<*« LJ i/mm 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: 5. Fengnan; 6. Ninghe; 7. Beitang; 8. Tanggukou; 9. Yangjiaogou: l(). Ycxian; II. ^antai; 12. Rongchen; 1.1. Dingzigang: 14. Shijiusuo: 15. Sheyang; 16. Jianggang Bay; 17. Rudong; 18. Huijiao: 19. Daishan: 2(1. Zhenhai: 21. Dinghai; 22. Meilin: 23. Sannien: 24. Wenling; 25. I.ei|ing Bay: 26. Wenzhou Bay: 27. Xiapu: 28. Ningde: 29. Luoyuan Bay: 3(1. Huian: 31. Tongan: 32. Xiamen: 33. I.onghai: 34. Haiclieng; 35. ^unxiao: 36. .Shantou: 37. Haimen: 38. Lanbiao. Huilai County: 39. ,)iazi: 4(1. .lieshi: 41. (iaoluo: 42. .Shanwei : 43. Qingcao: 44. Baoan: 45. \iangzhou: 46. Tangjiahuan: 47. Nanshui: 48. Hengshan: 49. Zhanjiang Bay: 50. Qinzhou\van(Longnien); 51. Baoping Bay: 52. Boao: 53. Qinglangang; 54. Qiongshan: 55. Lofu Shan: and 56. Deep Bay. 1986) or solitary (unattached) in the littoral zone (Awati & Rai 1931). The vertical range of C rivularis was described as the littoral zone (Awati & Rai 1931 ). sublittoral low waterline area or submerged offshore area (Durve I9K6). intertidal (Mahadevan 1987. Rao 1987) or tidal region (Patel & Jetani 1991 ) and also at 9-15 m depth (Durve 1986). In Pakistan, the preferred habitats of C. rivularis are the back- waters and creeks along the coast (Moazzam & Rizvi 1983). It seems that this species thrives in muddy environments (Ahmed 1971, Asif 1978b, Ahmed et al. 1987) and adheres to hard sub- strate such as stones (Ahmed et al. 1987). It occurs near the low water mark (Ahmed 1971, 1975, Ahmed et al. 1987, Barkati & Khan 1987) and the preferred tidal height for spat settlemeni is 0.5 ft mark (Ahmed et al. 1987). Predators, Harmful Organisms, and Diseases According to Zhang and Lou (1956b). in China, "led tide" is generally most hai-mful to oysters. It caused 509r mortality of 10 Zhou and Allen TABLE 3. Locations where C. (O.) riviilaris was reported in China. Province Locations Where C. (O.) riviilaris was reported Liaoning Gaiping. Andong (Dadonggou). Xindao. Zhuanghe ( Zhang & Lou 1959) Hebei Fengnan. Tanggukou. Beitaiig (Zhang & Lou 1959) Tianjin City Ninghe (Zhao et aL 1991) Shandong Rongchen (Zhang & Lou 1956b) Yangjiaogou. Dingzigang (Zhang & Lou I956h. 1959) Shijiusuo (Zhang & Lou 1959) Yantai. Yexian (Zhao et al. 1991 ) Jiangsu Sheyang, Rudong (Zhang & Lou 1959) Northern coast (north of Jianggang Bay; Chen 1991 ) Zhejiang Sanmen (Zhang & Lou 1956b) Zhenhai, Daishan, Huijiano. Dinghai, Meilin, Wenling (Zhang & Lou 1959) Wenzhou Bay (Huang et al. 1981) Leqing Bay (Zhou et al. 19821 Fujian Xiamen (Zhang & Lou 1956b. 1959) Tongan. Haieheng (Zhang & Lou 1959) Luoyuan Bay (.Xu el al. 1992) Yunxiao. Longhai. Huian. Ningde, Xiapu (Cai 1966) Guangdong Shanwei. Lanhiao (Zhang & Lou 1956b) Baoan. Tangjiahuan. Hengshan (Zhang & Lou 1956b. 1959) Shantou. Jiazi. Jieshi. Haimen, Nanshui (Zhang & Lou 1959) Qingcao. Gaoluo, Xiangzhou (Zhang et al. 1960) Zhanjiang Bay (Cai et al. 1992) Peal River estuary (Guan & Li 1986) Guangxi Longnien (Zhang & Lou 1959) Hainan Baoping Ba> (Zhang & Lou 1956b, 1959) Qiongshan. Qinglangang. Boao. (Zhang & Lou 1959) Hong Kong Lofu Shan (Ke & Wang 2001 ) Deep Bay (Mok 1974) cultured oysters in Baoan. Guangdong Province in 19.5.^. Red tide could be caused by Noctiluca sp. diatom or the more harmful Dityhun sp, The carnivorous oyster drills Thais gradata (known as "huluo," which means tiger snail in China) and Naticidae sp. (known as "yuluo," which means jade snail) are also very harmful to oysters. Tiger snail can drill through the shell of a spat in 3 min and in 8 h for a 3-y-old oyster (Wu et al. 1997). Beside these, carnivorous crabs, such as Scylla. Portunidae. Lithodidae. sea ur- chin Ecliiiioidea. and sea star Aseroidea. are also harmful to spat. Below we list the available reports on these subject areas by publication year. Harmful organisms to C. riviilaris cultured in Zhanjiang Bay, Guangdong Province, China (Cai et al. 1992) The effects of the predator T. gradata and Balanus spp. were reported in an important estuary for aquaculture. T. gradata was found harmful to l-y-old oysters. Its density on oyster cultch could be as high as seven individuals/m". Mortality caused by T. gradata could be as high as 31%, 14% on average. T. gradata preferred living in groups, usually hiding in the shaded area of concrete posts. Its reproductive season was from the beginning of April to the middle of June peaking from the beginning of April to the beginning of May. Each female carried .50-100 oospores, with about 100 eggs in each oospore. Hatchability was very high, al- most 100%. Incubation period was about 15-30 days. Barnacle Balanus spp. competed for setting space and food. In the worst situation, the oyster seed could be smothered with a total covering of Balanus spp. Balanus spp. set increased from the upper estua- rine area toward the lower saltier regions. Highest density occurred in the low intertidal area. Balanus spp. larvae preferred the sunny side of a setting place. Mass mortality putatively caused by Proroceiilnim sp. bloom in Zhanjiang, South China (Zhang et al. 1995) From late April to late May 1994, an episode of high mortality occurred at an O. rivularis farm close to the port of Zhanjiang. Fujian Province. South China. Mortality reached 98% o\er about 25 hectares. Water sampling and histopathological monitoring was conducted. During the outbreak, the water temperature increased from 18 to 30°C, pH fluctuated between 6.5 and 7.0. and salinity ranged 25.6-29.1 ppt. The water was blue-brown in color and all water samples revealed variable concentrations of phytoplankton. of which 96% were composed of Prorocentruin sp. with concen- trations of 201-667 cells/mL over the period of observation. The temporal association of the mass mortality and a Prorocentrum bloom suggested that the bloom was probably the cause of the mortality. This assumption is supported by the histopathological findings that suggest toxicosis. In particular, the observed lesions were acute and corresponded with the outbreak. Affected oysters were gray in color and had a softer than nor- mal texture. The most outstanding microscopic lesion was intense accumulation of hemocytes in and around hemolymph channels, especially in the Leydig tissue. Close examination of the larger vessels revealed that hemocytes were actively infiltrating the ves- sel walls, as well as involved in transmigration into the Leydig tissue and the formation of intravascular thrombi. A diffuse, and less intense, hemocytosis was present in the interstitium between the digestive tubules, while a mild hemocytosis was detected in the gills. Oedematous changes were prominent around the digestive tubules and in the Leydig tissues where they were accompanied by tissue necrosis/lysis. The digestive tubules were empty and their epithelia were dysplastic, varying from low columnar to cuboidal and in some instances there was necrosis of the tubular epithelium. Brown cells were pailicularly prominent in the intertubular tissues. The pathology was consistent with a systemic toxicosis resulting from absorption of toxins from the digestive gland. Bouamia-\\V.e parasite found in C riviilaris reared in France (Cochennec et al. 1998) C. rivularis was imported from the Haskin Shellfish Research Laboratory in New Jersey in 1994. Seven months after introduc- tion, some mortality occurred in quarantine. Histologic examina- tion revealed the presence of an intracellular protozoan parasite in the connective tissues of nine dead specimens. Ultrastructure analysis suggested that the protozoan might belong to the genus Bonamia. Bonamia was likely transmitted to the experimental oys- ters from neighboring waters, which are endemic for bonamiosis, possibly when inlet water treatment lapsed. An intracellular procaryotic micoorganism associated with lesions in C. ariakeiisis in Pearl River estuary. South China (Wu & Pan 2000) A series of mortalities of cultured oysters have occurred in Pearl River estuary since 1992. usually from February to May. The mortality peaks at 80-90%' during April and May. The diseased CRASSdSTREA ARIAKENSIS REVIEW 11 Figure 3. Luculions reported with ('. ariakensis populations in India and Paliistan. India: 1. Ratnagiri (I6N, 73E); 2. Balapur (not locatedl: 3. Porljander iPorbundar), Navibander (2IN, 69E|; 4. Dwarka (Gomati Creeli) (22N, 68El: 5. Oiiha. Aramda Creek, Posheira, Port Okha, Sikka (22N, 69E). Pakistan: 1. Korangi Creek (24N, 67E): 2. Karaclii (24N. 64E); and 3. Port Qasini (27N, 68E). oysters are generally aged 2-7 y. A rickettsia-like iiitracelliilar microorganism is present in the tissue of diseased oysters. PHYSIOLOGY Natural Reproduction Hermaphroditism and Sex Reversal Crcissostrea are oviparous and protrandric hermaphrodites (c./.. Coe 1934). The occurrence of true hermaphrodites (both sexes simultaneously) is rare. Hasan (1960) stated that hermaphrodites do not exist in O. discoidea ( = C. rividaris). In a study of her- maphroditism and sex reversal in C. rividaris from the coast of Karachi, Pakistan, true hermaphrodites were absent (Asif, 1979). Hermaphrodites observed were actually transitional stages of the sexes and used to study sex reversal. According to Asif, gonad generally appeared in C. rivukiris at the age of 2-3 mo at a length of 0.4-0.6 cm and 62* were male. Protandric hermaphrodites were found in summer and autumn, which indicates the time of sex reversal. The percentage of males declines gradually with increas- ing size as is true for other Cnissostrea spp. Cai et al. ( 1992) also claimed that sex ratio of C. riviiUiris had an obvious regular change during the reproductive season (usually summer and autumn) and the ratio of females to males increased as the oysters got older. Hasan (1960) also mentioned that individuals with undistinguish- able sex are fairly common throughout the spawning season. In Asif s study, the percentage of females increased over males be- yond the size class 5.0-5.9 cm. Spawning Importance of temperature in gonad maturity and spawning of oysters is well known. Temperature influences the development of gonad (Orton 1936, Spark 1925. Nelson 1928). Temperature also directly influences the abundance of food, which is necessary for the development of gonad (Loosanoff & Engle 1942. Loosanoff & Tomnier 1948). Periodic examinations of the gonad of O. dis- coidea showed that normal growth of the reproductive products was coincident with gradual rise of water temperature and food abundance in the summer months (Hasan 1960). The combined effect of temperature and salinity on the start of 12 Zhou and Allen spawning was discussed by Hornell (1910. cited from Hasan. 1960) and confirmed by Hasan (1960) through an experiment on O. discoidea in Pakistan. The rise in water temperature helps the development of gonad, while decrease in salinity stimulates the gonad for spawning. Cai et al. (1992) also mentioned that oyster reproduction is closely related to environmental conditions. High temperature and low salinity could cause mass spawning of C. rividaiis in Zhanjiang Bay. Guangdong province. Hu et al. (1994) presented a more detailed and slightly different discussion in his study of C. lividaris spat collection in Jioulong River estuary. Fujian province. He agreed that spawning is related to the change of water temperature and salinity. Water temperature could change with wind direction or strength. Salinity could be changed by precipitation, water current, and tides. However, he seemed to believe that simply a change of water temperature and salinity could be the trigger for spawning, whether an increase or decrease. According to his observation, whenever the tide changed from neap to spring, spring to neap, or during spring tide, oysters would spawn, as long as their gonad was well developed. If the wind direction happened to change from northeast to southwest, or cold air happened to pass by. spawning would increase. He explained that a temperature change of only about 1-2°C would stimulate C. rivularis to spawn. Hasan (1960) studied two natural O. discoidea beds at Wau- gudar Creek. Pakistan. Spawning starts by the first week of July when temperature was about 28-29°C and salinity about 24 ppt. Number of spawning individuals remains almost constant during August and September, much reduced in November and almost nil in December Several authors talked about reproduction of C. rividaiis from China. According to Zhang and Lou ( 1956a), the optimum salinity for reproduction of C. rivularis is 10-25 ppt in China. Hu et al. (1994) reported that in Jiulong River estuary. Fujian province, gonad maturity reaches its peak from the middle of April until mid-May. Oysters spawn twice each year: spring spawn is from May to June and fall spawn, from the end of October to the beginning of December. During spring spawn, water temperature fluctuated between 20 and 30°C, salinity 5-25 ppt. Guan and Li (1986) mentioned that in Zhujiang River estuary. Guangdong province, the reproductive season is from June to September. Spawning is mainly during June and July. There might be a second spawning if appropriate environmental conditions are available. Guan and Li did not report the environmental conditions associ- ated with spawning. Cai et al. ( 1992) reported that the reproductive season is generally from the beginning of April to the middle or end of June in Zhanjiang Bay, Guangdong. Environmental condi- tions in the study area (Shimen) are listed as follows: Annual water temperature ranged from 14 to 31.8°C. Daily water temperature changed 2 to 4°C. Water temperature was highest in June and lowest in January. Salinity ranged from 7.52 to 22.18 ppt in sum- mer (but could drop to 0.00 ppt when flooded). 18 to 30 ppt in winter. pH ranged from 7.1 to 7.9 in summer and 7.9 to 8.1 in winter. Zhang et al. (I960) mentioned that reproduction occurred year round in South China Sea area. The reproductive peak is from late May to eariy September. Zhang et al. did not report environ- mental conditions during this time period. According to Tanaka ( 1954). the spawning season of O. rivu- laris ranges from late May (20-22°C) to early September (28- 26.5°C) in Ariake Bay, Japan. There are three major spawning periods during this season: early June (22-23°C). late June to eariy July (24-26°C). and the beginning to middle of August (30- 28.5"C). The eggs of U. rividtiris measure 49-53 ixm in diameter. The relation between salinity and developmental condition is shown in Table 4. The temperature varied from 24 to 27°C (Amemiya 1928). The above results are neariy identical to those of Hu/iniori (1920. cited from Amemiya. 1928). Spalfall The preferred tidal height of settlement for C. rivularis spat was reported to be at the 0.5 ft mark in Pakistan (Ahmed et al. 1987). A broader range was reported from China by Nie ( 1991 ): from the low tide line to a depth of 10 m. with the maximum setting at ± 0.4 m low water mark. Hu et al. (1994) reported the optmial water depth for spat collection is from the low tide mark to a depth of 1 m in Jiulong River estuary. China. Larvae settle 12-18 days after spawning. In southern China, spatfall occurs from June to August, the period of highest temperature and lowest salinity (Nie 1991, Cai & Li 1990). Three reports on spatfall seasons from Pakistan are summarized below. One study was conducted at Paradise Point situated on the west coast of Karachi (Moazzam & Rizvi 1983). This is basically a rockv shore having frequent stretches of boulders and sand. The subtidal area along this shore is generally more deeply inclined than the rest of the coast. This is also a power plant site. C. rivularis occurs in the cooling system of the power plant, which has been made artificially "protected" and simulates conditions of a backwater environment. The enxironnient conditions were re- ported as follows. Temperature dropped to its minimum of 20- 22 C in December-January and reached its maximum of 28-30°C m June-July. Salinity remained fairly constant (35-36 ppt) except during the short spell of rains in July-August when salinity dropped to 28 ppt. The contents of suspended matter fluctuated between 0.003 mg/L in November and 0.1 16 mg/L in June. Trans- parency was less than 1 m in June-July. Maximum settlement of C. rivularis occurred in June and September-October. A consid- erable number were also observed in July-August. The second report came from two natural oyster beds (Hasan 1960). One is situated between Korangi and Kadero creeks, south of the village Vagudar and about 16 miles southeast of Karachi. The other one is about 6 miles south of Dhabeji. The temperature and salinity profile were reported from Vagudar creeks. Tempera- ture profile looks very similar to the one from the above report, except that it dropped even lower to 16-17°C in January. Salinity was reported only from April to September, with a maximum of 3(S-37 ppt in April-May and then dropped continuously to 21-22 ppt in September. The pattern of larval settlement of O. discoidea in this report is different from the one mentioned above. Settlement at Vagudar Creek occurred from July to December with mid- TABLE 4. Relationship between salinity and developmental condition, accordini> to .\meniiya 1928. Salinity ppt Sp. gr. at 0 C Condition ca. 7 ca. 1.0056 Minimum salinity S-14 1.0064-1.0112 Much too low salinity L'^-IX 1.0120-1.0144 Too low salinitv 1 9-25 1.0153-1.0200 Optimum salinity 26-30 1.0209-1.0241 Too high salinity 31-33 1.0249-1.0256 Much too high salinity ca. 34 L-a. L0273 Maximum salinity Crassostrea ariakensis Review 13 September being the peak permd. Moa//aiii and Ri/vi related setting failure to the presence of high contents of suspended matter in seawater during the southwest monsoon period (June- September). This high content of suspended matter is believed to interfere with larval settlement of many in\ertebrates in this area (Ahmed et al. 1978). The third report came form the Gharo-phitti saltwater creek system (Ahmed et al. 1987). Spat fall occurred from April to October with peak settlement from April to July. The maximum settlement occuned during the period June 24 to July 23. No environmental conditions were given in this report. Growth Growth Rate C. uriakeiisis is well known for fast growth. In Pakistan. C. rivularis spat reached the si/e of 0.5 mm in about one week and 2.0cm in about I mo (Ahmed et al. 1987). Hasan ( I960) found that a size of 3.0 cm was reached 2 mo after settlement. In about one and half years, they become ready for market. Temperature and salinity data of Hasan's study is shown in the spatfall section. In China. C. rivularis can growth to 10-16 cm in 2 to 3 y (Zhang & Lou 1956b). In Japan, it attains full size (20 cm) in 2 or 3 y (Amemiya 1928). The results of Fujiinori's study (1929) on the growth rate of O. rivularis was presented in two parts: spat / young oysters and the sexual adult. Fujimori found that the growth rate of the spat varies considerably according to their time of attachment. The size of adult O. rivularis in Kyushu was 5.5 cm shell height at I y. 9.7 cm at 2 y. 12.4 cm at 3 y. 15.2 cm at 4 y. 17.9 cm at 5 yr. and 19.7 cm at 6 y. In Japan, growth was most rapid in August and September (Cahn 1950). Environmental conditions were unavail- able for the above reports, if not mentioned. Shell Dimension C. ariakeiisis reaches a large size. As Cahn ( 1950) mentioned, the maximum size attained by this species according to the litera- ture is 257 mm with an estimated age of 20 y. The maximum length he recorded in Japan was 240 mm. A maximum shell height of about 200 mm was reported several times from Japan and the United States (Amemiya 1928. Hirase 1930. Coan et al. 1995). According to the growth rate of adult O. rivularis determined by Fujimori (1929). the estimated age of such size is more than 6 y old. Generally, adult specimens reach 6-7 inches (or 150-170 mm) in height, as reported from four countries (Hirase 1936. Galtsoff 1964. Ahmed 1971. Rao 1987). Allometric Growth A study of the allometric (relative growth) relationship between shells and tissues of C. rivularis was presented by Barkati and Khan (1987) from Pakistan. Shell length was defined as the maxi- mum distance between the tip of the anterior margin and the pos- terior margin. Shell width was defined as the maximum distance between the lateral maigins. The following points were reported. Shell width increased faster than shell length (/■ = 0.85). Shell length increased faster than dry tissue weight (/■ = 0.52). An exponential relationship exists between shell length and shell weight with faster growth in length compared with shell weight (/■ = 0.84). Dry tissue weight increased faster than shell weight (c = 0.74). Condition index (the proportion of dry tissue weight to total dry weight of shell and dry tissue) increased with increasing shell length (r = 0.41 ). No linear variable was useful to accurately predict other variables due to low coefficient of correlation (/). probably due to irregular growth in various shell dimensions (length and width). For example. Asif ( 1978b) reported variation in shell growth in two populations of C. rivularis caused by setting density in Pak- istan. One population in Korangi Creek was exploited and densi- ties were low. Another population in Sonari was crowded. In the Korangi Creek, the oysters are attached to rocks or stones hori- zontally, whereas those of Sonari grow upward with the umbo downwards. Generally, the wild stock of C. rivularis of the Kor- angi Creek are round and shallow whereas the Sonari population is elongated and deeply cupped. In the majority of the Korangi Creek population, height plus width varies closely with length of the shell while in the Sonari population, shell height plus width varies twice as much as the length. Feeding Food .Selectivity According to Cai et al. ( 1992). C. rivularis (collected in Zhan- jiang Bay, Guangdong Province. China) is a selective feeder. It prefened small articles to long-chain groups or large articles. The majority of its food is composed of phytoplankton such as Cosci- uihUscus sp., Nitzscliia sp. and Cyclotella sp. Feeding Habits Zhang et al. ( 1959) did an extensive study on the feeding habits of O. rivularis in relation to time, tides, season (change of tem- perature and salinity) and suspended particles. The experiment was conducted in the Pearl River estuary and some nearby bays. Most of the sampled oysters were 3 to 4 y old at the time of examination. These oysters were collected from the wild as spat and cultivated in oyster farms. The percent of O. rivularis that are feeding at any given time (incidence of feeding) was not related to periods of light and darkness, nor to the periods of tides, or the density of suspended particles. Salinity and temperature did have certain in- fluences, as summarized below. According to examinations at five different times of the year, the highest average incidence of feeding for O. rivularis was a little more than 80%. It was also found that feeding time of O. rivularis adds up to 16-19 h everyday with irregular intervals. Feeding habits of O. rivularis were not related to change of sea level or direction or speed of water flow caused by tidal change. In Pearl River estuary, feeding incidence of O. rivularis was highest from October to April (50-100%). when temperature ranges between 10 and 25°C and salinity between 15 and 30 ppt. During summer, the natural reproductive season of O. rivularis. when temperature is much higher (22-30°C) and salinity is much lower (3-26 ppt). feeding incidence is lower (0-70%). Feeding incidence seems to be more closely related to salinity according to monthly records. Although O. rivularis is known to tolerate low salinity, feeding rate was significantly retarded if salinity was lower than 5 ppt. Above 10 ppt, feeding was active. Increase in suspended particles in the seawater (higher turbid- ity) failed to influence feeding incidence of O. rivularis. In this case, the authors maintained that these suspended particles served as a food source for the oysters. Oxygen Consumption Guan and Li (1988) did an extensive study on oxygen con- sumption of C. rivularis. A Warburg manometer was used to mea- sure the oxygen consumption of dissected gill tissue of C rivularis taken from the Shenzhen Bay Oyster Fann. Oxygen consumption 14 Zhou and Allen varied with the change of seawater temperature. A negative cor- relation was found between oxygen consumption and the oyster age. Tlie older and heavier the oyster, the less oxygen was con- sumed by its gill tissue. Oxygen consumption differed significantly in different reproductive periods. BIOCHEMISTRY Biochemical composition Qasim et al. (1985) determined the following biochemical pa- rameters for C. hvidaris from Pakistan. Water contributes 787r of soft body wet weight. Of soft body dry weight, 35.7% was crude protein, 22.5% glycogen, 23% lipid, and 11.2% total inorganic substances. These are the averages from sampling over a period of time (sample interval was not stated in the article). Higher value for lipids (31%) was reported from India (Patel 1979. cited from Qasim et al. 1985). This difference is probably the result of geo- graphical variation, seasonal variation, or both. Qasim et al. ( 1985) mentioned that the ratio between glycogen and protein changes with reproductive state of an oyster (no spe- cific information available). Another report on biochemical in- dexes of C. rivularis from the Pearl River estuary. China (Guan & Li 1986) showed seasonal change of lipid content and its close relationship with reproductive physiology of the oysters. As the authors di.scussed, reproductive season in the Pearl River estuary is from June to September, of which June and July are primary spawning periods. There could be a second spawning in September if environmental conditions were appropriate. In their study, lipid content was highest in May (2.88% of wet weight), then dropped dramatically from June until it reached the lowest point 1 .06% in October, the end of the reproductive season. For protein, amino acid profile determines the nutritive quality of tissues. Such a profile of C. rivularis tissue protein has been reported from the Pearl River estuary. China (Guan & Li 1986) and Pakistan (Aftab 1988). There are only slight differences between the two reports. From China, specimens were tested in May, and the amino acid profiles are presented in Table 5 (Guan & Li 1986). Glutamine and asparagines are most abundant. From Pakistan, 14 amino acids were analyzed. Methionine and arginine were not detected. Glycine and aspartic acids were most abundant. Seasonal variation in bound amino acid content is shown in Table 6 (from Aftab 1988) The shells of O. rivularis have been used as traditional Chinese medicine. Zhao et al. (1991) examined the content of calcium carbonate, trace elements and amino acids in shells of O. rivularis collected from Tianjin. Shandong, Zhejiang, and Fujian provinces. Calcium carbonate in raw shells was 92.0-95.5% and in calcined shells, 96.4-96.9%. Calcined shells have had organic materials removed. The raw shells contain large amounts of Ca, small amounts of Mg, Na, Sr, Fe, Al, Si, and traces of Ti, Mn, Ba, Cu. etc. Shell decoctions (an extract obtained by boiling the shells) contain small amounts of Ca, Na, Mg. K. and trace element of Sr, P, Pb, Zn, Ni, V, Ba. Li. Mn, Ti, Cu, Cr, Mo. As, Hg, etc. The oyster shells contain 1 7 amino acids. Total amino acid content amounted to 0.16 to 0.24% in raw shells. Li et al. (1994) studied the medicinal value of "oyster complete nutritional tablet," a dietary supplement made from extracts of both shells and soft body of O, gigas and O. rivularis from South China Sea. The tablet contains a high content of eighteen amino acids, especially the eight essential to the human body. Putative benefits are attributed to the liver, kidney, spleen and intestine to a certain extent. TABLE 5. The amino acid compositions and their contents In C. rivularis sampled in May, 1984 (Guan & Li 1986). .\mino Acid Contents In Dried Samples ( % ) Alanine Arginine Asparagine Cystine Glutamine Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Serine Threonine Tyrosine Valine 2.04 L95 3.30 0.28 4.06 2.15 0.76 1.18 1.87 2.23 0.57 1.05 1.39 1.48 1.34 1.32 Heavy Metals and Toxins Lu ( 1994) did a preliminary study on the feasibility of using O. rivularis as a monitoring agent for heavy metals, like Cu, Zn, Cd, Pb, along the Guangdong coast, China. He found that profiles of Cu, Zn and Cd content in the oyster correlated with the distribution of industrial discharge along Guangdong province. Also see Ke and Wang 2001. Further investigations on the suitability of O. rivularis as a biomonitor of specific metals or other chemicals are presented below. Zn According to Lu et al. ( 1998a), Zn accumulated continuously in the tissues of the oyster through 12 days of exposure. Accumula- tion was linear with time. Loss of Zn from C. rivularis was not observed over 35 days of depuration. Zn accumulated less readily with increasing salinity. The author concluded that in general C. rivularis is a reliable indicator of Zn in marine systems. TABLE 6. Seasonal variation in the protein and amino acid composition of tissue protein hydrolysate of C. rivularis (Aftab 1988). Component February May August November Average Protein % d.v\. 40.36 41.25 52.50 55.00 47.33 Alanine 6.04 6.91 9.00 9.67 7.90 Aspartic acid 6.78 9.83 12.56 6.58 8.94 Glutamic acid 6.. 30 7.98 11.26 5.08 7.65 GIvcine 13.27 11.88 6.55 9.10 12.10 Histidine 2.07 1.87 2.72 1.91 2.14 Isoleucine 2.38 1.80 3.12 2.08 2.32 Leucine 3.98 2.89 5.34 3.63 3.96 Lysine 1 .59 1.74 1.44 1.28 1.51 Phenylalanine 1.73 1..3() 1.94 1 .55 1.63 Proline 0.46 2.05 0.79 2.40 1.43 Serme 4.07 6.01 7.82 3.51 5.35 Threonine 3.85 5.74 6.92 3.24 4.93 Tyrosine 0.85 0.60 0.91 0.79 0.79 Valine 3.64 3.75 5.55 2.98 3.98 CflASSOSTRKA AK/AKENSIS ReVIHW 15 Cd Lu et al. (1998b) studied Cd absdiplion in C. riviilaris. The content of Cd in body tissues of C hviilaris accumulates in linear proportion to Cd concentration in the water and to exposure time. Accumulated Cd attenuates slowly with a biologic half-life of 77 days. With increased salinity, rate of accumulation decreases while rate of Cd loss slows down. C. livuhiris seems to be a reliable bio-monitor of Cd pollution. Cu Cu absorption in C. rivuUiri.s was examined by Lu et al. (1998c). It continuously accumulated in the tissues of the oyster through the e.\posure to a concentration of 100 (J-g/L over 12 days. Accumulation was linear with time and decline of Cu concentra- tion was slow, with a half-life about 1.^1 days. Rate of Cu accu- mulation was significantly slower with increased salinity, but rate of decline in Cu concentration was not signiticantly related to salinity. Total Petroleum Hydrocarbons (TPHs) Lin et al. ( 1991 ) looked at concentration of TPHs in the Pearl River estuary. China. TPHs in C. rivularis tissues decreased with time during the period leading to sexual maturity. The rate of decrease was about 0.24 |jig/g, dry weight. The biologic half-life was 43 days. Aromatic hydrocarbon compounds with smaller mo- lecular weight were released sooner from oyster tissues than those with greater molecular weight. The concentrations of TPHs in oyster tissues were not significantly related to those in waters and sediments, and not clearly dependent on the contents of lipids in oyster tissues during the study period (September 1986 until Feb- ruary 1987). GENETICS Karyotype So far, research on the cupped oyster species of the genus Crassostrea shows a common diploid chromosome number of 2;; = 20, and their karyotypes include only metacentric and submeta- centric chromosomes. The proportion of these chromosome types can be different interspecifically (Leitao et al. 1999). Chromosome number of In = 20 was confirmed in C. aricik- eiisis (leyama 1975) and in C. rivularis from West Pakistan (Ahmed 1973) and China (Yu et al. 1993). Yu et al. reported the karyotype of C rivularis sampled in Southern China had 10 meta- centric pairs. A more recent karyological study (Leitiio et al. 1999) on an American population of C. ariakensis originally introduced from Japan shows that it consists of eight metacentric and two submetacentric (nos. 4 and 8) chromosome pairs. A variable num- ber of one to three Ag-NORs (nucleolus organizer regions) was observed terminally on the metacentric pairs 9 and 10. About 68'7f of the silver stained metaphases showed Ag-NORs only on pair 10. Polyploidy Rong et al. ( 1994) reported their attempts to produce tetraploid C. rivularis. Newly fertilized eggs of C. rivularis from south Chma were treated with physical and chemical methods in the first three minutes before the cleavage of zygotes or at the onset of first cleavage. Induction rates of tetraploids were 28% for heat shock, 30% for cold shock, 28% for chlorpromazinum treatment and 35,8% for "traditional Chinese medicine" treatment as indicated by chromosome spreads from larvae. Production of viable spat was not reported. Hyhridizaliun Gaftney and Allen ( 1993) reviewed previous hybridization re- ports among Crassostrea species and pointed out that most of reports of successful hybridization suffer from one or more of the following: I ) ambiguities in classification; 2) possible contamina- tion during spawning; 3) absence of experimental controls for assessing the quality of gametes as well as larval viabilities; and 4) the absence of genetic confirmation of hybrid status. They con- clude that there was virtually no unequivocal evidence for the formation of viable interspecific hybrids among Crassostrea spe- cies. Early studies on cross-fertilization between C. gigas and C rivularis gained little success (Miyazaki 1939, Imai & Sakai 1961), but was reported successful by Zhou et al. (1982) and Downing (I988a,b, 1991). Asif (1978a) reported successful pro- duction of trochophore larvae 4-5 h for the cross of C. rivularis with C. glomerata and Saccostrea cuccullata. For the reasons mentioned above, these should be viewed with caution. Hybridization of C gigas and C. rivularis was re-examined by using specimens originally introduced from Japan to the United States (Allen & Gaffney 1993). Such crosses are of interest be- cause of the disease resistant properties of these species (Calvo et al. 1999, 2001). In addition, the hardiness and apparent disease resistance of C. gigas and the high temperature, low salinity tol- erance of C. rivularis could lead to promising variants for aqua- culture, especially if the diploid is sterile. Three replicates of a 2 x 2 factorial mating of C. gigas and C. rivularis were produced to examine the viability of this cross. Fertilization rate, yield of 48- h-old larvae, and survival of fertilized eggs was lower in the hy- brids than in pure crosses. All crosses showed similar larval growth rates, except C. rivularis (female) x C. gigas, which grew more slowly. Isozyme electrophoresis and flow cytometry con- firmed hybridization. Triploid hybrids were produced using tetra- ploid C gigas and diploid C. ariakeusis (Que & Allen 2002). Hybridization between C. ariakensis and C. virginica failed (Alien et al. 1993). Cytogenetic and electrophoretic analysis re- vealed the formation of hybrid zygotes and larvae between C. virginica and C. rivularis. but larval survival was limited to a maximum of 10 days. Larvae stopped growing at about day 4, reaching a maximum length of about 80 um. Studies on larval feeding using fluorescent beads indicated that growth limitation apparently was not caused by an inability to feed. Induced triploidy did not rescue hybrid failure. Population Genetics A number of studies have used molecular markers of various sorts to distinguish among Crassostrea species, including C. ari- akensis. Among the earliest was work by Buroker et al. (1979) who estimated levels of genetic variation for six Crassostrea and three Saccostrea species based on electrophoretic variation in pro- teins in about 30 loci, C rivularis among them. Liu and Dai (1998) used RAPD techniques to differentiate C. talienwhanensis and C. plicatula froin C rivularis. Li et al. (1988) used electrophoretic markers to separate four Crassostrea species, and concluded that the "white oyster" was C. rivularis and the "red oyster," C. ired- iilai. C. rivularis was also among those used by Little wood (1994) to establish the first phylogenetic estimates for this species based on nuclear DNA. Since then, a number of other studies employing 16 Zhou and Allen molecular markers have been applied to C. ariakeiisis. mostly to discriminate among species (O'Foighil et al. 1995. GatTney & O'Biern 1996. Hedgecock et al. 1999. Francis et al. 20(X)). Hedge- cock et al.'s study confirmed the occurrence of C. ariakensis in the northern regions of the Ariake Sea and re-emphasi/cd the need for genetic confirmation for species identification. AQUACULTURE RefeiTences to aquaculture of C. ariakensis come mainly from Japan and China, and are discussed accordingly. Aquaculliire in Japan Of the five edible oysters species in Japan, only O. gii;as and O. rivuiaris were cultured commercially (Cahn 1950). O. i-i\'nlaris was second to O. gigas in commercial importance (Amemiya 1928) According to Amemiya (1928), cultivation of O. rivuiaris be- gan in Ariake Bay in the late 1890s and seed were later trans- planted to Kozima Bay in Okayama Prefecture around 1928. An even earlier report of cultivation in Ariake Bay in the 186{)s was given by Wakiya ( 1929). Both Wakiya and Langdon and Robinson (1996) mentioned that the culture of Suminoe oyster were con- ducted in the Suminoe river. Saga Prefecture from the beginning of the Meiji period in the mid- 19th century. Discrepancy between Cahn and Wakiya on the start of C. rivuiaris aquaculture might rest on their definition of cultivation. Cahn ( 1950) described two types of culture sy.stems at the mouth of the Suminoe-gawa ("gawa" in Japanese means river or stream). Ariake Bay. a primitive one and a more developed one. Cahn did not say when the primitive culture started, but he implied that the more sophisticated culture started after 1885. The primitive culture consisted simply of gathering natural oysters and storing the larger individuals for a short time on the bottom of the Sumino-gawa. later to be shipped to Nagasaki at the proper season for sale. Aquaculture of O. rivuiaris began fortuitously. For some rea- son during the winter of 1884 these oysters were not shipped for sale to Nagasaki. The ne.\t year they were considerably larger by size and weight. From this observation, a new type of culture evolved in the local area. Young oysters about 2.5 cm in length were gathered from every possible growing place from July until March and were placed on oyster beds at the mouth of the river. To prevent loss, they were heaped close together in masses. They were washed and cleaned twice or three times each month during low tide. In April individual oysters were stuck in the mud verti- cally, hinge down and ventral margins uppermost. As the mud was very firm, the oysters fared and grew well. As they grew, they were thinned and replanted to give them more growing space. Growth was most rapid in August and September. Aquaculture in China C. rivuiaris is the most economically important marine shell- fish species cultured in South China (Zhang et al. 1995), primarily in Fujian, Guangdong and Guangxi Province. The history of its culture in Guangdong is over .'^00 y old (Cai et al. 1979). The Pearl River (Zhujiang) estuary. Guangdong was considered the most famous cultivation site of this species (Zhang & Xie 1960). Some other places mentioned in the literature are Yangjiaogou, Shan- dong Province (Zhang et al. 1960), Leqing Bay, Zhejiang Province (Zhou et al. 1982) and in Deep Bay, Hong Kong (Mok 1974). In 1996. China produced 2.3 million tonnes of oysters from aquacul- ture, among which C rivuiaris accounts for 20-30% (Guo et al. 1999). In Guangdong province, C. rivuiaris production was about 40'>f of total sea culture production (Qiu & Li 1983). The primitive method of oyster culture was to improve growth and reproduction with procedures like fishing restrictions and pro- tection from diseases and predators (Zhang & Xie I960). The advanced method involves collecting natural spat and artificial grow-out. Modern oyster culture includes larval culture and breed- ing. Larval culture and breeding of C. rivuiaris larvae has been successfully accomplished on a research scale in South China (Li- ang et al. 1983. Cai et al. 1989) but has not been used in large-scale commercial culture. Hatchery production of seed is seen as a step to increase the reliability of seed production. Spat collection and artificial grow-out is still the most popular. This is composed of four steps: spat collection, grow-out, fatten- ing, and harvest. For spat collection, cultch material to collect spat was traditionally oyster shell and gravel (Nie 1991). Since the 1960s, cement plates (17-24 cm x 14-19 cm) or cement bars (40-80 cm long x 4-6 cm") reinforced with embedded bamboo stakes were used. Stakes are used increasingly since they are easier to handle, provide more surface area, and are not so readily cov- ered by silt. Season and location of spat fall is summarized in Physiology. Oyster larvae in the water are monitored to ensure the best time of planting the clutch. Spat collectors are placed in rows in rectangular blocks, usually 30 to 37.5 x lo' stakes or 100 to 135 X 10' plates per hectare. Further details follow below for specific culture techniques. The age of harvest is generally 3.5 to 4 y (Qiu & Li 1983). but \aries from 2 to 5 y depending on culture location where the environment, the specific culture technique, and even the expected market size could be different. For example, Guo et al. (1999) reported 2 to 3 y in Guangxi where oysters maintain rapid growth throughout the first 3 y and are usually harvested at a size of 10-15 cm. The culture technique used there is concrete bars or shell strings hanging on rafts and long lines. In Pearl River estuary, Guangdong, oysters were usually harvested at 3 y of age by bam- boo stake culture (Zhang & Xie I960). Cai and Li (1990) reported the period to be 3 to 5 y in Southern China. Cai and Li (1990) summarized oyster culture techniques in China. The ancient bottom culture techniques, including bamboo stake, stone and concrete culture, are still the major methods, but farmers are becoming increasingly aware of the advantage of off- bottom culture, like the rack and raft culture. The various tech- niques are described below (reproduced from Cai and Li's work, 1990). Rock (Stone) Culture Rock culture is usually applied in areas that have hard sub- strate. Marble flagstones approximately 90 cm x 25 cm wide and 10-cm thick are preferred for this method. Stones may be arranged one-by-one vertically, resembling tombstones or two stones may be aiTanged in an "A" shape. Three stones may be ananged to form a tripod. Average spacing between stone groups is 70 cm. Another choice of rock is irregularly shaped natural boulders of 4 to 5 kg. The traditional anangement of the boulders, called "stars in the sky," involves uniform distribution over the substrate. Two modi- fications were used along the coast of Guangdong and Hainan Provinces. One is called "plum blossom" with five or six boulders grouped together. Another is called "small house" with three flag- stones aiTanged to form a shed or an upside-down "U." Both kinds of rocks are thoroughly washed and then covered in limewash 10 davs before use. Crassostrea ariakensis Review 17 111 Guangdong and [■uiian Proxinces. the rocks are set out in early May to June or in November. Maxiniuni spatfall is expected in May. Spat collected in June is usually subject to heavy mortality due to high temperatures and strong sunlight during attachment. Spat collected late in the season usually grew poorly because of to low water temperatures. Oysters are grown to market size at the site of spat collection. Approximately 60,000 stones are required for one hectare, and C. rivularis may be harvested in 3 to 5 y. Production is moderate, ranging from 750 to 3000 kg per hectare. The oysters grown on rocks are more subject to predation by starfish and other organisms than are oysters grown on stakes, so considerable time must be invested in predator control. Concrete Culture Prefabricated posts or tiles are a derivative of the traditional rock culture technique for the culture of C. rivularis and has been used since 1930 in Guangdong Province. Spatfall occurs most of the year, but optimum periods are April and May. To prevent the tiles or posts from sinking into the mud, they are removed and reananged around May, September, and December. Concrete cul- ture requires a 4-y cycle. Spat collection and growth occupies the first year from June to April. The second and the third years involve a cultivation period yearly from May to August. Market size is attained in 2.5 to 3 y and involves a progressive increase in the spacing of the concrete tiles or posts. The cultivation cycle is completed by a fattening period extending from September to January. For fattening, oysters are transferred from the spat col- lection/grow-out area to prime growing grounds, usually in the low intertidal zone. For this culture method, in Guangdong, harvest generally occurs in February to April of the fourth year, when growth rates begin to decline sharply. Expected production from the concrete method is 7.5 to 15 tons of meat per hectare. Rack Culture Since 1965, rack culture has been used to cultivate C. rivularis in Guangdong Province. The racks may be constructed of bamboo, wood, stone or concrete. Because wood and bamboo are rapidly destroyed by shipworms and stone is heavy and awkward to handle, concrete is preferred. The forni of the rack varies greatly, but consists basically of members driven into the substrate to form a horizontal frame, which supports the oyster cultch 2.5 to 3 m above the substrate. Several types of material are used for spat collection. The most popular one is punched oyster shells, separated by 3 cm bamboo or plastic spacers, and strung on 2 m lengths of galvanized wire ox polypropylene line. Concrete tiles, approximately 10 cm" with a central hole, may be substituted for the oyster shell. Concrete poles between 70 and 130 cm in length may also be used. The cultch is suspended from the rack, with spacing proportional to the density of spat settlement and the character of the growing area. The number of racks accommodated varies widely between the grow- ing sites. Production is estimated at 10 to 20 tons per hectare. Raft Culture According to Qiu and Li ( 1983). raft culture started in Japan in 1950. Since 1979. the Fisheries Research Institute of the South China Sea has conducted experimental raft culture of C. rivularis in Guangdong Province. The fattening period lasts from September to May. and three crops may be harvested, because 2 mos are sufficient under optimal seasonal conditions. The ratio of meat production to shell is some 60'/^ higher in raft-fattened oysters than in oysters harvested directly from bottom culture. C. rivularis can be marketed in less than 3 y using rafts, and that the condition factor will be increased by more that 22% and the meat quality will be superior to oysters cultivated by the tra- ditiimal bottom methods (Qiu & Li 1983). Though initial costs are higher, the increased production and working advantages of float- ing raft culture are apparent, and it is expected that raft culture will account for a steadily increasing share of oyster production in China (Qiu & Li 1983). Nie ( 1991 ) also mentioned that raft culture gives faster growth and a higher yield. A raft of 84 n\' will produce in 2 y what 667 nr of bottom culture will in 4 y. Rafts seem to w ithstand typhoons better than originally thought. DISCUSSION C. ariakcusis shares many life history traits with other Cras- soslrea species. It is clearly an estuarine species v\ith salinity tolerances similar to C. virginica. Its occurrence in river systems and apparent responsiveness to salinity changes for spawning cues suggests that its reproductive strategy is somewhat different than C. virginica. There are indications that larval behavior differs from that of C. virginica (M. Luckenbach, VIMS, pers. comm.), perhaps an adaptation to fluvial existence. Many other questions about its ecology are unanswered or incomplete and a number of research priorities have been identified (Rickards & Ticco 2002). One of the principal problems with extrapolating life history from the avail- able literature is the uncertainty over species designation. Some reports are clearly referring to C. ariakensis. e.g.. those from southeast China where aquaculture activity is concentrated and there is a long history of working with this species. Other reports are not so clearly C. ariakensis, especially ones deriving from western India and Pakistan. Also because of likely morphologic confusion, the geographic range for C. ariakensis is incompletely described. For example, it seetns likely that its range should in- clude the coast of Vietnam, yet there seem to be no direct accounts of this. There are accounts of its occurrence as far as Borneo, the Philippines, and Thailand, but these are unconfirmed. Froin a prac- tical standpoint, C. ariakensis from China are probably an appro- priate starting stock for an introduction, should that proceed, be- cause of similarities in latitude. From that respect, this area seems a most appropriate focus for obtaining more information on the species. Korea and Japan are possible sources as well. We did not encounter reports of C. ariakensis from Korea except as casual remarks. Stocks in Japan seem to be limited in abundance. It is unclear whether C. ariakensis is a "reef-forming" oyster, depending on how you define "reef" Clearly, Crassostrea species, and oysters in general, benefit from aggregation and adults or their shells provide substrate for recruitment in subsequent generations. Some accounts of C. ariakensis describe "oyster hills" that would clearly qualify as reefs (Zhang & Lou 1956b. Zhang et al. I960). Apparently, it is common knowledge among fishermen in China that C. ariakensis forms reefs. Other accounts have C. ariakensis occurring as small aggregates and singles. In our travels to China, we encountered several sites that had "natural" populations of C. ariakensis (Allen et al. 2002). There seem to be natural popula- tions in proximity to Xiamen although we did not observe this first hand. They were available in the local market and reportedly from natural populations that were harvested. There are natural sets of r. ariakensis near Hong Kong on the shores of Deep Bay. but this 18 Zhou and Allen could be from culture activity in the area. Seed is imported froin the Pearl River estuary, so there are likely sources of ""natural" populations in the Pearl River delta system. We observed, first hand, collection (harvesting) of C. ariakensis adults from sections of the Shiman River near Guan Du in close proximity to Zhanjiang Ocean University. According to the diver on hand, they occur in various assemblages, mostly stuck onto available substrate such as large rocks. They also occur in the Dafeng River in Guangxi province near Beihai. There are probably many other natural popu- lations along the coast of China. By way of caveat, it is difficult to attest to the "naturalness" of resident C. ariakensis populations. That is, those that we observed or heard about first hand were populations that occurred relatively deep (3-10 m) in river sys- tems. Whether at some time in the past populations of C. ariak- ensis were distributed in higher reaches of the water column (i.e.. before they were exploited over the millennia) is difficult to es- tablish. It is also difficult to distinguish whether spat fall is from natural populations or from aquaculture operations. There are clearly big questions concerning basic physiology in the kind of detail that exists for other congeners. C. ariakensis seems to exhibit growth rates that are extraordinary in head to head trials with C. virginica. Yet, these trials have been carried out in disease endemic areas where C. virginica could be sick or dying. Growth rates of C. virginica in. for example, the Gulf of Mexico, approach those seen in trials of C. ariakensis in the Chesapeake Bay or reported growth rates from the literature. Similar knowl- edge gaps exist for larval biology, reproductive physiology, pre- dation. competition, etc. In our opinion. C. ariakensis is an underused resource around the world. It clearly has aquaculture applications in estuarine areas that are marginal or unsuitable to C. gigas. the most popular cul- ture species. It seems hearty, fast growing, and highly marketable. Of course, utilization of this species would require introduction, as in the Chesapeake Bay. From that perspective, it would be useful to have more basic research on C. ariakensis with which to guide decisions about movement of this potentially valuable oyster spe- cies. ACKNOWLEDGMENTS The authors thank our Chinese colleagues for their warm as- sistance in compiling many of the papers cited here, particularly. Dr. KE Cai-Huan, Professor LI Fu-xue, Dr. CAl Lizhe. Dr. WU Xinzhong, Dr. Catherine Lam. Dr. QILI Dequan. Dr YU Xiang- yong. Professor CAl Yao-Guo (retired). Director LAO Zan. and Dr. LIU Zhigang, among others. We also thank S. Shumway for early editorial assistance. This work was supported by the Camp- bell Foundation and an award to S. Allen, Jr. from the Virginia Center for Innovative Technology. Contribution number 2541 from the Virginia Institute of Marine Science, College of William and Marv. LITER,4TURE CITED Abbott, R. T. & S. P. Dance. 