the community find lost gear or clear
fouled propellers. The love of diving and natural history were combined when Mike was invited to spend nine days in the Underwater
Laboratory Helgoland in the Baltic Sea in 1974. He returned there as a scientific coordinator for a 14 day underwater mission in 1978.
In between (1976) he spent five days in a Hydro Laboratory off Freeport, Bahamas.
In 1962, Mike hired Paul Chanley and they began a series of investigations into bivalve natural history. This included providing
information on spawning times, salinity tolerance, larval development, and other aspects for over 60 species. By the end of Mike's tenure
as head of the laboratory. 55 species had been reared to setting and 26 species had been reared through their entire life cycle. Much of
this work was done in large garbage cans. Water was exchanged by siphons, but was, from time to time, carried in buckets across the
road by hand. When temperatures in the wet laboratory were not high enough to rear larvae, the "culture containers"" were placed on
wheeled cans and aligned down the hall between the offices. This Spartan setting was certainly indicative of funding limitations, but it
also reflected Mike's frugal, get-the-job-done approach.
As a direct result of the efforts to document the various life history parameters of bivalves. Mike developed expertise in hatchery
technology and aquaculture. This led to the development of a greenhouse for culturing large quantities of algae via the Wells-Glancy
technique and later, in a converted oyster shucking house, to a fairly large nursery for the hatchery output of bay scallops and hard clams.
Here again. Mike's ability to design and engineer simple, cost-effective solutions was critical. One of the most enduring images from
this hatchery was a heat exchanger crafted from an old whiskey ban-el and salvaged tubing. Mike often said, ""There's no reason to spend
$2 on a valve if pinching a hose will work just as well."
Already involved with maintaining a large number of oysters in trays, scattered throughout several bays, Mike was well aware of the
difficulties with field studies. This reality and the lack of seed caused his early focus on hatchery and nursery work with clams and
scallops. The success of this program provided burgeoning numbers of clams and scallops and he began to develop experimental field
plantings. Unless they were heavily protected in trays, the early clam plantings were nearly all consumed by crabs, and e\en modest size
grow-out experiments required tremendous effort. As an example of Mike's inventiveness, one fall, with a substantial number of clam
seed on hand, and the necessity of having to close down the seawater pumps for the winter, Mike happened to glance out the window.
Within the past week, the road had been taned and covered with gravel. Most of the gravel had been pushed to the side of the road. Mike
decided that, because of the well-documented association of clams with shell beds, gravel inight be a good shell substitute. The gravel
was swept from the road, loaded into a scow and placed on an intertidal mud flat of a marsh creek. Clams were planted in this gravel
and survival was excellent! Unfortunately, subsequent years' plantings did not survive as well. It took Mike, the Eastern Shore
Laboratory staff, input from various waterinen, many clams, a number of years and a lot of trial and error to develop the knowledge of
planting size and protective mechanisms to assure consistent results with seed planting. This effort, as with the innovative descriptive
work of Chanley and Castagna a decade earlier, established the Eastern Shore Laboratory as a premier place to do research on bivalve
shellfish. This reputation was enhanced by the development of a course to teach basic techniques in clam aquaculture, including how
to make the gear, to a cadre of individuals. Many of these individuals became leaders in the hard clam aquaculture industry that has
spread throughout the east and gulf coasts, now employs hundreds of indi\ iduals and is woilh tens of millions of dollars annually.
Mike has authored or co-authored >75 peer reviewed publications, many abstracts, served as editor for two books and was a co-author
on a host of reports — including one that has probably been read by more individuals than any work published in the peer-reviewed
literature. "'A manual for growing the hard clam Mercenaria mcrccnaria".
Field Trips
Because of his interest in natural history and his gregarious nature. Mike was always ready to lead a field trip. These were of two
types, those for fellow scientists visiting the Eastern Shore Laboratory and those for students.
Always the raconteur par excellence Mike had many tales to tell about visits from scientists. One that left a distinct impression was
a visit by a distinguished senior scientist from Europe. Mike was impressed by the scientist"s world reputation and wanted to provide
a grand tour, which included visiting the habitats on a nearby barrier island. The island had a few cabins that were used primarily on
summer weekends, and in the winter for hunting. Mike anchored the boat and indicated they would have to wade ashore. The senior
scientist had already figured this out and proceeded to disrobe — completely. Though there were seldom people on the island, passing
sport or commercial fishing boats were not uncommon. Mike, thinking that someone might pass by. and wanting to keep the situation
as decorous as possible for the laboratory's reputation, handed the individual a towel. The scientist thanked him and proceeded to wrap
the towel around his head as a turban and walked ashore.
A significant part of the program at the VIMS Eastern Shore Laboratory was the hosting of field trips for students from other
Honored Life Member Michael Castagna 617
institutions. This program, which) Mil<e enthusiastically instituted and formalized, required the maintenance and use of small boats.
Laboratory staff ran the boats and depending on the group size, availability of various personnel, Mike, or senior staff members were
often responsible for conducting the tour. During Mike's tenure, thousands of students from dozens of institutions of higher learning were
housed at the Eastern Shore Laboratory and given a first class "hands on" introduction to local habitats.
As might be expected at such a small laboratory, everyone on Mike's staff was expected to do a little of everything and to be on hand
to help everyone else. This expectation included a weekly. Friday afternoon general clean up of the laboratory and offices. Everyone was
expected to grab a broom or mop, haul out the trash and perform other janitorial duties. While this might seem like a waste of "valuable
staff time to some, the system worked well because Mike participated regularly. It also made everyone aware that if the laboratory was
kept clean all week, there was less to do on Friday afternoon. In addition, the "janitor" for the laboratory and dorm was also an individual
who helped run the hatchery, ran the nursery and helped in the field when needed. The entire laboratory staff also participated in building
the new shop, installing bulkheads, and refurbishing the seawater system and the "new dorm". In the more sophisticated environments
of today's laboratories, such a system might help reestablish the "hands on" and "everyone is responsible for the entire laboratory"
attitude that is so often lacking, but to do so requires commitment and leadership. This Friday clean up continued as Mike expanded the
laboratory's footprint by purchasing a complex of buildings including a former oyster shucking business and a house with a large lot
next door (a future dormitory). Also included in the laboratory administration and staff duties, with a few hired local hands during the
winter, was building the greenhouse mentioned above, the construction of a new shopAstorage complex, refurbishing of the bulkheads
along the entire property, and converting the shucking house to a wet-laboratory/bi\alve nursery.
Society Work
Leadership is a hallmark of Mike Castagna. While Mike's "aw shucks" demeanor might not lead one to conclude that he was leading,
he did so by example. This leadership quality has always been clearly evident to all who worked with him, and was recognized by his
peers. Evidence of this is his enormous efforts on the part of the Atlantic Estuarine Research Society, Estuarine Research Federation and
his beloved National Shellfisheries Association. In all three organizations he served as Secretary, Treasurer (or Secretary-Treasurer) and
President, and has been active on numerous committees and subcommittees, often for many years. Mike spent 16 y as Chair of the NSA
Publications Committee and almost single handedly rescued the Journal of Shellfish Research from near oblivion. For this and his
continued efforts on behalf of the NSA, he was recognized with an honorary award and a student endowment was established in his name
accompanying this honor. It was Mike who recruited Sandy Shumway as Editor, and thus he is directly responsible for the expansion
of the journal quality and quantity.
He has received honorary awards from the Atlantic Estuarine Research Society (1983) and the Estuarine Research Federation (1985).
He also became an Honorary Life Member of the Virginia Shellfish Growers Association (1992). The National Shellfisheries Association
honored him with the Wallace Award (1983). the Honored Life Member Award (1990). a special recognition in 1992, and lastly the
Society rewarded him for 16 y of service to the Publications Committee and the NSA with the establishment of the Castagna Student
Endowment, noting specifically that the award was to go to a student carrying out applied research.
In addition, Mike was an early enthusiastic supporter of the then fledgling Nature Conservancy. He particularlv liked the fact that
they didn't spend a lot of time litigating or trying to infringe on others land use, but simply bought the land and then tried to develop
appropriate management plans. Again this is a "hands on" approach and it earned Mike the Oak Leaf Award from the Nature
Conservancy as the Conservationist of the Year in 1974 for his efforts to preserve portions of the Eastern Shore for future generations.
In addition to these formal society activities, Mike also enjoyed the evening meeting socials, particularly if there was good music for
dancing. If there were music and willing partners. Mike would be on the dance floor until the music stopped, and then he would often
organize a group to go out and find a spot to continue the dancing. Somehow he always seemed to be ready for the first paper of the
meeting the next day.
A true love for the natural world and its mysteries, leadership coupled with humbleness, a "can do" spirit and interest in seeing these
combined and applied are the mark of someone who cares and makes a difference. These are the hallmarks of Mike's efforts for NSA,
Virginia, shellfish culturists, and science. We can all be Mike's students in this regard.
John N. Kraeuter
Haskin Shellfish Laboratory
IMCS
Rutgers University
Port Norris, NJ
Mark W. Luckenbach
Virginia Institute of Marine Science
Wachapreague. VA
.Icninial ofSlicllfixh Rcscunh. Vol. 22. No. 3, (i 19-620, 2003.
I
Dexter Stearns Haven
Honored Life Member
Dexter Stearns Haven may have officially "retired" from the Virginia Institute of Marine Science (VIMS) in 1984, but a quick look
at his publication list or curriculum vitae will illustrate that Dexter has far from actually retired. He has published and or coauthored over
1 1 papers in the intervening years. In addition, he has been involved as a Director of the York Chapter of the Chesapeake Bay
Foundation, and can be seen selling brooms to raise funds for the York Lions Club, of which he is a Charter Member. He volunteers
regularly as a docenl at the Watermen's Museum in Yorktown. where he shares his knowledge of the Chesapeake Bay. its resources,
and the men who harvest them with thousands of visitors. He assists the archaeologists of Jamestown by examining and dating old oyster
shells. In truth. Dexter is far from retired.
Fortunately for VIMS. Dexter is continuing his research by working with Bill Hargis. Helen E. Woods, and others on the ecology
of oyster bars (reefs) of the Chesapeake Bay. This research, aided by new technology, has resulted in a number of three-dimensional
posters on the reefs of the James, York. Rappahannock, and Potomac subestuaries of the Chesapeake system. Formal papers are expected
to follow.
Dexter Haven was bom in Lake Forrest. Illinois, on November 2, 1918. In 1942, he received a Bachelor of Science degree in
premedical subjects at the University of Rhode Island. After receiving his degree, he served his country in the U.S. Army Air Corps as
a weatherman in the 9th Weather Squadron.
At the end of his military service in 1946. he again enrolled at the University of Rht)de Island, receiving a Master of Science degree
in marine biology in 1948. After completing the masters degree. Dexter joined the U.S. Fish and Wildlife Service in 1948. In 1949, he
joined the staff of the Virginia Fisheries Laboratory at Yorktown, Virginia (predecessor of the VIMS of the College of William and Mary,
and now located at Gloucester Point. Virginia, just across the York River from Yorktown).
In addition to his research and teaching duties before retiring. Dexter served in several capacities at VIMS; as Senior Marine Scientist
and Head of the Department ot Applied Biology: and as Professor of Marine Science of the School of Marine Science. Upon officially
retiring in June of 1984. he became Professor Emeritus of the College of William and Mary.
During his .3."; years of service at the VIMS (and the School of Marine Science). Dexter worked primarily on the physiology and life
history of molluscs, and on the natural history and sedimentology of oyster bars, or reefs.
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His work resulted in over 50 formally published papers, including one paper on the precarious state of the Chesapeake public oyster
resource in 1995. and another on the oyster reefs of the Chesapeake, their destruction and possible restoration period. He also authored
a number (about 60) of research contract reports, some 20 to 30 VIMS papers, and over 30 VIMS data reports.
Dexter, an excellent field and laboratory scientist and teacher, worked with numerous graduate students while at the School of Marine
Science and with other members of the VIMS scientific staff, including Dr. J. D. Andrews, Curtis Leigh, and Reinaldo Morales-Alamo.
In addition to the National Shellfisheries Association (NSA). of which he continues to be a member, Dexter has belonged to the
American Society of Limnology and Oceanography, the Atlantic Estuarine Research Society, the Ecological Society of America, and the
Malacological Society. He is also a member of the Society of Cincinnati, a group whose members trace ancestry to persons who served
in the American Revolutionary War. During his membership in NSA. Dexter was President-Elect from 1974 to 1975, and President from
1975 to 1976.
Dexter and his wife. Doris Mills Haven, live in Yorktown. Virginia. They have been married since 1951 and have a daughter. Penny.
William J. Hargis, Jr.
Gloucester Point, Virginia
Journal ,<t Shellfish Rf.uaich. Vol. 22. No. .^. 621-6.11. 200.1.
STRATEGIES TO MITIGATE THE IMPACT OF CIONA INTESTINALIS (L.) BIOFOULING ON
SHELLFISH PRODUCTION
C. E. CARVER, A. CHISHOLM, AND A. L. MALLET*
Mallet Research Services Ltd.. 4 Columho Drive. Dartmouth. Nova Scotia. Canada B2X 3H3
ABSTRACT A sudden increase in the population of the solitary ascidian Ciona iiiteslinalis (L.) is causing serious biofouling
problems for shellfish growers on the Atlantic coast of Nova Scotia, Canada. The objective of the present study was to document the
growth, spawning, and recruitment patterns of this species, and to develop strategies to minimi/,e its impact on the culture of European
oysters at two locations in Lunenburg Bay, Nova Scotia. Profiles of condition index, which may be indicative of spawnmg activity,
suggested that the C. iiitesrimilis population at the Bayport site spawned from mid-May through June, whereas the population at
Mason's Beach spawned from mid-July to mid-August. Histological assessment of reproductive status indicated a period of gameto-
genesis in March-April |>.1°C) followed by spawning from mid-May to mid-August (>8'C). Although mature eggs were observed in
the ovary in July-August, spawning trials suggested a declme in the fecundity of the Bayport population during this period. Two main
recruitment events were observed at Mason's Beach (June and August), but only one at Bayport (June). From the data on fecundity
and settlement rates, it was estimated that a 100-mm long C. intestinalis (0.6 g dry weight) may produce 12.000 eggs in a season and
that recruitment intensity may reach 3.000 individuals m"-. Laboratory predation trials indicated that rock crabs (Cancer irroratus)
consumed significantly more C. inlestinalis than did green crabs (Carcinus muenas). A ma.ximum predation rate of 1 1 individuals per
day per rock crab (80 mm carapace width) was recorded at peak water temperatures of 18X. In a series of chemical width eradication
trials, exposure to 5'"* acetic acid was found to be a more effective strategy for eliminating C. inlestinalis than hydrated lime, saturated
brine, or hypochlorite solution. Total mortality was observed following exposure to 5% acetic acid for 15 to 30 s, with no corresponding
mortality in the control mussels or oysters. Initial field trials indicated that spraying with acetic acid might prove to be an effective
means of eliminating C. inlestinalis under commercial conditions.
KEY WORDS: Ciona inlestinalis. tunicates, biofouling, shellfish production, predation
INTRODUCTION
Ciona intestinalis is a solitary phleobranchiate ascidian, or tu-
nicate, which occurs on natural substrates such as rocky bottoms
and eelgrass beds, or on artificial structures such as aquaculture
gear, marker buoys, dock pilings, and boat hulls (Petersen & Riis-
gard 1992, Connell 2000, Ma/ouni et al, 2001), Although native to
the northern Atlantic Ocean (Van Name 1945, Plough 1978). this
species is now distributed worldwide, most likely as a result of
dispersion by shipping activities (Monniot & Monniot 1994, Lam-
bert & Lambert 1998). Published accounts indicate that C. intes-
tinalis has recently become a serious biofouling problem for many
shellfish culture operations including those in Scotland (Karayucel
1997), .South Africa (Hecht & Heasman 1999), and Chili (Uribe &
Etchepare 2002). In eastern Canada, the severe impact of C. in-
testinalis biofouling was first documented in 1997 at a mussel farm
in Lunenburg Bay, Nova Scotia (Cayer et al. 1999). In an unprec-
edented recruitment event, this tunicate species heavily colonized
the mussel sleeves, causing a substantial reduction in growth and
the eventual loss of the crop. Subsequent reports of significant C
intestinalis recruitment at several other shellfish growing sites in
Nova Scotia suggest that this species has become a widespread
biofouling problem. In a similar scenario, the nonindigenous club
tunicate Styela ckiva has recently infested several mussel fanns on
the eastern coast of Prince Edward Island and is now recognized as
a serious threat to the viability of the ttiussel industry (Boothrovd
etal. 2002).
Information on the basic life-history traits of C intestinalis
originates primarily from natural populations in northern European
*Corresponding author. E-mail: amalletcs'ns
>ympatico.ca
waters (Gulliksen 1972, Svane 198.3, Petersen et al. 1995, Petersen
et al. 1 997 ). Under these conditions, the life cycle of C. intestinalis
is reportedly 12 to 18 mo, with growth and longevity varying in
response to temperature and food levels (Millar 1952. Petersen et
al. 1995). Growth rates in terms of length are estimated at 1 to 3%
day"' or 10 to 20 mm mo ' (Dybern 1965. Petersen et al. 1995).
In contrast, reports from Japan indicate that C. intestinalis has a
life span of 3 mo in the summer at temperatures of 20 to 26°C, and
6 mo in the winter at 14"C (Yamaguchi 1975). The timing of
reproductive activity also varies depending on temperature. In
more northerly regions, such as in Sweden, reproductive activity
peaks in May and June, whereas in warmer zones, such as Britain,
gamete release may occur throughout the year (Dybern 1965, Gul-
liksen 1972). Given the various life-history strategies of this spe-
cies, it is important to document this basic information for C.
intestinalis populations in Atlantic Canada.
The primary objective of this study was to develop a strategy to
mitigate the impact of C. intestinalis on an oyster culture operation
in Lunenburg. Nova Scotia. In contrast to mussel culture, oysters
are contained in a cage from which the tunicates can be removed
without losing the inventory. Heavy infestations, however, have
the potential to depress shellfish growth, and to increase mortality
due to competition for food (Lesser et al. 1992) and obstruction of
water flow (Uribe & Etchepare 2002). The removal of these tuni-
cates from the grow-out structures and oyster inventory is labor
intensive, and, in .some cases, disposal of the waste biomass can be
costly, A series of field and laboratory experimental trials were
undertaken from November 1999 to Noveinber 2000 for the fol-
lowing purposes: (1) to document the local distribution of C in-
testinalis; (2) to investigate the growth, spawning, and recruitment
patterns of this species: and (3) to evaluate possible biological and
chemical strategies for eliminating this species from the culture
equipment and the oyster inventory.
621
622
Carver et al.
MATERIALS AND METHODS
Field Ecology
Distribution, Growtli, and Condition
Several exploratory dives aimed at documenting the local dis-
tribution of C. inlestinalis were carried out at the two field sites in
Lunenburg Bay, Bayport. and Mason's Beach, in the fall of 1999
and the fall of 2000 (Fig. 1).
Two experimental oyster tables with oyster bags containing
adult C. intestinalis (year 1999 class) were set up at each of the two
grow -out sites (i.e.. Mason's Beach and Bayport) on October 30.
1999 (Fig. 2). Temperature recorders were attached to the tables at
each site. The two experimental groups were sampled monthly
from November 1999 to May 2000 and then every 3 wk until
September 2000. On each occasion, a random sample of 10 indi-
viduals was collected from each site to evaluate their condition
index. Each individual was measured and dissected to obtain es-
timates of wet tunic and wet body weight, and then they were dried
overnight at 60°C for 24 h and reweighed. The condition index was
calculated as dry body weight divided by total dry weight.
In early June 2000. oyster bags with recently recruited indi-
viduals were transferred to the experimental tables. Growth in
terms of length, whole wet weight, and whole dry weight were
estimated for the newly settled year 2000 cohort. Ten individuals
from each site were measured, weighed, and then dried. Due to the
difficulty in obtaining measurements from individuals in a fully
e.xtended position, a relationship was derived between body diam-
eter when contracted and body length when alive and fully ex-
tended. This was used to estimate the mean body length of the
Figure 2. Pliotograph of an oyster table with oyster bags. Tunicates
are apparent on the lower side iif the oyster bags.
cohort over time. .\ final sample was collected in November 2000
to document the development of the year 2000 class.
Reproductive Status
Five individuals from each year class at each site were dis-
sected and weighed, and the body was fixed in 1% glutaraldehyde
and 47f formaldehyde. The samples were then sent to the Diag-
nostics Laboratory at the Atlantic Veterinary School (Prince Ed-
ward Island) for histological processing. The tissues were embed-
4 ^%^
K..
0 50 100
y
NoC: ~^-
44022 -
44°20'
64°20' 64° 16'
Figure I. Map of l,unenburg Bay showing the location of the mussel farm in Upper South Cove, the site of the initial C. intestinalis infestation,
and the two experimental sites, Bayport and Mason's Beach.
BlOFOULlNG OF CULTURED SHELLFISH BY ClONA
623
ded in paraffin and sectioned (6-fjLm thick), and the sections were
stained with hematoxylin and eosin. The histology sections were
assessed for reproductive status using a Weibel graticule (two
fields per slide). The contents of each field were assigned to five
categories: empty of follicle tissue; eggs in early development
stage: eggs in late development stage: mature eggs: and regressing
eggs. Mature eggs are surrounded by a thick layer called the vi-
telline coat, which clearly distinguishes them from immature or
developing eggs. These data were used to estimate the proportion
of the ovary that contained follicle tissue and the proportion of that
area occupied by eggs in various stages of development. The cross-
sectional area of the ovary was akso measured using an image
analyzer system.
Recruitment
Four recruitment plates (-200 cm") cut from clean but used
oyster bags (4-mm mesh) were attached to the lower side of each
oyster table on each sampling occasion. Plates deployed on the
previous sampling trip were retrieved and were placed in separate
plastic containers filled w ith seaw ater for transfer to the laboratory.
The plates were examined with a stereomicroscope to detect the
presence of newly recruited juvenile C. imestinalis. The plates
were then placed in flowing filtered seawater (50 |j,m) for 2 to 3
wk to allow for the development of very small individuals that may
not have been counted initially. The plates were then reassessed.
and the maximum of the two counts was retained. The final counts
for both sides of each plate were tallied and divided by the avail-
able solid area to estimate the intensity of settlement over the
previous sampling period. The data were plotted such that any
.settlement that was observed at the end of a particular interval was
assigned to the midpoint of that interval.
Laboratory Trials
Larval Development
The objective of the first .series of trials (January-May 2000)
was to induce natural spawning in the laboratory, to document the
various phases of larval development, and to devise a protocol for
rearing juveniles. Adult C. imestinalis from the 1999 cohort were
collected from the field populations at each sampling event and
were transferred to a fiow -through system running at ambient tem-
perature with unfiltered water. Spawning trials were undertaken on
January 19. February 16. February 28, March 13. March 30, April
18, and May 3. To determine whether the adults possessed com-
petent gametes, attempts were made to trigger spontaneous spawn-
ing by exposing individuals to a natural-light regimen for 24 h.
When this proved unsuccessful, adults were strip-spawned and
cross-fertilized to determine whether the eggs were competent.
Fertilization trials were conducted at ambient water temperatures
(0-6°C).
Fecundity
A series of five spawning trials were conducted in the quaran-
tine unit at the Bedford Institute of Oceanography from May 15 to
August 25, 2000, to estimate the fecundity of individuals obtained
from the 1999 C. intestinalis cohort at both sites. Several individu-
als from the newly recruited 2000 cohort were included in July and
August in an attempt to determine the minimum size at which
spawning was initiated. The first four trials each lasted from 14
tol8 d (May 15-June 2. June 8-26, July 4-21, and July 25-August
10), but the fifth trial (August 1-^25) was discontinued after 10
days because of technical problems with the water supply system.
Five individuals of various sizes from each site were placed in
separate 50()-mL Mason jars in a tank of ambient flowing seawa-
ter. The water was prefiltered through a 40-(xm mesh to remove
any risk of contamination from eggs originating outside the sys-
tem. The water level in the main reservoir was adjusted such that
the flowing water just cleared the top of each jar: the objective was
to allow sufficient flow for gas exchange and particle renewal but
not enough to entrain the eggs. Control jars were placed down-
stream in the tank to estimate whether eggs were being lost. No
eggs were retrieved from the control jars, and observations of fecal
deposition suggested that negatively buoyant particles, including
eggs, were retained inside their respective jars.
The experimental tank was set up approximately 3 m from an
east-facing window such that the dawning light each morning
would induce normal spawning behavior (Lambert & Brandt
1967). Every second or third day. the individual tunicates were
transferred to new jars, and the contents of each old jar were
screened through a 60-|xm mesh to retain any eggs ( 150 (xm size)
produced over the previous 48 to 72 h. The jar and the screen were
well rinsed with filtered seawater to remove any eggs stuck to the
surface and were then flushed with hot freshwater to avoid con-
tamination between samples. The eggs from each jar were col-
lected in a petri dish and were counted using a stereomicroscope.
Fecundity was estimated in terms of eggs produced per individual
per day over the duration of the trial. At the end of each trial, the
surviving individuals were dissected for assessment of dry body
weight.
Methods of Control
Natural Predation
A series of predation experiments were set up in flowing sea-
water tanks in the quarantine unit at the Bedford Institute of
Oceanography, Nova Scotia. Various sizes of C. intestinalis at-
tached to weighted pieces of oyster bag were offered to a range of
potential predators including starfish (Asterias vulgaris), green
crabs (Carcinus maenas), rock crabs {Cancer irroratus). and her-
mit crabs (Pagiints acadianus). The first three trials were con-
ducted in late January 2000 at water temperatures of 2 to 4°C. The
second series of five trials focused on assessing the predation
activity of rock crabs versus green crabs at a range of temperatures.
Trials were undertaken on February 4 to 14 (2°C), April 13 to May
3 (5°C), July 27 to 31 (15°C), August 8 to 10(18°C), and August
14 to 15 ( I8°C). The crabs ranged in carapace width (CW) from 40
to 100 mm, and the tunicate prey ranged in length from 15 to 125
mm. The duration of the experiments had to be reduced in the later
trials to ensure that the supply of prey was not exhausted prior to
the end of the trial. Predation rates were calculated in terms of
indi\idual tunicates consumed per crab per day.
Chemical Treatment
A series of physical/chemical eradication trials were under-
taken in the laboratory from February to August 2000. The chemi-
cals tested included sodium hypochlorite ( 10-60 parts per million),
hydrated lime ( 1—4%), saturated brine, freshwater, and acetic acid
( l-57f ). The effectiveness of heated freshwater (40°C and 60°C)
for eradicating C. intestinalis was also investigated. Various sizes
of tunicates were used in each trial to determine whether younge
624
Carver et al.
stages might be eliminated more easily than older stages. Mussels
and oysters were also included in the trials to ascertain whether the
treatment could potentially be used to remove tunicates from shell
surfaces or from gear containing shellfish.
RESULTS
Local Distribution and Conditions
Diving surveys carried out at both sites in the fall of 1999 and
the fall of 2000 did not identify any C. inlestinalis attached to
natural substrates, including rocks or eelgrass. None were ob-
served on local wharf pilings at Baypoit. but there was a substan-
tial population attached to the bottom of a floating dock at Mason's
Beach. Otherwise, C. intestinalis was only observed attached to
oyster tables or suspended culture gear such as mussel sleeves and
longlines. Both experimental sites typically have a lower incidence
of C. intestinalis than the more sheltered Upper South Cove, the
site of the original 1997 infestation, where the conditions tend to
be warmer and more productive (Mallet & Carver 1993). Tem-
perature profiles for the two e.xperimental sites were virtually iden-
tical (Fig. 3).
C. intestinalis: 1999 Year Class
Growth and Condition Index
Estimates of body length and total wet weight per individual for
the 1999 year class showed low variation over time or location
(mean values: November 1999 69 mm and 5.9 g. respectively;
September 2000 76 mm and 6.7 g. respectively). The mean dry
weight per individual remained at 0.3 to 0.4 g (range 0.1-0.9 g) for
the duration of the study, and the overall relationship between
whole dry weight (gl and body length (mm) was estimated as y =
0.0000106X-"* (r- = 0.91). Note that both primary tissues, the
outer tunic and the body, are composed of approximately 95%
water. Although the smaller individuals did grow from April to
September 2000. the mortality of the larger individuals during the
summer obscured any population growth trend.
During the colder months, the condition index (dry body
weight/total dry weight) declined slightly from 44% in November
1999 (6"C) to 40% in late February 2000 (0°C) (Fig. 4). The
condition index then increased sharply at both sites to a maximum
of 60% at Bayport in late April, and 55% at Mason's Beach in
80
20
Bayport
Masons Beach
1 — I — '
Feb
I — I — I
Apr
I — I — I
Jun
Dec Feb Apr Jun Aug Oct
1999-2000
Figure 4. Condition index (dry body weight/total dry weight) for the
year 1999 class of C. intestinalis at Bayport and Mason's Beach.
mid-May. At that lime, the ambient water temperature at both sites
was in the 6 to 9°C range (Fig. 3). The condition index of C.
inlestinalis at the Bayport site declined steadily from late April to
early August, stabilizing at 35%). This profile would suggest that
spawning started between April 20 and May 13. and continued
through June and July. In contrast, the 1999 cohort at Mason's
Beach exhibited a slight drop in condition in May but then main-
tained a condition index of >50% until mid-July, at which time
values declined sharply. If the condition index is related to repro-
ductive status, this profile suggests that the major spawning event
at Mason's Beach occurred after mid-July or later than at Bayport.
Reproductive Status
Data on the reproductive status of C. intestinalis were pooled
over the two sampling sites. The mean cross-sectional area of the
adult ovary increased from 10 mm" in November 1999 to 25 mm"
in late Januarv 2()()(). declined slightly in February-March, and
then rebounded in April-May to 24 mm". Between May 13 and
June 7. the mean size of the ovary fell to approximately 10 mm",
where it remained until September. Estimates of the proportion of
the ovary occupied by follicle tissue ranged from 55 to 70% from
November 1999 to March 2000, increased to 90% in April-May,
and then declined to 70% in July (Fig. 5). The follicle area occu-
Apr Jun
1999-2000
Figure 3. Temperature profiles for Bayport and Mason's Beach from
November 1999 to November 200(1.
too
80
60
CD
40
20-
D Early Dev Q Late Dev ■ Mature ■ Regressing
4,C
'iLU
6oC 9oC
0.C
•^ <$" $?
■•' ^<!f^ <,^ ^ ^
v4
^.Ay' „^'*' ^«<
'^ f V V 'i" 'V' N= Q~ "^ '^' o=
Figure 5. Reproductive status of the year 1999 class of C. intestinalis:
proportion of the ovary that contained follicle tissue with early devel-
opment, late development, mature, or regressing eggs.
BlOFOULlNG OF CULTURED SHELLFISH BY ClONA
625
pied by mature eggs increased from lO^r in November to SS'X in
December 1999. but then declined from January to late March
2000. By mid-May. the incidence of mature eggs had returned to
i09r and remained relatively stable until mid-September.
In summary, it would appear that egg development proceeded
during the late fall 1999 when water temperatures exceeded 2°C
but ceased when temperatures fell below 2''C in January-February
2000. During this latter period, there was an abundance of
hemocytes in the ovary, which suggested that resorption or regres-
sion may have been occurring. By late March (4°C). the follicle
area was starting to increase, as was the incidence of late devel-
opment and mature egg stages. The major period of gametogenesis
occurred in April through to mid-May (4-9''C). followed by the
initiation of spawning in mid-to-late May. This event was coinci-
dent with a significant decline in the size of the ovary as well as the
proportion of follicle tissue. From early June onward, the produc-
tion of mature eggs continued, but the size of the ovary remained
smaller than in April-May.
Fecundity
A total of five 2-wk spawning trials (May-July 2000) were
carried out at ambient temperature in the laboratory using indi-
viduals collected from the two field sites. The number of eggs
produced per individual varied widely from day to day. but there
was no consistent decline in the rate of egg production over time
within a trial. The maximum daily production estimated for a
single individual was 1 998 eggs day" ' . or a total of 5994 eggs over
3 days (May 19-22). (Table 1). The maximum fecundity for a
single individual averaged over one trial period was 53.3 eggs d"'.
Fecundity was positively correlated with whole dry weight
(Fig. 6). The results indicated that individuals with dry weights as
low as 0.1 g (40-mm long) could produce up to 200 eggs day"',
whereas individuals with dry weights of 0.9 g (120-mm long)
could produce as many as 500 eggs day"' (averaged over 10-18
days). In general, fecundity was higher for the individuals from
Mason's Beach than for those from Bayport. Estimates of mean
fecundity for Bayport individuals (Table 2) showed a steady de-
cline in egg production from May 15 onward. This was consistent
with the profile of condition index (Fig. 4). The data for Mason's
Beach suggest that the 1999 year class was producing >250 eggs
ind"' day"' in May-June. However, unlike the Bayport population,
the individuals at Mason's Beach continued to produce >100 eggs
TABLE 2.
Egg pniduction ratus for year 1999 class C. iiilesliiialis (eggs ind
day 'l from the two experimental sites overtime.
Trial
Bayport
Mason's Beach
Duration
(eggs Ind"' d"')
(eggs Ind ' d"')
May 15-June 2
183 ±80
221 ±98
June 8-June 26
172 ±71
257 ± 70
July 4-July 21
97 ± }4
160 ±40
July 25-August
10
35 ± 15
150 ±.30
Values given as mean ± SE.
day"' through July-August. This was consistent with the higher
condition index for this population.
Larval/Juvenile Development
From January 19 to March 30 2000. eggs were obtained by
dissection because of failures to trigger spontaneous spawnings.
Very few mature eggs were obtained from January through March,
and the sperm rapidly lost motility. In the few instances in which
mature eggs were obtained, fertilization was generally poor
(<10%), and development did not proceed to the larval stage. In
the April 18 and May 3 trials, however, larvae were successfully
produced both by spontaneous spawning and dissection. As in the
earlier trials, the eggs were fertilized at ambient temperature (6-
9°C in April-May) and then were allowed to gradually warm up to
15°C in the dark.
The development of C. inteslinalis eggs at 15'C typically took
24 to 36 h. hatching and growth of the tadpole larvae lasted 24 h,
followed by settlement and metamorphosis over another 12 h for
an approximate total of 3 days to the juvenile stage (see also Berrill
1947). Larvae were successfully settled on plastic petri dishes,
where they metamorphosed into juveniles. The dishes were sub-
merged in a 10-L tank, and the water was changed every 2 to 3
days. The juveniles proved to be remarkably resilient and survived
for weeks with minimal handling/feeding. A series of photos were
taken to document the development of C. intestinalis from the egg
to the juvenile phase (Fig. 7a, b, c, d. e, and f). It should be noted
that the species identity of C. intestinalis was confirmed by the
presence of single refringent bodies in the halo of follicle cells that
surround the egg (Byrd & Lambert 2000).
TABLE 1.
Results of first spawning trial (May 15-June 1) indicating the individual variability in daily egg production rate over time. C. intestinalis
individuals Here brought in from the t«o Held sites on May 13 and were held in flowing seawaler until Jun 2,
Ind
May
May
Mav
Mav
May
Mav
Mav
Mean Eggs
Length
Whole Drv
No.
15-17
17-19
19-22
22-24
24-26
26-29
29-31
May 31-.lun 2
day"'
(mm)
Weight (gl
Ml
7
292
91
378
615
206
717
37S
317
89
0.41
M2
2
1032
66
120
165
178
72
224
223
65
0.47
M3
0
0
0
0
0
0
54
0
6
56
0.27
M4
0
798
1998
363
0
398
0
44
533
89
o,m
M5
0
228
0
6
0
6
5
0
28
79
0.37
Bl
0
0
0
105
74
126
60
102
59
65
0.16
B2
0
3.W
IS
1158
453
416
140
1674
491
89
0.53
83
117
344
45
135
281
164
71
135
155
74
0.36
84
42
77
43
0
174
78
80
0
62
61
0.27
85
225
180
III
0
392
0
371
0
148
70
(1.29
Mean
44
329
237
226
215
157
157
256
202
74
0.40
Abbreviations: M = Mason's Beach; 8 = Bayport.
626
Carver et al.
D
■B
c
LLJ
BOOH
-•- Bayport
■
-o- Masons Beach
600-
o
■
•
400-
200-
0-
•
-1 — 1 1
1 1 ' 1 ' 1
0.2
0.4
0.6
1.0
Whole dry weight (g)
Figure 6. Fecundity (eggs ind"' day ') versus wliole dry weight of the
1999 C. iiilesliiialis from Mason's Beach and Bayport from May
through August 2000. Data from individuals that died during the trials
were not included.
C. intestinalis: 2000 Year Class
Recruitment Patterns
The observed recruitment profiles suggest one settlement event
at Bayport and two at Mason's Beach (Fig. 8). Estimates ot settle-
ment intensity ranged as high as 47 per 100 cm" of solid collector
area, but levels often varied substantially among replicate plates.
The timing of the settlement peak at Bayport (May 13-June 29) is
consistent with the condition index/spawning profile for the year
1999 class (Fig. 4). In the case of Mason's Beach, the timing of the
second recruitment peak (Aug 3-24) closely followed the decline
in condition observed at that site (Fig. 4). The absence of a decline
in condition in May-June may indicate that the first recruitment
event at Mason's Beach was related to an influx of larvae from
other areas such as Bayport or Upper South Cove. However, the
fecundity trials confirmed that Mason's Beach adults were capable
of producing eggs from mid-May onward. Recruitment plates de-
ployed from August 24 to September 19 exhibited some new
settlement at Mason's Beach, but no juveniles were observed on
the plates deployed from September 19 to November 29. 2000.
Growth Rate
The growth rate of the first year 2000 cohort in terms of body
length was relatively steady from mid-July through to mid-
September and then decreased, possibly due to declining water
temperature or the onset of maturity (Fig. 9). Continued growth in
terms of whole dry weight through October-November was appar-
ently related to an increase in body weight as opposed to length
(Fig. 10). Whereas profiles of mean body length were similar for
the two sites, estimates of whole dry weight were consistently
higher at Mason's Beach than at Bayport. At Mason's Beach in
November 2000, the mean body length was 96 mm. the whole wet
weight was 1 1 g, and the whole dry weight was 0.7 g. These values
were consistently higher than those for the year 1999 class the
previous November. Individuals from the second year 2000 cohort
at Mason's Beach had a body length of 36 mm. a mean wet weight
of 0.5 g, and a mean dry weight of 0.05 g on November 29, 2000.
The overall relationship between whole dry weight (g) and body
length (mm) for the year 2000 class was estimated as y =
0.0000080 lx--'(r- = 0.93).
Reproductive Status
Profiles of the percentage of follicle area as well as the pro-
portion of the follicle area occupied by mature eggs increased
rapidly between July 19 and August 3. 2000 (Fig. 1 1 ). At that time,
the tlrst year 2000 cohort had a mean length of 47 mm and a mean
whole dry weight of approximately 0.1 g. Although dry weight
continued to increase through the fall, gonad area increased only
slightly to approximately 10 mm", and follicle area remained at
70%. Over the same period, the proportion of mature eggs declined
from 40 to 20% in late November. Final values for all three re-
productive indices were slightly higher than those for the 1999
year class recorded 1 y previously.
Fecundity
A few individuals from the year 2000 class did produce eggs in
the July-August fecundity assessment trials. Estimates were typi-
cally <I0 eggs day"' in the July 27 trial but increased to as high as
460 eggs day"' for the largest individual in the August 14 trial. The
mean daily egg production was higher for the Mason's Beach
recruits (245 eggs day"') than for the Bayport recruits (25 eggs
day"'), which was consistent with the greater dry weight of the
former group (Fig. 10). The presence of mature eggs in the ovary
from early August onward (Fig. II) suggested that individuals
from the first year 2000 cohort were likely spawning during this
late summer period. However, given that there were 1999 indi-
viduals still spawning in August, the relative contribution of the
first 2000 cohort to the second year 2000 recruitment peak cannot
be ascertained.
Methods of Control
Natural Predators
A series of predation experiments were set up in tlow-through
seawater tanks at the Bedford Institute of Oceanography. Various
sizes of C. intestiiuiUs attached to weighted pieces of oyster bag
were offered to a range of potential predators including starfish [A.
vulgaris), green crabs (C. maenas)* rock crabs (C. irroratus), and
hermit crabs (P. acadiaims). In the first three trials, conducted in
late January 2000 at water temperatures of 2 to 4°C. only the green
crabs and. in particular, the rock crabs showed any feeding activ-
ity. The rock crabs were observed to use two different feeding
strategies, depending on the size of the prey. Small C. intestinalis
individuals (15-35 inm CW) were generally consumed whole,
although after extracting the body tissues the tunic was rejected.
Larger individuals (35-125 mm CW) were cut open with the
TABLE 3.
Effectiveness of various chemicals for the elimination of C.
intestinalis {% mortality) under laboratory conditions.
Duration
Mortality
Chemical Treatment
(min)
(%)
Sodium hypochlorite 60 ppm
20
0
Salt brine (saturated)
8
25
Hydraled lime (4%)
6
50
Fresh water (15°C)
1
10
Fresh water (40°C)
1
fi6
Acetic acid (5%)
0.5
y.^
BlOFOULING OF CULTURED SHELLFISH BY ClONA
bll
s
W:\ --
M ^.,
^ N,
^^^jStfHKJk
1 "'^-
Figure 7. Photographs of the various stages of development of C inteslinalis reared under laboratory conditions: (a I egg (150 urn) with vitelline
coat and follicle cells (protrusions) with distinctive refringent bodies (bright spots); (b) tadpole larvae with otolith (dark spot) and notochord
hatching from egg (250 jim): (c) tadpole larvae with notochord and adhesive papillae on head (800 pm long); (d) metamorphosing larvae (360
X 120 nm) developing peduncle for attachment and resorbing tail; (e) Juvenile (525 fim) with developing siphons; and (f) juvenile (1.3 mm) with
stigmata or slits evident in the branchial chamber (photo (f) courtesy of Dr. Uan Jackson, (Department of Fisheries and Oceans). Scale bars for (a)
to (e) are 50 pm; scale bar for (f) is 500 pm.
claws, anij the body tissues were dragged out and consumed, leav-
ing the empty tunic attached to the original substrate.
The predation trials undertaken at a range of temperatures in-
dicated that rock crabs (50-90 mm CW) may consume as many as
11 C. intestinaUs ind day"' (35-80 mm long) at 18 C (Fig. 12).
Predation rates were substantially lower at <6°C, but activity was
steady. The trials also suggested that the small to medium rock
crabs (<80 mm CW) tended to consume greater numbers of the
<35-m]n tunicates than did the larger crabs (Fig. 13). In general,
the green crabs showed less interest than the rock crabs in preying
on tunicates. There was also a tendency among the smaller green
crabs (50 mm CW) to consume the <80-mm tunicates and ignore
the larger individuals.
Physical/Chemical Eradication Trials
The results of the various eradication trials indicated that ex-
posure to 5% acetic acid was by far the most effective strategy
(Table 3). After the first trial, which indicated that a 1-min expo-
sure to 5% acetic acid was sufficient to cause 100% mortality,
further trials were carried out using shorter intervals. Exposure
times of 5 to 10 sec were found to be insufficient, but 30 sec was
generally 95% effective. The application of this cheinical treat-
ment by spraying or by immersing the tunicates proved equally
useful. Rinsing post-treatment was included in the protocol to
mimic conditions in the field where the acetic acid would be rap-
idly diluted by seawater. Oysters and mussels >20 mm in she!!
628
Carver et al.
50
40
e
" 30
o
o
20
10
0-t
Bayport
— Masons Beach
T 1 1 1 T"
Apr May Jun
Sep Oct
2000
Figure 8. Projected recruitment profiles for the two year 2000 cohorts
of C. intestinalis at Bayport and Mason's Beach.
length (SL) were typically uiiiilYected b> the acetic acid spray/dip.
but control mussels <10 mm SL died in one comparative trial.
Other chemical methods were consistently less effective at
eradicating tunicates. Exposure to hydrated lime for 8 min was
70% effective, whereas saturated brine was only 20% effective
over the same exposure time. Solutions of sodium hypochlorite at
concentrations up to 60 parts per million for as long as 20 min had
no impact on tunicate survival. Exposure to freshwater for 1 min
resulted in only 10% mortality. Longer exposure times may be
more effective, but under field conditions this may not be practical.
A 1-min exposure to 40°C freshwater was 100% effective at eradi-
cating C. intestinalis. but the European oyster (40 mm SL) and one
of the two mussels (50 mm SL) also died.
The second phase of the eradication trials was to test the ef-
fectiveness of acetic acid treatment on C. intestinalis attached to
oyster grow-out bags in the field. It should be noted that these trials
were preliminary and were only assessed at a qualitative level. To
administer the treatment, the acetic acid solution was placed in a
garden-spraying unit. Goggles, gloves, and appropriate clothing
were used, and care was taken to ensure that the bag being sprayed
was located downwind. Although there was a slight smell, the
fumes were rapidly dispersed in the open air. The treatment pro-
tocol followed was similar to that developed in the laboratory
trials: 5% acetic acid spray for .^0 s followed by air exposure for
-
Bayport
— Mason's
Beach
0 8 -
r 0 6 -
0
^"\^
5 04 -
0.2 -
^^
-
' 1 1
1 1
1 1 1 1 I 1 I -r- 1 1
Jun
Jul
Aug Sep
2000
Oct
Nov Dec
Figure 9. Increase in body length (mm) over lime for the year 2000
cohort of C. intestinalis at Bayport and Mason's Beach.
Figure 10. Increase in whole dry weight (g) over time for the year 2000
cohort of C. intestinalis at Bayport and Mason's Beach.
.^0 s. Note that the C. intestinalis individuals had ample time to
fully contract before being exposed to the treatment.
The preliminary trials were earned out on August 24 and Sep-
tember 19. 2000. The oyster bags were covered with a heavy
settlement of 70 to 80-mm-long year 2000 individuals. In each
case, individuals on one third of the bay were covered to act as
controls. It should be noted that the health of these control indi-
viduals was apparently not affected by either the nearby applica-
tion of the acetic acid or the subsequent mortality of their imme-
diate neighbors.
The effectiveness of the treatment varied from 60 to 100%,
depending largely on the density of the settlement (qualitative
assessment). In the second field trial (September 19). 10 European
oysters (40 mm SL) were placed in the bags while the acetic acid
treatment was administered. On average, 80% of the oysters were
alive when the bag was examined on November 29. Although
these small-scale experiments were in no way conclusive, the re-
sults were sufficiently promising to warrant further trials.
DISCUSSION
Natural Distribution
In northern Europe, substantial natural populations of C. intes-
tinalis are found in eelgrass beds or attached to rocky substrates
(Dybem 1965, Gulliksen 1973, Petersen & Riisgard 1992). Diving
surveys in 1999-2000 aimed at documenting the local distribution
of C. intestinalis at the two study sites in Lunenburg found no
individuals attached to natural substrates. Two independent sur-
veys of the Bayport area carried out in August 2000 and August
2001 also failed to locate any C. intestinalis on eelgrass or rocky
bottom areas (Barry MacDonald, Department of Fisheries and
Oceans, pers. comm.). It would appear that the distribution of this
species is generally restricted to man-made structures, such as
floating docks and aquaculture gear.
The sudden proliferation of the C. intestinalis population in
Lunenburg is a classic example of the potential impact of man-
made structures on the settlement and survival of sessile inverte-
brate species (Connell & Glasby 1999). Various Australian studies
comparing the species assemblages found on pier pilings and pon-
toons versus adjacent natural substrates have suggested that the
introduction of artificial structures may effectively increase local
species abundance and diversity (Butler & Connolly 1996, Glasby
BlOFOULING OF CULTURED SHELLFISH BY ClONA
629
Figure II. Roproductive status of the first year 2000 eiihort of C.
inlesiinalis: proportion of the ovary that contained follicle tissue with
early development, late development, or mature eggs.
1999. Connell & Glasby 1999. Connell :(l()0). In particular, soli-
tary ascidians such as C. intestinalis were typically more abundant
at marinas than at reference locations (Glasby 1997). Ascidians in
general have been recognized as the dominant biofouling organism
on oysters grown in rope culture in L'Etang de Thau (France)
(Mazouni et al. 2001).
The conspicuous absence of C. intestinalis from natural sub-
strates suggests that manmade stmctures may function as a refuge
from predation. Field studies in Denmark and Norway have re-
ported that variations in spatial abundance of this species are
linked to predation by sea stars {.Aslerias niljcns). plaice (Pleu-
ronectes platessa). and cod (Gctdus morhua) (Gulliksen 1972. Gul-
liksen & Skjaeveland 1973). Natural predators include jellyfish,
sea stars, rock crabs, hermit crabs, dog whelks, and surface-
feeding fish (Gulliksen 1972. Yamagiichi 197.'i. Svane 198.3. Ole-
,sen et al.l994). Recently settled juvenile stages may also be sus-
ceptible to dislodgment by surface grazers such as gastropods and
sea urchins (Svane 1983). Predation trials conducted in the present
study demonstrated that the rock crab. C. irroratus. can rapidly
excise the body tissues of C. intestinalis from the heavy tunic and
may consume as many as 1 1 ind day"' during the summer months.
M-\
10-
— 6
c
g
I 4
CD
Green Crab
Rock Crab
Temperature {°C)
Figure 12. Predation rates of two crab species (ind crab"' day"
intestinalis (35-80 mm long! for a range of temperatures.
20
) on C.
Field observations also suggested that predators, in particular
crabs, were actively reducing the abundance of C. intestinalis on
the upper surface of the oyster bags but were apparently unable to
access individuals attached to the underside of the bags (Fig. 2).
Other surface-feeding predators such as sea stars may also play a
role in controlling the distribution of small individuals, but. apart
from the crab activity, there was no indication of any significant
predation pressure on the larger lunicates.
Life History Traits
Field observations suggested that most of the individuals from
the 1 999 year class died prior to November 2000. This pattern of
mortality, apparently as a result of natural senescence, was con-
sistent with life span estimates of 12 to 18 mo for C. iniestiuatis in
Scandinavian waters (Petersen et al. 199.'i). Similar to Lunenburg,
reports from Sweden indicate that C. intestinalis. which settles in
the summer, spawns the following spring and dies during the win-
ter ( Dybern 1 965 ). Reports from Japan suggest the life span of this
species is apparently determined by cumulative environmental
temperature (Nomaguchi 1974). Thus, individuals that settle early
in the summer, such as those in the first year 2000 cohort at
Lunenburg, may die al a younger age than those that settle in late
summer.
Estimates of growth rate in terms of body length for the year
20(J0 cohort were approximately 20 mm mo"' from July through
September, which is similar to estimates from Swedish (Petersen et
ai. 1995) and Chilean waters al 12 to 21 mm mo"' (Uribe &
Etchepare 2002). Observations on maximum size in terms of body
length (100-140 mm) were higher than the 60 mm reported for
Japanese waters (Yamaguchi 1975). Perhaps this species can attain
a larger body size under colder conditions.
The results of the histological assessment and the spawning
trials indicated that individuals that settled in May-June were ca-
pable of initiating egg production and spawning by August of the
same year. This was consistent with observations from Sweden
where two breeding generations of C. intestinalis have been found
to co-occur in populations living close to the surface (Dybern
1965).
D 15-35 mm ■ 35-80 mm ■ 80-125 mm
50 70 90
Crab carapace width (mm)
Figure 13. Predation rates of various sizes of rock crabs (ind crab"
day"') on a range of sizes of C intestinalis.
630
Carver et al.
Yamaguchi ( 1975) also reported that C. intestiualis reached sexual
maturity within 2 mo of settlement in winter, and within 1 mo at
higher summer temperatures. This variability confirms that repro-
ductive capability is size-dependent rather than age-dependent (Pe-
tersen et al. 1995).
Gulliksen (1972) concluded that the lowest temperature for the
production of cionid larvae in Norwegian populations was in the
range of 6 to 8°C. or comparable to their deep water winter tem-
peratures. This was generally consistent with observations from
the present study, which indicated possible gonad regression in
January-February at <3°C, gametogenesis in March-April-May at
4 to 8°C. and production of competent gametes from mid-May
onward when ambient temperatures exceeded 8''C.
The juvenile settlement data indicated that C. intestiualis popu-
lations in adjacent inlets may differ in their spawning and recruit-
ment patterns. In Bayport. the recruitment peak was observed in
May-June, whereas at Mason's Beach recruitment peaks were re-
corded in May-June and again in early August. The liming of the
first peak was consistent with the histological data, indicating the
presence of mature eggs in both populations in early May. and the
spawning trials, suggesting that these two populations were ca-
pable of releasing eggs. However, unlike the Bayport population.
the condition index for the Mason's Beach population remained
relatively high until mid-July, suggesting that they did not spawn
in May-June. It should be noted that the use of the condition index
(body dry weight/total dry weight) as an index of spawning may be
misleading. Petersen et al. (1995) found that this index reflected
the level of growth but did not link it to spawning activity. It is
possible that the relatively high condition index values for the
Mason's Beach population in June-July were related to higher food
levels at that site rather than to a delay in the onset of spawning
activity. It remains unclear whether the first recruitment event at
Mason's Beach originated from larvae produced by the local popu-
lation or from other spawning populations such as those in Bayport
and Upper South Cove.
Unlike many shellfish species that spawn over an interval of
weeks, C. intestiualis can apparently spawn continuously over a
3-mo period (mid-May through mid-August). Information on re-
productive status combined with estimates of fecundity illustrated
the duration and intensity of the spawning events at the two sites.
On the basis of these data it was estimated that one adult tunicate
(100 mm long, 0.6 g dry weight) can produce on average 150 eggs
day"' for 60 days for a total of 12.000 eggs per year. This was
consistent with the estimate of Petersen and Svane (1995). who
suggested a conservative figure of 10.000 eggs ind^' over a sea-
son. In contrast, Yamaguchi (1975) reported that adult C. intesti-
nalis in Japanese populations released 2000 to 3000 eggs per night,
every other night, and that the total fecundity of a single specimen
was estimated conservatively at 100.000 eggs. At this stage, it is
impossible to estimate egg to juvenile survival rates, but observa-
tions of dense aggregations of tunicates on any floating surface, up
to 3000 ind m~-, suggest that the population has considerable
potential to expand.
Management Strategies
There are few published reports on strategies for controlling the
proliferation of tunicates on shellfish culture gear. In general,
growers who use nets recommend husbandry procedures such as
changing the gear more often, using power sprayers, or treating
bags with antifouling compounds. Other suggestions include ex-
posing tunicates to air. fresh water, lime, or saturated brine dips
(90 parts per thousand) followed by air (Shearer & Mackenzie
1997). Among mussel growers who use sleeving material rather
than nets, there are few cost-effective solutions. Suggestions from
anecdotal reports include air-drying lines overnight. //; situ pres-
sure washing with a bleach solution, and stabbing individual tu-
nicates. Although feasible in the case of small farms or low-level
infestations, these options are logistically impossible in the case of
large-scale operations.
An alternative strategy is to develop a site-specific manage-
ment plan for minimizing the level of settlement; for example,
growers in South Africa re-sleeve their mussels immediately fol-
lowing the recruitment of C. intestiualis (Hecht & Heasman 1999).
Based on the recommendations of the present study, the Nova
Scotian company involved in oyster culture adjusted its work
schedule to mitigate the impact of C. inlestinalis on its operation.
In particular, the major annual cleaning/changing of the shellfish
and the culture gear was postponed from May to September when
the heaviest settlement had passed; this strategy has since proved
to be a reasonably cost-effective option for the company. Because
C. intestinalis tends to occur in highly aggregated distributional
patterns (Havenhand & Svane 1991. Svane & Havenhand 1993,
Petersen & Svane 1995), the annual eradication of the broodstock
population from the culture gear may reduce future recruitiuent
levels.
Encouraging natural predation is always a preferred strategy for
pest management In aquaculture (e.g.. Enright et al. 1983) but may
only have a limited application in this instance. At an estimated
recruitment level of 25 ind 100 cm"~. 3000 tunicates may settle on
one oyster bag; even at a consumption rate of 1 1 tunicates day"' at
peak water temperatures, it would take one crab 273 days to clean
one bag. Moreover, tunicates that have settled directly on the shell-
fish inventory are not accessible. Field observations suggest, how-
ever, that natural predation, possibly by rock crabs, may play an
important role in reducing the abundance of tunicates during the
winter. Another potential control method that has yet to be inves-
tigated is based on the hypothesis that recently recruited C. intes-
tinalis may be vulnerable to dislodgement by surface grazers such
as periwinkles (Littorina littorea). Enright et al. (1983) reported
that the addition of periwinkles to lantern nets containing Euro-
pean oysters resulted in a significant reduction in biofouling levels.
Chemical treatment protocols including lime and brine immer-
sion have been developed for the purpose of eliminating the foul-
ing tunicate Molgida sp. from oyster spat collector units (MacNair
& Smith 1998). Laboratory trials undertaken in the present study
indicated that acetic acid was considerably more effective than
more traditional methods for eliminating C intestinalis. However,
it should be noted that the use of acetic acid dips or sprays under
field conditions should be carefully evaluated to ensure personal
safety as well as containment and/or neutralization of the chemical
so as to minimize any environmental impact.
This study has shown that the ascidian C. intestinalis is well
adapted to conditions on the Atlantic coast of Nova Scotia, being
capable of developing mature eggs and spawning at temperatures
upward of 8°C. It can apparently tolerate a wide range of envi-
ronmental conditions and has the potential to rapidly establish
substantial populations on floating structures. The presence of sus-
pended or off-bottom shellfish culture operations that offer refuge
from natural predators may inadvertently promote its survival.
Given the tendency of C. intestinalis to attach to the hulls of ships.
BlOFOULING OF CULTURED SHELLFISH BY ClONA
631
local maritime traffic will likely facilitate its dispersal to other sites
in Atlantic Canada over the next few years
ACKNOWLEDGMENTS
We would like to thank Dr. Ellen Kenchington [Department of
Fisheries and Oceans (DFO)] for providing access to laboratory
facilities for sample processing, and Dr. Ken Freeman (DFO) for
his advice on the design of the laboratory trials. Dr. Dan Jackson
(DFO) contributed one of the photographs and demonstrated the
use of the image analyzer system. Dr. Peter Strain (DFO) was very
helpful in as.sessing the potential environmental impact of certain
chemicals. Staff from Lunenburg Shellfish Inc. provided field sup-
port in sampling the experimental tables and conducting the pre-
liminary eradication trials. Special thanks are extended to Dr.
Donald Douglas. Industrial Technical Advisor, for his contribution
to the experimental design of this project, and to the National
Research Council Industrial Research Assistance Program for par-
tial funding.
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Jiiiiniiil of Shclljhli Rcscunh. Vol. 22. No. 3. 633-63!J. 2UU3.
FOULING ORGANISMS OF THE BLUE MUSSEL MYTILUS EDULIS: THEIR EFFECT ON
NUTRIENT UPTAKE AND RELEASE
A. R. LEBLANC,' T. LANDRY," AND G. MIRON '*
'Departciiwn! dc Biolofiie. Univcrsite dc Mancton. Moinion. NB. Canada. El A 3E9: 'DepartineiU of
Fisheries and Oceans Canada, Science Biancli Gnlf Fislieries Centre. Monctoii. NB. Canada EIC 9B6
ABSTRACT The effects of fouling organisms are a cause for concern among mussel growers. On Prince Edward Island. Canada,
mosi of the foulers are sedentary filter feeders, and are. therefore, potential competitors with cultured mussels for available resources.
This could translate into a reduction in meat yield in mussels. Laboratory experiments were carried out in July. September, and
December 2001 to determine the relative iinpact of fouling organisms on the uptake and release of nutrients. The uptake of chlorophyll
0, and the production of ammonium, phosphate, nitrate, nitrite, and organic matter were investigated. There were some significant
differences in chlorophyll a uptake between mussel/fouler units and mussels alone. The mean (±SE) chlorophyll a uptake by
mussel/fouler units was 5.05 ± 1.48. 8.53 ± 0.94. and 12.87 ± 1.03 L/h. respectively, for July. September, and December. The mean
consumption by mussels only was 5.37 ± 1.19. 8.72 ±0.83. and 9.64 ±0.97 L/h for each of the experiments. Foulers increased ammonia
production before water temperatures dropped in the fall (end of Septemher-early October). Mussel/fouler units released mean amounts
of ammonia of 58.42 ± 10.01 and 667.54 ± 252.69 L/h. respectively, in July and September, while mussels alone did not produce
ammonia in July, and in September they released 103.10 ± 13.25 L/h. There was a significant production of phosphate by mussels in
July (6.67 ± 2.96 L/h) and in December (46. 1 1 ± 3.02 L/h). and by the mus.sel/fouler units in December (27.95 ± 1 .8 /h). In the presence
of foulers. the nitrite production was 16.01 ± 6.53 L/h. In its absence, however, nitrite consumption was 17.09 ± 5.63 L/h. Mussel/fouler
units consumed nitrate (4.25 ± 1.47 L/h), however, there was no significant difference when foulers were absent (0.86 ± 1.44 L/h).
There was a significant consumption of organic matter by foulers in summer only (6.22 ± 1.38 L/h). Foulers have the potential to
prolong phytoplankton blooms by increasing the production of inorganic nutrients, especially ammonia. This study shows that the effect
of foulers on mussels may not be as great as previously thought, and it may not be profitable to invest time and money in trying to
reduce them.
A£) WORDS:
Tracadie Bay
Myiilus eciiilis. blue mussel, epifauna. fouling organisms, chlorophyll a. ammonia, phosphate, nitrate, nitrite, feces.
INTRODUCTION
Culture of the blue mussel. Mytiliis edidis Linneaus 1758. on
Prince Edward Island (PEI). Canada, began in the 1970s and grew
into a $25 million per year industry by 2002. Thi.s expansion can
be attributed to an increase in the number of mussel grow-out sites
accompanied by the rapid development of husbandry practices.
Presently, however, no new grow-out sites that can support mussel
culture are available on PEL and it seems that any further devel-
opment of a sustainable industry relies on the optimization of
productivity at the farm level (Thomas Landry, pers. comm.).
Mussel socks are fouled by different species of marine inver-
tebrates. Some of these foulers are filter feeders that compete with
mussels for resources and therefore represent an additional strain
on a system. In this context, the fouling of culture units by various
epifaunal species is an issue that warrants further attenti(m.
It is well-documented for molluscs such as scallops and oysters
grown in nets or cages that the settlement of fouling organisins can
restrict water flow to a point where both food availability and
growth are negatively affected (Claereboudt et al. 1994. Lodeiros
& Himmelman 1996. Taylor et al. 1997). PEI mussels, however,
are cultivated on longline systems, which consist of subsurface
buoyed backlines permanently anchored at each end. Mussels are
suspended in socks along the backlines. Little is known about the
effects of the fouling community on the growth of mussels on
longline systems. In theory, since the fouling organisms tend to
settle directly on mussel shells, they may obstruct the opening o'i
the valves, thereby interfering with feeding (Lesser et al. 1992.
Lodeiros & Himmelman 1996). Moreover, because most foulers
*Corresponding author. E-mail: mirongCs'umonclon.ca
are sedentary filter feeders (Arakawa 1990. Lesser et al. 1992,
Lodeiros & Himmelmann 1996. Taylor et al. 1997. Mazouni et al.
1998a. Mazouni et al. 1998b. Cayer et al. 1999. MacNair & Smith
1999. Uribe & Etchepare 1999), it is possible that they contribute
to the depletion of the phytoplankton biomass at culture sites.
Despite these possibilities, evidence suggests that foulers do not
significantly limit the yield of mussels cultured in suspension on
backlines (Beristain & Malouf 1988, Lesser et al. 1992). More-
over, it has been suggested that the fouling coinmunity may in fact
enhance seasonal phytoplankton blooms by altering nutrient fluxes
(Mazouni et al. 1998a. Mazouni et al. 1998b) in a favorable way.
Such an effect is plausible, given that the metabolic wastes re-
leased by fouling organisms may introduce nutrients into the water
column that would otherwise not be available to the phytoplankton
community (Kasparet al. 1985. Dame et al. 1991. Prins & Smaal
1994. Smaal & Zurburg 1997. Mazouni et al. 1998a. Mazouni et al.
1998b, Landry 2002).
The goal of this study was to investigate the relative uptake of
food and the release of nutrients by foulers commonly found on
cultured mussels in Tracadie Bay, PEI. Our experimental approach
was based on measuring food (i.e., seston and chlorophyll a) intake
and nutrient (i.e., ammonia, phosphate, and nitrate) relea.se in two
study groups (mussels and mussel/fouler units) during the ice-free
period. Results are compared with previous work on mussels, and
an attempt is made to relate laboratory findings to applied methods
used in mussel culture.
MATERIAL AND METHODS
Experimental Design
Mussel socks were collected from Tracadie Bay. PEI. Canada
in July. September, and November 2001. During each trip, four
633
634
LeBlanc et al.
socks were collected and transported to the Ellerslie Hatchery,
PEL At the hatchery, a small quantity [mean quantity (±SE) 30 ±
9 to 902 ± 82 mg ash-free dry weight] of foulers and 30 mussels
of approximately the same size were carefully removed by hand
from each of the four socks. Foulers and mussels were placed in
individual mesh (window screening) bags (four mussel/fouler
units) and were maintained alive using running water from the
Bideford Estuary. Water temperatures were 23°C in July, 16°C in
September, and 3°C in December, and salinity was 28 parts per
thousand for all experiments. After a short acclimation period (<1
wk), the experimental animals were transferred to 12-L flow-
through containers. Four containers with no animals were used as
controls. All containers were connected to a single supply tub that
was continuously fed sand-filtered seawater. Water tlow was set at
about 300 niL/min.
Immediately after the introduction of animals into the contain-
ers, 1-L water samples were taken from the supply tub (input) and
also from the output spout of all containers. Thereafter, additional
I -L samples were taken every hour during a 7-h period. At the end
of this time, feces were collected from the bottom of the contain-
ers. Foulers were separated from mussels and were frozen for
subsequent determination of weights (i.e.. ash-free dry weight
AFDW). Mussels (the same individuals as in the mussel/fouler
units), on the other hand, were subjected to a short reacclimatiza-
tion period (<l wk) and then were subjected to the same protocol
as the mussel/fouler units.
Laboratory Analyses
At the hatchery, suspended solids (500-mL water subsamples)
were filtered onto preashed and preweighed Whatman (Clifton.
NJ) GF/C 47-mm filters and were rinsed with 4% ammonium
fomiiate. Identical methods were used to filter feces samples. AH
filters were stored frozen. Filters were dried at 70°C for 24 h.
weighed, combusted at 500°C for 12 h, and reweighed. The results
are reported as total seston (i.e., organic + inorganic), organic
seston, total feces, and organic feces. Other water subsamples
(230 mL) were filtered through Whatman GF/F 23-mm filter for
chlorophyll a determination. The filters were immersed in 90Cf
acetone, frozen, and later analyzed using a Turner Design (Sunny-
vale, CA) fluorometer, as suggested by Parsons et al. ( 1984).
Ammonium concentrations were determined from frozen water
samples (20 mL) using the phenol method (Parsons et al. 1984)
and a spectrophotometer with a fiow-through 3-cm path length cell
at 640 nm. Phosphate concentrations were measured from frozen
water samples (10 mL) using either a spectrophotometer (5-cm
path length fiow-through cell at 885 nm: July 26 and December
experiments) or a YSI (Yellow Springs, OH) 9100 photometer
(July 30 and Sepletnber 29/October 2 experiments). Concentra-
tions were determined by using commercial Palintest kits (YSI,
Yellow Springs, OH) with the YSI photometer. All nitrate and
nitrite concentratio;? values were derived from the YSI 9100 pho-
tometer (Palintest kits).
Nutrient Uptake and Release
Nutrient budgets were calculated by subtracting the output nu-
trient concentration (container spout) from the input nutrient con-
centration (supply tub) at a corresponding time then dividing by
the input concentration. It was then multiplied by the water flow.
A positive value indicated an uptake of nutrients by the study
animals, while a negative value indicated nutrient release. Uptake
and release values were corrected for processes unrelated to animal
filtration (e.g., phytoplanklon reproduction or evaporation) using
data from the control containers.
Statistical Analyses
Paired sample t tests were used to compare the use of nutrients
by the mussel/fouler units and by the mussels. Each pair of ex-
periments was analyzed separately. The pairs were as follows; July
26 and 30. September 28 and October 2, and December 5 and 13.
All probability levels were fixed at 0.05. Statistical tests were
conducted with SPSS 10.0 for Windows (SPSS. Chicago. ID.
RESULTS
The ash-free weight proportion of foulers in the experiments
ranged from 0.4 to 9.0Vr. The experimental conditions were rep-
resentative of the naturally occurring changes in Tracadie Bay.
However, the proportion of foulers varied between 0.4 to 5.5% in the
bay. Therefore, the effect of foulers may have been overestimated in
our experiment in September when the foulers represented 9.09c of
the experimental animals. Table 1 shows the mean ash-free weight
and composition of the animals present in each experiment.
Food and inorganic nutrient concentrations varied throughout
the study (see Table 2). There were notable changes in food uptake
over the course of this study (Fig. la). Under stressful summer
conditions (temperature 23°C), the mussels consumed no organic
matter. However, when an epifaunal community, although low in
abundance (0.4%). was added to the experimental containers, the
uptake of organic matter became noticeable. In September, as the
water cooled to 16°C, mussels began assimilating organic matter,
and the effect of foulers on organic matter became insignificant. In
December, water temperatures dropped to near-freezing values,
TABLE L
Mean ash-free weights in mg (n = 4) of organisms in the four
experimental containers.
July
September
December
Temperature CC)
23
16
3
Organisms
M. eJulis
7000(300)
10,000(700)
17,000(400)
Foulers
30(9)
906(82)
548 (26)
Fouling community
Annelida
Scale worms
29
53(13)
150(26)
Arthropoda
Balanus crenatus
AB
3(4)
3 (0.4)
Caprellids
AB
45(15)
AB
Gainmarus sp.
AB
17(7)
AB
Isopods
AB
3
AB
Bryozoa
Bugula nirrita
AB
11
AB
Chordata
Molgula sp.
AB
452(88)
AB
Mollusca
Anomia sp
AB
AB
4(3)
Ciepiclitia foniicata
AB
90(13)
191 (22)
Mussel (M. edulis) spat
AB
40(16)
48(12)
Snails
AB
9(2)
AB
Plants
Red algae
23(12)
225 (60)
147 (40)
Abbreviation: AB = absent. Values in parentheses are the SE.
Effects of Mussels and Foulers on Nutrient Flows
635
TABLE 2.
Nutrient concentratiuns in input contalnir liir all experiments in = iH.
Chlorophyll a
.Xmmonium
Phosphate
Nitrite
Nitrate
Date
(pg/l-l
(pg N/IJ
(pg I'/L)
(pg N/L)
(mg N/L)
July 26
5.66 + 0.14
0.30 ± 0.03
1 .07 ± 0. 1 9
n/a
n/a
Julv M)
2.96 ± 0.77
2.36 ±0.1 8
37.50 + 8.57
n/a
n/a
Seplemher 29
7.42 ±0.31
0.32 ± 0.06
19.00 ±4.90
0.8 ± 0.4
0.07 ±0.01
October 2
11.01 ±0.80
0.18 ±0.04
33.40 ± 13.40
2.0 ± 0.5
0.11 ±0.02
December 5
8.09 + 0.38
0.51+0.14
0.20 ± 0.05
0.8 ± 0.4
0.1 3 ±0.02
December L^
6.27 ±0.18
0.26 ± 0.07
0.17 ±0.02
5.0 + 3.0
0.11 +0.01
Values given as mean ± SE. N/A = not available. N/L = pg of nitrogen per litre. P/L = pg of phosphorus per litre.
and all organisms ceased consuming organic matter. Therefore,
with respect to organic matter, the epifaunal effect was limited to
the summer period. However, the mussels consumed phytoplank-
ton continuously throughout the study (Fig. lb). It was at its high-
est in winter conditions. It is also only at this period that the effect
of foulers was visible.
Ammonium was released by mussels e.xcept under summer
conditions (Fig. 2a). Ammonium release was increased by almost
1009f when foulers were added. In September, ammonia release
was doubled by the presence of foulers. In cold water conditions,
the effect of foulers on ammonia release was insignificant.
Mussels did not consume or release phosphate e.xcept in De-
cember when they released it (Fig. 2b). When foulers where added
in summer conditions, there was a net release of phosphate, but it
remained significantly equal to mussels. In winter, when foulers
were more abundant than in summer (3.2% compared with 0.4%).
there was a release of phosphate, but it was smaller than when
S 7-
29 Sepi;2 Oct
Dale of experimeDIs
I mu&scls/foulers
i 600
S 400
? 200 (30)
(31)
26/30 July
28 Sept/2 Oct
Date of eiperimeot
I musselb'foulen D mussels
(29)
(25)
(23) (28) (32) (32)
r
26/30 July 28Scpt/2 0ct 5/13 Dec
Date of eiperiments
■ musseis/foulers D mussels
Figure L Food uptake hy the mussel/fouler units and mussels from
experiments carried out in summer and fall 20(11. (a) Organic matter
(L/h) and (b) chlorophyll a (L/h). Means are presented with SE as
error bars (h = 32) (b) 26/30 July in = 13). *0.01 <P< 0.05; **0.001 <
P < 0.01; ***p < 0.001 in I test comparing mussel/fouler units and
mussels; "significant positive and negative values represent an uptake
and a release, respectively.
26/30 July 29Sepl/2 0ct 5/1,1 Dec
Date of expeiiment
■ musseis/foulers G mussels
Figure 2. Inorganic nutrient fluxes by the mussel/fouler units and
mussels from experiments carried out in summer and fall 2001. la)
Ammonium (L/hl and (b) phosphate (L/h). Means are presented with
SE as error bars. Values in parentheses are the number of replicates.
*0.01 <P< 0.05; **0.001 <P< 0.01; ***P < 0.001 in t test comparing
mussel/fouler units and mussels: "significant positive and negative val-
ues represent an uptake and a release, respectively.
636
LeBlanc et al.
foulers were absent. In September, when foulers were most abun-
dant (9.09f). phosphate fluxes remained insignificant.
Mussels consumed nitrite in fall conditions (Fig. 3a). The ad-
dition of foulers resulted in a net release of nitrite. In winter.
neither mussels nor foulers had a significant effect on nitrite
fluxes. Mussels had no effect on nitrate fluxes. There was signifi-
cant consumption of nitrate (Fig. 3b) when foulers were added in
winter
Foulers increased total feces production in summer despite
their low abundance. They had no effect when their abundance was
higher. They did. however, reduce organic feces production in the
fall when they were the most abundant (Fig. 4).
DISCUSSION
Mussels are selective feeders. This selectivity is influenced by
the quality and quantity of the seston (Thompson & Bayne 1972.
Riisgard & Randlov 1981. Newell et al. 1989. Asmus & Asmus
1993, Bayne 1993. Prins et al. 1994. Hawkins et al. 1997). which
is composed of phytoplankton and other sources of organic and
inorganic matter. They choose food that is higher in nutrient con-
tent and that will, therefore, maximize growth. Phytoplankton is
the preferred food because of its higher nutritional value, therefore
26Sept/2 0cl S;l3Dec
Date of experimenl
■ mussels/foulers C mussels
'1
= -6 -
z
-8
-10-
-12 -
26 SepL'2 Oct
Dale of experimeDt
mussels/foulers D mussels
Figure 3. Inorganic nutrient fluxes by the mussel/fouler units and
mussels from experiments carried out in summer and fall 2001. (a)
Nitrite (L/h) and (b) nitrate (L/h). Means are presented with SE as
error bars in = 32). *0.01 < /' < 0.05: * (1.001 < /' < 0.01: ***P < 0.001 in
/ test comparing mussel/fouler units and mussels: •significant positive
and negati\e values represent an uptake and a release, respectively.
26/30 July 28 Sept/2 Oct S/I3D«
Date of experiitients
■ mussels/foulers □ mussels
Figure 4. Organic feces production (g) by the mussel/fouler units and
mussels from experiments carried out in summer-fall 2001. Means are
presented with SE as error bars in = 4). *0.01 < P < 0.05: **0.001 < P
< 0.01: ***P < 0.001 in I test comparing mussel/fouler units and mus-
sels: •significant positive and negative values represent an uptake and
a release, respectively.
when food is abundant and varied, mussels will consume more
phytoplankton than other types of organic matter. Foulers are com-
posed of filter feeders, predators (carnivores), and herbivores.
Their nutrition depends on the species composing the community.
In July, scale worms and red algae were the only fouling species
present. Neither one of these species consumes phytoplankton.
Scale worms, however, consume organic pseudofeces, thereby in-
creasing the available organic matter uptake by mussel/fouler
units. In fall conditions, when the abundance of filter feeders is
maximal, the consumption of phytoplankton and organic matter is
not increased by the presence of foulers. However, both food
sources are consumed. When the organic content of seston is high,
mussels become less selective (Bayne 1993, Dame 1996), which
may explain why both types of food sources are exploited. Filtra-
tion by ascidians is also influenced by seston concentrations
(Millar 1971, Holmes 1973, Fiala-Medioni 1979, Robbins 1984,
Riisgard 1988, Petersen & Riisgard 1992. Petersen et al. 1995).
When food is abundant and varied, competition between species is
insignificant (Zajac et al. 1989, Lesser et al. 1992). In winter,
mussel spat form part of the fouling community, thereby increas-
ing phytoplankton consumption.
There were problems during the spectrophotometric analyses
for phosphate in December and for ammonium. Blanks (deionized
water) were too high, and negative concentrations were obtained.
To obtain positive values, the lowest concentration of an experi-
ment was added to all concentrations of the same experiment, and
calculations were made from the adjusted concentrations. Foulers
increased ammonium release. This is consistent with the findings
of Mazouni et al. ( 1998a). who found that foulers increased am-
monium concentrations around oyster beds. Therefore, foulers
may have a beneficial effect on primary production by prolonging
blooms. Ammonium released by mussels is immediately available
to primary production, while other sources of nitrogen (e.g.. deni-
trification of sediments) are not as readily available (Kaspar et al.
1985. Dame & Dankers 1988, Mazouni et al. 1998a, Landry 2002).
The effect that foulers have on cultured mussels seems to be
limited to food uptake and ammonium production. Competition tor
food could be reduced when sources are diversified and abundant.
Many studies suggest that the main source of inorganic nutrients.
Effects of Mussels and Foulers on Nutrient Flows
637
excluding ammonia, is througli the remineralization of material
from sediments and biodeposition (Kaspar el al. 1985. Dame et al.
1991. Prins & Smaal 1994, Smaal & Ztirbui-g 1997. Mazouni et al.
1998a. Ma/oiini et al. 1998b). However, the presence of a diver-
sified community of foulers results in reduced levels of fecal pro-
duction. Certain species of foulers (e.g.. scale worms or caprellids)
consume organic fecal matter. They could, therefore, potentially
reduce the input of inorganic nutrients (other than ammonia) by
reducing the biodeposition of mussels.
This study suggests that foulers may not be as detrimental to
mussel aquaculture as previously thought. While they could in-
crease phytoplanklon consumption, they could also contribute to a
rise in ammonia levels, thereby counteracting the positive effect
they have on phytoplankton. In contrast, they can increa.se diver-
sity, which can prevent or reduce invasions (McGrady-Steed et al.
1997. Osman & Whitlatch 1999. Stachowicz et al. 1999) or popu-
lation explosions by specific species that can be potentially harm-
ful to mussel operations. In diverse environments, most ecological
niches are occupied, therefore, there is less potential for a new
species to invade.
ACKNOWLEDGMENTS
We would like to thank John MacLeod for providing and man-
aging the mussel lines used in this study. We would also like to
thank Kevin LeBlanc. Michelle Maillet. Jean-Fran(;ois Mallet,
Nathael Bergeron. Anne Page. Marc Ouellette. and Remi Sonier
for their help in the field and in the laboratory. We are especially
grateful to Paul Burleigh from the Ellerslie Hatchery, PEI, for all
his help and advice in the set up of the experiments. We would like
to thank Luc Comeau for his advice, which helped to clarify the
manuscript.
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Joiimul oj Shellfish Research. Vol. 22. No. 3. 6.^9-642. 2003.
AGE AND GROWTH OF THE MEDITERRANEAN SCALLOP PECTEN JACOBAEVS (LINNAEUS
1758) IN THE NORTHERN ADRIATIC SEA
MELITA PEHARDA,* ALEN SOLDO, ARMIN PALLAORO, SANJA MATIC, AND
PERiCA ce:tinic
Insiiuiic of Oceaiuii^niphy and Fisheries. P.O. Box 500. 21000 Split. Croalia
ABSTRACT Age and growth of the scallop Pecteu jacohaeus (Linnaeus I7.S,S) were investigated on specimens collected from a
commercial catch in the northern Adriatic during January and February 2003. Shells were aized based on licament scars and formation
of growth rings on the external shell surface, and data were fitted to the von Bertalanfly growth function L, = L^_ (l-e
"), In
addition, age and growth were estimated from growth increments using Gulland-Holt plot and relative growth function was constructed.
Length of analyzed Pecten jacohaeus ranged from 75.1 to 142.0 mm. while estimated age ranged from 2 to 13 y. With respect to two
methods applied, obtained von Bertalanffy equations for height were: H, = 108.79 ( l-e '
asymptotic shell lengths were estimated to be 127.93 and 127.3.'i mm. respectively.
KEY WORDS: age. growth, scallop. Pcclcn jacohaeus. Adriatic
'landH, = ll0.0S(l-e"
.The
INTRODUCTION
The scallop Pcclcn jactilnuiis (Liiitiaeiis 1758) lives on sand,
tiiud. and gra\el bottoms between 25 and 250 m depth (Poppe &
Goto 2000) and can grow up to 162 mm in diameter (Onofri &
Margus 1995). Although present throughout Mediterranean coastal
waters, P. jacohaeus occurs in commercial quantities only in the
Northern Adriatic, where it is highly prized at local markets and
restaurants (Mattel & Pelizzato 1996, Relini et al. 1999).
However, recent publications point out that population of P.
jacobaeiis in the western (Italian) part of the northern Adriatic
show obvious signs of overexploitation (Relini et al. 1999). This
species also is exploited in the eastern (Croatian) part of the north-
em Adriatic, but no signs of overexploitation of P. jacolnicus have
been recorded in that area (Cetinic & Soldo 1999). However, stud-
ies from that area have not been based on the age composition or
population structure of P. jacohaeus.
Previous studies in the region include bilateral research pro-
gram, between Italy and Croatia, conducted in 1980s in almost
entire Adriatic Sea, which only monitored and assessed the abun-
dance of P. jaciihaeus (Piccinetti et al. 1986). Other studies in the
Croatian part of the Adriatic investigated age cotnposition, distri-
bution, reproduction, and larval settletiient in the Krka river estu-
ary-middle Adriatic (Margus 1990. 1991. 1994. Margus et al.
1992. 1993). biometry and age composition in Mljet lakes-south
Adriatic (Onofri & Margus 1995). and dredge selectivity and some
biologic characteristics of P. jacohaeus in the northern Adriatic
(Cetinic & Soldo 1999).
Scallops are traditionally aged using growth rings on the ex-
ternal shell surface and ligament scars (Tang 1941. Mason 1957,
Merrill et al. 1961, Minchin & Mathers 1982. Richardson 2001).
Furthermore, a recent study has shown that distribution striae,
located on the external shell surface, can be used as a proxy for the
onset of winter growth and for distinguishing between disturbance
and truly annual surface rings (Owen et al. 2002).
The aim of this work is to present estimates of the age com-
position of the commercial catch and growth parameters of P.
jacohaeus in the Croatian part of the Northern Adriatic by using
*Correspondmg author. E-mail: melita@izor.hr
the analysis of the external shell surface, ligament scars, and
growth increment data.
MATERIALS AND METHODS
This study was conducted on western coast of the Istrian pen-
insula, which is, according to Croatian fishery legislation, the only
area where fishing of P. JMobaeus is allowed in Croatia (Fig. 1).
Commercial vessels with maximum breadth of dredges ranging
fiom 2.0 to 3.0 m have been used for fishing P. jacohaeus. Towing
speed varied from 3.0 to 3.8 knots, whereas sampling depth ranged
from 23 to 35 m. The study was conducted in January and Feb-
ruary of 2003. when lowest temperatures (~8°C) occur in northern
Adriatic Sea (Soldo pers. comm.). According to literature (Gibson
1953. Mason 1957) ring formation in a lelated species. P. uia.ximus
(Linnaeus 1758). occurs in U.K. waters at the beginning of spring,
follov\ing a period of coldest sea temperatures. Therefore, the edge
of the shell was treated as the last ring and growth increment
between previous ring and edge of the shell was treated as annual
growth increment. Shells of 292 specimens were set aside and
analyzed in the laboratory, including shells smaller than 100 mm
(;? = 70) that are usually returned back into the sea.
The length (anterior-posterior axis) and height (dorso- ventral
axis) of each specimen was measured, using vernier callipers, to
the 0.1 mm. Shells were aged based on ligament scars and the
number of growth rings on the external shell surface, taking into
account that first scars and rings are often missing (Tang 1941,
Mason 1957. Menill et al. 1961. Minchin & Mathers 1982. Rich-
ardson 2001 ). For observation of ligament scars, rubbery ligament
was first softened in water so that it could be easily rubbed away
and that the underlying growth lines could be reveled. Disturbance
rings on the external shell surface were distinguished from annual
rings based on distribution of striae; disturbance rings do no have
small crowded striae and striae on either side of disturbance ring
are equally spaced (Mason 1957. Owen et al. 2002). Length at age
data were fitted to the von Bertalanffy growth function L, = L^
d-e"'"'"""). where L, is shell length t. L^ is asymptotic shell
length, k is curvature parameter and t,, is theoretical age at zero
length (Beverton & Holt 1957).
Because of difficulties in detemiining marks on the shell re-
lated to the first year of life, a second method was applied to
confirm the shell growth rates. Distances between clearly visible
growth rings on the external shell surface were measured along t'l,.-
639
640
Peharda et al
Figure 1. Map of study area.
main growth axis of each sliell. These data were used for construc-
tion of a Gulland-Holt plot, where growth rates are plotted against
the mean height (GuUand & Holt 1959). Growth parameters were
estimated from a numerical value of the slope (k) and \-intercept
(L-J. Because it is not possible to calculate a value of to using this
method, this constant was omitted from the von Bertalanffy equa-
tion, and a relative growth curve was constructed (Spaire & Ven-
ema 1998).
RESULTS
The length of the analyzed Pecteii Jacohaeiis ranged from 75.1
to 142.0 mm (x = 108.73 + 13.19 mm). Smallest measured speci-
men had a height of 66.8 mm, while the height of the largest one
was 124.5 mm (x = 94.27 ± 10.96 mm). The relationship between
shell length and shell height can be described by the following
equation! = -2.54 -i- 1.18 H (;; = 292. r = 0.76. P < 0.001).
The first ring on the external shell surface was not clearly
visible in over 80% of the inspected shells. With respect to the
analyses of the ligament, it was also noticed that the first scar was
missing and that the ligament was less firm and flexible in animals
older than 5 y. The estimated age of the analyzed specimens
ranged from 2 to 13 y (x = 4.71 ± 2.27). Shells in age class three,
four and five constituted 22.6%, 26.7% and 19.9% of the total
sample, respectively (Fig. 2). Based on the observed shell height at
each scar and ring, asymptotic shell height (H,_) of P. jacohaeiis
was estimated at 108.79 mm. while the calculated curvature pa-
rameter (k) was 0.473 y"' {r = 0.803). With respect to length,
asymptotic value (L.,) was estimated at 127.93 mm and calculated
curvature parameter (k) was 0.420 y"' (r = 0.804). According to
the obtained von Bertalanffy growth equation, P. jacohaeiis
0 1 2 3 4 5 6 7 e 9 10 11 12 13
Age (number of external rings)
Figure 2. Histogram of P. jacohaeiis age frequencies, as determined
from external growth rings.
growth is intensive during the first four years of its life and growth
rate slows down considerably after the shell reaches 5 years of age
(Fig. 3). Shell reaches the legal catch length of oxer 100 mm after
its third year.
Results of the shell growth increment analysis using a Gulland-
Holt plot are similar to those obtained by the analyses of number
of rings and scars. The equation obtained in the plot was y =
-0.53 x -H 58.62 (n = 680. r = 0.768; Fig. 4A). The calculated
value of r indicates the degree of variation in shell growth. As-
ymptotic shell height (H,) and curvature parameter (k). obtained
from this equation, were 110.08 mm and 0.53 y"', respectively.
Based on these data a relative growth curve was constructed (Fig.
4B). From the previously established relationship between shell
length and shell height, the asymptotic shell length was estimated
to be 127.35 mm.
DISCUSSION
Previous researchers have shown that bivalve species in tem-
perate waters form annual surface rings and prismatic shell growth
lines as a result of reduced growth in winter caused by declining
seawater temperatures and decreased food availability (Richardson
2001). According to recent genetic work, Pecten jacohaeiis and
Pecten ma.xinuis are closely related, and may even be the same
species (Wilding el al. 1999). Taking into account Wilding et al.'s
5 6 7 8 9
Age (number ot external rings)
Figure 3. Growth curve for P. jacohaeiis fitted using the von Berta-
lanffy growth equation for height H, = 108.79 , )_£-" -i^'*" '") and length
L, = 127.93 d-e"" -■-'*"--).
Age and GROwifi of Pecten jacobaeus
641
60 80 100 120
Mean total shell height (mm)
120 T
100
80
60
40
20
1 2 3 4 5 6 7 8 9 10 11 12 13
Relative age (years)
Figure 4. A. (iulland - Holt plot for Feclen jacohaetis \ = -(1.53\ +
58.62 (n = 68(). r = 0.768), B. \on Btrtalanffy growth equation of
Pecten jacobaeus height based on results of the Gullland-Hold plot:
H, = ll(M)8 d-e"
').
study (1999) and the lack of growth data tor P. jacobaeus. we
compared the results of this study with results previously pub-
lished for both, the Mediterranean scallop P. jacobaeus and At-
lantic scallop P. iini.\i)iiiis.
Shell growth of the Atlantic scallop Pecten maxiimis. in Menai
Strait (Wales. U.K.) exhibits the slowest growth rates during Janu-
ary and February, when shell growth effectively ceases at water
temperatures below 8-9"C (Owen et al. 2002). According to Ma-
son (1957), rings in P. inaximiis are formed during a period when
the temperature starts rising and growth resumes. Sampling of P.
jacobaeus in the northern Adriatic was conducted in January and
February, during a period of the lowest sea water temperatures
(Soldo pers. comm.), and thus the shell margin was treated as the
beginning of a growth ring.
The largest P. jacobaeus recorded in this study was 142 mm in
length, whereas previous studies in the Croatian Adriatic recorded
specimens of 150 mm in the northern Adriatic (Cetinic & Soldo
1999), 146 mm in Krka river estuary (Margus et al. 1992), and 162
mm in Velikojezero. Island of Mljet (Onofri & Margus 1995). The
oldest shell in this study had 1 .^ grow th rings, whereas in samples
from Krka river estuary scallops attained an age up to 1 l-l- years
(Margus et al. 1992) and in Mljet lakes up to 17 y (Onofri &
Margus 1995). It is interesting to note that for P. inaxiiiius. Tang
{ 1941 ) noted that some scallop he collected had 22 growth rings,
whereas Mason (1957) found a shell with 18 rings. In this study,
shells in age classes three, four and five constituted 69.27f of the
total sample. In the Krka river estuary, around 70% of the analyzed
shells belonged to age categories from 4+ to 6-i- (Margus et al.
1992). while in the Irish Sea about 65'7f of P. imainnis belonged
to age categories 3. 4. and 5. Category 4-i- indicates that shell has
four rings, with new growth outside the fourth.
In Mljet lake, scallops attained the legal catch length of 1(10
mm after their fourth year (Onofri & Margus 1995). whereas in the
Krka river estuary shells reached 100 mm length after their fifth
year (Margus et al. 1992). According to Mattel & Pelizzato (1996),
P. jacobaeus is a fust-growing species that in the Italian part of the
northern Adriatic reaches a size of 100 mm or more in about 2
years. On average. P. niaxinuis attains a size of 100 mm after its
third growth ring and its growth rate decreases thereafter ( Mason
1957, Allison 1994). Our data indicate that P. jacobaeus collected
in the Croatian part of the northern Adriatic attains a length of over
100 mm after its third year and therefore appears to have a similar
growth rate to P. niaxiiuus. it is faster growing than P. jacobaeus
from Krka and Mljet and slower than specimens found in Italian
part of the northern Adriatic.
Variations in growth of bivalves, among other things, are lo-
cation dependent (Wilbur & Owen 1964). For example, it has been
shown for some other bivalve species, namely Area noae and
Pinna iiobilis. that their growth in Mljet lakes is slower than in
other regions in the Adriatic (Peharda et al. 2002, Peharda 2003).
Further more, higher concentrations of nutrients and hence greater
primary production in the northern Adriatic than in other areas in
the Adriatic Sea (Poulain et al. 2001). probably promoted faster
growth of this species.
Unfortunately. Margus et al. (1992). Mattel & Pelizzato ( 1996),
and Onofri & Margus ( 1995) did not calculate growth parameters
for P. jacobaeus. so further comparison is not possible. However,
growth data obtained in this study are similar to those obtained for
P. maxinuis by Mason ( 1957) and Allison ( 1994). as indicated by
values of o (o = Ln (k) -i- 2*Ln (L^)), calculated according to
Sparre & Venema ( 1992; Table 1 ).
According to Mason (1957) P. maxinuis has different growth
rates during it's first few years, depending whether they were the
spring (April-May) or fall (August-September) spawned. Spring-
spawned scallops grow faster in the first 2 years, whereas growth
of fall-spawned ones is greatest in the second and third annual
growth periods. In the western Mediterranean, P. jacobaeus settled
on artificial collectors from April to July, reaching a peak in June,
whereas some spat settled also in November and February (Pena et
al. 1996). In the Gulf of Trieste, gonads are mature from May until
July and a second maturation can be observed between November
and February (Renzoni et al. 1988). In the estuary of the river
Krka, one settlement peak of P. jacobaeus was recorded in a
period from April until July, with the highest values recorded in
June, while settlement did not occur in the fall or winter (Margus
1994). Based on shell length at second ring found in a present
study, the results of above-mentioned studies from the Adriatic and
findings of Mason (1957). majority of analyzed shells from the
northern Adriatic were probably spring spawned. However, ob-
served variations in growth during first few years suggest that a
certain number of fall settlers is also present in the population.
The observed age structure of P. jacobaeus from the Croatian
TABLE 1.
Values of asjmptotit length (I,,, in mm), curvature parameter (k)
and 0 for Pecten maximus (Mason 1957, Allison 1994) and Pecten
jacobaeus ( this study )
.Study
Location
L.
k
0
Ma.son(l957)
Isle of Man. spring spawned
I4fi.20
0.396
9.04
Mason (1 9.^7)
Isle of Man. fall spawned
146.96
0.371
8.99
Allison (1994)
Bradda. Isle of Man
133.68
0.466
9.03
Allison (1994)
SE Douglas. Isle of Man
133.92
0.329
8.68
This study
Northern .Adriatic
127.93
0.420
8.83
642
Peharda et al
part of the northern Adriatic and the presence of older individuals,
up to 13 y. indicates that overexploilation is probably not a prob-
lem under the current tlshing pressure. However, because of a
relatively long life span and slow growth of the species after it
reaches its fourth year, periodic monitoring of the stock age struc-
ture, rather than length frequency monitoring, should be conducted
in the future. This is necessary because of variations in shell
growth of individual specimens that were observed in this study
and previously noted by Mason (1957). who stated that a large
range of sizes is covered by each age-group, and that it is possible
to find a 4+ scallop larger than an 8+ scallop in the same sample.
ACKNOWLEDGMENTS
The authors express their gratitude to the Ministry of agricul-
ture and forestry. Fishery department, for funding this project.
Special thanks to N. Vrgoc for help with data analysis. A. Frankic
for help with literature search, and C. A. Richards<in for useful
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Jounuil ofShclll'ish Research. Vol. 22. No. 3. 643-646. 2003.
GEOGRAPHICAL PATTERNS IN GROWTH ESTIMATES OF THE SCALLOP ZYGOCHLAMYS
PAT AGONIC A, WITH EMPHASIS ON URUGUAYAN WATERS
OMAR DEFEO' -* AND NICOLAS GUTIERREZ"
'UNDECIMAR. Facitltad de Cienvuis. Igiid 4225. P.O. Bo.\ 10773. Montevideo 11400. Uniiiiuiy:
-CINVESTAV Unidad Merida. A.P. 73 Cordemex, 97310 Merida. Yucahm. Me.xico: ^Depto. Biologi'a
Pesqiwra. DINARA. Consiiluxcnle 1497. 1 1200 Montevideo, Uriigiuiy
ABSTRACT Growth parameters of the scallop Zygoclilcimys patagonica were estimated in Uruguayan waters of the southwestern
Atlantic Ocean. Data used to estimate growth were collected at latitudes SS^SO'S (northern end of the geographical di.stribution of the
species! and 36°40'S (southern end of Uruguayan waters). Scallop ages were estimated by couming external growth rings on the
left-hand valves. The von Bertalanffv function (VBGF) successfully explained some 93% (36°40'S) and 84% (35°50'S) of the
variance. A likelihood ratio test indicated that scallops grew significantly faster at latitude 36'40'S than at latitude 35°50'S, confirmmg
previous results showing large scale variation in density and indixidual muscle weight. Between-latitude differences were mainly
ascribed to variations in the parameter i^. which in turn could be explained by differences in observed lengths-at-age at earlier ages,
notably age 1. Information on growth parameters of Z. patagonica. extracted from published sources over a wide latitudinal range
(35°50'S-54°30'S), showed that asymptotic height H, and the index of growth pertbmiance <J.' were inversely correlated with latitude,
decreasing from north to south. The growth parameter estimates provided in this study are consistent with the pattern. Management
implications of these findings are discussed, placing special emphasis on the applicability of spatially explicit management tools.
KEY WORDS: scallops. Zygochlamy.',. growth, large-scale patterns. Uruguay
INTRODUCTION
The scallop Zyguclilainys patagonica (King and Broderip) is a
species widely distributed over the Magellanic Biogeographic
Province. In the Pacific it is found in the Chilean Channels, from
Puerto Montt (4r'25'S) to Magellan Strait (53=00'S). mainly in
shallow waters ranging from 5 to 25 m (Urban & Tesch 1996.
Valero 2002). In the southwestern Atlantic Ocean (SAO), it is
mainly found between ca. 35°50'S and 55°00'S. with largest scal-
lop beds occurring at a depth range of 70 to 120 m (Waloszek
1991. Defeo & Bra/eiro 1994. Gutierrez & Defeo 200.3). although
a few individuals have been found as deep as 960 m (Waloszek
1991). The discontinuous distribution and marked differences in
life history traits among beds have been considered in relation to
hydrographic features of the SAO. notably the presence of frontal
zones (Orensanz et ai. 2003). Density-dependence was also men-
tioned as a possible factor behind geographic variation (Ciocco et
al. 2003).
Several surveys undertaken between 1993 and 1994 identified
the stock of Z. patagonica from the Uruguayan shelf in this zone
as a potential fisheries resource (Defeo & Brazeiro 1994). Re-
cently. Gutierrez & Defeo (2003) documented the spatial structure
of scallop beds in Uruguayan waters of the SAO between latitudes
35°50'S and 36°50'S. of v\'hich the salient aspects are: (1) beds
close to the Uruguayan southern border (36°30'S) had densities 16
times higher than the northern border (35°50'S). (2) average indi-
vidual size increased southwards, and (3) muscle yield increased
northwards. In this article we provide first growth estimates for the
scallop Z. patagonica stock inhabiting Uruguayan waters of the
SAO, and analyze latitudinal patterns in growth parameters of the
species from a wide range of published sources. Implications for
management are discussed.
MATERIAL AND METHODS
*Corresponding author. E-mail odefeo&mda.cinvestav.mx
Data were collected at latitudes 35°50'S, close to the northern
limit of species distribution in the SAO, and 36°40'S, close to the
southern end of the Uruguayan shelf, during surveys conducted by
the Uruguayan RV "Aldebaran" (Gutierrez & Defeo 2003).
Each 15' tow was carried out at a mean trawling speed of 3.2
knots/h. Sampling gear was an otter trawl directly attached to the
doors (Otter boards) with a net opening of 9.5 m and a mesh size
of 5 cm. The total catch of scallops per tow was recorded, and a
subsample retained for processing. Scallops shell height (H) was
measured with 1 mm accuracy in the laboratory, from the umbo to
the ventral border of the shell. Some 270 and 96 individuals were
used for estimating growth at latitudes 35°50'S and 36°40'S,
respectively.
Age in each scallop was estimated by counting external growth
rings on the left-hand valves, assuming annual periodicity. The
latter was validated for scallop beds in contiguous Argentinean
waters (Waloszek & Waloszek 1986, Waloszek 1991, Lasta et al.
2001, Ciocco et al. 2003). The data was used to fit the von Ber-
talanffy growth function (VBGF; von Bertalanffy 1938):
where H, is shell height at age /. H, is the asymptotic height, K is
the curvature parameter and r,, is the estimated age at length zero.
A quasi-Newton method was used to estimate the parameters
(mean ± SE). Growth of scallops from both latitudes was com-
pared by likelihood ratio tests (Kitnura 1980. Cerrato 1990). under
different null hypotheses (Palacios 1994). First, we compared all
three parameters simultaneously under the null hypothesis H,,; H ,_,
= //-,,: K, = K-,: t„i = to2- Afterwards, the model selection
process was done by sequentially altering the number of param-
eters under comparison. We used raw data instead of mean length-
at-age information following Haddon (2001 ).
Latitudinal information on growth parameters of Z patagonica
came from published sources, and combined with our own results.
Estimates of H,. and K from 14 geographical sites between
35"50'S and 54=30'S were obtained: two from Uruguay (this
643
644
Defeo and Gutierrez
TABLE 1.
Sources of data used in regression analyses between latitude and growth parameters of Z. palagonica in Atlantic and Pacific scallop beds of
South America. The growth performance index <}>' was estimated in this study, using information of H^ and A'. Data used in all cases
are size-at-age.
Ocean
Country
Latitude
H^ (mm)
A' or-' I
Age Range
Source
Atlantic
Uruguay
30=50'
75.98
0.39
3.35
270
1-9
Atlantic
Uruguay
36=40'
81.15
0.31
3.31
96
1-7
Atlantic
Araentine
39=24'
74.70
0.42
3.37
197
1-9
Atlantic
Araentine
39°47'
68.69
0.50
3.37
75
1-8
Atlantic
Argentine
41°50'
74.18
0.38
3.32
83
1-10
Atlantic
Argentine
41°50'
69.93
0.37
3.25
87
1-8
Atlantic
Argentine
42°30'
59.76
0.49
3.25
152
1-7
Atlantic
Argentine
43°53'
66.32
0.50
3.34
79
1-8
Atlantic
Argentine
44°00'
75.59
0.54
3,49
124
1-8
Atlantic
Argentine
46°47'
65.67
0.63
3.43
65
1-7
Atlantic
Argentine
49°50'
62.65
0.40
3.19
89
1-8
Atlantic
Argentine
52=00'
54.66
0.58
3.24
91
1-8
Atlantic
Argentine
54=30'
54.90
0.39
3.07
90
1-7
Pacific
Chile
5}'00'
66.(10
0.14
2.78
95
1-11
This study
This study
Valero (2002: in Ciocco et al.. 2003)
Waloszek & Waloszek (1986)
Valero (2002: in Ciocco et al. 2003)
Waloszek & Waloszek (1986)
Waloszek & Waloszek (1986)
Waloszek & Waloszek (1986)
Waloszek & Waloszek (1986)
Waloszek & Waloszek (1986)
Waloszek & Waloszek ( 1986)
Waloszek & Waloszek (1986)
Waloszek & Waloszek (1986)
L'rban ct Tesch (1996)
study). II from Argentina an(j one from Chile (Table 1). The
growth inde.x (b' = 2\ogj„{L^^J log/,;A' (Pauly & Munro 1984) was
calculated and used to assess growth performance. The relation-
ships between growth parameters and latitude (centesimal units)
were tnodeled by linear and nonlinear fitting procedures, and the
model with the best goodness of fit selected.
RESULTS
Scallops at latitude 36°40'S grew significantly faster than at
latitude 35°50°S (Fig. 1). The non-linear fitting of the VBGF
explained 93% (36°40'S) and 84% (35°50'S) of the variance, and
all parameters were signitlcant. except /,, at latitude 35 = 50'S (Table
2). Results of likelihood ratio tests showed that the overall VBGF
significantly differed between latitudes (x" = 45.234. df = 3.
P < 0.0001 ). Testing of the remaining null hypotheses showed that
the H^ and K parameters did not differ significantly (x" test, df =
\.P> 0.05). Conversely, a significant difference between l,, values
was strongly indicated, either in isolation (x" test, df = 1, P <
0.01) or in combination v\ith the other two VBGF parameters
(Table 3). Marked differences in mean-length-at age at earlier
ages, notably age 1 (ANOVA test; p < 0.01; Cochran test for
35°50S
36°40'S
Figure
waters
4 5 6 7 8 9 10
AGE (years)
I, Growth models for the scallop Z. palagonica in Uruguayan
Details are provided in Table 2.
homoscedasticity C = 0.68; P = 0.49) could explain the observed
differences between curves (see Fig. I ).
The large-scale analysis showed that both H, and (}>' were
inversely correlated with latitude (/• = -0.80; P < 0.0006 for H.^
and r = -0.63: P < 0.015 for (j)'). but K was not (/■ = 0.03: P =
0.91) (Fig. 2). Values of H,- were in the range 55 to 81 mm, with
the lowest value at 54°30'S and the highest at 36'40'S (this study;
Table 1 ).
DISCUSSION
Scallops grew significantly faster at the southern limit of Uru-
guayan waters when compared to the northern border. Gutierrez &
Defeo (2003) also showed that muscle weight increased linearly
towards the southern end of Uruguayan waters, whereas the har-
vestable stock (ie, individuals >55 mm H). mean individual height
and maximum height increased asymptotically in the same direc-
tion. Defeo & Brazeiro ( 1994) found hardly any scallops north of
the range considered here (between 35=50'S and 35 'OO'S) and.
where few specimens were caught, individual height also tended
to be low. This is in agreement with the higher estimate of H-^
found at 36°40'S (81.15 mm H) when compared with that at
35°50'S (75.98 mm H). The occurrence of lower abundances and
sizes at the nonhem distribution end has been ascribed to habitat
TABLE 2.
Z. palagonica. Results of the >on Bertalanffy growth models fitted
by nonlinear least squares for scallops of Uruguayan waters.
Significant \alues {P < (I.OI) are highlighted in bold and italics.
Latitude 35°50'S
Parameters Estimate (SEl
Latitude 36°40'S
Estimate (SE) F
H,_ (mm)
A-(yr-')
'o (y)
R-
75.98(1.60)
0.39(0.03)
-0.01 (0.11)
0.84
0.0000
0.0000
0.9336
0.0000
81.15(2.81)
0.31 (0.04)
-0.65(0.19)
0.93
0.0000
0.0000
0.001 1
0.0000
Scallop Growth in the Southwestern Atlantic
645
TABLE 3.
LikelihiKid ratio tests comparing von Bcrtalanff) parameter estimates for the two scallop beds in Uruguayan waters. Results of the RSS
(residual sum of squaresl. the \" test and associate statistics are shown on the basis of the seven null hvpotheses tested (columns 3 to V).
assuming thai the listed parameter or a c<imbination of them do not differ between scallop beds. I'he second column refers to the
independent lltting of the two separate cur\es (see also Table 2). .Signillcanl \alues of the likelihood ratio test are highlighted in bold
and italics.
H„: = H,
= A
H„:=//.
Hyl - H^
H„: = A
Parameter
Independent
= 'o
H„:=//,
H„: = A
H,,: = '„
= A
= l„
= t„
Latitude 35 50'S
H^ (mm)
75.979
76.485
77.102
76.845
77.693
77.148
77.063
76.361
A-(yr-')
0.387
0.374
0.368
0.371
0.351
0.369
0.358
0.364
'o lyr)
-0.009
-0.200
-0.062
-0.062
-0.169
-0.053
-0. 1 70
-0.203
Latitude 35 50'S
//„ imm)
8L147
76.485
77.102
77.584
75,854
77.148
77.063
79.759
K()/T-')
0.312
0.374
0.375
0.371
0.420
0.369
0.404
0.364
'o (yr)
-0.647
-0.200
-0.411
-0.401
-0.169
-0.454
-0. 1 70
-0.203
RSS
5167.717
5848.974
5206.290
5190.891
5271.624
5199.697
5284.775
5379.007
X'
45.234
1.657
1.638
7.286
2.258
8.198
14.667
df
3
1
I
1
->
->
->
P
0.0000
0.1980
0.2007
0.0069
0.32.^4
0.0166
0.0007
unsuitability and scarcity of food (Gutierrez & Defeo 200.3). In
spite of tlie above, the likelihood ratio test indicated that major
between-latitude differences in the VBGF could be ascribed to
parameter /„. which jointly with H.^ strongly determines the form
of the VBGF (Haddon 2001 ). Differences between curves could be
attributed to \ariations in length-at-age at ages 1 to 5. notably age
1, where individual sizes-at-age at 36°40'S were greater than at
35°50'S (see Fig. 1).
Growth parameters of Z. patcifionica showed clear latitudinal
patterns: asymptotic height H , and the overall growth performance
4>' both significantly decreased from north to south in the SAO.
The results of our study are consistent with the pattern found at a
large-scale, and indirectly reaffirm the annual nature of growth
ring formation. These results pro\ide additional evidence for the
large-scale patterns found for Argentinean waters, where a signifi-
cant decrease in H^_ was estimated as latitude increases (Valero
2002. Ciocco et al. 2003). Between-latitude differences in growth
rate have been attributed to variations in environmental param-
eters, such as temperature and food availability (Ciocco et al.
2003 and references therein). Valero (2002) presented new evi-
dence on the effects of factors acting at small spatial scales (eg.
intrabed scale), seasonal cycles and year-to-year variation in
growth in this species, which were mainly related to variations in
temperature and oceanographic regimes. These factors seem to be
critical in explaining growth variations in other scallop populations
(see eg MacDonald & Thompson 1985, Schick et al. 1988 and
papers in Shumway 1991). Latitudinal differences in growth rate
could also be explained by density-dependence operating at the
scale of scallop beds, a mechanism already proposed by Orensanz
et al. (2003) to explain intra and inter-cohort variations in popu-
lation dynamics. Ciocco et al. (2003) showed that for high-density
patches occurring in Argentine waters, individuals at high concen-
trations are affected by density-dependence e\en in small areas
(Lasta & Bremec 1998). The low growth performance in high-
density beds in southern waters of the SAO (as denoted by <i>')
provides additional support to the density-dependence hypothesis,
which was also suggested by Gutierrez & Defeo (2003) for Uru-
guayan scallop grounds. Finally, differences between Atlantic and
Pacific estimates could be attributed to different environmental
scenarios and the fact that the species occurs in relatixely shallow
waters in the Pacific.
42 46
LATITUDE SOUTH
50
54
58
42 46
LATITUDE SOUTH
Figure 2. '/.. palagonica. Regression lines (±95'7f CI) between latitude
(centesimal units! and (al asymptotic height and (hi the growth per-
formance inde.v cj)'. (•) Lruguay; ( . ) Argentina and (■) Chile.
646
Defeo and Gutierrez
Managemenl Implications
The significant latitudinal gradient in growth rate detailed here
could have implications for fishery management, as spatial varia-
tion in population dynamics and life history traits are used to
provide area-based estimates of potential yield (Caddy 1975) and
to implement spatially explicit management measures (eg, rotation
of fishing areas and reproductive refugia: Orensanz & Jamieson
1998. Catilla & Defeo 2001). This should call for a spatially dis-
crete analysis of population dynamics and other life history traits,
the sun'ounding environment, and the fishery. In this setting, map-
ping of density and related population processes is worthwhile as
a way of forecasting the spatial features of the stock, and to assess
the economic potential of the fishery (Caddy 1989a. b).
ACKNOWLEDGMENTS
This paper was written during the M.Sc. thesis of Nicolas Gutie-
rrez. We are especially grateful to Dr. Raiil Palacios for his advice
on the application of the likelihood ratio test. Nestor Ciocco and
Juan Valero kindly provided us scallop growth estimates for Ar-
gentinean waters. Two referees gave us useful suggestions that
improved the paper. Financial support from DINARA and
PEDECIBA Uruguay is gratefully acknowledged.
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Caddy. J. F. 1989a. A perspective on the population dynamics and assess-
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vertebrate fisheries: their assessment and management. New York:
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Caddy. J. F. 1989b. Recent developments in research and management for
wild stocks of bivalves and gastropods. In: J. F. Caddy, editor. Marine
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Ciocco. N. F.. M. L. Lasta. M. Narvarte. C Bremec. E. Bogazzi. J. Valero
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gochlamys patagonica fishery in Uruguay: latitudinal and bathymetric
patterns in biomass and population structure. Fish. Res. 62:21-36.
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Lasta. M. & C. Bremec. 1998. Zygoclilaniys patagonica in the Argentine
sea: a new scallop fishery. J. Shellfish Res. 17:103-1 1 1.
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tagonica. Arch. Fish. Mar. Res. 49:125-137.
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J,,iinuil „f Shellfish Research. Vol. 22. No. 3. 647-654. 200.^.
INTERMEDIATE CULTURE OF KING SCALLOP {PECTEN MAXIAWS) IN SUSPENSION IN
CAGES: EFFECT OF STOCKING DENSITY AND DEPTH
G. ROMAN.* A. LOURO, AND J. P. DE LA ROCHE
lustinito Espcwol dc Ovcaiioiiiafia. Ministcrio clc Ciciicia y Tecnologia. Ccntro Ocecmogrcifico de A
Coniiui. P. O. Box 1.^0. 15080. A Coniila. Spain
ABSTRACT Scullop spal settled on collectors were grown ui suspended cages in O Grove. (Ria de Arousa. Galicia. nonhwest .Spain)
and in Fuengirola. (Malaga, Andaluci'a. southern Spain). Mean (±SDl spat heights of 20.4 ± 3.7 mm (Fuengirola. September 1998) and
26.6 ± 5.8 mm (O Grove. November 1998). were stocked al densities ranging between 25 and 2()0/cage"' (=200-1600 spat nV\. and
at depths of 6 and 10 m in O Grove, and between 13 and 25 m in Fuengirola. Even low stocking densities were found to affect scallop
growth, therefore juveniles (>35 mm) were used to set up new cultures at lower stocking densities (12 and 24 juveniles/cage"' ) at the
end of winter (Fuengirola) and at the beginning of spring (O Grove). The most rapid growth took place at Fuengirola, where the mean
height reached on May 19. 1999. was 63.9 ± 4.1 mm compared with a mean height of 51.2 + 4.5 mm for the O Grove spat on May
27. 1999.
KEY WORDS: Andalucia. density, depth. Galicia, intermediate culture. Peeten iiniMmus. suspension culture
INTRODUCTION
Attempts are currently being made in several European coun-
tries to cultivate Peeten maxinnis on a commercial scale (Fleury et
al. 1997), using both hatcheiy-produced spat (Norway and France)
and spat captured by natural settlement on collectors (Ireland and
Scotland). Pectinids (pectinoid form sensit Waller 1992) cultivated
in suspended cages grow slowly after a certain size (Slater 1995),
possibly because of the differences in the conditions in the cages
and in the natural habitat (recessed in sediment), although waves
have been observed to have a negative effect on Eitvola ziczac and
Nodipecten nodosus (Freites et al. 1999). Because of these diffi-
culties, the present trend in Europe is for intermediate culture,
usually in suspension, followed by the seeding of juveniles of
different sizes, depending on the conditions in each area, on the sea
floor. However, in certain areas the environmental conditions or
the techniques result in the successful use of suspended culture to
grow scallops to commercial size (Roman & Fernandez 1991.
Gallagher 1999, Cano et al. 2000).
The desired final size of the Juveniles maintained in interme-
diate culture will obviously depend on the method of on growing.
In the case of seabed culture, the size required depends on the
environmental conditions (i.e.. sediment, current, and predators)
and varies from region to region: 30 mm in France (Fleury et al.
1995); and 50 mm in Norway, Ireland, and Scotland (Fleury et al.
1997). The size required for ear-hanging culture is more stan-
dardized. Generally, scallops are not ear-hung until they have
reached at least 55 mm shell height (Ventilla 1982, Dadswell &
Parsons 1991, O'Connor et al. 1999). Gallagher (1999) considers
the minimum size for ear-hanging culture to be 50 mm, whereas
Cano et al. (2000) used juveniles of between 51.3 and 64.3 mm
height. Roman and Fernandez (1991 ) describe ear-hanging culture
in Galicia, where scallops of heights of between 60 and 70 mm are
used.
In order for the scallops to reach the size required for the
on-growing stage, they must undergo a period of intermediate
culture in mesh trays or cages, which are usually suspended in the
water, although cages are also placed on the seabed (Dao et al.
*Corresponding author. Fax: +34-981-229077: E-mail: guillermo.roman@
co.ieo.es
1996). Subsequently, scallops are seeded on the seabed (Fleury et
al. 1997), are suspended using the ear-hanging technique (Paul
1988, Roman & Fernandez 1991. Gallagher 1999). or are held
within lantern nets, cages, or other artifacts (Cano et al. 20(J0).
There are many reports in the literature on the effects on growth
and survival of the use of mesh enclosures (O'Connor et al. 1999)
and of the stocking density and depth at which pectinid spat are
cultured (Cote et al. 1993. Duggan et al. 1973. Leighton 1979,
Parsons & Dadswell 1992, Rhodes & Widman 1984. Wallace &
Reisnes 1984, 1985, Roman et al. 1999, Cano et al. 2000, Freites
et al. 1995). Most of these studies refer to aequipectinoid pectinids
(form sensu Waller. 1991 ). the natural habitat of which is similar
to the conditions of suspended culture.
Scallops are commercially produced in two areas of Spain:
Galicia (in northwest Spain): and in the province of Malaga (An-
daluci'a, in southern Spain). Cultivation on the seabed is not pos-
sible for legal and social reasons, and only suspension culture is
feasible. The aims of the present study were to determine, in each
area, the optimum conditions (in terms of depth and stocking den-
sity) required for the intermediate culture of spat and juveniles in
suspended cages to obtain scallops of a suitable size for ear-
hanging culture (=60 mm height), and to compare the growth and
survival of spat cultured in the different areas. In addition, the
possibility of cultivating scallops to commercial size in suspended
cages in Galicia was evaluated, which is an aspect that has been
studied previously in Malaga (Cano et al. 2000).
MATERIALS AND METHODS
Study Area
The study was carried out at two sites, in O Grove, Ria de
Arousa. in the Atlantic Ocean (Galicia, in northwest Spain), and in
Fuengirola. in the Alboran Sea (western Mediterranean, Malaga,
Andalucia, in southern Spain) (Fig. 1 ).
Environmental Conditions
The temperature and levels of chlorophyll (/ were measured,
using a conductivity-temperature-depth (CTD) recorder, every
week in O Grove and every fortnight in Fuengirola. Salinity also
was recorded in O Grove.
647
648
Roman et al.
O Grove (Galicia) , V
4»0'
-i_.
Atlantic f
>
Jy
Ocean > J
/
— 40° 0' / )
1 1
Spain
/ 40° 0' -
/< Mediterranean
Z' Sea
1 1
Fuengirola (Malaga)
lOOKms
Fijiure 1. Locations of the experimental intermediate culture of P. maxiiniis.
Animals
Scallops of up to 30 mm are considered to be spat, and between
30 and 60 mm they are considered to be juvenile. In both areas, the
spat was obtained by natural settlement on collectors.
Sampling
The cages were raised periodically so that the height of the
scallops (measured to the nearest millimeter using calipers) and the
number of dead could be recorded, and they then were resus-
pended.
Suspended Culture
The spat were cultured in circular rigid plastic cages (40 cm
diameter. 10 cm height. 10 mm mesh size). In O Gro\e. the cages
were hung from a raft, whereas in Fuengirola they were anchored,
following the scheme outlined by Cano et al. (2000). Spat growth
was greatly affected by stocking density, therefore new experi-
ments were started in the spring in both areas using juveniles at
lower stocking densities of 12 and 24 scallops/cage"'. The experi-
ments were carried out in duplicate (Table 1).
Statistical Methods
Mean sizes (height) were compared by factorial analysis of
variance (ANOVA) using stocking density and depth as factors.
Normality was previously checked using the Kolmogorov-
Smimov test, and the homogeneity of variance was checked by
Bartlett's test. The differences in size were compared a posteriori
using a Newman-Keuls test (a = 0.05). except when there was
interaction between factors. Comparisons between pairs of
samples were made using a Student's t test. Arcsin transformation
was used to compare percentage survival.
TABLE 1.
Description of experimental intermediate culture of P. maximus.
Date
Area
Start End
Initial Size (mm)"
(Mean ± sd)
Density Culture
n" Scallops Cage'
Initial Coverage ( Vc l''
Depth Culture (m)
Intermediate culture of spat
Fuengirola 9/24/98-2/24/99
O Grove 1 1 / 1 7/98-4/6/99
Intermediate culture of juvenile
Fuengirola 2/24/99-5/19/99
O Grove 4/6/99-.'S/.^0/99
204 ± 3.7
25/50/100/200
6.5/13.0/26.0/52.0%
13/18/23/26
26.6 ±5.8
25/50/100
11.1/22.1/44.2%
6/10
52.1 ±5.0
12/24
20.4/40.7%
13/18/23/26
39.9 ±3.2 (6 m)
12/24
11.9/23.9% (6m)
6/10
414 ±3.9 (10 m)
12.9/25.7% (10m)
' Presented as mean ± SD.
' Experimental design 2x2 factor.
Intermediate Culture of Pt:cT[-:N maximus
649
RESULTS
Envinnititfiiltil C 'omlitions
O Grove
There was a slight temperature inversion with depth in winter.
ho\\e\er. during the rest of the year the temperature was sHghtly
higher in the first 6 ni. In general, the temperatures were very
similar throughout the year, at both depths, ranging between 1 1°C
and 18°C, with only occasional differences of >1°C (Fig. 2). Sa-
linity ranged between M7co and .'^5.5^i throughout most of the
study period, except in May 2000. when minimum values of
32.0%f and 33.1%c were registered at depths of 6 and 10 m. re-
spectively. There was a trend toward slightly higher levels of
chlorophyll a in the surface layers of water from the end of autumn
until the beginning of spring, but from May onward increasing
levels were found at depth. There were large variations in the
le\els. with minimum \alues of approximately 1 p-g L~'. and
of 2 and 3 |a.g L at 6 and 10 m. respectively
maximum value:
(Fig. 3 1.
Fuengirola
High temperatures ( 17-18. 5°C) were registered at the begin-
ning of September, followed by a maximum of 2 1 "C at the end of
September. From the end of October until May. the temperature
varied between 14"C and 16"C (Fig. 2). The levels of chlorophyll
a observed were rarely < 1 (xg L" ' . with peaks of between 2 and ?
fj-g L"' in September. October, and February, and particularly
between April and May. As there was strong vertical mixing dur-
ing the period of the study, there was very little variation with
depth, with only a slight trend toward lower temperatures and
chlorophyll a levels at lower depths (Fig. 3).
Growth
O Grove
Spat culture. On March 2. 1949. the mean heights ranged
between 35.6 and 39.8 mm. depending on the culture conditions.
There were significant differences in the heights achieved at the
different stocking densities (25 scallops/cage"' > 50/cage"' > 100/
cage"'), but there were no significant differences associated with
depth (Fig. 4). One month later, on April 6. 1999. only very small
increases in size were registered, with mean heights ranging be-
tween 36.4 and 41.4 mm. depending on the culture conditions.
Again, growth was affected by stocking density but not by depth.
Scallops cultivated at a stocking density of 25/cage"' were sig-
nificantly larger than those cultivated at higher stocking densities,
whereas there was no difference in the size of scallops cultured at
the two higher stocking densities (50 and 100/cage"'l. The mean
coverage was 25.6% at 25/cage~'. 45.0% at 50/cage"'. and 86.0%
coverage at lOO/cage"'. The spat growth experiment finished on
this date, and a new experiment, using juveniles at lower stocking
densities, was started.
Juvenile culture. This experiment was begun on April 6. 1999,
using scallops previously grown at stocking densities of 25/cage~'
(the mean heights reached by scallops culti\ated at depths of 6 and
10 m were 39.9 ± 3.2 and 41.4 ± 3.9 mm. respectively). The new
stocking densities were 12 and 24/cage~', at the same depths as
before (i.e., 6 and 10 ni). The initial coverage was 11.9% and
23.9% and 12.9% and 25.7%, respectively, at 12 and 24 spat/
cace"' at 6 and 10 m.
22
20
s
16 -
14
12 -
10
-e — O Grove ( 1 Dm depth)
-^ — O Grove ( 6m depth)
-A- - - Fuengirola (13m depth)
24-07 12-09 01-11 21-12 09-02 31-03 20-05 09-07 28-08 17-10 06-12 25-01 15-03 04-05 23-06 12-08
1998 1999 2000
Figure 2. Interannual variation of temperature in U Grove and Fuengirola.
650
Roman et al.
et) 4
a.
1>3
£
■2 2
-3K — O Grove ( 6m depth)
-© — O Grove ( 10m depth)
■A- • - Fuengirola (13m depth)
0
24-07 12-09 01-11 21-12 09-02 31-03 20-05 09-07 28-08 17-10 06-12 25-01 15-03 04-05 23-06 12-08
1998 1999 2000
Figure 3. Interannual variation of Chlorophyll a in O Grove and Fuengirola.
45
40
?
E
- 35
en
6 m- 25/q
lOm-25/q
30
25
6 m- 50/q
10m- 50/q
6m-100/q
lOm-lOO/q
11/11/98 11/12/98 10/01/99 09/02/99 11/03/99 10/04/99
Figure 4. Growth of P. maxiimis spat on intermediate culture in O Grove.
-c^ 6 m- 24/q
-^-lOm-24/q
6 m- 12/q
10 m- 12/q
31/03/99 19/06/99 07/09/99 26/11/99 14/02/00 04/05/00
Figure 5. Growth of P. maximus juvenile on intermediate culture in O Grove.
Intermediate Culture of Pecten maximus
651
Depth affected the growth of the juveniles hetween May 27 and
September 28. 1999 (the jii\eniles maintained at a depth of 10 m
reached a larger size than that cultivated at 6 m; Fig. 5). On
February 1 7. 2000. there were no effects associated with depth, but
on May .■'0, 2000. a depth effect was once again observed
(ANOVA: Table 2).
The effect of stocking densit> on grov\th rate was obsersed
from September 28. 1999. until the end of the experiment on May
30, 2000. with larger scallops being obtained at the louer stocking
density (Fig. 5). Very little growth was registered between Febru-
ary 17 and May 30. 2000. On February 17. 2000. the mean height
reached ranged between 66.1 and 73.9 mm, depending on the
culture conditions, whereas on May 30. 2000, it ranged between
67.1 and 76.4 mm. In May. 9.2% of the scallops cultured under the
most favorable conditions (i.e., 12 scallops/cage^' at 10 m depth)
had reached commercial size. (ANOVA; Table 2).
T.4BI,E 2.
Effect of depth (6 and 10 m) and stocking density (25, 50, and 100
per cage"') on the growth of king scallop in O Grove. Ria
de .\rousa.
Fuengirola
Spat culture. Scallops were sampled on November 23. 1998.
Jantiary 27. 1999, and February 24. 1999. Faster growth rates
always were observed at lower stocking densities. The effect of
depth was not clear, although growth rates were generally higher
at shallower depths (Fig. 6). A posteriori analysis of data was not
carried out because there was interaction between factors each
month. At the end of the experiment, on February 24. 1999, the
mean heights of the spat at each stocking density (pooled for the
different depths) were 36.7 ± 5.9 {200/cage"'), 42.1 ± 6.6 (100/
cage-'). 48.2 ± 5.9 (50/cage-'). and 53.0 ± 6.2 mm (25/cage-').
Juvenile culture. A new experiment was started in February,
using stocking densities of 12 and 24 juveniles/cage"'. The initial
mean size was 52.1 ± 5.0 mm. Monthly sampling was carried out
on March 23, 1999, April 21. 1999. and finally on May 19, 1999,
when the scallops had reached a suitable size for ear-hanging
culture (overall mean height 63.9 mm I and the experiment was
finished (Fig. 7). Significant differences in size were observed
from March onward that were related to stocking density. Growth
was not affected by depth (ANOVA; Table 3).
Survival
Source of
F
P
Variation Df Ratio
Value
Newman-Keuls Test
March 2. 1999
Depth
0.04
0.852
D6 ni = D lOm
Stocking density I
42.34
0.003*
SD 25 cage"' > 50 cage"'
> lOO/cage"'
Density x depth 2
3.97
0.080
Apnl 6. 1999
Depth
1.80
0.229
D 6 m = D 10 m
Stocking density ;
14..S7
0.005*
SD 25cage"' > 50 cage"'
= 100 cage"'
Density x depth I
1.75
0.252
May 27. 1999-'
Depth
8.09
0.047*
D 6 ni< D 10 m
Stocking density
0.43
0.547
SD 12 cage-' = SD 24
cage"'
Density x depth
0.28
0.624
July 20. 1999
Depth
12.48
0.024*
D 6 m < D 10 m
Stocking density
2.44
0.193
SD 12 cage"' = SD 24
cage-'
Density x depth
0. 1 1
0.757
September 28. 1999
Depth 1
5.77
0.074
D 6 m = D 10 m
Stocking density 1
9.67
0.036*
SD 12 cage"' > SD 24
cage"'
Density x depth 1
0.00
0.996
February 17. 2000
Depth
5.26
0.084
D 6 m = D 10 m
Stocking density
25.69
0.007*
SD 12 cage-' > SD 24
cage"'
Density x depth
0.05
0.831
May 30, 2000
Depth
20.41
0.011*
D 6 m < D 10 m
Slocking density
129.58
0.000*
SD 12 cage"' > SD 24
cage"'
Density x depth
0.63
0.472
D. depth; SD. stocking density; DF, degrees of freedom.
"After April 6 new stocking densities were used (12 and 24 per cage"')
* Indicates a significant result. P < 0.05.
O Grove
Spat culture. The survival rates between November 1998 and
April 1999 were 100% at stocking densities of 25 and 50 spat/
cage-' at both depths, and 90% at a stocking density of 100 spat/
cage"' at both depths.
Juvenile culture. The survival rate ranged between 73.5% and
87.0% (Table 4). The multifactorial ANOVA revealed interaction
between factors, and a posteriori analysis was not carried out.
Fuengirola
Spat culture. The survival rate ranged between 91% and
100%'.
Juvenile culture. In May. the survival rate ranged between
83.3% and 95.8% (Table 5). There were no significant differences
in mortality associated with density or depth.
DISCUSSION
Effect of Depth
In Fuengirola. the environmental conditions showed very little
difference at the different depths tested. Possibly because of this.
-e— 25
-a— 50
-6—100
-o— 200
12/09/98
01/11/98
21/12/98
09/02/99
31/03/99
Figure 6. (irowlh of/", inaxiimis spat at four densities, depth pooled,
on intermediate culture in Fuengirola.
652
Roman et al.
-0—12
-B— 25
70 1
65
60
55
50
45
10/05/99 09/06/99
09/02/99 11/03/99 10/04/99
Figure 7. Growth of P. maximus ju\enile at two densities, depth
pooled, on intermediate culture in Fuengirola.
there was very little differenL-e in growth at different depths ot
either spat or juveniles.
In O Grove, during the period of spat culture no differences
were observed in temperature, levels of chlorophyll a. or growth at
the different depths. However, during the period of juvenile culture
in spring and summer of 1999. growth was faster at a depth of 10
m than at 6 m. possibly because of higher levels of chlorophyll a
TABLE 3.
Effect of depth (13, 18. 23, and 26 ni) and stocking density (25, 50,
100, and 200 per cage"') on the growth of king scallop in
Fuengirola, Malaga.
Source of
Variation
Df
F Ratio P Value
Newman-
Keuls Test
November 23. 1998
Stocking density
Depth
Density x depth
January 27. 1999
Stocking density
Depth
Density x depth
February 24. 1999
Stocking density
Depth
Density x depth
March 23. 1999»
Stocking density
Depth
Density x depth
April 21. 1999
Stocking density
Depth
Density x depth
May 19. 1999
Stocking density
Depth
Density x depth
3
3
9
3
3
9
3
3
9
1
3
3
I
3
3
]
3
3
64.06
10.11
7.47
790.28
331.28
24.12
1163.68
199.7?
8.11
6.37
1.35
0.23
5.43
1.05
1.49
43.46
1.13
3.41
0.000*
0.000*
0.000*
0.000*
0.000*
0.000*
0.000*
0.000*
0.000*
0.036*
0.326
0.876
0.048*
0.422
0.289
0.000*
()..W4
0.074
SD 12 > SD 24
SD 12 > SD 24
SD 12 > SD 24
D. depth; SD. stocking density.
" After February 24, new stocking densities were used (12 and 24 juveniles
per cage"').
