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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 
5735 Hitchner Hall 
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 

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 Kmgdom 

Leroy Creswell (2003) 
University of Florida/Sea Grant 
8400 Picos Road. Suite 101 
Fort Pierce, Florida 34945-3045 

Lou D'Abranio (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 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 
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 



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 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? 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 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 differences" 


2 


1 


1 





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 differences" 


2 


2 


1 





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 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 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 differences'' 







2 


3 





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 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 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 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 differences" 









2 


4 





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 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 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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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 


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1 




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, anhV?^*''"'" 


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UIAN 


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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 

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ariakenO 

virgl 



gigasl 

talienwl 

gigasO 

talienw2 

plical 

ariakenl 

ariaken2 

ariakenO 

virgl 



gigasl 

talienwl 

gigasO 

talienw2 

plical 

ariakenl 

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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 . . 
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.A . . 

.A . . 
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.T G. 



240 
AGATACCTTC ATTTAAAAGT TAGACAAAAA GACCCCGTGC AACTTTGAAA A--TTAACTT TATTCAGGAG TAAAAGATTT 



. .A. . 
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320 

TTAGGTGGGG CGCCTAGAAA GCAAG-TCTA ACCTTT-CTG AATAACT--A ACTCTTTCCG GATTTGACCC GATTATATTC 



. -C. 
. -C. 



. AA . T C . GT . 



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GT 

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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 

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virgl 

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talienwl 

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talienw2 

talienw3 

talienw4 C. .G. 



















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r 






















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talienwS C. .G. 

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ariakenl A G. . A. .G. 

plica2 A G.. A..G. 

plica3 A G.. A..G. 

plica4 A G. . A. . . . 

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240 


GTAACTGGCT 


TATCCCTTTG 


ATGCTTCTAG 


TAGCAGACAT 


GCAATTTCCT 


CGATTAAATG 


CATTTAGATT 


TTGAGTTTTG 


A ' '. . . 


_ A 






. . .T. 




A 






.A. . 






. .T. 









r 




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, . C 












c 






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ariaken3 A G. . A. .G. 

plica6 
plica7 
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virgl 

gigasl 

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gigasO 

taiienwZ 

taJ ienw3 

talienwj 

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plical 

ariakenl 

plica2 

plica3 

plica4 

ariaken2 

plicaS 

ariaken3 

plica6 

plica7 

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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()81 














0.0108 













C. plicanda 


0.0233 


0.0174 











0.1113 


0.1072 











C. ariakensis 


0.024? 


0.01 S5 


0.0013 









0.1143 


0.1 100 


0.0059 









C. ariakensisO 


0.0450 


0.04S7 


0.0444 


0.0462 







11.1619 


U.1639 


0.1652 


0.1691 







C. virginica 


0.1636 


0.1 60S 


0.1654 


0.1673 


0.1937 





0.2569 


0.2573 


0.2510 


0.2513 


0.2849 






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 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 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 (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 

38 
79 
45 




S 


307 


355 259 







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: 



= 



if V,* > and </, > 



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 



CO 


SI 




2 


3 



2 


4-» 


z 




^ 


4-1 
C 


U^ 


c 


3 






■t^ 


^mi 


u 





cd 


u 





«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 to 6 to indicate frequency where = 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, 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. 



LITERATURE CITED 



ABC News. 1998. Oysters Cause Illnesses. Retrieved July. 23, 2U01 from 
http://abcnews.go.com/sections/living/DailyNews/oysters980723.html. 

Billups. A.L. 2001. Seafood Safety. University of Florida, Research and 
Graduate Programs. Explore Magazine Vol. 2{ 1 ). March 3 1 . 

Center for Science in the Public Interest (CSPI). 2000. FDA Inaction on 
Raw Oysters Means More Deaths on the Half Shell. Retrieved May 10. 
2001 from http://www.cspinet.org/new/oysters.html. 