1986. Compendium of Seashells. Melbourne. FL: American Malacologists, Inc. Aftab, N. 1988. Amino acid composition of tissue protein from five species of oysters. Pakistan J. Sci. Ind. Res. 31:200-202. Ahmed. M. 1971. Oyster species of West Pakistan. Pakistan J. Zool. 3: 229-236. Ahmed, M. 1973. Cytogenetics of oysters. Cytologia. 38:337-346. Ahmed. M. 1975. Speciation in living oysters. Adv. Mar. Biol. 13:357-397. Ahmed, M.. S. Barkati & Sanaullah. 1987. Spalfall of oysters in the Gharo- Phitti salt-water creek system near Karachi (Pakistan). Pakistan J. Zool. 19:245-252. Ahmed, M.. S. H. Naiz Rizvi & M. Moazzam. 1978. Studies on the settle- ment and control of marine organisms in cooling systems of coastal installations. Final Tech. Rep. Institute of Marine Biology. University of Karachi. 234 pp. Allen, S. K., Jr. & P. M. Gatfney. 1993. Genetic confirmation of hybrid- ization between Crassostrea gigas (Thunberg) and Crassostrea rivii- laris (Gould). .Aquaculture 1 13:291-300. Allen. S. K.. Jr., P. M. Gaffney, J. Scarpa & D. Bushek. 1993. Inviable hybrids of Crassostrea virginica (Gmelin) with C. rivularis (Gould) and C. gigas (Thunberg). Aquaculture 113:269-289. Allen. S. K., Jr.. K. Sellner & M. Zhou. 2002. Recommendations to the Chesapeake Bay scientific community: Contacts and opportunitie^ for C. ariakensis research in China. A white paper available from Chesa- peake Research Consortium, sellner@serc.si.edu, 8 pp. Amemiya. I. 1928. Ecological studies of Japanese oyster, with special reference to the salinity of their habitats. Tokyo, [Impenal] University. / Coll. Agr. 9:333-382. Angell, C. L. 1986. The biology and culture of tropical oysters. Manila. Philippines: ICLARM (International Center for Living Aquatic Re- sources Management) Studies and Reviews 13. 42 pp. Anon. 1996. The geographical distribution of Crassostrea virginica. C gigas and C. rivularis. VA. Mar. Resour. Bull. 28:11. Asif M. 1978a. Some observations on interspecific cross in three species of oysters from the coast of Karachi. Pakistan J. Zool 10:217-221. Asif. M. 1978b. Variations in allometeric growth in the shells of Crassos- trea rivularis (Gould). Saccostrea glomerata (Gould) and S. cuccullata (Born) from the coast of Karachi. Pakistan J. Sci. Ind. Res. 23:46-49. .Asif. M. 1979. Hermaphroditism and sex reversal in the four common oviparous species of oysters from the coast of Karachi. Hydrobiologia 66:49-55. Avvati. P. R. & H. S, Rai. 1931. The Indian zoological memoirs on Indian animal types III. In: K. N. Bahl. editor. Ostrea cucullata (the Bombay Oyster). Lucknow, Methodist Publishing House, p. 7. Barkati. S. & R. M. Khan. 1987. Relative growth in three species of oysters. Pakistan J. Sci. Ind. Res 30:624-627. Buroker, N. E., W. K. Hershberger & K. K. Chew. 1979. Population genetics of the family Ostridae. II. Interspecific studies of the genera Crassostrea and Saccostrea. Mar. Biol. 54:171-184. Cahn. A. R. 1950. Oyster culture in Japan. United States Fish and Wildlife Service, fishery leaflet 383. 80 pp. Cai. v.. C. Deng & Z. Liu. 1992. Studies on the ecology of Crassostrea rivularis (Gould) in Zhanjiang Bay. Tropical Oceanol. 11:37—44. Cai. Y. 1966. A preliminary survey on bivalvia from Fujian coast. / Zool./ Dong Wu Xue Bao iChinese) 2:76-80. Cai. Y. & X. Li. 1990. Oyster culture in the People's Republic of China. World Aquaculture 21:67-72. Cai. Y.. Y. Zhang & R. Wei. 1979. Ostreacea. In Introduction of malacol- ogy. Shanghai Sci. and Tech. Press. Shanghai, pp. 216-217. Cai. Y.. Z. Liu & S. He. 1989. A study on the artificial rearing of spats of Ostrea rivularis Gould. Mar. Sci./Hai Yang Ke Xue (Chinese) 1:53-56. Calvo. G. W., M. W. Luckenbach & E. M. Burreson. 1999. Evaluating the performance of non-native oyster species in Virginia. / Shellfish Res. 18:303. Calvo, G. W., M. W. Luckenbach, S. K. Allen, Jr. & E. M. Burreson. 2001. A comparative field study of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin 1791) in relation to salinity in Virginia. J. Shellfish Res. 20:221-229. Carriker. M. R. & P. M. Gaffney. 1996. A catalogue of selected species of living oysters {Ostreacea) of the worid. In: V. S. Kennedy, R. I. E. Newell & A. F. Eble, editors. The eastern oyster: Crassostrea virginica. College Park. MD: Maryland Sea Grant College Publication, pp. 1-18. CHASSOSTHEA ARIAKENSIS REVIEW 19 Chen. H. 1941. The growth of oysters in Spuitina uni^Hca sea beach ni Northern Jiangsu. Mar. Sci. / Hai )V»is; Ke Xiic (Chiiuwcl 5:68-69. Coan. E. V.. P. V. Scott & F. R. Bernard. 199?. Bivalve seashells of western North America, marine bivalve mollusks from Arctic Alaska to Baja California. Santa Barbara: Santa Barbara Museum of Natural History, pp. 213. 215 and 217 . Cochennec. N.. T. Renault. P. Boudry. B. Chollet & A. Gerard. 1998. Bonamia-Wke parasite found in the Suminoe oyster Crassoslrea rivii- hiris reared in France. Dis. Aquatic Organisms 34:193-197. Coe, W. R. 1934. Alternation of se.xuality in oysters. /4m. Wa/ 68:236-251. Downing. S. L. 1988a. Comparing adult performance of diploid and trip- loid monospecific and interspecific Crassoslrea hybrids. J. Shellfish Res. 7:549. Downing. S. L. l98Sh. Triploid and diploid hybrids between the oysters Crassoslrea gigas and C. riviilaris: production, detection and potential. J. Shellfish Res. 7:156. Downing, S. L. 1991. Hybrids among the Pacific. American and Suminoe oysters: induction and aquaculture potential. / Shellfish Res. 10:514. Dunker, C. 1882. Index Molluscorum Maris Japonici. London. 301 pp. Durve. V. S. 1986. On the ancestry and distribution pathways of three species of Indian oysters. Ind. J. Mar. Sci. 15:56-58. Fei, H. 1928. Oyster industry. New construclion 5 Francis, E., K. S. Reece & S. K. Allen, Jr. 2000. Species designation among sympatric oysters Crassostrea ariakensis. C. giga.\. and C. .sika- mea. J. Shellfish. Res. 19:662-663. Fujimori. S. 1929. Studies on the marine aquiciilture of Ariake Bay. Fukuoka Fish Exp Sta. 75 pp. Fujita. I9I3. Nihon suisan dobutsugaku (Japanese aquatic fisheries ani- mals). Vol. 2. Tokyo: Shokabu. 292 pp. Gaffney, P. M. & S. K. Allen. Jr. 1993. Hybndization among Crassoslrea species: a review. AquaculUire 1 16:1-13. Gaffney. P. M. & F. X. O'Biern. 1996. Nuclear DNA markers for Cras- soslrea species identification. / Shellfish. Res. 15:510-511. Galstoff. P. S. 1964. The American Oyster. Crassostrea virginicu Gmelin. United States Depanment of the Interior. Fish and Wildlife Service. Fishery Bulletin 64. Washington. DC: United States Government Print- ing Office. 480 pp. Glude, J. B. 1971. Identification of oysters of the South Pacific Islands. National Marine Fisheries Service, Seattle, WA: Mimeo. Gould, A. A. 1861. Description of shells collected by the north Pacific exploring expedition. Proc. Boston Soc. Natural Histoiy 8:14-40. Guan, Y. & Y. Li. 1986. Comparative studies on some physiological and biochemical indexes of the cultured oysters in the Zhujiang River es- tuary. Proc. Conchol. 3:1 lO-l 16. Guan. Y. & Y. Li. 1988. Preliminary studies on the oxygen consumption of gill tissue separated the oyster, Crassostrea rivularis. Oceanol. Lim- nol. Sin./Hai Yang Yu Hu Zhao (Chinese) 19:210-214. Guan. Y. & Y. Zheng. 1990. A comparison study on the esterase isoen- zyme of cultured oysters in China. Soiiili China Sea Fisheries Res. 2:32-35. Guo. X.. S. E. Ford & F. Zhang. 1999. Molluscan aquaculture in China. / Shellfish Res. 18:19-31. Hallemian. E.. M. Leffler. S. Mills & S. K. Allen. Jr. 2002. Aquaculture of triploid Crassostrea ariakensis in Chesapeake Bay: A Symposium Re- port. Maryland Sea Grant Extension Publication UM-SG-TS-2002-OI and Virginia Sea Grant Publication VSG-02-03. 20 pp. Harry, H. W. 1981. Nominal species of living oysters proposed during the last fifty years. The Veliger 24:39-t5. Hasan. S. A. 1960. Oyster fishing resources of Pakistan. In: Proc. 4"' Congress. PIOSA. Karachi. Section B. pp. 167-171. Hedgecock. D.. G. Li. M. A. Banks & Z. Kain. 1999. Occurrence of the Kumamoto oyster Crassostrea sikainea in the Ariaki Sea. Japan. Mar. Biol. 133:65-68. Hirase. S. 1930. Transactions I. On the classification of Japanese oysters. Jap. J. Zool 3:1-65. Hirase. S. 1936. A collection of Japanese shells with illustrations in natural colors. Tokyo: M. Sanshodo. 217 pp. Honicll. J. 1910. The practice of oyster culture at Aroachon and its lessons for India. Madras Fisheries Bull. 5 Hu. J.. Y. Zhong & Y. Lin. 1994. Spat collection technique and spat collection forecast of Ostrea rivularis Gould. / Xiamen Fisheries Col- lege/Xiamen Shui Chan Xue Yuan Xue Bao I Chinese) 16:28-33. Huang. Z.. C. Li. L. Zhang. F. Li & C. Zheng. 1981. On the marine fouling and boring organisms off Zhejiang southern coast I. Notes on the fouling organisms of Wenzhou harbor. Acta Oceanologica Sinica/Hai Yang Xue Bao (Chinese) 3:634-638. Huzimori. S. 1920. Experiments on Oyster-culture. Hukuoka-Suisan- Sikenzyo-Gyomukotei-Hokoku. leyama. H. 1975. Chromosome numbers of three species in three families of Pteriomorpha. Venus. Jpn. J. Malacol. 34:26-32. Imai. T. 1978. The revolution of oyster culture. In: A. A. Balkema and Protterdam, editors. Aquaculture in shallow seas: progress in shallow sea culture. Translation of Senkai Kanzen Yoshoki. Tokyo: Koseisha Koseiiku Publishers. 1971. 614 pp. Imai. T. & M. Hatanaka. 1949. On the artificial propagation of Japanese common oyster. Ostrea gigas Thunberg by non-colored naked flagel- lates. Bull. Inst. Agr. Res. Tohoku Univ. 1:1-7. Imai. T. & S. Sakai. 1961. Study of breeding of Japanese Oyster Crassos- lrea gigas. Tohoku J. Agr. Res. 12:125-171. Ke. C. & W. Wang. 2001. Bioaccumulation of Cd. Se. and Zn in an estuarine oyster (Crassoslrea rivularis) and a coastal oyster (Saccos- trea glomerala). Aquat. Toxicol. 56:33-51. Kira. T. 1962. Shells of the Western Pacific in color. Osaka. Japan: Hoikusha Publishing Co.. Ltd. 224 pp. Kuroda. T. & T. Habe. 1952. Check List and Bibliography of the Recent Marine Mollusca of Japan. Tokyo: Leo W. Stach. 210 pp. Lamy. E. 1929-1930. Revision des Ostrea vivants du Museum National d'Histoire Naturelle de Paris. Journ. De conchyl. 73 (ser. 4, v. 27). No. 1 (30 April 1929): 1^6: 3 Figs; No. 2 (20 July 1929): 71-108: No. 3 (30 October 1929): 133-168; No. 4 (28 February 1930): 233-275; Pit. 1. Langdon. C. J. & A. M. Robinson. 1996, Aquaculture potential of the Suminoe Oyster (Crassostrea ariakensis Fugita 1913). Aquaculture 144:321-338. Leitao. A.. P. Boudry. J.-P. Labat & C. Thiriot-Quievreux. 1999, Com- parative karyological study of cupped oyster species. Malacologia 41: 175-186. Li. G., Y. Hu & N. Qing. 1988. Population gene pools of big-size cultivated oysters (Crassostrea) along the Guangdong and Fujian coast of China. In: Proceedings on marine biology of the South China Sea. Beijing: China Ocean Press, pp. 51-70. Li. K., L. Zou. J. Yang. P. Li. L. Yu. Z. Zheng, C. Chen & S. Wang. 1994. Experimental studies on nourishing Yin effect of oyster complete nu- tritional tablet. Chinese J. Mar. Drugs/Zhong Guo Hai Yang Yao \Vu (Chinese) 13:12-16. Li. X. 1989. A comparative morphology of mantle cavity in some Chinese oysters. Oceanol. Linmol. Sin./Hai Yang Yu Hu Zhao (Chinese) 20: 502-507. Li. X. & Z. Qi. 1994. Studies on the comparative anatomy, systematic classification and evolution of Chinese oysters. Studia Marina Sinica 35:143-177. Liang. G.. S. Chen & G. Xu. 1983. Report on artificial incubation of Ostrea rivularis Gould. Mar. Sci./Hui Yang Ke Xue (Chinese) 5:41^3. Lin. Q.. X. Jia & X. Lu. 1991. Petroleum hydrocarbons in oysters during the fattening period. In: Proceeding of the 4"' Chinese oceanological and limnological science conference (Qingdao. Shandong Province). Beijing: Science Press, pp. 135-141. Lischke. C. E. 1871. Japanische Meeres-conchylien. II. pp. 160-162. Littlewood, D. T. J. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Mol. Phylogenetics Evol. 3:221-229. Liu. B-Q. & J-X. Dai. 1998. Studies on genetic diversity in oysters. Cras- sostrea (in Chinese). / Fisheries China 3:193-198. 20 Zhou and Allen Loosanoff. V. L. & F. D. Tomnier, 1948. The effect of suspended silt and other substances on the rate of feeding of oysters. Science 107:69. Loosanoff, V. L. & J. B. Engle. 1 942. Effects of different concentrations of plankton forms upon shell movements, rate of water pumping and feeding and fattening of oysters. Anal. Rec. 84:86. Lu. C. 1994. Oyster Ostrea rivularis as an indicator of heavy metals pollution. J. Oceanogr. Taiwan Strait/Tai Wan Hai Xia (Chinese) 13: 14-20. Lu. C, W. Xie & G. Zhou. 1998a. Crassosnea rivularis as a biomonitor of zinc pollution of seawater. China Environ. Sci. 18:527-530. Lu. C. W. Xie & G. Zhou. 1998h. Studies on Crassnslrea rivularis as a biological indicator of cadmium pollution. J. Fish. Sci. Chimi/Zhon^ Guo Slmi Clian Ke Xiie (Chinese) 5:79-83. Lu. C, W. Xie & G. Zhou. 1998c. Studies on Crassoslrea rivularis as a biomonitor for copper pollution in seawater. Mar Environ. Sci./Hai i'ang Hiian Jing Ke Xue (Chinese) 17:17-23. Mahadevan, S. 1987. Oyster resources of India. In: K.N. Nayar and S. Mahadevan, editors. Oyster culture-status and prospects, central marine fisheries research institute bulletin 38. Cochin. India, pp. 14-16. Miyazaki, I. 1939. Some notes on the cross-fertilization of Japanese oys- ters. Bull. Jpn. Soc. Scientific Fisheries 7:257-261. Moazzam. M. & S. H. N. Rizvi. 1983. Settlement of oyster lanae in Pakistani waters and its possible implication for settnig up oyster culture. In: Pro- ceedings of the Symposium on Coastal Aquaculture. Part 2: Molluscan Culture. Marine Biological Association of India. Cochin. India. Mok. T. K. 1974. Observations on the growth of the oyster. "Crassoslrea gigas" Thunberg. in Deep Bay. Hong Kong. Honii Kong Fish. Bull. 4:45-53. Nelson. T. C. 1928. Relation of spawning of the oyster to temperature. Ecology 9:145. Nelson. T. C. 1938. The feeding mechanisms of the oyster I. On the pallium and branchial chambers of O. virginicu. O. edulis and O. un- gulala. J. Morphol. 63:1-61. Nie. Z. Q. 1991. The culture of marine bivalve moUusks in China. In: W. Menzel, editor. Estuarine and marine bivalve mollusk culture. Boston: CRC Press, pp. 261-276. O'Foighil. D.. P. M. Gaffney & T. J, Hilbish. 1995. Differences m mito- chondrial 165 ribosomal gene sequences allow discrimination among American [Crassoslrea virginica (Gmelin)] and Asian [C. gi.gas (Thun- berg) C. ariakensis Wakiya] oyster species. / &/). Mol. Biol. Ecol. 192:211-220. Orton. J. H. 1936. Observations and experiments on sex change in the european oyster (Ostrea edulis). Part 5. A simultaneous study of spawning in 1927 in two distinct geographical localities. Mem. Miis. Roy. Hist. Nat. Belg. Ser 2, 3. 977. Patel. B. V. 1979. Bionomical. biochemical and some pollution studies on oysters. Crassoslrea and Ostrea species. Ph.D. thesis submitted to Suarashtra University. Patel, S. K. & K. L. Jetani. 1991. Survey of edible oysters from the Saurashtra coast. / Curr. Biosci. 8:79-82. Qasim, R., N. Aftab & S. Barkati. 1985. Biochemical composition and caloric values of four species of oysters from Karachi Coast. / Phann. Univ. Kar. 3:51-55. Qiu. L. & Y. Li. 1983. A report on the raft culture of oyster (Ostrea rivularis Gould). Trans. Chinese Soc. Malacol. 1:149-156. Que. H. & S. K. Allen. Jr. 2002. Hybridization of tetraploid and diploid Crassoslrea gigas (Thunberg) with diploid C ariakensis (Fujila). J. Shellfish. Res. 27:137-143. Ranson, G. 1967. Les especies d'huitres vivants actuellement dans le monde, definies par leurs coquilles larvaires ou prodisso-conchs. Etud des collections de quelque,s-un de grand musees d'Histoire Naturell. Rev. Trav. Inst. Peche Maril. 31:127-199. Rao, K. S. 1987. Taxonomy of Indian oysters. In: K. N. Nayar and S. Mahadevan, editors. Oyster Culture-Status and Prospects. Central Ma- rine Fisheries Research Institute Bulletin 38. Cochin. India, pp. 1-6. Reeve, L. A. 1871. Conchiologica Iconica. London. Rickards. W. L. & P. C. Ticco. 2002. The Suminoe ovster, Crassoslrea ariakensis, in Chesapeake Bay: Current status and near-term research activities. Charlottesville. VA: Virginia Sea Grant. 6 pp. Rong. S.. S. Shi. Q. Mo. S. Liu, Z. Liang & X. Zhao. 1994. Tetraploid induced by physical and chemical methods in Jinjiang oyster (Cras- soslrea rivularis). Acta Oceanologica Sinica/ Hai Yang Xue Bao (Chi- nese) 13:275-283. Spark. R. 1925. Studies on the biology of the oyster (Ostrea edulis) in the Limfjord. with special reference to the influence of temperature on the sex change. Rep. Dan. Biol. Stat.. No. 30 Sparks. A. K. 1965. A report on the status and potential of the marine shellfisheries of Kenya. Stenzel. H. B. 1971. Oysters. In: K. K. Moore, editor. Treatise on inver- tebrate paleontology. Part N, vol. 3. Mollusca 6. Boulder. CO: Geol. America Inc. and the University of Kansas. Taki. I. W. 1933. On the report about the specific difference between Ostrea gigas and Ostrea rivularis in Ariake Bay by Mr. Fujimori. Venus 13:365. Tanaka. Y. 1954. Spawning season of important bivalves in Ariake Bay- II. Ostrea rivularis Gould and O. gigas Thunberg. Bidl. Jpn. Soc. Scien- tific Fisheries 19:1161-1164. Thompson. J.M. 1954. The genera of oysters and the Australian species. Aiist. J. Mar. Freshwater Res. 5:132-168. Thomson, J. M. 1954. The genera of oysters and the Australian species. Aiisl. J. Mar. Freshwater Res 5:132-168. Tongoe. K. 1981. Oysters in Japan. J. Sci Hiroshima Univ. Ser B. Div I (Zool) 29:291-118. Wakiya. Y. 1915. Oysters of Korea. Report of the Fish. Invest.. Govern- ment General of Korea. Wakiya. Y. 1929. Japanese food oysters. Jpn. J. Zool. 2:359-367. Wakiya. Y. 1930. Japanese food oyster. Proceedings of the Fouth Pacific Science Congress. Batavia. Java. 3:341-348 (Same as text part of Wak- iya. Y. 19.30) Wu. X. Z. & J. P. Pan. 2000. An intracellular prokaryotic microorganism associated with lesions in the oyster. Crassoslrea ariakensis Gould. J. Fi.^h. Dis. 23:409-414. Wu. X. Z.. J. Pan & J. Jiang. 1997. Advances in studies on marine mol- luscan diseases III. Pestology and neoplasia of marine moUusks. Mar. Sci. BulL/Hai Yang Tong Bao (Chinese) 16(4):82-87. Xu. Y.. Z. Gao. Y. Ji. X. Chen & C. Qin. 1992. Holocene oyster reef in Shangyuan-Kengyuan Luoyuan Bay. Fujian and sea level variation. J. Oceanogr. Taiwan Strail/Tai Wan Hai Xia (Chinese) 1 1:368-371. Yu. J.. Z. Xiong. W. Tong. S. Shi. X. Zhao & S. Rong. 1993. The Karyo- types of Jinjiang oyster Crassoslrea rivularis. J. Zhanjiang Fisheries College/Zhanjiang Shui Chan Xue Yuan Xue Bao (Chinese) 13:27-29. Zhang, X. & Y. Xie. I960. Culture of major bivalves in China. Bull. Biol./ Sheng Wu Xue Bao (Chinese) 5:197-201. Zhang. X. & Z. Lou. 1956a. A study on Chinese oysters. Acta Zool. Sin./ Dong Wu Xue Bao (Chinese) 8:65-94. Zhang. X. & Z. Lou. 1956b. Oyster. Bull. Biol./Sheng Wu Xue Bao (Chi- nese) 2:27-32. Zhang. X. & Z. Lou. 1959. Oyster. Beijing: Science Press. 156 pp. Zhang, X., Z. Qi & Y. Xie. 1959. Moeurs de s'alimenter chez VOstrea rivularis Gould. Oceunol. Limnol. Sin. /Hai Yang Yu Hu Zhao (Chinese) 2:163-179. Zhang, X.. Z. Qi. J. Li. X. Ma. Z. Wang. X. Huang & Q. Zhuang. 1960. Bivalves of South China Sea (Nanhai). Beijing: Science Press. 274 pp. Zhang, Y.. B. L. Munday & J. Handlinger. 1995. Mass mortality of flat oysters (Ostrea rivularis) associated with a bloom of Prorocenlrum sp. in the port of Zhanjiang. South China. Bull. Eur Ass. Fish Pathol 15:61-63. Zhao. Z.. P. Jiang & A. Li. 1991. Determination of calcium carbonate, trace elements and amino acids content in oyster shells. Chinese J. Mar Drugs/ Zhang Guo Hai Yang Yao Wu (Chinese) 10:11-14. Zhou. M.. Y. Kao & Y. Wu. 1982. Preliminary studies on hybridization of Crassoslrea gigas with Ostrea rivularis and Ostrea plicatida. J. Fish- eries China 6:235-241. Joiinial uj Shellfish Research. Vol. 22, No. 1, 21-3U, 20U3. CONSUMER RATINGS OF NON-NATIVE (CRASSOSTREA G/GAS AND CRASSOSTREA ARIAKENSIS) VS. NATIVE {CRASSOSTREA VIRGINICA) OYSTERS JONATHAN H. (JRABOWSKI.'* SEAN P. POWERS/t CHARLES H. PETERSON,' MONICA J. POWERS,' AND DAVID P. GREEN" ' University of North Carolina at Chapel Hill. Institute of Marine Sciences, Morehead City, North Carolina 28557 and 'North Carolina State University; Center for Marine Science and Technology, Morehead City, North Carolina 28557 ABSTRACT Given suggeslion^ that a non-native oyster be used to replace the depleted native oyster, consumer preference evalu- ations were conducted to determine how two non-native oysters, Crassostrea gigas and C. ariakensis, when grown in North Carolina estuaries, were rated by consumers. Tests compared the taste, appearance, and/or aroma of both raw and cooked non-native oysters to similarly prepared native oysters, C. virginica. In the first series of tests, consumers exhibited a slight preference for raw C. virginica over raw C. gigas. When cooked, both species were rated equal. In the second series of tests, a larger group of participants ranked the taste, appearance, and aroma of C. virginica. C. gigas, and C. ariakensis. Participants that tasted raw oysters collectn ely preferred C. virginica over both non-native species. This preference remained strong regardless of the frequency with which participants consumed oysters. Preferences for appearance and aroma varied; however, ratings never indicated a preference for either non-native species over C. virginica. Participants as a whole preferred the taste of cooked C. virginica better than C. gigas. whereas a taste preference did not exist between cooked C. virginica and C. ariakensisis. Given that participants collectively preferred the taste of both raw and cooked C virginica to C. gigas. the suitability of C. gigas for substitution in either the raw or steamed oyster market is questionable. For oysters of similar length (80 to 1 10 mm), dry tissue weight of C. ariakensis was twice that of C. virginica. This higher per-oyster yield suggests that C ariakensis might be more suitable for a steamed or packaged oyster market where oysters are sold by meat weight rather than by number. However, these markets often command much lower prices, perhaps rendering unfeasible the aquaculture of this introduced oyster. Before large-scale introduction of non-native oyster species occurs, consumer preferences should be incorporated into economic evaluations that include additional economic (oyster prices, market demand and supply functions) and biological information (growth and survivorship). Profitability expectations generated by the model then need to be weighed against the potential ecological risks and ecosystem benefits of aquaculture or introduction to the wild for each non-native oyster species. KEY WORDS: Crassostrea ariakensis. Crassostrea gigas. Crassostrea virginica. economic feasibility, native versus non-native oysters, raw versus cooked oysters, frequent versus inexperienced consumers, taste test INTRODUCTION Landings of the eastern oyster, Crassostrea virginica (Gmelin 1791 ), have declined by over 90'7c during the past century in the major estuaries of the eastern United Slates (MacKenzie 1983, Hargis & Haven 1988. Frankenberg 199.S). Habitat degradation from destructive harvesting techniques (Rothschild et al. 1994, Lenihan 1999) and mortality induced by bottom-water hypoxia/ anoxia, sedimentation, and parasitic diseases (Seliger et al. 1985, Ford & Tripp 1996, Lenihan & Peterson 1998, Lenihan et al. 1999) collectively have contributed to this decline. In North Carolina, efforts to sustain the oyster fishery over the past several decades through shell plantings have contributed to but not restored land- ings, which are less than K/r of historic maxima achieved in the late 1800s (Frankenberg 1995). Introduction of non-native species such as C. gigas (Thunberg 1793) or C. ariakensis (Fujita 1913) is a possible alternative or supplement to continued efforts to restore native populations, and could resuscitate the oyster industry in the eastern United States. The Pacific oyster, C. gigas. accounts for over 9,0'^i of the world's aquaculture production of oysters (Ayers 1991), and thrives in shallow, sub-tidal estuaries at higher salinities (Calvo et al. 1999). Native to Japan and the Korean peninsula (Mann et al. *Corresponding author. University of Maine at Orono, Darling Marine Center. 193 Clarks Cove Road. Walpole. ME 04S73. E-mail: jgrabow@ maine.edu tCurrent address: University of Southern Alabama. Dauphin Island ,Sea Lab. Dauphm Island. AL 36528 1991). it has been successfully introduced to France, Oregon, Washington, western Canada. Australia and New Zealand (Shatkin et al. 1997). C. gigas often establishes populations successfully when introduced and is successfully cultured in part because it is highly resistant to the protozoan diseases MSX. Haplosporidiuin nelsoiii. and dermo. Perkinsus inariiuis (Calvo et al. 1999). MSX and dermo continue to impede recovery of native oyster popula- tions along the eastern coast of the US (Ayers 1991. Mann et al. 1991 ). C. gigas also typically reaches harvest size more quickly than native oysters, leading many culturists to prefer growing C. gigas over native species (Pollard & Hutchings 1990. Ayers 1991, Parameswar 1991 ). In contrast to C. gigas. the Suminoe oyster, C. ariakensis, currently does not contribute substantially to oyster fisheries of the world. Despite some taxonomic confusion with C. riviilaris. the native distribution of C. ariakenis is thought to range from Paki- stan to Japan, and extends into quite low salinities within the estuaries that it inhabits (Breese & Malouf 1977, Langdon & Rob- inson 1996). Like C. gigas. C. ariakensis grows more quickly than most other oyster species (Byrne 1996. Calvo et al. 2001 ). partly explaining why many fishermen in North Carolina and Virginia are advocating its introduction. This species can be grown to mar- ket size in 12-18 mo in colder waters along the west coast of the U.S. (Langdon & Robinson 1996). Calvo et al, (2001) also dem- onstrated that C. ariakensis is resistant to MSX and dermo. Long- term failure of management to restore native oyster populations coupled with higher growth rates and disease-resistance of C. gi- gas and C. ariakensis have created the impetus within industry to promote triploid aquaculture of and even intentional introduction 21 22 Grabowski et al. of diploid non-native species along the Atlantic coast of North America. Previous intentional and accidental introductions of commer- cial fishery species have resulted in many well-documented nega- tive impacts (Naylor et al. 2001 ). For example, the predatory oys- ter drill, and both MSX and dermo. have been introduced unin- tentionally through oyster introductions (Carlton 1999, Burreson et al. 2000). Because of the risks associated with introducing a new fisheries species, including possible introduction of non-native dis- eases, competitors, and predators, importation of harmful mi- crobes, and induction of competition with native species (Ruiz et al, 2000, Naylor et al. 2001), assessing and contrasting the poten- tial risks and benefits associated with any proposed introduction should precede taking action. Here we present results of controlled trials assessing how oyster consumers rate the palatability of the two non-native species under consideration for introduction as compared with C. virginica. MATERIALS AND METHODS Two series of tests were conducted to determine consumer responses to non-native oysters grown in eastern North Carolina and to compare those responses to native oysters. In both series of tests, preferences among native, Crassostrea virginica (eastern oyster) and non-native species, Crassostrea gigas (Pacific oyster), and Crassostrea ariakensis (Suminoe oyster), were tested sepa- rately for raw and cooked oysters. Regulations set forth by the Shellfish Control Authorities in North Carolina mandated that we inform participants that they were consuming raw or steamed oys- ters, the location where oysters were grown (non-natives) or har- vested (natives), and the species of oysters that were being offered. Participants in the tests were drawn from the local coastal com- munity surrounding Morehead City, NC and represented a diverse range of ages (20-81 y old), professions, and knowledge of local fisheries. Of the 31 individuals that participated in the first taste test, a few also were among the 96 participants in the second. Each participant completed and signed a waiver form regarding risk of raw seafood consumption, completed a deinographic survey, and provided information on oyster consumption. Finally, participants were offered water and crackers to assist them to cleanse their pallets between tasting oysters. In the first series of tests (conducted on 21 August 2000), we compared consumer responses to taste and appearance of C vir- ginica to C. gigas. Triploid C. gigas (approx. 30 mm in length) obtained from the Virginia Institute of Marine Sciences (VIMS) had been cultured since February 2000 in plastic mesh vexar cages held on racks above the sea bottom in Chadwick Bay, Onslow County, North Carolina. C gigas achieved a length of approx. 80 mm by .August 2000 and were removed from the field and stored in upwellers at the Institute of Marine Sciences in Morehead City, North Carolina. Wild C. virginica oysters were harvested in Au- gust 2000 from both the Newport River and Bogue Sound (Cart- eret County, North Carolina). Participants were asked to rate un- labeled raw or cooked oysters in paired contrasts. Separate trials were performed for raw and cooked oysters. Some participants were involved in both trials. To begin a trial, two oysters (either raw or cooked) on the half-shell were presented to each participant, who then rated each oyster's appearance and (separately) taste on a scale of I (least desirable) to 5 (Fig. I). Each participant also specified whether either oyster tasted unappetizing, and, if any difference was perceived, which one tasted saltier, was more wa- tery, and was more preferable overall (including an explanation for any preference). A second pair of oysters was presented to each participant, who then answered the same set of questions. One of the pairs of oysters presented a contrast of the two species, whereas Circle the most appropriate response 1 . Have you eaten raw oyster before? Yes 2. Approximately how many times a year do you eat raw oysters? 0 1 2 1st Test Oyster # (A) vs. # JBL Yes No A B Yes No A B Yes No A B No 5 >6 1 . Rate tlie appearance of eacli oyster on a scale from 1 to 5 with 5 being the best and 1 being the worst. A=12 3 45 B=12 3 45 2. Rate the taste of each oyster on a scale from 1 to 5 with 5 being the best and ) being the worst. A= 12345 B= 12345 3. Did one or both of the oysters taste unappetizing? Ifso whichone(s): A B Both 4. Did one oyster taste saltier than the other? Ifso which one: 5. Did one oyster taste more watery than the other? Ifso winch one: 6. If you preferred one oyster over the other briefly explain why. Figure I, Survey form used in first taste test. Consumer Ratings oi- Oysthrs 23 OYSTER TASTE PANEL Panelist Code # Sample: Raw Steamed Date: Procedure: Three samples of oysters (either raw or steamed) will be placed in front of you. We would like for you to taste each of the oysters and evaluate them for their quality attributes by answenng the questions listed below. Please rate each sample accordini; to their four diait code by placin^ a mark across the unmarked line that best reflects your opinion, e.g.. like greatly (far right), neither like or dislike (middle) and dislike greatly (far left). Note that you are not required to chew or swallow the oyster samples. You may spit the sample out at any time you need to into the cup provided. You are expected to drink (wash mouth) between samples with water. If you feel a need to be less fatigued in terms of flavors, aromas and textures blending together between samples, then you should eat crackers and drink some water. Ql : When you eat oysters either at home or in a restaurant, what quality attributes are most important to you? Q2: How does the appearance of the samples appeal to you? What appearance characteristics do you like? Dislike? Dislike Greatly Like Greatly Q3: How does the aroma of the samples appeal to you? What aroma characteristics do you like? Dislike? Dislike Greatly Like Greatly Q4: How does the texture of the samples appeal to you? What texture characteristics do you like? Dislike? Dislike Greatly Like Greatly Q5: How does the flavor of the samples appeal to you? What flavor characteristics do you like? Dislike? Dislike Greatly Like Greatly Q6: What other attributes do you perceive in the samples? Please dispose of any left over samples in the appropriate trash container. Be sure to turn your sensory survey sheet to the project assistant when you leave the room. We appreciate your time in this study! Results will be available from the project coordinator. THANK YOU! Figure 2. Survey form used for second laste. the other presented two C. virginica with one from each site to ters (one of each species, either all raw or cooked) on the half- determine if grow-out location affected the test results. shell, and asked to rate the appearance, taste, texture and aroma of The second series of taste tests (conducted on 6 and 7 February each oyster. To quantify a participant's ratings of each oyster, we 2002) evaluated consumer responses to appearance, aroma and measured the distance of the mark along the line, creating a scale taste of C. virginica, C. gigas, and C. ariakensis. Triploid C. gigas from 0 cm (least desirable) to 10 cm (most desirable). We asked and C. ariakensis (approx. 30 mm in length) had been obtained participants to indicate profession, age group and the frequency from VIMS and planted at Chadwick's Bay (3 April 2001 ) and in with which they eat oysters (either raw or cooked, depending on the Newport River (23 March 2001). Oysters were cultured using whether they were tasting raw or cooked oysters) to determine if the cage and rack method and achieved harvestable size by January these factors influence their ratings. 2002. C. virginica was also harvested in January 2002 from Chad- We also quantified the wet and dry weights of ^0 replicate wick's Bay and the Newport River in close proximity to culture oysters (80-1 10 mm shell length) for each of the three species to operations. In this second set of taste tests, we requested more determine whether percent dry tissue or total dry tissue differed subtle distinctions by asking participants to rate each oyster tasted among the three species. We determined that the shell length of by placing a mark on a continuous line that ranged from least to oyster specimens did not vary among the three species with a most desirable (Fig. 2). Each participant was presented three oys- one-factor analysis of variance (ANOVA: F, ,47. 1.06. P = 0.35). TABLE 1. Results of Wilcoxon signed rank tests comparing consumer ratings for taste and appearance of Crassostrea virginica with C. gigas in the first series of taste tests. All Part cipants" Infrequent Oyster Consumers Taste .\ppearance Frequent 0\ Taste ster Consumers Oyster Feature Taste .\ppearance .\ppearance Raw oysters No. of 0 differences" 2 1 1 0 1 1 No. of ranlcs < 0" 4 11 3 8 1 3 No. of ranks > 0" 10 4 5 1 5 3 Z value -1.38 -1.2.^; -0.56 -2.19 -1.57 -0.63 P value (1.17 0.21 0.58 0.03 0.12 0.53 Cooked oysters No. of 0 differences" 2 2 1 0 1 2 No. of ranks < 0" 6 7 1 2 5 5 No. of ranks > 0" 7 6 3 3 4 3 Z value -0.21 -1.12 -0.37 -0.27 -0.06 -1.26 P value 0.S.3 0.26 0.72 0.79 0.9.S 0.21 ^ Raw data were analyzed collectively and then reanalyzed by subgroup to determine whether those participants who rarely eat oysters have preferences from those that frequently eat oysters. " No. of 0 differences indicates the number of participants that rated species equally, no. of ranks <0 indicate participants who rated C. gigns than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginicn as better than C. gigas. different as better 3 1 Participant Category b. Cooked Oysters 5 Appearance Participant Category Figure 3. Results from taste test 1. Taste and appearance ratings of (a) ra« and (b) cooked oysters (Crassostrea virginica vs. C. gigas) are presented for the following participant categories: I ) all participants, 2 1 infrequent consumers of raw oysters, and 3 1 frequent consumers of raw oysters. The test in which ('. virginica was ranked significantly lower than C. gigas is marked with an asterisk. Error bars indicate +1 SE. Consumer Ratings of Oysters 25 Soft tissue was removed from each oyster, placed in a pre-weighed aluminum pan. and weighed using a Mettler balance (0.001 g). Tissue was then dried at 60°C in a drying oven for 48 h. and weighed again to obtain a dry tissue weight (dry weight with pan minus pan weight). The proportion of each oyster's soft tissue that is biomass was calculated by dividing the dry weight (tissue weight minus water weight) by the initial wet weight. Slatisliial A luilyses Results from the first taste test were analyzed using the Wil- co.xon signed rank test. C. virginicci from Bogue Sound were first compared with C. viri^inica from the Newport River. Because rankings of native oysters from Bogue Sound and the Newport River did not differ from each other (in taste: P = 0.97; in ap- pearance: P = 0..^.^). we concluded that grow-out site did not affect the taste of native oysters in our study and we analyzed rankings for C. gigas versus C. virginica from both sites collec- tively. Separate C. virginica versus C. gigas tests were conducted for appearance and taste of raw and cooked oysters. Additional tests were conducted to determine if results varied between groups that ( 1 ) rarely and (2) frequently (three or more limes per year) eat oysters to determine if the frequency with which participants eat oysters affected preferences for native versus non-native oysters. Results from the second taste test were also analyzed using the Wilcoxon signed rank test to determine whether participants pre- ferred the taste, appearance, or aroma of raw and cooked C. vir- ginica better than C. gigas or C. ariakensis. Each measure of C. virginica quality was first compared with C. gigas and then to C. ariakensis for raw and cooked oysters. Two additional series of Wilcoxon signed rank tests were conducted on the results of the second series of taste tests (raw and cooked] to determine if rank- ings of people that eat oysters less frequently differ from those that often consume oysters. Finally, percent and mean dry tissue weights of all three species were coinpared using separate one- factor ANOVA tests. Cochran's test for homogeneity of variance was perfomied for both response variables (Underwood 1981). Student-Newman-Keuls (SNK) post hoc tests were conducted on significant ANOVA results {P < 0.05) to determine which of the three species differed from each other. The SNK test was selected because we conducted a balanced experiment with a priori pre- dictions and a fixed factor (Day and Quinn 1989). RESULTS First Series of Tests (C. virginica versus C. gigas) Collectively, survey participants ranked the taste of raw C. virginica slightly higher and its appearance slightly lower than C. gigas, but neither difference was significant (Table 1; Fig. 3). Of the 16 participants offered raw oysters, 10 preferred C. virginica. three preferred C. gigas, and three had no preference. Only two of the 16 considered C. gigas unappetizing and only one replied that C. virginica was unappetizing. Of the nine raw oyster tasters who rarely eat raw oysters, the appearance of C. gigas was ranked significantly higher than C. virginica. but the taste ratings were similar. Among the seven raw oyster tasters who frequently con- sume raw oysters, the taste of C. virginica was rated slightly higher than C. gigas: five of the seven preferred C. virginica. but low sample size more than likely rendered this difference non- significant (Table 1). Ratings of the appearance of the two species did not differ among this subgroup of tasters. Collectively, tasters of cooked oysters did not distinguish be- tween species in taste or appearance (Table 1; Fig. 3). Of the 15 TABLE 2. Results of Wilcoxon signed rank tests comparing consumer ratings for taste, appearance, and aroma of Crassostrea virginica h ith C. gigas and C. ariakensis during the second series of raw oyster taste tests. C". virginica vs. C. gigas C virginica vs. C ariakensis Oyster Feature Taste .Appearance .Aroma Taste Appearance .\ronia All participants'* No. of 0 differences" 2 3 15 5 2 16 No. of ranks < O" 22 35 31 32 40 35 No. of ranks > 0'' 64 53 44 51 49 39 2 value -4.75 -2.4 -0.88 -2.96 -0.14 -0.50 P value <0.0001 0.02 0.38 0.003 0.89 0.62 Infrequent consumers of raw oysters No. of 0 differences'' 0 2 3 0 1 5 No. of ranks < 0" 5 13 15 9 14 15 No. of ranks > O'' 24 17 14 20 17 12 Z value -3.43 -O.ftI -0.28 -2.32 -0.27 -0.99 P value 0.0006 0.54 0.78 0.02 0.79 0.32 Frequent consumers of raw oysters No. of 0 differences'' 2 1 12 5 1 11 No. of ranks < O" 16 T) 16 T") 26 20 No. of ranks > O'' 40 35 29 31 31 26 Z value -3.65 -2.48 -1.29 -2.07 -0.35 -1.22 P value 0.()()()3 (1.1)1 0.2(1 0.04 0.72 0.22 ■■ Raw data were analyzed collectively and then reanuly/ed by subgroup to delermuic v\hcther participants who rarely eat raw oysters liave dilferenl preferences from those who frequently eat them. No. of 0 differences indicates the number of participants who rated species equally, no. of ranks <0 indicate participants who rated the non-native species as better than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginica as better than the non-native species. 26 Grabowski et al. ■ C. virginica D C. gigas ■ C ariakensis All Participants Rarely Eat Oysters Frequently Eat Oysters b. Appearance 10 1 ■ C. virginica a C. gigas ■ C. ariakensis Al! Participants Rarely Eat Oysters Frequently Eat Oysters ■ C. virginica a C. gigas ■ C. ariakensis All Participants Rarely Eat Oysters Frequently Eat Oysters Figure 4. Results from taste test 2: raw oysters, (a) Taste, (b) appearance, and (c) aroma ratings of raw Crassostrea virginica. C. gigas, and C. ariakensis for the following participant groups: I) all participants. 