* Indicates a significant result. P < 0.05.
TABLE 4.
Survival rales of juveniles grown at different stocking densities and
depths in O Grove, Ria de Arousa.
6
m
10
m
12 juveniles
24 Juveniles
12 juveniles
24
juveniles
Date
cage"'
cage"'
cage"'
cage"'
5/27/1999
99.8
100
100
100
7/20/1999
96.9-'
100"
100"
100"
9/28/1999
92.7"
95.8"
100"
91.8"
2/17/2000
83.3
92.7
93.5
83.7
5/20/2000
78. l-''^
84.4"^
87,0"
73.5-'
Presented as ';;-. Values with a common superscript letter do not differ
significanlly in each month.
at that time, as the temperature varied very little at the different
depths. Similar results have been reported by Roman et al. ( 1999)
ior Aequipectcu openulahs. The results confirm those of Lodeiros
and Himmelman (1994). who observed that in temperate and
northern regions food availability is more important than tempera-
ture for the growth of pectinids. The results, however, contrast
with those of Laing and Utting (2001 ). who suggest that tempera-
ture is the most important factor for the growth of scallop seed in
the sea. However, they worked with a wider temperature range
(5-23°C) than that recorded in the present study (14-2rC in
Ftiengirola: 13-I8°C in O Grove).
From autumn until the following spring, the rate of growth of
the scallops maintained at 6 m was slightly higher than that of
scallops cultivated at 10 m. and there were no longer any signifi-
cant differences. The higher growth rate in the scallops maintained
at 6 111 may have been partly due to the sharp decrease in levels ol
chlorophyll a at 10 m between September and October, and partly
due to compensatory growth. In May 2000. the growth rate of the
scallops maintained at 10 m was again higher than that of the
scallops maintained at 6 m, although the reasons for this were not
clear. The slight decreases in salinity that were registered may
have been sufficient to produce differences in the giowth rates.
Fouling of the cages and of the scallops may be an important factor
affecting the growth rate, although no specific study has been
carried out to test this.
In O Grove, minimum temperatures (I2-13°C) and levels ot
chlorophyll a (1-1.5 [jlL"') were registered during the period of
spat culture (November 17. 1998. to April 6. 1999). However.
TABLE 5.
Surv ival rate of spat and juveniles grown at different stocking
densities, depths, and dates in Fuengirola, Malaga.
Stocking Density
(Spat/Cage)
26 m
23 m
18 ni
13 m
Spat. Febniary 1999
25
.SO
UK)
200
Juveniles. May 1999
24
12
100
100
96.0 ± 2.0
94.0 ± 2.0
100
94.0 ±2.0
100
100
100
100
9S.0 ± 2.0
92.0 + 2.0
92.0 ± 4.0
100
96.0 ± 2.0
9 1 .0 ± 4.0
93.8 ±6.3 92.8+1.0 89.6 ±2.1 88..? ± 5.2
93.8 ±2.1 95.8 ±0.0 83.3 ±16.7 95.8 ±4.2
Presented as mean (±SD) %.
Intermediati; Culture of Pixtkn maximus
653
growth rates of between 0.131 and 0.089 mm d~' were recorded
between November and March, when scallops were not handled,
and of only between 0.047 and 0.024 mm d~' between March and
April. The decrease in the growth rate may have been caused by
stress according to Laing et al. ( 1999). who observed an increasing
level of stress in scallops disturbed monthly during a period when
there was little food available for growth. In the previous winter
months, undisturbed scallops grew faster, even with lower or simi-
lar food availability and temperature levels.
Effect of Slocking Density and Areal Coverage
When spat reached a height of appro.ximately 35 mm. lower
growth rates were recorded in both areas at stocking densities of
50/cage"' and higher (corresponding to at least 38% areal cover-
age).
In O Grove, there were no significant differences in the growth
of juveniles (those of initial size range of 39.9—11.4 mm on April
6, 19991, until 6 mo after the .start of the experiment (September
28. 1999. height reached .57-65 mm), despite the different stocking
densities used ( 12-24 juveniles/cage"' ). However, in Fuengirola,
where growth was faster, significant differences in growth rates at
different stocking densities were observed in the first month after
the start of the cultivation period. Different growth rates were
apparent when areal coverage reached 23% and 47%. respectively,
for 12 and 24 scallops/cage"' (height, approximately 55 mm).
In O Grove, juveniles scallops maintained at 12/cage"' from
September onward showed a significantly higher growth rate than
those maintained at 24/cage^'. These densities corresponded to
mean pooled areal coverages of 29.7% and 54.6%. respectively.
Growth almost ceased between February and May 2000 at both
stocking densities, when areal coverage was 39.7% at 12 scallops/
cage''.
For the range of sizes used in this study. P. maxiiiuis showed
low growth rates at areal coverages of between 30% and 40%. This
is in accordance with the guidelines for growing scallops in net
culture (i.e., that the area of the floor space occupied by scallops
should not exceed 33%: Paul et al. 1981). When growing
aequipectenoid pectinids (A. operciilaris. Placopecten mageUani-
ciis. and Argopecten irradians). higher coverage rates can be used,
but the same is not true for pectinoid Pectinids. Perhaps because
they are not byssus-attached. there is a higher incidence of biting,
and they are more affected by sea swell.
Comparison Between Areas
In both regions, there was sustained growth of spat during the
winter. From November 1998 until May 1999. growth in the cul-
tures held under the most favorable conditions (i.e.. those held at
a stocking density of 25/cage"' as spat, and at 12/cage"' as juve-
niles) was higher in Fuengirola (final mean size 64.7 ± 4.5 mm)
than in O Grove (final mean size 52.6 + 4.1 mm; (P < 0.001 by /
test). The faster growth rates in Fuengirola may have been due to
the higher availability of food (measured as chlorophyll a). Fur-
thermore, the temperature in Fuengirola was higher, as during
most of the experimental period it ranged between 14°C and 16°C.
compared with between 12^0 and 13"C in O Grove.
In Fuengirola. during the first 2 mo of cultivation (September
24 to November 23. 1999) the spat increased in height from 20.4
to 34.9 mm (mean values), at the same time as the maximum
temperature was recorded {2rC). However, in natural populations
in Galicia. we have observed the formation of false growth rings
between September and October (Roman, unpub. results). These
rings are associated with an arrest in growth that coincides with the
maximum temperatures that occur during the year (~18.5-20°C). It
is therefore possible that the scallops in Malaga are genetically
adapted to the higher temperatures.
In O Grove, the rate of growth between February and April was
very slow; by this date scallops reach the size when juveniles
maintained in suspension stop growing, as has been described in
other areas. According to Slater (1995). the cultivation of P. iiia.xi-
iiiiis in baskets or cages is easily carried out until the spat reach a
size of 45 mm. but thereafter growth is retarded. Although Cano et
al. (2000) obtained scallops of commercial size (100 mm length)
after 18 mo of cage culture in Malaga, only a small proportion of
the spat culture in O Grove reached commercial size when main-
tained in cages. In O Grove, the scallops detached from collectors
in November and cultivated in cages had grown sufficiently (>60
mm) by the following September to be ear-hung; taking into ac-
count that in the latter months of cage culture (until May 2000)
growth was very slow, it may be advisable to begin ear-hanging
culture in September.
The culture of P. nni.xiiiuis in suspension is complicated and is
influenced by many factors, not all of which have been thoroughly
studied or are well understood. In addition to the factors usually
considered, such as food availability, temperature, stocking den-
sity, and depth, other factors such as handling frequency and foul-
ing of both shells and cages, and the interactions among these also
should be taken into account. This species lives recessed in the
sediment and under natural conditions is not usually heavily
fouled. However, when grown in suspension the animals are heav-
ily fouled, apparently more than other epifaunal pectinids. such as
A. operciilaris and Chlamys viiria. The effect of fouling should be
studied because, as well as the negative effects (i.e., competition
and reduction of water flow), there may be positive effects, such as
the prevention or reduction of mobility within the cages, thereby
reducing biting and malformations.
ACKNOWLEDGMENTS
This study was financed by Fondo Europeo para el Desarrollo
Regional (FEDER) grant IFD I997-020I-C03-01 in Galicia. by
the Junta de Andalucia in Fuengirola and by the Institute Espeiiol
de Oceanografia (lEO) in both areas. The CTD data for O Grove
were provided by the Centro de Control da Calidade do Medio
Marino. We also thank Recursos Mariuos Grovenses (REMAGRO)
for the loan of facilities, and for the help provided for Carmen
Presas. Carmen Vazquez. Juan Fernandez. Teresa Garcia, Lourdes
Fernandez, and the fishermen from Los Boliches.
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Jcniriial uj Skclljhh Research. Vol. 22. No. 3. 655-660. 2003.
INTRASPECIFIC GENETIC VARIATION IN MITOCHONDRIAL 16S RIBOSOMAL GENE OF
ZHIKONG SCALLOP CHLAMYS FARRERI
XIAOYU KONG,' ZINIU YU,' "* YAJUN LIU.' AND LINLIN CHEN'
College of Fisheries. Oeeaii University of China. Qingdiio 266003. Peoples Repuhlie of China: 'Haskin
Shellfish Research Laboratory. Institute of Marine and Coastal Sciences. Rnigers University.
Port Norris. New Jersey 08349
ABSTHACT A 592 base-pair fragment of the mitochondrial 16S ribosomal gene in 47 Zhikong scallop {Clilamys faireri) specimens
was sequenced to examine its intraspecific genetic variation and geographic structure. These samples were collected from six
populations [four from China, and one each from South Korea (SK) and Japan] across its range. Thirty-one nucleotide positions were
found variable, and twenty-three haplotypes were detected in all samples, which showed that more I6S rDNA variation existed in C.
farreii when compared with several other oyster species. Analysis at the intrapopulation level showed that the SK sample had the
richest sequence diversity. However, an analysis of haplotype frequency distribution and analysis of molecular variance indicated that
little geographic structure was present among all samples, and an absolute majority (99.659^) of the genetic variation was distributed
within populations, suggesting that the populations in this study may belong to a single panmictic unit. A relatively smaller distribution
range and various currents may account f(.>r sufficient gene flow am<ing these populations for this benthic species.
KEY WORDS: Clilaniy.s fiirreri. genetic variation, geographic structure I6S rRNA gene, scallop
INTRODUCTION
Distributed mainly along the coast of northern China. North
Korea. South Kotea (SK). and Japan (JP). the Zhikong scallop.
Chtamys farreri. has been one of the major species of the shellfish
aquaculture industry on the northern coast of China for several
decades (Qi 1984. Wang et al. 1993). This species comprises about
75 to 80% of the total production of scallops in China (other
species include bay scallop Art^oju'cten inadians. Japanese scallop
Patinopecteit yessoensis. and Chlaniys udhilis). In 1996. some
780.000 metric tons of the scallop C. farreri was produced in
China (Guo el al. 1999). In recent years, however, scallop culture
has been haunted by a high moilality problem. Mortality rates
varied from 20% to as high as 80% at a variety of areas in late
summer and early fall before harvest season. It is believed that the
problem was caused by a combination of overcrowding, high sum-
mer temperature, and deteriorating water quality. Additionally, one
more possible reason for the problem is that, to some extent, the
scallop stock may be deteriorating genetically. This is possible
because, although collected from the wild, most of the scallop seed
was primarily collected from restricted waters (Changdao. Yantai
district. Shandong province) where the wild population is believed
to have originated from hatchery production from the late 1970s to
the early 1980s (Guo et al. 1999). For this reason, refreshing the
scallop stock by introducing new .stocks from other populations
outside the coast of north China was considered. Consequently,
investigation and evaluation of its stock structure throughout its
geographic range are required.
It has been proposed that benthic marine species with pelagic
larvae have population genetic structures reflecting the dispersal
capacity of larvae. Most of them are thought to have little genetic
structure. Considerable work has been conducted to lest the hy-
pothesis on many species, including oysters (Buroker 1983, Reeb
& Avise 1990. Karl & Avise 1992. Small & Chapman 1997).
mussels (Karakousis & Skibinski 1992. Gelleret al. 1993). scallop
(Wilbur et al. 1997). gasptropods (Hoskin 1997. Kyle & Boulding
2000). abalone (Huang 2000). and many other invertebrates with
planktonic larvae (Palumbi c^ Wilson 1990. Amdt & Smith 1998.
Schizas et al.l999). Mitochondrial DNA sequences (including 16S
rDNA) were used for many of these studies. In the scallop C.
farreri. this hypothesis has almost never been checked. The in-
traspecific genetic variation of C. farreri was investigated using
allozyme frequency data with five populations along the northern
coast of China (Zhang & Zhang 1997). but the question of whether
significant geographic structure exists was not directly answered.
Moreover, as the results of other researchers have shown, when
different genetic systems are used in the same species, the resulting
population genetic structures may differ (Karl & Avise 1992). So,
this also led to our interest in examining the genetic structure of the
population using mitochondrial gene sequence data with samples
from the geographic range of the species.
MATERIALS AND METHODS
Sampling and Polymerase Chain Reaction .Amplifications
Scallop C. farreri samples were collected by scuba divers or
were dredged from Daliang (DL), Changdao, Yantai (YT),
Rongcheng (RC), and Qingdao (QD) along the northern coast of
China. Samples from SK and JP were obtained through commer-
*Corresponding author. E-mail: carlzyu@hsrl.rulgers.edu
Figure 1. A map of the sampling area for the Zhikong scallop C.
farreri. The numbers I. 2, ,3, 4, 5, and 6 represent, respectively, DL,
YT, RC, QU, SK, and JP.
655
656
Kong et al.
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Genetic Variation of 16S rDNA in Zhikong Scallop
657
TABLE 2.
The sequence indices of intrapopulatlun level of 16S rRNA gene in Zhikong scallop C.farreri.
DL
YT
RC
QD
SK
JP
No. polymorphic sites 3 8 7 7 14 6
No. haplotypes 4 7 6 6 8 6
Haplotype diversity 0.750 1.00(1 0.893 0.893 1. 000 0.929
Nucleotide diversity 0.0017.5 0.0()4IS 0.00296 0.00326 0.00.591 0.00314
Average No. nucleotide differences 1.0357 2.4762 1.7500 1.9286 3.5000 1. 857 1
cial catch practice in Gunsan. SK. and in Kana/awa. JP. respec-
tively (Fig. I ).
Total DNA was extracted from adductor muscle tissue using a
standard phenol/chlorofomi method (Sambrook et al. 1989). 16S
fragments of the 16S rDNA were amplified using a pair of uni-
versal primers: 16sar-L/16sbr-H: 5'-CGCCTGTTTATCAAAAA-
CAT-375'-CCGGTCTGAACTCAGATCACGT-3' (Palumbi
1991).
Amplification of the products was performed using a PTC- 100
thermal cycler (MJ Research. USA). The 100-(xL amplification
reaction contained the following: 2.0 mM MgCU; 200 (xM each
dNTP; 0.2 (jlM each primer: 2.3 (jlL of template DNA: 2.5 units of
Taq polymerase (Sangon. China) with supplied buffer. For all
amplifications, a hot-start polymerase chain reaction (PCR) was
initiated by the addition of polymerase and primers following an
initial 2-min denaturization at 80°C. The PCR cycling profile was
as follows: 35 cycles at 94°C for 45 sec. 50°C for 1 min. and at
72°C for 1 min: with a final extension at 72°C for 7 min.
C»
X 165
33
3^0
JO
/
M*-
82.
fas ,f-o
260
176
160 —
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Gc-
i' l' '
3J7
3^
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69
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Figure 2. \ median network diagram elucidating the relationship of
the 23 haplotypes of the 16S rRNA gene in the /Jiikong scallop ('.
farreri. Haplotype codes are dellned in Table 1.
Sequencing
PCR products were purified using UNIQ-5 Column PCR Prod-
uct Purification Kit (Sangon, China), were ligated into pMD18-T
vector by following the instruction of the Takara DNA Ligation
Kit. version 2 (Takara, Japan), and were used to transform com-
petent JM109 Escherichia coli cells using standard protocols. Re-
combinant colonies were identified by blue-white screening. In-
serts of the correct size were detected via restriction enzyme di-
gestion by EcoRl and HindUX. Vector DNA containing the desired
insert was further purified using the Pharmacia EasyPrep Kit
(Sweden). Sequencing was performed for both strands of every
sample on an ABI PRISM 377XL DNA Sequencer using ABI
PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit
with AmpHTaq DNA Polymerase, FS (Perkin Elmer).
DaKi Analysis
Initially. 16S sequences from individual specimens were
aligned with CLUSTAL X (Thompson et al. 1997) and then were
assigned a haplotype on the basis of discrete combinations of
nucleotide sites. Population-specific haplotypic diversity (Nei &
Tajima 1981) and nucleotide diversity (Nei 1987) were quantified,
respectively. A haplotype median network diagram describing the
relationships of observed haplotypes was built using Network
3.1.1.1 (Riihl 1999. Bandelt et al. 1999). All populations were
nested into three groups (China. SK. and JP), respectively, and
then analysis of molecular variance was conducted to determine
the genetic differentiation of the populations with ARLEQUIN
(Schneider et al. 1997). Haplotype frequency distributions also
were analyzed by exact test (Raymond & Rousset 1995) with the
same software. Genetic differentiation at different hierarchical lev-
els was assessed by <t> statistics (Weir & Cockerham 1984). A
pairwise matrix of interpopulation nucleotide divergences (Nei
1987) among all populations was calculated, and it was used to
construct an unweighted pair group method with arithmetic mean
(UPGMA) phenogram employing the NEIGHBOR program, and
the tree was drawn using the drawgr.-v.m program in the PHYLIP
package (version 3.56C: Felsenstein 1989).
RESULTS
Sequences of the 592-base pair 16S rRNA gene of all 47 speci-
mens were obtained, and 3 1 nucleotide positions were found vari-
able. Twenty-three haplotypes were detected among all samples,
and their frequencies are shown in Table 1 . Haplotype A and B
were the most common ones and were observed in all populations.
Their frequencies were 29.8% and 17.0%, respectively. Haplotype
C was shared by three populations (DL, YT, and RC), haplotype M
was present only in the SK and JP populations, and haplotype N
was observed in both the SK and RC piipulations. All others were
658
Kong et al.
TABLE 3.
Analysis of molecular variance of 16S rRNA gene haplotypcs in Zhikong scallop C.farreri.
Source of Variation
Df
% Total
* Statistics
P Value
Among groups
Among samples within groups
Within samples
3
41
2.00
0.00 (-1.67)
99.67
<i)cT: 0.0202
<I)sc: -0.0171
<f st: 0.0035
0.195
0.337
0.532
Df. dearee of freedom.
population-specific haplotypes. Haplotypic diversity, nucleotide
diversity, and other population-specific diversity indices are pre-
sented in Table 2. For all numbers. SK had the greatest value, with
YT the next greatest (also the greatest among the four populations
from China), and the DL population has the lowest frequency.
Construction of a median network based on nucleotide divergences
among the haplotypes detected in this study indicated that most
haplotypes were closely related, with the dominant haplotype (A)
as the center of radiation (Fig. 2). Many adjacent haplotypes dif-
fered from each other by one nucleotide, and some haplotypes
were two mutational steps removed from A.
The analysis of haplotype frequency distribution showed that
there were no significant differences among all samples iP =
0.92.5). and that there were none between any pairs of samples. The
analysis of the pailitioning of the haplotype diversity indicated that
an absolute majority (99.65%) of the genetic variation was distrib-
uted within populations (Table 3). No variance was attributable to
differentiation among populations within groups, and <29c could
be attributed to xariation among different geographic regions,
which was not significant (P = 0.124).
Interpopulation nucleotide divergences are presented in Table
4. While the greatest value of pairwise divergence among popu-
lations was observed between the SK and YT populations, the
smallest value was present between the DL and JP populations,
which are the most distant population pair in this study. The
UPMGA tree generated from these divergence data is shown in
Fig. 3. The tree clustered six populations included in this analysis
into one major branch that separated DL population from the other
five, and connected YT and SK first with other populations joining
sequentially in a semi-random pattern.
DISCUSSION
As Table I and 2 show. 16S rRNA gene sequences of C. fa ire li
presented a reasonable degree of variation, although it is usually a
low variation region in mitochondrial genome (Hixson & Brown
1986). The richest variation in YT population among the four
populations in China supported the fact that YT has been the center
of wild resources of this species in China. Considering this, it is
difficult to confirm that the YT stock is deteriorating genetically
based upon our results. With 14 polymorphic sites and 8 haplo-
types. the SK population showed the richest variation in all six
populations, which may support the idea of stock introduction
from SK. Zhang et al. (1997) studied genetic variation with five
populations from China in this species using allozyme starch gel
electrophoresis. Four of these populations were from the same
locations as ours (DL. YT. RC. and QD). Their results indicated
that YT samples showed highest heterozygosity (observed and
expected) among these populations, which matched our result from
the 16S sequence data.
When compared with oysters, the sequences of the scallop C.
farreri 16S rRNA gene seems more variable. In a 400-nucleotide
(nt) I6S rDNA sequence of Crassostrea gigas and Crassostrea
sikiimea. Banks et al. (1993) did not detect any polymorphism
from nine individuals. O'Foighil et al. (1995) found that both five
C. gigas and five Crassostrea ariakensis exhibited no variation in
a 443-nt 16S rDNA sequence, and only two nucleotide sites
showed polymorphisms among 20 specimens in Crassostrea vir-
ginica (five haplotypes). Similarly, just one. one. two. and two
haplotypes. respectively, were observed in the same 443-nt I6S
rDNA sequence for 8 C. gigas. 10 Crassostrea plicatiila. 7 C.
ariakensis. and 10 Cras.wstrea taliemvlianeiisis individuals in a
recent study (Yu et al. 2003). While a longer sequence (592 nt) of
scallop C. farreri was examined in this study than that of oysters
(400 or 443 nt) may account for part of the reason, species differ-
ence at the degree of sequence variation should be the greater part
of the explanation.
Although some degree of variation was observed in populations
of C. farreri. the results of a statistical analysis of 16S rDNA
haplotypes indicated that little geographic structure was present
among populations and regions. This lack of significant divergence
implied that there has been sufficient gene flow among these popu-
lations. It was supported by estimated rates of migration (Njn)
ranging from 15 to infinity per generation among populations.
Further evidence of the lack of population divergence is also in-
dicated by Table I and Fig. 2, which show that the most common
haplotypes. A and B. were shared by all populations, and that
TABLE 4.
Sequence divergences at interpopulation level of six populations of Zhikong scallop C.farreri.
DL
VT
RC
QD
SK
JP
DL
YT
RC
QD
SK
JP
0
0.00274
0.00250
0.00259
0.00385
0.00243
0
0.00348
0.00356
0.00477
0.00350
0
0.00332
0.00440
0.00326
0.00449
0
0.00327
0.00443
Genetic Variation of 16S rDNA in Zhikong Scallop
659
53
61
41
100
Figure 3. An 1"P(;NL\ tret' constructed Hith the interpopulation se-
quence divergences of 16S rRNA gene from six populations in the
Zhikong scallop C farreri. Numbers on the branches indicate the per-
centage of 200 bootstrap replications.
almost all other haplotypes were one or two steps removed from
haplotype A. This star phylogeny is generally viewed as a possible
sign of an expanding population.
Usually, much less or no genetic structure was found in many
marine species with longer periods of planktonic larvae than those
with short or no period of planktonic larvae (Hellberg 1996,
Hoskin 1997. Amdt & Smith 1998. Kyle & Boulding 2000). with
some exceptions. In molluscs, Wilbur et al. (1997) compared a
Siberian population with Japanese populations in the Japanese
scallop Patbuipecten yessoensis using PCR-restriction fragment
length polymorphism (RFLP) analysis of three mitochondrial cod-
ing regions. They found that there was not a significant variation
of restriction sites between these two regions, but haplotype fre-
quency distributions were found to be significantly different be-
tween regions. The Sea of Japan and the prevailing current patterns
between these populations were considered to be obstacles to gene
flow.
However, using the RFLP analysis of mitochondrial (mt) I6S
rDNA. Small and Chapman (1997) did not detect any significant
population structure for C. virginica sampling along the Atlantic
coast to the Gulf of Mexico, where a distinct genetic break along
the Florida coast was found by Reeb and Avise (1990) with RFLP
analysis of the whole mtDNA. and by Karl and Avise (1992) with
RFLPs of a few anonymous single-copy nuclear DNA sequences.
So. it was concluded that the restriction sites within I6S rRNA
gene were more conserved than other sites and other regions in the
mt genome of the species. We conducted restriction analyses of all
23 haplotypes with the set of 1 1 restriction endonucleases used in
the study of Wilbur et al. (1997) through WEBCUTTER 2.0
(http://www.firstmarket.com/cutter/cut2.html) and found that 4 of
16 restriction sites were among the .31 variable sites. The propor-
tion of polymorphic sites among all sites detected by these en-
zymes was 5.88% (4 of 68 sites), and that of sites detected by
sequencing was 5.247f (31 of 592 sites). It seems that restriction
sites are not necessarily more conserved than other sites w ithin this
gene in Zhikong scallop C. farreri.
Compared with the American oyster, C. farreri has a smaller
geographic range of distribution. Although collected from China,
SK. and JP. the sampling region actually is not very large. The
distances among the four samples next to each other in China are
<250 km, the RC and SK populations are around 400 km away
from each other, and only the JP is about 800 km from the SK
population. With the species having pelagic larvae for a few weeks
and with the various currents flowing among these regions, gene
flow among these populations seems not to be significantly
blocked in this relatively smaller range. It is difficult to deny that
these populations in this study belong to a single panmictic unit.
Some stocks from SK have been introduced into a few com-
mercial hatcheries in China. Initial efforts to produce seeds and
grow-out have been made. Faster growth rates and slightly lowers
mortality rates than those from China were observed (Dr. L. Song,
pers. Comni.). However, caution should be taken when compari-
sons are made between these data and those from other stocks in
China, because it is very possible that the SK stock and their
resultant seeds received better care or grew in better culture con-
ditions (e.g.. better nursery of seeds, lower density for the culture,
and better culture area provided) during the culture period. This
was normally the case for introduced stocks. Therefore, we may
not be able to say that the SK stock is not included in the same
panmictic unit.
Because of the nature of maternal inheritance and the ability to
reveal sequence variation to the highest degree, mitochondrial
DNA sequencing usually may not require as large a sample size as
other mitochondrial/nuclear techniques [e.g., PCR-RFLP, PCR-
SSCP, allozynie, microsatellites, and amplified fragment length
polymorphism ( AFLP)] of sequence variation do (a sample size as
small as 10 can also clearly detect genetic structure with nuclear
markers, as shown in Huang et al. 2000). However, since more
haplotypes were detected in this study when compared with oys-
ters, it may give a clearer picture of the stock structure in C. farreri
if the sampling size is somewhat larger.
ACKNOWLEDGMENTS
This work was financially supported by the 973 and 863 Pro-
grams (grants GI9990I2008 and 2()02AA626020) of the Ministry
of Science and Technology of China. The authors are grateful to
Dr. Patrick M. Gaffney for his critical review and help with con-
struction of the median network diagram.
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J<ninial of Slu-Ufish Research. VdI. 22. No. 3. 661-665. 2UU3.
PERKINSUS SP. INFFXTION RISK FOR MANILA CLAMS, VENERUPIS PHILIPPINARUM (A.
ADAMS AND REEVE, 1850) ON THE PACIFIC COAST OF NORTH AND CENTRAL AMERICA
RALPH A. ELSTON,'* CHRISTOPHER F. DUNCAN," THEODORE R. MEYERS,' AND
KIMBERLY S. REECE^
^AquaTedmics, PO Box 6H7. Carhborg, Washington 98324; -Maryland Department of Natural
Resources. Cooperative O.xford Laboratory. 904 S. Morris Street, Oxford. Maryland 21654: Alaska
Department of Fish and Game. PO Box 25526. Juneau. Alaska 99802: "^Virginia Institute of Marine
Science. PO Box 1346. College of William and Mary. Gloucester Point. Virginia 23062
ABSTRACT Manila clams ( Vcnenipis philippinciniin. A. Adams and Reeve 1850) are an impciruinl aquacultiire species on the west
coast of North America and are also cultured in Europe. Asia, and other locations. Clains cultured on the west coast of North America
are free of Perkinsus sp. infections, while clams from certain Asian and European sources are infected. Infection in Korean Manila
clams is reportedly associated with high morbidity and mortality. We evaluated the health status of readily accessible Manila clam
juveniles from Korea that were proposed for importation into Mexican waters where they would increase in size, and then be shipped
into the United States, either to market destinations or to receiving waters. The examination of the clams was performed as a
preliminary assessment for a producer considering the importation of Korean Manila clams. We report finding a high prevalence of
a Perkinsus sp. causing significant tissue damage in juvenile Korean Manila clams. Parasite taxonomic verification was made using
a ^enus-Perkinsiis SSUrRNA gene-specific DNA probe for in situ hybridizafion. The use of this probe is validated and reported for
the first time. As a result of this finding, no importation of this clam stock took place. It is urgently important to make widely know n
the risk of the spread of this disease into the clam stocks of the west coast of North and Central Amenca to prevent such an introduction.
In addition, we report new information regarding the prevalence and intensity of this disease in juvenile clams available tor export, as
well as pathologic features of the disease.
KEY WORDS: Veiieriipis iTcipes) plulippiiuiruiii. juvenile clam infection. Perkinsus sp.. DNA probe, in sitit hybridization
INTRODUCTION
Manila clams {Venerupis philippinarum, A. Adams and Reeve
1850) are an important aquaculture species on the west coast of
North America. More than 7 million pounds of littleneck clams,
predominantly V. philippinarwn. were produced in Washington.
California, and Oregon in 2000 (Pacific Coast Shellfish Growers
Association 2003), and additional production occurs in British
Columbia, Canada. Although Alaska produces native littleneck
clams, Protothaca staniiiiea (Conrad 1837). Manila clams are ex-
otic, and importation for aquaculture purposes is prohibited.
Venenipis philippinarum is also an important aquaculture species
in Europe and Asia, and is infected with Perkinsus sp. on both
continents. Specifically, Perkinsus atlanticus occurs in Europe
(Navas et al. 1992), a P. atlanlicus-Wkt parasite occurs in Japan
(Hamaguchi et al. 1998). and Perkinsus sp. occurs in Korea (Choi
& Park 1997) and China (Liang et al. 2001). Consistent with the
close homology noted between DNA sequences at several P. at-
lanticus and Perkinsus olscni loci by diverse investigators. Murrell
et al. (2002) assert these parasitic species to be synonymous, with
taxonomic priority to the P. olseni name.
In contrast, clams from the west coast of North America are
free of Perkinsus sp. infections. A survey of Manila clam health
and conditions on the west coast of North America (Pacific Shell-
fish Institute 2001), and the required examination of over 3000
clams for health certifications from 1991 to 2002, showed no
evidence of Perkinsus sp. infection. Moreover, such infections
have not been reported elsewhere on the west coast during routine
annual examinations and frequent health examinations of brood
stocks and seed clams since 1985. In addition, Perkinsus sp. in-
*Corresponding author. E-mail: aquatech@olypen.com
fection has not been reported in the native littleneck clam P. sta-
minea or any other bivalve species from the west coasts of North
or Central America.
Manila clams may be imported as a live market product from
Korea. Japan, or other Asian countries into North America. In
1998. we evaluated the health status of juvenile Manila clatns from
Korea that had been proposed for importation into Mexican waters,
where they would gain size before shipment to the United States,
either to market destinations or to receiving waters for further
grow out. The examination of clams was performed as a prelimi-
nary assessment for a producer considering the importation of
Korean Manila clams. We report the finding of a high prevalence
of a Perkinsus sp. causing significant tissue damage in juvenile
Korean Manila clams.
As a result of this finding, no importation of this clam stock
took place. It is urgently important to make widely known the risk
of the spread of this disease to west coast North American clam
stocks to prevent the introduction of this debilitating and lethal
clam parasite. In addition, we report here new information regard-
ing the prevalence and intensity of this disease in juvenile clams
that are available for export, as well as pathologic features of the
disease. Finally, a novel genus-Perkinsus DNA probe for /;; situ
hybridization (ISH) assays on histologic samples is described.
Taxonomic references to the Manila clam (also commonly re-
ferred to as the Japanese littleneck clam) in the scientific literature
are particularly confusing. We have designated the species as V.
philippinarum in accordance with the Committee on Scientific and
Vernacular Names of Molluscs within the Council of Systematic
Malacologists, Ainerican Malacological Union (American Fisher-
ies Society 1998). The common name Manila clam is also found in
the literature, apparently in reference to the same species, associ-
ated with scientific designations of Tapes philippinarum. Rudi-
lapes philippinarum. Tapes semidecussatus, and Tapes japonica.
661
662
Elston et al.
TABLE 1.
ISH assay results with genus-Perkinsus SSUrRNA probe, Perksp700DIG.
± Probe
Sample
Parasite
Host
Sample
Hybridization
Source
Reference
HiTkinstis sp.
V- philippimiruiii
q8-SH14-5
R. A. ElsKin
this article
Perkiii.siis sp.
V. pliilippinuruin
98051504-2
Y. Maeno
Maeno et al. 1999
P. atUinticus
R. clecussauis
685a
C. A/evedn
A/evedo 1989
P. olseni
H. laevigala
ST389-35
C. L. Goggin
Goggin etal. 1989
P. chesapeciki
M. arenaria
CHBRMa-14
C. Dungan
Dungan et al. 2002
P. cmdrewsi
M. balthica
MB3a2
F. G. Kern
Coss et al. 2001
P. marimis
C. virginica
221. 556-15
K. S. Reece
Maclan et al. 1950
P. meditcrnineii.s
0. edidis
08 and 016
A. Villalba
Casas et al. in press
PerkiiLSiis sp.
C. piicificiis
CH02882
C. L. Goggin
Goggin et al. 1989
P. (/»,i;u'i«//
P. yessoensis
6492-A5
-
S. M. Bower
Blackbourneet al. 1998
Haplospcruliimj nchoni
C. virginicii
201. 239
-
E. Biirreson
Haskin etal. 1966
H. costiilc
C. virginicci
196. 774
-
E. Biirreson
Couch 1967
haplosporidian-like sp.
P. platycews
90-568J
-
S. M. Bower
Bower & Meyer 2002
Heinatodiniiiiu sp.
C. sapidus
98-513
-
J. D. Shields
Shields 1994
Hemcitoclinhim sp.
N. uoncgitHs
990427Nnor- 1
-
G. Stent! ford
Field & Appleton 1995
MATERIALS AND METHODS
A total of 64 Manila clams [16-32 nini shell length (SL)l from
Inchon Bay, South Korea, were clinically examined in February
1998 and were fixed whole in Davidson's shellfish fixative (Shaw
& Battle 1937). These tissues were processed for routine histologic
examination.
A representative tissue section containing parasites was evalu-
ated by ISH. The genus-Perkinsus DNA probe was designed to
specifically target SSU rRNA sequences of Perkinsus species by
aligning the available SSU rRNA gene sequences, while not hy-
bridizing to the sequences of closely related parasite taxa including
dinoflagellates and apicomplexans. An SSU rRNA gene sequence
is not available for Perkinsus cju<;wacii. The resulting probe
Perksp700DIG (5'-CGCACAGTTAAGTRCGTGRGCACG-3')
was 5' end-labeled with digoxigenin (Sigma-Genosys, The Wood-
lands, TX). ISH assays were performed as previously described
(Stokes & Burreson 1995. Stokes & Burreson 2001). except that
125 [j-g/mL pronase was used for permeabli/.ation. instead of pro-
teinase K. for a 30-min digestion, and a probe concentration of 7
ng/p.1 was used for hybridization. The probe was tested on an array
of Perkinsus sp. -infected, paraffin-embedded tissues (Table 1 ).
including Perkinsus marinus in Crassostrea virginicii. P. atlanti-
cus in Rudimpes decussatus. P. olseni in Haliotis laevigata. Per-
kinsus andrewsi in Macoma balthica. Perkinsus sp. in Vereurupis
philippiiuirum from Japan. Perkinsus chesapeaki in Mya arenaria.
Perkinsus mediterraneus n. sp. in Ostrea edulis (Casas et al. in
press), Perkinsus sp. in Chama pacificus. and P. qugwadi in Pa-
linopecten yessoensis. Probe specificity was validated by testing
tissue sections of the blue crab Callinectes sapidus. which was
infected with the parasitic dinoflagellate Henialiuliniuiii sp.
(Shields 1994), Hematodiiiiuin sp. -infected Norway lobster A'c/)/;-
rops norvegicus (Field & Appleton 1995). Haplosp<nidum nel-
.w);;/-infected and Haplosporidunt cfwffl/f-infected C. virginica
oysters, and spot prawn Pandalus platyceros. infected by an un-
described haplosporidian-like protozoan parasite (Bower & Meyer
2002). Replicate sections of nonspecific ISH assay signal controls
of each sample were tested identically, except that they received
hybridization buffer without probe during the overnight hybridiza-
tion step.
RESULTS
Histologic Evaluation of Infected Clams
The prevalence of juvenile clams infected with the presumptive
Perkinsus sp.. was 59 of 64 (92%), based on histologic examina-
tion. The protozoa were systemically distributed in a variety of
organs, most typically in subepithelial areas of the gills, and fre-
Fifjure 1. Gill tissue of a juvenile Korean Manila clam infected with
Perkinsus sp. (arrows). Note the dense cellularity ( hemocytosis ) in the
vicinity ol the parasites. Bar, Id (ini, H&E.
Fijjure 2. Higher magnitkation of a cyst (tf Perkinsus sp. trophozoites
in the gill tissue of the Manila clam (arrow). Bar, 10 nm, H&F:.
P/iR/'./Nsus sp. IN Manila Clam Juveniles
663
i>Jf^
Figure i. Cyst of I'erkiiisus sp. trophozoites encapsulated by a
hemocytc within the jjill of a Manila clam. Bar, l(( |un\. H&E.
qiiciitiy III the mantle and labial palps. Parasites were ot'len asso-
ciated with tissue hemocytDsis (Fig. I) and occurred as single or
multiple trophozoites (Fig. 2). In severe infections, the parasites
were more abundantly distributed in the tissues, including the vas-
cular sinuses around the digestive diverticula. Broad areas of the
subepithelial connective tissues were composed of solid masses of
parasite cysts in the most severe infections. In many cases, the
parasites were contained within a thin-walled cyst formed by one
to several host cells (Fig. 3). Such encapsulations contained up to
10 protozoan cells and associated heniocytosis. The parasites were
often characterized by the presence of an eccentric vacuole (Fig. I
and ?i}. characteristic of Perkinsiis sp. trophozoites.
Confirmation of Perkinsus sp. by ISH
The genus-Perkinsiis SSUrRNA gene probe PerkspVOODIG
demonstrated strong hybridization to Perkinsus sp. cells in all of
the tissue sections, except those of P. qiigwadi infecting P. yes-
soeiisis (Table I and Fig. 4A-I). No hybridization to parasite cells
of other genera was observed. ISH of parasite cells in tissue sec-
tions of infected Korean Manila clams with this genus-Perkinsiis
probe confirmed the genus level affiliation of the parasites in our
sample of juvenile Korean Manila clams (Fig. 5).
DISCUSSION
We report the confirmation by ISH assays and histology of
Perkinsus sp. infections in Manila clam seed proposed for the
introduction into Mexican waters and the subsequent transport to
growout sites on the Pacific coast of the United States. This is the
first confirmation by a molecular diagnostic probe of Perkinsus sp.
infection of Korean Manila clams As a result of these findings, the
plan for importation of these clams was rejected by the shellfish
producer, and no Korean seed clams were imported to the west
coasts of Mexico or the United States. However, the ready avail-
-'■--vT";'''-
Uiy/'-ti^::.
■•.*V;
■?!»
v.v
2 »..
4G*
..v»
Figure 4. Tissues sections of host tissues reacted with the yenus-ZVrAmvHv probe Perksp7IIO b> ISH. Positi^ely stained l'crkinsu\ sp, parasites
are shown by arrows. (Al P. marinus in C. rirgiuica intestine (bar, 11) pni). (15) Perkinsus sp. in ('. paeijicus (bar. It) pnil. (Cl /'. atlanlieus in R.
deeussalus (bar, 10 pm). (D) P. olseni in //. laevigata gill and mantle (bar, 25 pm). (F) Perkinsus sp. in M. halthiea (bar 25 pml. (F) Perkinsus
sp, in Japanese V. pliilippiuarum (bar, II) pml. (G) P. ehesapeaki in M. tireuarin (bar It) pm), (Hi A", mediterraneus n. sp. in (). edulis (bar, 10 pm).
(Il /'. qugwadi in P. yessoensis (no hybridization observedl (bar. 25 pml.
664
Elston et al.
Figure 5. Tissue section ol Korean Muuilu ilani rtaitud witli gtnus-
Perkiiistis probe Perksp7()0DlG by ISH. Positively stained Perkiiisus
sp. parasites arc shown by arrows. Bar, Id pni.
ability ot such infected seed clams from Korean or Japanese pro-
ducers requires vigilance to ensure that no such importations take
place into areas thai are free of the pathogen, such as the west
coasts of North and Central America. Reports of lethal Perkiihsiis
sp. infections in European and eastern Asian Manila clams from
latitudes as far north as that of northern Oregon, confirm the high
likelihood that such infections, if introduced, could persist and be
transmitted, with damaging results to both wild and cultured clam
stocks along the Pacific coasts of North and Central America.
This study demonstrated that infection prevalence in seed
clams ranging from 16 to 32 mm SL can be nearly ]Q09r and that
high parasite intensities cause significant histologic damage to the
organs of infected clams, particularly the gills.
Choi and Park (1997) studied five species of Korean clams for
infections by Perkinsus sp. using Ray's fluid thioglycollate me-
dium (Ray 1966) and found infected Manila clams along the south
coast of Korea. While no infection occuned in clams of <15 mm
SL. nearly 100% infection prevalence occurred in clams of >20
mm SL. Park et al. ( 1999) reported mass mortality of Manila clams
along the west and south coasts of Korea over a period of several
years, which was associated with Perkinsus sp. infections. They
reported 100% infection prevalence in 142 clams from Komsoe
Bay on the west coast of Korea with moderately severe mean
parasite intensities of 2.87 based on the infection intensity scale of
Choi et al. ( 1989). A negative con-elation was found between the
intensity of Perkinsus sp. infections and the clam condition index,
while clam size was positively correlated with infection intensity.
Maeno et al. ( 1999) reported Perkinsus sp. parasites in Manila
clams from an inner bay of the western part of Japan in April 1998.
using genus-P(^/A/;i,v//.v-specific antibodies. These authors con-
cluded that the parasites were Perkinsus sp. based on a positive
reaction with both single and clustered trophozoites. Hamaguchi et
al. (1998) have reported the first detection of Perkinsus sp. in
Japanese Manila clams. Anecdotal information that we received
from the Korean supplier of the seed clams and their Japanese
customers indicated that the Manila clam seed had been trans-
ported from the Korean source to Japan for at least 20 y with no
unusual mortalities or loss of growth reported. This anecdotal re-
port and the multiple reports of the Perkinsus sp. parasite occuiring
about 1997 or 1998 in Japan and Korea suggest that it could have
been a new introduction to the Korean clams, as well as the Japa-
nese clams, at about this time.
Manila clams and other bivalve species from Europe reportedly
have been infected with Perkinsus sp., as follows: P. atlantieus
from the Mediterranean coast of Spain (region of the Ebro Delta,
Tarragona, Spain) infected R. plulippinarum (Sagrista et al. 1996);
Manila clams from the Lagoon of Venice in northeast Italy in-
fected with a Perkinsus sp. (DaRos et al. 1998): and P. atlantieus
infected the carpet shell clam (R. decussatus) from European lo-
cations (Ordas et al. 2000). Villalba et al. (2000) reported a sig-
nificant conelation between the SL of/?, deeussalus and P. atlan-
tieus infection intensity. No clams of <20 mm SL were infected,
and the highest seasonal parasite intensities occurred in spring and
late summer to early autumn.
The relationship of Perkinsus sp. in European waters to the
Perkinsus sp. found in Korea and Japan is unknown at this time.
Nonetheless, this and other studies cited in this report indicate the
presence of this damaging parasite in Korean and Japanese Manila
clams, confirmed first in this study by histology and then defini-
tively by the Perkinsus sp. -specific probe presented for the first
time in this article. This knowledge can be used to prevent the
iniintentional introduction of this parasite to west coast of North
and Central America. We urge that the science presented in this
article be applied by shellfish growers, and by natural resource and
conservation managers to prevent such a damaging introduction.
ACKNOWLEDGMENTS
N. A. Stokes, K. L. Hudson, K. Apakupakul, and R. M. Ham-
ilton provided expert technical assistance in the performance of
ISH assays. Perkinsus sp. -infected mollusc histologic samples
were generously provided by C. Azevedo. S. M. Bower, E. M.
Burreson, C. L. Goggin, F. G. Kern, and Y. Maeno. Parasitic
dinoflagellate-infected crustacean tissue samples were provided by
J. D. Shields and G. D. Stentiford. This work was supported in part
by National Oceanic and Atmospheric Administration (NOAA)
Sea Grant funding of project NA86RG0037 to CFD. This work is
also a result of research sponsored in part by NOAA Office of Sea
Grant, U.S. Department of Commerce, under grant No.
NA96RG0025 to the Virginia Graduate Marine Science Consor-
tium and the Virginia Sea Grant College Program, and under grant
No. NA016RG2207 to the Maryland Graduate Marine Science
Consortium and the Maryland Sea Grant College Program. The
U.S. Government is authorized to produce and distribute reprints
for governmental puiposes. notwithstanding any copyright nota-
tion that may appear hereon. VIMS contribution #2575.
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life cycle of the parasite, Perkinsus atlanticus (Apicomplexa). on the
clam, Riiditapes philippinaruiii. in the Mediterranean. Sci. Mar. Bare.
60:283-288.
Shaw, B. L. & H. I. Battle. 1957. The gross and microscopic anatomy of
the digestive tract of the oyster, Crassostrea virginiea (Gmelin). Can.
./. Zool. 35:325-347.
Shields. J. D. 1994. The parasitic dinonagellates of marine crustaceans.
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Stokes, N. A. & E. M. Burreson. 1995. A sensitive and specific DNA probe
for the oyster pathogen Haplosporidiiim nelsoni. J. Eiikaryot. Micro-
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Stokes, N. A. & E. M. Burreson. 2001, Differential diagnosis of mixed
Haplosporidiuni costale and Haplosporidiiim nelsoni infections in the
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Villalba, A., S. M. Casas, M. J. Carballal & C. Lopez. 2000. Effects of
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JcKriwI oj Shellfish Resenrch. Vol. 22, No. 3, 667-674. 2003.
TOLERANCE AND RESPONSE OF MANILA CLAMS, VENERUPIS PHIUPPINARVM
(A. ADAMS and REEVE, 1850) TO LOW SALINITY
RALPH A. ELSTON.'* DANIEL P. CHENEY." BRIAN F. MACDONALD,' AND
ANDREW D. SUHRBIER-
^Pacific Slu'lljisli liiMiiiite. PO Box 687. Cailsborg. Washiniito)i 98324: 'Pacific Shellfish Institute.
120 State Ave. N.E., No. 142. Olympia. Wa.shington 98501-0600; ^Washington Department of Fish and
Wildlife. 600 Capitol Wa\ North. Ohmpiu. Washington 98504-3200
ABSTR.ACT Til delerniine under what conditions winter mortalities of the Manila clam (Veiierupis philippiiianim. A. Adams and
Reeve. 1850) might be the result of excessive exposure to low salinities, a series of experiments was conducted. Clams were exposed
to various concentrations of salinity to determine their physiologic lower limit of tolerance to salinity concentration, the duration they
could withstand lethal or marginal low salinities through the mechanism of shell closure, and diagnostic structural changes in tissues
indicative of low salinity exposure. Salinities of ^ 10 parts per thousand (ppt) were not tolerated in long-term exposures of 13 groups
of clams. This lethal low salinity was also confirmed by the exposure of clams with a resection of a portion of shell. A salinity of 12.5
ppt was considered marginal, and various proportions of the different populations were able to tolerate this salinity, while no significant
mortality occurred at ^15 ppt. Clams could withstand lethal low salinities of 5 ppt and 10 ppt for between 6 and 8 days, but all
populations exposed to lethal low salinities for 14 days and then placed at high ambient salinity (-31 ppt) showed a high cumulative
mortality. Clams may not die until .several days after exposure to lethal low salinity followed by placement in a recovery tank at their
normally tolerated high salinity. We found no significant difference in the responses of several groups of clams to the marginal salinity
of 12.5 ppt when exposed at temperatures of 6°C, 12°C, and 18°C. Histologic examination showed that the following sequential
changes occurred in the digestive gland in clams exposed to 10 and 12.5 ppt for between 2 and 14 days: loss of granulation of the
digestive tubular absorptive cells: swelling of these cells and occlusion of the tubular lumina: and finally the shedding of necrotic
tubular epithelium into the digestive gland tubular lumina.
KEY WORDS: Venerupis {Tapes) iiluHi^pmanim. low salinity tolerance, Manila clam
INTRODUCTION
Over 3000 tons of Manila clams (Venenipis philippinaniin. A.
Adams and Reeve. 1850), valued at over $22 million (US dollars),
were produced on the west coast of the United States in 2000
(Pacific Coast Shellfish Growers Association 2003). Most produc-
tion occurs in Washington, but clams are also produced in Cali-
fornia. Oregon, and British Columbia. Canada. An unfilled domes-
tic and overseas demand is driving attempts to increase the pro-
duction of this clam. In addition, a significant Manila clam seed
production industry has developed, with production facilities in
Washington. Oregon. California, and Hawaii. Native littleneck
clams iProtothaca stainiiiea. Conrad 1837) are also produced in
Washington and Alaska, but production is limited due to a short
shelf life, a lower price for the producer, and the preference of
consumers for the Manila clam.
One constraint to the growth of the Manila clam industry on the
west coast of the United States is the occurrence of sporadic mor-
tality and poor growth due to unknown causes. With some excep-
tions, mortalities are usually reported between November and
March. Freezing damage may be a factor in Manila clam mortali-
ties during the winter (Bower 1992). No highly pathogenic infec-
tious diseases of Manila clams are known to occur on the west
coast of North America (Elston et al. 2003).
Clams may be reared in locations near freshwater streams or
rivers with occasional high outflows in winter. We therefore sus-
pected that at least some of the reported winter mortality events
could be the result of exposure to salinities below the physiologic
tolerance of the clam or from exposures to low salinity of duration
longer than that for which clams can maintain shell closure. The
clams burrow into the substrate, and clam deaths may only be
observed at some time after the mortalitv event. A sur\'ev of the
*Corresponding author. E-mail: aquatech (sHilypen.com
literature revealed limited information on the low-salinity toler-
ance of juvenile and adult Manila clams (Kim et al. 2001. Kurata
2000. Numaguchi 1998). Therefore, we conducted the studies re-
ported here (1) to determine the lowest salinity at which Manila
clams from several populations could survive over an extended
time period, (2) to determine the duration of exposure that adult
and juvenile clams can survive when exposed to lethal and mar-
ginal low salinities. (3) to determine the relationship of water
temperature to clam survival at a marginal low salinity, and (4) to
determine histologic changes that could be used to diagnose the
exposure of clams to low salinity.
Taxonomic references to the Manila clam (also commonly re-
ferred to as the Japanese littleneck clam) in the scientific literature
are particularly confusing. We have designated it Venenipis phil-
ippiiuirum in accordance with the Committee on Scientific and
Vernacular Names of Molluscs of the Council of Systematic Ma-
lacologists. American Malacological Union (American Fisheries
Society 1998). The common name of Manila clam is also found in
the literature, and. apparently in reference to the same species, the
clam is associated with scientific designations of Tapes philippi-
naniin. Riiditapes philippiiuinn. Tapes semidecussatus. and, less
recently, as Tapes japoniea.
MATERIALS AND METHODS
Apparatus for Low Salinity Exposure Assessment
We conducted initial salinity exposure experiments in static
aerated aquaria over a 3-day period. In these experiments, the
clams were not fed. However, the majority of experimental evalu-
ations of low-salinity effects were made in two flowing seawater
systems that we designed and built for this purpose, and that were
operated at a commercial shellfish hatchery facility in Quilcene,
Washington, where the ambient salinity ranged from 29 parts per
thousand (ppt) to 32 ppt. These systems provided several tlovv-
667
668
Elston et al.
through tanks capable of holding large numbers of test animals at
constant levels of reduced salinities (up to four treatments simul-
taneously) for extended periods of lime. This system was later
modified to allow for multiple temperature treatments across a
single salinity.
In the initial configuration of this system, sand-filtered seawa-
ter and unchlorinated fresh water were pumped into separate head-
tanks (-200 L) the levels of which were kept constant by stand-
pipes and float valves. A coiled length of vinyl tubing was used as
a heat exchanger for the fresh water line to help equalize the
temperature of the two water sources. Each of these two tanks fed
a manifold fitted with four outlets restricted by variously sized
orifices that flowed into mixing tanks. Each mixing tank (-2()L)
flowed in turn into a treatment tank (~40L) where the test animals
were held. Altering the sizes of each orifice feeding into the mix-
ing tanks thereby controlled the salinity of the water within each
treatment tank. A continuous flow of mixed algal food species
provided by the commercial hatchery production system was in-
troduced into the saline headtank at a rate sufficient to allow ex-
cess food in all treatments. Airstones were used in each mixing
tank to ensure the adequate mixing of the two water sources and to
maintain dissolved oxygen saturation prior to the water being al-
lowed to enter the treatment tanks. Salinity loggers and periodic
manual checks were used to track treatment salinity and tempera-
ture levels. Overall, the actual salinities varied no more that ±1.0
ppt from target salinities based on logger checks and spot manual
checks, with the exception of two instances where actual salinity
was 2.2 ppt higher than the target or 1 .2 ppt lower than the target.
Flow apertures were checked, and any salinity deviations ap-
proaching or greater than I ppt from target were corrected at least
twice per week during experiments.
Method of Testing Clams
Initially, we tried to maintain shell opening by inserting
wooden wedges between the \alves. but we abandoned this
method because the clams usually rejected the wedges, although
the method has been used successfully in other species such as
Mytilus edulis (Shumway 1977). Alternatively in the initial experi-
ment, we cut a wedge-shaped opening in the shell of clams (Fig. I )
to force exposure of tissues to the exposure salinities. While this
method appeared to have some utility for determining physiologic
tolerance to low salinity levels, it was time-consuming and success
required extensive operator practice to avoid damage to soft tis-
sues. Therefore, we abandoned this method in favor of long-term
exposures (4 wk) to evaluate physiologic adaptation or lack thereof
to various salinity concentrations.
Clams were obtained from locations in Washington. California,
and Hawaii, and were placed in trays in the flowing seawater tanks
without sand. Water temperatures were maintained at a constant
level within experiments but varied between experiments from
10.0 to I4.0°C, except for the trial in which we tested the effect of
temperature on tolerance to a marginal low salinity concentration.
Clams were removed from the experiments and considered
dead when their shells gaped and they were unresponsive to prob-
ing. Alternatively, clams that were counted as alive when returned
to recovery tanks at ambient salinity had active shell adduction and
extension of the siphons.
Two experiments were conducted to observe the histologic ef-
fects of low-salinity exposures on the gills and digestive gland of
clams, two organs that in preliminary experiments appeared sen-
sitive to low-salinity exposure. Adult clams [40-50 mm shell
length (SL)] were used in both experiments, which lasted 9 days
and 14 days, respectively, with samples collected at the initiation
of the study and at 2, 4, 7, 9, and 14 days of exposure to 10 ppt and
12.5 ppt, along with control clams at ambient (-30 ppt) salinity.
We compared the 4-wk mortality rate for the groups containing
two replicates (Table I) using the probability density function for
a binomial distribution (Samuels & Witmer 1999). Analysis of
variance was not used as it did not meet the requirements for
normality and sample size. The probability density function for the
binomial distribution is
,/'(.v)
/)'(l -/J)\.v = 0,l,2.
Fiyiiru 1. Manila ilani with portion ol shill resected to isolate the
physiologic response to low salinities.
where n is the number of trials and /) is the probability of "suc-
cess."" Applied to the data in Table I. there are only two outcomes
for each clam, dead or alive, with dead clams corresponding to the
success of a trial. For example, to test whether there is any sig-
nificant mortality difference between two locations (e.g., Stoney
Point-Willapa and Little Skookum Creek in the 10 ppt treatment),
the probability of success for the Stoney Point-Willapa site is
estimated as p,, = 13/30 = 0.4333. Our null hypothesis (/y„) is p
= /)||. And the alternative hypothesis (//,) is p > p^. The P value
for observing .v > 2 1 is P(X a 2 1 ) = 0.0002, where X is a random
variable following a binomial distribution with 30 trials and the
probability of success for each trial is 0.4333. Since the P value for
testing Hf, vs //, is so small, we reject the null hypothesis (at least
at a 5% level of significance). This means that there exists a
significant difference in the mortality rate at the two different
locations. Where applicable, results also were compared using t-
tests and determination of 95% confidence inter\ als for sequential
time points in serially sampled experiments.
RESULTS
Measurement of Physiological Tolerance to Low Salinity hy I'arliat
Shell Removal
Figure 2 shows the results when clams with a shell wedge
removed were exposed to six salinity levels for 3 days in a .static
aquarium at 10.5°C, followed by a 19-day recovery period in flow-
ing seawater. There was no mortality in either of the control groups
(shell cut or intact clams). Although the salinity treatment at 20 ppt
Manila Clam Low Salinity Tollrance
669
TABLE 1.
Cumulative mortality of Manila clams held in desi)>nated salinity concentrations for 4 \vk^
Replicates
H
Salinity
Concentrations Testedt
(%)
Clam Source?
25ppt
20 ppt
17.5
ppt
15 ppt
12.5 ppt
10 ppt
Oakland Bav
30
3
(1
0
97
Chelsea ground
30
0
0
7
67
Chelsea yearling
30
0
0
0
67
California nur.serv seed clams
30
20.0
13.3
66.7
100
Survivors of low-salinity event
30
6.7
1 0.0
16.7
90.0
Hawaii nursery seed clams
50
0
6
s:
100
Little Skookum Creek
2
15
0
3 ± 4.7
27 ± 9.4
73 ± 4.7
Little Skookum Slough
2
15
0
0
7±0
80 ± 6.7
Chelsea Seafarms Creek
2
15
0
0
27 ± 9.4
80 ±12.7
Chelsea Seafarms Beach-N
2
15
3%
±4.7
0
30 ± 4.7
93±4.1
Stoney Point-WiUapa
2
15
7%
±0
7 + 0
13 ±5.0
43 ± 2.0
Oakland Bay
2
15
0
0
80 ±18.9
97 ± 4.7
Thorndyke Bay
2
15
0
17±4.7
7 ±9.4
97 ± 4.7
* The results arc Ihe cuniulatn e mortality rate afier 4 u k exposure at the indicated salinity. Further mortality was observed in many groups within 7 days
after the 4-wk exposure, when the clams were placed in an ambient (30 ppt) salinity tank. Replicated treatment results are expressed as the a\'erage ± SD.
t Target salinity concentrations are shown.
+ All clam sources are from Washington except as noted and. except as noted, arc adult clams with 40 to 50 mm SL.
resulted in a cumulative mortality rate of 20Vf and the salinity
treatment at 1.5 ppt resulted in a 10% loss, these losses were
attributed to nontreatment effects. It was clear from this experi-
ment that 5 ppt and 10 ppt were lethal low salinities from which a
3-day exposure resulted in lOOVr mortality within 17 days postex-
posure. Most of the clanis in these two groups died between .3 and
6 days after removal from the static salinity treatment tanks.
Physiological Tolerance to Low Salinity Measure by Exposures of 4
Week Duration
Table 1 shows that there was relatively little mortality in any
group tested at 15 ppt or higher, in comparison with control group
mortalities, and no significant differences were found between the
tested groups at these higher salinities. At 12.5 ppt salinity, the
binomial distribution test showed a significant difference at the 5%
level between Stoney Point-Willapa and Little Skookum Creek,
and between Chelsea SeaFaniis Beach-N and Oakland Bay. In
terms of significant difference at the 5% level, the mortality of
clams at 1 2.5 ppt can be split into three groups that are different
100%
80%
60%
40%
_ 20%
0% »
—♦—5 5 ppt
_,_10 4ppt
_4_15 9ppt
_,_20 5ppt
^i(-25 1 ppt
-^ Shell cut •
30 1 ppt
-Shell intact -30,1 ppt
^t
12 14 17 18 19
Days post exposure (+) in recovery tank
after 3 day exposure to indicated salinity
Figure 2. Experiment 1 results showing the respon.se of Thorndyke
Bay adult clams with shell wedge removed to six salinity concentra-
tions in a 3-day static tank exposure in = 10 clams per group; 41 ± 1.9
mm mean SL; test temperature 10 to H C).
from each other: ( 1 ) Thorndyke Bay. Little Skookum Slough, and
Stoney Point Willapa: (2) Little Skookum Creek. Chelsea Sea-
Farms Creek, and Chelsea SeaFamisBeach-N; and (3) Oakland
Bay. At 10 ppt salinity, there is a significant difference (5% level)
in salinity between Stoney Point-Willapa and Little Skookum
Creek. Stoney Point-Willapa is significantly different from the rest
of the group. At the 57c level of significance for the 10 ppt expo-
sures, the seven locations can be split into three groups, which are
significantly different from one another: ( 1 ) Stoney Point-Willapa;
(2) Little SkookuiTi Creek, Little Skookum Slough, and Chelsea
SeaFarms Creek; and (3) Chelsea SeaFamis Beach-N. Thorndyke
Bay, and Oakland Bay,
Overall. Table 1 shows that, of the 13 groups of clams, in all
but one group very few clams could survive a 4-wk exposure to 10
ppt salinity. However, the ability to survive at a salinity of 12.5 ppt
varied greatly between groups of clams. Unlike Oakland Bay
clams, adult clams from Thorndyke Bay (Fig. 3) were tolerant to
salinity of 12.5 ppt. The highest mortality rate at 15 ppt was 17%
and apparently was due either to factors other than salinity or to
within-group variation since the mortality rate of the same group
of clams (Thorndyke Bay) at 12.5 ppt was only 7%. The clams
were removed from the .salinity exposures after 4 wk to ambient
salinity (-28 ppt) for I v\'k. during which additional mortality
occurred at 10 ppt and 12.5 ppt, bringing the mortality rate in all
10 ppt groups to nearly 100%, possibly as a result of partial ac-
climation to the lower salinity and the inability to readapt quickly
to the higher salinity. Only the 4-wk mortality rates are shown in
Table 1. The 4-wk exposure of the Thorndyke Bay clams demon-
strated that mortality reached nearly 100% after 3 wk of exposure
at a salinity of 10 ppt. Figure 3 also shows that differences in
mortality rate in clams from the Thorndyke Bay population at 12.5,
15.0. and 17.5 ppt were not statistically significant (P < 0.05),
We sampled two paired groups of clams (Chelsea Seafarms and
Little Skookum) from locations near intermittent high-flow
streams and paired locations distant from the freshwater sources.
Location near the creek outflows did not correspond to lower
mortality at 4 wk. and. in fact, the Little Skookum Creek clanis had
670
Elston et al.
ro
60%
5
3
40%
TO
-I
F
:^o%
D
o
0%
-♦-lOppt
_B_12 5ppt
r^-
_^15ppt
/
^i_17.5 ppt
/
y .^^
,, — ^
i^4^.^
1 00%
0
12 3 4 5
Four Weeks of Exposure and
One Week in Ambient Salinity Tank (30 ppt)
Figure 3. Cumulative mortality of Thorndyke Bay adult clams held at
four salinity levels for 4 \>k and a fifth week at ambient salinity (-30
ppt). Each treatment was replicated twice with 15 clams in each rep-
licate group (41 ± l.** mm SL; vertical bars are 95% confidence in-
tervals; test temperature 11-13'C).
TO
■e
o
>
E
O
1 3 5 7 9 11 13 15
Days Post Exposure
Figure 5. Thorndyke Bay adult clams exposed to the lethal low salinity
of 10 ppt for various durations show a graded mortality response that
is directly correlated with the duration of exposure and exhibit the
ability to survive longer than clams similarly exposed to the lethal low
salinity of 5 ppt (Fig. 5| (h = 15 clams per treatment and control at
ambient salinity: 41.7 ± 2.9 mm SL; test temperature 10-11 C).
a significantly higher mortality rate than did the Little Skookum
Slough clams.
Overall, the results establish that 12.5 ppt is a marginal salinity
for most populations of clams, with variable numbers of individu-
als able to survive this salinity concentration, and some popula-
tions, such as the Oakland Bay clams, contained very few clams
able to survive at 1 2.3 ppt.
Duration of Lethal and Marginal Salinities That Can Be Survived
Groups of 15 clams each |41.7 ± 2.9 mm average (±SD) SL]
from Thorndyke Bay were exposed to lethal low salinities in flow-
ing seawater of 5 ppt and 10 ppt for intervals ranging from 2 to 14
days and returns to ambient salinity (-28 ppt). The postexposure
mortality results (Figs. 4 and 5) showed that at 5 ppt a mortality
response occurred in all groups exposed for >8 days. The response
was graded, but there was nearly a 50% greater mortality rate at 12
days of exposure in comparison to 1 0 days of exposure. No mor-
tality was seen in clams exposed for 2. 4. or 6 days to 5 ppt. At 10
ppt a mortality response occurred in all groups exposed for SIO
days, and the response was graded from 10 to 14 days of exposure.
No mortality was seen in clams exposed for 2. 4. 6. or 8 days at 10
100%
ppt. The results demonstrate that a population containing a high
proportion of marginal ( 1 2.5 ppt) salinity-tolerant clams (Table 1 )
could withstand 5 ppt exposure for 6 days without losses, and
exposure to 10 ppt for 8 days without losses.
We conducted two additional experiments to evaluate the du-
ration of tolerance to lethal and marginal salinities. We compared
the response of two groups of adult clams to the lethal low salinity
concentration of 10 ppt followed by placement in an ambient sa-
linity (-28 ppt) tank. This included one population that showed a
low proportion of individuals with tolerance to marginal low sa-
linity (Oakland Bay clams) and another group of clams believed to
contain individuals with a high degree of tolerance to low salinity
(clams from Totten Inlet. Washington). Clams were exposed for
durations of betw een 1 and 1 4 days, and were observed over a total
of 4 wk, including the time in the low-salinity exposure tank and
the ambient salinity (-28 ppt) recovery tank (Figs. 6 and 7). These
results indicated that no mortality occurred in Totten Inlet clams
held for 7 days at 10 ppt but that an intermediate mortality rate of
40% occurred in Oakland Bay clams held for 7 days at 10 ppt.
Nearly 100% mortality occurred in both groups of clams held at 10
ppt for 14 days.
An experiment was conducted using juvenile clams to examine
their duration of tolerance to lethal (10 ppt) and marginal (12.5
ppt) low salinities, followed by a return to the ambient salinity.
The results showed that when exposed to salinity of 10 ppt (Fig. 8)
80%
60%
E
o
40%
20%
1 3 5
Figure 4. Thorndyke Bay adult clams exposed to the lethal low salinity
of 5 ppt for various durations show a graded mortality response that
is directly correlated with the duration of exposure (« = 15 clams per
treatment and control at ambient salinity: 41.7 ± 2.9 mm ,SL; test
temperature 10-11 C).
week 2 weeks 3 weeks 4 weeks
Weeks including exposure and recovery periods
Figure 6. Totten Inlet adult clams exposed to salinity of 10 ppt for
intervals ranging from 1 to 14 days followed by a recovery period in
ambient ( 30 ppt) salinity (;; = 2(1 clams per group: 46.1 ± 5.3 mm SL:
test temperature 10-11 C).
Manila Clam Low Salinity Tolerance
671
start 1 week 2 weeiss 3 weeks 4 weeks
Weeks including exposure and recovery periods
Figure 7. Oakland Bay adult clams txposed to salinity <>f 10 ppt for
intervals raufiiu); tnim 1 to 14 davs lolloHed In a recovery period in
anihient ( M) ppt) salinity (» = 20 clams per jjroup: 43.6 ± 3.'> mm SL;
test temperature 10-11 C(.
there was no significant difference in cumulative mortality be-
tween exposures for 1. 2. 4, and 7 days, which all had cumulative
mortality rates below 20%, but that a 14-day exposure resulted in
about a 90% cumulative mortality rate within 2 wk after placement
in the recovery tank. At 12.5 ppt, the mortality responses were
similar, but there was higher variability among replicate groups,
and the mortality rate was not statistically significantly different
between any of the treatment groups. The final mortality rates for
all treatments at 12.5 ppt were very similar to those resulting from
similar exposures of clams to salinities of 10 ppt. The high vari-
ability was not due to the smaller size clam replicate (5 mm SL
compared with 1."^ mm and 14 mm SL) being more sensitive to
12.5 ppt salinity, because one of the larger size groups showed
very high mortality compared with the other two groups.
Effect of Temperature on Tolerance to Marginal Salinity
We examined the effect of temperatures from 6 to 18 "C on the
survival of seed clams exposed to the marginal salinity of 1 2.5 ppt
(Fig. 9) There were no statistically significant differences in cu-
mulative mortality rate over the 4-wk exposure period. Mortality
was highest at 4 wk in the 6°C treatment, but this was due to a high
cumulative mortality rate in one of three replicate groups. The
maximum cumulative mortality rate was 20% over the 4-wk ex-
posure period.
Evaluation of Histological Changes at Lethal and Marginal Salinities
Although the histological observations were variable between
individuals, clear trends emerged that can be useful in the pre-
sumptive diagnosis of low salinity exposure. The following se-
quential changes occurred in the digestive glands of clams exposed
100% 1
-*- 1 Day J
.
f
80%
-B-2Day / •'
L
60%
-A— 4 I3ay /
40%
20%
-M-'Day /
-»— 14Day /
1
0%^
start 1 week 2 weeks 3 weeks 4 weeks
Time Including Exposure and Recovery
Figure S. Replicate groups of 20 juvenile clams, each exposed for
various durations to lethal salinity concentration of 10 ppt and a re-
covery period at ambient salinity i~M) pptl. Three seed replicate
groups measured 5 ± 1.5 mm, 1.' ± 1.,^ mm, and 14 ± 1.4 mm SI. each
(test temperature 10-11 C).
40%
r; 30%
20%
10%
-♦_Ambient(~28ppt)
_B_ 18 C
-*-12°C
_«_6°C 1
[
i 1
a — ""^ ^
F— '
L ^"T^ — i
0%_
0 12 3 4
Weeks of Exposure at 12 5 ppt
Figure 9. Evaluation of the effect of three temperatures on juvenile
clam survival at the marginal salinity of 12.5 ppt. Three clam groups:
group 1, two replicates of 22 clams each (5 ± 1.5 mm SL); group 2,
three replicates of 25 clams each ( 13 ± 1.3 mm SL); and group 3. three
replicates of 25 clams each (14 ± 1.4 mm SL).
to 10 ppt and 12.5 ppt: loss of granules in the absorptive cells
(presumably tVom lack of feeding) (Figs. 10 and 1 1 ); swelling of
the absorptive cells of the digestive gland so that the luminal
spaces of the terminal digestive tubules became occluded (Fig. 12);
and sloughing of absorptive cells of the digestive tubules into the
cell luinina where they appeared as necrotic cells and cellular
debris (Fig. 13). Loss of granules in the digestive tubular absorp-
tive cells occurred within 2 days of exposure to both 10 ppt and
12.5 ppt salinity. In the shorter experiment, the digestive tubule
absorptive cell luminu remained patent and normal-appearing
through 7 days of exposure to both 10 ppt and 12.5 ppt, but by 9
days of exposure the luminal spaces were occluded in all clams
exposed to 10 ppt salinity, and in about one half of the clams
exposed to 12.5 ppt salinity. At 9 days, mild evidence of digestive
absorptive cell sloughing was noted in a few clams exposed to 10
ppt only. In the 14-day experiment, using two different gioups of
:yS^'^'°^^
. i
I
■a... /Vk** -^^^ vGt^ - . ' .i'^ ^ . _ - -t
Figure 10. Histological section of normal Manila clam digestive gland
showing granules in absorptive cells and open digestive tubular lumina
(arrows). Bar, 2(1 pni, H&K.
672
Elston et al.
• './=)(;«•
w
' -J-** ' " •
o
'■J
' ^
^'- ^.^J
' .<:•
^^,
Figure 11. Histological section of normal Manila clam digestive gland
without granules in absorptive cells but with patent digestive tubular
lumina (arrows). Bar. 2(1 pni. H&E.
'^"' r- jp
«-»'
'A ®
.o
0
«^ • -•■'
©c
'#5
«'
J ^> ^.'N
/
»
•?.f
Figure 13. Histological section of digestive gland from Manila clam
exposed to 12.5 ppt salinity for 14 days showing shed necrotic absorp-
tive cells in the digestive tubular lumina (arrows). Bar, 20 |jm, H&E.
clams, about half of the clams exhibited swelling and luminal
occlusion of the digestive tubule absoiptive cells after 4 days of
exposure to both 10 and 12.5 ppt salinity. A similar proportion
showed these changes at 7 days as well as mild cell sloughing at
the lower salinity concentration. By 14 days of exposure, clams
from one group had uniformly sloughed and necrotic cells in the
tubular lumina. while in the other group about one half of indi-
viduals had occluded swollen luniina and one half had shed ne-
it;
;<3'
cv
-*ff
.rx.
... ^r,
1
1^
^'
'' .*/
.' 4 '
•'•'-r:^
3
^
7 ■■
(^^A
J
•^-'
r- 1 •.
iT ..
-
;.
> ! ^
fe-'.
•i-^
i-
'^.^^ .
: «
o ■
^0,
Q>
"©.
Figure 12. Histologk;il Mition of digestive gland from Manila clam
exposed to 10 ppt salinity for 4 days showing swelling of the digestive
tubular cells and occluded lumina (arrows). Bar, 20 (im, H&K.
erotic cells into tubular lumina at 10 ppt salinity. At salinity of 12.5
ppt and 14 days of exposure in both groups, about one half of the
clams had swollen occluded tubular lumina. and about one half had
sloughed necrotic cells in the tubular lumina.
The gills showed sloughing of epithelium in exposed clams,
but. due to the random planes of section typical in histological
preparations of the gill and the fact that at least mild epithelial loss
was observed in apparently normal clams, the gills were a less
reliable measure of low salinity exposure and were therefore not
systematically evaluated.
DISCUSSION
Other limited studies of juvenile or adult Manila clams have
shown similar low salinity tolerance. Kurata (2000) found that
when tested at a temperature of I °C. salinities below 15 ppt limited
the survival of Manila clams. Manila clam larvae have been found
to have an optimal salinity range of 20 to 30 ppt in hatchery studies
(Robinson & Breese 1984). Numaguchi (1998) reported that D-
hinge Manila clam larvae could survive for 72 h at 12 ppt but that
swimming was abnormal. Larvae did not survive at 8 ppt, but at
salinities of S|5.5 ppt survival and swimming behavior did not
differ from those of control larvae held at higher salinities. On the
other extreme of salinity, Shpigel and Fridman (1990) found that
Manila clams (referred to in the article as Tapes semidecussatiis)
grew well in a salinity of 41 ppt.
Lethal and Marginal Salinity Concentrations
These experiments clearly showed that salinity of slO ppt is a
lethal concentration for Manila clanis. at least for all of the popu-
lations of clams used in this study. Although we placed clams in a
recovery tank after the fourth week, we have reported only the
mortality that occurred after 4 wk at constant salinity. The fact that
additional mortality occurred in the fifth week may indicate at least
a partial adaptation to the lower salinity followed by an inability to
adapt quickly to the higher salinity experience in the fifth week.
Manila Clam Low Salinity Tolerance
673
This, in fact, represents likely environmental conditions that may
compound the mortality effect of long-term low-salinity exposure.
In fact, our other experiments showed that clams exposed to 10 ppt
for only 2 wk (Figs. 6 and 7) or less (Figs. 4 and 5) and then
removed to ambient high salinity (-28 ppt) succumbed at a high
rate. Therefore, the survival of clams held in low salinities (10 to
12.5 ppt for extended periods (e.g., 4 wk) may depend on the rate
at which they are reacclimated to higher salinities.
A salinity concentration of 12.3 ppt was shown to be a marginal
concentration in which the survival of clams over a 4-wk period
followed by 1 wk in a recovery tank was highly variable between
populations and even within replicated groups. The average per-
centage mortality rate at 12.5 ppt ranged from 7 to H2'/r. Standard
deviations were typically very high in replicated groups, indicating
the high variance within given populations for survival at 1 2.5 ppt.
The striking difference between two populations is demonstrated
by the Thorndyke Bay clams (tolerant to 12.5 ppt) and the Oakland
Bay clams (intolerant to 12.5 ppt). We were not able to statistically
link high survival at 12.5 ppt to specific locations wheie the clams
seemed likely to have adapted to low salinity due to freshwater
inflows near the clam beds. However, the Thorndyke Bay clams,
which had the greatest survival at prolonged exposure to 1 2.5 ppt.
are located near streams that may occasionally subject them to
low-salinity conditions. The results seem to indicate that most
clam populations contain some individuals with the ability to with-
stand 1 2.5 ppt for extended time periods. Clams from many of the
locations tested are the result of planting hatchery-produced juve-
nile clams and represent possibly mixed as well as undocumented
heritage, which may, in part, explain the variation in the propor-
tions of individuals that can survive at 12.5 ppt in various popu-
lations of Manila clams. However, if one had the objective of
selecting clams with resistance to low-salinity concentrations, it
would seem advisable to use a population such as the Thorndyke
Bay clams as a founder population, since it appears to be enriched
with individuals capable of withstanding a marginal salinity of
12.5 ppt.
Kim et al. (2001 ) reported that Manila clams recovered a typi-
cal endogenous circatidal rhythm of oxygen consumption when
placed in reduced salinity as low as 15 ppt but not at salinities
below 10 ppt. These authors concluded that Manila clams cannot
maintain normal metabolic activity below 15 ppt. They also re-
ported that all clams exposed to 5 ppt were dead within 7 days. The
authors apparently did not evaluate the metabolic activity of clams
at 12.5 ppt. The results of our study suggest that some clams may
be able to respire normally at 12.5 ppt, based on their long-term
survival at this salinity concentration.
Mechanism of Response lo Low-Saliiiily Concentration in
Manila Clams
In regard to low-salinity effects on Manila clams, our working
hypothesis was that resistance to low salinity consists of two fea-
tures: a physiological capacity of the tissues lo tolerate a particular
low salinity; and a survival response, consisting of the time for
which the clam can maintain a closed shell condition, thus exclud-
ing lethal low salinities, as has been shown to occur in other
bivalve species. For example. Shumway and Youngson (1979)
showed that shell closure oi Modiolus modiolus (Linnaeus 1758)
occurred at 60% seawater. Burrell (1977) hypothesized that the
greater resistance to low salinity in Merceiniria inercenaria (Lin-
naeus 1758) in comparison to Eastern oysters (Crassustrea vii-
ginica. Gemlin 1791) was due to the ability of clams to maintain
shell closure for a longer period of time. Clearly, the survival
response is complex, and depends both on aspects of the clam"s
metabolism (e.g., capacity for anaerobic metabolism) and possibly
on environmental factors that remain undelined, although, surpris-
ingly, temperature did not appear to affect the response, at least
within the parameters of the experiment conducted in this study.
Our results suggest that while Manila clams can successfully
resist lethal low salinities for a period of time, they are probably
constantly testing salinity either by active subtle valve opening or
seepage of low-.salinity seawater into the mantle cavity. The fact
that we observed swelling of digestive gland tubular absorptive
cells at 4 days of exposure to both 10 and 12.5 ppt salinity, com-
binations of exposure time and salinity that we also showed to be
clearly survivable. indicates that the clams do not totally exclude
lethal and marginal low salinities during exposure by shell closure,
although it is clear that they limit the exposure of their tissues to
the low salinities by shell closure.
Effect of Temperature on Tolerance to the Marginal Salinity of
12.5 ppt
We were not able to demonstrate any significant effect of tem-
perature on the tolerance of three groups of clams to the marginal
salinity of 12.5 ppt, even though the populations tested included
those that showed moderate to high mortality rates when exposed
to 12.5 ppt in earlier experiments. However, Cain ( 1973) reported
that survival was reduced in larval Rimgia cuneata at high tem-
perature-low salinity combinations. Laing and Child (1996)
showed that 6°C, the lowest temperature that we tested, was com-
patible with the growth of Manila clams, while Mann (1979)
showed that growth and spawning occurred at 18^C, the highest
temperature that we tested.
Structural Response of Tissues to Exposure to Lethal and Marginal
Low Salinities
These experiments provided data that can be used for the di-
agnosis or forensic evaluation of clams that are suspected of ex-
posure to lethal or marginal low salinities. Individual variation in
response is probably a result of the extent to which individuals
open and test the ambient salinity or, conversely, their ability to
remain tightly closed when they sen.se lethal or marginal salinities.
In either case, the results showed that relatively short-term expo-
sure (i.e., between 4 and 14 days) to salinity of 10 or 12.5 ppt
resulted in the swelling of the absorptive cells of the digestive
tubules, presumably from the absorption of hypoosmotic .seawater,
followed by the sloughing or loss of these cells into the digestive
tubular lumina.
Mortality Due to Lethal Low Salinity Exposure May Occur Over
Several Weeks
While our experiments on the duration of tolerance to 10 ppt,
a lethal low salinity, showed that only about 7 days of exposure
was required for a significant (approaching 100% in many cases)
mortality response, all of the experiments in which clams were
exposed and then placed in a recovery lank at ambient salinity
tended to show a sharp increase in obvious shell gaping (our cri-
teria of mortality) after placement in the recovery tank. The long-
term (4-wk) exposures, for example, showed that while some of
the clams were in the exposure tanks at 10 ppt they maintained
shell closure and appeared normal for up to 4 wk followed by a
674
Elston et al.
sharp rise in the mortality response during the fifth week when the
clams were placed in an ambient salinity recovery lank. The reason
for this is not known, but it may be due to the fact that the low
salinity stimulates a strong shell closure response that disappears
when the stimulus is removed (i.e.. the clams are placed in the
recovery tank). However, structural damage to tissues, as demon-
strated in this study, as well as stressful metabolic alterations (e.g..
depletion of free amino acids) are significant and. in fact, are
irreversible much earlier, although they are not manifested in the
obvious death of the clam at gaping, until it is returned to an
environment where the stimulus for protective shell closure is
removed. Whatever the basic underlying mechanism, the results
from this study show that the obvious mortality response to lethal
low-salinity exposure may be delayed, depending on the salinity
regimen and perhaps other factors. Therefore, as a practical appli-
cation of our results, it would be incorrect to assess a clam popu-
lation immediately after, for example, a 7-day exposure to salinity
of s 10 ppt and assume that clams showing tight shell closure were
unaffected. It would be more accurate to assess the clam popula-
tion several weeks later and. best of all. to additionally obtain
tissue samples for histological analysis during and at intervals after
the exposure to the low-salinity regimen.
Reasons for Control Mortality Losses
Control mortalities were generally <20'7f and often near zero.
However, a control mortality rate even approaching 20% is a vex-
ing issue and is one that will require further investigation to elu-
cidate the causes. Clearly, the clams were held in a somewhat
artificial environment in that a sedimentary substrate was not pro-
vided due to the necessity to evaluate their condition frequently
and could have contributed to the losses. We determined from an
extensive histological examination of the clam populations used in
this experiment and from other studies that there were no signifi-
cant known infectious diseases of Manila clams present.
ACKNOWLEDGMENTS
This work was supported in whole by a grant from the Salton-
stall-Kennedy Program, the National Marine Fisheries Service, the
U.S. Department of Commerce entitled "Manila Clam Mortality
and Health Evaluation" (grant number NA96FD0194). The assis-
tance of Mr. Kevin Ford in the administration of the grant is
gratefully acknowledged. The provision of space, water, and bi-
valve food supply to conduct these experiments by Taylor Re-
sources Company at their shellfish hatchery in Quilcene. Wash-
ington, made the study possible, and the cooperation of Mr. Paul
Taylor. Dr. Jonathan Davis, and Mr. Ed Jones in this endeavor is
appreciated. The assistance of Dr. Dane Wu with statistical analy-
sis is acknowledged. The efforts of Ms. Heidi Elston and Ms.
Kendra Kinnan in the maintenance of the clam tanks and the
enumeration of experimental clams is gratefully acknowledged.
LITERATURE CITED
American Fisheries Society. 1998. Common and scientific names of
aquatic invertebrates from the United States and Canada: mollusks. 2nd
ed. Special Publication 26.Bethesda. MD: American Fisheries Society.
Bower. S. M. 1992. Winter mortalities and histopathology in Japanese
littlenecks {Tapes philippiiuinim (A. Adams and Reeve. 1850) in Brit-
ish Columbia due to freezing temperatures. J. Shellfhli Res. 1 1:255-
26.1
Burrell, V. G. 1977. Mortalities of oysters and hard clams associated with
heavy runoff in the Santee River System. South Carolina in the spring
of 1975. Proc. Natl. Shellfish. As.wc. 67:35-13.
Cain. T. D. 1973. The combined effects of temperature and salinity on
embryos and larvae of the clam Rangia cuneala. Mar. Biol. 21:1-6.
Elston. R. A., C. Dungan. T. Meyers. & K. Reece. 2003. Perkinsus sp.
infection risk for manila clams. Venerupis philippinarum (A. Adams
and Reeve. 1850) on the Pacific Coast of North and Central America.
/ Shellfish Res. 22:661-665.
Kim. W. S.. T. H. Huh. S. H. Huh, & T. W. Lee. 2001 . Effects of salinity
on endogenous rhythm of the Manila clam, Ruditapes philippinarum
(Bivalvia: Veneridae). Mar. Biol. 138:157-162.
Kurata, M. 2000. Tolerance of the Japanese littleneck clam Riulilapes
phillipinarum to low salinity and dissolved oxygen at low temperatures,
(in Japanese). Sci. Rep. Hokkaido Fish. Exp. Stn. 58:17-2L
Laing. L & A. R. Child. 1996. Comparative tolerance of small juvenile
palourdes {Tapes descnssatus L.) and Manila clams {Tapes philippi-
narum Adams and Reeve) to low temperature. ./. £v/). Mar. Biol. Ecol.
195:267-285.
Mann, R. 1979. The effect of temperature on growth, physiology, and
gametogenesis in the Manila clam Tapes philippinarum (Adams &
Reeve, 1850). / £v/7. Mar. Biol. Ecol. 38:121-133.
Nuniaguchi. K. 1998. Preliminary experiments on the influence of water
temperature, salinity and air exposure on the mortality of Manila clam
larvae. Ac/i/acif/;. Int. 6:77-81.
Pacific Coast Shellfish Growers Association. 2003. West coast shellfish
production. Available at: www.pcsga.org. Olympia, WA: Pacific Coast
Shellfish Growers Association.
Robinson, A. M. & W. P. Breese. 1984. Gonadal developmeni and hatch-
ery rearing techniques for the Manila clam Tapes philippinarum (Ad-
ams and Reeve). / Shellfish Res. 4:161-163.
Samuels, M. L. & J. A. Witmer. 1999. Statistics for the life sciences.
Indianapolis, IN: Prentice Hall. 638 pp.
Shpigel, M, & R. Fridman. 1990. Propagation of the Manila clam {Tapes
semidecussatus) in the effluent of fish aquaculture ponds in Eilat, Is-
rael. Aquacidlure 90:1 13-122.
Shumway. S. E. 1977. Effect of salinity fiuciuation on the osmotic pressure
and Na*, Ca"*. and Mg"^* ion concentrations in the heniolymph of
bivalve mollusks. Mar. Biol. 41:153-177.
Shumway, S. E. & A. Youngson. 1979. The effects of fluctuating salinity
on the physiology of Modiolus demissus (Dillwyn). / Exp. Mar. Biol.
Ecol. 40:167-181.
.Itniniiil ,>f Shellfish Research. Vol. 22, No. 3. 675-6S0. 21)U3.
ON TWO NEW MACROSCOPIC INDEXES TO EVALUATE THE REPRODUCTIVE CYCLE OF
ENSIS MACHA (MOLINA, 1782)
OLGA L. ARACENA.* IRENE M. LEPEZ, JAVIER SANCHEZ, ANGELICA M. CARMONA,
LUCILA MEDINA, AND ALEJANDRA SAAVEDRA.
Deparkiiiicnti) dc Oceanoiinifia. L'nlvcrsickul clc Conccpcldn. Casilla 160-C. Concepciihi. Chile
ABSTRACT We describe the reproducllve cycle of razor clam Ensis maeha. during 1996 and 1997. in the Golfo de Arauco. Chile
(37°l4'S-73'2y'W) ba.sed in the variation of the monthly averages of common and new macroscopic and microscopic indexes and
scales. The common macroscopic indexes are weight ratio of soft tissues to valve weight or Somatic Valve Index and. weight ratio of
the soft tissues to total weight or Somatic Tissue Index. The new Macroscopic Index and scale are the quantification of the width of
the posterior foot or Morphometric Index and the quantification of digestive gland cover with ovary tissue plus the degree of ovary
development or mature morphometric scale. The microscopic indexes consist of the quantification of ripe gamete over the bulk of the
gonadic tissues, previously treated in formalin and without stain or gametic index and the same quantification over histologic
preparations or Gametic Histologic Index. The somatic valve index and somatic tissue index results are not adequate to describe the
reproductive cvcle of this specie; however, the Morphometric Index and Mature Morphometric Scale are very useful. These last two
methods, in addition to the Gametic Index and Gametic Histologic Index, show that the razor clam reproductive cycle, over these 2
years, is characterized by a resting period from March through July, and a progressive development of the gametes between August
and October. The spawn starts in November and is widespread until February. The index dropped abruptly during November 1 997,
showing a spawn rate more intensive than the previous year, which may be related to anomalous temperatures for the region. The
reproductive E. macha cycle described here, is similar to the Eiisis minor cycle in the Manfredonia Gulf, in Italy and the Eitsis siliqua
of Vilamoura on the southern coast of Portugal, but it is different to that observed for others authors in the Region X during 1 994, and
in the Golfo de Arauco and other locations of southern Chile during 1997.
KEY WORDS: gametic and gonadic indexes, reproductive cycle, razor clam. Ensis
INTRODUCTION
The razor clam Ensis macha (Molinu. 1782) is a bivalve shell-
fish distributed from Caldera to Magallanes in the Chilean coast
and up to San Mati'as Gulf in the Argentine coast. It is found in the
shallow sandy bottom living buried deeply in the sand favored by
its shape and a large foot.
Razor clam fishery became an important resource, exploited
mainly for exportation and includes activities of artisanal fisher-
man, mediators and canning enterprises that commercialize the
product mainly to Spain and Japan. The fishery started in south
Chile in 1988. with a catch reaching 1.741 tones from Region X
only. After that it reached a maximum landing of 8.617 tones in
1991 with contributions from the X and VIII Regions, but this year
the landings in Region X diminished while the landings from
Region VIII were increasing until 1993. Later on. the total land-
ings diminished to 6.1 15 tones in 1999 when 88.3% came from the
Region VIII, 10,1% from the Region X and the rest from the VII,
XII, and IV Regions which has been slowly incorporated but at
very small rate (Semapesca 2000). This reduction in the landings
led to additional fishery management and the support of the re-
search projects to cover the basic biology and fishery aspects of
this resource looking towards future aquaculture.
In this article, the background of the reproduction of this spe-
cies in the Golfo de Arauco, Region VIII, Chile, during 1996 and
1997 is given. It has been characterized through macroscopic and
microscopic methods in common use, plus two new methods not
described previously, here proposed as and easy application, vali-
dated with the histology of the female ovary. (Lepez et al, 1997a,
1997b, Aracenaet al. 1998a),
MATERIALS AND METHODS
Samples of about 60 adults of £. macha (S15 cm valve length)
were taken monthly during 1996 and 1997 for the study of scale
and macroscopic indexes. For microscopic indexes, monthly
samples of 20 adults were taken during 1 996, and monthly samples
of 30 adults during 1997. All samples taken during 1996 were
selected in the landing zone of Tubul, Golfo de Arauco (37°I4'W-
73 '29'W) and during 1997 were collected on board of artisanal
boats in the same Golfo,
E. macha is gonochoric. The males have white-grey gonads
with a homogeneous texture, while females show ovaries of a
white-cream color and granular texture, especially when they are
close to spawning. The sex was always corroborated with micro-
scopic observation of ovary tissue smears.
In the mature razor clams, as in many bivalves, the ovaries
extend dorsally over the digestive gland and the anterior adductor
muscle, showing a simple way to determine the sex and the de-
velopment stage. The ovaries invade the ventral zone of the vis-
ceral complex, the posterior part of the foot and form a cord in the
inner channel of the eatable foot.
To evaluate the mature stage, we applied the following mac-
roscopic indexes:
(i) Somatic Valve Index lS\T)
SVI--
DVV'S*IOO
DWV
vv here DWS is the dry weight of the soft body parts and DWV is
the dry weiahl of the valve.
(/(') Somatic Tissue Index iSTI)
STI = -
DVV.S*10()
*Corresponding author. E-mail: oaracena@udec.cl & ilepez@udec.cl
where DWS is the dry weight of the soft body parts and DWT is
the total dry weiuht.
675
676
Aracena et al.
(Hi) A new Macroscopic Maturity Scale (MMS)
Estimate the covering of the ovary over and around the diges-
tive gland, on a scale of 1 to 4:
1. Ovary covers 1/4 of the digestive gland.
2. Ovary covers 1/2 of the digestive gland.
3. Ovary covers 3/4 of the digestive gland.
4. Ovary covers totally the digestive gland.
Points were also assigned to the progressive development of the
gonadic tissue.
1. No development; no observable gonadic tissue or very
scarce and transparent.
2. hitermediate developmental stage; average bulk and granu-
late aspect.
3. Very developed; shows maximum bulk and granulate as-
pect.
Mixing these two scales, we obtain the mature stages from
Table I and the monthly average as follows:
1=1
Anterior adductor muscle
Left gill
2)mM5,
MMS =
where MMS, is the value of the scale assigned to the individual /.
and II is the total number of individuals counted every month. In
addition, the respective variance can be obtained from:
^(MMS, - MMS)-
VARiMMS)--
iiin- 1)
(iv) A new Morpliometric Index (MI)
The MI is obtained from the measureitient of the width of the
posterior area of the foot, under the visceral complex, showing the
degree of invasion of the gonad tissue. To this purpose, we made
a cut in the posterior area of the foot, as shown in Figure 1. to
measure its width. The monthly Ml is the average of the width
of each individual (MI/) on the total number of individual mea-
sured (/i)
.Ml.
Ml-
and its respective variance:
VAR (MI) :
^(M/, - A//)"
/)("- I)
TABLE 1.
Maturity stages of the razor clam: Macroscopic Maturity
Scale (MMS).
Covering
Developed
Foot
Cut to measure
foot width
Figure I. Ventral view of the soft body of £. madia showing the cut in
the posterior part of the foot where the width measure is made to
obtain the Morphometric Index.
Other tw(i microscopic indexes were;
)■; Gametic Index (Gl)
The Gl corresponds to the proportion of ripe gametes in rela-
tion to other kinds of cells in the bulk of the unstained ovary tissue
smeared on a slide.
The proportion of mature cells for each individual (Gl,) was
obtained by applying 10 times a microscope integration plate ot
10(_) points to the mass and quantifying;
Maturity Stage
(MMS)
Gl,
a,
iii
where, 01, is the proportion of ripe gametes for individual /. a, is
the number of ripe oocytes of the total elements in the individual
/. and III is the total number of quantified elements. With this, we
obtain the monthly average of Gl as follows:
Gl-
and its respective variance;
VAR(GI)-
I.O,.
2(G/,-G/)'
/!(/!- II
(>■/) Gametic Histologic Index (GHI)
The GHI is the same prior proportion but on histologic prepa-
rations stained with Gallegos embedding in Hystosec. previously
embedding in chloroform and mounted in Entellan (Con 1960,
modified by Delpi'n, personal communication). The proportion of
matures gametes for each individual (GHI,) was obtained applying
four times a integratiiin plate of 100 points on the histologic cuts
and quantifying;
GHI,
in
where GHI,. is the proportion of ripe gametes of each individual,
a, is the number of ripe oocytes of the total elements in the indi-
vidual / and m is the total number of quantified elements. With this
obtain the monthly average of GHI. following:
^GHI,
CHI--
and the variance is:
REPRODLicTivb Indexes for Ensis macha
677
(CHI-GHir
VAR[GHI] =
n{n- 1)
SVl ami STI. were used for the 1996 series on male and temale
razor clams plus GI only on female clams. MMS and MI were used
in the two series of samples, only on female. The GHl was used
onl\ ill 1997. for female clams.
All these inde.\es and scales were applied to individuals larger
than 15 cm \'al\e length, because Reyes et al. (1995) had previ-
ously defined the mean size of first sexual maturity al 14 cm salve
length for razor clam of the Region X.
To detect significant differences between the indexes and
scales and between males and females for 1996. a Kruskall-Wallis
test was carried out (P > 0.05 ). Same statistical test was carried out
for the 1997 informatiiin and also multiple comparisons test (Least
Significant Difference) to validate the macroscopic scale MMS
with the GHI index (Aracena el al. 1998b).
To explain the tendencies in the reproductive behavior of razor
clam described in this article, complementary oceanography infor-
mation available (temperature, salinity, density) for the Region
VIII was used (Salamanca. 1997).
RESULTS
From a total of 1.502 individuals analyzed during the years
1996 and 1997. the observed ratio of females to males was always
slightly smaller with 40.3% and 41.3%, respectively (Table 2).
although this difference was obviously never significant.
The Kruskall-Wallis test to detect differences between the in-
dexes and macroscopic scales was applied, among males and fe-
males for 1996. but it was not significant (P < 0.05). It was there-
fore possible to compare results of two years, even though in 1997
only females were considered.
Both SVI and STI showed similar tendencies during the year
1996 (Fig. 2c and d). being different to the other indexes and scales
applied in that same period. Maximum levels were observed from
March to May and low values from June through December. The
SVI oscillated between 34.34 and 50.16 with variance between
9.63 and 79.08. The STI o,scillated between 25.00 and 33.31. with
variance somewhat smaller, between 3.13 and 16.2S.
The GI is an index applied directly on gonadic tissues and it is
considered together with the GHI, that are the best to describe the
evolution of the gametes. From February to .lune the first index
stayed very low (Fig. 2e). with values between 0.02 and 0.04.
rising later on to a maximum of 0.21 in October and then down
again apparently through January or February of the next year. The
variances were always lower than 0.01 . except during December of
1995.
The MMS and the Ml followed similar lendencies in 1996 (Fig.
2a and b). The MMS fluctuated between 3.36 in March and 8.86
in October, with variances between 0.35 and 4.62. The MI have
TABLE 2.
Sexual proportions of the razor tlam: Golfo de Arauco.
Female
CXf )
Male
(%)
Total
(N)
1996
1947
40.3
41..^
.S.S.7
S34
(i6S
values from 3.98 mm (March) and 6.43 mm (September), with
variances between 0.39 and 1 .48.
During 1997 (Fig. 3), the GHI showed a very similar tendency
to the GI of the previous year, but the descent after the maximum
of October (0.253) it was very abrupt, falling to 0.017 in Decem-
ber, with equally smaller variances of 0.01. The same might be
said of the MMS and Ml of that year.
To validate the MMS. the average value of the GHI in 1997.
was calculated for each value of the scale, with its variance and
confidence intervals (Table 3) and then, the multiple comparisons
test (Table 4), indicated that stages 2, 3. and 4 of the MMS have
a very similar GHI and thus could only be considered one. The
stage 5 and 6 are very similar to each other but stage 7 is different.
In this way and for practical effects the MMS could be simplified
to the following three states:
1. 0 and '2 of the digestive gland is covered by the ovary;
volume and granulation are minimal or intermediate.
2. 1/2 of the digestive gland is covered by the ovary; voliuiie
intermediate and granulation are at their maximum.
3. The digestive gland is completely covered by the ovary;
volume and granulation are at tlieir maximum.
According to these data, the reproductive cycle of the razor
clam in the Golfo de Arauco. is characterized by a resting time
between March and June of every year (autumn and early winter),
a gradual increase of the maturity of the ovary starting in this last
month and reaching a maximum in October (spring), followed by
a single spawn period that begins in November and may finish in
December or be prolonged until February of the next year.
DISCUSSION AND CONCLUSION
Considering that the index GI and GHI. are the only ones that
represent the changes that happen at the level of the gametic tissue,
we may conclude that the index SVI and STI are not good to define
the reproductive cycle of this specie because through its evaluation
the gonadic tissue was not separated from the body tissue. In
experiments carried out by Sastry (1968). with Aequipeaen irra-
dians Lamarck, by Bayne (1975) with several species of bivalve
and for Lowe et al. (1982) with Mytilus ediilis L. among many
other authors, they showed that a nutritious transfer takes place
from the digestive gland into the ovary, or since nutritional tissue
from mantle toward the gametic tissue lowering the change of
volume and weight in the ovary when the total body weight is
considered.
However, the index MI and the simplified scale MMS are good
descriptors of the reproductive cycle of razor clam because they
are obtained from the observation and measurement of the ovary,
they are faster, easier and of low cost. Regarding the MMS. even
though the ovary cover on the digestive gland is a simple measure
to carry out and to be standardized, the gonadal volume and granu-
lation, are very subjective parameters. For this reason, between
these two indexes we recommend the MI index as a macro.scopic
index because is easy to obtain and quantify. The GI is a low cost
method as well, although not so simple or quick, but since it is a
direct quantification of the gonadic tissue it is more advisable than
the macroscopic index.
As observed in Figs. 2 and 3. the reproductive cycle of E.
macha during 1997 follows a similar tendency to the one observed
during 1996. but with a more marked fall between October and
December indicating a shorter, and more intense and synchronous
spawning than in the previous year. This may be associated with
salinity and temperature anomalies that affected the coastal areas
678
Aracena et al.
II -
10 -
9 -
8 -
7 -
4 -
3 -
2 -
a)
Die Jan Feb Mar AprMay Jun Jul Aug SepOct Nov
months
Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov
months
60
55
50
45 -;
>40
00
35 -
30
25
20
c)
d)
Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov
months
20
Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov
months
0.4
0.3
e)
0.2
0.1 -
0.0
-O.I
Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov
months
Figure 2. Monthly averages and variances of scale and maturity index of razor clam, Golfo de Arauco, 1995-1996. a, MMS = Macroscopic
Maturity Scale: b, Ml = Morphometric Index; c, SVI = SomaticA'alve Index; d. STI = Somatic/Total Weight Index; e, Gl = Gametic Index in
the bulk of gonadal tissue without stain.
of the Region VIII dtiring 1997 (Salamanca. 1997). Table 5 shows
that the spring-summer period 1997 was warmer and with less
saline water (on the average) than in a '"normal" year as the one
that was detected in 1981 for the same area (Llancamil 1982). The
winter conditions are similar among the two studies.
During the years 1996 and 1997 the razor clam of Golfo de
Arauco in the Region VIII, registered a cycle of annual maturity
with only one spawning period between November-December cor-
responding to the late spring early summer of the southern hemi-
spheie and this is very similar to the razor clams of the northern
hemisphere. Thus for Ensis minor (Chenu) of the Manfredonia
Gulf in Italy Casavola et al. (1994) describe a cycle of annual
maturity with a longer resting period between May and October.
The gametogenic activity starts in December and finish in March.
and spawning between these last months and April, spring of the
north hemisphere. Caspar & Monteiro ( 1998) point out that Ensis
siliqua (L.) from the south coast of Portugal has an annual gametic
cycle with an extended inactive period from June to October, the
gametogenesis activity starting in December with a maturity peak
ill March. Spawning starts in this last month and shows a maxi-
Reproductive Indexes for Ensis macha
679
TABIK 4.
Multipk' comparison tt-sls (LSD) lor seM'ii gonadit matiirily stages
(MMS) of £. macha compared with the Gametic Index (GHIl
during 1997.
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Die
Figure 3. Maturity index and scale of ra/.or clam. Golfo de Arauco.
1997. a, MMS = Macroscopic -Maturity Scale; b, Ml = Morfometric
Index: c. GHI = Gametic Histologic Index.
mum in April, which may be extended through May. They also add
that the males and females have a synchronous gonadal develop-
ment similar to E. iiuicha. In the Gormanstow bed of E. siliqiui in
Ireland. Fahy ( 1999) found that gonadal cycle are fairly similar to
the same species off the Portuguese coast, hut Ireland clams
spawns later in the year.
Reyes et al. (1995) found thai the largest evacuations of razor
clam gametes take place at the end of September. November.
February, and March in the Region X of Chile, which is in late
spring and throughout summer. This difference may be related to
the oceanographic conditions in the area because the fiords in the
Region X are very different to the Golfo de Arauco.
Urban (1996) describe an annual reproductive cycle with a
short spawning season in summer for E. macha. from Chile at 36'
S, very similar to our finding. However, Avellanal et al. (2002). in
a study of the reproductive cycle of £. macha in the south of Chile.
used a very different methodology consisting of the assigning of
six stages of gonadic organization to histologic preparations to
determine the reproductive cycle for this species at Tubul (Golfo
de Arauco) between November of 1996 and 1997. They found that
20% of the females presented ovaries partially spawned in Febru-
ary of 1997 and l()09f presented a partial spawning in March and
April. Between June and July, there was a quick recovery of the
ovaries and 40% of the samples presented partial spawning in
August, a percentage that increased to 100% in November and
December of the same year.
This apparent difference between the reproductive cycle de-
TABLE 3.
Statistics of GHI for each state of Macroscopic Maturity Scale
Maturity
Stage
(MMS)
2
3
4
5
6
7
T
0.40
0.37
0.00
0.00
0.00
3
0,40
0.9.5
0,011
0.00
0.00
4
0..^7
0.95
0.00
0.00
0.00
5
0.00
0.00
0.00
0.12
0.00
6
0.00
0.00
0.00
0.12
0.01
7
0.00
0,00
0.00
0.00
0.01
scribed by ,\vellanal et al. (2002) and this work, both on the same
population and period, probably would be due to different methods
for evaluating the state of development of the gametogenesis. In
both cases, a massive spawn between November and December of
1997 is described.
Other results by Avellanal et al. (2002) indicate that the spawn-
ing of Ensis macha in Corral (39°50'S-73''28'W) was similar to
thai one described by the same authors for Tubul. But. the cycle of
this species in Ancud (4r50'S; 73°47'W). was different because
the partial spawning started in January of 1997 and reached 100%
in April. June, and July. Later on a recovery of the ovary was
observed to reach 100% of mature individuals in December of that
year. These authors also found in Tubul and Corral a positive
conelation between the percentage of mature females and the chlo-
rophy 11 a and a negati\e correlation among the percentage spawn-
ing and the chlorophyll a. In Ancud, the percentage of mature
females was highest when the temperatures were increasing and
the spawning reached a maximum when the temperatures were
low. According to Avellanal et al. (2002). this relations points to
the important influence that temperature and the quantity of food
can have in the energy balance, transfer of nutrients and other
processes occurring during the gametogenesis.
Finally, we conclude that the quantification method of the stage
of maturity of the ovary of the razor clam, not described previ-
ously, which closely reflects the gametic cycle of Ensis macha are
MI and MMS. being easier, fast and of low cost. These two meth-
ods have also been used to define the reproductive cycle of Tage-
lus domlieii (Lamarck. 1818) (Lepez el al. 1997b) whose ovaries
are also diffuse in the visceral complex and may be adapted for
other similar species.
TABLE 5.
Comparative chart of oceanography parameters in Coliumo Bay
(average conditions).
(MMS) of razor
clam: Golfo de
.\rauc(i
.lanuary
-De
Lcmber 1997.
PlaceAVater Mass
Temperature
C
Salinity
xlO"^
GHI Averaged
GHI Variance
Confidence
Interval
Maturity
Scale (MMS)
Subantartic Water (SAW)
Subsurface Equatorial Water (SSEW)
>n.o
9^13
<34.3
>34.4
-)
0.063
0,0004
0.015
Coliumo Bay (Llancamil 1982)
3
0.07S
0.0010
0.008
Winter
12.07
32.76
4
0.078
0.0015
0.007
Spring
11.22
34.20
5
0.143
0.0075
0.019
Coliumo Bay (Salamanca 1997)
6
0,207
0.0031
0.016
Fall-Winter
12.57
33.44
7
0,262
0.0010
0.025
Spring-Summer
14.88
33.69
680
Aracena et al.
ACKNOWLEDGMENTS
We thank H. Moscoso for his help in fieldwork, M. Canales for
help in manuscript, and Dr. J. Stuardo for the review and sugges-
tions of an earlier version of this article. Financial suppon: Fondo
de Investigacion Pesquera FIP 95-20A and Fondo de Fomento al
Desarrollo CientiTico y Tecnologico. FONDEF D96/1095.
LITERATURE CITED
Aracena, O. L., A. Carmona & L. Medina. 19ySa. La navaja en la VIII
Region. Documento N°ldel Proyecto FONDEF D96/1095: Desarrollo
del cultivo de la navaja iEnsis macha) en la VIII Region. Universidad
de Concepcion-Instituto de Fomento Pesquero. 14 pp.
Aracena, O. L., A. Carmona, L. Medina & I. Lepez. 1998b. Ciclo repro-
ductive de Ensis macha (Molina, 1782) en base a indices gonadicos
macroscopicos y su validacion histologica. Resiimenes XVIII Congreso
de Ciencias del Mar, Iquique: 141 pp.
Avellanal, M. H.. E. Jaramillo, E. Clasing, P. Quijon & H Contreras. 2002.
Reproductive cycle of the bivalves Ensis macha (Molina, 1782) (So-
lenidae), Tagehis dombeii (Lamarck, 1818) (Solecurtidae) and Midinia
eiiiilis (King. 1831 ) (Mactridae) in southern Chile. The Veliger 44:33-
44.
Bayne, B. 1975. Reproduction in bivalves molluscs under environmental
stress. In: J. Vemberg, editor. Physiological ecology of estuanne or-
ganisms. Columbia: University of South Carolina Press, pp. 259-277.
Casavola, N., E. Rizzi. G. Marazo & C. Saracino. 1994. Ciclo nproductivo
e biometria di Ensis minor (Chenu) (Bivahia: Solenidael nel golfo di
Manfredonia. Oehatia 11:439-449.
Con, H. J. 1960. Staining Procedures. 2nd ed. Baltimore: Williams &
Wilkins, pp. 67-68.
Fahy, E. 1999. A new fishery for razor clams [Ensis silii/iia) on Ihe east
coast of Ireland. / Shellfish Res. 18:715.
Caspar, M. B. & C. C. Monteiro. 1998. Reproductive cycles of the razor
clam Ensis siliqua and the clam Venus striatula off Vilamoura. South-
em Portugal. J. Mar. Biol. Ass. U. K. 78:1247-1258.
Lepez, I, M., O. L. Aracena, A. Carmona. A. Espinoza, L. Fuentes, J.
Sanchez & A, Cerda. 1997a. Caracterizacion bioecondmica de las
pesqueri'as de huepo (Ensis macha) y navajuela (rage/iis dombeii) en la
VIII Region. Informe Final Proyecto F.I. P. 95-20A. Convenio U. de
Concepcidn- FEREPA Bio Bio: 87 pp.
Lepez, I. M., J. Sanchez, O. L. Aracena, M. A. Carmona & A. Saavedra.
1997b. Condicion reproductiva y sexual de los sUvks de navaja y
navajuela en Tubal y Lirquen, VIII Region. XVII Congreso de Ciencias
del Mar, Santiago. Resumen, 199 pp.
Llancamil. L. A. 1982. Variacion estacional inviemo-pnmavera de temper-
atura, salinidad y de oxi'geno disuelto en la Bahia Coliumo (36"32'S:
72°57'W). Tesis para optar al titulo de Biologo Marino. Universidad de
Concepcidn. Fotocopiado, pp. 1-94.
Lowe, D. M., M. N. Moore & B. L. Bayne. 1982, Aspects of the gamete-
genesis in the marine mussel Mytihis edulis L. J. Mar. Biol. Assoc.
U.K. 62:133-145.
Reyes, A., N. Barahona. A. Carmona, C. Rojas. E. Arias. V. Pezo, V.
Asencie & E. Lozada. 1995. Diagnostico de las Principales Pesqueri'as
Nacionales Bentiinicas. Ill, IV y X Region. 1994. Informe Tecnice
CORFO-IFOP. 96 pp. + Anexos.
Salamanca, M. A. 1997. Serie de liempo quincenal de las condiciones
oceanograficas en Bahia Coliumo. VIII Region, Chile. Informe interne,
U. de Concepcidn, Mimeografiado: 14 pp -i- Tablas y Anexos.
Sastry. A. N. 1968. The relationships among food, temperature, and gonad
development of the bay xaWops Aequipecten irradians Lamarck. Phys-
iol. Zool. 41:44-53,
SERNAPESCA. 2000. Anuario estadistico de pesca. Chile: Servicio Na-
cional de Pesca, Ministerio de Economia, Fomento y Reconstruccidn.
291 pp.
Urban. H.J. 1996. Population dynamic of the bivalves Venus annqua.
Tagelus dombeii. and Ensis macha from Chile at 36 degree S. / Shell-
fish Res. 15:719-727.
Journal ot Slwllflsli Rfsciiirli. Vol. 22. No. ,^. 6XI-fiSS. 2003.
POPULATION GENETICS OF TWO BIVALVE SPECIES (PROTOTHACA STAMINEA AND
MACOMA BALTHICA) IN PUGET SOUND, WASHINGTON
MICAELA SCHNITZLER PARKER.'* PETER A. JUMARS.' AND LARRY L. LECLAIR'
University of Wusliington School of Oceanogiapliy. Cumpiis Box 357940. Seattle. Washington
9S 195-7940; 'Darling Marine Center, University of Maine. 193 Clark's Cove Road. Walpolc. Maine
04573: and ^Washington Department of Fish and Wildlife. 600 Capitol Wax North. Olxmpia.
Washington 98501-1091. U.S.A.
ABSTRACT Allo/yme polymorphlsm.s from individuals ol Prototkuca slaminea and Mmoma balrliica were examined electropho-
retically and scored at five loci. Both species were sampled at three sites located in different hydrologically defined basins of Puget
Sound, Washington. Highly significant differences in allele frequencies among the three P. siumineci populations were found at all five
loci. Significant differences in allele frequencies were detected consistently at only one locus among the M. hcilthica populations.
Genetic distances between the three P. smminea populations, determined using both Cavalli-Sforza and Edwards ( 1967) chord distance
and Nei's (1972) genetic distance measures, revealed the South Sound population as the genetic outlier. This pattern is consistent with
the hydrology of the Puget Sound basins and the mixing that occurs at the sills between basins. Two to four of the allozyme loci
demonstrated heterozygote deficiencies in P. .stamiiieci. depending on population. Only one locus exhibited a heterozygote deficiency
in each of the three M Iniltliicu populations. Potential contributing factors to the heterozygote deficiencies include a temporal Wahlund
effect, selection, and null alleles. When data were corrected for the presence of a putative null allele, conclusions about population
differentiation did not change.
KEY WORDS:
population, genetics, allozymes, bivalves, Pioloiluua stumiiwa. Puget Sound, Macoina hallliica
INTRODUCTION
Early genetic studies of marine populations found little evi-
dence for genetic differentiation over large geographic distances. It
was generally believed that open aquatic environments permit ex-
tensive dispersal of planktonic larvae, i-esulting in little genetic
heterogeneity over wide spatial scales (e.g., Buroker et al. 1979,
Crisp 1978. Gooch et al. 1972). This notion was soon challenged,
however, by several studies presenting compelling evidence for
population structure even along open coastlines (Scheltema 1975,
Burton 1983). Increasingly, studies now find that any number of
factors can contribute to population differentiation in apparently
open systems. Populations may be defined not only by their re-
productive mode (Hellberg 1996), but by hydrological forcing
(Reeb & Avise 1990), chemical gradients (Koehn et al. 1976. Ma
et al. 2000), or changes in source populations (Kordos & Burton
1993), Population subdivision is evident even among the bivalves,
whose long-lived planktonic larvae might otherwise be equated
with high dispersal potential (e.g., Mariani et al. 2002). Other
examples of genetic differentiation in marine populations over
both small and large spatial scales are reviewed in Shaklee and
Bentzen (1998). Collectively, these studies demonstrate that re-
productive and dispersal strategies are not the only determinants of
genetic differentiation among marine populations.
In this study, we examined the potential for hydrological forc-
ing to promote differentiation of broadcast spawners with plank-
totrophic larvae in a small estuarine system, Puget Sound, Wash-
ington, is a fjord-like estuary composed of five contiguous basins
with constrictions and sills that strongly influence the tidally-
driven currents. The basins fall into two categories: well-mixed
with rapidly circulating water masses (Admiralty Inlet, Main ba-
sin, and Southern basin), or stratified with slow-moving water
masses (Hood Canal and Whidbey basin; Fig. 1 ). Ebbesmeyer et
al. ( 1988) proposed that as much as 50% of the water in each basin
■■"Corresponding author. E-mail: micaela@u. washington.edu
is recirculated back into the basin of origin because of intense
mixing at the sills. This recirculation includes the upper layer of
the water column (10-30 in deep) where planktonic larvae of
marine invertebrates are commonly found, possibly leading to par-
tial restriction of larvae to their basin of origin. Such a barrier to
dispersal could create genetically differentiated subpopulations
among basins.
Fevi' population genetic studies of marine invertebrates have
been conducted in Puget Sound despite the presence of many
managed commercial and recreational fisheries. Grant and Utter
(1988) examined allele frequencies from two polymorphic loci in
the intertidal gastropod NiicclUi [Thais ] lamellosu at several sites
within Puget Sound, adjacent waters and along the open coasts of
Oregon and Washington. They found evidence for population sub-
division at various geographic scales, however the differences
were attributed primarily to the nonplanktonic life history of this
species. A more limited study in Puget Sound involving a species
with a planktonic larval stage, the bivalve Saxidomus giganteus.
found a geographic dine in populations in one of the two allozyme
loci examined (Johnson & Utter 1973). Unfortunately, this study
did not investigate any differences that might be attributed to sepa-
ration by the hydrologically defined basins.
Our objective in this study was to test whether the bivalves
Protothaca staminca (Conrad) and Macoiiui balihica (L.) exhibit
evidence of genetic differentiation in Puget Sound consistent with
its unique hydrology. Both species broadcast spawn between April
and September with planktotrophic larvae that feed for weeks prior
to settlement. Given the long planktonic larval phase of the two
species and the small length scale of Puget Sound (on the order of
130 km), one might expect genetic homogeneity in the absence of
any physical baniers to dispersal. Differentiation of the popula-
tions inight suggest that recirculation of water masses at the sills
contributes to partial isolation of populations in Puget Sound. To
determine whether the hydrology of Puget Sound is the principle
mechanism for any observed differentiation we chose two species
that share similar reproductive and dispersal strategies yet have
681
682
Parker et al.
'^^^VvSkagit Bay
-, Wnidbey Basin
Admiralty Inlei
f'l ) -''^ "■ ^ r' Edmonds
/ \J. \ ^s^-j"^ ,■ Ma/r. Basin
Priest PtTo""*e
}
V
Figure 1. Sampling sites for Prnlnlhaca slamiiiea (Edmonds. Potlatch,
Priest Pt.) and Maconui hallhica (Skagit. Potlatch. Tolmiel in Puget
Sound, Washington.
disparate adult characteristics. Prointlnicti staininea occurs from
the Aleutian Islands of Alaska to Baja California; is a suspension
feeder preferring coarse sand to gravel substrate; attains a maxi-
mum valve length of around 7 cm; and is preyed upon primarily by
starfish, moonsnails, and octopuses. Macoma balthica. conversely,
is circumboreally distributed; may switch between surface-deposit
and suspension feeding; prefers muddy substrate; may inhabit
brackish waters; in Puget Sound, rarely exceeds 2 cm in length;
and is preyed upon primarily by flounder, crabs and sea birds. By
examining two species with similar reproductive and dispersal
strategies but with different adult characteristics, we hoped to as-
sess the influence hydrology may have on population distributions
of different species in this estuary.
We examined allozyme polymorphisms at five presumptive
gene loci in each of the two species of intertidal bivalve clanis:
Protothaca staininea and Macoma balthica. Genotype and allele
frequencies from each species were then compared among three of
the hydrologically defined basins of Puget Sound, Washington.
METHODS
Field Sampling
Protothaca staininea were collected at Potlatch (Hood Canal
Basin; /; = 94), Priest Point (Southern Basin; /; = 114). and
Edmonds (Main Basin; ;; = 114) between March 1 and Sept. 7.
1998. Macoma balthica were collected from Potlatch (n = 113).
Tolmie Park at Big Slough (Southern Basin; n = 1 16), and Skagit
Bay (Whidbey Basin; n = 132) between March 2, 1998 and June
29, 1999 (Fig. 1). All samples were obtained during low low tide
along 100- to 500-m transects running parallel to the shore. Care
was taken to sample individuals from the full extent of their range
in the intertidal zone as well as across size classes. The length of
the right valve of P. staininea specimens sampled ranged from 9
mm to 57 mm and for M. hallhica from 4.3 min to 17 mm. M.
balthica specimens included the white, pale pink, and dark pink
color morphologies. The clams were transferred live in ambient
seawater to the laboratory. Immediately upon arrival, foot muscle,
ctenidium, digestive gland, mantle, and adductor muscle were dis-
sected from each P. staininea. The tissues from each clam were
then combined in a single test tube. Because of the small size of the
Macoma clams, they were stored whole (minus shell) in indi\ idual
test tubes. All samples were stored at -80''C for subsequent elec-
trophoretic analysis.
Electrophoresis
Following the methods of LeClair and Phelps (1994), tissue
samples were homogenized in TC-1 gel buffer (Shaw & Prasad
1970) and centrifuged at 1.000 g for 5 min. Supernatants were
absorbed with filter-paper wicks (Schleicher & Schuell no. 470)
and used for starch gel electrophoresis. Details of the electropho-
retic method are described in Aebersold et al. (1987) and Harris &
Hopkinson ( 1976). Gels were run in a refrigerator at 8°C. Enzyme
and gene nomenclature follow the guidelines of Shaklee et al.
( 1990). Both species were assayed for allozyme polymorphisms on
four different buffer systems: CAME 6.8 (LeClair & Phelps 1994,
modified from Clayton & Tretiak 1972); LiOH-RW (Ridgeway et
al. 1970). TRIS-GLY (Holmes & Masters 1970); and TC-4 (buffer
"a" of Schaal & Anderson 1974). The following enzyme/buffer
combinations were tested: aspartate aminotransferase (AAT),
isocitrate dehydrogenase (IDHP), malate dehydrogenase (MDH).
malic enzyme (MEP). phosphogluconate dehydrogenase (PGDH).
and phosphoglycerate kinase (PGK) on CAME 6.8; esterase-D
(ESTD). formaldehyde dehydrogenase (FDHG). nucleoside-
triphosphate pyrophosphatase (NTP). octopine dehydrogenase
(OPDH). and strombine dehydrogenase (STDH) on LiOH-RW;
alanine aminotransferase (ALAT). arginine kinase (ARGK).
ESTD. glucose-6-phosphate isomerase (GPI). lactate dehydroge-
nase (LDH). mannose-6-phosphate dehydrogenase (MPI). cytosol
nonspecific dipeptidase (PEPA). tripeptide aminopeptidase
(PEPB). peptidase-S (PEPS), phosphoglucomutase (PGM). STDH.
and triose-phosphate isomerase (TPI) on TRIS-GLY; adenosine
deaminase (ADA), aconitate hydratase (AH), glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). PEPA. and proline dipepti-
dase (PEPD) on TC-4.
Of the 25 enzymes assayed, activity of six (AAT. ESTD. GPI.
IDHP. PGDH, PGM) were well resolved and indicated encoding
by polymorphic loci (more than one allelic form detected). These
enzymes were subsequently screened in all clams except AAT.
which was screened only in P. staininea. and IDHP, which was
screened only in M. balthica. Allelic variants are designated by
their electrophoretic mobility relative to the most frequent variant
encountered during the initial screening. Variants preceded by a
minus sign indicate cathodal migration.
Data .Analysis
The population genetics software GENEPOP version 1.2 (Ray-
mond & Rousset 1995a) was used to run analyses of population
differentiation and heterozygote deficiency or excess relative to
Hardy-Weinberg equilibrium. For testing population differentia-
tion, both "genie" and "genotypic" tests were run. The genie test is
used to determine whether allelic distributions are identical across
populations. Contingency tables for each locus were tested using
the R X C Fisher test to arrive at an unbiased estimate of the P
value (Raymond & Rousset 1995b). The genotypic test is used to
determine whether genotypic distributions are identical across
populations. Although less powerful, the genotypic test is more
appropriate when alleles within individuals are not independent,
which may occur when there is nonrandom mating (Goudet et al.
PopuLATKJN Genetics of Bivalves
683
19%). For this test, an unbiased estimate of the P value is achieved
by using the G-based test (Goudet et al. 1996) on contingency
tables for each locus. Tests for both heterozygote deficiency and
excess are concerned with the same H,,, random union of gametes.
For both tests, the unbiased P value was estimated using the score
test (U test: Rousset & Raymond 1995). Because of the presence
of rare alleles, defined as having frequencies <0.005 (Hartl &
Clark 1997). the exact tests used by GENEPOP are more appro-
priate than the comnmnly used x" test because the results will not
be biased b> rare alleles (Guo & Thompson 1992). Expected het-
erozygosities (//g), fixation indices (F,s) and the extent of popu-
lation divergence (F^-^) were also calculated for each locus in each
population using GENEPOP. The f-statistics used by GENEPOP
follow Weir & Cockerham (1984). GENEPOP was also used to
test for genotypic linkage disequilibria. The program BIOSYS-1
(Swofford & Selander 1981) was used to determine Cavalli-Sforza
and Edwards (1967) chord distances and Nei's (1972) genetic
distances. Finally, when individuals without a banding pattern are
observed, yet are not conclusively null homozygotes. the fre-
quency of a putative null allele can be estimated using (H^, -
Ho)KH^ + H(,). where Wj. and //<, refer to the expected and ob-
served heterozygosities, respectively (Brooktleld 1996). Using this
algorithm allele frequencies for the populations of both species
were corrected for the presence of a null allele.
RESULTS
Stains for GPI and PG.M were most successful on the Tris-Gly
buffer .system: PGDH. AAT. and IDHP on CAME 6.8: ESTD on
LiOH-RW. In each species, two private alleles (alleles only de-
tected in one population) were found: GPI*- 1 7 (P. staminea. Pot-
latch). AAT*-1500 {P. staminea. Edmonds). IDHP*I50 (M. bal-
thica. Skagit). PGDH* 1 14 (M. hallhica. Skagit). Four rare alleles
occurred in P. skimiiwa populations and eight in M. balthica popu-
lations (Table 1 ).
Expected heterozygosities (//p) and fixation indices {F^^) var-
ied widely in both species depending on the locus (Table 1 ). No-
tably. f,s values for the P. staminea population at Edmonds were
consistently higher than values for the population at Potlatch or.
with most loci, at Priest Point suggesting strong heterozygote de-
ficiencies in this population. The tests for Hardy-Weinberg equi-
librium revealed significant heterozygote deficiencies (P < O.O.S) in
up to four of the five loci in the P. staminea populations (Table 2).
Only at the ESTD* locus was a significant heterozygote deficiency
detected in the M. balthica populations (P < 0.001; Table 2). In
neither species was a heterozygote excess detected.
For both species, locus pairs were also tested for genotypic
linkage disequilibrium within each population. A significant link-
age disequilibrium suggests the genotypes at different loci are not
independent. Linked loci may be an indication of inbreeding. After
applying a sequential Bonferroni correction (Ury. 1976). only one
population (P. staminea. Edmonds) had loci with significant link-
age disequilibria. The two locus pairs demonstrating a significant
disequilibrium were: GPI* and AAT* (P < 0.001 ) and AAT* and
£5rD-2*(P< 0.005).
With both the genie and genotypic tests, we found strong evi-
dence for population differentiation among all three P. staminea
populations at all loci (P < 0.001; Table 3). Both chord and Nei's
distances indicated that the populations from Edmonds and Pot-
latch are more closely related than either is to the Priest Point
population (Table 4). When distances were determined locus by
locus, four of five loci were in agreement with this pattern. Fg^
\ alues for the P. staminea populations ranged from 0.07 {PGM*)
to 0.13 {ESTD*).
In the M. balthica populations, both the genie and genotypic
tests demonstrated differentiation at one of the five loci {PGDH*
Table 3). The genie test revealed an additional differentiation at the
ESTD* locus (Table 3). Ff^^ values for M. balthica ranged from
-0.002 {PGM*) to 0.009 {ESTD*). Because of the lack of differ-
entiation among M. balthica populations at most loci, distance
measures were not significant (data not shown).
To determine whether heterozygote deficiencies had any effect
on the population differentiation tests, the allele frequencies were
recalculated to account for the potential presence of a null allele.
An indication of null alleles is a null homozygote demonstrating
no banding pattern. In the P. staminea samples, absence of enzy-
matic activity occurred with only one individual from Priest Pt.
when stained for GPI and two individuals from Edmonds when
stained for ESTD and PGM. In the M. balthica samples, absence
of enzymatic activity occurred in three individuals from Skagit
Bay (all using the stain for IDHP. one additionally did not stain for
ESTD) and four individuals from Potlatch (all using the stain for
ESTD. one additionally did not stain for PGDH). Because this
absence of activity could also have been caused by tissue degra-
dation, staining inconsistencies, or tissue samples that are too
small (for M. balthica). we could not conclusively assign these
individuals as null homozygotes. It is possible to estimate the
frequency of a putative null allele based on the heterozygote de-
ficiency in a population. Following Brookfield (1996). allele fre-
quencies were corrected in each population to account for the
presence of a null allele and the genie and genotypic tests re-run.
The level of population differentiation observed did not decline for
either species. On the contrary, both the chord and Nei's genetic
distances increased slightly with the addition of the null allele
(between I and 30% increase, data not shown).
DISCUSSION
Evidence for Distinct Populatiinis of P. staminea But Mot M. balthica
Both Piotothaca staminea and Macoma balthica are free-
spawning bivalves, with feeding larvae that spend about 3—4 wk in
the plankton. These larvae are the dispersal propagules. largely at
the mercy of local horizontal currents. Given the similar reproduc-
tive and dispersal strategies of P. staminea and M. balthica. one
might expect consistency in the level of population differentiation
of these species when exposed to the same estuarine currents. The
population structure of these two species, however, is very differ-
ent in the complex estuarine system of Puget Sound. Washington.
Populations of P. .staminea were found to be highly differentiated
at all loci surveyed, whereas the M. balthica populations were
significantly different at only one locus using both the genie and
genotypic tests. While it is possible that allozymes are not variable
enough to detect differences between the populations of M. bal-
thica. it is likely that .species-specific selective pressures also play
a role in structuring these populations.
Piotothaca staminea and Macoma balthica occupy very differ-
ent ecological niches. It is possible that these two species experi-
ence different selective pressures in Puget Sound from the physical
environment or from local predators, including humans (van der
Veer et al. 1998. Ejdung & Elnigren 199S. Chew & Ma 1987). P.
684
Parker et al.
TABLE L
Allele frequencies at loci for Protothaca slaminea and Macoma balthica individuals from three locations in Puget Sound, \VA
Locus,
allele
Prololhaca slaminea
IjOcus.