Center for Disease Control and Prevention (CDC). 1993. Multistate Out- 
break of Viral Gastroenteritis Related to Consumption of Oysters — 
Louisiana, Maryland, Mississippi, and North Carolina. 1993. Morbidity 
and Morlality Weekly Report Series 42(49). 

Cheng, H. & O. Capps, Jr. 1988. Demand analysis for fresh and frozen 
finfish and shellfish in the United States. Am. J. Agri. Ecoii. 70:533- 
542. 

Cragg, J. 1971. Some statistical models for limited dependenl \ariahlcs 
with application to the demand for durable goods. Ecimimietrua 39: 
829-844. 

Gempesaw, C. M. 11. J. R. Bacon, C. R. Wessells & A. Manalo. 1995. 
Consumer perceptions of aquaculture products. Am. J. Agr. Econ. 77: 
1306-1312. 

Greene, W. 1995. Limdep version 7.0 user's manual. Econometric Soft- 
ware, Inc. 

Hanson, T.. L. House. S. Sureshwaran. B. Posadas & A. Liu. 2002. Opin- 
ions of U.S. Consumers Toward Oysters: Results of a 2000-2001 Sur- 
vey. Mississippi State University. Department of Agricultural Econom- 
ics, AEC Research Report 2002-005. 

Holland, D. & C. R. Wessells. 1998. Predicting consumer preferences for 
fresh salmon: the influence of safety inspection and production method 
attributes. Agr. and Res. Econ. Review 27:1-14. 



Manalo. A. B. & C. M. Gempesaw, li. 1997. Preferences for oyster attrib- 
utes by consumers in the U.S. Northeast. J. Food Dislrih. Res. 28:55- 
63. 

United States Census Bureau. U.S. Census 2000. Retrieved Februarv 4. 
2003 from http://www.census.gov/main/www/cen2000.htinl. 

United States Department of Agriculture. Economic Research Service. 
1999. Food Consumption. Prices, and Expenditures. Edited by J. Put- 
nam and J. Allshouse. 

United States Department of Commerce. National Oceanic Atmospheric 
Administration. National Marine Fishery Service. 2001. "Fisheries of 
the U.S., 2000." Current Fishery Statistics No. 2000. Silver Spring. 
MD. 

United States Department of Commerce. National Oceanic Atmospheric 
Administration, National Marine Fishery Service 2002. "Fisheries of 
the U.S.. 2001." CuiTent Fishery Statistics No. 2001. Silver Spring. 
MD. 

Wessells. C. R. & D. Holland. 1998. Predicting consumer choices for 
farmed and wild salmon. Aqtia. Eton, and Manag. 2:49-59. 

Wessells, C. R. & J. G. Anderson. 1995. Consumer willingness to pay for 
seafood safety assurances. J. Consiim. Affairs 29:85-107. 

Wessells. C. R., S. F. Morse, A. Manalo & C. M. Gempesaw. li. 1994. 
Consumer Preference for Northeastern Aquaculture Products: Repon 
on the Results from a Survey of Northeastern and Mid-Atlantic Con- 
sumers. Department of Resource Economics. University of Rhode Is- 
land. Rhode Island Experiment Station Pub. No. 3100. 

The Worid Almanac and Book of Facts. 1999. Mahwah. NJ: Wodd Al- 
manac Books. 

Yen, T. S. & L. C. Huang. 1996. Household demand for finfish: A gen- 
eralized double-hurdle model. / of Ag. and Res. Econ. 21:220-234. 



Juiinuil oj Shellfish Kcseanh. Vol. 22. No. 1. fil-67, 2U03. 

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 



























Recruits 




























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 





3 





3 


1 





1 




Recruits 


11 


1 





2 

















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 





1 


1 




Recruits 


8 


6 


2 


7 


2 


3 











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 





3 




Recruits 


5 


4 


6 


1 


5 


2 








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 





.^ 


"> 





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 







0.50 

0.53 

0.53 







High 


Low 


Control 


2.25 


0.75 




2.67 


0.80 




0.27 


0.53 


0.27 



















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 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- 
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Carriker. M. R. 1961. Interrelation of functional morphology, behavior, 
and autecology in early stages of the bivalve Mercenaria mercenaria. 
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Craig. M. A. & T. J. Bright. 1986. Abundance, age distributions and 
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Day, E. A. 1987. Substrate type and predatory risk: effects ot mud crah 
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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. 