2| infrequent consumers of raw oysters, and 3) frequent consumers of raw oysters. Tests in which C. virginica was ranked higher than non-nati\c oysters are marked with * for C. gigas and # for ('. ariakensis. Error bars indicate +1 SE. Consumer Ratings of Oysters 27 TAIU.E 3. Results of Wilcoxon signed rank tests coniparinj; consumer ratings lor taste, appearance, and uronia of Crassuslrea yirf;iiiica with C. gigas and C ariakeiisis during the second series of cooked oyster taste tests. lire C. virginica vs. C. gigas C. virginica vs. C. ariakensi. Oyster Feat Taste Appearance Aroma Taste Appearance Aroma All participants' No. of 0 differences'' 3 3 15 3 3 14 No. of ranks < 0" 33 49 37 38 40 36 No. of ranks > 0" 54 39 38 47 47 38 Z value -2.5.'i -0.89 -0.23 -0.68 -1.22 -0.003 P value (1.01 0.38 0.82 0.49 0.22 0.99 Infrequent consumers of cooked oysters No. of 0 differences" 0 2 4 0 2 3 No. of ranks < 0" 13 19 15 13 16 17 No. of ranks > 0'" 19 12 12 18 14 10 Z value -1.98 -1.53 -0.32 -0.36 -0.51 -1.59 P value 0.05 0.13 0.75 0.72 0.61 0.1 1 Frequent consumers of cooked oysters No. of 0 differences'' 3 1 11 3 1 11 No. of ranks < 0" 19 29 21 24 24 18 No. of ranks > 0" 35 27 26 29 32 28 Z value -1.83 -0.15 -0.57 -0.90 -1.82 -1.26 P value 0.07 0.88 0.57 0.37 0.07 0.21 " Cooked oyster data were analyzed collectively and then reanalyzed by subgroup to determine whether participants v\ ho rarely eat cooked oysters have different preferences from those that frequently eat them. '' No. of 0 differences indicates the number of participants who rated .species equally, no. of ranks <0 indicate participants who rated the non-native species as better than C. virginica. and the no. of ranks >0 indicates participants who rated C. virginica as better than the non-native species. participants tasting cooked oysters, seven preferred C. virginica. six preferred C. gigas. and two had no preference. Only one of the 1 .5 considered cooked C. gigas to be unappetizing, whereas two replied that C. virginica was unappetizing. Splitting participants out into inexperienced and frequent eaters of cooked oysters failed to detect any pattern of species preference in taste or appearance of the cooked oysters (Table 1; Fig. 3). Second Series of Tests (C. virginica versus C. gigas or C. ariakensis^ In the second taste test, raw oyster tasters collectively ranked the taste of C. virginica significantly higher than both C. gigas and C. ariakensis (Table 2: Fig. 4). Appearance of C. virginica was rated significantly above C. gigas but not above C. ariakensis (Table 2: Fig. 4). Neither of the paired species contrasts distin- gLushed native from non-native oysters by aroma. Infrequent oys- ter eaters ranked the taste of raw C. virginica significantly above both C. gigas and C. ariakensis. but rankings by appearance and aroma did not vary among the three species (Table 2; Fig. 4). Frequent oyster eaters ranked the taste of raw C. virginica signifi- cantly above both ni)n-native species and the appearance of C. virginica over C. gigas but not different from C. ariakensis. Aroma rankings did not differ in either contrast of pairs of oysters (Table 2; Fig. 4). Tasters of cooked oysters collectively rated the taste of cooked C. virginica significantly more than C. gigas (Table 3; Fig. 5) but did not distinguish between cooked C. virginica and C. ariakensis. Ratings of appearance and aroma did not differ between cooked native and non-native oysters in any contrast. The subgroup formed by infrequent consumers of cooked oysters also ranked the taste of cooked C. virginica significantly better than C. gigas but failed to distinguish between cooked C. virginica and C. ariakensis (Table 3; Fig. 5). These relatively inexperienced oyster eaters did not rate the appearance or aroma of native oysters differently from non-native species. Finally, frequent oyster eaters ranked the taste of C. virginica marginally above C. gigas but not significantly higher than C. ariakensis. For these experienced oyster eaters, aroma and appearance rankings did not differ significantly be- tween cooked native and non-native oysters, though the appear- ance of C. virginica was ranked marginally higher than C. aria- kensis (Table 3; Fig. 5). Dry Weight Percent dry weight of soft tissues (dry weight/wet weight) did not significantly differ among the three species (Table 4). Prior to this analysis, percent dry weight data were transformed using a square root transformation to remove heterogeneity among vari- ance groups. Total dry tissue weight (g) of C. ariakensis was significantly greater than that of C. gigas or C. virginica. and the dry tissue weight of C. gigas was greater than that of C. virginica (SNK post hoc comparisons; Fig. 6). Because average shell length did not differ among species, this analysis reflects biomass for oysters of a fixed range of harvestable lengths (80-1 10 mm). DISCUSSION As managers consider use of non-native species to enhance or restore fisheries, they should weigh carefully the risks and poten- tial benefits. Decisions on species introductions are driven by a variety of social and political pressures, often with insulTicient attention to potential ecological risks or economic benefits (An- drews 1980). In North Carolina, it is unclear, for example, how current market prices would adjust to an increase in oyster supply (Lipton & Kirkley 1994). Oyster and clam markets in the state have already endured low demand and reduced prices that threaten the economic viability of both culture operations and wild harvest 28 Grabowski et al. ■ C. virginica D C. gigas ■ C. ariakensis All Participants Rarely Eat Oysters Frequently Eat Oysters b. Appearance All Participants Rarely Eat Oysters Frequently Eat Oysters c. Aroma 10 o o ■ C. virginica ' D C. gigas \ ■ C. ariakensis All Participants Rarely Eat Oysters Frequently Eat Oysters Figure 5. Results from taste test 2: cooked oysters, (a) Taste, (b) appearance, and (c) aroma ratings of cooked Crassostrea virginica, C. gigas, and C. ariakensis for the following participant groups: I) all participants, 2) infrequent consumers of cooked oysters, and }) frequent consumers of cooked oysters. Tests in which C. virginica was ranked higher than non-nati\e oysters were marked with * for C gigas and # for C. ariakensis. Error bars indicate +1 SE. Consumer Ratings of Oysters TABLE 4. Ki'siills III ANOVA comparison of pcrctnl (lr\ tisMii' Hciyhl ol' Mif( tissiKs and tiilal dr> tissue weight for Crasso^lrcii \irf;iitica. C. giaas. and C. ariakeiisis. Source of Variance df MS F Value P Value Percent dry tissue weight Oyster species Residual Total dry tissue weight Oyster species Residual 2 0.001 1.3X 147 0.001 0.26 2 2.32 28.82y <0.()Oni 147 0.08 fisheries. Although the transport of C. ,?/,i;(i.v from the west coast for sale in the eastern United States has increased since the col- lapse of native stocks on the east coast, it is unclear whether consumers in the eastern United States prefer a particular oyster species (Lipton et al. 19921 and how such preferences may vary with targeted market (e.g.. raw on the half-shell versus steamed, etc.). In this study, we set out to identify (1) whether the taste of non-nati\e oysters is acceptable to oyster consutners in North Carolina and (2) whether consumer ratings differ and preferences exist among raw and cooked C. virginicci, C. gigas. and C. ciria- kcnsis. Our purpose was to begin the process of evaluating the market potential of the two non-native species of oyster in North Carolina and. by extension, the other east coast states where con- sumers are accustomed to eating native oysters. Although consumer ratings of taste and appearance provided no consistent pattern of preference in the first taste test (i.e.. for taste C. virginicci > C. gigiis. and for appearance C. gigas > C. vir- ginica). the majority preferred raw C. virginicii more than C. gi- gas. These findings suggest that consumer preference for raw oys- ters may be dictated more by taste than appearance. Cooking re- moved any indication of a difference between species in taste or appearance, indicating that non-native C. gigcis may be suitable for local cooked oyster markets. When asked, few participants con- sidered either C. gigas or C. virginica unappetizing regardless of C. ariakensis C. virginica C gigas Species Figure 6. Mean dry tissue weight (g) of Crassostrea virginica. C. gigas, and C. ariakensis. Error bars indicate +\ SE. Results of SNK post-hoc mean comparisons are Indicated with letters above the error bars, and species with different letters above them are signitkantl) different at P < (1.(15. preparation (raw or cooked), implying that non-native C. gigas might be acceptable. Fisheries managers may wish to assess next whether consumer demand exists for an acceptable but less pref- erable oyster and if lower preference implies a reduction in market price before allowing introduction of C. gigas to the east coast. The larger numbers of participants in the second series of tests pro\ided greater ability to resolve differences among oysters and included contrasts with the second non-native species. C. ariak- ensis. Participants in the raw oyster tests collectively indicated a strong taste preference for C. virginica over either non-native spe- cies. This preference held regardless of whether consumers rarely or frequently eat oysters. Because frequent consumers eat a dis- proportionately large amount of the raw oysters consumed in North Carolina, these results raise concern about the suitability of either non-native species for local raw oyster markets. Though appearance and aroma preferences were not as definitive, consum- ers collectively preferred the appearance of raw C. virginica to C. gigas. which raises further doubt about the marketability of raw C. gigas on the east coast. Tasters of cooked oysters in the second test exhibited weaker preferences among oysters. Yet participants collectively, as well as the subset who rarely consume oysters, preferred the taste of cooked C. virginica more than C. gigas. and frequent consumers of cooked oysters expressed a slight preference for the taste of cooked C. virginica more than C. gigas. Consumers as a whole, as well as the subset who frequently eat cooked oysters, did not exhibit a taste preference for cooked C. virginica or C. ariakensis. suggesting that C. ariakensis may be more suitable for steamed and packaged oyster markets. Because the weight of C. ariakensis oysters was double that of C. virginica of a given length and C. ariakensis grows to market size much more quickly than the native oyster (Calvo et al. 2001). the Suminoe oyster might be more successful in markets that sell by meat weight. However, the high costs of triploid aquaculture need to be considered in assessing the economic viability of this industry. On the other hand, our results show that the most widely marketed and consumed oyster in the world. C. gigas. is not rated as high by North Carolina consumers as the eastern native oyster. C. virginica. The alternative non- native oyster. C. ariakensis. is rated at least as high and in some contrasts higher than C. gigas. Thus, if the Suminoe oyster could be produced at sufficiently low cost, then it should compete fa- vorably with C. gigas for market share. Because of serious environmental risks associated with intro- ducing a non-native species as a self-replicating wild population or even for culture as triploids. we argue that an analysis of economic viability is necessary for responsible decision making by fisheries managers. Such an analysis would include our new information on consumer perceptions, ratings, and rankings of alternative species of oysters under consideration for use. A complete economic analysis to follow our study of consumer ratings and preferences would involve a model to convert these consumer ratings into prices. Additional costs of each type of culture and impacts on market supply and demand must also be assessed. Collapsing oys- ter fisheries along the Atlantic coast and declining water quality collectively have eroded consumer demand for oysters, such that current oyster markets are probably less elastic. Therefore, an in- crease in supply from successful introduction of non-native oysters in North Carolina could result in a corresponding decrease in oys- ter prices (Lipton & Kirkley 1994). especially within smaller raw oyster markets. Biological information on growth and mortality rates of non-native oyster species must be acquired and compared with nati\e oysters. Given that non-native oysters were generally 30 Grabowski et al. less preferable than the native eastern oyster in our study and that producing cultured oysters from triploid seed is expensive, suc- cessful culture of triploid oysters would require a substantial bio- logical benefit in the form of shorter time to market and/or higher survival. Inclusion of this information into a comprehensive eco- nomic analysis of potential benefits and costs of introduction would enable managers to assess whether the environmental risks are worth taking. Finally, restoration of any oyster will have posi- tive effects in restoring water quality and compensating for estua- rine eutrophication (Jackson et al. 2001. Newell et al. 2002), such that this ecosystem benefit should be included in a complete eco- nomic evaluation of any potential oyster introduction. If the intro- duced oyster were to form reefs, then further ecosystem benefits of habitat enhancement (Lenihan et al. 2001) should also be incor- porated. ACKNOWLEDGMENTS The authors thank Rachael Wagaman, Christina Tallent. David Gaskill, Hal Sumnierson. and Chris Stewart for culturing the oys- ters, assistance conducting the two food surveys and quantifying oyster tissue weights. Stan Allen, Jr., of the Virginia Institute of Marine Sciences provided disease-free triploid seed and much guidance. This research was supported by the North Carolina Gen- eral Assembly through the Rural Development Foundation and the Fishery Development Foundation and the North Carolina Depart- ment of Natural Resources. LITERATURE CITED Andrews. J. D. 1980. A review of introductions of exotic oysters and biologi- cal planning for new importations. Mar. Fisheries Rev. 42:1-11. Ayers. P. 1991. Introduced Pacific oysters in Australia. In: J. Sutherland and R. Osman. editors. The ecology of Crussosrreu gi.i^as in Australia. New Zealand. France, and Washington State. College Park. MD: Mary- land Sea Grant, pp. .V7. Breese, W. P. & R. E. Malouf. 1977. Hatchery rearing techniques for the oyster Crassostrea rivularis Gould. Aquaciitiiire 12:123-126. Burre.son. E. M.. N. A. Stokes & C. S. Friedman. 2000. Increased virulence in an introduced pathogen: Haplosporidiiim nelsoni (MSX) in the east- ern oyster Crassoslreu virt;inicu. J. Aqiianc Aiiim. Health 12:1-8. Byrne. R. J. editor. 1996. Strategic plan for molluscan shellfish research; including a rational plan for testing application of non-native oyster species. Richmond, VA: Report of the Virginia Institute of Marine Sciences. House Document No. 16. Report to the Governor and Gen- eral Assembly of Virginia. Commonwealth of Virginia. Calvo, G. W.. M. W. Luckenbach, S. K. Allen, Jr. & E. M. Burreson. 1999. Comparative field study of Crasso.strea git^as (Thunberg. 1793) and Crassostrea virginica (Gmelin. 1791) in relation to salinity in Virginia. / Shellfish Res. 18:465-473. Calvo. G. W.. M. W. Luckenbach. S. K. Allen. Jr. & E. M. Burreson. 2001. A comparative field study of Crassostrea ariakensis (Fujita 1913) and Crassostrea virginica (Gmelin 1791) in relation to salinity in Virginia. J. Shellfish Res. 20:221-229. Carlton, J. T. 1999. Molluscan invasions in marine and estuarine commu- nities. Malacologia 41:439^54. Day, R. W. & G. P. Quinn. 1989. Comparisons of treatments after an analysis of variance in ecology. Ecol. Monogr. 59:433—463. Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In: V. S. Kennedy. R. I. E. Newell & F. Ebele. editors. The eastern oyster, Crassostrea virginica. College Park. MD: Maryland Sea Grant, pp. 581-660. Frankenberg, D. 1995. Report of North Carolina Blue Ribbon Advisory Council on Oysters. Raleigh. NC: North Carolina Department of En- vironment, Health, and Natural Resources. Hargis. W. J.. Jr. & D. S. Haven. 1988. The imperiled oyster industry of Virginia: A critical analysis with recommendations for restoration. Spe- cial report Number 290 in applied marine science and ocean engineer- ing. Gloucester Point. VA: Virginia Institute of Marine Sciences. Jackson. J. B. C. M. X. Kirby. W. H. Berger. K. A. Bjomdal. L. W. Botsford, B. J. Bourque, R. H. Bradbury, R. Cooke, J. Erlandson, J. A. Estes, T. P. Hughes, S. Kidwell, C. B. Lange, H. S. Lenihan, J. M. Pandolfi, C. H. Peterson, R. S. Steneck, M. J. Tegner & R. R. Warner. 2001 . Historical overfishing and the recent collapse of coastal ecosys- tems. Science 293:629-638. Langdon, C. J. & A. M. Robinson. 1996. Aquaculture potential of the Suminoe oyster {.Crassostrea ariakensis Fugita 1913). Aqiiaciilliirc 144:321-338. Lenihan, H. S. 1999. Physical-biological coupling on oyster reefs: How habitat structure infiuences individual performance. Ecol. Monogr. 69: 254-275. Lenihan. H. S.. F. Micheli. S. W. Shelton & C. H. Peterson. 1999. The inlluence of multiple environmental stressors on susceptibility to para- sites: An experimental determination with oysters. Limnol. Oceanogr. 44:910-924. Lenihan. H. S. & C. H. Peterson. 1998. How habitat degradation through fishery disturbance enhances impacts of hypoxia on oyster reefs. Ecol. .Appl. 8:128-140. Lenihan, H. S., C. H. Peterson. J. E. Byers. J. H. Grabowski. G. W. Thayer & D. R. Colby. 2001. Cascading of habitat degradation: Oyster reefs invaded by refugee tlshes escaping stress. Ecol. Appl. 1 1:764-782. Lipton. D. W. & J. Kirkley. 1994. A profile of the oyster industry: North- eastern United States. Virginia Sea Grant Marine Resource Advi.sory No. 54. Gloucester Point, VA: Virginia Institute of Marine Sciences. Lipton. D. W., E. F. Lavan & 1. E. Strand. 1992. Economics of molluscan introductions and transfers: The Chesapeake Bay dilemma. J. Shellfish Res. 11:511-519. MacKenzie, C. L.. Jr. 1983. To increase oyster production in the north- eastern United States. Mar Fisheries Rev. 45:1-22. Mann, R.. E. M. Burreson & P. K. Baker 1991. The decline of the Virginia oyster fishery in Chesapeake Bay: Considerations for introduction of a non-endemic species. Crassostrea gigas (Thunberg. 1973). / Shellfish Re.'i. 10:379-388. Naylor. R. L., S. L. Williams & D. R. Strong. 2(J0I. Aquaculture— a gateway for exotic species. Science 294: 1 655- 1 656. Newell, R. 1. E., J. C. Comwell & M. S. Owens. 2002. Influence of simulated bivalve biodeposition and microphyrobenthos on sediment nitrogen dynamics: A laboratory study. Limnol. Oceanogr. 47:1367- 1379. Parameswar, D. 1991. Introduced Pacific oysters in New Zealand. In: J. Sutherland & R. Osman. editors. The ecology of Crassostrea gigas in Australia. New Zealand, France, and Washington State. College Park. MD: Maryland Sea Grant, pp. 9-11. Pollard, D. A. & P. A. Hatchings. 1990. A review of exotic marine organ- isms introduced to the Australia region. II. Invertebrates and algae. Asian Fisheries Sci. 3:223-250. Rothschild. B. J., J. S. Ault, P. Goulletquer & M. Herat. 1994, Decline of the Chesapeake Bay oyster population: A century of habitat destruction and overfishing. Mar Ecol. Prog. Ser. 1 1 1:29-39. Rui/. G. .M.. P. W. Fofonoff, J. T. Carlton, M. J. Wonham & A. H. Hines. 2000. Invasion of coastal marine communities in North America: Ap- parent patterns, processes, and biases. Ami. Rev. Ecol. Sysrematics 31: 481-531. Seliger, H. H.. J. A. Boggs & W. H. Biggley. 1985. Catastrophic anoxia in the Chesapeake Bay in 1984. Science 228:70-73. Shatkin, G., S. E. Shumway & R. Hawes. 1997. Considerations regarding the possible introduction of the Pacific Oyster [Crassostrea gigas) to the Gulf of Maine: A review of global experience. J. Shellfish Res. l6:46.^-+77. Underwood, A.J. 1981. Techniques of analysis of variance in experimental marine biology and ecology. Oceanogr. Mar Biol. Anna. Rev. 19:513- 605. Jo:inml of Slicll/isl, Rcscairh. Vol. 22. No. 1. .M-.^S. 20(13. TAXONOMIC STATUS OF FOUR CRASSOSTREA OYSTERS FROM CHINA AS INFERRED FROM MITOCHONDRIAL DNA SEQUENCES ZINIU YU,'"* XIAOYU KONG,' LIUSUO ZHANG,' XIMING GUO.- AND JIANHAI XIANG' ^College of Fisheries. Ocean University of Qingihio. Qingclao 266003. Peoples Republic of China: -Haskin Shellfish Research Laboratory. Institute of Marine ami Coastal Sciences. Riitiiers University. Port Norrls. New Jersex 0S.U9: and ''Institute of Oceanology. Chinese Academy of Sciences, Qingdao 266071. Peoples Republic of China ABSTRACT It has been presumed ihat there are tour eoaiiiion Cra\snstrea oyster species along the eoast ol China; the Pacitic oyster (Crassostrea gigas), Zhe oyster (C plicatula). Suminoe oyster (C ariakeiisis). and Dalianwan oyster (C. talienwbanensis). Classifi- cation and species identification of these Crassostrea oysters have been difficult because of morphologic plasticity. In this article, phylogenetic analysis was performed to clarify taxonomic status of these species using mitochondrial DNA sequence data. Nucleotide sequences of a 443-bp fragment of ribosomal RNA gene and a 579-bp segment of cytochrome c oxidase I gene were obtained through sequencing and used for analysis. Genetic distances among the four species, using C. virgiiiica as outgroup, were computed based on the sequence data, and phylogenetic trees for the five species were generated. The divergence between C. gigas and C. talienwhanensis was very low. as was that between C. pticaiula and C ariakeiisis. Phylogenetic analysis showed that haplotypes of C. gigas and C. lalieimhaiieiisis clustered in one clade and those of C. plicaluta and C ariakeiisis in another one. Our data suggest that C. gigas and C latieimlumensis may be the same species. However, the lack of divergence between C. plicaltila and C. ariakeiisis samples may indicate that the C. plicaliila specimen we sampled could actually be a morph of C. ariakeiisis living in high salinity habitats. More work is needed for confirmation. KEY WORDS: Crassostrea oysters, taxonomy, phylogenetic analysis, 16S rDNA, COI gene, nucleotide sequences INTRODUCTION Ainotig the over 20 species of oysters recorded in China, four Crasso.strea species are most cotnmon and of commercial impor- tance; the Pacific oyster (Crassostrea gigas), Zhe oyster (C. pli- catula). Suminoe oyster (C. ariakeiisis). and Dalianwan oyster (C talienwhanensis; Zhang et al. 1956, Qi I9S9). The Pacific oyster, which occurs naturally along the coast of China, is a well- recogni/ed species. However, most of the Pacific oysters cultured in China were originally introduced from Japan or Korea (Wang et al. 1993). The Zhe oyster is commonly found along the entire coast of China. It is relatively smaller in body size than the Pacific and Suminoe oysters and thin-shelled (Qi 1989, Guo et al. 1999). Suminoe oysters are also distributed along most of the coast of China with two major populations, one in the estuaries of Yellow river and the other in Guangxi and Guangdong in southern China. It can tolerate a wide range of salinity but prefers low-salinity estuaries and riverbeds (Torigoe 1981. Li & Qi 1994). The Dalian- wan oyster occurs mainly in areas along the coast of Liaoning and Shandong provinces in the North (Zhang et al. 1956, Qi I9S9). Because of the morphologic plasticity, there have been dis- agreements about the taxonomic status of the four Crassostrea types and difficulties in their identification. Some believed that the Pacific and Dalianwan oysters are different species (Zhang et al. 1956, Qi 1989), whereas others argued that the Dalianwan oyster. described by Zhang et al. ( 1956), is the Pacific oyster, or a variety of Pacific oyster (Torigoe 1981, Li & Qi 1994). In addition, some- times the discrimination of Pacific and Suminoe oysters was am- biguous with shell morphology, although it is distinguishable w ith some body anatomic features (Li & Qi 1994). The most common oysters found in the rocky intertidal zone and extensively cultured in the south are generally believed to be the Zhe oyster, although ♦Corresponding author. Tel: 856-785-0074; Fax: 856-7S5-I544; E-mail: carlzyu @ hsrl.rutgers.edu Li and Qi (1994) assumed it was the Pacific oyster. Liu et al. { 1 998 ) compared RAPD data from several Crassostrea species and concluded that the Dalianwan oyster, Zhe. and Pacific oysters were sister species with each other. Because of this confusion, further study, especially with DNA markers, is needed. DNA polymorphisms are useful tools for eco- logical, genetic, and evolutionary studies of both terrestrial and marine organisms, and DNA sequences can be used to detect dif- ferences among species, populations, or individuals. Proper iden- tification of oyster stocks will assist management, including con- servation and the sustainable use of these resources. Past efforts to investigate and identify differences among populations and species of oysters along the coast of China have provided useful but in- conclusive information {Liu et al. 1998, Yatig et al. 2000). Because of its fast sequence evolution and inaternal. nonrecom- bining nature of inheritance in animals, mitochondrial genes have proved a powerful tool in phylogenetic studies and species iden- tification (Banks et al. 1993, Littlewood 1994, Jozefowicz et al. 1998, Lapegue et al. 2002). The I6S rRNA and COI gene frag- ments are popular choices for phylogenetic analysis (O'Foighil et al. 1995, O'Foighil et al. 1998. Canapa et al. 2000). In this study, mitochondrial 1 6S rRNA and COI gene fragments from these four putative species were amplified and sequenced for phylogenetic analysis. MATERIALS AND METHODS Sampling and Polymerase Chain Reaction (PCR) Amplifications Crassostrea gigas samples (eight specimens) were obtained from a hatchery broodstock in Shandong province; C ariakeiisis samples (seven individuals) were collected from estuaries of the Yellow River, in Yantai, Shandong province, which is a typical habitat of this species in north China. C talienwhanensis was sampled from Dalian (five individuals). Liaoning province and Rongcheng (five individuals). Shandong province. C. plicatula 31 32 YU ET AL. samples were collected from Qingdao (five specimens). Shandong province and Wenzhou (five specimens). Zhejiang province. Sam- pling sites are showed in Figure 1 . C. virf^iiiica was collected from Delaware Bay in the United States. Morphologic identification was made according to that described in Zhang et al. (1956). Qi (1989), Torigoe (1981). and Li and Qi (1994). Total DNA was e,xtracted from mantle tissue using an extrac- tion kit (Pure Gene, Centra, USA). Fragments of the 16S rDNA and COI gene were amplified using two pairs of universal primers: 1 6sar-L/ 1 6sbr-H: 5 ' -GCCTGTTTATCA AAA ACAT-3 75 ' - CCGGTCTGAACTCAGATCACGT-3'(Palumbi 1991 ); COIL 1 490/CO1H2 1 98: 5 '-GGTCAACAAATCATAAAGATAT- TGG-37 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994). Amplification of the products was performed using a PTC- 100 thermal cycler (MJ Research. USA). The 100-p.L amplification reaction contained 2.0 niM MgCK; 200 (j.M of each dNTP: 0.2 (xM each primer; 2.5 p.L of template DNA; and 2.5 units of Taq poly- merase (Sangon. Canada) with supplied buffer. For all amplifica- tions, hot-start PCR was initiated by addition of polymerase and primers after an initial 2-min denaturization at 80°C. The PCR cycling profile was as follows: 35 cycles at 94'C/45 sec. 48°C (COI) or 50°C ( I6S)/1 min and at 72°C/1 mm. with a final exten- sion at 72"C for 7 min. Sequencing PCR products were purified using UNIQ-5 Column PCR Prod- uct Purification Kit (Sangon. Canada), ligated into pMD18-T Vec- tor by following instniction of Takara DNA Ligation Kit ver.2 (Takara. Japan) and used to transform competent JM109 Escheri- chia coli cells using standard protocols. Recombinant colonies were identified by blue-white screening. Inserts of the correct size jr^ VJliaoning J' 'NortK KorSa BEIJING^JJ (^^^ 'On Inn y "X /hebei /SHANDONG 1 j/ /fiurwo 1 o^^-i«'Ss4^ a fi y u 03 s cmj ^ r^JIANGSU , anhV?^*''"'" ^^/> '^'i/^-y ZHEJIANG UIAN K /^^ • IC hii IJ GXI^ ^/ Figure \. .V map nt sampling area »ith sampling sites underlined. were detected via restriction enzyme digestion by EcoRI and HiiicHU. Vector DNA containing the desired insert was further purified using Pharmacia EasyPrep Kit. Sequencing was per- fonned for both strands of every sample on an ABI PRISM 377XL DNA Sequencer using ABI PRISM BigDye"^"^ Terminator Cycle Sequencing Ready Reaction Kit w ith AmpliTaq DNA Polymerase. FS (Perkin-Elmer. USA). Dala Analysis The 16S and COI sample sequences, along with those already obtained for C. gigcis and C. ariakensis (0"Foighil et al. 1995. 1998; courtesy of Dr. D. OToighil) were aligned with CLUSTAL W (Thompson et al. 1994). For clarity and convenience in com- paring with other published sequences, the sequences were trimmed to the same length as published sequences after align- ment. Parsimony analysis was made with Phylip (Ver.3.56C. Felsenstein 1989) using the program DNAPARS with C. virginica as the out-group. Bootstrap analysis with 1000 replication was performed by the SEQBOOT and CONSENSE programs. Consen- sus phylogenetic trees were drawn with DRAWGRAM program in the Phylip package. Pair-wise sequence divergence between hap- lotypes and species were estimated by the DNADIST program of Phylip according to Kimura's two-parameter model (Kimura 1980). RESULTS A PCR fragment of 488 bp from the mitochondrial IdS ribo- sonial gene and a fragment of 649 bp from the mitochondrial COI gene were obtained and sequenced for 37 individuals of five spe- cies (including two C. virginica specimens). Figure 2 shows the alignment of 1 68 sequences of the seven haplotypes detected among all specimens in this study, along with those of C. gigas and C. ariakensis from O'Foighil's study. Eight specimens of C. gigas and 10 of C. plicatitla exhibited only one genotype, whereas seven C. ariakensis and 10 C. lalienwhanensis individuals had two hap- lotypes each. The two haplotypes of C. taliemvhanensis came from different sampling locations. Including the outgroup. 80 nticleotide positions were variable in the 16S data set. Six insertion/deletion sites were detected between C. virginica and all other haplotypes. Similarly, the alignment of the seventeen COI haplotypes de- tected in our study and those two of C. gigas and C. ariakensis from O'Foighil's study are shown in Figure 3. The 17 haplotypes in our study included one for C. gigas (gigas 1. 8 individuals), seven for C. plicatula (plical. 2. 3. 6 and 7. one individual for each; plica4. three individuals; plica5. two individuals), three for C. ariakensis (ariakenl. 4 individuals; ariaken2. two individuals and ariaken3. one individual), five for C. ralienwhanensis (talienwl. 2 and 3. one individual each; talienw4. four individuals; talienw5. three individuals), and one for C. virginica (virgl. two individuals). Including the outgroup. 170 positions are variable. No insertions/deletions were detected for this protein-coding gene fragment. Pair-wise genetic distances of 16S sequences among all nine haplotypes and those of COI sequences among all 19 haplotypes were computed, then the mean genetic distances were obtained (Table 1 ). In the 16S sequence, the genetic divergence between C. gigas and C. talienwhanensis was low. 0.81%, and so was that between C. ariakensis and C. plicatula. 0.13%. The sequence di- vergences between C. gigas or C. ralientvhanensis and C. aria- kensis or C. plicatula were higher, ranging from approx. 1.74 to Taxonomic Status of Cr.assostrea Oysters 33 gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl gigasl talienwl gigasO talienw2 plical ariakenl ariaken2 ariakenO virgl 80 GCAATACCTG CCCAGTGCGA AATATTACTG TAAACGGCCG CCCTAGCGTG AGGGTGCTAA GGTAGCGAAA TTCCTTGCCT C . ATAAGTC . . C T . 160 TTTGATTGTG GGCCTGCATG AATGGTTTAA CGAGGGTTTG ACTGTCTCTA AATTTTTTAT TGAAATTGTA CTGAAGGTGA .A . . .A . . .A . . .A . . .A . . .T G. 240 AGATACCTTC ATTTAAAAGT TAGACAAAAA GACCCCGTGC AACTTTGAAA A--TTAACTT TATTCAGGAG TAAAAGATTT . .A. . . .A. . . .A. . . .A. . . AAG. .GC.A.G. .G A. 320 TTAGGTGGGG CGCCTAGAAA GCAAG-TCTA ACCTTT-CTG AATAACT--A ACTCTTTCCG GATTTGACCC GATTATATTC . -C. . -C. . AA . T C . GT . . C.TT.--. . .T. .ATA. GT .T. . .AA.TA 400 GATCATAGGA GAAGTTACGC CGGGGATAAC AGGCTAATCC TTTAGTAGAG TTCGTATTGG CTAAAGGGAT TGGCACCTCG 443 ATGTTGAATC AGGGATAATA GCTTCAAGGC GTAGAGGCTT TGA (8) (5) (7) (5) (10) (5) (2) (5) (2) Figure 2. Mignnu'rit of seven oyster haplotypes of a 443-bp fragment of the mitochondrial I6S rDNA obtained in this study (C virgiiiica as oulgroup) «ith published sequences for (. gigas and ('. ariakensis (O'Foighil et al. 1995, 1998). gigasO and ariakenO designate the sequences of C. gigas and C. ariakensis from O'Foighil's study, respectively. Haplotype names are abbreviated as: gigas for C gigas. talienw for ('. talienwhaiiensis. plica for ('. plicaliilu. ariaken for C. ariakensis. and virg for C. rirginica. Additional haplotypes per species are numbered consecutively. Dots indicate nucleotide identity to the first sequence presented, gigasl. Dashes indicate inferred nucleotide indels relative to C. rirginica. The number of individuals observed for each haplotype is indicated in parentheses at the end of sequence. 2.45%. The same pattern appeared in the COI data set: the coire- sponding numbers were 1.08% between C. gigas and C. talien- whanensis. 0.59% between C. ariakensis and C. plicaluUi. and approx. 10.72 to 11.43% for the same comparisons mentioned above. It is worth noting that the COI sequence was more variable than the 16S sequence. Consensus phylogenelic trees based on a parsimony analysis of the 16S and COI fragments sequenced are presented in Figures 4 and 5. respectnely. Two groups (clades) in the 16S tree were clearly distinguishable: C. ariakensis and C. pliiatida vs. C. gigas and C. talienwhancnsis. whereas three groups (clades) were ap- parent in the COI tree: (1) C ariakensis and C. plicatula: (2) C. gigas and C. laliemvhanensis: (3) C. ariakensis from O'Eoighii's study. DISCUSSION Oysters are among the most extensively studied and morpho- logically variable marine invertebrates. However, our knowledge of oyster phylogeny and systematics is still limited. There had been over one hundred recorded species of oysters until 1970s, but two thirds of them could be synonymous w ith each other according to 34 YU ET AL. 80 gigasl GCTGTTCTTG CGGGAACTAG GTTTAGGTCT CTTATTCGTT GGAGACTTTA TAACCCTGGA GCTAAGTTTT TAGACCCCGT talienwl gigasO talienw2 talj.enw3 talienw4 talienw5 plical ariakenl plica2 plica3 plica4 ariaken2 plica5 ariakenS plica6 plical ariaKenO virgl gigasl talienwl gigasO talienw2 talienw3 talienw4 C. .G. G r G r -G A G .A. . r r G . .C. .G .A. G .A. . r r G . .C. .G A G .A. . r r G . .c. T p^ .G A G .A. . r r G . .c. T ft .G A G .A. . r r G . .c. T A .G C. . . . A G .A. . r T r G . .c. .G A G .A. . r r G . .c. .G .A. G .A. . r T c G c T ft .G A G .A. . r T r G . .c. .G A G .A. . r T r G . .c. . .c TA. .T. GCA . . .C. .A .A. TT _G. . r A G c T . .A. . .T.A. GACTTATAAT .G. .C. . GTTGTAA T CTAGGCATGC GTTGGTTATG .A. .T. .A. . ATTTTTTTCT . .CT G TTGTTATACC A TGTAATAATT G. .T. . 160 GGGGGGTTTG C talienwS C. .G. plical A G. . A. .G. ariakenl A G. . A. .G. plica2 A G.. A..G. plica3 A G.. A..G. plica4 A G. . A. . . . ariaken2 A G. . A..G. plicaS A G. . A. .G. .A, A. . .G. . A. .G. . . .A. A. . .G. . A. .G. . . A. .C. . . . .C .C. .A. A. .A. A C. ,T. G. . .C TGTG. . . . .c . .T. . ,G. . .G. .T. , .C. . .A. .A. . .C. . . .G. . A. .T. . 240 GTAACTGGCT TATCCCTTTG ATGCTTCTAG TAGCAGACAT GCAATTTCCT CGATTAAATG CATTTAGATT TTGAGTTTTG A ' '. . . _ A . . .T. A .A. . . .T. 0 r . . GC . . , . C c . . .T. A .A. . . .T. A r . . GC . . . .C . c .T. A .A. . . .T. B r . .GC. . . C c . . .T. .A .A. . . .T. A. . .GC. . . .c. .c. . . . . .T. A .A. . T A r . .GC. . . .c. .c. . . . . .T. A .A. . T A r . . GC. . . .c. .c. . . . . .T. A .A. . T A r . . GC. . . .c. .c. . . . . .T. A .A. . T A r . . GC . . . .c. .c. . . . . .T. .A .A. . A. .c. . .GC. . . .c. .c. . . . . .T. A .A. . . .T. A r . . GC . . . .c. c .A. .T. . .A. .A .A. . . .T. .G. . G. . . C. .G. T. . . . .T. .GC .T . .A, GA. . .G. .G. .0. .T. C. .A. . . ariaken3 A G. . A. .G. plica6 plica7 ariakenO virgl gigasl talienwl gigasO taiienwZ taJ ienw3 talienwj talienwS plical ariakenl plica2 plica3 plica4 ariaken2 plicaS ariaken3 plica6 plica7 ariakenO viryl 320 gigasl CCAGGGTCTC TTT.ATCTTAT GCTTATGTCT AACATTGTAG AAAACGGAGT TGGGGCAGGG TGAACAATTT ACCCTCCTTT talienwl gigasO talienw2 talienw3 talienw4 C G talienwS C plical A.. A T G. ariakenl A. .A T G. plica2 A. .A T G. plica3 A.. A T G. plica4 A. .A T G. ariaken2 A. .A T G. plicaS A. .A T G. ari3ken3 A.. A T G. plicae A. .A T G. plica7 A T G. ariakenO C..A TC GT..G.. C A virgl AT .GCTG..A.. AT . G A . . T . . . . CT . . G . GA T....A C GC . Figure 3. Alignment of 17 oyster haplot.vpe.s of a 579-bp fragment of the mtCOI gene obtained in this study (C virginica as outgroup) with published sequences for C. gigas and C. ariakensis (O'Foighil et al. 1995. 1998). gigasO and ariakenO designate the sequences of C. gigas and C. ariakensis from O'Foighil's study, respectively. Haplotype names are abbreviated as: gigas for C. gigas. talienvv for C. talienwhaneiisis, plica for C. plicalula, ariaken for C. ariakensis and virg for C. virginica. Additional haplotypes per species are numbered consecutively. Dots indicate nucleotide identity to the first sequence presented, gigasl. The number of individuals observed for each haplotype is indicated in parentheses at the end of sequence. GG C GG _ _ . . . r. _ GG GG . . .c GG C GG .... r GG GG GG GG Taxonomic Status of Crassustrea Oysters 35 400 gigasl ATCAACTTAC TCTTATCATG GAGTTTGTAT AGACCTTGCA ATTCTAAGCC TTCACCTTGC TGGTATTAGC TCTATTTTCA talienwl gigasO talienw2 talienw3 talienw4 C T C. talienwS C T C. plical G..G C. G T TT .A A.. .. ariakenl G..G C. G T....TT .A A plica2 G..G C. G T....TT .A A plica3 G..G C. G T....TT .A A plica4 G..G C. G T....TT .A A ariaken2 G..G C. G T....TT .A A pllcaS G..G C. G T....TT .A A ariakenB G..G C. G G T....TT .A A plicae G..G C. G T....TT .A A plica7 G..G C. G T....TT .A A ariakenO G..C..C TT.A A. virgl G TT C C.. G..TT....C . . . T . . . . GT .A...T.A.. A.... 480 gigasl GGTCAATTAA TTTCATAGTA ACGATTAGAA ATATGCGATC TGTTGGGGGC CATTTACTAG CACTATTCCC TTGATCTATT talienwl gigasO talienw2 talienw3 G talienw4 T T.. C talienwS T. plical T A. ariakenl T A A T.G. .G..G..T.. C plica2 T A A T.G. .G..G..T.. C plicaB T A A T.G. .G..G..T.. C pllca4 T A A T.G. .G..G..T.. C ariaken2 T A A T T.G. .G..G..T.. C plicaS T A A T.G. .G..G..T.. C ariakenS T A A T.G. .G..G..T.. C plicae T A A T.G. .G..G..T.. C plica7 T A A T.G. .G..G..T.. C ariakenO T C..T..G GT.G. .G T.. A..G C virgl ....T T C C T ..CA..T T G..A... 560 gigasl AAGGTTACTT CATTCTTGCT TTTGACTACT CTCCCAGTGT TAGCTGGAGG TCTTACTATA CTTTTGACTG ATCGTCATTT talienwl gigasO talienw2 talienwS talienw4 G talienwS G plical TC.A A T G.. C G C ariakenl TC.A A T G. . C G C plica2 TC.A A T G.. C G C plicaS TC.A A T G. . C G C plica4 TC.A A T G. . C G C ariaken2 TC.A A T G.. C G plicaS TC.A A T G. . C G ariakenB TC.A A T G.. C G plicae TC.A A T G.. C G plica7 TC.A A T G.. C G ariakenO ..A..C..A T..A A..A..C ..T..G..AC C..G C..G virgl ..A..G..A C... GC.T..C..G ..A..T..TC C. G G . . CC . T A 579 gigasl TAATACCTCT TTTTTTGAC (8) talienwl ( 1 ) gigasO (20) talienw2 ( 1 ) talienw3 (1) talienw4 C . . . ( 4 ) talienwS C. . . (3) plical . . .C. .G T (1) ariakenl ...C..G T (4) plica2 . . .0. .G T (1) plicaS . . .C. .G T (1) plica4 ...C..G T (3) ariaken2 ...C..G T (2) plicaS ...C..G T (2) ariaken3 ...C..G T (1) plicae . . .C. .G T (1) plica7 . . .C. .G T (1) ariakenO ...C..G T (5) virgl A. .G (2) Figure 3. (Continued) Harry ( 1985 ). The inability to clearly classify closely-related oys- proven to be a powerful tool for oyster identification and discrinii- ters has created problems for classification and species identifica- nation between closely related species or between nati\e and non- tion worldwide. native species. Banks et al. (1993) discriminated closely related Although morphologic identification of oysters often turned out oyster species, C. gigas and C. sikamea. via mitochondrial I6S to be unreliable or ambiguous. mtDNA sequence analysis has rRNA gene sequencing and PCR/RFLP analysis. O'Foighil et al. 36 YU ET AL. TABLE I. Pair-wise sequence divergence (mean genetic distances! according to Kiniura's two-parameter model iKimura 198(1) among the five species based on 443-nucieotide 16S rDNA and 579-nucleotide COI sequences. 16S COI Species 1 2 3 4 5 ft 1 2 3 4 5 6 C. gigas C. lalienwhanensis 0 0.0()81 0 0 0.0108 0 C. plicanda 0.0233 0.0174 0 0.1113 0.1072 0 C. ariakensis 0.024? 0.01 S5 0.0013 0 0.1143 0.1 100 0.0059 0 C. ariakensisO 0.0450 0.04S7 0.0444 0.0462 0 11.1619 U.1639 0.1652 0.1691 0 C. virginica 0.1636 0.1 60S 0.1654 0.1673 0.1937 0 0.2569 0.2573 0.2510 0.2513 0.2849 0 C. ariakensisO indicates S. ariakensis sequence from OToighil's studies (1995. 1998). Pair-wise comparisons yielding low genetic distances estimates are showed in boldface. (1995) succeeded in distinguishing C. viri>iiuco from two closely related oysters. C. gigas and C. ariakensis. and C. gigas from C. ariakensis by employing sequencing and PCR/RFLP analysis of pan of a fragment (443 bp) of the 16S rRNA gene. Sequence data revealed that C. gigas and C. ariakensis showed higher levels of similarity to each other (95%) than to C. virginica (84-86%). Comparison of a 579-nucleotide fragment of the COI between the Portuguese oyster. C. angiilala. and several Japanese oysters were made by OToighil et al. (1998). showing that Portuguese oyster haplotypes clustered firmly within a clade of Asian congeners and were closely related to C. gigas (but not identical). This result supports an Asian origin for the Portuguese oyster. Reportedly, there are over 20 recorded species of oysters oc- curring along the coast in China (Zhang et al. 1956, Qi 1989). and for some of them classification and identification have been prob- lematic or uncertain. Based upon extensive anatoinic studies of almost all oyster species in China. Li and Qi ( 1994) concluded that there were 15 species of oysters, and claimed that identification of a few oyster species was clarified. Most of the species are rare and found in South China Sea. However. Even for the four common species (the Zhe oyster. Pacific oyster. Suminoe oyster, and Dalianwan oyster), it is often not empirically easy even for marine zoologists sometimes, to distinguish them clearly. This has caused inconveniences and difficulties in broodstock management and aquaculture practices. If the Dalianwan oyster is a discrete species. ariakenO — ariakenl — ariaken2 — plical talienw2 talienwl gigasi — gigasO separate stock conservation and management should be applied. Accordingly, clarification of the Zhe oyster's status would also help oyster aquaculture practices. These are widespread concerns for the oyster fishery along the coast of China The molecular data provide some clarification on the species status and phylogenetic relationships of these four species. For Dalianwan and Pacific oysters, the 16S data show close similarity between the samples of these two species, and the haplotypes of Dalianwan and Pacific oyster formed a clear clade in the phylo- genetic tree. This relationship is strongly supported by the COI data set. in which all five haplotypes of the Dalianwan oyster and the only haplotype of the Pacific oyster clustered closely. This is also supported by the evident similarity in moi-phology between these two species. The Dalianwan oyster samples were collected from t> pical distribution areas, identified carefully according to the plica5 — virgl Figure 4. A consensus phylogenetic tree based on parsimony analysis of 443-nucleotide mt I6S rDNA fragment according to Kimura's model with C. virginica as an outgroup. talienw4 talienwS ■ virgl Figure 5. .\ consensus phylogenetic tree based on parsimony analysis of 579-nucleotide mt COI gene fragment according to Kimura's model with ('. virginica as an outgroup. Taxonomic Status of Chassostrea Ovstbrs 37 descriptions of Zhang et al. ( 1956) and Qi ( 1989). Although there are some morphologic differences compared with the Pacific oys- ter, Dalianwan oysters share some morphologic characteristics with Pacific oysters as described by Zhang et al. ( 1956) and Qi (1989). A similar situation exists in scallops Pecten imiximus and P. jacoheiis, where they share highly similar morphologic features but have a surprisingly close genetic distance based on 16S se- quences (Canapa et al. 2000). Our molecular data suggest that Dalianwan and Pacific oysters belong to the same species, which supports Li and Qi"s (1994) conclusion based on anatomy studies. Results for the Zhe and Suminoe oysters are rather surprising. The divergence between the two is much less than expected. The genetic distances between them are as low as 0.1 39i- (for 16S) and 0.59% (for COl), even lower than that between the Dalianwan and Pacific oysters (0.81 and 1.08'^H. They share a high degree of similarity in these two gene fragments. In contrast, they showed higher divergence from the Pacific and Dalianwan oysters in both the 16S and the COl sequence data, though more strongly in the latter. Also, haplotypes of the Zhe and Suminoe oysters clustered in a single clade in both trees. This result is different from that generally concluded from morphologic data. Morphologically, the Zhe and Suminoe oysters are easy to distinguish in most cases. Therefore, caution should be taken for the concern of status of these two species. A possible explanation could be as follow, the "Zhe oysters" we sampled could actually be a morph of Suminoe oysters living in high salinity habitats. Because ecologically the Suminoe oyster has a wide distribution and can tolerate a wide range of salinities, morphologies could vary in different habitats. Samples collected from the habitats other than an estuary may look different from the Suminoe oysters from a typical habitat. It is possible that Suminoe oysters from high salinity area and on rocky shores are mistakenly classified as Zhe oysters because of mor- phologic plasticity. It has been shown that the Zhe-like small oys- ters found in the rocky intertidal zones of northern coast, once removed to more productive waters, could grow to a bigger size, which resemble the Suminoe oysters from an estuary habitat (R. Wang, personal comm.). To confirm either of these possibilities, a more extensive sampling and sequence analysis throughout their natural range are needed. An interesting finding from this study is that O'Foighil's COl sequence of the Suminoe oyster showed a significant divergence not only from that of the Dalianwan-Pacific oysters, but also of the Suminoe-Zhe oysters. The divergence may be due to the fact that mt protein-coding genes like COl are usually more variable than iDNA (Hixson & Brown 1986) and the fact that OToichil's Sumi- noe oyster samples, which came from a hatchery stock originated from Japan, may represent a different population that is genetically isolated from the Chinese population (our samples). However, analysis of more specimens from Japan or other parts of their natural range is needed for confirmation. Li and Qi (1994) suggested that the Zhe-like oysters most com- monly found in the rocky intertidal zone were Pacific oysters instead of Zhe oysters as most people assumed. If so, the iiitDNA sequences of these (Zhe) oysters should have higher similarity to (or low divergence with) those of the Pacific oysters or Dalianwan oysters we presented here and that of O'Foighifs. Actually this is not the case. Our sequence data show that these smaller oysters from rocky shores could be Suminoe oysters, rather than Pacific oysters. Additionally, in this study the COl sequences showed more variations, as expected, than the 16S sequences. For instance, in the 16S data, we detected only one haplotype for Zhe oyster, two tor each of the Dalianwan and Suminoe oysters; but in COl data, the numbers of haplotype are seven, five and three for these three species, respectively. Also, the divergence between C. gigas and C. plicatida or C. ariakensis is three times as high as that between C. gigas and C. taUt'imhanensis in the 16S data, whereas the divergence is eleven times higher in the COl data. The COl se- quence is more sensitive in discriminating closely related species, supporting the observation by Boudry et al. ( 1998) where no vari- ability was detected with nine endonucleases among 253 individu- als of C. gigas and C. angiilata with 16S rDNA, but reasonable polymorphism was detected with four enzymes with COL Other works have also proved that CO! sequence is a good choice for similar purposes (Meyran et al. 1997. O'Foighil et al. 1998). In summary, the mtDNA sequence data strongly suggest that C. laliemvlianensis is not a discrete species and should be considered as synonymous with C. gigas. Our data also indicate that the "Zhe oyster" is different from the Pacific and Dalianwan oysters, but is genetically very close to Suminoe oyster, at least for the ones we sampled. ACKNOWLEDGMENTS This work was financially supported by National Science Foun- dation of China (39600113) and Research Foundation (2001) of Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P. R. China. Yu and Guo are partly supported by grants from US Sea Grant and New Jersey Commission on Science and Technology. LITERATURE CITED Banks. M. A., D. Hedgecock & C. Waters. 1993. Discrimination between closely related Pacific oyster spp. iCrassoslreu) via mitochondrial DNA sequences coding for large suhunit rRNA. Mol. Mar. Binl. Bio- techiwl. 2:129-136. Boudry, P.. S. Heurtebise, B. Collet, F. Comette & A. Gerard. 1998. Differentiation between population of the Portuguese oyster, Crasso- strea angiilata (Lamark) and the Pacific oyster, Crassostrea gigas (Thunberg), revealed by mtDN.^ RFLP analysis. J. Exp. Mar. Biol. Ecol. 226:279-291. Canapa. .A., M. Barucca, A. Marinelli & E. Olmo. 2000. Molecular data from the 16S rDNA gene for the phylogeny of Peclinldae (Mollusca: Bivalvia). ./. Mol. Evol. 50:93-97. Folmer. O.. M. Black, W. Hoeh. R. Lutz & R. Vnjenhoek. 1994. DNA primers for amplification of mitochondrial cytochrome c oxidase sub- unit I from diverse metazoan invertebrate. Mol. Mar. Biol. Biotechnol. 3:294-299. Felsenstein, J. 1989. PHYLIP-Phylogeny Inference Package (Version 3.2). Cladi.mcs. ."S: 164-166. Guo. X.. S. Ford & F. Zhang. 1999. Molluscan aquaculture in China. J. Shellfish Res 18:19-31. Harry, H. W. 1985. Synopsis of the supraspecific classification of living oysters. Vc/i^ijcr 23:121-158. Hixson, J. E. & W. M. Brown. 1986. A comparison of the small ribosomal RNA genes from the mitochondrial DNA of the great apes and humans: sequence, structure, evolution and phylogenetic implications. M. Biol. Evol. 3:1-18. Jozefowicz. C. J. & D. O'Foiahll. 1998. Phvlogenelic analysis of southern 38 YU ET AL. hemisphere flat oysters based on partial mitochondrial 16S rDNA gene sequences. Mol Phylogenet. Evot. 10:426 — 135. Kimura. M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide se- quences. / Mol. Evol. 16;1 11-120. Lapegue, S., I. Boutet, A. Leitao. S. Heurtebise, P. Garcia, C. Thiriot- Quievreux & P. Boudry. 2002. Trans-Atlantic distribution of a man- grove oyster species revealed by 16S mtDNA and karyological analy- sis. Biol. Bull. 202:232-242. Li, X. & Z. Qi. 1994. Studies on the comparative anatomy, systematic classification and evolution of Chinese oysters. Studia. Marina Sinica. 35:143-173. (In Chine.se). Liu, B. & J. Dai. 1998. Studies on genetic diversity of oysters of genus Crassostrea. J. Fish. China 22:193-198. (In Chinese). Littlewood, D. T. J. 1994. Molecular phylogenetics of cupped oysters based on partial 28S rRNA gene sequences. Mol. Phylogenetic. Evol. 3:221-229. Meyran, J. C, M. Monnerot & P. Taberlet. 1997. Ta,\onomic status and phylogenetic relationships of some species of the genus Gammarus (Crustacea. Amphipodal deduced from mitochondrial DNA sequences. Mol. Phylogenet. Evol. 8:1-10. O'Foighil. D., P. M. Gaffney & T. J. Hilhish. 1995. Differences in mito- chondrial 16S ribosomal gene sequences allow discrimination among American (Crassostrea virginica) and Asian (C gigas. C. ariakensis) oyster species. J. E.\p. Mar. Biol. Ecol. 192:211-220. O'Foighil D., P. M. Gaffney & W. A. Wilbur. 