Macimia balthica
Potlatch
Edmonds
Priest Pt.
allele
Potlach
Skagit
Tolmie
CPl
-17
0.011
0.000
0.000
-22
0.004*
0.004*
0.000
14
0.074
0.039
0.138
8
0.009
0.019
0.022
36
0.420
0.237
0.170
38
0.434
0.481
0.457
58
0.35 1
0.202
0.589
66
0.128
0.092
0.116
11
0.112
0.167
0.085
100
0.376
0.385
0.353
100
0.032
0.285
0.013
130
0.049
0.019
0.052
127
0.000
0.070
0.004*
(N)
(113)
(130)
(116)
(N)
(94)
(114)
(112)
«E
0.654
0.614
0.652
H^
0.685
0.791
0.600
f.s
-0.014
-0.027
-0.097
F,s
0.177
0.358
-0.012
PGM
66
0.101
0.108
0.090
38
0.018
0.008
0.030
85
0.261
0.171
0.232
62
0.159
0.129
0.156
100
0.314
0.230
0.602
86
0.053
0.057
0.065
ii:
0.245
0.387
0.076
100
0.611
0.621
0.593
132
0.080
0.104
0.000
124
0.124
0.159
0.113
(N)
(94)
(111)
(105)
154
0.035
0.027
0.043
«E
0.761
0.750
0.574
(N)
(113)
(132)
(115)
f.s
0.148
0.267
0.238
«E
0.585
0.570
0.609
fis
0.107
0.097
0.000
AAT
-1500
0.000
0.004*
0.000
70
0.004*
0.000
0.004*
-700
0.293
0.180
0.009
82
0.062
0.036
0,039
-100
0.670
0.798
0.947
94
0.013
0.008
0,000
500
0.021
0.004*
0.044
100
0.903
0.933
0,953
900
0,016
0.013
0.000
124
0,018
0.020
0.004^^'
(N)
(94)
(114)
(114)
150
0.000
0.004*
0.000
Hb
0.467
0.332
0.101
(N)
(113)
(126)
(116)
f.s
0.112
0.207
-0.043
He
0.182
0.129
0.091
Fis
0.124
0.017
0.152
PGDN
-1100
0.048
0.024
0.253
62
0.004*
0.004*
0.000
-600
0.425
0.524
0.552
74
0.022
0.068
0.030
-100
0.495
0.423
0.155
82
0.157
0.209
0.129
300
0.011
0.014
0.041
90
0.511
0..^92
0.500
1000
0.022
0.014
0.000
100
0.305
0.316
0.341
(N)
(93)
(104)
(97)
114
0.000
0.011
0.000
He
0.575
0.548
0.599
(N)
(111)
(131)
(116)
/^■s
0.178
0.299
0.333
He
0.625
0.700
0.619
F,s
0.063
0.052
0.025
ESTD-2
75
0.000
0.045
0.004*
90
0.081
0.116
0,078
84
0.202
0.134
0.425
100
0.720
0.633
0.759
92
0.069
0.290
0.294
106
0.199
0.251
0.164
100
0.723
0.513
0.276
(N)
(105)
(129)
(116)
111
0.005
0.018
0.000
He
0.443
0.526
0.393
(N)
(94)
(112)
(114)
f.s
0.448
0.485
0.519
«E
0.433
0.635
0.659
f,s
0.166
0.270
0.002
N = the number of clam.s scored in each collection. Frequencies in bold indicate priva(e alleles. Asterisks (*) indicate rare alleles (frequencies <0.005).
Hf = e.xpected heterozygosities; f,; = fixation index for individuals wi[hin each population.
sliiiiiiiwa has a larger adult si/,e and often occupies niueh more
sandy substrates than M. haltluca. .Sanche/.-Salazar et al. (1987a,
1987b) demonstrated the inllueiiee both tidal elevation and shore
crabs can have on the population structure of the bivalve. Ceras-
loilvniui I'l/iilc. The lecreational harvest of P. stamiiwa in Puget
Sound may also contribute to selective pressures in this species. In
addition, harvesting of P. staininea may reduce its effective popu-
lation size (yV(.). contributing to differentiation of populations
Population Genetics oi- Biv.xlves
685
TABI.K 2.
Probability values for the te.st of heterozjgote dcricienc) relative to Hardy-Wcinberg expectation.s at each locus for each population
Protolhaca slamiiiea
Locus
Macoma balthica
Locus
Potlach
Edmonds
Priest Pt.
Potlach
Skagit
Tolmie
GPI
0.096
<0.001*
0.347
GPI
0.333
0.835
0.942
PGM
0.084
<0.00l*
0.035*
PGM
0.100
0.403
0.173
PGDN
<0.001*
0.057
<0.001*
PGDN
0.123
0.097
0.474
AAT
0.019*
0.030*
1 .000
IDHP
0.087
0.447
0. 1 49
ESTD-:
0.008*
<0.00l*
0.383
ESTD
<().001*
<0.001*
<0.001*
Asterisks (*) indlcule signiricuiil hetero/ygole deliciencies (P < 0.05).
through genetic drift. M. halthicci is too small to attract recreational
or commercial interest and may therefore also have a much larger
A'^,. Additionally, neither selective pressures nor genetic drift may
be strong enough to dri\'e population differentiation of M. haltliica
if there are sufficient migrants to homogenize the populations
(Hartl & Clark 1997).
Exchange of individuals between populations may be facili-
tated by larval behavior. The planktonic larvae of many estuarinc
invertebrates do not behave as strictly passive particles, instead
exhibiting selective transport in horizontal currents mediated by
vertical migration (Morgan 1995). Although the most extensive
research has focused on crustaceans (e.g.. Sandifer 1975. Cronin
1982. Forward et al. 1995). a few studies have confirmed selective
larval transport among bivalves (Wood & Hargis 1971. Manuel et
al. 1997). It is possible that P. suiininea and M. balthica larvae
exhibit divergent swimming behaviors that could affect their trans-
port out of their respective estuarine basins of origin in Puget
Sound. Unfortunately, there have not been any studies investigat-
ing vertical migration behavior of P. staminea larvae. Work by
Roegner (2000) suggests that the larvae of M. balthica are pas-
sively distributed. However, there is evidence for selective post-
metamorphic drifting of M. balthica juveniles (Beukenia & de
Vlas 1989). Byssal threads attached to these post-larvae provide
drag and lift allowing transport on horizontal flow. A recent study
of Macoma spp. post-larval distributions in the York River estuary
of the Chesapeake Bay strongly suggests that this life-history stage
exerts a behavioral control over position in the water column (Gar-
rison & Morgan 1999). Because byssal thread-drifting has not been
demonstrated in P. staminea. one possibility is that M. balthica
populations in Puget Sound are less differentiated due to selecti\e
thread-drifting of the post-metamorphic juveniles.
TABLE 3.
Probability values for the genie and genotypic tests for population
differentiation of three Prolothaat ■ilominca and three
Macoma balthica populations
Protolhaca
staminea
Macoma balthica
Genie
Genotypic
Genie
Genotypic
Locus
test
test
Locus
test
test
GPI
<0.001*
<0.001*
GPI
0.467
0.524
PCM
<0.001*
<0.001*
PGM
0.589
0.681
AAT
<0.()01*
<0.001*
IDHP
0.273
0.320
PGDN
<0.001*
<0.001*
PGDH
0.011*
0.007*
ESTD-2
<0.()01*
<0.()01*
ESTD
0.043*
0.140
P. staminea Populations May Be Constrained by Puget
Sound Hydrology
Because we found substantial differentiation among Pro-
tolhaca staminea populations, we hypothesize that gene flow be-
tween these populations may indeed be restricted. The chord dis-
tances as well as Nei's genetic distances suggest that the popula-
tions of P. staminea in Hood Canal and the Main Basin are more
similar to each other than either is to the South Sound population
(Table 4). Hydrology of the Puget Sound estuary supports the
hypothesis of South Sound isolation. Cokelet et al. (1991 ) deter-
mined that as much as 52% of the water entering Admiralty Inlet
from Puget Sound is recycled back through mixing at the sill (Fig.
1). This retluxing coupled with their proximity suggests a large
potential for exchange between Hood Canal and the Main Basin.
Cokelet et al. ( 1991 ) also estimated that the longest residence times
in Puget Sound are for waters originating in the southernmost
reaches of the Sound. Populations from the South Sound and Main
Basin might therefore be restricted in their ability to exchange
indi\'iduals. In fact, there are two minor sills and one major sill (at
Tacoma Narrows) between the Priest Point population in South
Sound and the Edmonds population in the Main Basin. Recently,
the slow flushing times of South Sound have been implicated in
the die-off of a number of benthic species, perhaps due to pollutant
retention (Ebbesmeyer et al.. 1998). It remains to be seen whether
the refluxing of South Sound waters is directly preventing dis-
persal of P. staminea larvae. There is. however, a correlation be-
tween the observed genetic pattern and the expected circulation
pattern of Puget Sound.
Deviations from Hardy-Weinberg Equilibrium
Heterozygote deficiencies are commonly found with a variety
of molecular methods, especially in marine bivalve populations
(Raymond et al. 1997. Gaffney 1994. Zouros & Foltz 1984. Singh
TABLE 4.
(Jenetic distance measures for the three Protolhaca staminea
populations (Edni = Edmonds; Pot = Potlatch: PPt = Priest Pt.)
Protolhaca staminea.
all loci
Edm-Pot
Edm-PPt
Pot-PPt
CSE
NEI
0.1997
0.0723
0.3141
0.1947
0.2858
0.2085
Asterisks (*) indicate significant differences [P < 0.05).
CSE = Cavalli-Sforza and Edwards (19671 chord distance; NEI = Nei's
distance from Nei (1972).
686
Parker et al.
& Green 1984). Often, heterozygote deficiencies are indicative of
reproductive isolation resulting in inbreeding. Additional causes
ascribed to heterozygote deficiencies are wide ranging but may
include aneuploidy. molecular imprinting, genotype-dependent
spawning, selection, population mixing, null alleles, scoring bias,
and tissue degradation. Aneuploidy. molecular imprinting, and ge-
notype-dependent spawning have not been reported for either of
these species and there is little evidence to support these phenom-
ena in bivalves. Heterozygote deficiencies resulting from spatial
population mixing do not seem likely either, given the large sam-
pling area (100- to 500-m transects), high abundances, and long
pelagic phases of these two species in Puget Sound.
However, it is possible that we encountered temporal popula-
tion mixing since we likely sampled over several generations by
sampling over a wide range of sizes. It has been hypothesized that
the chance reproductive success of free-spawners may lead to large
variances in the genetic composition of each successive generation
due to random drift (Hedgecock 1994). The result is a small num-
ber of individuals contributing disproportionately to the next gen-
eration. Sampling across these generations may lead to temporal
population mixing, also know as a temporal Wahlund effect. To
maintain differences between year-classes, selection and/or assor-
tative mating may also be occurring (HartI & Clark 1997). Similar
to Ruzzante et al. (1996). we investigated the effect of pooled
age-classes on Hardy-Weinberg equilibrium by dividing the P.
staminea individuals into large- and small-size classes and re-
testing for heterozygote deficiencies. For all populations, the num-
ber of loci with heterozygote deficiencies decreased in both size
classes except one (small class, Potlatch) compared with popula-
tions that had both size classes pooled (data not shown). This
suggests that pooling the size classes may have contributed to the
observed heterozygote deficiencies.
Selection may also act to reduce the number of heterozygotes in
a population. An ongoing debate in bivalve genetics is the apparent
paradox between observations of hybrid vigor and heterozygote
deficiencies. Individuals in both the laboratory and natural field
populations demonstrate strong correlations between heterozygos-
ity and fitness-related traits, e.g.. size, growth rate, and reproduc-
tive capacity (Hedgecock et al. 1996, Zouros 1987). Yet field
populations of many bivalve species are heterozygote deficient.
One possible explanation is genotype-dependent larval mortality
(Singh & Green 1984, Zouros & Foltz. 1984). Investigating the
timing of the heterozygote deficit, Fairbrother and Beaumont
(1993) found heterozygote deficiencies in a cohort of newly settled
mussel (Mytilus edulis) spat, concluding that the loss of heterozy-
gotes must have occurred during the larval stage or early settle-
ment. Singh (1982) suggested that selection might act against the
more heterozygous, faster-growing larvae because of their in-
creased food requirements during the critical period of larval de-
velopment. If plankton abundances are not high during this period.
these larvae face a greater mortality. This phenomenon has yet to
be investigated in either P. staminea or M. balthica.
Finally, the presence of null alleles may also contribute to the
observed deficiencies. It is possible that either true null alleles or
artifacts, such as insufficient tissue or staining inconsistencies,
caused the deficiency in the one locus (ESTD) across all Macoma
halthica populations. However, heterozygote deficiencies occuiTed
in most loci and in all populations of Protolhaca staminea. sug-
gesting null alleles are not sufficient to explain the observed de-
ficiencies in this species. For these populations, selection and in-
breeding due to partial reproductive isolation could explain the
deficiencies we observed. In addition, it is possible that we en-
countered a temporal Wahlund effect in the P. staminea popula-
tions. Importantly, when all other allele frequencies were con'ected
for the presence of a null allele and the analytical tests re-run. the
population differentiation conclusions did not change.
CONCLUSIONS
Many factors may contribute to population differentiation of
marine invertebrates in Puget Sound. To prevent genetic homoge-
neity over such a small geographic scale, however, selective forces
must be strong, gene flow must be restricted, and/or temporal
variance of the populations must be extreme. Environmental fluc-
tuations can be dramatic in the estuarine ecosystem. Extremes of
salinity and temperature can be found over small spatial scales. In
such a heterogeneous environment, selection may take the form of
both physical and biological constraints. They may act in concert
to vary pressures on adult clams or the recruiting larvae. Popula-
tions may vary from generation to generation simply due to pulsed
recruitment or sweepstakes sampling from the previous generation.
Factors that might limit gene flow between populations in this
estuary include large-scale retlux via mixing at sills, larval behav-
ior, or small-scale circulation patterns such as nearshore eddies.
We have demonstrated a correlation between the population dif-
ferentiation of P. staminea and the circulation pattern of Puget
Sound warranting further study of the effects of Puget Sound hy-
drology on larval dispersal. The hydrology of Puget Sound, how-
ever, does not ensure differentiation in every species. In stark
contrast to P. staminea. we have shown that M. luiltliica popula-
tions reveal little differentiation among the same basins. The
amount of differentiation between sites is highly species depen-
dent, and therefore population dynamics should not be generalized
based on reproductive characters alone.
ACKNOWLEDGMENTS
The authors thank Paul Bentzen for his insights and expertise
with the population genetic analyses and Fred Utter and Tatiana
Rynearson for their helpful comments in reviewing the manuscript.
They also thank Cherril Bowman and Norm Switzler for assisting
with the preliminary phase of the lab analysis. This work was
funded by National Science Foundation Grant OCE 9617701.
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Jmirmil oj Shcttfish Hcscanh. Vol. 22. No. 3. bii9~Mb. 2()()3.
SHELL REPAIR OF MECHANICALLY INDUCED FRACTURES IN MERCENARIA
MERCENARIA UNDER EXPERIMENTALLY SUBOPTIMUM CONDITIONS
RICHARD R. ALEXANDER' AND ROBERT M. BARON'
Department of Geological and Marine Sciences. Rider University. Lawrenceville. New Jersey: and
-/nsitute of Marine and Coastal Studies. NOVA Southeastern University. Fort Lauderdale. Flmida
ABSTRACT Sixty hand-tonged, harvested specimens of Mencnariii nierceiuirici from wild stocl< in Raritan Bay. New Jersey,
measuring 34 to 43 mm in dorsal venlrul length, were apportioned among buckets of sediments submerged in predator-e.xcluded
flow -through tanks. Experimental sediments simulate substrata found native to hard clams and included: ( 1 ) well-sorted sand. 1 2) pure
mud. (3l. an admixture of equal volume of shell-free sand and Jiiud. (4) an admixture of 759c sand and 25'7f .shell hash, and (5) an
admixture of ISVc mud and 25'y'r shell hash. Hand-excavated clams reburrowed monthly for one year. Progressively dysoxic interstitial
pore water beneath the sediment interface mediated burrowing conditions. Shells of live specimens in progressively blackened sands
became chalky in appearance with ornamentation completely abraded and/or etched away. Upon sacrifice, 30 (50%) specimens
revealed fractures in the valve interior that radiated from the ventral (24), posterior (four), and anterior (two) margins, whereas only
five of 36 (\49c) specimens in the unburrowed "control" group showed anthropogenically (harvesting and machine-sorting) induced
microfractures at the ventral margin. Mean annual dorsal-ventral shell accretion was negligible under these experimentallv suboptimal
conditions. Distribution of fractured specimens among the five experimental substrata is statistically random, although, paradoxically,
more clams that reburrowed in mud than sand-shell hash had internally repaired valves. Severity of fractures is evidenced by stuccoed
cracks that encroached within a cm of the dorsal hinge and others that bifurcated and deflected though the adductor inuscle scars.
Converged fractures in one rebunowed specimen removed a large triangular wedge of shell that proved lethal. Nevertheless, repaired
fractures did not fail under the strain of repeated re-burrowing.
KEY WORDS: Mcrct'imria menenoria. burrowing, fracture, repair, abrasion
INTRODUCTION
Lethal and sublethal shell fractures in Mercenaria mercenaria
have been primarily atttibuted to durophagous predators. The toll
these molluscivores inflict on this commercially valuable species
has been reviewed by Krauter (2001 ). Dredging activity also may
sublethally fracture shells of commercially valuable clams as ob-
served in commercially harvested Glyeymeris s^lycymeris (Ramsey
et al. 2000). Ensi.s silitjua (Caspar et al. 1994), Solen sp. (Bergman
& Hup 1992). and Arctica islandica (Wilbaard & Klein 1994).
Scar frequencies have been used to attempt reconstruction of the
history of past shellfishing pressure. Another possible non-
predatory cause of shell fracture in bivalves is burrowing, although
such shell-fracturing mechanical processes have been infrequently
investigated experimentally. Checa (1993) illustrated specimens of
the thin-shelled deep bivalve.? Lutraria liitraria. Panopea gylcy-
meris. and Soleciirtus strigalatits with scars of repaired cracks
induced by reburrowing by individuals that were prone to exca-
vation by winter storm waves.
However, repair of burrowing-induced fractures and its fre-
quency has not been documented in a shallow-burrowing, thicker-
shelled, and coiTimercially harvested clam, such as Mercenaria
mercenaria. The extent to which such mechanically induced frac-
tures can be repaired is unreported. Appreciable abrasion of the
ventral margin of M. mercenaria has been documented in trans-
plant experiments (Pannella & MacClintock 1968, Rhoads &
Panella 1970. Kennish 1978). Repeated burrowing may chip the
commissure margin of some young adults of M. mercenaria.
thereby providing a site for initiation of dorsally propagated frac-
tures. Conceivably, sediment texture and cohesiveness could in-
fluence both sediment loading against the valves (Checa 1993)
and/or the likelihood that shell shards become occluded between
the valves during the repeatedly opening and adduction of the
valves. Reburrowing may provide the additional stress on valves
marginally chipped by the commercial excavation, handling, and
sorting processes. Raked specimens, jostling against each other in
transport and sorted by conveyor-belt into bags of commercially
graded sizes may bear very slightly chipped margins that could
become the initiation sites of fractures if the clams are afforded an
opportunity to reburrow. Conceivably, sediment texture may be
causally related to frequencies of (1) anthropogenically induced
microfractures that are propagated through the valve during rebur-
rowing, and/or (2) burrowing-induced microfractures that are fur-
ther expressed during repeated penetration of the sediment. Sedi-
ment texture may also influence interstitial water chemistry medi-
ated by sediment porosity and permeability. Substrates of different
mean grain sizes and degree of sorting have different porosity and
permeability properties. Suboptimum interstitial conditions be-
neath the sediment surface where the clam bun'ows also may in-
fluence both shell fracture propagation and the ability of the
mantle to repair cracks.
Accordingly, this investigation experimentally focuses on the
repair of nonpredatory shell fractures in young adults of M. mer-
cenaria that repeatedly burrow into various textured sediments.
The testable, refutable null hypotheses are { 1 ) that microfractures
possibly initiated by anthropogenic excavation and handling are
repaired prior to or during reburrowing activity. (2) that the bur-
rowing process also initiates microfractures that are repairable, (3)
repaired fractures withstand the strain induced by reburrowing, (4)
that no significant difference in the frequency of fractures results
from reburrowing in different textured sediments, and (5) that no
significant change in valve thickness and external ornamentation
resulted from re-burrowing in different textured sediments.
METHODS AND MATERIALS
Within Raritan Bay. New Jersey-New York, commercial shell-
fish beds, some situated in depths above effective storm wave
base, include sediments characterized as mud, shell, gravel, sand,
and sand-mud that host \arying densities of M mercenaria. To test
689
690
Alexander and Baron
the effect that sediment texture has on shell abrasion, chipping, and
fracture-initiation or propagation in M. mercenaria. fivel2-L
buckets of sediment were submerged in each of two 690-L flow
through tanks at the NOAA Laboratory at Sandy Hook. New Jer-
sey, which pumps in water from Raritan Bay. Each bucket was
filled with a substratum to a 14 cm depth, resulting in the sediment
surface recessed about 4 cm form the top of the bucket. Enclosed
substrate included one of five types of sediments and shell hash
native to Raritan Bay to simulate the various substrata naturally
occupied by M. mercenaria. The five sediment categories included
( I ) sieved, intertidal sand void of any gravel size grains and shell
fragments. (2) pure mud. (3). an admixture of shell-free intertidal
sand (50% by volume) and mud (507f by volume). (4) an admix-
ture of 75% by volume of beach sand and 25% by volume of shell
hash, and (5) an admixture of 75% by volume of mud and 25%
by volume shell hash. Shell hash included shards of razor clams
{Eiisis direcliis). blue mussels [Myliiiis cjiilis). surfclams iSpisiila
solidissima), and hard clams (M. mercenaria) created by mortar
and pestol. The longest dimension of any shell shard did not ex-
ceed 4 mm. Admixtures of sediment types were thoroughly mixed
with a trowel to homogenize the substrates. Two replicates of each
substratum were created, one for each flow through tank.
Sixty hand-raked, machine-sorted, specimens of /W. mercenaria
obtained from a depuration plant operating in Raritan Bay were
measured dorsal-ventrally ( = shell length), and perpendicular to
the hinge line at the point of maximum curvature or maximum
cross-sectional height ( = shell height) to the nearest 0. 1 mm by
means of electronic vernier calipers. All specimens ranged from 34
to 43 mm in dorsal-ventral length. Initial scrutiny of the specimens
revealed no hairline fractures expressed on the valve exteriors. A
separate batch of 36 machine-sorted specimens from the depura-
tion plant, measuring 34^1 mm in DV length, were held in an
aquarium without sediment for four weeks and then sacrificed to
determine if commercial har\esting and handling could have ini-
tiated any interior fractures in the shells prior to reburrowing.
Among the 60 experimental clams, six specimens were assigned to
each of the 10 buckets of substrata and placed reclining on one
valve in a clockwork arrangement (12. 3. 6. and 9 o'clock with two
specimens at the center) on the sediment in May 1998. Acclima-
tion to the conditions in the tanks occurred during the ensuing
summer months. Monitoring of changes in the shell dimensions
and external surface appearance commenced in October 1998 and
lasted through October 1999.
The flow of water discharged into each tank was maintained at
nearly 20 cm/s. Discharge occurred from eight 3-mm diameter
perforations along the length of 30-mm diameter pipe that jetted
water into the tank. These perforations are too narrow to allow
metamorphosed clam predators to enter the tanks. Nevertheless.
tanks were checked monthly for incidental invasions. None were
found. Water exited the tanks from two vertical oriented, overflow
drains at each end of the 70-cm deep tank. Twice a month the
dissolved oxygen, salinity, temperature, and pH were recorded for
each tank by means of a portable hydrolab. A Marsh McBimey
current meter checked the flow velocity jetting from perforations
in the tube in the tank twice a month. The tanks were not dosed
with any algal extract to enhance clam growth during the experi-
ments.
The clams were excavated by hand from their buckets monthly,
and their shell length, and height recorded after any adhering sedi-
ment was washed off from the valve exteriors. This procedure was
followed monthly from October 1998 until October 1999 when the
clams were sacrificed. No data were collected in May 1999. Dead
specimens were cleaned and examined for abrasion, fracture, and
repair. No specimen showed infestation with the boring sponge
Cliona sp.
Fractures and repairs among M. mercenaria at the end of ex-
perimental interval were described and categorized as to ( I ) frac-
ture expression (crack visible on interior or exterior of valve, or
both). (2) valves affected by fracture (right, left, or both). (3)
number of fractures per valve. (4) length of fractures. (5) fracture
initiation site at, or very near the valve margin (ventral, posterior,
anterior). (6) fracture propagation inward from the valve margin
(diagonal, curved, right angled deflections, merging and/or bifur-
cating), and (7) state of fracture repair (internally stuccoed cracks
or unrepaired). A Goodness of fit test determines (1) if sublethal
fractures occur randomly among specimens in different textured
sediments. (2) if fractures occur randomly around the shell margin
(posteriorly, ventrally. or anteriorly), and (3) if fractures propagate
in a restricted pathway. A t test determined ( I ) if fractured and
iinfractured specimens differ according to valve thickness at the
ventral margin and (2) if repair condition (stuccoed vs. unrepaired)
differs according to fracture length.
Additionally, at the conclusion of the 12-month monitoring
period, the valve suiface of each surviving clam was examined
under magnification and the degree of shell abrasion and/or surfi-
cial etching categorized according to the relief of the concentric
lamellae as ( 1 1 abrasion-negligible. (2) abrasion/etching — slight;
wear restricted to ventral area. (3| abrasion/etching — moderate;
shiny, bare patches over central and ventral valve area, and (4)
abrasion/etching — extensive; obliteration of concentric lamellae
over most of valve surface area. It should be noted that abrasion
and etching must be distinguished from ontogenetic changes in
shell micro-ornament over the valve surface. A swath of the central
valve of M. mercenaria inherently lacks micro-ornamentation in
adulthood, although the entire valve surface of many juveniles to
young adults possess fine concentric ribbing.
RESULTS AND ANALYSIS
Among the 36 "control" specimens held in an aquarium and
sacrificed after 4 weeks, tlve showed microfractures radiating dor-
sally from the ventral margin that were most probably induced
anthropogenically during raking, transport, and/or machine-
sorting. None showed any signs of repair.
The pH in the experimental tanks holding the sixty specimens
fluctuated from 7.0 to 8.0 during the 16 mo interval (Fig. I). The
dissolved oxygen ranged from 3.4 to 6.7 mg/L o\er the same time
frame (Fig. 1). Temperature changed seasonally, peaking in the
summer at 25°C. and dropping to a low of 8°C in the winter
months (Fig. 1 ). Salinity fluctuated (sub) parallel with temperature,
ranging from a high of 28 ppt in November 98 to a low of 22 ppt
in March 99 (Fig. I). Current velocity from the pipe perforations
ranged between 16 cm/s and 24 cm/s over the 16-month interval
(Fig. I)
Of the sixty experimental specimens. 30 were fractured sub-
lethally (Fig. 2A-G) and one lethally (Fig. 2H). Of those fractured
sublethally. the crack was visible on the valve interior exclusively
in 20 specimens. For 10 specimens, the fracture was evident on
both the valve interior and, faintly, on the exterior (Fig. 2A; Table
I ). In 12 specimens, the fracture occurred in both opposing valves
(Table 1; Fig. 2A). Eleven specimens had a crack in the right valve
only and seven had a fracture in the left valve only (Table I).
Shhll Repair in Reburrowlu Hard Clams
691
— A —
Temperature (deg. C)
Current Velocity (cm/sec)
Salinity (ppt)
DO (ppm)
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28
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25
24
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1 I I r
Month and Year
Figure I. Monthly fluctiiutions in miinitiiri-d ahlolic variables in flo»
tlirouHh tanks with \l. iiurciiiaria at NOAA laboratory. Sandy Hook,
New Jersey.
Fracture length varied from 4 mm to 38 mm. Very long, stuccoed
fractures propagated to within 5 mm of the dorsal margin of the
clam (Fig. 2B). One specimen had three fractures in one valve and
five specimens had two fractures in one valve. Short cracks (.'i-lO
mm) that extended dorsally from or near the ventral margin to the
pallial line, but not beyond, were unrepaired ( = unstuccoed; Table
1). However, cracks longer than l.'imm. extending dorsally beyond
the pallial line, were repaired by the mantle (Table 1 ).
Fractures were initiated at or very near the ventral margin (Fig.
2A and B) in 24 specimens, at the posterior margin (Fig. 2C) in
four specimens, and the anterior margin in two specimens. Good-
ness of fit test revealed that this distribution is nonrandom (Table
1 ). Thirteen fractures radiated inward on a diagonal from the ven-
tral valve margin, slightly oblique to the dorsal-ventral axis (Fig.
2D; Table I ). Six fractures curved and three had sharp right angle
deflections cutting through the edge of the adductor muscle scars
in two instances (Fig. 2E-F; Table 1 ). Two fractures bifurcate near
the center of the valve (Fig. 2G). Two cracks converge inward
from the ventral margin. One convergence of cracks resulted in a
lethal fracture (Fig. 2H).
Thirty-six specimens (62'?^) have chipped ventral margins.
Nevertheless, only 13 of the 30 fractures radiated dorsally from a
chipped point on a valve margin (Fig. 3A and B). Resecretion of
a small, v-shaped wedge to fill in the chipped ventral margin
accoinpanied mortaring of the fracture in one specimen (Fig. 3C).
Seventeen fractures became fainter between the pallial line and the
ventral margin, and cannot be traced to the very edge of the shell.
Although the highest frequency of sublethal shell breakage oc-
curred in clams burrowing into pure mud (9 of 12), the distribution
of fractures is statistically randoin (Fig. 4). Ventral margin thick-
ness had no bearing on which valves fractured (Table 1 ). Fractures
were just as likely to be confined to one valve as to be mirrored in
both valves (Table 1). Furthermore, valve fractures occurred
mostly in mud-burrowing specimens (Fig. 4). and appears to be to
be independent of the degree of external valve abrasion, which is
most severe in sand-shell burrowing specimens (Fig. 5). Two
thirds of the specimens that bunowed in sand had the concentric
lamellae obliterated on all areas of the valves (Fig. 3D), whereas
18 specimens of the 24 that burrowed into mud and mud-shell had
only slight ventral abrasion (Fig. 5).
Accretion along the ventral margin was suppressed under these
experimental conditions. Mean annual increa.se in dorsal-ventral
shell length varied from only 0.45 mm in clams kept in sand to 1.3
mm for clams kept in mud (Fig. 6). Clams reared in sand and
shell-sand showed an annual decrease (<0.05 mm) in cross-
sectional shell height whereas clams reared in mud and shell-mud
showed an annual increase of -0.3 mm in cross-sectional shell
height (Fig. 7).
DISCUSSION
These experiments indicate that monthly reburrowing by young
adults increases the risk of either self-induced shell-breakage or
the propagation of fractures induced anthropogenically. The fact
that 30% of the experimental clams had fractures when sacrificed
but only 14% of the control group had fractures indicates that the
burrowing process is responsible for initiation and propagation for
many, if not the majority of fractures. These experiments do not.
however, indicate a threshold of reburrowing frequencies at which
fracturing is likely to be initiated or expressed.
The rate of repair of the fractures also cannot be precisely
established, although repair of fractures induced by burrowing
possibly occuiTed between inonthly rebunowing episodes. In a
separate study, seed of M. merceiiaria 15-25 mm in dorsal-ventral
length were able to resecrete 2- to 3-mm long notches beveled by
a high-speed Dremel in the anterior, posterior, and ventral valve
margins within 2 weeks while living caged on an intertidal flat in
North Carolina (Fig. 8; Alexander c& Dietl. in preparation). Serra-
tions or contiguous scallops in the valve posterior (Fig. 3E) may be
lethally inflicted on young adult, intertidal M. merceiiaria by wad-
ing birds (Krauter 2001). Conditions on the North Carolina mud-
flat subjected to tidal flushing facilitated rapid repair with preclu-
sion of predators. The shell repair processes may have been re-
tarded under suboptimum laboratory conditions at Sandy Hook,
New Jersey. Furthermore, internal fractures inay be stuccoed (Fig.
2) at a different rate than notches of the valve margin are filled in
by resecreted shell (Fig. 8). Nevertheless, the repair of the internal
fractures within one month, i.e.. between reburrowing sequences,
is a realistic estimate given the much shiirter time it takes to repair
notches around the valve inargin.
Regardless of the timing of initiation of the fractures, or their
692
Alexander and Baron
Figure 2. Expression of internal, stuccoed, sublethal fractures, and lethal breakage in valves of M. mercenaria that repeated!) reburrowed in
sediment. Width of bar = one cm. A, External and internal expression of sublethal fractures in opposing valves. B, Stuccoed fracture radiating
from posterior-ventral margin to within 5 mm of dorsal hinge. C, Stuccoed fracture radiating from posterior margin. D, Stuccoed Hnear fracture
from ventral margin. E, Stuccoed ventral fractures that merge and dellect through edge of adductor muscle scar. F. Stuccoed fracture that makes
right angle deflection through adductor muscle scar. G, Bifurcating, stuccoed fracture radiating fnmi ventral margin. H, Lethal fractures that
merged, resulting in removal of large triangular piece of valve.
propagation, during the months of reburrovving. these experiments
complement the experimental results of Checa (1993) who dem-
onstrated that reburrowing only onee fractured the valves of the
deep-burrowing Solecurtus strii^akinis. However, fractures in the
shallow-buiTowing M. mercenaria were not necessarily invariably
induced by sediment-loading against the hardclam valve exteriors
as advocated by Checa (1993) for S. sirigalatus. First, the experi-
mental hard clam specimens never burrowed deeper than 10 cm
(maximum sediment depth 14 cm) in contrast to the deep burrow-
ing (>40 mm beneath the sediment surface), thin shelled clams
studied by Checa (1993). Second, three times as many fractures are
visible on the in the interior of the valve rather than the exterior of
M. mercenaria. Yet all of Checa's (1993) illustrated examples
show external expression of the fractures. Eschewing those speci-
mens fractured by anthropogenic handling, these observations are
congruent with the argument that closure of the valves on sediment
grains or shell shards introduced between the valves fractured the
ventral margin and valve interior of many if not most of the speci-
mens.
The high percentage of cracks (677f ) that did not propagate
from the valve interior to be expressed on the valve exterior indi-
cates that fracture propagation was halted at the annual growth
increment discontinuities in the shell microstructure of M. merce-
naria. The valve microstructure consists of overlapping layers of
crossed lamellar aragonite (Boggild 1930) bounded by organic
films (Pannella & Maclintock 1968. Rhoads & Pannella 1970.
Kennish 1980). Although all but six of the fractures were initiated
near the ventral margin, the fact that 1 7 of the 30 cracks did not
radiate from a chipped point on the valve margins, but instead
disappear within 1 to 2 mm of ventral margin, suggests that chip-
ping of the margin is not invariably the progenitor of fractures. The
faint expression of the fractures in the area between the pallial line
and the ventral margin coincides with the thicker part of the shell
relative to shell thickness dorsal to the pallial line. Fractures may
have originated dorsal to the pallial line, dissipating before crack-
ing the entire thicker area between the pallial line and the ventral
margin.
Although contrasting sediment textures did not statistically sig-
nificantly differentiate the frequency of fractures among this
sample of M. mercenaria (Fig. 4). the greater frequency of sub-
lethal fractures among clams that burrowed in mud (nine) vs. sand
(three) and shell-mud (six) is counterintuitive. If adduction of the
valves upon clasts introduced between the valves during burrow-
ing caused the fractures, the probability of encountering shell
Shell Repair ln Reburrowed Hard Clams
693
TABLK 1.
Distribuliun uiid iiiurphulogy of fractures induced b) burrowing of 60 specimens of Merceitaria merceiiaria.
Mean ventral valve thickness of
fractured vs. unfractured shells
Mean length for stuccoed
vs. unplastered shell fractures
Location of fracture initiation
on valve marsin
Expression of Valve interior Valve exterior Both sides of
fracture only = 20 only = 0 valve = 10
Fractures =
Unfractured
=
1.5 mm
1.5 mm
Stuccoed =
Unplastered
=
20 mm
12 mm
Posterior
Anterior
Ventral
margin =
= 4
margin =
■>
margin =
■)^
Fracture-affected Right = 1 1
valves
Left
Both
12
Propagation of Dorsal-xentrally Dorsal-ventrally Rt. angle
fracture straiizht = 5 curved = 7 deflection = 3*
Merging and
branching =
Diagonal to
dorsal-ventral
axis = 13
r test; P = 0.71; Accept Ho
(means are equal)
; test; P < 0.001; Reject Ho
(means are unequal)
Goodness of Fit; x" = 29.6 with
2 df; Reject Ho at P = 0.01
(nonrandom distribution)
Goodness of Fit; x" = 20.0 with
2 df; Reject Ho at /> = 0.01
(nonrandom distribution)
Goodness of Fit; x" = 1- ^''h
2 df; Accept Ho
(distribution random)
Goodness of Fit; x' = 10.22 with
4 df; Reject Ho at P = 0.05
(nonrandom distribution)
* Two stuccoed fractures cut across muscle scar area.
shards during burrowing would be highest in the sediment admix-
tures with ly/c by volume shell hush. Furthermore. buiTowing in
sand increased external shell abrasion, including the ventral mar-
gin. (Fig. 5). but any ensuing chipping of the ventral margin did
not increase the frequency of fractures propagated dorsally. As
previously noted, only 13 of the 30 fractures can be traced from a
chipped point on the posterior-ventral margin. One possible ex-
planation is that the initial commercial excavation and handling of
the specimens induced the fractures, and more specimens with
microfractures were fortuitously placed on the muddy versus the
sandy susbstrata. Given the probability of the low percentage
(14%) of specimens with fractures induced before the bun'owing
experiment commenced, based on extrapolation from the control
group, it is unlikely that a preponderance of the lew clams frac-
tured before commencement of the experiments were experimen-
tally placed on mud.
It should be noted that the interstitial water in the sand and
sand-shell hash had become blackened during the experiments
with accumulated fecal tnatter in the sediment interstices a few cm
beneath the sediment surface before the conclusion of the experi-
ments. This accumulation of organic matter occurred despite hand-
tilling of these sediments each month during excavation of the
specimens. Valve surfaces became slightly chalky in appearance,
but if the valve skeletal microstructure was altered and mechani-
Figure ,1. \ alves of A/, merceiiaria with chipped margins, resecreled val>e wedges, and degree of abrasion following miinlhjy reburrowing into
sediment. \\ idth of bar = one cm. .\, Specimen with chipped ventral margin from which crack radiates dorsallv. B, \ enlrallv chipped margin
with faint expression of dorsally radiating fracture. Note also abrasion of concentric micni-ornament limited to ventral margin of specimen. C,
Specimen with secreted wedge at ventral margin filling In small triangular piece of shell removed bv cracks. Internally, fractures are stuccoed.
D, Complete obliteration of micro-ornament on valve exterior of specimen that monthly reburrowed into sand. K, I'redator-lnduced. contiguous
divots at posterior shell margin
694
Alexander and Baron
Reburrowed Mercenaria mercenaria
[3 Repaired Fracture; mean valve thickness = 1.5 mm
H Lethal Fracture; mean valve thickness = 1.5 mm
■ Unfractured; mean valve thickness = 1.5 mm
Experimental Sediment Substratum
Figure 4. Frequency of fractures among specimens of M. mercenaria
that reburrowed monthly in various sediment textures. Distribution Is
random according to Goodness of Fit test (x" = 4.52 with 4 df).
cally weakened by the change in interstitial water chemistry, it
didn't facilitate the initiation of more fractures than specimens that
rebuiTOvved in muds (Fig. 4). Clams that repeatedly reburrowed in
mud did not show the same degree of loss of surface ornament
(Fig. 5). Reburrowing in abrasive sand, accompanied by etching
of the shell exterior by the interstitial water did significantly re-
tard the expected annual increase in cross-sectional shell height
relative to that shown by clams burrowed in mud and mud-shell
hash (Fig. 7).
A valve thickness threshold may exist at which shell fracture
due to burrowing does not occur (Table 1 ). but it could not be
unequivocally established by this investigation. All of the speci-
mens in this investigation that cracked had a valve margin thick-
ness along the dorsal-ventral axis of less than 2.0 mm just ventral
to the pallial line. The four specimens with a ventral margin valve
thickness greater than 2.0 mm did not bear fractures. This inves-
tigation deliberately used similar size young adults (mean DV
length 37 mm; std dev. 4 mm) to minimize ontogenetic (age)
effects on experimental results. Expanded experiments should use
a wide range of hard clam sizes to determine if a size threshold for
burrow ing-induced fracture exists.
The question can be raised as to whether young adult (30—40
min in dorsal-ventral length) hard clams show such reburrowing-
induced fractures naturally in their native substrata, or if the fre-
quency of repair in the experimental clams is merely an artifact of
shell fatigue under suboptimum conditions in sediments in holding
tanks where they reburrowed monthly. A specimen of A/, merce-
naria collected from the field shows very siinilar internal fractures
to Figure 3A, but this is only one individual out of .'iOO specimens
re-examined from a collection analyzed for repair scars from
Tuckerton NJ (Alexander & Dietl, 2001 ). Se\eral specimens have
fractures similar to those in Figure 2, but they lack the stuccoed
thread-like ridge o\er the crack. Without the stuccoed repair ridge,
it cannot be determined if the crack occuued during the life of the
clam or during its post-mortem, transportational history. Greg
Dietl (personal communication) forwarded a photograph of a farm-
raised hard clam from North Carolina that has an internal fracture
and repair in both valves similar to Figure 2B. These anecdotal
occurrences of internal, stuccoed fractures from field collections
belie the high frequency of fracture and repair found in the ex-
periments. The disparity suggests that the anthropogenic handling
of the specimens and/or the strain induced by monthly reburrowing
contributed to internal fracturing of the shell in the experiments.
Regardless, these artificial experimental stresses did not pre-
clude repair of the fractures by the mantle tissue. Given that many
repairs were probably followed by re-burrowing episodes, the stuc-
coed repair process is sufficiently strong to enable the overwhelm-
ing majority of clams to repeatedly stress the valves during rebur-
rowing without a repaired fracture failing lethally. Whatever the
percentage of fractures induced anthropogenically before the re-
burrowing experiments commenced, which based on the control
group could be approximately \4'7c. or 7 specimens, the repairs
withstood the repeated strain in the shell due to reburrowing as
many as 1 2 times. Thirty specimens had fractures in the valves, but
only one specimen fatally cracked its valves during monthly re-
burrowing over a 12-month period (Fig. 2H). The dysoxic pore
water beneath the sediment surface, and diminished supply of
plankton flowing through the holding tanks may have contributed
to the severely retarded accretionary growth (Fig. 6), but these
DEGREE OF VALVE ABRASION
03 negligible
Q slight - ventral
[D moderate - ventral & central
■ extensive - all surface area
12 n
Experimental Sediment Substratum
Figure 5. Frequency of various degrees of valve abrasion for speci-
mens that reburro\>ed monthly into various sediment textures.
Shi I L Rfpair in Rkburrowed Hard Clams
695
Mercenaria mercenaria
One Way ANOVA
p < 0.079
1.4
1.2
ti
t« . 4
c .2
■a
■o
c
■o
3
s
c
n
(0
c
V)
E
V)
1
3
T3
V
E
3
E
Experimental Sediment Substratum
Figure 6. Mean increase (delta mm) in dorsal-ventral length (mm I of
specimens of M. mercenaria that reburrowed monthly in various tex-
tured sediments. **Mean increase is signillcantlv greater tha// value
for sand (P = O.Oll and sand-shell (/' = ().02l according to Fisher's
PSLD test. Mean ventral margin thicknesses are not signit'icantly dif-
ferent among specimens reburrovving into different sediments based
on analysis of variance (ANOVA; P = 0.067). Sample size = 60.
suboptimum conditions did not prevent the mantle from stuccoing
the fractures.
This investigation on hardshell clams also shows that Checa's
(1993) investigation on burrow ing-induced fractures and repair is
not necessarily a phenomenon restricted to thin-valved, deep-
burrowing clams, although the frequency of reburrowing necessary
to fiacture the valves may be an order of magnitude higher for
thick shelled clams and less likely to occur naturally in their native
habitats. This investigation should prompt bivalve functional inor-
phologists interested in shell biomechanics to search for internally
stuccoed fractures in field surveys of shells of a variety of ven-
erids, not just M. niercciniria. Just as external shell repairs in
commercially valuable clams may be an indicator of shellfishing
pressure (Bergman & Hup 1992, Caspar et al. 1994, Witbaard &
Klein 1994, Ramsey et al. 2000), frequency of stuccoed micro-
fractures expressed on the interior of valves may indicate the his-
tory of both naturally and anthropogenically caused excavations
and reburrowing episodes experienced by a clam population. Re-
pair frequencies also may reflect the physiochemical conditions
beneath the sediment surface in which commercially valuable
clams reburrowed. Fracture repair may have impact on accretion-
ary growth rates of hardclams yet to reach harvestable sizes.
CONCLUSIONS
Reburrowing into the substrata by M. mercenaria may either
induce sublethal shell fractures, or further propagate fractures in-
duced by anthropogenic excavation and handling processes. Tex-
ture of the sediment (sand, mud. sandy mud, shelly sand, shelly
mud) may not necessarily differentiate frequencies of burrowing-
Mercenaria mercenaria
One way ANOVA
P< 0.0018
E
E,
£
'5
I
Q)
w
<
c'
(0
0)
S
.35
.3
.25
.2
.15
.1
.05
0
05
T3
C
(0
w
TJ
3
E
■a
c
(S
u
I
■a
3
E
■o
c
10
in
•a
3
E
Experimental Sediment Substratum
Figure 7. Mean net change (delta mm) in shell height in cross-
sectional, lateral profile for M. mercenaria that reburrowed monthly in
different textured sediments. **Mea« value significantly greater vs.
sand and sand-shell, (P < 0.01), as well as mud-sand iP = 0.025) ac-
cording to Fisher's PSLD test. ##MeaH values significantly greater vs.
sand, shell-sand, and mud-sand (P = or < 0.01) according to Fisher's
PSLD test. Sample sizes = 60.
propagated fractures. Nevertheless, adduction of valves on sedi-
ment grains and shell shards can induce the strain that initiates or
propagates fractures; sediment-loading against the valve exterior is
not the likely culprit of fracture propagation in the shallow-
bunowing Mercenaria mercenaria. Shell surface micro-ornament
may be completely abraded and/or corroded away by repeated
reburrowing in organic-rich sands with dysoxic pore water condi-
tions beneath the sediment surface. Nevertheless, such abraded and
etched shells are no more susceptible to fractures than shells of
clams that repeatedly reburrowed in muds.
Fractures are most often initiated at or very near the ventral
margin, rather than shell posterior or anterior margin. Fractures
that do not extend dorsal to the pallial line are not likely to be
repaired. Fractures may extend beneath the adductor muscle and be
B
\
Figure 8. Resecreted shell in notched anterior (A), ventral (B), and
posterior (C) margins of seed of .1/. mercenaria after 2 weeks while
kept caged on a muddy sand tidal fiat near Masonboro Inlet, North
Carolina. Notches created by a Dremel in early October 2001. Width
of line is 2 cm.
696
Alexander and Baron
repaired. Fractures are mostly likely repaired (stuccoed) between
monthly burrowing episodes, given rates of shell regeneration in
marginally notched specimens. These repaired fractures withstand
the strain induced by repeated burrowing as evidenced by the fact
that only one of 30 fractures failed lethally.
ACKNOWLEDGMENTS
We thank Barb Boyd and Bruce Boyd of the Marine Academy
of Science and Technology of Mommouth County. New Jersey.
for dredging of the sediment samples, collection of shells for cre-
ation of shell hash, periodic monitoring of the abiotic conditions in the
flow through tank, as well as providing access to the NOAA labora-
tory at Sandy Hook. Jonathan Radcliffe and Daniela Zima. students of
the MAST, assisted in the measurements of the clams and monitoinng
of the abiotic conditions in the flow- through tanks. We appreciate the
ciitical suggestions of Greg Dietl. which greatly improved the manu-
script. Finally, thanks to the New Jersey Baymens Association for
donating the hardshell clams used in these experiments.
LITERATURE CITED
Ale.xander. R. R. & G. P. Dietl. 2001. Shell repair frei.|uencies in Neu
Jersey bivalves: A Recent baseline for tests of escalation with Tertiary
mid-Atlantic congeners. Pcilnios 16:354-371.
Bergman. M. J. N. & M. Hup. 1992. Direct effect of beanitrawling on
macrofauna in a sandy sediment in the southern North Sea. ICES. 7.
Mm: Sci. 49:5-11.
Boggild, O. B. 1930. The shell structure of mollusks. Del Kaiigelifit'
Danske Viedenskabemes Selskab Skrifter 9 raekke bind II. part 2.
Math. Afd 9 Raekke 223- 326.
Checa, A. 1993. Non-predatory shell damage in Recent deep-endobenthic
bivalves from Spain. Pohieoiieogv PciUu'tHlinitilol. PnUieoccol. 100:
309-331.
Caspar. M. B.. C. A. Richardson & C. C. Monteiro. 1994. The effects of
dredging on shell fonnation in the razor clam Eiisis siliqiia from Bar-
rinha. southern Portugal. J. Mar. Biological Assoc. U. K. 74:927-938.
Kennish, M. 1 978. Effects of thermal discharge on mortality of Mercenaria
mercenaria in Barnegat Bay. New Jersey. Environ. Geol. 2:223-254.
Kennish. M. 1980. Shell microgrowth analysis. Mercenaria mercenaria as
a type e.xample for research in population dynamics. In: D. C. Rhoads
& R. Lutz. editors. Skeletal growth of aquatic organisms. Topics In
geobiology. New York: Plenum, pp. 255-294
Kraeuter. J. N. 2001. Predators and predation. In: J. N. Kraeuler & M.
Castagna. editors. Biology of the hard clam. Developments in aqua-
culture and fisheries science. Amsterdam: Elsevier Science, pp. 441-
589
Pannella. G. & C. MacClintock. 1968. Biological and environmental
rhythms reflected in molluscan shell growth. In.- D. B. Macurda. editor.
Paleobiological aspects of growth and development: a symposium. Pn-
leomol. Soc. Mem 2. J. Paleonlol. 42(Suppl):64-80.
Ramsey. K.. M. J. Kaiser. C. A. Richardson. L. O. Veale & A. R. Brand.
2000. Can shell scars on dog cockles iCIycymeris glycymeris L.) be
used as an indicator of fishing disturbance? J. Sea Res. 43:167-176.
Rhoads. D. C. & G. Panella. 1970. The use of molluscan shell growth
patterns in ecology and paleoecology. Lethaia 3:143-161.
Witbaard, R. & R. Klein. 1994. Long-term trends on the effects of the
southern North Sea beamtrawl fishery on the bivalve mollusk Arctica
islandica L. (Mollusca. bivalvia). ICES. J. Mar. Sci. 51:99-105.
JiniriHil ol Shell full Rcseciivli. Vol. 22, No. 3. 697-703. 2UU3.
IDENTIFICATION AND INCORPORATION OF GROWTH AND SURVIVAL BOTTLENECKS IN
ECONOMIC MODELS OF NORTHERN QUAHOG (HARD CLAM), MERCENARIA
MERCENARIA MARICULTURE
JONATHAN H. GRABOWSKI,'* SEAN P. P0VVP:RS,- ' AND MARK HOOPER^
'University of North Carolina at Chapel Hill. Insiitiiie of Marine Sciences. Morehead City. North
Carolina 2S557: 'Department of Marine Sciences. University of South Alabama, Mobile. Alabama
36688: 'Dauphin Island Sea Lab. Dauphin Island, Alabama 36528; ^Hooper Family Seafoods.
Smyrna, North Carolina 28579
ABSTRACT Research ihal identifies potential bottlenecks in survival and growth penalties during the different phases of clam
grow-out is necessary to iiia.\imize the profitability of clam aquaculture and reduce pressure on already threatened wild stocks along
the Atlantic coast of the Eastern United States. In this study, initial planting density (489. 729. and 972 clams m"-) did not affect
survival (64.8-77.5%) during the first year of clam grow-out. Clams planted at the lowest density outgrew (greater final shell length.
SL, and individual clam volume) those planted at higher densities: therefore, clam growth was density dependent during the first year
of grow-out. In the second experiment, size of clams (26.2, 32.5. 37.7. and 42.(.) mm SLi planted after year one did not affect
survivorship (92.3-96.6%) or growth (36.2, 41.7. 45.1, and 49.2 mm SL, respectively). Evaluations of the economic feasibility of clam
culture demonstrated that clams planted at intermediate densities would result in the greatest return on the initial investment. To
increa.se the robustness of our economic feasibility analysis to interannual variations in clam survivorship and growth during the initial
year of grow-out. we pert'ormed an identical analysis with data from an eariier study. Taken together, these studies bracket a realistic
range of survivorship and growth during the initial year of clam grow-out: low survivorship and growth (this study) and high
survivorship and growth (earlier study). Based on this range, the estimated expected profitability ranged from $4893 to $7717 per
100.000 seed clams. In contra.st to aquaculture of other bivalve species (e.g., oysters), our analysis demonstrates that the profitability
of clam aquaculture is fairiy robust to substantial variations in market prices primarily as a result of the development of methods over
the last decade that enable relatively high survivorship with moderate growth penalties.
KEY' WORDS: Mfrcunariu nwicenaria. northern qiiahog. h;ud cUim. aquaculture. survivorship, growth, density dependence, eco-
nomic feasibility
INTRODUCTION
Bivalve aquaculture holds great promise in contributing to the
goal of sustainable and dependable production of seafood.
Whereas aquaculture of some marine species, primarily fish and
shrimp, is associated with a host of negative environmental effects
(e.g.. increased biological oxygen demand as a result of fecal
production (Silver! & Sowles 1996. Paez-Osuna et al. 1998. Tovar
et al. 2000), habitat loss associated with construction of shoreline
aquaculture facilities (Hopkins et al. 1995, Paez-Osuna 2001 ), and
introduction and propagation of pathogens (HaiA-ell el al. 1999)),
negative environmental effects of bottom or near-bottom culture of
bivalves are relatively minor (Kaiser et al. 1998. Nay lor et al.
2000). In fact, aquaculture of bivalves may contribute positively to
the local environment. Removal of phytoplankton as a result of
filter feeding may improve water clarity in coastal areas, thus
promoting the growth of sea grasses, which serve as essential
habitat for fish and crabs. Because many coastal estuaries have
experienced increased eutrophication in recent decades (Paerl et al.
1998), bivalve aquaculture could assist wild populations of filter
feeders remove excess nutrient loading.
Despite a relatively reliable market for northern quahog (hard
clam). Mercenaria mercenaria and the minimal environmental ef-
fects of bivalve aquaculture, hard clam aquaculture in many areas,
including North Carolina, has yet to reach its potential (Diaby
1997). Two of the primary obstacles hindering establishment and
* Corresponding author. Present address: University of Maine at Orono.
Darting Marine Center. 193 Clarks Cove Road. Walpole. Maine 04573.
E-mail: jgrabow@maine.edu
expansion of economically viable hard clam aquaculture are ( 1 )
restrictive regulations by states and (2) low and/or unpredictable
yields of clams on leases. The latter obstacle largely results from
heavy predation of seed clams (Carriker 1959. Castagna & Kraeu-
ter 1981. Peterson et al. 1995. Kraeuter et al. 1998), lower grovvth
rates of clams associated with many practices adopted to exclude
predators (Sumnierson et al. 1995. Grabowski et al, 2000). mor-
tality induced by clam diseases, variation in the quality of lease
sites for clam growth, and the frequency of natural perturbations
(e.g.. hurricanes and floods). Further hindering the development of
successful aquaculture initiatives is the relative paucity of eco-
nomic feasibility models that couple relevant biological informa-
tion with econoiTiic assessments. Specifically, bioeconomic mod-
els that identify and incorporate major survival and/or growth
bottlenecks during the entire grow-out phase while allowing for
fluctuations in market price are of critical importance in develop-
ing an industry that is competitive to wild harvest.
Profitable clam culture requires planting clams at densities far
above those found under natural conditions. If not mitigated, such
aggregations of potential prey items can greatly increase predator
efficiencies, resulting in severely reduced clam survival (Carriker
1959, Eldridge et al. 1976). Methods to reduce clam mortality rates
have involved identifying threshold seed sizes for planting and
appropriate times to plant seed clams in the field (Menzel et al.
1976. Whetstone and Eversole 1978. Manzi et al. 1986. Peterson et
al. 1995. Marelli & Arnold 1996. Grabowski et al. 2000). Further
reductions in predation have been achieved by planting clams in
gravel, nylon-mesh bags, or cages and possibly by using biological
controls (Castagna & Kraeuter 1977. Eldridge et al. 1979, Walker
1984, Bisker & Castagna 1989, Summerson et al. 1995, Kraeuter
et al. 1998, Fernandez et al. 1999). Because most of these protec-
697
698
Grabowski et al.
tive measures typically reduce clam growth (Grabowski et al.
2000). effective grow-out requires balancing increased sur\ivor-
ship with subsequent growth penalties. In a previous study, we
quantified clam growth and survivorship in bottom beds versus
tented bags and determined that tented bags increased survivorship
but reduced growth rates of clams (Grabowski et al. 2000). We
also determined that expected additional revenue from increasing
survivorship should more than compensate for potential lost rev-
enue as a consequence of slower growth rates during the first year
in tented bags. Eldridge et al. (19791 noted that survival rates were
greater for clams planted at higher initial densities. Eldridge et al.
( 1979) also found that clams planted at higher densities can take up
to an extra 12 mo to achieve legal size in South Carolina, which
could jeopardize the economic feasibility of clam aquaculture. Yet
it is uncertain whether increasing planting density during the first
year will affect survivorship or growth (i.e.. if these processes are
density dependent) enough to counterbalance associated reduc-
tions in costs of clam grow-out.
Aquaculture research has traditionally focused on the early
stages of clam grow-out (Peterson et al. 1995). .Although clam
mortality in the wild and in culture operations is typically greatest
during postlarval and early juvenile life history stages, survivor-
ship and growth rates of larger clams may be size or density
dependent (Eldridge et al. 1979). Further empirical tests are nec-
essary to determine whether the size of larger clams will affect
growth rates when planted at intermediate densities. Culture stud-
ies often assume mortality is extremely low after the initial stages
and use estimated mortality rates for the final stages when evalu-
ating the protltability of differing types of clam grow-out. Quan-
tifying survivorship and growth during the later stages of clam
grow-out is necessary to evaluate whether these assumptions are
valid and to enhance the reliability of economic models that proj-
ect the profitability of clam culture. Even if mortality is relatively
minor after the initial hatchery phase, small differences in survi-
vorship and growth at later stages may be critical in determining
profitability under marginal market conditions. Consequently, ef-
fective crop management requires identifying culture techniques
during each phase of grow-out that increase revenues relative to
costs.
In this study, we examined potential growth penalties and/or
survival bottlenecks within the first two years of clam grow-out.
Included in this effort were experiments designed to quantify the
relationships between seed clam planting density and growth w hen
using methods that offer substantial predator protection. In par-
ticular, we tested whether clam planting density during the first
year of grow-out in nylon bags, a widely used predator exclusion
technique in bivalve aquaculture. influences clam survivorship,
individual growth, and total yield. Further, we examined whether
differences in growth after one year of grow-out are propagated
throughout the second year or if compensatory growth reduces size
variation in older clams (Peterson 1979). Finally, results from both
of these experiments were incorporated into a cost-benefit analysis
designed to examine the profitability of manipulating planting den-
sities within a range of empirically derived survivorship and
growth conditions under varying market conditions.
MATERIALS AND METHODS
Experimental Grow-Out
In August 2000, seed clams (4—6 mm) were obtained from
Atlantic Farms, Inc.. Charleston, South Carolina, and placed into a
nursery system on the premises of Hooper Family Seafood.
Smyrna, North Carolina. In October 2002. seed clams were sieved
on a 10 mm screen to obtain clams of mean 13.7 mm shell length
(SL), with SL being the maximum measurement along the anteri-
or-posterior axis. Seed clams were planted at three densities (700,
1 050, and 1 400 clams per bag ) in three sets of 1 0 nylon bags, mesh
size 9.4 mm (stretch) and measuring 1.2 x 1.2 m. Each of the three
sets of nylon bags corresponded to one of the three densities of
clams. We planted seed clams in nylon-mesh bags because this
method resulted in greater survivorship and was more viable eco-
nomicallv than bottom beds (Grabowski et al. 2000). A random
sample of 50 clams w as measured for SL from four of the 1 0 nylon
bags of each initial density at the inception of the experiment. A
one-factor ANOVA confirmed that the initial SL of the three den-
sity classes was not significantly different (F,„ = 1.2: P = 0.35).
Nylon bags were interdispersed randomly on North Carolina shell-
fish lease 570 D in Midden's Creek, Smyrna, North Carolina. Each
nylon bag was sealed with a cable tie, staked down on each comer,
and raised in the center with a 30-cm-long PVC stake that pro-
jected 20 cm above the substrate surface. In January of 200 1 . the
center stake in each nylon bag was removed.
In October of 2000, one-year-old clams grown out under simi-
lar methods and at the same lease site as described in the previous
paragraph were collected. Clams were graded by shell thickness
using slotted graders (1.5. 1.9, 2.2, and 2.5 cm bar spacing) into
four distinct size classes (small, mean SL = 26.2 mm; medium,
mean SL = 32.5 mm; large, mean SL = 37.7 mm; and extra large,
mean SL = 42.0 mm). We then planted six sets of 500 clams of
each size class in 1.2 x 1.2 m bottom beds (24 total beds) and
covered the beds with 7 mm polypropylene mesh. Random
samples of 50 clams were measured for SL from three of the six
bottom beds for each size class. A one-factor ANOV.A confirmed
that the initial SL of the four size classes were significantly dif-
ferent (F3S = 129.2; P < 0.0001). The clams were planted in
shallow water (<I m below mean low water [MLW]), sandy sub-
strate. The 24 bottom beds were interdispersed on a subplot of
North Carolina shellfish lease 9102 near the premises of Hooper
Family Seafood.
In October of 2001, all nylon bags and bottom beds were har-
vested. Bottom beds were raked and then checked by hand to
ensure that all surviving clams were harvested. Every bag and bed
was sampled by counting all surviving clams, measuring a random
sample of 50 clams for length, and grading the clams using the
slotted grading system mentioned previously. The number of
clams in each grade was counted, and the displaced water volume
of a random sample of 50 clams from each graded size class was
quantified to estimate the entire volume of each replicate.
Statistical Analyses
Data were analyzed using separate one-factor ANOVAs for
clam survivorship and size of seed clams (density experiment) and
one-year-old clams (size class experiment). A one-factor ANOVA
was conducted to assess whether initial planting density influenced
clam survivorship. One-factor ANOVAs were also used to deter-
mine the effect of initial planting density on the following size
parameters; individual SL. individual volume of clams, and total
V olume of clams. A second set of one-factor ANOVAs were con-
ducted to determine the effect of initial clam size of planted one-
year-old clams on percent survivorship and all three size param-
eters. Prior to any of these analyses, data were tested for homo-
Economic Viabilit'i' of Hard Clam Culture
699
geneity of variance using Cochran's test (Underwood 1981). The
analysis of the effects of planting density during the first year of
grow-out on the individual volume of clams required square-root
transformation to remove the heterogeneity of variance. Post hoc
contrasts were performed on all significant effects detected by the
ANOVAs using Fisher's protected least significant difference
(PLSD) test (Day & Quinn 1989).
Economic Analyses
Cost-benefit analysis was conducted to assess the economic
implications of differing culture methods used in our study. We
first evaluated whether reduced revenues from any survival bottle-
necks or growth penalties from planting seed at higher density
during the first year of clam culture outweigh the reduced cost
created by planting clams at higher density. Projection of revenues
from this size of operation was achieved using the results of the
first two years of clam grow-out to estimate the number of clams
that would survive to be harvested in subsequent years. From data
collected in the first experiment, we determined the proportion of
clams that grew to each size grade (<1.5 cm. 1.5-1.9 cm. 1.9-2.2
cm. and 2.2-2.5 cm shell thickness) after one year for all three
planting densities. From data collected in the second experiment,
we could then determine the proportion of clams from each of
these size categories that attained legal size (>2.5 cm shell thick-
ness) after one additional year of grow-out. For those clams that
did not attain legal size after two years, we estimated the time to
legal size by ( 1) determining which size category they grew into
after the second year of growth and (2) projecting future growth by
determining the proportion of two-year-old clams in each of the
size classes that would grow to legal size after one or more addi-
tional years of grow-out. Using this series of calculations, we were
able to project the time duration of clam grow-out and the timeline
of harvests for clams planted at each density during the first year
of grow-out (75% legal after 48 months). Clams in North Carolina
typically grow to legal size in two to four years depending on
several physical and biological factors associated with grow-out
location. Clams that achieved legal size in each projected year of
grow-out were multiplied by a price of IS? per clam, the average
market price in North Carolina for clams at or just above the legal
size over 1998-2001. and discounted at an annual rate of 39r.
The costs (i.e.. labor, disposable supplies, equipment, bottom-
water lease, electricity, and seed clams) of planting 100.000 seeds
were estimated from records of Mark Hooper's clam culture op-
erations over the past half-decade. Based on informal surveys of
other clam culturists in North Carolina, we are confident that
Hooper's operations are representative of hard clam culture in the
region. Costs of equipment such as nylon bags and bottom-bed
materials were factored in under two scenarios; ( 1 ) actual, all costs
incurred and (2) annualized, equipment costs projected over a
five-year lifespan (i.e.. equipment would be used for future crops).
To evaluate the robustness of hard clam aquaculture to fluctuation
in market price, we calculated the break-even clam price at which
revenues still could meet or exceed expected costs given the pro-
jected streamline of clam harvests. The break-even price (P^) was
calculated as follows:
P.=
Costs
^H*\/i\ +d)'
(1)
We next compared results frotii the first year of grow-out to
those from our previous study (Grabowski et al. 2000) to deter-
mine how variability in growth and survival in the first year of
grow-out influences the profitability of clam aquaculture. In 1999.
seed clams were planted at a density of 700 clams per bag using a
similar range of seed sizes (Grabowski et al. 2000); therefore, we
compared economic estimates derived from survivorship and
growth parameters of the first year of grow-out in 1999 to tho.se in
2000 (this study). For each year, actual and annualized costs were
subtracted from revenues, which were calculated using a price of
18e and a discount rate of 3%. Finally, we compared the profit-
ability and break-even price of 1999 versus 2000 operations.
RESULTS
Experimental Grow-Out
Initial planting density did not affect percent survivorship
among the three density treatments (F,,, = 1.6. P = 0.23). and
survivorship ranged from 71. 291- (low density) to 77.5% (medium
density) and 64.8% (high density). Initial planting density did af-
fect SL after one year of grow-out (Fig. I; one-factor ANOVA
F2.27 = 8.7. P = 0.001). Shell length in low-density bags was
significantly greater than SL for both mediuin- and high-density
clam bags {P < 0.05 for both comparisons), but medium- and
high-density treatments did not differ (P = 0.59). Initial planting
density also influenced individual clam volume (one-factor
ANOVA F-, ,7 = 6.9. P = 0.004). which was also significantly
greater in low-density bags than in either medium- or high-density
clam bags (P < 0.05 for both comparisons). Individual clam vol-
ume for medium- and high-density clam bags did not significantly
differ (P = 0.26). Finally, initial planting density did not affect the
total clam volume per sample (one-factor ANOVA F, n-, = 1.5, P
= 0.25). which ranged from 2034 mL/replicate (low density) to
2500 mL/replicate (medium density) and 2559 mL/replicate (high
density).
In the second experiment, we tested w hether clam planting size
after one year of growth affects survivorship and growth during the
second year of clam grow-out. Clam planting size marginally af-
fected survivorship after the second year of grow-out (Fig. 2;
one-factor ANOVA F, ,(, = 2.8, P = 0.06). Post hoc comparisons
indicated that the small size class had significantly lower survival
28.0 -
I 27.0 -
£ 26.0 -
ot
S 25.0
.J
=5 24.0
23.0
22.0
21.0
I
where H, is the number of clams harvested in year i. d is the
discount rate (3%), and costs are as mentioned previously.
Low Medium High
Initial Planting Densit>-
Figure I. Final clam shell lengtii after 1 y of grow-out in nylon bags:
low density, 26.6 mm; medium density, 24.3 mm; high density, 23.2
mm. Frror bars are +1 SE in = 10 for each planting density). Letters
above bars signify post hoc results (different letters denote significant
differences al P < 0.05, Fisher's PLSD).
700
Grabowski et al.
50
Small
(26.2 mm)
Medium
(32.5 mm)
Large
(37.7 mm)
Extra Large
(42.0 mm)
Initial Clam Size
Figure 2. Clam survivorship after the second year of clam grow-out:
small, 92.3%; medium, 96.5%; large, 96.6%; extra large, 95.9%. Er-
ror bars are -fl SE (;i = 6 for each clam size). Letters above bars signify
post hoc results (different letters denote significant differences at P <
0.05, Fisher's PLSD).
than the other three size classes (P < 0.05 for all three compari-
sons), and that the three larger sizes did not differ from each other
{P > 0.05 for all three comparisons). Clam planting size signifi-
cantly affected clam SL after the second year of grow-out (Fig. 3;
one-factor ANOVA F, ,o = 160.1. P < 0.0001). The ranking of
final SL was consistent with differences in initial clam planting
size (P < 0.0001 for all comparisons). The results of both volume
measurements were consistent with the results from the analysis of
final clam size (SL). Clam planting size also influenced individual
volume per surviving clam (one-factor ANOVA F, ,0 = 160.6, P
< 0.0001 ) and total volume of surviving clams per replicate (one-
factor ANOVA F, -
139.3, P< 0.0001).
Economic Analyses
Cost-benefit analysis determined that planting clams at an in-
termediate density of 1050 clams per bag during the first year of
grow-out resulted in the greatest projected return on the invest-
ment. Clams planted at the intermediate density were 25.1% and
33.2% more profitable than clanis planted at the high ( 1400 clams
per bag) and low (700 clams per bag) densities, respectively (Table
1 ). Annualizing equipment expenses over a more realistic time
period of five years increased overall profits by an average of
18.9% in 2000. After annualizing equipment costs, cost-benefit
analysis again determined that profits were greatest from clams
planted at the intermediate density (Table I ). Under this scenario,
profits from clams raised at low densities were slightly greater than
profits of clams at high densities (Table I ). The break-even price
ranged from I l.Otf (low) to 9.2«! (medium) on the actual expenses
and 9.2v^ (low) to 7.9v; (medium) when expenses were annualized
(Table I ), which was substantially lower than the price ( 18^^) used
to calculate projected revenues. Projected profits after the initial
year of grow-out in 1999 (high survivorship and growth) were
72.3% higher for actual expenses and 57.7% higher for annualized
expenses than profits based on data from 2000 (poor survivorship
and growth).
a
1
Small
(26.2 mm)
(37.7 mm)
Extra Large
(42.0 mm)
Initial Clam Size
Figure 3. Final clam size after the second year of clam grow-out:
small, .16.2 mm; medium, 41.7 mm; large, 49.2 mm; extra large, 45.1
mm. P>ror bars are -i-l SE in = 6 for each clam size). Letters above
bars signify post hoc results (different letters denote significant differ-
ences at P < 0.05, Fisher's PLSD).
DISCUSSION
Empirical assessments of clam aquaculture have attempted to
identify the magnitude and scope of survival bottlenecks and
growth penalties associated with differing culture methods and
techniques. Unfortunately, methods that increase survivorship of-
ten are associated with subsequent growth penalties (Grabowski et
al. 2000). Assessment of the economic consequences of survival
bottlenecks and growth penalties associated with each culture
method is necessary to maximize the profitability of hard clam
culture ventures and to determine the price levels where revenues
exceed costs. In this study, we determined the degree to which
survivorship and growth were affected by ( I ) seed planting density
during the first year of grow-out and (2) size of 1-y old clams in
the second year of grow-out. Both of these experiments wei'e in-
corporated into cost-benefit analysis of hard clam culture to iden-
tify if any reductions in survivorship or growth penalties associ-
ated with planting density during the first year of grow-out would
impact profitability.
Although survivorship did not vary statistically with planting
density, planting clams at the higher density ( 1400 clams per bag)
reduced survivorship by 12.7% and 6.4%. in comparison to me-
dium- (1050) and low-density (700) bags, respectively. Similar
results (i.e., clam survivorship is unaffected by planting density
during the first year of grow-out if clams are protected from pre-
dation) have been shown by other studies (Summerson et al. 1995,
Fernandez et al. 1999). Eldridge et al. (1979) found in South
Carolina that seed clams (13.0 mm SL) planted at low densities
experienced higher mortality rates, which they attributed to pre-
dation rather than competitive exclusion. These studies suggest
that if survival bottlenecks in clam culture exist as a consequence
of clam density, they occur at smaller sizes and during the hatchery
or nursery phases of grow-out. Furthermore, choosing an appro-
priate method of grow-out that protects clams against local preda-
tors might be more important to patterns of survivorship than
initial planting density (Summerson et al. 1995). It is important to
note that although these relatively small differences in survivor-
ship rarely meet a formal statistical threshold for detecting differ-
Economic Viability of Hard Clam Culture
701
TABLE 1.
Kcunumic cvuluuliun of (A) pluntin;; clams at difterent dcnsitits and (H) tariatiun in the first year of grow-out ( IW) vs. 2000 results).
(A) Planting Clams at Different Densities in the First Year of Grow-out (700, 1050, and 1400 per bag)
Actual
Annaulized"
Planting!
J Density:"
Low ($1
Medium ($)
High 1$)
Low ($)
Medium ($)
High ($)
Cosloflabor (SlO/h)
3,453
3.145
2.561
3,453
3.145
2.561
Supplies
243
195
171
243
195
171
Equipment
1.236
983
849
247
197
170
Electricity
\5i)
150
150
150
150
150
Lease price
10
10
in
10
10
10
Clam seed
1,000
1.000
1.000
1,000
1,000
1.000
Total costs
6.042
5.483
4.741
5,103
4.697
4.062
Projected revenues
9,995
10.683
8,897
9,995
10.683
8.897
Net present
value (NPV)
3.904
5.200
4.156
4,893
5,987
4,835
Break-even
price
0.110
0.092
0.096
0.092
0.079
0.082
(B) Comparison of Projected Range of Earnings from 1999 and 2000 Data
(irovvth and .Survivorship Comparison
Grow-Out Year 1999 2000
Initial planting size (SL)
Survivorship after 1 y
Mean size (SL) after I y
13.9 mm
90. 1 '7r
32.4 mm
13.9 mm
71.2%
26.6 mm
Actual
1999 ($1
2000 ($)
Total costs
Projected revenue''
Net present value (NPV)
Break-even price
6.599
6,092
13.326
9,995
6.728
3.904
0.089
O.IIO
Annualized"
1999 ($)
2000 ($1
5,610
5,103
13,326
9,995
7,717
4,893
0.076
0.092
" Annualized costs accounts for equipment expenditures that were spread over 5 y.
"Clams were planted at 700 per bag in 1999; therefore, 1999 results are compared to the low density irealnient in 200(1.
" Revenues were estimated at a price of $0. 18/clam and 3% annual discount rate.
ences, the economic impact of these declines still affect profitabil-
ity of clam culture, particularly in years of poor growth and sur-
vivorship.
Increasing clam density did negatively impact clam growth
(both in terms of SL and volume): clams planted at the low (700
clams per bag or 489 clams m~") density grew larger than clattis
planted at either of the higher ( 1 050 and 1400 clams per bag or 729
and 972 clams m"") densities. Total clam yield at the end of the
first year of grow-out did not differ among the three treatments
because clams at the low density grew larger than clams at either
of the higher densities, thus providing further evidence that clam
growth was density dependent. Eldridge et al. (1979) showed in
South Carolina that seed clams planted in oyster trays protected
with 9.0 mm mesh cloth at a density of 290 clams m"" were
significantly larger than similar-size clams planted at 869 or 1 1.59
clams m""". Fernandez et al. ( 1999) planted larger (21.1 mm) seed
clams in 10.5 mm nylon mesh bags (1.2 x 1.2 m) at Oak Hill.
Florida, at densities of 520, 694, and 866 clams m"" and found no
difference in SL among the density treatments after nine months of
growth. However, they did find a greater proportion of legal-size
clams in the low-density treatment than in the medium and high
densities. Differences in findings between Fernandez et al. ( 1999)
and our study could be explained by their use of a narrower density
range, larger initial seed size, or protective-mesh size. More plau-
sibly, differences between the quality of grow-out conditions (e.g..
physical/chemical variables, phytoplankton supply, abundance of
fouling organisms) between areas used by Fernandez et al. ( 1999)
and our study explain the dichotomy in findings. In areas that
experience marginal growth conditions, clam cultures would ex-
perience more pronounced density-dependent growth penalties
(Powers & Peterson 2000).
Our findings demonstrate that methods which effectively re-
duce clam predation not only reduce clam growth but also result in
more pronounced growth penalties at higher densities where food
limitation is presumed to be more inten.se. If risk of clam kiss to
theft or from hurricane damage is great, the interest rates and thus
the rate of inflation are high, or \olatility in clam prices is con-
siderable, growth penalties associated with planting clams at high
densities may decrease overall profitability and add substantially
more risk to grow-out success.
Results from the second experiment suggest that survival is
very high and largely independent of size during the second year
of clam grow-out. Eldridge et al. (1979) also reported high survi-
vorship of clams after the initial six months of grow-out. Thus,
clam mortality is predominately an issue for clam growers during
the nursery phase and the first year of grow-out. Differences in
individual clam size and total clam yield in volume after two years
of grow-out were in direct proportion to differences after one year
of growth. Therefore, growth penalties resulting from culture tech-
niques in year I are propagated unmodified through the additional
702
Grabowski et al.
years of grow-out. The results of our study as well as that of
Eldridge et al. (1979) demonstrate that investigation of the tem-
poral sequence of growth penalties and survivorship can produce
more precise crop management and further the profitability of
aquaculture endeavors.
Economic analyses suggest that planting clams at intermediate
(729 clams/m~") densities should increase the profitability of clam
culture operations. Therefore, the reductions in operating costs
associated with planting at higher densities are initially greater
than the lost revenues from growth penalties and lower clam sur-
vivorship. However, increasing clam densities even further (972
clams/m"-) during the first year of growth eventually results in
reduced profit margins as a consequence of survivorship and
growth penalties. If market demand for clams does not meet recent
increases in clam production from several southern coastal states
within the eastern United States, clam prices could continue to fall
and threaten the viability of aquaculture operations. Planting clams
at intermediate densities should reduce the threat of clam prices
dropping beneath the break-even price, which should be of great
concern to potential clam growers.
Given the fairly modest value of our calculated break-even
price (7.6 to 9.2c per clam with annualized costs), our analysis
demonstrates that profitability of clam aquaculture could be robust
to substantial variations in market prices. The relatively low cost
per clam produced results primarily from the development of
methods over the last decade that enables relatively high surxivor-
ship with moderate growth penalties. Further identification of and
reductions in the magnitude of growth penalties and survival
bottlenecks in clam aquaculture may lead to additional increases in
the profitability of these operations. These enhancements coupled
with realistic economic models for clam aquaculture operations
should continue to stimulate expansion of bivalve mariculture. In
turn, further expansions may indirectly reduce harvest pressure and
thus provide greater opportunity for natural recovery of wild
stocks.
ACKNOWLEDGMENTS
The authors gratefully acknowledge the assistance of B. Wood-
ward. M. Dolan. D. Kimbro. A. Baukus, K. Sullivan, and R. Wa-
gaman in the field. The manuscript benefited from comments pro-
vided by S. E. Shumway and anonymous reviewers. Support for
this research was provided by the North Carolina Fisheries Re-
source Grant Program administered by the North Carolina Sea
Grant College Program and by the state of North Carolina.
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Joiinuil of SlwUthh Research. Vol. 22. No. 3, 703-709. 20(13.
A STUDY OF THE NOAH'S ARK SHELL {ARCA NOAE LINNAEUS 1758) IN MALI STON BAY,
ADRIATIC SEA
MELITA PEHARDA.' JAKSA BOLOTIN,' NEDO VRGOC,' NENAD JASPRICA,"
ANA BRATOS/ AND BOSKO SKARAMUCA"
Uwititutc of Occaiioi^rciphy and Fisheries. S. I. Mcstrovica 63. 21 000 Split. Croatia: -Institute of
Oceanoiiraphv and Fisheries. D. Jiide 12. 20000 Dubrovnik. Croatia: ^Collesiitin Rafiiisiiuon. Cira
Carica 4. 20000 Dubrovnik. Croatia
ABSTRACT A Mud\ of the Noah's Arks (Aicu none) was conducted in Mah .Slon Bay. between Novemher 20111 and Novenitier
2002. Noah's Arks were collected monthly for the analysis of the condition index (CI I. and every 2 mo for biometric measurements.
CI was related to seawater temperature, salinity, and chlorophyl a levels, which were measured every 2 wk. Throughout the study, 39%
of the ,4. mnw were >50 mm in size. Based on length frequency distribution, a modified Von Bertalanffy growth equation was
constructed: L, = 79.19 [1-e"" '■"'''"']. Using the modal sizes estimated from the length frequency distributions, the estimated
population growth rates of the shell were greater than the individual growth rates estimated from shell sections. Low values for the CI
were recorded m December and January, and also in the period from .luly to October. The highest condition values were recorded from
April until June.
KEY WORDS: Bi\alvia. Ann noae. biometrics, condition index
INTRODUCTION
In recent years, an increase in the collection and aqiiactillure of
bivalves from the family Arcidae has occurred (Food and Agri-
culture Organization 2002a. Food and Agriculture Organization
2002b). In 1991. a total of 69.700 metric tons (MT) of bivalves
from the Arcidae fainily were collected from natural populations
around the world, while in 2000 94,518 MT were landed in Cuba.
"Venezuela, Korea, Mexico, Japan. Indonesia, Fiji, and the Philip-
pines (Food and Agriculture Organization 2002a). In addition,
>33O,00O MT of Scapluirca l>roiigluonii and Anadara i-ramdosa
was cultured in 2000. mostly in China, Malaysia, Thailand, and
Korea (Food and Agriculture Organization 2002b). Arcidae spe-
cies are also fast becoming important fished species in some new
regions, such as along the eastern coast of the United States
(McGraw et al. 2001, Power & Walker 2002).
The Noah's Ark shell {Area noae Linnaeus 1758) is a coin-
mercially important bivalve that is distributed in the eastern At-
lantic Ocean, the Mediterranean Sea. the Black Sea, and the West
Indies (Nordsieck 1969), It lives attached with a solid byssus on
rocks or shells, and is widely distributed and locally common in
the Adriatic Sea (Hrs-Brenko & Legac 1996). The species is com-
mercially exploited and, until the end of the Second World War,
constituted an important component of the diet of local populations
(Hrs-Brenko 1979, Zavodnik 1997). In the late 1940s, due to a
catastrophic mortality caused by an unknown agent, the A. noae
fishery in the Adriatic Sea collapsed (Hrs-Brenko 1980). Although
the fishery has never returned to the annual catch rate of >600 MT
of the 1940s (Hrs-Brenko 1980), it is still one of four major com-
mercially exploited bivalves in the eastern Adriatic (Benovic
1997).
Due to an increasing number of tourists and a subsequent in-
crease in demand for seafood products, the A. noae fishery re-
ported in this article has recently intensified in the Croatian part of
the Adriatic. In a lecent study of A. noae shell sections (Peharda et
al. 2002). it was found to be a slow-growing bivalve. .4. noae can
live for >I6 y. a feature that makes it potentially susceptible to
overfishing. However, the research conducted by Peharda et al.
*Corresponding author. E-mail; melita@izor.hr
(2002) investigated only the growth of the shell, but did not in-
vesdgate the population structure of this species. The research
undertaken in this article had the objective of gaining a better
understanding of seasonal changes in the A. noae population struc-
ture and condition index (CI), data that are crucial for monitoring
the sustainability of the A. noae fishery in the Adriatic.
MATERIALS AND METHODS
Mali Ston Bay is an extended bay located in the southeastern
Adriatic Sea (Fig. 1 ). It is characterized by strong marine currents,
underwater freshwater springs, and abundant and constant sedi-
mentation that influences the formation of soft-mud sediments
(Simunovic 1981 ). The concentration of nutrients is high due to the
high freshwater input (Vukadin 1981. Caric et al. 1992). Analysis
of phytoplankton abundance and zooplankton community structure
indicate that the bay is a naturally moderately eutrophic ecosystem
(Vilicic 1989. Lucie & Krsinic 1998). The sampling station for the
study was located in part of Mali Ston Bay called Bistrina.
The study was based at Bistrina marine station between No-
vember 2001 and November 2002. Noah's Arks were collected
from the seabed by scuba divers at depths of between 2 and 4 m.
Sampling was conducted once a month for the analysis of CI (;; =
30) and every 2 mo for biometric measurements.
The following parameters were measured for each specimen:
length (L). height (H). and width (Wd) in millimeters: and dry
flesh weight and wet weight of shell in grams. Flesh was dried at
60°C to constant weight, and the following CI was calculated
according to the method of Davenport and Chen ( 1987):
C.I. = Dry flesh weight/Shell weight x 100
Temperature and salinity were measured at a depth of 2 m twice a
month with a WTW (Ft. Myers, FL) multiline hydrographic probe.
Seawater samples for chlorophyll a (Chi a) analysis were collected
twice a month at the same depth using Niskin (General Oceanics,
Miami, FL) water bottles. Samples of 0.5 dnv^ were filtered using
Whatman (Kent, U.K.) GF/F glass-fiber filters and were subse-
quently stored at -20°C. The Chi a level was determined fluoro-
metrically using a Turner TD-70() Laboratory Fluorometer
(Sunnyvale. CA), and was calibrated with pure Chi a (Sigma
Chemical. St. Louis. MO) after homogenization and 90'7f acetone
705
706
Peharda et al.
Figure I. Location of Mali Ston Bay and Bistrina.
extraction (24 li at room temperature) of filters, following the
method of Strickland and Parsons ( 1972).
Spearman's correlation analysis and regression were applied to
describe the biometric characteristics of the shell and body tissue.
and to determine the degree of association with the CI and envi-
ronmental conditions. A nonparametric Kruskal-Wallis test was
used to examine monthly changes in CI. Length frequency data
were analyzed using the FiSAT statistical package (Food and Ag-
riculture Organization-The International Center for Living Aquatic
Resources Management (ICLARM). Rome. Italy). Data were
smoothed using the running average of three classes, and the Pow-
ell-Welherall method (Wetherall 1986) was applied to estimate
asymptotic length {LJ. The method of Bhattacharya (1967) was
used to separate a composite distribution into separate cohorts,
while the method of the sum of squared errors was used to deter-
mine the curvature parameter (k) of the modified von Bertalanffy
growth equation L, = L^ [ 1 -e
(SpaiTC & Venema 1992).
RESULTS
The seawater temperatures ranged from 7.2'C (January 2002)
to 25.8°C (June 2002). Seawater temperatures >20°C were re-
corded between June and mid-September (Fig. 2). The lowest sa-
linity values were recorded during sampling in July |26.9 practical
salinity units (psu)| and October 2002 (28.8 psu). The highest
D. 3
c Q.
1 E
40
35
30
25
20
15
10
5
0
V
■0.22
.♦ -.^ + 0-20
0.18
0 16
r 0 14
0.12
0.10
- 0-08
0.06
0.04
0.02
0.00
N D
2001
J F M A M J J
A S 0
2002
Chi a
N D
♦ Salinity ■ Temperature
Fiuure 2. Seasonal variation in the salinity (psu), temperature ("O.
and Chi a levels (mg m"') in Mali Ston Bay.
salinity value recorded in this study was .^7.1 psu (March 2002).
Chi (( values ranged from 0 mg m"' (December 2001 ) to 0.094 mg
nr' (April 2002).
The minimum shell length recorded during the 1-y study was 6
mm. while the maximum was 80 mm [mean (±SD) length 4.'i.04 ±
13.68). Only 1% {n = 14) of measured individuals were longer
than 70 mm. 6% in = 96) were longer than 60 mm. and 39%
{II = 589) were longer than 50 mm. Shell height values ranged
from 3 to 44 mm (mean 23. 1 7 ± 6.59 mm), and shell width ranged
from 3 to 51 mm (24.81 ± 7.24 mm). Length frequency histo-
grams, according to sampling months, are shown in Figure 3. The
polynomial type of length distribution is visible in all the presented
graphs, indicating the presence of several age classes.
Using the length frequency distributions, up to eight cohorts
were separated according to the method of Bhattacharya (1967)
(Table 1). The asymptotic length (L^j of A. noae was estimated at
79.91 mm. while the calculated curvature parameter (k) was 0.342
y"' (r" = 0.992). According to the von Bertalanffy growth equa-
tion obtained. .4. iiocic reaches a length of 60 mm in its 5th year
of life, while it takes over 10 y to grow to its asymptotic length
(Fig. 4).
The relationship between length and height could be described
using the following equation: H = 4.33 -I- 0.418 L (/( = 1531;
r- = 0.75; P < 0.001 ). while the equation Wd = 3.32 + 0.477 L
{n = 1531; r- = 0.82; P < 0.001) described the relationship
between shell L and Wd. The calculated values of r"^ indicate the
degree of variation in the shape of the shells. The relationship
between shell weight and length could be described using the
following equation W = 0.01* L' "" (;; = 390; r" = 0.79: P <
0.001 ). A. noae has negative allometric growth, meaning it grows
proportionally more in length than in H. Wd. total weight, or flesh
weight with increase in age (Table 2.)
Seasonal differences in body CI are shown in Figure 5. Low
mean ratios of dry flesh weight and shell weight (<9) were re-
corded in December and January, and also in the period from July
to October. The highest ratio values (-11) were recorded from
April until June. A sharp decrease in CI was noted between June
and July. Observed monthly changes were statistically significant
(Kruskal Wallis H = 126.95; P < 0.001 ). There were no statisti-
cally significant correlation between CI and temperature (r =
-0.369; P = 0.468) and Chi (( (r = 0.036: P = 0.477). while a
negative correlation was found between CI and salinity (r =
0.137; P = 0.007).
DISCUSSION
The Bay of Mali Ston is the largest bivalve aquaculture area in
the eastern Adriatic Sea. with a long tradition of collecting marine
organisms and their aquaculture over several centuries, and. ac-
cording to some authors, even from the time of the Roman Empire
(Basioli 1968). Although A. noae is one of the main bivalve spe-
cies traditionally collected in this area, there are no data on its
biometry, population structure, or seasonal changes in CI at this
location. The current study confirms previous observations that A.
noae is variable in shape (Valli & Paro\el. 1981. Poppe & Goto
2000. Peharda et al. 2002). Negative allometric growth noted for
A. noae in the Gulf of Trieste (Valli & Parovel 1981) also was
confirmed in this study.
According to the literature. A. none can grow up to 90 mm, hut
usually it grows up to 70 mm in size (Parenzan 1974. Hrs-Brenko
1980. Poppe & Goto 2000, Peharda et al. 2002). In this study, the
A Stud\' of Arca noae in the Adriatic Sea
707
(a) November
(d) May
N=269
X=42.0+10.4
Length (mm
(b) January
_ 6
N=85
c
X=48.3±13.0
.il.lJ
_ 6
-S s
>. 4
o
10 20 30 40 50 60 70 80
10 20 30 40 50 60 70 80
Length (mm)
(c) March
N=200
X=40.0±15.0
_ 6
>, 4
0) '^
== 2
J? 1
^ 0
0 10 20 30 40 50 60 70 80
Length (mm)
(e) July
N=200
X=44.7±16,2
10 20 30 40 50 60 70 80
Length (mm)
(f) September
>. 4
N=188
X=40.6±13.0
10 20 30 40 50 60 70 80
Length (mm)
o- 2
N=219
X=45.3±12.6
0 10 20
30 40 50
Length (mm)
60 70 80
Figure 3. Length-frequency histograms for A. iioae collected in (al November 2001, (b) January 2002. (cl March 20(12. (d) May 2002, (el July
2002. and (f) September 2002.
largest indi\idual had a length of 80 iiini. but \ery few animals
longer than 70 mm were recorded. Length frequency distributions
recorded in 1977 and 1978 on the west coast of the Istria peninsula
(Hrs-Brenko 1980) are similar to that recorded in this study. The
relatively high percentage of .4. noae longer than 50 mm indicates
good survival, a stable population, and a potentially good spawn-
ing stock (Hrs-Brenko 1980).
The parameters of the \on Bertalanffy growth equation ob-
tained in this study show that it takes >10 y for A. noae to grow to
its asymptotic length. Mistri et al. (1988) found similar results for
a related species. Scapluiira inaequivalvis, which was introduced
by ballast water into the northern Adriatic. S. iiiaec/iiivtilvis grows
slowly and needs >10 y to reach its maximum theoretical length
(Mistri et al. 1988). A slow growth rate was also shown for the ark
TABLE L
Cohorts split using Battacharya's method, according to sampling months.
Cohort
November
January
March
May
July
September
1st
10.89 (4.474)
16.09 (3.323)
20.43 (2.829)
10.00(3.101)
13.19 (3.3S5)
16.34(2.447)
2nd
21.37 (3.122)
25.95 (4.085)
28.28(3.711)
18.67 (2.717)
22.87 (2.997)
24.96 (2.980)
.^rd
31.78 (3.585)
33.45 (2.085)
38.68 (3.640)
28.50 (3.926)
30.25 (3.694)
34.63 (4.276)
4ili
41.31 (4.243)
42.93 (4.052)
47.06 (2.959)
39.68(5.951)
39.70 (4.639)
44.50 (5.219)
.^ih
50.33 (3.358)
53.18 (2.812)
53.47 (2.898)
49.38 (3.510)
48.95 (2.104)
52.95 (5.6,39)
fith
57.54 (2.686)
58.27 (2.190)
62.63(3.921)
56.79 (4.219)
58.71 (5.702)
64.36 (2.579)
7tli
64.65 (3.934)
63.38 (3.157)
64.81 (3.256)
74.94 (3.44S)
Sth
71.75(1.779)
Values given as mean length (SD).
708
Peharda et al.
90
80
70
"E 60
^50
O) 40
-I 30
20
10
0
TABLE 2.
Parameters of log,,, regressions of biometric parameters.
012345678
Relative age (years)
Figure 4. (Jrowth eur>e for A. none fitted using the von Bertalanffy
growth equation L, = 79.19 [i_e" '^"-'"'].
shell Noctia poiulcrosa, which was shown to attain a market size
when it is 8+ years old and could li\'c tor 15 y (McGraw et al.
2001).
The presented resuUs in this study indicate that .4. iioae might
be a somewhat faster growing species than previously found using
shell sections (Peharda et al. 2002). According to this study, the
asymptotic length of A. none is larger (80 mm) than the asymptotic
length that was obtained from the analysis of shell sections (6.5
mm). Similarly, the calculated curvature parameter (k) was also
larger (0.34), than the k value (0.17) in Pehai'da et al. (2002).
which indicates a faster growth rate. However, it is important to
keep in mind that the Bhattacharya method applied in this study is
useful for splitting a composite distribution into separate normal
distributions in cases in which there are several age groups (co-
horts) and is less reliable for longer living species (Gayanilo &
Pauly 1997). On the other hand, the study of shell sections is
usually limited to a smaller number of specimens (Richardson
2001), and it is possible that the earlier study by Peharda et al.
Dependent
Independent
\ ariable
\ariable
a
b
r-
11
H
L
-0.04
0.85 ±0.010*
(I.S30
\5}\
Wd
L
-0.09
0.90 ± 0.009*
0.864
I5,M
Total weiaht
L
-2.04
1 .92 ± 0.050*
0.794
390
Flesh weight
L
-2.61
1.94 ±(1.(165*
0.695
390
* Values given as mean ± SE (departure from isometry at P < 0.01 ).
(2002) was somewhat infiuenced by sample size. Fuilher research
should compare these two methods and examine the possibility of
determining growth parameters using mark-recapture techniques.
The results obtained for the CI show that there are seasonal
changes in body weight. The ma.Kimum values were recorded dur-
ing the period from April until .lune. The minimum values of CIs
were recorded in December and January, at the end of the sumtner,
and at the beginning of the fall. The first minimum value is prob-
ably related to temperature stress and a period of reduced feeding,
as it is a period when we also recorded the lowest seawater tem-
peratures. The later minimal value might be attributed to a period
following spawning. Reduction in body CI in September and Oc-
tober was attributed to a summer spawning in related species. S.
hroiiiihroitii (Park et al. 2001 ) and .S". imictiuivalvis (Mistri et al.
1988). respectively.
According to Graeffe (190.3) and Vatova (1928. 1949). the
spawning of A. none in the Adriatic Sea occurs in May and June.
In the Gulf of Trieste, this species has a prolonged spawning
season, with peaks occurring in March and September (Valli &
Parovel 1981). Our data on seasonal changes in body weight in-
dicate that the spawning activity of A. noae in Mali Ston Bay might
be the most intense in June. This is further supported by the ob-
servation of small individuals in the November samples.
D
13
0
Q t
B
Q 5
R
B
Q ^
B
R
Nov Dec Jan Feb l^ar Apr IVIay Jun Jul Aug Sep Oct Nov
2002
Figure 5. Monthly variations in CI of .1. iwac. CI = ratio between dry llesh weight and shell weight, with values given as percentages.
A Study of Arca noah in thf, Adriatic Sea
709
According to our results, salinity is the only environmental
factor that correlates with CI throughout the year. This result is
similar to the findings of Park et al. (2001 ), who did not record a
coiTclation between the CI of S. hnnighloiiii and temperature and
Chi (( level, and have recorded a positive correlation between CI
and salinity. Although during our study period Chi a concentra-
tions were about 10 times lower than the lowest values recorded by
Jasprica and Caric (1997), previously established patterns of sea-
sonal variation of phytoplankton in the semi-closed bays along the
eastern Adriatic Sea and our values are in agreement with the
presented data (Vilicic & Stojanoski 1987). Increased temperature
values, in addition to available nutrients (Caric pers. comm.), fa-
\i)red phytoplankton development in April, when an increase in A.
iKhw CI was registered. According to the thermohaline conditions
and Chi a levels analyzed, Mali Ston Bay may be considered to be
an ecologically stable location suitable for the growth of ,4. iiocw.
ACKNOWLEDGMENT
The authors express their gratitude to the Ministry of Science
and Technology of the Republic of Croatia for funding this project,
and to Zeljko Bace and Vladimir Onofri for providing technical
support. Special thanks to C. A. Richardson for valuable assistance
with data analysis and preparation of the manuscript.
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Jiiiiiiuil iif Shell fish RiSi-anh. Vol. 22. No. 3, 711-714. 2()(U.
PRESENCE OF GIANT POLYMORPHIC CELLS IN CRASSOSTREA GIGAS CULTURED IN
BAHIA FALSA, BAJA CALIFORNIA NW MEXICO
JORGE CACERES-MARTiNEZ* AND REBECA VASQUEZ-YEOMANS
Laboratoiio cle Biologi'a v Patologia tie Organisnio.s Aciidticos del Departumento de Aciiiculliiia. Cciitro
de Investigcicii'm Cienlifica v de Ediicacldn Superior de Emcmidu. Apdo. Posnd 2732. 22800. Ensenada
Baja Ccdifornia. Mexico
ABSTRACT The culture of the Japanese oyster Crassoslrea aigas is a successful commercial activity In Northwest Mexico. Since
1997. hi!;h mortality outbreaks have occurred in the area without apparent reasons. In thi.s study we found gill erosions during clinical
observations, hemocyle infiltration into the tis.sues at the histopathological level, and in some cases we detected the presence of giant
polymorphic cells. In general, conditions mentioned above, including the presence of Trichodiiia sp. and especially the presence of
giant polymorphic cells matches with the signs of the gill disease caused by an icosahedral DNA virus (Gill Necrosis Virus infection)
first recorded in the Portuguese oyster Crassoslrea angidala and in the Japanese oyster C. ninas in Europe. However, the Transmission
Electron Microscopy (TEM) analysis of damaged tissues did not reveal the presence of viral particles.
KEY WORDS: Crassosrrea i^lsas. giant polymorphic cells, mortality, gill necrosis \irus infection (GNV). trichodines.
INTRODUCTION
The Japanese oyster Crassostreci gigas is one of the most
widely cultured mollusks in the world. This species has been in-
troduced from its original area in Japan, to countries such as Aus-
tralia. France. Holland, Spain, Portugal, Thailand, to the Pacific
coast of the United Sates, and United Kingdom (Bardach et al.,
1982: Edwards, 1997). In 1973, C. gigas was introduced in several
coastal lagoons in the states of Sonora, Baja California Sur, and
Baja California, in Northwest Me.xico. including Bahi'a Falsa. B.C.
The oyster seed was obtained from The Laboratory of the Lumi
Indians in Marietta, Washington U.S.A. (Islas Olivares. 1975). In
Bahi'a Falsa, this species was cultured in floating rafts. At present,
the culture is produced in racks, bags, and occasionally some
stocks are maintained directly on the bottom. Currently, the annual
production in the region is around 1,622 metric tons with a value
of 2,4 million dollars. Around 1.800 workers are involved in this
activity and Bahi'a Falsa contributes \6Vr of this figure (Anuario
Estadistico de Pesca. 2001). The industry depends on the impor-
tation of oyster spat from Oregon. Washington, and California,
USA.
Since 1997, several high mortality outbreaks of C. gigas. in-
cluding seed, juveniles, and adults, have occurred in Sonora and
Baja California Sur. In April 1998, mortality outbreaks began to be
recorded in Bahi'a Falsa, B.C. Unusual inortality has remained in
the region. Several causes have been attributed to these mortalities:
1 ) unusual high temperatures and conditions produced by El Nino
in 1997 and 1998. 2) the presence of toxins in the environment
produced by microalgae or other organisms, 3) pollution, 4) the
quality and quantity of food (phytoplankton), and 5) pathogens, or
synergic conditions by the joint action of two or more of the above
factors (Caceres-Martinez 2000, Hoyos 2000). This work presents
the results of a clinical and histopathological survey of C. gigas
cultured in Bahia Falsa. B. C. and the possible relation of the
observations and parasites with mortality outbreaks present in the
bay.
MATERIALS AND METHODS
The study was conducted in Bahi'a Falsa. Baja California,
Mexico from July 1997 to June 1998 (monthly samplings). Two
localities. Agromarinos. in the middle area of the bay. and Alfon-
sos, in the inner area of the bay, were sampled (Fig. 1 1. Rack and
bag cultures are used in Agromarinos, whereas bottom culture is
used in Alfonsos. In each locality and culture condition, 30 com-
mercial size oysters were collected (mean total shell length, 12.45
cm ± 5.5 in the Agromarinos" racks, mean total shell length 10.46
cm ± 5.5 in the Agromarinos" bags and Alfonsos"). In all three
culture conditions, oysters are exposed to air during low tide. Live
oysters were transported to the laboratory and all fouling organ-
isms were removed with a brush and a stream of seawater. Oysters
were placed in a Petri dish, opened, and the intervalvar water and
oyster flesh were examined for the presence of parasites under a
dissecting microscope. The soft body of the oysters were removed
from the shell and fixed whole in Davidson"s fixative (Shaw &
Battle 1957) for at least 24 h An anterior transverse section in-
cluding the digestive gland, mantle, and gills was taken. Tissue
samples were embedded in paraffin wax and were sectioned and
stained with hematoxylin and eosin (Shaw & Battle 1957). Tissue
analysis and measurements were made with a micrometer placed
in an optical microscope. Prevalence of parasites and lesions were
considered as the number of infested or wounded oysters/number
of oysters examined xlOO. Intensity was considered as the number
of lesions or parasites per damaged or infected oysters in the
Present address of both authors: Instituto de Sanidad Acui'cola. Calle 9na
y Gastelum No. 468 Local 14. Zona Centro. C.P. 22800. En.senada, Baja
California, Mexico.
*Corresponding author. E-mail jcaceres@cicese.mx
V ..' 1 1
\- Baja California
\ ^^. Falsa
\ AlfoniLH ■( ^-\
A^^-T^
\\ j^ 30^5 >
/ ^^ 1 \^ Sonora ^
/ /^ \mexico
/ / I \
/ Baja CaliTornia Sur ^-j ^v^.
c?
Figure 1. Map showing the region, where Crassoslrea gigas culture is
conducted in North Western Mexico, and the Bahia Falsa, where the
study was performed. Black dots indicate the sampling sites.
711
712
Caceres-Martinez and Vasquez-Yeomans
B
50
p
r
40
e
V
3 30-
1
e
n 20.
c
e
10-
Gill inflamation
1-30
1 r^ r
Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun
50-
Trichodina sp.
40_ Salinity ppt
30-
20-
1-40
• 30
• 20
Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun
1997 1998
30-
40-1
Figure 2. A, The combined mean values and standard error of prevalence and intensity of gill intlammaticm of C. gigas from all three cultures
conditions during the study period. The dark triangle indicates when the oyster culturist noted the mortality outbreak. B, Mean values and
standard error of prevalence and intensity of Trichodina sp. in C. gigas from the three cultures conditions during the study. Temperature and
salinity values are shown as a line on the top of A and B, respectively, and their scale \alues are placed in the right side of the graphics.
il
:aw
tf»
% ,.
* ♦Pn *
♦ ,fe*
♦ ' ^ .
"*
Figure i. A. Strong gill inllammation in C. gigas with destruction of gill tllaments. tissue rupture with massi\e inllltration of heniocytes (h) and
the presence of some giant polymorphic hypertrophic cells (gc), scale bar = KM) (jm. B, Detail of a gill lesion showing the polymorphic
hypertrophic cells (gc) with basophilic inclusion (bi), picnotic nucleus (pnl and heniocytes around the lesion (h). Haematoxylin and eosin, scale
bar = 2U fim.
Giant Polymorphic Chlls in Crassostrea g/gas
713
sample. For the estimation of the gill lesions the following scale
was used: (1) light gill intlammation, low infiltration of hemocytes
and the gill structure unaltered; (2) medium gill intlammation.
infiltration of hemocytes within the gill filaments and interlamellar
septum; (3) Strong gill inflammation. massi\e infiltration of
hemocytes. swelling of gill filaments and necrosis of the gill tissue.
After this study period, two additional samplings were earned
out. one after a mortality episode occurred in the Bahia Falsa, in
June 20(10. During this sampling 30 surviving oysters were col-
lected, opened in the field, and the condition of the gills was
observed. Photographs of the gills were taken with a camera placed
on a stereomicroscope and the degree of gill damage was deter-
minate. All gills were fixed in Davidson's fluid and processed for
histopathological analysis as mentioned above. The last sampling
was carried out in Nov ember 2000. Ten oysters from an area of the
bay where mortalities were common were reviewed and those
showing symptoms of gill erosion were separated and a small
pieces from the eroded area of the gill was removed and fixed in
3% glutaraldehyde in 0.1 M sodium cacodylate buffer. pH 7.8. for
4 h at 4"C. Fixed tissues were washed for 1 2 h at 4'"C in the same
buffer and cut into 1 mm' pieces. These pieces were then postfixed
in buffered Wr OsOj for 4 h at the same temperature, dehydrated
through a series of ethanols, and embedded m Epon. Sections (90
nni thickness) were cut and stained with 5% uranyl acetate for 30
min and lead citrate for 2 min and observed with a Transmission
Electron Microscope (TEM) operated at 75 kV at the Instituto de
Investigaciones Marinas. Vigo. Spain.
RESULTS
Histologic slides showed from light to strong gill intlammation.
There were no differences among the prevalence and intensity of
gill intlammation of oysters collected from different culture con-
ditions (Kruskal-Wallis test. H = 2.42. P = 0.29 and H = 0.97.
P = 0.61, respectively). The combined mean values of prevalence
and intensity of gill inflammation from the three culture conditions
are shown in Figure 2. There was a positive correlation between
the prevalence and intensity of gill intlammation (r = 0.72. P =
0.007) and there was a general increase in the prevalence and
intensity of gill inflammation from the beginning to the end of the
study period (Fig. 2). In two cases. April in the Alfonsos" and June
in the Agromarinos" rack, we detected cellular lesions reminiscent
of the gill necrosis virus infection (GNV) caused by an icosahedral
cytoplasmic deoxyribovirus (Comps. 1988): occun'ence of giant
polymorphic cells (ranging from 25 to 30 |xm in diameter) con-
taining basophilic granules and some oval hypertrophied nucleus
and hemocytes accumulation in the lesion (Fig. 3). Also. Tri-
chodina sp. were detected in the mantle cavity and gills of C.
gigcis. Their prevalence and intensity was similar in oysters taken
from the three different culture conditions (Kruskal-Wallis test.
H = 0.9. P = 0.63 and H = 0.77. P = 0.67. respectively). The
combined mean values of prevalence and intensity of Trichodimt
sp. from the three culture conditions are shown in Fig. 2. There
was a positive correlation between the prevalence and intensity of
Triclwdina sp. (r = 0.77, P = 0.003) and there was a general
increase in the prevalence and intensity of Trichodina sp. from the
beginning to the end of the study period (Fig. 2). Temperature and
salinity remained between the tolerance limits of this oyster spe-
cies (Pauley et al. 1988; Fig. 2). There was no statistical correlation
between gill lesions and Trichodina sp. presence; however, there
was a trend of an increase in gill lesions and Tricliodina sp. pres-
ence from the beginning to the end of the study period (Fig. 2).
Figure 4 shows a varying degree of gill erosion in surviving
oysters sampled in June 2000, from normal appearance (0 de-
grees), to very eroded appearance (4 degrees). Histologic analysis
revealed the presence of picnotic nuclei in cells at the distal edge
of the gill filaments where erosion occurred but no Trichodine
presence. Curiously, no evidence of necrotic areas was observed in
the eroded areas of gill filaments; however, deformations of the
distal edge of the gill filaments were evident (Fig. 4). The preva-
lence of the lesions was 1009f and its intensity value was medium.
These lesions showed inflammation of tissue and cicatrisation The
TEM study did not reveal the presence of giant polymorphic cells
and viral particles.
DISCUSSION
According to Comps (1988), virus infection has been associ-
ated with major diseases of oysters of the genus Crassostrea.
These infections include the GNV affecting the Portuguese oyster
and. to a lesser degree, the Japanese oyster cultured in Europe.
Figure 4. DiUcrinl (k};riis of «!!! inisioii in surviving C. gigas. A,
Normal appearance (n). light eroded appearance (lei. B, Medium
eroded appearance (me). C Highly eroded appearance ihe).
714
Caceres-Martinez and Vasquez-Yeomans
This author remarked that the gill necrosis virus causes, mainly in
the Portuguese oyster, an evolutive ulceration of the gills, includ-
ing cellular hypertrophy and severe hemocyte infiltration. Mortali-
ties have been observed in the most serious cases. In this study, we
found clear histologic evidence of giant polymorphic cells, which
have been associated with GNV infection; however, we did not
detect characteristic damage of GNV on the gills because we failed
to look for it. It is important to mention two points: 1 ) this study
was concluded three months after an unusual mortality in the bay
was recorded, and 2) no moribund oysters were sampled. How-
ever, in the second sampling (June 2000) when we went specifi-
cally looking for gross signs of the GNV infection; we detected the
erosion of the gill filaments, which is the characteristic gross sign
of the GNV infection. In spite of the failure to reveal viral particles
by TEM possibly because of the sample process, the status of the
gill tissues of surviving oysters (cicatrisation), and the difficulties
in finding those particles in fixed tissues, evidence of giant poly-
moiphic cells in C. g/go.v cultured in the bay and region showed an
obligated line of research and the possibility that a virus may be
involved in oyster mortality outbreaks. It is known that herpes-like
viruses in several oyster species have been associated with high
mortality rates (Le Deuff & Renault 1999). However, the presence
of Trichodina sp. was observed in a similar condition when the
unusual mortality of C. angiilata occurred in France. On that oc-
casion, it was thought that Trichodina sp. could be the cause of
oyster gill lesions (Besse 1968), Afterwards, it was found that
Trichodina sp. might have been a secondary invader of oysters that
were suffering from virus-caused gill necrosis (Bower et al. 1994).
The trend of the increase in prevalence and intensity of Triclwdina
sp., from the beginning to the end of the study period, is consistent
with the increase of gill lesion observed and a secondary invasion
of the protistan. Oyster culturists from Bahia Falsa first reported a
mortality outbreak in April 1998. and gill inflammation increased
from November 1 997 to the end of the study period. It is possible
that the presence of giant polymorphic cells or the gill inflamma-
tion condition could have been present in the oysters of the bay
earlier; however, an unknown factor may favor the increase of gill
lesions at the end of the study period. Temperature and salinity
remained between the tolerance limits of the oyster species and the
culture technique seems to be independent of gill lesions, and the
parasite prevalence and intensity observed. Further studies using
molecular tools are being conducted to confirm the presence of
virus in oysters from the region and its possible relationship with
mortality outbreaks.
ACKNOWLEDGMENTS
The authors thank M.C. Jose Angel Olivas Valdez and Oc.
Sergio Curiel Ramirez for sample processing; Dr. Antonio
Figueras from Instituto de Investigaciones Marinas de Vigo. Spain,
for TEM analysis; and Consejo Nacional de Ciencia y Tecnologia
from Mexico for financial support throughout the project number
39.V^P-B.
LITERATURE CITED
SAGARPA. Gobierno de Mexico.
Anuano Estadistico de Pesca. 2001.
Poder Ejecutivo Federal.
Bardach, J. E.. J. H. Ryther & W. O. Mclarney. 1982. Aquaculture. John
Wiley & Sons. Inc., 741 pp.
Besse, P. 1968. Resultats des quelques observations sur une affection bran-
chiale des huitres [Crassosuea an;iidaut Lrnk). Bull. Acad. Veterinmri'
de France 41:87-91.
Bower, S., S. E. McGladdery &. I. M. Price. 1994. Synopsis of infectious
diseases and parasites of commercially exploited shellfish, Ann. Rev.
Fish Dis. 4:1-199.
Caceres-Martinez. J. 2000. Resultados de los analisis patoliigicos efectua-
dos a ostiones del Pacifico relacionados con mortalidades masivas.
Foro Regional Sobre la Problematica del Cultivo de Moluscos Bivalvos
en el Noroeste de Mexico. Veiliuno de enero del 2000. Hemiosillo.
Son. Mexico.
Comps, M. 1988. Epizootic diseases of oysters associated with viral in-
fections. In: W. S. Fisher, editor. Disease processes in marine bivalve
molluscs. Bethesda, MD: American Fisheries Society, Special publi-
cation 18, pp. 23-37.
Edwards, E. 1997. Molluscan fisheries in Britam. In: C. L. MacKenzie. Jr..
V. G. Burrell. Jr.. A. Rosenfield. W. L. Hobart. editors.. The history.
present condition, and future of the molluscan fisheries of North and
Central America and Europe. Volume 3, Europe. U.S. Depl. Commer..
NOAA Tech. Rep. 129. 240 p.
Hoyos. C. F. J. 2000. Antecedentes de las mortalidades masivas de ostion
Japones y otros bivalvos en 1997-1999. Foro Regional Sobre la Prob-
lematica del Cultivo de Moluscos Bivalvos en el Noroeste de Mexico.
Veiliuno de enero del 2000, Hermosillo, Son. Mexico.
Islas Olivares. R. 1975. El o.stion japones Cra.ssoslrea gigas en Baja Cali-
fornia. Cienc. Mar. 15:21-38.
Le Deuff. R. M. & T. Renault. 1999. Purification and partial genome
characterization of a herpes-like virus infecting the Japanese oyster,
Crassostrea gigas. J. Gen. Vir. 80:1317-1322.
Pauley. G. B.. B. Van Der Raay & D. Troutt. 1988. Pacific oyster. Bio-
logical Report 82 (11.85). Species profiles: life histories and environ-
menlal requirements of coastal fishes and invertebrates (Pacific North-
west). Washington. DC: Fish and Wildlife service. U.S. Department of
the Interior. 28 p.
Shaw. B. L. & I. H. Battle. 1957. The gross microscopic anatomy of the
digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J.
Zoul. 35:325-346.
Joiirmil ofShrlllhli Risciinh. Vol. 22, No. 3, 715-720, 2003.
IN VIVO AND IN VITRO APPROACHES TO THE ANALYSIS OF GLYCOGEN METABOLISM IN
THE PACIFIC OYSTER, CRASSOSTREA GIGAS
CLOTHILDE HEUDE BERTHELIN.'* BRUNO FIEVETr GAEL LECLERC,'
PIERRE GERMAIN,- KRISTELL KELLNER,' AND MICHEL MATHIEU'
'Laboraloire de Biologic et Biotechnologies Marines. UMR IFREMER "Phxsiologie et Ecophysiologie
des Mollusques marins. " Univcrsite de Caen Basse-Noniiandie. 14 032 Caen cedex. France:
-Laboraloire d'Etndes Radioecologiqnes de la Fa(,adc Atlantiqne. Institnt de Radioprolection et Si'irete
Nucleaire, BPIO. Rue Max Pol Foncliet. 501 M) Chcrhowg-Octeville. France
ABSTRACT Seasonal variations of glycogen and prolein metabolism m the Pacific oyster Cnissastmi f;iiiii\ were investigated in
vivo using a radiolabeled glucose injection technique and were compared with in vitro experiments on vesicular cells. Protein
metabolism appeared stable during a gametogenetic cycle, whereas glycogen metabolism in low was found to be clearly dependent on
the sexual cycle, with decreasing incorporation during gonadal tubule development. The in vivo results correlated well with data from
in viti-o experiments on vesicular cells, which correspond to the animal's glycogen storage compartment,
KEY WORDS: Pacific oyster. Crassa\lrca fiiK'ts. gametogenesis. glycogen, storage tissue. //; vivo, bioassay
INTRODUCTION
111 the Pacific oyster. Crassostrea gigas. as in mosl bivalves,
glycogen is one of the major energetic fuels for gametogenesis
(Bayne et al, 1982. Gabbott & Whittle 1983, Ruiz et al. 1992;
Mathieu & Lubet 1993), On the west coast of Europe, gametoge-
nesis in C. gigas follows an annual cycle: gonial mitosis occurs in
autumn and early winter in the gonadal tubules: the gonad devel-
ops in winter and spring: and in summer, the ripe gonad is ready
for sequential spawning in July or August, depending on the rear-
ing site. The biochemical composition of the whole animal and
isolated organs was previously studied, and glycogen levels were
determined (Walne & Mann 1975, Robert et al. 1993, Berthelin et
al, 2000b), Glycogen storage and mobilization activities were
tightly correlated to the reproductive cycle. Histologic studies
showed a seasonal inverse relationship between the increase of the
gonadal tubules and the regression of the storage tissue in the
gonadal area. Moreover, glycogen was stored during autumn and
early winter while gonadal tubules regressed, and was subse-
quently mobilized during active gametogenesis (Berthelin el al.
2000a. 2000b).
In the oyster, the biochemical mechanisms of glycogen storage
and mobili/ation in relation to reproductive activity remain poorly
documented in comparison with other models like the marine mus-
sel. Mytihis ediilis (Houtteville 1974. Pipe 1987. Lenoir 1989).
Recently, an in vitro bioassay was developed for C. gigas to mea-
sure glucose incorporation into glycogen in vesicular cells, the
glycogen storage compartment (Berthelin et al. 2(.)0()a). The //;
vitro approach provided some valuable information on the cellular
mechanisms of glycogen metabolism in vesicular cells, but it may
not reflect the true metabolism of reserve cells in vivo. First, cel-
lular dissociation could damage receptor protein structure leading
to glucose uptake modifications, coinpared with physiologic con-
ditions in the whole animal. Second, the incubation conditions may
not reflect the seasonal variations in the natural environment.
For these reasons, the glycogen metabolism of cupped oysters
was investigated using an in vivo approach based on injections of
'■*C-labeled clucose into the adductor muscle. After defining the
*Corresponding author. E-mail: heude(3>ibfa.unicaen,fr
optimal experimental conditions, glucose incorporation was stud-
ied in the whole animal in relation to the annual sexual cycle.
Finally, in vivo results were matched with in vitro data obtained
from isolated vesicular cells.
MATERIAL AND METHODS
Animals
Pacific oysters (C gigas. 3 years old) were obtained from a
commercial oyster farm in Saint-Vaast-La-Hougue. Normandy.
France. The animals were kept in aerated seawater at 13°C
throughout each experiment. The animals were starved for 24 h
prior to all in vivo experiments.
In vivo Bioassay
Conditions of Injection
Two days before injection, both valves of the oyster were
notched beside the adductor muscle, paying attention not to dam-
age the muscle, A preliminary experiment was conducted to evalu-
ate the diffusion of the injected solution into the animal tissues:
200 ^^.L of neutral red in sterile seawater was injected into the
adductor muscle. The oysters, kept in 0.5 L tanks, were dissected
45 min or 3.5 h after injection. Seawater coloration in each tank
was controlled, and neutral red diffusion was observed in each
animal.
[U-'^C] Glucose Injection
For labeling experiments, the injected solution included 50 (xL
of |LI- '""Cl glucose and 1 50 |jlL of D-glucose ( 1 3 niM). resulting in
a final glucose concentration increase of approximately 1 mM in
the hemolymph. according to the standard of Livingstone and
Claike ( 1983). For each condition, six animals were injected and
were kept in aerated seawater at I3°C. Control animals were ana-
lyzed just after injection to estimate nonspecific radioactivity. Af-
ter incubation, soft parts were separated from shells that had been
individually collected in 50-mL tubes and blended (ultra-turrax,
Labosi. France). Animal tissues were stored at -20'C before
sample treatment. For each animal, the blended tissue sample vol-
ume was adjusted to 40 niL with sodium hydroxide (0,006 N) and
thoroughly homogenized.
Different [U-'''C1 glucose doses were tested (0,5. I, 5. and 10
(iCi). Kinetic measurements of incorporation in protein and gly-
715
716
Berthelin et al.
cogen also were performed (after 0. 7.5. 16, 24, 48, and 72 h of
incubation). Seasonal variations of incorporation were studied.
Total Radioactivity Determination
Fi\e hundred microliters of potassium hydro.xide (0.3 N) was
mixed with 500 jjlL of a blended tissue sample, and 250 p.L of this
mixture was diluted in 4 mL of scintillation fluid (Optiphase.
Hisafe II 2*51 Wallac. France, EG and G division instruments)
were analyzed for '"'C activity (Packard scintillation counter.
France).
[U-"C] Glycogen Content
Five hundred microliters of a blended tissue sample was mixed
with 500 p.L of 10% trichloroacetic acid, and precipitated proteins
were discarded by centrifugation (8000 t;, 10 min, 4'C). Seven
hundred microliters of the supernatant was transferred to a 5-mL
tube containing 10 mg of unlabeled oyster glycogen (Sigma-
Aldrich. France) as a carrier and 4 mL of absolute ethanol. After
overnight precipitation at 4°C. glycogen was collected by centrifu-
gation (2500 i;. 10 min, 4°C), and the pellets were washed three
times with absolute ethanol containing D-glucose (0.1 M). Glyco-
gen pellets then were dried and resuspended in 500 p.L ol potas-
sium hydroxide (0.3 N). Radioactivity was determined in 250 |xL
of glycogen suspension diluted in 4 mL of scintillation ftuid.
[U-^CI Protein Content
Protein content also was determined in each tissue sample as
follows: 500 |j.L of oyster extract was mixed with I mL of potas-
sium hydroxide (0.3 N) and 3 mL of 10% trichloroacetic acid.
After overnight precipitation at 4°C, protein pellets were collected
by centrifugation (3000 g, 10 min. 4°C), were washed three times
in trichloroacetic acid, and were resuspended in potassium hydrox-
ide (1 niL, 0.3 N). Radioactivity was determined in 250 |jlL of
protein suspension diluted in 4 iiiL ol scintillation fluid.
In Vitro Approach
Preparation of Vesicular Cell Suspension
Oysters were maintained on ice during the dissection, were
opened by sectioning the adductor muscle, and were rinsed thor-
oughly with sterile seawater. The labial palps were dissected,
rinsed three times in sterile sea water, and decontaminated for one
night in 50 niL of Leibovitz culture medium (Leibovitz LI 5; NaCI
340 mM, KCl 50 mM. Hepes 20 mM (pH 7.4). 1100 mOsm,
filtered on a Millipore 0.22-|j.m filter] supplemented with antibi-
otics (streptomycin 100 mg L~'. penicillin 60 mg L~'. gentamycin
50 mg L~', and nystatin 8.2 mg L"').
Vesicular cell isolation was performed as previously described
by Berthelin et al. (2000a). Dissociated cells were diluted in Lei-
bovitz. culture medium to obtain 3 x 10"' cells mL"' and were
distributed into 24-well culture plates. Significant survival was
evaluated with the MTT [3-(5.5- dimethyl-thiazol-2-yl)-2.5-
diphenyl tetrazolium bromide] reduction assay (Mosmann 1983,
Coulon 1993).
[U-'"'C] Glucose Incorporation into Glycogen by Vesicular Cells
Glucose incorporation measurement was derived from Berthe-
lin et al. (2000a). In each well, 500 \xL of vesicular cell suspension
(3 X 10'' cells mL"') was mixed with 50 |jiL of (U-'-'Cj glucose
(0.5 (xCi. specific activity: 150-260 mCi mmoP') and 50 |jiL of
unlabeled D-glucose (1.5 mM). After incubation (7 h, I5"C), the
well contents were transferred into 1.5-mL microtubes and centri-
fuged (8000 ^. 10 min. 4"C), resulting in cell disruption. Three
hundred microliters of supernatant was transferred into a 5-mL
tube containing unlabeled oyster glycogen (10 mg) and then was
mixed with 4 mL of cold absolute ethanol. Glycogen was precipi-
tated o\ eniight and rinsed three times with a solution of D-glucose
in absolute ethanol (0.1 IVI). Glycogen pellets were dried and di-
luted in 500 p,L of distilled water. An analysis of radioactivity was
performed on 200 |j.L of glycogen suspension diluted in 4 mL of
scintillation fluid.
Blanks without cells or without radioactive labeled glucose
were tested, and control samples were prepared by stopping the
incubation immediately after [U-'"'C] glucose addition.
Data Analysis
Results were expressed as the mean ± SD. Each value is the
mean of six replicates. A nonparametric test (Kruskall-Wallis test)
followed by a multiple comparison test (Newnian-Keuls test) also
was performed to determine the significant differences between
samples (Scherrer 1984).
RESULTS
Injection Into the Adductor Muscle
A preliminary experiment using neutral red as a visual tracer
led to an estimate of its diffusion in seawater. After injection, no
seawater coloration was observed. Moreover, after 45 min, the
digestive cardiac sinus, the adductor muscle, and the gills were
stained red, whereas the palps and mantle appeared colorless.
Three hours after injection, the gills were still stained, and the
palps and mantle also appeared red. However, the digestive cardiac
sinus was faded.
Reco>ered Radioactivity
It represented 35 to 85*5^ of injected radioactivity with large
individual variations due to injection efficiency. Because of these
variations, [U-'''C] glucose incorporation into protein and glyco-
gen were expressed as the percentage of recovered radioactivity
for each animal.
[U-'''C] Glucose Incorporation
Four doses of [U-'"*C] glucose were tested: 0.5 \x.Ci (0.0185
MBq) and 1.0 p.Ci (0.037 MBq) values were chosen by reference
to previous in vitro experiments (Berthelin et al. 2000a); and 5 p.Ci
(0.185 MBq) and 10 fjiCi (0.37 MBq) were tested considering the
potential dilution of radioactive material in the whole animal. After
24 h of incubation, irrespective of the amount of injected labeled
glucose, 8 to 10% of recovered radioactive carbon was found in the
protein fraction, and about 2% was incorporated into glycogen
(Fig. I). Ten microcuries (0.37 MBq) of radioactivity was used
routinely in all subsequent experiments to keep sensitivity as high
as possible, since low levels of labeled glycogen were expected at
certain periods of the year.
The kinetics of radioactive carbon incorporation into proteins is
presented in Fig. 2a. The incorporation rate was maximal during
the first 16 h of incubation. After 48 and 72 h, I 1% and 12.2%,
respectively, of radioactive carbon was incorporated into proteins.
For the same animals, glucose incorporation into glycogen in-
creased linearly during the first 24 h of incubation (Fig. 2b) and
reached a maximal value of 1.6% of recovered radioactive carbon
Analysis of Glycogen Metabolism in Crassostrea gigas
717
JL.
O.SmCi
■ Protein
D Glycogen
i i
A.
Figure I. Fraction of recovered radioactivity ineasured in protein and glycogen depending on the quantity of radioactivity used (^C'il. Results
are expressed as tfie percentage of recovered radioactivity in oyster ±SD.
after 4S h (this iiicuhatinn time was chosen for further experi-
ments). This maximal value \ aried in the range of 1 .6 to 29c for the
eight experiments performed.
Seasonal Variations of /;/ l7i'o Carbon Incorporation into Proteins
The radioactive carbon fraction incorporated into proteins was
measured over the annual cycle with an incubation time of 48 h.
The radiolabeled protein fraction was found to represent 7 to 1 I'/r
of recovered radioactivity according to month (Fig. 3). With re-
spect to protein metabolism, two statistically different groups were
observed: one from January 1 to October 1; and the second, with
reduced metabolism, from November 1 to March 2 [P < 0.05
(Kniskall-Wallis and Newman-Keuls tests)].
Ill Vivo and In Vitro Glucose Incorporation into Glycogen
In vivo results (Fig. 4) showed that glycogen represented be-
tween 0.6% and 1.9% of recovered glucose incorporation in the
oyster, depending on the season (injected amount ()..17 MBq; in-
>-<
o
>
o
o
O
•5
to
S?
a
10
20
30
40
hours
50
60
0
3n
b
overed
tivity
2-
1 -
■
■
r
1- o
.-^^^^^^
>
0<
, *^^
10 20 30 40 50 60 70
hours
Figure 2. Kinetics of '^C incorporation, (a) Incorporation of '''C' into proteins, (bl Incorporation of '''C into glycogen. Results are expressed as
the percentage of recovered radioactivity ±SD.
718
Berthelin et al.
14 n
12
10
"?
£
a.
0
I
1
1
A
JL
B
B
I
B
M
JFMAMJJASONDJ__
Figure 3. Seasonal variation of carbon incorporation in protein fraction. Results are expressed as the percentage of recovered radioactivity ±SD.
Groups A and B are statistically different.
cubatioii time 41S h). In the first year, labeling was maximal in
February ( 1.9%). decreasing progressively to 0.6% in July, before
increasing during the autumn and returning to the maximal value
the following March (with a lower value in February of the second
year) (P < 0.03). The stages of development of the gonadal and
storage tissues in the gonadal area (Heude Berthelin et al. 2001)
are overlaid on Fig. 4.
In vitro measurements were performed on vesicular cells from
February 1 to October 1 (Fig. 5). These values show that ;;; vitrn
incorporation was maximal in February (1.5 nmol per 1.5 x 10''
cells), decreasing to an undetectable level in July and August, and
finally rising again during the following autumn (P < 0.05).
DISCUSSION
The investigation of different metabolic pathway.s in bivalves
has been based mainly on //( vitro techniques, due to the anatomic
characteristics of these animals. These in vitro approaches have
o
r.
T3
on
o
T3
o
»
>.
o
3,0 -
2,5
2,0
1 ,5 -
I ,0
0,5
0,0
I
I
I
I
I
M
M
O N
M
ST
developed
regression
regressed
development
developed
regression
GT
gonial mitoses development
regression rest gonial mitoses development
spawn
Figure 4. (top) Seasonal variation of carbon incorporation in glycogen fraction. Results are expressed as the percentage of recovered radioac-
tivity ±SD. (bottom) Gametogenesis and storage tissue development in oysters (Saint Vaast la Hougue, France) |redra\vn from histologic data
published In Heude Berthelin et al. (20111 )|.
Analysis of Glycogen Metabolism in Crassostrea gigas
719
3.0 n
f= 2,5 -
F M A M J J A S O
Figure 5. Changes of [l'-'''C| glucose incorpr>rati()n into gl>cogen iiieasurtd both in vitro and in vivo. The results are expressed in nanomoles of
glucose incorporated per 1.5 x 10'' cells for in ritru experiments, and as a percentage of recovered radioactivity for in viro experiments (mean
± SD; n = 61.
revealed specific information about metabolism at the scale of
isolated cells or specialized tissue samples maintained in strictly
controlled conditions. However, tissue or cell preparation steps
associated with the chosen artificial conditions may disturb the
metabolic activity of the sample relative to its true state in the
whole animal.
The present study aimed to compare seasonal variations of
glucose incorporation into glycogen measured by in vitro bioassay
in C. gigas (Bertheiin et al. 2000b) with in vivo levels of incor-
poration. This incorporation was measured after the injection of
'""C glucose into the adductor muscle using an experimental pro-
cedure originally used for the artificial infection of oysters with
pathogens (Hervio et al. 1992).
Protein metabolism was first analyzed by measuring the incor-
poration of radioactive carbon into proteins. Radiolabeled proteins
represented 7 to I l^f of recovered radioactivity. Whereas experi-
mental conditions were significantly different (i.e.. tracer concen-
tration, incubation time, and injection procedure), these data may
be compared with the results reported by de Zwaan et al. ( 1975) for
the mussel Mxtilus edulis in aerobic conditions: these authors
found that proteins accounted for ll^r of radioactivity. In the
oyster, the annual pattern of '"'C incorporation into proteins ap-
peared rather stable throughout the complete gametogenetic cycle
(from January 1 to October 1 ). Following the gametogenetic cycle
(from November I ) incorporation was also stable, but slightly
lower (1%).
By comparison with this relative stability, glycogen metabo-
lism shows some significant variations: the ''^C incorporation into
glycogen ranged from 0.6 to 1.9'7f of the radioactivity. Glycogen
storage decreased during gonadal development from February to
July and increased after the spawning event when the oysters were
in the sexual resting stage. These results were then compared with
in vitro data obtained during a previous gametogenetic cycle (Ber-
theiin et al. 2000b). Because of possible interannual bias, addi-
tional in vitro measurements also were performed within the same
year as the /;; i'/i(; experiments.