LITERATURE CITED 



Aller. R. C. & R. E. Dodge. 1974. Animal-sediment relations in a tropical 
lagoon. Discovery Bay. Jamaica. / Mar. Res. 32:209-232. 

Bock, M. J. & D. C. Miller. 1995. Storm effects on particulate food re- 
sources on an intertidal sandflat. J. E.xp. Mar. Biol. Ecol. 187:81-101. 

Boschi. E. E. 1964. Los Crustaceos Decapodos Brachyura del litoral bo- 
naerense (R. Argentina). Bol. Inst. Biol. Mar. Mar del Plata 6:1-99. 

Botto. F. & O. O. Iribarne. 1999. The effect of the burrowing crab Cluis- 
magnathus granulata on the benthic community of a Southwestern 
Atlantic lagoon. J. Exp. Mar. Biol. Ecol. 241:263-284. 

Botto. F. & O. O. Iribarne. 2000. Contrasting effects of two burrowing 
crabs {Chasmagnatlms granulata and ilea uniguayensis) on sediment 
composition and transport in estuarine environments. Est. Coast. Shelf 
Sci. 51:141-151. 

Brenchley, G. A. 1981. Disturbance and community structure: an experi- 
mental study of bioturbation in marine soft-bottom communities. / 
Mar. Res. 39:767-790. 

Brenchley. G. A. 1982. Mechanisms of spatial competition in marine soft- 
bottom communities. J. E.vp. Mar. Biol. Ecol. 60:17-33. 

Grant, J. 1983. The relative magnitude of biological and physical sediment 
reworking in an intertidal community. ./. Mar. Res. 41:673-689. 

Gutierrez. J. L. & O. O. Iribarne. 1998. The occurrence of juveniles of the 



grapsid crab Chasmagnatlms granulata in siphon holes of the stout 
razor clam Tagelus pleheius. J. Shellfish. Res. 17:925-929. 

Gutierrez. J. L. & O. O. Iribarne. 1999. Role of Holocene beds of the stout 
razor clam Tagelus pleheius in structuring present benthic communi- 
ties. Mar. Ecol. Prog. Ser. 185:213-228. 

Gutierrez. J. L. & J. L. Valero. 2001. La almeja navaja y su partlcipacion 
en mecanismos ecologicos de comunidades intermareales mediante la 
produccion de valvas. In: O. O. Iribarne. editor. Reserva de Biosfera 
Mar Chiquita: Caracteristicas fi'sicas. biologicas y ecologicas. Mar del 
Plata, Argentina: Editorial Martin, pp. 121-128 

Holland. A. F. & J. Dean. 1977a. The biology of the stout razor clam 
Tagelus pleheius. 1. Animal-sediment relationships, feeding mecha- 
nism and community biology. Chesapeake Sci. 18:58-66. 

Holland. A. F. & J. Dean. 1977b. The biology of the stout razor clam 
Tagelus pleheius. 2. Some aspects of the population dynamics. Chesa- 
peake Sci. 18:188-196. 

Iribarne. O. O.. A. Bortolus & F. Botto. 1997. Between-habitat differences 
in bunow characteristics and trophic modes in the southwestern At- 
lantic burrowing crab Chasinagnathus granulata. Mar. Ecol. Prog. Ser. 
155:137-145. 

Murphy, R. C. 1985. Factors affecting the distribution of the introduced 



Spatial Variation in Body Mass of Tagelus plehfjus 



73 



bivalve, Mercenaria mercenaria, in a California lagdon The impor- 
tance of bioturbation. J. Mar. Res. 43:673-692. 
Newell. C. R. & H. Hidu. 1982. The effect of sediment type on growth rate 

and shell allometry in the soft-shelled clam Mya aremirki L. J. E.xp. 