1998. Mitochondrial cyto- chrome oxidase I gene sequence support an Asian origin for the Por- tuguese oyster Crassostrea angiilara. Mar. Biol. 131:497-503. Palumbi. S. R., A. Martin & S. Romano. 1991. The simple fool's guide to PCR. Honolulu, Hawaii: University of Hawaii Press. Qi, Z. 1989. Mollusk of Yellow Sea and Bohai Sea. Beijing: Agricultural Publishing House, pp. 176-180. (In Chinese). Thompson, J. D., D. G. Higgins & T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through .sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4672^680. Torigoe. K. 1981. Oysters in Japan. J. Sci. of Hiroshima Univ.. Ser. B. Div. 1 (Zool). 29:291^19. Wang, R., Z. Wang & J. Zhang. 1993. Marine molluscan culture. Qingdao, China: Qingdao Ocean University Press. (In Chinese). Yang. R. & Z. Yu. 2000. AUozyme variation within oysters Crassostrea plicatiila and C. gigas from Shandong coastal waters. / Fish. China 24:130-133. (In Chinese). Zhang, X. & Z. Lou. 1956. Studies on oysters in China. Acta. Zool. 8:65- 94. (In Chinese). Jniiniul ,>f Shclirish Rcsi-anh. Vol. 22, No. I. 34-19. 2()().V INCREASED BIOMASS YIELD FROM DELAWARE BAY OYSTERS (CRASSOSTREA VIRGINICA) BY ALTERNATION OF PLANTING SEASON JOHN N. KRAEUTER,' SUSAN FORD,' AND WALTER CANZONIER" ^Haskin Shellfish Research Lahnniiory. Iiistiliite of Marine and Cixistal Sciences. Rutgers University, 6959 Miller Avenue. Port Norris. New Jersey US349: and 'Aquarius Associates. Manasijuan. New Jersey ABSTRACT The practice of moving oysters from low-salinity to high-salinity areas for improving growth and meat quality has been practiced for well over a century. In the Delaware Bay. the practice was abruptly changed when MSX [Haplosporidium nelsoiii) caused large-scale oyster mortality in the higher salinity portions of the bay. Similar disruptions occurred in Chesapeake Bay and other areas. In lime the Delaware Bay. the oyster industry learned how to operate around the disease, but in early 1990s. Dermo (Pert^innis mariims) began to cause serious mortality on transplanted oysters. Despite the historic and continuing movement of oysters within and between estuaries, there is little published scientific literature indicating optimum conditions for transplantation. We investigated the effects of transplantation from a low-salinity seed bed to a typical higher salinity leased ground. The transplants were designed to evaluate an early, the traditional spring, and two fall transplant dates on the subsequent disease levels, growth, and survival of the oysters in three size classes: market, submarket. and small. Environmental and oyster disease data suggest we conducted the experiment under nearly worse-case conditions, high Dermo. and low food (chlorophyll). There were no significant differences associated with the timing of transplant. We did not record significant growth on any size oyster and disease caused mortality exceeded 50% for early transplants. Smaller oysters experienced greater mortality than market size individuals. Despite these conditions, meat dry weight nearly doubled within 1 to 2 mo after transplant in all but the March transplant. Under these di.sease and environmental conditions the only economic gain would be from the doubling of the meat weight and associated better meat quality. No gain can be expected from submarket oysters growing into the market size classes. KEY WORDS: oyster. Cnisso.strea. Delaware Bay. season, disease, growth INTRODUCTION In the Delaware Bay oysters have been transplanted from upper bay low-salinity seed producing areas to lower bay higher-salinity growing beds for more than 150 years (Ford 1997; Fig. 1 ). Similar transplantation strategies have been used by oyster growers in Chesapeake Bay (Andrews & McHugh 1957) and New England (Ingersoll 1881, Goode 1887). Further, to increase production and/ or to supplement local seed as resources became depleted, oysters were imported from distant sources. Despite these historic and continuing large scale movement of oysters within and between systems, there is little scientific literature indicating the optimum conditions for transplantation. Hopkins and Menzel ( 1952) developed a framework for study- ing the transplantation of oysters based on the biomass yield of the product, and Andrews and McHugh (1957) used biomass yield estimates from trays of oysters to evaluate the effectiveness of transplantation strategies. Reliance on biomass as a means of as- sessment in both of these studies was based on the assumption that the majority of oysters were destined to be shucked, and thus meat yield was the most important aspect of production. This may not be the case for those oysters that are grown to be sold for the half- shell trade. In this latter case, assuming adequate meat quality, numbers at market size are more important than total bicmiass. Haskin et al. ( 1983) and Hargis and Haven (1988) both indicate that the oyster planting industry in the Delaware Bay and the Virginia portion of Chesapeake Bay, respectively, operated under the assumption that transplanting was profitable if one bushel of seed oysters yielded one bushel of market oysters. In the late 1950s, the parasite MSX, Haplosporidium nelsoni. caused epi- zootic mortalities in both estuaries and forced major changes in oyster industry practices. In the Virginia portion of Chesapeake Bay, growers abandoned higher salinity grounds and concentrated efforts in areas that historically produced higher than the 1:1 yield (Hargis & Haven 1988), Despite H. nclsoni-c-Msed losses, the Delaware Bay oyster industry continued to transplant oysters based on the system developed in the KSOOs. Oysters were left on the planted grounds, where high salinity favored the H, nelsoni parasite, but for no more than 1 y (Ford 1997), and yields contin- ued to be about 1:1 (Haskin & Ford 1983). After the 1950s H. nelsoni epizootic, the importation of seed from out of state into the New Jersey portion of Delaware Bay was banned. In 1990, an outbreak of Dermo disease caused by Perkinsiis niarlniis prompted a further change in strategy by the Delaware Bay oyster industry. After 1990. P. inarinns infected most of the oysters in the seed bed areas (Ford 1997), and oysters planted in the spring of 1991 suffered high mortality in the late summer. The oyster industry and the State of New Jersey responded by devel- oping a program to market oysters directly from the seed beds. This strategy produced oysters that had poorer meat quality and a lower value than those from higher salinity waters. At the same time, it was realized that although Powell et al. ( 1997) modeled the effect of transplant time, disease, and preda- tion on market oyster populations, there were no real data on which to base transplantation decisions in the presence of this new para- site. The model predicted that fall (November) transplants left for 1 y yielded the best survival of market oysters compared with transplants in January, March, or May that were harvested in No- vember. In all cases the number of market oysters declined from July to November. The model did not include an August transplant with immediate harvest that fall, a strategy that would minimize disease-caused mortalities while still taking advantage of typically good fall "fattening" conditions. The industry requested data on the following: 1) the best time of the year to transplant oysters; 2) the survival of transplanted oysters at various times after trans- plant; 3) the numbers of market oysters expected from the net result of growth and mortality; and 4) the gains that could be made in iTieat quality and the length of time after transplant this gain might take. The industry, through a nonprofit foundation, collaborated with 39 40 Kraeuter et al. NEW JERSEY DELAWARE CAPE HENLOPEN Figure 1. Delaware Bay showing locations of the seed beds and Shell Rock bed, leased grounds, and the ground used for transplant studies. state New Jersey Department of En\ironmental Protection (NJDEP) and Haskin Shellfish Research Laboratory (HSRLl per- sonnel to conduct an initial test of alternative planting dates. This study (Canzonier 1998) moved oysters from the Shell Rock seed bed to higher salinity grounds (527 D) in December. February. May, and August. The effort clearly established that transplanting in months different from the historical spring period was economi- cally feasible, but cautioned that a single year's result could not provide sufficient background for assessing year-to-year variation. In addition, all months but the traditional spring transplant period, represented by the May transplant, gave nearly identical results. The May transplant had significantly less market oysters produced than the other months (Canzonier 1998). The information at the onset of the current study suggested that transplantation strategy would depend on several factors: oyster population size frequency distribution, source stock disease level, seed bed used as a source, environment of the planted ground, disease pressure, and harvest timing. In addition to biological vari- ables, market factors, and industry seasonal work cycles affect the economic impact of alternative planting seasons. The present study builds upon earlier efforts and evaluates the effects of varying the timing of transplanting oysters from one seed bed to a lower bay planting ground. MATERIALS AND METHODS Experimental Design Oysters from Shell Rock Bed were transplanted to ground 354 D (Fig. 1 ) in March, May, September, and October of 1999. Shell Rock was selected because it represented a central seed bed source, had a significant number nearly market size oysters, and pro\ ided the oysters for the Can/onier ( 1998) study. The transplant ground was subdivided into experimental plots. each marked with navigation coordinates. A preliminary sampling indicated that only a small number of large residual oysters (mean 99 mm) were present (mean 2.4 oysters bu~' from 8 one-bushel samples). Approximately 1800 US Standard bushels (36.4 L; herein after referred to as bushels or abbreviated as bu.) of oysters were planted on each 24.4 x 91.4 m plot each transplant time (3.200 bu.acre"' or 90.000 oysters hectare"'). At each transplant time, triplicate bushels of oysters were re- moved from the deck load of the boat and analyzed in a manner similar to the techniques used for the subsequent monthly samples (see below). In addition, oysters were processed for disease diag- nosis. After planting, at least three dredge samples were collected each month from each planting. All material was placed in the bushels so that triplicate composite bushel samples of material were examined from each planting each inonth. These were ex- amined in the same manner as the source oysters, but with special attention to growth, meat condition. P. inarintis level, and mortal- ity (apportioned by oyster size). In the latter months, additional oysters were set aside after the samples had been collected to be sure enough material was available in all size classes to process P. marinus and condition index samples. H. nelsoni levels were not detemiined on the monthly samples, but were evaluated on the fmal samples from each plot in No\'ember, as well as on the initial transplants. Sample Processing All live oysters >20 mm, old, new boxes, and gapers in the entire sample were counted. All oysters >20 mm were measured and divided into market (>76 mm) and submarket (35-73 mm) and small (<55 mm) classes. All parameters were normalized to a standard bushel for comparison with other samples. Mortality was estimated by calculating the percentage of new boxes and gapers in each sample. This was considered recent mortality. Recent mor- talities were accumulated to provide an estimated cumulative mor- tality at the end of the study (Ford & Haskin, 1982). Twenty oysters (six or seven from each of the 3 bu.) of each size class were set aside for evaluation of condition index and an additional group of similar size was examined for P. inariiuis infection. Condition index was derived from the ratio of meat dried at 50°C, and greatest shell dimension (height). P. marinus was diagnosed after incubation of the rectum and a piece of mantle in Ray's fluid thioglycollate medium. Infection intensity was scored from 0 to 5 (Ray 1954) and a weighted prevalence calculated as the mean intensity, including zeros, of all oysters in a sample. Oysters in the initial planting and final sampling were diagnosed for H. nelsoni by tissue section histology. Infection intensities were rated from 0 to 4 (Ford 1983) and a weighted prevalence calculated as for P. marinus. Individual Oyster Growth and Mortality Study To evaluate production requires size-class-specific growth and mortality data. This was approximated from the bushel samples, but a second method was utilized to provide a more precise evalu- ation of individual oysters. A group of experimental oysters rep- resentative of the source bed was deployed at the time of trans- plant. This group consisted of five replicates of 20 oysters from each of three size classes (63.5 to 69.9 mm, 70 to 75.9 mm, and >76 mm) for a total of 300 oysters. Fishing leader tethers were glued to the top valve of each oyster with Marine Tex. The tethers Increased Biomass Yield ok Oysters 41 were then attached with cable ties along the side of a square reint'orcing rod frame square (~l m on each side) that was held approximately 5 cm above the bottom by a centrally located ce- ment anchor. The entire array was attached to a surface lloat. Each individually identified oyster was measured (height) and the array deployed so that the oysters would lie on the bottom. Each month each oyster was measured and mortality or loss noted. In this instance, mortality was calculated directly because the history of each oyster was known. Environmental Data The following environiuenlal data were collected on bottom water on at least an every other week basis: temperature, salinity, dissolved oxygen, pH, total suspended solids. Chlorophyll a. and suspended organic material. In addition, temperature was moni- tored continuously with an electronic recorder. Salinity was ob- tained with a refractometer. All grab sample temperature and dis- solved oxygen data were measured with a YSI oxygen meter, and pH data were obtained with an electronic pH meter. Suspended solids, chlorophyll and particulate nitrogen samples were obtained from at least 500 ml of water filtered through Whatman GF/C glass fiber filters, which were stored on ice until they were returned to the laboratory. Chlorophyll samples were immediately placed in buffered acetone and refrigerated. Particulate samples were dried at 50°C. All en\ ironmental data were analyzed according to Strick- land and Parsons ( 1968). Data Analysis Size frequency data were normalized by adjusting the base live and recent dead (gapers and new boxes) frequency distributions from all individuals collected in the three bushel samples (in 5-mm increments) to 100 individuals. These frequencies were then ad- justed to the number of live or dead bu."' by multiplying the frequency of occurrence in all sizes by the average number of live or dead bu."' Data were summarized and significant tests were run using one-way analysis of variance, I tests, or other descriptive techniques. Percentages were transformed using an arc-sine trans- formadon before performing analysis. RESULTS Envirnnnicntal linla Temperature on the transplant ground was 3.5°C in March, at the beginning of the study, and peaked in August at 27.5"C. Sa- linity was generally between 21 and 23 ppt.. with a low of 19 ppt in April and a high of 26 ppt in October and December. pH remained relatively stable, ranging from 7.8 to 8.6 with the excep- tion of a low value of 6.9 on September I. Dissolved oxygen ranged from a high of 13.5 mg L"' in March to a low of 5.6 mg L^' on July 14. In general dissolved oxygen levels remained near or above saturation at temperatures below 2()"C and near or slightly below saturation above those temperatures. Total suspended solids were typically between 30 and 55 mg L"'. with highest and lowest values of 86 and 1 8 mg L~ ' on August 1 8 and May 5, respectively. Chlorophyll a showed a typical spring (late March to early April) bloom followed by generally lower vales in summer (Fig. 2). There was an increase in Chlorophyll a in fall (October to early November). Highest Chlorophyll a levels were found March 25, April I, May 18 and November 5 with values of 54, 46, 38 and 39 mg m~' respectively. 60 50 ^40 30 D- O U 20 10 Mar 9 Apr 1 May 5 May 18 Jun 18 July 14 Aug 18 Sept 23 Oct 8 Oct 29 Dec 15 Mar 25 Apr 18 May 1 1 Jun 7 Jun. 30 Aug 4 Sept I Oct 5 Oct 22 Nov 5 1999 1996/1997 Figure 2. Buttuni water chlorophyll a In samples taken from bottom water over ground 554 1) in Delaware liay in 1999 compared with similar data taken over ground 527 D In Delaware Bay in 1997. Data are in mg per m'. 1997 data from Canzonier (1998). 42 Kraeuter et al. Oyster Data Because the samples taken at the time of transplant represented the source bed and culHng machinery on the boat, not the ground to which the oysters were transplanted and monitored, time 0 (7",,) for subsequent analyses was the first sample after transplant. The samples taken from the deck at the time of transplant were utilized to estimate the size, condition and numbers of oysters transplanted. Numbers of Live and Dead Oysters The numbers of oysters being transplanted, based on the initial samples for each transplant period, suggests that all groups, with the exception of the October transplant, received uppro.ximately the same number of individuals per unit volume of material moved. The October samples had fewer oysters than those groups transplanted in March and September, but was equivalent to the May transplant (Table I ). It seems likely that more live oysters were moved in the May transplant than in October, but the high variance in May precludes making a definite statement. The total numbers of live oysters significantly decreased from Tf, to the fmal samples {T,) in November. The numbers in the March and May transplants fell approximately 50* from 200 in initial post-planting samples to 20 mm bu."' by month with 95% conndence limits (h = 3 for each monthly sample). March May September October Mean 95% Conf . Limits Mean 95% Conf. Limits Mean 95% Conf. Limits Mean 95% Conf. Limits M 323 363 283 A 212 124 156 105 119 80 108 76 230 189 209 155 144 120 131 87 195 59 103 56 93 40 84 65 M 296 403 188 J J A I8,S 121 76 90 92 79 239 169 154 143 104 113 137 73 0 38 79 45 S 307 355 259 0 169* 146 203 136 205 87 243 254 232 N 78 101 56 Bold numbers indicate a significant difference from the prior month. The area in gray indicates samples removed from the deck of the transplant vessel. These were not used in subsequent calculations. * Significantly more oysters than in other transplants during the sample period. Increased Biomass Yield of Oysters 43 I ABIE 2. Mean number of live market (>7f) ninil, subniarket (75-55 mm), and small (55-20 mm) oysters bu. ' of dredfjcd material from transplants in March, May, September, and October 1999. March 1999 May 1999 September 1999 October 1999 Market Submark Small Market Submark Small Market Submark Small Market Submark Small M 58 115 150 (^ 63 48 61 24 40 30 32 76 32 42 37 41 30 38 29 75 45 54 40 38 21 38 15 M 78 104 114 .1 J 34 38 23 25 38 34 64 34 25 37 33 33 87 49 28 39 21 16 s 56 86 167 54 70 o 29 27 47 35 94 84 120 N 25 26 28 Oys(ers were transplanted from Shell Rock to Ground 554D on the Delaware Bay leased grounds. Areas of gray indicate samples from deck loads of transplanted oysters. All other samples were dredged from transplant plots. Submark = submarket. this linic. heavy (iiortality was observed in the March transplants to those transplanted earher. but, unhke the former, infections only (Fig. 3) and the drop was probably the beginning of the retnained at very high levels in these oysteis into November. The overwinter loss of infections (Bushek el al. 1994). Oysters trans- persistence of high infection levels was as.sociated with low mor- planted in September and October had weighted prevalence siniilar tality in both fall groups. Mar >75mm Mar 55 to 74mm Mar <55mm I Li .....III! Mil Apt Mav June Iul> Aug May >75mm Scpl Oci Nov *7 1 30-L-- 1 1 1" 1 1 1 ■ ■ - ■■ " Apt Miy June July Aug Sept Do Nov Sept >75nun Hi Mm Apr May June July Aug Sep( Oci Nov Oct >75mm II Apr May June July Aug Sep! Ocl Nov Sept 55 to 74 mm li Apt M»y June July Aug Sept Oct Nov Oct 55 to 74 mm h Mai Api Mdy June July Aug Scpi Oct Nov May <55mm Mar Apr May June July Aug Sepi Oct Nov Sept <55 mm ■ I Ms Apr Miy tunc luly Aug Sepi Oa Nav Oct <::55 mm Apr Miy June July Aug Sept CJti Nov Mat Apt Miy June July Aug Sepi Oci Nov Mat Apt Miy June July Aug Sept Oct Nov Figure 3. Interval percent mortality by month of market (>75 mm), submarket (55 to 74 mm), and small {<55 mm) oysters transplanted from Shell Rock to Delaware Bay ground 554 D in 1999. Transplant months were March {top graphs), May (middle top graphs), September (middle bottom graphs), and October (bottom graphs). 44 Kraeuter et al. TABLE 3. Estimated cumulative percent mortality, from plantin}< to November 19')9. by size category of dredged oyster samples collected in Delaware Bay by transplant month. March 1999 May 1999 September 1999 October 1999 Market Submark Small Market Submark Small Market Submark Small Market Submark Small 46 48 65 45 4,S XS 19 16 14 11 11 5 Market (>76 iiini). Mibniarket (75-55 mm), and small (55-20 mml. Growth and Condition With the exception of the March transplants, there were no differences in the sizes of oysters in the subniarket and small categories through time. Mean dry meat weight of market oysters for the March and May transplants increased significantly in June, after .^ and I mo, respectively (Table 4). That of markel-si/e Sep- tember and October transplants rose in November after 2 and I mo. respectively. There were no significant differences in meat weight among any of the transplanted groups by November. While not statistically significant, there was a consistent increase in meat weight in all transplants of market-si/e oysters between October and November. In general, meat weight increases of submarket and small oysters mirrored those of the market-size individuals. Reflecting the increase in meat weight without increased shell size in market oysters, the condition index increased during the study period. With the exception of the March transplants, oysters required one month after transplant to the lower bay to improve condition, and they typically retained this condition throughout the summer and into the fall. While not statistically .significant, there Mar > 75 mm Mar 55-75 mm March April May June July Aug Sept Oct Nov irxll March April May June July Aug SepI Oct Nov Mar < 55 mm 4- 3- 2- 0- Mafch April May June July Aug SepI Oct Nov May > 75 mm 5 : llili mil T r March April May June July Aug Sept Ocl Nov May 55-75 mm III T . illi 1 Oct nil i JUU 1 March April May June July Aug Sept Nov May < 55 mm March April May June July Aug SepI Ocl Nov Sept > 75 mm March April May June July Aug Sept Ocl Nov 4—] Sept 55-75 mm T i [ 1 1 I 1 1 ! March April May June July Aug 1 1 1 ^ept Oct Nov Sept < 55 mm i i™i™i ™i — I — i — I — I — March April May June July Aug Sept Oct Nov Oct > 75 mm 5„ 1 1 1 1 1 1 r March April May June July Aug Sept Oct i Oct 55-75 mm 1 1 1 1 1 1 r March April May June July Aug Sepl Ocl I Oct < 55 mm t 1 1 1 1 1 1 r March April May June July Aug SepI Oct i Figure 4. Monthly weighted prevalence of Dermo (/'. nuirinu\) infections in market (>75 mini, subniarket (55 to 74 mini, and small (<55 mml oysters transplanted from Shell Rock to Delaware Bay ground 554 D in 1999. Transplant groups were March (top graphs). May (middle top graphs), September (middle bottom graphs) and October (bottom graphs). For each transplant group, the llrst sample represents that on Shell Rock bed when the oysters were moved. All subsetpient samples represent infection levels on ground 554 D. Error bars represent 95 Cr confidence interval. Increased Biomass Yield of Oysters 45 TABI.K 4. Mean dry meal Heijjht (jjl of markel-size (ijsterv b> month with 'tS'c conlldence Mmit.s. March May September October Mean 95 "/f Conf. Limits Mean 95% Conf. Limits Mean 95% Conf. Limits Mean 95% Conf. Limits M 1,1 1.2 0.9 A 1..^ 1..5 1.2 M 1,? 1..S 1,2 l.s 1,7 1.3 .1 2.4 2.8 2.0 2..^ 2.6 1.9 J 2.5 2.8 T 1 2.3 2.7 2.0 A -> 1 2.4 2.0 1 T 2.4 2.0 S 2.4 2.7 2.1 2.5 2.8 T 1 1.6 1.8 1.4 O 2 2 2.6 1.8 2.3 2.7 2.0 1.9 2.3 1.6 I.I 1.3 1,0 N 2.9 .V4 T 1^ 3.1 3.6 2.6 2..S 2.9 2.1 2.7 3.2 2.2 Bold niinihers indicate a significant difference from the previous month. was a general trend for market oysters to improve in condition from October to November. Condition index for submarket and small oysters generally fol- lowed the same trends as for the market oysters with no significant change from June to Nmember. In general, there was a significant increase in condition w ithin 1 mo after transplant for all submarket and stnal! oysters with the exception of the March transplants and small oysters transplanted in October. By November, the meat condition index of all si/e classes in the March and September transplants was statistically the same. Among the October transplants, condition of submarket and mar- ket oysters was the statistically similar, and greater than that of small oysters, while the condition of market oysters in the May transplants was greater than that of either submarket or small oys- ters. (iriiHlli ami Mdilalily of hulividually Marked Oysters For calculations of mortality, the data from the tethered oysters were corrected for oysters lost during the experiment by reducing the numbers of oysters present from the initial counts. A few oysters were lost because of detachment of the adhesive, but one entire rack was lost. Mortality of tethered oysters mirrored that of oysters trans- planted at similar times, with a few notable exceptions (Fig. 3). It IS evident from the cumulative mortality data (Table 5) that the tethered oysters (particularly those put out in May and September) liad substantially more mortality than that estimated from exami- nation of boxes and gapers in dredged samples. At times, shells on one section of an airay were observed to have become blackened. This suggests that some silting had taken place around these oys- ters and may have elevated the mortality above that experienced by the planted oysters, but we have no independent measure to evalu- ate if some planted oysters were silted in and not adequately TABLE 5. Cumulative percent mortality of tethered oysters, and oysters in dredged samples as a function of transplant time. Month of Transplant Method March May September October Tethered Dredged 76 ,54 93 55 59 15 38 9 sampled with the dredge. There were no significant differences in recent or cumulative mortality based on si/e of the tethered oys- ters. Because all tethered oysters were large and the growth incre- ment was small relative to the potential error, the monthly growth increment of tethered oysters was difficult to measure. This diffi- culty is evident in the fluctuations in increment growth for the various size classes (Fig. .5) and the negative growth measured for some months. Growth, as indicated by new shell being accreted to the oysters, was observed on some oysters in all but the coldest months. Because individual oysters were followed, cumulative growth is the difference between the initial measurement and the measure- ment of surviving oysters at any time period (Fig. 5). Because not all oysters survived through all time periods, cumulative growth reflects both survival and growth of individuals. By November there were no differences in growth of surviving tethered oysters classed as market-sized in March and May, but individuals in both groups had grown more than those tethered in September and October. There was no statistically significant growth for either of these latter two periods. Growth of submarket size oysters was also at the limits of detection. The 70- to 75-mm size class showed >0 growth only for the May and September groups when the mean were 4.8 and 1.8 mm. respectively. With the exception of the March tethered individual (only one oyster survived to October) oyster classed as small did not show tnea- surable growth. DISCUSSION Hopkins and Men/el ( 1952) indicated that the major difficulty in deriving estimates of production was not related to measurement of growth, but to measurement of losses due to mortality. In our case, where only large oysters were being evaluated and growth was poor; it was also difficult to assess growth. The dominant themes of Delaware Bay oyster transplantation in 1999 were related to high Dermo (P. niciriiiiis) levels and the associated high mortality and low chlorophyll and the associated poor growth. There is a general hypothesis that mortality of trans- planted, market-sized oysters, due to disease or other factors, can be made up for by oysters growing from smaller sizes to the market classes during the year Powell et al. (1997). This can hap- pen in some years (Canzonier 1998), but in periods such as 1999 with high P. iiiarinus levels and relatively low food, growth may 46 Kraeuter et al. Mar 70-75 mm Apnl Nby June July Aug Sepi Oct Nov Dec May 70*75 mm -\ 1 ! ! ! '■ \ \ 1 Apnl May June July Aug Scpi Ocl Nov Dec Sept 70-75 mm 1^1 May lunc July Aug SqX On Nov Dec Oct >75 mm i 1 ■ 1 ■ 1 1 1 1 Alril Mv luoe July Am Sq« On Nov Dec Oct 70-75 mm 7J " — 1 S i; 1 1 III ■ ! ' 1 1 Mar 63-70 mm 1 a E g ,5 ■ ■ - m ■■ ^Hll ■ 1 1 1 1 1 1 1 1 i Apnl my June July Aug Sept May 63-70 mm Oci Nov ! Dee 1 g jj^ ■ ■ 3 .II.B [ ; \ t Apnl Miv June July Aug Scpl Sept 63-70 mm OCL Nov Dec ! 1 = 2S s ■ t ----- - ■ -"' Ap«l Miy 1 1 ! June July Aug Sept Oct 63-70 mm Oci Nov Dee i i ^ ! ! 1 1 i ^ 1 } 1 ! ! - ^-4—1 ! I I H ! I I Apnl May June luly Aug Scpi Oci Nov Doc Apnl May lunc luly /wg Scpl Oci Nov Dec Apnl May lunc July Aug Scpl Oci Nov Dk Figure 5. Cumulative growtli of >75 nini, 7(1-75 mm, and 63- to 7(l-mni tetiiered oysters transplanted from Shell Rock to Delaware Bay ground 554 D in 1999. Transplant months Here March (top graphs), Ma_\ (middle top graphs), Septemher (middle hottom graphs), and October (bottom graphs). Negative growth is due to measurement error. All oysters were followed as individuals and growth is the summation of all oysters alive in that size class at the time of measurement. be reduced to the point that this hypothesis is not valid. Neither the tethered oysters nor the transplanted oysters in the dredged samples, of any size class, in the present study showed statistically significant growth. The data did not show statistically sigiuficant differences in numbers, based on month of transplant, of market, submarket. or total oysters per bushel in final sampling in November. This sug- gests that in periods of high P. imiriinis. high i-i-)ortality. and low food the timing of transplantation is not a major consideration from the point of view of the numerical yield of market oysters. In addition to the nearly 50% losses of submarket and market oysters, losses of si-nall oysters exceeding 65% suggest that transplantation of small oysters with the expectation that they will grow into the market-size category is not an efficient use of the resource under high P. mariiuis conditions. In view of lack of significant differences in the numbers of i-i-)arketable oysters associated with transplant month, possible dif- ferences in meat quantity need to be considered. In all cases (ex- cept the March transplants when water temperatures were low) total meat weight improved within one month following transplan- tation (Table 4). Beyond this initial improvement in there was no change durinc the summer months, but in all cases there was a trend (not statistically significant) toward further improvement in between the October and the November samples. Clearly the im- provement in meat weight in the May to June period could be due to the increase in gonadal tissue, but the weight did not decrease in the summer or fall, after the spawning period, indicating that some of this weight gain was more than gonadal production. The im- provement in meat quality occurred in 1999 despite the high dis- ease levels, high mortality and lack of shell growth. Comparison with Previous Studies Powell et al. (1997) modeled the effect of transplanting Dela- ware Bay seed bed oysters in November. January. March. April, and May on the number of market size oysters available the fol- lowing July to November. The model predicted that a November transplant with a November harvest provided the best yields, and that growth of submarket sized oysters compensated for the losses of market sized individuals. Mortality of submarket oysters was less than for larger ones because the added scope-for-growth offers these individuals some disease protection. Simulated P. nmrinus levels peaked slightly above four weighted prevalence a level nearly reached in the present study. The model simulated that Increased Biomass Yield of Oysters 47 TABLE 6. Comparison of niimbcrs of marktl and siihniarkil ovslurs hu. plantt'd on leastd fjrounds in IMMft to IW7 and IWy. Year of Transplant Market 95 '7r Confidence Limit Submarket Confidence Limit 1999 1996/97 62 ?6 tl3 232 576 ±5? tl05 Data from 1996 to 1997 are from Canzonier (199S). Data arc from samples removed from the deck of the transplant vessels. submarket size oysters were less susceptible to mortality from P. mciriiuis than the market-sized oysters, which allowed them to grow to market size and replace larger, individuals with lethal infections. This simulation was not verified in the present studies. One reason is that, in contrast with the model simulation, the smaller oysters did not grow. Thus, they did not increase in bio- mass fast enough to "outgrow" the parasite and maintain parasite burdens below lethal levels. It is important to emphasize that the food present in 1999. as indicated by Chlorophyll a. was lower than that used in the model of Powell et al. ( 1997). It seems likely that the low food concentrations in 1999 reduced the potential for compensatory growth of submarket oysters to replace market oys- ters that died during the study period. The lack of growth may also have been a consequence of high disease levels (Men/el & Hop- kins 19.'i.'i. Paynter 1996). Further, many of the assumptions of the Powell et al. ( 1997) simulations were based on age/size relation- ships observed in the Gulf of Mexico, which do not apply to Delaware Bay. In Delaware Bay. for instance, submarket-sized oysters (35-75 mm) obtained from seed beds are at least 3 years old and many of the small oysters (<55 mm) are at least 2 y old. All sampling of oysters in the Bay indicate that by age 2, oysters have P. marimis infection levels that are equal to that of older oysters. Thus, it is not surprising that cumulative mortality for our submarket and small oy.sters was equal to. or greater than, that of market-sized oysters. A second major difference between our study and the model simulations is that significant numbers of submarket oysters did not grow into market individuals in 1999. Canzonier et al. (1998) reported on a similar transplant. He moved oysters from the same seed bed (Shell Rock) in December 1996, and February, May and late August 1997. and sampled them until November 1997. Growth of oysters into the market size cat- egory was clearly evident in the 1996 to 1997 period (Canzonier 1998). The number of oysters bu. ' transplanted differed signifi- cantly between this study and the present one (Table 6). There were no differences (P = 0.43) in the numbers of market oysters bu. ' from the deck loads of the two studies, but there were neariy twice as many submarket oysters in the earlier trial (Table 6). In 1996 to 1997. the percentage of market oysters bu. ' ranged from 8 to 10% whereas in 1999 market oysters were between 18 to 26% of the total. Canzonier (1998) found the number of market oysters from dredge samples remained relatively constant throughout the test period in spite of the substantial mortality. Thus despite twice as many submarket size oysters and growing conditions that were better than in 1999. there were no changes in the number of market size oysters in any month of transplant in 1996 to 1997. Growth of submarket oysters made up for the loss of older oysters. As opposed to the 1999 results, in which a 21% decrease in the numbers of market oysters was observed in all transplants. Can- zonier ( 1998) reported an insignificant 4% decrease in the number of market-size oysters at the end of the experiment in November. P. mariniis levels were generally lower in 1996/97 when compared to both the model and the 1999 data (Table 7). Cumulative mor- tality was less for December and February transplants but appar- ently higher for May and August transplants in 1996/97 when compared with roughly similar transplant months in 1999 (Table 8). Chlorophyll ii in 1997 showed a slight peak in the spring, a second peak in June and continued high levels (relative to 1999) throughout the summer, but a general decline from late August to November (Fig. 2). In this latter condition. Chlorophyll a in the earlier period was similar to those in the Powell et al. (1997) model. The presence in 1996 to 1997 of high summer food con- centrations, lower P. mariiuis. and consequently lower moitality than in 1999 suggests that the 1999 conditions may be nearly a worst-case representation. The only exception would be the pres- ence of the fall bloom in 1999 that would have allowed the oysters to enter the winter in better condition. This may or may not be important because there was no difference between the dry meat weights in 1996 to 1997 when there was no fall bloom and 1999. Canzonier (1998) reported that market oysters moved from Shell Rock in December. February. May, and August averaged the same dry meat weight (1.2 to 1.3 g) as those at the time of trans- plant in the present study. His final product in November had a meat weight of 2.8 g. the same weight as oysters in 1999. How the increase in meat quality in transplanted oysters, vs. those marketed directly Iriim the seed beds, would affect profit- TABLE 7. Initial and selected months. December February May .August Market Submark Market Submark Market Submark Market Submark D l.S 1.3 F 0.8 0.7 A 0.2 0.1 0.1 0.1 M 0.1 0.1 A 2.1 1.4 1.2 1.3 0.7'' 1.1 1.0 0.6 S 1.2 1.3 1.9 1.3 1.3 2.3 1.7 1.8 N 0.4 0.6 0.2 1.2 0.5 1.2 0.5 0.9 Weighted prevalence oi P. marimts (Dermo) in oysters transplanted from Shell Rock to 527D m 1996 to 1997. Market >75 mm. Submark = Submarket (55-75 mm). (From Canzonier 1998). 48 Kraeuter et al. TABLE 8. Cumulative percent mortality from planting to November of oysters from Can/.onier (IWSt and present study. Study Month of Transplant Present study March 54 May 55 September 15 October 4 Canzonier (I99SI Decemtier 43 February 45 May 30 August 15 ability is dependent on the relationship among the following pa- rameters: 1 ) the number of market oysters bu.~' and/or the amount of meat bu."' that could have been harvested directly from the seed beds; 2) the number of market oysters and/or the amount of meat bur' that could have been harvested from the transplanted oysters; 3) the cost of re-harvesting the transplanted oysters; 4) the added value that is derived from post-shucking processing (washing with fresh water and blowing with air to help remove shell materials) a higher salinity oyster; and 5) the value of the bushel of oysters to the market. The latter \ alue is dependent on the season of harvest, competing product and whether the oysters are shucked or sold in the shell. If oysters are used as shell stock, there would be little gain in value to the harvester from an increase in meat yield, because in current conditions, there is little chance (hat additional price would be paid (S. Fleetwood. Bivahe Packing, pers. comm.). The best that could be expected would be a longer term value increase because of better market acceptance. Before the disease infesta- tions. Delaware Bay oysters received a premium price because of their high meat yields. Thus for shell stock oysters, in years of high or moderately high P. marinus disease-caused mortality, there would be little to gain from transplantation. For oysters that are to be shucked, results of both the 1996 to 1997 and 1999 studies indicate a significant increase in meat yield after transplantation. It is important to note that the meat yield increase, during months with warm water, can be obtained in one or at most two months. In 1999 the average meat yield increase by November was about 1 13'-^. and in 1996 to 1997 the meat yield increased by about 1339}- (Table 9). Given that there was no difference in the number of oysters available for market in November (Table 9) associated with trans- plantation time, it would appear that there was no value added from transplantation in any month or tor the average of all months. It should be emphasized that under current conditions, market oysters are culled on board. This means that nearly equal numbers of oysters bu."' would be delivered to the packing house from both the seed beds and the planted grounds. Under these conditions the meat from oysters harvested from the planted grounds in both trial periods would weigh approximately 1249^ more that of oysters from the seed beds. In both cases the use of oysters for shucking stock would result in increased yields. The higher salinity on the planted grounds and the added meat weight, will provide addi- tional gains during the washing and blowing of the meats during processing. CONCLUSIONS When combined with the Canzonier (1998) study the data cover two of a myriad of possible cases. In 1996 to 1997 there were slightly elevated summer chlorophyll levels, moderate growth and moderate P. marinus. whereas in 1999 there were low or typical Delaware Bay sunmier chlorophyll levels, no growth and high P. iiniriiuis. The month of transplant did not have a significant effect on the numbers of market oysters available at the end of the year. When P. marinus levels were elevated and food supply was low. transplanted small oysters were lost at a higher rate than market or submarket oysters. The data from both studies suggest that food levels on the planted grounds in the warmer part of the year are generally sufficient to support increases in meat yield 1 to 2 mo after transplant, but may not be sufficiently high to support shell growth in all years. Under high to moderate P. marinus conditions, exclusive of tiiarkel timing, meat weight or shucked meat volume gain were the most important factors for economic comparison of market oysters between the seed beds and the planted grounds. TABLE 9. Estimated dry meat yield (g) of market oysters (>76 mm) bu. ' of dredged material at time of transplant (Shell Rock) and in November 1997 and 1999. Shell Rock Transplants Transplant Month Oyster/bu. Dry Meat Wt Dry Meat/bu. Oyster/bu. Dry Meat Wt Dry Meat/bu. 1999 March 99 63 1.1 69 32 2.9 92 May 99 34 1.5 51 34 3.1 105 September 99 29 1.6 44 27 2.5 68 October 99 25 1.1 28 25 2.7 68 Average 38 1.3 49 30 2.8 84 1996/1997 December 96 110 1.1 121 108 2.8 302 February 97 92 1.2 110 110 2.5 275 May 97 95 1.5 143 93 2.7 251 August 97 133 1.3 174 106 3.0 318 Average 108 1.2 130 104 2.8 291 Oyster numbers for Shell Rock have been adjusted by using data from the first month of post transplant sampling to accommodate for differences culled deck load samples and dredge samples. Oysters transplanted from Shell Rock by month of transplant. Increasbu BioMASS Yield of Oysters 49 ACKNOWLEDGMENTS The study was funded through funds supphed by the State of New Jersey for evaluation of the Delaware Bay oyster resources, and allocated through the Oyster Industry Science Committee of the Delaware Bay Shellfish Council. The present study could not have been completed without the on-the-water efforts of Royce Reed and Russell Babb of NJDEP— Shellfisheries. Staff of the Haskin Shellfish Research Laboratory (Bob Barber. Beth Brewster and Meagan Cummings) were instrumental in carrying out much of the sampling and sample processing efforts. The NJ Agriculture Experiment Station also pro\ided support. LITERATURE CITED Andrews. J. D. & J. L. McHugli. \95T. The sLir\i\al and giowlh of South Carolina seed oysters in Virginia waters. Pidc. Nur. Shclljlsh As.s .Authority. Unnersity of Delaware. Lewes. Delaware. 326 pp. Haskin. H. H. & S. Ford. 1983. Quantitative effects of MSX disease iha- plosporidium nelsoni) on production of the New Jersey oyster beds in Delaware Bay. USA. Int. Counc. E,\plor. Sea. CM 1983/Gen:7/Mini Symp.. Goteborg. Sweden. Hopkins. S. & R. W. Menzel. 1952. Methods for the study of oyster plant- ings. Convention Addresses NaL Shellfish. Assoe. 1952:108-112. IngersoU. E. 1881. The oyster industry. In: The history and present con- dition of the fishery industries: Tenth Census of the United States. Department of the Interior. Washington. DC 251 pp. Menzel. R. W. & S. H. Hopkins. 1955. Growth of oysters parasitized by the fungus Dermocystidium marinum and by the trematode Bucephalus cueiihis. J. Parasitol. 41:333-342. Paynter. K. T. 1996. The effects of Perkinsus mariniis infection on physi- ological processes in the eastern oyster. Cnissosrren virginicn. J. Shell- fish Res. 15:119-125. Powell. E. N.. J. M. Klinck. E. E. Hoffman & S. Ford. 1997. Varying the timing of oyster transplant: implications for management from simu- lation studies. Fish. Oceanogr. 6:4. 213-237. Ray. S. M. 1954. Biological studies of Dermocystidium mariiuim. Rice Institute Pamphlet. Special Issue. (The Rice Institute. Houston. Texas). Strickland. J. D. H. & T. R. Parsons. 1968. A practical handbook of sea- water analysis. Fish. Res. Bd. Canada. Bull. 167. 311 pp. .loiinuil oj Slu'ltfisk Rcscanh. Veil. 22. No. I, 51-59. 200.^. U.S. CONSUMERS: EXAMINING THE DECISION TO CONSUME OYSTERS AND THE DECISION OE HOW FREQUENTLY TO CONSUME OYSTERS LISA HOUSE,'* TERRILL R. HANSON," AND S. SURESHWARAN' ^ Fo(xl and Resource Economics Di'purtnwnt. University of Florida. P.O. Box 1J024U, Gainesville. Florida 32611: 'Department of Agricultural Economics. Mississippi State University, PO Box 5187. Mississippi State, Mississippi 39762: and Higher Education Programs, Cooperative State Research, Education and Extension Sen'ice, USDA, Mail Stop 2251, 1400 Independence Ave, SW, Washington, DC 20250-2250 ABSTRACT Oyster consumption has been decreasing in the United States. Investigating consumer attitudes and preferences can help identify factors involved in this decrease. This study used data obtained through a nationwide survey in a douhle-hurdle regression model to determine factors that influence both the decision to consume oysters and frequency of consumption. Results uidicate there is a significant difference in the reasons people choose to eat oysters or not and the reasons oyster consumers choose how frequently to eat oysters. Concern for product safety significantly influenced the decision of how frequently to consume but not whether to consume oysters. Consumers also indicated a potential willingness to pay for measures that would increase product safety. KEY WORDS: consumer preference, double-hurdle model, food satety . marketing, oyster industry INTRODUCTION METHODS Overall per capita fresh shellfish consumption in the United States has increased from 2.5 pounds in 1980 to a high of 4.7 pounds in 2000 (Fig. 1). Per capita consumption of oysters, how- ever, has decreased from an average of 0.35 pounds per year (average of 1980-1989) to 0.25 pounds in 1990 to 0.20 pounds in 1999 and 2001 (USDOC 2001; Fig. 2). Food safety is a factor often blamed for decreases in consump- tion of oysters. In a 1993 news release, a multi-state outbreak of viral gastroenteritis related to consumption of oysters occurred iti Louisiana. Maryland, Mississippi, and North Carolina (Centers for Disease Control and Prevention 1993). In 1998. bacteria-tainted oysters from Texas were identified as the cause of sickness for 368 people, and in the preceding summer, 209 laboratory-confirmed cases of illnesses were linked to consumption of raw oysters har- vested in the Pacific Northwest (ABC News 1998). The Center for Science in the Public Interest has asked FDA "to take immediate action to protect consumers from raw oysters contaminated with deadly bacteria" (Center for Science in the Public Interest 2000). They cite 36 deaths in the previous 2 years and 1 19 deaths since 1989 associated with Vibrio viiliiifuus — contaminated raw oysters and other shellfish. In 1990. Billups (2001) showed only 9% of respondents considered oysters "not at all safe" compared with 31% rate in a similar survey conducted 5 years later. Although food safety is suspected to be a major factor in the decision to consume oysters, other factors may be involved. Re- gional and national oyster consumption can be affected by many determinants that may vary across geographical region, ethnicity, income levels, and perceptions of nutritiim (Wessells et al. 1994. Gempesaw et al. 1995. Wessells & Anderson 1995. Manalo & Gempesaw 1997, Wessells & Holland 1998, Holland & Wessells 1998). The goal of this study was to investigate the decision to consume oysters and the decision of frequency of oyster consump- tion. *Corresponding author. E-mail: lahouse@'utl.edu The data for this study was obtained through a mail survey. After conducting a number of focus groups of seafood consumers and nonconsumers (in three locations in the United States), and conducting survey pretests, a questionnaire designed to elicit in- formation on seafood consumption, specifically consumption of oysters, shrimp, tuna, and catfish, was mailed to a sample of 9(J00 households in the United States, with 1000 mailed to each of the nine major census regions (shown in Fig. 3: Hanson et al. 2002). The stratified sample was chosen as the region is expected to be a significant determinant of both the choice to consume and the choice of how often to consume oysters. The surveys were mailed in late 2000 and early 2001, with households receiving a second copy of the survey if they did not return the first. This approach resulted in a return of 1 790 surveys or a response rate of 20. 