Whatever the approach. '""C glucose incorporation into glyco-
gen presents the same annual pattern. Indeed, the observed varia-
tions correlated with seasonal changes in glycogen content re-
ported in C. gigas (Mann 1979. Robert et al. 199.^. Almeida et al.
1997) and confirm that glycogen storage occurs during eariy ga-
metogenesis to support the energetic cost of the reproductive effort
(Ruiz et al. 1992. Mathieu & Lubet 1993. BertheUn et al. 2000b.
Heude Bertheiin et al. 2001). The mobilization of glycogen also
was observed in vivo in early spring and autumn. This matching
between in vivo and in vitro data is essential to verify the previous
(/; vitro approach to the study of seasonal variations in glycogen
metabolism: the \n vitro bioassay should be considered as an ad-
justed technical approach to investigate the cellular mechanisms
involved in the regulation of these changes of metabolism.
Moreover, the correlation between the pattern of glycogen me-
tabolism in the whole animal (i.e.. the in vivo approach) and the
gonadal vesicular cells suggests that in the oyster glycogen me-
tabolism occurs mainly in the specialized storage tissue located in
the gonadal area (Bertheiin et al. 2000a), and that this metabolism
is the main source of energy for reproductive effort. Further ex-
periments should now be carried out to improve different technical
aspects of the /;/ vivo procedure. With the current in vivo proce-
dure, glucose is supplied directly into the hemolymph sinus with-
out taking into account digestive assimilation or possible short-
term storage in the digestive gland (Bertheiin et al. 2000b). Im-
provement may result from the use of labeled microalgae or coated
beads. In addition, organ dissection should be considered to quan-
tify the respective role of each coinpartinent involved in glucose
metabolism.
ACKNOWLEDGMENTS
The authors would like to thank C. Costil for essential ad\ ice
on all the statistical aspects of this study, and I. Probert for his
expert linguistic guidance.
LITERATURE CITED
Almeida. M. J., J. Machado & J. Coimbra. 1997. Growth and biochemichal Bayne. B. L.. A. Bubel, P. A. Gahbon, D. R. Livingstone. D. M. Lowe &
composition of Crassostrea gigas (Thunberg) at three fishtarm earthen
ponds. J. Shellfisli Res. 16:455-462.
M. N. Moore. 1982. Glycogen utilisation and ganietogenesis in Mytilus
edulis (L.). Mar. Biol. ten. .^:89-I05.
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Berthelin, C K. Kellner & M. Mathieii. 2000a. Histological cliaracteriza-
tion and glucose incorporation into glycogen of the Pacific oyster Cra.s-
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Berthelin. C, K. Kellner & M. Mathieu. 2000b. Storage metabolism m the
Pacific oyster (Crassoslrea gigas) in relation to summer mortalities and
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Coulon, I. 1993. Mise au point d'un systeme controle de culture de cellules
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De Zwaan. A.. A. De Bont & J. Kluytmans. 1975. Metabolic adaptation on
the aerobic-anaerobic transition in the sea mussel MmHiis ciliitis (L.l.
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138.
Gabbott. P. A. & M. A. Whittle. 1985. Glycogen synthetase in the sea
mussel Myiiliis ediilis L: II. Seasonal changes in glycogen content and
glycogen synthetase activity in the mantle tissue. Comp. Biochcni.
Physiol. 836:197-207.
Hervio. D. 1 992. Contribution a Tetude de Boiuiiniu oslreae [Ascctospora I.
protozoaire parasite de I'huitre Osriea edulis (Bivalvia). et a I'analyse
des interactions hote-parasite. These de doctorat. Clermond-Ferrand.
France: Universite de Clermond-Ferrand.
Heude Berthelin. C. J. Laisney. J. Espinosa, O. Martin. G. Hernandez, M.
Mathieu & K. Kellner. 2001. Storage and reproductive strategy in
Crassostreu gigas from two different growing areas (Normandy and
the Atlantic coast. France). Invert. Reprod. Dev. 40:79-86.
Houtteville, P. 1974. Contribution a I'etude cytologique et experimentale du
cycle annuel du tissu de reserve du manteau de Myrilas cdidis (L.).
These de Doctorat. Caen. France: Universite de Caen.
Lenoir, F. 1989. Mise au point de techniques de dissociation, de purifica-
tion et de culture cellulaires chez la moule Myriliis edidis (L.). Appli-
cation a I'etude des regulations du metabolisme du glucose et du gly-
cogene dans les cellules a glycogene ( = cellules vesiculeuses). These
de Doctorat. Caen. France: Universite de Caen.
Livingstone, D. R. & K. R. Clarke. 1983. Seasonal changes in hexokinase
from the mantle tissue of the common mussel Myrilus edulis (L.).
Comp. Biochem. Physiol. 746:691-702.
Mann. R. 1979. Some biochemical and physiological aspect of growth and
gametogenesis in Ciassosrrea gigas and Osirea edulis at sustained
elevated temperatures, J. Mar. Biol. Ass. U. K. 59:95-100.
Mathieu. M. & P. Lubet. 1993. Storage tissue metabolism and reproduction
in inarine bivalves: A brief review. Invert. Reprod. Dev. 23:123-129.
Mosmann. T. 1983. Rapid colorimetric assay for cellular growth and sur-
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Methods 65:55-63.
Pipe, R. K. 1987. Ultrastructural and cytochemical study on interactions
between nutrient storage cells and gametogenesis in the mussel Mytilus
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Robert. R.. G. Trut. M. Borel, M. & D. Maurer 1993. Growth, fatness and
gross biochemical composition of the Japanese oyster Crassostrea gi-
gas in stanway cylinders m the bay of Arcachon. France. Aijuaculture
110:249-261.
Ruiz. C. D. Martinez. G. Mosquera, M. Abad & J. L. Sanchez. 1992.
Seasonal variations in condition, reproductive activity and biochemical
composition of the flat oyster. Osirea edulis. from San Cibran (Galicia.
Spain). Mar. Biol. 112:67-74.
Scherrer. 6. 1984. 6iostatistiques. Quebec. Canada: Gaetan Morin. 850 pp.
Walne. P. R. & R. Mann. 1975. Growth and biochemical composition in
Ostrea edulis and Crassostrea gigas. In: H. 6arnes. editor. Proceedings
of the 9th European Marine Biology Symposium. Aberdeen. Scotland.
UK: Aberdeen University Press, pp. 587-607.
Journal of Shcllfisl, Rcsccuih. Vol. 22. No. 3. 721-731, 2003.
EFFECTS OF TEMPERATURE AND FEEDING REGIMES ON GAMETOGENESIS AND
LARVAL PRODUCTION IN THE OYSTER CRASSOSTREA GIGAS
JORGE CHAVP:Z-VILLALBA,"* JEAN-CLAUDE COCHARD/ MARCEL LE FENNEC,"
JEAN BARRET,- MARTHA ENRIQUEZ-DIAZ,' AND CARLOS CACERPIS-MARTINEZ'^
^ Unite Mixte de Recherche (U.M.R.) Centre National de Reclierclw Scientifiqne (C.N.R.S.) 6539:
Institiit Universitaire Europeen de la Mer. 292H0: Ploiizane. France: 'Laboratoirc de Physiologie des
Inrertehres. Institute Fram^ais de Recherche poin- L' exploitation de la Mer (IFREMER) Centre de Brest.
BP 70. 29280 Ploiizane. France: ^Centre de Investii^aciones Bioldgicas del Noroeste (CIBNORj.
GiiaYinas Unit. AP 349. Giiuynuis. .Sonora H5465. Mexico: ^Universidad Aittononui de Baja California
Sitr. AP I9-B, La Paz. Baja California Snr (B.C.S.) 23080, Mexico
ABSTRACT The effect of feeding regimes and temperature on the beginning of gametogenesis in the Pacific oyster Crassoslrea
,i;/,i;c/.v (Thunherg) was examined under laboratory conditions. Oysters from two different culture sites in France. Baie des Veys
(Department Manche) and La Tremhlade (Department Charente-Maritime). were collected in January 2000 and exposed to four
treatments, nivolving a period of maintenance at lO'C with or without feeding followed by a conditioning period at I9°C with or with
feeding. Routine conditioning procedures at 19°C (direct conditioning), with or without food, were performed at the same time and
were used as controls. Oocyte size was used to describe the evolution of gametogenesis in all treatments. Contrasting responses were
noticed between samples from Baie des Veys (B V-oysters) and La Tremblade (LT-oysters). BV-oysters containing more tissue reserves
than specimens from the other location used carbohydrates to support gametogenesis. while LT-oysters used proteins to fuel oocyte
development. During the initial period at 10°C, fed BV-oysters began gametogenesis and produced mature oocytes, while unfed
BV-oysters began gametogenesis, but at a slower rate. Fed LT-oysters began gametogenesis at 10°C. whereas unfed LT-oysters
remained unchanged (early gametogenesis stage) during the cold phase and only initiated gametogenesis when the temperature was
increased. Oysters conditioned without food produced significantly less oocytes than specimens conditioned with food, but no
differences in larval yield (D-larvae) were detected amongst the different conditions and sampling locations. Only LT-oysters kept
without food throughout the experiment did not produce oocytes at the end of the conditioning period. These experiments demonstrate
that oocyte production in C. )>igas is dependent upon food supply and temperature, but that oocyte quality under controlled conditions
appears to be related to stored reserves in natural settings.
KEY WORDS: conditioning. Crassostivci gigiis. food, gametogenesis, temperature
INTRODUCTION
Acciinnilation of reserves in the Pacific oyster Crassoslrea gi-
gas (Thtinhcrg 179.3) take.s place in autumn ancJ winter. an(J the
first signs of the beginning of gametogenesis are observed in Janu-
ary when temperature is still descencjing (Chavez-Villalba et al,
2002a). The influence of food supply and tetnperature on the re-
production cycle of bivalves has been noted by many authors
(DinamanI 1987, Ruiz et al. 1992). However, there are few data on
the influence of environmental factors on gametogenesis in C.
gigas. The studies on this topic for the scallop Aeqiiipecten irra-
dians concenlriciis (Sastry 1979) and eastern oyster Crassoslrea
virginica (Thompson et al. 1996) suggest that in the early phase of
gametogenesis, bivalves require an adequate food supply, as well
as a suitable temperature to stimulate gonad growth. These authors
proposed that, under inadequate food conditions, tissue reserves
are used for maintenance metabolism rather than gametogenesis.
and that food supply appears to be less critical after certain mini-
mum reserves have accumulated in the gonad. Gonad maturation
then occurs at a rate that is dependent on temperature. Gametoge-
nesis in oysters is directly coiTelated with water temperature (Mu-
ranaka & Lannan 1984). However. Sastry (1968) found that low
temperature can be inhibitory in well-fed scallops held at I5^C that
already had started early gametogenesis. but oocytes did not enter
into normal growth until exposed to higher temperatures (20°C).
*CorTespondmg author. E-mail; jechavezCs'cibnor.mx
Thus, normal reproductive development requires a minimum tem-
perature and an adequate food supply
Temperate bivalves exhibit a marked seasonal cycle in the syn-
thesis, accumulation, and use of biochetiiical energy reserves. In
general, reserves are stored during periods of high food availability
(late summer and fall), at which time the major energy require-
ments for somatic and germinal growth have already been satis-
fied. Stored reserves are used to initiate gametogenesis and to
maintain metabolism during periods of low food availability
(Thompson et al. 1996). Berthelin et al. (2000) found that glycogen
accumulation in the gonad of C. gigas occurs in fall and winter,
and this compound serves as a substrate to support gametogenesis.
In this way. oysters can partially uncouple temporal food avail-
ability with gamete production, allowing gametogenesis to start
when food supply is at a miiiiminii (winter).
The accumulation and use of stored reserves in bivalves depend
on the state of gonad development, the influence of en\ ironinental
parameters on metabolic activities, and the nutritional value of
food supplied during conditioning. In French hatcheries, the be-
ginning of broodstock conditioning of C. gigas starts in December
to obtain viable gametes and larvae by the end of January (Chavez-
Villalba et al. 2002b). Conditioning procedures consist of feeding
abundantly the oysters for 7 wk at a warm temperature ( 19°C). The
use of this technique allows animals to be conditioned from De-
cember until April, producing an increase of viable gametes and
larvae with time (Chavez-Villalba 2001). Additionally, it was
found that there were sotne groups of oysters that can produce
721
722
Chavez-Villalba et al.
viable oocytes without being fed during the conditioning proce-
dures, and that larval hatching rates from unfed oysters were not
significantly different, compared with fed animals. However, the
nutritional stress produced by partial or complete deprivation of
food can substantially alter the biochemical composition of bi-
valves. Whyte et al. ( 1990) demonstrated that oysters exhibit dif-
ferent biochemical composition if deprived of food.
Different experiments in our laboratory (Chavez-Villalba 2001.
Chavez-Villalba et al. 2002a. Chavez-Villalba et al. 2002b.
Chavez-Villaba et al. 2003) have shown that gametogenesis in C.
gigas seems to be an integrated response to different environmen-
tal factors, in which temperature and food supply play significant
roles. In this study, the effects of food and temperature in the
period at the beginning of gametogenesis. as well as the biochemi-
cal changes in soft tissues produced by the effect of these param-
eters, were investigated. To achieve these objectives, we studied
two oyster populations: one from Baie des Veys (BV) where oys-
ters have low spawn rates as a result of low summer temperatures;
and the other from La Tremblade (LT) where en\ironmental con-
ditions allow full spawning in summer.
MATERIAL AND METHODS
Experimental Conditions
At the end of January 2000. two 600-oyster samples were taken
from two different culture sites in France where they had been
raised in plastic mesh bags on iron tables. These animals were
initially collected from the Bassin d'Arcachon (44''41.8'N.
I°8.3'W). In the Bassin. the water temperature varies from 7.5°C
in January to 22°C in August, and salinity records go from 26 to
33i7(:c during the year. Juveniles then were grown at Le Morbihan
(47°35.5'N, 3°I.3'W) until they were 18 mo old and subsequently
were dispatched to one of two culture sites, where they were raised
for about I y (Fig. I). In Le Morbihan. water temperature fluctu-
ates from 3 to 5°C in the winter to 20 to 22^C in the summer, and
salinity values remain stable throughout the year (34. 8-35. 4*^*^?).
One of the culture sites is on the Baie de la Seine in the BV in the
Departments of Manche and Calvados (49°2I.5'N. r6.9'W). In
this zone, the average temperature in January is about 6°C. about
I7°C in August, and during spring and summer temperature in-
creases on the surface. Salinity records in the bay are always under
34.5'/rc. showing a decreasing pattern toward the coast (30^f ). The
other site is located on the Atlantic coast in the estuary of the
Seudre River at LT (45°3I.6N. I°I.7W) in the Department of
Charente-Maritime (Fig. I). In this area, the temperature varies
from 7°C in winter to 22°C during the summer, and salinity
records are closer to I5%f (www.ifrenier.fr).
Oyster samples were transported from the culture sites to the
Brest-IFREMER center where the samples from the two sites were
divided into several groups. Fifty animals from each site were
exposed to standard conditioning (I9°C with ample food). Two
more 50-oyster groups from each site were exposed to standard
conditioning (I9°C) but without food. These groups were condi-
tioned (direct conditioning) from February 8 to March 30, 2000,
and were used as controls.
Four groups of 2.'iO-oysters each (two from BV and two from
LT) were placed in maintenance tanks at IO''C. One group from
each site was fed continuously with the same diet as that used for
conditioning, and one group from each site was maintained with-
out food. These maintenance treatments were sustained for 60 days
(8 February-9 April. 2000). At the end of this period, each group
was divided into two subgroups that were conditioned under two
different treatments, with and without food (Fig. 2).
For the conditioning experiments, seawater temperature in the
tanks was increased IC per day until it reached I9°C (heating
period), and the photoperiod was adjusted to 16 h of daylight and
8 h of night (spring conditions). Oysters were fed a diet coiumonly
used in experimental hatcheries for conditioning: a mixture of two
micro-algae species ( 10" cells of each species per day per animal)
from monospecific cultures of hiichrysis aff. galbana Green
(Clone T-Iso: Tahiti Isnchnsis) and Clnwroceros calcitnuis Ta-
kano.
Sampling
English Channel^ _,_^
r^
"' r
Baie de^^ys
^1
^ ■ — ^
Manche
O ^-
-48°
*"^ Baden
(Intermediate culture)V^^
FRANCE
Atlantic \^^
Ocean '^
<
La Tremblade)
i Charente-
-Maritime
Bassin d Arcachon
k
— 44°
(Spat collection)!
0°
1
Figure 1. Location of C. gigas sites.
During the direct conditioning, the groups were sampled (20
oysters per site) two times: the first sample was obtained before the
heating period ( 10°C), and the second sample was taken after 6 wk
at I9°C. For the four treatments, oyster samples (20 from each
group) were taken at the beginning, in the iniddle. and at the end
of the period at IO°C. The last samples were obtained at the end of
the four conditioning procedures (Fig. 2). Froiri each sample (20
oysters). 10 specimens were used for histologic examination, and
the other 10 were used for biochemical analyses.
Semi-Quantitative Histology
Procedures in this part of the study generally followed the
methods of Chavez-Villalba et al. (2002a, 2002b). Oysters used for
histology were opened, and a section of approximately 1 cm^ of
visceral mass was taken from above the pericardial area and fixed
in Bouin's solution for at least 48 h. Samples were dehydrated with
a series of ethanol treatments of increasing concentration, cleared
in toluene, and embedded in paraffin following a standard proce-
dure. Sections 3 p-m wide were cut. mounted on glass slides, and
colored with Groat's hematoxylin and eosin Y solution (Martoja
and Martoja-Pierson 1967). The histology slides were examined
under a microscope connected to a video camera to determine
Gametogenesis and Larval Production in Chassostrea cncAS
20 -, Conditioning at 19 °C
723
16
12
8
4
0
-
f
•
o
o
o
-s.
a
-a
I
c
G
o
With food
^« ^^
>
w
WHh food ^^ ^ ^
,r<
o
?
0)
#
•Without food ^* ^ ^ft y^
T°
^k Winter conditions (10 °C)
....4
^
Q.
Witfifood
^^ ^t"
E
^^
L
w
(U
Wittiout food ^* ^
.,<
^
Wittiout food
^B .^t*
^^ Oyster groups
^ Samplin
g
1 1— 1 1 1 1
0 20 40
1 1 1
60 80
1 1 1
100
1
120
Days
Figure 2. Experimental conditions of the four treatments (black circles = samples taken for histologic and hiochemical studies). B = BV
specimens; T = I,T specimens. Starting date is Kebruarj 8, 2(((M).
oocyte size and frequency, and gametogenic activity. Recorded
images were processed by digital image analysis.
Oocytes were measured and histology classified following the
description by Lango-Reynoso et al. (2000). These operations
were conducted on 1 00 randomly chosen oocytes per oyster, and
measurements followed a standard bias reduction procedure for
selecting measurement fields. Transects of gonad preparations
were oriented to maximize coverage of the larger vertical or hori-
zontal oocyte field axis. All oocytes with a well-defined germinal
vesicle in a field were measured, and every oocyte measured was
assigned to a reproductive stage based on the diameter and histo-
locic characteristics of the sonad (Table 1 1. In the case of male
oysters, the evolution of the spermatogenesis was described ac-
cording to the histologic characteristics of the gonad. Three de-
velopmental stages were recognized (Table 1).
Biochemical Analyses
We used 10 oysters per sample for biochemical analyses.
Specimens were dissected, and soft tissues were divided into two
sections: gonad-digestive gland portion (called "gonad"); and the
remaining tissue (called "meat"). All samples were ground by
adding 1 niL of distilled water per gram of tissue at 5°C in an ice
bath. A 400-|a.L aliquot was used for lipid detennination using the
TABLE 1.
Reproductive stages in the female and male oyster C gigas: Cytologic characteristics corresponding to each stage arc included.
.Stages
Stage Interval
(Mm)
Histologic Description
Females
Early gametogenesis
Growing
Mature
Degenerating
Males
Early gametogenesis
Growing
Mature
,^.0± 12.0 Follicles are elongated and often isolated in the abundant connective tissue, with walls
consisting of primary oocytes of homogeneous size.
I2.1-.^0.0 Start of oocyte growth. A large range of oocyte size at all gametogenic stages can be
observed, including some free oocytes. Interfollicular connective tissue disappears.
30. 1— H.O Follicles of relatively homogeneous size completely filled with mature oocytes with distinct
nucleus.
41.1-60.0 Follicles containing degenerating oocytes, often elongated in shape, sometimes broken.
Obvious redevelopment indicated by increased number of primary oocytes.
Abundant connective tissue containing elongated follicles with walls consisting of germinal
epithelium with some spermatogonia and spermatocytes
Connective tissue is reduced, follicles become larger, and normal sequences of
spermatogenesis are observable with spermatocytes 1 and 11. spermatides. and some
spermatozoids organized in the lumen
ConnecUve tissue almost disappeared. Follicles tilled with packages of spermatozoids oriented
with tails toward the follicle lumen
Reproductive stages in female specimens are based on an oocyte diameter (p.m) interval (Lango-Reynoso et al. 2000).
724
Chavez-Villalba et al.
Bligh and Dyer (1959) method. Carbohydrates were analyzed in a
300-|xL sample by the method of Dubois et al. ( 1956). and proteins
were analyzed in a 300-(j.L aliquot by the method of Lowry et al.
(1951). Tissue dry weights were calculated from the macerate of
each sample; 2 niL were emptied into preweighed aluminum con-
tainers and dried in an oven at 80°C for 48 h. Finally, aluminum
containers were reweighed after cooling in a desiccator.
Given that no significant differences were observed between
total dry weights of oysters (from BV and LT locations) at the
beginning and by the end of the experiments, the dry weight per-
centages of each biochemical compound were multiplied by the
total dry weight of each tissue sample to express the results in
milligram equivalence of each biochemical compound per total dry
weight of the tissue (gonad and meat).
Oocyte PrDcluclioii and Larval Yield Estimation (I)-Lanae)
Oocyte production and larval yield estimations followed the
recommendations of Chavez-Villalba et al. (2()()2a). Oysters from
each group were taken from the experimental tanks at the end of
conditioning procedures; 20 animals per group were opened, and
their sex was determined by observing a fresh smear sample from
the gonad under a microscope. After this procedure, females and
males were separated, and gametes from both sexes were recov-
ered using the scarification technique described by Allen and
Bushek (1992). The gonads of all oysters were scarified by a light
incision of the gonadal tegument. Oocytes were collected in bea-
kers by rinsing the gonad with filtered seawater. The oocytes were
passed through a 60-|xm sieve to eliminate undesirable material.
Mature oocytes were retained in a 20-|jim sieve. These were rinsed
several times and placed in 2- or 5-L beakers. To determine oocyte
production, three 50-|xL samples per group were examined and
counted under a profile projector. Males underwent the same pro-
cedure, but spermatozoa suspensions were examined under a mi-
croscope for motility. Batches of spermatozoa of low motility were
discarded. A minimum of three batches was mixed together and 10
to 20 niL were used for fertilization. Oocytes were fertilized in 5-L
beakers and checked for normal progress about 0.5 to 1 h later
(Robert and Gerard 1999).
After fertilization, an equal number of embryos from all oysters
of each group were pooled together and placed, one group per
tank, in 150-L tanks at a 33 embryos mL"' concentration. After 48
h. the tanks were emptied, and the larvae were recovered by siev-
ing. Three 50-(j.L larvae samples from each tank were taken for
larval yield estimation (number of D-larvae after 48 h of culture/
initial number of embryos).
Data Analysis
The oocyte proportion corresponding to each reproductive
stage was calculated according to Lango-Reynoso et al. (2000).
and the arcsine was transformed (Snedecor and Cochran 1972) for
each oyster. The logarithms of oocyte production data were cal-
culated. The transformed proportions and logarithms were com-
pared using the Kruskal-Wallis test. A two-way analysis of vari-
ance (ANOVA) test was used to examine the effect of origin and
feeding regimen on early, growing, and mature oocyte categories
for the direct conditioning. A three-way ANOVA test was run to
analyze the following: ( 1 ) the effect of time (between days 30 and
60). origin, and feeding regimen on the different oocyte categories
during the period at 10°C; (2) the effect of time (beginning and end
of conditioning), origin, and the four tested conditions on the oo-
TABLE 2.
Mean proportion" of different oocyte stages: early gametogenesis, growing, and mature in C. gigas under direct conditioning «ith and
without food, and four treatments during a second phase of conditioning.
Day (I
End of Conditioning
(Day 56)
Oyster Sample
M
M
Characteristics
Direct conditioning
BV
LT
69
hi
31
M
6
.^0
7 \\k at I9°C
51
41
4h
72
19
38
Conditioning WF
Conditioning WOF
Conditioning WF
Conditioning WOF
Period at IOC
Day 0
Day 3(1
Day 6()
Oyster Sample
M
M
M
Knd of Conditioning
(Day 110)
E G M
Characteristics
Four treatments
BV
LT
69
63
37
58
42
0
33
66
1
-)
84
16
0
(lU
40
t)
0
11
VU
10
u
38
62
0
5
96
4
0
87
Li
0
10
57
It
26
12
76
28
27
34
43
76
73
88
13
67
72
56
0
Treatment
Treatment
Treatment
Treatment
Treatment
Treatment
Treatment
Treatment
1 (WFAVF)
2 (WFAVOF)
3 (WOFAVF)
4 (WOFAVOF)
1 (WFAVF)
2 (WF/WOF)
3 (WOFAVF)
4 (WOFAVOF)
Presented as %. E, early gametogenesis; G, growing; M. mature; WF. with food; WO. without food.
" Mean of values found in a 10-oyster sample.
Gametogenesis and Larval Production in Crassosthha gigas
725
cytes (early, growing, and mature) during the conditioning pliase;
(3) the effect of origin, time, and feeding regimen on the lipid,
protein, and carbohydrate content in the gonad and ineat of oysters
maintained at lO'C for 60 days; and (4) the effect of origin, time,
and four tested conditions on the lipid, protein, and carbohydrate
content in the gonad and meat of oysters during the conditioning
procedures. Statistics were analyzed at a significance level a =
0.03.
RESULTS
Oogenesis
Direct Conditioning
The proportions of the different oocyte categories tound at the
beginning and the end of direct conditionings are presented in
Table 2. Mature oocytes were observed in all BV and LT groups
at the end of conditioning, but statistical results (Table 3) showed
a significant effect of feeding regimen and place of origin. There
was a higher proportion of mature oocytes in specimens condi-
tioned with food, and BV oysters produced a higher proportion of
mature oocytes than LT specimens.
Four Tested Conditions
The proportions of the oocyte categories found during the cold
phase and at the end of conditioning in both BV and LT oysters for
TABLE 3.
Results of two-Hav and three-way ANOVA tests for early, groHing,
and mature oocyte categories, and for biochemical content in the
gonad and meat of oysters C. gigas, respectively (direct
conditioning).
Source of Variation Df MS
F
P
Oogenesis
Early gametogenesis stage
Factor A (feeding regime) 1
1840.1
8.53
0.0079
Factor B (origin) I
1144.1
5.54
0.0280
Interaction (A x B) I
111
0.05
0.8225
Growing stage
Factor A (feeding regime) 1
475.2
345
0.0768
Factor B (origin) 1
68.5
0.5
0.4882
Interaction (A x B)
360.2
2.61
0.1202
Mature stage
Factor A (feeding regime) 1
6201, fs
.34.8
().()()()()
Factor B (origin)
1615.2
9.05
0.0065
Interaction (A x B) I
78.9
0.44
0.5128
Biochemistry
Meat
Factor A (origin)
1 .03E6
,54.2
0.0000
Factor B (feeding regime)
936.7
0.05
0.8246
Factor C (time)
433395
22.7
0.0000
Interaction (A x B)
814.4
0.04
0.8.366
Interaction (A x C)
28714
1.5
0.2217
Interaction (B x C)
1179.6
0.06
0.8040
Gonad
Factor A (origin)
2.25E6
126
0.0000
Factor B (feeding regime)
324793
18.2
0.0000
Factor C (time)
349092
19.5
0.0000
Interaction (A x B)
7675.7
0.43
0.5132
Interaction (A x C)
1445.8
0.08
0.7765
Interaction (B x C)
339690
18.9
0.0000
DF. degrees of freedom; MS, mean .square: F, ratio; and P, probability.
all conditions are summarized in Table 2. Statistical analyses
(Table 4) for the period at lO'C showed a significant effect of
feeding regimen, place of origin, and time. In the treatment with
food, there was a higher proportion of growing oocytes than in the
treatment without food. The production of growing oocytes was
significantly higher in BV oysters, and there was a significant
increase of growing oocytes over time. For the conditioning pe-
riod, we found a significant effect of treatment (the proportion of
mature oocytes was significantly lower in treatment 4) and time
(mature oocytes increased significantly with time). Nevertheless,
there was not a significant effect of place of origin for tiiature
oocytes (Table 4).
Spermatogenesis
The number of male oysters found in this study did not allow
observation of any pattern of change in spermatogenesis. The
number of males detected in the four treatments is presented in
Table 5.
Biochemistry
Direct conditioning. Place of origin and time had a significant
effect on the biochemical content in both the gonad and meat of
oysters (Table 3). Specimens from BV had a higher content of
biochemical compounds in the gonad and meat coirtpared with
oysters from LT, and the biochemical content increased signifi-
cantly over time in the conditioning procedures. Feeding regitnen
also had a significant effect but only on the biochemical content
in the gonad. There was a higher content of proteins, lipids,
and carbohydrates in the gonad of oysters conditioned with food
(Table 6).
Four tested conditions. Three-way ANOVA during the phase
at 10°C (Table 4) showed a significant effect of feeding regimen,
place of origin, and time on the biochemical content of the gonad,
and a significant effect of place of origin and time on the bio-
chemical content in the meat. A significantly higher biochemical
content was found in the gonads of oysters maintained with food.
No significant differences were noted relating biochemical content
in the meat of animals kept with or without food. Significant
biochemical differences in meat and gonad tissue favored the BV
oysters. The biochemical content increased significantly over time.
Statistical analyses (Table 4) during the conditioning procedures
showed a significant effect of treatment, place of origin, and time
on the biochemical content of oyster gonads. For meat, a signifi-
cant effect of place of origin favored the BV specimens. Concern-
ing conditioning effects on the biochemical content of gonads, the
highest content was found in treatment I (BV-1 and LT-1) com-
pared with the other treatments, and there were no significant
differences between treatments 2 and 3. The lowest concentration
was observed in treatment 4. Finally, the biochemical content was
significantly higher in oysters from BV. and compound concen-
trations increased significantly over time (Figs. 3 and 4).
Oocyte Production and Larval Yield
Direct conditioning. LT oysters conditioned without food did
not produce any oocytes at the end of the direct-conditioning
phase. In the other three treatments, BV oysters produced more
oocytes than LT specimens. Concerning the yield of larvae (D-
larvae), similar values were obtained (85% and 78%) for fed oys-
ters coming from both sources and a yield of larvae (62%) in BV
.specimens conditioned without food. There were no significant
726
Chavez-Villalba et al.
TABLE 4.
Results of three-way ANOVA tests for early, growing, and mature
oocyte categories, and for biochemical content in the gonad and
meat of oysters C. gigas in the period at WC and conditioning.
TABLE 4.
continued
Source of Variation
Df
MS
Oogenesis
Period at 10°C
Early gametogenesis stage
Factor A (origin)
Factor B (feeding regime)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Growing stage
Factor A (origin)
Factor B (feeding regime)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Mature stage
Factor A (origin)
Factor B (feeding regime)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Conditioning
Early gametogenesis stage
Factor A (treatment)
Factor B (origin)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Growing stage
Factor A (treatment)
Factor B (origin)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Mature stage
Factor A (treatment)
Factor B (origin)
Factor C (time)
Interaction (A x Bl
Interaction (A x C)
Interaction (B x C)
Biochemistry
Period at 10°C
Meat
Factor A (origin)
Factor B (feeding regime)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Gonad
Factor A (origin)
Factor B (feeding regime)
Factor C (lime)
1 1016.3
4.36
0.0491
1 1S08.2
7.76
0.0111
1 2378.y
10.2
0.0043
1 21.03
0.09
0.7668
1 7.38
0.03
0.8604
1 131.58
0.56
0.4607
1 993.1
4.33
0.0499
1 1 768.2
7.71
0.0113
1 2343.1
10.2
0.0043
1 25.6
(J. 11
0.7417
1 9.51
0.04
0.8406
1 120.9
0.53
0.4758
1 2.57
0.45
0.5079
1 4.3
0.76
0.3938
1 2.57
0.45
0.5079
1 4.3
0.76
0.3938
1 2.57
0.45
0.5079
1 4.3
0.76
0.3938
3 2193.3
16.9
0.0000
1 2152.8
16.6
0.0000
1 16.M0
126
0.0000
3 353.2
2.73
0.0523
3 552.8
4.27
0.0087
2 39.2
0.3
0.5842
3 546.5
2.97
0.0395
1 354
1.92
0.1711
1 561.4
3.05
0.0862
3 340.6
1.85
0.1486
3 2121.5
11.5
0.0000
1 560.7
3.04
0.0864
3 2396.8
26.7
0.0000
1 701.3
7.81
().()()71
1 29084
324
0.0000
3 55.9
0.02
0.6030
3 2337.6
26 1
0.0000
1 614.6
6.85
0.0114
1 2.73E6
100
0.0000
1 21011
0.77
0.3816
1 224169
8.22
0.0048
1 8410.4
0.31
0.5796
1 5842.8
0.21
0.6442
I 2307.9
0.08
0.7716
1 1.9E6
229
0.0000
1 149781
18
0.0000
1 1270S6
15.3
0.0008
c
tnlinucd
Source of Variation
Df
MS
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Conditioning
Meat
Factor A (treatment)
Factor B (origin)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
Gonad
Factor A (treatment)
Factor B (origin)
Factor C (time)
Interaction (A x B)
Interaction (A x C)
Interaction (B x C)
1
39084
4.7
0.0318
1
20100
2.42
0.1222
1
96709
11.6
0.0008
3
32563
1.02
0.3829
1
5.02E6
158
0.0000
1
147545
4.63
0.0321
3
651.59
2.14
0.0950
3
10153
0.32
0.8119
1
236027
7.41
0.0068
3
492786
17.9
0.0000
I
5.5 1E6
201
0.0000
1
308652
11.3
0.0009
1
32763
1.2
0.3113
3
307844
11.2
0.0000
1
324.5
0.01
0.9134
Df, degrees of freedom; MS, mean square; F, ratio; and P. probability.
differences in oocyte production among the three treatments in this
part of the experiment (Fig. 5).
Four tested conditions. The highest oocyte production oc-
curred in oysters fed during the experiment, in particular in speci-
mens under conditions BV-I and BV-3. Specimens raised under
condition LT-4 (without food) did not produce any oocytes by the
end of the e.\periment. The highest yield of larvae was detected in
BV and LT oysters that were not fed during the cold phase and in
oysters raised with food during conditioning. The lowest yield of
larvae was observed in specimens from BV maintained under
treatment 1, even though the mean oocyte production in these
oysters was 42.5 million. The statistical analysis showed that treat-
ments involving feeding produced significantly more oocytes than
did treatments involving oysters kept without food, with the high-
est values favoring BV oysters. There are no significant differ-
ences in oocyte production among unfed oysters (Fig. 5).
DISCUSSION
Oocytes in early gametogenesis and growing stages in BV oys-
ter samples were observed at the beginning of February in our
laboratory in previous experiments (1999). These oocyte catego-
ries were detected later in LT oysters (Chavez-Villalba et al.
2002), which is in agreement with the results of Lango-Reynoso
(1999), who found oocytes in the growing stage in oysters from
Marennes-Oleron (near LT) by the end of February 1998. In this
study, oocytes in early gametogenesis and growing stages in both
oyster samples were detected at the beginning of the direct con-
ditioning (February 2000), showing that the oysters in LT began
gametogenesis earlier in the year. Even though similar proportions
of early gametogenesis and growing oocytes were measured at the
beginning of conditioning in both samples, by the end of condi-
tioning lower proportions of mature oocytes were found in LT
oysters in both treatments, indicating differences in the environ-
mental patterns regulating the beginning of gametogenesis be-
tween these samples.
With decreasing latitude, the temperature rec^uired for the ini-
Gametogenesis and Larval Production in Ch-^ssostrea gigas
727
TABI.K 5.
Number of male oysters (C. gigas) and their respective developmental stage found during the four treatments.
10 C
0 Davs
30 Davs
60 Days
BV
LT
B\
LT
BV
LT
End C
1 10 Days
BV
LT
Treatment 1
Early gametogenesis
Growing
Mature
Treatment 2
Early gametogenesis
Growing
Mature
—
Treatment 3
Early gametogenesis
Growing
Mature
Treatment 4
Early gametogenesis
Growing
Mature
End C. end ot conditioning.
tiation of the ganietogenic cycle increases, and. as a result, repro-
ductive cycles occur later in the year (Barber and Blake 1983).
This study showed different responses between northern and
southern oysters to food and temperature during the period con-
sidered, such as the beginning of gametogenesis in C. gigas. Oys-
ters from BV (in the north of France), within a high productivity
ecosystem (Goulletquer et ul. 1996). acclimated to colder water
than southern populations, developed mature oocytes after 60 days
TABLE 6.
Protein, carbohvdrate. and lipid content (ing/e(|ui\alent tissue") in the gonad and meat of C. gigas samples from t"o culture sites at the
beginning and by the end of the direct conditioning experiment conducted under two types of conditions: with and without food.
Conditioning
Oyster Sample
Tissue
Compound
Beginning
End
Conditioning
Characteristics
BV
Gonad
Meat
LT
Gonad
Meat
Carbohydrates
Proteins
Lipids
Carbohydrates
Proteins
Lipids
Carbohydrates
Proteins
Lipids
Carbohydrates
Proteins
Lipids
283 ± 27
264 ± 1 1
113 ±7
195 ± 2:1
253 ± 17
45 ±2
5+1.5
47+12
8±2
15 ±3.5
93 ± 1 1
3 1 + 4.5
211 + 16
With lood
256 ± 37
Without food
732 ± 58
With food
307 + 28
Without food
278 ± 30
With lood
119 ±23
Without food
144 ±28
With food
206 ± 32
Without food
557 ± 34
With food
502 ±31
Without food
128 ±16
With food
106 ±20
Without food
70 ±9
With food
16 ±5
Without lood
341 ±.%
With food
63 ±9
Without food
73±1I
With food
66 ±37
Without food
36 ±6
With food
I8±7
Without food
235 ± 30
With food
262 ± 47
Without food
40 ±5
With food
53 ± 20
Without food
" Presented as mean ± SE in a sample size of 10 oysters.
728
Chavez-Villalba et al.
0)
cn
E
0)
*-»
c
o
u
15
o
E
O
o
£0
••♦■ Carbohydrates
Meat
Proteins
Treatment 1
4- Lipids
Gonad
Treatment 2
— «
Treatment 3
Treatment 4
120
30
60
90
120
Time (days)
Figure i. Protein, carbohydrate, and lipid content in the meat and gonad of C gigas from the BV site during the four treatments (10°C for the
period of O-fit) days, and conditioning at 19 C for the period of 6(>-110 days).
at 10°C when maintained with food. When l<ept without food,
oysters produced a large proportion of growing oocytes during this
period. In contrast, the LT oysters (cuhured in a central coastal
bay), where conditions included a limited food supply (Pas-
toureaud et al. 1996), produced some growing oocytes when raised
with food during the cold phase. LT oysters kept without food
remain blocked in the early gametogenesis stage. Considering that
BV oysters can initiate gametogenesis and continue oocyte devel-
opment even if winter conditions are artificially extended, this
eliminates temperature as the principal regulator of gametogenesis
in C. gigas. Bivalves require sufficient energy to meet mainte-
nance and reproductive requirements during their gametogenesis
cycles. The differences found in this study are probably a result of
northern oysters having a better storage reserve than animals from
southern locations. Moreover, since both samples of oysters were
collected from the same more southern location (Bassin dArca-
chon). no genetic diversity is considered.
Lubet (1976) found that the initiation of gametogenesis in Mvti-
liis edtdis and Mytilus galloprorincinlis is not dependent on ther-
mal conditions. This author emphasizes that populations of C.
gigas and Ostrea edtdis in the English Channel confirmed similar
results, since the reinitiation of gametogenic activity coincides
with low temperatures (8-9°C). It seems that these facts are valid
for the oysters in BV. but in the case of oysters in LT it appears
that if enough food is available to build a reserve, these oysters can
initiate gametogenesis under low-temperature conditions. Never-
theless, LT oysters kept without food initiated gamete develop-
ment until the temperature increased, but the mature stage was not
reached after conditioning, probably because reserves were used to
assure maintenance instead of gametogenesis. Lubet (1976) hy-
Gametogenesis and Larval Productkw in Crassostrea gigas
ll")
D
(0
cr
0)
£
c
0)
'c
o
o
ro
o
E
0)
o
o
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♦■ Carbohydrates
Meat
600 ■
500 ■
400
300
200
100
0
600 1
500
400 ■
300 ■
200 •
100
0
Treatment 2
Treatment 3
600
500 .
400 .
300
200 ■
100
0
Treatment 4
30
60
90
120 0
Time (days)
30
60
90
120
Figure 4. Protein, larbolivdrate, and lipid content in the meat and gonad of C. gigas from the LT site during the four treatments (10 C for the
period of 0-60 days, and amditioning at 19 C for the period of 60-110 days).
pothesized that the beginning of gametogenie activity is under the
influence of a neurosecretion internal clock that determines the
initiation and extent of the sexual cycle, and this clock may be
modified by external factors, of which temperature would play an
essential role. Nevertheless, the results of this study showed that,
apart of temperature, the beginning of gametogenesis is also de-
pendent on reserves accumulated during the previous year. As a
consequence, we can hypothesize that, even if the beginning of
gametogenesis is dependent upon temperature, oocyte develop-
ment will not occur in conditions of low temperature unless a
minimum reserve stock had been accumulated. Thompson et al.
( 1496) pointed out that food supply seems to be less critical once
a minimum quantity of reserves is accumulated in the bivalve
gonad. Some studies have demonstrated a site-specific variation of
the gametogenesis cycle associated with phenotypic adaptations to
local food supply variations (MacDonald & Thompson 1988).
Therefore, we assume that oocyte growth in C. gigcis is dependent
upon food supply and a certain minimum temperature that varies
with geographic location. This minimum temperature of about
10°C for BV oysters accords with the observations of Lubet
(1976). The higher water temperature for LT oysters takes into
consideration environmental conditions in that geographic loca-
tion.
It is known that there is a relationship between the proximate
biochemical composition of oysters and the gametogenie cycle
(Deslous-Paoli & Heral 1988). According to Berthelin et al.
(2000). glycogen concentration in C. gigcis is minimal immediately
after spawning, and increases during fall and early winter, reaching
maximum values prior to gametogenesis. Protein and lipid con-
centrations follow a similar pattern, w ith a fairly uniform percent-
730
Chavez-Villalba et al.
100
ss
HO
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60
(D
40
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40
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o
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o.
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O
10
-II-
T
T
T
T
r^
rn
L "^ 1
//-
w wo w wo
BV LT
Direct conditioning
BV
LT
Treatments
Figure 5. Oocyte production and larval yield (D-larvae) of C f^igas conditioned in February to March 2000 (direct conditioning at 19°C: W ■■
with food; WO = s>ithout food).
age composition during autumn and winter, but with the highest
amounts in the gonad from April to June. Even though BV oysters
were exposed to conditions that affected physiologic activities,
they have an important quantity of reserves, using carbohydrates as
the principal source to support gametogenesis under all conditions.
In contrast. LT oysters used proteins to fuel gamete development.
Probably, southern oysters used proteins because they have a net
loss of glycogen during the winter, when it may be catabolized to
meet maintenance requirements during poor food conditions
(Deslous-Paoli & Heral 1988). Whyte et al. (1990) found that
protein can contribute more than carbohydrates for the metabolic
processes of oysters maintained in unfavorable food conditions.
Moreover, Barber and Blake (1983) found that the source of re-
productive energy for Argopccten imuUans over its latitudinal
range could be affected by food supply and metabolic rates. These
authors suggest that, with decreasing latitude, the bay scallop has
a greater metabolic rate as well as a smaller food supply, with less
energy available for reproduction. This concept may be true for
this study if northern oysters are in more favorable food conditions
than southern populations, and that metabolic rates in this species
are influenced by temperature (Bougrier et al. 1995)
BV and LT oysters conditioned with food produced more oo-
cytes by the end of the conditioning period than those conditioned
without food. Similar results in C. gi^os were obtained by Rob-
inson (1992). These results indicate that the oocyte quantity pro-
duced under controlled conditions is dependent on the food offered
during conditioning, since the oysters fed during the phase at lO^C.
but conditioned without food, have produced significantly fewer
oocytes than animals conditioned with food, but maintained with-
out food during the cold phase. Oocyte production was signifi-
cantly lower in the oysters kept with food during the direct-
conditioning procedure than in oysters maintained in treatments 1
and 3 (BV-1, BV-3 and LT-1, LT-3, respectively). If it is consid-
ered that oysters in treatments 1 and 3 were maintained for 60 days
at the same temperature as that at the beginning of the experi-
ments (10°C), then the difference in terms of oocyte production
may indicate that animals during the cold phase continue their
oogonie multiplication with or without the influence of food. It
would be interesting to study changes at the cellular level and try
to quantify oogonie multiplication under similar experimental con-
ditions.
Le Pennec et al. ( 1990) found a significant relationship between
the lipid index of oocytes in Pecieii nicLxiiniis and the parameters
involved in the endotrophic phase of larval rearing. They empha-
size that D-larvae and the anomaly rates of prodisoconch I are
strongly related to the mean lipid index; the greater the lipid con-
tent in the oocytes, the greater the quality of larval rearing required
in the first 2 days of culture. We observed that lipids accumulated
in the gonads of oysters conditioned with food, and these animals
produced more oocytes than oysters maintained without food, but
the larval yield of the two groups was similar. Muranaka and
Lannan (1984) observed higher fecundity rates in oysters condi-
tioned with food when compared with oysters conditioned without
food. Nevertheless, the results of this study did not show signifi-
cant differences in larval yields between samples conditioned with
or without food. On the contrary, the lowest larval yield was found
in the oysters kept in condition 1 . These observations suggest that
lipid reserves in unfed oysters are maintained even in conditions of
the absence of food, and, although there are few oocytes in these
animals, these gametes will yield good quality D-larvae. In previ-
ous experitnents, Chavez- Villalba et al. (2003) observed that unfed
oysters not only yielded good-quality D-larvae but that larvae pre-
sented similar growth and survival patterns as larvae from fed
animals throughout 19 days of trials. Thus, oocyte quality seems to
be related not only to food quality during conditioning, but also to
reserves accumulated in nature prior to experiments.
Gametogenesis and Larval Production in Chassostrea gigas
731
In this study, oysters having the same place of origin show
flexible reproductive patterns that are responses to \arying envi-
ronmental factors, most notably food availability. Northern oys-
ters, having a larger reserve stock than southern oysters, initiate
gamete development in conditions of low temperature, which con-
firms that the beginning of gametogenesis is not dependent on
thermal conditions. The amount of gametogenic material is also
dependent on food supply, but oocyte quality seems to depend, to
a large extent, on accumulated reserves. The differences found in
this study are that the stored reserves in BV oysters are used to
initiate gametogenesis and to maintain metabolism under low food
a\ ailability. while LT oysters operate closer to their energetic limit
at the production site, and require supplementary energy from
spring planktonic blooms to continue gametogenesis and to pro-
duce \ lable oocytes and larvae.
ACKNOWLEDGMENTS
We thank Consejo Nacional de Ciencia y Tecnologi'a (Mexico)
for a scholarship to Jorge Chavez-Villalba for doctoral studies at
the Universite de Brelagne Occidentale. France. Experimental
work was supported by IFREMER/Contrat Universitaire Univer-
site de Bretagne Occidentale (UBO) project No. 98/2.'i21426. We
are grateful to Christian Mingant for \ery helpful technical assis-
tance during the experiments. The editing staff at Centro de In-
vestigaciones Biologicas del Noroeste (CIBNOR) reviewed and
improved the English text.
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J.mriHil of Shclljhl' Research. Vol. 22. No. 3. 733-73&. 2003.
TWO SPECIES OF OYSTER LARVAE SHOW DIFFERENT DEPTH DISTRIBUTIONS IN A
SHALLOW, WELL-MIXED ESTUARY
PATRICK BAKER*
Viri^iiila Institute oj Murine Science. College of William and Mary. Gloucester Point. Virginia. 23062
ABSTRACT The vertical distribution of late stage, or pediveliger. larvae of several bivalve mollusks was examined in a west Florida
estuary. The study site was an artificial canal, and the water was shallow (1.5 m) and well mixed, with only modest cuncnts.
Pediveligers of three bivalve taxa were collected: the eastern oyster Crassostrea virginica: the crested oyster OsHea ec/iu'stris: and
unidentified shipworms (Teredinidae). Despite the shallow and well-mixed water column, larvae exhibited vertical zonation. with most
larvae of all three species collected from lower in the water column. The larvae of C viri^inicu and shipworms showed no significant
effect of time of day. but larvae of O. eqiiestris reversed their distribution pattern at night, with most larvae being near the surface.
Pediveliger larvae were not behaving as neutrally buoyant particles but appeared to regulate their depth even in this well-mixed and
shallow water column. Given that the larvae of the two oyster species were probably coinpelent to settle, their vertical distribution
patterns do not fit what has been reported about their adult depth distribution.
KEY WORDS: Crassostrea viri;iiuca. estuary, larvae. Oslrea cc/iwslris. pediveliger. plankton. Teredinidae
INTRODUCTION
A variety of studies over the years have attempted to address
the issue of whether larval distribution in estuaries is controlled
mainly by hydrologic forces, or whether there is a significant larval
behavioral component that also affects distribution. For some crus-
tacean larvae, the case seems to be fairly well made that behavior
plays a large part in planktonic distribution, usually (but not al-
ways) for late-stage larvae or post-larvae (.Shanks 1986. 1995.
Benfield & Aldrich 1992. Gherardi 1995).
Bivalve mollusks also have been the focus of studies on larval
distribution in estuaries, but there is no consensus in the literature
on whether bi\alve veligers are distributed as neutrally buoyant
particles or whether behavior significantly affects their distribu-
tion. Like crustacean larvae, bivalve larvae clearly exhibit oriented
swimming, at least in the laboratory (Feeny 1984, Hidu & Haskin
1978). .Some field studies have appeared to show nonrandom bi-
valve larval distribution, relative to hydrodynamic processes
(Tremblay & Sinclair 1990, Shanks et al. 2002. Baker & Mann
200.^). Compared with crustacean postlarvae. however, bivalve
pediveligers are small and slow swimming, and Banse (1986)
questioned whether the weak swimming rates observed for these
larvae are sufficient to produce distribution patterns. The distribu-
don of bivalve larvae in estuaries may be attributed to hydrody-
namic processes alone in some cases, if larvae are treated as neu-
trally buoyant particles (Wood & Hargis 1971, Mann 1988).
This author examined the above question (i.e.. does bivalve
larval distribution in an estuary have a behavioral component?)
under the most restrictive conditions possible for an estuarine sys-
tem. The estuarine system in question was simple in shape (an
artificial inlet), very shallow, and well mixed throughout the study,
although it was a low-energy system. Only late-.stage bivalve lar-
vae were included in the study. If bivalve larvae behave as neu-
trally buoyant particles, their distribution should be fairly even
throughout the water column (allowing for boundary-layer ef-
fects), and the species should have similar distributions.
'Present address: Department of Fishenes and Aquatic Sciences. Institute
of Food and Agricultural Sciences. University of Florida. P.O. Box
110600. Gainesville. FL 32611. E-mail: pbaker@mail.ifas.un.edu
MATERIALS AND METHODS
Research was conducted at the Harbor Branch Oceanographic
Institute, near Foil Pierce. FL. in May 1993. The study site was
about halfway along a I -km artificial canal that opened into the
Indian River Lagoon. The sides of the canal were concrete and
steel seawalls, heavily fouled by eastern oysters. Crassostrea rir-
ginica. and the mean water depth at the wall were about 1 m.
gradually increasing toward the center of the canal. The observed
currents were mostly tidal, with velocities near the seawalls of 1 to
.^ cm s"'. and the tidal range was up to 0.5 m.
Plankton was sampled with two modified 12-V bilge pumps,
each rated at 1 800 L h~ ' . Power came from a standard 1 1 0- V outlet
with a transformer to regulate voltage. Pumps were suspended
about 2 m out from the canal wall, where the mean water depth
was about 1.5 m. One pump was maintained at a depth of about 20
cm above the bottom, which was determined by preliminary
samples to be the maximum depth achievable without entraining
significant quantities of sediment. The other pump was adjusted
for each sampling episode to a depth of about 20 cm below the
surface. Mann (1986) and Mohlenberg (1987) found no avoidance
of a plankton pump intake by bivalve mollusk larvae, which swim
slowly compared with many /ooplankton.
Water from each pump was delivered by a garden hose to a
separate sieve on the banks of the canal. Each sieve consisted of a
400-|ji,m coarse filter and a 150-|xm final filter on which the sample
was retained. Plankton was sampled twice daily, at mid-morning
(full daylight) and mid-evening (after nightfall), for about 2 h at a
time. The volume sampled at each depth was calculated from the
time, to the nearest minute, multiplied by the mean pumping rate.
The pumping rate was estimated before and after each sample, for
each pump, by the time required to fill a 20-L container. (If sam-
pling episodes included high or low water, the pumping rate mea-
surements also were taken then and factored into volume calcula-
tions.) Samples were taken into the laboratory, and bivalve larvae
were counted and identified to the lowest possible taxonomic level.
The identification of oyster pediveligers (C virgiiiica and Os-
trea eqiiestris) was verified by collecting newly settled juveniles
on shell-strings (Haven & Fritz 1985) that had been immersed at
the study site for <24 h. marking individuals, and letting them
grow in the canal for .several weeks. By the end of this time. O.
73.3
734
Baker
ecjuestris shells had developed the diagnostic dorsal-marginal den-
tition, or chomata (Galtsoff & Merrill 1962).
Only samples that had six or more pediveligers of a given taxa
from the two pumps combined were used in the analysis. Data for
each pump were converted to proportions of total larvae of a given
species collected in a sampling episode and were arcsine-square
root-transformed prior to statistical analysis (Zar 1996). Analysis
of variance tests were used to test null hypotheses of equal pro-
portions of larvae collected by either pump (top vs. bottom) at
either time of day (morning vs. evening), with no interaction (Zar
1996).
RESULTS
Two species of oyster larvae were collected in plankton
samples on the majority of days sampled: the eastern oyster. C.
virgiiiica: and the crested oyster, O. equvstris. Pediveligers.
or late-stage larvae, of these species could be distinguished on
the basis of shape (O. equestris pediveligers were nearly identical
to those of C. virguiica in size but were more rounded, with a
broader, less pronounced umbo). Living pediveliger larvae were
clearly distinguishable on the basis of color. C virgiiiica pedive-
ligers at this site were tan to brown and opaque, while O. equestris
pediveligers were transparent except for their visceral masses,
which were green to brown. The only other common bivalve lar-
vae were shipworms (Teredinidae) of unknown species, which
were treated in this study as if they were a single taxon. Uniden-
tified pediveligers of other bivalve taxa were occasionally col-
lected.
The abundance of all three species was highly variable, but
fairly low. C. virginica and O. equestris reached peak densities of
just over 1 2 per m \ but teredinids peaked at less than half of that.
All three taxa showed peak densities near the beginning of the
study. Density data for all three taxa from the lower intake are
shown in Fig. 1.
The plankton pumps at the two sample depths did not collect
equal densities of larvae, for any species. About 85% of C. vir-
giiiica pediveligers and 759f of teredinid pediveligers were col-
lected from the bottom pump, and time of day had no significant
effect. During the day. the distribution patterns for O. equestris
pediveliger larvae appeared to be similar to the above taxa. but at
night 61% of O. equestris pediveligers were collected by the near-
surface pump. Thus, for O. equestris. abundance differed signifi-
cantly for neither time of day nor depth, but the interaction of
depth and time of day was significant at a = 0.05. The proportions
for each species collected for each time and daylight treatment are
presented in Table 1. and the results of the analysis of variance are
presented in Table 2.
DISCUSSION
The above study ro.se serendipitously from an attempt to locate
an estuarine environment in which oyster pediveliger larvae (C
virginica) were randomly distributed throughout the water column,
for a separate study (Baker 1993). Clearly, nonrandom distribution
complicates the effort to quantify the larval supply. Yet. even in
this highly simplified estuarine environment, in <2 m of water, all
three bivalve taxa exhibited strong vertical distribution patterns.
The vertical distribution patterns from this study were similar to
those observed for C. virginica and teredinid larvae in a more
complex estuarine environment in Virginia (Baker 1993). The ma-
jor difference noted from that prior study was the effect of time of
day on the distribution of O. equestris larvae; no effects of time of
14
12
10
8
6
4
2
Crassostrea virginica
* • Sti
• •> ♦«
100
200
300
400
500
600
700
(0
c
O
0.
16
14
12
10
8
6
4
2
«
Ostrea equestris
♦
♦
♦
«
♦
♦
#
♦
• X
*
♦
♦ ♦ •
♦ ♦ ♦
♦
-•-
_^ ♦
♦^»
♦
100
200
300
400
500
600
700
Teredinidae
0 . — • #4. * •>»...
• •♦♦ ♦
100
200
300
400
500
600
700
Elapsed Time (h)
Figure 1. .\bundance (density per cubic meter) of three taxa of bivalve
pediveligers at the Harbor Branch Oceanographic Institute canal dur-
ing May 1993. from the near-bottom plankton pump.
day were reported for any species in the Virginia study. The
sparseness of pediveliger larvae also was noted by Carriker ( 1 95 1 ),
who collected only 56 pediveligers from > 14.500 C. virginica
larvae across six samples.
TABLE 1.
Mean proportional ( % ) abundances of three taxa of bivalve
pediveligers at two times (morning vs. evening) and two depths (top
vs. bottom! in the Harbor Branch Oceanographic Institute canal
during May 1993.
Taxon
Depth Morning Evening All Times
C. vir^intLii
Top
14.6 (18.8)
16.8 (30.3)
15.5 (24.5)
Biittom
85.4 (18.8)
83.2 (30.3)
84.5 (24.5)
(H = 19)
(" = 13)
in = 32)
O. equestris
Top
18.6(29.4)
61.0(41.9)
33.2 (38.6)
Bottom
814(29.4)
39.0(41.9)
66.8 (38.6)
(" = 19)
Ui = 10)
(« = 29)
Unidentitled teredinids
Top
23.2 (39.1)
26.8 (334)
24.2 (38,3)
Bottom
76.8 (.39.1)
73.2 (33.4)
75.8 (38.3)
(» = 20)
(n = 8)
(n = 28)
SDs are given in parentheses.
Bivalve Larval Depth Distribution in an Estuary
735
TABLE 2.
Summary of analyses of variance for the effects of time of day (morning vs. evening) and depth (lop \s. bottomi on proportional abundance
of three ta\a of bivalve pediveligers in the Harbor Branch Oceanographic Institute canal during May I'n^.
Source
DF
Seq SS
Adj SS
Adj MS
F Value
P Value
Analysis of variance
Time of day
for C.
vlrfiinica
1
7.779
7.779
7.779
1.89
0.174
Depth
1
46.685
36.225
36.225
8.81
0.004
Time x depth
EiTor
1
58
7.967
238.417
7.967
238.417
7.967
4.111
1.94
0.169
Total
61
300.848
Analysis of variance
Time of day
Depth
for O,
ecjitestris
1
1
10.701
41.905
10.701
15.269
10.701
15.269
1.39
1.98
0.244
0.165
Time x depth
E[Tor
1
54
52.386
416.069
52.386
416.069
52.386
7.705
6.80
0.012
Total
57
521.062
Analysis of variance
Time of day
Dep(h
for teredinids
1
1
0.0161
16.6334
0.0161
10.9201
0.0161
10.9201
0.02
11.26
0.898
0.001
Time X depth
Error
1
52
0.7875
50.4180
0.7875
50.4180
0.7875
9.9696
0.81
0.372
Total
55
67.8550
Seq SS = sequential
sum 1
>r squares; Adj SS
= adjusted
sum of squares;
Adj MS = adjusted
mean square.
Several authors have reported the vertical stratification of bi-
valve larvae in estuaries (Nelson 1927. Perkins 1932. Wood &
Hargis 1971. Sekiguchi et al. 1991 ), although they did not attempt
to demonstrate that this was due to larval behavior. Vertical strati-
fication or the migration of bivalve larvae also has been observed
in the absence of estuarine stratification (Tremblay & Sinclair
1990. Raby et al. 1994), but those studies were in systems signifi-
canlK deeper than l.S in.
Dekshenieks et al. { 1996) modeled C. virt^inica larval distribu-
tion in the water column of a well-mixed estuary, and predicted, as
observed here, that the majority of late-stage larvae would be
within a meter of the benthos. As larvae grow, they sink faster (due
to an increased shell/cilia ratio), and the swim-sink behavioral
pattern observed for this species by Hidu and Haskins (1978)
would result in a net sinking rate for older larvae, according to the
model (Dekshenieks et al. 1996). The above model, however, does
not include bottom avoidance; larvae must either increase swim-
ming rates in response to the benthos or spend a certain amount of
time resting on the benthos. The latter behavior (except for benthic
explorations by competent-to-settle larvae; Prytherch 1934. Cran-
field 1973) has not been reported, and increased contact with the
benthos also exposes the larva to a new guild of predators (Breese
& Phibbs 1972. Steinberg & Kennedy 1979. Cowden et al. 1984.
Osman et al. 1989. Andre et al. 1993). It is likely, therefore, that
size-related sinking/swimming ratios provide only a partial expla-
nation for pediveliger distribution in C. virginica. O. equestris
pediveligers. which in this study were about the same size as C.
virginica pediveligers. were not constrained to the lower reaches of
the water column by the weight of their shell, at least not during
the night.
If pediveliger larvae were no more than negatively buoyant
particles, they could not remain in the water column in a low-
energy environment. If they were neutrally buoyant particles, they
would be distributed evenly in a well-mixed water column. None
of the species observed in this study were evenly distributed, and
one species (O. equestris) differed from the others, altering its
depth distribution on a diurnal cycle. Thus, while neutral buoyant
(nodels may be sufficient to describe broad distribution patterns
(Wood & Hargis 1971. Mann 1988). ciliated larvae are clearly not
inert particles, and species-specific larval behavior must be in-
voked to describe at least some scales of distribution.
ACKNOWLEDGMENTS
Funding for this study was provided by the Commonwealth of
Virginia through the Virginia Institute of Marine Science Bivalve
Ecology program. The Smithsonian Marine Station (then at Link-
port) and the Harbor Branch Oceanographic Institute (HBOI) gra-
ciously provided us with the use of their facilities for this study.
Technical assistance was pro\ ided by Sherry Reed and other mem-
bers of the Smithsonian Marine Station staff. Gratitude is also
expressed to the alligators in the HBOI canal for restraining their
territorial and predatory tendencies when I had to enter the water
at night to service equipment.
Andre, C, P. R. Jonsson & M. Lindegarth. 1993. Predation on settling
bivalve larvae by benthic suspension feeders: The role of hydrodynam-
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Baker. P. 1993. Quantification of settlement and recruitment processes in
bivalve mollusks. Ph.D. Thesis. Williamsburg. VA: College of Vv'illiam
and Mary. 381 pp.
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Joiinml of Shellfish Research. Vol. 22. No. 3. 737-746. 2(XI3.
DIOXIN/FURAN AND POLYCHLORINATED BIPHENYL CONCENTRATIONS IN EASTERN
OYSTER {CRASSOSTREA VIRGINICA, GMELIN) TISSUES AND THE EFFECTS ON EGG
FERTILIZATION AND DEVELOPMENT
M. L. VVINTERMYER* AND K. R. COOPER
Rutgers, The Stare University of New Jersey. Joint Groclinite Proi;nini In Toxicology.
Pi.scataway. New Jersey
ABSTItACT A 10-mo field study was conducted to evaluate the bioaccumulatioii of dioxins/furans and polychlorinated biphenyls
(PCBs) in transplanted adult eastern oysters {Crassoslrea virginica. Gemliii) to Newark Bay and the Raritan Complex. New Jersey.
Adult oysters (mean size 86.4 ± 14.2 mm) were deployed from September 2000 until June 2001. Oysters transplanted to Newark Bay,
Anhur Kill, and Sandy Hook. NJ. accumulated 3.2/2.1. 1.3/1.7. and 0.15/2.3 parts per trillion (pptr) of 2.3.7.8-Tetrachlorodibenzo-
/)-dioxin (TCDD)/2.3.7.8- Tetrachlorodibenzo-p-furan. respectively. In addition, oysters transplanted to Newark Bay. Arthur Kill, and
Sandy Hook. NJ. had bioaccumulation levels of 68.6. 64.5. and 35.3 parts per billion total PCBs. respectively. The number of fertilized
eggs (±SD) from strip spawned transplanted oysters from Newark Bay. Arthur Kill, and Sandy Hook. NJ. was 107 (±6.00). 54 (±36.1 1),
and 1 13 (±13.61 ). respectively, and the number of unfertilized eggs was 164 (±25.6). 178 (±15.9). and 97 (±39.9). respectively. The
number of veliger larvae that resulted from fertilized eggs ((7 = 100) was 3 (±1.7). 4 (±2.31), and 82 (±12.2). respectively, for Newark
Bay. Arthur Kill, and Sandy Hook. NJ. Survival data from a laboratory study using an acute static 48-h hi vivo and ex vivo exposure
regiment to 2.3.7.8-TCDD showed that exposure to 2 pptr dioxin caused adverse effects on egg fertilization and development. Exposure
to dioxin-like compounds at the low parts per trillion ranges can result in altered gonadal development and altered embryonic
development.
AT;)' WORDS: Crassoslrea virginica. dioxins/furans. egg fertilization, polychlorinated biphenyls. transplant study
INTRODUCTION
Since the early 1970s there has been concern about the impacts
of 2,3,7,8-Tetrachlorodibenzo-/)-dioxin (TCDD) and related com-
pounds because of their potential hazard to humans and animals.
TCDD is a byproduct of anthropogenic processes such as paper
and chemical manufacturing, incineration, the manufacturing of
pesticides and herbicides, the production of iron and steel, and
enzymatic reactions in sewage sludge (Rappe 1992. Alonso et al.
1996, Poland et al. 1982). The most important source of TCDD for
humans is food, especially diary products, meat, and fish (Pohja-
virtaet al. 1994. EPA 2000).
Concern about TCDD stimulated numerous studies to assess its
behavior in the environment and its effects on living organisms.
Studies conducted in contaminated areas have shown a positive
correlation between dioxin levels in animals and their soil contact
(Pohjavirta et al. 1994). Studies in aquatic model ecosysteins also
have shown that TCDD and other organochlorine pollutants bio-
accumulate in organisms in concentrations approximately equal to
those in the sediment (Isensee et al. 1975. Chen et al. 2002). The
effects of TCDD on feeding, growth, and development are most
pronounced in young, growing organisms compared with adults
(ASTM 1994. Davis & Herber 1969. Calabrese et al. 1973.
Capuzzo. 1989, Capuzzo 1996). Because of the lipophilicity of
these compounds, they are associated with lipid stores and high
lipid-containing tissues (Cooper 1989. EPA 2000). Prior to spawn-
ing, bivalves have a high lipid and glycogen content in gonadal
tissue. Therefore, the spawning status of the bivalve would affect
the amount of dioxin present over the spawning season in a similar
fashion to that observed in fish (Capuzzo 1989. Vashchenko et al.
1993. Bayne et al. 1972, Bayne et al. 1978).
Oysters release their gametes into the water column: therefore,
planktonic lar\ae will have limited exposure to TCDD via water
*Corresponding author. E-mail: margyw@eden.rutgers.edu
due to the low water solubility of dioxin (EPA 2000.). Newly
settled bivalve spat and adult bivalve molluscs may be exposed to
TCDD through their sediment contact and feeding on resuspended
materials, while the developing eggs would receive the inajority of
exposure from the adult female (Cooper 1989). Bivalve embryos
begin to accumulate TCDD at the two-cell embryonic stage
(ASTM 1994). This may explain the sensitivity of young, growing
organisms to low-level concentrations of dioxins.
There has been limited work on the bioaccumulation of dioxin
in the eggs of aquatic organisms. Isensee and Jones (1975) re-
ported no effect of 2,3,7,8-TCDD exposures on snail egg survival,
but there was a reduction in the number of viable eggs. There have
been several studies on both resident and migratorv species of fish
and crustaceans in New Jersey. Aquatic organisms in the tidal
Passaic River were found to contain elevated levels of TCDD in
the edible tissue, ranging from 38 parts per trillion (pptr) in the
American eel (Angiiilla rostrata) to 476 pptr in the blue crab
{Callinectes scipiihis) hepatopancreas (Tucker and Prince 1993).
Cooper et al. (1993) found that (he TCDD levels in the .Arthur Kill
organisms accumulated within higher trophic levels. For example,
the soft-shell clam {Mya arenaria) contained 6.9 pptr TCDD, and
the killifish (Fimdiilus heteroclitiis) contained 100 pptr TCDD,
total body burden.
Changes in the gonadal tissue of bivalves after exposure to a
wide variety of pollutants such as oil, heavy metals, and lipophilic
organic compounds have been reported (Vashchenko et al. 1993,
Capuzzo 1996, Moore et al. 1980. Gardner et al. 1991. Lowe &
Pipe 1985. 1986. 1987: Capuzzo & Leavitt 1988: Lowe. 1988;
Moore 1988. Widdows & Johnson 1988). For instance, oocyte
mass resorption observed in the sea urchin as well as other inver-
tebrates at prespawning is considered to be a reaction to pollution
(Vashchenko ct al. 1993, Lowe & Pipe 1985. 1986. 1987. Capuzzo
1996). The abnormal development of oocytes, and altered egg
shape and size have been correlated with polluted sites (Winter-
myer 1998, Lowe & Pipe 1985). The accumulation of pollutants in
737
738
WiNTERMYER AND CoOPER
bivalves can cause stress. Capuzzo (1996) reported that pollution-
induced sites can lower biochemical reserve, and contribute to
poor egg quality and fertilization rates in bivalves. Bayne et al.
(1972. 1978) similarity reported that under stressful conditions the
mussel (Mytiliis ediilis) produced fewer and smaller eggs, and that
larvae that developed from the gametes of stressed adults had a
lower growth rate. In a study comparing egg size and larval sur-
vival of the hard-shell clam (Mercenario mercenaria) and the bay
scallop (Argopecten irradians). Kraeuter et al. ( 1982) reported that
for both species, smaller eggs (20-25 jjim) had a significantly less
than expected survival rate, while larger eggs (35—14 |j.m) had a
significantly greater than expected survival rate. Intermediate size
eggs (25-35 \i.m) showed no difference between the expected and
observed survival rates.
The objectives of this study were to transplant adult oysters into
sites contaminated with different levels of dioxin and dioxin-like
compounds to measure the effects on egg development and fertil-
ization, and to evaluate the potential for restoring oyster popula-
tions into the New Jersey bay area.
METHOD AND MATERIALS
Deployment
Adult eastern oysters (n = 180) were purchased from Prince
Edward Island, Canada, and were transplanted in September 2000
at three study sites (n = 60 per site): Newark Bay, NJ; Arthur Kill,
NJ; and Sandy Hook Bay, NJ (reference site). The oysters v\ere
determined to be disease free by histologic examination prior to
deployment. Oyster bags (/i = 2) were suspended in the water
column in Sandy Hook Bay located north of the bridge connecting
the Highlands entrance to Sandy Hook State Park. For the Arthur
Kill site, oyster bags (/; = 2) were suspended in the water column
from General Anline Works building dock (longitude 74"12.312W.
latitude 40°36.647N) in Elizabeth, NJ. For the Newark Bay site,
oyster bags (/) = 2) were suspended in the water column from
an abandoned dock on Shooter's Island (longitude 74°09.7S8W,
latitude 40 38.482N) in Newark, NJ (Fig. 1).
Each oyster was filed, numbered (1-60), and weighed (in
grams), and the dimensions were measured [i.e., length, width, and
height (in millimeters)] prior to being placed into marked, mesh
polyethylene bags (0.5 x 0.5 inch mesh). Each site was equipped
with two bags containing 30 oysters each suspended into the water
column 1.8 to 2.4 m (6-8 feet) below the water surface. The depth
was selected to avoid low-tide exposure and icing during the win-
ter. Oyster bags were collected in June 2001. terminating the 10-
mo field study. Oysters were wet weighed immediately upon col-
lection, and were prepared for tissue chemical analysis, histologic
evaluation, and fertilization assays.
Chemical Analysis
Samples of shucked oysters (50 g, /( = 7) from each site were
sent to Triangle Laboratories (Research Triangle Park, NC) for
dioxin, furan. and polychlorinated biphenyl (PCB) tissue analysis.
Samples were analyzed by high-resolution chromatography and
high-resolution mass spectrometry [method 1613B (9/97) and
modified method 680 (11/85), Triangle Laboratories], Tissues
were sent in labeled amber-colored jars and were frozen during
shipment.
Histologic Evaluation
Oysters from each site (/; = 15) were selected randomly for
histologic evaluation. Shucked oyster samples were preserved in a
10% phosphate formalin buffer for several days followed by 70%
ethanol. Transverse cuts were made with a scalpel through the
mid-visceral region of the oyster to obtain a segment approxi-
mately 5 mm thick. Segments were embedded in paraffin after
processing (i.e., dehydration and clearance through an alcohohxy-
lene series). Sections (6 |j.m) were cut and stained with Harris'
hematoxylin and eosin. Histologic grading was based on a scale
New Jersey
Atlantic
Ocean
Sandy Hook Site
Figure 1. Locations of New Jersey field study sites in the Newark/Raritan Bay Complex.
Dioxin/Furan and Polychlorinated BiPHENYL Concentrations in Eastern Oyster Tissues
739
from mild (T) to severe (TTT) for lesions. iiinaniin;Uion-likc re-
sponses, and infectious diseases.
Gonad condition was graded according to Kennedy (1977):
Stage 0 = resting stage
Stage 1 = early development
Stage II = later development
Stage III = sexual maturity
Ilia = maturity
Illb = spawning
IIIc = redevelopment
Hid = recently spent
Tissues evaluated were gills, mantle, adductor muscle, kidney/
heart, digestive gland, and gonadal condition.
Fertilization Assay: Strip Spawning
Field Study
A total of six ripe oysters from each site were strip spawned
(male = 3, female = 3). Eggs and sperm were extracted from the
gonadal region using a scalpel and lightly lacerating the gonad
(Allen et al. 1989). Collected eggs were sieved on a 25-|ji.m screen
and were washed with seawater collected from the respective site.
Eggs were viewed under a microscope for maturation before being
fertilized with the collected oyster sperm (sperm was diluted to 50
mL). Once sperm (1 niL) was added to the egg suspension (200
eggs per mL), the eggs were set aside for 1 h before being assayed
to allow for fertilization. The total number of fertilized and unfer-
tilized eggs, in three 1-mL replicate samples, was ascertained be-
fore eggs were dispensed into petri dishes. To each 10-mL glass
petri dish (/; = 3 per site). 10 mL of the site-collected water and
fertilized eggs (« = 100) from each site were dispensed into the
appropriate petri dish. Fertilized eggs were allowed to develop for
48 h at room temperature without aeration or food. After 48 h, the
larvae were sieved on a 53-p.m screen, and the number of larvae
that had developed to the straight hinge stage was counted.
(i.e., control. 2.0 pptr. and 20.0 pptr groups) were placed into
separate recirculating seawater systems 24 h after the injections.
All oysters were reinjected on day 14 of the study according to the
procedure described above. This procedure was performed to
maintain dioxin concentrations in the oysters over 28 days (Win-
termyer 1998). Treatment groups were strip spawned on day 28
according to the procedure described above (field study). Eggs (10
eggs per mL) from each treatment group were fertilized with
sperm (1 mL; sperm was diluted to 100 mL) collected from the
corresponding treatment group.
Ex vivo. The 48-h static ex vivo assay consisted of control eggs
(9 eggs per mL) fertilized with control sperm (1 mL: sperm was
diluted to 100 mL). Glass exposure beakers (150 mL) (n = 3)
consisted of 0.1 mL of nominal 2.0 pptr TCDD. and 0.1 mL of
nominal 20.0 pptr TCDD and 0.0 pptr TCDD. respectively. To
each treatment beaker, a 10-mL egg suspension and a 2-mL sperm
suspension were added, and allowed to set for 2 h for fertilization.
Both 48-h //) vivo and ex vivo assays were conducted in 20-mL
glass petri dishes. Fertilized eggs (10 mL) from each treatment
group was pipetted into individual petri dishes (/; = 20 per group)
and were incubated at 22°C for 48 h. After 48 h, each petri dish in
both the in vivo and e.x vivo assays was examined for the number
of fertilized and unfertilized eggs, as well as for the number of
living and dead larvae and their development stages.
Radiolabeled Compounds
-'[HI 2,3,7,8-TCDD (34.7 Ci/mM, 98% pure by high-
performance liquid chromatography, with carbons 1 and 6 radio-
labeled) was purchased from Chemsyn Science Laboratories (Le-
nexa. KA). Oysters were exposed to 0.996 pg/g (2 pptr) or 27.7
pg/g (20.0 pptr) of -'[Hj-TCDD via adductor muscle injection. All
""[Hj-TCDD values were based on equivalents.
RESULTS
Laboratory Study
In vivo. Adult eastern oysters (Crassostrea virginica) were
purchased from Haskin Shellfish Research Laboratory (Rutgers
University, Piscataway, NJ). Oysters (/( = 32) were exposed to
two treatments of tritium-labeled 2.3.7..S-TCDD via adductor
muscle injections. The study was conducted for 28 days to allow
the circulation and distribution of dioxin throughout the oyster.
This time period was selected based on results obtained from a
distribution study using 2,3,7,8-TCDD (Wintermyer 1998). Oys-
ters (/! = 48) were weighed (mean weight 50 g), numbered and
notched, and their dimensions were measured (i.e., height, length,
and width). Oysters were notched on the left side of the valves for
access to the adductor muscle. Control oysters (n = 16) were
injected (via adductor muscle) with 100 p.L (0.1 mL) of 20 parts
per thousand filtered seawater. The nominal 2.0 pptr treatment
group {n = 16) was injected with 100 |xL (0.1 mL) of 0.996 pg/g
^[H]-TCDD. The nominal 20.0 pptr treatment group (;; = 16) was
injected with 100 \xL (0.1 niL) of 27.7 pg/g '[HJ-TCDD. '|H]-
TCDD equivalents were based on radioactivity in 0. 1-mL injection
volumes in a 50-g oyster (pg/g) in = 3). All oysters were placed
on absorbent paper for 1 h before being put into 76-L aquarium
tanks for 24 h. This procedure was performed to allow the dis-
charging and recirculation of dioxin by the oysters. Oysters were
not fed 24 h before or 24 h after the injections. Treatment groups
Deployment and Retrieval
In this study, a total of six bags containing eastern oysters was
transplanted to the Newark Bay and the Raritan Bay Complex
from September 2000 until June 2001. Oysters transplanted to
Newark Bay for 10 mo had the second highest increase in total
weight gain (-1-6 g). Oysters transplanted to Arthur Kill had a
decrease in total weight gain (-10.9 g), and oysters transplanted to
Sandy Hook Bay had the highest increase in weight gain (-t-10.3 g).
There was not a significant difference in shell growth among the
Newark Bay. Arthur Kill, or Sandy Hook transplanted oysters over
the 10-mo field study (Table 1).
Tissue Analysis
Oyster tissues were analyzed for dioxin. furan, and PCB ana-
lytes. Newark Bay oysters had the highest tissue levels of 2,3,7,8-
TCDD (3.2 pptr), total TCDD (16.5 pptr). total TCDF (93.8 pptr).
and total PCBs [1.7 parts per billion (ppb)]. Arthur Kill trans-
planted oysters had the second highest tissue levels of 2,3,7,8-
TCDD (1,3 pptr), total TCDD ( 13.3 pptr), total TCDF (56.7 pptr),
and total PCB (64.5 ppb). Sandy Hook oysters had the lowest
levels of 2.3.7.8-TCDD (0.15 pptr), total dioxin (2.5 pptr). total
furan (47.6 pptr). and total PCBs (35.3 ppb) (Tables 2 and 3).
740
WiNTERMYER AND COOPER
TABLE 1.
Deployment and retrie^al data from C. virginica transplanted to Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ, field sites.'
Temp.
Salinity
Sites
Date
No. of Oysters'"
(X)
(ppt)
Weight (g)
H (mm)
1, (mm)
\\ (mm)
Deployment
Newark Bay
9/12/00
60
18.5
20
57.5 ± 15.3
81.4 ± 13.6
45.8 ±5.0
19.8 ±2.8
Anhur Kill
9/12/00
60
19.5
20
66.8 ± 19.9
88.7 ± 14.0
46.9 + 7.4
20.5 + 2.9
Sandy Hook
9/12/00
60
18
23
68. 1 ± 25
89.7 ± 15.0
46.6 ± 5.0
20.8 ±4.0
Retrieval
Newark Bay
6/1/01
47/13'' (2 bags recovered)
14.3
16
63.5+18.0
81.7 ± 13.2
45.4 ±4.8
19.5 ±2.6
Arthur Kill
6/1/01
45/15'^ (2 bags recovered)
17.3
16
55.9 ± 13
88.2+ 13.8
46.2 ±7.7
20.8 ± 3.0
Sandy Hook
6/1/01
25/5' (1 bag recovered)
14.6
20
78.4 ±26
89.4 ± 14.7
46.1 ±5.5
20.5+4.2
Presented as mean + Sd, unless otherwise indicated.
" H, height; L. length; W, width; ppt. pans per thousand.
'' Number of oysters per site; two bags per site.
" Number of live oysters/number of dead oysters.
Histologic Evaluation
Oysters transplanted to Newark Bay showed moderate signs of
epithelial-severe hyperplasia, while oysters transplanted to Arthur
Kill showed signs of severe epithelial-severe hyperplasia with
some cells (>4) showing mitotic division, and connective tissue
displaying areas of focal fibrosis. Oysters transplanted to Sandy
Hook showed signs of slight epithelial-severe hyperplasia. Only
the transplanted oy.sters to Arthur Kill were observed to have a
haplospoiidiuin nelsoni (MSX) infection in the digestive gland and
mantle tissues (Table 4). All transplanted oysters showed slight-
to-moderate gill hyperplasia ("clubbing"). Oysters transplanted to
Newark Bay and Arthur Kill showed an alteration in gill cilia
shape, size, and orientation. The cilia had a thickened appearance
and an alteration in cilia length resulting in a distinct whip-like
appearance (approximately six times the length of normal gill
cilia).
Gross Body Evaluation
Oysters transplanted to Newark Bay had semi-developed go-
nadal tissue. The gonadal area had a slightly cream-colored ap-
pearance, and the oysters appeared to be of moderate health and
were plump. The shell interior had a white, iridescent color and
had no obvious scarring or discoloration. Oysters transplanted to
TABLE 2.
Oyster tissue analysis for dioxins/furans at Newark Bay, N,I, Arthur Kill, NJ. and Sandy Hook Bay, NJ, during a 10-mo water suspension
field study.
Analytes
Newark Bay Concentration (pptr)"
Arthur Kill Concentration (pptr)'' Sandy Hook Concentration (pptr)'
2,3,7.8-TCDD
1,2,3.7.8-PeCDD
1,2.3.4.7.8-HxCDD
1,2.3,6,7,8-HxCDD
1.2,3.7,8.9-HxCDD
1.2,3.4,6.7,8-HpCDD
1.2,3.4,6.7,8.9-OCDD
2.3,7,8-TCDF
1.2.3.7.8-PeCDF
2,3,4,7,8-PeCDF
1,2,3.4,7,8-HxCDF
1,2,3.6.7.8-HxCDF
2.3,4.6.7.8-HxCDF
1.2,3.7.8.9-HxCDF
1.2,3.4,6.7,8-HpCDF
1, 2,3.4,7,8,9- HpCDF
1,2,3.4.6.7.8.9-OCDF
Total TEFs'
<DL (0.3f
<DL (0.3)
<DL (0.3)
<DL(0.3)
0.59
1.8
6.5
<DL (0.2)
0.93
<DL (0.2)
<DL (0.2)
<DL (0.2)
<DL (0.2)
<DL (0.2)
<DL (0.4)
<DL(0.5)
4.3
1.3
<DL (0.3)
<DL(0.3)
<DL (0.3)
<DL (0.3)
1.0
4.8
4.3
<DL (0.2)
0.73
<DL(0.2)
<DL(0.2)
<DL(0.2)
<DL (0.2)
<DL(0.2)
<DL (0.3)
<DL (0.4)
0.15''
<DL(0.2)
<DL(0.1)
<DL(0.1)
<DL(0.1)
0.43
2.3
<DL(2.5)
<DL(0.1)
<DL(0.1)
<DL (0.08)
<DL (0.07)
<DL (0.09)
<DL(0.1)
<DL(0.1)
<DL(0.2)
**0.47
0.6
DL. detection limit; TEF, total equivalent factor.
■■ Sample size, 25.15 g; 0.3% lipids.
" Sample .size, 25.17 g; 0.2% lipids.
■^Sample size; 25.1 g; 0.6% lipids (shown in parentheses).
"* Concentration is below the calibration curve. Value is an estimate only.
' Values are less than the detection limit.
'^Environmental Protection Agency (19X9a).
DIOXIN/FURAN and POLYCHLORINATED BiPHENYL CONCENTRATIONS IN EASTERN OvSTER TISSUES
741
TABLE 3.
Oyster lissuf analysis for total PCBs at Ne"ark Bay. N.I, Arthur Kill, N.I. and Sandy Hook Bay. N.I. durinv; a l(l-mo water suspension
field study.
Analvtes
Newark Bay Concentration (ppb)"
Arthur Kill Concentration (pph)'
Sandy Hook Concentration (ppb)'
Total MonoCB
Total DICE
Total TriCB
Total TetraCB
Total PentaCB
Total HexaCB
Total HeptaCB
Total OctaCB
Total NonaCB
DecaCB (#209)
Total PCB'
Total PCB + EMPC
<DL(0.()9r'
<DL(0.1)
5.6
24.2
22.8
14.2
1.7
<DL(0.8l
<DL(1.2l
<DL(2.2)
68.6
71.6
<DL(0.1)
<DL(().ll
4.5
23.5
21.6
13.0
1.9
<DL (0.7)
<DL(1.0)
<DL(1.9)
64.5
69.4
<DL (,0.08j
0.54
2.1
12.2
14.9
4.9
0.68
<DL(0.6)
<DL(0.9)
<DL(1.6)
35.3
35.3
DL, detection limit; EMPC, estimated maximum possible concemration.
" Sample size, 30.0 g; 0.3% lipids.
" Sample size, 20.0 g; 0.2% lipids.
' Sample size, 24.0 g; 0.6% lipids.
'' Values are below the DL.
"Newark Bay and Arthur Kill total PCB was approximately two times that of Sandy Hook.
'if matched sets of peaks in the time window do not ha\e the appropriate ion mass ratios for a true PCB. the EMPC is calculated using the sum of the
observed peaks (Triangle Laboratories. Inc.. modified method 680 ( 1 1/85).
Arthur Kill had underdeveloped gonads, and were easily shucked
and watery. The gonadal area had a vein-like appearance and a
gray coloration. The shell interior had a white, iridescent color and
had no obvious scarring or discoloration. Oysters transplanted to
Sandy Hook were plump, had a whitish-cream coloration and well-
developed gonads, and were in a prespawning state (Table 4).
Field Study Strip Spawning Assay
Results from the strip-spawning assay using oysters trans-
planted to Newark Bay, Arthur Kill, and Sandy Hook, NJ, showed
that the majority of eggs collected from female oysters at the
Newark Bay and Arthur Kill sites were not viable. There was not
a difference in fertilized egg size (64 |j.m) among the transplanted
oysters, however, oysters transplanted to Arthur Kill had a smaller
unfertilized egg size (48 (xm) compared with oysters transplanted
to Newark Bay or Sandy Hook. This study shows that 60. .59^ and
76.7%, respectively, of eggs collected from oysters transplanted to
Newark Bay and Arthur Kill were not fertilized, and of the eggs
that were fertilized (.39.5% and 23.3%) only 0.03% and 0.04%,
respectively, of the eggs developed to the straight-hinge stage.
TABLE 4.
C. virginica histological evaluation for 10-mo field study (September, 2000-June, 2(101)
Gill
Adductor Muscle
Kidnev/Heart
Digestive Gland (Midgut)
Gonad^
Condition
Newark Bay T Hyperplasia (80%) TT Epithelial severe T Brown cell ( 10% )-f
(n = 15) hyperplasia (70%)
T Inflam. (IOO'!'c)t T InHam. (100% ) T Inflam. (10%)
TT-TTT Dysplasia (100%) TT-TTT Dysplasia (100% ) TT-TTT Dysplasia
(100%)
Arthur Kill TT-TTT Inflam. (100%) TTT Epithelial severe T Inflam. (30%)
(n = 15) hyperplasia (100%)
TT-TTT Inflam. (100%)
T Hyperplasia (100%) TT-TTT Dysplasia (100%) TT-TTT Dysplasia
(100%)
T Brown cell (100%)
T Brown cell ( 100%) TT-TTT MSX (70%)
Sandy Hooli T Dysplasia and tilameiit T Epithelial severe
(n = 15) fusion (100%) hyperplasia (40%)
T Brown cell (100%)
T Hyperplasia (100%) T Dysplasia (100%)
T Dysplasia (100%)
TT-TTT Brown cell
(70%)
T Inflam. (70%)
TT-TTT Dysplasia
(100%))
T Inflam. (60%)
TT-TTT dysplasia
(100%)
TT-TTT Brown cell
(60%)
T Dy.splasia (100%)
TT Epithelial severe Stage 2 and 3
hyperplasia (80%j) (80%)
T Inflam. (100%) Stage 1 (20%)
TT-TTT Dysplasia (100%)
TTT Epithelial severe Stage 1 and 2
hyperplasia (100%) (40%)
TT-TTT Inflam. (100%)) Stage 3 (60%j)
TT-TTT Dysplasia (100%)
T Brown cell (100%o)
TT-TTT MSX (70%)
T Epithelial severe
hyperplasia (40%)
T Brown cell (100%)
T Dysplasia (100%)
Stage .la and 3b
(80%)
Stage 0 (0.01%)
Numbers in parentheses are % of oysters. Lesion grading definitions: (-), absent; (T|, slight; (TTi, moderate; (TTT), severe: inflam.. inflammatory like response; hrown cell,
brown cell accumulation.
"Gonad grading: stage 0. resting stage; stage 1. early development; stage 2. later development; stage 3. sexual inaiuniy; stage 3a. maturity; stage 3b. spawning: stage .3c,
redevelopment; stage d, recently spent (Kennedy 1977),
742
WiNTERMYER AND COOPER
Most fertilized eggs did not develop beyond the zygote stage. The
strip-spawning assay from oysters transplanted to Sandy Hook
showed that 53.7% of the eggs were fertilized, and of those eggs
84% developed to the straight-hinge stage (Table 5. Fig. 2).
Acute Static 48-li In Vivo and Ex Vivo Assays
In this study using C. virgiiiicch there was an observable de-
crease in the number of fertilized eggs in the 2 and 20 pptr TCDD
groups. In Table 6, controls for the in vivo and e.\ vivo assays had
high rates of egg fertilization and larvae development to the
straight hinge stage (80.3%). The 2.0 pptr in vivo assay had 48%'
egg fertilization, but 100% mortality at the zygote development
stage. In the 20.0 pptr in vivo assay, to which viable control eggs
were fertilized with 20.0 pptr sperm, there was very little fertil-
ization (0.9%). which resulted in a high egg mortality rate (99.%).
The 2.0 pptr ex vivo and 20.0 pptr e.x vivo assays also had low
fertilization rates (3% and 2%, respectively), which resulted in
high egg mortality rates (97% and 98%. respectively). In both the
48-h acute in vivo and e.x vivo studies, there were large decreases
in the number of veliger larvae compared with the controls. Within
treatment groups (nominal 2.0 pptr TCDD and 20.0 pptr TCDD).
there were 52 to 99% unfertilized eggs. Eggs that were fertilized
had a 98 to 100% mortality rate and did not develop beyond the
zygote stage. In contrast, the control eggs had an 80% survival rate
to the straight-hinge stage (Table 6. Fig. 3).
DISCUSSION
PCBs were first commercially produced in 1929 (NJDEP
1993). PCBs were commonly used in transformer oils and electri-
cal products. In 1977, the U.S. Environmental Protection Agency
banned the production of PCBs. However, many PCB-laden trans-
formers, capacitors, and other electrical equipment remain in ser-
vice (NJDEP 1993). PCBs have been and continue to be dispersed
throughout the environment through spills, effluent discharges,
and incineration.
In the 1970s and 1980s, the levels of TCDD in Newark. NJ. and
Arthur Kill. NJ. shellfish approached the no-consumption advisory
level suggested by the U.S. Food and Drug Administration of 25
pptr (Belton et al. 1985). The levels of other isomers such as PCBs.
polychlorinate dibenzo-p-dioxin (PCDDs), and polychlorinated
dibenzo-p-furan (PCDFs) found in aquatic organisms (striped bass
and blue crab) in Newark Bay and Arthur Kill resulted in the
closing of the waterways to fishing beginning in 1984 (NJDEP
1990). Extensive soil contamination with dioxin, specifically
2,3.7.8-TCDD. discovered at a site adjacent to the Passaic River in
Newark. NJ. prompted an intensive study of dioxin le\ els in sedi-
ments and biota in 1983 and 1984 (NJDEP 1990).
In this study, a total of six bags were deployed in the field in
September 2000. The field sites were selected based on historical
data about the bay system and accessibility via boat. Sandy Hook.
NJ. was selected as the reference site, and Arthur Kill and Newark
Bay. NJ. were selected as the exposure sites due to the high level
of industrialization along the waterways. The approximate distance
between the Newark site and the Arthur Kill site is 5 miles. The
distance between the Sandy Hook site and the Newark-Arthur Kill
site is approximately 32 miles. Oysters were put in the field at the
completion of the 2000 spawning season and were collected prior
to the 2001 spawning season to ensure bioaccumulation levels
prior to and during gametogenesis. Oyster tissues were analyzed
for dioxin. furan. and PCB analytes. Newark Bay oysters had the
highest tissue levels of 2.3,7,8-TCDD (3.2 pptr), total TCDD ( 16.5
pptr). total TCDF (93.8 pptr). and total PCBs (68.6 ppb). Oysters
transplanted to Arthur Kill had slightly lower tissue levels of
2.3.7.8-TCDD (1.3 pptr). total TCDD levels (13.3 pptr). total
TCDF levels (56.7 pptr). and a slightly lower total PCB level (64.5
ppb) than those of the Newark Bay oysters (Tables 3 and 4). Sandy
Hook oysters had the lowest levels of 2,3,7.8-TCDD (0.15 pptr),
total dioxin (2.5 pptr), total furan (47.6 pptr). and total PCBs (35.3
ppb) (Tables 2 and 3).
Oysters transplanted to Newark Bay showed moderate signs of
epithelial-severe hyperplasia, and oysters transplanted to Arthur
Kill showed signs of severe epithelial-severe hyperplasia, with
some cells (>4) showing mitotic division and connective tissue
displaying areas of focal fibrosis. Oysters transplanted to Sandy
Hook showed signs of slight epithelial-severe hyperplasia. The
epithelial-severe hyperplasia could be interpreted as preneoplastic
in nature, however, further research is needed to verify that these
lesions can progress to a neoplastic condition. Only the Arthur Kill
oysters were observed to have a moderate-to-severe MSX infec-
tion in the digestive gland and mantle tissues (Table 4). Sandy
TABLE 5.
Summary of the strip-spawning assay from Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ, l(l-mo field study (September,
2000-June, 2001 ).
Newark Bay, NJ
Arthur Kill, NJ
Sandy Hook. NJ
Weight of oysters at time of
deployment (g) (9/00)
Weight of oysters at termination
of study (g) (6/01)
% lipid (6/01)
Egg size fertilized vs. unfertili/ed
(|x at 40x) (n = 5)
Total number of fertilized eggs'
Total number of unfertilized eggs"
Number of veligar larvae after
48 h"
57.5 ± 15.3 (n = 60)
63.5+ lS.4(n = 45)
0.3
64 (xm fertilized
56 (Jim unfertilized
107 ±6.00
164 + 25.6
3± 1.7
66.8 ± 19.9 (n = 60)
55.9+ 13.1 (n = 47)
0.2
64 |j.m fertilized
48 fjim unfertilized
54 ± 30. 1 1
178+ 15.9
4 ± 2.3 1
68.1 ±24.4(11 = 60)
78.4 ±25.6 I n
25)
0.6
64 (xm fertilized
56 (im unfertilized
113± 13.61
97 ± 39.9
82 ± 12.2
Presented as mean + SD, unless otherwise indicated.
"Numbers repre,sent the average of 1-mL replicate samples (n = 3).
'' Number of veligar larvae resulting from approximately 100 fertilized eggs (n
3 replicates).
Dio.xin/Furan and Polychlorinated Biphenyl Concentrations in Eastern Oyster Tissues
743
■ fertilized eggs
0 unfertilized eggs
= veliger larvae
Transplant sites
Figure 2. The percentage of fertilized eggs, unfertilized eggs, and veliger lar\ae resulting from the strip-spawning assay using transplanted
ii>sttrs from Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ ( lO-mo field study, Septemher 2000-June 2001 1. Numher of fertilized
and unfertilized eggs are averages of 1-niL replicates (three per site). Numbers of veliger larvae are those resulting frcmi 100 fertilized eggs after
4X h (three sites). #* (light), fertilized egg groups that are significantly different (/' < 0.05: ANON A); #* (dark), unfertilized egg groups that are
significantly different [P < 0.05; ANOVA); a, veliger larvae groups that are significantly different (P < 0.05; ANOVA); NB, Newark Bay; AK,
Arthur Kill; SH, Sandy Hook.
Hook oysters had fully developed gonads and were in a prespawn-
ing state. Newark Bay and Arthur Kill oysters were slightly mod-
erately underdeveloped due to a lack of gonadal development com-
pared with Sandy Hook oysters at the time of collection (Table 4).
All transplanted oysters showed slight-to-moderate gill hypeijila-
sia (clubbing). Oysters transplanted to Newark Bay and Arthur Kill
showed an alteration in gill cilia shape, size, and orientation. The
cilia had a thickened appearance and an alteration in cilia length
resulting in a distinct whip-like appearance (approximately six
times the length of normal gill cilia). This alteration in gill cilia
could be a result of chronic exposure over time. The lesions ob-
served in the transplanted oysters would be consistent with those
resulting from chronic exposure to chemicals. The lesions are not
pathoneumonic but are consistent with a wide variety of chemical
and physical irritants.
Oysters transplanted to the Newark Bay site had the second
highest increase in weight gain (-F 6 g). percentage of lipids (0.3%),
egg fertilization (39.5%). and larval development (0.03%). Oysters
transplanted to the Arthur Kill site had a decrease in weight over
the 10-mo study (-10.9 g). the lowest percentage of lipid content
(0.2%), the lowest percentage of egg fertilization (23.3%). and a
decrease in larval development (0.04%). Oysters transplanted to
the Sandy Hook site had the greatest increase in weight gain (-1-10.3
g). the highest percentage of lipids (0.6%). the highest percentage
of egg fertilization (53.770). and the highest percentage of larval
development (84%) (Table 5, Fig. 2). Weight gain and the per-
centage of lipid content of the oyster contribute greatly to egg
development and production, egg fertilization success, and larval
development (Capuzzo 1996, Capuzzo & Leavitt 1988, Lowe
1988, Moore 1988). Oysters transplanted to Sandy Hook had the
highest level of fitness followed by oysters transplanted to Newark
Bay and Arthur Kill, based on lesion grading, intlammatory-like
responses, infectious disease states, weight gain/loss, and the de-
gree of gonadal development.
Results from the strip-spawning assay using oysters trans-
planted to Newark Bay. Arthur Kill, and Sandy Hook. NJ. showed
TABLE 6.
Summary of an acute static 48-h in vivo and ex vivo strip-spawning bioassay for C. virginica exposed to 2 and 20 pptr 2,3,7,8-TCDD.
Initial (Egg)
.After 48 h (Veliger Larvae)
Number of
Fertilized Eggs
Number of
Unfertilized Eggs
Number Dead
after 48 h
Stage of Development
Number Alive
after 48 h
Stage of Development"
Control in vivn 19h ± 143 2 ± U.63 39 ± 1.45 Trochophore. egg. and D-stage
2 pptr in vivo 1 .^2 ± 3. 1 2 1 66 ± 3.80 3 1 X ± 3.45 Egg
20 pptr »! i/\fi'' 6 ±0.801 660 ±16,2 663+17.94 Egg
Control t-A vivo 1 94 ± 2. 1 7 4 + 0.84 48 + 2. 1 0 Trochophore. egg, and D-stage
2 pptr ex vivo 13 + 0.489 420 ± 10.8 423 ± 12.0 Egg and D-stage
20 pptr o- VIVO 16 ±0.410 803 ±27.3 810 ±27.6 Egg and D-stage
1 5y + 1 .66
0
3 + 0.52
150 + 2.36
D-stage
NA
D-stage
D-stage
10 ±0.513 Trochophore and D-stage
9 + 0.510 D-stage
Presented as mean + SD. unless otherwise indicated. N.A. not applicable.
In vivo represents eggs expo.sed to TCDD during gametogenesis and ex vivo represent eggs exposed to TCDD in petri dishes during fertilization (n
20 for each group). Table taken from Wintermyer ( 1998).
" Stage of fertilized egg (Loosanoff and Davis 1963).
Viable control eggs were fertilized with 20 pptr spemi.
744
WlNTERMYER AND COOPER
•a
a>
N
s
c
3
«o"
O)
O)
0)
■o
o
_N
t
.(1*
c
o
0)
Q.
120
100
80
g»
"35
>
■o
c
re
w
O)
a>
60
40
20
in VIVO
^
a'
^
ex VIVO
I
/\^\.'''
■ fertilized eggs
s unfertilized eggs
Qveliger lar\«e
Figure 3. The percentage of fertilized eggs, unfertilized eggs, and veliger larvae resulting from an acute static 4S-h /'/; vivo and f.v vivo
strip-spawning assay using C. virgiiika exposed to 2 and 20 pptr 2,3,7,8-TCDD. "in vivo, eggs exposed to TCDD during gametogenesis; *ex iii'o,
eggs exposed to TCDD in petri dishes during fertilization in = 20 for each group). Table from Winterniyer (1998).
that the majority of eggs collected from female oysters at the
Newark Bay and Arthur Kill sites were not viable (Fig. 2). This
study shows that 60.59r and 76.7%. respectively, of eggs collected
from Newark Bay and Arthur Kill transplanted oysters were not
fertilized, and of the eggs that were fertilized (39.5% and 23.3%,
respectively) only 0.03% and 0.04%. respectively, of the eggs
developed to the straight-hinge stage. Most fertilized eggs did not
develop beyond the zygote stage. The strip-spawning assay for
oysters transplanted to Sandy Hook showed that .'i3.7% of the eggs
were fertilized, and of those eggs 84% developed to the straight-
hinge stage (Fig. 2). This study was perfonned to evaluate the
potential for restoring oysters in to the bay area. Based on the field
study and strip-spawning assay, transplanting oysters into the
Newark bay and Arthur Kill sites at this time would not result in
successful recruitment of the bay area. However, the Sandy Hook
site would be an ideal area for oyster restoration.
In the laboratory studies, the 2.0 pptr and 20.0 pptr treatment
concentrations of 2,3,7, 8-TCDD used in the 48-h acute in vivo and
ex vivo studies were based on tissue concentrations that were re-
ported from the soft-shelled clam (Mya arenaria) living in New-
ark. NJ. (11-20 pptr TCDD) and Tuckerton. NJ. (0.1-0.6 pptr)
(Brown et al. 1993) and on sediment samples from Newark Bay
(20 pptr). Arthur Kill (10 pptr), and Tuckerton (0.5 to 1.0 pptr)
(Brown et al. 1993). In this study using C. virgiiiica. there was an
observable decrease in the number of fertilized eggs within the 2
and 20 pptr TCDD treatment groups. In Fig. 3. controls for the in
vivo and e.x vivo assays had high rates of egg fertilization and
larvae development to the straight-hinge stage (80.3%). The 2.0
pptr ill vivo assay had a 47.8% egg fertilization rate, but a 100%
mortality rate at the zygote development stage. In the 20.0 pptr //;
vivo assay to which viable control eggs were fertilized with 20.0
pptr sperm, there was very little fertilization (0.901%), which re-
sulted in a high egg mortality rate (99.6%). The 20-pptr treatment
group did not have any female oysters remaining due to toxicant-
induced stress and mortality by the end of the 28-day period. The
2.0 pptr e.x vivo and 20.0 pptr e.x vivo assays also had low fertil-
ization, which resulted in high egg mortality (Fig. 3). In both the
48-h acute in vivo and e.x vivo studies, there was a large decrease
in the number of fertilized eggs respective to treatment group
compared with the controls. Within treatment groups (nominal 2.0
pptr TCDD and 20.0 pptr TCDD), there were 52 to 99% unfertil-
ized eggs. Eggs that were fertilized had a 98 to 100% mortality rate
and did not develop beyond the zygote stage. In contrast, the
control eggs had an 80% survival rate to the straight-hinge stage
(Table 6, Fig. 3). This laboratory study is important in understand-
ing the effects of 2,3, 7, 8-TCDD independent of other lipophilic
compounds on oyster gametogenesis and egg fertilization. We can-
not state that the field study results were solely due to 2,3,7,8-
TCDD, but laboratory studies demonstrate that TCDD can result in
a significant decrease in gametogenesis and egg viability.
CONCLUSION
In conclusion, this study was designed to investigate two points
of interest: (1) the dioxin/furan and PCB concentrations in the
eastern oyster during gametogenesis and the effects on egg fertil-
ization and development; and (2) to evaluate the potential for
restoring oysters back into the New Jersey bay area. Oysters trans-
planted to Sandy Hook, NJ, had the greatest weight gain, percent-
age of lipid content, percentage of egg fertilization, and percentage
of larval development to the straight-hinge stage, followed by
oysters transplanted to Newark Bay and Arthur Kill. NJ. The labo-
ratory in vivo and e.x vivo strip-spawning assays showed that ex-
posure to compounds such as dioxin can accumulate in animal
tissues and can interfere with normal metabolic processes that
affect gonadal development and egg fertilization. While we cannot
separate the effects of different gonadal development on strip-
spawning fertilization and larval development, the laboratory stud-
ies support the effect of 2,3, 7, 8-TCDD on gonadal development at
levels observed in the field.
This study demonstrated that dioxins. furans. and PCBs are still
bioavailable in the Newark Bay estuary. The levels approach con-
centrations that in the laboratory result in impacts on gonadal
development and egg viability. This study clearly demonstrates
that 2. 3. 7. 8-TCDD effects gonadal development and egg viability
in the eastern oyster in a similar fashion to fish species.
ACKNOWLEDGMENTS
The authors would like to thank Michael Stringer and the NY/
NJ Baykeeper Program for helping with the deployment and re-
trieval of the oyster bags for the field study reported in this article.
Dioxin/Furan and Polychlorinated Biphenyl Concentrations in Eastern Oyster Tissues
743
Allen, S.. S. Downing & K. Chew. 1989. Hatchery manual for producing
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WiNTERMYER AND COOPER
Tucker. B. & R. Prince. (1993). Draft: Issues paper-dioxin and related
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I'dulis: Scope for growth. Mar. Ecol. Prog. Ser. 46:1 13-121.
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NJ: Rutgers University.
Journal of Shellfish Research. Vol. 22, No. 3, 747-752. 2003.
AN IMPROVEMENT TO THE DETERMINATION OF MEAT CONDITION INDEX FOR THE
EASTERN OYSTER CRASSOSTREA VIRGINICA (GMELIN 1791)
GEORGE R. ABBE* AND BRIAN W. ALBRIGHT
Academy of Natural Sciences Exliiarine Research Center, 10545 Mackall Road,
St. Leonard, Maryland 2()6S5
ABSTRACT The meat condition index (MCI) of a bivalve is a numerical representation of the quality of its soft tissue. Based on
the percentage of the internal shell volume occupied by a bivalve's soft body tissue, the enumeration of a quantitative index is possible.
Early methods sought to measure the shell cavity volumetrically; however, this technique is both slow and difficult to perform
accurately. In 1982. Lawrence and Scott developed a method to determine the MCI for oysters gravimetrically, in which shell cavity
capacity was determined by the difference between whole oyster weight and empty shell weight after drying for 24 h This technique
was not only as accurate as the volumetric method, but it was faster and easier. However, since any water contained within the shells
themselves (not between them) was included in the initial whole oyster weight, it seemed logical that this should be included in the
weight of empty shells as well. Drying shells for 24 h could inake the calculated shell cavity appear larger, resulting in reduced meat
condition. To determine the significance of weighing shells after 0 h versus 24 h of drying, the MCls were determined by shell cavity
casts and were compared with MCIs determined by the two gravimetric methods. Weighing shells immediately after processing (0 h)
was determined to more accurately estimate cavity volume whenever shells lost >3% of their weight due to drying. Of 1749 oysters
examined from the Patuxent River. Maryland, over 3 y, 74% lost >3'7f of their shell weight. Several other sites in the Chesapeake Bay
were also exainined. yielding similar results. Weighing shells at 0 h not only increased accuracy for most of the oysters examined, but
also saved time, as shells did not need to be held for an additional 24 h. Differences in shell morphology and fouling community
structure may intluence shell porosity, favoring one technique over the other.
KEY WORDS: oyster. Crassosrrea virgiiiicti. meal condition. Chesapeake Bay
INTRODUCTION
The meat condition index (MCI) of a bivalve i.s a iiunierical
representation of the quality (i.e., nutritive status or "fatness") of
its soft tissue. Quantitative methods of determining the meat con-
dition of bivalves have been conducted by various researchers as
far back as the early 1900s. Crosby and Gale ( 1990) presented a
brief history of the development of condition indices, which men-
tioned the works of Moore (1908), Milroy (1909), Grave (1912).
Higgins (1938), Haven (1962), Walne (1970), Lawrence and Scott
(1982). and Hawkins et al. (1987). Mo.st of these indices were
calculated using a formula that relates the weight of the soft tissue
to the shell cavity volume. The Hopkins formula (Higgins 1938)
used dry tissue weight (g) x lOO/intemal cavity volume (cm'). In
many of the earlier methods, shell cavity volume was determined
by displacemetit. until Lawrence and Scott ( 1982) showed that the
weight of the whole oyster in air (g). less the weight of the etnpty
valves in air (g). gave a very close approximation of cavity volutne
(cm ). Their fomiula for determining MCI became:
MCL
dry soft tissue wt (g) X 100
internal shell cavity capacity (g)
Although some authors prefer to use dry soft tissue weight x
1000 (Hawkins et al. 1987, Crosby & Gale 1990). this formula
simply results in a condition inde.x value an order of magnitude
larger than that of Lawrence and Scott ( 1 982). Internal shell cavity
volume (cm') and shell cavity capacity (g) are the same when the
cavity contents are assumed to have a density of 1 g cm"'.
This index represents meat quality or nutritive status while not
necessarily reflecting the health of the indiv idual. A fat oyster or
one with a high MCI generally has a rich creatiiy color due to
stored glycogen reserves and shows little of the internal organs
beneath the mantle (Fig. I A). An oyster of lesser qualilv will show
*Corresponding author. E-mail: abbeCs'acnatsci.org
some internal structure because the mantle is thinner and more
transparent (Fig. IB). A very poor quality oyster will be watery
and almost entirely clear.
Condition indices of oysters (Crassostrea viri^inicii) in the
Chesapeake Bay normally display u cyclical pattern, with the high-
est levels occurring in late fall and winter, and the lowest levels
occurring in late summer after spawning is completed, but low
conditions may occur at any time of year, possibly indicating a
disease problem or unfavorable environmental conditions (Haven
1962. Abbe & Sanders 1988). In fact. Scott and Middaugh (1978),
Scott and Vernberg (1979), and Lawrence and Scott (1982) all
suggested the use of an MCI to monitor the effects of waterborne
pollutants. Low salinity may have the opposite effect on condition
since gametogenesis may be depressed or halted at salinities be-
tween 5 and 7.5%c (Butler 1949. Loosanoff 1953). Oysters that do
not ripen their gonads, and thus fail to spawn, may attain higher
condition indices than would otherwise be observed if gametes are
resorbed or if the energy intended for gamete production is tun-
neled into glycogen production. A visual assessment of oyster
meats, at least in some primitive form, has probably been con-
ducted almost as long as humans have consumed oysters. As an
index based on opacity due to glycogen content, however, visual
condition indices are overly subjective and somewhat impractical.
Lawrence and Scott (1982) determined shell cavity capacity by
weighing the whole oyster after air drying for 4.*^ to 60 min. and
then subtracting the weight of the valves after air drying for an
additional 24 to 30 h After using this method for more than 15 y
to detertnine the MCIs of the eastern oyster C. virginiea (Gmelin.
1791 ). we questioned its accuracy when shells were dried for 24 h.
We suspected that the elapsed titne betv\een when the oyster is
shucked and when the valves are weighed might have a major
effect on the calculated value of shell cavity volume. When the
whole oyster is weighed, there is a certain amount of water within
the shells (not between them) in the spaces created by shell-boring
animals. Since this weight is included in the initial weight of the
747
748
Abbe and Albright
Figure 1. A fat oyster (A) with high MCI and one of lower meat
condition (B). Note that the mantle along the left edge of B is trans-
parent allowing shell structure to be observed beneath it.
whole oyster, it should also be included in the weight of the empty
valves because it does not represent any part of the internal shell
cavity. Drying shells for 24 h could make the calculated shell
cavity volume appear larger, resulting in a reduced meat condition.
We expected that valves might continue to lose weight as they
dried over time for up to several days, although most of the weight
loss would probably occur during the first 24 h. We suspected,
therefore, thai it might be necessary to weigh the empty valves
immediately after the oyster is opened and the soft tissue removed
to obtain a more accurate determination of shell cavity capacity or
volume.
The amount of water weight within the valves also depends on
the size (age) of the oyster as well as the number and size of the
organisms living in (not on) the shells, which may include boring
sponge (Cliona sp.) and mud worms (Polydora sp.) Young oysters
have smaller shells to be inhabited by boring organisms and less
time for their shells to be colonized. Older and larger oysters have
more shell surface area to hold boring organisms and more time for
their shells to be colonized. Regardless of size, however, if shells
were weighed immediately after the meat was removed, the water
weight in the shells should have a minimal effect on the calculated
cavity volume, and thus on the condition index. We conducted
several experiments (one that examined 1749 oysters over 36 mo)
to investigate and quantify the effect of water-weight loss and
drying time on oyster MCI.
MATERIALS AND METHODS
Drying Time
Thirty oysters were collected from the Holland Point oyster bar
in the Patuxent River (Fig. 2 1 near Benedict. Maryland, in March
1997. They were cleaned of external fouling organisms and
scrubbed with a nylon brush in the field. They were held in water
until they were returned to the laboratory, where they were held in
a raceway of running filtered river water at ambient salinity (10-14
parts per thousand). They were kept in water until processed, since
it is critical that they release no cavity fluid when weights are used
to determine cavity volume. Oysters were removed from water,
rinsed, blotted dry, numbered, measured for right valve length
(height), and weighed before they could gape and lose fluid. Once
weights were determined, the loss of some cavity fluid by animals
that gaped had no effect on subsequent measurements. Oysters
were then shucked into preweighed beakers and dried to constant
Patuxent
River
# Monthly Sampling
■ Annual Sampling
10 km
v^
Figure 2. Locations of 12 natural oyster bars in the Patuxent River
where oysters were collected during 1997-2(100: Teague Point (TP);
Holland Point (HP): Macks Hallow (MH); Broad Neck (BN); Jacks
Marsh (JM); Gatton (GAT); Peterson (PT); Hellen (HEL); Hawks
Nest (HN); Town Creek (TO: Southeast Middleground (SM): and
Little Cove Point (LCP).
weight at 60 to 70^C (5-7 days). Dry meat weight was measured
to the nearest 0.001 g. After shucking, the internal surfaces of the
valves of each oyster were wiped dry. and the valves were weighed
immediately to the nearest 0.001 g. They were weighed again after
6. 24. 48. and 72 h
Weight Loss
To determine whether valves weighed immediately after oys-
ters were shucked (0 h) or after drying for 24 h resulted in the
closest estimate to true cavity volume, it was necessary to first
determine the true volume as accurately as possible. Several meth-
ods were tried, all of which were discussed by Crosby and Gale
(1990), but most gave highly variable results and were time-
consuming. The best technique that we found used a liquid casting
medium of known density that was poured into each valve until
slightly overfilled. As the liquid began to harden, the two valves
were realigned and pressed tightly together so that excess casting
material was squeezed out. Valves were banded to keep them
tightly together until the cast was solid, which took only a few
minutes. When the cast was hard, it was removed from the shells,
the flashing was trimmed from the edges, and the volume of the
cast was then determined volumetrically by displacement or gravi-
metrically by weighing and dividing by the density of the casting
medium. MCIs were determined for 169 oysters using 0 and 24 h
valve weights and cavity volumes using casts.
From March 1997 to February 2000, monthly sampling of oys-
ters from four beds (Holland Point, Gatton, Hellen, and Southeast
Middleground) and annual sampling from eight additional beds
(Teague Point. Macks Hollow. Broad Neck, .lacks Marsh. Peter-
son, Hawks Nest, Town Creek, and Little Cove Point; in the Patux-
Improvement in Determining Oyster Condition Index
749
ent River (Fig. 2) was conducted witfi MCIs determined for 1 749
oysters using shell weights at both 0 h and after drying for 24 h.
Following the analysis of Patuxent River oysters, oysters were
examined from two sites in the upper Chesapeake Bay (Eastern
Bay and near Shady Side) and from two tributaries of the Potomac
River (Wicomico and St. Mary's Rivers) (Fig. 3) to determine
whether oysters from these areas were similar in weight loss to
those from the Patuxent River. Approximately 50 oysters were
collected and analyzed from each of these four areas.
RESULTS
Drying Time
Oysters used to determine drying time ranged from 72 to iS9
mm shell length (SL), with a mean (±SD) SL of 80.4 ± 4.1 mm.
Whole weights were 89.6 to 163.0 g, with a mean weight of 128.2
± 23.2 g. The mean shell weight at 0 h was 104.4 g. and 101.5 g
after drying for 6 h (a loss of 2.8'7r). Shells continued to lose
weight out to 72 h when they averaged 100.0 g (Fig. 4A), although
the rate of decrease declined over time. As shell weight decreased
o\er 72 h. calculated cavity volume increased from 23.8 to 28.2
cm', resulting in a decrease in mean condition from 9.5 at 0 h to
8.1 after 72 h (Fig. 4B). However, since the mean condition had
already decreased to 8.2 after just 24 h, the decrease over the next
48 h was minimal.
Weight Loss
When the percentage shell weight losses were averaged by
2-mni SL increments, the means ransied from 2.87r for oysters of
Figure 3, Other sites in Maryland from which oysters were collected
during 20nO including Kastern Bay (EB), near Shady Side (SS), the
Wicomicd Ri\er (W K), and the St, Mary's River (SMR).
I-
I
LU
X
w
<
LU
X
LU
Q
z
z
o
Q
z
o
o
I-
<
LU
<
LU
1 lU
A
105
n
100
-
1 11
1 \
-|
95
-
9.5
- r-|
B
9.0
-
8.5
r-|
8.0
-1
0 6
72
24 48
HOURS
Figure 4. Mean shell weight over time as shells dried (A) and resulting
loss in condition index (B). Drying was nearly complete after 24 h. hut
continued to 72 h,
62 mm SL to 5.09? for those of 110 mm SL, and they exhibited a
highly significant relationship (P < 0.001 ) between SL and weight
loss (Fig. 5). Figure 5 also shows that the three smallest groups
were well below the regression line. The individual percentage
weight loss for all 1749 oysters, however, ranged from as little as
1.2% to as much as 13.0%, with distribution skewed to the right,
although most were in the 2 to 8% range (Fig. 6).
Shells were weighed at 0 and 24 h, and calculated cavity vol-
O
X
in
if)
UJ
o
cr
UJ
Q.
Y = 0899 +
R-' = 0 760
p<0001
0.039 X
60 70 80 90 100 110
SHELL LENGTH (mm)
Figure 5, Linear regression of SL (hy 2-mni increments) and shell
weight loss after drying for 24 h.
750
Abbe and Albright
2 3 4 5 6 7 8 9 10 11 12
PERCENT SHELL WEIGHT LOSS
Figure 6. Percent weight loss frequency for sliells dried for 24 h. More
tlian 74% of tlie 1749 oysters examined lost at least 3^} of their initial
shell weight (darker bars).
umes were compared, with "true" volumes determined by casts for
169 oysters (Fig. 7). Shells weighed at 0 h underestimated true
volume by about 5.8% and remained nearly unchanged as shell
weight loss increased from 1 to 12% (Fig. 7A). Shells weighed at
24 h. however, estimated a cavity volume that was fairly close to
true volume when shell weight loss was 1 to 2%, but as weight loss
increased to 12%. cavity volume was overestimated by as much as
20% (Fig. 7B). The point at which the underestimation of cavity
volume by 0-h shell weight equals the overestimate of cavity vol-
ume by the 24-h weight was at a shell weight loss of slightly <3%.
Thus. 3% allowed a fairly conservative breakeven point, below
which the 24-h weight gave better estimates of cavity volume and
above which the 0-h weight gave better estimates.
Since cavity volume is the denominator in the MCI equation.
o
>
LU
o
o
>
10
-10
t 30
>
<
a:
O
a:
o
a:
20
10
Shells Weighed at 0 Mr
Y = -6 310* 0 257X
R' = 0,054
Shells Weighed after 24 Hr
B
Y = -0 451 ♦2 423X
R' = 0,591
p < 0.001
23456789 10 11
PERCENT SHELL WEIGHT LOSS
12
Figure 7. Linear regressions of the percentage error in cavity volume
and the percentage shell weight loss for shells weighed at II h (A) and
after 24 h (B).
overestimated cavity volume results in underestimated meat con-
dition by an equal ainount. Although 0-h weights overestimated
meat condition by about 5.8% (range -2 to 15%), they remained
relatively constant over the range of weight loss from 1 to 12%.
However, the 24-h shell weights underestimated meat condition by
about 7.5% (range 10 to -20%). and the estitnates became worse
as the pei'centage of shell weight loss increased.
Meat condition determined gravimetrically was correlated with
meat condition determined by casts for 169 oysters after 0 h (Fig.
8A) and after 24 h drying (Fig. 8B). and both gave significant
correlations. The 24-h shell weights had a highly significant r" of
0.952 {P < 0.001). but the 0-h shell weights were slightly better
with an r' of 0.979 (P< 0.001).
Because the oysters used in this analysis were all native to the
Paluxent River, the possibility existed that oysters from elsewhere
in the Chesapeake Bay might show different weight loss properties
after drying for 24 h and might reduce the validity of using 0-h
shell cavity volumes. In order for this technique to be valid else-
where, the average shell weight loss after drying for 24 h should
exceed 3%. For those areas examined. 24-h weight losses were
>3% in all cases. Eastern Bay oysters lost 4.6%, Shady Side oys-
ters lost 5.3%, the Wicomico River oysters lost 6.1%, and the St.
Mary's River oysters lost 8.3%. All of these were greater than the
4.3% for Patuxent oysters, indicating that this technique would be
valid in at least those areas, but probably in many other areas of the
state, and perhaps other areas of the east coast, as well.
DISCUSSION
The value of gravimetric meat condition ineasurements has
been demonstrated by Lawrence and Scott ( 19S2) and Crosby and
Gale (1990). Dry weight measured at 24 h provides an excellent
estimate of cavity volume with comparisons between cavity ca-
pacity (in mL) by water displacement and cavity volume (in cm'')
16
SHELLS WEIGHED AT 0 HR
14
, -j^v '
i 12
. A
^J^
_l
< 10
.sir
Q^ 8
ol^
H
• Jtf^
LU ^
^
-> b
^
>^ Y = 0,203 + 1,032 X
y^ R- = 0,979
y p< 0.001
o ^
- ^
Q
1 1 1 1,1.1
^ 14
SHELLS WEIGHED AFTER 24 HR ^
^ 19
' ^' '
^ 10
. B
1' 'tS^ '
lU
VJ* ■ ' '
e 8
-
y^ '
o
^yfT '
^ 6
-
•^'
4
^"^ Y = 0.122 + 0.910 X
y^ R' = 0.952
2
: f^
^ p< 0,001
1 . > . 1 , 1 , 1 . 1
2 4 6 8 10 12
MCI DETERMINED BY CASTS
14
Figure 8. Linear regressions of MCI determined gravimetrically and
by cast. For shells weighed at 0 h (A) the points fit the line slightly
better than for shells weighed at 24 h (B).
Improvement in Determining Oyster Condition Index
751
Figure 9. An oyster with a smooth shell typical of one that will lose
<iVc of its shell weight after drying for 24 h. Insets show enlarged
detail.
Figure Id. An oyster with a rough shell typical of one that will lose
>i9c of its shell weight after drying for 24 h. Insets show enlarged
detail.
yielding correlation coefficients of 0.93 to 0.98 for oysters from
three sites in South Carolina (Lawrence & Scott 1982). Our 24-h
dry weights also yielded a correlation coefficient of 0.98, but 0-h
dry weight coefficients reached 0.99 (Fig. 8A and B). Either
method appears to yield a good estimate of meat condition, but
since the water in the shell pores was weighed initially, there is no
reason to exclude it in the second weight. The arbitrary removal of
a variable amount of weight (water) artificially increases the vol-
ume (capacity) of the shell cavity, which in turn artificially and
unnecessarily decreases the meat condition.
In addition to slightly increased accuracy, the use of 0-h
weights means that all the shell weighing is completed within
minutes. There is no need to allow shells to dry overnight and to
return the next day to weigh them again.
While this technique appears to be an improved method for
estimating the meat condition of C. virginica in much of the Mary-
land Chesapeake Bay. in terms of time and accuracy, it has not
been tested elsewhere. The amount of water in the shell depends on
porosity, which can be a function of oyster size, rate of growth,
boring organisms, and shell structure. Small oysters generally lost
less weight after 24 h. as a percentage of 0-h weight, than larger
oysters because their shells had less time for shell-boring organ-
isms to inhabit them. Oysters with smooth shells (Fig. 9) will often
lose ^3% because there are few places for water to enter the shell.
Oysters with rough shells (Fig. 10) will generally lose >?>% and
sometimes a great deal more (up to IjVr). If the shells lose an
average of just <3% of their weight from shell water, then a 24-h
shell weight is the best estimator of cavity volume and thus of
MCI. However, if shells lose >y/( . then the 0-h weight proves to
be the best estimator. It should be relatively easy to determine
whether oysters from any particular area lose (on average) >3% or
<3% of their shell weight upon drying for 24 h, and thus determine
which method is more accurate for that site. Investigations of
condition index have been conducted with Cnissostrea gigas on
the west coast (Schumacker et al. 1998, Brett Dumbauld, Wash-
ington Department of Fish and Wildlife, pers. comm.l. and since
the shell structure and porosity of C. gigas may differ from that of
C. rilginica. we await the results of these investigations.
ACKNOWLEDGMENTS
Funding for this project was provided by the Academy of Natu-
ral Sciences. An early draft was reviewed by B. Dumbauld. and the
authors thank him for his helpful suggestions. We also appreciate
the critical comments of an anonymous reviewer. The senior au-
thor would like to acknowledge the efforts of junior author Brian
Albright who was instrumental in the design and management of
much of this project from the beginning. Brian passed away fol-
lowing surgery in October 2001. at the age of 36, before we com-
pleted this manuscript.
LITERATURE CITED
Abbe. G. R. & J. G. Sanders. 1988. Rapid decline in oyster condition in Ihe
Patuxent River. Maryland. J. Shellfish Res. Ti.iT-.Sg.
Butler, P. A. 1949. Gametogenesis in the oyster under conditions ot de-
pressed salinity. Biol. Bull. 96:26.V269.
Crosby. M. P. & L. D. Gale. 1990. A review and evaluation of bivalve
condition index methodologies with a suggested standard method. J.
Shellfish Res. 9:233-237.
Grave. C. 1912. A manual of oyster culture in Maryland. Board of Shellfish
Commis-sioners of Maryland. 4th Report, pp. 279-348.
Haven. D. 1962. Seasonal cycle of condition index of oysters in the York
and Rappahannock Rivers. Proc. Nat. Shellfish. A.-isoc. 51:42-66.
Hawkins. C. M.. T. W. Rowell &. P. Woo. 1987. The importance of cleans-
ing in the calculation of condition index in the soft-shell clam. M\a
arenaria (L.). / Shellfish Res. 6:29-36.
752
Abbe and Albright
Higgins. E. 1938. Progress in biological inquiries, 1937. Bulletin of the
U. S. Bureau of Fisheries Administration Report No. 3(1. Washington.
DC: U.S. Government Printing Office, pp. 1-70.
Lawrence, D. R. & G. L Scott. 1982. The determination and use of con-
dition index of oysters. Estuaries 5:23-21 .
Loosanoff. V. L. 1953. Behavior of oysters in water of low salinities. Proc.
Nat. Shellfish. Assn. 1952:135-131.
Milroy. J. A. 1909. Seasonal variations in the quantity of glycogen present
in samples of oysters. Sci. Invest. Land. Sen 2. 17(6).
Moore. H. F. 1908. Volumetric studies of the food and feeding of oysters.
Bull. U. S. Bur. Fish. 28:1297-1308.
Schumacker. E. J.. B. R. Dumhauld & B. E. Kauffman. 1998. Investiga-
tions using oy.ster condition index to monitor the aquatic environment
of Willapa Bay Washington. J. Shellfish Res. 17:338-339.
Scott. G. L & D. P. Middaugh. 1978. Seasonal chronic toxicity of chlori-
nation to the American oyster. Crassastreu virgiuica. In: Water chlo-
nnation. vol. 2: Environmental impact and health effects. Ann Arbor.
Ml: Ann Arbor Science Publishers, Inc., pp. 31 1-328.
Scott. G. I. & W. B. Vemberg. 1979. Co-occurring chlorine produced oxi-
dants in seawater and their effect on the growth, survival and physiol-
ogy of the American oyster. Crassostrea virginica (Gmelin): evidence
for synergistic effects with seasonal temperature stress. In: W. B. Vem-
berg. F. Thurberg. A. Calabreese & F. J. Vemberg. editors. Marine
pollution: functional responses. New York: Acadeinic Press, pp. 4L1-
435.
Walne. P. R. 1970. The seasonal variation of meat and glycogen content of
seven populations of oysters Osirea edulis L. and a review of the
literature. Miu. Agric. Fish. Food. Fish. Invest. Ser. 2. 26(3).
Journal of Shctlfi.sh Rcseanh. Vol. 22. No. 7,. 15i-lbl. 2003.
EFFECTS OF OYSTER REEFS ON WATER QUALITY IN A TIDAL CREEK ESTUARY
KIMBERLY A. CHESSMAN, MARTIN H. POSEY,* MICHAEL A. MALLIN,
LYNN A. LEONARD, AND TROY D. ALPHIN
University of North Carolina at Wilmiiii^ton Center for Marine Science. 5600 Marvin K. Moss Lane.
Wilniini^ton. Norlli Carolina 2,^409
ABSTRACT The importance of oyster filtering in moderating aspects of water quality has received increased attention over the past
several years. This sttidy examined the intluence of intertidal oyster reefs on chlorophyll a. fecal coliform bacteria, and total suspended
solid concentrations under field conditions in a tidal creek estuary. Oyster reefs of varying live oyster density were sampled during
summer 2002, winter 200.1. and spring 201B. Water samples were taken upstream and downstream of each reef as well as over a mud
Oat control area on an ebb tide and analyzed for concentrations of these water column constituents. Summer data showed consistent,
significant decreases in chlorophyll a concentrations as water moved over the reefs, usually by 10-25%. Fecal coliform counts were
frequently lower downstream, by up to 45%. but were much more variable and not statistically different in most cases. Data taken in
winter, when temperatures and oyster feeding rates were lower, showed less consistency in upstream versus downstream patterns. In
spring, chlorophyll « decreases were less frequent than in summer, but significant fecal coliform decreases were more frequent. Total
suspended solid concentrations were not changed by the presence of oyster reefs during any season. Data from this study indicate that
feeding by oysters and changes in water How caused by the presence of reefs may both play a role in reducing chloi-ophyll a and
bacterial concentrations in the water column.