Mar. Biol. Ecol. 65:285-295. 
Posey. M. H. 1989. Functional approaches to soft substrate coninmiiities: 

how useful are they? Rev. Aqiiat. Sci. 4:2-4-41. 
Rhoads. D. C. & D. K. Young. 1970. The influence of deposit-feeding 

organisms on sediment stability and eonimunils trophic structure. J. 

Mar. Res. 28:150-178. 
Spivak, E., K. Anger, T. Luppi. C. Bas & D. Ismael. 1994. Distribution and 

habitat preferences of two grapsid crab species in Mar Chiquita lagoon 

lPro\ince of Buenos Aires. Argentina). Helaolaiuler Meeresimlcrs 48: 

59-78. 
Swan. E. F. 1952. The growth of the clam M\a arcinina as affected by the 

substratum. Ecology 33:530-534. 
Swinbanks. D. D. & J. L. Lutemauer. 1987. Burrow distribution of 



thalassinidean shrimp on a Fraser delta tidal Hat, British Columbia. J. 
Paleoinol. 61:315-332. 

Turner, E. J. & D. C. Miller. 1991. Behavior and growth of Mercenaria 
mercenaria during simulated storms events. Mar. Biol. 1 1 1:55-64. 

Vaugelas, J. V. 1984. Preliminary observations on two types of calli- 
anassids (Crustacea: Thalassinidea) burrows. Gulf of Aqaba. Red Sea. 
Proc. 1st Int. Symp. Coral Reef Environ. Red Sea 1:520-539 

V'legas. O. 1981. Dinamica populacional e produ^ao de Tagelus plebeiiis 
no canal do Calunga. Maceio-Alagoas. M.Sc. thesis. Departamento de 
Biologia Vegetal, Universidade de Brasilia, 86 pp. 

Witbaard. R. & G. C. A. Duineveld. 1989. Some aspect of the biology and 
ecology of the burrowing shrimp Callianassa siibterranea (Montagu) 
(Thalassinidea) from the southern North Sea. Sarsia 74:209-219. 

Woodin. S. A. 1976. Adult larval interactions in dense infaunal assem- 
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Zar. J. H. 1984. Biostatistical analysis. Englewood Cliffs. NJ: Prentice- 
Hall. 718 pp. 



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 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 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 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^^ = 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- 

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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Joiinuil of Shellfish Resfunh. Vol. 22. No. 1. S5-y(). 2003. 

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 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 




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 = 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 



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 



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 







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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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 





0.2 


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 





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 








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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Ray, S. M. 1952. A culture technique for the diagnosis of infections with 
Dermocystidium marinum. Mackin. Owen and Collier, in oysters. Sci- 
ence 166:360-361. 

Roberts, M. H., Jr.. R. J. Diaz. M. E. Bender & R. J. Huggett. 1975. Acute 
toxicity of chlorine to selected estuarine species. J. Fish. Res. Canada 
32:2525-2528. 

Roberts, M. H and R. A. Gleeson. 1978. Acute toxicity of bromochlori- 
nated seawater to selected estuarine species with a comparison lo chlo- 
rinated seawater toxicity. Mar. Environ. Res. 1:19-30. 

Stewart, M. E., W. J. Blogoslawski, R. Y. Hsu & G. R. Hetz. 1979. 
By-products of oxidative biocides: Toxicity to oyster larvae. Mar. Poll. 
Bull. 10:166-169. 

Tsao. T.. D. E. Williams. C. G. Worley & S. D. Worley. 1991. Novel 
N-halamaine disinfectant compounds. Biotechnol. Prog. 7:60-66. 

White, M. E.. E. N. Powell, S. M. Ray & E. A, Wilson. 1987. Host-to-host 
transmission of Perkinsus marinus in oyster {Crassostrea virginica) 
populations by the ectoparasitic snail Boonea impressa (Pyramedel- 
lidae). J. Shellfish Res. 6:1-5. 