1 % (after accounting for "return-to-sender" surveys). Because of the length and complexity of the survey, a large number of respon- dents did not answer all of the questions in the survey, therefore, a total of 874 observations are included in this study. Table 1 shows descriptive statistics for the responses used in this study. Compared with U.S. Census data (United States Census Bureau 2000), the results showed a larger percent of Caucasians responded to the survey (89% in the survey compared with 75% in the 2000 US Census). The survey results also contained a sample slightly older than the US population, with 69% of survey respon- dents over the age of 45. compared with 53% of the US adult (over 25) population. The tnean response for income in the survey was in the S50,000-$59,999 category, compared with a US mean of $42,148. Religious composition of the survey respondents corre- sponds to that presented in the World Almanac and Book of Facts (1999), i.e., 85% of the US population practices Christianity, in- cluding 23% Catholic, and approximately 2% and 1% of the US population practices Judaism and Islam, respectively. Our survey results indicated 83% Christianity with 25% Catholic, and 3%' practicing Judaism. In a series of six questions, respondents were asked to indicate how often they consumed oysters for breakfast, lunch, and dinner, both at home and away from home. This differs from most previ- ous studies (including Cheng & Capps 1988. Yen & Huang 1996) 52 House et al. o> o ^— CN CO ■V lO CD h- oo CJ) o ^— oo O) CD cn cn O) O) CT) cn o> cn o o a> o> O) O) cn OJ cn <3) cn cn cn o CM o CM Figure 1. I'liited States per capita fresh and liozen shellllsh consiimplicin (Source: IISDA, ERS. 1999). that analyze at-home consumption only. Overall. 56.9% of the respondents indicated that they never ate oysters. The means and ranges of the responses are shown in Table 2. As expected, con- sumption of oysters, as well as other seafood products, differed by region of the respondent's residence (Fig, 3), Additionally, respondents were asked to identify and rank the top three reasons they consumed and did not consume oysters. Results from the question on reasons nonconsumers do not con- sume oysters and why consumers do not consume more oysters provide an interesting insight into the data (Fig. 4). Visual inspec- tion of the results from this question may provide support for a double-hurdle regression model because it appears nonconsumers have different reasons for not consuming compared with consum- ers decision on frequency of consumption. A number of factors were hypothesized to be relevant to the consumption and frequency of consumption decisions. The same set of variables was used as regressors in both equations as theory provides no guidance for differences and to allow for a specifica- tion test. The dependent variable was constructed from responses to a set of six questions regarding frequency of consumption of oysters for breakfast, lunch, and dinner at-home and away-from- home. If a respondent indicated they never consumed oysters for each of the six questions, the value of the dependent variable was set to zero. For the sample, 56,9% of the responses were zero. For the remainder of the sample, the responses were summed to de- termine the frequency of consumption in one month. For example, if a respondent answered they consumed oysters once per month for dinner at home and once per month for dinner away from home, but never for lunches and breakfasts, their frequency of consumption for the month was two. Those who did eat oysters consumed oysters on an average of 2.2 times per month. Quantity of oyster consumption was not obtained in this survey because respondents were not asked how much was consumed (or by how many in the household) because of time and space limitations of the survey. Additionally, because the survey was asking for all consumption, including away from home and recreational catch, it was determined from the focus groups and test surveys that re- spondents were having difficulty answering in terms of quantity (i.e,, pounds or ounces — other quantities, such as number of oys- ters, were not considered because of the fact other species were considered and did not have comparable measures). Independent variables included demographic variables (age, gender, ethnicity, religion, household income), variables relating to the respondents geographic location and variables relating to slated preference. For geographic location, a dummy variable was included representing the census region the respondent belonged to, as well as one variable that represented how close the respon- dent currently lives to a coast. It was hypothesized that persons li\'ing closer to the coast would have a higher probability of con- suming shellfish. Other expected explanatory variables included perceptions of safety and top reasons for eating and not eating oysters as indicated by the respondent. Descriptive statistics for all variables are shown in Tables 1 (demographic) and 3 (other). Model Cheng and Capps (1988) and Yen and Huang (1996) recog- nized the restrictions of using a tobit model in demand analysis for finfish and shellfish. The tobit model assumes the factors that affect level of consumption are the same as those that determine the probability of consumption. Cheng and Capps (1988) used a Heckman two-step procedure and Yen and Huang (1996) used a generalized double hurdle model to analyze household demand for finfish. As a result of information obtained in focus groups and the preliminary visual appearance of the data, we have chosen to use Cragg's ( 1971 ) double-hurdle model, similar to the model u.sed by Yen and Huang (1996). The double-hurdle model has separate participation and con- sumption equations that are related in the following manner: = 0 if V,* > 0 and 0 otherwise (1) (2) U.S. 0\sTtR C0N.SUMPT10N - To Eat or Not to Eat 53 0.30 1 0.20 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 Figure 2. I'nited Slates per capita consumption of oysters (Source: L'SDOC7N0.4.4/NMFS, 'Fisheries of the L.S., 2001,' September 2002). where v,* represents the consumption decision and i/, is a latent variable describing participation as shown below: = .V,'P + £, (3) the same explanatory variables appear in all three equations, the following value will be distributed as a x" random variable with degrees of freedom equal to the number of explanatory variables under the null hypothesis that the Tobit specification is correct: a + Tii (4) where .v, and -, are vectors of explanatory variables and \i and a are vectors of parameters. Estimation o( the double-hurdle model is straightforward. Maximum likelihood estimation of a probit equa- tion is used to evaluate the censoring rule (r,'a). whereas maxi- mum likelihood estimates that account for a truncated normal dis- tribution are used for the subsample of uncensored obser\ alions. A specification test that evaluates the restrictions imposed by the tobit specification (assumption that the decisions are based on the same parameters) is obtained through a comparison of the log- likelihood function \ alues of the tobit. probit. and truncated nor- mal regression models (Greene 1993). Specifically, assuming that '^ — -VTdhit ./pr.ihil ./Truncalcd'- ^-'j where the /,s represent the respective log-likelihood function val- ues. RESULTS Using the double-hurdle model with frequency of oyster con- sumption as the dependent \ ariable. the model was estimated with the variables described in Table 4. The coefficients from the probit and truncated tobit equations, as well as the marginal effects (cal- culated at the means) are reported in Table 5. The probit model correctly predicted a consumer's likelihood to consume or not 1.00 0.80 0.60 0.40 0.20 0.00 T3 n I III I I I IIJUULIJ -a c ^1 (/I CS C 0 CO SI 0 2 3 0 2 4-» z ^ 4-1 C U^ c 3 ■t^ ^mi u 0 cd u 0 «a U UJ ^ UL) o 2 1) (J c 2 c 3 O 2 a. Figure 3. Percent consumption of oysters by region. 54 House et al. TABLE 1. Summary of demographics. Oyster Nonconsumers ( % ) Oyster Consumers ( % ) Overall Sample (%) Age of Respondent Greater than 6? Between 50 and 65 Between 35 and 50 Under 35 Gender Percent female Household Income Less than $29,999 Between $30,000 and $59,999 Between $60,000 and $99,999 100.000 or greater Region of Residence New England Mid-Atlantic Southeast Atlantic East North Central East South Central West North Central West South Central Mountain Pacific Lives within 50 miles of Coast Religion Catholic Christian Other Ethnicity Caucasian Noncaucasian Education High school or less Some College College degree(s) 17.3 34.0 39.4 9.3 52.7 16.3 37.2 29.2 17.3 13.1 10.7 9.3 14.7 8.2 13.9 7,0 13.5 9.7 29.4 26.4 56.1 17.5 90.5 9.5 17.1 32.2 50.7 19.6 39.3 33.7 7.4 67.4 11.4 34.0 28.1 26.5 9.8 8.8 14.6 8.0 12.2 9.3 13.8 13.0 10.6 30.0 23.6 59.4 17.0 87.3 12.7 14.9 30.0 18.3 37.0 36.3 8.5 59.0 14.2 35.8 28.7 21.3 11.7 9.8 11.6 n.8 10.0 11.9 10.0 13.3 10.1 29.6 25.2 57.6 17.3 89.1 10.9 16.1 31.2 52.6 consume oysters 87% of the time (incorrectly predicted consump- tion 49c of the time and no consumption 9% of the time). The results of the test shown in equation (5) indicate the double-hurdle model is a better specification than the traditional tobit (\ = 264.9, df = 431. The results indicated that different variables affected the decision to consume versus the decision of frequency of consumption, as expected. A set of variables was included to determine if the location of purchase of seafood affected either decision. Results indicated that if a person bought seafood (any seafood, not just oysters) at grocery stores (GRSOURCE) or spe- cialty stores (OTHERCS; such as fish markets or gourmet stores), they were more likely to be oyster consumers. However, these variables did not significantly influence frequency of consumption. The variables indicating if a person consumed seafood purchased from restaurants (RESTSC) or obtained through recreational catch (RECCATCH) were not significant in determining if a person would consume oysters, but significantly decreased the frequency of consumption. A potential explanation for these results is that if a person purchases seafood (again, any seafood) from grocery stores or specialty stores, they are a different type of seafood consumer than someone who purchases from a restaurant or eats recreational catch. Perhaps they are more "dedicated" seafood con- sumers than those who eat at restaurants, hence more likely to eat oysters, as well as consume different types of seafood than those who eat recreational catch (unlikely to be oysters). Following this line, a person who does eat oysters, but is a restaurant or recre- ational catch consumer is likely to consume oysters less frequently. Our results indicate the average oyster consumer consumes oysters 2.21 times per month. Respondents who purchased seafood from restaurants were likely to consume oysters 1.16 times per month and those who indicated recreational catch as a source of seafood were likely to consume 1.84 times per month. Respondents were asked to identify the top three reasons they consumed oysters. These reasons give insight to the type of person that both consumes oysters and what influences a person to con- sume more or less frequently. If the person indicated they enjoyed the flavor (FLAVOR) of oysters, as expected, they were both more likely to consume oysters (66.5% more likely) and consume oys- ters more frequently (0.46 more times per month). Tradition (TRAD) plays a part in determining how frequently a consumer eats oysters, but did not influence whether the person was a con- sumer. In other words, those who indicated they eat oysters out of tradition, or habit, were likely to eat oysters 0.62 times more often per month. Importance of availability was shown in the probit. but U.S. OvsThR Consumption - To Eat or Not to Eat 55 TABLE 2. Statistics on frfqutiK> of ojsttr consumpliun (H = 1(167). Mean Mode (Times Consiinii dAIonthl (% Frequency) Range Breakfast at home (1.0.^ Never (93.0%) Never to less than weekly Breakfast away from home (1.01 Never (97.1%) Never to less than 1 /month Lunch at home (1.14 Never (84.0%) Never to 1/week Lunch away from home 11.2(1 Never (74.8%) Never to 1/week Dinner at home (1.21 Never (73.8%) Never to 1/week Dinner away from home (1.34 Never (63.0%) Never to 1/week Respondents used a scale of 0 to 6 to indicate frequency where 0 = Never; 1 = Infrequently ( Prepar Know 3 u D H o o H S C o a. 0- CO X tj INon- Consumers D Consumers Figure 4. Reasons given for not consuming oysters or not consuming more oysters. 56 House et al. TABLE 3. Statistics on factors included in the double-hurdle model. Mean. Mean. Overall Nonconsumers Consumers Mean Frequency of oyster consumption (dependent variable) U/monlh (4M7 observations) 2.21/month (377 observations) U.y5/month Indicated oysters were the least safe of all shellfish and fintlsh products 34.6% 44.5% 39.0% Indicated the following was a source of seafood for con^ iumption: Grocery store 86.1% 89.4% 87.5% Restaurant 86.3% 90.7% 88.2% Recreational catch or fish farms 15.7% 27.1% 20.6% Fish market or gourmet store 17.5% 37.1% 26.0% Indicated the following was one of the top three reasons for consuming oysters Enjoy flavor 4.4% 65.6% 31.8% Variety in diet 2.2% 31.6% 15.3% Availability 1.5% 21.9% 10.6% Tradition/habit 2.2% 16.6% 8.6% Health/nutrition 1.0% 16.4% 7.9% Know how to prepare 0.5% 8.2% 3.9% Convenience 0.5% 7.2% 3.5% Price 1.0% 5.9% 3.2% Aphrodisiac properties 0.3% 4.8% 2.3% Other 0.3% 4.0% 2.0% Indicated the followmg was one of the lop three reasons for not consuming oysters Taste 49.7% 8.8% 31.5% Texture 43.8% 10.1% 29.1% Smell 26.7% 5.5% 17.2% product safety concerns 20.9% 25.3% 22.9% Price 12.7% 37.9% 23.9% Fresh not available 5.1% 20.4% 11.9% Lack of preparation know ledge 9.8% 12.0% 10.8% Custom 4.2% 4,4% 4.3% Health/nutrition 2.5% 6.3% 4.2% Too time consuming to prepare 3.0% 5.9% 4.3% Other 8.2% 3.2% 5.8% consumption for oyster consumers, from the average of 2.21 to 1.63. a 0.58 per month decrease. Demographics did have an effect on both the choice to con- sume and the frequency decision. Persons living in the Southeast Atlantic (SEATL) and West South Central (WSC) regions of the country were more likely (17.891- and 33.29f respectively) to con- sume oysters than persons living in New England. Other regions did not significantly differ from the New England region. Persons in the East South Central (ESC). West South Central (WSC). and Pacific (PACIFIC) regions were likely to consume irtore fre- quently (0.90. 1.08. and 0.80 times per month, respectively) than those in the New England region. In the United States. 67% of oyster landings come from the Gulf of Mexico and 23% from the Pacific region (USDOC 2002). Given the three regions that con- suined oysters significantly more frequently are closest to oyster production, these results make intuitive sense. All income categories above the base category of $30,000 or less consumed oysters significantly more frequently. However, income was not a factor in the decision to consume. Birlhdate (BD) was a factor in both decisions, with younger ages significantly less likely to consume oysters, or if they were oyster consumers, sig- nificantly likely to consume less frequently. Education levels, re- ligion, gender, and ethnicity did not significantly infiuence either the participation or consumption decisions in this study. However, the sample did not include a representative portion of the nonCau- casian population in the United States. Future studies might benefit from specifically targeting these populations for information on seafood consumption. DISCUSSION The two main goals of this study were to determine whether the factors that infiuenced the decision to consume oysters differed from the factors that influenced the decision of how often to con- sume oyster and to see what factors were significant that could be used to develop marketing strategies for the oyster industry. Re- sults showed that the two decisions were based on significantly different factors, as suspected. Though food safety is often credited as a reason why people do not consume oysters, this was not. in fact, the case. Concerns about food safety did influence how often oyster consumers ate oysters, but did not significantly influence whether a person was an oyster consumer. In fact, the belief that oysters are the least safe of all fish and seafood products did not influence this decision either. Somewhat surprisingly, nearly 45% of oyster consumers identified oysters as the least safe of all sea- food products, while only 35% of nonconsumers identified oysters. U.S. Oyster Consumption - To Eat or Not to Eat 57 \ariate Source cil purchase Reasims lor eating oysters Reasons tor not eating oysters, or not consuming oysters more frequentls TABI.F, 4. Description ol independent \ariables. \ ariable Name Safety perception Region of residence (U.S. Census GRSOURCE RESTSC RECCATCH OTHERSC FLAVOR HEALTH TRAD PRICE AVAIL CONV VDIET KNOWHOVV APHROD NOPRICE NOFPAVAI NOCUSTOM LPKLDGE TOOTIME TEXTURE SMELL TASTE TRAUMA PRODSAFE ALLERGY UNSAFE Description I if seafood is purchased at a grocery store 1 if seafood is purchased at a restaurant I if seafood is from recreational catch I if seafood is purchased at specialty fish markets or gourmet stores The following variables are 1 if this reason was listed as one of the top three reasons for consuming oysters: Enjoy flavor Health/nutrition Tradition Price Availability Convenience Variety in diet Know ledge of how to prepare Aphrodisiac properties The following variables are I if this reason was listed as one of the top three reasons for NOT consuming oysters, or not consuming MORE oysters: Price Lack of availability of fresh products Custom Lack of preparation knowledge Too time consuming to prepare Dislike texture Dislike smell Dislike taste Traumatic experience Product safety concerns Allergic reaction I if respondent believes oysters are the least safe of all seafood products Religion Race/Ethnicity Income Education Proximity to Coast Age Gender NEWENG New England (omitted category) MIDATL Mid-Atlantic SEATL Southeasit Atlantic ENC East North Central ESC East South Central WNC West Nonh Central WSC West South Central MOUNTAIN Mountain PACIFIC Pacific CHRISTIA Christian (omitted category) CATHOLIC Catholic OTHERREL Other religions CAUC 1 if Caucasian, 0 otherwise EMCI <$30.000 (omitted category) INC2 $30.000-$.59.999 INC3 $60.000-S99.999 INC4 SIOO.OOO or above EDUCATI High School degree or less EDUCAT2 Some College EDUC.AT3 At least one degree from College PROXCST I if currently lives within 50 miles of a coast BD Birth date GENDER 1 if female However, 25% of oyster consLimers indicated they ate oysters less frequently due to product safety concerns. Results indicated that people did not consume oysters, and did not consume oysters as frequently, if they indicated price was an inhibiting factor. Future studies are needed to address the issue of willingness to pay for safer oyster products. Consumers who in- dicated price was a reason they did not consume oysters more frequently were likely to consume oysters 0.39 times per month 58 House et al. TABLE 5. Empirical results from double-hurdle model. Variable Probit Truncated Name Coefficient F(z)/X Coefficient E(Y*)/X Source of seafood for consumption GRSOURCE 0.391**" (0.197)" 0.155 1.949(2.054) 0.263 RESTSC 0.005(0.196) 0.002 -7.783* (2.142) -1.050 RECCATCH 0.249(0.164) 0.099 -2.711*** (1.509) -0.366 OTHERSC 0.699* (0.155) 0.277 2.039(1.362) 0.275 Top three reasons for consuming oysters FLAVOR 1.682* (0.181) 0.665 3.434*** (2.016) 0.463 HEALTH 0.155(0.324) 0.061 -2.771 (2.968) -0.374 TRAD -0.223(0.241) -0.088 4.579* (1.746) 0.618 PRICE -0.201 (0.340) -0.080 3.124(2.134) 0.422 AVAIL 0.566** (0.261) 0.224 -0.821 (1.445) -0.111 CONV 0.411(0.467) 0.163 1.210(2.034) 0.163 VDIET 0.766* (0.223) 0.303 -1.576(1.361) -0.213 KNOWHOW 0.164(0.417) 0.065 1.819(2.147) 0.245 APHROD 0.569(0.517) 0.225 -2.500 (3.286) -0.337 Top three reasons for not consuming oysters, or not consuming more oysters NOPRICE 0.454* (0.155) 0.179 -2.852** (1.473) -0.385 NOFPAVAI 0.172(0.209) 0.068 0.402(1.749) 0.054 NOCUSTOM -0.217(0.296) -0.086 -3.530(3.464) -0.476 LPKLDGE 0.065(0.184) 0.026 -4.618** (2.170) -0.623 TOOTIME -0.314(0.307) -0.124 0.008 (2.556) 0.001 TEXTURE -0.030(0.175) -0.012 3.312(2.523) 0.447 SMELL -0.215(0.192) -0.085 -3.531 (3.511) -0.477 TASTE -0.412** (0.169) -0.163 -5.850** (3.054) -0.790 TRAUMA -0.727(0.519) -0.288 14.509(9.523) 1.958 PRODSAFE -0.145(0.152) -0.057 -4.311* (1.708) -0.582 ALLERGY -0.977** (0.589) -0.387 -4.596(7.728) -0.620 BeMe\'ed oysters to be least safe of all seafood products UNSAFE -0.048(0.1.%) -0.190 1.889(1.354) 0.255 Demographics MIDATL 0.152 (0.279) 0.060 3.535 (3.207) 0.477 SEATL 0.450** (0.270) 0.178 2.263(2.918) 0.305 ENC -0.118(0.290) -0.047 4.071 (3.470) 0.549 ESC 0.480 (0.299) 0.190 6.632** (3.211) 0.895 WNC 0.040 (0.297) 0.016 3.991 (3.438) 0.539 WSC 0.840* (0.308) 0.332 8.017* (3.151) 1.082 MOUNTAIN 0.246(0.290) 0.097 3.851 (3.367) 0.520 PACIFIC 0.139(0.274) 0.055 5.927** (3.044) 0.800 CATHOLIC 0.039(0.150) 0.015 -1.259(1.538) -0.170 OTHERREL -0.008(0.168) -0.003 1.183(1.688) 0.160 CAUC -0.266(0.190) -0.105 -0.944(1.796) -0.127 INC2 0.151 (0.193) 0.060 7.973* (2.601) 1.076 INC3 0.099(0.210) 0.039 6.859* (2.634) 0.926 INC4 0.224 (0.229) 0.089 6.105** (2.701) 0.824 EDUCAT2 0.081 (0.190) 0.032 2.545(2.070) 0.343 EDUCAT3 -0.077(0.191) -0.031 -0.831 (2.014) -0.112 PROXCST -0.220(0.185) -0.087 2.112(1.705) 0.285 BD -0.008* (0.0002) -0.003 -0.007* (0.003) -0.001 GENDER 0.106(0.130) 0.042 1.461 (1.453) 0.197 Log-likelihood function -281.04 -635.67 Percent of correct predictions in prohit model 87.1% ■" One. two, and three asterisks indicate significance at the 0.01, '' Standard errors of the coefficients are reported in parentheses. 0.05. and 0.10 levels, respectively. less frequently than the average oyster consumer. However, con- sumers who indicated concern for product safety was a reason for not consuming were likely to consume oysters 0.38 titties per month less frequently. The tradeoff between an increased price due to increases in costs of implementing safety programs and in- creases in consumption if consumers believe oysters to be safer is an area for future investigation. Overall, this study does identify characteristics that the oyster industry can use to segment consumers for marketing purposes. As expected, people living in regions nearest to oyster production are U.S. O'l'STBR Consumption - To E.m or Not to Eat 59 more likely to consume oysters and more likely to consume more oysters. Avuilubility ot fresh products also significantly increased the likelihood of the respondent to consume oysters. Consumers who purchase seafood products at grocery stores or specialty stores may be a segment that could be targeted, as they are more likely to consume oysters. ACKNOWLEDGMENTS This research was supported by the Florida Agricultural Ex- periment Station and the following grants and approved for pub- lication as Journal Series No. R-09388. This work is a result of research sponsored in part by the National Oceanic and Atmo- spheric Administration, U.S. Department of Commerce under Grant #GMO-99-24. the Mississippi-Alabama Sea Grant Consor- tium. Mississippi State University, and University of Florida. The U.S. Government and the Mississippi-Alabama Sea Grant Consor- tium are authorized to produce and distribute reprints notwith- standing any copyright notation that may appear hereon. The views expressed herein are those of the author(s) and do not necessarily reflect the views of NOAA or any of its subagencies. This material is based upon work supported by the Cooperative State Research. Education and Extension Service, U.S. Department of Agriculture, under Agreement No. 99-.388 1 4-8202. Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture. 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 0 0 0 0 0 0 0 Recruits 0 0 0 0 0 0 0 0 1992 # replicates 6 6 6 5 5 8 5 5 4 Mean Shell DW 4994 1676 59.9 1059 0.2 1315 409 2.5 14.6 Total Clams 13 1 0 3 0 3 1 0 1 Recruits 11 1 0 2 0 0 0 0 0 1993 # replicates 5 5 5 5 5 .s 5 5 5 Mean Shell DW 5892 5305 3904 1463 4256 1538 89 27 23 Total Clams 10 6 2 9 3 5 0 1 1 Recruits 8 6 2 7 2 3 0 0 0 1996 # replicates 5 5 5 5 5 5 5 5 5 Mean Shell DW 6279 3966 4264 312 2986 1893 10 56 154 Total Clams 5 4 7 2 11 2 1 0 3 Recruits 5 4 6 1 5 2 0 0 2 1998 # replicates Mean Shell DW Total Clams Recruits 13 4031 11 10 10 288 5 1 10 1004 4 4 2001 # replicates 10 10 10 10 10 10 10 10 10 Mean Shell DW 3285 2358 5095 639 1104 1039 24 ->1 280 Total Clams 15 12 20 5 1 4 2 1 4 Recruits 12 11 IS 3 0 .^ "> 0 1 H = High density shell. L = low density shell, and C = control. In 1991 replicates were 0.(143 m" all subsequent samples were 0.25 ni- ls in grams. Clams and recruits are the totals for all samples. . Shell drv wei.sht 64 Kraeuter et al. TABLE 2. Intercept, regression coefficient and correlation coefficient for the effects of shell density (g) on total clams and those that have recruited since 1990 (clams 0.25 m""). Year Intercept Regression IT 1992 Total Clams 0.005 NS 4.028 E-04*** 0.47 Recruited Clams -0.082 NS 3.338 E-04*** 0.48 1993 Total Clams 0.297 NS 2.152 E-04** 0.18 Recruited Clams 0.1 IONS 2.080 E-04*** 0.26 1996 Total Clams 0.363 NS 1.827 E-04** 0.19 Recruited Clams 0.118NS 1.876 E-04*** 0.31 1998 Total Clams 0.314 NS 1.476 E-04* 0.14 Recruited Clams 0.073 NS 1.624 E-04** 0.26 2001 Total Clams 0.129 NS 3.713 E-04*** 0.45 Recruited Clams -0.003 NS 3.703 E-04*** 0.53 NS = not sisnificant. *0.05. **0.01. *0.001. both cases the results with respect to treatments were similar, and we have presented the as-sampled data (Table 3). High and low shell density plots had similar numbers of total clams and seed; both had significantly more total clams and seed than the controls. We arrayed the data according to shell density and used linear regression. Both total numbers of clams and seed clams (Table 2) were significantly correlated with shell density. Latin Square analysis of the 1996 data on shell weight indicated that column 2 had significantly less shell than the other two. These differences negated further use of the Latin Square. We evaluated the total clams and recruited clams with ANOVA based on the three treatments (high density shell, low density shell, and con- trol): a linear regression for all satiiples (total clams and recruited clams vs. shell weight) was then computed. There were no sig- nificant differences in total clams with treatment (Table 3); how- ever, the regression line showed a significant positive effect of shell density (Table 2). In contrast, the ANOVA analyzing the effect of shell on clams that had recruited since 1990 was signifi- cant. A Tukey (HSD) test found that clam density in high-density shell and low density shell were not significantly different, and low TABLE 3. Tukey (HSD) results (number 0.25 m"" for total number (Total) of hard clams {Mercenaria mercenaria) and those that had recruited to the population (Recruit) since the beginning of the experiment ( 1990). 1993 High Low Control 1996 High Low Control Total Recruit 1948 Total 1.29 1.14 High 0.85 Recruit 0.69 1.13 0.73 Low 0.50 0.30 0.07 0.00 Control 0.40 0.10 Total Recruit 2001 Total Recruit 1 .07 1.00 High 1.53 1 .00 0.53 Low 0.33 1,40 0.20 0.27 0.67 Control 0.23 0.10 High = those areas covered with high density of shell, low = those areas covered with low density shell, control = those areas that did not receive shell. Underlines indicate those treatments that were not significantly dif- ferent a (a = 0.05). density shell and control areas had similar clam density (Table 3). High density shelling increased clam recruitment over that ob- served in the control areas. In 1998, only 3 plots were sampled, and ANOVA results were similar to 1996. There was no difference in total clams between treatments, but the clams that had recruited since 1990 were more abundant in high shell plots. There were no significant differences between low shell and control (Table 3). Again, linear regression indicated a positive effect of shell density on total and recruited clams (Table 2). In 2001. as with previous sampling, Latin Square analysis of the shell distribution revealed significant differences between all columns and some rows. The total numbers of clams and clam recruitment were evaluated relative to shell weight and treatment type with general ANOVA and linear regression techniques. After I \+ years, most plots remained intact, but the increasing differ- ences between rows and columns suggest that the shell is gradually being dispersed. In contrast to 1996, when total clams were not significantly different by treatment, both the total and recruiting clams since 1990 exhibited significant differences by treatment. In both the total clams and recruited clams, the Tukey (HSD) test found that high shell density plots had significantly more clams than either the low-density shell or the control. The latter two treatments were not significantly different from each other. The similarity between total and recruiting clams after I l-l- years may have been greater than indicated by the base data. We were unable to distinguish ages of clams >10 y. Thus, some of the clams in this class may have recruited to the area since the shell was placed on the bottom. In 2001. 20.7% of the sampled clams were in the age 10 or older category. As a comparison in 1996, 31.4% of the clams were from classes that had recruited before the shell was placed on the bottom). Recruitment We considered clams <15 mm in shell length to be seed clams. Relatively few of these clatns were found (Table 4), and never in the control areas. In some years, seed can be as large as 20 mm. We found only one clam of this size in a control plot (Table 4). We have attempted to evaluate annual recruitment (long-term survival) of clams at this site by back calculating from the age data to determine when particular clams had set (Fig. I). We have averaged the data from the 1996, 1998, and 2001 samples, but, because so few animals were obtained by sampling, have not at- tempted to place error bars around these estimates. With the ex- ception of 1993, there is a relatively good correspondence between the back calculated data and that from animals recovered. The TABLE 4. Mean number of seed clams m'" bv treatment. .Seed <15.1 mm Seed <20.1 mm Year High Low Control 1992 1993 1996 1998 2001 2.00 2.67 (1 0 0 0.50 0.53 0.53 0 0 High Low Control 2.25 0.75 2.67 0.80 0.27 0.53 0.27 0 0 0 0 Seed = <15.1 nimor<20.1 mm Shell Length. Numher of 0.25 nr samples is given in Table 1. Rehabilitation of Mercenaria mercenaria 65 1.40 -High Shell -Low Shell -Control Figure 1. RiTiuitnieiit of hard clams {Mercenaria mercenaria) into hi)>h-densit> shell. l10 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 - 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- blages: patterns of abundance. J. Mar. Res. 34:25^1. 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 0 to 4 cm/sec (Grizzle et al. 1994). Growth response in the soft-shell clams was similar to that ob- served in hard clams with a proportional increase in shell length for 4 y old, 40 mm clams with flow and no evidence of growth inhibition between free-streatn velocities of 0.1 to 5.8 cin/sec (Em- erson 1990), For epifauna species, it has been speculated that growth is maximized at water flows that match inhalant pumping speed. Mussels grown in multiple flume trials at flow velocities of 0. I. 2. 4. and 8 cm/sec had a statistically nonsignificant increase in shell growth at a flow of 2 cm/sec. which matched the approximate inhalant pumping speed (Grizzle et al, 1994). Eastern oysters in- creased growth at a flow of 1 cm/sec relative to tlows of 0 and >1 cm (Grizzle et al. 1992). The constant flow in flume studies, however, is different from tidal currents, which vary in magnitude and direction. Flume experiments with ascending and descending flows have found clearing or grazing rates of scallops differed by 307f (Pilditch & Grant 1999). Currents may affect shellfish growth, but estimating current velocities can be difficult. A device coinmonly used to determine flow rates is a current meter. However, collecting time series ve- locity profiles with current meters over large areas is time con- suming with conventional instrumentation, particularly in inter- tidal waters. When current rates and flow patterns are needed for large regions being considered as potential shellfish grow-out sites, the use of current meters becomes impractical. Two- and three-dimensional numerical computer models can be used to describe the direction and magnitude of currents for individual cells in grids covering coastal areas. The output data from numerical models can then be used to create thematic maps for Geographical Information System (GIS) coverages (Congleton 75 76 CONGLETON ET AL. et al. 1999). Numerical models are supported by data for bottom elevations for each cell in the grid and tidal amplitude at the ocean boundaries of the models. They simulate time series estimates of velocity vectors for grid cells covering the model domain. Veloc- ities may be estimated for discrete layers in individual grid cells or may be vertically averaged, as in this study. Model output can be analyzed in the GIS to identify sites with optimum conditions for shellfish growth. The major drawback, however, is the difficulty of initializing and running a numerical model. An alternative method for estimating currents is by measuring a process, which is affected by the current magnitude. A physical analog measurement of current velocity is the dissolution of cal- cium sulfate (Plaster of Paris or gypsum) blocks or hemispheres, called clod cards, placed in moving water (Muus 1968. Doty 1971, Peterson & Skilleter 1994). Thompson and Glenn (1994) devel- oped an equation for calculating mean water speed from field deployed clod cards using clod cards from the same batch for laboratory calibration in quiescent water of the same salinity tem- perature as in the field. They concluded that proper execution of field and calibration tests result in a simple and practical method for measuring water motion over a wide range of temperatures, salinities, and current speeds. Clod cards are inexpensive and simple to construct, but the difficulty of deploying large numbers limits their usefulness for estimating cunent magnitudes o\ er large areas. The objective of this study is to evaluate the relationship be- tween ( 1 ) field measurements of tidal currents made with clod cards; (2) average current estimates generated by a numerical flow model; and (3) growth of soft-shell clams on a mudfiat in Eastern Maine. The appropriateness of incorporating current estimates from a numerical model into a GIS for the selection of sites for grow-out of juvenile shellfish will then be considered. METHODS The study was conducted in Mason Bay in Eastern Maine on the western side of Englishman Bay, which bounds the Gulf of Maine. The bay (Fig. I A) is 2.39 km long by 1.03 km wide, oriented in an east-west direction, and is located 9.7 km north of Jonesport, Maine (44°61.80'N, 67°56.23"W). At low tide (mean low water = -1.875 m nisi), mudflats are exposed along the entire length of the bay with two channel inlets from Englishman Bay joining on the west side of Spar Island and running the length of the bay (Fig. IB). Water temperatures vary from 5°C in April to I6'=C in September (Beal et al. 2001). Soft-shell clams were collected at 15 sites at the same water line spaced 40 m apart to the south of Spar Island and west of Flake Point Bar (Fig. IB) at an elevation of -2.0 m msl in Spring. 1 996. These sample sites were close to one of the inlets of the bay with a maximum separation of 485 m from the most easterly site to the most westerly site. A sixteenth site between the tip of Flake Point Bar and Spar Island was sampled during spring of 2000 to increase the range of water velocities sampled. One of the low flow sites in the center of the earlier sampling array was also sampled the second year. Sites were relocated in the second year using their global positioning system (GPS) coordinates. Location of the 16 sites was determined by caiTier-phase GPS measurements made with a Trimble GeoExplorer™ GPS receiver, and post-processed. Carrier-phase GPS is commonly used for sur- veying with sub-decimeter accuracy for measurements in the ho- rizon plane. Measurements in the vertical plane are less accurate. The range in the elevation measurements for the 10-min carrier- phase GPS readings at the 16 sites was -1.5 to -2.7 m msl with 95% confidence range of ±0.55 m for individual measurements (Congleton et al. 1999). Because inaccuracy in GPS measurements alone could have resulted in a difference in elevation between sites, locations were selected with simultaneous flooding and dry- ing times. Sample site coordinates were then imported into the Maplnfo'^' GIS creating a layer of sampling site locations (Fig. IB ). Sediment cores from four of the sites were analyzed for composition by the Analytical Laboratory of the Maine Soil Testing Service using the hydrometer method for particle size and 1050'"C combustion ana- lyzer for total carbon. Fifty clams were dug with a clam rake at the 1 5 sites South of Spar Island at the end of the first growing season. A single low flow site (sixth site counting from the most easterly) and the high flow site SE of Spar Island were sampled in the second growing season. External annual rings were used to determine the increase in shell length during the preceding summer. Brousseau ( 1979) found winter rings to be a reliable method of determining age in soft- shell clams from Gloucester. Massachusetts. However. Mac- Donald and Thomas ( 1980) found external growth rings to be less reliable for age determination than thin shell sections, and Lewis and CeiTato (1997) found shell increment might be temporarily decoupled from soft-tissue growth by high temperature or starva- tion. However, external growth rings have been used for long-term estimation of growth (Kube et al. 1996) and growth and age (Jacques et al. 1984. Evans & Tallmark 1977) of /;; situ Mya arenaria. Because of limitations of using growth rings for measuring age. length between the last shell check marks were used to measure the size at the beginning of last growing season. Initial size was then used as a covariate in the statistical analysis instead of age. Annual growth increment was then calculated by subtracting the final shell length from the initial size. The problem of lengthy shell abrasion limiting the usefulness of external rings in aging was minimized by taking measurements of growth only in the last growing season. NUMERICAL MODEL OF TIDAL CURRENTS Estimated currents for Mason Bay were obtained from the Ma- son Bay Model (MBM). which is an adaptation of Princeton Ocean Model (Mellor 1992. Blumberg & Mellor 1987) modified to de- scribe intertidal areas (Congleton et al. 1999). Input bathymetry data for the model were processed in the Maplnfo GIS including sublidal depths from NOAA nautical chart no. 13325, the shoreline boundary traced frotn an aerial photograph and 27 high accuracy canier phase GPS measurements made at the waterline near low water on a single Spring tide. To increase the accuracy of the description of the bottom in the study area, fourteen of the GPS measureinents were in the region, which is enlarged in Figure lb. These data were used to generate a 100 by 76 grid covering the bay composed of square cells with 36.125 m sides. The 7600 cells in the grid gave increased resolution of depths between points with known elevations without unnecessarily increasing computing time for a run describing a tidal cycle. Grid cells (36 m sides) were smaller than the distance between the clam sampling locations (40 m) resulting in a different estimate of current velocity at each sample site. The model generated estimates of vertically averaged cuirent velocities for each grid cell flooded by the tide at one-second Tidal Currents and Clam Growth 77 O Sample site + High flow. - Low flow i^^°del H High flowj _ L Low flow* Water displacement per minute -2 3 Depth m msl Shoreline mean high water Figure 1. (A) Location of Mason Bay in Eastern Main near Jonesport. Maine with Englisliman Ba> and the Atlantic Ocean to the east connected by channels north and south of Dunn Island. Lines and labels show locations and extent of 7.5 niin L'S(;S quadrangles. (Bl Aerial photos of Mason Bay. Right image is the rectangular area in SE image of the entire Bay. The array of sample locations (-2 m msl) are spaced 40 m apart except for the site nearest Spar Island. Vectors show water displacement/minute at maximum flood tide. 78 CONGLETON ET AL. intervals for an average 2 m amplitude tide (Congleton et al. 1999). A vertical average of the current velocity for each time step was used because tidal amplitude and shallow water depths would in- hibit stratification. Bottom friction was proportional to the square of the veilically averaged bulk flow. Vectors showing the current magnitude and direction estimated by the model for each grid cell were imported into the GIS. Layers of cuirent vectors at different times in a tide cycle described flow throughout the bay. For the statistical analysis of clam growth, the time series of velocities were averaged over the flood phase. The layer showing the sample site locations was placed over a layer of average cuirent velocities to estimate velocities at each site. Because the sample locations were not centered on the grid used by the numerical model, the mean velocity of adjacent grid cells with the same approximate elevation were averaged. FIELD MEASUREMENT OF CURRENTS— CLOD CARDS Plaster of Paris hemispheres (clod cards) were used for mea- suring relative water motion at each of the fifteen sampling sites. In previous studies, rectangular clod cards were used (Doty 1971, Thompson & Glenn 1994). Clod cards used in this study (Fig. 2) were molded in hemispheric plastic capsules (32.36 cm'), creating a uniform surface area exposed to the current regardless of card orientation. Commercial Plaster of Paris or gypsum was mixed two parts powder to one part water. The slurry was poured into the capsules and leveled off with a straightedge and left at room temperature for a week to insure thorough drying. After attachment to a 9 x 6.5 cm sheet of plastic with silicone epoxy. initial dry weights for each of the clod cards were measured and recorded. For field deployment, the backing sheet of each clod card was attached to a brick with rubber bands. One clod card was placed at each of the 16 clam sample sites (Fig. IB), and a total submersion time was estimated for the period that included air exposure at low tide. Because all clod cards were deployed on a spring tide in April (-0.7 m mllw), they were recovered after 4 days while the loca- tions were still accessible at low tide. After recovery, cards were lightly rinsed to remove mud and were left to dry at room tem- perature for one week and weighed. The percentage loss and the change in weight were calculated. The calibration of clod cards in quiescent water or under free convection conditions is necessary for the overall calculation of integrated field water speed. Four clod cards from the same lot as those used in the field trial were suspended 5 cm below the surface of a 22-1 cylindrical container containing seawater (30-32 ppt salinity). The container was placed in a larger recirculating tank maintained at 7''C. which corresponded to the average water tem- perature during the field trial. Every 24 h, the water inside the container was replaced with fresh salt water and the dissolved Plaster of Paris on the bottom of the container discarded. After the calibration period of four days, each card was dried at room temperature for a week and then weighed. The average of the initial weights and average of the final weights for the four calibration cards were used in the water ve- locity calculations. The scalar arithmetic mean velocity of the water in the field (V) was estimated for the 16 sites following the methods of Thompson and Glenn (1994): V = 4.31 (W,„„„„/A,„,„,,)"-^(S,„,'-^/S,,„„„„,,„) (1) where W,,,,,, ,, is the initial clod card weight of the field deployed card: Ai,,,,,.,, is the initial exposed surface area: Si,^,,,, and S^^,|,„„on are calculated as |i-(W,-„,„/w„„„.