KEY WORDS: Cnissoxtrcii virginicu. fecal coliform bacteria, chlorophyll a. tidal creek
INTRODUCTION
Increasing coastal papulations and watershed development
have led to concerns over water quality for both shellfishing and
human contact waters. Among the water quality concerns in
coastal areas are water-borne pathogens, eutrophication, increased
turbidity, and sediment loads. Nutrients, sediments, and pathogens
enter natural water bodies through runoff and can have both human
health and ecosystem-level impacts.
Microbial pathogens, particularly those from human and animal
feces, can pose concerns for human health (Grimes 1991). Fecal
coliform bacteria, indicators of pathogens associated with human
and animal wastes, have been shown to be positively conelated
with impervious surface cover in a watershed as well as with
nitrate and orthophosphate concentrations (Mallin et al. 2000) and
turbidity (Pommepuy et al. 1992, Mallin et al. 2000), and inversely
correlated with salinity (Goyal et al. 1977, Mallin et al. 1999,
Mallin et al. 2000). Suspended solids and turbidity can contribute
to survival and even growth of fecal coliform bacteria by providing
protection from light, an organic substrate, and a mechanism for
transport downstream (Gerba & McLeod 1976, Pommepuy et al.
1992. .Sayler et al. 1975). Rainfall events have also been coiTelated
with increases in fecal coliform concentrations (Goyal et al. 1977.
Struck 1988. Howell et al. 1995) due to runoff inputs.
Increasing sedimentation and turbidity are concerns not only
for their role in the survival of fecal coliforms, but also because of
their effects on water column irradiance. Suspended solids and
turbidity can prevent light from penetrating the water column and
thus can negatively impact the growth of primary producers such
as rooted aquatic macrophytes. benthic microalgae. and phy-
toplankton (Cordone & Kelley 1961). Benthic cominunity struc-
ture, including the occurrence of shellfish beds, can be affected
through burial by sediments and interference with filter feeding
(Loosanoff & Tommers 1948, Posey 1990, Shumway 1996).
Eutrophication. caused mainly by nutrient loading, can also
'Corresponding author. E-mail: poseymCs'uncw.edu
ha\e detrimental effects on ecosystems (Nixon 1995. Brickeret al.
1999). Direct effects of eutrt)phication include initial increases in
chlorophyll and primary production, changes in phytoplankton and
macroalgal communities, and loss of seagrass (Burkholder 2001,
Cloern 2001). Indirect effects include changes in water transpar-
ency, nutrient cycling, benthic communities, and food web struc-
ture (Cloern 2001. Posey et al. 2002). The.se effects are moderated
by system attributes, with some areas being more sensitive to
nutrient loading than others (Cloern 2001. Posey et al. 2002).
In response to the potential deterioration of water quality as-
sociated with watershed development, natural measures are being
examined as possible remediation techniques. Several recent stud-
ies have concentrated on the role of bivalves in regulating sus-
pended particulate loads in estuarine systems. Models based on
laboratory studies of bivalve filtration rates predict that bivalves,
when sufficiently abundant in shallow waters, can control phy-
toplankton biomass (Cloern 1982. Officer et al. 1982, Gerritsen et
al. 1994). These models, however, are often based on high esti-
mates of feeding rates from laboratory trials and fail to take into
account variability in bivalve feeding rates under field conditions
or bivtilves' release of nutrients, which could actually stimulate
phytoplankton growth. Oyster feeding rates can be affected by
temperature, salinity, suspended solid concentrations, and other
factors (Shumway 1996). While filter feeding is hypothesized to
remove substantial amounts of particulate matter, removal may
also be caused by physical effects of oyster reefs on water flow
(Dame 1987). The presence of reefs can cause eddies and turbu-
lence, which lead to the settling of fine particles.
Field studies regarding removal of particulate matter by oyster
reefs are somewhat limited. Dame et al. (1984, 1985. 1989) and
Dame & Dankers (1988) found significant decreases in total or-
ganic carbon, particulate organic carbon, total suspended solids,
nitrite-i-nitrate. and chlorophyll a. Ammonium concentrations in-
creased downstream of oyster reefs, suggesting a role for oyster
reefs in nutrient cycling (Dame et al. 1984. 1985. 1989: Dame &
Dankers 1988; Nelson et al. 2003). In one sitidy. tidal creeks with
oysters did not show significantly lower chlorophyll a levels than
753
754
Cressman et al.
creeks without oysters (Dame & Libes 1993); however, another
study found significantly lower chlorophyll a (especially pho-
totrophic flagellates I in creeks with oysters (Wetz et al. 2002).
The eastern oyster. Crassostrea virginica (Gmelin). is a filter
feeder that is widely believed to reduce the amount of particulate
matter in the water column. Field evidence to support this idea is
limited, however, and no field tests of fecal coliform reductions
over oyster reefs have been published. The research described here
assessed the impacts of intertidal oyster reefs on suspended solids,
chlorophyll a. and fecal coliform bacteria in a human-impacted
tidal creek and also examined whether live oyster density over
natural ranges influenced rates of seston removal.
MATERIALS AND METHODS
Study Site
Six natural, intertidal oyster reefs were examined in Hewletts
Creek, southeastern North Carolina. Hewletts Creek is an anthro-
pogenically impacted tidal creek with a watershed that is approxi-
mately TC/f developed, with 18% impervious surface coverage
(Mallin et al. 2000). The reefs used in this study were bar reefs
approximately 10 m wide and were selected to provide a gradient
of ambient live oyster density from "low'" (79 live oysters m~") to
"high"" (167 live oysters m~"-. Table 1) based on live densities
available in the study area. Because the amount of shell hash
covering oyster reefs may contribute to physical effects on water
flow, reefs with different amounts of shell cover were used. Two
of the reefs had low dead shell cover (approximately 60-80% of
the reef consisted of live oysters, and the rest of the substrate was
exposed sediment); the others were completely covered by live and
dead shell. All reefs were located near a channel in the creek to
ensure sufficient flow and were at least 5 m distant from other
reefs. Reefs were not located immediately adjacent to marsh, thus
reducing potential effects of sedimentation associated with
marshes. A mud flat area immediately upstream of the selected
reefs was used as a no-oyster control. The mud flat area lacked any
shell cover, was more than 20 m distant from oyster reefs, and was
dominated by sediment of similar grain size (fine sands) as that
adjacent to the studied oyster reefs. The vertical height and vertical
complexity of each reef were measured, as they may impact physi-
cal effects such as flow velocity (Lenihan 1999, Posey & Alphin.
unpubl. Table 1 ). Reef height was measured while water covered
the crest of the reef by recording the depth of water over the crest
and subtracting this from the depth of water covering the edges of
the reef. Vertical complexity was calculated by allowing a 1 m
long chain to conform to the vertical contours of the reef and
measuring the actual horizontal distance covered by the chain. Com-
plexity was quantified as the ratio of straight distance after conform-
ing to the contours divided by 1 m. Values for complexity range from
0 to 1. with smaller values indicative of higher complexity.
Because flow speed can affect bivalve growth and filtration
(Lenihan et al. 19961 as well as sediment deposition, it was im-
portant to characterize the flow regimen of each reef in this study.
Flow measurements were taken with a Marsh McBirney, Inc.,
(Frederick. MD) Flo-mate Model 2000 handheld current meter
once in the summer and during sample collection in winter and
spring. Further, because oyster reefs may cause settling of fine
particles, it was desirable to determine whether sediment compo-
sition was different upstream versus downstream of the reefs in
this study. Sediment samples were taken at approximate upstream
and downstream water column sampling locations during a low
tide in June 2003, and grain size fractions were determined using
a Beckman LS Coulter Counter (Miami. FL).
Sampling
Fecal coliform and chlorophyll a concentrations in tidal creeks
have been shown to be highest at approximately mid-to-low tide
(Mallin et al. 1999). Additionally, significant decreases in chloro-
phyll a concentrations downstream of a created oyster reef near the
study area were observed 3 h after high tide (Nelson et al. 2003).
To increase the likelihood of detecting effects, water samples were
taken as close as possible to mid-ebb tide (generally about 2 h after
high tide). Samples were taken from a canoe to avoid disturbing
sediment. All sampling was conducted on ebb tides with a pre-
dicted range of 0.9-1.1 m after a high tide of approximately 1 m.
Water depth was less than 35 cm on the upstream and downstream
sides of the reef at the time of sampling and only a few cm of water
were present over the crest, thereby maximizing the amount of
water that came into contact with the oysters.
Samples were taken at two locations upstream and two loca-
tions downstream of each reef. The two upstream samples were
approximately 1 m apart from each other, as were the downstream
samples. Upstream samples were taken at mid-depth in the water
column. Because dye studies conducted prior to sampling showed
that water from mid-depth flowed up over the crest of the reef and
stayed near the surface, downstream samples were taken just under
the surface of the water. Downstream samples were taken before
upstream samples to avoid the collection of sediments that had
been stiired up by prior sampling. For the same reason, the first
reef sampled in a day was downstream of the second reef.
Sampling of the six reefs, as well as a mud-bottom control area.
TABLE 1.
Physical characteristics of oyster reefs used in the study. Live oyster densities (m"") were measured in Summer 2002 and Spring 2003. Also
indicated is ''i shell cover, which is indicative of the amount of dead sliell covering the reef. Width is the distance water traveled over the
reef between upstream and downstream sampling locations; height is the vertical difference between the crest and base of the reef.
Sunmier Density
Spring Density
% Shell
Length
Width
Height
Vertical
Reef
(per m -)
(per m~')
Cover
(m)
(m)
(m)
Complexity
1
79
132
100
14.5
13.5
0.29
0.68
2
113
129
100
10.0
15.0
0.15
0.64
3
114
150
60
13.0
8.0
0.40
0,68
4
116
163
80
13.0
9.5
0.50
0.75
5
129
176
100
13.0
S.O
0.30
0.70
fi
167
IS3
100
17.7
5.5
0.65
0.73
Oyster Reef Effects on Water Quality
755
was accomplished over a period of three days during each sam-
pHng period, with two reefs sampled per day. Sampling was con-
ducted twice per season during summer 2002 (once in July and
once in August) and spring 2003 (twice in May. approximately two
weeks apart). Due to low concentrations of water column constitu-
ents as well as weather limitations, only one sampling set was
conducted in winter 2003 (February). Sampling within 24 h of ruin
was avoided due to potential effects of storm water runoff on water
column constituents. In winter, however, there were such low con-
centrations of the water column constituents of interest that it was
necessary to sample after a rain event, in addition to the scheduled
sampling period, to have sufficiently high chlorophyll a and fecal
coliform concentrations to allow detection of potential effects. The
two reefs with the highest live-oyster density and the mud tlat
control area were all sampled the day after a rainfall of approxi-
mately 3 cm in February 2003.
Chlorophyll ci samples were taken in triplicate into 125-mL
opaque plastic bottles. A fourth bottle was used to ensure collec-
tion of enough water for total suspended solids (TSS) analysis.
Fecal coliform samples were collected using autocluved 50()-mL
glass bottles. All samples were kept on ice until they were filtered.
Water remaining after filtration of fecal coliforms and chlorophyll
a was combined and stored at 4 C until it could be used in analysis
of TSS. Originally, this project was intended to focus on changes
in turbidity rather than TSS. However, initial attempts to measure
turbidity met with methodologic difficulties, and TSS analysis was
added to the study in the second summer sampling period.
Sample Processing
Fecal coliform and chlorophyll a samples were filtered upon
return to the laboratory and within 6 h of collection. Fecal coliform
bacteria concentrations were determined according to the mem-
brane filter procedure, using niFC medium (Sparks, MD) (APHA
1995). Chlorophyll a samples were filtered through Gelman
(Clifton, NJ) A/E glass fiber filters with 1.0 jxm pore size. The
filters were wrapped individually in aluminum foil and frozen in a
sealed container with desiccant. Concentrations were determined
tluorometrically (Welschmeyer 1994) within three weeks. TSS
were analyzed gravimetrically (APHA 1995) using 500 mL of
water from each sampling location. TSS were filtered through
predried Gelman A/E 47 mm diameter glass fiber filters with 1.0
p.m pore size.
Statistical Analysis
The parameters of chlorophyll ci and fecal coliform concentra-
tions were tested for normality and nonheterogeneity of variances.
Variances upstream and downstream of reefs were nonheteroge-
neous for both parameters. However, neither showed a normal
distribution, even after standard transformations, leading to the use
of nonparametric tests. Kruskal-Wallis tests were used (Sokal &
Rohlf 1445) to test upstream versus downstream concentrations of
the sampled variables and to determine whether they were signifi-
cantly different across each individual reef for each sampling pe-
riod. In all other analyses, which involved concentration changes
of variables and not the non-normally distributed concentrations
themselves, parametric methods were used. Multiple regression
was used to determine whether the concentration changes of the
studied variables were related to live oyster density, mean up-
stream fiow speed, tidal range, and the time elapsed between high
tide and actual sampling. An ANOVA was used to test for differ-
ences between the high-shell and low-shell reefs of the same live
oyster density. A /-test was used to test for overall reef effects
within a season (i.e., did the reefs show consistently decreased
concentrations downstream?). All analyses used SAS (SAS Insti-
tute. Inc. 1989).
RESULTS
SniniNvr
Mean chlorophyll a concentrations ranged from 2.3-10.6 p-g
L"' over the reefs and mud tlat during the summer sampling pe-
riods. Mean fecal coliform concentrations ranged from 1 .3-54.8
colony forming units (CFU) 100 mL"'. Total suspended solid
concentrations ranged from 10-27 mg L"'. Temperature was ap-
proxiinately 25-27°C and salinity ranged from 30-36 ppt at the
study site during these sampling periods.
Chlorophyll ci was significuntly lower downstream of reefs than
upstream in summer for 9 of 1 2 comparisons (two comparisons for
each of the six reefs: Table 2). This overall reef effect was sig-
nificant for all reefs combined {P = 0.002), for high-shell-cover
reefs (P = 0.023) and for low-shell-cover reefs (P = 0.053). Each
reef demonstrated a significant decrease in chlorophyll a at least
one of the two times it was sampled over the summer. There was
no significant difference in percent removal of chlorophyll a be-
tween the high-shell-cover and low-shell-cover reefs of the same
live oyster density (P = 0.52). The control was sampled only once
during suinmer, and at that lime chlorophyll a was significantly
lower downstream than upstream (P = 0.010). Changes in chlo-
rophyll a concentrations were not significantly related to live oys-
ter density (Fig. lA) or tidal range.
Fecal coliform concentrations were often lower dow nstream of
reefs than upstream (8 out of 12 comparisons), although only two
differences were statistically significant and there was not a sig-
nificant overall reef effect (P = 0.22). Fecal coliform concentra-
tions were higher downstream on the mud fiat than upstream, but
this difference was not significant. Changes in fecal coliform con-
centrations were not significantly related to live oyster density
(Fig. IB) or tidal range. There was no significant difference in
percent fecal coliform removal between the high-shell and low-
shell reefs of the same live oyster density iP = 0.86).
Because of difficulties encountered when measuring turbidity.
TSS concentrations were added to sampling during the second
summer sampling period. There were three instances of lower
(24-38%) TSS concentrations downstream of reefs, two instances
of higher (25^3%) concentrations downstream, and one instance
with very little change. The mud tlat showed no change in TSS
concentration. Due to a lack of replication (only two samples
upstream and two downstream), no statistical test could be run on
the differences across each reef or the mud tlat. There was no
significant overall reef effect on TSS concentrations {P = 0.44).
Changes in TSS concentrations were not significantly related to
live oyster density (Fig. IC) or tidal range, and percent change was
not significantly different between the high-shell and low-shell
reefs of the same live oyster density {P = 0.80).
Winter
Mean chlorophyll a concentrations ranged from 0.3-1 .5 p.g L"'
over the reefs and control during the winter sampling period. Mean
fecal coliform concentrations ranged from 0.2-8.0 CFU 100 mL '
over the reefs and 22.5-36.7 CFU 100 mL ' over the nonreef mud
flat area (control). Temperature was approximately 4 C and salin-
756
Cressman et al.
TABLE 2.
Results of Kruskal-Wallis ttsts on upstream vs. downstream
concentrations of chlorophyll a (chl) and fecal coliform hacteria (fc)
concentrations. Significant differences are in bold. All sinnificant
changes were reductions (lower downstream) evcept for one,
designated with a. Each reef was sampled twice in summer 2002
and spring 2003 and once in winter 2003. The mudHat was sampled
only once in summer, and reef 6 was sampled tw ice in winter.
Reef
TABLE 2.
(Continued!.
K-W
Season
Parameter
df
Chi-square
/•-Value
Summer 1
chl
10
5.SI0
0.016
Summer 2
chl
10
0.SI9
0.366
Winter
chl
5
.V667
0.056
Spring 1
chl
10
0.85(1
0.357
Spring 2
chl
10
9.000
0.003
Summer 1
fc
9
3.427
0.064
Summer 2
fc
10
0.315
0.575
Winter
fc
10
4.046
0.044
Spring 1
fc
8
0.099
0.753
Spring 2
fc
10
0.660
0.417
Summer 1
chl
9
0. 1 38
0.711
Summer 2
chl
10
8.768
0.003
Winter
chl
10
1.000
0.317
Spring 1
chl
III
1.169
0.280
Spring 2
chl
10
4.373
0.037
Summer 1
fc
10
6.322
0.012
Summer 2
fc
10
1.664
0.197
Winter
fc
9
0.222
0.637
Spring 1
fc
10
0.523
0.470
Spring 2
fc
10
0.241
0.624
Summer 1
chl
10
8.366
0.004
Summer 2
chl
7
5.492
0.019
Winter
chl
10
4.083
0.043
Spring 1
chl
III
8.366
0.004
Spring 2
chl
9
5.307
0.021
Summer 1
fc
10
0.007
0.934
Summer 2
fc
8
0.702
0.402
Winter
fc
10
2.898
0.089
Spring 1
fc
10
6.657
0.010
Spring 2
fc
10
3.718
0.054
Summer 1
chl
10
8.426
0.004
Summer 2
chl
10
4.790
0.029
Winter
chl
10
5,978
0.015
Spring 1
chl
10
4.333
0.037
Spring 2
chl
10
4.889
0.027*
Summer 1
fc
10
0.058
0.810
Summer 2
fc
9
0.533
0.465
Winter
fc
10
0.946
0.331
Spring 1
fc
10
0.410
0.522
Spring 2
fc
10
0.235
0.628
ity ranged IVotii 17-35 ppt ;tl the stridy site during this sampling
period. Turbidity was very low, ranging frotn 1.5-5.0 NTU. and
TSS concentrations ranged from 1.8-7.5 mg L"'.
Because concentrations of the studied water column constitu-
ents were so low, the two highest live-oyster-density reefs (both
with high dead shell cover) and the mud flat were also sampled
after approximately 3 cm of rain, when the creek water level was
higher than normal. After this rain event, mean chlorophyll ci con-
centrations ranged from 1.8-2.6 |jLg L"' and mean fecal coliform
concentrations were approxitnately 146-516 CFU 100 tiiL"'. Tem-
perature was 4°C and salinity ranged from 15-29 ppt among sites
Reef
Season
Parameter
df
Chi-square
K-W
P-Value
Mudnm
Summer I
Summer 2
Winter
Spring 1
Spring 2
Summer 1
Summer 2
Winter
Spring 1
Spring 2
Summer 1
Suminer 2
Winter 1
Winter 2
Spring 1
Spring 2
Summer 1
SuiTimer 2
Winter 1
Winter 2
Spring 1
Spring 2
Summer
Winter
Spring 1
Spring 2
Summer 2
Winter
Spring 1
Spring 2
chl
chl
fc
fc
fc
fc
fc
chl
chl
chl
chl
chl
chl
fc
fc
fc
fc
fc
fc
chl
chl
chl
chl
fc
fc
fc
fc
10
8.396
10
8.640
10
0.000
10
1.331
10
5.843
10
2.857
10
0.103
10
0.244
8
2.455
10
0.026
10
2.929
10
5.810
10
3.008
10
1.637
10
0.232
10
0.164
9
1 .656
9
7.569
10
5.507
10
0.007
10
6.564
10
1.713
10
6.610
10
3.209
10
0.058
10
3.274
9
0.307
10
6.587
10
5.043
10
0.000
0.004
0.003
1 .000
0.249
0.016
0.091
0.749
0.622
0.1 17
0.871
0.087
0.016
0.083
0.201
0.630
0.686
0.198
0.006
0.019
0.933
0.010
0,191
0.010
0.073
0.810
0.070
0.580
0.010
0.025
1 .000
on the same day. Water flow speed was higher than normal after
the rain event. This was due partly to a larger tidal range than was
normally sampled ( 1.5 tn versus a usual range of 0.9-1.1 ni) as
well as flow effects from storm water runoff. Turbidity was com-
parable to spring and suinmer turbidity, ranging from 7.8-12.5
NTU. TSS concentrations were 9.0-15.4 mg L"'.
During the regular winter sampling period, there were 2 sig-
nificant decreases {P < 0.05) in chlorophyll ci concentrations over
the reefs (Table 2). These differences were observed over low-
shell-cover reefs, but there was not a significant difference be-
tween these reefs and the high-shell-cover-reef of the same density
(P = 0.564). A /-test did not show a significant overall reef effect
on this variable for all reefs combined (P = 0.691), for high-shell-
cover reefs (P = 0,582), or for low-shell-cover reefs (P = 0,323).
0\er the mud flat, there was no significant change in chlorophyll
II. Changes in chlorophyll a in winter were not significantly telated
to live oyster density (Fig. 2A), mean flow speed upstream of the
reefs, or change in flow speed. After the rain event, both reefs and
the nuid flat showed slight, nonsignificant increases in chlorophyll a.
In the regular winter sampling period, fecal coliforms were
lower downstream than upstream five times (out of seven coin-
parisons; the highest-density reef was sampled twice in winter),
but this overall reef effect was not significant for all reefs com-
bined (P = 0.26), for high-shell-cover reefs (P = 0,22), or for
low-shell-cover reefs (P = 0,86). Two of the fecal coliform de-
creases were significant, and these occurred over the highest-
Oyster Reef Effects on Water Quality
737
c
o
o
o
c
03
o
O
0)
O)
c
CO
c
o
o
o
0)
en
c
10 1
0
60
-10
-20
-30
°~ -40
o
I 20
c
-20
-40
-60
60
40
20
60
-20
-40
-60
60
Chlorophyll a
♦
80
100
120
140
160
180
♦
♦
♦
Live Oyster Density (m'-)
Fecal Conforms
80
100
►120
140
160
180
Live Oyster Density (m"')
TSS
80
♦
100
120
140
160 180
♦
Live Oyster Density (m"-)
Figure 1. Water column constituents as related to live ovster density, summer 2(HI2: Percent changes in chlorophyll a, fecal coliforms. and TSS.
Negative numbers represent a lower concentration downstream of the reef than upstream.
density reef (/> = 0.019) and the lowest-density reet'(P = 0.044;
Table 2). Fecal coliform concentrations significantly decreased
over the mud flat {P = 0.010) during this sampling period.
Changes in fecal coliform concentrations were not correlated with
live oyster density (Fig. 2B). upstream flow speeds, or changes in
flow. There was no significant difference between percent change
in fecal coliform concentrations between the high-shell and low-
shell reefs of the same live oyster density (P = 0.67).
After the rain event, fecal coliform concentrations were el-
evated above nonrain conditions. Due to crowding of the petri
dishes on which the bacteria were grown, the counts were not
considered reliable enough for statistical analysis. However, it was
apparent that fecal coliform concentrations were highest over the
mud flat (approximately 400 CFU 100 mL"'). lower over the
highest-density reef, which was slightly downstream of and adja-
cent to the mud flat (approximately 360 CFU 100 mL"'), and
lowest over the most downstream reef (approximately ISO CFU
100 niL"').
During the regular winter samplmg period, TSS concentrations
were higher (25-36*^) downstream of reefs as compared with
upstream on three occasions. TSS concentrations were moderately
lower {\0%) once, and twice were only slightly (<5'^»-) lower
downstream. Given the low TSS concentrations during this sam-
pling period, however, an increase of <1 mg L"' could translate to
a 30% change. There was no significant overall reef effect on
concentration changes {P = 0.252). Upstream to downstream
changes in TSS concentrations were not significantly related to
live oyster density (Fig. 2C), flow speed of water upstream of the
reefs, or changes in flow .speed during the winter sampling period.
There was no significant difference in TSS change between high-
shell and low-shell reefs of the same live oyster density {P =
0.744). TSS concentrations were 0J9r higher downstream than
758
Cressman et al.
g
CO
Chlorophyll a
c
70 1
o
ro
50 -
c
o
o
o
30 -
O
c
<u
10 1
O)
-10120
-30 J
100
50 -
130
140 150
160
170
180^ 190
Live Oyster Density (nr^)
Fecal Coliforms
a>
o
c
o
O
0
c
o
O)
c
-50
to
^
O
S5
-100
i;!0
130
♦
140
150
♦
160
170
180
190
Live Oyster Density (m'^
TSS
40
30
c
20
o
o
10
c
o
n
D)
C
-10
U
s«
-20
120
f30
140
150
160
170
180
190
Live Oyster Density (m"^)
Figure 2. Water column constituents as related to live oyster densit>, winter 20IL^: Percent changes in chlorophyll a, fecal coliforms, and TSS.
Negative numbers represent a lower concentration downstream of the reef than upstream.
upstream over the liighest-density reef after the rain event, but
were 30% higher over the second-highest-density reef. On the mud
flat. TSS concentrations were approximately \\% lower down-
stream.
Spring
Mean chlorophyll a concentrations ranged from 1.3-7. 1 |j.g L"'
over the reefs and 2.0-12.2 |xg L~' over the mud flat during the
spring sampling period. Mean fecal coliform concentrations
ranged from 8-330 CFU 100 niL"' over the reefs and mud flat.
Fecal coliform counts were higher during the first spring sampling
period than the second due to a long rainy period preceding sam-
pling. Samples were not taken within 24 h of rain, but the earlier
rain did affect the water column. Temperature was approximately
24°C, and salinity ranged from 19-2.'i ppt during the flrst spring
sampling and 30-34 ppt during the second spring sampling period.
Turbidity ranged from 5.8-9.8 NTU over both spring sampling
periods.
In spring, there were six significant decreases and one signifi-
cant increase in chlorophyll (/ concentrations across the reefs
(Table 2). There was not an overall reef effect on chlorophyll fl
concentrations for all reefs combined {P = 0.18), for high-shell-
cover reefs (P = 0.19), or for low-shell-cover reefs (P = 0.28).
Chlorophyll ii changes also were not significantly related to live
oyster density (Fig. 3A). flow speed upstream of the reefs, change
in flow speed, or how long after the high tide samples were taken.
There was no significant difference in percent removal of chloro-
phyll (( between high- and low-shell-cover reefs of similar live
oyster density (P = 0.45).
Ten of 12 comparisons showed fecal coliform concentrations
that were lower downstream than upstream in spring. Three of
these decreases were significant (Table 2). as was the overall reef
effect (P = 0.009). The mud flat showed a significant {P = 0.025)
downstream decrease in fecal coliforms during one of the two
spring sampling periods. Changes in fecal coliform concentrations
were not significantly related to live oyster density (Fig. 3B). flow
o
O
O
10
0
-10
-20 -
-30
-40
-50
120
Oyster Reef Effects on Water Quality
A Chlorophyll a
759
130>
♦
140
150
♦
160
170 ♦ 180*
190
Live Oyster Density (m"-)
Fecal Conforms
10
c
o
2
0
c
OJ
o
c
o
-10
O
c
(n
-20
C31
c
CO
-30
O
oS
-40
o
O
g
O)
c
CO
120
130
♦ ♦
140 150
♦
160^ 170 180 190
♦ ♦
Live Oyster Density (nr^)
TSS
15
10
5
0
-5
-10
-15 J
♦
♦
♦
120
130
140 150 160' 170 A 180 190
Live Oyster Density (m"-')
Figure 3. Water column constituents as related to live oyster density, spring 2003: Percent changes in chloropli>ll a. fecal coliforms, and TSS.
Negative numbers represent a lower concentration downstream of the reef than upstream.
speed upstream of reefs, or changes in flow. A /-test did show
significantly decreased fecal coliform concentrations downstream
of oyster reefs in spring for all reefs combined (P = 0.009).
High-shell-cover reefs did not show this overall effect (P = 0.10);
the pattern was driven by the low-shell-cover reefs (P = 0.012).
However, high- and low-shell-cover reefs of similar live oyster
density did not show significantly different patterns of fecal
coliform removal in spring (P = 0.16).
TSS did not exhibit a significant pattern with respect to the
variables examined in spring. Out of 12 comparisons, downstream
TSS concentrations were higher seven times, lower three times,
and unchanged twice. There was not a significant overall reef
effect on TSS concentration changes (P = 0.29). TSS concentra-
tions were higher downstream once over the mud flat and re-
mained unchanged during the other spring sampling period. The
observed changes in TSS concentrations were not significantly
related to live oyster density (Fig. 3C). water flow speed upstream
of the reefs, or changes in flow. Percent removal of TSS was not
significantly different between high- and low-shell-cover reefs of
the same live oyster density (P = ().5A).
Overall
During the warm seasons of summer and spring, chlorophyll a
was significantly lower downstream of reefs than upstream a total
of I .^ times (out of 24 observations). Only once was it significantly
higher. In summer, chlorophyll a concentrations were significantly
lower downstream of oyster reefs than upstream (P = 0.002)
overall. In spring, however, there was no significant overall reef
effect. Fecal coliforms were reduced the majority of the time dur-
ing the warm seasons ( 18 of 24 comparisons), but only 4 of these
decreases were statisticallv sisznificant. In summer, this overall reef
760
Cressman et al.
effect was not statistically significant, but it was significant in
spring {P = 0.009).
Water flow varied somewhat from reef to reef. The lowest
observed flow over the parts of the reef from which samples were
taken was 6 cm s' . Flow velocity reached 22 cm s" ' over the other
reefs. The three-dimensional cuirent study showed increases in
flow speed over the crest of three of the reefs and decreases over
the crests of the other three reefs. However, differences in flow
speeds between reefs were not significantly related to changes in
the water column constituents. Vertical complexity did not differ
among the reefs (Table 1 ). Over five of the six reefs, downstream
sediments contained a greater amount of coarse sediment than
upstream (by S-H'/f; Table }•). The mud flat did not exhibit the
same distribution of sediment texture.
DISCUSSION
The presence of oyster reefs caused significant reductions in
chlorophyll a and fecal coliform bacteria concentrations in this
study. Effects on chlorophyll were greatest in summer, whereas
effects on fecal coliforms were strongest in spring when bacterial
counts were highest. The decreases in chlorophyll concentrations
were consistent with previous studies showing that bivalve beds
can have significant effects on the overlying water column (Dame
et al. 1984. 1985. 1989: Asmus & Asmus 1991 ). and there has not
been any previous investigation regarding effects of oyster reefs on
fecal coliform concentrations. In this study, oyster reefs did not
have any clear, consistent effects on TSS concentrations.
Haven and Morales-Alamo ( 1970) found that a doubling of the
number of oysters in an experimental tank led to an approximate
doubling of removal rates of particulate matter. Changes in sus-
pended particulate concentrations, then, should be significantly
related to live oyster density if oyster feeding is the sole or over-
riding factor in particulate removal. In this study, such a relation-
ship was not observed. One possible explanation for this observa-
tion is a threshold effect, some critical density of live oysters at
which a measurable effect can be detected. Alternatively, the re-
lationship between changes in seston and live oyster densities may
be detected only over a greater density range and spatial scale. The
oyster reefs used in this study provided only a small range of live
oyster densities, especially after a large spatfall in summer 2002
(Posey & Alphin. unpubl.). Thus, the examined range of live oys-
ter densities may have been too narrow for a density relationship
to be detected. Because the changes in concentrations of the stud-
ied water column constituents were not significantly related to
flow speeds or changes in flow speed across the reefs, it is
TABLE 3.
Sediment eomposition. as '7t fine sediment (detlned as less than
63.41 fini diameter), upstream and downstream (el)b tide) of the
oyster reefs.
Reef
% Fine
Upstream
'7c Fine
Downstream
1
2
3
4
5
6
Mudflat
40
38
72
85
41
64
40
32
21
70
78
30
51
41
unlikely that the observed changes were due solely to flow speed.
Live oyster lengths near the study site averaged 65 mm (Har-
well. Posey & Alphin, unpubl.). Using the methods of Dame
( 1972). the mean dry weight for these oysters was calculated to be
1.33 g. NewelFs (1988) estimate of oyster clearance rates of 5 L
h~' g"' were used to calculate the potential volume of water that
could be cleared by each oyster reef in this study. In summer, flow
velocities upstream of the oyster reefs ranged from 6-21 cm s"',
and the oysters on the reefs could potentially clear only 5-15% of
the water moving over them. Many of the observed chlorophyll a
differences in summer were greater than the potential filtration
capacity of the oysters on the reefs based on these estimates (up to
30% removal), suggesting that either oyster feeding rates are
higher than NewelTs (1988) estimate or that factors other than
oyster feeding (i.e.. other filter feeders or physical effects) are
important in particulate removal.
Additional calculations of approximate clearance rates, assum-
ing 100% efficiency of particle removal, were made using the
observed summer decreases in chlorophyll a concentrations. These
rates ranged from 3-18 L h~' g"' across the reefs. The mean was
10 L h~' g~', which is consistent with Jordan's ( 1987) laboratory
estimate. Oysters do not remove all particles from water with
100% efficiency, however, so this estimate may be conservative.
Other filter feeders, such as mussels, were not abundant on
these oyster reefs and therefore cannot account for the larger than
expected effects. Even though flow velocities did not decrease
downstream of the reefs, particle trapping within the reef crest may
have occuned in flow shadow /ones between oyster clumps. This
explanation is consistent with chlorophyll a and fecal coliform
data in that the reefs that consistently showed significant decreases
in chlorophyll it and fecal coliform concentrations were the reefs
with low shell cover (i.e., low areas floored by mud). These were
also the reefs with the lowest flow velocities (approximately 8 cm
s"' ). Dame et al. (1985) and Dame (1987) found that most material
uptake over an oyster reef in North Inlet occurred when flow was
less than 15 cm s"' and attributed this to a combination of biofil-
tration and sedimentation. Lower flow speeds could contribute to
removal of particles by increasing the time water is in contact with
the oysters and thus increasing their ability to filter particulates; it
could also be that particles settled out of the water at these lower
speeds. Preferential ingestion of chlorophyll (microalgae) by oys-
ters (Ward et al. 2000, Wetz et al. 2002) may interact with low
flow velocities to produce the strongest effects on this parameter,
consistent with the significant reduction in chlorophyll concentra-
tions over these reefs but low influence on TSS concentrations.
Oyster reefs ha\e been shown to play a role in nutrient cycling
in tidal creeks by releasing NH/ (Dame et al. 1984, 1985, 1989;
Dame & Dankers 1988; Nelson et al. 2003). As such, it could be
argued that chlorophyll a concentrations should actually be higher
downstream of reefs than upstream. Ammonium released by bi-
valves can be taken up by phytoplankton and lead to increased
phytoplankton biomass. Asmus and Asmus (1991) made this ar-
gument for sy.stems impacted by a mussel bed, though their field
study showed significant decreases in phytoplankton biomass
across the bed. Increased phytoplankton production due to nutrient
release is also a possibility for oyster reefs. However, there is a lag
time of a few hours before the ammonium shows up as primary
production in the water column, and any increased production may
be appearing further downstream of the reefs than the location of
sample collection for this study. In terms of the parameters exam-
ined in this study, the only change that would be immediate
Oysti;r Rhhf Effects on Water Quality
761
enough lo detect as water Hows over the oyster reefs is particle
remo\al.
hccal colit'orni concentrations v\ere often lower downstream of
reefs than upstream, but the differences were rarely significant.
The overall reef effect of decreased fecal coliform concentrations
was significant in spring but not suinmer, the opposite of the effect
for chlorophyll a. Fecal coliform counts are e.\tremely variable,
necessitating large changes before a significant effect can be de-
tected. Because fecal coliform concentrations were higher in
spring than in summer and winter, differences were slightly more
detectable.
C. virgiitka filters unattached bacteria with an efficiency of
only 5% {Langdon & Newell 1990). However, fecal coliforms
have been associated with turbidity and suspended sediments in
the water column (Sayler et al. 1975. Pommepuy et al. 1992.
Mallin et al. 2000) and inay be removed with suspended particulate
matter through either filtration or settling. In this study, fecal
coliform counts did not have consistent relationships with either
turbidity or TSS. Changes in fecal coliform concentrations were
not significantly related to live oyster density, fiow speeds, or
changes in flow speed across the reefs. None of these factors is
readily apparent as the most influential one. and changes in fecal
coliform concentrations are likely due to a combination of factors.
Changes in TSS concentrations did not exhibit any significant
patterns relative to the variables examined in this study. Due to a
lack of replication, statistical tests could not be used to determine
whether changes across a single reef were significant. However,
tests could be run to detect overall reef effects within a season, and
none of these were significant for TSS. Changes in TSS were not
consistently positive or negative in any season.
Water temperature in winter was 4°C, lower than the minimum
temperature (5°C) at which oysters typically feed (Galtsoff 1928.
in Shumway 1996). Chlorophyll a and fecal coliforms were con-
sistently decreased in the warm seasons of summer and spring, but
neither showed a consistent effect in winter. Feeding effects are
suggested by a lack of consistent change in water column con-
stituents during winter, even when concentrations were high
enough to detect a difference (after the rain event).
The fact that the presence of oyster reefs frequently led to
significant decreases in chlorophyll ci and fecal coliform concen-
trations, but rarely reduced total suspended solids, leads to specu-
lation that selective feeding by oysters occurred. In laboratory
experiments, oysters have been shown to feed selectively on high
quality food particles (Loosanoff 1949, Newell & Jordan 1983). In
South Carolina tidal creeks. Wetz et al. (2002) found preferential
feeding on phototrophic fiagellates. but not heterotrophic flagel-
lates, bacterioplankton, or cyanobacteria. Although the current
study was not designed to investigate selectivity, these results do
suggest that it occurs to a degree in these systems.
Flow conditions may also have contributed to changes in water
column constituents; particles may have settled over the crest of
the reefs (also suggested bv Dame 1987). Differences in bottom
sediment composition, however, may be due to larger-scale flow
patterns. Sediments were finer on the sides of the reefs that were
upstream during ebb tide (downstream during flood tide). Faster
flow during ebb tide than flood tide would lead to greater depo-
sition of fine particles during flood tide than ebb (as suggested by
Dame 1987), which could explain the observed differences in sedi-
ment texture. In Bradley Creek, a tidal creek in southeastern North
Carolina, current velocities were 14—55% higher on flood than ebb
tides (Angelidaki 1997). Howexer. high velocities lasted longer on
the ebb tide than flood tide (Angelidaki 1997). possibly causing
more sediment to settle on the flood tides. This study did not
examine effects of oyster reefs during flood tides because chloro-
phyll (/ and fecal coliform concentrations are highest during ebb
tides (Mallin et al. 1999). reflecting upland drainage influences.
While there was never a significant difference for changes of
chlorophyll ii. fecal coliform. or TSS concentrations between high-
shell-cover and low-shell-cover reefs, the reefs themselves showed
different patterns of effect. The reefs with low shell cover were
also the reefs with lowest flow velocities and showed consistent
removal of fecal coliforms in spring, whereas the other reefs did
not. Vertical complexity was approximately equal between all
reefs, and complexity may be a more important component in flow
effects than the presence of shell itself. Multiple factors could be
responsible for the observed effects on chlorophyll a. fecal
coliform, and TSS concentrations. Both filtration by oysters and
flow patterns over oyster reefs could contribute to particle removal
in tidal creek ecosystems.
CONCLUSIONS
Significant changes in concentrations of chlorophyll a and fecal
coliform bacteria were detected during warm seasons, even when
effects on TSS concentrations were not observed. None of the
examined variables were significantly related to live oyster den-
sity, flow speed, or change in flow speed across reefs, suggesting
possible threshold effects. Oyster reefs do have detectable effects
on chlorophyll a and fecal coliform concentrations under field
conditions, though effects vary temporally. The degree of removal
suggests physical mechanisms for removal in addition to filtration
effects.
ACKNOWLEDGMENTS
This work was supported by North Carolina Sea Grant (R/MER
46 to M. Posey andT. Alphin and R/MG 0213 toT. Alphin and M.
Posey), the New Hanover County Tidal Creeks Program and the
new center for marine science. The authors thank the Benthic
Ecology Lab (B. Allen. M. Anderson. R. Barbour, B. Boutin. H.
Harwell, T. Molesky, B. Noller, M. Owens, and J. Vinson) and the
Aquatic Ecology Lab (H. CoVan, V. Johnson, T. MacPherson, M.
Mclver, D. Parsons, and D. Wells) at the UNCW Center for Ma-
rine Science for assistance in the field and laboratory. Additional
thanks go to A. Croft and J. O'Reilly.
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Joimuil oj Slu'llfisli Research. Vol. 22. No. 3, Ity-lUh. 2003.
EXPRESSION OF HSP 70 IN EXPERIMENTALLY METAL-EXPOSED EUROPEAN FLAT
OYSTERS OSTREA EDULIS
ISABELLE BOUTET.' ARNAUD TANGUY," MICHEL AUFFRET,' NEDZAD MUJDZIC.' AND
DARIO MORAGA'*
'Lahonitoire des Sciences cle I'Emirowwiuent Marin (LEMAR), UMR-CNRS 6539. Insiimt Univer.sitaire
Eiiropeen de la Men Universire de Bretagne Occidentale. Place Nicolas Copemic. 292iS(). PUnizane.
France: and 'Ha.-ikin Shellfish Research Lahoraloiy. 6959 Miller Avenue. Port Norris. New Jersey 0SJ49
.\BSTR.\CT The heat shock protein 70 family is eomposed of both environmentally inducible (Hsp) and constitutively expressed
(Hsc) members. The expression of Hsp70 was investigated in the European tlat oyster Ostrca cdulis exposed to different metal
concentrations. By using a polyclonal antibody developed in our laboratory for a recombmant HspVO of the oyster Crassostrea gigas.
the soluble HspVO level in O. editlis was found metal dose dependent. An exposure to copper did not induce Hsp70 synthesis in either
gills or digestive gland. A decrease of Hsp70 was observed in gill from cadmium-exposed animals, whereas digestive gland tissue
showed an increase.
KEY WORDS: heat shock protein 70. 0\lrfci eihitis. ELISA. expression, quantification, metal accumulation.
INTRODUCTION
The cellular stress response is involved in protecting organisms
from damage caused by e.xposure to a great variety of stressors,
including temperature, heavy metals, and other xenobiotics. The
stress response entails the rapid synthesis of heat shock proteins
(HSPs) to protect the proteins against denaturation (Lindqiiist &
Craig 1988. Sanders 1993). HSPs were first described in Droso-
phila husckii (Ritossa 1962) and the genes encoding the Droso-
phila Hsp were among the first eucaryotic gene to be cloned (Craig
et al. 1979). The major and the most highly conserved and studied
of the HSPs in all organisms is the 70-kDa protein family (HSP70)
because ot its implication in protein chaperoning (Gething & Sam-
brook 1992) and acquired tolerance processes (Lindquist & Craig
1988, Clegg et al. 1998). The genes encoding Hsp70 are highly
conserved in evolution and contain both heat-inducible (Hsp) and
constitutive genes (Hsc). both of which encode stress proteins
under nomial conditions (Hightower 1993, Wood et al. 1998).
The types of studies conducted on stress proteins in aquatic
organisms are highly variable (Sanders 1993, Gourdon et al. 1998).
The synthesis of Hsp70 and induction of thermo-tolerance has
been demonstrated in the Pacific oyster, Crassostrea giiius iSham-
seldin et al. 1997, Clegg et al. 1998, Gourdon et al. 2000) and in
the mussels Mytiliis ediilis and Mytilus galioproviiicialis ( Sanders
1988. Snyder et al. 2001 ). Piano et al. (2002) showed a rapid and
significant synthesis of the inducible Hsp69 in thermal stressed flat
oyster Ostrea edulis, but no significant variations in the constitu-
tive isoforms level (Hsp72 and Hsp77). Recently, we characterized
two HSPVO genes and quantified soluble HSPVO by enzyme-linked
immunosorbent assay (ELISA) in C. gigas exposed to metals in the
laboratory (Boutet et al. 2003). In this previous study, we showed
that soluble HSP70 level decreased in tissues of experimentally
metals-exposed oysters.
In the present work, the expression of HspVO and Hsc70 pro-
teins in different organs of the European flat oyster, Osirca edulis.
exposed to a concentration gradient of metals under experimental
conditions was quantified by ELISA, using a polyclonal antibody
♦Corresponding author. E-mail: Dario.MoragaCsHiniv-brest.fr
for a recombinant HscV2 of C. gigas developed in the laboratory
(Boutet et al. 2003).
MATERIALS AND METHODS
Oyster Collection and Maintenance
Adult European tlat oysters. O. edulis (3 years old; 7-8 cm),
were purchased from an oyster farm of Mont Saint-Michel Bay
(France) and maintained for one week in aerated 0.22-|j.m filtered
seawater before experimentation. All the experiments were con-
ducted in a temperature-controlled rooin (15°C) at a salinity of
349fc. Groups of 25 oysters were exposed to two metals, one es-
sential (Cu-*) and the other toxic (Cd"""). Each metal was applied
from a stock solution ( 100 mM) at each of two final concentrations
(0.4 \xM and 4 p.M) and also in a mixture (0.2 (jiM each) for 15
days. The metal doses were chosen according to those found in
most contaminated French estuaries. A group of 25 oysters was
maintained in seawater. without metals, as a control. Seawater was
renewed every day and oysters were fed with microalgae (Isoch-
n-sis galbaiui) every two days. The metals were reapplied to the
appropriate concentrations after every water change.
Protein Extraction from Oyster Tissues
On days 0. 1.2. 3, 5, V, and 15 of the experiment, gills and
digestive gland from exposed and control oysters (/! = 3 for all
samples) was harvested after oyster killing and homogenized in
protein extraction buffer (150 mM NaCl, 10 mM NaH^POj, I mM
phenylmethanesulfonyl fluoride, pH V.2) according to the protocol
described by Tedengren et al. (1999). Samples were then centri-
fuged at 12.000 g for 10 min at 4''C and supernatant fractions
containing soluble proteins were collected in fresh tubes. Total
soluble proteins were quantified using the D^ Protein Assay kit
(Bio-Rad) with dilutions of Bovine Serum Albumin (Sigma) as the
standai'd. Optical density was measured at 620 nm using a micro-
plate reader.
Metal .Analysis
Pools of soft body excised from three oysters per sample day
were mineralized with suprapure nitric acid. Concentrations of
763
764
BOUTET ET AL.
cadmium and copper were measured in each tissue sample using
tlie potentiometric stripping metliod (Riso et al. 1997. Boutet et al.
2002).
66.2 kDa
Weslern Biol Analysis
The cross-reactivity of the anti-CgHsc72 IgG antibody de\el-
oped in our laboratory (Boutet et al. 2003) was tested by Western
blot as follows. Samples of O. edidis (control and cadmium-
exposed) proteins were electrophoresed on 12% SDS-
polyacrylamide gel and electrotransferred to PVDF-membrane
(Bio-Rad). The membrane was blocked for Ih with blocking buffer
(0.1 M Tris. 5% nonfat dry milk) and then incubated with Tris
buffer containing anti-CgHsc72 antibody (1/125 diluted) for 1 h
with gentle agitation at room temperature. The membrane was
washed twice for 10 min with washing buffer (0.1 M Tris. 0.02%
Tween 20) and incubated with Tris buffer containing 1/1.000 di-
luted polyclonal anti-rabbit IgG horseradish peroxidase-
conjugated (Sigma) for 1 h with gentle agitation at room tempera-
ture. Again the membrane was washed twice with washing buffer,
the reactive band was visualized by staining with 2.4 mM ot
3-amino-9-ethyl-carbazole (Sigma) dissolved in 50 mM acetate
buffer (0.2 M acetic acid. 0.2 M sodium acetate, pH 5) containing
5% of MW-dimethyl Formamide (Sigma) and 12%f of H,Oo.
ELISA
Microtiter plates were coated with 20 |xg per well of total
proteins extracted from the digestive gland and gills of control and
experimentally exposed oysters. HSP70 concentrations were quan-
tified by ELISA developed in C. gigas using rabbit anti-CgHsc72
IgG and recombinant CgHsc72 as a standard (Boutet et al. 200.^).
Statistical Analysis
The variations in metal and Hsp le\el during the experiment
were analyzed by analysis of covariance (a = 0.05) using CSS
Statistica (Statsoft).
RESULTS
Metal Quantification in Oyster Tissues
Copper and cadmium concentrations in tissues of oysters ex-
perimentally exposed to Cu"* and Cd"* showed a significant time-
dependent increase (compared with controls) during the 15 days of
the experiment. Copper concentrations in the tissues of oysters
exposed to 4 \iM or 0.4 |xM of Cu"* increased from 0.17 to
0.73.10"^ M/g wet weight tissue (M/gwwt) and 0.17 to 0.37. 10^^"
M/gwwt. respectively. Dosing with 4 (jlM or 0.4 (xM of Cd"*
resulted in an increase of Cd concentration in the gills from less
than 0.01 to 0.31. lO"*" M/gwwt and 0.01 to 0.075.10"" M/gwwt.
The concentration of metals in tissues of oy.sters exposed to a
mixture of the two metals increased from O.OI to 0.025.10 '
M/gwwt for Cd. while copper concentration did not vary.
Cross-Reactivity of Anti-CgHsc72 Antibody With O. eduih Proteins
The Western blot revealed a high cross-reactivity of our anti-
CgHsc72 antibody with O. cdidis HSP70 (Fig. 1). Two bands
appeared on the membrane at a molecular weight of 68 and 70
kDa, confirming the specificity of the antibody with Heat Shock
Protein 70 of this oyster species.
M 1 2
Figure 1. Western Ulot (((digestive gland protein sample from control
(lane 1) and cadmium-exposed oyster (lane 2) electrophoresed and
probed with anti-Cghsc72 antibody. Marker (M) is SDS-PAGE Stan-
dard broad range (Bio-Rad Laboratories, Hercules, CA),
Quantification of Heat Shock Proteins 70 by ELISA
Application of the ELISA to protein samples extracted from
gill and digestive glands of control oysters showed significant
differences between these tissues in basal level of Hsp70. Quan-
tities of 46.5 ± 2.6 and 59.3 ± 3.4 mg Hsp/g"' protein, correspond-
ing to approximately 4.7 and 5.9% of total proteins, were measured
respectively in the gills and the digestive gland of control oysters.
Hsp levels decreased significantly (compared with the control,
a = 0.05) in the gill of oysters exposed to a mixture of the two
metal and ^^^x.M of Cd (Fig. 2. A and B). A decrease (not signifi-
cant) of Hsp70 levels in the gill of oysters exposed to copper was
also observed. In contrast, a significant increase of Hsp concen-
tration occurred in the digestive gland of animals exposed to 0.4
|jiM of Cd (Fig. 2E). No differences were observed in gills of
individuals exposed to 0.4 p.M of Cd (Fig. 2B) or to Cu (Fig. 2C)
and in digestive gland of oysters exposed to a mixture of metals
(Fig. 2D), to 4|j.M Cd (Fig. 2E) or to Cu (Fig. 2F). A stronger
dosage-effect of cadmium was observed as either a decrease or
increase of Hsp levels in the two organs. No dosage-effect of
copper could be demonstrated in either organ.
DISCUSSION
In this study, we quantified soluble HSP70 by ELISA in ex-
perimentally metal-exposed O. edulis. The cross-reactivity of
the purified rabbit anti-CgHsc72 IgG demonstrated here with
OeHspJO supported the suitability of using these reagents to quan-
tify HSP70. An increase in intensity of the 70 kDa bands was also
observed in digestive gland of a cadmium-exposed oyster, in
agreement with measurement of Hsp70 by ELISA. Now. our re-
sults showed that HSP70 level is different in gill and digestive
gland (4.7 vs. 5.9% of total protein). We previously reported a
concentration of about 6% in the oyster C. giga.s (Boutet et al.
2003). and Feige and Polla (1994) observed a general HSP level of
about 5% under normal conditions (without stress) in other organ-
isms. In comparison to these basal levels, the quantification of
soluble HSP70 in experimentally exposed O. edulis showed a
metal-dosage response. A decrease of soluble HSP70 was ob-
served in gills of oysters exposed to the highest concentration of
cadmium or to a mixture of the two metals, in spite of a significant
increase of metal concentration in the tissues. In contrast, an ex-
posure to the lowest cadmium concentration induced an increase of
HSP7() in digestive gland. Furthermore copper did not modify
HSP70 levels in oyster tissues. We previously showed that metal
exposure induced a significant decrease of HSP70 in tissues of C.
gigas with the same treatments (Boutet et al. 2(X)3). Veldhuizen-
Tsoerkan et al. (1991) found no variation in HSP70 in M. edulis
caged in seawater with various concentrations of cadmium, like the
response in copper-exposed O. edulis. In contrast. Lewis et al.
(2001 ) showed an inhibitory effect of metals, particularly copper.
Hsp70 Expression in Metal-Exposed Ostrea edulis
765
a.
ai
a.
V)
I
5 10
exposure duration (days)
D 100
- - -a ■ - control
— «^ 0.2mMCu
+0.2 \iM Cd
5 10
exposure duration (days)
B
5 10
exposure duration (days)
control
4pMCd
0.4 pM Cd
5 10
exposure duration (days)
S
Q.
a.
M
X
5 10
exposure duration (days)
-^■- control
— • — 4 pM Cu
-a-- 0 4pMCu
S 10
exposure duration (days)
Figure 2. QiiantifKation of HSP70 (mean ± SKl by KLIS,\ in the nills l.\. B, and t) and in the digestive gland (D. E, and F) of O. edulis exposed
to copper and cadmium l.\ and Dl, cadmium (B and K), and copper (C and F).
in the seaweed Eiiternmorplici liilestiiuilis. These aulhors observed
that high levels of copper appeared to damage protein synthesis.
therefore impairing the HSP70 response. In our experiment, a de-
cline of HSP70 was observed in cadmium- and a cadmium-copper
mixture exposed oysters. A similar HSP70 synthesis inhibition was
observed in earthworms. Liimhriats terrcstris. exposed to a variety
of metals (lead, cadmium, and copper; Nadeau et al. 2001 ). When
exposure approaches lethal levels, such as 4 p,M in our experi-
ments, the average degradation rate of HSP70 will exceed its syn-
thesis rate because of cytopathologic damage, such as ruptured
766
BOUTET ET AL.
membranes, in many cells (Triebskorn & Kohler 1996. Quig
199S). The fact that the gills are the first barrier to metals could
explain why this organ was more affected by the toxic effect of
metals and showed a higher decrease in HSP70 concentration. At
sub-lethal levels, our work showed an increase of HSP70 in re-
sponse to exposure, in agreement with results described by Snyder
et al. (2001). These authors showed a significant increase of
HSP70 in cadmium-contaminated mussels. M. editlis. and limpets.
Collisella peltci. and in heat-shocked and oil-exposed mussel. M.
galloprovincialis. and abalone. Hidiotis riifescens.
The ELISA developed in a previous study in C. gigas allowed
us to specifically and rapidly quantify HSP70 proteins in tissues
from marine mollusks. This immunologic method has the advan-
tage of quantifying the protein of interest, unlike the commonly
used Western blot analysis (Clegg et al. 1998. Nadeau et al. 2001 ),
which gives only a semi-quantitative estimation of HSP70
amounts. Furthermore, this study showed that O. edulis displayed
a differential response to the level of metal contamination. Ac-
cording to the present study and a previous work on metallothio-
nein in this species (Tanguy et al. 2003), the oyster O. edulis do not
seem to be an appropriate indicator for studying environmental
contamination.
ACKNOWLEDGMENTS
This research program was supported by the Region Bretagne
and the CE program FAIR DISENV CT98-4129: "Environmental
factors and shellfish diseases." We are grateful to Jean-Michel
Escoubas who purchased the Crassostrea gigas hsc72 cDNA
clone. Thanks are also due to Dr. Ricardo Riso for metal analysis
in oyster tissues, to Brenda J. Landau for useful English correc-
tions, to Dr. Louis Quiniou for his help with use of the CSS
Statistica. and to Monique Briand for editing the figures.
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Jonnuil ,ij Shellfish Research. VoL 22. No. 3. Ibl-llX. 2003.
IS BEAUTY IN THE EYE OF THE BEHOLDER? DEVELOPMENT OE A SIMPLE METHOD TO
DESCRIBE DESIRABLE SHELL SHAPE FOR THE PACIFIC OYSTER INDUSTRY
JOHN BRAKE. FORD EVANS, AND CHRIS LANGDON*
Coastal Oregon Marine Experiment Station and Department of Fislieries and Wildlife, Hatfield Marine
Scienee Center. Oregon State University. Newport. Oregon 97365
ABSTRACT Shell samples of Pacific oysters (Crfi.v.vo.vfcco ,t;',i;'") were evaluated from three different U.S. West coast farms. Industry
experts described each shell sampled as being either desirable (good) or undesirable (bad). There were slight differences in the
categorization of good and bad oysters among farms, but common trends were evident. The ratio of greatest shell depth to greatest shell
length (D/L) was found to be more effective in categorizing good and bad shell shapes compared with other descriptors. Good oysters
had a mean D/L of 0.316. whereas the bad oysters had a significantly lower mean of 0.219 iP < 0.001). Using the threshold value of
D/L > 0.25 for good oysters. 85.6% of all sampled oysters were correctly assigned to good and bad categories, as defined by industry
participants. The use of D/L and greatest shell width to greatest shell length (W/L) may be beneficial in distinguishing shell .shape
quality and allow for the rapid assessment of many sampled oysters. These findings have implications in the development of industry
standards for shell shape; furthermore, such standards would be useful in designing oyster breeding programs to improve shell shape.
KHY WORDS: shell, shape, oyster. Crassosirea gigas. standards, industry
INTRODUCTION
Product quality is becoming more important as production of
Pacific oysters (Crassostrea gigas) increases and competition for
lucrative markets rise. Shell morphology often provides consumers
w ith their first impression of product quality. Many shellfish in-
dustries recognize shape as a valuable marketing tool. For the
Atlantic Canadian oyster industry. Section 65 of the Canadian
Food Inspection Agency's Fish Inspection Regulations outlines
four different shape classes (differentiated by length to width ra-
tios) by which Eastern oysters. Crassostrea virginica. are to be
sold. In certain regions of France, growers have to sign a contrac-
tual agreement with the Shellfish Professional Organization in
which they agree to not sell oysters (Crassosirea gigas) of a cer-
tain shape (determined by a previously reported forinula; Galtsoff
1964). In exchange, these growers are able to market oysters using
that region's trademark (Goulletquer, personal communication).
Such industry quality control has provided successful and favor-
able product label identification within the inarketplace. In con-
trast, classification of desirable and undesirable shell shapes has
not been objectively defined by the U.S. West coast oyster indus-
try.
The development of industry standards by which shape can be
objectively defined would be of use to the West coast oyster in-
dustry in assessing the effects of different culture practices and
genetic stocks on shell shape. In addition, shell shape may become
increasingly important in product label identification and industry
quality assurance. The objective of this research was to use simple
linear measurements to characterize the shape of Pacific oyster
[Crassosirea gi.qas) shells, classified as being either desirable
(good) or undesirable (bad) in appearance by industry experts.
MATERIALS AND METHODS
Experimental oysters (commercially farmed oysters) were
sampled from three (A. B. and C) commercial oyster farms and
divided into two groups (approximately 50 per group) of either
good or bad shell shape by each farmer. Oysters provided by farms
A and C were grown intertidally in mesh oyster bags, while
*Corresponding author. E-mail: chris.langdon@oregonstate.edu
samples provided by farm B were grown subtidally in lantern nets.
All shell measurements were performed at the Hatfield Marine
Science Center in Newport. OR. Sample oysters were shucked to
obtain the left valve, or "halfshell." Greatest shell length, greatest
shell width, and greatest shell depth were measured for all oysters.
All size measurements were made using Vernier calipers to the
nearest 0.1 mm.
Analysis of variance was used to determine whether good- and
bad-shaped oysters differed for any of the three linear measure-
ments (greatest shell length, width, and depth). Analyses were
performed using data from within each farm site. Normality was
tested using the Kolmogorov-Smirnov method (SPSS Inc.. 2000.
Chicago. IL). These analyses were repeated for data pooled across
all farms.
Absolute measures, such as greatest shell length, width, or
depth, are less useful in categorizing oysters by shape because of
variation in oyster size at harvest. To eliminate the confounding
effects of size on shape it is usual to apply proportional measures
(Reist 1985). As a result, two descriptors were generated to char-
acterize shell shape: ratios of depth to length (D/L) and width to
length (W/L). Normality of D/L and W/L values were determined
using the Kolmogorov-Smirnov method (SPSS Inc.. 2000, Chi-
cago, IL). The ability of each variable to discriininate good from
bad oysters was determined using data collected froin each farm
site separately and with data pooled across all farm sites. A com-
parison of the total percentage of correct assignments of sampled
oysters (into good or bad groups) was then used to determine
which of the descriptors (D/L, W/L, and three previously described
formulae: Wada (1986); Galtsoff (1964): Imai and Sakai (1961)
resulted in the most accurate discrimination between good and bad
shaped oysters.
RESULTS
All variables were normally distributed after log transformation
(Kolmogorov-Smirnov. P > 0.05), except for shell depth measured
in the sample from farm B (P = 0.014). However, due to robust-
ness of ANOVA and the large sample size (ii = 99), this departure
from normality may be ignored for the purpose of analysis (Ram-
sey & Schaffer 2002). Good and bad shaped oysters differed in
length, width, and depth at all farm sites (P < 0.05) except at farm
767
768
Brake et al.
C. where the two groups of oysters only differed in length (Table
1 ). When significant differences occurred, good oysters tended to
be both deeper and wider than bad oysters. Good oysters were
significantly shorter in length (P < 0.051 than bad oysters at farms
B and C: however, good oysters were significantly longer than bad
oysters at farm A. Table 2 lists the means and standard deviations
of D/L and W/L ratios for each individual farm, and for the pooled
good and bad oyster samples. Based on the ratio D/L. good oysters
were significantly deeper than bad oysters at all farms {P < 0.05;
Table 2). Good oysters were 1 .35, 1 .88, and 1 .36 times as deep, per
unit length, as bad oysters at farms A. B, and C. respectively. Good
oysters were 1.06, 1.10, and 1.22 times as wide, per unit length, as
bad oysters at farms A, B, and C, respectively (P < 0.05). Although
there were differences in mean D/L and W/L ratios among farms,
the trends (larger values for good oysters than bad oysters) within
sites were similar. To obtain a robust sample that would best
represent different farms across industry, the data from all farms
were pooled to evaluate the average differences between good and
bad shell groups across all sites. Good oysters had a mean W/L of
0.689 and D/L of 0.316, whereas bad oysters had significantly
lower means of 0.597 and 0.219 respectively (P < 0.001 ).
Table 3 lists reported shape descriptors for assignment of oys-
ters into good and bad groups. Previously used thresholds for
separating good and bad shapes, as well as the values maximizing
the percent coixect assignment in the current study are given. Cor-
rect assignment was maximized for D/L at 0.25, with 92.6% of all
good oysters falling above the value of 0.25 (depth was at least
0.25 of shell length), while 78.8% of all bad oysters fell below this
value. Using the Atlantic Canadian shell shape guidelines, shells
with a value of length / width <1.75 would be termed as either
■■fancy" or ""choice", the top two of four possible categories ot
oyster shells. Table 3 shows the percentage of oysters correctly
assigned to their proper good or bad groups by using D/L > 0.25,
W/L, and the Atlantic Canadian threshold of good >1.75 (as well
as the value for which percent correct assignment was maximized,
1 . 1 2). In addition, a formula (Galtsoff 1964) used as a standard for
the Irish and French industry (using a threshold value for good
shell shape of >3, and the value of maximum correct assignment of
3.5) was compared along with a previously described formula for
shell convexity (Wada 1986). Percent correct assignment for con-
vexity was maximized at a value of 0.3 1 5 when this descriptor was
applied to the current data. The index of shell depth described by
Imai and Sakai (1961) maximized the percentage of oysters cor-
rectly assigned to both good and bad groups at a value of 3 1 .6.
TABLE 1.
Shell length, width, and depth measurements of good- and bad
shell-shaped oysters from three commercial U.S. West coast
oyster farms.
TABLE 2.
Observed ratios of depth/length (D/L) and width/length (W/L) of
good- and bad shaped-oyster shells from three commercial U.S.
West coast oyster farms.
Length (
Good
mm)
Bad
Depth (
Good
mm)
Bad
Width (mm)
Farm
Good
Bad
A
Mean
101. LS*
95.99
30.41*
21.64
71.50*
61.56
SD
7.10
8.35
3.39
3.89
5.78
7.09
B
Mean
77.62*
90.86
27.15*
17.29
52.82*
50.61
SD
5.00
13.32
3.96
3.41
4.71
6.49
C
Mean
90.66*
117.12
27.07
26.57
60.37
61.33
SD
7.L^4
23.58
4.33
6.52
8.58
6.35
Sample
D/L
W/I.
Standard
Standard
Farm
Type
Mean
Deyiation
Mean
Deviation
A
Good
0..W2
0.037
0.711
0.084
Bad
0.226
0.040
0.646
0.092
B
Good
0.351
0.052
0.683
0.072
Bad
0.194
0.046
0.571
0.125
C
Good
(1..^0I
0.055
0.669
0.097
Bad
0.235
0.079
0.547
0.136
Pooled
Good
0.316
0.053
0.689
0.087
Bad
0.219
0.057
0.597
0.123
* Means were significandy different (P < 0.05: ANOVAi.
All mean good-shaped samples within a site were statistically larger than
bad-shaped samples (P < 0.05, ANOVA).
A threshold value of D/L > 0.25 (for good oysters) was the
most effective at correctly assigning oysters to their respective
categories, with 85.6% of all oysters being correctly assigned
(Table 4). This threshold was more effective at correctly assigning
good oysters to good groups (90.9% correct) than bad oysters to
bad groups (77.8% correct). The index of shell depth (Imai &
Sakai 1961) was also effective at correctly assigning good oysters
(84.2%) and bad oysters (82.0%). correctly assigning 84.4% of the
total oysters sampled. The ratio of W/L, using the threshold value
of >0.63 (for good oysters) correctly assigned 70.6% of all oysters.
When the ratios of D/L and W/L were applied simultaneously,
only 30.0% of all oysters were correctly assigned by both ratios
(Table 4, Fig. I ). The Atlantic Canadian guideline was less effec-
tive in discriminating good from bad oysters using the threshold
value for good oysters of >1.75 (65.7%), resulting in an overall
maximum correct assignment of 70.6%. The measure of convexity
(Wada. 1986) was effective at correctly assigning good oysters
(86.1%). but not bad oysters (35.9%). correctly assigning 61.8% of
the total oysters sampled. Using the previously applied threshold
and the value of percent maximum assignment, the Galtsott for-
mula was effective at correctly assigning bad oysters (96.4% and
98.8% respectively), but ineffective at assigning good oysters (0%
and 0%). and only assigned 49.2% and 50.4% of all oysters cor-
rectly.
DISCUSSION
Members of the U.S. West coast oyster industry have subjec-
tively identified shell depth and width, relative to length, as the
two most important factors in determining the quality ot an oyster
halfshell. Most oyster growers identified a long and skinny shape
(typically called "'rabbit ears") as being undesirable, with a deep
and wide halfshell being more desirable. These distinctions, when
relative shell size is considered, are described by the ratios of D/L
and W/L.
Previous work has shown the abUity to categorize bivalve
shape regardless of absolute size. Day et al. (2000) used stepwise
discriminant analysis and principal component analysis to show
that relative size of the umbo cavity was the most useful character
for identification of sympatric Saccostrea species. The authors
reported success in identifying species using the non-lethal mea-
Method to Describe Desirable Shell Shape
769
TABLE 3.
Descriptors used in the current study to compare percent correct assignment of oysters into farm-specified good and bad shell-shape groups.
Previously
\ alue Maximizing
Employed
Percent Correct
Descriptor
Expression
Species
Threshold
Assignment
Reference
D/L
Depth/length
Crassoslrea
gigus
na
0.25
Current study
W/L
Width/length
Crassoslrea
gigas
NA
0.63
Current study
Atlantic
Leneth/width
Crassoslrea
1.75'
1.58
Section 65 of CHA's
Canadian
viri^inica
Fish Inspection
Regulations
Galtsoff
(Length depth i/width
Crassoslrea
Crassoslrea
virginica
3
3.5
Galtsoff 1 964.
Heath & Wilson 1999
Convexity
Width/( length width
depth)
Pinclada
fucala
marlensii
NA
0.315
Wada 1986
Index of
(Depth/mean of width and
Crassoslrea
NA
.M,6
Iniai & Sakai 1961
Shell Depth
length) X 100
gigas
■■ Separates top two of four possible shape categories; details in results section.
sures of total oyster depth, right valve length, and right valve
width. In addition. Wilding et al. (1998) reported that hinge length
was the only one of four investigated shell measures that provided
a clear distinction among groups of the scallop, Pecten maximus.
The various formulae (Tables 3 and 4) used to separate good
froin bad oysters confirm the importance of depth in determining
optimal oyster shape. The two relationships that were inost effec-
tive at correctly assigning good and bad oysters (D/L and an index
of shell depth; Imai & Sakai 1961 ) had only depth in the numera-
tor. These descriptors were more effective than the ratio of W/L. a
previously described shell convexity assessment method (Wada
1986). the formula used as a guideline in the Irish and French
industry (Galtsoff 1964). and the Atlantic Canadian guideline
(Table 3).
Culture environment has an important effect on shell shape
(Carriker 1996, Boulding & Hay 1993, Seed 1968). It is commonly
believed by growers that oysters subjected to movement by fre-
quent disturbance tend to grow deeper halfshells. The outer edge of
the shell is repeatedly broken off and subsequently grown back
with the net result being that the oyster grows more quickly in
terms of depth than length. This process is commonly referred to
as "pruning."
Few attempts have been made to investigate genetic effects on
shell shape or to improve bivalve shell shape using selective breed-
ing in aquaculture. Wada ( 1986) reported on first, second and third
generation responses to selection for shell width and shell convex-
ity for the Japanese pearl oyster, Pictada fucata martensii. Selec-
tion for shell convexity and shell width was effective and Wada
obtained a realized heritability of 0.467 for shell convexity after
two generations of selection, pro\ iding evidence that shell shape in
the Japanese pearl oyster may be improved by selective breeding.
The current study suggests that width may not be as important
as depth in determining quality. It is conceivable, however, that if
a breeding program were to select oysters without consideration of
width, the result could be a less desirable deep and narrow oyster.
As both depth and width have been anecdotally described as being
important (by industry) to the quality of a halfshell, it may be
prudent to consider both relationships to determine the true quality
TABLE 4.
Percent correct assignment of good and bad oysters based on D/L, W/L, and Atlantic Canadian Measure, the Galtsoff measure, an index of
shell depth, and a measure of shell convexity using values for maximum percent correct assignment, unless stated otherwise.
Percent Correct .Assignment
Sample
Type
Depth/I.ength
(D/L)
Index of
Shell
Depth
Width/I.ength
(W/I.)
D/L
+
W/L°
.Atlantic
Canadian''
Atlantic
Canadian
Convexity
Galtsoff*
Galtsoff
Good oysters
Bad oysters
Good -f bad oysters
90.9
77.8
85.6
84.2
84.5
84.4
74.5
64.7
70.6
56.4
3.1
30.0
89.1
42.0
65.7
74.5
64.7
70.6
86.1
35.9
61.8
0
96.4
49.2
0
98.8
50.4
A description of the measures and the maximum percent correct assignment values for each are given in Table 3.
' Maximum percent correctly assigned by both D/L and W/L.
''Threshold value described in the literature; not the value maximizing percent correct assignment given in Table 3,
770
Brake et al.
0.6
0.5
0.4
0.3
0.2
0.1
0
■ Good
° Bad
■
mg
■■*- o I* "■a* ^ ' ■ ■
D W OP D D D C*, g'
i'
_ a 4 o
D D
D O
0.2
0.4
0.6
0.8
1.2
L/W
Figure 1. Ratios of depth/length (D/L) and width/length (\V/L) of good- and bad-shaped oyster shells from three commercial West coast U.S.
oyster farms. Lines represent threshold values of VV/L > 0.63 and D/I. > 0.25 maximizing percent correct assignment.
of an oyster halfshell. The use of the D/L and W/L thresholds simul-
taneously was ineffective in correctly assigning oysters to good or bad
groups. The index of shell depth (lamai & Sakai 1961) might be
useful in this regard as it incorporates shell width, and should there-
fore exclude any abnormally deep and narrow oyster shells.
A method to evaluate shell shape quality that is both simple and
reliable could be of great value to the U.S. West coast oyster
industry. Growers could objectively compare practices to find
which culture methods tend to influence shell shape in a positive
way. Oysters grown in different areas commonly have different
shape characteristics. Using an objective method, site differences
could also be as.sessed, with a grower being able to determine
whether particular sites produce oysters with a better shape. An-
other possible long-term benefit of having an objective comparison
could be the establishment of industry shape standards. This would
allow producers and consumers to use a common scale of shell
shape measurement. The Atlantic Canadian oyster industry has
realized the benefits of such a set of standards. For example.
"fancy" oysters are defined in section 65 of the Canadian Food
Inspection Agency's Fish Inspection Regulations as having a
length not exceeding one and one-half times its greatest width, and
as not being abnormally flat, thin-lipped, or malformed. Consum-
ers can therefore go to several different famis or retailers and pur-
chase "fancy" oysters, knowing that they share a common shape.
An important consideration in a method to characterize shell
shape is the practicality of the methodology. Heath and Wilson
(1999) used computer assisted image analysis to assess shell shape
and size in Crassostrea gigas. Although they demonstrated that
this method could be used to separate oysters into categories ac-
cording to general shape specifications, the required equipment
might be cost prohibitive and this method does not allow for as-
sessment of oysters in the field. The ratio of D/L and the index of
shell depth used in the current study would, therefore, be more
practical to separate good from bad shells compared with using
image analysis.
In summary, industrial-scale assessment and selective breeding
programs both require methods to efficiently determine the value
of an oyster in terms of shell shape. The D/L ratio and the index
of shell depth show promise in this regard. The D/L threshold of
<0.25 separates most of the good and bad oyster halfshells, while
requiring only two simple linear measurements. The index ot shell
depth (lamai & Sakai 1961) was nearly as effective and has the
advantage of incorporating width, which might eliminate any ab-
normally deep and narrow oysters. The current data suggests that
depth is likely the most important measure to evaluate oyster shell
shape quality. Consideration of W/L might also be prudent in shell
shape assessments to avoid narrow deep oysters. The current study
only investigated differences between good and bad shell samples
from three farms; therefore, future work should include samples
from more industry participants.
ACKNOWLEDGMENTS
The authors thank Ebru Onal. David Stick. Drew Mosher, Sean
Matson, Salina Gaskill, and Dave Jacobson, for technical assis-
Method to Describe Desirable Shell Shape
771
tance during the project. Thanks also to Olivier Drean for help in
measuring many sample shells. Special thanks are given to the
generous support afforded by the donor farms. Taylor Shellfish
Farms Inc., Westcott Bay Sea Farms Inc., and Oregon Oyster Farm
Inc. This project was funded by a special L'SDA-CSREES grant to
the Molluscan Broodstock Program, Oregon State University.
LITERATURE CITED
Boulding, E. G. & T. K. Hay. 199.^ Quanlitalive genetics of shell form of
an intertidal snail: constraints on short-term response to selection. £10-
Uuum 47:576-592.
Carriker. M. R. 1996. The shell and ligament. In: V. S. Kennedy. R. E. I.
Newell, and A. F. Eble, editors. The eastern oyster, Crassostrca vii-
ginica. pp. 75-168. College Park. MD: Maryland Sea Grant College
Publication, pp. 75-168.
Day, A. J.. A. J. S. Hawkins & P. Visootiviseth. 2000. The use of al-
lozymes and shell moqihology to distinguish among sympatric species
of the rock oyster Saccostrea in Thailand. Aquaculmre 187:51-72.
Galtsoff. P. S. 1964. The American oyster, Crassoslrea virginica (Gmelinl.
U.S. Fish. Wildl. Sen: Fish. Bull. 64:1^80.
Heath, P. L. & J. H. Wilson. 1999. Assessment of Pacific oyster. Cms-
sotrea gigas (Thunberg), size and quality using a computer-based shape
analysis technique. Aquae. Res. 30:299-303.
Imai. T. & S. Sakai. 1961. Study of Japanese oyster, Crassoslrea gigas.
TohokuJ. Agric. Res. 12:W1-\12.
Ramsey, F. L. & D. W. Schaffer. 2002. The statistical sleuth: A course in
methods of data analysis. Pacific Grove. CA: Duxbury, 742 pp.
Reist. J. D. 1985. An empirical evaluation of several univariate methods
that adjust for size variation in morphometric data. Can. J. Zool. 63:
1429-1439.
Seed, R. 1968. Factors influencing shell shape in the mussel Mxtihis eilulis.
J. Mar. Biol. Ass. U. K. 48:561-584.
Wada, K. T 1986. Genetic selection for shell traits in the Japanese pearl
oyster, Pinctada fucada martensii. Aquaculture 57:171-176.
Wilding, C. S., J. W. Latchford & A. R. Beaumont. 1998, An investigation
of possible stock structure in Pecten maximus (L.) using multivariate
morphometries, allozyme electrophoresis and mitochondrial DNA
polymerase chain reaction-restriction fragment length poymorphism.
J. Slwllfish Res. 17:131-139.
JoKinal oj Shellfish Research, Vol. 22, No. 3, 773-77.'i. 2003.
SHOULD SLOW GROWING PEARL OYSTER {PINCTADA MARGARITIFERA) SPAT ("RUNTS")
BE DISCARDED?
JOSIAH H. PIT* AND PAUL C. SOUTHGATE
Pearl Oyster Researeh Group School of Murine Biology and Aquacultiire. James Cook University.
Townsville. Qiieenshiud 4HI I . Australia
ABSTRACT In this laboratory, hatchery-produced Piiictiula luariinntiferd ju\endes are routinely graded at 3.5 mo of age. when .spat
of <5 mm ("runts") are generally discarded. This anicle reports on an experiment to assess the relative growth rates of three size classes
(<5, 5-10, and >10 mm) of hatchery-produced blacklip pearl oyster (P. iimrftaritifeni) spat from the same cohort. The three size classes
were classified as runts, normal growers, and fast growers, and had mean (±SE; n = 30) dorso-venlral shell heights (DVHs) of 4.5
± 0. 1 . 8.6 ± 0.3, and 1 2.8 ± 0.2 mm, respectively, at the start of the 4-mo experiment. The mean DVH at completion of the study for
each initial size cla.ss (<5, 5-10, and >10 mm) was 24,6 ± 0.4, 32.3 ± 0.4, and 35.6 ± 0.4 mm, respectively. All differed significantly
from each other {P < 0.001 ). The mean incremental increases in DVH for each size class (<5. 5-10, and >I0 mm) over the 4-mo period
was greatest in oysters from the 5-10-mm size cla.ss (mean DVH 23.3 ± 0.4 mm) and lowest in oysters from the <5-mm size class (mean
DVH 20.0 ± 0.5 mm). Incremental increases in DVH were significantly different between oysters from the <5-iTim size class and those
from the larger size classes. The mean (±SE) percentage increase in DVH was greatest in oysters from the <5-mm size class (448 ±
17%) and lowest in oysters from the >10-mm size class (178 ± 7%). A number of oysters in the <5-nim size class grew very rapidly
during the experiment and reached the same DVH as oysters in the larger size classes. This study shows that, given appropriate
conditions, runts are capable of similar growth rates as larger spat. It may therefore be inappropriate to discard pearl oysters, which
are classed as runts (<5 mm) at grading (3.5 mo). Furthermore, it is suggested that grading be delayed until 5 to 6 mo when a greater
proportion of oysters are likely to be in the larger size classes.
A'£>' WORDS: pearl oyster. Pimlada inuri;aritifcra. spat, runts, growth
INTRODUCTION
The growth of cultured bivalve molluscs is highly variable
during hatchery and nursery culture, and variation in growth can
occur among individuals of the same age reared under identical
conditions (Newkirk 1981), Small differences in the size of spat
can become large differences in juvenile size (Mason et al. 1998),
and the greater the time required by slow growers to reach com-
mercial size increases costs and reduces profitability (Askew
1978), Pearl oysters need to reach a minimum shell size before
being used for pearl production. This size is generally reached at
appro.ximately 2 y of age. As such, maximizing growth rate and
minimizing growth variation are important factors in pearl oyster
cultivation,
A large variation in growth rate is evident for pearl oysters
reared under identical conditions. For example, 43-day-old black-
lip pearl oyster (Pinctada margarilifera) spat have been reported
to range in size from 1 to 5 mm in dorso-ventral shell height
(DVH) (Pit & Southgate 2000). and from <2 to 23 mm DVH at 3.5
mo of age (Southgate & Beer 1997), To minitnize the size varia-
tion in pearl oyster spat. Rose (1990) recommended continual
grading to separate fast growers frotn slow growers. Slow-growing
pearl oyster spat are often discarded. In this laboratory, hatchery-
produced P. margarilifera are routinely graded at 3.5 mo of age,
when spat <5 mm ("runts'") are generally discarded. "Runting"
may result from unfavorable culture conditions, and, if this is the
case, runts may be capable of good growth rates if provided with
appropriate culture conditions. Given the high cost of hatchery
production and the high value of pearl oyster spat, it is in the
interest of pearl oyster fanners to maximize the number of spat
from a given cohort that are eventually used for pearl production.
The aim of this study was to determine whether slow-growing P.
margaritifera spat remained as runts or whether they are capable
*Corresponding author. E-mail: Josiah.Pit@jcu.edu.au
of similar growth rates as normal spat when provided with appro-
priate conditions.
MATERIALS AND METHODS
This study was conducted at the Orpheus Island Research Sta-
tion of James Cook University, north Queensland, Australia
( 180°35' 146°29'E), and larvae and spat were cultured according to
the methods described by Southgate and Beer ( 1997) and Pit and
Southgate (2()()()). At 43 days of age, when spat had a mean (±SE)
DVH of 2.8 ± 0. 1 ttim (range 1-5 mm), they were transferred from
the hatchery to the ocean where they were held in suspended mesh
trays at a depth of 6 ni (Southgate & Beer 1997).
Spat were graded at 3.5 mo of age into three different size
classes. <5, 5 to 10, and >I0 mm, which, for the purpose of this
study, were classified as runts, normal growers, and fast growers,
respectively. The mean DVH in = 30) of P. margaritifera in the
<5-. 5-to-lO- and >10-mm size classes were 4.5 ± 0.1. 8.6 ± 0.3.
and 12,8 ± 0.2 mm. respectively, and these differed significantly
from each other (F,;,? = 285.42; P < 0.001). Thirty P. marga-
ritifera spat from each size class were individually fixed to the
bottoms of each of three replicate plastic mesh trays (60 x 35 x 10
cm) using a waterproof cyanoacrylate adhesive (Loctite 454 gel.
Loctite Australia, Caringbah, New South Wales. Australia). This
minimizes oyster aggregation (Friedman 1999. Pit 1998). which
can significantly affect growth (Friedman & Southgate 1999). To
minimi/.e the disturbance to spat and to maximize growth rates,
oysters were not measured during the 4-n)o study; however, trays
were cleaned in situ every month to remove external fouling or-
ganisms (Pit & Southgate in press). Cleaning involved the manual
scrubbing of the outside surfaces of the trays. Trays were not
cleaned internally, but were moved gently up and down in the
water column to remove any silt and mud that had accumulated
inside the trays. Oysters from each tray were measured for DVH at
the end of the study.
Data were analyzed using a one-way analysis of variance to
773
774
Pit and Southgate
determine whether P. margarilifeni from different size classes
differed in size (DVH) at the completion of the study. Assumptions
of homogeneity and normality were met (Zar 1984). The rates of
growth among the three size classes were also assessed using
nonparametric analyses to determine whether differences existed.
Significant differences were identified using the Tukey"s test and
the Dunnett's T.^ for the parametric and nonparametric tests, re-
spectively (Zar I4S4).
RESULTS
On completion of the study, the mean (±SE) DVH for each
initial size classes (<5, 5-10, and >I0 mm) were 24.6 ± 0.4, .^2..^
± 0.4. and 35.6 ± 0.4 mm, respectively. All differed significantly
from each other (F, ^-, = 167.67; P < 0.001 ) (Fig. 1 ). The mean
incremental growth in DVH (n = 30) for each size class «5.
5-10, and >10 mm) over the 4-mo period was greatest in oysters
from the 5-IO-nim size class (23.3 ± 0.4 mm) and was lowest in
oysters from the <5-mm size class (20.0 ± 0.5 mm). Incremental
shell growth was significantly greater in the two larger size classes
(F2 87 = •5-99; P < 0.001) (Fig. 2). Weekly growth rates averaged
1.25, 1.42, and 1.48 mm, respectively, for the <5-. 5-I0-, and
>10-mni size classes. However, the mean percentage increases in
DVH for each size class (<5. 5-10, and >I0 mm) over the 4-mo
period was greatest in oysters from the <5-mm size class (448 ±
17%) and lowest in oysters from the >IO-mm size class (178 ±
7%), while oysters in the 5-IO-mm size class increased by 308 ±
12%. A number of oysters in the <5-mm size class grew very
rapidly and achieved DVH measurements within the ranges of
those shown by oysters in the two larger size classes.
DISCUSSION
P. nuiriiariufera used in this study were hatchery-reared ani-
mals of the same age that were cultured under identical conditions.
However, when oysters were transferred from the hatchery to the
nursery at 6 wk of age, their DVH ranged from 1 to 5 mm (mean
2.8 ± 0.1 mm). It is unclear whether such size variation resulted
from environmental factors, genetic factors, or a combination of
both. Factors that have previously been suggested to cause such
size variation in bivalves include fluctuations in water quality and
food quality (environmental), as well as egg and larval quality
(genetic) (Gallager & Mann 1986, Rose 1990. Mason et al. 1998,
Devakie & Ah 2000, Nicolas & Robert 2001 ).
Size variation was also evident during early nursery culture
prior to grading when oysters ranged in size from 2 to 23 mm.
Again, it is unclear whether size variation at grading reflected a
continuation of llic size variability observed in the hatchery, or
4U -
a
F ^U-
E
" S^'^^ ■ '^c
— 20
^ ^
^"^ , - '
X
, -^
>
^- ^^
a 10 -
0 -
--■•'
-1 ^
<5 mm
5-10 mm
— - — >10 mm
3.5 75
Age (months)
Figure 1. Changes in mean (±SK: n = M)) DVH of P. margarilifera
juveniles In different size classes l<5, 5-10, and >l(l mm) culliired for
4 mo at Orpheus Island. Means with the same superscript are not
significantly different (P > 0.05).
E
24 1
E.
23 -
X
>
22 -
Q
21 -
c
«
20 -
O)
c
19 -
re
r
1 0
0
<5 mm
>10 mm
5-10 mm
Oyster size class
Figure 2. Mean (±SF) change in DVH of P. iiiari;ahlifera juveniles in
different size classes (<5, 5-10, and >1() mm) cultured for 4 months at
Orpheus Island. Means with the same superscript arc not significantly
different [P > 0.05).
whether subsequent environmental factors were also involved. The
negative impacts of poor growing conditions on pearl oyster
growth rates during nursery culture are well documented. For ex-
ample, pearl oysters aggregate to form clumps in culture units
(Southgate & Beer 1997, Friedman & Southgate 1999). This re-
sults in a greater size range of individuals within a cohort and a
higher proportion of smaller oysters when compared with oysters
grown in conditions that prevent clumping (Friedman & Southgate
1999. Southgate & Beer 2000). The smaller oysters in the former
group are thought to be those that are bound into clumps of oysters,
and, as a result, have impaired access to good water flow and food
availability (Friedman & Southgate 1999).
The growth rates of P. niariiaririfeni spat recorded in this study
were clearly influenced by initial size class, suggesting that genetic
factors were more influential on initial spat size than were envi-
ronmental factors. In a similar study with Pacific oysters. Collet et
al. (1999) demonstrated a positive relationship between larval and
postmetumorphic growth, indicating a genetic rather than environ-
mental basis for slower growth in postmetamoi-phic bivalves. In
contrast. Mason et al. (1998) reported that growth variation in
Sydney rock oyster spat was not affected by initial size class and
suggested that initial differences in size resulted from "temporary
environmental stunting" rather than from genetic factors. Similar
findings have been reported for edible oysters (Newkirk 1981,
Newkirk & Haley 1982)
Hatchery production of pearl oysters is expensive, and it is
clearly in the interest of pearl oyster farmers to maximize the
number of spat from a given cohort that can be used for pearl
production. However, the use of smaller spat, which take a longer
time to reach a size suitable for pearl production, becomes an
economic issue. Pearl farmers must consider the benefits of maxi-
mizing the number of usable oysters from a cohort of spat, against
the increased time required for slower growers to reach pearl pro-
duction size. Prior research at the culture site used in this study
reported growth rates for P. maiiiaritifera during nursery culture
ranging from 3.66 mm mo'' (in trays) to 4.86 mm mo"' (in pocket
nets) (Southgate & Beer 2000). Assuming similar subsequent
growth rates for the three size classes of oysters used in this study,
it is possible to estimate the time required for each size class to
reach a pearl production size of 110 mm DVH. On this basis,
oysters in the 5-10- and >10-mm size classes would reach I 10 mm
at 19 to 24 mo and 19 to 23 mo of age, respectively. However,
oysters in the <5-mm size class would require 2 1 to 27 mo to reach
this size (110 mm DVH). The costs involved in culluring oysters
from the smaller size class for this additional time, however, may
outweigh the costs of increasing oyster numbers by additional
Slow Growinc; Pbarl Oyster Spats
775
hatchery production or the purchase ot'juveniles. hi a similar study
with Crassostrea virginica. O'Beirn and Luckenbach (2000) noted
that the use of runts for the oyster industry would be feasible, given
good growing conditions, but that it may not warrant the invest-
ment of extra time and resources.
When provided with good growing conditions, oysters in the
<5-mm size class grew at a significantly slower rate than those in
larger size classes. Nevertheless, certain individuals from the <5-
mm size class did attain sizes within the overall si/e ranges of
oysters in the larger size classes. This suggests that some runts may
not always remain runts and indicates that such individuals are
likely to have been affected by environmental stunting. Clearly, at
first grading (3.5 mo of age), it is not possible to identify those P.
margaritifera individuals in the <5-mm size class that are capable
of growth rates allowing them to catch up to larger individuals
within a cohort. Culling runt oysters at this stage would result in
the loss of oysters that could subsequently be used for pearl pro-
duction. A second grading at approximately 5 to 6 mo of age.
however, would allow such individuals to be identified. This
would maximize the number of oysters used for pearl production
from a given cohort of juveniles. A similar outcome might also be
achieved through more appropriate spat collector design. .Spat are
generally transferred from the hatchery to the field on spat collec-
tors and remain on them until grading (.Southgate & Beer 19^7).
Spat collectors that provide more uniform environmental condi-
tions are likely to result in a more tiniform size range of spat at
grading.
Hatchery production of P. imirgaiilijcni in many developing
Pacific nations is often constrained by limited resources (South-
gate & Beer 1997) and cannot be conducted on a routine basis. In
these cases, it is preferable to use as many oysters as possible from
each cohort of hatchery-produced spat. The results of this study
indicate that modifications to the current protocols may allow in-
creases in the number of P. margunlifera from a given cohort that
can be used for pearl production.
ACKNOWLEDGMENTS
This study was conducted as part of project FIS 97.^1. "Pearl
Oyster Resource Development in the Pacific Islands," which was
funded by the Australian Centre for International Agricultural Re-
search. The authors thank the staff at the Orpheus Island Research
Station of James Cook University for technical assistance during
the study.
LITERATURE CITED
Askew. C. G. 1978. A generalised gniwlh and nuirtalily niddcl lor assess-
ing the economics bivalve culture. Aqmuultiirc 14:9 1-104.
Collet. B.. P. Boudry, A. Thebault. S. Heurtebise. B. Morand. & A. Gerard.
1999. Relationship between pre- and post-metamorphic growth in the
Pacific oyster Crasso.slrea ,?/,?a.s (Thunberg). Aciiuiculliirc 17,'i:21.'i-
226.
Devakie. M. N. &. A. B. Ali. 2U00. Salinity-temperature and natnlional
effects on the setting rate of larvae of the tropical oyster, CiiissDstreu
iredalei (Faustino). Acjiiaciihure 184:105-1 14.
Friedman. K. J. 1999. Pearl culture using wild-caught spat of hlacklip
oysters (Piiichula mcirgariiifera) in Solomon Islands. PhD Thesis,
School of Marine Biology & Aquaculture. James Cook University.
Townsville, Australia.
Friedman. K. J. & P. C. Southgate. 1999. Growout of blacklip peari oys-
ters. Piucliuhi ntargarinfera collected as wild spat in the Solomon
Islands. / Shellfish Res. 18:159-167.
Gallager. S. M. & R. Mann. 1986. Growth and survival ot larvae of A/cr-
cenuiia inerceiuiria (L. ) and Crassosrreu viii;iiiiai (Gmelin) relative to
broodstock conditioning and lipid content of eggs. Aiiiiaiiilturc 56:
10.5-121.
Mason, C. J., D. D. Reid, & J. A. Nell. 1998. Growth characlerislics of
Sydney rock oysters, Saccostrea commercialis in relation to si/e and
temperature. J. Exp. Men: Biol. Ecol. 27:155-168.
Newkirk, G. F. 1981. On the unpredictability of bivalve growth rates: is a
slow growing juvenile oyster a runt for life. In: C. Claus. N. De Pauw.
& E. Jaspers, editors. Nursery culture of bivalve molluscs. Proceedings
of the International Workshop on Nursery Culturing of Bivalve Mol-
luscs, February 24-26, Ghent, Belgium, pp. 211-218.
Newkirk, G. F. & L. E. Haley. 1982. Phenotypic analysis of the European
oyster Oslrca echilis L: relationship between larval period and postset-
ting growth rate. J. Exp. Mar. Biol. Ecol. 59:177-184.
Nicolas. L. & R. Robert. 200 1 . The effect of food supply on metamorphosis
and post-larval development in hatchery reared Peclen iiiumiiiiis. .Aqua-
culture 192:347- .159.
O'Beirn. F. X. & M. W. Luckenbach. 2000. A study investigating the
potential of an alternate seed source for Virginia aquaculturists. /
Shellfish Res. 19:653-654.
Pit. J. H. 1998. Factors affecting growth and survival of the blacklip pearl
oyster (Pinctada margaritifera. Linneaus) during early nursery culture.
Honours thesis. School of Marine Biology & Aquaculture. James Cook
Univershy, Townsville, Australia.
Pit. J. H. & P. C. Southgate. 2000. When should pearl oyster, Pincuula
margaritifera (L.). spat be transferred from the hatchery to the ocean?
Aquaculture Res. 3\:71i-nH.
Pit. J. H. & P. C. Southgate. In press. Fouling and predation: how do they
affect growth and survival of the blacklip pearl oyster. Pinctuda mar-
garitifera, during nursery culture? Ac/uaculture Int. 1 1:545-555.
Rose, R. A. 1990. A manual for the artificial propagation of the silverlip or
goldlip pearl oyster, Pinctada maxima, (Jameson) from Western Aus-
tralia. Fisheries Department Western Australian Marine Research
Laboratories. North Beach. Western Australia. 41 pp.
Southgate. P. C. & A. C. Beer. 1997. Hatchery and eariy nursery culture of
the blacklip pearl oyster (Pinctada margaritifera L.). J. Shellfish Res.
16:561-567.
Southgate. P. C. & A. C. Beer. 2000. Growth of blacklip pearl oyster
{Pinctada margaritifera L.) juveniles using different nursery culture
techniques. Aqiuicullure 187:97-104.
Zar. J. H. 1984. Biostatistical analysis. 2nd ed. Upper Saddle River. NJ:
Prentice Hall. 718 pp.
Journal ,<) Shellfish Research. Vol. 22. N(i. 3. 111-11^). 2003.
CORROSION CASTING OF THE DIGESTIVE DIVERTICULA OF THE PEARL OYSTER,
PINCTADA FUCATA MARTENSII (MOLLUSCA: BIVALVIA)
TAKESHI HANDA* AND KEN-ICHI YAMAMOTO
Department of Applied Aqiiabiology. National Fisheries University. 2-7-1 Nagata-honinachi.
Shiinonoseki. )'aiihii;uchi 759-6595. .lapan
ABSTRACT We examined corrosion casting as a means of studying the digestive organ in the pearl oyster Pinctada fiieaui nuinensii
and other molluscs. The cast was made with resin that mixed hardener (Mercox MA) and prepolymerization methyl methacrylate
(MercoxCL-2R). In pearl oysters, the resin was injected through the polyethylene tubing within 3 min, after the animal sufficiently
relaxed in 0.4 niM MgCl, solution. It was left at least I h in the .seawater and hardened. Then, it was treated with 20% NaOH for I
day at room temperature. As a result, it was po.ssible to cast from the mouth to the anus, including the ducts and tubules of the digestive
diverticula. Using the same method, the castings of digestive organ in other molluscs, Scapharca broughtonii. Crassoslrea gigcis.
Meretrix hisoria (Bivalvia), and Haliotis discus (Gastropoda), were completed as well as the pearl oyster.
KEY WORDS: corrosion cast, digestive organ, digestive diverticula, duct, tubule
INTRODUCTION
Molluscs absorb food and nutrients, secrete digestive enzymes,
and store the nutrients in the digestive diverticula that develops at
the circumference of the stomach. The digestive diverticula is
connected with the stomach by ducts (Owen I95fia. 1995b, Pur-
chon 1957. 1958. I960). The structure of the tip of the digestive
diverticula is shown as a terminal vesicle (Owen 1955a, 1995b.
Nakajima 1956). Yonge (1926) demonstrated the structure of the
stomach and the main ducts of the digestive diverticula with a cast
made in gelatine in the Pacific oyster. Cras.wstrfa gigas. but the
secondary ducts and tubules were not cast.
Corrosion casting is well suited to study the three-dimensional
structure of the cardiovascular-respiratory system; however, there
is little information provided on the structure of the branch and
connection in the secondary ducts and tubules with casting. Infor-
mation on the structure of digestive diverticula will be useful for
the research of taxonomy and the function of digestive diverticula.
This study examined corrosion casting as a means of charac-
terizing the whole digestive organ, especially digestive diverticula,
with prepolymerization methacrylate in some molluscs with spe-
cial emphasis on the pearl oyster, Pinctada fucaia martensii.
MATERIALS AND METHODS
Pearl oysters were obtained from a farm in Tsushima, Na-
gasaki prefecture. After cleaning the shell valves, they were reared
for 5-10 days in running seawater filtered to remove particles >0.5
[j.m. The experiments were conducted in 60 pearl oysters (mean
shell length; 64.4 ± 5.8 mm (SD), shell height; 72.8 ± 4.3 tnm,
shell width: 24.6 ± 1.6 mm, and total wet weight; 40.3 ± 6.3 g).
The resin used was red prepolymerization methyl methacrylate
(Mercox CL-R. Oken Shoji) and hardener (Mercox MA, Oken
*Corresponding author. E-mail: handat@nsh-u.ac.jp
Shoji). When both reagents were mixed at 5-20%, the resin started
to gradually solidify after about 5 min. Therefore, they were mixed
just before injection to the digestive organ. After the pearl oyster
was relaxed enough in 0.4 mM MgCl, solution (Nainba et al.
1995), the left shell valve was removed and the mantle was dis-
sected to expose the labial palp. Polyethylene tubing ( 1 inm in
outer diameter, 20 cm length, Hibiki No. 3), which inflated the tip
spherically in order to prevent the counterflow of the resin, was
inserted about 5 mm from the mouth to the esophagus. Then, 4 niL
of resin were injected within 3 min w ith a plastic syringe of 5 mL
capacity. The tubing was sealed with the flame to .stop the resin
overflowing, and the injected pearl oyster was returned to the
seawater. After it was left at least for 1 h and the resin hardened,
the pearl oyster was immersed in 20% NaOH solution for I day at
room temperature, and then washed with tap water. The completed
corrosion castings were preserved in the 0.1% sodium azide.
We also examined the injection of the resin from the anus, the
casting to the pearl oyster which was preserved in formalin, and
the addition of methyl methacrylate (Nisshin EM) in order to lower
the viscosity of the resin. We also cast other mollusks; ark shell,
Scapharca broughtonii. Pacific oyster C. gigas. clam. Meretrix
hisoria (Bivalvia). and abalone. Haliotis discus (Gastropoda) using
this method.
RESULTS AND DISCUSSION
The cast was easily made from the mouth to the anus (Fig. 1 ),
Within the digestive diverticula, various features of the casting
were observed, such as the tubule that surrounded the stomach
(Fig. I A), the main duct (Fig. IB), the ducts of digestive diver-
ticula without tubules (Fig. IC). and the ducts and tubtiles (Fig.
ID, E). The cast was also showed the stomach and the orifice of
the ducts of the digestive diverticula that were illustrated by Yonge
(1926). The ducts and the tubules, which Owen (1955a, 1955b)
and Nakajima (1956) showed, were also observed (Fig, IE).
777
778
Handa and Yamamoto
Figure 1. The corrosion cast of the digestive organ in the pearl oyster, Pinctada fiicala martensii. A, the whole of digestive organ; B, the main
duct; C, the stomach and the main duct without the tubule; D, the main duct and the tubule; E, the secondary duct and the tubule. The a and
b represent the right and left aspects, respectively. Ksophagus I O), digestive diverticula (D), stomach (Si, Intestine (I), anus (AN I, main duct (MD),
secondary duct (SD), and tubule (T). Bars in A, B, and C = I mm, bar in D = l«(l (jm, and bar in E = 1 \im.
Injecting the resin wliicli contained the hardener at 20%. it
always filled the ducts and tubules, terminal spaces (Fig. lA). In
low concentration at 5*^. the resin reached the ducts (Fig. IB and
C). After casted, the observation was not often easy because there
was hardly contrast on the castings. Then, they were immersed in
20%^ NaOH solution at 60°C. As a result, many contrasts (e.g., red
to pink) emerged (Fig. IB).
When the methyl methacrylate was added to the resin to lower
the viscosity, the castings come apart to pieces in 20% NaOH
solution for the proteolysis and could not be completely made.
Previous studies have demonstrated the opening and closing of
the tubule in the digestive diverticula synchronized with tidal pe-
riods (Morton 19.S6. Morton 1970. Owen 1972). circadian rhythms
(McQuiston 1969, Morton & McQuiston 1974, Robinson & Lang-
ton 1980), and food intake (Morton 1969, McQuiston 1969, Mor-
ton 1979. Robinson & Langton 1980). In this study, the castings
seems to be not inlluenced the conditions of the tubule, because
they were made regardless of them.
The food particles are transported to the tubules by the ciliary
movement in the stomach (Owen 1955a, 1955b, Purchon 1957,
1958, 1960), and ciliary and/or muscular movement of the diges-
tive diverticula (Owen 1955a, 1995b). Castings were similar
whether resin was injected from the anus or the mouth. It was not
possible to make casts with animals preserved in formalin. Thus
the resin is probably transported to the tubules by not only the
injected pressure but also similar functions of sending the particles
from the stomach to tubules.
We also examined the corrosion casting of the digestive organs
in 4 molluscs. As the results, it was possible to cast and observe the
ducts and tubules in ark shell 5. broughtonii (Fig. 2A). Pacific
oyster C. gigas (Fig. 2B), clam M. htsoria (Fig. 2C), and abalone
H. discus (Fig. 2D). Therefore, these methods are applicable to
cast the digestive organ in molluscs.
The detailed structure of digestive diverticula is suggested by
the histological method, but it is very difficult to indicate the
distribution of ducts and tubules, or to grasp a sense of the three-
dimensional aspects of the structures. This casting method accu-
rately shows the whole structure of digestive diverticula, for ex-
ample, the positional relation to the stomach, and the features of
branch and connection of ducts and tubules. Information on the
digestive diverticula map will be very important for the criteria for
classification, and also useful to investigate the function of diges-
tion and absorption through the digestive canal, especially in the
digestive diverticula and stomach.
Corrosion Casting of thf Digestive Divertici'La
119
Figure 2. The casting preparation of the digesti\e organ of four molluscs. A, ark shell Scapharca hroiighloiiii: B. pacific oyster Crassostrea gigas:
C, clam Meretrix liisoria (Bivalvial; D. abalone Haliolis discus (Gastropoda). The a and b represent the right and left aspects, respectively. The
c and d represent the dorsal and ventral views. rcspecti>ely. Anus lANl. stomach (S). digestive diverticula (DD). Oesophagus (O). intestine (1).
The cannula (Cnl is polyethylene tubing which was used for the injection of resin. Bars = 1 mm.
McQuiston, R. W. 1969. Cyclic activity in the digestive diverticula of
Lusaea rubra (Montagu) (Bivalvia: Eulamellibranchia). Proc. Malm:
Sac. Land. 38:483-192.
Morton. B. 1969. Studies on the biology of Drcissena pah inorplia Pall. 11.
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excretion. Proc. Malac. Soc. Land. 38:401-414.
Morton, B. 1970. A note on the cytological structure and function of the
digestive diverticula of Mocoma ballhica cortelaled with the rhythm of
the tide. Malac. Rev. 3:115-119.
Morton. B. 1979. The biology, ecology and functional aspects of the organ
feeding and digestion of the S.E. Asian mangrove bivalve. Enigmonia
aenigmatica (Mollusca: Anomiacea). J. Zaol. Ltvid. 179:437—466.
Morton, B. & R. W. McQuiston. 1974. The daily rhythm of activity in
Teredo navalis linnaeus correlated with the functioning of the digesti\ e
system. Forma et functio 7:59-80.
Morton. J. E. 1956. The tidal rhythm and action of the digestive system of
the lamellibranch Lasaea rubra. J. Mar. Biol. Ass. U. K. 35:503-586.
Nakajima. M. 1956. On the structure and function of the mid-gut gland of
mollusca with a general consideration of the feeding habitats and sys-
tematic relation. Jpn. J. Zool. 1 1 :469-566.
Naiiiba, K., M. Kobayashi.. S. Aida., K. Uematsu.. M. Yoshida.. Y. Kondo
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& Y. Miyata. 1995. Persistent relaxation of the adductor muscle of
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Owen. G. 1955a. Observations on the stomach and digestive diverticula of
the lamellibranchia I. The Anisomyaria and Eulamellibranchia. Quart.
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Owen. G. 1955b. Observations on the stomach and digestive diverticula of
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Owen, G. 1972. Lysosomes. peroxisomes and bivalves. Sci. Prog. O.xf.
60:299-318.
Purchon. R. D. 1957. The stomach in the lllibranchia and pseudolamelli-
branchia. Proc. Zool. Soc. Land. 129:27-60.
Purchon. R. D. 1958. The stomach in the Eulamellibranchia; Stomach type
IV. Proc. Zool. Soc. Umd 131:487-525.
Purchon. R. D. 1960. The stomach in the Eulamellibranchia: Stomach type
IV and V. Proc. Zool. Soc. Land. 135:431-489.
Robinson. W. E. & R. W. Langton. 1980. Digestive in a subtidal popula-
tion of Mercenaria iiiercenaria (Bivalvia). Mar. Biol. 58:173-179.
Yonge. C. M. 1926. Structure and physiology of the organs of feeding and
digestion in 0.s7/-<'« <'(/»//,v. J. Mar. Biol. Ass. U. K. 14:295-386.
.loiiriuil nfSJii-Ufish RcKcavch. Vol. 22, No. 3. 7S1-7S7. 2(K).V
MITOCHONDRIAL DNA REVEALS GENETIC DIFFERENTIATION BETWEEN AUSTRALIAN
AND INDONESIAN PEARL OYSTER PINCTADA MAXIMA (JAMESON 1901) POPULATIONS
JOHN A. H. BENZIE,"* CAROLYN SMITH,' AND KETUT SUGAMA'
^Australian Institute of Marine Science. PMB No 3. Tonnsville, Queensland 4810. Australia; 'Centre for
Marine and Coastal Studies. The University of New South Wales. Sydney. NSW 2052. Australia: and
^Gondol Research Institute for Aquaculture. PO Bo.x 140. Singaraja. Bali. Indonesia
ABSTRACT A total of 234 individual silver-lipped pearl oyster (Pinclada maxima) from six populations in Australia and two
populations in Indonesia were analyzed for genetic variation within a 680-base pair region of the mitochondrial DNA COI gene using
restriction fragment length polymorphism analysis. The Indonesian populations were markedly different from all Australian popula-
tions examined, and the differences were greater than that expected on the basis of their geographical separation. In contrast with this
broader regional pattern of genetic differentiation, the Australian populations sampled were not significantly differentiated from one
another, and a high degree of connectivity was observed among Western Australian pearl oyster populations. In addition, these genetic
data show that Western Australian P. maxima populations have a closer tie to those from the northern Australian coast than with
populations in Indonesia. This regional pattern of genetic separation is evident despite the proximity of Indonesia to the eastern Indian
Ocean locations sampled and the potential for dispersal afforded by the southward currents of the Indonesian throughflow.
KEY WORDS: aquaculture, biogeography. fisheries
population genetics.
INTRODUCTION
gement, Indo-Pacific. mitochondrial DNA. pearl oyster. Pmctada maxima.
The silver-lipped pearl oyster. PiiiclmUi maxiina (Jameson
1901) is found in Southeast Asia and northern Australia and pro-
vides the basis for the strong south sea pearling industry (Shirui
1994). Although there is increasing use of hatchery stock, the
industry in Western Australia is still dependent upon the collection
of wild shell and upon the effective management of wild stocks.
Early work by Johnson and JoU (1993) showed marked differences
in allozyme frequencies in pearl oyster populations collected from
northern and Western Australia (WA), suggesting that these
needed to be managed separately. Despite significant genetic dif-
ferences detected between two northern populations of P. maxima.
which are separated by as little as 320 km. Johnson and Joll ( 1993)
found no differentiation between the two WA populations that
were sampled some several hundred kilometers apart. On the basis
of this limited sampling of just two populations, the pearl oyster
stocks within this important pearl producing region were consid-
ered essentially panmictic.
Significant genetic differences have also been detected between
populations of several other species of pearl oyster over a range of
spatial scales. Pinclada fiicala (Gould 1850). Pinclada albimi
(Lamarck 1819), and Pinclada macidala (Gould 1830) were
shown to be differentiated between sites less than 100 km apart
(Wada 1982), Small genetic differences have also been observed
between both widespread (Durand ct Blanc 1986. 1989) and geo-
graphically closer populations (Benzie & Bailment 1994) of the
black lip pearl oyster Pinclada margaritifera (Linnaeus 1758).
Finally a genetic study of Pinclada radiata (Leach 1814) revealed
significant differentiation between sites less than 33 km apart
(Beaumont & Khamdan 1991 ). This collection of studies suggests
the potential for population substructure within the P. maxima
pearl oyster stocks, which extend for thousands of kilometers
along the WA coast.
The maternally inherited mitochondrial DNA (mtDNA) ana-
*Corresponding author. E-mail: j.benzie@unsw.edu, au
lyzed in the present study has a smaller effective population size
than the allozyme genetic markers used by Johnson and Joll in
their 1993 study and. as such, is inore sensitive to the effects of
genetic drift and consequently often affords greater sensitivity for
detecting genetic differences in population studies. Given the im-
portance of the WA pearl oyster stocks to the Australian pearling
industry, a sensitive genetic study of WA population substructure
with more extensive spatial coverage than that of Johnson and Joll
(1993) was undertaken. In addition, the analysis of collections
from both Indonesia and northern Australia, two potential long-
distance sources of recruits, allowed larger scale connectivity to be
assessed.
Populations of a number of marine species from northwest
Australia have closer genetic affinities with Pacific rather than
Indian Ocean populations despite being situated geographically in
the Indian Ocean (Benzie 1999), These results are consistent with
a connection via the strong cutxents of the Indonesian throughflow
which move south from Indonesia towards Australia. Given that P.
maxima is a broadcast spawner with a larval life of 2 to 3 wk
(Shirai 1994). this species is potentially capable of dispersal over
long distances. For this reason the present study examines the
extent to which WA populations may derive recruits from both
Indonesia and northern Australia.
The present article reports the genetic structure of P. maxima
stocks using mtDNA to determine local population structure
within WA and the extent of connectivity to both Indonesian and
northern Australian populations.
MATERIALS AND METHODS
Sample Collection
Between 27 and 30 adult P. maxima were analyzed from each
of six populations in Australia and two populations in Indonesia.
Samples of adductor muscle were collected from P. maxima oys-
ters aboard pearling industry vessels between February 1998 and
November 1999, Samples were obtained in Northern Australia to
the west of Darwin and in Western Australia from the Lacepede
Islands. 80 Mile Beach (shallow water). 80 Mile Beach (deep
781
782
Benzie et al.
water). Port Hedland, and Exmouth Gulf (Fig, 1). The 80 Mile
Shallow collections were made inshore at less than a 10 m depth
from the Northern end of 80 Mile beach. The 80 Mile Deep col-
lections were made at a similar latitude but from a more offshore
site at -30 m depth. The two Indonesian populations, Madura and
Sumbawa Island, were collected in November 1999. Live animals
were delivered by road to Gondol Fisheries Station and held in
flowing sea water tanks before dissection. Adductor muscle
samples were immediately snap frozen in liquid nitrogen after
collection.
DNA Extraction and Polymerase Chain Reaction (PCR)
DNA was e.xtracted from using a CTAB extraction procedure
modified from Adamkewicz and Harasewych (1996) in which
small cubes of frozen muscle (-0.5 cm^) were ground in pre-
warmed (60°C) CTAB extraction buffer (29^ CTAB. 27c polyvi-
nylpyrrolidone. 100 niM Tris-HCl pH 8.0. 1.4 M sodium chloride,
20 niM EDTA) to which proteinase K was added to a final con-
centration of 0.5 mg/niL. After overnight incubation at 60°C,
samples were heated to 90°C for 20 min before addition of RNase
A (0.1 mg/mL) and a 1-h incubation at 37°C. DNA was then
extracted and precipitated using standard phenolxhloroform:
isoamyl alcohol methods as per Sambrook et al. (1989).
Echinoderm universal primers for the Cytochrome Oxidase I
(COD gene (Col, fwd: 5' ATA ATG ATA GGA GGR TTT GG 3'
and Col. Rev: 5' GCT CGT GTR CTA CRT CCA T 3' (Williams
1997) were used to amplify a 680-base pair segment of that gene.
PCR reactions were conducted with 2 ng/p.L DNA in a IX PCR
buffer containing 1.5 mM MgCK. 0.03 units/jiL Taq DNA poly-
merase (Qiagen. Australia). 200 |jiM dNTPs, and 0.5 |jlM each
primer. Thermocycler conditions were 94°C for I min (one cycle),
followed by 94°C for 1 min. 45°C for 1 min. 72°C for 1 min, 30
s (30 cycles), with a final 4°C hold. Fifty-microliter PCRs were
performed in a Perkin-Elmer 9700 thermocycler.
Restriction Fragment Length Polymorphism (RFLP) Analysis
Of the 39 restriction enzymes tested, only fi\e (D/))j1I. Eco0\90
I. Fokl. HcicUl. and NlaW) produced polymorphic fragment pat-
terns, and these were used to survey RFLP variation within the
amplified region of the COI gene. Overnight digest reactions con-
tained 5 [X.L of PCR product and -0.03 units/ |jiL restriction enzyme
(New England Biolabs. Beverley, MA) in a 15-|jlL reaction with
IX buffer as per the enzyme manufacturer's instructions. Digest
fragments were separated on 3% agarose gels (2% GibcoBRL
agarose- 1 000, F/r Progen DNA grade agarose) at 4-5 volts/cm for
up to 5 h with repeated photography of ethidium bromide-stained
gels throughout the running period. Fragment sizes were estimated
by regression against standard size markers and for each restriction
enzyme the unique fragment patterns were given an alphabetical
assignation (Table 1 ). The position of each restriction enzyme site
producing the unique fragment patterns was identified by DNA
sequencing of several individuals and a composite profile of the
<:>Qr^=^''CZ:x^''^
s^
Lacepedes =^7v
, ^tj-- WESTERN
\> AUSTRALIA
500km
Port Hedland
Haplotype 1
K'-'J Haplotype 2
^H Shared
Private to Indonesia
Private to Australia
Figure 1. Pie diagrams illustrating the frequencies of the major haplotype or haplotype groups differentiating the eight P. maxima populations.
Pearl Oystkr COI Genetic Structure
783
TABLE 1.
Mitochondrial DNA restriction fragment sizes observed anions 234
f'iiiclada maxima from Australia and Indonesia
Enzvme
Haplotvpe
Fragment Sizes (bp)
DpnW
EciM\m\
lok\
HiiA\
MalV
454.226
263.226.14?
226,158.145.105.45
408.226.45
680
590,93
603,77
680
353.250.77
448.155.77
299.29 1 ,5 1 .42
590.5 1 .42
632.51
388.249.43
388.155.94.43
43 1 .249
543,94.43
presence/absence of each site was constructed tor each animal tor
all restriction enzymes (Table 2).
Statistical Analyses
The DA program in REAP (McElroy et al. 1992) was used to
estimate haplotvpe diversity (h) and nucleotide diversity (tt)
within populations and nucleotide divergence (d^-, ) among popu-
lations (Nei & Tajima 1981 ). Spatial structuring of the populations
was investigated using programs in ARLEQUIN (Schneider et al.
2000). AMOVA (Excoffier et al. 1992) was used to calculate <i>sT
(analogous to F^i )• N^.m. and to perform hierarchical analysis of
(bsT- The MXCOMP program in NTSYS (Rohlf 1997) was used to
calculate the Mantel test (Mantel 1967) to measure the degree of
association between the matrix of pairwise <i>^-^ comparisons and
the geographic distance between populations. Significance levels
for simultaneous multiple tests were adjusted following Rice
(1989). Further analysis, such as mismatch distributions, tests of
neutrality, and timing of population expansion were not conducted
because the small number of sites covered by the RFLP data would
result in large errors and low statistical power.
A character state matrix showing the presence or absence of
presumptive restriction sites created using programs in REAP
(McElroy et al. 1992) was used to construct unrooted, phylogenies
using the maximum likelihood method in the RESTML program in
PHYLIP, which assumes a Jukes-Cantor model of evolution
(Felsenstein 1993). and the parsimony method implemented in
PAUP (Beta Version 4.0b2; Swofford 19901. RESTML was set to
find the best tree with global rearrangement of subtrees and input
order of the haplotypes jumbled three times. In PAUP. restriction
sites were treated as relaxed Dollo characters with gains weighted
twice as heavily as losses (McMillan & Bermingham 1996). One
thousand optimal trees were found using a heuristic search with the
tree bisection and reconnection branch swapping algorithm and the
50% majority rule consensus was applied to obtain a single con-
sensus tree.
RESULTS
The survey identified 16 composite haplotypes among the 234
samples (Table 2. Fig. 1). Two haplotypes (12.59^ accounted for
91% of the individuals assayed (213 individuals). Nine haplotypes
(56.3%) were unique, accounting for 3.9%- of the animals. The
other five haplotypes (31.3%) were each represented by only two
or three animals. At the population level, nine haplotypes were
private (i.e.. occuired in only one population) while at the regional
level, eight haplotypes were private to Australia (62% of all hap-
lotypes found in Australia) and three private to Indonesia (38%).
Genetic Diversity Within I'opulalians
On average, the Indonesian populations had higher levels of
genetic diversity than the Australian ones, with Darwin having the
lowest level of all (Table 3). The pattern was seen most clearly in
the data for haplotype diversity (h). which was two times greater
in the Indonesian populations (mean h = 0.520) than in the Aus-
tralian populations (mean h = 0.246). Nucleotide diversity (tt)
was also two times greater in the Indonesian populations (mean -n
= 0.0097) than in the Australian populations (mean tt = 0.0046).
Genetic Differentiation Among Populations
The most common haplotype ( 1 ) was more frequent in Austra-
lian populations, where it comprised 79-93% of the individuals
assayed compared with 17-27% in the Indonesian populations.
The next most common haplotype (2) was more frequent in Indo-
TABLE 2.
Composite mtDNA haplotypes observed among 234 Pinctada
maxima from Australia and Indonesia
Composite
Haplotvpe
Number
DpnW
£foOI09l
Fokl
Haem
iV/olV
abed
c
Igh
,Jk
Inin
1
1011
0
001
111
Oil
"1
1011
1
001
Ml
111
3
1011
1
000
111
111
4
1011
0
001
101
on
5
1011
0
Oil
111
oil
6
1111
1
001
111
111
7
1011
0
001
1 1 1
111
8
1011
0
000
111
oil
9
0011
0
001
111
oil
10
0001
0
001
111
oil
11
1011
1
101
111
111
12
1011
0
(101
111
010
13
1011
1
001
101
111
14
1011
1
001
1 1 1
101
15
1011
i
001
111
(III
16
1011
0
001
100
OKI
1110
1
112
031
211
Presence (1) or absence (Ol of restriction sites was inferred from banding
paltems obtained from single digestions of extracted total DNA with each
of the five restriction enzymes (Table 1 ). Bold numbers at the base of the
table indicate the number of times a cutting site was lost in the maximum
likelihood phylogeny. The 16 haplotypes represented 14 putative cutting
sites, two of which were present in all animals surveyed. In the maximum
likelihood phylogeny one site (j) was lost three times and two (h. 1) were
lost two times. The remainder were lost once (nine sites).
784
Benzie et al.
TABLE 3.
Measures of genetic diversity within pupubtions: number of haplotypes (h,,), the ratio of (h,,) to the number of individuals sampled («,):
[(H|,/«i)]. haplotype diversity I//), and nucleotide diversity (71) within each of eight populations of the pearl oyster Pinctada maxima
Population
II
"h
njn,
h (±)SE)
7T
Madura
29
3
0.17
0.458 (±0.102)
0.(.)()S6
Sumbawa
30
5
0.17
0.582 (±0.079)
0.0107
Darwin
30
2
0.07
0.129 (±0.(.)79)
0.0026
Lacepedes
30
4
0.13
0.251 (±0.102)
().()()5I
80 Mile Deep
29
4
0.14
0.200 (± 0.098)
0.0028
80 Mile Shallow
29
6
0.21
0.374 (±0.1 13)
0.0078
Port Hedland
27
3
0.11
0.271 (±0.105)
0.0048
Exmouth
30
5
0.17
0.253 (±0.104)
0.0045
Average
4.25 (±0.49)
0.15 (±0.02)
0.315 (±0.056)
0.0059 (±0.0011)
All populations
234
16
0.07
nesian populations where it comprised 60-72% of individuals
compared with 3-11% in Australian populations. These data, and
the fact that three haplotypes were private to Indonesia and eight
were private to Australia, suggest considerable regional differen-
tiation among populations (Fig. 1 ). There were highly significant
pairwise cts^ values between the two Indonesian populations and
all the Australian populations, the mean 4>sr heing 0.562 (Table 4).
There was no significant differentiation among populations within
Australia (with the exception of Darwin and some Western Aus-
tralian sites) or among populations within Indonesia. The signifi-
cant differentiation of Darwin and some Western Australian popu-
lations (mean 4)^^ = 0.038) was an order of magnitude less than
that for the Indonesian-Australian comparisons. A hierarchical
AMOVA analysis, partitioning variation within populations, be-
tween populations within regions (Indonesia and Australia), and
between regions, confirmed that all of the genetic variation oc-
curred within populations (47%). and between regions (33%: P <
0.05).
When pairwise <bsT values were plotted as a function of the
geographical separation of the populations, the Australian-
Indonesian comparisons formed a separate group whose degree of
genetic differentiation was far greater than those comparisons
within regions (Fig. 2). The Mantel test of <i>^-j- against distance in
km for the total data set was significant (r = 0.69. P < 0.001).
However, when the data were decomposed into comparisons either
between or within regions, there was no significant relationship
between <i>^y and geographical separation among Australian popu-
lations (;■ = 0.56. P = 0. 156). There was not enough data to allow
a test within Indonesia. The pattern of connectivity among popu-
lations (using the effective number of migrants per generation
(N^,,„) as the measure of exchange) emphasizes the strong connec-
tion within regions and the limited exchange between regions
(Fig. 3).
Haplotype Phylogeiiy
The 50% consensus tree based on parsimony analysis showed
little structure and no deep relationship between haplotype group-
ings related to their geographical distribution (Fig. 4). The maxi-
mum likelihood network (not illustrated) was dominated by two
star-like nodes each centered on one of the two most common
haplotypes (haplotypes I. 2). Both stars included haplotypes found
in either Australian and Indonesia or both regions, and most hap-
lotypes differed by only one restriction site change from the dom-
inant haplotypes.
DISCUSSION
RFLP analysis of a portion of the mitochondrial COI gene has
provided strong evidence for high levels of dispersal among WA
populations of P. nuixinia confirming the findings of Johnson and
Joll (1993) based on nuclear markers (allozymes). The analysis
also showed clearly that the WA populations weie more closely
connected to northern Australian populations than to Indonesian
ones. There has been movement of pearl oysters by the cultured
pearl industry, largely from some WA wild sites to farms in the
Northern Territory, but it is highly unlikely that the pattern ob-
TABI.E 4.
Pairwise F.,, among eight populations of the pearl oyster Pinctada maxima
Madura
Sumbawa
Darwin
Lacepedes
80 Mile
Deep
80 Mile
Shallow
Port
Hedland
Sumbawa -().()23"'
—
Darwin 0.684***
0.617***
—
Lacepedes 0.569***
0.497***
0.046"-
—
80 Mile Deep 0.657***
0.585***
0.000"-
-0.000"-
—
80 Mile Shallow 0.521***
0.456***
0.050***
-0.008"-
0.010"-
—
Port Hedland 0.560***
0.487***
0,057*
-0.028"-
0.005"-
-0.024"-
Exmouth 0.592***
0.521***
0.038"-
-0.022"-
-0.013"-
-0.012"-
* P < 0.05; *** P < 0.001 : "' not significant.
-0.024"-
Pearl Oyster COI Genetic Structure
785
0.9
0.7 -
0.5
0.3 ^
0.1
-0.1
0
500
2500
1000 1500 2000
Dista nee (km )
Figure 2. F^, graphed as a function of tlie geographical separation of
the population pairs. Comparisons with Indonesian populations have
been given a different symbol (triangles).
served in this study is related to those stock movements. Using
allozymes. Johnson and Joll (1993) noted clear genetic differences
between the WA populations and those from Oxley Island in the
Northern Territory despite large numbers of WA animals having
been introduced to a farm within HO km of Oxley Island. The
present study sampled wild populations in the Darwin region that
were geographically more distant from the nearest farm supporting
the suggestion that the connectivity observed between the Darwin
and WA populations is unlikely to be a reflection of stock transfer.
The le\el of di\ergence of P. nuixiina populations using the
mitochondrial COI gene RFLP data (assuming a 2% divergence
per million years) suggests present day mixing between all Aus-
tralian populations but isolation of Indonesian and Australian
populations during the Pleistocene at least 100.000 years ago. Cau-
tion needs to be applied to these interpretations because of the
limited data available in the RFLP analysis and the large errors
inherent in these types of estimates in any case (Edwards & Beerli
2000, Kishino et al. 2001, Zhivotovsky 2001). Nevertheless, these
data present a consistent picture of divergence between Indonesian
and Australian populations well before the last low sea level stand
around 12,000 years ago. Therefore, despite the apparent possibil-
ity for migration of marine invertebrate larvae from Indonesia on
strong southerly flowing currents the limited gene exchange ob-
served between Indonesian and WA populations of P. maxinui is
consistent with the strong westward deflection of the Indonesian
4'5^ ,'^
_^^-r^-C-''
Nem (mtPNA)
<1
1-15
15-100
>100
Figure 3. .Map illustrating the le\els of gene flow between P. maxima
populations estimated from mtDNA.
throughflow just south of the Indonesian arc which makes it un-
likely that this cuiTent would reach the coastal regions of Australia.
In population genetic analysis of the giant tiger prawn Penaeus
moiiodon (Fabricius 1798) also using RFLP analysis of mtDNA,
Benzie et al. (2002) showed a closer relationship between the WA
population of P. monodon and those from northern and eastern
Australia, and a clear distinction from Indonesian and Philippines
populations. The Philippines sample was to some extent interme-
diate between Indonesian and Australian samples, suggesting links
to Southeast Asia primarily via eastern Southeast Asian and east-
ern Australian populations and also linkages to WA via northern
Australia. There were no samples of P. maxima available from the
Philippines or elsewhere in Southeast Asia or eastern Australia for
the present study but the pattern observed in P. maxima over the
range surveyed is consistent with that for P. moiwd(m.
A strong genetic divide between the Pacific and Indian Ocean
populations found in several species of marine invertebrates may
involve considerable shifts in gene frequency as well as deep di-
visions in haplotype phylogeny (Benzie 1999, Barber et al. 2000).
In contrast, genetic distances between the Pacific and western
Australian populations of marine invertebrate species may be an
order of magnitude less (Benzie 1999) and in mtDNA markers
may involve differences in the frequency of relatively closely re-
lated haplotypes (Williams & Benzie 1997, 1998, Benzie et al.
2002). The fact that Indonesian and northernAVA populations off.
maxima did not show deep divergence of COI haplotypes associ-
ated with geographical region is consistent with these studies. The
fact that genetic diversity is higher in southeast Asian populations
of P. maxima than in Australian populations is also consistent with
a general trend of decreasing genetic diversity outwards from
southeast Asia to more geographically distant sites (Benzie et al.
2002).
Marked genetic differences between WA and Indonesian pearl
oyster stocks contrasts with considerable gene exchange over thou-
sands of kilometers among WesteiTi Australian populations. The
mtDNA variation in P. maxima populations between Indonesia and
Australia suggests a strong influence of biogeographical events at
the regional scale. Future assessments of larger scale patterns of
dispersal of this species should include samples from elsewhere in
the Indian Ocean and from additional locations in Southeast Asia
and from eastern Australia.
ACKNOWLEDGMENTS
This work was supported by grant 97/.344 from the Fishing
Research and De\elopment Corporation (FRDC) in Australia. We
thank the Arrow Pearling Company, Broome Pearls, Maxima
Pearling Company, Morgan and Co. Pty Ltd, Norwest Pearling,
Paspaley Pearling Co.. Pearl Coast Di\ers Pty Ltd.. The Gun Char-
ter Fishing, and the Pearl Producer's Association for their assis-
tance with the project. Thanks also to Serena Sanders, Rick
Scoones, Mick Buckley, Helen O'Donogahue, and officers from
Western Australian Fisheries and Northern Territory Fisheries for
their assistance. We also thank the staff of the Gondol Fisheries
Research Centre of the Indonesian Government, particularly Dr
Haryanti and Sari Budi Moria, and E. Bailment and S. Uthicke
from AIMS, for their collaboration in sampling pearl oysters from
Indonesia. We thank Megan Johnson, Lesa Peplow. Christine
Clegg. and Melissa Merrit for technical assistance and Lee Ann
Rollins for assistance with statistical analysis of the results. In part
this work made use of the bioinformatics facilities of the Austra-
lian National Genomic Information Service (ANGIS).
786
Benzie et al.
M S D L 8D 8S PH E All Rank
(29) (30) (30) (30) (29) (29) (27) (30) (234)
3.5
□ 17.2 26.7 93.3 86.7 89.7 79.3 85.2 86.7 70.5 1
3.5 3.7
• 8 □ 3.5
3.5
1.3 3
3.3 0.4 5
0.4 5
.2 □ 72.4 60.0 6.7 3.5 6.9 II. I 3.3 20.5
□ 3.3
3.3 0.8 4
6 □ 6.7
3.5
0.4 5
0.4 5
3.3 0.4 5
n Haplotype shared by Australia and Indonesia
■ Haplotype private to Indonesia
Figure 4. Percentage frequencies of tlie 16 composite mtDNA haplotypes observed among P. maxima individuals, and the 50''f majority rule
con-sensus tree (from KMM) maximum parsimony networks) indicating the relationships among the haplotypes. Numbers in parentheses imme-
diately below the location codes indicate the number of individuals assayed. The column of numbers immediately to the right of the tips of the
branches of the tree is the composite haplotypes listed in Table 2. The columns on the far right give the percentage of each haplotype in the total
population, and their rank abundance, respectively. Locations are as follows: M. Madura; S, Sumbawa; D. Darwin; L, Lacepedes; 8D, 8(( Mile
Beach deep: 8S. 80 Mile Beach shallow; PH. Port Hedland; E, Exniouth Gulf.
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Jcmnuil of Shellfish Research, Vol. 22, No. 3. 789-794, 2U03.