Wong. G. T. F. & J. A. Davidson. 1977. The fate of chlorine in sea water. 
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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en batea en Galicia (NO de Espana). .Actas IV Congreso Nac. Acuicult. 
317-322. 

Pearce. M. & E. Bourget. 1996. Settlement of larvae of the giant scallop. 
Placopecten inagellanicus (Gmeiin) on various artificial and natural 
substrata under hatchery-type conditions. Aquaculture 141:201-221. 

Ramonell, R., G. Roman, C. P. Acosta & M. Malvar. 1990. Captacion de 
semilla de pectfnidos en colectores: resultados de la campafia de 
prospeccidn en Bueu (Ria de Pontevedra, Galicia) en 1988. In: A. 
Landi'n & A. Cervino, editors. Actus III Congreso Nac. Acuicult pp. 
439-444. 

Reddiah, K, 1962. The sexuality and spawning of manx pectinids. J. Mar. 
Biol. Ass. UK. 42:683-703. 

Rodhouse. P. G. & G. M. Burnell, 1979. In situ studies on the scallop 
Chlamys varia. In: J. C. Gamble & J. D. George, editors. Progress in 
underwater science. 4 (NS). England, Pentech Press Ltd,, pp, 87-97, 

Roman. G. & A. Perez. 1979. Cultivo de larvas de vieira. Pecten maximus 
(L.) en laboratorio, Boletin del Inst. Espa. Oceano. Num 223. 

Roman. G. 1986. Larvae rearing of bivalve molluscs. In; B. Loix, editor. 
Production in marine hatcheries. Rovinj-Zadar (Yugoslavia): 10-28 
Fef. 1986 MEDRAP, pp. 10-28. 

Roman, G., F. Fernandez-Cortes. C. P. Acosta & E. Rodriguez-Moscoso. 
1987. Primeras experiencias con colectores de pectfnidos en las rias de 
Arosa y Aldan. In: A. Landi'n & A. Cervino. editors. Actas II Congreso 
Nac. Acuic. Cuademos marisqueros: Santiago de Compostela, pp. 375- 
380. 

Roman, G. & I. Fernandez. 1990. Metodos de obtencion de gametos de 
pectinidos para su cultivo larvario. In: A. Landi'n & A. Cervirio. editors, 
Actas III Congreso Nac. Acuicult.: 433-438. 

Roman, G. 1991. Fisheries and aquaculture. Spain. In: S. Shuniway, editor. 
Scallops: biology, ecology and aquaculture New York: Elsevier, pp. 
753-762. 

Roman, G.. M. J. Campos, C. P. Acosta & J. Cano. 1999. Growth of the 
queen scallop (Aequipecten opercularis) in suspended culture: influ- 
ence of density and depth. Aquaculture 178:43-62. 

Ruiz. C. M. 1996. Ecologia microbiana en cultivos larvarios de Pecten 
ma.ximus (L.) Tesis doctoral. Univ. Santiago de Compostela. 

Samain. J. F., C. Seguineau. J.-C. Cochard. F. Delaunay. J. L. Nicholas. Y, 
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variability for Pecten ma.ximus production? Oceanis 18:49-66. 

Torkildsen, L., O. B. Samuelsen, B. T. Lunestad & 0. Bergh. 2000. Mini- 
mum concentrations of chloramphenicol, tlorfenicol. trimethoprim/ 
sulfadiazine and tlumequine in seawater of bacteria associated with 
scallop [Pecten ma.ximus) larvae. Aquaculture 185:1-12. 

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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 ■ 
■ 


'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 
20 
10 




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 




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B. 




a 


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IS 






c 


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Jul Jul Aug Aug Sep Sep Sep Oct Oct Nov 



=5. 

C 
O 



o 

c 
o 
U 



20 
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• 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 













• 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 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. 