„)"-'i/e (2) where W,,,,^^, and W„„„_,| are the final and initial weights of the field and calibration tests, and H is time submerged in the field and calibration tests. Theta for the field trial 0 was total time between deployment and recovery even though the clod cards in the field experienced air exposure during low tides. During periods of air exposure, field clod cards remained wet and continued to dissolve. On average, the cards were subjected to aerial exposure for approximately 1 h during each low tide. STATISTICAL ANALYSIS Tidal velocities for each site were categorized as high or low using estimates from the clod cards and numerical model. Because the mean (±SD) velocity estimated using the clod cards (4.96 ± 0.88 cm/s) was higher than the mean estimated by the numerical model (3.85 ± 1 .34 cm/s), high flow sites were identified as having flow s greater than 5 cm/s using clod cards and 4 cm/s the numeri- cal model. Mean flow at the seven high flow sites identified using clod cards a\eraged 5.87 ± 0.63 cm/s and at the five high flow sites identified using the numerical model averaged 5.85 ± 0.47 cm/s. Mean flow at the nine low flow sites identified by clod cards averaged 4,35 ± 0.37 cm/s and at the 1 1 low flow sites identified by the numerical model averaged 3.02 ± 0.33 cm/s. High and low flow means categorized using either clod cards or the numerical model were statistically different {P < 0.001) using pooled vari- ance f-tests. Variability in shell growth increment during the preceding growth season was analyzed by analysis of variance (ANOVA) Plastic backing sheet Figure 2. (Jypsum clod cards constructed from a plastic mold cemented to a 9 x 6.5 cm backing stieet of plastic. Tidal Currents and Clam Growth 79 using the GLM procedure in SYSTAT v. 10. Shell length at the beginning of the growing season (distance between the last most anterior and posterior margins of the last growth check), years of sampling (1996 and 2000), and water velocity category of either high or low as indicated by either clod card weight loss or the numerical model were the independent factors. The initial model included main effects and all interactions. y, = B,, + BiX, + B.X, + B^X, + B4.V1.Y, + fijA-iX, + B,,^,^, + fiyXiA'.A', + e Where y, is the annual growth increment; B„ is the intercept; B,X, are the coefficient and categorical variable for year; fi-,X, are co- efficient and value for initial size and 5,^", are the coefficient and categorical current estiinate (H,L) either from clod cards or the model: B,X,X„ S^X^X, and fi^XiX^X, are coefficients for two and three way interactions; and e is the random error term. Terms that were statistically insignificant iP > 0.05) were de- leted from the model using the backward elimination procedure (Draper & Smith 1466). To ensure independence of residual errors in predicting growth increment of spatially proximate observations, the Durbin- Watson Test Statistic was used to test for the existence of autocorrelated errors. Because 50 clams were collected at each location, residual eiTor terms remaining after fitting the GLM model might not be independent if there is a site effect independent of local cun-ents. First-order autocorrelation (lag = 1 ) results in the error term con- sisting of a fraction of the previous error term plus a new random disturbance tenn (Neter et al. 1996). Error terms are uncorrelated only at the time the autocorrelation term (p) is statistically equal to zero. RESULTS Composition of the sediments at the 16 sites ranged from 47- 55% sand. 29-tl<7f silt. 12-16% clay and 1.15 to 1.27% carbon. Shell lengths at the start of the two years ranged from 6.9 mm to 55.7 mm. with average (±SD) of 23.1 ± 10.8 mm. After excluding the juveniles or individuals without a growth check mark, sample size was 724 with an average (±SD) growth increment of 9.4 ± 4,0 mm with clams sampled once. Trends in estimated water velocities from the numerical model and clod cards were similar. Velocities estimated with clod cards were highest at the site nearest Spar Island and at the sites near Flake Piiint Bar. Velocities decreased at the sites near the center and increased at the western end of the cove (Fig. IB). Estimates from the numerical model displayed a similar trend generally de- creasing moving westward from Flake Bar. but without the in- crease at the most western locations. The correlation coefficient between the 16 estimates of current velocity from the numerical model and Eq. I was 0.74 (P < 0.05). Velocity averages over the flood tide at the sixteen sites ranged from 2.2 cm/sec to 7.14 cm/sec as estimated by the numerical model and ranged from 3.8 cm/sec to 7.52 cm/sec as estimated by Eq. I. The estimation of current velocities by a numerical com- puter model and Eq. 1 were similar although the estimates from the numerical model were lower. The velocities estimated by the clod cards were during a spring tide, which would be expected to be higher than the velocities predicted by the numerical model during an average tide. Clod card measurements, however, were near the bottom where velocities are decreased by bottom shear. The maximum water speed on the flood tide was also estimated for the sixteen sites. Maximum velocities predicted by the numeri- cal model ranged from 4.0 cm/sec for some of the western and central sites to 21.4 cm/sec at the site closest to Flake Point Bar. All linear models used growth increment as the dependent vari- able, year ( 1996 vs. 2000) and flow (high vs. low as categorized by either clod cards or the model) as categorical variables and in- cluded initial size as a continuous variable. The Durban-Watson Statistic indicated that the GLM models had statistically signifi- cant first order autocorrelations. An inspection of autocorrelation plots of correlation versus lag indicated significant but diminishing positive autocorrelations up to lag 10 (Fig. 3). Autoconelation significance (P < 0.05) was deterniined from the 95% confidence interval for the sampling distribution of the autocorrelation of lag k or i-f.. which is normal with (x^^ = 0 and a^^- = l/n"" with a sample size of n (Lin et al. 1995). A difference transformation replaced values for the dependent variable (growth increment) with the difference between it and the preceding value. Differencing is a popular and effective method of removing trend from spatial (location effect) and time series (tem- poral effect) data. Autoconelation plots following the transforma- tion had no trend because as lag increased there was a random distribution of positive and negative autocorrelations (Fig. 3). To ensure validity of significance tests using the transformed data, a linear regression with a hierarchical layout with clams (or trial) nested or stacked within site was used. The trial or clam within site effects was insignificant for hierarchical models tested (P = 0.87). Consequently, independence of error terms could be assumed and significance tests based on the diffei-ence-transformed data would be valid. The ANOVA tables for the difference transformed growth in- crement as the dependent variable and high-low current category estimated by clod cards or the numerical model are in Tables 1 and 2. Both models explained 57-589^ of the variability in growth increment. Estimates from both models indicated clams grew slower the first year of sampling ( 1996) and that larger clams grew less with -0.26 mm and -0,28 mm decrease in the growth incre- ment for each mm increase in initial size depending on whether 1.0 0.5- 0 0 Jljrthm..Tnrrnii 0.0 -0.5 +0.5 0,0 -0,5 - -1.0 f 10 20 30 Lag 40 50 60 Figure 3. Autocorrelations between residual linear model errors with lags from 1 to 50 for predicting shell increment (top) and difference transformed measurements of shell increment ( bottom ). Lines above and below zero baselines are 95% amlldence Intervals for autocorre- lation = 0.0. so CONGLETON ET AL. TABLE 1. ANOVA of growth increment with a difference transformation resulting from fitting a complete model reduced until only statistically significant effects remain. Current categories were average current <5 cm/s or average current 55 cm/s as estimated from clod cards using Eq. 1. R- of 58%. Source Sum-of-Squares df Mean-Square F-Ratio Year Initial size Clod card current Current * size Current * year Error 39:.378 513S.024 143.122 33.430 77.765 435.^,1 OQ 392.378 513S.024 143.122 33.430 77.765 6,032 65.049 851.793 23.727 5..542 12.892 0.000 0.000 0.000 0.019 0.000 cuiTents were described with the numerical model or clod cards (Fig. 4). Larger average currents also stimulated growth although the effect on growth increment was less than that of year or initial size (Table .3). The adjusted least squares mean (±SE) for the growth increment at the sites identified by clod cards as low flow was 9.6 ± 0.25 and at the high flow sites was 1 1.9 ± 0.32 (Table .3). The least squares means (±SE) for growth increment at sites identified by the numerical model as low flow was 9.51 ± 0.274 cm/s and at the high flow sites was 1 1.70 ± 0.33 cm/s. There was a significant interaction between year and current (Tables 1 and 2l. Increased growth for high flow was expected during the second year because the highest flow site was only sampled in the second year. There were also significant two-way (clod card analysis) and three-way (numerical model analysis) in- teractions involving the effect of initial size indicating an incon- sistent stimulatory effect of current on growth for animals of dif- ferent size. However, interaction terms involving initial size made the smallest contribution to the model Sum of Squares or R". DISCUSSION A previous study (Congleton et al. 1999) also reported general agreement between water velocities estimated by the numerical model and measured by a portable current meter. The conelation between flows estimated by the numerical model and Eq. 1 in this study were lower than reported in Congleton et al. 1999. The 16 sites in this study, however, were a subset of the 25 sites in the previous study and had a smaller range of current velocities. Numerous factors affect the accuracy of using clod dissolution in measuring currents. Mean current velocities estimated with the clod cards were higher than the velocities estimated using the model (Table 3). As previously noted, cards were deployed during a Spring tide when cunents were stronger than an average tide that is simulated by the model. High estimates of currents using clod cards compared with other techniques ha\e been previously re- ported with dissolution rates in field experiments 16-18% high (Porter et al. 2000) compared with measured flows. Although flow estimates using cards in this study were higher than estimates using the model, there should also be some negative bias in the clod card estimated flows because 9 in Eq. 1 included the time when the cards were air exposed at low tide while the H used for calibration was total emersion time. Clod card accuracy could be increased by calibration in known steady flows rather than using a diffusion index factor as in this study (Porter et al. 2000). Flows were anticipated to be greatest at the most easterly and most westerly sample locations because the flood tide entered the cove on either side of Spar Island. This anticipated pattern was seen in the flow rates estimated by the clod cards, but not the numerical model. The failure of the numerical model to predict increased currents west of Spar Island may be caused by the av- eraging of flow rates of the surrounding grid cells, because sample sites were not centered on the grid. Also, velocity estimates were an average for a cell with an area of 1305 m". A model with greater spatial resolution would show flow patterns in greater detail. With a significant correlation between the current velocities estimated by the clod cards and numerical model, the similarity in the statistical analysis for the two sets of current measurements was not unforeseen. As expected, initial size had a significant effect on the grow th increment of M. arenaria. resulting in slower growth in larger individuals (Fig. 4). In an earlier study (Beal et al. 2001 ) placed clams at the same intertidal locations in Mason Bay and measured increment in shell length between time of removal from the hatchery and seeding on the flats in April and removal from the flats at monthly intervals until December. Mean shell length increased from 14.1 mm to 21.9 mm resulting in a 7.S mm increase between June and August to December. Growth increment for the entire srowins season was TABLE 2. ANOV.\ of growth increment with a difference transformation resulting from fitting a complete model reduced until only statistically significant effects remain. Current categories were average current <4 cm/s or average current >5 cm/s as estimated from the numerical model. R' »{S19c. Source Sum-of-Squares df Mean-Square F-Ratio Year Initial size Model current Current * year Current * year * size Error 304.81(1 3524.739 155.673 142.192 62.972 4458.564 I 1 1 1 1 722 ,W4.810 3524.739 155.673 142.192 62.97 6. 1 75 49.360 570.781 25.209 23.026 10.197 0.000 0.000 0.000 0.000 0,001 Tidal Currents and Clam Growth 81 Annual Shell Increment E E. *•> c 0) E S. u c 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. LITERATURE CITED Bayne. B. L.. R. J. Thompson & J. Widdows. 1976. Physiology I. In: B. L. Bayiie, editor. Marine mussels: their ecology and physiology. Cam- bridge: Cambridge University Press, pp. 121-206. Beal. B. P.. M. R. Parker & K. W. Vencile. 2001. Seasonal effects of intraspecific density and predator exclusion along a shore-level gradi- ent on survival and growtli of the solt-shell clam, Mya arenaria L., in Maine. USA. J. Exp. Mar. Biol. Ecol. 264:133-169. Blumberg, A. F. & G. L. Mellor. 1987. Three-Dimensional Coastal Ocean Models. In: N. Heaps, editor. A description of a three-dimensional coastal ocean circulation model. Americal Geophysical Union. 208 pp. Brousseau, D. J. 1979. Analysis of growth rate in Myii arenaria using the Von Bertalanffy equation. Mar. Biol. .sl(no. 31:221-227. Brousseau, D. J. 1981 . Use of internal growth bands tor age determination in a population of Geuken.sia demi.'i.ui (ribbed mussel). Biol. Bull. 161: 323-324. Cahalan, A., S. E. Siddall & M. W. Lukcnback. 1989. Effects of flow velocity, food concentration and particle flux on growth rates of juve- nile bay scallops .Argopecren irradiuns. J. Exp. Mar. Biol. Ecol. 129: 45-60. Congleton, W. R., Jr.. B. Peaice. M. Parker & B. F. Beal. 1999. Mariculture Siting: a GIS description of intertidal aras. Ecol. Model. 1 16:63-75. Congleton. W. R.. Jr., B. R. Pearce & B. F. Beal. 1997. A C++ Imple- mentation of mdividual/landscape model. Ecol. Model. 103:1-17. Draper. N. R. & H. Smith. 1966. Applied Regression Analysis. New York: John Wiley. DMR. 1997. Fisheries landings statistics for Maine. Maine Department of Marine Resources. Augusta: State House Station #21. 1 p. Doty. M. S. 1971. Measurement of water movement in reference to benthic algal growth. Botanica. Marina. 14:32-35. Emerson, C. W. 1990. Influence of sediment disturbance and water flow on the growth of the soft-shell clam. Mya arenaria L. Can. J. Fish. Aqual. ScL 47:1655-1663. Evans. S. & B. Tallmark. 1977. Growth and biomass of bivalve mollusks on a shallow, sandy bottom in Gullmar Fjord (Sweden). Zoon-.ii-iH. Fernandez. E. M., J. Lin & J. Scarpa. 1999. Culture of .Mercenaria Mere- cenaria (Linneaeus): Effects of Density, predator exclusion device and bag inversion. J. Shellfish Res. 18:77-83. Frechette, M., C. A. Butman & W. R. Geyer 1989. The importance of boundary-layer flows in supplying phytoplankton lo the benthic sus- pension feeder. Mylihis edulis L. Limnol. & Oceanogr. 341:19-36. Grant, J.. P. Cranford & C. Emerson. 1997. Sediment resuspension rates, organic matter quality and food utilization by sea scallops {Placopecten magellanicus) on Georges Bank. / Mari. Res. 55:965-994. TiuAL Currents and Clam Growth 83 Grizzle. R. E.. R. Langan & W. H. Howell. 1992. Growth of suspension- fceding bivalve molluscs to changes in water tlow : differences between siphonate and nonsiphonate taxa. J. Exp. Mar. Biol. Ecol. 162:213-228. Grizzle. R. E.. R. Langan & W. H. Howell. 1994. Growth responses of Cni.s.so.srmi virgiiiica. Mercenaria merceiniria. tniil Mylilus eilulis to changes in water flow: A test for the "inhalant pumping speed hypoth- esis." 7. Shellfish Res. 13:315. Grizzle. R. E. & P. J. Morin. 1989. Effect of tidal currents, .seston. and bottom sediments on growth of Mercenaria nurcciiaria: Results of a field experiniem. Mar. Biol. 102:85-93. Jacques, A.. J-CF. Brethes & G. Desrosiers. 19X4. Growth o( Mya arcnaria in relation with sedimentological characteristics and immersion period on the Rimouski tidal flat. Sci. Tech. Eaii. 17:95-100. Jorgensen. C. B. 1990. Bivahe Filter Feeding: Hydrodynamics. Bioener- getics. Physiology and Ecology. Denmark: Olsen & Olsen. 140 pp. Lewis. D. E. & R. M. Cerrato. 1997. Growth uncoupling and the relation- ship between shell growth and metabolism in the soft shell-clam Mya arenaria. Mar. Ecol. Prog. Ser. 158:177-189. Lin. F.. X. H. Yu. S. Gregor & R. Irons. 1995. Time Series Forecasting with Neural Network. Complexity International 2: www.csu.edu.au/ci/ vol02/ci2.hlml. Kowalik. Z. & T. S. Murty. 1993. Numrerical Modeling of Ocean Dynam- ics. World Scientific. Singapore. Kirby-Smith. W. W. 1972. Growth of the bay scallop: the influence of experimental water currents. / E.\p. Mar. Biol. Ecol. 8:7-18. MacDonald. B. A. & M. L. H. Thomas. 1980. Age determination of the soft-shell clam Mya arenaria using internal growth lines. Mar. Biol. 58:105-109. Kube. J.. C. Peters & M. Powilleit. 1996. Spatial variation in growth of Macoma balthica and Mya arenaria (Mollusca. Bivalvia) in relation to environmental gradients in the Pomeranian Bay (southern Baltic Sea). Arch. Fish. Mar. Res./Arch. Fisch. Meereforsch. 44:1-2; 81-93. Malouf. R. E. 1989. Clam culture as a resource management tool. In: J. J. Manzi & M. Castagna. editors. Clam Mariculture in North America. Amsterdam: Elsevier, pp. 427-447. Mellor, G. L. 1992. User's Guide for a three-dimensional, primitive equa- tion, nuinerical ocean model. Progress in Atmosphere and Ocean. Sci- ence, Princeton University. 35 pp. Muus. B. J. 1968. Field measuring "exposure" by means of plaster balls — a preliminary account. Sarsia 34:61-68. Neter. J.. M. H. Kulner. C. J. Nachtsheim & W. Wassennan. 1996. Applied Linear Statistical Models. 4th ed. Chicago: Irwin Inc. 1408 pp. Newell. C. R. 1990. The effects of mussel {Mxtiliis cdiilis. Linneaeus. 1758) position in seeded bottom patches on growth at subtidal lease sites in Maine. / Shellfish Res. 9:1 13-1 18. Newell. C.R. 1996. Currents, food critical to shellfish farm siting. Fish Farm. News 2:6-7. Peterson. C. H. & G. .A. Skilleter. 1994. Control of foraging behavior of individuals within an ecosystem context: the clam Macoma halihica. flow environmenl. and siphon-cropping fishes. Oecologia 100:256- 267. Peterson. C. H.. H. C. Siimmcrson & J. Huber. 1995. Replenishment of hard clam stocks using hatchery seed: combined importance of bottom type, seed size, planting season and density. J. Shellfish Res. 14:293- 300. Pilditch, C. A. & J. Grant. 1999. Effect of variation in flow velocity and phytoplankton concentration on sea scallop (Placopeclen magellani- cus) grazing rates. J Exp. Mar. Biol. & Ecol. 240:1 1 1-136. Porter, E. T., L. P. Sanford & S. E. Suttles. 2000. Gypsum dissolution is not a universal integrator of "water motion." Limnol. & Oceanogr. 45: 145- 158. Ramming. H. & Z. Kowalik. 1980. Numerical Modeling of Marine Hy- drodynamics Applications to Dynamic Physical Processes. Amster- dam: Elsevier. Sucsy. P. v., B. R. Pearce & V. G. Panchang. Comparison of two- and three-dimensional simulation of the effect of a tidal barrier on the Gulf of Maine tides. / Phys. Ocean. 23:1231-1248. Thompson. T. L. & E. P. Glenn. 1994. Plaster .standards to measure water motion. Limnol. & Oceanogr. 39:1768-1779. Turner. E. J. 1991. A new method for directly measuring bivalve pumping. J. Shellfish Res. 10:275. Vincent. B.. G. Desrosiers & Y. Gratton. 1988. Orientation of the infaunal bivalve Mya arenaria L. in relation to local current direction on a tidal flat. J. Exp. Mar. Biol. Ecol. 124:205-214. Walne. P. R. 1972. The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves. J. Mar. Biol. Assoc. 52:345-374. Wildish. D. J. & M. P. Miyares. 1990. Filtration rate of blue mussels as a function of flow velocity: preliminary experiments. J. Exp. Mar. Biol. Ecol. 142:213-219. Wildish. D. J. & A. M. Saulnier. 1992. The effect of velocity and flow- direction on the growth of juvenile and adult giant scallops. J. of Exp. J. E.\p. Mar. Biol. Ecol. 133:133-143. Wildish. D. J., Kristmanson. D. D.. Hoar. R. L., DeCoste. A. M., McCor- mick, S. D. & A. W. White. 1987. Giant scallop feeding and growth responses to flow. J. Exp. Mar. Biol. & Ecol. 113:207-220. 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 0 was immature (no differentiation in gonadal tissue: loose vesicular connective tissue in gonad). The other stages were for mature geoduck (connective tissue well developed, primary cells evident on follicle walls or eggs or sperm development evi- dent) and classified as: ( 1 ) early active: (2) late active: (3) ripe: (4) partially spent: and (5) spent. Average von Bertalanfy growth curves were fitted to all data points of size at age using the equation; L, = L fl ') where t is age in years. L, is shell length (mm) at age t, L,, is theoretical maximum size, k is a constant, determining rate of increase or decrease in length increments, t„ is the hypothetical age at which the organism would have been at zero length. The pa- rameters L^ . k, and t^, were estimated using a non-linear Gauss- Newton least squares method (SYSTAT 2000). The proportion of mature geoduck (P) at shell length or age (X) was estimated using the equation; Px = X/(X -t- e'*-''^') where A and B are parameters estimated using a non-linear Gauss- Newton least squares method (SYSTAT 2000). Data for both sexes were combined for each of the growth and maturity curve analyses since sex could not be distinguished in the immature sizes. 85 86 I- O LU 200 150- 100- LU ^ 50H Campbell and Ming 200 n o o O O On OO OCPO (5> n oO Q?' O O o 20 40 60 AGE (YEARS) 80 100 )J!i^ >^ T T 20 40 60 80 AGE (YEARS) 100 Figure I. Growth curves for P. abnipla collected from (Al Gabriola Island, and (Bl Yellow Bank. Curves calculated from the von Bertalanfy growth parameters (Table ll. RESULTS Growth The oldest P. ahruphi collected was 77 y (146 mm SL| from Gahriola Island, and 1 17 y ( 154 mm SL) from Yellow Bank. The smallest and largest geoduck, respectively, was 10 mm SL (age unknown, probably 1 y) and 163 mm SL (42 y) from Gabriola Island, and 43 mm SL (2 y) and 180 mm SL (58 y) from Yellow Bank. Growth was fastest in the first 10 y followed by slow growth thereafter for geoduck from both areas (Fig. I). There was con- siderable variability of size within each age group. Growth rates of P. ahrupui from Gabriola Island were slower than those from Yellow Bank (Fig. 1. Table I). Gonadal Condition Immature gonads comprised 10.85% and 12.10% of the total geoduck gonads sampled from Gabriola Island {n = 129) and Yellow Bank (n = 124, includes three individuals without SL measurements), respectively (Fig. 2). The largest immature geo- duck was 80 mm SL (5 y) and 72 mm SL (4 y) from Gabriola Island and Yellow Bank, respectively. There were Insufficient data to determine spawning periods because seasonal monthly samples were not collected. However, most mature gonads were in the ripe or partially spent condition for geoduck collected from both areas (Fig. 2). There were no gonads that were spent (gonadal TABLE I. Von Bertalanfy growth parameters for P. abnipla from Ciabriola Island and Yellow Bank during May 1991. Values in brackets are approximate 95% confidence intervals. Area Lx Gabriola Island Yellow Bank 129.6 (±4,1) 147.7 (±5. Si 0.146 (±0.020) 0.189 (±0.055) -1.02 (±0.951 -1.42 (±1.17) 120 108 condition 5). This suggested that geoduck spawning had begun at both areas during mid to late May 1991. Sex Ratio For geoduck <90 mm SL. in both areas combined. 41.1 8% were immature, and 54.41% were males (Table 2). The sex ratio for mature geoduck <90 mm SL was predoniinantls (92.5%) male 70 n 0 12 3 4 GONADAL CONDITION Figure 2. Frequency of gonadal condition stages found in gonads of all /'. ahrupta collected from Gabriola (black bars) and Yellow Bank (hatched bars). Gonads classified as 0 = immature, and mature stages that are I = early active: 2 = late active: 3 = ripe: and 4 = partially .spent. Geoduck Maturity 87 TABLE 2. Pericnl of total gonads differentiated into mature males and females and immature /'. ahnipla from (iahriola Island and \ eiloM Bank during .Ma> IVMI. One 91 mm SI, hermaphrodite was found. N = total nuniher. Includes onlv individuals with SL measurements. Percent of Total Area Male Female Immature Hermaphrodite N 90 mm SL Gabriola Island 57.61 42..^9 92 Yellow Bank 45.56 53..^-^ 1.11 90 Total 5 1 .65 47.80 0.55 182 uith few (7. 3%) females for both areas combined. In contrast. geoduck s90 mm SL had generally a more equal se,\ ratio, al- though males were slightly more abundant than females in the Gabriola Island sample, whereas there were slightly more females than males in the Yellow Bank sample (Table 2), Figure 3. Ph()liiriiicni;;ra|)lis iil /' ahnipla gonadal tissue cross- sections of (.\l Male (x4(Mt magnilkation) showing spermatozoa-filled follicle surrounded by connective tissue, (B) Female (x4()0) showing oocyte-filled follicle surrounded bv connective tissue. Hermaphroditism .Although most of the histological material of mature P. ahnipta gonads allowed differentiation between females (follicles with oo- cytes) and males (follicles with .spermatozoa) (Fig. 3) there was one individual that was a hermaphrodite, with a gonad showing both male and female characteristics (Fig. 4). This gonad had some follicles containing only either female or male gametocytes per follicle, and other follicles, which contained spermatozoa and oo- cytes in the same follicle. The geoduck was 91 mm SL (age was not determined). Malurity Mean size at 50% maturity was similar for geoduck from Gabriola Island, 58.3 mm SL (55.2-59.4 mm SL, lower and upper 95% confidence intervals, CI), and Yellow Bank, 60.5 mm SL (51.1-64.0 mm SL. 95% CI) (Fig. 5, Table 3). Mean age at 50% maturity was about 1 y slower for geoduck from Gabriola Island, 3.09 y (2.68-3.25 y, 95% CI), than at Yellow Bank, 2.04 y ( 1 .72- 2.16 y. 95% CI) for Yellow Bank geoduck (Fig. 6. Table 3). The smallest mature male was 45 mm SL (2 y) and 60 mm SL (2 y), the smallest mature female was 59 mm SL (4 y) and 88 mm SL (2 y), and the largest immature geoduck was 80 mm SL (5 y) and 72 mm SL (4 y), respectively, in the samples from Gabriola Island and Yellow Bank. B *■ ■ . *• s- Figure 4. Photomicrographs of hermaphrodite I', ahnipta gonadal tis- sue cross-sections of (.\) (x250 magnification), and (B) (xl60) showing single follicles containing oocytes and spermatozoa. 88 Campbell and Ming 1.0 UJ Q: 0.8H Z) 0.6- 01 0.4 O CL o a: 0.2H Q_ 0.0- I I I I T 0 50 100 150 SHELL LENGTH (MM) 200 1.0 LU Qi 0.8- I- < ^ 0.6- z o fe 0.4- O Q. o 01 0.2- CL 0.0- O OC OO QCO 0 50 100 150 SHELL LENGTH (MM) 200 Figure 5. Size at maturity curves for P. abntpta collected from (A) Gabriola Island, and (B) Yellow Bank. Symbols indicate number of individuals per shell length: "O" = I; "X" = 2; "+" = 3. See text for equation for the predictive curve and Table 3 for parameter values. DISCUSSION Our findings indicated that growtli rales were faster for geo- duck from Yellow Bank than those from Gabriola. Results were similar to those of Burger et al. (1998) and Bureau et al. (2002) who found that geoduck from Georgia Strait were generally smaller than those from the west coast of Vancouver Island. Rea- sons for the differences in P. abntpta growth rates between areas could be attributed to a variety of environmental and biological factors associated with different habitats (e.g.. substrate type, tem- perature, exposure to water surge activity, pollution, food avail- ability, and geoduck density or genetic characteristics) (Breen & Shields 1983. Harbo et al. 1983, Goodwin & Shaul 1984, Goodwin & Pease 1991, Noakes & Campbell 1992. Hoffman et al. 2()0(). Bureau et al. 2(X)2). Our examination of gonadal condition suggested thai the spawning period for geoduck from both study areas was just be- ginning in mid to late May 1991. Results agree with other gonadal studies of geoduck, which found the main spawning period was TABLE 3. Parameter estimates for equation indicating relationships betv\een proportion that are mature with shell length (SI. in mm) or age (years! of P. abriipta from (iabriola Island and Yellow Bank during May 1991. See text for equation formula. \ alues in brackets are approximate 95% confidence intervals. Parameter Estimates Variable .\rea X Gabriola Island SL Yellow Bank SL Gabriola Island Age Yellow Bank Age 8.512 (±2.741) 0.076 (±0.044) 79 7.224 (±2.. ^14) 0.052 (±0.0.^3) 80 2.956 ( ± 1 .55 1 ) 0.59 1 ( ±0.435 ) 1 5 2..397 (±1.540) 0.828 (±0.644) 14 during June and July (.Andersen 1971. Goodwin 1976. Sloan & Robinson 1984). The male:female sex ratio of mature P. abntpta found in this study (52:48) was similar to that reported by Goodwin (1976) (.53:47) and Sloan and Robinson ( 1984) (37:43). The high percent- age of males in the small sizes (young ages) in this study was 1.0- LU cn O.BH 3 0.6- q: 0.4- O Q. o q: 0.2 Q. 0.0- 6 6 5 12 2 1 3 XM^^:^!^'^ ig^ O » O , ' " "^/Al 4 5 6 3 11 -1 1 1 \ 1 1 1 r 0 5 10 AGE (YEARS) 15 Figure 6. Age at maturity curves for P. abriipta collected from Gabriola Island ("O" solid curve), and bellow Bank ("X" and dashed curve). Number by each symbol indicates numlier of individuals per age group. See text for equation for the predictive curve and Table 3 for parameter values. Geoduck Maturity 89 similar to Andersen's ( 1971 ) findings of94.4'7i- males among geo- duck with <100 mm SL. Our findings indicated the first recording of a P. cibnipui her- maphrodite. Most bivahe species are dioecious (sexes are sepa- rate) although hermaphroditism does occur in some species of this group (Coe 1943, Coan et al. 2000). Factors causing hermaphro- ditism in P. ubnipia are unknown. Whether the "simultaneou.s" hermaphroditism (Coe 1943. Eversole 1989) in this geoduck was fully functional in producing viable eggs and sperm is unknown. However, sexuality of different sizes (or ages) In F. nhntpta has not been studied extensively. We estimated thai only -1.200 indi- vidual gonads have been histologically examined to date from mature P. dhniprn sampled in Washington State and British Co- lumbia (Andersen 1971. Goodwin 1976. Sloan & Robinson 1984. this study). Andersen (1971) and Goodwin (1976) suggested that P. ahnipia might be gonochoristic where sex is determined by development with males maturing at a smaller size (earlier age) than females. Although we suspect that hermaphroditism is rare in P. ahriipta. the probability that some level of protandry. sex re- versal, or "simultaneous"" hermaphroditism in P. nhnipla (espe- cially for sizes / Shellfish Ri'search. Vol. 22, No. I. 91-94. 2(K).\ THE EFFECTIVENESS OF N-HALAMINE DISINFECTANT COMPOUNDS ON PERKINSUS MARINUS, A PARASITE OF THE EASTERN OYSTER CRASSOSTREA VIRGINICA M. A. DELANEY,'* Y. J. BRADY,- S. D. WORLEY,' AND K. L. HUELS- ^ Aquatic Animal Health Research Laboratory. USDA-ARS. P.O. Bo.\ 952. Auburn. Alabama 36831: 'Department of Fisheries and Allied Aquaeultures. Auburn University. Auburn. Alabanui 36H49: Department of Chemistry. Auburn University, Auburn, Alabama 36849 ABSTRACT The pathogenic protozoan Perkinsus marinus (Mackin. Owen and ColHer) is the cause of extensive mortalities in Eastern oyster. Cwssosirea virgiiiicci. populations along the Gulf and East Coasts of the United .States. A series of experiments was undertaken to determine the effect of N-hakutiine disinfectants on this protozoan parasite. The organic N-halamine disinfectants. 1.3-dichloro-2.2.5.5-tetramethyl-4-imidazolidinone (DC) and l-chloro-2,2.5,5-tetramethyl-4-imidazolidinone (MC). apparently dam- age the permeability of the parasites outer membrane and alter the osmoregulatory functions of the cell. Damaged parasites were unable to reproduce at concentrations as low as 14.9 mg/L DC at 8 h exposure, or for the chemical MC at 24.9 mg/L at 12 h exposure. The chemical compounds appear to lyse the larger meronts first, followed by lysis of the daughter spores. These studies strongly suggest that the chemical compounds DC and MC can be u.sed to disinfect seawater allowing the production of specific pathogen-free stock in oy.ster hatcheries, and having the potential to prevent the spread of these parasites froin contaminated oysters to uninfected oysters. KEY WORDS: ovster. Pcikinsii.\ marimis. disease, disinfection. N-halamine INTRODUCTION The Eastern oyster. Crassostrea vir^inica (Gmelin 1791 ) natu- rally occurs in North America frotn the Gulf of St. Lawrence in Canada to the Gulf of Mexico. It is common in estuaries in coastal areas of reduced salinity, and is an important commercial species. Once considered the most abundant source of oysters in the world, eutrophication, overharvesting and the parasites Haplosporidiiim nelsoiii and P. nuu'iiuis have caused the Chesapeake Bay oyster population to be reduced to a critically low level (Andrews 1988. Haskin & Andrews 1988, Hargis & Haven 1988). The parasites inhibit growth, reduce fecundity, and lower the oyster's condition and glycogen content (Menzel & Hopkins 1953, Newell 198.3, Barber et al. 1988. Crosby & Roberts 1990). Oyster populations that have incurred high infection prevalence and intensities typi- cally have low mortalities during their first year, but suffer higher mortalities in the following years (Paynter & Buneson 1991 ). The parasite does not have the same drastic effects on the oyster popu- lation in the Gulf of Mexico as it does in the Chesapeake Bay. An oyster requires three or tnore years to reach marketable size in the cooler waters of the Atlantic; however, only two years are required in the warmer waters of the Gulf of Mexico. In the Gulf of Mexico, this parasite infects over 80% of Eastern oysters with annual mor- talities typically 50% of the adult oyster population. Transmission of the parasite occurs through the water by release of infective stages from the feces of living oysters, the tissues of dead oysters (Ray 1932. Mackin & Hopkins 1962). and by the gastropod ecto- parasitic snail, Boonea impressa (White et al. 1987). Perkin.sus marinus has several life stages in the host oyster (Mackin & Boswell 1956. Perkins 1969). These include immature thalli. mature unicellular thalli (trophozoites), and presporangia. When released into seawater. presporangia develop a resistant cell wall, and then enlarge to become hypnospores. Under aerobic conditions, hypnospores differentiate into sporangia and produce *Corresponding author: E-mail: mdelaney@vetmed.auburn.edu This study was funded by Mississippi-Alabama Sea Grant Consortiimi. motile zoospores (aplanospores in Mackin & Boswell 1956) within the hypnospores cell wall. One sporangium of P. marinus is ca- pable of releasing approximately 354.700 zoospores (Chu & Greene 1989). Zoospores are released from hypnospores and un- dergo free-living stages in seawater. Eradication of these pathogens in the wild is not possible be- cause of the widespread nature of the diseases and the lack of knowledge regarding other species that might carry the disease (Elston 1990). Resistance to H. nelstmi. but not to P. marimis (Barber & Mann 1991 ) has been achieved through selective breed- ing of C. virgiiiicci (Ford & Haskin 1987. Foid et al. 1990. Bur- reson 1991). Developing and maintaining hatcheries to produce larval oys- ters for grow out for co)nmercial production or to repopulate de- pleted areas is one approach to alleviate the lack of natural repro- duction. This method, however, requires the incoming seawater to be specific pathogen free. The traditional methods of using ozone and ultrafiltration are expensive for continuous production. Chlo- rine is an inexpensive alternative for water disinfection; however. Its chemistry changes when combined with seawater. Observations of oyster larvae exposed to chlorine-treated sea- water indicate a lethal concentration for 50% of the test organisms (LC 50) for C. virgiiiica larvae of 0.005 mg/L free chlorine (CI+), regardless of whether static or intermittent addition of chlorine was used (Roberts et al. 1975, Bellanca & Bailey 1977. Roberts & Gleeson 1978). Concentrations as low as 0.05 mg/L of bromate, broiTtoform and chloroform caused some C. virgiiiica 48 h larval mortality (Stewart et al. 1979). Galtsoff (1946) noted a 46% de- crease in pumping action at a dose of 0.2 mg/L chlorine. He and other workers concluded, however, that chlorine was an effective means for disinfecting shells of contaminated oysters and that the oxidant would not interfere with depuration if chlorine levels were kept at a minimum. Later studies agreed with this finding but cautioned that oysters reduce pumping when chlorine concentra- tions exceed 0.01 mg/L. At chlorine concentrations above 1.0 ing/L. pumping cannot be maintained; thus, the use of chlorine as an effective means of depuration is limited by the tolerance of the species. The ability of adult shellfish to respond to low concen- trations of total residual oxidant and to cease pumping may be 91 Delaney et al. beneficial because it allows the animal to survive chlorine- produced oxidant (CPO) concentrations as high as 10 mg/L for 30 days (Galtsoff 1964). The corresponding decrease or cessation, however, of shell growth and feeding is disadvantageous. The most severe restrictions to chlorine use arise from the formation of chemical compounds from adding this to seawater. Halogenated organic compounds are formed that display complex chemistry. The products of chlorination of seawater are complex and not fully understood (Carpenter & Macalady 1973. Davis & Middaugh 1977, Wong & Davidson 1977, Carpenter et al. 1980). In seawater and brackish water, chlorine replaces some of the bromine in hy- pobromous acid releasing the bromine cation that is considered the disinfecting compound. Full strength seawater has a bromide ion concentration of 65 mg/L, and chlorine reacts with it to produce hypobromous acid and hypobromite ion. Bromamines and chloramines may be formed in the presence of ammonium ion. For normal seawater of pH 8, the initial products of chlorination are a mixture of hypobromous acid and hypobromite ion that are un- stable with respect to decomposition and disproportionation (Macalady et al. 1977). The N-halamine compounds used in this study were 1,3- dicliloro-2,2,3.5-tetramethyl-4-imidazolidinone ( DC; dichloro) and 1 -chloro-2,2,5,5-tetramethyl-4-imidazolidinone (MC; monochloro). Both compounds were synthesized at Auburn Uni- versity in the laboratory of S. D. Worley. Department of Chemis- try. The compound MC can be produced in the laboratory and as a result of the hydrolysis of the compound DC. The compounds will be marketed by Vanson/HaloSource Corporation, Seattle WA'. These compounds are more stable in water and dry storage than free chkirine and other commercial products, such as the hydantoins and isocyanurates (Tsao et al. 1991). The N-halamine compounds do not produce trihalomethanes or react with bromide in seawater and should be more stable and more effective than free chlorine. The compound DC is the faster acting compound and the amide N-Cl moiety is more labile than the amine N-Cl group, providing a small amount of free chlorine. The hydrolysis decom- position product MC. having only the more stable amine N-Cl moiety, acts more slowly as a disinfectant. In this series of experiments, the parasites were exposed to the chemical compounds in sterile artificial seawater (SASW) to de- termine the effectivity of the compounds. A related compound, 3-chloro-4,4-dimethyl-2-oxa/olidinone, has been shown to kill Giaidia lanihlia more effectively than free chlorine (Kong el al. 1988), and it was speculated that DC or MC would penetrate oyster tissues and the thick parasite walls at a reduced level of chlorine. A previous study using Anadara trapezia (blood cockles) and Haliotis laevii-ata (greenlip abalone) showed that free prezoospo- rangia of Peikinsus sp. (remo\ed from oyster tissues) died within 30 min in chlorine solutions of 40 mg/L (Goggin et al. 1990); however, within tissues the parasites presumably are more pro- tected and survived at least 2 h. Their study was concerned pri- marily with disinfecting meats of abalone. The objective of this study is to determine if the parasite P. iiniriiuis could be eliminated in the water column. The possibility of controlling P. inariiuis in an oyster hatchery by treating incoming water, or as an interim control preventing the spread of the parasite between oysters, could mean economic gains associated with increased health and growth characteristics. Use of trade or manufacture's name does tuh imply endorsement. METHODS A series of three experiments were conducted to evaluate the effectiveness of these compounds on P. marinus. Perkinsiis mannus cultures were obtained from the American Type Culture Collection (ATCC), and cultured according to La Peyre and Faisal (1995). In experiment one, an aliquot was re- moved from culture, vortexed briefly to break up cell clumps, and then centrifuged at 5(.)0i; for 5 min. These cells were rinsed twice with 15 ppt sterile artificial seawater (SASW), then resuspended in SASW at a concentration of approximately 5 x lO'' cells niL"'. The chemicals DC and MC, which were synthesized according to the method of Tsao et al. (1991), were prepared in three concen- trations: 0.3. 14.9 and 29.8 mg/L and 0.5, 24.9 and 49.8 mg/L, respectively. These concentrations are based on molar equivalents of chlorine. Four replications of each chemical at each concentra- tion were prepared in sterile. 50 niL. polypropylene centrifuge tubes. Approximately 5000 parasites were added to tubes contain- ing 50 mL of each chemical concentration. The same amount of SASW with and without parasites served as the positive and nega- tive controls. Contact time consisted of eight time intervals: 0.5. 1, 2, 4, 8, 12. 18. 24. and 48 h. At the appropriate time, the samples were mixed and I niL removed from each tube. Sodium thiosulfate (0.02 N) was added to neutralize the chlorine (i.e., to quench disinfecting action! and the cells were observed microscopically at xlOO with and without staining with Lugols Iodine. A second experiment was initiated to determine the percent mortality at \ arious concentrations and time intervals using a vital dye, trypan blue, which distinguishes between living and dead cells. This viability test evaluates the breakdown of membrane integrity determined by the uptake of the dye to which the cell is normally impermeable. Cell and chemical preparation was the same as previously described. Contact time consisted of three time intervals: 1. 2. and 8 h. At the appropriate time, the samples were mixed and 1 niL removed from each tube. The cells were washed with Hanks Balanced Salts Solution (HBSS) (Sigma, St. Louis, MO) and resuspended in 0.3 niL HBSS to which 0.5 niL trypan blue was added. The cell suspension was mixed and allowed to stand at room temperature for 5-15 min. Living and dead cells were counted and enumerated using a hemacytometer at xlOO. Dead cells stained a dark blue, but living cells were able to exclude the dye. Cells with an intermediate blue color stain were consid- ered dead. A third experiment was performed to detemiine the viability of the cells after exposure to the two chemicals, targeting the cells that lightly stained indicating damage to the membrane. It was important to know whether these damaged cells would be able to recover and initiate a new infection. Cells were removed from culture, centrifuged to pellet the para- sites then resuspended in SASW. Four concentrations of DC (7.4. 14.9. 29.8. 44.6 mg/L) and 4 concentrations of MC (12.9. 24.9. 49.8. 76.6 mg/L) v\ere prepared in sterile, polypropylene centri- fuge tubes, and then 2 niL were transferred to individual wells of tissue culture plates. Three replications of each chemical con- centration were prepared. Approximately 20 |j.L of the P. marinus (4.5 X lO'* parasites mL~') cell suspension were added to each disinfectant chemical. The same amount of SASW with and with- out parasites was added to the positive and negative controls. Contact time consisted of four time intervals: 1. 2. 8. and 12 h. At the appropriate time, the chlorine in the samples was neutralized with 20 |jiL of 0.02 N sodium thiosulfate and the cells resuspended Effectiveness of N-Halamine Compounds 93 TABLE 1. Experiment 2: the etTect of DC and MC concentration and exposure time on mortality of P. mariniis. TABLE 2. Experiment 3: the effect of DC and MC concentration and exposure time on mortality and replication of P. mariniis. 'Jc Staining '?c Staining mg/L I hour 2 hours 8 hours DC 0..^ 0.3 1.3 DC 14.9 11.4 77.4 DC 29.8 80.0 80.8 MC0.5 0 0.2 MC 24.9 .VI 16.3 MC 49.S 19.2 22.9 in 3 mL of culture media. A portion of the cell suspension was removed and evaluated with typan blue staining as previously described. The remainder of the samples were incubated in the dark at 25°C and evaluated at 24 and 48 h. RESULTS In the first experiment, no visible effects on P. marimis were observed for DC or MC treatments at any tested concentration up to 4 h. At 8 h exposure to either DC or MC. all parasite cells appeared to decrease in size, and at 18 h all cells were completely lysed at all concentrations. The negative controls appeared free of debris and bacterial contamination during the test. The positive controls appeared unchanged and did not exhibit any decrease in size, nor did they lyse. The second experiment attempted to refine the earlier one by determining viability at various contact times. The viability of the cells exposed to DC has been reduced by 80% at I h at a concen- tration of 29.8 mg/L (Table 1). At a concentration of 49.8 mg/L MC at 1 h, a reduction of only 19.2% was observed. At the end of 8 h. 99.8% mortality was observed at 29.8 mg/L DC. as compared with 25% with 49.8% MC. In the study addressing the viability and the ability of the para- site to recover from exposure to the DC and MC compounds showed a trend towards more rapid deactivation of the parasites by DC as compared with MC. at similar concentrations (Table 2). Cells in the positive control treatment exhibited normal growth and development. DISCUSSION Results of this study demonstrated that the compounds MC and DC eliminated the pathogen P. mariniis in 15 ppt seawater under laboratory conditions. It is important to kill all parasites because a single sporangium of P. mariniis is capable of releasing approxi- mately 354.70(J zoospores (Chu & Greene 1989). Mortalities of 100% of P. mariniis can be achieved using the faster acting chemical DC at concentrations of 14.9 mg/ for 8 or 12 h. 29.8 mg/L for 8-12 h or 44.6 mg/L for a minimum of I hour. mg/L I hour 2 hours 8 hours 12 hours 12.9 DC 7.4 4.1 11.0 6.9 13,4 88.2 DC 14.9 12.7 34.8 83.0" 33.0" 99.8 DC 29.8 10.4 29.8 36.0" 98.6" 0.2 DC 44.6 98.1" 99.6-' 100-' 100" 16.0 MC 12.4 0 3.4 6.2 7.1 25.0 MC 24.9 MC 49.8 10.9 17.2 14.3 14.6 20.7 82.0" 70.0" 87.2" 1 was MC 76.6 0 0 98.6" 90.3" " Indicates cultures in which all parasites died without producing viable offspring when observed 48 hours after chemical treatment. The slower acting chemical MC can achie\e 100% mortality at concentrations of 24.9 mg/L for 12 h, 49.8 mg/L for 8 or 12 h. or 76.6 mg/L for 8-12 h. Additional testing would be desirable to determine lower concentration effectivity against this pathogen. Both DC and MC are effective against the oyster parasite P. mariniis in vitro at concentrations less than the estimated LDg,, of the oyster larvae exposed to these same chemicals (Delaney et al. 2002). Histologic and physiologic information would be required on the long term effects of chemical exposure to oyster larvae; however, either compound has the potential to be used in oyster hatcheries to prevent infections of P. marimis from occurring, or to prevent the spread of the disease through the water column if the contact time is sufficient. Electron microscopy would provide ad- ditional insight on the mechanism of damage to the parasite's cell walls at different stages in the life cycle of the parasite. N-halamines DC and MC at concentrations of total chlorine within the lar\ al and adult oysters range of tolerance, are effective for the control of a protozoan pathogen. P. marimis. of Eastern oysters. These compounds have the potential to be used in oyster hatcheries and in recirculating based systems to produce specific pathogen free oysters. The use of these compounds as a substitute for free chlorine or chloramines would mitigate deleterious physi- ologic effects currently observed on oyster recruitment and sur- vival in estuaries receiving chlorinated discharges. ACKNOWLEDGMENTS The authors thank Dr. David D. Rouse, Dr. Sharon R. Roberts, and Dr. George W. Folkerts for their technical assistance and Dr. Thomas McCaskey, for his attention to details which improved this manuscript. Additional thanks to Dr. Jeffrey Williams of the Vanson/HaloSource Company for providing the chemicals used in this study and technical assistance, and Dr. John Supan for pro- viding larval oysters. LITERATURE CITED Andrews. J. D. 1988. Epizootiology of the disease caused by the oyster pathogen Perkinsus marimis and its effect on the oyster industry. Am. Fisli. Soc. Special Pulylication 18:47-63. Barber. B. J., S. E. Ford & H. H. Haskin. 1988. Effects of the parasite MSX {Haplosporidium nelsoni) on oyster (Crassostrea virginica) energy me- tabolism. 1. Condition index and relative fecunditv. J. Slwllfisli Res. 7:25-31. Barber. B. & R. Mann. 1991. Triploid Crassostrea virginica (Gmelin, 1 791 ) grow faster than diploids but are equally susceptible to Perl10 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 LITERATURE CITED AL'Osta. C. P. & M. J. Alvarez. 1990. Cullivo de /.amburina. I. Experiencias en cultivo larvario. Adas III Congreso Nac. Acuicult. 445^M9. Acosta, C. P., G. Roman & M. J. Alvarez. 1990. Cultivo de zamburina (Chlamys varia). IL Preengorde en batea. Adiis III C(yiii;re.\o Ncic. Acuicul. 533-538. An.sell, A.. J. Mason & J. C. Dao. 1991. Three European scallops: Pcctcn maxiiiiKs. Aequipeclen nperculuiis and Chlamys varia. In: S. Shum- way. editor. Scallops: biology, ecology and aquaculture. New York: Elsevier, pp. 715-751. Brand, A. 1991. Scallop ecology: distributions and behaMoiir, In: S, Shuni- way. editor. ScaUop.s: biology, ecology and aquaculture. New Y'ork: Elsevier, pp. 517-579. Bourne, N. F. 2000. The potential for scallop culture- the ne\t niillcnniuni. Aquacidlure Int. 8:113-122. Burnell. G. M. 1983. Growth and reproduction ot the scallop Chlamys varia (L.) on the west of Ireland. Ph.D. Thesis. National University of Ireland, Galway. 295 pp. Gonzalez, G. & G. Roman. 1983. Larval culture of the scallop {Peclen ma.ximtis). 4"' Pectiniil Workshop. Aberdeen, Scotland (mimeo). Gruffydd, LL. D. & A. R. Beaumont. 1970. Determination of the optimum concentration of eggs and spermatozoa for the production of normal larvae in Pecten maximus (Mollusc, Lamellibranchia). Hflf^oUinder wiss, Meeresunters. 20:486—497. Le Pennec, M. & B. Diss-Mengus. 1985. Rearing of Chlamys varia in commercial hatchery. 5''' Interiuilional Puciiiuil Wurk.shop. A Coruna, Spain (mimeo). Le Pennec, M. & B. Diss-Mengus. 1987. Aquaculture de Chlamys varia (Ly. donnees sur la biologic de la larve et de la posllarve. Vie Marine 8:37-42. Lubet. P. 1956. Recherches sur le cycle sexuel et laemission des gametes chez les mytilides et les pectinides (Mollusques Bivalves). Rev. Trav. Inst. Peches Marit. 23:387-548. Lucas, A. 1965. Recherche sur la sexualite des mollusques bivalves. Thesis d'Etat. Universite de Bretagne Occidentale, Brest. 136 pp. Mendes de Bern, M., G. S. Rupp. A. Pereira & C. R. Poll. 2001. Effect of antibiotics on larval survival and microbial levels during larval rearing of the tropical scallop Nodipecten nodosus. 13"' International Pectinid Workshop. Coquimbo. Chile. Book of Abstracts. O'Connor, W. A., M. P. Heasman, A. W. Frazer & J. J. Taylor. 1994. Hatchery rearing the doughboy scallop, Chlamys asperriiiia (Laniark). Memoirs Queensland Museum 36:357-360. O'Connor, W. A. & M. P. Heasman, 1995. Spawning induction and fer- tilisation in the doughboy scallop, Chlamvs asperrimu. Aipiiuiiliure 136:117-129. Parada, J. M., M. J. Cancelo, A. Fernandez & A. V. Guerra. 1993. Com- portamiento reproductivo de la zamburifia {ChUunys varia L.) cultivada 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, Marty, R. Galois, M. Mathieu & J. Moal. 1992. What about growth 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. Uriarte. I.. A. Farias & J. C, Castilla. 