SHALLOW-WATER DISTRIBUTION AND POPULATION CHARACTERISTICS OF STROMBUS
GIGAS AND S. COSTATUS (GASTROPODA: STROMBIDAE) IN BOCAS DEL TORO, PANAMA
ALEXANDER TEWFIK ' AND HECTOR M. GUZMAN "*
^ Dcpariiiu'iit i>f Bioloi^x. McGIII University. 1205 Ave. Dr. Penfield. Montreal, Canada. H3A IBl:
-Smithsonian Tropical Research Institute, Unit (ms. APO AA 34002. USA
.ABSTRACT Extensive visual surveys for the economically and ecologically significant queen conch iSlroiiilnis gigas) and milk
conch (Stromlms cosralus) were conducted within the Bocas del Toro archipelago. Overall population densities are among the lowest
recorded in the region (S. gigas 1.43 conch ha"': S. costatus 1.27 conch ha"'), and are likely the result of overexploitation by both
commercial and subsistence fishing. The very low adult densities (S. gigas 0,30 conch ha"') and the lack of reproductive behaviors
observed are a serious concern when one considers the "Allee effect" and the resultant negative per capita population growth rates
reported elsewhere in the literature. This information has provided some of the rationale for establishing the recently announced 5-y
ban on conch exploitation on the Caribbean coast of Panama.
KEY WORDS: queen conch, stock assessment, overfishing, Panama, Allee effect, Siromlms
INTRODUCTION
StroiiihKs f^igds Linnaeus, 1758, and Strombus costatus Gme-
lin, 1791. are two herbivorous gastropods of the family Stromhidae
that inhabit shallow seagrass meadows (SGs). sand beds, and algal
flats throughout the Caribbean. Queen conchs have long been val-
ued for their meat and shell, and were first harvested in the Ca-
ribbean by the Lucayans and Arawaks during pre-Columbian tunes
(Brownell & Stevely 1981, Berg & Olsen 1989). Local commer-
cial and subsistence use of both conch species has continued to this
day and on occa.sion still provide a primary source of protein in
some fishing communities.
During the last 30 y, the overall harvest of queen conch has
increased substantially, driven largely by international export as
well as growing resident populations and increasing tourism in the
Caribbean region (Berg & Olsen 1989. Tewfik 1997). Conch is
commercially exploited in at least 22 countries throughout the
region, and is often consumed only as a luxury food item due to its
relative rarity and high market value (Mulliken 1996. Theile
2001 ). The shell products of several strombids are also sought after
and are well recognized in the tourist industry of many Caribbean
nations. Present landings of conch meat in the region are now in
excess of 13.000 metric tons (Food and Agriculture Organization
of the United Nations 2000). However, it should be noted that
Food and Agriculture Organization landings are for all "Strombid
conchs" and may therefore include several species. Significant
landings of other strombids. including S. costatus. are likely to be
occurring in places such as Mexico (Gil 1994. Theile 2001). The
fear of the disappearance of commercial Queen conch fisheries has
prompted 5. gigas to be included under appendi.x 2 of the Con-
vention for the International Trade of Endangered Species
(CITES) in 1992. Most recently. CITES has initiated a "significant
trade review" for the species (Theile 2001 ).
The San Bias and Bocas del Toro archipelagos are the main
areas of conch fishing in Panama (Marians 1997). Limited data are
available for the total number of conch landings in Panama, such
landings being considered incidental to the spiny lobster harvest,
with the latest figure being 1 16 metric tons in 199S (Martans 1997.
Autoridad Maritima de Panama 1999). No specific regulations
exist for the harvest of either 5. gigas or S. coslalus in Panama.
*Corresponding author. E-mail address: gu/.manh@naos.si.edu
however, the use of scuba gear is prohibited for the harvest of any
marine resource (Martans 1997). Aside from the role that conchs
serve in both local and regional economies, their populations pro-
vide critical links between primary producers and higher-level
consumers within near-shore marine communities throughout their
range (Stoner & Waite 1991, Stoner et al. 1995).
The following article will describe the abundance, population
structure, morphology, and spatial distribution of S. gigas and 5.
costatus, which have been heavily exploited over the last few
decades in the Bocas del Toro archipelago. The consequences of
this exploitation on future recruitment will also be discussed. Fi-
nally, some brief comments will be made regarding the potential
interaction that may exist between the two strombids defined here,
with special attention to the spatial partitioning of these species
over shallow, near-shore seagrass-sand-algal complexes that are
typical of many areas of the Caribbean.
MATERIALS AND METHODS
The study was conducted over a 47,158-ha area of shallow
water (<10 m) habitats in the Bocas del Toro archipelago between
February and September 2000, A comprehensive description of the
sea bottom topography, climate, geology, and reef distribution of
the archipelago are available in several other publications (Rod-
riguez et al, 1993. Greb et al. 1996, Guzman & Guevara 1998).
The entire shallow (<10 m) coastal zone, inespective of habitat
type, was divided into 240 2 x 2-km grid squares, of which 120
grids or sites were randomly selected and surveyed. Within each
site, three replicate belt transects (100 x 6 m) were surveyed by
two divers (width 3 x 3 m each) at each of two different depth
strata (0.5-5 and 5-10 m). In total, each site had 1800 m" per depth
strata or 3600 m" in total area surveyed.
All strombids located within a transect were counted and mea-
sured for total shell (siphonal) length (SL), maximum shell width,
and lip thickness (at mid-lateral region approximately 40 mm from
the edge) to the nearest millimeter using a caliper. Adult status was
assigned to all conchs with a lip thickness >4 mm (Appeldoorn
1988). The depth and major substrate/habitat type where the strom-
bids were located was also noted. The substrate/habitat types were
classified according to a predefined typology that included only
the most common habitats: algal plain (AP); SG; sand plain (SP);
and coral rubble (CR) (Table 1 ). All data sets were analyzed using
789
790
Tewfik and Guzman
TABLE 1.
Substrate/habitat categories used in characterizing all sites surveyed within the Bocas del Toro archipelago, Panama.
Habitat
Code
Description
Algal plain
AP
Seagrass meadow
SG
Sand plain
SP
Coral rubble
CR
Fine mud. coarse sand, rubble, shell bottom dominated by benthic algal cover {Pi'iiicilliis spp.. Caulcr/ui spp..
Dasyclaihis spp.. Halimedii spp.. LUlorea spp.. Puilina spp.. Luureiiciu spp.)
Coarse sand bottom dominated by Turtle {Thalassia tesmdinum.) and Manatee {Syriiigocliiiiu filifiinne.) grass.
Coarse sand bottom with sparse or no benthic algae or seagrass cover.
Rubble, shell fragment bottom with sparse cover of macro and encrusting algae.
parametric statistics in .SYSTAT. version 10.2 {Systat Software
Inc.. Richtiiond. CA).
The density distribution of the two species was mapped using
Geographical Information System. A digital classification for the
area of study was based on a combination of digital images frotn
three sources: topographic maps at a scale of 1 :50,0()0; color aerial
photographs at a scale of 1:25.000; and LANSAT TM-.'i satellite
images (Guzman & Guevara 2002). Density data were integrated
using the programs MIP (Micro Images Inc.. Lincoln. NE), ver-
sion ."^.1 (Map and Image Processing System), and ArcView, ver-
sion .^.0.
RESULTS
Shallow marine environments (<I0 m) covered approximately
47.138 ha of the archipelago. A total of 432,000 m" (43.2 ha) was
surveyed during the course of the study using 720 transects. A total
of 45 S. gigas (SO'/r juveniles) and 48 S. costatus {4,2% juveniles)
were found during the entire S-mo survey (February-September
2000). SL and shell lip thickness distributions occurred over the
40 60 80 100 120 140 160 180 200 220 240 260 280
Shell Length (mm)
6 8 10 12 14 16 18 20 22
Shell Lip Thickness (mm)
Figure 1. SL (A) and shell lip (B) frequency distributions of S. gigas
{II = 45) and S. costatus {it = 48) in Bocas del Toro, Panama.
norma! ranges reported in the literature for both species (Fig. la,
b). Significant correlations were made between total SL and shell
width (S. gigas R- = 0.933. P < 0.05; S. costatus R- = 0.874,
P < 0.05) (Fig. 2a). Although coiTelation coefficients were much
lower for total SL versus lip thickness (S. gigas R" = 0.202;
S. costatus R" = 0.533). the relationships were still significant
{P < 0.05) (Fig. 2b). The lower correlation coefficients were to be
expected, given the cessation of SL growth at sexual maturity
(3.5-4.5 y old) followed by only lip-thickness growth during adult-
hood, which is typical for this group of mollusks (Alcolado 1976,
Appeldoorn 1988).
S. gigas occurred at 20'7f of the 1 20 sites surveyed, with den-
sities ranging from 0 to 27.8 conch ha"', a tiiedian of zero, and a
mean (±SE) total density of 1.43 ± 0.37 conch ha"' (adults 0.30 ±
0.11 conch ha"'; juveniles 1.13 ± 0.31 conch ha"') (Fig. 3). S.
costatus occuiTcd at 1 1.7% of sites surveyed, with densities rang-
ing from 0 to 58.3 conch ha"', a median of zero, and a mean total
density of 1.27 ± 0.55 conch ha"' (adults 1.23 ± 0.53 conch ha"';
juveniles 0.05 ± 0.03 conch ha"') (Fig. 3). The highest densities
for 5. gigas (21-30 conch ha"') were observed in two regions
— 250-
E
.§.200-
Sl50-
= 100-
^ 50-
n -1
A
• S costatus V
V S. gigas ^ "
V
1 1 1 I 1 >
E
E, 25-
S 20-
■§ 15-
i-
9- 10-
—1
0 5-
sz
CO
n-
B
•
50 100 150 200 250
Shell Length (mm)
300
350
Figure 2. SL versus shell width (A) of S. gigas (R' = O.'J.V') and
.V. costatus {R- = (I.KVJl, and SL >ersus shell width (B) of ,S'. gigas
(R- = 0.202) and S. costatus (R- = 0.533) in Bocas del Toro, Panama.
Distribution and Ahiindance of Strombus in Panama
791
Figure 3. Density distribution (individuals/ha) of S. gigas (A) and
S. costaliis (B) over the shallow water (<10 m) in Bocas del Toro,
Panama. The MPA is denoted by the polygon.
encompasMng 1.7% (805 ha) of the total area. The lowest densities
( 1-10 conch ha~') were found in 19 scattered areas: two inside the
marine protected area (MPA), Cayos Zapatillas (474 ha with 6
conch ha~' ), and near the southwest side of the park (480 ha with
3 conch ha~') (Fig. 3a). The highest densities for S. costalus (41-
50 conch ha"' ) were located northwest of Bastimentos Island in an
area of 125 ha (0.3%) (Fig. 3b). The lowest densities for this
species (1-20 conch ha"') were observed in 11 relatively small
areas (7.5%), one of which occurred inside the MPA (761 ha with
14 conch ha"') (Fig. 3b).
The distribution of strombids by the two depth strata vastly
favored the shallower of the two (0-5.0 m) with 84% and 98%,
respectively, of S. gigas and S. costatus being found in these areas.
The most favored habitat/substrate type for both species was SGs
(>70%i), with a relatively even distribution of the remaining indi-
viduals among AP, CR, and SP areas (Fig. 4). When examining
site occupation among the two species, S. gigas appears to have a
broader distribution than S. costatus (24 vs. 14 sites), and the
number of co-occupied sites was limited to just 4.2%', or 5 sites of
the total 120 sites surveyed (Fig. 3).
AP CR SG
Habitat / Substrate Type
Figure 4. Habitats occupied by S. gigas and S. coslaliis in Bocas del
Toro. Panama. See Table 1 for habitat descriptions.
DISCUSSION
The long-term, heavy exploitation of strombid populations
within the shallow water habitats of the Bocas del Toro archi-
pelago have likely contributed to the overall densities of S. gigas
(1.43 conch ha"'), which are among the lowest reported in the
region (Table 2). Considerably less information is available for S.
costatus in the literature, however, the densities observed here
(1.27 conch ha"') are considered low when compared with that of
Bermuda (2.6 conch ha"') (Berg et al. 1992) and the .Southwest
Dominican Republic (50-200 conch ha"') (Tewfik, unpubl. data).
It is suspected that the densities of S. costatus began to decline
only after the populations of the larger and more valuable fisheries
species, S. gigas. were already at low levels.
Although this study has no information available on conch
densities of <10 m, it did intensively survey habitats that are
known to be important for conch as nursery and breeding areas
throughout the region (Randall 1964, Sloner & Ray 1996. Tewfik
et al. 1998. Stoner 2003). We suspect that areas down to >20 m
may also have low densities, given the considerable capabilities of
artisanal free divers that have been observed in Panama and other
areas of the Caribbean (Martans 1997, Bene & Tewfik 2001 ). this
despite the refuge that deeper waters might provide for adults. The
low densities of conch and the lack of reproductive activity ob-
served during this study become quite serious when one considers
the "Allee effect," as described by Stoner and Ray-Culp (2000).
Negative rates of per capita population growth were shown to
occur below critical population levels. Specifically, mating (pair-
ing and copulating) never occurred when adult densities fell below
56 conch ha"', and spawning never occurred with densities below
48 conch ha"'. Again, no such reproductive activities were ob-
served during the entire 8 mo (February-September) of this study,
which covered the intense spring and summer reproductive period
for conch (Randall 1964, Buckland 1989. Stoner et al. 1992, Tew-
fik et al. 1998). This has serious implications for the future levels
of local recruitment and rebuilding of depicted populations, even
with the establishment of MPAs and strict enforcement of fisheries
regulations.
The spatial distribution of the two-strombid species was con-
centrated in the shallow (<5 m) SGs and is slightly surprising,
given that these areas are the most accessible to local fishers.
Another interesting element of the spatial distribution is that there
was relatively little overlap (5 sites) out of the 33 sites occupied by
either species (Fig. 3). This begs the question of whether there may
792
Tewfik and Guzman
TABLE 2.
Comparison of mean densities of S. gigas in tiie Caribbean determined by visual surveys.
Location
Conch ha
Reference
Antigua and Barbuda
Bahamas
Little Bahamas Bank
Great Bahamas Bank
Bermuda
Belize
Dominican RepubMc
Florida Keys
Haiti
Honduras
Cayos Cohinos
Jamaica
Pedro Bank (1994)
Pedro Bank (1997)
Morant Bank (1996)
Mexico
Panama
Puerto Rico
US Virein Islands
Juveniles
Adults (lip >4 mm)
1983/83
Unprotected Bank (1983/1983)
Protected Bank (1991/1994)
Protected Shelf ( 1991/1994)
1988
1989
Sub-legal (<15 cm)
Legal (>15 cm)
Juvenile (del Este 1996)
Adults (del Este 1996)
Juvenile (del Este 1997)
Adults (del Este 1997)
Juvenile (del Este)
Adults (del Este)
Juvenile (Jaragua)
Adults (Jaragua)
1987-1988
1990
Juveniles (Gonave Island)
Adults (Gonave Island)
Rochelios Bank
Western end
Juveniles
Adults
Juveniles (Artisanal Zone)
Adults (Artisanal Zone)
Juveniles (10-20 m)
Adults (10-20 m)
Juveniles (20-30 m)
Adults (20-30 m)
Juveniles (Artisanal Zone)
Adults (Artisanal Zone)
Juveniles (10-20 m)
Aduhs (10-20 m)
Juveniles (0-10 m)
Adults (O-IO m)
Juveniles (10-20 m)
Adults (10-20 m)
Juveniles (20-30 ni)
Adults (20-30 m)
Cozumel (1989)
Cozuniel (1995. after closure)
Bocas del Toro (0-10 m)
Southwest (1985/1986)
West (1995)
East (1996)
St. Croix (1981)
St. Thomas/St. John (1981)
St. Thomas/St. John (1990)
13.5
3.7
28.5
20.8
53.6
96.0
0.5
2.9
14.4
14.9
283.0
4.5
22.5
1.6
14.4
0.6
53.0
0.6
2.4
1.5
10.0
0.0
15.0
160.0
7.3
7.3
15.0
73.6
51.2
152.3
73.7
202.9
221.0
93.0
466.0
48.0
482.1
10.9
59.9
101.1
31.8
214.5
89.0
830.0
1.4
8.1
4.2
7.2
7.6
9.7
12.3
Tewtlk et al. (2001)
Tewfik et al. (2001)
Smith & Neirop (1984)
Smith & Neirop (1984)
Stoner & Ray (1996)
Stoner & Ray (1996)
Berg et al. (1992)
Berg et al. (1993)
Appeldoom & Roike (1996)
Appeldoorn & RoIke (1996)
Delgadoet al. (1998)
Delgado et al. (1998)
Delgado et al. (1998)
Delgadoet al. (1998)
Torres & SuUivan-Sealy (2000)
Torres & Sullivan-Sealy (2000)
Posada etal. (1999)
Posada et al. (1999)
Berg & Glazer (1995)
Berg & Glazer (1995)
Haitian Fisheries Division (pers. com.)
Haitian Fisheries Division (pers. com.)
Haitian Fisheries Division (pers. com.)
Haitian Fisheries Division (pers. com.)
Tewfik etal. (1998)
Tewfik etal. (1998)
Tewfik (1996)
Tewfik (1996)
Tewfik (1996)
Tewfik (1996)
Tewfik (1996)
Tewfik (1996)
Tewfik & Appeldoom (1998)
Tewfik & Appeldoom (1998)
Tewfik & Appeldoom (1998:
Tewfik & Appeldoom (1998)
Stephens (1997)
Stephens (1997)
Stephens (1997)
Stephens (1997)
Stephens (1997)
Stephens (1997)
Martinez Vasquez (1995)
Martinez Vasquez (1995)
This study
Torres Rosado (1987)
Mateo et al. (1998)
Mateo etal. (1998)
Wood & Olsen (1983)
Friedlander et al. (1994)
Friedlander et al. (1994)
be a true partitioning of suitable habitats and resources between the
two congeneric herbivores due to some form of competitive inter-
action. Berg et al. (1992) explained the differences in population
distribution between the two species as being due to differences in
habitat preference, and to the processes of larval dispersion, reten-
tion, and recruitment (see Stoner 2003). However, a true competi-
tive interaction (exploitative or interference) may also be possible,
as has been investigated for other groups of trophically similar
benthic plants (Williams 1987) and animals (Williams 1981, Teg-
ner& Levin 1982, Keller 1983).
Distributiiiii and Abundance of StronihK.s in Panama
793
In summary, this study concentrated on the population charac-
teristics of two common strombids o\er their critical shallow water
nursery and breeding habitats. Both species appear to be severely
overexploited within the archipelago. The present low densities,
combined with the suspected Allee effect, ultimately resulting in
decreased recruitment levels, could severely restrict recovery. In-
formation from this study will be combined with other surveys of
macrophyte (see Stoner 2003). algal, and other invertebrate distri-
butions to begin to understand the overall benthic community dy-
namics within the archipelago and elsewhere. Finally, it is hoped
that this baseline information mav also be useful in assessing the
success of the nationw ide 5-y ban on conch harvest that is under
consideration by the Panamanian government.
ACKNOWLEDGMENTS
This research was partially funded by the Fundacicin
Natura, the Fundacion Proteccion del Mar (PROMAR). and the
Smithsonian Tropical Research Institute. A. Domingo. C. Guevara,
L. Partridge, and W. Pomaire provided invaluable assistance in the
field. C. Mufioz developed the map in Geographical Information
System. The authors thank the Government of Panama for provid-
ing all necessary permits to work in the country.
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Joimml of Shclljhh Research. Vol. 22. N,.. 3. 79S-S()0. 2003,
WHEN IS THE ABALONE HALIOTIS DISCUS HANNAI INO 1953 FIRST ABLE TO USE
BROWN MACROALGAE?
HIDEKI TAKAMI.' ' DAISUKE MURAOKA,' TOMOHIKO KAWAMURAr AND
YOH YAMASHITA'
' Tohoku National Fisheries Research Instiliiie. Fisheries Research Agency, Shinhama, Shiogama. Miyagi
9S5-00U1. Japan: -Ocean Research Institute. The University of Tokyo. Minawiclai. Nakano. Tokyo
164-8639. Japan: and ^ Kyoto University Graduate School of Agriculture. Fisheries Research Station.
Nagahaina. Maizuru. Kyoto 624-0R31. Japan
ABSTRACT The dietary value of microscopic algal stages (gametophyte and juvenile sporophyte) of a brown alga Laminaria
jiiprmica. Areschoug 1851 and of the benthic diatoms Cyliiulniilwcii closlehiim (Ehrenberg) Reimann and Lewin 1964. andAclimmtlws
langipes Agardh 1824 were examined for different developmental stages of Huliotis discus lumniii Ino 1953 (0.4-2.9 mm shell length
(SL)| to determine the size at which abalone begin to use macroalgae efficiently. Most individual abalone showed active feeding
behavior, but there was considerable variation in growth of abalone between different algae and developmental stages of abalone. The
growth rates of smaller post-larvae (0.4-1.2 mm SL) fed gametophytes and juvenile sporophytes of L. japonka. or A. longipes were
significantly lower than those fed C closteriwn. In contrast, juvenile sporophytes of L. juponica and A. longipes produced significantly
faster growth in larger postlarval abalone (>1.8 mm SL) than gametophytes of L. japcmica or C. closterium. Postlarvae in all
developmental stages fed C. closterium actively grazed and efficiently ingested diatom cells. However, the relative dietary value of C.
closterium decreased as abalone grew, probably because feeding efficiency on this diatom decreased because of its low cell volume
and thin film-like colonies. Smaller post-larvae (0.4-1.2 mm SL) grazed repeatedly on the same .surface of gametophytes. juvenile
sporophytes of L. juponica. or on A. longipes without detaching these algae, whereas larger post-larvae (>1.8 mm SL) detached and
ingested large amounts of whole cells of these algae. Postlarval abalone 01.8 mm SL) began to use L. japonica gametophytes and
juvenile sporophytes at approximately the same size at which morphologic changes occurred in their radulae, which enabled the
ingestion of macroalgae.
KEY WORDS:
phyte
benthic dialoni. brown alga, dietary value, gametophyte. growth. Haliotis discus liannin. postlarval abalone. sporo-
INTRODIICTION
Survival and growth rates in early life stages of abalone Hali-
otis discus banned Ino 1953 are considerably affected by food type
and the ability of individuals to use available food (Kawainura &
Takami 199.^. Kavvamura et al. 1995, Seki 1997. Takami et al.
1997a. 1997b. Takami et al, 2000. Sasaki & Shepherd 2001,
Takami 2002). Understanding the abalone' s early life feeding hab-
its considered to be important in improving the rearing techniques
in abalone hatcheries and also in understanding the factors con-
trolling natural recruitment (Kawaniura et al. 1998a. Sasaki &
Shepherd 2001, Takami 2002).
As young of//, discus liannai grow, the main food sources shift
frotn benthic diatoms to macroalgae (Kawamura et al. 1998a,
Takami 2002). For postlarval abalone. benthic diatoms are the
principal foods. The dietary value of diatoms for postlarvae is
significantly different between diatom species or strains and is
controlled largely by the ingestibility and digestibility of diatoms.
Limited diatoms produce high digestion efficiencies and thus rela-
tively rapid postlarval growth (Kawamura & Takami 1995. Kawa-
mura et al. 1995, 1998a, 1998b, Roberts et al. 1999a). Attachment
strength of diatoms is one of the factors that affects diatom digest-
ibility for postlarval abalone (Kawamura et al. 1995. 1998a.
1998b. Roberts et al. 1999a). Very tightly attached diatoms, such
as Cocconeis spp. and Achnanlhes spp.. require considerable force
to be detached from substrata and are usually ruptured if dislodged.
In contrast, many diatoms with low adhesive strength are ingested
without cell rupture, and the majority of ingested cells pass
*Corresponding author. E-mail: htakaiiii@affrc.go.jp
through the gut alive and unbroken. There are some exceptional
diatom species, such as Cylindnitheca closterium (Ehrenberg) Rei-
mann and Lewin 1964. which has low attachment strength but is
subject to high digestion efficiencies and supports rapid growth of
postlarvae. probably because of its weak silica frustule. which is
easily broken (Kawamura et al. 1995. 1998a. 1998bl. Cocconeis
spp.. which have a high attachment strength and a relatively high
dietary value for postlarval H. discus hannai larger than -0.8 mm
shell length (SL; Kawamura et al. 1995. Takami et al. 1997a), are
often dominant in the habitat of postlarval abalone in the natural
environment (Kawamura et al. 1992. Takami 2002) and are used
for rearing postlarvae in abalone hatcheries (loriya & Suzuki 1987,
Suzuki et al. 1987). Benthic diatoms, such as Cocconeis spp., are
probably one of the important diets for postlarval abalone in their
natural habitat. In contrast, it has been suggested that juvenile
abalone of more than 10 mm SL do not graze Cocconeis species if
more favorable foods are available (loriya & Suzuki 1987. Suzuki
et al. 1987). This is because Cocconeis spp. are not efficient food
sources for these larger juveniles because their low-volume cells
and prostrate growth form provides little energy (Takami et al.
1996).
Large juveniles (>10 mm SL) and adult H. discus hannai prefer
to feed on brown macroalgae especially Laminaria spp. (Sakai
1962. Kikuchi et al. 1967. Uki 1981 ) and show rapid growth rates
when fed these algal species (Kikuchi et al. 1967. Uki 1981. Uki
et al. 1986). Evidence from natural habitats suggests that the diet
of abalone becomes dominated by macroalgae as juveniles grow
(Tomita & Tazawa 1971. Shepherd & Cannon 1988). However, it
is not clear at what size H. discus hannai begin to use macroalgae.
Moreover, most of the food value experiments of brown macroal-
gae for abalone have been conducted with mature algae whose
795
796
Takami et al.
tolerance to herbivory may be different from juvenile algae (Van
Alstyne et al. 1999, 2001). From the standpoint of physical as-
pects, small abalone may be able to ingest juvenile macroalgae
more easily than mature macroalgae.
In this study, we compared the dietary value of microscopic
algal stages (gametophyte and juvenile sporophyte) of Lciminaria
japonica Areschoug 1851 and benthic diatoms for different devel-
opmental stages of W. discus hannai to determine the size at which
abalone begin to use macroalgae efficiently.
MATERIALS AND METHODS
Reproductive fronds of L. japonica were collected from the
subtidal zone. Hokkaido Japan in October 2000. To obtain
zoospores, fragments (2-3 cm~) of reproductive fronds were rinsed
with sterilized seawater and placed separately in glass culture ves-
sels containing sterilized seawater. To obtain zoospores, fertile
fragments (2-3 cm~) of the desired algae were rinsed with steril-
ized seawater and placed separately in 200-mL glass beakers con-
taining sterilized seawater. Newly liberated zoospores were pipet-
ted to 50-niL polystyrene or 200-mL glass beakers containing
PESI medium (Tatewaki 1966). Beakers were kept in a growth
chamber at I5°C and 43-1 13 nE/m'/s on a 12:12 LD cycle, and
zoospores were allowed to settle on to the surface of the beaker.
The settlement density was 25-30 zoospores/mm~. Any diatom
contaminants were not observed in the beakers. After 5-8 days of
incubation, morphologic differences were observed between fe-
male and male plantules. Two types of microscopic algal stages of
L. japonica (haploid gametophytes and diploid sporophytes) were
used for the experiments. Gametophytes were kept in a growth
chamber at 25°C to inhibit maturation, whereas the sporophytes
were kept at 15°C to promote maturation (Yabu 1964). Juvenile
sporophytes were allowed to grow until the size of thalli reached
0.5-1 mm in length. Gametophytes grew prostrate across the sur-
face of the vessel, whereas juvenile sporophytes grew erect and
formed three-dimensional colonies.
Benthic diatoms Cylindrotlieca closteriuni and Achnanlhes lon-
gipes Agardh 1 824 were also used as food items for abalone. These
benthic diatoms were isolated from an abalone nursery tank at
Tohoku National Fisheries Research Institute, Miyagi Japan, and
were grown following the methods of Kawaniura et al. ( 1995).
Larval abalone were hatched m May and October 2000 at the
Yamagata Sea Farming Association (Yamagata. Japan) and reared
using the method of Uki and Kikuchi (1984). Four days after
fertilization at 20'C, the veliger larvae were transported to Tohoku
National Fisheries Research Institute within 4 h. Competent larvae
were transferred to 200-mL glass beakers with 150 mL of au-
tocaved filtered (0.45 (jim: Millipore HA) natural seawater (FSW)
containing 150 (xg/mL each of penicillin G sodium and strepto-
mycin sulphate BP. These larvae were induced to metamorphose
by the addition of I jjlM 7-amino butyric acid (Takami et al. 2000).
Four days after metamorphosis induction, an adequate number of
C. closteriuin cells were added as a food supply. The rearing
beakers were incubated in light at 31-53 jiE/m'/sec on a 12:12 LD
cycle. These abalone were maintained as a source of experimental
animals, by adding supplementary C. closteriuin cells and replac-
ing the water every 3—4 days with new FSW without antibiotics.
All chemicals were obtained from Wako Pure Chemical Industries
(Osaka, Japan).
Si.\ experiments were conducted using different size classes of
abalone. Detailed information on the experiments is presented in
Table I . Before each experiment, abalone were dislodged with a
fine needle from the stock beakers and placed into a 50-mL poly-
styrene dish with 25 mL of FSW containing 6 mg/L of GeO,
without food for a period of 2 days in the dark. GeO, effectively
inhibits the proliferation of diatoms attached to abalone and does
not affect the survival and growth of animals (Takami et al.,
1997b). Most C. closteriuni cells ingested by abalone were di-
gested, so any contamination by live diatom cells from abalone
feces was negligible (Kawamura & Takami 1995, Kawaniura et al.
1995, Roberts et al. 1999a).
Active postlarval abalone were placed into 50-mL polystyrene
(Exp. I-IV) or 200-mL glass beakers (Exp. V, VI) in which each
algal diet was available (Table I ). Beakers were submerged in a
35-L tank. Beakers containing experimental animals and algal di-
TABLE 1.
Details of experimental treatments.
Initial Shell Length
Rearing
Duration of
Exp.
of .\balone
Temperature
Experiment
Number
Number of
No.
Algal Species
Algal Type
((jm, mean ± SE)
( C)
(days!
of Rearing
.Abalone per Beaker
I
Liiimiiiina japonica
Gametophyte
458 ± 6.4
20 ±1
7
3
5
Cylimiroiheca closteriiim
Benthic diatom
447 ± 6.9
20 ±1
7
3
5
U
Achmmlhes loiigipes
Benthic diatom
674 ± 5.4
20 ± 1
7
5
10
Cylimiroiheca closteriiim
Benthic diatom
657 ± 5.5
20 ± I
7
5
10-12
III
Laminaria japonica
Gametophyte
898 ±21
20 ±1
8
3
5
Unninaria japonica
Juvenile sporophyte
860 ± 17
20 ±1
8
3
5
Cylindrotheca closteriuin
Benthic diatom
854 ± 23
20 ±1
8
3
5
IV
Laminaria japonica
Gametophyte
1092+14.7
20 ±1
7
3
5
Laminaria japonica
Juvenile sporophyte
1152 ±28.0
20 ± 1
7
3
5
Cylindrotheca closteriuin
Benthic diatom
n21±21.2
20 ± 1
7
3
5
V
Unninaria japonica
Gametophyte
2008 ±51.6
17+ 1
10
3
5
Laminaria japonica
Juvenile sporophyte
2106 + 81.4
17± 1
10
3
5
Cylindrotheca closteriuin
Benthic diatom
1874 ±78.7
17± 1
10
3
5
VI
Laminaria japonica
Juvenile sporophyte
2894 ±153
17± 1
10
3
2
Achnanthes longipes
Benthic diatom
2809+ 137
17± 1
10
3
2
Cylindrotheca closteriuin
Benthic diatom
2711 ±90
17+ 1
10
3
-)
When Does an Abalone Begin to Use Macroalgae?
797
ets were covered with a 200 (Exp. I-IV)- or a 600 (Exp. V, VlVixni
nylon mesh to allow water exchange. Incoming filtered (1 |xm)
natural seawater was maintained at a flow rate of approximately
2.4 L/min into the tanks. The rearing temperatures were set at a
temperature that abalone of specific developmental stages encoun-
ter in the Miyagi coast. Because the spawning season of H. discus
hamuli in the area is from late summer to mid autumn, postlarvae
encounter decreasing temperature as they age. SL of live individu-
als in each experiment was measured to the nearest 10 [ji,m using
a monitor and video camera system with an image analyzer, con-
nected to an inverted microscope (Exp. I-Vl) or a dissecting mi-
croscope (Exp. V. VI) at the beginning and at the end of the
experiment. The feeding behavior of abalone was observed at in-
tervals of 1-3 days using an inverted microscope.
The differences between survival and growth rates of treat-
ments were tested using Student's r test (Exp. I. II) or Tukey-
Kramer multiple comparison test (Exp. III. VI). Sur\ i\al data were
arcsine-transformed before analysis to normalize the data.
RESULTS
In all experiments, considerable variation was found in the
growth rates of abalone between both algal types and developmen-
tal stages of abalone (Fig. 1 ). even though most individuals were
observed actively feeding. The growth rates of postlarval abalone
of 0.4— 1.2 mm SL that were fed gametophytes and juvenile sporo-
phytes of L. japonica were significantly lower (14-17 (j.m/day)
Exp.l
(0.4-0.£
T '
mm)
n
T
=3
■
1
Exp.ll (0.4-0.7 mm)
n=5
b
•
n=5
.
1
LiG
Cc
Exp III (0 80-9 mm) "=3
b
■
— 1 —
m
LjG
IjS
Cc
120-
Exp, VI (2-8-2,9
r=3
mm)
ion •
T"
i
n=3
80 ■
60 ■
1
r4
40 •
20
n-
— 1—
■
— 1 —
Ij S Al Cc
Figure 1. Growth of six developmental stages of postlarval Haliolis
discus hannai Ipm per day) fed ganietophjles of iMiniitaria japonica
(Lj (;), juvenile sporophytes of L. japonica (Lj Si, the benthic diatom
Achnanllies longipes (,\ll, or the benthic diatom Cylindrolhcca cluste-
riuni (C'cl. Numbers in parentheses indicate the range of Initial shell
length of abalone used for each experiment. Each bar represents mean
± SE with the number of replicates. Letters on the top of each column
indicate the results of Student's / test (Exp, I, II) or Tukey Kramer
multiple comparison (Exp, III-VIl tests; columns with different letters
represent means that are statistically different (/' < 0,(15).
than those of postlarvae fed the benthic diatom C. closterium (21-
44 jim/day; Fig. 1 ; Exp. I. III. IV. P < 0.05). Differences in growth
rates between postlarvae fed C. closterium and gametophytes or
juvenile sporophytes were larger for postlarvae of 0.8-1 .2 mm SL
(Fig. 1: Exp. Ill, IV) than of 0.4-0.5 mm SL (Fig. 1: Exp. I).
Growth rates were not significantly different between postlarvae
fed gametophytes and juvenile sporophytes in Exp. I. 111. and IV
{P > 0.05). For larger postlarvae (> 1.8 mm SL), juvenile sporo-
phytes produced significantly faster mean growth (81-95 jxm/day)
than gametophytes (41 (xm/day, Exp. V, P < 0.05) or C. closterium
(58 |xm/day. Exp. VI. P < 0.05).
Postlarvae of 0.6-0.7 mm SL (Fig. 1; Exp. II) fed A. longipes
showed significantly lower growth rates (9 (xm/day) than those fed
C. closterium (33 p.m/day. P < 0.05). In contrast, the mean growth
rate of post-larvae of 2.8-2.9 mm SL (Fig. 1: Exp. VI) fed A.
loiii;ipes was significantly higher ( 100 ixm/day) than that of post-
larvae fed C. closterium (58 (a.m/day. P < 0.05).
The postlarvae that were fed C. closterium actively grazed and
efficiently ingested diatom cells in all the experiments. Smaller
postlarvae <1.2 mm SL (Exp. I-IV) grazed repeatedly on the same
area of gametophytes. juvenile sporophytes. or A. longipes uithout
detaching these algae. We could not directly observe the ingestion
of algal diets by larger postlarvae (>1.8 mm SL; Exp. V, VI)
ingested algal diet or not because most of the abalone stopped
feeding when we tried to observe them under the microscope.
However, we concluded that larger postlarvae ingested large
amounts of gametophytes and sporophytes of L. japonica because
these algae were almost completely cleared from the substratum
and many feces remained. Ruptured cells of A. longipes were
observed in the fecal pellets of postlarvae in Exp. VI but not in
Exp II.
Significantly lower survival rates were detected when smaller
postlarvae were fed L japonica gametophyte (Exp. I) and A. lon-
gipes (Exp. II: Fig. 2. P < 0.05) rather than C. closterium. In Exp.
III-VI, the survival rates of individuals were generally high (80-
100%) except for Exp. IV (Fig. 2) when many contaminant pro-
tozoans were observed in all rearing beakers.
DISCUSSION
The results of this study show that dietary values of gameto-
phytes and juvenile sporophytes of a brown alga, L japonica, vary
depending on the developmental stage of abalone. Most smaller
postlarvae (<1.2 mm SL) could not etTiciently detach either ga-
metophytes and sporophytes when feeding. In contrast, larger post-
larvae (> 1 .8 mm SL) detached and ingested these brown algae and
showed comparable or faster growth rates than those fed a benthic
diatom. C. closterium (Fig. 1 ).
In the experiments using smaller postlarvae < 1.2 mm SL (Exp.
1-IV). abalone fed C. closterium showed the highest growth rates.
This diatom species has a weak silica frustule and low attachment
strength; therefore, abalone can ingest and break the diatom cells
resulting in high ingestion and digestion etTiciencies of abalone.
The differences in growth rates between postlarvae fed L. japonica
and C closterium were more marked on animals of 0.8-1.2 mm SL
(Exp. III. IV) than those of 0.4-0.5 mm SL (Exp. I). These results
correspond to the changes in abalone feeding (Kawamura et al.
1998a). The energy source of postlarvae is graduall) transferred
from yolk supply to particulate food after metamorphosis at a size
of -0.4—0.5 mm SL. Young postlarvae can grow using mucus
materials secreted from diatoms (Kawamura & Takami 1995) and
798
Takami et al.
100 '
Exp.
(0.4.0,
T °
S mm)
T '
3
3
50 •
0 •
1
LjG
Exp.lll (0.8-0.9 mm)
n.3 n=3
L
o f *■ [ ^H
■
0 1 1 P — ""i 1 1 1
loo-
se ■
(1 ■
Exp. IV (1.0-1.2
— 1 1 — 1 —
mm)
=3
Tn=3
o
II
LjG
lis
Cc
L|G
US
Cc
100 ■
Exp.V(1.8-2 2mm)
n=3
100 •
Exp VI (2 8-2.9 mm)
n=3 1
o
0
° ■"
w*
50 H
50 -
0 •
— 1 —
0 -
— 1 —
£^
— 1 —
LiG
US
LjS
Figure 2. Survival rates of six developmental stages of postlarval Hali-
Otis discus hannai fed ganietophytes of Laminaria japonica (Lj G),
juvenile sporophytes o{ L. japonica (Lj S). the benthic diatom Achnan-
thcs longipes (Al), or the benthic diatom Cylindrolheca closterium (Cc).
Numbers in parentheses indicate the range of initial shell length of
abalone used for each experiment. Each bar represents mean ± SE
with the number of replicates. Letters on the top of each column
indicate the results of Student's / test (Exp. \. II) or Tukey Kramer
multiple comparison (Exp. III-VI) tests; columns with different letters
represent means that are statistically different (/• < 0.05).
macroalgae, such as crustose coralline algae (CCA; Daume et al.
1997, Kitting & Morse 1997, Takami et al. 1997b) supplemented
by residual yolk supply fTakami et al. 2000. Roberts et al. 2001 )
and possibly absorption of dissolved organic matter (Shilling et al.
1996). At around 0.6-0.8 mm SL, postlarvae become responsive to
the digestibility of diatom diets and grow more rapidly on effi-
ciently digested diatoms (Kawamura et al. 1995. Takami et al.
1997a, Kawamura et al. 1998b, Roberts et al. 1999a). Dietary
benefits are size dependent in postlarval abalone (Roberts et al.
1999a). Postlarvae of 0.6-1-2 mm SL who were fed ganietophytes,
juvenile sporophytes, and A. longipes could not get adequate en-
ergy sources for rapid growth because of the difficulty in ingestion
of these algae and possibly insufficient amount of secreted mucus
(Kawamura & Takami 1995, Kawamura et al. 1998a).
The growth of postlarval H. discus hannai of 1.3 mm SL fed
thinly sliced fronds of brown alga ihuhiria pinnatifula was com-
parable to that of abalone fed diatoms (Sakai 1976). H. discus
discus of 3— i mm SL fed softened fronds of U. pinnatifula (Fujii
& Yotsui 1989) and germlings of macroalgae (Maesako et al.
1984) also showed good growth rate (72-1 10 (im/day). Takami et
al. (1998) reported that postlarval H. discus hannai of 1 mm SL
had a suite of enzymes useful for digesting brown algal polysac-
charides and these enzyme activities increased rapidly from ap-
proximately 2 mm SL, suggesting they could use macroalgae if
they could ingest the algal fronds. The ingestion efficiency of
postlarvae on algal diets is determined by the radula morphology
(Roberts et al. 1999a, 1999b, Kawamura et al. 2001). The major
ontogenetic changes in the radula structure occur around 1-2 mm
SL for postlarval H. discus hannai (Kawamura et al. 2001). For
example, the adult complement of five pairs of lateral teeth was
completed by 1 .9 mm SL. A rapid increase in the clearance angle
of the radula (Padilla 1985) was observed in postlarval H. discus
hannai between 1-2 mm SL (Kawamura et al. 2001). Postlarvae
>1 mm SL develop radula teeth w ith positive clearance angles that
are more suitable for cutting rather than just sliding across the
substratum. Larger post-larvae have well-developed outer lateral
teeth (L3-L5 teeth), which appear to be used to cut the elastic
macroalgae and three-dimensional growth forms of benthic diatom
such as A. Umgipcs. In H. discus liannai larger than 1 .5 mm SL, the
L3-L5 teeth become longer and more pointed (Kawamura et al.
2001). The results of this study show that postlarval H. discus
liannai begin using ganietophytes and juvenile sporophytes almost
at the same size at which major morphologic changes in radula
occur.
The relative dietary value of C. closterium decreased as post-
larvae grew (Fig. 1). Three-dimensional A. longipes colonies and
juvenile L. japonica sporophytes provide a much higher biomass
per unit area than low volume, two-dimensional C. closterium
films, once post-larvae are able to detach and ingest them. The
significant difference in growth rate between post-larvae fed ju-
venile sporophytes and ganietophytes (Fig. 1; Exp. V) might be
caused by differences in the algal growth forms, because gameto-
phytes also show prostrate growth form.
In Exp. I and IL the survival rates of postlarvae fed ganieto-
phytes of L japonica (66.7%) and A. longipes (65.6%) were sig-
nificantly lower than those fed C. closterium (92.7-93.3%; Fig. 2).
This low survival was not considered to be caused directly by
starvation because abalone at these stages could survive more than
15 days of food deprivation (Takami & Kawamura. unpubl.).
There is a possibility that the diffusive boundary layer (DBL),
where diffusion dominates molecular transport, severely affects
survival of post-larval abalone because oxygen concentrations in
the dark may be reduced whereas algal secondary metabolites
increase affecting water quality (Searcy-Bemal 1996, Roberts et
al. 2000). The DBL "water quality" probably depends upon culture
condition, and the algal strains and species used. Algal species
with three-dimensional growth forms have a thicker DBL than the
diatoms that form flat film-like colonies (Roberts et al., 2000);
therefore, the DBL produced by A. longipes may have had reduced
"water quality" and affected survival of the smaller postlarvae.
Another possibility is that the combination of nutritional stress and
water quality stress causes mortality more quickly than seen from
starvation in clean containers.
Larval H. discus hannai settle preferentially on CCA in the
natural environment, and grow on CCA for several months (Saito
1981, Sasaki & Shepherd 1995, 2001, Takami 2002). Food sources
from CCA include the alga's surface polysaccharides and epithe-
lial cell contents, which can keep postlarvae alive but are not
adequate to support rapid growth (Garland et al. 1985. Daume et
al. 1997. Kitting & Morse 1997, Takami et al. 1997b). CCA rely
on grazing by herbivores to prevent their surfaces from being
covered with competitively superior algae (Paine 1980. Steneck
1982). Grazing-resistant algae with strongly adhesive prostrate
forms such as benthic diatoms Cocconeis spp. tend to dominate
under high grazing pressures by relatively large gastropods (Kesler
1981, loriya & Suzuki 1987, Suzuki et al. 1987, Steinman et al.
1989, Kawamura et al. 1992). Because grazing gastropods occur at
high densities on CCA (Ayling 1981, Choat & Schiel 1982, Kawa-
When Does an Abalone Begin to Use Macroalgae?
799
mura et al. 1992. Takami. 2002). Cocconeis spp. are ot'leii domi-
nant and appear to be the main food sources for early life stages of
abalone on CCA (Kawamura 1994. Takami 2002). However. Coc-
coneis films probably become energetically inadequate as juvenile
grow, and juvenile abalone come to rely on three-dimensional
algal populations for food (Takami et al. 1996. Kawamura el al.
1998a).
The germling or ju\enile stage of macroalgae is generally sus-
ceptible to grazing by herbivores (Lubchenco 197S. Robles &
Cubit 1981. Lubchenco 1983. Dayton 1983. Dean et al.. 1989.
Asano et al. 1990. Paine 1992. Martinez & Santelices 1998, Van
Alstyne et al. 1999. 2001). Therefore, newly recruited juvenile
algae may find it difficult to grow on CCA surfaces. However,
northern Japanese Laminarian species have prodigious reproduc-
tive output, consequently they have considerable potential tor
dense recruitment if grazing pressure is low (Yendo 1911. 1919).
The season of se.xual reproductive in L. jciponica in Miyagi is from
late autumn to mid winter when grazers' activities are relatively
low due to the low water temperature. By this tiine. most of the
0-y-old abalone are more than 2 mm SL (Sasaki & Shepherd 1995,
2001. Takami 2002). a size at which they can efficiently ingest
ju\ enile sporophytes of L. japonica. Juvenile L. japonica may be
an important food source for these abalone at this early life stage.
ACKNOWLEDGMENTS
We thank Hiroyuki Kawakami of Yamagata Sea Farming As-
sociation for providing the hir\al abalone. The critical readings of
this manuscript by Christopher Clarke and Rodney Roberts are
gratefully acknowledged. This study was supported in part by a
grant-in-aid (Development of seed production and releasing tech-
niques for stock enhancement of marine resources considering the
conservation of ecosystem) from the Ministry of Agriculture. For-
estry and Fisheries. Japan.
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Joiimul of Shellfish Research. Vol. 22. No. 3, 8UI. 2003.
PROCEEDINGS OF
WORKSHOP ON REBUILDING TECHNIQUES FOR ABALONE IN
BRITISH COLUMBIA
Nanaimo, B.C. Canada
January 14-16. 2003
Guest Editor
Alan Campbell
Department of Fisheries and Oceans
Pacific Biological Station
Nanaimo. British Columbia V9T 6N7
CANADA
801
Journal nf Slwllfish Rcscanh. Vol. 22. No. 3. S()3. 2003.
PREFACE
The lolknving 6 papers and 1 1 abstracts published in this issue
of the Journal of Shellfish Research are part of 17 presentations
delivered at an international workshop on rebuilding techniques
for abalone in British Columbia (BC) held at Nanaimo. BC.
Canada. January 14 to 16. 2003 (Campbell & Heimstra 200.^). The
decline of northern (pinto) abalone {Hulinlis kanitscliatkaua)
stocks since the late 1970s has prompted fishery closure since
1990. listing this species as "threatened" by the Committee on the
Status of Endangered Wildlife in Canada in 1999. two interna-
tional workshops (Campbell 2(X)0. Campbell & Heimstra 2003).
and development of a national recovery strategy for H. kamtwhcil-
kana in BC (Toole et al. 2002).
Over exploitation and declines in wild abalone populations
base occurred m mam parts of the world and the methods for suc-
cessfully rebuilding wild stocks are still in the developmental stage.
The workshop discussed community stewardship projects, aquacul-
ture. out planting and restocking, wild stock manipulation, and moni-
toring tools and evaluation performance indicators as methods for
abalone rebuilding. The 6 papers published in this volume represent
some of the presentations at the workshop. The papers underwent
the stringent refereeing and review process required by this jour-
nal. I thank the authors and the many referees for their efforts and
co-operation for reviewing and revising the manuscripts.
ALAN CAMPBELL
Editor
LITERATURE CITED
Campbell. A., editor. 2000. Workshop on rebuilding abalone stocks in
British Columbia. Can. Spec. Publ. Fish. Aquat. Sci. 130. 158 pp.
Campbell. A. & L. Hiemstra.. editors. 2003. Proceedings of the workshop
on rebuilding techniques for abalone in British Columbia. Can. Tech.
Rep. Fish. Aquat. Sci. (In press)
Toole. J.. B. Adkins. E. Bomhold. J. Boutillier. G. Caine, A. Campbell, A.
Castledine. L. Convey. C. Cote. P. Coulson, T. Down. K. Francis. H. Gill.
R. Harbo. H. Holmes. B. Jubinville. D. Lawseth, B. G. Lucas, A. Morgan, G.
Parker & J. Rogers. 2002. National Recovery Strategy for the Northem Aba-
lone {Haliotis kamtschatkana) in British Columbia Fisheries and Oceans
Canada, (http://www-conim.pac.dfo-mpo.gc.ca/pages/consultations/
fisheriesmgmt/abalone/AhaloneRecovStrategy_e.html. 22 pp.
803
Joiinnil ofSluilfish Hcscunh. Vol. 22. No. 3, iS()5-SI0. 2U0.i.
UPDATE ON EMERGING ABALONE DISEASES AND TECHNIQUES FOR
HEALTH ASSESSMENT
SUSAN M. BOWER
Departiuciii of Fislieries and Oceans. Pacific Biologic Station. Nanaimo. British Cohiiuhia. \VR 5K6
ABSTRACT This article presents a review of new diseases and additional information on known pathogens of abalone that were
encountered in the last few years as a result of increasing efforts towards the culture of abalone around the world and concurrent
investigations into abalone health. A novel haplosporidian was associated with high mortalities (82. ,^-90%) of cultured juvenile paua
t.Hiiliotis iris) in New Zealand. Disease outbreaks among cultured abalone in Tasmania. Australia were associated with two species of
Viljrio {V. Imn-eyi and V. splciulidiis I) and a Flciv(ilnicterium-\ike bacterium with stress factors precipitating the diseases in most cases.
The agent of withering syndrome responsible for mass mortalities of black abalone (W. cracherodii) in southern California was
identified as the Rickettsiales-Iike prokaryote "Camiuiatus Xenohaliotis califomiensis". The exotic sabellid polychaete that seriously
impacted abalone culture in California was named Terehrasalyelta heterouncinuta and experimentally found to reproduce at low
temperatures but with a significantly temperature-dependant generation time (a developmental cycle of 298 days at ll.2°C in
comparison to 165 days at 15.6°C). To assess the health of cultured abalone. histologic examinations are essential. For histology, tissue
samples (less than 1 cm thick) should be fixed in Davidson's solution or 10"^ formalin in seawater such that there is at least 10 parts
fixative to 1 part tissue. Histopalhology will not only indicate the presence of infectious agents but can be useful for monitoring the
suitability of the diet and aquaculture environment. These assessments will benefit abalone aquaculture and provide assurance that only
healthy animals are used in stock rehabilitation programs.
KEY WORDS: disease, parasites, abalone. Huliotis
INTRODUCTION
The decline tif wild stocks of abalone around the world and the
increasing demand for this product in the market place has in-
creased efforts in the culture of various abalone species and inter-
est in rehabilitating wild stocks. Concurrent with this increased
attention to abalone, awareness of the various infectious diseases
of abalone has arisen. Prior to 2000, six severe diseases associated
with mortality in various species of abalone had been reported in
the literature (Table I Bower 2000). Since thai lime, knowledge of
.some of these diseases has increased and other infectious diseases
have been detected.
Various investigations have revealed that infectious diseases
can have a significant negative impact on the aquaculture of aba-
lone (Elston & Loekwood 1983. Oakes & Fields 1996, Bower
1987a, Li et al. 1998. Lizarraga-Partida 1998, Nishimori et al.
1998. Ruck & Cook 1998, Kuris & Culver 1999. Caceres Martinez
et al. 2000. Moore et al. 2000b. Diggles el al. 2002). Because
infectious diseases can be equally disastrous if inadvertently in-
troduced into new locations by stock rehabilitation efforts that
involve the translocation of abalone, it is important to be aware of
the available information on diseases to circumvent complications
(Sinderman 1988). The current low abundance of wild stocks of
northern abalone [Haliotis kamtschatkaiun in British Columbia
has stimulated the culture of this species and the development of
plans for rehabilitation. In conjunction with this effort, abalone
will be examined for infectious diseases. Thus, it is prudent to have
information on diseases that have affected abalone around the
world and on procedures used to assess abalone health at hand.
This paper presents information on abalone diseases that was pub-
lished since the review by Bower (2000) and describes techniques
that can be implemented to examine abalone for infectious disease.
Although directed towards concerns for northern abalone. the in-
Phone: E-mail: BowerSCa'dfo-mpo.gc.ca
formation provided herein is directly applicable to all abalone
species regardless of locatiini.
UPDATE ON ABALONE DISEASES
.Since 2000. new significant diseases of abalone have been
encountered during efforts to culture abalone in various parts of
the world and new information has been published on previously
known diseases.
In New Zealand, a novel haplosporidian was associated with
high mortalities (82.5-90'7f) of cultured juvenile paua {Haliotis
iris) at one farm in the eastern Bay of Plenty (Diggles et al. 2002).
Runts were more severely affected but. all infected abalone
showed weak adherence to the substrate, had a shriveled foot with
pale blister-like lesions on the foot and mantle, and failed to right
themselves when turned over. In lightly infected abalone. uni- to
multinucleate plasmodia (up to 1.3.5 (xm in length) occurred in the
connective tissue surrounding the gut, amongst glial cells adjacent
to the nerves of the mantle and foot and within the gill lamellae. In
heavy infections, numerous plasmodia were present in the
hemolyinph, gills, heart, kidneys, mantle, foot, epipodium, and
connective tissue of the digestive gland. Spore formation was not
observed but sporocyst-like bodies were found amongst plasmodia
in the right kidney of an adult paua collected from the wild (Hine
et al. 2002. Diggles et al. 2002). Research into this new pathogen
is on going in New Zealand.
Various bacteria have also been isolated from cultured abalone
experiencing disease and mortalities. In Tasmania. Australia, dis-
ease outbreaks among cultured abalone {Haliotis rubra. H. laevi-
gata and their hybrids) were associated with two species of Vibrio
(V. haireyi & V. splendidiis I) and /•7<nv)/x/(7fni»«-like bacterium.
In most cases, stress factors (e.g., high temperatures, grading
trauma, anesthetics, gradual increase in salinity in the recirculation
system, etc.) were reported to have precipitated the diseases ( Hand-
linger etal. 2001,Handlingeretal. 2002). In Kanagawa Prefecture,
Japan, Vibrio carchariae (possibly a junior synonym of V';7)nV)
han'eyi) was isolated from cultured abalone {Haliotis ( = Sulculus)
diversicolor supratexta) experiencing a mass mortal-
805
806
Bower
TABLE 1.
Sunimary of diseases reported from abalone prior to 2000. For details and references sec Bower (2000).
Category
Pathogen/Disease
Known Distribution
I. Cause severe disease and mortality
2. Parasites of lesser concern
3. Detrimental under adverse conditions
Vibrio fluviuli.s Il/pustule disease
Lahyrintliiiloides haliolidis
Perkinsus olseni
Sabellid polycheate
Withering foot syndrome
Amyotrophia
Ciliates
Margolisiethi halioiis
Echinoceplmhis psciidoiincinauis
Trematode parasitism
Ubiquitous opportunistic organisms
Shell-boring organisms
vicinity of Dalian. China
British Columbia. Canada
South Australia
California. USA; Baja California, Mexico; southern Africa
California. USA
western Japan
Global-specific studies from southern Africa
California. USA
Southern California. USA; Gulf of California. Mexico
Global
Global
Global
ity. In this case, white spots consisting of necrotic muscle fibers
and bacteria on the abalone foot accompanied by high mortalities
were characteristic of the disease (Nishimori et al. 1998). Vihria
canhariae was also identified as the probable cause of mass mor-
talities of HtiliDtis tiibcniilata in the natural environment along the
Brittany and Nortiiandy coasts of France and in a land-based aba-
lone farm in Normandy (Nicolas et al. 2002). Dixon et al. ( 1991 )
repotted that exposure to ozonated water and treatment (bath and
injection) with a broad spectrum antibiotic (sulphadimidine so-
dium) was effective against bacterial infections (caused by
Clostridium iitiiseberense or Vibrio ali;inolyticiis) in some abalone
(Haliotis midae) in a South African experimental facility. How-
ever. Handlinger et al. (2002) found antibiotic use to give equiv-
ocal results on bacterial infections in Tasmanian fanned abalone.
In addition to the newly encountered diseases, new information
has been published on previously known diseases (Table 1 ). Tai-
wu et al. (2000) reported that phage particles isolated from Vibrio
fhivialis-ll and inoculated into Haliotis discus hannai suffering
from pustule disease caused by this bacterium in China raised
abalone survival rates by up to 50%. In Australia, field studies
using tnolecular detection techniques indicated that infections with
the protistan Perliiiisiis olsciii in wild Haliotis rubra at Taylor
Island. South Australia, was positively correlated with both water
temperature and size of abalone. Also, the parasite was being
maintained by H. rubra with negligible contributions from other
su.sceptible abalone species or other mollusks (Lester et al. 2001 ).
Subsequent data and analysis by Hayward et al. (2002) indicated
that the transmission of P. olseiii among the wild H. rubra ap-
peared to be reduced and infections were less severe in 2002. This
apparent reduction in disease was attributed to lower maximuin
summer sea surface temperatures (cooling of almost 3°C to below
20°C).
The exotic sabellid polychaete that seriously impacted abalone
culture in California was natned Terebrasabella hetcrouncinala by
Fitzhugh and Rouse (1999). Experimental studies indicated that
one T. heterouuciiuita is capable of self-fertilization for the pro-
duction of fully functional oiganisms and that generation time was
highly temperatuie dependent. Although this polychaete is capable
of completing its life cycle at cold temperatures (ll.2°C). the
complete developmental cycle was slow requiring 298 days in
comparison to 165 days at I5.6°C and 1 1 1 days at 20.9°C (Finley
et al. 2001 ). Also. T. heteroiuicinata is capable of infecting a wide
host of gastropod species native to California (Kuris & Culver
1999).
Shell mineral deposition in Haliotis rufescens was exploited by
T. heterouncinata resulting in the formation of a protective bun'ow
around the polychaete. Heavy infestations caused downward ori-
entation of the shell margin, shell deformation and .stunted abalone
growth (Day et al. 2000). Ultrasound micro-cavitation was found
to destroy the feeding crown of this sabellid and improved the
growth of treated H. midae. However, the abalone showed severe
stress behavior during the ultrasound treatments and a second
treatment may be required to destroy new sabellid infestations
recruited from larvae and eggs that were protected by the abalone
shell during the first treattiient (Loubser & Dormehl 2000).
The agent of withering syndrome responsible for mass mortali-
ties of Haliotis cracherodii in southern California was identified as
a Rickettsia-like prokaryote in the class Proteobacteria and was
given the provisional name of "Candidatus Xenohaliotis califomi-
ensis" because of the inability to culture the organism in vitro
(Friedman et al. 2000a). A polymerase chain reaction (PCR) assay
and in situ hybridization (ISH) test have been developed for the
detection of this pathogen (Andree et al. 2000. Antonio et al.
2000). "Candidatus Xenohaliotis californiensis" can also be rap-
idly detected in tissue squashes of infected gastrointestinal epithe-
lium using a nucleic acid fluorochrome stain (Moore et al. 2001a).
In surveys of abalone from Baja California. Mexico, this pathogen
was detected in high prevalences in symptomatic and non-
symptomatic cultured and natural populations of H. rufescens, H.
fiilgeiis. and H. corriigata (Caceres Martinez et al. 2000, Caceres
Martinez & Tinoco-Orta 2001. Alvarez-Tinajero et al. 2002).
Also, infected H. rufescens have been detected as far north as San
Francisco. California (Finley & Friedman 2000). An unidentified
Rickettsia-like prokaryote was also detected in the digestive gland
of H. midae from South Africa (Mouton 2000).
Warm temperatures (>I8°C) seem to be required for the de-
velopment of withering syndrome in abalone exposed to "Caiuli-
datiis .Xenohaliotis californiensis" (Moore et al. 2000a. 2000b).
This pathogen was transmitted between abalone by injection and
bath exposure to post-esophagus homogenates prepai'ed from in-
fected abalone and by cohabitation with abalone exhibiting with-
ering syndrome (Moore et al. 2001b. Friedman et al. 2002). Ex-
amination of hemocyte activity indicated that the hemocytes of
infected H. cracherodii were more chemotactic but were less able
to engulf and destroy foreign particles, which may contribute to the
mortality associated with withering syndrome (Friedman et al.
2000b). Intramuscular injection and oral administration of oxytet-
racycline was effective in reducing the losses of infected abalone
Disease update and screening of abalone
807
(Friedman et al. 2003). However, other antimicrobials (chloram-
plienicol. clarithromycin, and sarafloxicin) had no measurable af-
fect on the disease (Friedman et al. 2000a).
In addition to the new information on severe diseases of aba-
lone, observations on other parasites were also published since the
previous review by Bower (2000V A renal coccidia was reported
from H. midae in South Africa (Mouton 2000) and Murgolisii'lla
haliotis was detected in H. nifesceiis from Baja California, Mexico
(Caceres Martinez & Tinoco-Orta 2001). Two species of spionid
polychaetes (mudworms). Boccardia bwxi and Polydora hopluni.
were associated with severe blistering in the shell and 50% or
greater mortality among cultured abalone at several sea-based fa-
cilities in southern Tasmania. Australia (Lleonail et al. 200.^).
Three species of shell boring clams (Lilhophaga arishihi. Lirlm-
phaga pliinnda. and PcuitcUa caiimdi) were found boring in tlic
shell of H. fiilgens and two of these species (L. uiistata and L.
phiiiiuhi) were also observed infesting the shells of H. cornigata
from the vicinity of Isla de Cedros on the west coast of Baja
California, Mexico (Alvarez-Tinajero et al, 2001). Nollens et al.
(2002) reported that endoscopy applied to anesthetized H. iris was
more accurate than radiography and ultrasonography for the de-
tection of the shell lesions caused by the invasion of a fungus
(described by Grindley et al. 1998), Although endoscopy was in-
vasive, apparently no discernible effects on survival of the aba-
lone, attributable to the procedure, were observed 8 months after
screening (Nollens et al. 2002).
ABALONE DISEASE CONCERNS IN BRITISH COLUMBIA
Although some and possibly all of the diseases of abalone
detected in other parts of the world have the potential of occurring
in British Columbia, to date, only one infectious disease of concern
has been detected. The protistan Lidnriiuhidoides ludiotidis was
involved in the demise of an attempt to culture northern abalone.
H. kamtschatkana. in British Columbia in the early 198()s (Bower
1987a. 1987b). This parasite is only known to be lethal for abalone
smaller than 5 mm in shell length. Because of the relatively large
size of this parasite (about 10 jxm in diameter) and the translucent
nature of the tissues of small abalone {<3 mm shell length), in-
fected abalone can easily be detected by examination w ith a com-
pound light microscope.
Detection of L. ludiotidis in small abalone begins when the
culture containers are being cleaned. Moribund abalone that are
either weakly attached to the substrate or have fallen to the bottom
and are not attempting to right themselves should be sampled. The
foot and head of infected abalone will have lost tissue integrity and
appear swollen (look puffy). Squash the abalone between a glass
slide and coverslip. and examine squashed tissues under a com-
pound microscope. In comparison to normal/health abalone (Fig.
la), the tissues of infected abalone will be filled with stationary
spherical protists (-10 |xni in diameter, see Fig, lb) many of which
may be undergoing binary fission (semispherical specimens with a
clear central dividing line, see Fig Ic). If infection with L. haliotis
is suspected, samples should be submitted to a competent authority
for confirmation. Prior to submission, abalone should be preserved
for histologic examination as described later. Also, immediate
steps should be taken to mitigate potential spread of the disease.
The spread of L ludiotidis within an abalone culture facility or
between facilities can be mitigated by applying good husbandry
techniques. Essentially, abalone and equipment should not be
transferred between tanks, water exchange between tanks should
be avoided and personnel must be careful to not facilitate cross
contamination. Although L. ludiotidis is resistant to many disin-
fectants, it can be destroyed by a 20 min exposure to 23 ing sodium
hypochlorite (chlorine) per liter of sea water (Bower 1989). Treat-
ments applied to infected abalone in the past (Bower 1989) have
proven problematic. If this parasite should again appear in an
/
E
4
?
4-\ -
b'
Figure 1, Unstained wet mount squashes of Juvenile abalone, Haliotis kamlsihalkaiui. about 2 mm in shell length, (a) Head region of a normal
uninfected specimen showing the eye (El and shell (Si. Scale bar = KtO jim, (bl .Same magnilkation of a specimen heavily infected with numerous
iMbyrinlhyUndes haliotidis (P) liberated from and embedded in tissues of the head region— the eye is swollen due to loss of tissue integrity caused
by the parasite. Scale bar = 1(10 |im. (c) Magnification of infected foot muscle showing numerous L. haliotidis some of which are in the process
of dividing by binary fission (I)). Scale bar = 25 jim.
808
Bower
abalone culture facility, research will be required to identify effi-
cacious methods of control.
ASSESSMENT OF ABALONE HEALTH
As for all other mollusks. infectious diseases of ahalone are
becoming more evident with increased efforts towards the culture
of various species of abalone around the world. Disease can often
be circumvented in the culture environment by implementing
knowledge gained from research on the cause of the disease. Also,
when cultured abalone are to be used in rehabilitation efforts,
specimens placed into the natural environment must be in good
health for the endeavor to have a chance of success. Because few
specific diagnostic tools are available for detecting diseases of
abalone and assessing abalone health, standard procedures of his-
tologic examination must be used. Although the microscopic ex-
amination and interpretation of histologic preparations of abalone
tissues requires extensive specific knowledge and experience, the
preparation of the tissues for histology can be performed with
minimal training and equipment. Following is a brief description
of the procedures and materials required to prepare abalone for
histologic examination.
Abalone for histologic examination should first be examined
fresh, all abnormalities noted, and shell length measured. For his-
tology, appropriate tissue samples must be chemically preserved
(Table 2). Regardless of the preservative used, it is critical that
tissue samples are less than 1 cm thick, that there is at least 10 parts
preservative to 1 part tissue and that the tissues are placed in the
preservative as soon as possible after collection. After 24 to 48 h
in the preservative, tissues should be transfen'ed to 70% ethanol for
storage until further processing or shipping to a pathologist for
examination.
Abalone of small size and some organs in larger abalone should
not be dissected because of damage caused to the tissue in the
dissection process. As a general guideline, abalone less than 5 mm
in shell length should be preserved intact. Abalone from about 5
mm to 3 cm in shell length should be removed from the shell prior
to being preserved whole (ie. with no further dissection). Abalone
greater than 3 cm in shell length must be removed from the shell
and tissues dissected (Fig. 2). The region of the digestive gland,
stomach, crop, and heart kidney complex (Fig. 2b.c) should not be
dissected prior to fixation because of damage to these delicate
tissues caused by the dissection process. The part of the abalone
consisting mainly of the foot muscle can be discarded unless le-
TABLE 2.
Formulation of two preservatives used to fix abalone tissues for
histological examination. Tissues must be fixed as soon as possible
after the abalone is removed from the water. Prior to fixation.
record all lesions observed on each specimen and confirm that there
is at least 10 parts preservative to every 1 part of abalone tissue.
-•~%'*isf>.
Davidson's Solution
(Shaw & Battle 19571
10'7r Formalin
in Sea Water
400 niL glycerin, and
800 niL formaldehyde
1 200 mL 95?^ ethanol
1 200 mL sea water
Just prior to use add I part
glacial acetic acid to every
9 parts of the above mixiuie.
1 part fomialdehyde
9 parts sea water
i
■9:0gr
%
iK
\"^<^ epipodial
' tentacles
-digestive gland stomat
C
Figure 1. Dorsal views of an abalone. (a) Orientation of features on
outer surface of shell, (b) Body parts .A (head) and B (viscera) should
be preserved for histologic examination and part C (foot I can be dis-
carded. The lines indicate where the tissues should be cut to avoid
unnecessary disruption to the delicate visceral organs, (c) Major or-
gans underlying the shell, (dl Major organs underlying the respirato-
ry, excretory, and reproductive organs. Images b, c and d were modi-
fied from Bullough (1958).
sions are detected or withering syndrome is suspected. Lesions or
other tissue abnormalities should be described and location noted
because they are usually not evident after the tissue has been
chemically preserved. If the lesion is on the foot (part of the
abalone usually not preserved for histologic examinations), a rep-
resentative sample, which is no greater than a I cm cube, should be
added to the preservative. If withering syndrome is suspected, a
3-5 mm cross section of the foot muscle should be preserved.
Preserved tissues can be stored for prolonged periods (years).
However, histologic examinations should be conduced as soon as
possible to expedite the use of resulting interpretations. For histo-
logic examination, the tissues must be processed and stained for
microscopic examination as described and illustrated by Howard
and Smith (1983). In brief, the fluids in the tissues must be re-
moved and replaced with paraffin wax. The wax embedded tissues
are then cut into about 6-p.m thin slices (sections) and the sections
mounted on a glass slide and stained. The cells in the resulting
stained histologic sections are then examined microscopically for
abnoniialities and obser\ations correlated with the notes taken
prior to sample preservation.
Histologic examinations will usually reveal the presence of
synibionts and parasites. Knowledge and experience on the iden-
tification of these organisms is used to determine which can cause
diseases of concern. Once an infectious agent has been identified,
steps to prevent further spread within a culture facility or to the
natural environment can be implemented. In addition to detecting
pathogens, histologic examinations can also be useful in assessing
Disease update and screening of abalone
809
the suitability of the culture environment. The morphology of tis-
sue cells can indicate the suitability of the diet or an increased
intensity of synibionts (either bacteria or protists) can signify un-
suitable parameters in the habitat. Until other more specific and
sensitive assays are available to detect disease agents and assess
abalone health, histologic examination will ser\e as a valuable tool
for optimizing abalone culture conditions and avoiding unneces-
sary losses during rehabilitation efforts.
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carchariae, a pathogen of the abalone Haliotis tuherculata. Dis. Aqiial.
Org 50:35-43.
Nishimori, E., O. Hasegawa, T. Numata & H. Wakabayashi. 1998, Vibrio
carchariae causes mass mortalities in Japanese abalone, Sulcuhts di-
versicolor supratexia. Fish Pathol. 33:495-502.
Nollens, H. H., J. C. Schofield, J. A. Keogh & P. K. Probert. 2002. Evalu-
ation of radiography, ultrasonography and endoscopy for detection of
shell lesions in live abalone Haliotis iris (MoUusca: Gastropoda). Dis.
Aquat. Org. 50:145-152.
Oakes, F. R. & R. C. Fields. 1996. Infestation of Haliotis nifescens .shells
by a sabellid polychaete. Aquaciihure 140:139-143.
Ruck, K. R. & P. A. Cook, 1998. Sabellid infestations in the shells of South
.African molluscs: implications for abalone mariculture. J. Shellfish
Res. 17:693-699,
Shaw, B. L. & H. I. Battle. 1957. The gross and microscopic anatomy of
the digestive tract of the oyster Crassosirea virginica (Gmelin). Can. J.
Zool. 35:325-347.
Sindemiann, C. J. 1988. Disease problems created by introduced species.
In: C. J. Sindermann & D. V. Lightner, editors. Disease diagnosis and
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Tai-wu, L., J. Xiang & R. Liu. 2000. Studies on phage control of pustule
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19:535.
Jimnml of Slwllfi.sh Rcicunh. Vol. 22, No. 3. Sll-SIH. 2003.
FECUNDITY AND SEASONAL REPRODUCTION OF NORTHERN ABALONE, HALIOTIS
KAMTSCHATKANA, IN BARKLEY SOUND, CANADA
A. CAMPBELL, J. LESSARD, AND G. S. JAMIESON
Fisheries and Oceans Canada, Science Branch, Pacific Bialoi^ical Slatimi. Nanainio.
B.C. V9T6N7, Canada
ABSTRACT Fecundity, si/e at maturity and seasonal reproduction of noilliem or "pinto" abalone, Halious kamischatkana. from
exposed "surf"' areas and more sheltered, productive abalone habitat were investigated in Barkley Sound. Examination of histologic
sections of gonads indicated that si^e at maturity occurred at a smaller size for the stunted 'surf ' abalone than for abalone from more
sheltered areas. Gonad index and stages showed that gonads were mainly ripe and that most abalone spawned during April to July.
Although there were smaller abalone with ripe eggs from the "surf area than those from the sheltered area, abalone females of
comparable si/e from both areas had similar egg numbers. However, there were larger females with considerably higher fecundity from
the sheltered areas than from the "surf' areas. Implications of transplanting "surf"' abalone to productive habitats to increase growth
and fecundity rates are discussed in the context of population rebuilding attempts for H. kamtschaikana. which is listed by the
Committee on the Status of Endangered Wildlife in Canada as a "threatened" species in Canada.
KEY WORDS: abalone, Huliotis kainisciuitkaihi. fecundity, reproduction, size at maturity
INTRODUCTION
The nofthern or "pinto" abalone. Huliotis I<am1sclwtkana J. H.
Jonas. 1845 (Gastropoda), which occurs from Sitka Island. Alaska,
to Baja California, is found on rocky habitats from the intertidal to
subtidal depths of 100 m. with most adults found at less than 10 m
in British Colutnhia (BO (Sloan & Breen 19881, Declines of
northern abalone abundance in the 1980s resulted in an abalone
fishery closure in December 1990. and with continued poor re-
cruilrnent (Campbell 2000. Jamieson 20011 this species was des-
ignated as "threatened" in April 1999 by the Committee on the
Status of Endangered Wildlife in Canada (COSEWIC). Reproduc-
tive characteristics of northern abalone are considered important in
understanding the population biology of this species in BC, Early
studies indicated that northern abalone became sexually tnature at
about 50 mm shell length (SL) (Quayle 1971. Paul & Paul 1981 ),
Eggs released by captive spawning females in sea water were
estimated at 2,3 million over 2.3 h by a 135 mm SL female and
33.700 over 1.5 h by a 101 mm SL female (Olson 1980. Caldwell
1981 ). Campbell et al, ( 1992) found SC/r size at tnaturity to be 55
mm SL and fecundity to range from thousands to millions of eggs
per fetnale for northern abalone collected from southeastern Queen
Charlotte Islands (QCI) in June 1990, Spawning generally oc-
curred frorn April to July (Quayle 1971, Breen & Adkins 1980.
Sloan & Breen 1988).
Spatial variation in demography of abalone populations is com-
mon throughout the world (Shepherd et al. 1992. Worthington &
Andrew 1998). Abalone populations that have small or stunted
individuals typically occur in locations having less than optimal
environmental conditions. In poor habitats, individuals generally
have slower growth and reach a smaller maxitiiutii size than aba-
lone found in more optimal habitats (Shepherd 1988. Sloan &
Breen 1988. Etnmett & Jamieson 1988. Nash 1992. Wells & Mul-
vay 1995). In BC. transplanting slow growing northern abalone
from high wave exposure ("surf") areas to sheltered locations was
shown to increase individual growth rates in both the QCI (Breen
1986) and in Barkley Sound (Emmett & Jatnieson 1988). Surf
abalone in exposed areas are slower growing individuals that fail
to reach sizes greater than 100 mm SL (Sloan & Breen 1988).
Factors such as abalone density, genetics, quantity and quality of
available food, predator activity, substrate type (rugosity), and
v/ave action may all influence growth and survival rates (Sloan &
Breen 1988), Donovan and Carefoot (1997, 1998) found respira-
tion activity and locomotion (mucus production) were a major part
of the energy budget of northern abalone. Predator presence, high
wave action, lack of food (Sloan & Breen 1988), and lack of
suitable crevices (Shepherd 1986) for shelter from predators or
wave action inay increase abalone activity and respiration, reduc-
ing energy available for growth and reproduction.
Little comparative information on the reproductive capabilities
between abalone frotn poor and optimal habitats is available for
different areas of BC. The purpose of this article is to provide
seasonal patterns of reproduction, sizes at maturity, and fecundity
levels for northern abalone from both exposed "surf" and moder-
ately sheltered abalone habitats in Barkley Sound on the west coast
of Vancouver Island. Size-specific sexual maturity and fecundity
are itnportant in determining the relative potential egg production
of northern abalone populations from poor and good areas,
MATERIALS AND METHODS
Northern abalone were collected by SCUBA divers at two sites
in Barkley Sound: ( I ) an exposed area east of an unnamed island
with a height of 39 m indicated on chart 3670 (Canadian Hydro-
graphic Service) referred to in this article as Island 39 (Lat.
48=51.550'N. Long. 125°19.048'W) where "surf" abalone were
found, and (2) a moderately sheltered area southwest of Willis
Island (Lat. 48°54,907'N, Long, 125°20.873'W) where a large size
range of abalone was found. Representative size ranges of abalone
at an equal sex ratio were collected on several occasions from both
sites during 1991 and 1992 (see Table 1 for sample dates), Itnme-
diately after collection, shell length (SL) in mrn. total wet weight
(g). sex (by gonad color: beige for males and green for females)
were recorded for each abalone. Random transect surveys (for
the method see Cripps & Campbell 1998. Lessard et al, 2002)
to determine density and size of emergent northern abalone,
were conducted along approximately 80 m of the east side of
Island 39 (n = 3) on 4 July, 2002, and along approxiinately 300
m of the east side of Hankin Island (Lat. 48 55.266'N, Long,
125°21,946'W) in = 5) on 6 June 2002, Substrate type for Island
39 was bedrock with a few crevices and rocks. Substrate type for
both Willis and Hankin Islands was bedrock with many small and
811
812
Campbell et al.
TABLE 1.
Seasonal gonad stages of H. kamtschalkaiia sampled from Willis
Island and Island 39 during 1991-1992.
Gonad Stages i
in of nl
Date
1
2
3
4
5
n
Willis Islan
id
18 June.
1991
42
45
3
10
0
60
5 July
100
0
0
0
0
20
7 Augusl
0
0
0
75
25
20
10 October
l.'^
0
8
69
8
13
22 Jan..
1992
3
0
7
90
0
29
6 April
8
50
4
17
21
24
21 April
5
50
10
20
15
20
5 May
35
30
0
25
10
20
20 May
15
45
0
40
0
20
6 June
5
40
5
35
15
20
20 June
28
56
(1
0
17
18
2 July
35
50
0
0
15
20
Island 39
18 June.
1991
16
45
0
30
9
44
6 April.
1992
0
50
0
21
29
24
21 April
15
50
0
5
30
20
5 May
33
29
0
10
29
21
20 May
10
55
0
30
5
20
6 June
5
48
II
48
0
-) 1
20 June
35
40
(1
15
10
211
2 July
20
20
II
25
35
211
The 5 stages of mature gonads were classified as 1 = npe; 2 = partly
spawned; 3 = fully spawned: 4 = recovery stage 1; 5 = recovery stage
2. n = sample size.
large boulders. Daily sea surface water temperatures were obtained
from the Amphitrite Point {north west outer tip of Barkley Sound)
database during 1991 to 1992 to compare mean monthly tempera-
tures. Mean annual and mean first 6 mo surface temperatures were
calculated from monthly mean temperatures for each year.
To determine seasonal reproductive condition and maturity of
each male and female abalone. the conical appendage containing
the gonad sheath located over the hepatic gland (Poore 1973) was
removed and preserved in Davidson's Solution (Shaw & Battle
1957). The fixed gonad was cut about midway between the apex of
the shell and the tip of the conical appendage and cross sections of
the gonad and hepatic gland were traced on transparent plastic.
Relative areas of hepatic gland and gonad in cross section were
obtained by weighing the plastic outlines. The gonad index (I) was
calculated as I = 100 G/T. where G is the cross-sectional area
(weight of plastic) of the gonad, and T is the total cross-sectional
area (weight of plastic) of both the gonad and hepatic gland. His-
tologic slides were prepared by staining sections of gonad with
hematoxylin-eosin. Histologic sections of gonad were classified
into 6 stages. Immature individuals (Stage 0) were characterized
by no differentiation in gonadal tissue, or where there was small
gonadal bulk for (a) males that was comprised mostly of primary
or secondary spermatocytes with no spermatozoa present and (b)
for females that was comprised mostly of primary oocytes and
some stalked oocytes, but no mature or degenerating oocytes
present. The other stages were for mature abalone gonads that had
well developed spermatazoa or oocytes usually in abundance, or
where there were few mature oocytes, or degenerating unspawned
oocytes were present. The 5 stages of mature gonads were: ( 1 )
ripe, (2) partly spawned. (3) fully spawned, (4) recovery stage 1;
and (5) were recovery stage 2 (Wells & Keesing 1989). This
pattern of gametogenesis is similar for several different abalone
species (eg, Newman 1967, Young & DeMartini 1970, Giorgi &
DeMartini 1977, Mottet 1978, Tutschulte & Connell 1981).
To determine the relationship between the proportion of mature
northern abalone and shell length, data were combined by 5-mm
SL classes for both males and females (since the curve for each sex
was similar) and was estimated for each study area using the
equation:
P=L,/{L, + e''
'}
m
where P, is the proportion of the mature abalone in the /th 3-iiim
SL interval, Z., is the shell length in the /th 5-mm SL interval, and
the coefficients A and B were estimated with a non-linear (sim-
plex) Marquardt least squares method (SYSTAT 2000). Only data
from northern abalone collected during June 1991 and June 1992
were used in estimating proportion maturity values, since this re-
productive time period provided the maximum opportunity to dis-
tinguish between immature and mature individuals.
Potential fecundity was determined from ripe gonads represent-
ing the full size range of mature H. kanitscluiikiiiui collected during
June 1991. just prior to spawning, by placing whole females in
1 Wc formalsaline. Internal organs of individuals more than 90 mm
SL were injected with formalsaline to help accelerate fixation and
hardening of the ovaries. Each ovary was then removed, the gonad
index measured, the hepatic organ excised, and the drained wet
weight of each ovary recorded. Three small subsamples, each
weighing approximately 0.006 g (range ± 0.003 g) wet weight,
were removed randomly from each ovary, weighed, and the mature
eggs (oocytes) in each ovary freed from ovarian connective tissue
with fine dissecting forceps and a small paint brush. Freed eggs
were counted under a dissecting microscope. Diameters of 10 oo-
cytes per female were measured from a selection of mature fe-
males over a wide range of sizes (59.8-125 mm SL and 40.5-64.4
mm SL, respectively) from Willis Island and Island 39. Mean egg
density per gram was determined from the subsainples. Initial tests
indicated no differences (/-test, P > 0.05) in mean egg density
between different locations on the ovaries of five northern abalone.
Other studies (eg. Giorgi & DeMartini 1977. Wells & Keesing
1989) found eggs homogeneously distributed throughout the ova-
ries in other abalone species. Fecundity, or total number of eggs
per female, was estimated as the product of mean egg density and
total ovary weight.
The relation between fecundity (F) and shell length (L) was
expressed with the natural log transformed linear regression equa-
tion:
loe,. F = log,. A -I- B loa,, L
(2)
where the coefficients A and B were estimated using the least
squares method. Analysis of covariance (ANCOVA) was used to
test for the homogeneity of slopes and elevation coefficients of the
log transformed data regressions (Zar 1984) of fecundity of north-
em abalone collected from different areas. All ANCOVA com-
parisons for fecundity and SL, in similar size groups, between
areas Willis Island. Island 39, and south east QCI (Campbell et al.
1992) indicated there were no differences [P < 0.05) between
slopes or elevations, so size at fecundity data were combined into
one equation. The relation between fecundity (F) and total abalone
drained wet weight (W) was expressed with the linear equation
NORTHHRN ABALONB REPRODUCTION
813
F = A + B W. where the coefficients A and B were estimated
using the least squares method.
Potential total egg production per m" (E) was estimated as:
E = 2f,P,S,(N,/N)</
(3)
where F, is the fecundity (eggs/female) at shell-length increment i
(mid point of each 5 mm SL size class increment was used). P, is
the proportion mature at i. S, is the sex ratio of mature individuals,
assumed to be i).5 for all i (this study. Sloan & Breen 1988), N; is
the number of abalone measured in the i"th size, and N is the total
number of abalone measured. Estimated mean density, cl (number
/ m"). of all emergent abalone for a number of transects was
calculated as:
(4)
Ev,
where A', is the number of abalone counted in transect /. .v, is the
length of transect t (i.e.. area in square meters since each transect
was one meter wide).
Cumulated total potential egg production was estimated from
H. kamtschatkana size frequency and mean density estimates of
0.750 and 0.404 abalone/m". obtained at Island 39 and Hankin
Island, respectively, during June to July 2002.
RESULTS
Shell Lengths
From biologic samples collected during 1991 to 1992. the me-
dian SL was larger for abalone from Willis Island. 87.0 mm (range
44.0-125 mm SL, n = ?•]?•). than abalone from Island 39, 67.0
mm (range 32.4-87.0 mm SL. ;; = 217). Of the total abalone
collected, 16% and 0% were 100 mm SL or greater, from Willis
Island and Island 39, respectively.
From the random transect survey completed during June to July
2002. the median SL was larger for abalone from Hankin Island.
79.0 mm (range 18.0-1 15 mm SL. n = 37), than for abalone from
Island 39, 61.0 mm (range 23.0-98.0 mm SL.n = 27). Of the total
abalone collected. 13.5% and 0% were 100 mm SL or greater,
from Willis Island and Island 39, respectively.
Seasonal Reproduction
The gonad index was high for H. kamtsclialkuna from both
locations during May to July 1991 to 1992, but was low at Willis
Island during August 1991 to January 1992 (Fig. 1). Histologic
examination of gonad sections indicated a similar reproductive
pattern for most abalone from both locations (Table 1 ). During
June 1991 and April to June 1992. gonads were either ripe or partly
spawned. Abalone from Willis Island had ripe gonads during July
1991 and mostly recovery stages during August 1991 to January
1992 (Table 1). Results indicated that gonads were ripe mainly
during the summer months of May to early July, with spawning
occurring mainly during May to July. A few abalone had ripe to
spawned gonads in April 1992. A few abalone from Willis Island
had ripe gonads and may have spawned from October 1991 to
April 1992 (Table I).
The mean annual sea surface temperature was 10.4'C for 1991
and 11.3"C for 1992 in Barkley Sound (Amphitrite Point). Mean
sea surface temperatures were warmer (by 1 .4°C) for the first 6 mo
90 n
80
X
S 70H
2
Q 60
<
O 50H
O
40
30
j'j'a's'o'n'd'j'f'm'a'm'j'j'
1991
1992
MONTH
Figure 1. Seasonal changes in mean gonad index (percent) of H.
kuintscluilkanu from Willis Island (()) and Island 39 (X) from June
1991 to Julj 1992. Horizontal line about each mean is ±1 standard
error. See Table 1 for sample sizes.
of 1992 (10.7''C) than that of 1991 (9.3°C). During the main re-
productive period of northern abalone, May, June, and July mean
monthly sea surface temperatures were 10.7. 1 1.4. and 12.5°C for
1991 and 12.1. 13.0. and I4.4^C for 1992, respectively.
Size at Maturity
Although there was considerable variation, gonad indices in-
creased with increasing SL (Fig. 2). Gonad indices were larger at
smaller sizes of abalone from Island 39 than at Willis Island. Size
at 50% maturity was lower for abalone from Island 39 (44 mm SL)
lOOn
80
60
40
20
X
LU
Q
Q
<
O
o
0-
X S>
X X
o
n — ' — I — ' — I — ' — \ — ' — T"
0 20 40 60 80
SHELL LENGTH (MM)
100
Figure 2. Size specific mean gonad index (percent) of H. kamtschat-
kana from Willis Island (()) and Island 39 (Xl during June 1991.
814
Campbell et al.
than those from Willis Island (.'50 mm SL) (Fig. 3. Table 2). Ex-
amination of histologic sections indicated that the smallest mature
abaione were 42.6 and 49.4 mm SL and the largest immature
individuals were 50.8 and 57.0 mm SL at Island 39 and Willis
Island, respectively, during June 1991 to 1992. Maturity occurred
for most abaione at sizes greater than 65 mm SL. Although the
color of whole gonads could be differentiated to determine sex,
histologic examination indicated that not all gonads were mature in
the 32 to 57 mm SL range.
Fecundity
For females with ripe gonads, both mean oocyte diameter (231
|jLm ± 2 SE, (1 = 6) and mean density or number of eggs per g
(178.386 ± 3,360 SE, /; = 33) did not significantly differ (two way
7 9 14 10
5 10 14 7
08
1
LLI
-
a:
Z)
0.8-
1-
<
^
0.6-
z
o
-
1-
0.4-
o
a.
O
a:
0.2-
a.
-
0.0-
0
— 1 — ' — I — ' \ ' I ' I
20 40 60 80 100
SHELL LENGTH (MM)
1.0n
LU
§ 0.8
0.6
S 0
O
a.
O
a:
Q.
4-
0.2-
0.0
9 18 10 2
~i — ' — \ — ' — I — ' — r~
20 40 60 80
SHELL LENGTH (MM)
100
Figure 3. Proportion mature at mid point of each 5-mni shell length
class of//. kanUschatkaim (sexes combined I from (.\l Willis Island and
(B) Island .W. Numhers next to s>mbols are sample sizes. See Tahle 2
and text for information on predicted equations for size at maturitj
curves
TABLE 2.
Equation coefficients for size at maturity equation from //.
kamtschatkana sampled in Barkley Sound. See text for equation
details and Fig. 3 for data. Values in brackets are approximate 95%
confidence intervals. R" = coefficient of determination.
Equation
Coefficients
Site
A
B
R-
Willis Island
Island -^9
:?. SO? (±6.475)
12.S63(±4.973)
0.438 (±0.129)
0.204 (±0.1 ID
0.987
0.943
ANOVA. P > 0.05) between abaione of different sizes or between
those from Willis Island and Island 39. ANCOVA comparisons
indicated there was no difference (P > 0.05) in slopes or intercepts
in the linear relation of log^, transformed gonad weight (g) and SL
between Willis Island and Island 39. Therefore, data from both
locations were combined into one equation:
log^. (gonad weight, g) = -13.4807 + 3.48 log,. (SL)
(R- = 0.82, P<0.01. n = 33)
estimated by the least squares method. Given that ( 1 ) egg densities
were independent of abaione size and eggs were distributed ho-
mogeneousl) throughout the ovai^. and (2) there was a relation-
ship between the preserved ovary wet weight and SL. the fecundity
of H. kamtschatkana could be related to SL or total abaione weight
(Table 3). Although there was considerable variation in fecundity
between indi\'iduals of similar size, the increase in fecundity in
relation to increases in SL and weight of H. kamtschatkana was
highly correlated (Fig. 4, .see Table 3). The smallest female (40
mm SL) from Island 39 had 90,594 eggs, the largest female (125
mm SL) from Willis Island had 3.0 million eggs, and the largest
female (144 mm SL) from southeast QCI had 11.3 million eggs
(Campbell et al. 1992).
Potential Population Egg Production
The cumulated proportion of sizes was higher for small size
classes of H. kamtschatkana from Island 39 than from Hankin
Island (Fig. 5). This was reflected in a higher cumulated potential
egg production from the 50 to 80 mm SL class from Island 39 than
from Hankin Island (Fig. 6).
TABLE 3.
Equation coefficients for the fecundity (F, number of eggs per
female) and shell length (I. in mm) log transformed equation log.F =
log,..\ Blog^L for //. kamtschatkana. and fecundit> and total weight
(\\ in g) linear equation F = A BW. Areas: I = Willis Island and
Island .'9 combined (this study), 2 = Willis Island and Island 39
(this study) combined with south east Queen Charlotte Islands
(Campbell et al. 1992).
Independent
\ ariable
-Areas
Equation Coefficients
B
R-
n
Shell length
Total weight
-1.5203 3.5137
-2.2152 3.6789
108.463 14588.5
-411.590 25179.5
0.843 33
0.915 .56
0.803 33
0.910 .56
R- = coefficient of detennination, n = sample size.
Northern Abalone Reproduction
815
UJ
q:
LU
Q.
CO
o
CD
LU
Ll_
O
(/)
10-
-
+-f
10-
-
5-
/
-
w
o
-
^^%
^^
0-
1 , , 1 r-rf^^^^
' ' 1
III.
0 50 100
SHELL LENGTH (MM)
150
Figure 4. Kccundit> at shell leiislh of feniule //. kumlsclialkaiia from
Willis Island (Ol and Island 39 (X) durinK .June 1991 (this study), and
from Queen Charlotte Islands l + l during June I99(t (after Campbell et
al. 1992). See Table 3 for regression equation coefficients.
Despite meun ahaloiie density estimates being higher at Island
39 (0.750/nr) than at Hankin Island (0.4()4/m-) dunng June to July
2002, the estimated total potential egg production per unit area
(millions of eggs per m~) was higher at Hankin Island (0.26) than
at Island .^9 (0.20) (Fig. 6) due to higher fecundity of large abalone
(2100 mm SL) present at Hankin Island. In contrast, if we as-
sumed a similar hypothetical abalone density of l.O/iir for both
areas, estimated total potential egg production (millions of eggs
I.U-|
z
o
1-
oa-
q:
o
-
CL
O
OR-
ir
Q.
_
Q
111
04-
H
5
-
-)
^
0.2-
3
O
"
0.0-
0 50 100
SHELL LENGTH (MM)
150
Figure 5. Cumulated proportion size frequency by 5-mm .SL classes of
H. kamtsvhalkanu from Hankin Island )()) and Island .W (X) during
June 2002. Lines fitted to data by a spline smoother with cubic equa-
tions (SYSTAT 2000).
HI
Q^
1-
Lli
0 3^
^
LU
DC
<
3
O
CO
0.2-
a:
LU
Q.
-
CO
CD
CD
0 1-
LU
Ll_
o
.
CO
z
O
00-
_j
50 100
SHELL LENGTH (MM)
150
Figure 6. Cumulated total potential egg production (millions per nr)
by 5-mm SL classes of H. kamlschalkaiia from Hankin Island (()) and
Island 39 (X) based on mean density estimate of 0.404 and 0.750 aba-
lone/m", respectively, during June to July 2002. See text for method to
calculate potential total egg production. Lines fitted to data by a spline
smoother with cubic equations (SYSTAT 2000).
per m") would be niore than twice as much for Hankin Island
{0.65) than for Island .^9 (0.27).
DISCUSSION
Shell lengths of H. kmntschatkana sampled from Island 39
were all less than 100 mm, typical of abalone from a "surf area
with less than optimal habitat conditions (Sloan & Breen 1988,
Emniett & Jamieson 1989). In contrast, there was a larger range of
sizes of abalone from the moderately exposed areas of Willis and
Hankin Islands. The largest abalone encountered in this study was
a 12."^ iT)m SL female from Willis Island. Low numbers of large
abalone (5100 mm SL) found in this study and in other recent
surveys (eg. Lucas et al. 2002) are in sharp contrast to the many
(51%) large H. kaintschatkana (maximum 146 mm SL, /; = 1305)
sampled by Quayle (1971 ) in Barkley Sound during 1963 to 1964.
Seasonal reproduction of H. kunuschatkana occurred mainly
from April to June at Willis Island and Island 39 in our study,
similar to that found by Quayle ( 1971 ) in other islands in Barkley
Sound. Warmer conditions in January to June 1992 than in 1991
may have caused abalone to spawn earlier in 1992 than in 1991.
Our study also confirms that there may be a few abalone that are
ripe throughout the year that may be able to spawn (see review by
Sloan & Breen 1988). Intra- and inter-specific variability in ga-
metogenesis and annual spawning periods is cornmon for many
abalone species and spawning may partly depend on local condi-
tions (eg, temperature, storms, food quality, and abundance) (New-
iT)an 1967. Webber & Giese 1969. Shepherd & Laws 1974. Paul et
al. 1977. Hayashi 1980, Sloan & Breen 1988, Wells & Keesing
1989, Stekoll & Shirley 1993. Hooker & Creese 1995, Sasaki &
Shepherd 1995, Wilson & Schiel 1995).
816
Campbell et al.
This study recorded the lowest 50% size at maturity (44 mm SL
from Island 39) to date for wild H. kaintschatkana. The 50%
maturity at 50 mm SL at Willis Island was similar to that found by
Quay le { 1 97 1 ) for abalone generally in Barkley Sound. Maturity of
wild H. kamtschatkana further north of Barkley Sound (eg, QCI
and Alaska) was found to vary between 50 to 64 mm SL (Larson
& Blankenbeckler 1980 referenced in Sloan & Breen 1988. Paul &
Paul 1981, Campbell et al. 1992). We confirm Quayle's (1971)
observation that although sexes could be differentiated by color of
gonads at small sizes (e.g., 32 mm SL) the start of sexual maturity
was observed at larger sizes (smallest mature individual we ob-
served was 42.6 mm SL). Size at sexual maturity for an abalone
species can vary between locations (Shepherd & Laws 1974) de-
pending on various factors such as food quality and availability
and different temperature regimes (Kikuchi & Uki 1974a. Kikuchi &
Uki 1974c, Kikuchi & Uki 1975, Paul et al. 1977, Paul & Paul 198 1 ).
Fecundity estimates were similar for H. kamtschatkana of
equivalent shell lengths from Willis Island, Island 39 and southeast
QCI, and the number of eggs increased exponentially with in-
creases in shell size. Fecundity estimates of//, kaml.schalkana (this
study. Campbell et al. 1992) are within the range reported for other
abalone species. The largest number of eggs reported for a H.
kamtschatkana was 11.56 million eggs for a 139-mm SL female
(Campbell et al. 19921. High fecundity has been reported for other
abalone species — 25.4 million eggs for a 175-mm SL //. midae
(Newman 1967), 12.6 million eggs for a 190.5-mm SL H. rufe-
scens (Giorgi & De Martini 1977). Methods to estimate fecundity
vary from estimating the total number of eggs in ovaries by weight
(Newman 1967, Poore 1973. Giorgi & DeMartini 1977, Hayashi
1980, Wells & Keesing 1989, this study) and by volume (Sains-
bury 1982, Prince et al. 1987, McShane et al. 1988) to counting the
number of eggs spawned (Kikuchi & Uki 1974a. Kikuchi & Uki
1974b, Kikuchi & Uki 1974c. Kikuchi & Uki 1975, Hayashi 1980,
Olson 1980. Caldwell 1981, Tutschulte & Council 1981, Ault
1985, Clavier 1992). Although fecundity was estimated as the total
eggs present in an ovary prior to spawning in this study, probably
not all eggs may be spawned within a spawning event (Poore 1973,
Giorgi & DeMartini 1977. Ault 1985). Caldwell ( 1981 ) found that
H. kamtschatkana females at 101 and 135 mm SL spawned an
estimated 0.03 and 2.3 million eggs, respectively, in 1.5 to 2.5 h in
the laboratory, which were lower than the mean total eggs (2.6 and
7.5 million eggs) estimated in ovaries of individuals of the same
size from our study. The relationship between fecundity and SL for
various abalone species has been described either as curvilinear
(Newman 1967, Poore 1973, Giorgi & DeMartini 1977, Hayashi
1980. Wells & Keesing 1989. Wilson & Schiel 1995, Litaay & De
Silva 2001. this study) or as linear (Poore 1973. Sainsbury 1982.
Prince et. al. 1987, McShane et al. 1988). The relationship between
fecundity and weight of the whole abalone has been considered as
curvilinear (Ault 1985. Litaay & De Silva 2001) or as linear (New-
man 1967. Teener etal. 1989, Shepherd et al. 1991, Shepherd et al.
1995).
Northern abalone from exposed "surt" areas were capable of
reproducing and, at the equivalent size, potentially have similar
fecundity per unit area as abalone from more sheltered areas. Size
at maturity and fecundity, size composition and density are im-
portant in determining the total potential contribution of an aba-
lone population in an area. Given similar densities, larger abalone
in moderately sheltered areas have a potentially higher reproduc-
tive contribution than smaller, slower growing abalone in high
■'surf' exposed areas. The importance of having sufficient numbers
of large abalone for reproductive output has been emphasized in
the fishery context (Breen 1986, Tegner et al. 1989, Shepherd &
Baker 1998) and for evaluating marine protected areas as conser-
vation tools for abalone (Edgar & Barrett 1999. Wallace 1999,
Rogers-Bennett et al. 2002). Fertilization and recruitment success
of various abalone species may also be density dependent (Clavier
1992. McShane 1995a. McShane 1995b. Babcock & Keesing
1999).
Implications of transplanting abalone from "poor" to "good"
habitats to increase survival, growth, and reproductive potential, as
a rebuilding technique for H. kamtschatkana in BC. are compli-
cated. Emniett and Jamieson (1988) concluded that transplanting
large sizes of "surf' northern abalone from exposed sites to more
productive areas was biologically and economically feasible if
survival of the transplanted abalone was reasonably high. They did
not evaluate methods to potentially enhance reproductive output or
recruitment in the transplant areas. Tegner (1992. 1993. 2000)
reported on a transplant of reproductively mature green abalone,
H. fiilgens. in California with subsequent strong evidence of suc-
cessful local recruitment until the brood stock were poached. The
long-term success of a brood stock transplant is dependent on adult
survival and density for fertilization success, and local hydrody-
namics for larval settlement (Babcock & Keesing 1999). Future
attempts to rehabilitate H. kamtschatkana in BC by transplanting
"surf' abalone will require pilot experiments to test transplant
methods for their feasibility to determine measurable success in
increased population survival, growth, reproduction, and recruit-
ment. The choice of recipient "good" sites would include criteria
such as identifying locations with complex substrates, moderate to
low exposure, and availability of suitable algal food for optimal
abalone growth and survival. Abundance and distribution of H.
kamtschatkana in exposed and moderately sheltered areas are not
well known and need to be estimated prior to large scale rebuilding
efforts in local areas of coastal BC.
ACKNOWLEDGMENTS
The authors thank J. Bagshaw. D. Brouwer. W. Carolsfeld. B.
Clapp. D. Cooper. G. Dovey, S. Gazetas. W. Harling. S. Head, P.
Ladynian, J. Lash, L. Lee, B. Lunn, P. Menning, F. Merilees. A.
Phillips, J. Rogers, J. Whang, T. White, for technical assistance.
Parks Canada personnel for logistical support. W. Hajas for sta-
tistical advice, and F. Wells and G. Gillespie for providing helpful
comments to improve earlier drafts of this paper. Partial funding
was provided by the Species at Risk Interdepartmental Recovery
Fund.
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PP-
Joiirihil of Slicllfiih Research. Vol. 22, No. 3. 819-823. 2(103.
ESTIMATING JUVENILE NORTHERN AB ALONE {HALIOTIS KAMTSCHATKANA)
ABUNDANCE USING ARTIFICIAL HABITATS
BART DEFREITAS
HaUla Fisheries Program. P.O. Box 87. Ma.sset, B.C. VOT I MO Canada
ABSTRACT This, .study asscsse;. the use of anificial concrete block habitats that provide standardized sample areas for measuring the
abundance of northern abalone (Haliotis kamtschalkana Jonas) in comparison to 10 randomly selected l-m" quadrant samples where
all movable rocks were examined for cryptic abalone. A total of 278 abalone were measured within artificial structures and juvenile
abalone (£50 mm shell length. SL) were the most abundant size class. Juvenile abalone used artificial structures at greater mean
densities (abalone/nr) than nearby natural habitat (1,27 ± 0,25 SE versus 0,07 ± 0,09 SE) and emergent abalone (>50 mm SL) used
artiUcial habitats at similar densities as they did in nearby natural habitats (0,38 ± 0,09 SE versus 0,44 ± 0,10 SE). Juvenile abalone
abundance was significantly different between sites but not within sites, suggesting artitlcial structures showed promise in their ability
to detect area specific differences in recniitment and to easily measure juvenile abalone abundance,
KEY WORDS: ju\enile, abalone. cryptic, artificial habitat, recruitment. Hulioln kaiiilschalkaiui
INTRODUCTION
Northern abalone [Hidiotis kannsclnilkiinii) fisheries in British
Columbia (BC) remain closed to commercial, recreational, and
First Nations groups since 199(3 due to conservation concerns
(Campbell 2000), Dive surveys conducted by Fisheries & Oceans
Canada (DFO) at inde.x sites in BC estimated that northern abalone
abundance had declined by more than 75% during 1978 to 1984
and continue to remain low (Breen & Adkins 1979 1981. Winther
et al, 1995. Campbell et al, 2000). In April 1999. the Committee on
the Status of Endangered Wildlife in Canada (COSEWIC) listed
northern abalone as "threatened", meaning Hkely to become en-
dangered if limiting factors are not reversed.
The most significant factors inhibiting northern abalone recov-
ery are illegal harvests and poor recruitment (Campbell 2000),
Recruitment, defined as the number of juvenile abalone growing
and surviving to the adult population each year, may be insuffi-
cient as a result of critically low adult densities (Shepherd &
Brown 1993, Shepherd & Partington 1995) that reduce reproduc-
tive success due to low fertilization of gametes (Alice et al, 1949),
Other processes that may reduce abalone recruitment include
variation in timing and intensity of gamete production, larval pre-
dation, and post-larval mortality (McShane 1992. 1995), Recruit-
ment processes for northern abalone are not well understood
(Breen 1986. Sloan & Breen 1988),
Increasing the abundance of existing wild northern abalone
populations in BC is the long-term goal of the northern abalone
national recovery strategy (Toole et al. 2002). One component of
the strategy is to conduct abalone research and rebuilding experi-
ments that inay lead to increased breeding success, recruitment,
and population densities. To evaluate the success of various re-
building experiments, it will be necessary to measure changes in
abalone recruitment by quantifying the abundance of juveniles.
Artificial collectors have been successful at measuring the in-
tensity of abalone larval settlement (Keesing et al, 1995. Nash et
al, 1995) but require high maintenance, a considerable titne in-
vestment to sort samples and appropriate larval identification ex-
pertise. Other larval settlement survey techniques such as under-
water magnification (Shepherd & Turner 1985). anesthesia (Prince
& Ford 1985). and suction (McShane & Smith 1988) also require
great diving and sample sorting efforts. In California. Davis ( 1995)
used artificial concrete block habitats that provided standardized
sample areas to monitor juvenile abalone recruitment. Coiuparing
results from previous juvenile abalone surveys that required the
destruction of natural habitat {Tegner et al, 1989). Davis (1995)
was able to provide surrogate juvenile abalone habitat and produce
an index of abalone recruitment.
This article describes the design and testing of artificial con-
crete block habitats over a 12-month period at 6 sites in Haida
Gwaii (Queen Charlotte Islands), BC, The objectives were to de-
termine if concrete block habitats provided surrogate habitat for
juvenile northern abalone and if so. the ability of artificial habitats
to quantify juvenile ubalone abundance in different locations. To
determine if juvenile abalone abundance within artificial habitats
was representative of nearby natural habitats, invasive surveys of
natural abalone habitats during the same time period were com-
pared,
MATERIALS AND METHODS
Twenty-four artificial concrete block habitats were tested at 6
sites located at Lyell, Faraday and Murchison Islands (Fig, 1),
These sites are within the Haida Gwaii Juan Perez Sound abalone
stewardship area, where annual ecological assessments, abalone
population surveys, and mark-recapture monitoring were con-
ducted during 1998 to 2003 (Jones et al, 2003), The general area
currently supports average densities of 0.35 emergent abalone/m"
and 0,17 emergent youth (<70 mm shell length. SL) abalone/m"
(Campbell et al, 2000),
The artificial habitat design used is a modification of that de-
scribed by Davis (1995), Each habitat provides about 3,5 m~ of
surface area and consists of 24 concrete mini-blocks haphazardly
oriented within a modified commercial crab trap (Fig, 2), Standard
20 cm X 20 cm X 40 cm concrete blocks were cut into quarters
longitudinally to produce four individual mini-blocks. Discarded
commercial crab traps measuring approximately 1 m in diameter
and 0,3 m in height were altered by removing the central "fishing"
component, leaving a structurally effective frame of corrosive re-
sistant metal enclosed with stainless steel mesh. Diamond-shaped
openings within the wire mesh frames were approximately 66 mm
X 91 mm and tested with empty shells to confirm their permeabil-
ity to abalone measuring less than 66 mm SL, Each structure also
possessed a prefabricated entry or exit hole measuring 102 mm in
diameter that was permeable to all abalone sizes and a hinged lid
that allowed access to load, remove, and examine concrete mini-
blocks during artificial habitat deployment and sampling.
In July 2001. 24 habitats were deployed by belaying each intact
819
820
DeFreitas
LEGEND
• Artificial Habitat
Sites
■ Natural Habitat
Sites
•■
Hecate
Strait
•■
^ •
^1
Lyell I.
Ramsay I.
Figure 1. Artificial and
Columbia.
natural abalone habitat study sites in northern Juan Perez Sound abalone stewardship area. Haida Gvvaii, British
unit from the dive support vessel to the ocean floor. Divers repo-
sitioned each structure with an industrial airlift bag. Within a site,
4 habitats were oriented parallel to shore in depths of 4 to 9 m and
from 7 to 30 m apart. The habitats were randomly located within
areas dominated by small boulders and cobble encrusted with red
coralline algae. No anchoring mechanisnts were used to secure the
units in place, because each unit weighed approximately 120 kg
and possessed a stable base.
Divers visually inspected artificial concrete habitats for struc-
tural integrity in February 2002 and thoroughly surveyed each unit
in situ during May and July 2002. A pair of divers sampled arti-
ficial habitats by removing and examining each concrete mini-
brick for abalone. All abalone found were measured for maximum
SL to the nearest millimeter and empty abalone shells were also
measured and removed. After all bricks were examined, they were
haphazardly repositioned within the metal frame. No special effort
^i
TABLE 1.
Total number of abalone found in 4 artificial habitats at each of 6
sites during surveys in May and July 2002.
Kigure 2. Artificial habitat design.
May
July
Site
Alive
Dead
Alive
Dead
1
17
1
24
1
">
44
1
38
4
3
5
2
3
2
4
12
0
10
1
5
37
3
31
0
6
37
3
20
1
Totiil
152
10
126
y
Estimating Juvenile Hauot/s kamtschatkana Abundance
821
to
60-
50-
40
30-
20-
0
ll
Mean = 42.6 mm
SE = O.S mm
n =278
ll..l ..
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Shell Length (mm)
Figure 3. Size-frequency distribution of :ib;ilone measured within artitlcial habitats during May and July 2002.
was made to remove or monitor abaloiie adhering to bricks as the
bricks were replaced back into the wire mesh containers.
To estimate the abundance of juvenile abalone occupying natu-
ral habitats, sampling was conducted within 10 iir of area at 4
artificial habitat sites and 4 additional random sites (see Fig. 1 ). At
randomly selected locations throughout the available abalone habi-
tat at each study site, divers invasively searched 10 l-m"^ quadrats
for all hidden and exposed abalone. This method in\i)lved looking
on the undersides of all movable rocks but did not include any
destruction of natural habitat as care was taken to return any dis-
turbed rocks to their original position. Diver efficiency in search-
ing natural habitats was not measured.
RESULTS
All 24 artificial habitats contained abalone (/; = 152. mea/( =
6.3 ± 0.95 SE abalone/container) during the first survey in May
2002. ten months after installation. During the second survey in
July 2002, all but 2 artificial habitats contained abalone (n = 126,
mean = 5.3 ± 0.87 SE abalone/container). There was no signifi-
cant difference in mean abalone/container for either total abalone
abundance (/-test, t = -0.84, d.f. = 46, P > 0.406) or total juve-
nile abalone (s50 mm SL) abundance {/-test, t = -0.47. d.f. =
34, P > 0.643) between the two sample periods.
A total of 278 abalone and 19 empty shells were counted and
measured within artificial habitats during the study (Table I ). Ju-
venile abalone (S50 mm SL) accounted for 75.4% (n = 224) of
all those measured, while only 3.6% (n = 15) were more than 70
mm SL and considered to be mature. The smallest and largest
abalone found in artitlcial strtictures were 15 mm and 100 mm SL.
The average abalone size was 42.6 mm SL (Fig. 3) and 56.4 mm
SL for all empty shells. On average, each artificial habitat required
12.5 min for a pair of divers to completely survey.
The mean density of juvenile, mature, and all-si/ed abalone
within artificial habitats was 1.27, 0.06, and 1.65 abalone/m". re-
spectively (Table 2). Juvenile abalone densities in artificial habi-
tats were significantly different between sites (one-way ANOVA,
F = 8.409. d.L = 5,35. P < 0.001 ). but not within sites, suggest-
ing differential recruitment to these locations.
A total of 82 abalone were counted and measured within natu-
ral habitat samples. Juvenile abalone accounted for 13.4% {ii =
I I ) of all those measured, while 64.6% (n = 53) were mature. The
smallest and largest abalone found in natural habitats were 14 mm
and 124 mm SL. The average abalone was 79.3 mm SL (Fig. 4)
and 75.2 mm SL for empty shells. Mean abalone densities during
the May and July surveys were similar (ANOVA, F = 0.819, d.f.
= 1,15. P = 0.38) and there was no difference in total abalone
densities between artificial habitat sites (sites 1-6) and additional
random sites (sites 7-10). The mean density of juvenile, mature
and all-sized abalone measured with natural habitats were 0.07.
0.33, and 0.51 abalone/m", respectively, (Table 3). Natural habitat
samples were located at a mean depth of 3.08 ± 0.08 m datum (min
= -0.4 m, max = 4.8 m).
Juvenile abalone densities measured within artificial habitats
were compared with natural habitat samples. At sites 1 to 4. where
TABLE 2.
Mean number and densities (#/m") of abalone in 4 artificial habitats
at each of 6 sites surveyed in Mav and ,lulv 2002.
No. of
Abalone
Abalone Density (#/nr)
Site
<50 mm
SL
>70 mm
SL
All Sizes
1
2().-S
1.00
0.00
1.46
1
41.0
2.14
0.14
2.93
3
4.0
0.29
0.00
0.29
4
II.O
0..39
0.18
0.79
5
.14.0
2.25
0.00
2.43
6
28.5
1.64
0.04
2.04
Mean
23.2
1.27
0.06
1.65
SE
4.1
0.25
0.03
0.29
Standard errors shown are for site groups (/i = 12).
822
DeFreitas
12 -I
10 -
8 -
u
1 '
t
to
Mean = 79.3 mm
SE = 2.9 mm
n =82
4 -
2 -
0 -
II .. ■ 1
il
III.
0 10 20 30 40 50 60 70 80 90 100 110 120 130
Shell Length (mm)
Figure 4. Size-frequency distribution of abalone measured wittiin natural habitat samples during May and Jul> 2002.
both artificial and natural habitat samples were conducted within
an area greater than 10.000 m", juvenile abalone densities mea-
sured within artificial habitats were significantly greater than those
within natural habitat samples (r-test. t = 3.049. d.f. = 14. P =
0.009). When all locations were included in the comparison, ju-
venile abalone densities measured within artificial habitats re-
mained significantly greater than those within natural habitat
samples (Mest. t = 5.597, d.f. = 26. P < 0.001). There was no
significant difference in juvenile abalone densities measured in
natural habitats at sites 1 to 4 when compared with sites 7 to 10.
indicating that the presence of artificial structures at sites 1 to 4 did
not influence juvenile abalone abundance in surrounding natural
habitats.
DISCUSSION
The artificial habitat design tested in this study provided sur-
rogate habitat for both juvenile and mature northern abalone.
Within 10 months of installation, native abalone had discovered
and occupied each of the 24 artificial habitats. The similar number
TABLE 3.
Mean number and density (#/ni-) of abalone in 10 natural habitat
samples at each of 8 sites surveyed in May and July 2002.
Site
No. of
Abalone
Abalone Density (#/m-)
<50 mm SL
>70 mm SL
All Sizes
1
8.0
2
1.0
3
6.5
4
3.0
7
3.5
8
6.0
9
6.0
10
7.0
Mean
5.1
SE
2.99
0.15
0.05
0.05
0.00
0.05
0.05
0.20
().()()
I). 07
0.09
0.45
0.45
0.10
0.25
0.55
(1.40
0.35
0.10
0.33
0.07
0.80
0.65
0.30
0.35
0.60
0.60
0.70
0.10
0.51
0.10
Standard errors shown are for site groups (/i
16).
of abaUme occupying artificial habitats in July suggested the con-
crete blocks continued to provide preferred shelter throughout the
summer months when surrounding food abundance is high and
good quality alternative natural habitats were available. The spe-
cific length of time required for artificial materials to condition and
attract abalone was difficult to determine due to the limited num-
ber of sample periods. The concrete materials appeared to be suit-
able for northern abalone within 7 months based on observations
of abalone occupying most artificial habitats during structural in-
spections in February 2002. The conditioning time of this material
was consistent with Davis (1995) who found "juvenile native H.
nifescens and H. comigata inhabited artificial habitats within 4
months of deploymenf" in California.
As indicated in Figure 3, juvenile abalone were the most abun-
dant size class occupying artificial habitats. Both the small mesh
size and high substrate complexity may have contributed to the
size selectivity by limiting access and suitable shelter for abalone
greater than 70 mm SL. Juvenile abalone densities measured
within artificial habitats were significantly different between sites
but similar within sites. This apparent ability of artificial structures
to quantify juvenile abalone abundance within standardized
sample areas at different locations may provide the feedback re-
quired to gauge the success of future stock restoration experiments.
Benefits of the modular artificial habitat design tested here in-
cluded the low cost of construction, ease of deployment, durability
within high energy subtidal environments, and most importantly,
their ease of being dismantled and reconstructed by divers in situ.
without the destruction of natural habitat.
In this study, juvenile abalone recruitment measured within
artificial habitats was not representative of recruitment measured
within nearby natural habitat samples. At sites 1 to 4, juvenile
abalone abundance measured within artificial habitats was signifi-
cantly greater than natural habitat samples, ranging in magnitude
from 4.3 times greater at site 3 to 42.9 times greater at site 2.
Although each natural habitat sample was randomly located within
good quality juvenile abalone habitat and the mean juvenile aba-
lone density found within natural habitats was similar to Campbell
et al. (2000), natural habitats provided little consistency with sub-
Estimating Juvknile Haliotis kamtschatkana Abundance
823
strate composition and hence, the lower abundance of sheltered
habitat. Specific factors that made the artificial structures attractive
to abalone were not investigated experimentally but were likely
due to the consistent and availability of good quality sheltered
habitat provided by the concrete blocks. Based on observations,
additional factors that may have influenced the abundance of aba-
lone in artificial habitats included easily accessed algal food grow-
ing on concrete bricks and a mesh frame that may have excluded
large predators such as Sunflower seastars (PYCiiopciiia hcliiiii-
thoides).
The measured abundance of juvenile abalone within artificial
habitats may haxe been at their annual spring peak, as surveys
were only conducted during May and July, a similar time of year
that Davis (1995) measured a peak in abalone recruitment. To
calibrate artificial habitats into better juvenile abalone abundance
instruments, it will be necessary increase the number of surveys
and monitor fluctuations in abalone abundance throughout the
year. Only by comparing the changes in abalone abundance from
winter to summer can the magnitude of localized recruitment
events be determined.
The use of artificial habitats as a standardized sampling instru-
ment to estimate the abundance of cryptic juvenile abalone was
supported by this research. The haphazardly oriented concrete
blocks provided preferred habitat for juvenile abalone and the
metal frame covered with wire mesh provides structural integrity
and allowed each sampling unit to be quickly deployed or reposi-
tioned. A pair of divers could easily sample units in situ, with no
destruction to either natural habitat or abalone adhering to concrete
bricks. For proposed abalone rebuilding experiments, artificial
habitats of this design can be used as an initial release site for
cultured juveniles, as an affordable method of determining base-
line juvenile abundance along coastlines of interest, and as a means
to quantify changes in juvenile recruitment that may be due to
experimental stock enhancement.
ACKNOWLEDGMENTS
The author thanks the many divers who participated during
field activities. Alan Campbell. Russ Jones, and Ron Ydenberg
who provided helpful comments during the experimental design
and analysis stages. This work was financially supported by the
Haida Tribal Society. Fisheries and Oceans Canada's Subvention
Grants Research Program, Environment Canada's Habitat Stew-
ardship Program for Species at Risk, and the Centre for Wildlife
Ecology at Simon Eraser University.
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Breen. P. A. 1986. Management of the British Columbia fishery for nonh-
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Sci. 92:300-312.
Breen, P. A. & B. E. Adkins. 1979. A survey of abalone populations on the
east coast of the Queen Charlotte Islands. August 1978. Fi.\li. Mar.
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Campbell. A. 2000. Review of northern abalone. Haliotis icamischulkana
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Campbell, A.. D. Brouwer. J. Rogers & D. Miller. 2000. Abalone resurvey
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Jones. R.. B. DeFreitas. N. A. Sloan. L. Lee. K. Von Boetticher & G.
Martin. 2003. Abalone stewardship in Haida Gwaii: forging a long-
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Keesing, J. K.. R. Grove-Jones & P. Tagg. 1995. Measuring settlement
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539-543.
McShane, P. E. 1992. Early life history of abalone: a review, pp. 120-138.
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McShane, P. E. & M. G. Smith. 1988. Measuring recruitment of Haliotis
rubra Leach (Gastropoda:HaIiotidae): comparison of a novel method
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Post-larval recruitment of blacklip abalone (Haliotis rubra) on artificial
collectors in southern Tasmania. Mar. Freshwater Res. 46:531-538.
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abalone (genus Haliotis). VI. Habitat preferences and abundance and
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295-339.
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Castledine. L. Convey. C. Cote. P. Coulson. T. Down. K. Francis. H.
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Joiimal of Shellfish Research. Vt.l, 22, No. 3, 825-829. 2003.
EARLY REPRODUCTION IN HATCHERY-RAISED WHITE ABALONE, HAUOTIS SORENSENI,
BARTSCH, 1940
THOMAS B. MCCORMICK'* AND JENNIFER L. BROGAN^
'Cluiniifl Islands Marine Resource Institute. P.O. Box 1627. Port Hiienenie. California. 93024;
'California State University. Nortliriili;c. Department of Biology. I SI] I Nordhoff Street.
Northridgc. California 91330
ABSTRACT While abalone. Hiilioiis sorenseiii. is the first marine invertebrate species to be placed on the endangered species list
in the United States. A controlled breeding program is underway to provide .stocks to reestablish wild populations ol this species. There
is no knowledge of the reproductive potential of early adults. This study examines the onset of gonad maturation and spawning
capability of one-year-old abalone. Both hydrogen peroxide and ultra-violet irradiated seawater induced spawning in males and females
as small as 25 to 35 mm in shell length. More males spawned in each treatment than females (P < 0.01 ). The onset of gonad maturation
is much smaller for white abalone than for other sympatric abalone in California. The implications for restocking strategies are
discussed.
KEY WORDS: abalone, endangered species. Halioris .wreiiscni. reproduction, niaturalion, hatchery
INTRODUCTION
The white abalone [Haliotis sorenseni) is one of seven species
of a large marine gastropod inhabiting the waters off the west coast
of North America. The deepest li\ ing of these species, the white
abalone was historically found between depths of 20 to 60 m and
was most abundant between 25 to 30 m (Cox I960, Tutschulte
1976). The range of the white abalone extended from Point Con-
ception in California to Punta Abreojos, Baja California, Mexico,
with an historical center of abundance in southern California
around the Channel Islands (Cox I960). Like other abalone, this
species was targeted by sport and commercial fishermen; 95% of
white abalone landings occurred between 1969 and 1977 yielding
268 metric tons. Such exploitation was not sustainable and resulted
in the dramatic decline of this species (Haaker 1994, Davis et al.
1996. Tegner et al. 1996. and Davis et al. 1998). With a life span
of approximately 30 to 40 y, it is estimated that the last successful
recruitment of the species was in 1966 (Hobday et al. 2000). Hob-
day and Tegner (2000) concluded that the population density of the
surviving animals was too low to permit successful recovery and.
without intervention the species may become extinct by 2010. In
2001 the white abalone was listed as endangered under Endan-
gered Species Act (Anon 2001). For the first time in the United
States, over-exploitation had pushed a marine invertebrate to the
brink of extinction.
Artificial propagation is one method that has been explored to
restore or re-establish species. Methods for the large-scale culti-
vation of abalone were first developed in Japan and have been
adapted for a variety of species within this genus (McCormick
2000) with the intent of enhancing natural productivity. Hatchery-
raised abalone from larvae to adults 80 mm in shell length (SL)
adults have been out-planted with varying success (McCormick et
al. 1994). To avoid costs associated with long-term hatchery cul-
tivation of juvenile and adult abalone. the release of larvae has also
been undertaken for a number of species. This approach has been
shown to increase the number of newly recruited juveniles (Tong
et al. 1987). however, the long-term impact of this method is
difficult to assess. Schiel (1992) compared the costs of releasing
larval and juvenile abalone. and concluded that the higher survival
*Corresponding author: P.O. Box 1528. Ojai. CA 93023
of juvenile abalone more than offset the additional cultivation
costs. Up to a point, increased survival has been correlated with
increased size for some species of abalone. Inoue ( 1 976) found that
survival for//, giganlca increased from 10-70% after I y when the
size of the abalone planted was increased from 10 mm to 30 mm
SL. Saito (1979) found that survivorship of transplanted H. discus
luiiiiiai reached a maximum when animals were 34 to 36 mm SL.
Survival rates declined for both smaller and larger animals.
Successful enhancement programs require not only that the
hatchery-reared animals survive in the wild, but reproduce as well.
Rearing the abalone in a protected laboratory setting allows for the
reintroduction of larger specimens, thus reducing losses resulting
from natural mortality prior to sexual maturity. The age at which
male and female abalone first reproduce and their fecundity will
partially detennine their contribution to the population after out-
planting. Studies of several abalone species revealed that the mini-
mum age of sexual maturity was found to be between 3 and 5 y.
ranging from 29 mm SL for female H. coccinea canariensis (Pena
1986), and 40-120 mm SL for 14 other species. Field studies of
abalone in southern California indicate that the age of sexual ma-
turity for pink abalone {Halialis corrui^atu) was 3 to 4 y (39 to 44
mm SL), for green abalone [Haliolis fuli;ens) was 5 to 7 y (61 to
89 mm SL), and for white abalone was 4 to 6 y (93 to 88 mm SL)
(Tutschulte 1976), Ault (1985) found that wild red abalone {Hali-
oris rufescens) could be induced to spawn at sizes of 65 mm SL for
males and 1 10 mm SL for females. Observations of a crop of white
abalone grown in our hatchery indicated that these animals exhib-
ited signs of sexual maturity at an age significantly younger than
previously reported. Sexual maturation was quantified and several
experiments were conducted to measure maturation, spawning re-
sponse, and fecundity.
MATERIALS AND METHODS
.Ahaloiie Ciiltivatinn
In November 2000, wild adult white abalone were collected by
the California Department of Fish and Game and transported to the
Channel Islands Marine Resource Institute (CIMRl) in Port Huen-
eme for culture. In April 2001. two females and one male were
successfully spawned using methods described by Morse et al.
(1977). Sperm from the male was used to fertilize eggs from both
825
826
McCORMICK AND BROGAN
females to create two half-sib families. Larvae were raised in
flow-through systems (Tong & Moss 1992) and settled on plastic
plates covered with cultures of micro-algae, diatoms and bacteria
(Seki & Kanno 1980). Outdoor tanks received filtered sunlight and
sand filtered UV sterilized seawater at amhieni temperatures.
Minimum and maximum temperatures ranged from 12°C to 20°C
during the first 15 mo of culture. After 6 mo, cultivated Pacific
dulse (Palmaria mollis) (Levin 1991. Evans & Langdon 2000) and
wild giant kelp (Macrocystis pyriferia) were added to the culture
tanks to supplement the micro-algae feeds. Tanks were periodi-
cally cleaned by siphoning detritus from the bottom.
Abaloiie Growth
Starting 79 days after spawning, the shell length of 50 abalone
from each of 1 1 tanks was measured to the nearest 0.1 mm. Aba-
lone were also periodically weighed to determine weight-length
relationships. Normality of the lengths of the sampled individuals
was first determined using the Kolmogorov-Smirnov test, and then
data was subjected to ANOVA. which revealed no significant
difference in growth between the two half-sib families or among
the 1 1 cultivation tanks. A growth curve was then constructed
using the von Bertalanffy growth function (Bertalanffy 1960)
based on the mean lengths of the abalone within the 1 1 tanks.
Sex Determination
During routine measurement of shell length, abalone were in-
spected externally for presence of gonads. Histologic exaininations
could not be performed since such studies would require sacrifice
of the animals. We used a non-lethal method developed by Uki and
Kikuchi ( 1982). This method is used in many hatcheries to assess
the spawning readiness of the broodstock. The Gonad Index (GI)
ranking is as follows:
Gonad
Index Description of (ionad and .SpaHninjj .Activity
0 No gonad observed. Not possible to determine sex. Abalone
will not spawn.
1 Small volume of gonad observed. Possible to determine sex of
abalone by gonad color. Abalone will not spawn.
2 Larger volume of gonad covers the conical appendage of the
digestive gland. Easy to determine sexes. Gonad hulk
visible. Abalone may spawn.
y Volume of gonad quite large, may extend below the plane of
the shell opening. Abalone will usually spawn.
Pluses and minuses attached to these values were sometimes
used to designate a gonad index that fell between the index num-
bers. Sex was distinguished by color: the gonad of the males was
a cream color while that of the females was green-grey.
All of the abalone examined were from the same family but
were reared in two separate tanks. The GI and shell length of each
animal were determined and recorded. From one tank. 146 animals
were examined, from another, 255 for a total of 401 abalone (Table
1 ). From the two tanks examined, 1 15 abalone with a GI of "2" or
higher were sequestered for the spawning study.
Spawning Methods
Two spawn inducing treatments, ultraviolet (UV) inadiated
seawater (Kikuchi & Uki 1974) and hydrogen peroxide, H^O,.
(Morse et al. 1977). were tested to determine if these methods
resulted in differences in the number of animals that spawned or in
the quantity of gametes released. In both treatments, individual
abalone were placed in 260-itiL plastic containers. This prevented
early spawners from stimulating neighboring animals and enabled
us to isolate and count gametes from each animal. For the UV
spawning method. 4.8 L/min seawater was filtered to 5 p.m and
passed through four 30-watt UV sterilizers. Two of the units failed
during the experiment, resulting in a dosage of approximately 200
milli-Watt h/L. This is lower than the 800 milli-Watt h/L optimal
dosage suggested by Kikuchi and Uki (1974), but still within the
effective range. Abalone subjected to the UV irradiated water were
also given a thermal shock by rapidly increased water temperature
from I5°C to 2\°C followed by gradual cooling back to 15 C (Ino
1952).
Hydrogen peroxide is commonly used in hatcheries to induce
spawning in abalone. To test this method on white abalone, hy-
drogen peroxide and tris-(hydroxymethylamino) methane solu-
tions were added to filtered, UV irradiated seawater at 15'^C. Each
spawning container received 200 mL each of hydrogen peroxide
and Iris solutions. When the abalone began to release gametes, or
if the suggested 2.5 h had elapsed since initial exposure (Morse et
al. 1978), the solutions were decanted and replaced with filtered,
UV-irradiated seawater.
Both UV and H,Ot treatments were tested with a minimum of
25 females and 24 males. Table 1 shows the sex, GI, and, number
of abalone used in this study. More males with a GI of 3 were used
in the H2O2 treatments to determine the frequency of males able to
spawn, and the correlation between the amount of gametes pro-
duced and shell length. In cases where there were an uneven num-
ber of individuals with a particular GI (for example, there were
nine females with a GI of 3). the extra individual was randomly
assigned to either the H,0; or the ultraviolet (UV) spawning treat-
ment. Because of a shortage of ripe females, five additional fe-
males were taken from the second tank used in the sex determi-
nation study previously mentioned. After both treatments had been
completed, gametes were examined under x400 magnification for
visual identification of any defects in the gametes. None of the
eggs were fertilized since our permit under the Endangered Spe-
cies Act of 1976 was pending. Subsequently all gametes were
destroyed and abalone were returned to their respective tanks,
unharmed.
RESULTS
Abalone Growth
Growth of individuals within different tanks were compared,
and found to be .statistically similar (Anova, P = 0.76). Mean
lengths were calculated for all 1 1 tanks, and then compared with
lengths predicted by the von Bertalanffy growth function (L, =
L„(l-e''"); L., = 231.70, k = 0.065, t = age of abalone. Total
body weight was also measured for individuals of different lengths
(/) = 317) and fitted to a non-linear least squares regression, which
denotes weight (W in g) as a function of shell length (L in mm) (R~
= 0.9957). W = 0.0001 L' '*"' (Fig. 1).