199 





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. 













495 





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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Journal of Shellfish l<,:s<;urh. Vol. 22, Nu. I. I 19-123, 2003. 

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 





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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 


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o 


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200 



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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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Gardner, J. P. A. 1992. Mytilus galloprovincialis (Lmk) (Bivalvia. Mol- 
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Gardner, J. P. A. 1996. The Mytilus edulis species complex in southwest 
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Gilg, M. R. & T. J. Hilbish. 2000. The relationship between allele fre- 
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Gray. P. 1954. The microtomist's fomiulary and guide. New York: The 
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Hilbish. T. J.. B. L. Bayne & A. Day. 1994. Genetics of physiological 
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286. 

Hilbish, T. J.. E. W. Carson. J. R. Plante. L. A. Weaver & M. R. Gilg. 2002. 
Distribution of Mylilus edulis. M. galloprovincialis. and their hybrids 
in open-coast populations of mussels in southwestern England. Mar. 
Biol. 140:137-142. 

Howard. D. W. & C. S. Smith 1983. Histological techniques for marine 
bivalve mollusks. NOAA technical memorandum NMFS-F/NEC-25:9. 

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Koehn. R. K.. R. Milkman & J. B. Mitton. 1976. Population genetics of 



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Koehn. R. K., J. G. Hall. D. J. Innes & A. J. Zera. 1984. Genetic differ- 
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117-126. 

Mallet, A. L. & C. E. Carver 1995. Comparative growth and survival of 
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McDonald, J. H., R. Seed & R. K. Koehn. 1991. Allozyme and morpho- 
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ti.^h. Res. 20:31-38. 

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hybridize. Genetics 143:1359-1367. 

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710-712. 

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reproducti\e output in two sympatric mussel species {Mytilus edulis. 
M. Irossulus) and their hybrids from Newfoundland. Mar Biol. 141: 
897-909. 

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between Baltic and North Sea Mytilus populations (Mytilidae: Mol- 
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51-60. 



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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fraction) and average total particulate mass (TP^L mg L"') of seston 
s\ithin the experhnentai feeding conditions. Data are the mean of three 
replicate deternilnatiuns per condition. Reler to Table 3 for equation. 



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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 = 

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 = 
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 
08 - 

I"' 

o 

m 

^ 04 

UJ 

to 

O 02 

00 



E, 
a: 40 



TPM (mg I"') 



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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effect of diets of phytoplankton and suspended bottom material on 
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1991. The physiological energetics of mussels iMyriliis galloprovin- 
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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 


-2 

4 



■ ■ 

^ TtTT ▼ 



t'V 



▼ ' T 



-10 



-5 



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. 



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ogy of cultivated mussel (Mytilus edulis) stocks cross-planted between 
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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 






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 





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 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 







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 

+ 









^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 





^^ 















100| 

8a 

60 

4a 

20 

'' 


































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 



2 4 6 8 1012 1416 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 





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 


* 









n 




n 



P Cirr Phrag. Other 



50 

100 

100 

50 



P. Cirr Phrag. Other 

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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.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 




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 . 











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 





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 




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. - 







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. 



LITERATURE CITED 



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Coote, T. A. 1998. The protein, energy and lysine requirements of greenlip 
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Ousel. G.. H. Kluge. K. Glaser, O. Simon, G. Harmann. J. v. Lengerken & 
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Elyakova, L. A.. N. M. Shevchenko & S. M. Avaeva. 1981. A comparative 
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Fleming. A. E. 1995. Growth, intake, feed conversion efficiency and 
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Hardy, R. E. 1997. Understanding and using apparent digestibility coeffi- 
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Hillebrand, W. F., G. E. R Lundell. H. A. Bright & J. 1. Hoffman. 1953. 
Applied inorganic analysis. New York: Wiley. 1034 pp. 

Ikeda. K. & T. Kusano. 1983. In vitro inhibition of digestive enzymes by 
indigestible polysaccharides. Cereal Chem. 60:260-263. 