2001. Effect of antibiotic treatment during larval development of the Chilean scallop Argopecten purpuru- tus. Aquacult. Eng. 25:139-147. Journal of Shellfish Research. Vol. 22. No. I. I()1-I(W, 2()(1.V EFFECT OF DEPLOYMENT DATE AND ENVIRONMENTAL CONDITIONS ON GROWTH RATE AND RETRIEVAL OF HATCHERY-REARED SEA SCALLOPS, PLACOPECTEN MAGELLANICUS (GMELIN, 1791), AT A SEA-BASED NURSERY LORELEI A. GRECIAN,' G. JAY PARSONS,'* PATRICK DABINETT,^ AND CYR COUTURIER' 'Fisheries and Mciriiw Inslittite. Memorial University of Newfoundland. P.O. Bo.x 492U. St. John's, Newfoundland, Canada AIC 5R3 and 'Department of Biology. Memorial University of Newfoundland, St. John's. Newfoundland. Canada AIC 5S7 ABSTRACT The effect of date of deployment on jti'owth ;ind subsequent retrieval of hatchery-reared scallop spat from a land-based hatchery to a sea-based nursery was studied to provide information for management of juvenile-size scallops, ranging from 1.4-7.0 mm ui shell height. The objective of this study was to determine the optimal time period for spat deployment to a sea-based nursery to yield commercially acceptable growth rates and retrieval (scallops reinaining after mortality and loss through nets). Spat of the same size class and stocking density were deployed over five consecutive 16-23 day intervals beginning in August 19^7. Environmental factors were monitored weekly. Scallops were sampled after each deployment period for determination of shell height and retrieval. Scallops were then re-deployed and sampled before (November! and after (June! the winter season. Results demonstrated that there were significant differences in scallop growth and retrieval among the t~ive consecutive deployments. Only scallops that had been deployed in August were greater than 7 mm by November and could be sorted and transferred to larger mesh equipment for ongrowing prior to winter. The findings of this study demonstrated that early deployment (August! to sea-based nursery yielded high growth rates and retrieval. Deployment later than eariy September required over-wintering in nursery culture before transfer to ongrowing. Significant correlations were found between both growth rates and retrieval and some of the environmental parameters (e.g., temperature, chlorophyll-a, particulate organic matter!. Acclimation to the new farm conditions inay be necessary for nursery-sized .scallops to adjust physiologically without a major lag in growth following transfer from the hatchery to the sea. KEY WORDS: growth, nursery culture, Phuupeelen mai;ellanieus. scallop spat, sea star INTRODUCTION The aim of a nursery stage in bivalve aquaculture is to foster the development of young postmetamorphic settled animals to an optimal size for ongrowing and handling. For scallops, the nursery stage starts with the transitional period between a planktonic larval phase in a well-maintained hatchery setting and a benthic postlar- val phase where the settled spat are deployed to a sea-based nurs- ery or to a semicontrolled land-based growout environment. Sea- based nursery culture can be improved by determining the varia- tion of environmental factors at the nursery site and by manipulating the liming of the deployment of spat to nursery cul- ture to coincide with optimal conditions. Determining the timing of deployment at the sea-based nui'sery is necessary to optimize growth rates of hatchery-reared Pati- nopecleii yessoensis (Bourne & Hodgson 1991 1. Spat deployed during optimal food density and temperatures have higher growth rates and survival. The window of opportunity of deployment to the sea-based nursery can be assessed by determining growth rates and retrieval as functions of measurable natural factors, such as water quality, food availability, and the presence of potential predators over time. When adequate nursery conditions are provided, growth rates and survival are maximal, and the time scallops spend in the nursery stage exposed to other risk factors decreases. Growth rates of scallops vary seasonally as a result of tluctua- tions in food supply and temperature (Kirby-Smith & Barber 1974, Vahl 1980, Grecian et al. 2000). Growth rates of cultured P. ma- gellanicus are highest in the summer and lowest in the winter •■^Corresponding author. Tel: 709-778-0331; Fax: 709-778-053.'^; E-mail: Jay.Parsons@mi.mun.ca (Dadswell & Parsons 1991. 1992, Cote et al. 1993. Kleinman et al. 1996. Parsons et al. 2002) and show no increase during the autumn bloom compared with summer (Emerson et al. 1994). Sea scallops in some areas of Atlantic Canada are able to naturally produce two cohorts annually of which the summer (June to July) cohort grows faster than the autumn (September to October) cohort over the entire culture period (Dadswell & Parsons 1992). Dadswell and Parsons (1992) proposed that the higher growth rates of the first cohort were caused by the initial exposure of spat to the summer food conditions in the water column and a longer, more favorable period of warmer water. Thus, in bi\al\e hatcheries and nurseries, the early production of scallop spat is important for deployment to nursery culture early in the summer, as is the practice for oysters. This may result in the growth of scallop spat to a size of 7 mm or greater by the autumn, at which time spat would be large enough to transfer to intermediate culture gear as well as for sale to com- mercial growers. This growing period is much shorter than waiting until the following summer, which is the current protocol in the sea scallop industry (Dadswell & Parsons 1991, Couturier et al. 1995). Salinity, temperature, and predation impact survival of scal- lops. Salinity concentrations below 13 psu and 18 psu cause mass mortality in scallops in short-term and long-term exposures, re- spectively (Bergman et al. 1996, Frenette & Parsons 2001). As well, sea star predation on scallops can be significant in wild or bottom seeded scallops (Dickie & Medcof 1963, Scheibling et al. 1991, Barbeau & Scheibling 1994a). Sea star predation on scallops is limited in suspended nursery culture gear, unless the nursery gear is deployed prior to the settlement and growth of sea stars (Dadswell & Parsons 1992, Parsons 1994). Survival of post larval scallops, Pecten ma.ximus. transferred from hatchery to nursery was dependent on the immersion time during transfer, temperature differential and spat acclimation to the thermal regimen of the 101 102 Grecian et al. sea-based nursery (Christophersen 2000. Christophersen & Magnesen 2001). Timing of deployment of nursery-sized spat at the sea-based nursery is critical for optimizing growth rates and survival. The objective of this study was to determine the window of opportunity for deployment of hatchery-reared sea scallops at a sea-based nurs- ery that enhances growth rates and retrieval and provides avail- ability of spat for intermediate grow-out. Based on previous re- search on sea scallops, the hypotheses for this study are: ( 1 ) growth will be highest in scallops deployed earliest in the summer (Au- gust) when temperature and food availability are highest and (2) retrieval of scallops will decline with the onset of sea star settle- ment. MATERIALS AND METHODS Study Site Scallops were deployed on a scallop farm. Shell Fresh Farms Ltd.. based in Poole's Cove. Newfoundland. Canada. The inain study site was located in North Bay, head of Fortune Bay. NL at the Ladder Garden lease (47°42'N, 55°26'W). Experimental Design and Sampling Protocol This experiment was designed to determnie the optimal period for the deployment of nursery-size, post larval scallops at a sea- based nursery. Scallops were deployed over consecutive treatment intervals from the time they were first available from the hatchery and were large enough to be handled Ol .4 mm shell height) until no new cohorts of spat were available in the autumn. The spat were reared at 15°C from several spawnings undertaken at the Belleo- ram Sea Scallop Hatchery. Belleoram, Newfoundland (47^32 'N. 55°25'W). Spat were sorted by screening and those between 1.4 and 2.0 mm in shell height were used in the study. A sample of spat was obtained for initial shell height measurements {n = 30) for each deployment. Scallops were counted and deployed on five occasions at 500 spat/collector in 1.2-mm-mesh collector bags on August 4, August 22. September 7. September 26. and October 19. 1997. Two col- lector bags, each filled with 1 m of NetronT*' (34 g). were held in individual plastic bread trays (69 cm x .'i7 cm x 15 cm) at a 5 m depth (Grecian et al. 2000). The number of replicate bags varied from two to four depending on scallop spat availability. The initial "short-term" interval duration between successive deployment and retrieval dates ranged from 16 to 23 days and depended on site accessibility. Each short-term deployment interval ended when the next set of collector bags was deployed and the final short-term deployment interval ended on November 8, 1997. Scallop retrieval (defined as number remaining after mortality and any potential loss through the mesh of the nets) was assessed by counting scallops remaining at the end of each interval and scallops were measured for shell height (/; = 30). All scallop treatments were then redeployed and again counted and measured for shell height before and after the winter season on November 8. 1997 and June 24, 1998, respectively. During the experiment, all scallop treatments were handled in a similar manner. Water samples were pumped from a 5-m depth for phytoplank- ton identification, density and determination of total particulate matter (TPM), particulate inorganic matter (PIM). particulate or- ganic matter (POM), and chlorophyll-iv concentration. Tempera- ture and salinity were measured through the water column to a depth of 10-m using a YSI Model No. 30 S-C-T meter. Sea star settlement was also determined (see below). Each parameter was sampled approximately weekly during the short-term intervals (August to November). Immediately after water samples were collected, the phy- toplankton samples were fixed with Lugol's Iodine and 1% form- aldehyde. These samples then sat undisturbed for at least two weeks to allow the seston particles to settle. The top 909^ of water was siphoned off and its volume was measured. The remaining volume, which contained all settled algal particles, was also mea- sured. This concentrated volume was mixed thoroughly and 10 mL were transferred to a 10-mL Utermohl settling chamber for over- night settlement. The sample was analyzed visually for total num- ber of cells and species composition using a Zeiss Axiovert 35 microscope under phase contrast at 400x magnification. The total plankton assemblage was categorized into 8 major groups (McKenzie. 1997). Seven of these were on the basis of size while the final group comprised "unidentified species." The size categories included microzooplankton including tintinnids and ciliates (>20 iJim in diameter), autotrophic and heterotrophic di- notlagellates (12 to 60 |jLm). prymnesiophytes comprising small (2 to 12 (xm in diameter) spherical nanofiagellates. auto-nano- flagellates comprising spherical flagellates from 2 to 20 |j.m in diameter, cryptophytes comprising small (8 to 18 |j.in in length) tear-drop shaped biflagellates. centric diatoms (12 to 30 (jim in diameter, connected in long chains), and pelagic pennate diatoms (30 (jLin in length, single cells). Phytoplankton were identified according to Rott (1981). For TPM and chlorophyll-a samples, 15 L of seawater were pimiped from a depth of 5 m and pre-screened at 300 p.m into separate 20-L buckets and taken to the hatchery. Water samples (4 L) for TPM were filtered onto Whatman GF/C 45-mm diameter glass microfiber filters, which had been previously combusted in a muffle furnace at 500°C for 4 h to remove organic matter and were then weighed. The filters were then stored frozen at -20'C and ultimately oven-dried at 80°C for 24 h, weighed for TPM, trans- ferred to a muffle furnace for 4 h at 500°C, and reweighed to determine PIM. From these weights, ash-free dry weight or POM was calculated according to the formula TPM = POM + PIM. An additional 4 L of seawater was filtered onto Whatman GF/C filters for chlorophyll-n and pheopigment determination. Filters were frozen (-20°C) for later processing according to the fluoro- metric methods of Strickland and Parsons ( 1968) and Parrish et al. (1995). Sea star settlement was monitored weekly from July 15 to November 8, 1997. by deploying strings of eight empty pearl nets (34-cm X 34-cm square base pyramidal-shaped nets. 6-mm mesh) weekly at the farm with retrieval after approximately two weeks. Individual pearl nets were washed and all material greater than 250 (xm was collected on a mesh screen and preserved in 40% metha- nol. Samples were analyzed using a dissecting microscope for determination of numbers of sea stars present. Data Analysis Data were analyzed using the SPSS statistical package (Version 8.0). All percent data were arcsine-square-root transformed before statistical analysis (Sokal & Rohlf. 1995). Differences in growth rates and retrieval were analyzed using an analysis of variance (ANOVA) and the post hoc Tukey's b test was used to test for differences among treatments. Equality of means was analyzed Effect of Deployment Time on Sea Scallops 103 using an Independent sample Mest. Pearson correlation analyses were also performed on growth and retrieval data with the envi- ronmental parameters. The le\el of sisznificance was set at a = 0.05. RESULTS Grow til Rales Initial shell height among the replicates was not significantly different for all dates {P > 0.01) except September 7 (One-way ANOVA;F = 9.735. df= 2, 87, P< 0.001). This was because the scallops in one of the replicates were from a slow-growing batch of larvae and they were not randomly assigned among the repli- cates for that date, hence this replicate was not used for further analysis or in figures. The initial mean size ranged from 1.41 to 1.62 mm shell height (Fig. I). The mean shell heights of .spat at the end of each short-temi deployment interval were significantly different from the initial mean shell heights (Hests. P < 0.05. Fig. I). As well, mean shell heights at the end of each short-term interval were significantly different among the different deployment dates and decreased from 3.54 mm to 1.51 mm shell height (one-way ANOVA; F = 556.621, df = 4. 445. P < 0.001 ). Growth rates declined over the short-term intervals (Fig. 2). Significant differences were found among growth rates for the different intervals (one-way ANOVA: F = 95.162; df = 4. 1 1. P < 0.001). Highest growth rates occurred during the first deploy- ment interval at I 18 |jLm d"' (SE ± 1 .3). whereas the lowest growth rates occurred during the last interval at 3.3 p.m d"' (SE ± 0.7). The mean growth rate of all spat deployed between August 4 to November 8. 1997 was 43.2 pim d"' (SE ± 0.8). Growth rates of scallops from the earliest deployment were higher in the autumn and over winter than those from the subse- quent deployments (Fig. 3). For scallops deployed on August 4 and 22. growth rates were high until November 8. For the same scal- lops, growth rates from November to June 24. 1998. declined to a level similar to that of scallops deployed from September 7. 1997. Scallops deployed on September 26 and October 19. had lower overall giowth rates to November [1 1.4 |xm d"' (SE± 1.1) and 3.3 p.m d"' (SE ± 0.7). respectively] and to June |2I.5 [xm d~' (SE ± 1.4) and 7.2 fjim d~' (SE ± 1.3). respectively]. 04-Aug 22-Aug 07-Sep 26-Sep Initial deployment date Figure 1. Mean shell height of scallops deployed over five consecutive 2-Heek intervals in 1997 and on November 8, 1997, and June 24. 1998, at Shell Fresh Farms Ltd., Poole's Cove, NL. The initial date of an interval was the final date of the previous short-term interval, t'oni- mon letter denotes no significant difference among mean shell heights for each sample period iTukey's b test). Vertical bars are ±SE. I5U 120 - 90 ■ 60 ■ 30 ■ 0 ■ 'd -■■■■'-1. ^^* Short-term Growth i ""■•■"' Short-term Retneval 1 -3 5. 1 "■•-.T i 2 M li b r * 1 n a 100 90 80 70 ' 60 S 50 1 40 i 30 0 20 10 0 04-Aug :-Aus; 07-Scp 2b-Sep 19-Ocl Initial deployment date Figure 2. .Mean growth rates and retrieval of scallops over consecutive deployment intervals at Shell Fresh Farms Ltd., Poole's Cove, NL. The initial date of an interval is the final date of the previous interval. Common letter denotes no significant difference in growth rates or retrieval among intervals (Tukey's b test). \ ertical bars are ±SE. Retrieval Retrieval of spat at the end of each deployment interval (num- ber remaining after mortality and loss) declined over time (Fig. 2) and was significantly different among the different short-term de- ployment intervals (one-way ANOVA; f = 47.129, df = 4. I I, P < 0.001 ). Highest retrieval was obtained from spat deployed during the first interval (97%), whereas lowest retrieval was for spat deployed on September 26 (53%). Retrieval of spat from their initial deployment to November 8 was not significantly different than their retrieval after the short-term intervals (Paired t-test; t = 0.013. df = \4.P = 0.990; Fig. 3). En vironmeiital Characteristics Water temperature declined over the deployment periods (Au- gust-November. Fig. 4A). Mean temperatures for the five con- secutive deployment intervals were 14.7, 13.6. 11.3. 11.2. and 7.9°C. respectively. Spat were not acclimated from 15 C in the hatchery to ambient seawater temperatures before sea-based de- ployment. Salinity increased over the study period (Fig. 4A). Mean salinity was 28.3 psu whereas the range was from 26.5 to 31.5 psu. Chlorophyll-fl concentrations (one-way ANOVA; F = 0.544. df = 14. 24. P = 0.881). pheopigment concentrations (one-way -^ .Autumn 'Spring " November Retneval 04-Aug 22-Aug 07.Scp 26.Sep 19-Ocl Initial deployment date Figure 3. Mean growth rates and retrieval of scallops deployed at a sea-based nursery at Shell Fresh Farms Ltd.. Poole's Cove. NL, on five dates in 1997 and sampled on November 8. 1997, and June 24, 1998. Common letter denotes no significant difference in growth rates or retrieval among intervals (Tukey's b test). Vertical bars are ±SE. 104 Grecian et al. u 3 « u u a. 20 16 12 4 ■ ■»- *- Jul 22- 5- 19- 2- 16- 30- 14- 28- Jul Aug Aug Sep Sep Sep Oct Oct 35 30 ""i B. a 71) ,>i IS c lU "3 5 C/2 Nov 8- 22- 5- 19- 2- 16- 30- 14- 28- 11- Jul Jul Aug Aug Sep Sep Sep Oct Oct Nov =5. C O o c o U 20 16 12 8 4 0 8- Jui • Chlorophyll " Phaeopignients A. A-A A- Jul 5- Aug 19- Auti 2- 16- Sep Sep Date 30- Sep 14- Oct Oct 11- Nov Kisure 4. Water quality at Ladder Garden site. Shell Fresli Farms Ltd., Poole's Cove, NL, from July IS to November 8, 1997. A) Temperature and salinity (±SE: ;i = 3), Bl seston, C) chlorophyll and pheopigments at 5 m. (TPM, total particulate matter; POM, particulate organic matter). ANOVA; F = 0.500. df = 14. 24. P = 0.910), and POM (one- way ANOVA; F = 0.71.5. df = 14. 21. P = 0.737) were not significantly different o\er the duration of the study. TPM remained constant al Ladder Garden (Fig. 4B) with weekly mean TPM being 5.6 mg L '. POM was also constant at Ladder Garden with a mean of 1.9 mg L '. Chlorophyll-o and pheopigments averaged 2.4 and 10.1 mg L"', respectively (Fig. 4C). There was a significant difference in total phytoplankton den- sity among the weekly samples (one-way ANOVA; F = 7.084. df = 13. 28. P < 0.001; Fig. 5). The total phytoplankton density peaked around the middle of August, followed by a decline. The decline was also evident when the mean total phytoplankton den- sity was calculated for each interval (Fig. 6). The autotrophic nanoflagellates, pelagic pennate diatoms, and dinotlagellates were the numerically dominant groups present (Fig. 7A and B). The species that contributed to the peak abundance were Naviciila sp.. Cliluinydoinoiuis sp.. Ochronxnias sp.. Microinonas sp. (Fig. 8A and B). Percent abundance of phytoplankton size groups indicated that species <5 p,m had the greatest contribution to phytoplankton biovolume (Fig. 9). Sea star settlement at the Ladder Garden site peaked between September 19 and October 23 (Fig. 10). There were significant differences in sea star settlement over the different sampling dates (ANOVA; F = 99.674. df = 13. 336. P < 0.001). Maxiinum i S-Jiil 2:-Jul 5-Aug 19-Aiig 2-Sep 16-Sep 30-Sep 14-Oct 28-Ocl 1 1-Nov Date Figure 5. Total phytoplankton density at Ladder Garden site of Shell Fresh Farm, Poole's Cove, NL, from ,luly 15 to November 8, 1997. Effect of Deployment Time on Sea Scaelops 105 Deploynicnt inlenai Fij"ure 6. Mtun density of total phvtoplankton over five intervals of scallo|) deplovMunt on a sea-based nursery at Shell Fresh Farm, Poole's Cove, Nl.. Intervals hejjan on Ausiust 4 and ended on Novem- ber S. IW7. \ertieal bars are ±SE. setllenieiit was 311) sea stars per collector per day ami mean sea star settlenienl was 79 sea stars per collector per day. Most environmental factors were highly correlated with growth rates and retrieval (Tables 1 and 2). TPM and dinoflagellates were not correlated with growth rates and TPM and PIM were not correlated with retrievals. DISCUSSION Effects of Deploymiiil Dale on Growth Rates anil Retrieval The date of transfer or deployment of scallop spat from hatch- ery to nttrsery was a useful predictor of growth and retrieval. The higher growth rates and retrievals in the earlier deployments were related to several parameters in this study, where ambient tem- 5- 19- T. 16- 30- 14- 28- 11- Aug Aug Sep Sep Sep Oct Oct Nov A 2500 ■ 2000 ■ 1500 ■ 1000 ■ 500 ■ ri ■ / .♦■■♦■ --■•■- " Pelagic pennale diatoms ~ Dinoflagellates ~ Unidentified phytoplankton ~ Autotrophic nanoflagellates U — e— Q V ^fe *f»==«^«-,.>-*.T^, 8- 22- 5- ly- 2- 16- 30- 14- 2S- 11- Jul Jul Aug Aug Sep Sep Sep Oct Ocl Nov J 80 60 4U 1 20 c D 0 • Microzooplankton * Prymnesiophytes - - o - - Centric diatoms /\ .'^ A /^^^:^:i\ 8- Jul 22- 5- 19- 2- 16- 30- 14- 2S- 11- .lul Aug Aug Sep Sep Sep Oct Oct Nov Date Figure 7. Mean density of "(.V)" four dominant and "(B)" three less dominant groups of major plankton at Shell Fresh Farms Ltd., Poole's Cove, NL, from July 15 to November 8, 1997. ■ Rluzosolenia sp. - Coccolithophore sp. Prorocentruin sp. Choanoflagellate sp. Sirohilidnim innninuin Dinophysis non'egica -J C Q 8- 22- 5- 19- 2- 16- 30- 14- 28- 11- Jul .lul Aug Aug Sep Sep Sep Oct Oct Nov Date Figure 8. Mean den.sity of "(A)" dominant and "(B)" less dominant plankton species that showed a declining trend over intervals of scallop deployment at a sea-based nursery at Shell Fresh Farms Ltd., Poole's Cove, NL, from .luly 15 to November 8. 1997. perature and food availability and quality (species composition, organic content and lipid characteristics inferred from literature reports) were higher initially, then declined after early August. Predator (sea star) abundance peaked near the second deployment date before declining. Spat growth and retrieval from the initial deployment demonstrated that there is an optiinum time or window of opportunity, which could be used to maximize nursery growth. After this period, scallops face increasing adversity in terms of declining temperature and food quantity and quality, and increas- ing predation and temperature shock (the difference between hatchery and ambient temperatures). In a similar study in Southern Norway, Pecten inaximus spat transferred from hatchery to sea- based nursery from March to August showed increased growth and survival during the summer when water temperatures were >10°C and when temperature differences between the hatchery and nurs- ery were minimal (Christophersen & Magnesen, 2001). Temperature and food availability declined from August to November while sea star settlement began in mid-September. Variations in temperature and food availability were similar to those found in other areas of Atlantic Canada, including Concep- tion Bay, NL, and Bedford Basin, NS (Mayzaud et al. 1989, Na- varro & Thompson 1995). In earlier studies of sea scallop aqua- culture, temperature and food availability were the main predictors of growth (Parsons & Dadswell 1992, Cote et al. 1993. Emerson et al. 1994. Kleinman et ul. 1996). Likewise, sea stars are a signifi- cant predator of scallops (Barbeau & Scheibling 1994a. b). Changes in these parameters may best explain the variation in growth and retrieval of the scallops over the different deployment intervals. A negative correlation of salinity with growth and retrieval of scallops in the deployinent study was probably a coincidence as 106 Grecian et al. 04-Aug 22-Aug 07-Sep 26-Sep Initial deployment date 19-Oct H <5 Hill ■ <10nm D <20 urn I D<50nm O<100nm ■ <2()0 urn D >200 urn B Unidentified Figure 9. Particle size frequency distribution of planlvton at Ladder Garden, Sliell Fresh Farms Ltd., Poole's Cove, NL, over five consecutive deployment intervals of scallops at a sea-based nursery. the salinity tolerance range for wild juvenile sea scallops is >25 psu (Frenette & Parsons 2001), which is lower than the salinity during the present study. The increase in salinity over the study period reflects the decreased runoff and the increased upwelling that occurs in the autumn in this area. Decreases in metabolic processes due to declining temperature may explain why reduced growth rates were observed in scallops deployed on different dates in this study as has been found for Pecteii fiimatiis (Cropp & Hortle 1992). Respiration rates in sea scallops decrease with declining temperature (Shumway et al. 1988), but clearance rates are coirelated with ambient temperature in sea scallops (MacDonald & Thompson 1986) as well as in the eastern oyster, Crassostrea virginica and the bay scallop. Ar- gopecleii irradians (Rheault & Rice 1996). In the present context, reduced clearance rates would be expected to decrease food intake and result in reduced growth. Declining retrieval over time was correlated with deployment temperature. This however, does not indicate that scallops died as a direct result of decreasing temperature. Scallops are able to live within a temperature range of-2' C to 22"C (Dickie 1958). Hence their survival should not have been influenced by decreasing tem- peratures per se. Christophersen and Magnesen (2001 ) found that when Pecten maximus spat were deployed at water temperatures >10°C, spat had up to a 4-fold increase in survival compared with scallops deployed at temperatures <10°C. The sea scallops were likely influenced more by the temperature difference from the hatchery to the sea-based nursery environment than their physi- ologic condition or predation by sea stars. Effects of Food Variation on Growth Rates and Retrieval Scallops deployed when Prnrocciilninu DiiuipltYsis and Nav- icitla spp. densities were elevated exhibited higher growth rates than scallops deployed when densities of these phyloplankton spe- cies had declined. All these mircoalgae have been found in gut analyses of adult scallops (Shumway et al. 1987). We found all three species in high abundance and the first two species are con- sidered to add greatly to the energy uptake of scallops (Shumway et al. 1987). Cryptophyte densities also peaked during August when growth rates were highest. Cryptophytes are rich in the fatty acids. 22:6w3 and 20:5w3 (Volkman et al. 1989. Viso & Marty 1993) and are important for a good diet and membrane fluidity in bivalves (Enright et al. 1986, Napolitano et al. 1992). Crypto- phytes are a preferred alga in mixed diets and are related to growth 350 8-Jul 22-Jul 5-Aug 19-Aug 2-Sep 16-Sep 30-Sep 14-Oct 2S-0ct 11-Nov Date Figure 10, Mean sea star settlement al Ladder Garden lease of Shell Fresh Farms Ltd., Poole's Cove, NL, from .July 15 to November 8. 1997 (;i = 8). Vertical bars are ±.SH Effect of Deployment Time on Sea Scallops 107 TABLE 1. Pearson's ciirrelalion coefficients of shorl-lcrni };ro"th rates and retrieval of nursery-size scallops «ilh mean water quality parameters at a sea-based nursery at Shell Fresh Farms ltd.. I'oolc's Cove, NL. from August 4 to November 8, 1997. % Sea Star Temperature Salinity Chlorophyll-a Phaeopigments TPM PIM POM POM Settlement Growth rate /■ value 0.840 -0.826 0.901 0.940 -0.043 -0.573 0.700 0.773 -0.796 Signifieancc (two-tailed) <0.001 <0.0()1 0.001 <0.001 0.4.19 0.013 0.002 0.001 <0.001 Retrieval /• value 0.828 -0.698 0.849 0.870 0.2.1.1 -0.358 0.714 0.644 -0.890 Significance (two-tailed) <0.001 0.002 <().()01 <0.001 0.201 0.095 0.001 0.005 <0.0()1 15 for all parameters. in sea scallops (Shumway et al. 1985. Pairish et al. 199.5). It is expected that scalkips exposed to a higher quality diet allowitig adaptation to declining conditions would peifomi better than scal- lops exposed to a lower quality diet (see Shunnvay et al. 1997). MacDonald and Thotnpson ( 1985) found that shell growth was higher under favorable conditions of food and tetiiperature, and that this was site specific. Location of sea-based nursery sites should consider food quantity and quality. However, because there have been so few growth studies of juvenile bivalves with respect to natural phytoplankton composition, the actual quantity and qual- ity of food required is not l. and K are mean values ± standard deviations. ingly. all standard measures of genetic diversity were conipura- tively high (e.g., haplotype diversity ranged 0.94-0.99). Twenty-four wild-individual haplotypes matched five 1997 original-broodstock haplotypes for segment 2. However, none of the individuals that matched original-broodstock segment-2 hap- lotypes also matched the same broodstock individual for seg- ment 1. No wild individuals collected in 1998 matched any of the original-broodstock segment-2 haplotypes. If our assumptions as- sociated with Equation I were vahd, we could expect to obtain a match between a wild-individual haplotype and an original- broodstock haplotype if the broodstock haplotype was present in our wild-population sample at a frequency of approximately \% or greater {MDF,,^ = 0.00917). Thus, the estimated prerestoration frequency of each of the 1997 and 1998 broodstock haplotypes in the wild population probably was less than 1%. Ten of the assessment scallops collected in 1999 matched three of the 1997, segment-2, original-broodstock haplotypes. Eight of those were identical to the single original-broodstock scallop with the haplotype that was the second most common in the wild popu- lation. However, the haplotypes of all of those individuals differed from that original-broodstock individual's segment 1 haplotype. No segment-2 haplotypes from assessment bay scallops collected in 2000, and only one segment-2 haplotype from an assessment bay scallop collected in 2001, matched any 1998, original- broodstock, segment-2 haplotype. That individual did not match for segment 1 the original-broodstock individual that it matched for segment 2. Thus, our collective sample size of 694 individuals gave no indication that the bay scallop restoration project contrib- uted to the local Homosassa bay scallop population during 1999-2001. The MDF^,^ for detection of an original 1997 or 1998 brood- stock haplotype in the appropriate assessment collection(s) was, respectively 0.015 (1999 assessment collection) or 0.0060 (2000 + 2001 assessment collections). Original-broodstock haplotypes that were present in the putative admixed Homosassa population at frequencies near or below the MDFg^s were at statistical risk of not being detected. However, these frequencies were so low that stock restoration contributions at or below these levels may essentially be inconsequential. Although haplotype diversity and nucleotide diversity in our hypothetical assessment of stock restoration contribution were pro- portionally reduced with increasing stock restoration contribution, they were not as sensitive to the input of stock restoration contri- bution as was the percentage of different haplotypes (Table 2). Nevertheless, our simulations indicate that a stock restoration con- tribution of at least 15% in the 1999 assessment collection and \0% in the 2000-2001 combined assessment collection would be needed to generate a significant difference between those assess- ment collections with versus without stock restoration contribu- tions. Genetic Tags and Molluscan Stock Restoration The general strategy in a stock restoration program is to collect animals from the targeted restoration site, produce large quantities of aquaculture-reared or. in the case of our bay scallop program, aquaculture-derived (one generation removed 1 individuals, and use them to supplement or replenish the population at the same site. Determining the success of such an effort depends on the ability to detect the contribution (in numbers or percentages) of hatchery- reared or hatchery-derived offspring in the post-restoration re- cruits. In supplemented populations, the frequency of aquaculture- generated individuals can range from undetectable to a complete swamping of the admixed population. A single-gene genetic tag such as ours can indicate whether restoration effort has resulted in essentially undetectable input, substantial input, or a complete swamping of the local population. However, the capacity of this tag to estimate the contribution of the stock restoration effort be- tween the extremes of essentially no input and very high input is 116 Seyoum et al. TABLE 2. Hypothetical analysis of stock restoration contribution in the assessment collections from Honiosassa with levels of contribution varying from 0% (original assessment collection) to 25% (see Materials and Methods for method of simulating stock restoration contributions). (A) 1999 assessment collection (A' = 199 individuals). (B) 2I)(II) + 20(11 combined assessment collections (A' = 495 individuals). SRC(%) Nl N2 HNl HN2 A. 0 199 0 0.72 0.72 5 189 10 0.73 0.70 10 179 20 0.73 0.66 15 169 30 0.72 0.62 20 159 40 0.75 0.60 25 149 50 0.77 0.5S B. 0 495 0 0.69 0.69 5 470 35 0.69 0.66 10 445 69 0.69 0.62 15 421 104 0.70 0.73 20 396 139 0.72 0.57 25 370 174 0.73 0.55 Abbreviations: SRC = hypothetical stock restoration contribution; Nl = number of individuals taken from the specified year assessment collection; N2 = hypothetical number of individuals contributed from the stock res- toration program (within a single percentage, all of which were taken from a single, randomly chosen broodstock individual); HNl = percentage of individuals with different haplotypes without stock restoration contribution (calculated based on Nl only); HN2 = percentage of individuals with different haplotypes with stock restoration contribution (calculated on Nl + N2). related to the degree of statistical uniqueness, as measured by statistical probability, of the tag in each application. To precisely define an intermediate-level contribution from a stock restoration effort, the assessment collection must consist of a very high num- ber of individuals; the genetic tag must be complex (e.g., com- posed of our compound mtDNA genetic tag plus several micro- satellite loci), or. if it is a single-gene tag. extremely variable; or the method for determining the contribution must differ from ours. Because we found no original-broodstock haplotypes in either the wild population or the assessment collections, we can combine all of these collections to estimate the uniqueness of our original- broodstock haplotypes and calculate the MDF above which we might expect to encounter one of these haplotypes. We can esti- mate with 95'7r probability that v\e would have detected at least one original-broodstock haplotype in this combined sample ( 1,019 individuals) if the frequency of any of these haplotypes was 0.003 or greater. Clearly, frequencies below this MDF would represent inconsequential contributions from a stock restoration effort. Thus, our single-gene genetic tag should be useful for assessing the success of our entire bay scallop restoration effort. In many cases, a single-locus, preliminary genetic tag such as ours could be useful in assessing the contribution of stock resto- ration efforts. Multi-locus genetic tags can be laborious, time- consuming, and expensive to develop, test, and apply. Fuilher- more. in our case, the potential for reproductive mixing between restoration broodstock and wild scallops limits the ability for nuclear DNA-based assignment of individuals to either the brood generation or to the wild population. Our genetic tag can be used to preliminarily evaluate the success of a bay scallop stock en- hancement or restoration effort and thereby to evaluate whether it is worth the expense and effort to develop a more definitive ge- netic tag. Then, if it appears that the stock restoration effort may have contributed a potentially significant fraction of the recruits to an area, a high-resolution, multi-gene tag can be developed. How- ever, under certain conditions, the type of genetic tag presented here may be sufficient for an entire study. The advantages of using a single-gene genetic tag composed of more than one hypervariable segment and in which the segments can be used sequentially are increased resolution and reduced ef- fort. In our genetic tag. both segment 1 and segment 2 had ample and nearly equivalent variation. By sequencing first for segment 2. the expense and time required were reduced significantly because only the individuals that had segment 2 haplotypes identical to those of the original-broodstock haplotypes also needed to be se- quenced for Segment 1 , The utility of a single-gene genetic tag such as that presented here is enhanced if the broodstock used possesses essentially unique haplotypes or genotypes. However, there are limitations to this type of approach. A large number of wild individuals or a high percentage of the wild population must be assayed to establish the frequencies of the genetic-tag haplotypes in the pre-restoration population, and individuals with "unique" haplotypes should be used as broodstock. Threatened or depleted populations can be further endangered if they are flooded with aquaculture-derived individuals that collectively possess only a few naturally rare genotypes or haplotypes, if those individuals interbreed exten- sively and successfully with the remnant wild population. Never- theless, for some applications, the procedure that we described here provides researchers with a method for finding an mtDNA genetic tag in organisms for which little is known about their mtDNA. This type of genetic tag can be used to screen individuals and derive parentage or group associations for stock restoration efforts, conservation biology, or other suitable applications. ACKNOWLEDGMENTS We thank M. Tringali for assistance in the designing of the primers and notable suggestions in many aspects of the analysis. We also appreciate the assistance of D, Marelli. M. Parker, M. Harrison, and S. Peters with the field collections and C. Lund, T. Thompson, and D. Warner for various types of assistance. We additionally thank M. Tringali, A. McMillen-Jackson, and two reviewers for valuable comments on our manuscript. This study was funded by a grant from the National Oceanic and Atmospheric Administration (NOAA), grant NA76FK0426 and project FWC 2234 and by the state of Florida. The views expressed herein are those of the authors and do not necessarily reflect the views of NOAA or any of its sub-agencies. LITERATURE CITED Arnold. W. S. 2001. Bivalve enhancement and restoration strategies in Florida, USA. Hydmbiologia 465:7-19. Arnold, W. S., D. C. Marelli. C. P. Bray & M. M. Harrison. 1998. Re- cruitment of bay scallops Argopccteii irradimis in Ploridan Gulf of Mexico waters: scales of coherence. Mar. Ecol. Prof;. Scr 170:143-157. Barber, B. J. & N. J. Blake. 1983. Growth and reproduction of the bay Mitochondrial DNA Genetic Tag for Bay Scallop 117 scallop, Argopecten iirmlians (Lamarck) al it^ southern distributional limit. J. Exp. Mar. Biol. Ecol. 66:247-25fi. Bert, T. M., M. D. Tringali & S. Seyoum. 2002. De\elopnient and appli- cation of genetic tags for ecological aquaculture. In: B. Costa-Pierce, editor. Ecological aquaculture. Oxford. UK: Blackwell Science, Ltd.. pp. 47-76. Blake, N. J. 1998. The potential for reestablishing bay scallops to the estuaries of the west coast of Florida. Trans. 63"' No. Am. Wildlife Natural Re.murces Conf. 63:184-189. Blake, N. J.. Y. Lu & M. Mover. 1993. Evaluation of Tampa Bay waters for the survival and growth of southern bay scallop larvae and juve- niles. Final Report. Tampa Bay National Estuary Program. Tech, Pub. #04-93. p. 18. Boore, J. L. & W. M. Brown. 1994. Complete DNA sequence of the mitochondrial genome of the black chiton, Kallniriiia Uinicala. Gener- ics 38:423-+43. Boore. J. L.. T. M. Collins. D. Stanton. L. L. Daehlcr & W. M. Brown. 1995. Deducing the pattern of arthropod phylogeny from mitochondrial DNA rearrangements. Naliire 376:163-165. DeSalle, R.. A. Templton. I. Mori, S. Pletscher & J. S. Johnston. 1987. Temporal and spatial heterogeneity of intDNA polymorphisms in natu- ral populations of Drosophila merealonim. Genetics 1 16:215-223. Haddad. K. 1988. Habitat trends and fisheries in Tampa and Sarasota Bays. NCAA Estuary of the Month Series #11, Tampa and Sarasota Bays. pp. 113-128. Hargis, W. J., Jr. 1999. The evolution of the Chesapeake oyster reef system during the Holocene epoch. In: M. W. Luckenbach. R. Mann & J. A. Wesson, editors. Oyster Reef Habitat Restoration: A Synopsis and Synthesis of Approaches. Proceedings from the Symposium, Williams- burg. Virginia, pp. 5-23. Hoffmann, R. J., J. L. Boore & W. M. Brown. 1992. A novel inilochondrial genome organization for the blue mussel. Mxlilus ediilis. Genetics 131: 397^12. Hutchings, J. A. 2000. Collapse and recovery of marine fishes. Nature 406:882-885. Lansman, R. A., R. O. Shade. J. F. Shapira & J. C. Avise, 1981. The use of restriction enzymes to measure mitochondrial DNA sequence relat- edness in natural populations. / Mol. Evol. 17:214-226. Liu, H.-P., J. B. Mitton & S.-K. Wu. 1996. Paternal mitochondrial DNA differentiation far exceeds maternal mitochondrial DNA and allozyme differentiation in the freshwater mussel. .Anadimta grandis grandis. Emiuium 50:952-957. Marchuk. D.. M. Drumm, A. Saulino & E. S. Collins. 1990. Construction of T-vectors, a rapid and general systein for direct cloning of unmodi- fied PCR products. Nucl. Acid Res. 19: 1 154, Marelli, D. C, W. S. Arnold & C. Bray. 1999. Levels of recruitment and adult abundance in a collapsed population of bay scallops {Argopecten irradiuns) in Florida. / Shellfish Res. 18:393-399. Meyer, A. 1993. Evolution of mitochondrial DNA in fishes. In: P. W. Hochachka & T. P. Mommsen. editors. Biochemistry and Molecular Biology of Fishes, Vol. 2. Amsterdam: Elsevier, pp. 1-38. Orensanz, J. M., A. M. Parma & O. O. Iribarne. 1991. Population dynamics and management of natural stocks. In: S. E. Shumway, editor. Scallops: Biology, Ecology, and Aquaculture. New- York: Elsevier, pp. 625-713. Palsboll, P. J. 1999. Genetic tagging: contemporary molecular ecology. Biol. J. Linnean Soc. 68:3-22. Palsboll. P. J., J. Allen. M. Berube. P. J. Clapham, T. P. Feddeersen. P. S. Hammond, R. R. Hudson, H. Jorgensen, S. Katona, A. H. Larsen, F. Larsen. J. Lien, D. K. Maltila, J. Sigurjonsson, R. Sears R, R. Smith, R, Sponer. P. Stevick & N. Oien 1997. Genetic tagging of humpback whales. Nature },U:161 -1 b^ . Palumhi, S. R. 1996. Nucleic acids II: The polymerase chain reaction. In: D. M. Hillis. C. Moritz & B. K. Mable editors. Molecular Systematics, 2nd ed. Sunderland, MA: Sinauer Associates, Inc., pp. 205-247. Peterson, C. H., H. C. Summerson & J, Huber. 1995. Replenishment of hard clam stock using hatchery seed: combined importance of bottom type, seed size, planting season, and density. / Shellfish Res. 14:293- 300. Sambrook. J., E. F. Frit.sch & T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press. Schneider, S., D. Roessli & L. Excoffier. 2000. Arlequin: A .software for population genetics data analysis. Ver. 2.000. Geneva: Genetics and Biometry Lab, Dept. of Anthropology, University of Geneva. Simon, C, F. F. Frati. A. Beckenbach. B. Crespi. H. Liu & P. Flook. 1994. Evolution, weighting, and phylogenetic utility of mitochondrial gene sequences and a compilation of conserved polymerase chain reaction primers. Ann. Entoino. Soc. Am. 87:651-701. Southworth. M. & R. Mann. 1998. Oyster reef broodstock enhancement in the Great Wicomico River, Virginia. / Shellfish Res. 17:1101-1114. Svasand, T., T. S. Kristiansen, T. Pedersen, A. G. V. Salvanes, R. En- gelsen, G. Naevdal & M. Nodtvedt. 2000. The enhancement of cod stocks. Fish & Fr-^heries 1:173-205. Tettelbach, S. T, & P, Wenczel. 1993. Reseeding efforts and the status of bay scallop Argopecten irradians (Lamarck 1819) populations in New York following the occurrence of "brown tide" algal blooms. / Shell- fish Res. 12:423-431. Wilding, C. S., P. J. Mill & J. Grahame. 1999. Partial sequence of the mitochondrial genome of Littorina sa.xatilis: relevance to gastropod phylogenetics. J. Mol. Evol 48:348-359. Williams, J. G. K., A. R. Kubelik, J. J. Livak, J. A. Rafalski & S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acid Res. 18:6531-6535. Zouros, E., A. O. Ball, K. R. Freeman & C. Saavedra. 1994. An unusual type of mitochondrial DNA inheritance in the blue mussel Mytilis. Proc. Natl. Acad. Sci. USA 91:7463-7467. 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 l.U - T - ■■ 1 c 0.9 - - isc- o /- > — 1) s - •/ \ u 7 \ II 7 • T ^ ■'' \. ^ 1 U. . \ — *— V/ fduIlK Ol 0.6 ■ 'r L \ - -c- -M imsMilu'' b ' 3 U.S- ' 1 , . / \ O y ^^^ \ > 114 ■ ■/ » \ Ol , \ u ,' s / ^ " \ s y. ] ' 'S o (1 : - 4 J .: ... - (1.0 - 1 1 1 1 1 1 1 1 1 1 1 1 1 1—^' — r— — p^ ^-1 u! 5 < 6 _1 r^ s s Sample Date E O 10 0.4 0.8 0.7 0.6 (1..^ ■ (14 - Male Female ZIt jij 01) 3 3 3 < < < # B Sample Date o > O 1 0 y ■ ^ ;^-JHM /:--^■--^--J\ O.S ■ /- \ : \ 0.7- / A /^ A —•—Male / ;\ - - - • Female 0 5 ■ / ■' \ 04 - r / .- J \ n 1 - / .,' ; , V T \ _/ ' \ II : - I.'"': ' 1 ^' 'N ^ / II 1 - »--' ■• ';. " ■ ' 110 - ^ ^ , 1 , , — , — 1 , _: "7 s Sample Date 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, (J o o 300 200 — ■ — M edlilis T*" M :rossiiltis C -= OU OU 5JJ C- T1 = ^ 3 3 3 O ^ g -g I £. ^ ^ g V "r 5 < 5 S 3 2 c - r- ■ ' T ^ - _ _ r^ o • - r- •-.. — r. ^, _ -f =c — ^, _ „ — — _ Sample Date Figure 3. Mytiliis ediilis. Mytilus troxsiiliis. Mean (±1 SD) oocyte area for mussels from East Bav, Maine. significant decreases between dates were slightly out of phase, with the mean oocyte area of M. edulis decreasing from 14 Sep- tember to 15 October and that of A^. trossidits from 15 October to 17 November. A significant difference in oocyte area (t = 7.24, P < 0.001) was observed just prior to spawning on 17 July indi- cating that M. edulis spawned larger eggs than did M. trossiihis. Mean shell length of females sampled on this date was not sig- nificantly different (t = 1.29, P = 0.220). DISCUSSION AND CONCLUSIONS The reproductive cycle of mussels in this population was highly seasonal which is typical of many benthic marine invertebrates in northern temperate zones. In this study, gonadal development was minimal through the winter as indicated by low GVF and small oocyte diameters. Increased gametogenic activity in spring corre- sponded to increasing water temperature and presumably food a\'ailability. A significant decrease in GVF and oocyte diameters, indicative of a major spawning event, took place in late July and involved a large proportion of the population. Interestingly, GVF for females increased slightly in samples collected after this initial spawning event. Such an increase could be caused by redevelop- ment of the gonad in preparation for a second spawning. However, we observed little histologic evidence of redevelopment in indi- vidual mussels that had already spawned. The predominant histo- logic feature at this time was empty follicles containing a few refractory oocytes. Thus, the few individuals that did not spawn or had only partially spawned after the peak-spawning event in late July were responsible for the observed increase in GVF. TABLE 3. Mean oocyte area of Mytilus edulis and Mytilus trossulus: results of a two-way ANOVA testing the effects of date and species on the gametogenic cycle. Source df Mean Square F Value Date 16 1.6398 53.67*** Species 1 0.1236 4.04* Date X species 16 0.0665 2.18** Error 285 0.0306 * P < 0.05. **P < 0.01, ***P < 0.001, More importantly, the reproductive cycles of Myliliis edulis and M. trossulus sampled from this population were highly synchro- nous. For the year 2000 at the East Bay site, the results of this study indicate that interspecific fertilization between M. edulis and M. trossulus is possible based on spawning times. Similar findings have been reported elsewhere. Freeman et al. (1992) and Mallet and Carver ( 1995) observed synchronous reproductive patterns in populations of M. edulis and M. trossulus from Lunenburg Bay, Nova Scotia. Additionally, Toro et al. (2002) found that the ini- tiation of spawning was coincident between these species and their hybrids in Trinity Bay, Newfoundland; although M. trossulus dis- played a more protracted period of spawning at this location the variation alone was not sufficient to explain the limited numbers of hybrids observed. Thus, four studies covering a wide geographic region from Maine to Newfoundland have observed similar results all suggesting that hybridization is not limited solely by species- specific differences in spawning times. It is possible that genetic identity is maintained between M. edulis and M. trossulus by a factor other than different spawning periods. Gamete recognition proteins have been shown to drasti- cally reduce the hybridization potential between closely related taxa of marine invertebrates. Interestingly, molecular phylogenies suggest that M. trossulus is the most divergent of the blue mussel taxa (Rawson & Hilbish 1995). It has been recently shown that M. edulis and M. trossulus have also diverged significantly with re- spect to amino acid sequence at a spenn lysin locus (C. Riginos. pers comm). Divergence in gamete recognition proteins such as sperm lysin could act to limit hybridization between M. trossulus and other blue mussel taxa. Though no evidence of functional differentiation has been documented as yet, preliminary data indi- cates that cross-fertilization of M, edulis and M. trossulus is lim- ited except at very high sperm concentrations (Rawson unpub- lished). Thus, future effoils should focus on more detailed obser- vations of the spawning behavior of these two species as well as the potential for functional variation in gamete recognition pro- teins. The present study found that M. trossulus had smaller mean oocyte size at maturity and presumably spawned smaller eggs than M. edulis. Given that M. trossulus has a higher reproductive output (Toro et al. 2002), it follows that similarly sized M. trossulus produced more (but smaller) eggs than M. edulis. which might provide a selective advantage for the more fecund M. trossulus. Similarly, M. galloproviiicialis has a higher fecundity per unit length than M. edulis at Croyde in S.W. England, but genotypic ratios between these two species have not changed over time be- cause of large numbers of small M, edulis (Gardner & Skibinski 1990). Smaller oocytes may also represent a response to environ- mental stress. Cobscook Bay in eastern Maine is near the southern distributional limit of M. trossulus (Rawson et al. 2001) and as such, may be a less than optimal environment for this species. However, M. tros.sulus from Newfoundland also produces smaller eggs, has a smaller size at first maturity than M. edulis (Toro et al, 2(J02), as well as a population structure containing a higher fre- quency of small M. trossulus individuals (Comesana et al. 1999). Given that a difference in oocyte size has been observed in both Maine and Newfoundland it is more likely that this difference is the result of a difference in life history strategy rather than a response to environmental stress. Additional data are needed on extrinsic factors such as population structure, size at first maturity, reproductive output, and size dependent mortality to draw coiiclu- Gametogenesis in Sympatric Blue Mussels 123 sions concerning the intrinsic factors sinaping (he lite history evo- lution of M. cdiilis and M. trossiiliis. ACKNOWLEDGMENTS Funding for this project was provided through a Maine Aqua- cuhure Innmation Center crant to B. J. Barber and P. D. Rawson. Maine Sea Grant, and Experiinent Station Hatch Funds to P.D. Rawson. We are also grateful to D. Beane for histologic preparations, and S. R. Fegley and P. A. Haye for helpful comments on earlier versions of this manuscript. This is Maine Agricultural and Forest Experiment Station external publication #2627. LITERATURE CITED Bates. J. .A. & D.J. Iniies. 