Sex Determination
Of the 401 individuals surveyed for sex determination, the
lower limit on males exhibiting gonads was 15.9 mm SL, but this
was an outlier. The next lowest was 20.8 mm SL. For females, the
lower limit was 20.9 mm SL. A Kruskal Wallace test showed that
Earl>' RhPRouucTiuN IN Hatchery-Raised White Abalonh
827
Female
Male
TABLE I.
Percentage of abalone spawned and gamete production using ultra>iolet and hydrogen peroxide.
GI
2
2+
3
2
2+
3
Ultraviolet + Temp. Shock
Number
Tested
9
11
5
11
5
Percent
.Spawned
11
46
70
63
IIKI
SO
Number of
CJametes
X 10'
1.3
0.2-10.2
1.3
76-3.210
16.3-1,686
924-7.018
Hydrogen Peroxide
Number
Tested
10
11
4
9
11
20
Percent
Spawned
70
36
50
67
64
95
Number of
Gametes
X 10'
0.07-1.1
0.-39-1.3
0.33-0.90
no significant difference existed in the SL of the males and females
(P = 0.43). Also, Pearson's x" tests revealed that there was no
significant difference in the number of males in the population
compared with the number of females (P = 0.09).
Spawning
The smallest female that spawned measured 25.6 mm SL,
whereas the smallest male was 23.1 mm SL. There was no differ-
ence among different size classes of individuals with regard to the
occuiTence of spawning (Pearson's x" test, P = 0.42). Greater
than 95% of the eggs and sperm examined appeared normal, with
no obvious defects. Significantly more males than females
spawned in both treatments (Pearson's x" test. P < 0.001 under UV
treatment, P = 0.009 under H,Oo treatment) (See Table I ). How-
ever, there was no significant difference in the number of males
that spawned in either UV or H-,0, (P = 0.07), nor was there a
difference between the number of females that spawned in either
of these treatments (Pearson's x" test, P = 0.08). During the
course of this study, males began releasing sperm early in the
hydrogen peroxide treatment. The seawater solution was immedi-
ately decanted to minimize exposure to the hydrogen peroxide. In
doing so, some sperm were lost and thus it was impossible to
obtain accurate gamete counts.
For males in the UV treatment, no correlation existed between
size and number of sperm released (Pearson correlation coeffi-
cient, r = 0.306). The largest amount of sperm (5.6 and 7 x lO'')
were released by abalone that measured 27.8 and 31.2 mm SL.
respectively, while an intermediately sized male released only 7.6
X lO' cametes. An average number was 1 .5 x lO'' gametes released
0 10 20 30
Shell Length (mm)
Figure 1. Shell length — whole wet live weight relation for white aba-
lone. Kquation for power curve: Weight = (1.00(11 * Shell Length-''"'''^
(R- = 0.9957, » = 317).
per male abalone. Similarly, for females no correlation existed
between shell length and the number of eggs released, which
ranged between 65 and 10,237 eggs per individual (Pearson cor-
relation coefficient, r = 0.079). Because of large variation within
treatments, a Mann-Whitney t/-test indicated that there was no
difference in the amount of eggs released between treatments (P =
0.09), although the average number of eggs released in the UV and
H-,0, treatments was 2,880 and 673, respectively.
DISCUSSION
Growth of white abalone for the first year in the hatchery
averaged 30.0 to 43.4 |j.ni/day, yielding an average 16 mm SL.
(minimum 7.0 mm SL to maximum 30.3 mm SL). This was some-
what less than hatchery growth rates expected for red and green
abalone but was similar to that of pink abalone (McCormick, pers.
obse.). No significant difference was observed in the frequency of
males and females in the population of I -year-old abalone exam-
ined (Pearson's x" test, P = 0.502). This trend is considered
common in organisms of many phyla, including invertebrates,
where males and females reach maturation at approximately the
same size and age. Analysis of 600 legal adult white abalone from
the Channel Islands yielded a sex ration of 1 : 1 over all size classes
(Tutschulte 1976, Tutschulte & Connell 1981).
The present work shows the onset of sexual maturation is sig-
nificantly earlier than previously suggested, and hatchery-raised
abalone, as young as 1 y in age (23-25 mm SL), are capable of
spawning. Tutschulte and Connell (1981) proposed that the mini-
mum age required for white abalone to reproduce is 4 y. However,
the estimate was based on a sample of only three individuals
smaller than 130 mm SL (Tutschulte 1976). The red abalone is a
close relative to white abalone (Yang et al. 2000). Studies of
sexual maturation of red abalone in northern California, (Giorgi &
DeMartini 1977) have shown that wild red females matured at a
minimum of 39.5 mm SL, whereas males were at least 84 mm SL
before reaching sexual maturity. They also found that the onset of
sexual maturation precedes that indicated by visual observation of
the gonad. When examined histologically, animals as small as
25-50 mm SL were found to contain spermatozoa or oocytes. A
similar phenomenon was found in the ormer. (//. tiibenulara)
where spermatozoa were present in animals as small as 28 mm SL
but were not observable until the animals reached a size of 40 mm
SL (Pena 1986). The smallest red abalone spawned by Auit (1985)
were 65 mm SL for males and I 10 mm SL for females.
828
McCORMICK .\ND BROGAN
Hatcher\-raised white abalone mature at sizes smaller than
their wild coiniterparts. Hatcher>' conditions, most notably the con-
tinuous presence of abundant food sources, no doubt promote the
early sexual maturation of white and some other species of aba-
lone. Ault (1985) demonstrated this effect with red abalone and
after conditioning them in the laboratory for 90 days. Conditioned
animals spawned at minimum sizes of 55 mm and 60 mm SL for
males and females, respectively. These sizes were 85% and 55%
the length of the smallest wild spawners (see earlier). Ault (1985)
also found that the improved diet of laboratory conditioned aba-
lone increased fecundity. Laboratory conditioned red abalone less
than 100 mm SL were as fecund as wild animals up to 140 mm SL.
Over the course of the last 2 decades, we have noted that crops
of hatchery-raised red. green, and pink abalone start to mature at
about 50 mm SL. This was less than wild animals but still twice the
size of spawning white abalone in the present study (T. McCor-
mick, pers. obse.). Tutschulte (1976) found that large (88 to 159
mm SL) wild white abalone were more fecund than either pink or
green abalone. We now know that white abalone in the hatchery
also mature at a smaller size. Mature gonads were observed in
laboratory populations in animals as young as 10 months in age.
Although the gametes were not tested for viability (due to permit
constraints) no obvious defects were observed and we expected
that the eggs and sperm would be as viable as those of older
animals.
The implied higher fecundity of hatchery-raised white abalone
may have an impact on enhancement efforts for this species. When
hatchery-raised white abalone are used for enhancement efforts the
number of animals that ultimately make a reproductive contribu-
tion depends upon the natural annual mortality rate (M) and the
time required to reach sexual maturity. Even in natural communi-
ties, M is high for newly recruited abalone and may vary from
1-10 between populations (Schiel 1992. Shepherd & Breen 1992).
For hatchery-raised abalone M is often higher than that of wild
populations (Rogers-Bennett & Pearse 1998). Survival and con-
sequent reproductive contribution may be improved by increasing
the size of abalone at release. To date, many enhancement efforts
utilizing hatchery-raised abalone have focused on abalone averag-
ing 20-30 mm SL, with larger animals having greater survival (see
summary in McCormick et al. 1994). Production of larger animals
requires longer growout times and increased cultivation expense.
Earlier sexual maturity and higher fecundity rates of hatchery-
raised abalone may be another way of increasing reproductive
contribution. We have shown that hatchery-reared white abalone
mature at a much younger age and smaller size than anticipated.
Providing white abalone with abundant food sources in the hatch-
ery could make their initial reproductive contribution equivalent to
that of animals much larger, as Ault (1985) observed.
Observations in our laboratory indicated that, unlike their wild
counterparts that have highly synchronized maturation and spawn-
ing cycles, small hatchery-raised adults were apparently capable of
spawning for much of the year. After first maturing in the late
winter and early spring, the present crop of young adults remained
gravid for over a year. Large wild adult abalone synchronize gonad
maturation, culminating in a short spawning period in late winter
(Tutschulte 1976. Tutschulte & Connell 1981 ). As with some other
abalone species (Tutschulte & Connell 1981, Newman 1967,
Poore 1974, and Shepherd & Laws 1974), food availability may
regulate periodicity of the reproductive cycle.
CONCLUSIONS
This work defines the lower limit of sexual maturation in cul-
tured white abalone. Field studies suggested that wild white aba-
lone matured at 4 to 6 years in age at a size 80 mm SL or greater
(Tuschulte 1976). At 25 mm SL. white abalone in our hatchery
were sexually mature at a much smaller size than other species
reared in North American hatcheries. Ault (19851 noted the same
phenomena for red abalone in which wild abalone were approxi-
mately twice as large when sexually mature than hatchery condi-
tioned animals. If the same relationship holds true for white aba-
lone, we expect that wild white abalone could begin spawning at
50 mm SL. Tutschulte (1976) noted that white abalone have
greater variation in reproductive success than do pink or green
abalone and would benefit from an early age at sexual maturity and
long life span. This would give them more opportunities for suc-
cessful spawning. The present data seems to support this argument.
Studies quantifying survivorship of a range (25-100 mm SL) of
young adult white abalone after outplanting into the marine habitat
are necessary. Field research will be needed to document survival
and changes in gonad maturation as young hatchery-raised adult
white abalone acclimate to seasonal cycles of temperature and
food abundance in their natural environment. Recruitment events
and seasonal changes in gonad bulk will provide additional infor-
mation on long-term impact of food resources on the abalone.
ACKNOWLEDGMENTS
The authors thank Peter Haaker. Ian Taniguchi and other per-
sonnel from the California Department of Fish and Game for col-
lection of adult white abalone for CIMRI. Carl Demetropouolos
cultivated red algae used as one of the feeds for the abalone. We
also thank Carolyn Friedman and Alan Campbell for valuable
suggestions and criticism of the manuscript. This work was sup-
ported, in part, by grants from Reliant Resources, Inc. and the
Ventura County Fish and Game Commission. The opinions pre-
sented in this article are those of the authors and not the funding
agencies.
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Jiniriuil of ShcUfish Rcscanh. Vol. 22, No. 3. 831-838. 2003.
DISTRIBUTION AND ABUNDANCE OF HAUOTIS KAMTSCHATKANA IN RELATION TO
HABITAT, COMPETITORS AND PREDATORS IN THE BROKEN GROUP ISLANDS, PACIFIC
RIM NATIONAL PARK RESERVE OF CANADA
T. TOMASCIK' AND H. HOLMES^
^ Parks Canada. Western Canada Serx'ice Centre. 300-300 West Georgia Street, Vancouver. BC. Canada
V6B 684: -Parks Canada. Pacific Rim National Park Reserve. Box 280 Uchielet. BC. Canada VOR 3A0
ABSTR.ACT Ba.seline mfomiation on the distrihutioii and abundance of Hiiliolis himlsclmlkwm wa.s obtained throughout the Broken
Group Islands (BGI) in shallow- (2-5 m) and deep-water (6-9 m) habitats. The study demonstrates that abundance of northern (pinto)
abalone varied spatially throughout the area and with depth. The shallow habitats in the study area supported significantly higher
densities (0.18 abalone/m" ± 0.02 5£) of northern abalone when compared with deep habitats (0.10 abalone/m" ± 0.02 Sf). Maximum
and minimum sizes of northern abalone measured in BGI were 132 and 4 mm shell length (SL). respectively. There were significant
differences in abalone SL among the 5 island groups and the 2 depth /ones. Juvenile abalones were more abundant in the deep habitat
than in the shallow habitat. A significant correlation was detected between abalone densities and the relative index of exposure. There
was a positive correlation between abalone size and the abundance of benthic macroalgae and an inverse relationship between abalone
size and the abundance of red sea urchins {StnmgylocentiDtus fiimci.scaniis). A positive correlation between abalone and red sea urchin
densities was observed. Seven percent of juvenile abalone (<45 mm SL) was found under the red sea urchins" spine canopy.
Distribution and abundance of selected invertebrate species associated with northern abalone including its known predators (ie. sea
stars, crabs, octopuses) were assessed. The abundance of northern abalone was inversely correlated with predator abundance and
density of benthic macroalgae. Detailed surveys of associated organisms and substrate types suggest that the distnbulion and abundance
of northern abalone is a complex function of community interactions and substrate habitat characteristics.
KEY WORDS: Northern (Pinto) abalone. Halititis kaiiil.'.clhilkami. red sea urchins, competitors, predators, habitat, distribution
INTRODUCTION
Large, mobile invertebrates, such as abalone and sea urchins,
are an important component on subtidal rocky reefs in the coastal
waters of British Columbia. The northern (or pinto) abalone iHali-
Otis kamtscliatkana Jonas. 1845) is found distributed from Alaska
(Paul & Paul 1981) to California (Sloan & Breen 1988) along the
west coast of North America. Historically. H. kamtschatkana was
widely distributed in British Columbia with preference to semi-
exposed to exposed coastal habitats where they graze mainly on
attached or drift macroalgae and diatoms. Abalone are slow grow-
ing and long-lived gastropods, characterized by patchy distribu-
tion, sporadic recruitment, density dependent reproduction and
short larval period (Hobday et al. 2001 ). They are dioecious broad-
cast spawners with peak reproductive activity during the summer
(Breen & Adkins 1980). During spawning events abalone aggre-
gate in shallow subtidal areas to maximize fertilization success,
which depends on their aggregation density (Babcock & Keesing
1999). It is now recognized that northern abalone is particularly
vulnerable to overexploitation because of this life history strategy.
The coastal First Nations of British Columbia have a long
history of harvesting northern abalone for a wide range of uses,
ranging from subsistence harvests to use in native art and cultural
activities (Stewart 1977). The first record of modern commercial
abalone fishery in British Columbia dates to the early 1900s (Sloan
& Breen 1988). Prior to the invention of SCUBA, the abalone
fishery targeted mainly the intertidal populations; thus subtidal
areas were in effect natural refugia. The use of SCUBA to harvest
abalone started in the 1950s, but was generally restricted to few
operators. Abalone commercial landings in British Columbia
peaked in 1977 to 1978 (428-433 tons, respectively) and then
continued to decline. Northern abalone was targeted by recre-
ational and commercial dive fisheries until 1990, when the fishery
was closed due to major stock declines (Campbell et al. 2000). The
purpose of the 1990 commercial fishery closure in British Colum-
bia was to allow the abalone populations to rebuild. However.
numerous stock assessment surveys by Department of Fisheries
and Oceans Canada (DFO) during the 1990s have shown no evi-
dence of recovery (Campbell 2000). As a result. H. kamt.schatkana
was designated as threatened in 1999 by the Committee on the
Status of Endangered Wildlife in Canada (COSEWIC). Recovery
strategy and action plans are now in place to assist in rebuilding
the northern abalone population to sustainable levels. This study is
part of that strategy.
The present study was conducted in the Broken Group Islands
(BGI). which are part of the Pacific Rim National Park Reserve of
Canada (PRNPR). A recent DFO survey of abalone populations in
southeast Barkley Sound, adjacent to BGI, provided no evidence
of recovery of abalone populations since the province-wide closure
in 1990 (Lucas et al. 2002). The mean reported density of 0.1
abalone/m~ is significantly lower than the mean density of 0.52
abalone/m" reported by Emmett and Janiieson (1988) from the
same area prior to the 1990 closure. The objective of the present
study is to provide baseline information for the managers of
PRNPR on the distribution and abundance of northern abalone
throughout the BGI at two depth zones (shallow: 2-5 m below
chart datum: and deep: 6-9 m below chart datum). The study was
designed to explore the association of abalone with other compo-
nents of their subtidal habitats, by providing key information on
the distribution and abundance of organisms associated with the
species, including its major known predators. The study forms the
baseline against which to compare future response of abalone
populations to sea otter {EnlnJni lutris Linnaeus, 1758). recolo-
nization of BGI, and to the expected increase in climate variability
associated with climate change.
MATERIALS AND METHODS
Description of Study Sites
The BGI Archipelago is located on the Pacific coast of Van-
couver Island in Barkley Sound, roughly between latitudes
831
832
TOMASCIK AND HOLMES
48°57.683'N and 48°50.233'N, and longitudes 125°12.700'W and
125°24.700'W (Fig. 1). Based on geographic and oceanographic
features, the BGI were sub-divided into 5 island groups and strati-
tied in two depths. The 5 island groups were: Group I. Hand:
Group 2, Doddi Group 3. Clark; Group 4, Wouwer: and Group 5.
Gibraltar (see Fig. 1). The tidal range within the BGI is approxi-
mately 3.8 m. Based on past studies by DFO. each of these island-
groups was stratified into shallow (2-3 m below chart datum) and
deep (6-9 ni below chart datum) zones reflecting the distribution
of northern abalone populations (eg, McShane & Naylor 1995,
Sloan & Breen 1988. Campbell et al. 1998. Campbell et al. 2000,
Lucas et al. 2002).
SAMPLING PROTOCOL
A 200 X 200 m geo-referenced grid was laid over each of the
5 island groups using ArcGIS 8.x software. All 200 x 200 m blocks
that intersected a shoreline or offshore reefs within each island
group were sequentially numbered. The number of blocks that
intersected a shoreline or an offshore reef ranged from 58 in Group
3 to 295 in Group 5. Random selection without replacement was
used to select 4 sampling locations (ie, blocks) in Group 1. 5
locations in Group 2. 4 locations in Group 3. 4 locations in Group
4. and 5 locations in Group 5. for a total of 22 sampling locations
(see Fig. I ). A relative index of exposure was computed for each
site following procedures described by Ekebom et al. (2002). At
each location, 2 sites approximately 30 m apart were sampled.
Sampling was conducted by 2 dive teams.
Sampling at each site was conducted by randotnly placing 1 nr
quadrats along 25 m virtual transects that were laid randomly
parallel to the depth contour. Two transects were sampled within
each depth zone at each site. The position of transects within each
depth zone was determined by randomly selecting two specific
Figure \. Map of the Broken Croup Islands within the Pacific RIni National Park Reserve located on the west coast of Vancouver Island. British
Columbia, Canada. Dark lines represent (he rough boundaries of the 5 groups in which randomly chosen study locations were set up. Black dots
and associated numbers indicate the number and position of each study location in the survey. The 5 geographic groups were: Group I. Hand;
Group 2, Dodd; Group 3, Clark; Group 4, Wouwer; and Group 5, Gibraltar.
Distribution and Abundance of H. kamtschatkana
833
depths within each zone (shallow zone: 2. 3. 4, and 5 m; deep zone:
6, 7, 8, and 9 m). Ten random 1 m" quadrats were sampled along
each 25 m virtual transect. The positions of the 10 random quadrats
along each transect were determined by randomly selecting 10
numbers between 1 and 25. The random quadrat selection was
conducted prior to the survey and marked on underwater recording
sheets that were specific for each sampling transect. The starting
point of each transect was selected haphazardly by swimming
along the depth contour and dropping the quadrat after about a 1 to
2 min swim. Once the starting point was determined the divers
proceeded to flip the 1 m~ quadrat along the virtual transect until
they reached the first predetermined randomly selected position.
Quadrat sampling included divers carefully lifting up (but not re-
moving) all large macrophytes from the quadrat area to facilitate
the systematic search for both emergent and cryptic specimens.
Rocks were not removed or turned over in this survey. Once sam-
pling of the quadrat was completed divers proceeded to flip the
quadrat to the next randomly selected position along the virtual
transect. This procedure was repeated until all 10 quadrats were
sampled, or divers were forced to surface due to safety consider-
ations.
Abalone and red sea urchin (Su-ongylocen1rotus franciscanus
Agassiz. 18631 counts, including maximum shell length (SL) and
test diameter (TD) measurements in mm. were recorded in all 10
quadrats m each transect. The green sea urchin. Slrongyhicentiotus
droebachiensis (O.F. Miiller, 1776) and the purple sea urchin.
Strongylocentrotiis inirpitnitiis (Stimpson. 1857) were also re-
corded. The number and size of juvenile abalone found under the
red sea urchin spine canopy were also recorded in each quadrat.
Predator densities, including dungeness crab (Cancer magistcr
Dana, 1852), red rock crab {Cancer productiis Randall, 1839),
octopus (Enlcroctopiis clofleiiu Wiilker. 1910), and sunflower sea
star (Pycnopodia heliantlwides (Brandt, 1835)), were recorded in
all quadrats along each transect. For octopuses, either individuals
or inhabited dens were counted. Sea otters were not observed in the
study area.
Densities of benthic macroalgae were estimated in 5 randomly
selected 1-m" quadrats (from the original 10 quadrats) along each
transect. Because of time constraint, ease of taxonomic identifica-
tion and reporting efficiency, the following macroalgae were in-
cluded: (1) Macrocystis inlegrifolia Bory, 1826; (2) Nereocyslis
luetkeana (Mertensi Postels et Ruprecht. 1840; (3) Fnciis spp.; (4)
Eisenia arborea Areschoug, 1876; (5) Hedopliylluni sessile (C.
Agardh) Setchell, 1901; (6) Aganiiu cliitluarmii Dumortier, 1822;
(7) Pteiygophora californica (Ruprecht, 1852); (8) other browns;
and (9) green algae. In each quadrat, the macroalgae were identi-
fied and counted. Algal holdfasts were counted for all species
except M. inlegrifolia for which the number of stipes was used.
The following substrate cover types were defined in the present
study: (1) encrusting coralline algae, (2) articulated coralline algae.
(3) brown algae, (4) green algae, (5) bryozoans, (6) sponges. (7)
other invertebrates, and (8) sand. The percentage cover of each
substrate type was quantified in 3 randomly selected 1 ni~ quadrats
(from the original 10 quadrats) in each transect using a point-
intersect method. This method involved the use of a quadrat, which
was permanently marked along one side with 20 points (5 cm
apart), and a I m PVC bar that was permanently marked with 5
random points. Sampling involved placing the 1 m PVC bar across
the quadrat from 3 randomly chosen points along the side of the
quadrat and recording the substrate type that was found under each
of the 5 points on the PVC bar. The three random points along the
side of the quadrat were chosen earlier and were marked on re-
cording sheets. Each quadrat was sampled with 15 points (45
points per transect ).
STATISTICAL ANALYSES
All data analyses were conducted using the NCSS statistical
package (Hint/e 2001). Tests of normality and homogeneity of
variance were performed on all data sets using normal probability
plots and modified Levene equal-variance test, respectively.
Square root (SQRT) and ARCSINE(SQRT(X)) transformations
were performed as appropriate (Zar 1996) and the assumptions
were tested again on the transformed data sets to verify the success
of the transformations.
A nonparametric Kruskal-Wallis one-way ANOVA on ranks
was used to compare abalone and red sea urchin densities, as well
as red sea urchin test diameters, among the island groups and depth
zones, since no transformation was able to normalize the data. This
non-parametric procedure tests the null hypothesis that all medians
are equal and is an accepted substitute for one-way ANOVA.
Where significant (P < 0.05) among group differences were indi-
cated, we used the Kruskal-Wallis multiple-comparison Z-value
test to find specific among group differences. The Z-values are
appropriate for testing whether the medians of any two groups are
significantly different.
One-way ANOVA (fixed model) was used to compare abalone
shell lengths (untransformed) among the 5 island groups and be-
tween the two depth zones. To identify specific among group
difference we used the Tukey-Kramer multiple-comparison test,
which examines all pairs of group means. The Kolmogorov-
Smirnov goodness of fit test was used to compare abalone and red
sea urchin size frequency distributions between the shallow and
deep zones. The nonparametric Spearman Rank Correlation was
used to assess the relationship between the relative exposure index
and abalone densities, since transformations failed to normalize the
data. The test produces a correlation coefficient (/\), which may
range from -1 to 1, and it has no units (Zar 1996). The parametric
Pearson Product-moment Correlation analysis was used to identify
significant relationships between the abundance of northern aba-
lone (SQRT transformed) and the various substrate cover types
(ARCS1NE(SQRT(X) transformed). This procedure was also used
to examine relationships among abalone. red sea urchin, predator,
and macroalgae densities (SQRT transformed). The test produces
a simple correlation coefficient (/), which is unitless and ranges
from -1 to 1. Simple linear regression analyses were used to assess
the relationships between abalone size (untransformed), red sea
urchin densities (SQRT transformed), and benthic macroalgae den-
sities (untransformed). One location (group 1. location 2l was
excluded from these regression analyses because no abalone were
found at this location.
RESULTS
Dislribution and Abundance of Abalone
Northern abalone were present at all island groups surveyed in
this study, although in varying densities (Table 1 ). The result of the
Kruskal-Wallis one-way ANOVA on ranks indicated significant
differences (f < 0.05) in abalone densities among the 5 island
groups. The Kruskal-Wallis multiple-comparison Z-value test re-
vealed that abalone densities in Group 3 were significantly higher
(P < 0.05) than at all other groups. There were no significant
834
TOMASCIK AND HOLMES
TABLE 1.
Northern abalone, red sea urchin, predator and macroalgae densities (mean ± SE) at the 5 island groups and 2 depth zones in the study.
The numbers in parentheses are sample size - n (ie. number of quadrats), ii for red sea urchins and predators is same as for abalone.
Island
Group
Abalone
(Nuniber/ni")
Red Sea Urchin
(Number/m")
Predators
(Number/m")
Macroalgae,
(Number/m")
Group 1
Shallow
Deep
Group 2
Shallow
Deep
Group 3
Shallow
Deep
Group 4
Shallow
Deep
Group 5
Shallow
Deep
Total
Shallow
Deep
0.143 H
0.163 d
0.117 d
0.131 d
0.16.';d
0.044 d
0.266 d
0.288 d
0.240 d
0.104 d
0. 1 54 d
0.045 d
0.085 d
0.135 d
0.025 :
0.147 d
0.180:
0.100:
0.03 (280)
0.05(160)
0.04(120)
0.03 (320)
0.03 (230)
0.03 (90)
0.04 (320)
0.06(170)
0.05(150)
0.02 (240)
0.03(130)
0.02(110)
0.02 (353)
0.03(193)
0.01 (160)
0.01 (1513)
0.02 (883)
0.02 (630)
0.596 :
0.700 :
0.458 :
0.456 :
0.370 :
0.678 :
2.538 :
3.059 :
1.947:
1.242:
0.915:
1.627:
0.244:
0.264 :
0.219:
0.997 :
1.005:
0.987 :
0.06
0.12
0.08
0.07
0.07
0.17
0.13
0.19
0.16
0.12
0.14
0.20
0.06
0.10
0.06
0.05
0.07
0.07
0.200 d
0.225 d
0.167:
0.228 :
0.261 :
0.144:
0.166:
0.141 :
0.193:
0.208 :
0.208 :
0.209 :
0.241 :
0.306 :
0.163:
0.210:
0.233 :
0.176:
0.03
0.04
0.04
0.03
0.03
0.04
0.03
0.03
0.04
0.04
0.05
0.05
0.03
0.04
0.04
0.01
0.02
0.02
0.943 :
0.725 :
1 .233 :
4.176:
5.042 :
1.867:
0:
0:
0:
2.565 :
3.072 :
1.927:
4.486 :
4.367 :
4.627 :
2.534 :
2.883:
2.038 :
0.17(140)
0.21 (80)
0.29(60)
0.58(165)
0.76(120)
0.51 (45)
0(160)
0(85)
0(75)
0.43(124)
0.62(69)
0.58(55)
0.42(181)
0.55(98)
0.66(83)
: 0.19 (770)
: 0.27 (452)
; 0.24 (318)
differences in abalone abundance among the other 4 groups {P >
0.05). Mean abalone densities in the shallow zone (0.18 ± 0.02 SE)
were almost twice as high than in the deep zone (0.10 ± 0.02 SE).
The Kruskal-Wallis Z-value test revealed significant differences
(P < 0.05) in northern abalone densities between the shallow and
deep zones.
The mean SL of H. kamtschatkana measured in this study was
59.4 mm (± 2.0 SE\ n = 222). The results of one-way ANOVA
revealed significant differences in the mean SL of northern aba-
lone among the 5 island groups. The mean SL of abalone in Group
3 was significantly smaller {P < 0.05) than those of Groups 2. 4.
and 5 (Table 2 and Table 3). Largest abalone were found in Group
2 followed by Group 4. No differences (P > 0.05) in abalone size
were found between groups 1 & 3. 1 & 4, 2 & 5, and 4 & 5. The
area with the highest densities of abalone (i.e.. Group 3) was also
the area with the smallest abalone. In general, the mean SL of
abalone in the shallow depth zone (0 = 64.7 mm ± 2.4 SE) was
significantly larger than in the deep-water habitat (0 = 46.3 mm
± 3.2 SE) (one-way ANOVA; F = 18.1; P < 0.001). The results
of the Kolmogorov-Smimov test indicated that differences in aba-
lone size frequency distributions between the shallow and deep
zones were statistically significant (P < 0.001) (Fig. 2).
Distribution and Abundance of Red Sea Urchins
The red sea urchin iS. franciscaiuis) was the most abundant
echinoid in the study area. The abundance of both green (S. droe-
bacliiensis) and puiple (5. piirpiirains) sea urchins was so low (i.e..
17 and 5 individuals, respectively) that they were left out of the
analysis. Red sea urchins were found in all island groups (Table 1 ).
Significantly higher mean red sea urchin densities (urchins/m")
were found in Group 3 than anywhere else in the study area (Table
4). No significant differences in red uichin densities were observed
between groups I & 2 and groups 2 & 5. For all areas combined,
red sea urchin mean densities were not different between the shal-
low-water zone (1.01 ±0.07 SE, n = 833 ) and the deep-water zone
(0.99 ± 0.07 SE. II = 630) (Kiiiskal-Wallis Z-test; ; = 1.936; P
>0.05).
The results of the Kruskal-Wallis Z-value test revealed signifi-
cant differences {P < 0.05) in red sea urchin TD among the 5 island
groups (see Table 2; Table 5). The mean TD of red sea urchins in
Group 3 was significantly smaller (80.4 mm ± 1.0 SE) when com-
pared with other groups, with the exception of Group 5. Red sea
urchins in Group 4 had largest mean TD (91.3 mm ±1.1 SE). No
TABLE 2.
Summary statistics (mean ± SE) for maximum shell length (mm) of
northern abalone and maximum test diameter (mm) of red sea
urchin at the 5 island groups and 2 depth zones in the study.
Sample size (ie, number of individuals measured) in parentheses.
Island
Abalone
Red Sea I'rchin
Group
Shell Length (mm)
Test Diameter (mm)
Group 1
51.2 ±4.3 (38)
87.5 ± 4.0 (li56)
Shallow
57.5 ±6. 1(24)
82.0 ±5.5 (82)
Deep
40.4 ±3.6 (14)
95.8 ±5.3 (54)
Group 2
83.0 ±4.7 (42)
88.9 ±2.7 (143)
Shallow
84.7 ±4.6 (38)
92.4 ± 3.4 (84)
Deep
66.3 ± 23.5 (4)
83.9 ±4.3 (59)
Group 3
46.1 ±2.5 (87)
80.4 ± 1.0(811)
Shallow
49.9 ± 3.2 (50)
82.8± 1.2(517)
Deep
41.0 ±3.8 (37)
76.0 ±1.5 (294)
Group 4
62.7 ±6.1 (25)
91.3 ±1.1 (301)
Shallow
61.3 ±7.0 (20)
91. 8± 1.6(179)
Deep
68.4 ± 13.4 (5)
90.4 ± 1.4(122)
Group 5
72.4 ± 4.4 (30)
83.6 ±6.0 (75)
Shallow
73.0 ±4.9 (26)
96.0 ±8.5 (35)
Deep
68.5 ±10.0 (4)
72.8 ±8.3 (40)
Total
59.4 ±2.0 (222)
84.2 ±0.8 (1466)
Shallow
64.7 ±2.4 (158)
84.3 ± 1.1 (845)
Deep
46.3 ±3.2 (64)
84. 1 ±1.2(621)
DiSTRlBLTION AND ABL'NDANCE OF H. KAMTSCHATKANA
835
TABLE 3.
Results of one-way ANOVA and the Tuke\-Kranier
multipk'-comparison test to discern statistically si^nltlcant
differences in the shell length Imnu of northern abalone amon;; the
5 island groups in the study, (iroup designation as in Figure I.
Source
Term
Sum of
DF Squares
Mean
Square
F-Ratio
A: Group
S(A)
Total (Adjusted)
Total
4
217
221
46536.75
I4SS23.5
195360.2
11634.19
6S5.S224
16.96
NS
NS
NS
NS
I'rob
Level
(1.01 )0*
* Term significant a( alpha = 0.05
Tukey-Kramcr Multiple Comparison Test
Group 12 3 4
5
Represents significant difference at least at P < 0.05 level. NS indicates no
significant difference between groups. This report provides multiple com-
parison tests for all pairwise differences between the means.
relative index of exposure was not correlated with macroaigae
densities and other substrate cover types. Encrusting coralline al-
gae were a dominant substrate cover type in all groups, ranging
from 50% to 867f (Table 6). In Groups 1. 3, and 5 encrusting
coralline algae occupied more than H)% of the available substrate.
The highest percent cover by encrusting coralline algae was mea-
sured m Group 3, where they covered 85.9% of the substrate. The
percent cover of encrusting coralline algae in Group 3 was sig-
nificantly higher that in any other group (Kruskal-Wallis multiple
comparison Z- value test; P < 0.05). Articulated coralline algae
represented relatively low percentage of substrate throughout the
study area, ranging between 2% to 6%.
0 20 40 60 80 100 120 140
significant differences in the TD of red urchins were found be-
tween groups 1 ct 2. 1 & 4. 1 c% 5. 2 & 4. and 3 & 5. There were
no differences in red sea urchin TD between the two depth /ones
(Kruskal-Wallis Z-test: ; = 0.443; P > 0.05). The si/.e frequency
distribution of red sea urchins in BGI for both depth zones were
combined (Fig. 3), since the Kolmogorov-Smirnov goodness of fit
test indicated no differences (Dinn = 0.06, P > 0.05) in size
frequency distribution between the two depth zones.
Habitat Relationships
The relative index of exposure was positively correlated with
abalone densities (r, = 0.61, P < 0.003: n = 22). red sea urchin
densities (r, = 0.54. P < 0.01; ;; = 22) and with encrusting
coralline algae (i\ = 0.44. P < 0.05; n = 22), but was inversely
related to predator abundance (r, = -0.45; P < 0.05; /; = 22). The
TABLE 4.
Kruskal-Wallis multiple comparisons Z-value test to discern
statistically significant differences of red sea urchin {S.
franciscanus). densities (# individuals/nr) among 5 island groups.
Numbers represent '/.■values for the Bonferroni Test (Hintze, 20(11).
Bold numbers indicate significant differences among groups at /* <
0.1(5. Group designation as in Figure 1.
Group 1 2 3 4 5
1
—
2
2.29
—
3
13.80
16.65
—
4
7.78
6.09
9.33
5
3.9(1
1.62
18.67
7.71
Bonferroni Test: Medians significantly {P < 0.05) different if Z-valiw
2.81
LU
m
0 20 40 60 80 100 120 140
30i
25
2&
15
ia
5
JlllhJ ■
0 20 40 60 80 100 120 140
SHELL LENGTH (MM)
Figure 2. Size frequency distributions of northern abalone {H. ka-
mlschatkana) from BtJI measured during the study. (.\) .\ll abalone
measured during the study in BGI at both shallow and deep zones, (B)
abalone measured in shallow zones, (C) abalone measured in deep
zones. The size frequency distributions at the shallow (B) and deep (C)
zones were significantly different (Kolmogorov-Smirnov goodness of
fit test Dmii = 0.32: P < 0.001 ). The vertical axes represent number of
abalone per size class.
836
TOMASCIK AND HOLMES
TABLE 5.
Kruskal-VVallis multiple comparisons Z-ralue test to discern
statistically significant differences in the test diameter (mml of red
sea urchin iS. fraiuiscaniis) among 5 island groups. Numbers
represent Z-raliies for the Bonferroni Test iHintze, 2001). Bold
numbers indicate significant differences among groups at P < 0.05.
Group designation as in Figure 1.
Group 1 2 3 4 5
1
—
2
0.61
—
3
3.83
4.73
—
4
0.32
0.40
5.75
5
2.68
3.22
0.25
3.24 —
Bonferroni Test: Medians significantly different if Z-iuluf > 2.8070
Community Relationships
The results of simple linear correlation analysis revealed a sig-
nificant positive relationship between abalone and red sea urchin
densities (r = 0.48. P < 0.05; n = 22). A significant inverse
relationship was found between abalone and predator densities ir
= -0.61; P < 0.01; /? = 20). as well as between abalone and
benthic macroalgae densities (r = -0.43; P < 0.05; n = 22). A
significant negative correlation was also found between red sea
urchin and benthic macroalgae densities (/■ = -0.69; P < 0.001; n
= 22). While encrusting coralline algae showed a strong positive
correlation with red sea urchin densities (/■ = 0.75; P < 0.001; /; =
22), they showed no correlation with abalone densities {P > 0.05).
The results of simple linear regression analysis levealed a signifi-
cant inverse relationship between abalone size and red sea urchin
densities (r = 0.33, P < 0.001; n = 21). Furthermore, simple
linear regression found a significant positive relationship between
abalone size and abundance of benthic macroalgae (r = 0.54, P
< 0.001; n = 21).
DISCUSSION
The results of this study concur with recent surveys by DFO
(Lucas et al. 2002). The estimated mean density of abalone in this
study (0.15/m") is similar to the mean abalone density (O.lO/m^)
a:
LU
OQ
30(>i
250^
20a
150
ioa
5a
Jlj
I
I..
0 30 60 90 120 150 180
TEST DIAMETER (MM)
Figure 3. Size frequency distribution of red sea urchins iS. fruncisca-
;i»v) from B(;i measured during the study. Test diameter frei|uencies
from shallow and deep zones were combined. The vertical axis repre-
sents number of red sea urchins per size class.
reported by Lucas et al. (2002) from an adjacent area only a few
kilometers away. These values are in sharp contrast to mean den-
sity values reported from the north coast of British Columbia be-
tween 1978 and 1984 (0.65 to 2.86 abalone/nr, .Sloan & Breen
1988). Surveys conducted in the Queen Charlotte Islands in 1978
reported densities of up to 28 abalone/m" (Breen & Adkins 1979).
The size range of abalone in Barkley Sound changed from 51 to
146 mm SL in 1964 (Quayle 1971) to 38 to 119 mm SL in 2000
(Lucas et al. 2002). The size range recorded in this study was 4 to
132 mm SL, with a mean of 59.4 mm SL. Roughly 10% of the
abalone population measured in this study was more than 100 mm
SL, while 58% of the sampled abalone population was more than
50 mm SL (Fig. 2A). Although northeni abalone reach sexual
maturity between 50 to 55 mm SL (Sloan & Breen 1988). juvenile
abalone represented 42% of the sampled population. This sug-
gested that abalone recruitment was occurring, albeit at relatively
low numbers.
The low densities of abalone. as well as low abundance of large
size indi\iduals recorded in this study may be related to several
Island
Group
TABLE 6.
Summary statistics for percentage cover of eight (8) substrate cover types measured during the study.
EC
AC
BA
75.7
4.7
0.4
(±1.9)
(±1.0)
53.3
4.9
(±2.2)
(±0.9)
(±3.8)
(±0.6)
85.9
0
(±1.3)
(±0.7)
(±0)
50.;
5.2
10.0
(±2.8)
70.2
(±1.9)
(±1.2)
(±0.9)
(±1.6)
2.8
(±0.5)
Substrate Types
GA
BR
SP
0.6
11.0
0.2
tO)
(±0)
(±0.4)
(±1.4)
(±0.1)
0.7
22.5
0.1
(±0.3)
:1.9)
(±0.1)
0.1
6.3
0
(±0.1)
(±0.1)
(±0.9)
(±0)
0.1
4.2
9.0
(±0.1)
:1.1)
(±1.5)
(±0)
0
1.8
12.0
0.3
(±0)
(±0.4)
(±1.4)
5.8
(±1.1)
15.6
(±1.8)
3.7
(±0.7)
13.7
(±1.9)
(±0.9)
252
279
288
219
309
First number is the mean % cover; second number ui brackets is ± standard error (.SE). Acronyms; EC. encrusting coralline algae; AC, articulated coralline
algae; BA. brown macroalgae; GA, green macroalgae; BR. bryozoans; S. sand: SP. sponges (Porifera); O. other invertebrates. /;. sample size (# of
quadrats). Island Group designation as shown in Figure 1.
Distribution and Abundance of H. kamtschath\na
837
factors, such as human exploitation (i.e.. poaching), competition,
predation, starvation, disease, ditferenlial mortality, or environ-
mental factors. Ocean-climate variability may also play a role in
keeping abalone populations at their current low levels. Tegner et
al. (2001 ) demonstrated a strong link between declines in landing
of red abalone (Hiiliotis nifesccns Swainson. 1822) in southern
California and increased variability in sea surface temperatures
(SSTs) associated with El Nino events that affect kelp abundance.
However, the inverse relationship between abalone and predator
densities found in this study suggests that predation may be an
important factor contributing to present day structure of abalone
populations. The low abundance of large abalone (>nO mm SL)
suggests that large abalone may be more susceptible to predation
than small individuals (<70 mm SL). Predators may be preferen-
tially selecting larger individuals, or as abalone reach larger size
they may loose the ability to either outrun or hide from the preda-
tors. In contrast, Watson (2000). citing Sloan & Breen (1988),
suggested that only sea otters and human exploitation seem to have
a significant impact on the abundance and size of abalone popu-
lations.
There was a significant positive conelation between abalone
and red sea urchin densities. This is in contrast to studies con-
ducted in California, where consistent negative correlations be-
tween H. nifescens and red sea urchins were found, suggesting
spatial inter-specitlc exclusion between these two species (Karpov
et al. 2001). The red sea urchin is viewed as perhaps abalone's
most important competitor for space and food, since both species
are grazers competing for the same food resource and space. The
positive relationship therefore suggests that competition for food
and space may not be direct, and that abalone may be in some way
benefiting from their association with the red sea urchin. The
positive relationship between abalone and red sea urchins in this
study may be partly a function of lower population densities than
those reported previously. For example, Watson (1993) reported
that mean red sea urchin densities in Barkley Sound between 1988
to 1989 were about 6.9 urchins/m", which is about 7 fold higher
than at present. Current abalone densities in the BGI are about 4
times lower that pre-closure (Emmett & Jamieson 1988). At these
low densities direct competition between abalone and red sea ur-
chins may not be apparent. However, we also found a significant
negative correlation between abalone size and red sea urchin abun-
dance. This negative relationship suggests that the urchins may be
exerting .some degree of influence on abalone populations through
their effect on either encrusting coralline algae or benthic mac-
roalgae. Densities of both species exhibited a significant negative
correlation with macroalgae abundance. Although abalone size
also showed a strong positive relationship with benthic algal abun-
dance, food availability may have played a key role in this inter-
relationship.
The significant positive relationship between red urchins and
encrusting coralline algae and the negative relationship with
benthic macrophytes suggests that the presence of sea urchins may
in some way benefit abalone through their maintenance of a habitat
that is preferred by Juvenile abalone. Sloan & Breen (1988) sug-
gested that abalone settlement occurs on encrusting corallines in
deeper water and that juveniles and adults migrate upwards as they
grow. Sasaki & Shepherd (2001) showed that ezo abalone (Hali-
olis discus himinii Ino, 19.'i2) settled on encrusting coralline algae
and moved into shallow Eisenia forest as they aged. Several other
studies have also suggested that abalone prefer to settle on sub-
strates dominated by encrusting coralline algae (Shepherd &
Turner 198.'>, McShane 1995). However, the primary food source
for abalone, essential for rapid growth of post-larval abalone, may
be the associated diatoms rather than the encrusting coralline algae
themselves (Takami et al. 1997). Encrusting corallines were found
to occupy about 71% of the substrate in the deep zone (6-9 m),
which was statistically higher than the 66% cover in the shallow
zone (2-5 m).
Vance (1979) suggested that echinoids play a key role in struc-
turing algal turf communities by removing encrusting inverte-
brates, thus promoting the growth of encrusting coralline algae.
This study found a strong negative correlation between encrusting
coralline algae and "other inveilebrates". By maintaining encrust-
ing coralline algae free of other invertebrates, the red sea urchin
may indirectly influence abalone settlement rates and perhaps post
settlement survivorship. However, the present study supports ear-
lier studies in British Columbia that found no significant associa-
tion of juvenile abalone with red sea urchin spine canopy (Sloan &
Breen 1988), even though there was a positive correlation in the
densities of these two species. In contrast, several recent studies
around the world have shown that juveniles of some Hiiliotis spe-
cies have a strong association with the sea urchin spine canopy (eg.
Day & Branch 2002). We found only six juvenile abalones (<45
mm SL) under the red sea urchin spine canopy, which represents
only 7% of all juvenile abalones recorded in this study. Rogers-
Bennett & Pearse (2001) reported that one third of juvenile aba-
lone inside a marine protected area was found under the spine
canopy of sea urchins.
ACKNOWLEDGMENTS
The authors thank Rick Holmes, Pete Clarkson, Bob Hansen,
Sebastian Marcoux, and Angus Simpson (Pacific Rim National
Park Reserve of Canada, PRNPR), as well as, Doug Brouwer and
James Pegg (Pacific Biologic Station [PBS]. Nanaimo. DEO) for
conducting abalone surveys and field support. Joanne Lessard and
Ian Muifitt (PBS) for cotiiputing the index of exposure, and Greg
MacMillan and Steve Lobay (Western Canada Service Centre.
Parks Canada) for their technical support. The authors thank Alan
Campbell (PBS) and Larry Harbidge (Chief of Resource Conser-
vation PRNPR) for their continuous support in this interdepart-
mental research project. This manuscript was greatly improved by
comments from three reviewers and A. Campbell. This project was
funded by the Species at Risk Interdepartmental Recovery Fund
Prottrani.
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Jiiiinial Hi Slit'llfish Rc.sccinh. Vol. 22. No. .\ 839-847. 2003.
IMPLICATIONS OF HIGH LEVELS OF GENETIC DIVERSITY AND WEAK POPULATION
STRUCTURE FOR THE REBUILDING OF NORTHERN ABALONE IN BRITISH
COLUMBIA, CANADA
RUTH E. WITHLER, ALAN CAMPBELL. SHAORONG LI, DOUG BROUWER,
K. JANINE SUPERNAULT, AND KRISTINA M. MILLER
Fisheries and Oceans Canada. Science Brancli. Pacific Biolo^iic Station. 3190 Hannnand Rax Road.
Nanaimo. BC. Canada V9T 6N7
ABSTRACT In the past 25 year.s. the abundance of northern ubalone (Haliotis kaimschatkana) has dechned by 80% in British
Columbia (BC). leading to concern over a possible loss of genetic diversity and fragmentation of breeding aggregates in the species.
Abalone from 31 sites in BC and one site in southeastern Alaska were surveyed for variation at eight polymorphic inicrosatellite loci.
The high level of Hf. characterizing all samples resulted in a large estimated effective population size for northern abalone l>3.5().00()).
consistent with high estimates for the historical average number of migrants entering abalone aggregations each generation (-20-125).
Hierarchical analysis of gene diversity revealed that 99.6% of genetic variation was contained within abalone samples and only 0.4%
partitioned among samples. Approximately half of the variation was accounted lor by differences between abalone of the Queen
Charlotte Islands. Alaska and those from central, southern British Columbia while the other half was caused by differences among
samples within the two regions. Little allele frequency variation was observed among size classes or between repeat samples from sites
sampled in more than 1 year. The results indicated that, historically, northern abalone aggregations did not represent isolated breeding
units and any disruption of gene flow that may have been caused by recent low abundance levels cannot yet be detected. These results
are discussed with respect to rebuilding efforts to be undertaken for northern abalone within BC.
KEY WORDS: Halioiis kuintscluukaiHi. genetic variation, microsatellite. gene tlow. inbreeding, population structure
INTRODUCTION
Exploited species of abalone throughout the world have suf-
fered severe declines in abundance (Davis et al. 1992. Prince &
Shepherd 1992) and some are close to extinction (Davis et al.
1998). The northern or pinto abalone {Halicitis kainischatkuna).
which inhabits shallow coastal waters of the northeastern Pacific
Ocean from southern California to Alaska, declined in abundance
by 75% to 80% in British Colutiibia (BC) between 1978 and 1990
(Campbell 2000). Abalone abundance did not increase with imple-
mentation of a complete harvest closure in 1990 and northern
abalone was listed as a "threatened" species (i.e.. one likely to
become in imminent danger of extinction or extirpation if limiting
factors are not reversed) by the Comtnittee on the .Status of En-
dangered Wildlife in Canada in 1999 (COSEWIC 2(.)(J0). Two
factors identified as major threats to northern abalone recovery
were low recruitment levels and continued (illegal) harvest. In this
study, we undertake a genetic assessment of population structure in
northern abalone as an element of a comprehensive recovery plan
for the species in BC.
Northern abalone are distributed in patches on exposed and
semi-exposed rocky coastal areas in BC. Species at low abundance
partitioned into isolated small populations are at risk for extirpa-
tion and extinction from stochastic demographic, environmental,
and genetic factors. The biology of northern abalone makes it
vulnerable to all three types of processes. Spawning is usually
restricted to summer months (i.e., May to August), and the pelagic
larval stage is of short duration, varying frotn 4 to 8 days with local
factors such as teinperature ( 14'-'C to 1()°C) (Sloan & Breen 1988).
The current low abundance and low densities of mature abalone
(Campbell 2000) may rellect not only the historical commercial
harvest but also adverse environmental conditions likely to have
hindered successful recruitment over the period 1975 to 198.3
(Breen 1986). In turn, low abalone abundance hinders successful
spawning because external fertilization requires high-density ag-
gregations of tiiature individuals (Babcock & Keesing 1999). Fi-
nally, reduced spawning success may lead to disruption of the
larval-mediated gene flow among spawning aggregates that typi-
cally offsets a loss of diversity within local populations. Expected
results would include increased inbreeding within, and genetic
drift among, local populations.
The level and distance of larval dispersal mediate both detno-
graphic and genetic processes in sedentary marine organisms. Lar-
val dispersal levels for abalone species are generally not known
but are apparently sufficiently low to ensure that demographic
processes occur on a local scale (i.e.. recruitment is primarily local,
ranging from a few meters or kilometers) and sufficiently high
enough to prevent strong genetic differentiation over large geo-
graphic ranges (Brown 1991, Hamm & Burton 2000, Huang et al.
2000). Nevertheless, the genetic studies have provided evidence
for different scales of population structure in abalone species, even
those that are sympatric. This indicates factors such as habitat
utilization, spawning season, and larval duration may also intlu-
eiice abalone population structure.
For blacklip abalone. H. rubra, sampled along the southern
coastline of Australia, genetic data indicated there was "isolation
by distance", but even the most geographically distant (>1000 ktii)
populations were genetically similar. The F^^ value for this spe-
cies estimated frotn allozymes was 0.022 (Brown 1991 ) and from
microsatellltes was 0.077 (Huang el al. 2000). Greater microspatial
genetic heterogeneity was observed in Australian greenlip aba-
lone, H. laevigata, a species with a more patchy distribution than
H. rulnii. but the estimated F^x value (0.014) was not greater than
that of the blacklip abalone (Brown & Murray 1992, Shepherd ct
Brown 1993). Microspatial variability contrasting with genetic ho-
mogeneity was also evident in the sympatric Roe's abalone. H.
roci. for which an F^x value of 0.009 was estiinated from samples
collected over almost .3000 km of coastline (Hancock 2000).
Differences in population structure have also been observed in
two sympatric abalone species of California. Red abalone, H. rufe-
seens, from northern and southern California were little differen-
839
840
WlTHLER ET AL
tiated at allozyme loci, in mitochondrial DNA sequence, or at a
single microsatellite locus, with the F<;t value of 0.012 estimated
from allozyme data not significantly different from zero (Gaffney
et al. 1996. Kirby et al. 1998, Burton & Tegner 2000). In contrast,
significant genetic differentiation among samples of black abalone.
H. cracherodii. in central California (F<.
0.039) was attributed
to a restricted spawning season that limits larval dispersal (Hanim
& Burton 2000). These results indicated that immigration from
distant sources was unlikely to be sufficiently great to accelerate
recovery in the depleted black abalone populations of southern
California, estimated to have declined in abundance by as much as
97% (Altstatt et al. 1996).
Low levels of intraspecific variation may make the partitioning
of genetic diversity within abalone species most amenable to ex-
amination with highly polymorphic, rapidly evolving microsatel-
lite loci (Huang et al. 2000. Withler 2000). In the present study, we
survey variation at eight polymorphic microsatellite loci in north-
ern abalone collected from 3 1 sites in BC and one site in southeast
Alaska. The objectives of this study are to determine levels of
genetic variation within and amona aggregations of northern aba-
lone in BC, and to estimate effective population sizes and inbreed-
ing levels for the species. We examine the genetic data for evi-
dence of recent bottlenecks in population abundance that might
have reduced genetic variation within, or increased variation
among, extant abalone aggregations and incorporate the genetic
data into recommendations for conservation efforts likely to ben-
efit the northern abalone of BC.
MATERIALS AND METHODS
Epipodial tissue samples from adult abalone were collected
from 31 sites within BC and one site in southeast Alaska between
1998 and 2002 (Table 1, Fig. 1). Abalone were collected in 2
different years at six sites. SCUBA dive teams searched for emer-
gent or exposed (visible on rocks) individuals because most are
easily found, whereas immature abalone tend to be cryptic (Camp-
bell 1996). Samples from abalone within 10 to 200 m were used to
represent each collection area. The small epidodial tissue sample
removal from each abalone was considered non-destructive, caus-
ing no mortality to the abalone (A. Campbell unpublished data on
TABLE L
Locations, years and sample sizes of Haliotis kamtschatkana collections for microsatellite DNA analysis.
Site
Latitude
Longitude
Year
West coast Vancouver Island
Elbow Island
Vargas Island
Dempster Island
Hankin Island
Turret Island
Austin Island
Deer Group Islands
Bamfield Inlet
Georgia Strait
Denman Island
Queen Charlotte Strait
Alert Bay
BC central coast
Cranstown Point
Nalau Passage
Simonds Group
Iroquois Island
Stryker Island
Nowish Islands
Higgins Passage
Lotbiniere Bay
Hankin Point
Freeman Passage
Kitasu Bay
Mosquito Island
Rennison Island
Kingkown Inlet
Queen Charlotte Islands
Louscoone Inlet
Montserrat Bay
Skincuttle Inlet
Faraday Island
Virago Sound
Bruin Bay
Carpenter Bay
Alaska
Sitka Sound
48 -S4.060
49 09.429
48 54.000
48 ?.S.000
48 54.000
48 51.370
48 53.000
48 49.000
49 28.883
50 35.000
51 22.500
51 47.000
51 57.800
52 02.895
52 05.990
52 3 1 .000
52 28.500
53 01.372
53 42.400
53 49.300
52 32.500
51 50.195
52 51.308
53 29.685
52 07.692
52 06.227
52 20.780
52 .36.770
54 04.000
54 10.017
52 13..M1
57 03.100
125 16.556
125 57.729
125 16.000
125 22.000
125 20.000
125 19.100
1 25 08.000
125 08.000
124 41.209
126 55.000
127 46.500
1 28 06.500
128 16.700
128 19.445
123 23.207
128 26.000
128 45.500
129 31.770
130 24.610
130 31.600
128 49.300
1 28 09.900
129 20.540
130 27.559
131 14.127
130 59.170
131 14.260
131 27.800
1 32 3 1 .000
132 58.752
131 03.260
135 20.500
2000
2000
2002
2002
2002
2002
2002
2001
1999. 2000
2000
1999
1999
1999.2001
1999
1999.2001
1999
1999
2000, 2001
2000, 2001
2000, 2001
2001
2001
2001
2001
1999
1998
1998
1998
1998
1999
2002
1999
45
70
170
170
180
ISO
3(1
90
45, 85
40
110
115
80,70
no
90, 20
112
90
28. 90
55. 25
75. 85
35
110
95
85
130
70
73
72
70
90
90
95
Genetic Diversity in Northern Abalone
841
Figure 1. Map showing locations of Haliotis kamtschatkana sample collections made in British Columbia and southeast Alaska between 1998 and
2002.
a laboratory experiment). Samples were stored in 95% ethanol
prior to DNA extraction using DNeasy kits (Qiagen. Valencia.
CA).
Variation at eight microsatellite loci isolated from northern
abalone (Hka\2, Hka2%, Hka40, Hka4?,, Hka4S, Hka56, Hka65.
Hka&5) was surveyed using the primers and protocols outlined by
Miller et al. (2001 ). The microsatellite loci consisted of di-. tri- and
tetra-nucleotide repeat sequences (Table 2). Alleles at each locus
were generally differentiated by the number of basepairs (bp) of
the predominant repeat unit, but alleles differentiated by a single
base pair were observed and scored without binning at two imper-
fect dinucleotide loci (WAy(48 and Hkii65). Allele frequencies for
all samples surveyed in this study are available at <http://
www,pac.dfo-nipo.gc.ca/sci/aqua/bgsid_e.htm>.
Analysis of the allelic and genotypic frequency data was car-
ried out using the Genetic Data Analysis (GDAl program of Lewis
& Zaykin (2000). GENEPOP version 3. Id (Raymond & Rousset
1995) and FSTAT version 2.9,3.2 (Goudet 2001). Genotypic
frequencies at each locus in each sample were tested for conform-
ance to Hardy Weinberg equilibrium (HWE) distributions in
GENEPOP. Weir & CockerhanVs (1984) F^t values were com-
puted over all samples and on a pairwise basis between samples
using FSTAT. The significance of the multilocus F^,- value over
all samples was determined by jackknifing over loci. FSTAT was
used to measure the "allelic richness" (allelic diversity standard-
ized to a sample size of 15) for each sample and to perform
Mantel's (1967) regression of the pairwise F^.^ values on geo-
graphic distance to test for "isolation by distance" among abalone
samples. Geographic distances were measured as the shortest di-
rect distances between sites.
Pairwise F^-y values were clustered with the neighbor-joining
algorithm to provide a dendrogram of the genetic relationships
among abalone samples. The pairwise average number of migrants
(N»;) between samples was estimated by the private alleles method
of Barton and Slatkin (1986) using GENEPOP and with the expres-
sion F^T = l/(4N»i -t- I), a relationship based on the assumption
of island model of population structure (Whitlock & McCauley
1999). The effective population size (N^) for northern abalone was
calculated from expected heterozygosity (H^) values for the eight
microsatellite loci using the relationship N^, = (l/[l-Hg]^-l)/8ti„
842
WiTHLER ET AL
TABLE 2.
Microsatellite loci examined in samples of Haliotis kamtschatkana from 32 locations In British Columbia and Alaska. The total numher (A)
and size range (in base pairs) of alleles, the expected (H^ ) and observed (H„) heterozygosity values, the Fs, value and the inbreeding
coefficient (f,,) calculated over all samples for each locus are shown. The effective population size (N^) estimated from H^ is also shown.
Size Range
Locus
Repeat
A
(bpl
Hp
H„
FsT
fi.
N^IOOO
//fa; 12
di
S2
171-377
0.92
0.89
0.000
0.03
19(1
Hka2S
di
37
183-271
0.94
0.57
0.001
0.40
350
HkaAO
di
37
112-210
0.91
0.85
0.001
0.07
150
Hka43
tetra
24
163-263
0.88
0.87
0.005*
0.01
90
HkaAS
di
68
93-250
0.97
0.71
0.002*
0.27
1390
Hka56
di
35
93-164
0.92
0.86
0.001*
0.07
190
Mr/65
di
58
115-250
0.95
0.87
0.001
0.09
500
HkaS5
tri
49
122-390
0.89
0.49
0.000
0.45
100
Mean
-
49
-
0.92
(1.76
0.002*
0.17
370
' P < 0.05.
where yi is the mutation rate for the microsatellite loci (Lehmann
et al. 1998). Little is known of the mutation rate of microsatellite
loci in invertebrate organisms except Drosophila. in which the
observed rate (-lO"*"! is much lower than in mammals (-lO'^'l. N^.
for northern abalone was estimated in this study using the conser-
vative assumption that p. = 10""*. with recognition that N^, values
are 100 times greater if the true value is 10""^.
Hierarchical analyses of allele frequency variation were carried
out with nested ANOVA (random effects model) as described by
Weir ( 1996) using GDA. The significance of differences in allele
frequencies between pairs of samples collected in different years at
each of six sites was examined. Similarly, the significance of allele
frequency differences attributable to two geographic regions iden-
tified in the dendrogram based on genetic distances (the Queen
Charlotte Island [QCI| and southeastern Alaskan sites versus re-
maining sites from coastal BC) was tested in a hierarchical model
with sample sites nested within regions.
Heterogeneity among size classes within samples was investi-
gated in abalone from 16 sites (within year samples). Abalone
from each site were divided among 4 size classes based on shell
length; up to 50 mm. immature; 51 to 69 mm, transition of imma-
ture to mature; 70 to 99 mm. mature; and more than 99 mm.
fishery; defined by size at maturity estimates by Campbell et al.
(1992). For each site, abalone from between 2 and 4 of the size
classes were obtained. Each of the size groups contained a range of
ages whose growth rates could have been influenced by local
environmental conditions; less than or equal to 2 to less than or
equal to 4 y (<50 mm SL). between 2 and 7 y (51-69 mm SL).
between 3 and 14 y (70-99 mm SL) and more than 6 or more than
14 y (>99 mm SL) estimated from (Fig. 8 in Sloan & Breen
1988). The maximum age of H. kamtschatkana is not known, but
individuals reach ages of 30 y and older (Breen 1980). Thus, the
potential number of cohorts contained within each size class in-
creases with size class. Allele frequencies in the two or three size
classes containing the most abalone at each site were analyzed by
ANOVA to examine the possibility that small numbers of adults
contribute to recruitment in individual cohorts of northern abalone.
leading to low genetic variability within cohorts and significant
variation among cohorts within abalone aggregations. The allelic
richness and inbreeding coefficient was estimated using FSTAT
for each size group containing at least 20 abalone from each site.
RESULTS
Genetic Variation Within Populations
All microsatellite loci examined were highly polymorphic, ex-
hibiting high numbers of alleles and high values of both observed
(Hq) and expected (H^.) heterozygosities (Table 2). Genotypes at
all eight loci showed an excess of homozygotes in comparison to
those expected under HWE, but the level of heterozygote defi-
ciency varied greatly among loci (Table 2). Estimates of f,„ (the
level of population subdivision and inbreeding if the excess of
homozygotes was due entirely to assortative mating) ranged from
0.01 at HkaAi to 0.45 at Hka^5.
Differences in allele frequencies between pairs of samples col-
lected in 2 different years from each of six sites were not signifi-
canKFs 7,,,, = 1.75. P> 0.10 1. The Fj.;- ^'^lues between the sample
pairs ranged from 0 to 0.003. with an average value of 0.001. In
each case, samples from the same site were combined for further
analysis.
All 32 samples of northern abalone displayed high levels of
allelic diversity (mean numbers of alleles observed over all loci)
and the standardized number of alleles, termed allelic richness,
averaged 14.4 over all samples and did not differ among samples
(f>0.10) (Table 3). Each locus was characterized by between two
and seven common alleles, with frequencies of common alleles
rarely exceeding 0.25 in a sample. None of the loci possessed a
single allele that was present at the highest frequency in all
samples. Although allelic diversity was high, private alleles (those
observed in a single sample) were rare. Of the 390 alleles observed
over all eight loci, only 30 were private and each was present at a
frequency of less than 0.025 in the single sample in which it was
observed. Average Hq by sample ranged from 0.73 to 0.79 (mean
of 0.76). but in all cases was less than the Hg. which was essen-
tially 0.92 for all samples (Table 3). Thus, the estimated f,„ value
varied much less among samples (from 0.14-0.21) than among
loci. The great range of f,,. values among loci and the consistency
of the f|,. values for a given locus among samples indicate that
population structure was not the sole explanation for the large
observed heterozygote deficits at HkalS. Hka4^, and HkaiiS.
Using the mammalian microsatellite mutation rate (lO"'^) and
H[. values estimated for the abalone microsatellite loci of this
study, we obtained locus-specific estimates of effective population
Genetic Diversity in Northern Abalone
843
TABLE 3.
(lenetic >ariation within samples of Haliotis kamlschalkana sampled from locations in British Columbia and southeast Alaska. The average
number of alleles (A,,), standardized allelic richness (Aj,), and expected (H^ ) and observed (H,,) levels of heterozygosity are shown for each
sample. The inbreeding coefficient calculated over all loci (f,^ — all loci) and over the five loci at which there was no evidence of
non-amplifying alleles (f,., — 5 loci) are also shown.
fu (All
fi, (5
Site
N
Ap
Ar
Hr
H„
loci)
loci)
West coast Vancouver Island
Elbow Island
45
22.6
14..^
0.92
0.78
0.15
0.08
Vargas Island
70
25.9
14,1
0.91
0.74
0.19
0.09
Dempster Island
170
32.4
14. .S
0.92
0.76
0.17
0.06
Hankin Island
170
32.0
14.3
0.92
0.76
0.18
0.07
Turret Island
180
31.9
14.4
0.92
0.73
0.21
0.08
Austin Island
180
31.5
143
0.92
0.74
0.19
0.06
Deer Group Islands
30
19.0
14.4
0.93
0.74
0.20
0.06
Bamfield
90
27.1
14.6
0.92
0.77
0.17
0.02
Georgia Strait
Denman Island
130
29.9
14.4
0.92
0.77
0.17
0.03
Queen Charlotte Strait
Alert Bay
40
21.5
14.3
0.92
0.78
0.16
0.06
BC central coast
Cranstown Point
110
29.6
14.9
0.93
0.79
0.15
0.04
Nalau Passage
115
30.9
14.6
0.92
0.78
0.16
0.06
Simonds Group
150
31.9
14.5
0.92
0.74
0.20
0.08
Iroquois Island
110
31.6
14.8
0.92
0.78
0.16
0.04
Stryker Island
110
28.1
14.2
0.92
0.77
0.16
0.04
Nov\ish Islands
112
28.9
14.3
0.92
0.73
0.20
0.08
Higgins Passage
90
27.1
14.2
0.92
0.76
0.18
0.05
Lotbiniere Bay
118
29.9
14.3
0.92
0.76
0.17
0.07
Hankin Point
80
27.5
14.2
0.92
0.77
0.16
0.05
Freeman Passage
160
32.8
14.7
0.92
0.79
0.14
0.04
Kitasu Bay
35
20.0
14.2
0.92
0.76
0.17
0.01
Mosquito Island
110
30.5
14.6
0.92
0.77
0.17
0.06
Rennison Island
95
28.1
14.3
0.92
0.78
0.16
0.06
Kingkown Inlet
85
26.3
13.9
0.92
0.74
0.19
0.05
Queen Charlotte Islands
Louscoone Inlet
130
32.4
14.8
0.92
0.77
0.17
0.05
Montserrat Bay
70
28.0
14.7
0.92
0.76
0.17
0.04
Skincuttle Inlet
73
27.6
14.3
0.92
0.75
0.18
0.02
Faraday Island
72
26.4
14.2
0.92
0.74
0.19
0.04
Virago Sound
70
26.4
14.2
0.92
0.79
0.14
0.06
Bruin Bay
90
29.6
14.6
0.92
0.78
0.14
0.02
Carpenter Bay
90
29.9
l?.l
0.93
0.76
0.18
0.06
Alaska
Sitka Sound
95
28.5
145
0.92
0.76
0.17
0.05
Total/Mean
3345
2S.3
14.4
0.42
0.76
0.17
0.05
size (N^.1 ranging from 90.000 to 1 .390.000 and a mean value of
370.000 (Table 2). Use of the possibly more realistic mutation rate
of 10"'' provides estimates 100 times larger.
Genetic Variation Among Size Groups Within Samples
Allele frequencies did not differ significantly among size
classes within each site (Foy ,200 = 1-39. P > 0.05) and size
accounted for none of the variation observed within and among the
samples of abalone subdivided into size classes. The mean allelic
richness of the individual size samples (14.3 alleles) was the same
as that of the total samples indicating that there was not reduced
di\ersity within cohorts. Fewer ages (year classes) contributed to
the smaller (immature and transition) than the larger (mature and
fishery) abalone size classes. However, neither allelic richness nor
the inbreeding coefficient varied among size classes (both P >
0.05). providing little evidence that individual cohorts were the
products of small numbers of or highly related abalone parents.
The lack of allele frequency variation among size groups also
indicated that the number of abalone participating in individual
spawning events was not extremely low.
Genetic Variation Among Samples
The Fsy value calculated over the eight loci among all samples
was low but significantly greater than zero (0.002; SE 0.000).
E.xamined on a single locus basis. Fjy values ranged from 0.000 to
0.003. and were significantly greater than 0 for three of the eight
loci examined (P < 0.05) (Table 2). There was no strong
geographic clustering of samples apparent in the dendrogram (Fig.
844
WiTHLER ET AL
2). The seven QCI and single Alaskan samples clustered together,
but the central coast, Georgia Strait and west coast Vancouver
Island samples did not cluster geographically. The hierarchical
analyses of gene diversity indicated that 99.6% of the observed
genetic variation occurred within samples and only 0.4% was at-
tributable to differentiation among samples. Of the differentiation
among samples, approximately half (0.2% ) was due to differences
between the two regions (QCI/Alaska vs. coastal BC) and the other
half to differences among samples within regions. The effect of
region was not highly significant (F,
3.46, 0.05 <P< 0.10)
and there was no significant differentiation among samples within
region (F, „oo = 1-39, P > 0.05)
The regression of all pairwise ¥^y values on geographic dis-
tance was significant (P < 0.05), but geographic distance ac-
counted for very little of the observed variation in F^t values (r" =
0.11) (Fig. 3A). The distinctiveness of the QCI and Alaskan
samples and their relatively great geographic distance from many
of the remaining samples accounted for the relationship between
geographic and genetic differentiation. With the QCI and Alaskan
samples removed from the data set, there was no relationship (P =
0.67) and distance accounted for less than 1% of the observed
variation in F^-^ values (Fig. 3B). For this set of coastal BC popu-
lations, pairwise Fs-p values did not exceed 0.005 (note the change
in the F^^ scale between Figs. 3 A and 3B ), and F^,- values of 0 were
t^
Bruin Bay
Sitka Sound
Faraday Island
Louscoone Inlet
I Virago Sound
Montserrat Bay
J Skincuttle Inlet
Carpenter Bay
J Freeman Passage
Deer Group Islands
Lotbinlere Bay
I Elbow Island
Alert Bay
I Denman Island
Austin Island
Rennison Island
Nalau Passage
KItasu Bay
Turret Island
HIgglns Passage
Iroquois Island
Vargas Island
_r" Cranstown Point
\ I Nowjsh Islands
KIngkown Inlet
Dempster Island
Hankin Point
Mosquito Island
Bamfield Inlet
Hankin Island
Stryker Island
Simonds Group
0.001
Figure 2. Neighbor-jolninR dendrogram of relationships among Hali-
otis kamtschatkana samples based on pairwise F^, values. Samples
from the Queen Charlotte Islands and southeast Alaska cluster inde-
pendently from samples from coastal British Columbia locations.
0.01 1
0 008 -
0.006
•
0004
. i':t
0002
£<&^^
0
€^S^
200 400 600 800 1000 1200 1400
DISTANCE (km)
B
0005
0.004
800
DISTANCE (km)
Figure 3. Regression of pairwise values of genetic ditferentiation (F^p)
on geographic distance for (Al all Haliolis kamtscltalkana samples and
(B) coastal British Columbia samples only (excluding samples from the
Queen Charlotte Islands and southeast Alaska). Note the difference in
y-axis scale between A and B.
observed between pairs of samples over the entire range of geo-
graphic separation from 1 to 700 km.
Using the entire data set, the regression of Fj^- on geographic
distance intersected the average F^-j value between repeat samples
from the same geographic location at the y-axis intercept, a value
of 0 km in geographic distance. This would suggest a neighbor-
hood size of less than 1 km, the smallest distance by which
samples in this study were separated. However, using the data set
for coastal BC sites only, the regression of F^-r on distance was
essentially a straight line (slope = 1.2 x lO"'), indicating an
average pairwise Fj^ of 0.0008 over the entire 700 km range. This
line coincided with the F^-^ value of 0.001 obtained between re-
peated samples from the same site and suggested that the entire
coastal range of BC sampled in this study, exclusive of the QCI,
constituted a single genetic neighborhood.
The average number of migrants per generation into the aba-
lone aggregations represented by each sample was estimated by
the private alleles method as 18.7, a number consistent with the
observed lack of genetic differentiation among samples. This value
changed little when only QCI/Alaskan samples (22.5) or only non-
QCI samples (22.6) were considered. Calculating the average
number of migrants using the standard expectation for the rela-
tionship between F<;-p and Niii provided an estimated 125 migrants
entering abalone aggregations each generation.
DISCUSSION
Northern abalone throughout BC and southeast Alaska were
characterized by very high levels of microsatellite DNA variation
J
Genetic Diversity in Northern Abalone
845
within and very low levels of differentiation among spawning
aggregates. Less than 1% of genetic variation was attributable to
differences among samples and little geographic structure was ob-
served. The lack of strong differentiation among sample locations.
between repeated samples from single locations and among aba-
lone of different size classes within samples suggested that gene
How among abalone breeding aggregations throughout BC has
been extensive. If the recent low abundance of abalone has dis-
rupted historical patterns of gene flow, it is not yet evident among
abalone of the age groups encompassed in this study.
During the last Cordilleran glaciation of North America, which
ended approximately 12.000 y ago, the QCI and Alaskan coastal
regions may have provided refugial habitat for terrestrial and ma-
rine organisms (Warner et al. 1982). Thus, northern abalone
throughout much of coastal BC and those of the QCI (and perhaps
northern BC and southeast Alaska) may be descendants of differ-
ent refugial populations. Two distinctive clades in mitochondrial
DNA sequences of the littorinid snail Littorina subronindata
throughout BC and Washington have been attributed to dispersal
from separate glacial refugia (Kyle & Boulding 1998, Kyle &
Boulding 2000). The small differences in microsatellite allele fre-
quencies between coastal and QCI samples of northern abalone
may reflect either historical isolation in separate refugia or more
recent restrictions of gene flow between coastal and QCI habitats.
Even if extant abalone are descendants of different refugial popu-
lations, the high level of intraspecific variability and low level of
Hitersample differentiation indicated that refugial population sizes
were large and limited genetic divergence occurred during isola-
tion, or that gene flow has occurred since the glacial period.
All of the microsatellite loci examined in this study exhibited
an excess of homozygosity such as that observed in surveys of
other mollusks, including abalone species (Brown 1991, Hara &
Kikuchi 1992, Beaumont et al. 1993, Huang et al. 2000, Perez-
Losado et al. 2002). For abalone, the deficiencies generally have
been attributed to inbreeding. In northern abalone. more variation
was observed among loci than among samples in the level of
heterozygote deficiency, indicating that locus-specific factors such
as non-amplifying alleles were also involved. Thus, some level of
inbreeding may have occurred in northern abalone. as in other
abalone species, the level of which is best estimated by those loci
showing the least evidence of non-amplifying alleles (i.e. those
loci with genotypic frequencies closest to HWE). The average
inbreeding coefficient over all samples for the five loci closest to
HWE was O.O.S. This may represent the typical level of inbreeding
in northern abalone populations.
High levels of local larval recruitment or asynchronous spawn-
ing on a small geographic scale may have contributed to inbreed-
ing in H. kamtschalkana. as suggested for blacklip abalone (Huang
et al. 2000). However, the analysis of allelic differentiation among
size classes of northern abalone within samples provided no evi-
dence of the increased genetic differentiation among cohorts and
the reduced genetic diversity within cohorts expected under
"sweepstakes-style" recruitment success (Hedgeeock 1994). Ac-
cording lo this model, spatial and temporal variability in recruit-
ment success may lead to detectable genetic drift among cohorts
and to "chaotic genetic patchiness", in which samples in very close
proximity are as genetically differentiated as ones very far apart
(Larson & Julian 19991. Although proximal samples of northern
abalone in coastal waters were as different as distal ones, all
samples were highly polymorphic and little differentiated. Samples
representing individual si/e and restricted age groups were as al-
lelically "rich" as samples containing all size classes from a single
location. Moreover, the average F^x value between size classes
within sites (0.001 ) was the same as that between repeat samples
from the same site and that between coastal sites. Thus there was
no evidence that the successful spawners at any given time were
sufficiently small in number or closely related to result in accel-
erated genetic drift. Instead, the coastal abalone populations in this
study could be considered to form a single genetic neighborhood,
with genotype distributions showing no departure from those ex-
pected under panmictic mating. The abalone of the QCI and south-
eastern Alaska may constitute a second, only slightly differentiated
neighborhood.
The high abundance of rare alleles in all northern abalone
samples (>809'f of alleles were present at frequencies <0. 1) sug-
gested that populations have existed at long-term stable sizes (ie.
not suffered recent bottlenecks) (Luikart et al. 1998). This obser-
vation and the high estimates of effective population size indicated
that the small local aggregations of mature abalone observed in
census studies (Wallace 1999, Campbell 2000) did not represent
genetically isolated breeding units. "Cryptic" abalone. not recently
included in census counts, possibly also contributed to reproduc-
tion in northern abalone. However, it is evident that local northern
abalone aggregations have been connected by gene flow as the
result of larval dispersal.
The strong genetic homogeneity of northern abalone, a seasonal
spawner, contrasts with results obtained for the black abalone. in
which higher levels of genetic differentiation were attributed at
least in part to the limited spawning season and strong seasonal
differences in oceanographic patterns in the coastal waters of Cali-
fornia (Hamm & Burton 2000). The lack of genetic structure in
northern abalone is more similar to the low level of genetic dif-
ferentiation observed in the red abalone of California, which
spawns throughout the year (Burton & Tegner 2000) and in three
sympatric abalone species inhabiting the waters of southern Aus-
tralia. The Australian blacklip, greenlip. and Roe's abalone all
show low levels of genetic differentiation over spatial scales as
large or larger than those encompassed in the present study (Brown
1991, Brown & Mun-ay 1992, Hancock 2000).
Small-scale genetic heterogeneity coupled with large-scale ho-
mogeneity in Roe's abalone was attributed to predominantly local
recruitment, with the high gene flow resulting more from large
effective population sizes than from large migration rates (Han-
cock 2000). Hancock also suggested that rare cases of successful
long-distance dispersal might play a role in maintaining the ob-
served large-scale genetic homogeneity. Little small-scale hetero-
geneity was observed among samples of northern abalone. The
lower Fsy values observed over short distances in northern abalone
suggest that the N^ of this species is larger, or that larval dispersal
is greater, than that observed for Roe's abalone. Given the high
densities observed for Roe's abalone (Hancock 2000). it seems
unlikely that the N^. for northern abalone exceeded that for Roe's
abalone even before the recent decreases in abundance. The large
estimated numbers of successful migrants among the samples in
this study support the idea that dispersal may contribute more to
the low observed levels of differentiation in northern abalone than
in many other species. Whether successful larval dispersal in
northern abalone occurs on a regular basis or is predominantly the
result of rare, but highly effective, long distance dispersal events
is not known.
Marine species with extended longevity possess a "storage ca-
pacity" for genetic variation in the face of fluctuating environments
846
WlTHLER ET AL
in the large cohort of adults produced from each strong recruitment
(Warner & Chesson 1985, Ellner & Hairston 1994. Ellner 1996,
Gaggiotti & Vetter 1999). Each large cohort effectively "stores"
many genotypes within the reproductive population over many
spawning periods that are capable of contributing to both popula-
tion size and genetic diversity when favorable spawning and re-
cruitment conditions return. However, extended periods of low
reproductive or recruitment success may be masked in genetic
surveys heavily influenced by the genetic variability being stored
in. but not transmitted from, the older age groups. The analysis of
genetic variation in different size classes of abalone at several sites
in this study provided no indication that younger abalone were less
diverse than older ones, but sampling of the younger ages did not
include newly recruited "cryptic" individuals. In the black abalone
of southern California, recruitment failure was observed after aba-
lone abundance dropped by approximately 509c (Richards & Davis
1993). Because of the longevity of northern abalone individuals, it
is essential that recruitment be measured to determine current lev-
els of reproductive success. Long-term genetic monitoring of
newly recruited abalone would reveal the loss of genetic diversity
and population fragmentation that might follow a disruption of
gene tlow at lov\' abundances, but only some years after the fact.
Options for rebuilding abalone abundance in BC include main-
taining fishery prohibitions, aggregation of reproductive adult aba-
lone in the wild to increase density and improve reproductive
success, and out-planting of hatchery-raised juvenile abalone to the
wild to enhance recruitment. The possibility of disrupting natural
population structure in northern abalone by aggregating adults or
out-planting juveniles over geographic areas larger than the small
aggregations monitored for stock assessment purposes appears un-
likely given the low level of microsatellite differentiation observed
in this study. However, studies on other marine molluscan organ-
isms have provided indications that both adaptive genetic and non-
genetic inducible phenolypic changes may be typical responses to
different environments (Kim et al. 2003. Trussell & Smith 2000).
Rearing abalone in "common-garden" conditions to assess differ-
ences in fitness-related traits may be required to determine at what
geographic scale, if any. adaptive differences occur, but it seems
likely that transplanting northern abalone will be limited more by
disease transfer than by genetic concerns.
Two other concerns associated with the out-planting of hatch-
ery produced organisms are the random loss of genetic diversity
due to a limited number of spawners and, if the broodstock is
maintained in the hatchery over generations, the de\ elopment of a
strain that is not well adapted to survival and reproduction in the
wild. Hatchery strains that are intended for reseeding into natural
populations should be carefully monitored to ensure that high lev-
els of genetic variation are maintained, and should be open popu-
lations that incorporate naturally produced individuals on a regular
basis. Genetic monitoring may also contribute to evaluation of the
success of enhancement efforts (Burton & Tegner 2000). This
study has indicated that, in a genetic sense, northern abalone in BC
are poised for recovery under favorable environmental circum-
stances. Whether or not active intervention in abalone reproduction
is undertaken, prudent management activities would include the
identification, protection, and monitoring of spawning aggregates
(and recruits) on a regional basis to examine both demographic and
genetic parameters for signs of population recovery or decline.
ACKNOWLEDGMENTS
The authors thank B. Lucas. S. Carignan. B. DeFrietas, J. Dis-
brow, R. Gurr. J. Harding. M. McNab, T. Norgard, D. Miller, and
D. Woodby for help with sample collections. Drs. Ellen Kench-
ington and Nicholas Elliott provided helpful suggestions for im-
provement of the paper.
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Workshop on Rebuilding Techniques tor Abulone in British Columbia
Abstracts
849
STATUS OF STEWARDSHIP PROJECTS
ABALONE STEWARDSHIP IN HAIDA GWAII: FORGING
A LONG-TERM COMMITMENT. Russ Jones and Bart I)e-
Freitas, Haida Fisheries Program, PO Box 9S, Skidegate, Haida
Gwaii. BC Canada VOX ISO; Norm Sloan. Gwaii Haanas National
Park Reserve / Haida Heritage Site. PO Box 37. Queen Charlotte
City, Haida Gwaii. BC Canada VOX ISO; Lynn Lee. World Wild-
life Fund. PO Box 74. Xlcll. Haida Gwaii. BC Canada VOX lYO;
Kimiku von Boetticher. Haida Gwaii Marine Resource Group
Association. P.O. Box 6S0. Massett. Haida Gwaii, BC Canada
VOX IMO: and Greg Martin. Laskeek Bay Conservation Society.
PO Box 867; Queen Charlotte City. Haida Gwaii. BC Canada VOX
ISO.
Local stewardship is a possible solution to the vexing problem
of rebuilding over fished northern abalone (Halioti.s kaintschat-
kana) stocks. Northern abalone fisheries in British Columbia were
closed coastwide in 1990 but stocks have failed to rebuild and the
species became federally listed as "threatened" in 1999. We de-
scribe 3 years of community-based stewardship effort in Haida
Gwaii to rebuild abalone and prospects for recovery over the long-
term. Steps taken include forging a community partnership through
regular meetings of a core group and development of a Community
Action Plan. Xhe Action Plan's goal is to rebuild abalone popula-
tions sufficiently to support both Haida traditional and recreational
food fisheries. Specific initiatives include public education, cur-
ricula development, establishment of two large abalone steward-
ship areas and a research area, creation of an Abalone Watch
(coastal surveillance) program, and research diving to test rebuild-
ing approaches and monitor recovery. The community response
has been positive, but it is too soon to confirm whether there have
been changes in human attitude and increases in abalone popula-
tions. Xhe challenge is to maintain community interest and com-
mitment over the long-term to allow results to be manifested.
Much will depend on setting achievable stock rebuilding reference
points both for the stewardship areas and Haida Gwaii. As well,
the prospect of the return of the sea otter (also listed as a "threat-
ened" species) that is a keystone species in the kelp forest ecosys-
tem and a predator of northern abalone could result in reduced
abalone abundance despite stewardship efforts.
THE KITASOO ABALONE STEWARDSHIP PROJECT:
SMALL PROJECT, BIG HOPES. Joel Harding, Kitasoo Fish-
eries Program. 95.'i Comox Rd.. Nanaimo, BC V9R 3J7 Canada.
Xhe Kitasoo Fisheries Program (KFP), operating out of the
remote coastal First Nations community at Klemtu, has been work-
ing since 199.5 to gain an understanding of northern abalone (Hcili-
oti.s kamischatkaiia) population demographics within the Kila.soo/
Xaixais traditional teiTitory. Xhe traditional territory is large with
a ont small village of 3.50 people. Xhe objective of the KFP is to
develop capacity within the Kitasoo/Xaixais Nation, to foster ac-
tive community participation in the conservation and management
of the fisheries resources in the region. Xhe program covers manv
species, including salmon, herring, manila clams, urchin, prawn,
sea cucumber Porphyra. and abalone. Ov er exploitation and deple-
tion of northern abalone stocks has brought this species to the
tbrefroiit of the program. A significant portion of the Kitasoo/
.Xaixais traditional territory has been surveyed for remnant abalone
populations and information shared with Fisheries and Oceans
Canada has helped to document the post-closure distribution of
abalone. Inventory surveys were initiated in 1995 at sites through-
out the area. A study site was established in south Nowish Inlet
during 1999. Since then, data on abalone growth from tagging,
habitat requirements, predator, and competitor relationships to
abalone abundance, have been recorded.
In 2001, the KFP and the Habitat Stewardship Program initi-
ated the Kitasoo Abalone Stewardship Project, with the purpose of
expanding the scope and capacity of abalone rebuilding efforts
while joining with local stewardship initiatives, such as education
and monitoring campaigns. Xhe main objective of this program is
to rehabilitate local abalone populations to self-sustaining levels
within the Kitasoo/Xaixais traditional territory. Xhe level of com-
munity support and participation will determine the success of the
program. Community workshops and follow-up meetings, since
2002. have raised awareness and encouraged local participation in
project initiatives. Ongoing outreach efforts include project up-
dates on the community radio channel, distribution of material to
visitors and tourists, and youth education activities. Xhe project
has promoted participation by supporting those able to combine
local food fishing activities with voluntary monitoring. Xhe KFP
used local knowledge and past survey infiirmation to establish two
new stewardship areas to provide sites for evaluation of wild stock
manipulation as a means to increase abalone densities and repro-
ductive success. In addition, artificial cement habitats (condos) are
being evaluated as an index tool to monitor juvenile recruitment
and abundance. Xo date, juvenile abalone have been found in the
condos. but whether the abalone density data from the condos are
representative of wild resident juvenile densities or are useful as an
index tool to monitor changes in juvenile abundance over time is
still unclear.
Xhe negative effect of illegal harvesting on recovery efforts is
likely substantial. Increased local monitoring and decentralization
of enforcement power, from Fisheries and Oceans Canada to com-
munity-based programs, would benefit the abalone resource, stew-
ardship programs, enforcement agencies, and communities in-
volved. Xhe KFP is a strong proponent of information exchange on
this project and is eager to develop working partnerships with
other abalone stewardship groups. Xhe project is taking an eco-
system-based approach to abalone recovery where all work under-
taken is within the constraints of the natural environment.
850 Abstracts
Workshop on Rebuilding Techniques for Abalone in British Columbia
ABSOLUTELY ABALONE: HABITAT STEWARDSHIP
PROGRAM FOR THE PINTO ABALONE ON THE WEST
COAST OF VANCOUVER ISLAND. Anne Stewart. Banitield
Huu-ay-aht Community Abalone Project. Bamfield Marine Sci-
ences Center. Bamfield. British Columbia. Canada, VOR IBO
Pinto or northern abalone {Haliotis kamtschatkana) were har-
vested traditionally at low tide, for millennia on the west coast of
Canada. After intense diving harvests and an inability of manage-
ment strategies to control harvests, the pinto abalone was desig-
nated as a threatened species in 1999. The Bamfield Huu ay aht
Community Abalone Project (BHCAP) was fomied in response to
a request for proposals, to work on abalone recovery on the west
coast of Vancouver Island. The two key elements of the project
strategy are to engage the community in abalone recovery and to
operate a successful abalone hatchery for out-planting abalone.
The objective of the project is the promotion of Pinto abalone
recovery through conservation, education, and community engage-
ment. Collaborations have been established with Fisheries and
Oceans Canada. Canadian uni\ersities, the Nuu chah nulth Tribal
Council and the Pacific Rim National Park Reserve.
Members of BHCAP are the Huu ay aht First Nations. Bam-
field Community School Association and the Bamfield Marine
Sciences Center. The Huu ay aht First Nations have a goal of
restoring abalone to the point where they can harvest for food and
ceremonial use. The Community School Association is invoh ed in
building capacity. The Bamfield Marine Sciences Center, a non-
profit society with five western Canadian universities as members,
has a mandate for research and education in marine sciences. They
provide a base of operation for both the abalone education and
research programs and the co-ordination of the dive program for
abalone surveys, collections, and out-planting.
Public education is a major component of the program and
3.500 students per year, including students from school, college
and universities, adult programs, and the Community School learn
about abalone conservation biology. Raising the profile of the
stewardship project at public events also inspires concern for aba-
lone habitat and the kelp forest ecosystem for thousands of people.
The Ocean Link website (www.oceanlink@island.net) provides a
wealth of information on abalone and this project and had over 8
million visits during 2001.
To reduce abalone poaching, the Huu ay aht First Nations
crews patrol traditiimal territories and Coast Watch members keep
a look out for poaching. Fishers, boaters, crews, lodge operators,
and dive operators are also part of the Abalone Coast Watch.
Future plans include out-planting projects in conjunction with
Fisheries and Oceans Canada, sourcing funding options and con-
tinuation of education, outreach and community engagement to
strengthen community involvement. This last aspect is especially
important to reduce illegal harvesting. This project is a fine ex-
ample of First Nations and non-First Nations groups working to-
gether. With hard work and co-operation, the future of a healthy
and sustainable abalone community and ecosystem is possible.
STATUS OF ENFORCEMENT
HAVE WE GOT PROBLEMS. Bryan Jubinville. Conservation
Protection Branch, Fisheries and Oceans Canada. Labieux St..
Nanaimo, BC V9R 5¥.b Canada
Do we have problems? Yes we do — on 2 fronts: (1 ) the con-
tinued illegal harvesting of northern abalone [Haliotis kaiulschat-
kiiini) and (2) the reduction of reports on illegal activity. Essen-
tially the same core group of fisheries officers has been enforcing
the abalone fishery closure and has advocated protection of north-
ern abalone in British Columbia (BC) since the closure started in
1990. We have received considerable support from other jurisdic-
tions both within and outside the province, from stakeholders, the
public, and federal Science and Fish Management branches. In the
early 1990s, the conservation and protection of abalone started
slowly, with the development of public and infor)nant contacts, the
creation of an awareness campaign using multi-lingual posters,
media contacts, court cases, impact statements, convictions, imagi-
native sentencing efforts by lawyers and judges, and video clips.
Pi'otectio)! has piogressed which includes the ability to identify the
species DNA footprints, the development of abalone stewardship
groups, and the fostering of contacts with the public. Public sup-
port in enforcement is critical. When the public observes, records
and reports poaching activity to enforcement officers, all reports
are examined and the infor)iiation provided assists in developing a
file and an investigation. Although some of the infoiniation re-
ceived may prove to be of little value, some can be of significant
value resulting in a conviction.
Six years ago. fisheries officers would receive numerous calls
each year, which they would investigate. A file would be created
and, if possible, poachers prosecuted with success. I do not know
of a file that we have failed on when we have had the accused in
possession of northern abalone. However, recently important in-
formation being provided has diminished. Is it because the amount
and the quality of effort by the fishery officers in the field have
increased resulting in an exceptional job of enforcing the closure in
BC? Aie the illegal harvesters now more reluctant to poach aba-
lone because of the increased detenence created'.' I would like to
think that these are the answers, but I am realistic enough to know
there is a bigger picture in terms of the global problem of poaching
abalone. Recently, South Africa reported that the annual seizure of
illegal abalone had exceeded the legal harvest. I have to conclude
that our northern abalone is under similar pressure. So where are
the general public reports of illegal activity? I believe that we need
another method of getting the message out to the public to increase
the information being sent to us. The Haida Gwaii stewardship
emphasizes that "e\ery tip counts'" and the guardians will be docu-
menting and sending reports. This should foster credibility for
Fisheries and Oceans Canada and the guardians in the communi-
ties. Perhaps in addition to the telephone, word of mouth, and other
workshop on Rebuilding Techniques for Abalone in Britisli Columbia
Abstracts
851
means of communicalnit;. such as an Internet tip hue. would be
helpful. Do we have a problem? Yes, we do and one aspect oi the
problem is the recent reduced information provided on illegal aba-
lone harvesting actisity. We need the assistance of the public and
from communities throughout coastal BC in observing and report-
ing poaching incidents.
AQUACULTURE
RECENT PROGRESS IN HATCHERY PRODUCTION OF
PINTO ABALONE. HALIOTIS KAMTSCHATKASA, IN
BRITISH COLUMBIA, CANADA. Christopher M. Pearce.
Fisheries and Oceans Canada, Pacific Biologic Station. 3 1 90 Ham-
mond Bay Road. Nanaimo. BC V9T 6N7. Canada; Pelle Agerup.
Malcolm Island Shellfish Co-operative. 430 First Street. Box 229.
Sointula, BC VON 3E0. Canada; Abayomi Alabi, Probiotic Solu-
tions. 7143 Blackjack Drive. Lantzville. BC VOR 2H0. Canada;
Davvn Renfrew, Bamfield Huu-ay-aht Community Abalone Proj-
ect. Bamfield Marine Sciences Center. Bamfield. BC VOR 1 BO.
Canada; John Rosser. Malcolm Island Shellfish Co-operative. 430
First Street. Box 229. Sointula. BC VON 3E0. Canada; Guy
Whyte, Bamfield Huu-ay-aht Community Abalone Project. Bam-
field Marine Sciences Center. Bamfield, BC VOR IBO. Canada;
and Fu Yuan. Island Scallops Ltd.. 5552 West Island Highway.
Qualicum Beach. BC V9K 2C8. Canada.
In July 1999. Fisheries and Oceans Canada issued a Request for
Proposals for 18-mo pilot projects that would develop land-based
hatchery rearing techniques for the pinto abalone. Hciliaiis ka-
mtschatkana. A percentage of the cultured juveniles produced
were to be utilized for wild stock rebuilding. Six projects were
initially appro\'ed and fi\e proceeded with the collection of wild
broodstock for the purpose of developing hatchery techniques. Of
these projects, three were successful at rearing substantial numbers
of juveniles (ie. Bamfield Huu-ay-aht Community Abalone Proj-
ect, Island Scallops Ltd., and Malcolm Island Shellfish Coopera-
tive). Their techniques for broodstock conditioning, spawning, lar-
val rearing, larval settlement, and early juvenile grow out are sum-
marized in this review paper. Adult broodstock were conditioned
with wild kelp (Lximinaria saccluiriiia. Macrocystis integrifolia.
Nerencystis hietkeana) and spawned using hydrogen peroxide,
temperature shock, and/or UV-treated seawater. Larvae were
reared in tlow-through or static systems at 1I°C to I5°C at a
density of 1 to 9 larvae ml"' and settled on wavy or flat plastic
sheets covered with natural biofilms of various ages. Early juve-
niles fed on benthic diatoms and were later converted to kelp
and/or prepared diets. Grow out time to commercial size is pre-
dicted to be 4 to 6 years. To date, these three projects have pro-
duced approximately 170.000 juvenile abalone of various sizes.
FIELD RESEARCH
NIGHT AND DAY SURVEYS OF A NORTHERN ABA-
LONE, HALIOTIS KAMrSCHAThA.\A. POPl'LATION IN
EAGLE BAY, BRITISH COLUMBIA. James P. Mortimor,
Caitlin R. Henderson, Bamfield Marine Sciences Center, Bamfield
B.C. VOR IBO Canada; and Glen R. D. Elliott. Bamfield Huu ay
aht Community Abalone Project. Bamfield B.C. VOR 1 BO Canada.
This study, initiated by the Bamfield Huu ay aht Community
Abalone Project, attempted to establish characteristics of behavior
and site selection, for possible out-planting of northern abalone
{Haliotis kiiinlsclmikami). Diurnal and nocturnal surveys were un-
dertaken to determine population estimates of emergent juvenile
and adult abalone at one small area in Barkley Sound. While using
conventional methodology the survey conducted was intensive and
small scale in nature, contrasting with previous studies that estab-
lished abalone population estimates over broader areas. No clear
community association was identified, however, behavioral and
physical constraints were established. Recommendations for in-
creasing out-planting effectiveness include out-planting juvenile
abalone at night, between 4 and 6 m below chart datum, on struc-
turally complex substrates.
TRENDS IN PINTO ABALONE {HALIOTIS KAMTSCHAT-
KAN A) ABUNDANCE IN THE SAN JUAN ISLANDS AND
MANAGEMENT OF ABALONE IN WASHINGTON
STATE. D. P. Rothaus. Washington Department of Fish and
Wildlife, Marine Resources. 16018 Mill Creek Blvd. Mill Creek.
WA 98012-1296 USA; and C. S. Friedman. School of Aquatic
and Fishery Sciences. University of Washington, Box 355020 Se-
attle, WA 98195 USA,
Northern abalone are contagiously distributed in shallow,
rocky, exposed, and kelp covered habitats from Sitka Alaska to
Monterey California. In Washington State, abalone are found in
the San Juan Islands. Strait of Juan de Fuca, and northern coastal
waters. They are a slow growing species, reproductively mature at
25 to 50 mm shell length (SL) depending on location. In 1984. the
sport harvest was estimated at 38.200 abalone annually and by
1991 this had increased to 40.934. Before 1992. regulations al-
lowed a sport fishery for abalone of 90 mm SL or greater with a
harvest limit of 5 abalone per day. and an abalone iron (for re-
moval of abalone from rocks) was required. From 1992 to 1 994, the
allowable harvest was 3 abalone per day. minimum size 102 mm
SL. and an abalone iron and calipers were required. A total closure
was instituted in August of 1994. Stocks have declined in both
British Columbia and Washington State. leading to the listing of
northern abalone as a "Threatened Species" in Canada and as a
"Species of Concent" in the USA. Common concerns and potential
852 Abstracts
Workshop on Rebuilding Techniques for Abalone in British Columbia
trans-boundary issues suggest co-operative restoration efforts be-
tween BC and Washington State may be valuable.
Surveys conducted throughout the San Juan Islands, 1979 to
1982. with timed 15-min dives, have provided baseline informa-
tion on abalone abundance and size for this area. Twenty-three of
these sites were again surveyed between 1990 and 1991. Abalone
density at 1 site increased, at 4 sites stayed the same, at 9 sites
decreased, and at 9 sites no abalone were found. The overall den-
sity decrease was appro.ximately 50%. Locating the original 23
sites was problematic and may have been a factor in the dramatic
decrease observed. Even with the potential problems with this
comparison, the magnitude of the apparent decline, combined with
the anecdotal information from sport divers and University of
Washington researchers, raised serious concerns about the health
of abalone stocks in Washington. Further surveys were required to
adequately evaluate the apparent trend in abalone abundance. As
with other areas of the world, illegal harvesting is considered to
have a major impact on the abalone stocks in Washington State.
In 1992, 10 permanent abalone index dive stations were estab-
lished around the San Juan Islands. The sites ranged in size from
50 m" to .^80 ni", averaging 220 ni" with depths between 0 to .^0
ft MLLW. Abundance and size of emergent abalone were deter-
mined over the whole site (census) with dives of 180 to 340 min
bottom time. A declining trend in total abundance for all sites was
observed 1992 to 1994 (/i = 351 to n = 288), with no statistical
difference. Following the fishery closure in August of 1994.
the 1996 survey results were n = 297. Additionally, no significant
difference in the mean shell length over time was observed. The
average density of half the sites surveyed in 1996 was less than
0.15 abalone/nr. Research indicates that sedentary invertebrates,
such as abalone. must be within 1 .0 to 2.0 m of one another
(0.33-0.15 abalone/m') for successful fertilization. Therefore, low
population levels can lead to inability for gametes to cross-fertilize
resulting in recruitment failure. In Washington State, data shows
that half of the index stations have abalone densities below the
level for successful recruitment.
Recovery efforts include a captive broodstock project initiated
in 2002 for the development of hatchery techniques using 80 aba-
lone collected from Lopez Island. Sixteen percent mortality in the
broodstock has occurred over the subsequent 5 months of captiv-
ity.
Anecdotal information and quantitative survey data suggest a
decline in abalone populations in Washington State. Data from
index stations show a gradual decrease in abundance at 6 of 10
sites but no overall significant change in abundance from 1992 to
1996. Some of the current index stations report abalone densities
below the minimum density levels that are needed for successful
recruitment.
Additional stock assessment studies will include the re-
evaluation of the 10 index sites in February 2003, more frequent
(yearly) assessment of abalone abundance at the 10 index sites,
development of a better survey method si) that population esti-
mates can be obtained, creation of additional index sites in the
Strait of Juan de Fuca. and initiation of juvenile abundance sur-
veys. Genetics studies, in collaboration with Canadian scientists,
will include analysis of relatedness between sites and between
individuals within a site. The captive broodstock project will con-
tinue to culture abalone using techniques to maximize genetic
diversity and to compare behavior of hatchery reared animals in
normal versus "natural" tanks.
Should surveys show continued instability in abalone popula-
tions, management plans would be developed for abalone stock
restoration, potentially including out-planting of hatchery raised
juveniles and aggregation of adults. Public information meetings
and scientific workshops will be held in co-operation with the
Puget Sound Restoration Fund to raise public awareness.
REHABILITATION METHODS
OVERVIEW OF ABALONE STOCK ENHANCEMENT IN
NEW ZEALAND AND LESSONS FROM LABORATORY
STUDIES OF ABALONE LARVAL SETTLEMENT AND
POST-LARVAL FEEDING. R. Roberts, Cawthron Institute.
Prisate Bag 2, Nelson. New Zealand. E-mail:nxlneyCs'cawthron.org.nz;
and N. Andrew. NIWA, PC Box 14-901, Kilbirnie. Wellington,
New Zealand, E-mail:andrew@niwa.cri.nz
In New Zealand, abalone catch from commercial, recreational,
traditional, and illegal harvest is approximately 1700 t per year.
Fishing effort is controlled by catch limits, minimum size ( 1 25 mm
shell length. SL). and method restrictions. Since 1999. the com-
mercial fishery has been reduced in several main fishing areas
through quota cuts and voluntary reduction in catch entitlement.
Inxesligations have begun that may provide alternatives to further
quota cuts including temporary closures, larger minimum harvest
size, and release of hatchery reared juveniles or larvae. Catch
reporting has been modified to provide data at high spatial reso-
lution, as population dynamics in abalone can vary over small
spatial scales and recruitment may be localized. A project under-
way is to determine the reason(s) for the large number of stunted,
sub-legal size animals. (<I25 mm SL) in certain areas. Abalone
less than 1 10 mm SL in these areas were removed and ongoing
surveys will determine if this improves the growth rate of the
remaining animals and if the transplanted animals reach minimum
legal size.
The commercial fishery is dominated by Haliotis iris, a large
abalone up to 180 mm SL. which is also the only species currently
farmed in New Zealand. A minor fishery exists for Haliotis aiis-
inilis. a small (<1 10 mm SL) abalone. Haliotis vir)>inea is a small
(<70 mm SL) cryptic species that is not landed in the coiinnercial
harvest.
The most substantial study of abalone reseeding in New
Workshop on Rebuilding Techniques tor Abalone in British Columbia
Abstracts 853
Zealand produced very promising results at some of 8 sites, with
the best site showing 54'* sur\i\'al of lO.OOO of 7 to 12 mm SL
seed, 2 years after release. Apparent survival was higher at 2 y than
at 1 y, illustrating the difficulty in obtaining accurate survival
estimates for cryptic life-stages.
In New Zealand reseeding studies, a large proportion of juve-
nile mortality often occurred soon after release. Burial by sand was
a major cause of mortality in three of four studies. Lower sur\ i\ al
and naive behavior from hatchery seed compared with wild juve-
niles was observed in each of .^ studies. Predation was considered
in only I studs and found to be minor. Naive beha\ ior of hatchery
abalone may be reduced if the hatchery encouraged appropriate
abalone beha\ior, (eg. by providing shelters to maintain cryptic
behavior) using strong light cycles to encourage feeding at night,
and exposing abalone to predators periodically to maintain defense
responses.
Two small trials of larval release have been carried out in New
Zealand. In the first trial, 300,000 larvae were released in a 50-m2
gully. Minimum survival was 0.4'v'f after 3 months and the calcu-
lated cost of each surviving animal was USS0.I4. indicating this
method could be economically viable. In a second trial, mesh-
tented seatloor areas of I m" were seeded with 20,000 larvae. Only
10% of larvae settled and minimum survival after 5 months was
0.06% resulting in the cost of each surviving abalone of US SO. 80,
indicating this method would be uneconomic.
Laboratory studies on larval settlement and post-larval feeding
have provided insights into larval reseeding and natural recruit-
ment. Abalone larvae are capable of attaching and crawling prior
to metamorphosis. Haliotis iris will attach from 4 days of age and
metamorphose at 7 to 8 days at 17 °C. Abalone can delay meta-
morphosis for 2 to 3 weeks at 17°C to 20°C. In cold water, both the
pre-competent period, and the ability to delay metamorphosis
would be extended. Hence, potential larval dispersal may be wider
than previously assumed for abalone.
Larvae become increasingly responsive to metamorphosis cues
as they age, so older larvae are more likely to metamorphose close
to the point of release. Crustose coralline algae are the most ef-
fective settlement-inducer for most abalone species, but larvae
often resume swimming after landing on corallines, particularly
less preferred species. Resumption of swimming could lead to
transport out of the study area with consequences for survival
estimates.
Abalone of less than 5 mm SL consume the biofilm on coralline
algae. Abalone less than 0.8 mm SL will scoop up loose diatoms,
bacteria, and coralline secretions, competing with many generalist
grazers. Abalone of 0.8 to 5.0 mm SL develop radula teeth spe-
cialized for gouging, increasing their grazing capability and reduc-
ing competition. In animals more than 5 mm SL, the radula is
further specialized and the diet expands to coralline crusts, macro-
algae, sea grasses, and drift particles, further reducing competition.
The modest carrying capacity of corallines for young abalone
should be taken into account when deciding release densities in
larval reseeding. Visible signs of star\ation have been described
from laboratory studies and reported in post-larvae from natural
habitat.
Areas with good recruitment are not necessarily recruitment
saturated. However, reseeding may not be successful if there is
strongly density-dependant mortality at some stage of life. Little is
known about the prevalence or intensity of density-dependant mor-
tality in abalone — whether it can be strong enough to negate re-
seeding returns, or how it may vary spatially, temporally, or be-
tween species. Sites that previously had good recruitment and a
strong fishery but cuiTcntly suffer from recruitment failure should
be ideal for reseeding. Though the results of New Zealand seeding
studies are encouraging, more research is needed, especially to
determine the factors conlrollina survival after release.
A REVIEW OF ABALONE ENHANCEMENT AND REHA-
BILITATION IN SOUTH AFRICA. Peter Cook, Zoology De-
partment, University of Cape Town, South Africa. Current ad-
dress: Center of Excellence in Natural Resources Management,
Albany WA 6330, Australia.
The South African coastline is approximately 3,000 km long
with very few bays, inlets or sheltered areas. The exposed coastline
limits opportunities for mariculture. However, the commercial har-
vest of abalone is threatened by a high level of illegal poaching and
this situation has provided support for a successful farming indus-
try for Haliotis midae. Abalone farming has expanded rapidly in
South Africa and, by 2004, annual production is expected to ex-
ceed 600 tonnes per year. Most farms have hatcheries and this
leads to excess production of juveniles that could be used for
enhancement or ranching. Wild populations, consisting of six spe-
cies of abalone, occur on the southwest, south and southeast coasts
of South Africa. Although the west coast is a highly productive
area with extensive kelp beds and high wave action, abalone occur
naturally only in the southernmost sections.
Experiments to determine the feasibility of abalone enhance-
ment and ranching in South Africa were carried out at Port Nol-
loth, on the northwest coast. This site was chosen due to the
presence of abalone fossils, the presence of high densities of ur-
chins— both indicating appropriate environmental conditions for
abalone — and the availability of security from a diamond mine in
the vicinity. The site was over 300 km from any natural abalone
population, assuring that any animals in the area were froin the
ranching experiments.
Anticipated problems with the sea ranching included release
mechanisms that could cause mortalities, predation after release,
monitoring success (% survival), and assessing economic viability.
To reduce the mortality caused by handing during the transporta-
tion and release of abalone, special release mechanisms were de-
vised. These devises consisted of PVC pipes halved lengthwise
and glued to a Perspex sheet. Both ends of the PVC covered with
mesh after the abalone entered the devices naturally. The devices
854 Abstracts
Workshop on Rebuilding Tecliniques for Abalone in British Columbia
were transferred intact without handling the individual animals.
The devices were attached to concrete blocks at the experimental
site in the late afternoon. Twenty-four hours later, the mesh was
removed and the abalone were allowed to exit at will. This pro-
cedure also provided protection from predators for the first 24 h
while the abalone became acclimatized to the environment.
At four experimental sites, 500 or 800 abalone of approxi-
mately 14 mm shell length were released per site. Growth and
survival were monitored over 24 months and there was signifi-
cantly slower growth through the summer compared with the win-
ter months suggesting that the timing of the release is important to
both survival and growth. Survival is reported in Table 1.
The maximum mortality occurred during the first twenty-four
hours after release and many remained in the transport devices for
extended periods. Even at good sites there was little dispersal from
the release area. Abalone appeared to seek out urchins for protec-
tion and were often found under the urchin spines. However, the
presence of urchins did not appear to ensure a high survival rate
since at one site no surviving abalone were found in March 1W7.
Factors affecting survival are complex, variable, and inter-related,
displaying a variable hierarchy of importance.
For ranching to be economically \ iable a survival rate of 5'7f
tol0% for out-planted juveniles is required. Preliminary results
suggested that, in certain circumstances, survival rates in excess of
30% could be obtained with seeded animals with particularly good
survival being obtained when precautions are taken to reduce han-
dling stress and at sites where sea urchins were present. Later work
at similar sites, however, produced contrasting results and it was
concluded that an extremely coinplex interplay between many dif-
ferent factors affected survival.
Of these, the presence of sea urchnis was only important at
certain sites, whilst, at other sites, the present of optimum-si/ed
boulders seemed to replace that requirement. Overall, the size of
seeded animals was the most important factor that influences sur-
vival, larger seed having better survival rates. Following the dem-
onstration that, under certain circumstances, ranching could be
economically feasible, genetic implications of this operation were
investigated. Using mt-DNA markers, it was shown that, not only
could animals from dilferent geographic regions be shown to be
genetically differentiated but, in addition, distinct genetic differ-
TABLE 1.
Survi>al of released abalone.
Simple
April
September
March
Survival to 6
Sites
Released
1996
1996
1997
Months
Site A
.soo
97
40
0
27.4%
SiteB
500
117
70
19
39.2%
SiteC
800
-
124
99
27.8%
SiteD
800
-
145
60
25.6%
Average 307r
ences between naturally occuiTing and hatchery reared animals
was also apparent.
REBUILDING CALIFORNIA ABALONE POPULATIONS.
Haaker, P. L., I. Taniguchi. California Department of Fish and
Game. 4665 Lampson Avenue. Suite C. Los Alamitos. CA. 90720
USA; J. Butler. NOAA Fisheries. Southwest Fisheries Science
Center (NWFS). La Jolla. CA. USA; and N. Wright. California
Department of Fish and Game. 4665 Lampson Avenue. Suite C.
Los Alamitos, CA. 90720 USA.
Since 1997. all seven California abalone species have been
closed to commercial and recreational fishing south of San Fran-
cisco Bay. California abalone occur throughout the coastal marine
environment from the intertidal zone to deep offshore reefs, pre-
senting a challenge for the development of an abalone recovery
plan. A successful plan needs to address the various characteristics
of abalone and include an approach that can be applied as broadly
as possible using available resources and a method for evaluation
of progress.
Assessment of remaining abalone stocks is of immediate im-
portance. Assessment goals have been established to address and
prevent extinction of the abalone. to restore resource sustainability.
and to rebuild resources to fishery sustainability levels. Assess-
ment must include the identification of remnant populations, es-
tablishment of locations for further evaluation, and determination
of locations for enhancement.
The range of abalone is often specific, extensive, and with
patterns of distribution. For each species, information on landings
from fishing effort is accumulated into catch block areas which are
used to determine the most extensive and the best habitat for
abalone and used to determine research index sites. Population
density is an essential element of the assessment; however, when
numbers are low standard density surveys are uninformative. Free-
form searches can yield more individuals than transect constrained
surveys and data on individuals, such as size, can be collected.
Shell length (SL) is a population indicator, where the occur-
rence of a broad size range, even at low numbers, is evidence of
reproduction and growth. Size surveys are conducted using 3 size
categories. 0 to 100 mm SL. 100 mm SL to minimum legal size
(MLS), and MLS to maximum size. Since the 0 to 100 mm SL
category includes the cryptic population, which is difficult and
destructive to assess, it is only used for occasional determination of
settlement. When a broad range of sizes is present, quantitative
surveys are used to determine emergent abalone densities. A mini-
mum viable population target size is an emergent population of
2,000 abalone per hectare, at all index locations. When an average
of 6,600 abalone per hectare at three out of four of the index
locations is reached, a fishery could be considered.
Survey criteria and modifications should be made according to
species characteristics. For example, surveying intertidal popula-
tions with a GPS can provide the position of each abalone. White
abalone, currently surveyed using free form dives, could be sur-
Workshop on Rebuilding Techniques for Abalone in British Columbia
Abslraets 855
veyed using an ROV or submarine to pro\ ide geographic position.
Multi-beam sonar sea tloor maps are being generated by Depart-
ment of Fish and Game and. together with data from a ROV. could
be used to construct benthic habitat maps.
Abalone populations are at extremely low levels throughout
southern California. Some remaining populations are so dispersed
that successful natural reproduction is unlikely and enhancement
may be the only remaining method of intervention. Enhancement
techniques include aggregation of abalone, translocation of indi-
viduals from remote source populations, and out-planting of cul-
tured juveniles and competent larvae. At the present, aggregation
is seen as the only viable method of enhancement.
In abalone recovery, there are several challenges that must be
addressed as part of the recovery process. Some challenges must
be considered before the recovery process can proceed. The pres-
ence of sea otters precludes a fishery of abalone and most other
marine invertebrates. In the abalone recovery plan, any areas either
currently or previously occupied by sea otters are excluded from
assessment.
Disease has severely affected abalone stocks. The black aba-
lone was virtually extirpated from southern California by wither-
ing syndrome. Warmer seawater temperatures enhance withering
syndrome, which is a concern for translocation projects. A para-
sitic sabellid worm, which causes shell growth disruption and de-
formity, was introduced into California aquaculture facilities and
currently a prohibition exists on the out-planting of abalone from
non-certified facilities.
Genetic questions need to be addressed, prior to translocation
of abalone from different bio-geographic zones and using cultured
animals for enhancement of natural populations.
Poaching of abalone is a serious problem in California. Recov-
ery site criteria should include a low likelihood of illegal activity.
Part of recovery is establishing large, dense populations and
groups of individuals to facilitate reproduction. It is precisely those
conditions that are good for poaching. Optimal locations would
include remote islands, and mainland locations within limited ac-
cess reserves.
Marine Protected Areas. MPAs. offer one of the best opportu-
nities for abalone restoration activities. Recently. California estab-
lished a number of MPAs at the Channel Islands National Marine
Sanctuary and most include ongoing abalone study sites and ap-
propriate abalone habitat.
Each abalone species has specific environmental requirements,
which must be addressed to optimize successful recovery. South-
ern California is at the northern end of the range of pink, green,
and white abalone, and the southern end of red abalone range. Red
abalone growth and reproduction is depressed during warm water
periods, but they can survive until temperatures decline. Pink and
green abalone prefer warmer water which may allow their popu-
lations to extend farther northward with increasing sea water tem-
peratures. Environmental effects complicate other factors. If sea-
water temperatures increase farther north, withering syndrome
may become infectious in northern populations.
Our major challenge in rebuilding abalone stocks is to return at
least part of the abalone population to a natural situation, where
bio-diversity and natural selection can be effective.
Jounuil of Shellfish Research. Vol. 22. No. 3. 857-863. 2003.
SIZE AT MATURITY OF FEMALE AMERICAN LOBSTERS FROM AN ESTUARINE AND
COASTAL POPULATION
SUSAN A. LITTLE* AND WINSOR H. WATSON, III
Zooloiiy Departineut & Center for Marine Bioloi^y. University of New Hunipshire.
Durham. New Hampshire 03824
ABSTRACT The size at which female lobsters reach sexual maturity was determined for two populations that inhabit waters along
the coast of New Hampshire. One group was captured in the Great Bay estuary, where water temperatures in the summer typically
average between 1 7°C and 20°C. The other group of lobsters resided in coastal waters, near the Isles of Shoals, where the water
temperature was much colder during the summer ( 1 1-15"C). Maturity was assessed using criteria that included the following: ovarian
classification; abdominal width/carapace length (CL) ratio; and the size frequency distribution of berried females. All the techniques
yielded similar results and consistently demonstrated that female lobsters in the estuary matured at a smaller size than those in colder
coastal waters. The smallest mature females from Great Bay were 72 mm in CL. with iWr reaching se.xual maturity by 83 mm CL
and all beconung mature by 89 mm CL. The smallest mature female from the Isles of Shoals area was 77 mm CL, with 50% mature
by 86 mm CL and all mature by 93 mm CL. The difference in the proportion of mature lobsters in the estuarine versus coastal
populations was much greater in the smaller size classes than in the larger size classes, suggesting a mi.xing of the two populations,
most likely due to females from Great Bay migrating into coastal waters.
KEY WORDS: cslu.irv. Hoiiniiiis itmericiiniis. lobster, sexual maturitv
INTRODUCTION
The American lobster. Hoinunts anicncanus (Milne-Edwards)
is the most commercially valuable species harvested in the north-
west Atlantic Ocean (NMFS 2002). Although lobsters are most
abundant in coastal waters, estuarine populations are common and
have been investigated from Canada to Massachusetts (Thomas
iy6S. Thomas & White 1969. Munro & Theriiaull 19S-3. Reynolds
& Casterlin 1985. Jury et al. 1995: Howell et al. 1999; Watson et
al. 1999). One population that has received considerable attention
is located in the Great Bay estuary in New Hampshire. Howell et
al. (1999) have demonstrated that, like the lobsters in the Iles-de-
l-Madeleine in Canada (Munro & Therriault 19S.3). the sex ratio is
skewed toward males throughout the estuary, with the greatest
proportion of male lobsters found in the portions of the estuary
furthest from the coast. It has been proposed that the skewed sex
ratio in the estuary is the result of the differential seasonal migra-
tion of mature female lobsters out of the estuary (Watson et al.
1999).
To ensure that there are enough mature females in a given
lobster population, a minimum legal size has been established.
This allows a given proportion of the females to reach sexual
maturity and reproduce at least once befoi-e they are landed. The
size at which 50% of the females from an area are mature (50%
maturity) is often used as a reference point because most models
indicate that when the minimum size is set at this value sufficient
recruits will be produced to sustain the fishery. Currently, the
minimum size limit in the inshore waters of New Hampshire is 83
mm carapace length (CL).
There is a wide range of sizes over which female lobsters reach
maturity. The smallest size at 50% maturity. 70 to 74 mm CL. is
found in western Long Island Sound (Briggs & Mushacke 1979),
and the largest size. 110 to 120 mm CL. is found in the Bay of
Fundy (Templeman 1936. Groom 1977. Campbell 1983). It has
been suggested that a number of different factors infiuence the size
*Corresponding author. E-mail: slittle (sunh.edu
at which female lobsters mature, including nutrient availability
(Lawton & Lavalli 1995). fishing pressure (Polovina 1989.
Landers et al. 2001 ). and temperature (Templeman 1936. Temple-
man 1944. Aiken & Waddy 1980. 1986. Estrella & McKiernan
1989. Fogarty 1995). Increases in all. or any, of these factors
results in a decrease in the size at which females reach sexual
maturity.
Temperature is thought to be the most influential of these
factors because it is known to directly affect the growth rates
of lobsters, with development occurring more quickly with
increased temperature (Aiken & Waddy 1976). The rate of
ovarian development is primarily controlled by summer water
temperature, with little development occurring throughout the
winter months (Templeman 1936). Thus, in areas with warmer
water in the summer, lobsters reach sexual maturity at smaller
sizes.
Estuaries, such as the Great Bay estuary in New Hampshire, are
characterized by large daily and seasonal fluctuations in tempera-
ture and salinity. In the Great Bay estuary, the water temperature
in the summer is approximately IO°C higher than in New Hamp-
shire coastal waters (Short 1992). Given the apparent influence of
water temperature on the rate of inaturation of female lobsters, we
hypothesized that female lobsters in the Great Bay estuary would
reach sexual maturity at a smaller size than those in coastal waters,
such as near the Isles of Shoals, which are located 1 1 km away
from where the Great Bay estuary empties into the Gulf of Maine
(Fig. 1).
To test our hypothesis, we determined the size at maturity for
92 lobsters collected in the Great Bay estuary with 106 lobsters
collected near the Isles of Shoals. A comparison of the results
yielded by analyzing (1) the size distribution of berried females,
(2) the size of female abdomens relative to their length, and (3) the
stage of eggs removed from the ovaries yielded the same pattern.
Female lobsters from the estuarine site matured at a smaller size
than those from the coastal site, probably due to the influence of
warmer summer water temperatures on their growth and develop-
ment.
857
858
Little and Watson
43 ID-
70° «■
Figure 1. The two study sites are marl\ed witli an X [Great Bay Es-
tuary and Isleof Slioals (II l<m off liie iitast of New Hampsiiirel]. Sites
of temperature data collection for the (ireal Bay Estuary are: A, Jack-
son Estuarine Laboratory; B, Fox Point; and C. Upper Piscataqua
River. Lobsters were obtained from the Great Bay estuary within the
area indicated by shading.
MATERIALS AND METHODS
Temperature
Bottom temperatures were collected in the waters surrounding
the Isles of Shoals from 1997 to 2001 at depths of approximately
8 to 10 m using HOBOTemp temperature data loggers (Onset
Computer. Falmouth. MA) thai recorded water temperature at 2-h
intervals for 5 to 6 mo at a time. Bottom temperature data for Great
Bay was collected from 1997 to 2001 at three different locations
that spanned the area where lobsters were collected (Fig. 1). The
most consistent data set were obtained from a location near the
University of New Hampshire Jackson Estuarine Laboratory, at a
depth of approximately 3 to 5 m. using a YSI multiparameter 6600
datalogger (YSI Inc.. Marion. MA) that recorded the water tem-
perature every 30 min. Water temperature also was recorded near
Fox Point and along the Piscataqua River in 1990 and 1993. using
a YSI meter model 33 attached to a probe that was lowered to a
point near the bottom. Data were obtained from these two sites
approximately every other day while hauling some of the traps
used to collect lobsters for this study. Data from all three sites were
averaged from all available years to yield a temperature profile of
the area from which lobsters were collected. The mean monthly
temperature then was calculated, and the total annual degree-days
>8°C were summed for each location by adding together the num-
ber of degrees that exceeded S' C for each day of the year and
summing them for the entire year.
Maturity Assessments
Dissections
Lobsters were collected from two areas (Fig. i ) by commercial
fishermen and by University of New Hampshire personnel using
standard traps. The first site consisted of the upper region of the
Great Bay estuary (i.e.. Great Bay. Little Bay. and the upper Pis-
cataqua River), and the second site included waters near the Isles
of Shoals.
Lobsters were collected in 1991. 1992. 1994. and 2002. The
lobsters from each site were divided into l-mm size classes rang-
ing from 66 to 110 mm CL. A total of 92 lobsters were dissected
from Great Bay. and a total of 106 from Isles of Shoals.
Female, nonovigerous. lobsters were examined, using multiple
criteria, to determine whether they were sexually mature. For each
animal, the CL and the width of the second abdominal segment
were measured in millimeters, and the molt stage was recorded by
examining the carapace and pleopods. One pair of pleopods then
was removed for examination under a dissecting microscope to
determine the cement gland stage (Aiken & Waddy 1982) and
whether lobsters were in a premolt condition (Aiken 1973). A
small circular incision then was made just behind the eye socket to
access the anterior end of one of the ovaries. Several eggs were
removed, and their size range and color were recorded. An egg
stage was assigned to each lobster based on criteria established by
Aiken and Waddy (1980).
Whether a female was sexually mature, or not. was determined
using a combination of criteria, with ovarian stage as the primary
tool. Any females with resorbed oocytes were considered to be
mature, as these are an indication of prior spawning. Of the fe-
males without resorbed oocytes, those with ovaries that were at
stage 4 and higher were also considered to be mature. The size
range for stage 4 ovaries is different in the spring (stage 4b) than
in the fall (stage 4a) due to the timing of development, and this was
taken into account. Those females with ovaries at stage 2 and
below were considered to be immature. To determine the maturity
of those with stage 3 ovaries, we considered cement gland stage as
well as egg stage. If a female lobster with stage 3 ovaries had
cement glands that were at stage 3 or greater, then the lobster was
considered to be mature.
To determine the size at which SC/c of the females from each
area were mature, a nonlinear regression of percent mature for
each l-mm CL size class was carried out using the statistical
program, SYSTAT. The following equation was used:
p = (1/(1 -I- exp(-bO*(L-hl )))
where p is the proportion mature, bO is the curve shape parameter,
L is the carapace length, and bl is the size at 50% maturity (es-
timated as a starting point for calculations by the user). The pro-
gram estimated values of bO, based on the data set. until it found
the best-fit curve. This resulted in sigmoid curve from which bl
could be calculated with a 95% confidence interval. A statistical
comparison of the regression lines that resulted from each popu-
lation of lobsters was made to determine whether they were sig-
nificantly different from each other.
Sea Sampling Data
Sea-sampling data were obtained from LIniversity of New
Hampshire research traps, and during trips on commercial lobster
boats in 1990 to 1993 and 2002 at each location. The data collected
included CL. width of the second abdominal segment, sex. and
whether females were ovigerous. A total of 8199 lobsters were
examined during these sea-sampling trips.
Abdominal Width
A ratio of abdomen width to CL (ABD/CL ratio) was calcu-
lated for each female, and these were averaged for each l-mm CL
Size at Maturity of Femalh American Lobsters
859
size class. A plot then was made of CL versus this ratio for each
size class. A nonlinear polynomial regression of these data was
created for each site using SYSTAT. The following equation was
used: ABD/CL = a + bx + cx'^2 + d\'^3. where x = CL. SYSTAT
then estimated the values of a. b. c. and d to most closely fit the
curve to the data. To determine the inflection point of the curve,
which represents the point at which the rate of change in the
ABD/CL ratio is greatest, and therefore approximates the size at
which SO'/c of the feinales have reached maturity, the second de-
rivative of the original equation, y = 2cx + 6dx. was calculated.
That equation was then set to equal zero and was solved for x.
yielding the equation x = -2c/6d. Then, the c and d values from
SYSTAT were used to solve for x (the CL at 50% maturity)
(Landers et al. 2001 ). The size at 50% maturity that was estimated
by this method was compared with that obtained by dissection for
the estuarine and coastal lobster populations to determine whether
the abdominal width estimates fell within the 95% confidence
intervals of the dissection estimates.
months (June-August; Great Bay 995; Isles of Shoals 404). The
difference in degree-days between the two sites for these 3 mo
accounted for 75% of the difference in degree-days for the entire
year. During this period, the mean water temperature averaged
12.5°C at Isles of Shoals and 19=C in Great Bay.
Maturity Assessments
Dissections
Nonlinear regressions of CL versus percent mature, as deter-
mined by dissections, were used to calculate the size at 50% ma-
turity for each site (Fig. 3a). The size at 50% maturity for females
obtained from waters near the Isles of Shoals was 85.9 mm CL
(95% confidence interval 85.3-86.5; n = 106). Fifty percent of
females from Great Bay were mature at 83 mm CL (95% confi-
dence interval 80.6-85.4 mm; ;; = 92). A comparison of the two
regressions showed that they were significantly different from
each other (P < 0.001 ). The smallest mature female captured near
Berried Female Size Frequency Distributions
From the sea-sampling data, a size frequency distribution of
berried females, as well as a plot of the overall size frequency
distribution of the population was made for each area. The plots of
overall size frequency were divided into the proportions that were
male and female in each size class so that the proportion that was
female at a given size class could be compared with the proportion
of females that were berried at that same size class. For each plot
the average size, the SEM. size range, and sex ratio were calcu-
lated for comparison. The size distributions for the overall popu-
lation and for only berried females were compared between sites
using a x" test of independence.
RESULTS
A Comparison oj tlsluarine Versus Coastal Water Degree-Days
There was a large difference between the number of annual
degree-days (>8°C) in the Great Bay estuary (1532) compared to
those in the waters near the Isles of Shoals (738) (Fig. 2). The
greatest difference in temperature occun'ed during the summer
25 n
A.
.-^
?0
u
01
73
lb
m
Cl
10
E
,<i^
.■^
-B
JB
s^-
I ^^
^x
I [■ 1 1 ! 1 r—
— f 1 1 1 1
9 10 11 12
Month
Figure 2. Mean monthly bottom temperatures (°C), with SE bars, for
water in the (Ireat Bay estuary (open circlel and near the Isles of
Shoals (solid circles! ( 1^97-2(1(11 ). W a(er temperature for (ireat Bay is
an average of three sites that encompass the area from w hich lobsters
were collected.
2
13
TO
C
g
o
Q.
o
B.
1
0,8
0.6
0.4
0.2
0
0.8
0)
h_
E 0 7
c
g
■■c
o
S-0.6
0 5
■crg^
1 1 ^1 M J»I J^ — 1 1 1 1 1 1
30 40 50 60 70 80 90 100 110 120 130
Carapace length (mm)
30 40 50 60 70 80 90 100 110 120 130
Carapace length (mm)
Figure 3. (A) Maturity ogiyes estimated by nonlinear regressions
based on dissection data from 1-mm size classes from Great Bay
(dashed line! and Isles of Shoals (solid line): CJreat Bay 50% maturity
= S3 mm CL (95% confidence interyal 8(1.6-85.4: n = 92l; Isles of
Shoals 5(1% maturity = 85.9 mm CL (95'/^ confidence interval 85.3-
86.5: n = 106). .\ctual values are plotted for each 5-mm size class. (B)
Polynomial regression estimated from abdominal width measurements
for I-nim size classes from (Jreat Bay (dashed line) and Isles of Shoals
(solid line): (;reat Bay •^tt"( maturity = 81.5 mm CL (h = 1613): Isles
of Shoals 50% maturity = 86.9 {n = 1699). Actual values are plotted for
each 5-mm size class.
860
Little and Watson
the Isles of Shoals was 80 mm CL. while in the estuary a 72-mm
CL mature female was captured. All females were mature by 93
mm CL at the Isles of Shoals study site, and by 89 mm CL in the
Great Bay estuary.
Abdominal width: CL ratios
Nonlinear regressions of ABD/CL ratios were fitted to the data
to calculate size at 50% maturity (Fig. 3b). The resulting curves
indicated that half the females from Isles of Shoals were mature by
86.9 mm (/; = 1699), while the size at 50% mature for lobsters
captured in the estuary was 81 .5 mm (/; = 1613). The estimate for
the Isles of Shoals lobsters did not fall within the 95% confidence
interval generated from the dissection data (85.3-86.5). but was
very close. The estimate for the Great Bay estuary lobsters fell
within the 95% confidence interval (80.6-85.4).
Size frequency distributions
The size range of berried females collected near the Isles of
Shoals was 77 to 138 mm CL. with an average (±SEM) size of 92
± 1.0 mm CL (« = 152; Fig. 4b). The size range of berried females
from the Great Bay estuary was 72 to 107 mm CL, with an average
size of 85 ± 0.6 mm CL {n = 98; Fig. 4a). These means were
significantly different from each other (P < 0.001 two-tailed / test).