Mercer, J. P., K. S. Mai & J. Donlon. 1993. Comparative studies on the 
nutrition of two species of abalone. Haliolis niberculala Linnaeus and 
Haliolis discus hannai Ino I. Effects of algal diets on growth and 
biochemical composition. Imerlebrale Reprod. Dew 23:75-88. 

Cshima. K. 1931. Digestive enzymes appeared in abalone viscera. J. Ag- 
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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 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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Joiinial of Slwllfish Rcsi-anli. Vol. 2:, No. I. 1X5-192. 2()().V 

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. 



LITER.\TURE CITED 



Bernard, F. R. 1969. The parasitic copepod Myulicola oricnkilis in British 
Columbia Bivalves. J. Fish. Res. Bd. Can. 26:190-191. 

Bower. S. M., S. E. Mc Gladdery & I. M. Price. 1994. Synopsis of infec- 
tious diseases and parasites of commercially exploited shellfish. .-A;;". 
Re\: Fish Dis. 4:1-199. 

Bower. S. M.. J. Blackbourn & G. R. Meyer. 1992. Parasitic and symhiont 
fauna of Japanese littlenecks. Tapes plulippinarwn (Adams and Reeve. 
1850). in British Columbia. J. Shellfish Res. 11:13-19. 

Bourne. N. 19S2. Distnhution. reproduction and growth of Manila clam. 
Tapes plulippinanini ( Adams and Reeve ). in British Columbia. ,/. Shell- 
fi.sh Re.^. 2:47-54. 

Coan, E. V., P. Valentich Scott & F. R. Bernard. 2000. Bivalve seashells 
of Western North America. Marine bivalve molluscs from Arctic 
Alaska to Baja California. Santa Barbara. CA: Santa Barbara Museum 
of Natural History, 764 pp. 

Chew. K. K.. .\. K. Sparks & S. C. Katkansky. 1965. Preliminary results 
on the seasonal distribution of Myiilicoki orientulis and the effect of 
this parasite on the condition of the Pacific oyster Crassoslrea !iij>as. J. 
Fish Res. Bd. Can. 22:1099-1101. 

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 
revision of the coccidian parasites of bivalves (Mollusca: Bivalvial. 
Folia Parasilol. 44:241-247. 

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- 

ales-like infection associated with a mass mortality of sea scallop 

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. 
Leger. L. & O. Duboscq. 1915. Pseudoklossia glomenila n. g. n. sp.. 

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. 

Otto. S. v.. J. C. Harshharger & S. C. Chang. 1979. Status of selected 
unicellular eucaryote pathogens, and prevalence and histopathology of 
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 
Columbia waters. J. Fish. Res. Bd. Canada 21:1155-1181. 

Sparks. A. K. 1962. Metaplasia of the gut of the oyster Crassosirea gigas 
(Thunberg) caused by infection with the copepod Mytilicola orientalis 
Mori. J. Insect Pathol. 4:57-62. 

Sprague, V. & P. E. Orr, Jr. 1955. Nematopsis ostreuni and N. prytherchi 
(Eugregarinina: Porosporidae) with special reference to the host para- 
site relations. / Parasitol. 41:89-104. 

Sugiura, Y.. A. Sugita & M. Kihara. 1960. The ecology of pinnotherid 
clams as pest in culture of Tapes japonica-l. Pinnotheres sinensis living 
in Tapes japonicu and the influence of the crab on the weight of the 
host's flesh. Bull. Jpn. Soc. Sci. Fish. 26:89-94. 



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 




68 ±22 

225 ±118 


124+56 
1,828+622 
1,522 ±565 



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 






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 


42 ±26 




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 





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 





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 (- - - -) 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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CORBICVLA IN THE LOWER CONNECTICUT RiVER 



203 



1976. The invasion of the Asiatic clam (Corbicula munilensis Philippi) 
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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 



-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 



-25 

-50 H 

-75 

■100 



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 







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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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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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 si