Only a small portion (30%) of berried females from near the Isles
of Shoals were smaller than 85 mm CL, whereas 50% of the
berried females from the estuary were <85 mm CL. In contrast,
very few berried females (1%) from the Great Bay estuary were
>100 mm CL, while 20% of berried females from waters near the
Isles of Shoals were >100 mm CL. Nevertheless, despite these
differences, the distribution of sizes of berried females was not
sianificant between the two sites (P = 0.067).
A 10
o
Si
E
Great Bay Berried Females
8
6
4 ^
2
0 II I
n=98
avg. slze=84.8 ± 0.6
size range=72-107
OLnoinoLOOLnomomom
i^r^coooairooo^^csicNcoco
B 14
12
O 8
•5 6
J3 •*
12
Carapace length (mm)
Isles of Shoals Berried Females
n=152
avg. size=91.7 ±1.0
size range=77-138
llllilllllllllllllll
o
U-)
n
in
o
in
o
in
CO
O)
O)
CT)
o
o
'-
Cv] Csl CO CO
Carapace length (mm)
Figure 4. Size frequency histoHranis of berried females from ( A ) Great
Bay and (B) Isles of Shoals (/' = 0.1)67 x" test of independence).
The size range of the overall lobster population at the Isles of
Shoals site was 48 to 144 mm with a mean size of 8 1 ± 0. 1 mm CL
in = 3337; Fig. 5b). while the size range of the population from
the Great Bay site was 38 to 1 13 mm CL, with an average size of
78 ± 0.1 mm CL (;; = 4862; Fig. 5a). The size frequency distri-
bution of all lobsters was significantly different between the two
sites (P < 0.05). The Great Bay population includes more small
lobsters <65 mm CL (6%) than the Isles of Shoals population
(3%), and the Isles of Shoals site has more legal lobsters >83 mm
CL (277(1) than the Great Bay estuary (18%), particularly those
>I00 mm CL (2% at Isles of Shoals, <1% at Great Bay). The most
striking difference between these sites is the sex ratio, as reported
by Howell and Watson (1999). The overall proportion of females
at the Isles of Shoals site (64 ^c) was much larger than that in the
Great Bay estuary population (35%), and this was increasingly true
at larger sizes. The percentage of females in the Great Bay estuary
fluctuated between 30% and 40% but dropped to <30% at sizes
>82 mm CL, and no females >96 mm CL were captured in the
Great Bay estuary. In contrast, the proportion of females near the
Isles of Shoals increased with size class, so that 80% of the lobsters
>96 mm CL were female.
DISCUSSION
All three methods used to assess the size at maturity of female
American lobsters (i.e., egg stage, ABD/CL ratios, and benied
female size frequency distributions) indicate that female lobsters
from the Isles of Shoals mature at a larger size (50% = 85.9 mm
CL) than those from the Great Bay estuary (50% = 83 mm CL).
even though the two populations are <14 km apart. One of the
major differences between these two locations is water tempera-
ture. The Great Bay estuary (1532 annual degree-days) is signifi-
cantly warmer than the Isles of Shoals study site (738 degree-
days), with the greatest difference in temperature (74% of the total
difference in degree-days) occurring in the summer months. We
conclude that this increased temperature accelerates the rate of
development of females in the Great Bay estuary, thereby causing
them to reach sexual maturity at a smaller size. This finding once
again supports the theory first put forth by Templeman (1936) that
summer water temperatures determine size at maturity. The small
difference in size at maturity reported is similar to a larger scale
pattern observed along the entire range of the American lobster.
For example, 50%' of female lobsters from Long Island Sound
reach maturity at 70 to 74 mm CL (Briggs & Mushacke 1979),
while those from the Bay of Fundy do not reach maturity until 1 10
to 120 mm CL (Templeman 1936, Groom 1977, Campbell 1983).
While the size at 50% maturity for female lobsters from Great
Bay is significantly different (P < 0.001 ) than that of females from
Isles of Shoals, it is clear from the maturity ogives (Fig. 3) that the
greatest difference in the two populations exists in the smaller size
classes. This may be due to the mixing of mature females from
Great Bay with those from the coast, as mature females migrate out
of the estuary. As reported by Howell et al. ( 1999), the proportion
of females in Great Bay (35%) is much smaller than that near the
Isles of Shoals (64%), and this difference is most pronounced in
the larger size classes. In fact, the proportion of females in Great
Bay begins to decline above the 82-mm CL size class (Fig. 4),
which is approximately the size at which lobsters are reaching
maturity. As proposed by Watson et al. (1999) and Howell et al.
(1999), it would be advantageous for females to move out of the
estuary for optimal egg development and survival of larvae. While
Size at Maturity of Female American Lobsters
861
500
w 400 -
B
o 300
o 200
100
35
500
(/I
400 -
<D
tn
o
300 1
.,
o
Q)
?nn
n
fc
3
Z
100
Great Bay
45
..Illllllol
55
IJaDlliifl..ii .
65
D males
■ females
n=4862
avg. slze=78.3 ± 0.1
size range=38-113
75 85 95 105 115
Carapace length (mm)
Isles of Shoals
125
135
145
D males
■ females
n=3337
javg. slze=80.5±0.1
size range=48-144
35 45 55 65 75 85 95 105 115 125 135 145
Carapace length (mm)
Figure 5. Sizt frequency histograms of the overall catch from (A) Great Bay and (B) Isles of Shoals, divided into proportions of males and
females (f < 0.05 \' test of independence).
there is a greater tendency for lobsters to leave the estuary, a
number of coastal lobsters also move into the estuary, especially in
the summer, presumably to take advantage of the warmer tempera-
tures (Watson et al. 1999). Therefore, while there is a clear dif-
ference in the size at maturity of female lobsters from the two
populations, the mixing of the coastal and estuarine lobsters due to
seasonal migrations may be responsible for making this difference
less evident, especially in the larger size classes.
Although warmer summer water temperature appears to be the
most likely factor causing lobsters in the estuary to mature at a
smaller size than New Hampshire coastal lobsters, another possi-
bility is that berried females from offshore waters migrate inshore
to the waters near Isles of Shoals and skew the size frequency
of berried females there toward larger sizes. Berried females
often migrate inshore to complete their reproductive cycle because
the warm temperature inshore speeds their development (Cooper
& L'zmann 1971. Uzmann et al. 1977. Cooper & Uzmann l9Sf),
Fogaity et al. 1980, Campbell et al. 1984. Campbell & Stasko
1986). Seasonal concentrations of large berried females in inshore
areas off Cape Cod. MA (Estrella & McKieman 1989), and Long
Island, NY (Briggs & Mushacke 1979). are thought to be the result
of berried females from offshore migrating shoreward. Berried
females from offshore in both of these areas are larger than those
inshore, and thus the mixing of offshore berried females with the
local inshore populations would distort the apparent size frequen-
cies. This remains a viable explanation for the size at maturity
differences that we have observed.
Analyses of both egg stage data and ABD/CL ratios yielded
similar results, in ternis of size at maturity. Based in egg stages.
50% of females from the waters off the Isles of Shoals were mature
at 85.9 mm CL. while, according to ABD/CL ratios, 50% were
mature at 86.9 mm CL. In Great Bay. the values were 83 and 81.5
mm CL. respectively. The value based on ABD/CL ratios for the
estuarine lobsters fell within the 95% confidence interval gener-
ated from egg stage data, and, while the estimate based on ABD/
CL ratios from Isles of Shoals lobsters did not fall within the 95%
confidence interval (85.3-86.5) generated from dissection data, it
was very close. Thus, it seems that ABD/CL ratios provide a
reasonably good estimate of size at maturity, as indicated in sev-
eral previous studies (Skud & Perkins 1969, Krouse 1973, Briggs
& Mushacke 1979, 1980, Ennis 1980).
The size ranges of berried females from both sites were very
similar to what one would predict from analyses of the egg stages
of dissected lobsters. In the population near the Isles of Shoals, the
smallest mature female was 80 mm CL. while the smallest berried
female captured was 77 mm CL. Likewise, the smallest mature
Great Bay female was 72 mm CL. which was the same size as the
smallest berried female observed while sea sampling. This sug-
gests that it might be possible to construct a fairly accurate matu-
rity ogive using a combination of two noninvasive methods: the
size range of berried females and ABD/CL ratios. Measurements
of berried females are useful in defining the size range of mature
females in a population and can serve as a good indication of the
size at which the smallest females become mature. However, these
measurements do not indicate what proportion of the females at a
given size are mature, and these data could be derived from mea-
surements of the ABD/CL ratios over a range of relevant size
classes.
862
Little and Watson
While the size frequency distributions of berried females from
the two sites were not significantly different (P = 0.067). there
were clearly more large beri'ied females near the Isles of Shoals
(20% > 1 00 mm CL at Isles of Shoals vs. 1 % > 1 00 mm CL in Great
Bay) and more small berried females in Great Bay (SOVr <85 mm
CL in Great Bay vs. 10% >85 mm CL near the Isles of Shoals).
Therefore, it is likely that the size frequency distributions of ber-
ried females in both study sites were not significantly different due
to the low sample size of berried females in the Great Bay estuary
{J! = 98). This assumption is supported, in part, by the fact that the
size frequency distributions of the overall populations (;; = 4862
for the estuary) at the two sites were significantly different (P <
0.05). As with the berried female size frequency distributions, the
bulk of this difference can be accounted for by the lack of large
lobsters in the Great Bay estuary (<l% were >100 mm). As dis-
cussed earlier, these data support the hypothesis that as lobsters
reach se.xual maturity they migrate out of the estuary into deeper
water (Watson et al. 1999, Howell et al. 1999). While mature
females probably undergo this migration shortly after reaching
sexual maturity, giving rise to the skewed sex ratios observed in
the estuary in size classes >80 mm CL and the low number of large
berried females, male lobsters eventually move into coastal waters
as well, as indicated by the scarcity of any lobsters >I00 mm CL
in the Great Bay estuary.
Our results indicate that while there is a small difference in the
size at which females from the two sites reach maturity, that dif-
ference is small, suggesting that these are not two distinct popu-
lations. There appears to be mixing between the two areas, par-
ticularly among the sexually mature lobsters. Thus, despite the
small differences in size at maturity, it is probably not necessary to
implement different management measures for each area. The size
at which half of the females mature from both sites approximates
the minimum size limit, and thus it appears to be appropriate to
maintain adequate egg production and recruitment to satisfy the
FIO requirement.
ACKNOWLEDGMENTS
We are deeply indebted to Dr. Michael Lesser for providing us
with water temperature data for the Isles of Shoals; Jaimie Wolf
for helping access the National Estuarine Research Reserve Sys-
tem (NERRS) water temperature database for the Great Bay estu-
ary: Chris Becker, for her help with some of the maturity dissec-
tions and; Dr. Chris Neefus for his assistance with constructing the
ogives and clarifying other statistical analyses. We would like to
offer special thanks to both Al Vetrovs and Dr. Hunt Howell for
their help collecting so much of the data, and Ed Heaphy for
allowing us to collect sea sampling data aboard his vessel the Lady
Martha. Finally, as with so many of our projects, we would like to
thank all the students who helped collect data over the course of
this project. This work was made possible as a result of grants from
National Oceanic and Atmospheric Administration (Sea Grant)
and the Northeast Consortium to W. H. W. It is contribution num-
ber 408 in the Center for Marine Biology/Jackson Estuarine Labo-
ratory series.
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GREEN CRAB (CARCINUS MAENAS LINNAEUS) CONSUMPTION RATES ON AND PREY
PREFERENCES AMONG FOUR BIVALVE PREY SPECIES
KELLY C. FALACIOS'* AND STEVEN P. FERRARO" t
College of Oceanic and Atmospheric Sciences. Oregon Stale University. 104 Ocean Admin Bidg..
Corvallis. Oregon 97331: and 'U.S. Enviroiuuental Protection Agency. 2111 S.E. Marine Science Drive.
Newport. Oregon 97365-5260
ABSTRACT Laboratory experiments were conducted to determine green crab. Carciinis maenas. consumption rates on and prey
preferences among lour bivalve species: Olympia oysters (Ostrea concbaphila Carpenter), Japanese littleneck clams {Veiienipis
philippinanim A. Adams and Reeve), bent-nosed macoma clams (Macoina iiasiiia Conrad), and California softshell clams {Crypiomya
californicu Conrad) of different sizes. The bivalve size classes tested ranged in length from 10-14 mm to 33-37 mm. Consumption
rate and prey preference experiments were conducted by allowing one starved (48 h) green crab (55-75 mm carapace width) to feed
ad libitum on bivalve prey for 16 h. All tests were conducted in 38-L aquaria containing sand substrate 13 cm deep. A total of either
60 or 30 individuals of each prey species were offered without replacement in each test. Mean green crab consumption rates varied
depending upon the prey species and size class. For bivalve prey of similar size, Olympia oysters were consumed at a higher rate than
bent-nosed macoma clams and Japanese littleneck clams, while Olympia oysters and California softshell clams were consumed at about
the same rate. Green crabs preferred Olympia oysters to both bent-nosed macoma clams and Japanese littleneck clams by ratios ranging
from 2:1 to 28:1. depending upon the prey size. Small California softshell clams were preferred to small bent-nosed macoma clams
by a ratio of 8:1. The mean total biomass of Olympia oysters and bent-nosed macoma clams eaten was 2.31 g • d~'. Our results show
that green crabs are capable of consuming large quantities of all four bivalve species tested, and that on bare sand substrate Olympia
oysters are at greater risk of green crab predation than bent-nosed macoma clams and Japanese littleneck clams, and California
softshell clams are at greater risk than bent-nosed macoma clams.
KEY WORDS: Carciiuis inaeiia.'i: consumption rates: Ciyptonna californiea: Macoma na.siiki: Ostrea coiichaphila; prey preference;
Venerupis plulippinanim.
INTRODUCTION
The green crab, Curciniis maenas. a species native to Europe,
has recently invaded Pacific Northwest (PNWl estuaries (Dum-
bauld & Kauffman 1998, Hunt et al. 1998, Yumada 2001 ). Green
crabs prey heavily upon bivalves (Ropes 1968. Davies et al. 1980,
Parache 1980, Dare & Edwards l98l,Elner 1981, Dare et al. 1983,
Grosholz & Ruiz 1995. Mascaro & Seed 2000). Ropes (1968) and
EIner (1981) attributed the decline in the softshell clam {Mya
areuaria Linnaeus) fishery along the northeastern coast of the
United States to invasive green crabs. Studies by Grosholz and
Ruiz (1995. 1996) suggest and Jamieson et al. (1998) have pre-
dicted that invasive green crabs could impact bivalve populations
in PNW estuaries.
The objectives of this study were to estimate green crab con-
sumption rates on four bivalve prey species inhabiting PNW es-
tuaries and to determine green crab prey preferences among these
prey species under controlled laboratory conditions. Consumption
rate experiments were conducted on one to three size classes of the
four bivalve species to determine the effect of prey species and
prey size on consumption rates. Prey preference experiments were
conducted with two or three bivalve species of similar size. The
bivalve species tested were the Olympia oyster {Ostrea con-
chaphila). the Japanese littleneck clam [Venerupis plulippinanim).
the bent-nosed macoma clam (Macoma nasiita). and the California
softshell clam iCrypiomya californiea). Olympia oysters are native
to and were once widely distributed throughout PNW estuaries but
now. probably primarily because of overharvesting (Baker 1995,
Robinson 1997. Cook et al. 2000). only remnant natural and
*Current address: Elkhorn Slough Foundation. P.O. Box 267, Moss
ing. CA 95039.
tCorresponding author.
Land-
culture populations remain. Bent-nosed macoma clams and Cali-
fornia softshell clams are common native PNW bivalves. The
Japanese littleneck clam is a nonindigenous species that has been
naturalized and is cultured in PNW estuaries for its commercial
value.
MATERIALS AND METHODS
Green crabs used in our experiments were collected from
Yaquina Bay. OR (44 °37 'N, 124 °02 'W) with crab traps de-
ployed subtidally and baited with salmon scraps. Prior to their use
in an experiment, the crabs were fed a standardized diet of squid
while being held submerged in individual containers in now-
through water tables in the U.S. Environmental Protection Agency
Laboratory at the Hatfield Marine Science Center. Newport. OR.
The flow-through water system supplies fresh filtered or unfiltered
seawater from Yaquina Bay. Only intermolt crabs were used as
experimental subjects to avoid possible behavioral differences as-
sociated with molting. The size range of green crabs in our ex-
periments (55- to 75-mm carapace width. CW) reflected the size
range of crabs collected in the field.
Olympia oysters and Japanese littleneck clams used in our ex-
periments were obtained from the Olympia Oyster Company. Shel-
ton. WA. Bent-nosed macoma clams and California softshell
clams used in our experiments were collected from Yaquina Bay.
Experimental bivalves were measured and divided into size classes
(Table I ). Shell length was measured as the distance from the
hinge (umbo) to the furthest edge of the shell. Bivalves were held
in the laboratory in water tables supplied with unfiltered. flow-
through seawater prior to their use in our experiments. The bi-
valves appeared healthy and did not lose weight or die while being
held.
865
866
Palacios and Ferraro
TABLE 1.
Bivalve species and prey size classes used in the consumption rate" and prey preference'' experiments
Size (Class)
Oljmpia Oyster
19-23 mm'
California Softshell Clam
Bent-Nosed Macoma Clam
Japanese Littleneck Clam
Small (1)
Medium (II)
Large (111)
1(;H4 mm"'
26-30 mm""
33-37 mm
12-15 mm-""
18-21 mm""
,ih
14-18 mm"
22-26 mm '"
Consumption Rate and Prey Preference Experimental Protocol and
Data Analysis
Both consumption rate and prey preference laboratory experi-
ments were performed in 38-L (50 cm x 25 cm x 30 cm) glass
aquaria placed in flow-through water tables. Sand, that had been
air-dried for at least five days and sieved through a 1 .0-mm mesh
screen, was placed in each aquarium providing 13 cm of substrate
depth. Each aquarium was continuously supplied with fresh, fil-
tered Hatfield Marine Science Center seawater with out flow near
the top through a mesh cover. At the short (25 cm) end of each
aquarium a clear plastic partition was installed about 10 cm into
the aquaria to separate the green grab predator from the bivalve
prey during a 24-h pre-e.xperimental acclimation period. This setup
created staging and feeding areas in each aquarium. Semiopaque
visual barriers were placed on the vertical sides of each aquarium
to minimize external influences on predator and prey behavior.
Seawater temperature (range, I2-16°C) and salinity (range, 32-33
ppt) were monitored during every experiment. The light regimen
was fixed using a timer and matched the natural daylight regimen
(I4L:I0D). The consumption rate and prey preference tests were
each of 16-h duration, beginning with 10 h of darkness followed by
6 h of light. Prior to each experiment, each experimental crab was
starved for a total of 48 h: 24 h in its holding container plus 24 h
in the staging area. The bivalve prey were measured and placed in
the feeding area of the aquaria and allowed to acclimate to the test
conditions at least 18 h before the beginning of each test.
The same basic protocol was used in both the consumption rate
and prey preference experiments. One experimental crab was used
in each test, and each crab was used only once. After 24 h in the
staging area, partitions between the staging and feeding area were
removed and crabs were allowed to feed acl libitum on one bivalve
prey species (consumption experiments) or two or three bivalve
prey species (preference experiments) without replacement for 16
h. At the end of each test all bivalves were removed from each
aquarium and whole, live bivalves were counted and remeasured.
The number of individuals of each species eaten was determined as
the number originally available minus the number of whole, live
individuals remaining at the end of each test. In the consumption
rate tests, the feeding area originally contained 60 bivalves (prey)
of the same species and the same size class. In the prey preference
tests, the feeding area originally contained either 60 or 30 bi\ alves
of similar size of each of two or three species. The total number of
tests performed was constrained by bivalve prey availability. Due
to laboratory space limitations or prey availability, a maximum of
twelve tests could be run at the same time. Tests were randomly
assigned among aquaria, and each experiment was completed
within one month. Seven replicated (;i = -1-8) consumption rate
experiments and five replicated (;; = 3-8) pairwise and one rep-
licated {n = 4) three-way prey preference experiments were con-
ducted (Table 1).
Differences in mean consumption rates (number bivalves eaten
in 16 h) between two prey species or size classes were tested by
/-tests after confirming the parametric assumptions of normality
and homogeneity of variances (Sokal & Rohlf 1995). Differences
in mean consumption rates among three prey species or three size
classes were tested by analysis of variance and Tukey's test. or.
when the data failed to meet the parametric assumptions, by an
approximate test of the equality of means using the Games and
Howell method (Sokal & Rohlf 1995). Prey preference was in-
ferred using single classification G-tests with Williams" correction
(Sokal & Rohlf 1995) by determining if the observed proportion of
prey species eaten differed from the expected ratio (1:1 and 1:1:1
for two and three prey species, respectively) if there was no pref-
erence.
Bivalve Biomass Estimates
Meat weight-length relationship models for Olympia oysters
and bent-nosed macoma clams were developed by regressing the
logarithms of the biomass (g. flesh dry wt) of 50 individual Olym-
pia oysters (18-38 mm shell length) and 30 individual bent-nosed
macoma clams (12-22 mm shell length) on shell length (mm). We
did not have a sufficient number of indi\ iduals of different shell
lengths to generate biomass-length relationships for Japanese
littleneck clams and California softshell clams. The flesh of each
bivalve was removed from the shell, placed in a pre-weighed dry-
ing tin. and dried in an oven for 48 h at 70°C. Upon removal from
the oven, the tins were kept in a dessicator. allowed to cool, and
re-weighed. Flesh dry weight was determined by subtracting the
weight of the drying tin from the total weight (dried flesh -i- drying
tin).
Biomass-length regression models were used to convert the
known length of individual Olympia oysters and bent-nosed ma-
coma clams eaten in our consumption rate and prey preference
experiments to biomass. The individual biomass estimates were
summed to estimate the total bivalve biomass of each species
consumed in each test. ANOVA was used to test for differences
among the mean total bivalve biomass eaten in our Olympia oyster
and bent-nosed macoma clam consumption rate and prey prefer-
ence experiments.
RESULTS
Consumption Rate Experiments
The number of bivalve prey eaten in our consumption rate tests
ranged from zero large Japanese littleneck clams to fifty-four small
California softshell clams. Mean (SE) green crab consumption
rates and results of analysis of variance comparing mean consump-
tion rates across prey species within a size class and across dif-
ferent size classes within prey species are presented in Table 2.
The rank order of green crab mean consumption rates for bivalve
Green Crab Feeding on Four Bivalves
867
TABLE 2.
Prey species," prey size class,'' sample size (;;). and mean (SK) number of bivalves eaten by one 48-h starved green crab in 16 h and ANOVA
results in tests of five hypotheses of no sifinillcanl ditTirences betHeen/among mean consumption rates for different prey species of similar
size [H|, ( la-lc)l and for different size classes of the same prey species [H,, (2a and 2b)|
Prey Species
Size Class
OO
I
00
II
00
ni
BN
I
BN
n
cs
I
JL
in
No. Consumed
Mean (SE)
Consumption
Rate (per day)
H„
(la)
H„
H„
H„
H„
(lb)
do
(2a)
(2bl
A
A
A
A
B
8
41.5(5,24)
62.3
A
17.4 (L74)
26.1
10.3 (0.82)
15.5
17.7(2.61)
26.6
B
7.4(1.56)
11. 1
43.4 (2.99)
65.1
A
1.8(0.49)
2.7
B
A
B
Different letters (A. B) in the columns nidicate statistically significant different iP < 0.05) means.
°00 = Olympia oyster; BN = bent-nosed macoma clam: CS = California softshell clam; JL = Japanese littleneck clam.
"See Table 1.
species by size class (I, II, III; see Table 1 ) was Olympia oyster (I)
= California softshell clam (I) > bent-nosed macoma clam (I),
Olympia oyster (II) > bent-nosed macoma clam (II). and Olympia
oyster (III) > Japanese littleneck clam (III). The rank order of
green crab mean consumption rates for different size classes of the
same bivalve species were Olympia oyster (1) = Olympia oy.ster
(11) > Olympia oyster (III), and bent-nosed macoma clam (I) >
bent-nosed macoma clam (II).
Prey Preference Experiments
When two bivalve prey species were present, green crabs ate,
on average. 16x more small Olympia oysters than small bent-
nosed macoma clams. 2x more small Olympia oysters than small
Japanese littleneck clams. 8x more small California softshell
clams than small bent-nosed macoma clams. 3x more medium
Olympia oysters than medium bent-nosed macoma clams, and 28x
more large Olympia oysters than large Japanese littleneck clams
(Table 3). When three bivalve prey species were present, green
crabs ate, on average, small California softshell clams, small
Olympia oysters, and small bent-nosed macoma clams in a 6:4:1
ratio (Table 3). The proportions of the prey species eaten were all
significantly different from 1:1 or 1:1:1 (Table 3). indicating strong
green crab prey preferences among the bivalve species tested.
Bivalve Biomass Estimates
Regressions of the logarithm of Olympia oyster and bent-nosed
macoma clam flesh dry weight on their shell lengths (mm) were:
log (Olympia oyster dry wt. g) = -2.40 + 0.048 Olympia oyster
shell length, r = 0.79. P < 0.001. and log (bent-nosed macoma
clam dry wt, g) = -2.26 -I- 0.077 bent-nosed macoma clam shell
length, ;- = 0.95. P < 0.001.
Using the regression equations above, we converted the shell
lengths of the Olympia oysters and bent-nosed macoma clams
eaten in our consumption rate and prey preference tests to indi-
vidual oyster or clam biomass. Individual biomass estimates of
consumed prey were then summed to estimate the total biomass of
Olympia oysters and bent-nosed macoma clams eaten in each test.
There were no significant differences (ANOVA. P > 0.05) among
the mean total biomass of bivalves eaten in our Olympia oyster and
bent-nosed macoma clam consumption rate and prey preference
experiments (Table 4). The grand mean total biomass of Olympia
oysters and bent-nosed macoma clams eaten in these experiments
was 1.54 (±0.10) g • 16 h"'. which extrapolates to 2.31 g ■ d"'.
DISCUSSION
Consumption Rates
This is the first published report of green crab consumption
rates on Olympia oysters, bent-nosed macoma clams, and Califor-
nia softshell clams. Parache ( 1980) previously reported green crab
consumption rates on Japanese littleneck clams. For a given bi-
valve prey size, green crab (55-75 mm CW) consumption rates
were highest for California softshell clams and Olympia oysters,
intennediate for bent-nosed macoma clams, and lowest for Japa-
nese littleneck clams (Table 2). smaller individuals of each prey
species were consumed at a faster rate than larger individuals
(Table 2), and the mean total biomass of Olympia oysters and
bent-nosed macoma clams consumed was 2.31 g • d~' (Table 4).
Crab consumption rates on bivalves can vary depending on the
crab (species, size, hunger level, and health), the bivalve (species,
TABLE 3.
Prey preference ratios of green crabs for t«o or three bivalve prey
species" of comparable size and results of G-tests comparing the
observed versus the expected ratios if there was no prey preference
Observed
Size
Preference
Expected Ratio If
G
Class"
Ratio
n
No Preference
Statistic
P
16 00:1 BN
3
1:1
94.5
<0.001
2 OO: 1 JL
5"
1:1
29.7
<0,001
8CS:1 BN
6
1:1
118
<0.001
II
3 OO: 1 BN
4
1:1
34.9
<().001
111
28 00:1 JL
8
1:1
82.4
<0,001
6CS:4 00:1 BN
4
1:1:1
93.2
<().()01
All tests were replicated (/i) and 16-h duration.
" OO = Olympia oyster; BN = bent-nosed macoma clam; CS = Cali-
fornia softshell clam; JL = Japanese littleneck clam.
" See Table 1 .
■^^ Thirty individuals of each bivalve prey species were originally available
in each replicate test. In all other experiments. 60 individuals of each
bivalve prey species were originally available in each replicate test.
868
Palacios and Ferraro
TABLE 4.
Mean total bivalve biomass (g) consumed b> one 48-h starved green
crab in 16 h in consumption rate and prev preference experiments
with Olympia oysters (OO) and bent-nose macoma clams (BNl
Biomass
Prey
Size
Consumed (gl
Species
Class''
Exp
eriment""
/(
Mean (SE)
OO
I
C
4
1.70(0.183)
OO
n
C
8
1.54(0.350)
OO
m
C
8
2.01 (0.235)
BN
I
C
8
1.10(0.116)
BN
II
C
8
1.35(0.759)
00 + BN
1
P
3
1.55(0.124)
00 + BN
II
P
4
1.63(1.067)
"See Table 1.
•"C = Consumption; P =
Prefere
nee.
size, density, siiell strength, and morphology), and the experimen-
tal conditions (water temperature, duration, with or without prey
refuge, with or without prey replacement, etc) under which the
rates are measured (Jubb et al. 1983, Arnold 1984, Sanchez-
Salazar et al. 1987. Juanes 1992. Ebersole & Kennedy 1995, Mas-
caro & Seed 2000. Yamada 2001). Our experiments were con-
ducted in aquaria with sand substrate to approximate prime oyster
and clam culture habitat in the field. Further research is needed to
estimate green crab consumption rates and prey preferences on
bivalves in other PNW estuarine habitats (e.g.. salt marsh, eelgrass,
burrowing shrimp). We attempted to minimize potential confound-
ing variables in our experiments, and to obtain near maximum
estimates of average green crab consumption rates under environ-
mental conditions as similar as possible to those in the field. We
only experimentally varied the bivalve prey species and size
(Table 1 ). The predator crab species, size (55-75 mm CW), num-
ber (one), and initial hunger level (48 h starved) were constant, and
environmental conditions (water temperature, salinity, photope-
riod, etc.) were held constant at levels matching local field condi-
tions. The bivalve prey were placed on sand substrate ( 1 3 cm deep)
and given time ( 18 h) to acclimate and orient themselves naturally
on and in the sediment. Thus relative differences in predator for-
aging times for the different prey species are subsumed in our
consumption rates. Sixty bivalve prey were available at the begin-
ning of each consumption rate test, and. on average, twenty whole,
live bivalve prey remained at the end. Mean bivalve prey densities,
therefore, decreased but remained high (446-131 m"") throughout
the tests, thus minimizing the effect of decreasing prey density on
crab consumption rates. Bivalves eaten during the experiments
were not replaced as newly introduced bivalves would tend to be
more vulnerable to predation than the original bivalves that had
time to acclimate and bury. Since starved green crabs consume
prey more rapidly in the first three feeding hours (Jubb et al. 1983).
our tests were run for 16 h to better refiect longer term, average
rates. Our experimental light regimen ( 10D:6L) approximates the
green crab's natural foraging cycle (Klein Breteler 1976, Elner
1981). In Table 5 we summarize the experimental conditions and
results of this and other green crab consumption rate studies.
In Parache"s (1980) laboratory experiments, green crabs (50-
69 mm CW) consumed 0.2-0.7 Japanese littleneck clams (23.5
mm) • d"', whereas in our experiments green crabs (55-75 mm
CW) consumed Japanese littleneck clams (22-26 mm) at an aver-
age rate of 2.1 clams ■ d"'. We used one crab in each test as
compared with Parache's three, and our prey densities were higher
(Table 5). Aggressive competition is high among green crabs,
especially in the presence of food (Kaiser et al. 1990; Sneddon et
al. 1997). and green crab consumption rates decrease with decreas-
ing prey density (Walne & Dean 1972). Our estimates of green
crab consumption rates on Japanese littleneck clams, therefore,
better reflect rates when green crab densities are low and clam
densities are high, whereas Parache's ( 1980) estimates may reflect
rates when green crab densities are higher and clam densities are
somewhat lower.
Green crabs consumed 19-37 mm Olympia oysters in our ex-
periments al an average rate of 15-62 d"' (Tables 2 and 5). This
consumption rate is much higher than the £ 2.75 d"' reported by
Dare et al. ( 1983) for 19-37 mm Pacific oysters (Cnissostrea gigas
Thunberg) and the 1.1 d"' reported by Mascaro and Seed (2000)
for 5—40 mm edible oysters iOstrea edulis Linnaeus) (Table 5).
Differences in the experimental conditions (Table 5) preclude di-
rect comparisons of these results. Nevertheless, such large differ-
ences in consumption rates suggests that green crabs can eat Olym-
pia oysters at a faster rate than other oysters, perhaps due to dif-
ferences in shell strength or morphology (Mascaro & Seed 2000).
Green crabs consumed bent-nosed macoma clams and Japanese
littleneck clams at a slower rate than similar sized Olympia oysters
and California softshell clams (Table 2). Bent-nosed macoma
clams and Japanese littleneck clams in our experiments buried into
the sediment, some along the sides of the aquaria where they were
observed at the maximum sediment depth of 13 cm. California
softshell clams buried just below the surface, and Olympia oysters
remained on the surface. Slower green crab consumption of deeper
burying bivalve species supports the premise that burying provides
greater refuge from predation. Blue crab (Callinecles sapidiis
Rathbun) consumption rates were also less on deeper-burying bi-
valves (Blundon & Kennedy 1982, Ebersole & Kennedy 1995).
Green crab consumption rates on bent-nosed macoma clams
were less than those on similar size Olympia oysters on a numeri-
cal basis (Table 2), but not significantly different on a total bio-
mass basis (Table 4). These results suggest that crab consumption
rales, measured as number of prey eaten per hour, may have been
largely a function of the crab's hunger level. Initial hunger levels
of our experimental crabs were the same (48-h starved). But as
crabs ate prey, their hunger levels must have decreased, and, logi-
cally, the rate of decrease would be more closely related to bio-
mass of prey than number of prey consumed. Our length-biomass
regressions (see Results. Bivalve Biomass Estimates) show that
bent-nosed macoma clams have a greater flesh biomass than
Olvmpia oysters of the same length. The hunger level of a starved
green crab feeding on bent-nosed macoma clams, therefore, would
decrease at a faster rate than if the same crab fed on the same
number of similar size Olympia oysters. The total biomass of
Olympia oysters and bent-nosed macoma clams eaten in our ex-
periments (Table 4) exceeded the approx. 0.8 g of dry blue mussel
(Mytiliis edulis Linnaeus) flesh required to satiate green crabs (70-
75 cm CW) (Jubb et al. 1983). It, therefore, appears that the crabs
in our consumption rate experiments ate to satiation, but that more
individual Olympia oysters than bent-nosed macoma clams of the
same size had to be eaten to reach satiation and to maintain ap-
proximately the same hunger level thereafter.
Green crab consumption rates on larger Olympia oysters were
less than those on smaller Olympia oysters, and green crab con-
sumption rates on larger bent-nosed macoma clams were less than
Green Crab Feeding on Four Bivalves
869
TABLE 5.
(ireen crab consumption rate studies on bivalve prey \\ith rales standardized tu mean number consumed in 24 h
Crab Size
Prey
Prey Size
Consumption
Tank Size
Number Prey
Prey
Time
Sediment Hepth
Citation
(mm)
Species"
(mm)
Rale (24 h ')
(cm)
Offered
Replaced
Id)
( cm )
Palacios & Ferraro
55-75
Oc
19-23
62.3
50 X 25
60
No
0.7
13
(This study)
55-75
Oc
26-30
26.1
50x25
60
N(i
0.7
13
55-75
Oc
33-37
15.5
50x25
60
No
0.7
13
55-75
Mn
12-15
26.6
50x25
60
No
0.7
13
55-75
Mn
18-21
11.1
50x25
60
No
0.7
13
55-75
Cc
10-14
65.1
50x25
60
No
0.7
13
55-75
Vp
22-26
2.7
50 X 25
60
No
0.7
13
Walne & Dean (1972)
60-69
Mm
14-20
3.22
27 X 18
15
Yes
7
0
60-69
Me
24-32
4.83
27 X 18
15
Yes
7
0
Elner & Hughes (1978)
60-65
Me
5-35
13
43x23
90
Yes
11
0
Parache (1980)''
50-59
Vp
8
1.71
50 X 50
50
No
8
8-10
50-59
Vp
14
2.78
50x50
50
No
8
8-10
50-59
Vp
23.5
0.17
50x50
30
No
8
8-10
60-69
Vp
8
2.88
50x50
50
No
8
8-10
60-69
Vp
14
5.88
50x50
50
No
8
8-10
60-69
Vp
23.5
0.71
50x50
30
No
8
8-10
Dareetal. (1983)
65
Cg
19-23
2.75
28x18
2-10
Yes
4-10
0
65
Cg
26-30
1.75
28x18
2-10
Yes
4-10
0
65
Cg
34-37
1.00
28x18
2-10
Yes
4-10
0
Jensen & Jensen (1985)
6
Ce
2-6
7.00
7x7
30
No
1
3
Sanchez-Salazar
65-70
Ce
13
3 @ 9°C
50x30
40
Yes
5-10
5
et al. (1987)
9 @ 15°C
Mascaro and Seed
55-70
Me
5-+0
12.0
30 X 20
35
Yes
-10
0
(2000)
55-70
Oe
5-40
1.1
30x20
35
Yes
-10
0
55-70
Cg
5-40
2.1
30 x 20
35
Yes
-10
0
55-70
Ce
5-40
10.1
30 X 20
35
Yes
-10
0
^ Oc = Ostrea conchaphila: Mn = Macoma nasuta: Cc =Ciyptomya califonica; Vp =Venerupis (previously, Ruditapes) phUippinarum): Mm =
Mercenaria mercenaria (Linnaeus); Me = Mytilus ediilis: Cg = Crassosirea gigas: Ce = Cerastodenna ediile: Oe = Ostra eduHs.
^ Parache's experiments had three crab predators per tank. All other reported experiments had one crab per tank.
those on smaller bent-nosed macoma clams (Table 2). There was
no significant difference, however, in the mean total biomass of
larger and smaller prey consumed (Table 4). Dare et al. (1983).
Jubb et al. (1983), and Sanchez-Salazar et al. (1987) also found
prey species specific, inverse relationships between green crab
consumption rates and bivalve prey size within the range of con-
sumable prey sizes. Such relationships probably hold generally
because more time, on average, is needed to handle and eat larger
bivalve prey (Jubb et al. 1983; Sanchez-Salazar et al. 1987). while
crabs need to eat fewer individuals to reach satiation when con-
suming larger prey.
Green Crab Prey Preferences
Green crabs exhibited prey species preferences based on the
proportional number of similar size individuals eaten in our ex-
periments. In tests with two prey species, Olympia oysters were
preferred to bent-nosed macoma clams and Japanese littleneck
clams of similar size, and small California softshell clams were
preferred to small bent-nosed macoma clams (Table 3). In a three-
way test, small California softshell clams, Olympia oysters, and
bent-nosed macoma clams were preferred in a ratio of 6:4; 1 (Table
3). These results indicate that, on bare sand substrate, Olympia
oysters are more susceptible to green crab predation than bent-
nosed macoma clams and Japanese littleneck clams, and California
softshell clams are more susceptible to green crab predation than
bent-nosed macoma clams.
Factors that influence crab prey preferences include the prey
encounter rate, the time and energy the crabs expend to handle and
eat the prey, and the nutrient and energetic value of the prey (Elner
& Hughes 1978; Ebersole & Kennedy 1994). Our study was not
designed to determine the relative importance of these factors.
However, because green crabs are tactile and chemosensory hunt-
ers (Cohen et al. 1995), and their prey preference ratios (Table 3)
were almost always consistent with the bivalve prey burial depths
observed in our experiments (Olympia oysters < California soft-
shell clams < bent-nosed macoma clams = Japanese littleneck
clams), prey encounter rates were probably an important factor.
A summary of the results and experimental conditions under
which our study and other studies on green crab prey preferences
on bivalves is presented in Table 6. Jensen and Jensen (1985)
found that juvenile green crabs preferred small cockles (Cerasto-
denna ediile Linnaeus) to small Baltic macoma clams (Macoma
balthica Linnaeus), and they concluded that juvenile green crabs
could be responsible for the decline of small cockles and changes
in benthic macrofaunal diversity in the Wadden Sea. Cohen et al.
(1995) found that green crabs preferred brackish-water corbula
clams (Potainocorbula amurensis Schrenck) to Japanese littleneck
clams and mussels (Mytilus spp.) of similar size. Cohen et al.
(1995) speculated that green crab predation might lead to a de-
crease in brackish-water corbula clams and an increase in benthic
diversity in San Francisco Bay. Grosholz and Ruiz ( 1995) showed
that green crabs preferred larger individuals of two Niitricola (pre-
viously, Transennella) clam species, and they predicted that green
870
Palacios and Ferraro
TABLE 6.
Green crab prey preference studies with bivalve prey and results presented as ratios
Citation
Crab Size
(mm)
Prey
Species"
Prey Size
(mm I
Preference
Ratio
Tank Size
(cm or L)
Number Prey
Offered"
Time
(days I
Sediment Depth
( cm )
Palacios & Ferraro
55-75
Oc:Mn
12-23
16:1
50 X 25
(This study)
55-75
Oc:Mn
18-30
3:1
50 X 25
55-75
Oc:Vp
14-23
2:1
50 X 25
55-75
Oc:Vp
22-37
28:1
50 X 25
55-75
Cc:Mn
10-15
8:1
50 X 25
55-75
Cc:Oc:Mn
10-23
6:4:1
50x25
Jensen & Jensen (1985)
11
Ce:Mb
2-6
7:1
7x7
Cohen et al. (1995)
55-60
Pa:Ms
10-20
1:1
25 X 25
55-60
Pa:Vp
10-20
3:1
25 X 25
55-60
Ms:Vp
1(3-20
16:1
25x25
55-60
Pa:Vp
10-20
8:1
25 X 25
55-60
Pa:Ms
10-20
5:1
25x25
Grosholz & Ruiz (1995)
44-61
Nc«l&>3):
<1 & >3
52(Nc&Nt>3):
40L
Nt«l cS:>3)
1 (Nc&Nt
<1)
120
120
60
120
120
180
30
30
30
30
30
30
40
0.7
0.7
0.7
0.7
0.7
0.7
1
0.08
0.08
0,08
0.08
0.08
2
13
13
13
13
13
13
3
0
0
0
6
6
0
"00 = Osrrea concluiphilir. Mn = Macomu nasuur. Vp = Venerupis (previously, Riiditapes) philippinarum: Cc = Cnproiiixa culifitnica: Ce =
Cerasroderma editle: Mb = Macoiiw halrhica: Pa = Potamocorbula amuiensis: M^ = A/\7(/h,v spp.; Nc = Niitrlcola {pre\iouf,\y TranseuelUi) ccinfiisa
(S. Gray). Nt = Niitricola (previously. Tmnsenella) kinrilla (Gould).
" Numbers are totals for all hivaUe prey species, and all studies were conducted without prey replacement.
crabs will impact Niiuicohi clain populations and benthic commu-
nities in West Coast embayments.
The impact of green crabs on bivalve populations in PNW
estuaries will depend upon niany factors, including green crab
abundance, distribution, predators, competitors, and recruitment
success. Currently, poor recruitment appears to be the main factor
limiting green crab abundances in PNW estuaries (Yamada 2001 ),
Intra- and interspecific predation and competition for food and/or
shelter could also limit their abundance and spatial distribution
(McDonald et al, 1998. Moksnes et al, 1998, Yamada 2001. Jensen
et al. 2002). If green crab populations increase, however, their
potential direct impact is high, as they are capable of consuming
large quantities of ecologically and economicall\ important PNW
bivalve species (Tables 2 and 4), In bare sand habitat, at or near
surface dwelling bivalve species (e,g,, Olympia oysters. California
softshell clams) are probably at greater risk of green crab predation
than deeper dwelling species (e.g.. bent-nosed macoma clams.
Japanese littleneck clams; Table 3), Heavy green crab predation on
bivalves could also have substantial indirect effects on benthic
niacrofaunal community structure and composition (Griisholz et al.
2000).
ACKNOWLEDGMENTS
This research was conducted under a U.S. EPA National Net-
work for Environmental Management Studies Fellowship (No,
U-9 1 529 1 -01-0) to KCP, The authors thank C, Hunt, D. Berube.
and Z. Bassett for field and lab assistance, the Olympia Oyster
Company for donating Japanese littleneck clams, and S, Yamada
for reviewing an earlier draft of the manuscript. The U.S. EPA,
Office of Research and Development funded this research, which
has been subjected to agency review and approved for publication.
Mention of trade names or commercial products does not consti-
tute endorsement or recommendation for use.
LITERATURE CITED
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HISTOPATHOLOGY AND PREVALENCE OF THE PARASITIC DINOFLAGELLATE,
HEMATODINWM SP, IN CRABS {CALLINECTES SAPIDUS, CALLINECTES SIMILIS,
NEOPANOPE SAYl, LIBINIA EMARGINATA, MENIPPE MERCENARIA) FROM A
GEORGIA ESTUARY
MICHAEL SHEPPARD," ANNA WALKER.- MARC E. FRISCHER,' AND RICHARD F. LEE'*
'Skuhnvay Institute of Occauoiiwphy. 10 Ocean Science Circle. Savannah. Georgia 31411: -Department
of Pathology. Mercer University School of Medicine. Macon. GA 31207: ^Murine Science Program.
Savannah State University. Savannah. Georgia 31404
ABSTRACT This study reports on seasonal vanations in the prevalence and intensity of HemiUodin'mm sp. infection in the blue crab
{Callinectes sapidus). spider crab {Lilvnia eimirgimilu). a xanthid crab (Neopanope sayi). stone crab {Mcnippe mercenaria). and lesser
blue crab (Callinectes i»n(7/.!) collected from Wassaw Sound on the Georgia (USA) coast. During the fall of each year there has been
a peak in the prevalence of Hematodinium in L emarginata and N. sayi. while in C. sapidus there have been infection peaks in both
fall and spring. There was a much lower frequency of infection in M. mercenaria and C. sinnlis. Based on comparisons of I8S rRNA
gene sequences of Hemaiodiniiim sp.. it appears that the Hematodinium sp. found in spider and stone crabs are the same or very closely
related to the Hematodinium isolated earlier from the blue crab. Morphologically, most parasites were in the mononuclear trophont
form, although occasional binucleated and multinucleated forms were observed. The highest numbers of Hematodinium sp. were found
in the gills where parasites were present extracellularly within vascular spaces. The parasite infiltrated cardiac and skeletal muscle in
an interstitial pattern, but did not invade individual myofibers. Our findings suggest that Hemalndinium sp. is impacting the blue crab
population in Wassaw Sound and is responsible for the disappearance of C. sapidus in the summer months, allowing other opportunistic
crab species to invade the niche vacated by C. sapidus.
KEY WORDS: prevalence, disease. Hcnuiunlummh crab, intensity, estuary. Georgia
INTRODUCTION
Henuiliitliniiiiii pcrezi is a parasitic dinotlagellate that was first
reported in 1931 in two crab species, the green shore crab, Carci-
nus maenas, and the harbor crab. Liocarcinus depurator. along the
French coast (Chatton & Poisson. 1931 ). Infection with this para-
site has since been shown to produce a spectrtim of disease ranging
from asymptomatic carriage to death. The parasite proliferates in
crustacean hemolymph, consuming hemocyanin, along with other
hemolymph proteins and possibly hemocytes (Love et al. 1993,
Field & Applcton 1995. Field et al. 1992). Hemolymph taken from
heavily infected animals subsequently does not clot. The parasite
also infiltrates other tissues, including cardiac and skeletal muscle
(Hudson & Shields 1994, Shields & Squyars 2000), Morbidity
appears to depend on the burden of organisms. Heavily infected
crabs become lethargic, possibly due to hypoxemia and compro-
mise of cardiac and skeletal muscle. If not preyed upon, they often
succumb to the overwhelming infection.
Since the work of Chatton and Poisson (1931) on diseased
crabs in France, there have been reports of crustaceans infected
with Hematodinium sp. in Australia (Australian blue crab, Piirlii-
niis pelagiciis: sand crab, Portimus pelagicits: mud crab, Scylla
serrata; coral crab. Trapezia aerolata [Hudson & Lester 1994,
Hudson & Shields 1994, Shields 1992, Hudson et al. 1993]).
Alaska (Tanner crab. Chionoecetes IniinI (Meyers et al. 1987.
1994]), Scotland (Norway lobster, Ncphraps nonegiciis (Field et
al. 1992]) eastern Canada (snow crab, Chionoecetes opilio [Taylor
& Khan 1995]) and the eastern United States (blue crab, Calli-
nectes sapidus: rock crab, Cancer irroratus: Jonah crab. Cancer
borealis: lady crab, Ovalipes ocellatus: amphipods, Leptocheinis
pinguis, Ampelisca vadorum [Johnson 1986, MacLean & Rudell
*Corresponding author: Richard F. Lee, Skidaway Institute of Oceanog-
raphy. 10 Ocean Science Circle. Savannah. GA 31411. E-mail: dickt*
skio.peachnet.edu
1978. Messick 1994, Newman & Johnson 1975 j). The life cycle of
Hematodinium sp. in blue crabs is complex and involves several
different stages, including dinospores, prespores, trophonts, and
Plasmodia (Messick 1994, Shields 1994).
While Hematodinium sp. has been found in blue crabs, C.
sapidus. collected on both the Atlantic and Gulf coasts of the
United States (Messick 1994. Messick & Shields 2000. Messick et
al. 1999, Newman & Johnson 1975, Shields & Squyars 2000),
there have been few reports of this parasite in other crab species
from the south Atlantic coast of the United States. The present
study repoils on seasonal variations in the prevalence and intensity
of Hematodinium sp. infection among the blue crab {Callinectes
sapidus], spider crab (Libinia emarginata), xanthid crab (Neopan-
ope sayi), stone crab (Menippe mercenaria). and lesser blue crab
{Callinectes similis) collected from a coastal Georgia estuary
(Wassaw Sound. Fig. I). Histologic examination of tissues from
diseased blue, spider and stone crabs was peiformed to study the
pattern of the infection and immune response of the different hosts.
The parasites from each of the three crab species were morpho-
logically very similar. The genetic similarity of the parasites in the
three crab species was confirmed by sequencing the I8S rRNA
gene.
MATERIALS AND METHODS
Collection. Preparation. Fixing, and Staining of Hemolymph
Crabs were collected in the spring and fall from the Wassaw
Sound estuary by trawling or with traps baited with menhaden.
Crabs were bled at the hemal sinus with a 1-ml syringe.
Hemolymph samples were applied to poly-L-Iysine-coated micro-
scope slides as described by Messick (1995). fixed in Bouin's
fluid, and stained with Mayer's hematoxylin and eosin (Luna
1968). Fixed and stained slides were examined at xlOOO with a
Nikon Eclipse 6400 microscope equipped with a Nikon xlOO
I.3NA oil objective. Hematodinium sp. was identified based on
873
874
Sheppard et al.
31 »
31 s^
31 x^<
31-5
-81.3
-HI 2
-Hll -«ll) -80.9
Figure 1. Study site, Wassaw Sound in coastal Georgia.
moijihologic similarities to blue crab HemaUHlinium sp. on slides
authenticated by G. Messick (NOAA. Oxford. MD). Prevalence,
expressed as a percentage, using the definition for this term given
by Margolis et al. (1982), was the number of crabs infected with
Hfiiuitodiniiim sp. divided by the number of crabs examined times
one hundred. Infection intensity was the percentage of Hemato-
diwn sp. cells counted ainong a total of 300 cells from the
hemolymph from an individual crab. Average intensity for a sam-
pling period was the sum of the intensities of infected crabs di-
vided by the number of infected crabs.
Fixing and Staining of Tissues
Representative portions of tissues were dissected for histologic
examination from 10 infected blue crabs, 3 spider crabs, and 1
stone crab. Tissues were fixed in zinc formalin, processed for
routine light microscopy and embedded in paraffin. Five-
micrometer sections were cut. mounted on glass slides, stained
with hematoxylin and eosin, coverslipped and examined by one of
us (ANW).
Stages of Hematodinium SP.
Identification of the different forms of Hcinaliuliniiim sp. was
based on our own observations and the observations of others,
including Appleton and Vickerman (1998). Hudson and Shields
(1984), and Shields and Squyars (2000).
The trophont or vegetative form oi Hcinatodiniuin sp. is 8 to 12
p.m in diameter. It has a fairly high nuclear cytoplasmic ratio with
the nucleus 7 to 9 |j.m in diameter. Nuclear chromatin varies from
appearing rather homogenously dispersed throughout the nucleus
to being condensed into structures that resemble chromosomes at
metaphase. Trophonts generally possess a single nucleus, but oc-
casional, otherwise typical forms appeared to have two nuclei.
The Plasmodium is larger than the trophont form ranging in
size from 20 to 50 p.m in its longest dimension. Plasmodia are
characteristically multinucleated. An elongated, slipper-shape
form is referred to as a vermiform Plasmodium; the nuclei in this
form are usually arranged in a single file along the long axis of the
parasite. There are also more rounded forms that resemble tro-
phonts, but have much greater cytoplasmic volumes and are mul-
tinucleated.
Dinospores are notably smaller than trophont forms, 3 to 6 |i.m
in diameter, and are uninucleate.
Molecular Identification and Detection of Hematodinium in
Crab Hemolymph
The specific diagnosis of Hematodinium sp. in crabs was rou-
tinely made using a recently developed Polymerase Chain Reac-
tion (PCR) assay (Gruebl et al. 2002). Hemolymph (O.-'i-LO riiL)
was collected as described above using a sterile chilled syringe and
transferred to sterile 1.5-ml microfuge tubes. Anticoagulant was
not required if the hemolymph was kept cool. Total DNA was
extracted and purified from hemolymph samples as previously
described by Gruebl et al. (2002) using the DNeasy^^^' Tissue Kit
(Qiagen) and the Heitiatodiniimi-specific primers Hemat-F-1487
(5'-cct ggc teg ata gag ttg) and Hemat-R-I654 (5'-ggc tgc cgt ccg
aat tat tea c) to detect Hematodinium. These primers specifically
amplify a 195 bp fragment of the 18S rRNA gene from Hemato-
dinium. PCR was performed using GenAMP 97(X) or 2400 PCR
thermal cycler systems (Perkin Elmer). Amplified gene fragments
were visualized and sized by agarose gel electrophoresis in 1.2%
gels stained with GelStari® nucleic acid stain (Cambrex). The pres-
ence of the correct sized amplicon was routinely taken as evidence
of Hematodinium infection.
To confirm the identity of the parasites detected in each crab
species, representative 195 bp PCR amplicons were sequenced. In
addition, nearly the complete 18S rRNA gene sequence ( 1682 bp)
from the parasite detected in the spider crab was sequenced and
compared with the known Hematodinium 1 8S rDNA fragment that
was amplified from DNA purified from a highly infected spider
crab (95-98% intensity) using the previously described primers
Univ-F-15 (5'-ctg cca gta gtc ata tgc) and Hemat-R-i6.S4 (5'-ggc
tgc cgt ccg aat tat tea c) (Gruebl et al, 2002). Sequencing was
facilitated by cloning the amplified 18S rRNA gene fragments into
the PCR 2.1-TOPO cloning vector using a TOPO^"^' Cloning Kit,
Hematodinium Infection in Georgia Crabs
875
Version J (Invitrogen) following the manufacturer's instructions.
The plasmid was isolated and purified from E. coli using the High
Pure Plasmid Isolation Kit iBoehringer Mannheim) following the
manufacturer's instructions. Plasmid concentrations were esti-
nialed by fluorometry after staining with PicoGreen® (Molecular
Probes) using a TD-700 tluorometer (Turner Designs). Sequencing
was accomplished by automated sequencing using the sequencing
primers described in Griiebl et al. (2002) with a Beckman CEQ
2000XL DNA Analysis System. Sequencing reactions were facili-
tated by using a CEQ DTCS dye terminator cycle sequencing
quick start kit, following the protocols recommended by the manu-
facturer (Beckman Coulter). Sequence analysis was accomplished
using the Beckman CEQ 2000XL Sequence Analysis software,
version 4.3.9.
RESULTS
I'rivak'iicc and Intensity of Hematodinium sp. in Crabs from
Wttssaw Sound
The prevalence and intensity of Heinatocliiiiiim sp. infection
were determined in five crab species collected in Wassaw Sound
during different seasons over several years (Table I. Figs. 2. 3).
Prevalence at a time period, expressed as a percentage, is defined
as the number of crabs infected with Hematodinium sp. divided by
the number of crabs examined times 100. Intensity in a crab was
the percentage of Hematadinium sp. cells in the hemolymph. Av-
erage intensity for a sampling period was the sum of the intensities
of infected crabs divided by the number of infected crabs. The
TABLE L
Prevalence and intensity of Hematodinium sp. in Callineetes similis,
Neopanope suyi, and Menippe mereenaria.
Average
Collection
Number
Prevalence
Intensity"
Species
Data
of Crabs
(%)
(%)
C. similis
May. 200U
15
0
—
Aug.. 2000
12
0
—
Oct., 2000
17
0
—
June. 2001
12
0
—
Oct., 2001
14
7
11
June, 2002
18
0
—
N. sa\i
March. 2000
5
0
—
Aug. 2000
4
0
—
Sept.. 2000
8
6.^
32
Oct.. 2000
5
40
22
March. 2001
3
0
—
Oct.. 2001
7
43
12
March. 2002
4
0
—
Oct.. 2002
6
33
26
M. mereenaria
March, 2000
4
0
—
Aug., 2000
5
0
—
Oct.. 2000
10
0
—
May, 2001
4
0
—
June, 2001
8
13
29
Oct., 2001
7
0
—
June, 2002
16
0
—
" Average intensity for sampling period was the sum of the intensities of
infected crabs divided hy the number of infected crabs. Infection intensity
was the percentage of Henniiodium sp. cells counted among a total of 300
cells from the hemolymph from an individual crab.
1999 I 2000 I 2001 I
Figure 2. Monthly crab catches and the prevalence and average in-
tensity of Hematodinium infection in blue crabs. Callineetes sapidus.
collected during l'W9-20(t2 in Wassaw Sound, .\sterisks indicate that
infected crabs were not detected in that sampling period.
average intensities of Callineetes similis. Neopanope sayi. and
Menipppe mereenaria are reported in Table 1 . for Callineetes sapi-
dus in Figure 2 and for Lihinia etnarginata in Figure 3. Among the
crab species collected, highest prevalences were found in C. sapi-
dus. L. emarginata and A', sayi.
In C. .sapidns. infection peaks occurred in late spring and fall of
each year; moreover, there was an almost complete disappearance
of crabs during the summer (Fig. 2). Crabs collected in the winter
months of 1999 to 2001 were not infected, but the disease was
found in crabs collected during the unusually warm winter of 2001
to 2002. During peak infection periods, prevalence reached 40%
with average intensity as high as 80%.
Heavily infected L. emarginata were collected each fall for 3
years, but only during one spring (spring 2002) were infected crabs
found (Fig. 3). L emarginata normally enter Wassaw Sound in the
fall and are common throughout the winter and early spring, and
then retreat into cooler, deeper waters in the late spring and sum-
Prevalance and intensity of hematodinium infection in
spider crabs collected during 2000 - 2002 in Wassaw Sound.
a ■»"■
I
•g
Pwvjbncc(%>
Figure 3. Prey alence and ay erage intensity of Hematodinium infection
in spider crabs. Lihinia emarginata. collected during 21)110-21102 in
Wassayy Sound, .\sterisks indicate that infected crabs were not de-
tected in that sampling period.
876
Sheppard et al.
men Infected N. sayi were only found in the fall even though this
species is a year round resident of Wassaw Sound (Table 1 ).
In contrast to the high prevalences and intensities of Hemato-
diniwn sp. found in C. sapidus. L. emarginaui. and N. sayi. only
one infected Menippe mercenaha and one infected CalUnectes
similis were found during the study (Table 1 ). The trophont form
was the only form observed in the hemolymph from infected L
emarginata and M. mercenaria. While the trophont was the most
common form in C. sapidus. the plasmodia form was regularly
seen in C. sapidus during peak infection periods. Dinospores were
observed in three infected C. sapidus and one infected TV. sayi.
Molecular Identification o/Heniatodiniuni
Representative 195 bp 18S rRNA gene fragments amplified
from both L. emarginata and M. iiwi-cenaria had a 1 00% sequence
similarity to comparable gene fragment of the Heinatodinium sp.
found in C. sapidus. Based on these comparisons, the parasite
identified in these species was confirmed to be Heinatodinium sp.
and is likely the same species that occurs in the blue crab. To
confirm to the species level the identity of the Heinatodinium sp.
found in the spider crab, a larger 18S rRNA gene fragment ( 1682
bp) was amplified, cloned, and sequenced. This resulting sequence
exhibited a 99.6'7f base pair similarity to the previously sequenced
Heinatodinium sp. (Genbank Accession #AF286023) isolated from
the blue crab. By convention, sequence similarities in the 18S
rRNA gene greater than 98% are indicative of the same species
(Hillis & Dixon 1991). Therefore it can be concluded from these
observations that the same species of Heinatodinium occurs in M.
mercenaria. L. emarginata. and C. sapidus.
Pathologic Findings in Infected Crabs
Libinia emarginata
The three crabs examined varied in their burden of organisms
from light to heavy. In the lightly infected crab, the gills contained
occasional trophont forms intermixed with equal numbers of
granulocytes. There were occasional mononuclear and multinucle-
Figure 4, Gill from a heavily infected spider crab, Libinia emarginata.
The vascular spaces of the gills contain many trophont forms of the
parasite and a few host hcmocytes, (Hematoxylin and eosin: original
magnification: xKMHI).
ated trophont forms on the abluminal side of the hepatopancreas.
In the heart there were rare mononuclear trophonts and multinu-
cleated forms. The skeletal muscle was largely spared. In the mod-
erately infected crab, the gill tissues demonstrated mononuclear
trophonts in the larger vascular spaces at the base of the gills.
There were scattered granular and agranular hemocytes present,
but these were considerably outnumbered by parasites. In the heav-
ily infected spider crab, there were numerous mononuclear and
multinucleated trophont forms dispersed along the vascular spaces
of the gills: few hemocytes were present (Fig. 4). Some skeletal
muscle fibers appeared fragmented; there were also interstitial
clusters and infiltrates of parasites and foci of myofiber necrosis
(Fig. 5A). The hepatopancreas was heavily infected with the tro-
phont forms on the abluminal side of the tubules and in vascular
spaces (Fig. 5B). There were no parasites within the hepatopan-
creatic cells or within the tubular lumina.
Menippe mercenaria
The crab examined was heavily infected. Most of the parasites
were in the mononuclear trophont form, although occasional bi-
.4^
V
.*
25(1
^0
y>
■»V
••fS ••*
»v
,»:-^*> i»l> • '
■it
25(1
Figure 5. Heavily infected spider crab, Libinia emarginata. i\) Inter-
stitial infiltrates of the parasite in the skeletal muscle. The heniocytic
response is minimal. Some muscle fibers lack nuclei: there are foci of
apparent destruction in association with the parasites (arrow). (B)
Hepatopancreatic vascular spaces are filled with parasites, but no in-
filtration of the glandular epithelium is seen. (Hematoxylin and eosin:
original magnifications: x40(>).
Hematodin/um Infection in Georgia Crabs
877
nucleated and multinucleated forms were observed. The highest
concentration of Heiitcitodiiuitm sp. was in the gills where the
parasites were dispersed along the vascular spaces (Fig, 6A). Crab
hemocytes. primarily granulocytes, were present in these vascular
spaces although they were far outnumbered by the parasite. He-
matodiniiinm sp. were concentrated on the abluminal sides of the
hepatopancreas and in its vascular spaces. Both granular and
agranular hemocytes were present in the heart; some had infiltrated
the cardiac muscle along with Hematodiniuin sp. (Fig. 6B). There
was, in addition, a single focal plaque-like aggregate of parasites
and granulocytes on the surface of cardiac muscle. Skeletal muscle
contained only a few Hematodiniuin sp. in connective tissue ex-
ternal to muscle fibers. Gonadal tissue appeared to be free of the
parasite.
Callinecles sapidus
In lightly infected crabs (less than 2% of hemolymph cells were
parasites) there was a strong cellular response to Hematodinium
sp., as evidenced by scattered aggregates of granulocytes, which
formed encapsulating nodules in gill, hepatopancreas. and cardiac
muscle (Fig. 7A,B). The nodules in the hepatopancreas were found
l\
10(1
ll.
m - -
% 0
d
%-**3^
■^
L
0 ^
<
Figure 6. I.Al Gill troiii an Infected stone crab. Meiiippe mercenariu.
Trophont forms of the parasite and host hemocytes are present « ithin
the vascular space. (Bl Cardiac tissue from the same crab. Interstitial
inflltrate of trophonts and host hemocytes. (Hematoxylin and eosin;
original magnifications: xlOOO).
Figure 7. Lightly infected blue crab, Callinecles sapidus. (A) The vas-
cular spaces of the gills contain abundant granular and agranular
hemocytes and occasional hemocylic nodules. A few trophont forms
are present ( arrow). (Bl A cluster of hemocylic nodules on the ablu-
minal side of the hepatopancreas. (Hematoxylin and eosin; original
magnifications: A. x400; B, xlOOO)
on the abluminal side and there was no invasion by parasites of the
hepatopancreatic glandular epithelium or tubular lumina. Only
mononuclear trophont forms were observed.
In heavily infected crabs, several parasites but few hemocytes
were found in vascular spaces and within tissues (Figs. 8, 9).
Plasmodia (Fig. 8A). mononuclear and binuclear trophonts were
noted in both the hemolymph and cardiac muscle. Dense infiltrates
of parasites were noted on the abluminal side of the hepatopan-
creas (Fig. 8B). but even with heavy infection there was no evi-
dence of hepatopancreatic epithelial or tubular luminal invasion by
(he parasite. In the hepatopancreatic region of one moribund ani-
mal, there were large numbers of a smaller parasite form that
possessed a polymorphic nucleus; these may have been dinospores
(Fig. 9A).
In addition to high concentrations of parasites in bivascular
spaces, parasites infiltrated cardiac and skeletal muscle. Focal
muscle necrosis was present (Fig. 9B); hemocyte nodules were
rare or absent. Parasites were present in the tissues adjacent to the
gonads, but not within gonadal tissues.
878
Sheppard et al.
#
V.
9^ "■'.,•.*.
«^
10i
W 9
9 "» ^ I"
... ^
,'■?
-<*■■
^.,
'%'
*5#
^ ..
M
^♦% *■'
^ .,
10 u
Figure 8. Heavily infected blue crab, ( iillinccles sapidus. (Al The >as-
cular spaces of the gills of this crab contain many Plasmodia; few
hemocytes are present. (B) Hepatopancreatic tissue from another
heavily infected animal. This vascular space is filled with parasites,
mainly in the trophont form. (Hematoxylin and eosin; original mag-
nifications: A, x400; B, xlOOO)
DISCUSSION
Prior to 1999, Wassaw Sound on the Georgia coast supported
a robust, year-round commercial blue crab fishery. Since the stud-
ies began in 1999. there have been high Hciiuiiocliniiiiii sp. preva-
lences in Ccillliu'iles sapidus. Lihiiiia I'nhiri^iinilu. and Neopanope
sayi. In addition to a peak each fall, a peak in HematocUnium sp.
prevalence in C. sapidus also occurred during the spring months.
Associated with the increased prevalence of Hematodiniuin sp. in
the spring was the disappearance of C. sapidus from Wassaw
Sound during the summers of 3 successive years (Fig. 2). These
observations suggest that high mortality secondary to Hemato-
diniuin infection in the spring led to the near absence of C. sapidus
in the summer. During the summer months, blue crabs were
abundant in low salinity areas near freshwater rivers in coastal
Georgia (Lee. unpubl.). We hypothesize that female blue crabs
found each fall for the past 4 years in Wassaw Sound were return-
ing through Wassaw Sound from low salinity areas to spawn in the
ocean.
Other seasonal studies on prevalence o\' Hcnuitodiniiini sp. have
Figure 9. Heavily infected blue crab, Callinecles sapidus. (A)
Hemocytic nodules were rare in most of the heavily infected animals.
This one is on the abluminal side of the hepatopancreas. Also present
are thousands of small forms of the parasite, possibly dinospores. (Bl
Infiltrates of the parasite in cardiac muscle. Both trophont and Plas-
modia forms are presenl. Focal coagulative muscle necrosis (arrows)
has occurred. (Hematoxylin and eosin; original magnit'ications: x400)
been conducted on crabs in different coastal areas. A seasonal
study of Hematodinium sp. infection in C. sapidus collected from
coastal bays of Maryland showed a peak of infection each fall.
Prevalences reached 80'7f at this time, while the disease was almost
undetectable from March thru May (Messick & Shields 2000).
Seasonal studies of Henialodiniuni sp. were conducted in the Nor-
way lobster (Nephwps norvegicus) off Scotland (Field et al. 1998)
and the tanner crab {Cluonoectes hairdi) off Alaska (Eaton et al.
1991, Love et al. 1993). The peaks oi Hematodinium sp. infection
in both species occurred in the late spring and summer, with de-
clines in infection noted during the fall and winter. These studies,
along with our own findings, indicate that the seasonality of He-
matodinium infection can vary among different crustacean species
in the same area and among species from different areas.
We have shown that Hematodinium sp. can be transmitted
when an uninfected crab feeds on an infected crab (Lee et al.
unpubl.). Both C. sapidus and L. emarginata are aggressively
cannibalistic. We noted a much lower frequency of infection in
Menippe mercenaria and Callinecles similis. We speculate that
the indolent feeding behavior of M. mercenaria and C. similis
Hematodinium Infection in Georgia Crabs
879
account for their low Heinutodiniuin sp. prevalence during periods
when there is both high prevalence and intensity of HcnuiUhliiiiimi
sp. among other crab species. Other explanations for the varying
prevalence of Hematodinium sp. in different crab species include
the possibility that Hematodinium sp is more virulent for certain
species, possesses tropism for particular crab species, or that the
immune systems of C. similis and M. mercenaria are more effec-
tive in limiting Hematodinium sp. infection. Another important
factor may be crab densities, since we find that Hematodinium
epidemics occur in areas where there are high densities of either C.
sapidus or L. emarginata (Sheppard. Lee, and Fischer, unpubl.).
Some marine diseases are well correlated with host densities
(Richardson et al. 1998), but in other diseases there is no relation-
ship (Powell et al. 1999).
Only two Hematodinium spp.. H. perezi (Chatton & Poisson
1931 ), and H. australis (Hudson & Shields 1994), have been char-
acterized. While the parasite in C. sapidus has been referred to as
Hematodinium perezi (Messick 1994. Shields & Squyars 2000),
Messick and Shields (2000) suggest that the parasite in C. sapidus
be referred to as Hematodinium sp. until more comparisons have
been made with the type species. Based on the sequence of frag-
ments of the 18S rRNA gene, it appears that the Hematodinium sp.
found in L. emarginata and M. nwrcenaria are the same or very
closely related to Hematodinium sp. isolated from C. sapidus
(Gruebl et al. 2002). It thus appears likely that the infection can be
readily transmitted among various crab species in our study area.
Histopathologic studies of Hematodinium sp. infections include
('. sapidus from coastal bays of Maryland (Messick 1994), Por-
tunus pelagicus from the eastern seaboard of Australia (Hudson &
Shields 1994) and Cliionoeeetes luiirdi from southeast Alaska
(Meyers et al. 1987). The histologic changes described in infected
gill and muscle tissues of the animals in tho.se studies are similar
to those seen in the tissues of the infected crabs in our studies.
Hematodinium sp. was present extracellularly within the vascular
spaces of gills. The parasite produced interstitial infiltrates in car-
diac and skeletal muscle but did not invade individual myofibers
(Figs. 4-5. Hudson & Shields 1994, Meyers et al. 1987). Focal
muscle necrosis was apparent in some of our infected crabs. Myers
et al. (1987) noted pathologic changes in muscle cells of heavily
infected Tanner crabs, including loss of cross striations and cyto-
plasmic eosinophilia. Parasitic infiltrates and muscle necrosis
would likely compromise the structure and function of these or-
gans and thereby contribute, along with the hemocyanin depletion,
to the lethargic behavior exhibited by heavily infected animals.
The presence of encapsulating nodules in lightly infected C.
sapidus and their absence in non-infected crabs is of interest since
the response of crustaceans to large foreign bodies is encapsulation
by circulating hemocytes (Galloway & Depledge 2001. Holmblad
& Soderhiill 1999). In heavily infected animals, the hemocyte
population appeared depleted, suggesting that large numbers of
parasites can overwhelm the host's ability to contain the infection.
Whether such animals are immunocompromised by pre-existing
conditions or the parasites gain a proliferative advantage due to
environmental circumstances awaits further study. In addition, we
have found that bacteria often colonize the hemolymph of heavily
parasitized animals (Sheppard, unpublished data). Such secondary
invaders may hasten the demise of these impaired hosts, since they
cannot mount an adequate hemocyte response.
Our results suggest that Hematodinium sp. is impacting the blue
crab populations in Wassaw Sound and is largely responsible for
the disappearance of C. sapidus during the summer months. As the
population of C. sapidus in Wassaw Sound has decreased there
have been increases in the populations of other crab species, such
as C. similis. Ovalipes ocellalus, Petrolisthes armatus. and Are-
naeus cribrarius (Sheppard, unpubl.).
ACKNOWLEDGMENT
These studies were supported by the NOAA National Sea Grant
College Marine Environmental Biotechnology Program (Grant
NA06RG0029).
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Jourmil of Shflljhh Kcsi'unh. Vol. 2:, No. 3, 88l-88fi, 2003.
THE ROLE OF MACROALGAL BEDS AS NURSERY HABITAT FOR JUVENILE BLUE CRABS,
CALLINECTES SAPIDUS
CHARLES E. EPIFANIO,* ANA I. DITTEL. RAYMOND A. RODRIGUEZ, AND
TIMOTHY E. TARGETT
Graduate College of Marine SmJIes. Uiilversin of Delaware. 700 Pilottowu Road. Lewes.
Delaware 19958
ABSTRACT We itnestigaled the role of macroalgal beds as juvenile habitat for the blue crab CalUnectes supiihis. A 2-year study
was conducted in Rehoboth Bay. a lagoonal estuary in the Middle Atlantic Bight along the east coast of North America. Sea grass
meadows do not occur in Rehoboth Bay. and submersed aquatic vegetation consists entirely of macroalgae. Quantitative samples were
collected from both vegetated and open (unvegetated) habitat with a throw trap. Results indicate that inacroalgal beds provide important
habitat for juvenile blue crabs, beginning at settlement and continuing until the crabs reach a carapace width of about 30 mm. Average
abundance of juveniles in macroalgal beds was 7 times greater than in adjacent open habitat, and maximum abundance in the beds
reached weekly mea;i values >90 crabs m"^ during periods of high recruitment in early autumn. Mean size of individual crabs was 15
inm carapace width when sampling began in May. These crabs had settled the previous autumn and had over-wintered in the bay. Mean
size continued to increase through early summer, and the crabs had reached a inean carapace width >30 mm by August. These 30-mm
crabs disappeared frotii the beds in inid-August and were replaced by newly metamorphosed juveniles <I0 mm in carapace width. Very
small crabs were common in the beds throughout September and October. Results of gut-content analysis imply a direct trophic linkage
between indigenous macroalgal production and juvenile crabs collected from the beds. This putative linkage involves various species
of amphipods that graze directly on the macroalgae and constitute over 25* (by volume) of the gut contents of juvenile crabs collected
from macroalgal habitat.
KEY WORDS: juvenile, blue crab. Callincctex \iipidiis. macroalgae. nursery habitat
INTRODUCTION
The preservation of plant-based habitats such as sea grass
meadows has become a lynchpin of international marine conser-
vation policy, but regardless of conservation efforts, there has been
a general decline in the extent of this habitat woridwide (e.g..
Giesen et al. 1990. Dennison et al. 1993, Heyman & Kjerfve
1999). This problem has been studied intensively in estuaries along
the east coast of North America, where sea grass provides nursery
area for many species of fish and invertebrates (Orth & Moore
1983. Shepherd et al. 1989. Moore et al. 2000). The value of sea
grass beds as nursery grounds has been attributed to the provision
of complex bottom topography that reduces the extent of predation
on juvenile stages (Orth & van Montfrans 1987. Wilson et al.
1990). However, sea grass meadows are also the sites of high
indigenous primary production (e.g.. Duarte & Chiscano 1999),
and the role of this production in supporting the growth and de-
velopment of juveniles is less clear (Fry & Parker 1979, Hughes &
Sherr 1983).
In areas where sea grass is in decline, newly available bottom
often has been colonized by benthic macroalgae (Valiela et al.
1997). But unlike sea grass meadows, the nursery role of these
macroalgal beds has not been well studied. For example, there
have been only a few experimental investigations of the role of
macroalgal beds as refugia from predation (e.g., Wilson et al.
1990. Dittel et al. 1996), and the number field surveys of juvenile
fomis of fish and inveilebrates occupying this habitat is commen-
surately low (Sogard & Able 1991, Sogard 1992. Szedlmayer &
Able 1996). Moreover, the role of indigenous primary production
in supporting the growth of juveniles within macroalgal nurseries
is virtuallv unknown.
*Corresponding author: E-mail: epics' udel.edu
Growth of macroalgae is maximized under eutrophic condi-
tions typical of poorly flushed lagoonal estuaries (Lavery et al.
1991. Duarte 199.5). One such estuary is Rehoboth Bay. which is
located in the Middle Atlantic Bight along the east coast of the
USA (ca. 38.5°N. 77.1°W). Although historical accounts indicate
that areas of sea grass meadow occurred in Rehoboth Bay as
recently as the 1960s, submersed aquatic vegetation now consists
entirely of macroalgae (Price 1998). The dominant macroalgae
occurring in Rehoboth Bay are the green alga Ulva Icictiica and the
red algae Agardhiella tenera and Gracilaria spp. (Timmons &
Price 1996). Macroalgal beds are patchily distributed on sandy
bottom throughout the bay. and typical patches are on the order of
10'' to lO"* m-^. Macroalgal beds in estuaries like Rehoboth Bay
often occur as drift algae (i.e., not attached to the bottom). Thus,
the location of patches changes as a function of winds and currents.
We have used Rehoboth Bay as a case study in which we
investigated the extent to which one of the dominant invertebrate
species in the region (the blue crab. CalUnectes sapidiis) uses
macroalgal beds as nursery habitat. Although several types of bot-
tom have been identified as nurseries for blue crabs (Szedlmayer &
Able 1996). maximum abundance of juveniles typically occurs in
vegetated areas, and sea grass meadows are generally considered
critical nursery habitat for the species (Pardieck et al. 1999). The
utility of macroalgal beds as surrogates for sea grass has been
generally established (e.g.. Sogard & Able 1991 ). but details of the
association between macroalgae and juvenile blue crabs (including
possible trophic linkages) have not been determined. The study
described in this paper addresses this gap and provides data on
seasonal changes in the abundance of different life history stages
in macroalgal beds and the relationships between the abundance of
juveniles and the standing crop of macroalgae. The investigation
involved extensive field collections and included comparative
analysis of gut contents of Juvenile blue crabs collected from mac-
roalaal beds and from two alternative nursery habitats.
881
882
Epifanio et al.
METHODS
Study Location
Rehoboth Bay is a small lagoonal estuary located in the Middle
Atlantic Bight (Fig. 1). Mean depth is about 1.7 m with a tidal
range < 0.5 m. Rehoboth Bay has no direct connection to the
coastal ocean. At its northward end. Rehoboth Bay adjoins the
Lewes & Rehoboth Canal, which eventually reaches Delaware
Bay, approximately 12 km to the north. At its opposite end. Re-
hoboth Bay connects through several shallow channels to Indian
River Bay, immediately to the south. Indian River Bay communi-
cates with the coastal ocean through an inlet at its eastern terminus.
Total surface area ot the Rehoboth-lndian Riser system is approxi-
mately 75 km~.
Comparison of Vegetated and Open Hahilal
In the first year of the in\estigalion ( 1998). we compared the
abundance of juvenile blue crabs in macroalgal beds to abundance
at open bottom sites. Crabs were collected using a throw trap that
allowed quantitative sampling of a confined area of bottom (.see
Sogard & Able 1991). The base of the throw trap was an open
aluminum box ( 1 m x 1 m x 0..^ m) with a bund of fine-mesh (0.5
cm) nylon netting (1.5 m high) attached around the entire perim-
eter. The upper edge of the netting was lashed to a buoyant, frame
(1 m X 1 m). and the remaining seam was sewn together to com-
plete the trap. The device was deployed froin a small boat in water
<1.5 m deep. Upon deployment, the base of the trap penetrated
several cm into the sediment, and the upper section extended all
the way to the surface. Thus, a l-m~ quadrate of bottom was
segregated from the surrounding environment and could be
sampled quantitatively.
Sampling was conducted every 2 wk. starting in late June and
continuing through the end of September. Collections were gen-
erally made at three stations during each sampling week. However,
inclement weather occasionally restricted effort, resulting in a total
of 18 stations sampled over the entire period. Stations were located
in shallow water around the periphery of the bay (Fig. 1). The
exact location of the stations varied from week to week, depending
on the availability of macroalgal beds and adjacent open habitat.
Throw trap sampling was performed in conjunction with a beam-
trawl survey of the deeper 01.5 m) parts of Rehoboth Bay. Abun-
dance of juvenile crabs was considerably lower in deep water than
in the shallow water sampled with throw traps (Targett et al. 1999).
At each station the trap was deployed once in a macroalgal bed
and once on the adjacent open bottom. The two sampling locations
were always within 50 m of each other, and the exact site within
each habitat was chosen haphazardly. Temperature, salinity, and
dissolved oxygen were measured in conjunction with each deploy-
ment. Crabs and macroalgae were remo\ed from the trap with a
3-nini mesh dip net. The rectangular frame of the dip net was
designed to allow maximum coverage of the area within the throw
trap with each sweep of the net. Dip-net sweeps were made along
the bottom until 3 consecutive sweeps produced no organisms.
Earlier work with similar gear has shown that efficiency of sam-
pling approaches 100% with this technique (Kushlan 1981. Pihl &
Rosenberg 1982).
Juvenile blue crabs were returned to the laboratory where cara-
pace width was determined to the nearest mm. Volume of mac-
roalgae in each sample was determined in the field. This involved
removal of extraneous water by blotting the sample on paper tow-
Fijjure I. .Map of study area. Insert shows location of Rehoboth Bay in
Middle Atlantic Bight.
els. followed by measurement of the respective volumes of green
and red algae in a large graduated beaker. These values were
converted to their gravimetric equivalents using regression equa-
tions relating volume to dry weight (green algae: r = 0.95, P <
0.001, n = 26; red algae: r = 0.97, P < 0.001, n = 29). The
regression equations were ba.sed on volume measurements and
dry-weight determinations (60°C, 48 h) for representative mac-
roalgal samples collected from Rehoboth Bay.
Seasonality of Habitat Use
In the .second year of the study (1999), we investigated detailed
seasonal patterns in use of macroalgal beds by juvenile crabs.
Early season sampling (mid-May through early August) was con-
ducted every two weeks and targeted crabs that had .settled during
the previous autumn and had over-wintered in the bay (i.e., the
1998 y-class). Late season sampling (mid-August to early Novem-
ber) occurred weekly and concentrated on newly settled juveniles
(i.e., the 1999 y class). As in the first year of the investigation,
stations were located in shallow water around the periphery of the
bay, but in this case sampling was restricted entirely to vegetated
areas. Again, the exact location of stations varied from week to
week, depending on the availability of macroalgal beds.
Macroalgal Beds as Juvenile Habitat for C. sm'idus
883
Sampling was generally conducted at five stations during each
sampling week. However, inclement weather occasionally re-
stricted effort, resulting in a total of 81 stations sampled from May
through October. Deployment of the throw trap and analysis of
samples were the same as in year 1.
Analysis of Giil Contents
Crabs for gut-content analysis were collected as part of routine
throw trap sampling in 1999. Sampling areas were always located
in macroalgal beds and typically encompassed stands of both red
and green niacroalgae. A total of eight throw trap samples were
collected for gut-content analysis between July and September,
resulting in 52 individual crabs (<30 mm carapace width). Com-
parative samples were collected from two alternative blue crab
nursery areas (one a marsh tidal creek and the other an open water
tide flat) in nearby Delaware Bay. Marsh-fringe samples were
collected with a dip net from a tidal creek within an extensive salt
marsh; tide Hat samples were obtained with a beach seine from an
open water site a few km away (Fig. 1 ). Sediment at the tide flat
site consisted of coarse sand, cobble, and shell fragments — thus
providing a modicum of structured nursery habitat for juvenile
crabs. Sampling was conducted a total of 12 times at the marsh site
between July and October, yielding 73 individual crabs for subse-
quent analysis. An additional nine sampling efforts at the tide flat
site between August and October resulted in 47 individual crabs
for analysis.
In each case, crabs were placed on ice while still in the field to
minimize digestion of food. Upon return to the laboratory, the
crabs were frozen (-20°C) for later analysis. Gut contents of
thawed specimens were determined using standard dissection and
microscopy techniques (e.g.. Dittel 1993). Separate analysis was
conducted for each of the 172 crabs in the samples. The occurrence
of each food item was represented as a proportion of the total
volume of food in the stomach, and mea;i values were determined
for each of the 3 sample groups (Hines et al. 1987).
Statistical Analysis
Mean abundance of crabs in sea grass beds was compared with
abundance on open bottom by a two tailed f-test (a = 0.05). Data
were log-transformed to meet assumptions of the r-test model.
Relationships between crab abundance and standing crop of mac-
roalgae were determined by Pearson product-moment correlation
analysis. All correlations were done using pooled data from both
years. Separate analyses were conducted for the entire data set. for
early-season data, and for late-season data. A similar approach was
used to examine correlations between crab abundance and the
respective proportions of green and red algae in each sample.
Significance of all correlations was determined at a = 0.05.
RESULTS
Hydrographic conditions were similar during the two years of
the study, and any differences in the overall range of values were
attributable to the broader seasonal coverage during 1999. Salinity
ranged from approximately 23-32 %o over the period of the in-
vestigation, with a median value around 29%c. Temperature
reached maximum values in August (>25°C), and was minimum
(<15°C) in early November 1999. Water was typically well oxy-
genated, and levels rarely fell below 5 mg L '. Supersaturated
values as high as 15 mg L"' were occasionally measured in mac-
roalgal beds on calm, sunny days.
Abundance of juvenile blue crabs was significantly greater in
inacroalgal beds than in adjacent open bottom (/-test, tij = 3.1,
P < 0.001). When calculated for all stations in 1998, mean abun-
dance in niacroalgae was 7.3 m"" (±10.1 ), while open areas had an
abundance of 1.0 m"" (± 1.9). The large standard deviations re-
sulted from seasonal differences wherein the abundance of crabs in
macroalgal beds increased markedly in September (Fig. 2).
Results from 1999 showed strong seasonal variation in both
numbers and size of juveniles in macroalgal beds. Mean abun-
dance from May through early .August was always <10 crabs in"",
but increased to levels >20 crabs m"" by mid-August and reached
values >90 m'" in September and October (Fig. 3). The mean
carapace width of juveniles was somewhat less than 15 mm when
sampling began in May and approached 30 mm by late July (Fig.
4). The large juveniles disappeared from the beds in mid-August
and were replaced much smaller crabs (<10 mm). These small
individuals dominated the population throughout the remainder of
the study period and were still abundant when sampling ended in
early November.
Macroalgal beds were well developed during both years of the
investigation, with a median standing crop of niacroalgae of ap-
proximately 150 g m"". This is within the range of values typical
for niacroalgae at high nutrient levels (e.g., Schneider & Searles
1977, De Busk et al. I9S6). Of the 99 vegetated stations sampled
over the two years of the study. 40 had a greater proportion of
green algae and 59 a greater proportion of red. Among these, 1 8
stations were pure stands of red species, while only 3 stations were
pure stands of green forms.
Analysis of all stations pooled over the 2 y of the investigation
showed a significant positive correlation between abundance of
crabs and total standing crop of macroalgae (Table 1). However,
there was no correlation between abundance of crabs and the dry-
weight ratio of green to red algae at the respective stations. Sepa-
rate analysis of early-season data and late-season data gave results
that were similar to those for the entire data set.
As expected, the gut contents of crabs collected from all three
habitats showed a wide variety of prey items (Table 2). These
included a number of crustacean groups, bivalve and gastropod
mollusks, polychaetes, vascular and macroalgal plant material, and
considerable amounts of highly digested tissue that we were un-
able to assign to any particular taxonomic group. Regardless of this
July
August
September
Figure 2. Abundance of juvenile blue crabs Callinectes sapidus in mac-
roai^al beds in Reiiobotli Bay, Delaware. Solid bars are weekly mean
abundance in 1998. Error bars = one standard deviation.
884
Epifanio et al.
200
120 -
Figure 3. Abundance of juvenile blue crabs Calliiucles sapidus in niac-
roalgal beds in Rehoboth Bay, Delaware. Solid bars are weekly mean
abundance in 1999. Error bars = one standard deviation.
taxonomic diversity, crustaceans were the dominant stomach com-
ponent in crabs from each of the three sampUng sites. However,
the taxonomic groups comprising this crustacean component var-
ied greatly among crabs from the three respective habitats. For
example, crab body parts accounted for nearly 30% (by volume) of
the stomach contents of juvenile C. siipidus collected from marsh
habitat adjacent to Delaware Bay. but never exceeded 13% in
either of the other two habitats. In contrast, a miscellaneous group
that we called "other crustaceans" composed almost 50% of the
stomach contents of crabs collected from tide flat habitat in Dela-
ware Bay. This group consisted of harpacticoid copepods, palae-
monid and crangronid shrimp, and crustacean body parts that could
not be assigned to any particular taxon. Crabs collected from mac-
roalgal beds in Rehoboth Bay differed most notably from the other
two habitats in the very low proportion of crab body parts in their
gut contents and in the high proportion of amphipods in their
ID
n
o
May
June
July Aug
Sept
Oct
Figure 4. Size of juvenile blue crabs {Callinectes sapidus) in seaweed
beds in Rehoboth Bay. Solid bars are weekly mean carapace width in
1999. Error bars = one standard deviation.
stomachs (>30%). This was remarkable because amphipods were
entirely absent from the identifiable gut contents of crabs from the
other two sampling sites.
DISCUSSION
Results of our investigation indicate that macroalgal beds pro\'ide
important habitat for juvenile blue crabs, beginning at settlement and
continuing until the crabs reach a c;irapace width >30 mm. Average
abundance of juveniles in macroalgal beds was approximately 7 times
greater than on adjacent open bottom, and maximum abundance in
the beds reached weekly mea;; values >90 crabs nr^ during periods
of high recruitment in early autumn. Mean size of indi\ idual crabs
was about 15 mm in carapace width when sampling began in May.
Because settlement of blue crabs in this region occurs almost exclu-
sively in late summer and autumn (Jones & Epifanio 1995). the crabs
collected in May apparently had settled during the previous autumn
and had over-wintered in Rehoboth Bay. Mean size continued to
increa.se through early summer, and the crabs had reached a mean
carapace width >30 mm by mid-summer. The 30-mm crabs disap-
peared from the beds in mid-August and were replaced by newly
metamorphosed juveniles <10 mm in carapace width. These small
crabs had probably settled in the beds as megalopae and had under-
gone metamorphosis soon thereafter (Orth & van Montfrans 1987,
Jones & Epifanio 1995). Very small crabs were common in the beds
throughout September and were still abundant when sampling was
completed at the end of October. Mean size of the crabs did not
increase during this period, probably a result of overlapping cohorts
of new recruits. However, there was considerable variation in abun-
dance among stations (note the high standard deviations in Fig. 4).
which undoubtedly reflects the patchy nature of settlement in the bay.
This was probably a result of the patchy distribution of megalopae in
the water column (Natunewicz & Epifanio 2001), rather than some
difference in the attractiveness among beds (Brumbaugh & McCon-
naugha 1995).
Earlier work in the Little Egg Harbor-Great Bay system along
the coast of New Jersey (100 km noilh of Rehoboth Bay) also
addressed the importance of macroalgal beds as juvenile habitat
(Wilson et al. 1990). This system is similar to our study site, but
has ample sea grass meadow in addition to macroalgal beds (Sog-
ard & Able 1991). As in our investigation, early-season abundance
at the New Jersey site was on the order of 5-10 crabs m"" in
vegetated habitat and considerably lower on open bottom. More-
over, the general pattern of seasonal abundance of different size
classes was similar to that in Rehoboth Bay. However, late-season
sampling in New Jersey did not find the extremely high abundance
of newly settled crabs seen at our study site, perhaps reflecting a
greater distance from the very large spawning stock of blue crabs
in Delaware Bay (Garvine et al. 1997).
This difference aside, it appears that macroalgal beds generally
pro\ ide nursery habitat for blue crabs that is comparable to that of
sea grass meadows. For example, there was little difference in
mean abundance of juveniles in macroalgal and sea grass habitats
in the Little Egg Harbor-Great Bay system; in fact, the abundance
of crabs was slightly higher in macroalgal habitat (Sogard & Able
1991). Likewise, mean abundance in macroalgal beds at our study
site in Rehoboth Bay was sitnilar to that in sea grass meadows in
Chesapeake Bay, and general patterns in seasonal occuixence were
nearly identical (e.g., Orth & van Montfrans 1987).
The present investigation has provided a much more detailed de-
scription of the utilization of macroalgal habitat than was previously
Macroalgal Bkds as Juvenile Habitat eor C. sah/dus
885
T.ABLE 1.
Correlations betHutn abundunce of ju\eiiilt bluu crabs {C'alliiiecles sapidiis) and two proptrtits (algal standing crop and Iht ratio of green
to red macroalgae) of macroalgal beds in Rehoboth Bay, Delaware, USA.
Full Season
Earl) Season
Late Season
r
/'
n
r
P
n
r
P
n
[nx.4
[QxR
0.379
-0.103
<0.001
0.3 1 3
99
99
0.443
-0.034
0.002
0.824
45
45
0.575
-0.184
<0.001
0.183
54
54
Data were analyzed separately for full season, early season, and late season (see text). Correlalon statistics: /■ = Pearson product-moment correlation
coefficient. P = probability of rejecting a correct null hypothesis, n = number of coordinate observations. Variables: [C] = crab abundance. A = algal
standing crop. R = ratio of green to red algae (dry weight).
avuilable. Fur example, our results inipl\ iIkiI iiiacmalgai beds are
important settlement sites blue crab megalopae in autumn and further
de[ini[istrate the consequent role of the beds as nurseries for the ear-
liest juvenile stages. In addition, our analysis shows that juvenile blue
crabs use beds of red and green macroalgae with equal propensity and
that abundance of crabs in a bed increases in propoition to the stand-
ing crop of macroalgae. Moreover, these relationships are eqtially
valid during early season when the population is dominated by over-
wintered crabs or later in the season when beds are poptilated entirely
by newly settled juveniles.
Supplemental to their provision of complex benthic structure,
macroalgal beds may also be important as a source of primary
production that supports growth of juvenile blue crabs. Results of
our analysis indicate that crustacean body parts dominated the gut
contents of crabs collected from all three nursery habitats consid-
ered in this investigation. However, the taxonomic groups repre-
sented within this dietary category varied considerably among
habitats. Of primary relevance is the fact that amphipods were the
dominant component in the stomach contents of crabs collected
from macroalgal beds, but were entirely absent from the identifi-
able gut contents of crabs from marsh or tide flat environments.
Available evidence in the present investigation is restricted en-
TABLE 2.
Mean percentage by volume of prey items in gut contents of juvenile
blue crabs iCallinccles 'iapidiis) collected from three different
nursery habitats.
Gut Contents
Macroalgae
Marsh
Tide Flat
Amphipods
31.3
0,0
0,0
Crab body parts
3.3
27.4
12.9
Other crustaceans
13.0
19.0
46.1
Bivalves mollusks
2.8
3.5
0.0
Gastropod mollusks
9.7
1.5
15.7
Polychaeles
1.1
6.1
4.2
Foraminiferans
0.8
0.0
0.0
Macroalgal material
4.3
3.7
1.5
Vascular plant material
0.8
8.3
1.0
Highly digested material
32.3
30.2
16.8
Sand grains
0.4
0.3
1.8
Macroalgal beds were located in Rehoboth Bay. Delaware, USA. Marsh
and tide tlal habitats were located in nearby Delaware Bay. Explanation of
selected gut-content categories: Crab Body Parts = items identified to the
infraorder Brachyura; Other Crustaceans = items identified as harpacti-
coid copepods, palaemonid and crangonid shrimp, or simply to the sub-
phylum Crustacea; Highly Digested Material = organic material uniden-
tifiable to a taxonomic group.
tirely to gut-content analysis, which has an inherent bias in favor
of material that is refractory to digestion. Nevertheless, the high
proportion of amphipods in the guts of crabs from macroalgal beds
is in striking opposition to the complete lack of this taxon in the
gut contents of juveniles from the other two environments and
strongly suggests a major difference in diet between crabs from
macroalgal habitat and either of the other habitats.
Because of the very high abundance of amphipods in macroal-
gal beds in Rehoboth Bay (Timmons & Price 1996). it is reason-
able to conclude that the amphipods found in crab stomachs ana-
lyzed in our study originated in the beds themselves. Moreover, the
common taxa of amphipods found within these macroalgal beds
(various species in the families Gammaridae, Amphitoidae, and
Bateidae) graze directly on macroalgae, which constitute the main
portion of their diets (Watling & Maurer 1972, Macko et al. 1983,
Parker et al. 1993, Lotze & Worm 2000, Kamermans et al. 2002).
Thus, it is likely that macroalgal production is an important com-
ponent of the food web supporting juvenile blue crabs in estuarine
systems like Rehoboth Bay.
This is in contradiction to results of earlier work with juvenile
shrimp in mangrove nurseries, which has shown a link with pri-
mary production originating in benthic diatoms, rather than with
production emanating from the inangroves (Stoner & Zimmerman
1988, Newell el al. 1995. Dittel et al. 1997). Likewise, previous
work with juvenile blue crabs in salt marsh environments has
demonstrated at least partial dependence on benthic diatom pro-
duction and only indirect linkage to production by emergent marsh
plants (Dittel et al. 2000). Investigations of sea grass systems have
come to varying conclusions concerning the role of indigenous
primary production in supporting growth of juvenile crabs (e.g..
Fry & Parker 1979, Hughes & Sherr 1983), but a recent review
finds little evidence that sea grass productit)n per se is a major
contributor (France 1996).
When considered as a whole, the results of our investigation
provide credible evidence that macroalgae beds constitute critical
nursery habitat for juvenile blue crabs in areas where seagrass beds
are lacking. Moreover, the value of this habitat may include a
direct trophic linkage between primary production originating in
the macroalgae; this has not been demonstrated in other plant-
based nursery habitats used by juvenile blue crabs.
ACKNOWLEDGMENTS
The research was supported by funds from the Di\ ision of .Soil
& Water Conservation and the Division of Fish and Wildlife.
Delaware Department of Natural Resources and Environmental
Control, from the Wallop-Breaux Program of USF^'. and from the
Marsh Ecology Research Program (MERP) (no. G98-04A).
886
Epifanio et al.
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Journal of Slwllji.^h Research. Vol. 22, No. 3, 8S7-S92, 2003.
ISOLATION AND MOLFXULAR CHARACTERIZATION OF VITELLIN FROM THE MATURE
OVARIES OF THE PRAWN LITOPENAEUS VANNAMEI
CELIA VAZQUKZ-BOUCARD,'* HUMBERTO MEJIA-RUIZ,' FERNANDO ZAMUDIO,"
VANIA SERRANO-PINTO,' AND HECTOR NOLASCO-SORIA'
'CIBNOR-Centro de Investigaciones Biologicas del Noroeste. S.C. P.O. Box 128. La Paz 23000, BCS.
Me.xico; and ~lns1ituto de Biotecnohgia-UNAM. Aveuida Universidad #2001. Col. Chamilpa C.P. 62210.
Ciieniavaca Morelos. Me.xico
ABSTRACT Vjtellins from ovaries in shrimp Litopenaeus vannamei were examined by polyacrylamide gel electrophoresis, sodium
dodecyl sulfate polyacrylamide gel electrophoresis, crossed- Immunoelectrophoresis, chromatography (Sepliarose CL 2B and hydrox-
ylapatite columns), and high-performance liquid chromatography. Using these methods, two forms of vitellin (Vtl and Vt2) were
observed in ovaries (oocyte 1 10 |j.ml. The vitellins identified appear to be lipoglycoproteins. Similar vitellin polypeptide composition
was observed in the two forms of vitellin. with molecular weights of approximately 60. 90, 95, 100, 140, and 160 kDa. Policlonal
antibodies against the two forms of purified protein were prepared, and their specificity was demonstrated by radial immunoprecipi-
tation and Western blotting analysis. The PI and P2 peptides from N-terminal 100 kDa and 60 kDa polypeptides were highly similar
to regions of proline 20 and glycine 63.5 residues of crustacean vitellogenins.
KEY WORDS: ovary, shrimp, vitellin. lipovilellin. vitellogenesis. L vannamei
INTRODUCTION
Vitellin is the major yolk protein accumulated in developing
oocytes of a female crustacean. Yolk protein is the source of
nutrition for development of embryos and larvae. The vitellin from
ovaries and vitellogenin from the hemolymph have been charac-
terized for several species of penaeids [Penaeiis japonicus.
Vazquez Boucard et al. 1986; P. monodon. Quinitio et al. 1990; P.
semisidcatiis. Browdy et al. 1990; Tom et al. 1992 and Lubzens et
al. 1997; P. monodon, Chen & Chen 1993; Chang et al. 1993 and
Chang et al. 1994; P. chinen.sis. Chang & Jena 1995; Chang et al.
1996).
In vertebrates and several invertebrates, vitellogenin trans-
ported into the blood or hemolymph is considered the precursor of
vitellin. In the Crustacea, it is still uncertain whether vitellogenin
is the precursor of vitellin, even though intraovarian synthesis has
been demonstrated by Yano and Chin/ei (1987), Rankin el al.
(1989), Fainzilber et al. (1992), and Khayat et al. (1994). Recent
molecular studies showed that specific vitellogenin niRNA was
expressed in both the ovary follicle cells and the hepalopancreas
parenchymal cells of penaeid shrimp P. japouicas (Tsutsui et al.
2000) and Macrohrachiiim rosenbergii (Soroka et al. 2000). Vi-
tellin has been found in subepidermal adipose tissue of penaeids P.
japonicus (Vazquez Boucard 1985), P. longiro.stris (Tom et al.
1987a), and P. semi.\itkatus (Fainzilber et al. 1992), but their role
in active synthesis of these compounds is not confirmed. Fainzil-
bert et al. (1992) confirmed the double synthesis of vitellin. in the
hepalopancreas and the ovary, but in different proportions depend-
ing on ovarian maturity. Khayat et al. (1994) suggested that ovarian
vitellin and hepalopancreas vitellogenin are the products of one gene.
Litopenaeus vannamei is an important commercial species in
Mexico and other countries. The failure of ovarian maturation is an
obstacle for reproduction control. Accordingly, purification and
characterization of vitellin from mature ovaries of L. vannamei
were the objectives of this study.
Using this information, we will be able to undertake molecular
studies in vitellogenin gene expression by several tissues of
*Corresponding author. E-mail: cboucardCacibnor.mx
Litopenaeus vannantei. The complete primary structure of vitello-
genin has been elucidated for several crustaceans. The vitellogenin
amino acid sequences of Marsupenaeus japonicus (Tsutsui et al.
2000), Metapenaeus ensis (Tsang et al. 2003), Penaeus semisul-
catus (GenBank accession number AY05I3I8). Chera.x cjuadri-
carinatus (Abdu et al. 2002), and Macrobrachiimx rosenbergii
(Yang et al. 2002) share several conserved regions. These are irtore
than 2,500 residues long, and vitellins are derived from each
vitellogenin.
MATERULS AND METHODS
Preparation of Ovarian Homogenale
Mature (110 |j.m oocyte) and immature female prawns (35 |jim
oocyte) were obtained from Acuacultura Mar, La Paz, B.C.S.,
Mexico, Ovaries were rinsed and homogenized in glassware at 4°C
with 0.05 M Tris. 0.5 M NaCl, and 5 niM EDTA (pH 7.0). Pro-
lease inhibitor cocktail (Sigma P-2714) was added (0.005%) to the
extraction buffer, just before use. The homogenate was centrifuged
at 10.000 g for 15 min at 4"C (Beckman ultracentrifuge, Pasadena,
CA). The supernatant was frozen at -70°C until analysis.
Electrophoresis
For identification of vitellins in the \itellogenic female, the
ovary homogenates were separated by native PAGE on 6% poly-
acrylamide gel in TRlS-glycine buffer (pH 8.8). The vitellin frac-
tion was characterized by sodium dodecyl sulfate polyacrylainide
gel electrophoresis (SDS-PAGE; 7,5% polyacrylamide gel). A so-
lution of 0.5 M TRIS-HCI (pH 6.8). 1% SDS. 1% 2-mercaptoeth-
anol, I09f glycerol, and 0.05% bromophenol blue was used as
dissociation buffer. Molecular masses of native proteins and dis-
sociated subunit polypeptides were determined by comparison of
the relative mobility of molecules to those of molecular mass
markers. The molecular masses of polypeptides were determined
by native PAGE (precast gel gradient polyacrylamide 4-20% Bio-
Rad) with a kit containing midrange protein molecular mass stan-
dards: p-thyroglobulin (669 kDa), ferritin (440 kDa). catalase
(232 kDa). lactate dehydrogenase (140 kDa). and albumin (67
kDa; Pharmacia Fine Chemical. Uppsala. Sweden). The molecular
887
BOUCARD ET AL.
masses of standard proteins on SDS-PAGE were myosin (200
kDa). p-galactosidase (1 16 kDa). phosphorylase (97 kDa). serum
albumin (66 kDa). and ovalbumin (45 kDa; Bio-Rad. Richmond.
CA). The gel was stained with Coomassie brilliant blue R-2.5() and
Silver Stain Plus kit (Bio-Rad) for proteins. Sudan black B for
lipids, and periodic acid-Schiff s reagent for carbohydrates.
Preparation of Aniisera
Rabbits were immunized with 6'7r page purified L. vannamei
specific ovarian yolk polypeptides. Small gel portions were cut
vertically from both extremes and stained with Coomassie brilliant
blue R-250 to reveal the migration distance of proteins. These
portions were placed next to the rest of the gel without stain at the
same level, and the gel vitellin band was cut horizontally. The two
proteins (50 |jLg) separated from the polyacrylamide gel were ho-
mogenized with NaCl (0.9%), emulsified with complete Freund's
adjuvant, and injected at multiple sites on the backs of rabbits.
Boosters of 120 |xg of antigen emulsified with incomplete Fre-
und's adjuvant were injected at intervals of two weeks.
Purification of Vitellin
Litopenaeus vannamei vitellin was purified according to Chang
et al. (1996. 1993). The ovarian extracts were gel filtered in a
Sepharose CL-2B column (Pharmacia Fine Chemicals. Uppsala.
Sweden: 100 cm x 1.8 cm i.d.) equilibrated in 0.01 M TRIS buffer
with 2 mM phenylmethylsulphonyl tfuoride (pH 7.0). and eluted in
the same buffer at flow rate 18 niL/h. Effluent was collected in
2.4-mL fractions, and the absorbance of each fraction was mea-
sured at 280 nm. Each concentrated peak (PM 10 membrane. Ami-
con. Danvers. MA) was analyzed by immunodiffusion precipita-
tion and PAGE {5% gel). The vitellin peak was applied to a hy-
droxylapatite column (Bio-Rad. Richmond. CA. #732-0085) using
a 0.01 M potassium phosphate buffer (PPB), pH 7.0, with 2 mM
phenylmethylsulphonyl fluoride with stepwise gradients of 0.01
M. 0.10 M. 0.20 M. and 0.35 M. The flow rate was 18 mL/h and
the fraction size was 2.4 mL. Immunoprecipitation and PAGE of
concentrated peaks (PM 10 membrane. Amicon, Danvers, MA)
were also analyzed. The concentrated vitellin peak was further
separated by high-performance liquid chromatography (HPLC.
Beckman Spherisorb) equilibrated with 0.2 M sodium sulfate in
0.1 M sodium phosphate pH 6.5. The tlow rate was 1 mL/min.
Immunologic Procedures
Immunodiffusion precipitation proceeded according to Outch-
terlony (1948). Agar gel (1%, 2 mm thick) was prepared on a glass
slide. Vitellin antisera and samples were put in separate wells 0.9 cm
apart and put in a humid chamber (4"C) for 48 h. After washing (3 x
6 h) in 0.9% NaCl, the gel was stained with Coomassie blue R250.
For crossed Immunoelectrophoresis analysis of vitellins in the
vitellogenic female, the homogenates of ovaries were separated by
agarose gel (1%) in 0.02 M veronal buffer (pH 8.6). The gel portion
enclosing the antigen was cut and placed on a glass slide (6.5 x 10
X 0.1 cm). The slide was then covered with 6.5 ml of \% agarose
in veronal buffer and 1% anti-vitellin antibodies of L. vannamei.
After 18 h of migration at 2 volts/cm. the slide was washed with
9% NaCl and colored with Coomassie blue.
The immunoreactivity of the subunits of vitellin with vitellin
antisera was examined by Western blotting. After purification of
vitellin in a hydroxylapatite column, effluent containing the fourth
peak was analyzed by SDS-PAGE (7.5% polyacrylamide gel in
TRIS-glycine buffer. pH 7.2, 1% SDS). Proteins in the polyacryl-
amide gel were transferred to polyvinylidene diflouride (PVDF.
Immobilon transfer membranes. Bio-Rad. Richmond. CA) with a
mini transblot electrophoretic transfer cell (Bio-Rad #170-3930)
using 25 mM TRIS, 192 mM glycine, 20% methanol buffer. Ni-
trocellulose paper was immersed in the following solutions: 5%
blotting grade blocker (Bio-Rad # 170-6404) in TBS buffer (0.15
M NaCl. 10 niM. TRIS). antisera against vitellin (1/3000). and
goat anti-rabbit IgG-alkaline phosphatase conjugate (1/3000).
Color was developed using diaminobenzidine in TBS buffer.
N-Tenninal Amino Acid Sequence
After purification, the vitellin was analyzed by SDS-PAGE:
7.5% polyacrylamide gel in Tris-glycine buffer. 1.5 M. thioglyco-
lateO.l M(pH7.2). 10% SDS. A solution of Tris-Hcl. 312.5 mM;
Na2 EDTA. 10 mM (pH 6.9): 15% SDS: and 0.5 M sucrose was
used as dis.sociation buffer at 37°C for 10 min. The proteins in the
polyacrylamide gel were transferred to Sequi Blot PVDF (polyvi-
nylidene diflouride) membrane (Bio-Rad. Richmond. CA) with a
mini transblot electrophoretic transfer cell (Bio-Rad #170-3930)
using a 25 niM TRIS. 192 M glycine. 20% methanol buffer. The
membrane was stained for 5 min with PVDF Coomassie blue
R-250. and destained for 10-15 min with PVDF destain solution.
The 100 and 60 kDa bands were cut and N-terminal sequenced in
a protein/peptide sequencer at the Molecular Medicine Laboratory
Biotechnology Institute UNAM, Mexico (DF).
RESULTS
The native gel electrophoresis patterns of ovarian extracts (pre-
cast gel gradient polyacrylamide 4-20%) of mature and non vitel-
logenic L. vannamei females showed a specific protein from ma-
ture females with an apparent molecular mass of 500 kDa. The
protein contained carbohydrates and lipids, based on staining by
Sudan black B (Fig. 1) and periodic acid Schiff s reagent, respec-
tively. However, when the sample was centrifuged before electro-
phoresis, and the gel was stained with Sudan black B, we observed
two female-specific lipoproteins of nearly the same molecular size
(Fig. 1 ). The crossed immunoelectrophoretic pattern of the ovarian
extract of mature L. vannamei females with antiserum against
ovarian extract of the same species is shown in Fig. 2. There were
two precipitation lines in the ovarian extracts of vitellogenic
shrimp (Vtl and Vt2).
Two proteins peaks in ovarian homogenate of mature females
were obtained from gel filtration chromatography in a Sepharose
CL-2B column (Fig. 3). The concentrated second peak showed a
single band considered vitellin (500 kDa) and another more nega-
tively charged non-vitellogenic band. The second peak from gel
filtration chromatography had a specific immunodiffusion precipi-
tation line that reacted with antisera against vitellin of L. van-
namei. but the first peak did not (results not shown). The concen-
trated second peak was separated into four peak fractions, by hy-
droxylapatite column chromatography. Electrophoretic analysis
revealed two proteins with very approximate migration coefficient
(Fig. 4). The third (Vtl) and fourth (Vt2) peaks separated by
hydroxylapatite column chromatography, the mature female
hemolymph. and vitellogenic ovarian extracts were recognized by
polyclonal anti-Vt antibodies raised against L. vannamei (Fig. 5).
To confirm these results, the third and fourth peaks were com-
bined, concentrated, and separated by reverse-phase chromatogra-
VlTELLINS FROM OVARIES IN SHRIMP L VANNAMEI
889
Vt
kDa
669
440
232
140
67
12 3 4
Figure 1. Native-PAGE of Litopenaeus mnnamei o\arits. Precast gel
gradient 4-20'7f. (B) Lane 1: silver stain ovarian extract mature
females: Lane 2: silver stain ovarian extract nonvitellogenic females;
Lane 3: molecular marker: Lane 4: Sudan black B stained ovarian
extract mature females after centrifugation.
phy (HPLC). revealing two peaks with retention times of 14.53
and 19.55 niin (Fig. 6).
Characterization of Purified Vitellin
The third (Vtl ) and fourth (Vt2l peaks from the hydroxylapa-
tite column, analyzed by SDS-PAGE, both showed six polypeptide
subunits. The molecular weights of the subunits were estimated at
60. 90. 95. 100. 140. and 160 kDa. To confirm the subunits cor-
responding to each vitellin. Western-blotting was conducted with
the anti-Vt antibodies raised against L. vaniiamei. thus confirming
that they were molecules composed of six similar polypeptide
subunits (Fig. 7).
Protein Sequencing
We sequenced the N-terminal ends of the 100 kDa and 60 kDa
subunits, and two amino acid residue sequences. PI (GQVSLA-
Vt1
Vt2
Figure 2. Crossed immunoelectrophoretic pattern of ovarian extract
L. vannamei using vitellin specific polyclonal antibodies. (\ tl and V't2
vitellins)
Fraction number
Figure 3. Elution profiles of ovarian homogenates from a Sepharose
CL-2B gel filtration column equilibrated and eluted with 0.01 M Tris
buffer. Flow rate: 18 niL/h. Fraction size: 2.4 ml. P.4GE (5% gel). Blue
Coomassie stain. Peak 1: High molecular weight proteins. Peak 2:
Vitellin (Vt) and contaminate proteins.
PEFALGXTVE) and P2 (APXGADVPSKG) respectively, were
obtained. PI and P2 were aligned to each vitellogenin reported
(Fig. 8), and similarity specific to two regions was observed. The
conserved residues ""P and ^"S aligned with P2. and the conserved
residues """^G and "'"E aligned with PI. This suggests that PI and
P2 are derived from a vitellogenin, as in M. japonicus (Tsutsui et
al. 2000) and Metapenaeus ensis (Tsang et al. 2003).
DISCUSSION
One specific protein, with an approximate molecular weight of
500 kDa, was identified by electrophoresis (PAGE 6% and gradi-
ent gel 4-20% ) in the ovaries of Litopenaeus vannamei females in
vitellogenesis. The characteristics of this fraction (lipo-glyco-
protein) were similar to those of penaeid vitellins. but did not exist
100
50
l»M
t4-t6
Fraction number
Figure 4. The second peak fraction from Sepharose column was frac-
tioned in a hydroxylapatite column equilibrated with 0.01 M PPB
buffer. Step-wise gradients of 0.01 M, O.IOM, 0.20.M. and 0.35\I PPB
buffer. Flow rate: 18 mL/h. Fraction size: 2.4 ml. Peak 1 and 2: con-
taminate proteins. Peak 3 (Vtl) and 4 (Vt2l.
890
BOUCARD ET AL.
A
Figure 5. Inimunoprecipitation of mature female ovary (11 mature
female lieinolymph (2, 6| hemolvmph male {^) third peak hvdroxyl-
apatite column (4) fourth peak hydroxylapatite column (5) reacted to
specific antiserum against vitellin (7) from L. vanmiiiiei.
in the ovaries of immature females. Only one form of vitellin has
been detected in Panipeiiaeiis langimstris (Tom et al. 1987b). P.
monodon (Quinitio et al. 1990, Chang et al. 1993), P. semisidcatus
(Tom et al. 1992, Lubsenz et al. 1997), Metapenaeiis ensis (Qiu et
al. 1997). P. Japonicus (Vazquez Boucard 1986, Kawazoe et al.
2000), and P. vaiiinimei (Tom et al. 1992, Garcia Orozco et al.
2002).
However, when we centiifuged the mature female ovary
sample before loading the electrophoretic gel (gradient 4/30%),
and stained it with Sudan black B, we confirmed two lipoproteins.
Absorbance
0.0400 -
0.0300 -
Vt2
\
vtl
/
^vti
\Vt2
0.0200 -
i
0.0100-
0.0000
_J
u
1
10.00
1
20.00
1
30.00
Minute
Figure 6. Analytical HPLC of combined third Vtl and fourth Vt2
peak obtained from a hydroxylapatite column. Flow rate, absorbance
at 280 nm.
B
kDa
200
'*«-'=?^!»tl"-
:,,i;:^«.j..i :;*.;,' ftjf.
-116
■a, .,, . 97
-66
>iV;v*,ii':vi;;K;
,-^n'iiVf;,-'V':>''SJ;i
1 2
Figure 7. \. Western blotting analysis of vitellins purified by hydrox-
ylapatite column (third and fourth peakl; B. SD.S-PAGE (7.5 "7, ) of (I)
third and fourth peak of hydroxylapatite column. (2| molecular
marker
Similarly, the crossed immunoelectrophoresis showed two lines of
precipitation with L. vaniuwiei anti-vitellin antibodies. After fur-
ther separation by hydroxylapatite column and HPLC, two main
vitellin peaks were seen also, which might correspond to the vi-
tellins detected by native gradient gel electrophoresis and crossed-
immunoclectrophoresis. Denatured SDS gel electrophoresis of the
native vitellin isolated (Vtl and Vt2) showed six subunits with
VTG_
VTG^
vtg'
vtg]
vtg'
Pi'
MARJA:
PENSM;
'CHRQU :
^METEN :
^MACRO :
LPEVA :
LPEVA:
20
JL
;£NGADLPRCSrE.
;f NISADLPRCSrE.
I P FGOTTPVC S IE .
;PElEDEAPRCSrE.
^JHPSGTNLCSKE.
;PXGADVP. .SKG.
635
.AFAF(a<!GAD]l£
.apafg*;gagi£
.spsag\gagie
. SAAFEb :G KD\ E
.APTFG\
.GQVSL. .APEFAIGJCT\'E
■GA<3M
Figure 8. Alignment of crustacean vitellogenins. VTGM.ARJA: Mar-
mpenaem japonicus, GB AB0337I9 (BAB(I1568) (Tsutsui et al.. 2(I(MI);
VTG.PENSM: Penaeiis iemisulcaliis .Gii AV(I513I8 (AAL12620);
VTG_CHRQll: Cherax quadrkarinalus. GB AF306784 (AAGI7936)
(Abdu et al., 21)02): VTG^IETEN: Metapenaeiis ensis, GB AF548364
(AAN4(I701) (Tsang et al., 2003); VTG_MACRO: Macrohrachiiim
rosenbergii. GB AB056458 (BAB69831 ) (Yang et al.. 2000);. PI and P2
LPEVA: peptides 1 and 2 n{ Litopenaeiis vannamei
VlTELLINS FROM OVARIl-S IN SHRIMP L. VANNAMF.I
891
molecular weights of 60. 90. 95. 100. 140. and 160 kDa. The
polypeptide subunits of each vitellin presented similar molecular
masses. Chang et al. (1996). using our purification methods, de-
tected two forms of native vitellin in P. chineiisis also. The mo-
lecular mas.ses they reported were 380 and 500 kDa. However, the
authors miscalculated the molecular masses because they used
native molecular weight markers designed for gradient gel elec-
trophoresis in an S^r native PAGE gel. where proteins are sepa-
rated by differences in charge, not by weight. Serrano Pinto et al.
(2003) observed two forms of vitellin in ovaries and eggs at dif-
ferent stages of development in freshwater crayfish Cherax cjiicul-
rkaiiniitus with a similar \itellin polypeptide composition.
Electrophoresis, analytical ultracentrifugalion. and chromatog-
raphy have been used for the isolation and purification of vitellins
of penaeid shrimp. The common goal was to increase vitellin
purity, and prevent or retard its degradation. Vitellins are very
susceptible to dissociation during and after isolation, so it is im-
portant to treat sample with protease inhibitors, and keep them at
low temperature during extraction, purification, and storage to de-
lay degradation. In this study, besides adding protease inhibitors to
the tissue extracts and chromatography buffers, we purified and
concentrated the vitellin under refrigeration (4"C).
Sixmajorvitellinsubunitsof60, 90, 95, 100, 140, and 160 kDa
were observed in L. vannamei during the present study. Tom et al.
(1992) separated ovary proteins in same species by filtration gel
and ion exchange chromatography, and concluded that native vi-
tellin is present in this species in one form with a molecular weight
of 289 kDa. Only two subunits were detected in this study, and
their molecular weights were not determined. Tom et al. used
proteolytic inhibitors neither in the preparation of extracts, nor
during separation of proteins by chromatography, and this could
have caused protein hydrolysis. Garcia Orozco et al. (2002) de-
tected one native vitellin in L vannamei with an apparent molecu-
lar weight of 388 kDa. The method used by these authors to
homogenize the tissues (30,000 rpm for 25 s. three times), and to
concentrate the elutions separated by chromatography (ultrafiltra-
tion, PM 100 membrane, Amicon) could have caused degradation.
Using western blotting, we were able to identify the inimuno-
reactivity of the six subunits using the antivitellin antibodies of L.
vannamei ovaries. The PI and P2 peptides from N-terminal 60 kDa
and 100 kDa polypeptides were highly similar to regions of proline
20 and glycine 635 residues of crustacean vitellogenins. Both re-
gions were highly conserved, which suggests specific processing
sites to produce vitellin subunits. More molecular analysis is
needed to corroborate this hypothesis.
ACKNOWLEDGMENTS
This research was supported by grants from CONACYT (Proj-
ect # 32597-N/2000). We thank Ariel Cruz Ramirez. M. C. Martin
Ramirez Orozco. and B. Mario Burgos Aceves for assistance with
chromatography and electrophoretic analysis. Dr. L. Possani
Postay for invaluable help in the sequence analysis.
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genin and its e.xpression in the hepatopancreas and ovary during vitel-
logenesis in the kuruma prawn Penaeus japonicus. Zoolog. Sci. 17:
651-660.
Vazquez Boucard. C. G. 1985. Identification preliminaire du tissu adipeux
chez le crustace Decapode Penaeus japonicus Bate, a I'aide d'anticorps
antilipovitelline. C.R. Acad. Sci. Sec. Ill 300:95-97.
Vazquez Boucard. C. G.. H. J. Ceccaldi. Y. Benyamin & C. Roustan. 1986.
Identification, purification et caracterisation de la lipovitelline chez un
Crustace Decapode Natantia Penaeus japonicus (Bate). J. E.xp. Mar.
Biol. Ecol. 97:37-50.
Yang. W. J.. T. Ohira. N. Tsutsui, T. Subramoniam, D. T. Huong. K. Aida
& M. N. Wilder. 2000. Determination of amino acid sequence and site
of mRNA expression of four vitellins in the giant freshwater prawn
Macrohrachiuni rosenbergii. J. E.xp. Zool. 287:413—422.
Yano. 1. & Y. Chinzei. 1987. Ovary is the site of vitellogenin synthesis in
kuruni prawn Penaeus japonicus. Comp. Biochem. Physiol. 868:213-
21S.
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The National Shellfisheries Association (NSA) is an international organization of scientists, manage-
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Watts. R. J.. M. S. Johnson & R. Black. 1990. Effects of re-
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lates. Amsterdam: Elsevier, pp. 343-348.
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John Brake. Ford Evans, and Chris Langdon
Is beauty in the eye of the beholder? Development of a simple method to desciibe desirable shell shape lor the Pacillc
oyster industry 767
Josiah H. Pit and Paul C. Souihgate
Should slow growing pearl oyster (Pimludu margarilifera) Spat ("Runts") be discarded? 773
Takeshi Handa and Ken-ichi Yamamoto
Corrosion casting of the digestive diverticula of the pearl oyster. Pimuida fucalii marleiisii ( Mollusca: Bivalvia) 777
John A. H. Benzie, Carolyn Smith, and Ketut Sugama
Mitochondrial DNA reveals genetic differentiation between Australian and Indonesian pearl oyster PinctuJa maxima (Jameson
1 90 1 ) populations 781
Alexander Tewjlk and Hector M. Guzman
Shallow-water distribution and population characteristics of Sirombus gigas and S. coslatus
(Gastropoda: Siromhidae) in Bocas del Toro. Panama 789
Hideki Takami, Daisuke Muraoka, Tomohiko Kawamura, and Yoh Yamashita
When is the abalone Hiilioiis discus luinnai Ino 1953 first able to use brown macroalgae? 795
PROCEEDINGS OF WORKSHOP ON REBUILDING TECHNIQUES FOR ABALONE IN BRITISH COLUMBIA
Preface 803
Susan M. Bower
Update on emerging abalone diseases and techniques for health assessment 805
A. Campbell, J. Lessard, and G. S. Jamieson
Fecundity and seasonal reproduction of northern abalone. Halialis kumlschatkumi. in Barkley Sound. Canada 811
Bart Defreitas
Estimating juvenile northern abalone {Halioiis kamtschaikana) abundance using artificial habitats 819
Thomas B. McCormick and Jennifer L. Brogan
Early reproduction in hatchery-raised white abalone. Haliotis sorenseni. Bartsch. 1940 825
T. Tomascik and H. Holmes
Distribution and abundance of Haliotis kamtsclwtkcoiu in relation to habitat, competitors and predators in the Broken Group Islands.
Pacific Rim National Park Reserve of Canada 83 1
Ruth E. Withler, Akin Campbell, Shaorong Li, Doug Brouwer, K. Janine Supernault, and Kristina M. Miller
Implications of high levels of genetic diversity and weak population structure for the rebuilding of northern abalone in British
Columbia. Canada 839
Status of Stewardship Projects 849
Susan A. Little and Winsor H. Watson, UI
Size at maturity of female American lobsters from an estuarine and coastal population 857
Kelly C. Palacios and Steven P. Ferraro
Green crab tCuniiuis mueiuis Linnaeus) consumption rates on and prey preferences among four bivalve prey species 865
Michael Sheppard, Anna Walker, Marc E. Frischer, and Richard F. Lee
Histopathology and prevalence of the parasitic dinoflagellate, Hemalodinium sp. in crabs iCallinecles sapidus. Callinectes similis.
Neopanope sayi. Libinia emarginata. Menippe mercenaria) from a Georgia estuary 873
Charles E. Epifanio, Ana L Dittel, Raymond A. Rodriguez, and Timothy E. Targetl
The role of macroalgal beds as nursery habitat for juvenile blue crabs. Callinecles sapidus 881
Celia Vazquez-Boucard, Humberto Mejia-Ruiz, Fernando Zamudio, Vania Serrano-Pinto, and Hector Nolasco-Soria
Isolation and molecular characterization of vitellin from the mature ovaries of the prawn Litopenaeus vannumei 887
COVER PHOTO: Fouling ot scallop [Placopecten magelhmicus) cages in Bayporl, Nova Scotia. Growth of scallops in those cages
was reduced to 60% of normal. See paper by Carver and Mallet (p. 619). Photo courtesy of Carver and Mallett.
2128 056
The Journal of Shellfish Research is indexed in the following: Science Citation Index®, Sci Search®, Research Alert®, Current
Contents®/Agriculture, Biology and Environmental Sciences, Biological Abstracts, Chemical Abstracts. Nutrition Abstracts, Current
Advances in Ecological Sciences. Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography,
Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts.
JOURNAL OF SHELLFISH RESEARCH
Vol. 22, No. 3 December 2003
CONTENTS
J. Evan Ward
Honored Life Member: Melbourne Romaine Carriker 611
John N. Kraeuter and Mark H'. Luckenhach
Honored Life Member: Michael Castagna 615
William H. Hargis, Jr.
Honored Life Member: Dexter Steams Haven 614
C. E. Carver, A. Chisholm. and A. L. Mallet
Strategies to iinligalc the mipact of Ciima intestinalis (L.) biofouling on shellfish production 621
A. R. LeBlanc, T. Landry, and G. Miron
Fouling organisms of the blue mussel Myiiliis ediilis: Their effect on nutrient uptake and release 633
Melita Peharda, Alen Soldo, Armin Pallaoro. Sanja Matte, and Perica Cetinic
Age and growth of the Mediterranean scallop Pecteii jacohaciis (Lmnaeus 1 758) in the northern Adriatic Sea 639
Omar Defeo and Nicolas Gutierrez
Geographical patterns in growth estimates of the scallop. Zygochhimys pataKonica. with emphasis on Uruguayan waters 643
G. Roman, A. Louro, and J. P. de la Roche
Intermediate culture of king scallop {Pecien miLximiis) in suspension in cages: Effect of slocking density and depth 647
Xiaoyu Kong, Ziniu Yu, Yajun Liu, and Linlin Chen
Inlraspecific genetic variation ni mitochondrial I6S nbosomal gene of zhikong scallop Chlaiiixs farreri 655
Ralph A. Elston, Christopher F. Dungan, Theodore R. Meyers, and Kimberly S. Reece
Perkinsus sp. infection risk for Manila clams. Venenipis philippiiuinoii (A. Adams and Reeve, 1850) on the Pacific coast of North
and Central America ^6 1
Ralph A. Elston. Daniel P. Cheney, Brian F. MacDonald, and .Andrew I). Suhrhier
Tolerance and response of Manila clams. Vencrupis philippiiuintm (A. Adams and Reeve. 1850) to low salinity 667
Olga L. Aracena, Irene M. IJpez, Javier Sanchez, Angelica M. Carmona, Lucila Medina, and Alejandro Saavedra
On two new macroscopic indexes to evaluate the reproductive cycle of Ensis machu ( Molina, 1 782) 675
Micaela Schnitzler Parker, Peter A. Jumars, and Larry L. LeClair
Population genetics of two bivalve species {Prutolhuca stiiniinca and Mac<niui hallhua) in Puget Sound, Washington 681
Richard R. Alexander and Robert M. Baron
Shell repair of mechanically induced fractures in Mercenaria mercenaria under experimentally suboptimum conditions 689
Jonathan H. Grabowski, Sean P. Powers, and Mark Hooper
Identification and incorporation of growth and sur\i\al bottlenecks in economic models of northern quahog (Hard clam), Mercenaria
mercenaria mariculture "^'
Melita Peharda, Jaksa Bolotin, Nedo Vrgoc, Nenad Jasprica, Ana Bratos, and Bosko Skaramuca
A study of the Noah's ark shell {Area noae Linnaeus 1758) in Mali Ston Bay, Adriatic Sea 705
Jorge Cdceres-Martinez and Rebeca Vdsquez- Yeomans
Presence of giant polymorphic cells in Cnissustrea gigas cultured in Bahia Falsa. Baja California. NW Mexico 711
Clothilde Heude Berthelin, Bruno Fievet, Gael Leclerc, Pierre Germain, Kristell Kellner, and Michel Mathieu
In vivo and in vitro approaches to the analysis of glycogen metabolism in the Pacific oyster, Crassostreci gigas 715
Jorge Chdvez-Villalba, Jean-Claude Cochard, Marcel le Pennec, Jean Barret, Martha Enriquez-Diaz, and Carlos Cdceres-Martinez
Effects of temperature and feeding regimes on gametogenesis and larval production in the oyster, Crussostrea gigas 721
Patrick Baker
Two species of oyster larvae show different depth distributions in a shallow, well-mixed estuary 733
M. L. Wintermyer and K. R. Cooper
Dioxin/Furan and polychlorinated biphenyl concentrations in eastern oyster iCnissoslrea virginica. Gmelin) tissues and the effects of
EGG fertilization and development ^- '
George R. Abbe and Brian W. Albright
An improvement to the determination of meat condition index for the eastern oyster, Cra.'^soslred virginica {Gmelin 1 79 1 ) 747
Kimberly A. Cressman, Martin H. Posey, Michael A. Mallin, Lynn A. Leonard, and Troy D. Alphin
Effects of oyster reefs on water quality in a tidal creek estuary '-'-
Isabelle Boutet, Arnaud Tanguy, Michel Auffret, Nedzad Mujdzic, and Dario Moraga
Expression of HSP 70 in experimentally metal-exposed European fiat oysters Ostrea cdulis ^63
CONTENTS CONTINUED ON INSIDE BACK COVER
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