1995. Genetic variation amcmg popiikitidiis of Myliliis Slip, in Eastern Newfoundland. Mar. Biol. 124:417—424. Bayne. B. L.. D. L. Holland. M. N. Moore. D. M. Lowe & J. Widdows. 1978. Further studies on the effects of stress in the adult on the eggs of Mylilus ediilis. J. Mar. Biol. Ass. U.K. 58:825-841. Comesatia. A. S. & A. Sanjuan. 1997. Microgeographic allozyme differ- entiation in the hybrid zone of Mylilus galloprnvincialis Lmk. and M. ediilis L. on the continental European coast. Helgol. Meeresunters 5 1 : 107-124. Comesana. A. S.. J. E. Toro. D. J. Innes & R. J. Thompson. 1999. A molecular approach to the ecology of a mussel {Mylilus edulis-Mylitus irossulus) hybrid zone on the east coast of Newfoundland. Mar. Biol. 13.1:213-221. Freeman, K. R.. K. L. Perry & T. G. DiBacco. 1992. Morphology, condi- tion and Reproduction of two co-occurring species of Mytihis at a Nova Scotia mussel farm. Bull, Aquacul, Assoc. Canada 92-3:810. Gardner, J. P. A. 1992. Mytilus galloprovincialis (Lmk) (Bivalvia. Mol- lusca): The taxonomic status of the Mediterranean mussel. Ophelia 35:219-243. Gardner, J. P. A. 1996. The Mytilus edulis species complex in southwest England: Effects of hybridization and introgression upon interiocus associations and morphometric variation. Mar. Biol. 125:385-399. Garrido, C. L. & B. J. Barber. 2001. Effects of temperature and food ration on growth and oogenesis of the green sea urchin, Strongylocentrotus droebachieiisis. Mar Biol 138:447^58. Gardner. J. P. A. & D. O. F. Skibinski. 1990. Genotype-dependent fecun- dity and temporal variation in spawning in hybrid mussels {Mytilus) populations. Mar Biol. 105:153-162. Gilg, M. R. & T. J. Hilbish. 2000. The relationship between allele fre- quency and tidal height in a mussel hybrid zone: a test of the differ- ential settlement hypothesis. Mar. Biol. 137:378. Gosling. E. M. 1984. The systematic status of Mytilus galloprovincialis in western Europe: A review. Malacologia 25:551-568. Gosling, E. M. 1992. Systematics and geographical distribution oi Mytilus. In: E. M. Gosling (ed.). The mussel Mytilus: Ecology, physiology, genetics and culture. Amsterdam: Elsevier, pp. 1-20. Gray. P. 1954. The microtomist's fomiulary and guide. New York: The Blakiston Co Inc.. pp. 108. Hilbish. T. J.. B. L. Bayne & A. Day. 1994. Genetics of physiological differentiation within the marine mussel genus Mylilus. Evol. 48:267- 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. Koehn. R. K. 1991. The genetics and taxonomy of the species in the genus Mytilus. Aquiu-ulmre 94:125-145. Koehn. R. K.. R. Milkman & J. B. Mitton. 1976. Population genetics of marine pelecypods. IV. Selection, migration, and genetic differentia- tion in the blue mussel Mytilus edulis. Evol. 30:335-342. Koehn. R. K., J. G. Hall. D. J. Innes & A. J. Zera. 1984. Genetic differ- entiation of Mytilus edulis in eastern North America. Mar Biol. 79: 117-126. Mallet, A. L. & C. E. Carver 1995. Comparative growth and survival of Mytilus trossulus and Mytilus edulis in Atlantic Canada. Can. J. Fish. Aquat. Sci. 52:1873-1880. McDonald, J. H., R. Seed & R. K. Koehn. 1991. Allozyme and morpho- metric characters of three species of Mytilus in the northern and south- ern hemispheres. Mar. Biol. 111:323-333. Palumhi. S. R. 1994. Genetic divergence, reproductive isolation, and ma- rine speciation. Annu. Rev. Ecol. Sysl. 25:547-572. Palumhi. S. R. 1999. All males are not created equal: Fertihty differences depend on gamete recognition polymorphisms in sea urchins. Proc. Natl. Acad. Sci. USA 96:12632-12637. Rawson, P. D. & T. J. Hilbish 1995. Evolutionary relationships among the male and female mitochondrial DNA lineages in the Mytilus edulis species complex. Mol. Biol. Evol. 12:893-901. Rawson. P. D.. S. Hayhurst & B. VanScoyoc. 2001. Species composition of blue mussel populations in the northeastern Gulf of Maine. / Shell- ti.^h. Res. 20:31-38. Saavedra, C. D. T. Stewart. R. B. Stanwood & E. Zouros. 1996. Species- specific segregation of gender-associated mitochondrial DNA types in an area where two mussel species {Mytilus edulis and M. trossulus) hybridize. Genetics 143:1359-1367. Sanjuan. A., C. Zapata & G. Alvarez. 1997. Genetic differentiation in Mytilus galloprovincialis Lmk. throughout the world. Ophelia 47:13- 31. Sarver. S. K. & D. W. Foltz. 1993. Genetic population structure of a species' complex of blue mussels {Mytilus spp.) Mar. Biol. 117:105- 117. Seed. R. 1992. Systematics evolution and distribution of mussels belonging to the genus Mytilus: An overview. Am. Malac. Bull. 9:123-137. Suchanek. T. H., J. B. Geller, B. R. Kresier & J. B. Mitton. 1997. Zoo- geographical distribution of the sibling species Mytilus galloprovincia- lis and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the North Pacific. Biol. Bull. 193:187-194. Swanson. W. J. & V. D. Vacqier. 1998. Concerted evolution in an egg receptor for a rapidly evolving abalone sperm protein. Science. 281: 710-712. Toro. J. E.. R. J. Thompson & D. J. Innes. 2002. Reproductive isolation and reproducti\e output in two sympatric mussel species {Mytilus edulis. M. Irossulus) and their hybrids from Newfoundland. Mar Biol. 141: 897-909. Vainola, R. & M. M. Hvilsom. 1 99 1 . Genetic divergence and a hybrid zone between Baltic and North Sea Mytilus populations (Mytilidae: Mol- lusca). Biol. J. Linn. Soc. 43:127-148. Varvio. S. L.. R. K. Koehn & R. Vainola. 1988. Evolutionary genetics of the Mytilus edulis complex in the North Atlantic Region. Mar. Biol. 98: 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) 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). O .J en c o « ■n = ^ 3 =U Of c 2 f^l ■n v: n c CO n E < £ r" Oi c c £ "^r ^ c ■? L. at a> .C C s2 r — = = = 5= 5=: 5= 5c' X. 3 ■ ■ — ■ __^- __; _■ _■ __■ _; ■ — ■ ■ — ' ^ — ■ — ' ■ — ■ ■ — ■ ■ — ■ ■ — ■ ■^ \/ M — V V V V V V V V n, n, V a. O. D- n, n. U- c Dh n o yr, C2 c: oc 00 c «M ri o oo ■y~i ct ^ ^t E _ ■^■ ■^■ -^■ •^ -1- T ^ -t II II L. II 11 II II II II II II -3 ■^ T3 ~o T3 "3 ■a ■a -f — .' -3 i/^, tr. ^. r^. in or) r- r*", Tf oc- r- oo CO oc '^ "O f*-! ^P ^o -t in r-i r^i m r*~i r-l r-l ON r^ Yi r- •rt m r\ r- — ^ ri t/"^. II II II II II 11 II II 11 II II t/j U. U- U- U- O. tJ- U. U. tl. u. U, r- r- o r\ '^■ r^ O m, r-i o ri r-, (N r- r- iri r- -i- r- ON ON Ti- O o o o O c o o o o o II II II II II 11 II II II 11 II ^1 ^1 ^J J., fi ri Al ri ri r-l "- ■- '- ■- *- *- "- '- *- c o _ r-l r^. -t +1 + +1 3^ 1 + + + o 1 (-1 S ^ 2 rr" a. s a. a. + n s a. 1 H o d + S a. 5 -T r^, '^ f- ,:;^ Q. p =;■ — ■ ^ ■ isC -t o t- +1 Tl +1 +1 Tl Tl 5C 04 ^ £ — ri O' r^ o O H — . C: + -H o O o +1 + t/5 8 + -1- t/1 O -1- + fll q n -n ( ) ( ) s f/^ n O X y O O X n n X o O X X X z X X r^ ^o o . , ^ ,-, 00 ' ' rr +1 +1 +1 +1 +1 c +1 +1 r~- r^ r\ r^l p d d -H 8 -i- . + o: vC a. u. ol 2 0- f- 1 0- H X 2 D. t- CL f- X o +1 oT X X 3C X 00 X X o 2 X s d +1 Ol d -1- a. d +1 ^1 -r o +1 +1 ? ? ol O S H U", ri [^ +1 ri +1 s rj ri U n t + +1 T in. -t ri +1 o +1 o J, n '^ ri +1 00 o o +1 d c Ti d +1 00 ri OO r- 'rf '■~^ ^ — II g 11 II oo II II II 1 II II c/1 O II II II a: < u n< rv lY. £ Di O U O o < o U U- a < Z o ^ z z •?■ u 8 8 C/5 8 00 U n u o O 0- c O Z c ^ -o ■a c a. -a ^ r: „^ a. ^ r5 S E S S a. f- U O u o t— a. r- C/1 H oi OC < CJ K Di ^ Oi O U O u < o U u, a: < ^ O Z z z Modeling Feeding Behavior in Perna perna 129 of eiivironmeiits. Model predictions and observed data of FR. RR. IR, NOSE. OCI. and AR of mussels in a range of TPM between 2 and 40 mg L '. are shown in Fig. 7 A. B. C, D. E. and F. respec- tively, showing that predicted values satisfactorily reproduce the main trends of feeding behavior observed in P. perna. As bivalve feeding behavior is mainly controlled by concen- tration and organic content of seston (Hawkins et al. 1998b). it is likely that this model is sensitive to these forcing functions (TPM and OCS). To verify the model sensitivity to changes in the coef- ficients of the equation that predicts OCS as a function of TPM. we ran the model three times, varying the coefficients values. Each coefficient (EQ. ( I ). Table -■^. Fig. 2| was varied by ±10"^* from its standard value, and the sensitivity was measured by the following equation: S = [x/x|/IP/P] where (S) is a measure of sensitivity, x refers to model outputs at the end of the integration period in the standard model, and r'»x is the change in the value of x brought about by varying the model Figure 3. Perna perna. The relationship between total particulate mat- ter (TPM, mg !,"') and organic content of seston (OCS, fraction I and (Al clearance rates (C'R I h '). (I?) filtration rate (FR, mg h"'), (C) rejection rate (RR. mg h 'l, (I)) Ingestion rate (IR. mg h"'l, (E) net organic absorption rate (N().\R, mg h 'l. Net organic selection effi- ciency (NOSE, fraction) is plotted against particulated organic and inorganic matter (PIM and POM, mg L"') (F). Refer to Table 3 for equations and statistics. Both the net absorption efficiency of ingested organics (NAEIO, fraction) and the net organic absorption rate (NOAR. mg h'') had a hyperbolic relationship with the organic content of ingested matter (Fig. 4C and 5. Table ?}. NOAR was essentially controlled by quantity (filtration rate) and quality (OCI) of food passing through the digestive system (Fig. 4C, Table 3). The ab- sorption rate across the experiments varied from 21.84 mg h" (TPM .3.3.18 mg L '. OCS 0.18) to -0.69 mg h"' (TPM 10.09 mg L"'. OCS 0.10). The differential equations, logical functions, and starting values of the state variables used to couple the equations describing the filter-feeding processes for P. perna in STELLA are listed on Table 4. We produced a robust model with relatively low com- plexity and specificity. Figure 6A depicts the conceptual diagram of the P. perna feeding process as a function of TPM and OCS. The sub-model inserted inside the "ingested matter" variable (Fig. 6B ) reproduces the absorption of organic matter and the passage of inorganic matter as inert material through the gut. As the model was based on natural seston in both turbid and clear environments and feeding rates measured in these environments, we believe that it has incorporated feeding adaptations by P. perna for both kinds B Figure 4. Perna perna. The relationship between (.A I net organic se- lection elTiciency (NOSE, fraction), total particulate matter (TPM. mg L"') and organic content of ingested (OCI. fraction): (B) ingestion rate (IR, mg h"'), TPM and net organic ingestion rate (NOIR. mg h'); (C) net organic absorption rate (NO.\R, mg h"'), filtration rate (mg h"') and OCI. Refer to Table 3 for equations and statistics. 130 SUPLICY ET AL. O • • -0,2 0.0 02 1.0 12 1.4 04 06 08 OCI (fraction) Figure 5. Penia perna. The relationship between the organic content of ingested (OCI, fraction) and the net absorption efficiency from ingested organics (NAEIO, fraction). Refer to Table 3 for equaliiins and statistic. coefficient. Similarly, the denominator measures the variation in the coefficient of interest divided by its standard value. This equa- tion compares the percentage change in the model outputs with a given percentage change in one of the model parameters. The value of (S) was averaged for positive and negative variations and the results of the model outputs (absorbed matter, pseudofeces, and feces produced) for the coefficients relating TPM and OCS are shown in Table 5. The output most sensitive to variation in the relationship between seston TPM and OCS was pseudofeces pro- duction, as a result of increased or decreased rejection rate. DISCUSSION This study showed that P. perna, like other mussels, controlled its feeding mechanisms to achieve an optimum organic absorption rate independent of fluctuations in seston concentration and qual- ity. It is important to note that the range of TPM recorded was within normal values during the year for other bivalve aquaculture locations in Southern Brazil (Suplicy, unpub. data). Therefore, the TPM range experienced in the experiments and included in the model are directly applicable to Brazilian shellfish famis condi- tions. Although seasonal changes in feeding physiology were not examined in this study, time series data of TPM, POM, and OCS from 1998 to 2002 do not suggest strong seasonal changes in food availability in the sub-tropical waters of Santa Catarina, (Suplicy et al. unpublished data). Similarly, the condition inde.x of P. perna does not follow a seasonal trend, as seen in Mylilus echtlis (Navanxi & Iglesias 1995), because spawning occurs throughout the year with small peaks in summer, autumn and spring (Marques et al. 1991 ). Therefore, we believe that the findings reported here can be used to predict feeding physiology throughout the year. Food availability (TPM and OCS) was the main forcing func- tion of the models produced, therefore characterizing the available seston is of primary importance to generate a model to predict food uptake by P. perna. Data for Southern Brazil showed that the organic content of available food decreased as TPM increased, a common pattern in many estuaries and sheltered bays both in teiTiperate and tropical waters (Hawkins et al. 1996a, 1998b). This reduction of the organic proportion is a function of the dilution of organic particles when resuspended silt increases particulate inor- ganic matter on the water column (Frechette & Grant 1991. Wid- dows et al. 1979) The methods used in this study to estimate clearance rates of filter feeders were less accurate than the methodology proposed by Hawkins et al. (1998a, 1999) for measurements using natural seston. The most appropriate method to accurately measure clear- ance rates by bivalves is controversial (Cranford 2(M1. Riisgard 2001. Widdows 2001 ). As new methods are being developed, new models about how these animals control their food uptake are being produced. It is agreed that mussels do not always filter at their maximal rate in their natural environment (Riisgard 2001. Widdows 2001 ). This may be due to a regulation of feeding pro- cesses in response to changes in quantity and quality of suspended particles, salinity, temperature, and the presence of pollutants in the water (Widdows 2001 ). In this study only ll^c of the variation clearance rates of mussels using TPM and OCS as independent variables was explained, and the significant proportion of the re- maining variance in clearance rate in POM was not. In their ex- periments, however, Hawkins et al. (1999) increased the amount of the variability in clearance rate explained from 13-,').^'/f when they included Chi and TPM as independent variables instead of only POM. Although all precautions proposed by Iglesias et al. (1998) in the use of the biodeposition method for suspension-feeding TABLE 4. Equations used in the formulation of feeding physiology model in STELLA. TPM = GRAPH (time-series) OCS = 1/(2.55 -hO.47 * TPM) PIM = 0.22 -1-0.81 * TPM POM = TPM-PIM PR = 68.77-0.12 TPM-370.10 OCS -i- 0.07 TPM" -i- 565.80 OCS" Fillered matter (t) = Filtered matter (t - dt) + (FR - RR - IR) * dt INIT Filtered matter = 219.81 RR = 52.43 + 0.97 TPM-362.47 OCS -i- 0.02 TPM" + 589.79 OCS" Pseudofeces (5) = pseudofeces (t - dt) -f (rejection) * dt IR = filtration-rejection Ingested (t) = ingested (t - dt) + (ingestion' - NIIR - NOIR) * dt INIT ingested - 36.46 NOIR = 1.37 - (.1.23 TPM -i- 0.1 1 IR -i- 0.01 TPM- + 0.004 IR" NIIR = mgested-NOIR Inorganic (t) = inorganic (t - dt) ■(- (NIIR - IM on gut) * dt Organic (t) = organic (t - dt) + (NOIR - OM on gut) * dt OM on gut = organic Im on gut = inorganic INIT organic = 13.17 INIT inorganic = 23.29 Ingested matter = food on gut + organic + ingested -i- inorganic Food on gut (1) = food on gut (t - dt) -i- (OM on gut -i- IM on gut - absorption' - egestion') * dt INIT food on gut = 0 NOSE = 0.30 - 0.21 PIM -i- 1.03 POM + 0.01 PIM" - 0.20 POM- OCI = 0.13 - 0.001 TPM + 0.27 NOSE -t- 0.0002 TPM" -i- 0.19 NOSE- NOAR = -2.62 -I- 0.012 RF -i- 15,73 OCI -i- 0.0001 FR- - 9.22 OCI" Ahsorption' = NOAR Absorption = absoiption' Absorbed matter (t) = absorbed matter (t - dt) + (absorption) * dt INIT absorbed matter = 0 Egestion' = IM on gut + (NOIR-NOAR) Egestion = egestion' Feces (t) = feces il - do -i- (cgestioni * dt Modeling Feeding Behavior in Perna perna 131 absofbed matter -^ pseudofaeces B organic organic matter Figure 6. (A) Diagram of the feeding processes of a general niter-fceding bivalve, used on the modeling of P. perna feeding physiology. (B) Diagram of the sub-model of a mussel gut showing the absorption of organic matter and feces production. Refer to Tables 2 and 3 for variables and acronyms and Table 4 for logical and differential equations. measurements were taken in this study, it seems that the new methodology proposed by Hawkins et ai. (1998b, 1999) is more appropriate for studies using natural seston. It seems that qualita- tive features of seston may be just as important as availability of food in mediating feeding responses (Hawkins et al. 1998b). The general trend for decreasing clearance rates as seston concentra- tions increase, however, is seen in other studies (Hawkins et al. 1999, Hawkins et al. 1998b, Wong & Cheung 2001). There are many methods to quantify concentration and organic content of seston in feeding experiments. Most use mass measurements of total particulate matter available in the seston (TPM, mg L"'), pailiculate organic matter available in the seston (POM, mg L"'), and the ratio between these two variables, which is the organic content of seston (OCS. fraction). Recent findings suggest that clearance rate is primarily dependent on seston availability mea- sured in terms of total volume, rather than mass. This helps to explain the confusing variation in clearance rate reported by many studies and stresses a need to consider volumetric constrains in bivalve feeding studies (Hawkins et al. 2001 ). More detail about the seston organic fraction can be obtained if the carbon;nitrogen ratio is measured, which can vary from <4 to >26 (Bayne & Hawkins 1990). The measurement of the biologically available 132 SUPLICY ET AL. 160 140 -. 120 E — 80 cr ^ 60 1 0 08 - I"' o m ^ 04 UJ to O 02 00 E, a: 40 TPM (mg I"') 0 10 20 30 40 50 TPM (mgr') Figure 7. Predictions of the P. pcnia filter-feediii}; model produced on STELLA. (A» nitration rate IFR, nig h '), (B) rejection rate (RR, nig h"'). (CI ingestion rate (IR. mg h '), ID) selection efficiency (NOSE, fraction), (E) organic content of ingested matter (OCL fraction), and (F) net organic absorption rate (NOAR, mg h~'), in the range of total particulate matter (TPNL mg L ') observed in this study. organic carbon and nitrogen in the water and in associated biode- posits can provide, not only more accurate measurements of the clearance rate, but also important information about the absorption of these elements by filter feeders. The biodeposiljon approach demands that the gut residence time is correctly calculated to generate accurate physiologic feed- ing rates. As starved animals were used to estimate gut passage time this may have over-estimated the normal passage time. How- ever, our estimates are comparable to those from other biodepo- sition studies using Penui canaliculus, in which the gut passage time for non-starved mussels was 80 min. and no delay time was assumed for Perna viridis (Hawkins et al. 1998al. Perna perna appeared to selectively enrich the organic content of ingested matter by rejecting particles of higher inorganic con- TABLE 5. Sensitivity analysis of absorbed matter, pseudofeces and feces production for the coefficients a and h in the equation OCS = l/(a + b * TPM). Absorbed Matter Pseudofeces Feces 0.2(12 0.2-^2 0.(1.^7 0.7-14 0.118 0. 1 36 tent before ingestion. This selection efficiency was a function both of filtration rate and the proportion between inorganic and organic particulated matter available in the water. The increase in selection efficiency at higher filtration rates is important, because this helps to maintain nutrient acquisition independent of fluctuations in seston organic content (Hawkins et al. 1998a). Extreme values of net organic selection efficiency measured in this study (NOSE >l or <0) must be considered with caution as they are probably mea- surement errors associated inadvertently with collecting settled sediment when collecting biodeposits. This would effectively alter the organic ratio of pseudofeces. Extreme values were observed in 15% of measurements. Nevertheless, NOSE values recorded in this study (>0.7) suggest that P. perna is efficient in selecting organic particles available in the seston. Hawkins et al. (1996a) recorded NOSE values of up to 0.5 in M. edulis. and Hawkins et al. ( 1998b) report maximum NOSE of 0.7 for P. viridis. Maximum net organic ingestion rate (NOIR) recorded for P. perna was 24.05 mg h"' and occurred when TPM was 33.93 mg L"' and OCS was 0.18. This is similar to values obtained for P. canaliculus in New Zealand, that showed maximum organic in- gestion rate of 27.3 ± 6.3 mg h"' (Hawkins et al. 1999), and for P. viridis in Malaysia with a recorded rate of 24.8 ± 3.6 mg h"' (Hawkins et al. 1998a). These rates are considerably higher than the maximum organic ingestion rate of 6.5 mg h"' reported for M. edulis (Hawkins et al. 1997). The growth rates of P. perna in southern Brazil are among the fastest reported for mussels in the Perna genus, reaching commercial size (80 mm) in 8-10 mo (Su- plicy. unpub. data). This rapid growth is probably related to higher weight-specific rates of energy acquisition and higher water tem- peratures in the sub-tropical waters of southern Brazil. Data from this study suggested that P. perna takes advantage of the abundant organically rich seston available in Brazilian waters throughout the year by maintaining high ingestion rates. There is evidence that when ingestion rate is high absorption efficiency is high and gut residence time is short (Bayne et al. 1988). Fuilher- more, the proportion of gut volume occupied by ingesta may vary, thereby facilitating an increase in absorption efficiency with little change in the gut passage time (Bayne et al. 1987). Widdows et al. ( 1979) report that absorption efficiency declines as ingestion rate increases and food progresses from the digestive gland to the in- testine. However, this pattern may be counterbalanced by elevated organic content of ingested matter due to selection processes (this study, Hawkins et al. 1999) that positively increase the absorption efficiency and ultimately the absorption rate. Similarly to the con- siderations raised for NOSE values, negative absorption rate val- ues are not biologically meaningful and must be considered with caution as these could be caused by collection of inorganic sedi- mented material together with mussel feces. Negative absorption rates were measured in 7% of measurements. The integration of all equations from Table 4 with STELLA software resulted in a reductionistic and deterministic non-linear model that reproduces the feeding processes of P. perna in both clear and turbid environments. The general conceptualization of the diagram was based on the description of the bivalve filter- feeding process provided at the TROPHEE workshop (Bayne 1998. Hawkins et al. 1998b), and final equations were based on intensive measurements that enabled calibration of the outputs. This feeding model may not be a perfect reproduction of the bi- valve feeding process, but the objective is to provide a useful tool to understand and predict feeding processes of this species. The model includes a complete sequence of steps in the feeding process Modeling Feeding Behavior in Pekna perna 133 that may cause an accumulation of predictive error (Grant & Baciier 1998). Its value lies in the ability to provide an understand- ing of the interaction between a mussel farm and the environment, for example, the amount and organic content of biodeposits re- leased into the water column and sediment beneath the farm. Sensitivitv analysis indicated that model predictions of ab- sorbed matter and feces production were less affected by changes in the relationship between TPM and OCS than model prediction of pseudofeces production. This analysis suggests that predicted absorption would stay reasonably invariable if the model is applied to environments with different seston concentration and organic content. Therefore, mussels maintain a reasonably constant or- ganic ingestion rate in varying seston conditions by compensating for low organic content of the seston through adjusting selection efficiency and rejection of inorganic matter as pseudofeces. This feeding model can be used as an important tool for the understanding of how P. pcniu interact with the culture environ- ment. Current studies are under way to integrate this feeding model w ith energy budget and population dynamics of P. perna. Further coupling of the P. perna biologic models with physical models of seston hvdrodynaniics and models of primary production are also planned, and this approach will allow the development of cairying capacity analysis for suspended mussel culture in sub-tropical en- vironments like the southern Brazilian coast. ACKNOWLEDGMENTS The research was supported by CNPq, a Brazilian govcrnnienl agency for scientific and technologic development. The authors thank two anonymous reviewers for their valuable criticism and comments of the original manuscript. LITERATURE CITED Bayne. B. L. & A. J. S. Hawkins. 1990. Filter-feeding in bivalve molluscs: control of energy balance. In: J. Mellinger. editor. Animal nutrition and transport processes. I. Nutrition in wild and domestic aniniaK. Karger. Basel, pp. 70-8.^. Bayne. B. L. & R. C. Newell. I9S3. Physiological energetics of marine molluscs. In: K. M. Wilbur. A. S. Saleuddin. editors. The Mollusca. Vol. 4. New York: Academic Press, pp. 407-5 Li. Bayne. B. L. 1993. Feeding physiology of the bivalves: lime-dependence and compensation for changes in food availability. In: R. Dame, editor. Bivalve filter feeders in estuarine and coastal ecosystems processes. NATO ASI Series. G 33 Berlin: Springer-Verlag. pp. 1-24. Bayne. B. L. 199X, The physiology of suspension feeding by bivalve mol- luscs: an introduction to the Plymouth 'TROPHEE" workshop. J. Exp. Mar. Biol. Ecol. 219:1-19. Bayne. B. L.. A. J. S. Hawkins & E. Navarro. 1987. Feeding and digestion by the mussel Myliliis edulis L. (Bivalvia: Mollusca) in mixtures of silt and algal cells at low concentrations. / Exp. Mar. Biol. Ecol. 1 1 1 : 1-22. Bayne. B. L., A. J. S. Hawkins & E. Navarro. 1988. Feeding and digestion in suspension-feeding bivalve molluscs: the relevance of physiological compensations. Am. Zool. 28:147-159. Bayne. B. L.. J. I. P. Iglesias. A. J. S. Hawkins. E. Navarro. M. Heral & J. M. Deslous-Paoli. 1993. Feeding behaviour of the mussel. Mylilus (■(/»/«: respon,ses to variations in quantity and organic content of the seston. J. Mar. Biol. Ass. U.K. 73:813-829. Bayne. B. L., D. W. Klumpp & K. R. Clarke. 1984. Aspects of feeding, including estimates of gut residence time, in three mytilid species (Bi- valvia, Mollusca) at two contrasdng sites in the Cape Peninsula. .South Africa. Oecologia 64:26-33. Berry, P. F. & M. H. Schleyer. 1983. The brown mussel Perna pcnia on the Natal coast. South Africa: utilization of available food and energy budget. Mar. Ecol. Prog. Ser. 13:201-210. Brylinski. M. & T. W. Sephton. 1991. Development of a computer simu- lation model of a cultured blue mussel {.ifyiilns edulis) population. Can. Tech. Rep. Fish, and .\qualic Sci. 1805 (vii + 88 pp.). Cranford. P. J. 2001. Evaluating the "reliability" of filtration rate measure- ments in bivalves. Mar. Ecol. Prog. Ser 215:303-305. Frechette. M. & J. Grant. 1991. An in situ estimation of the effect of the wind-driven resuspension on the growth of the mussel Mytihis edulis L. / E.xp. Mar. Biol. Ecol. 148:201-213. Gardner. J. P. A. & R. J. Thompson. 2001. Naturally low seston concen- tration and the energy balance of the Greenshell mussel (Perna canali- cuius) at Island Bay. Cook Strait. New Zealand. N.Z. J. Mar. Frcsliw. Res. 35:457-^68. Gardner. J. P. A. 2002. Effects of seston variability on the clearance rate and absorption efficiency of the mussel Aulacomya maoriami. Mylilus galloprovincialis and Perna canaliculus from New Zealand. / Exp. Mar. Biol. Ecol. 268:83-101. Grant. J. & C. Bacher. 1998. Comparative models of mussel bioenergetics and their validation at field culture sites. / Exp. Mar. Biol. Ecol. 219:2I-t4. Grant. J. 1993. Working group report: modelling. In: R. Dame, editor. Bivalve filter feeders in estuarine and coastal ecosystems processes. NATO ASI Series, G 33 Beriin: Springer-Verlag. pp. 549-555. Griffiths. C. L. & R. J. Griffiths. 1987. Bivalvia. In: T.J. Pandian. F.J. Vemberg. editor. Animal energetics. Vol. 2: Bivalvia through Reptilia. New York: Academic Press, pp. 1-88. Hawkins. A. J. S., B. L. Bayne, S. Bougrier, M. Herat. J. I. P. Iglesias. E. Navarro. R. F. M. Smith & M. B. Urrutia. 1998a. Some general rela- tionships in comparing the feeding physiology of suspension-feeding bivalve molluscs. J. E.xp. Mar. Biol. Ecol. 219:87-103. Hawkins. A. J. S.. J. G. Fang, P. L. Pascoe. J. H. Zhang. X. L. Zhang & M. Y. Zhu. 2001a. Modelling short-term responsive adjustments in particle clearance rate among bivalve suspension-feeders: separate unimodal effects of seston volume and composition in the scallop Chlamys fur- reri. J. Exp. Mar. Biol. Ecol. 262:61-73. Hawkins, A. J. S.. J. G. Fang, P. L. Pa.scoe, J. H. Zhang. X. L. Zhang & M. Y. Zhu. 2001b. Modelling shon-term responsive adjustments in par- ticle clearance rate among bivalve suspension-feeders: separate unimo- dal effects of seston volume and composition in the scallop Chlamys farreri. J. E.xp Mar. Biol. Ecol. 262:61-73. Hawkins. A. J. S.. M. R. James, R. W. Hickman. S. Hatton & M. Weath- erhead. 1999. Modelling of suspension-feeding and growth in the green-lipped mussel Perna canaliculus exposed to natural and experi- mental variations of seston availability in the Marlborough Sounds. New Zealand. Mar Ecol. Prog. Ser. 191:217-232. Hawkins, A. J. S., P. N. Salkeld, B. L. Bayne & E. Gnaigeri. 1996a. Novel observations underlying the fast growth of suspension-feeding shellfish in the turbid en\lronments: Mylilus edulis. Mar. Ecol. Prog. Ser. 131: 179-190. Hawkins, A. J. S.. R. F. M. Smith. B. L. Bayne & M. Heral. 1996b. Novel observations underlying the fast growth of suspension-feeding shellfish in the turbid environments: Mylilus edulis. Mar. Ecol. Prog. Ser. 131: 179-190. Hawkins, A. J. S., R. F. M. Smith, S. Bougrier. B. L. Bayne & M. Heral. 1997. Manipulation of dietary conditions for maximal growth in mus- sels. Mylilus edulis L., from the Marennes-Oleron Bay, France. Aquat. Living Resour. 10:13-22. Hawkins. A. J. S.. R. F. M. Smith. S. H. Tan & Z. B. Yasin. 1998b. Suspension-feeding behaviour in tropical bivalve molluscs: Perna viri- dis. Crassotrea helcheri. Crassostrea iradelei. Saccostrea cucculala and Pinctada margariferu. Mar. Ecol. Prog. Ser. 166:173-185. Iglesias. J. I. P.. M. B. Urrutia, E. Navarro & I. Ibarrola. 1998. Measuring feeding and absorption in suspension-feeding bivalves: an appraisal of the biodeposition method. J. E.xp. Mar. Biol. Ecol. 219:71-86. 134 SUPLICY ET AL. James, M. R.. M. A. Weatherhead & A. H. Ross. 2001. Size-specific clearance, excretion and respiration rates, and phytoplankton selectivity for the mussel Penia caiuiliciihis at low le\els of natural food. N.Z. J. Mar. Fresim: Res 35:73-86. Marques. H. L. de A.. R. T. L. Pereira & B. C. Correa. 1991. Study on reproductive and settlement cycles of Perna pema (Bivalvia: Myril- idae) at natural beds in Ubatuba .shore — Sao Paulo Stale. Brazil. Bo- letim Institulo Pesca Sao Paulo. 18:73-81. Navarro, E. & J. 1. P. Iglesias. 1995. Energetics of reproduction related to environmental variability in bivalve molluscs. Haliotis 24:43-55. Navarro, E., J. 1. P. Iglesias, A. Perez Camacho & U. Labarta. 1996. The effect of diets of phytoplankton and suspended bottom material on feeding and absorption of raft mussels {Mylihis gallopioviiiciali.s Lmk.). J. Exp. Mar. Biol. Ecol. 198:175-189. Navarro, E., J. I. P. Iglesias, A. Perez Camacho. U. Labarta & R. Beiras. 1991. The physiological energetics of mussels iMyriliis galloprovin- cialis Lmk) from different cultivation rafts in the Ria de Arosa (Galicia. NW Spain). Aquaculnire 94:197-212. Newell. C. R. & D. E. Campbell. 1998, MUSMOD©, a production model for bottom culture of the blue mussel, Mytilus edulis. J. Exp. Mar. Biol. Ecol. 219:171-203. Perez Camacho, A. & R. Gonzales. 1984. La filtracion del mejillon {Myri- lus edulis L.) en laboratorio. In: Actas do Primeiro Seminario de Cien- cias do Mar. As Rias Galegas, Cuadernos da Area de Ciencias Mannas, Seminario de Estudos Galeaos, 1:427—437. Riisgard, H. U. 2001. On measurements of filtration rates in bivalves — the stony road to reliable data: review and interpretation. Mar. Ecol. Prog. Ser. 211:275-29L Scholten. H. & A. C. Smaal. 1998. Responses of Mytilii.^ edulis L. to varying food concentrations: testing EMMY, an ecophysiological model. J. Exp. Mar. Biol. Ecol. 219:217-239. Smaal. A. C. & H. A. Haas. 1997. Seston dynamics and food availability on mussel and cockle beds. Esruarine. Coastal Shelf Science 45:247- 259. Smaal. A. C, T. C. Prins, N. Dankers & B. Ball. 1998. Minimum require- ments for modelling bivalve carrying capacity. Aijuai. Ecol. 31:423- 428. van Erkon Schurink, C. & C. L. Griffiths. 1992. Physiological energetics of four South African mussel species in relation to body size, ration and temperature. Coinp. Biochem. Physiology 10IA:779-789. Widdows, J. 2001. Bivalve clearance rates: inaccurate measurements or inaccurate reviews and misrepresentation? Mar. Ecol. Prog. Ser. 221; 303-305. Widdows, J., P. Fieth & C. M. Worral. 1979. Relationships between seston, available food and feeding activity in the common mussel Mytilus edulis. Mar Biol. 50:195-207. Wong, W. H. & S. G. Cheung. 2001. Feeding rhythms of the green-lipped mussel, Perna viridis (Linnaeus. 1758) (Bivalvia: Mytilidae) during spring and neap tidal cycles. / E.\p. Mar. Biol. Ecol. 257:13-36. Joiinuil „f Shflllhh Research. Vol. 22. No. 1, 135-140. 200.^. PHENOTYPES OF THE CALIFORNIA MUSSEL, MYTILUS CAUFORNIANVS, CONRAD (1837) JORGE CACERES-MARTINEZ,'* MIGUEL A. DEL RIO-PORTILLA.' SERGIO CURIEL-RWIIREZ GUTIERREZ,' AND IGNACIO MENDEZ GOMEZ HUMARAN" ^ Departamento de Aciiicultura del Centra de Investigacion Cientifica y de Ediicacion, Superior de Ensenada. A. P. 2732 C. P. 22860 Ensenada. Bajci California. Mexico: 'Instiruto Nacional de la Pesca, Pitdi^oras 1320 6° Piso, Col. Sia. Cruz Aroyac. C.P. 03310. Mexico D.F. ABSTRACT The morphological variability of Mytilii.s ediilis complex species has been the subject of a variety of studies. However, the morphological variability of Mytiliis califomiumis has not been studied. We found that there are some M. californiamis without some of the shell characteristics mentioned by Conrad ( 1837) in the original description of this species. The most remarkable difference was the absence of radial ribs on the exterior of the shell: thus, we tested the presence of at least two phenotypes in M. ealiforniainis. Six hundred ninety five M. ealiforniainis of different sizes were collected from the locations La Mina del Fraile. La Bufadora, and La Salina in Baja California. For comparison, 58 M. i^atloprovincialis were collected from an aquaculture facility at Rincon de Ballenas in Bahi'a de Todos Santos. Baja California. Fourteen morphometric measures and the weight of the shell were measured and a principal coinponent analysis (PCA) and a logistic regression (LR) were carried out to tlnd differences between mussels studied and for obtaining a prediction to assign the phenotypes. The presence of ribs, small ligament margin, a narrow posterior byssal retractor muscle scar, and shell weight were the discriminating characters between two groups in M. californiamis. These findings confirm the presence of at least two phenotypes in this species, in all mussel sizes and the studied locations. The LR correctly assigned 99.28% of the shells to each phenotype. and it considered only eight out of the fifteen morphometric measures. The PCA showed a clear morphologic difference between both phenotypes of A/, ealifornianiis and A/, gulloprovineialis. The original description of this species by Conrad in 1837 was done taking into account only the phenotype with ribs. KEY WORDS: Mytilus ealifornianiis. Mytiliis eihilis complex species, morphological variability, phenotypes INTRODUCTION MATERIALS AND METHODS The marine mussels of the genus Mytilus are widely distributed in boreal and temperate waters of the Northern and Southern Hemispheres (Soot-Ryen 1955), Prior to protein separation and molecular genetics, about nine species of the genus Mytilus were recognized (Gosling 1992). Today, about five species are consid- ered belonging to this genus: Mytilus californiamis. Mytilus cor- ».«■;(.? (Gould 1861), Mytilus edulis (Linne 1758). Mytilus gallo- provincialis and Mytilus. trossulus (Gould 1850) (Seed 1992), The three later species are considered to be the M. edulis complex species because they are very close in their external shell mor- phology. These species have caused a variety of studies for their differentiation, taking into account shell morphology, allozyme, and molecular genetics (Beaumont et al, 1989. Figueras & Figueras 1983, McDonald & Koehn 1988. Koehn 1991, McDonald etal. l99l,Gelleretal. 1994. Inoue et al, 1995. Rawson & Hilbish 1995. Ohresser et al, 1997), Mytilus californianus has never been questioned as a separate species from the Mytilus edulis-comp\ex because of its characteristic radiating ribs, strong growth lines, and heavy shell in larger specimens: these characters allow easy dif- ferentiation from the other species in adult stage (Soot-Ryen 1955. Koehn 1991). During a field study of Mytilus californianus in an exposed rocky shore of the West Coast of Baja California, Mexico, we found some specimens with typical external characteristics of the shell described by Conrad in 1837. Other individuals, however, showed a smooth shell without coarse ribs, similar to the M. edulis complex form, but with heavy shells. A question arises from this observation, are there two or more phenotypes of M. califor- nianus'l This study focused on answering this question. *Corresponding author. Mailing address: Department of Aquaculture. CICESE. PO Box 434844, San Diego, CA 92143 In March 1997, 129 M. californianus (size range from 16,8- 1 13,5 mm, mean size 59,1 mm) were collected from an exposed rocky shore along the intertidul zone during low tide in La Mina del Fraile. B. C, Mexico, In August 2()(X), 278 mussels were col- lected from La Salina (size range from 27.6-98.1 mm, mean size 56.9 mm) and 288 from La Bufadora (size range from 44.7-88.1 mm. mean size 54.1 mm). B. C. Mexico, both areas exposed rocky shores, and the mussels were collected during low tide along the intertidal zone. Additionally. 58 M. galloprovincialis were ob- tained from culture long-lines placed at Bahi'a de Todos Santos, B,C. (size range from 47.2-85.3 mm. mean size 61.4 mm) and they were used to compare the morphological characteristics with M. californianus (Fig. 1 ). The shells of ail mussels were cleaned with a brush and water stream and dried in an oven at 40"C overnight. The following morphometric dimensions were measured for differentiation among mussel groups and species (Fig. 2): number of ribs on the external shell (rib), maximum shell length (si), height (sh) and width (sw), the position of maximum shell width (a) along the dorso- ventral axis, the maximum dimensions of the anterior (aams) and posterior (pam) adductor muscle scars, the maximum length (Ibr) and width (wbrs) of the posterior byssal retractor muscle scar, the location of the center of the posterior adductor muscle scar along both the anterior-posterior (pam-pm) and dorso ventral (pam-vm) axes, the size of the hinge plate (hp) and number of hinge teeth, the distance between the palial line and the \entral shell margin (pl-vm) midway along the shell, and ligamentary margin (Im), All measurements were taken with an electronic digi- tal caliper to the nearest 0. 1 mm and were in accordance with those taken by Beaumont et al, ( 1989), The dry shell weight (w) was also measured for all mussels and it was included in the analyses, A principal component analysis (PCA) was carried out to discrimi- nate between phenotypes, followed by a logistic regression (LR) 135 136 Caceres-Martinez et al. _32"3r Pacific Ocean V La Salina \ Baja California _32« ^^ <^\ Ensenada Baja V California' Mussel culture facilityN. • a] La Bufadora \ _30°3r ^- — f 1 1 ,„ /La mina del Fraile 1 4 Figure 1. Map showing the three exposed rod^y shore localities where Mytihis californiaiius was collected: La Mina del Fraile. La Bufadora. and La Salina. The blue mussel yfyliliis f;allopr(niiiciali\ was collected from a culture facility at Bahia de Todos Santos, Baja California. Mexico. (Sokal & Rohlf 1995) to fit the mussel phenotypes. A two way ANOVA was used to determine possible differences between mus- sel size among locations and phenotypes. A comparison through the PCA between both phenotypes of M. culifonmimis with M. gaUoprovincialis was carried out. These analyses were done using the JMP statistical package by SAS Institute Inc. RESULTS Fifteen piincipal axes were extracted from the morphological and shell weight data of M. califomiamts (Table I). The first component explained 16% of total variance and was considered as a size axis. A low correlation of size with number of ribs sug- gests that the number of ribs does not change w ith mussel si/e. The second component accounted for 1% of the variation indicating morphological differences. Mussels with a high number of ribs were correlated with this second component separating two groups (Fig. 3). Also, in the second component, mussels with higher shell weight (w). but with small ligament margin dm) and a naiTow posterior byssal retractor muscle scar (wbrs) were coiTelated. The rest of the components had eigen values smaller than the unit accounting for about 139<^ of the total observed variance and thus, no further explanation is necessary (Table 1 ). These data provide statistical support to validate the presence of two phenotypes in M. californiwms: A (with ribs) and B (without ribs), and they were visually differentiated in mussels of different sizes (Fig. 4). After separating both groups in all locations. 689f of the total mussels belong to phenotype A and the rest to phenotype B. Both phenotypes of M. caUfornianus were present in the three locations. The two way ANOVA showed size differences among mussel from different locations. (F -.f,^^ = 6.58. P = 0.001 ). but the phenotype mean size was similar (F , ^^1) ~ 0.02. P = 0.892) without interaction (F ,f,gg = 2.11. P = 0.122). Once the PCA differentiated two phenotypes. the LR (Sokal & Rohlf 1995) was used to determine whether it was possible to assign any M. ctiUfonuanus to a particular phenotype. taking into account morphological \ariables. excluding the number of ribs. The LR considered only eight morphological measures from the original fifteen to assign any mussel to a particular phenotype. (X- 1 84.73. P < 0.0001 ; Lack of fit: x" 684.5. P = 0.51). The coefficients of the eight morphometrical variables were positive for: shell length (si = 0.104) and height (sh = 0.247). posterior adductor muscle scar (pain = 0.483). the dis- tance between the palial line and the ventral shell margin (pl-vm = 0.708). and weight (wO. 145); while the shell width (sw = -0.281). the position of maximum shell width (a = -0.333). and the ligamentary margin (Im = -0.348) were negative. After ap- plying the LR we found that 99.28% were correctly assigned to each phenotype. Thus, the visual. PCA and LR confirm the pres- ence of two phenotypes in the Californian mussel. Results of the PCA between morphometric data and v\eight of Figure 2. Morphometric dimensions measured for Mytilus califoniiamis and Myliliix galldprovincialis: number of ribs on the external shell (ribi, maximum shell length (si), height (sh) and width (sw), the position of maximum shell width (al along the dorso-ventral axis, the maximum dimensions of the anterior (aams) and posterior (pam) adductor muscle scars, the maximum length (Ibrl and width (wbrs) of the posterior byssal retractor muscle scar, the location of the center of the posterior adductor muscle scar along both the anterior-posterior (pam-pm) and dorso ventral (pam-vm) axes, the size of the hinge plate (hp), and number of hinge teeth, the distance between the palial line and the ventral shell margin (pl-vm) midway along the shell and ligamentary margin (Im). Phenotypes of the California Mussel 137 TABLE 1. Eigenvalues, explained variance (^rl. cumulative explained variance I "^i- 1 and eigenvectors (rounded to luo decimal places) from the principal component analysis of Myliltis califoniUiiiiis niorphometric data from the Facillc coast of Baja California. Principal Components 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Eigenvalue 11.45 1.07 0.59 0.45 0.29 0.25 0.22 O.IS 0,14 0,11 0.08 0.08 0.05 0.03 0.02 Variance!*) 76.31 7.12 3.93 3.02 1.94 1 .69 1.46 1.17 0.96 0,71 0.51 0.50 0.35 0.21 0.12 Cum. var.(%) 76.31 83.43 87.36 90.37 92.31 94.00 95.46 96.64 97.59 98.3 98.81 99.32 99.67 99.88 100.00 Eigenvectors rib 0.02 0.95 0.17 -0.02 0.02 -0.02 -0.06 0.06 0.20 0,07 0,04 0,09 0.04 -0.03 0.01 si 0.29 -0.04 0.02 -0.15 -0.06 -0.10 -0.10 -0.03 0.20 0.03 -0. 1 3 -0. 1 2 0.19 0.16 -0.86 sh 0.28 0.05 -0.11 -0.01 0.04 0.01 0.03 0.32 0.07 -0.23 -0.16 -0.12 -0.82 0.15 -0.03 sw 0.29 -0.06 0.04 -0.09 0,02 -0.12 -0(16 -0.12 0,04 -0,01 0,24 0, 1 7 -0.17 -0.86 -0.11 a 0.27 -0.06 -0.27 0.09 0.14 -0.09 0.15 0.62 0.22 -0.30 0.07 0.26 0.42 -0.02 0.12 aams 0.26 0.01 0.05 -0. 1 1 -0.04 0.92 -0.14 0.03 -0.13 -0.09 0.06 0.01 0.10 -0.04 -0.01 pam 0.27 0.05 0.14 -0.33 0.29 -0.08 0.48 0.17 -0.39 0.34 0.24 -0.35 0.06 0.05 0.04 Ibr 0.28 -0.04 0.00 0.03 -0.08 -0.05 -0.20 -0.13 0.45 -0.04 0.00 -0.70 0.16 -0.07 0.35 wbrs 0.21 -0.13 0.76 0.59 0.04 -0.01 0.07 0.10 0.00 0.01 -0.05 0.06 0.00 0.04 -0.01 pam-pni 0.28 0.04 0.14 -0.31 0.00 -0.13 0.03 -0.17 -0.24 -0.20 -0.75 0. 1 3 0. 1 5 -0.08 0.22 pam-vm 0.28 0.00 0.15 -0.22 -0.04 -0.21 -0.17 -0.33 -0.12 -0.45 0.5 1 0.20 -0.01 0.38 0.11 hp 0.25 0.05 -0.34 0.35 0.61 0.09 0.16 -0.48 0.11 0.09 -0.07 0. 1 3 -0.02 0.10 -0.01 Im 0.27 -0. 1 8 0.04 -0.23 -0.16 0.00 -0. 1 3 0.04 0.33 0.64 -0.01 0,41 -0.10 0.21 0.23 pl-\iii 0.26 0.10 -0.27 0.32 -0.07 -0.17 -0,57 0.16 -0.54 0.23 0.00 -0.08 0.06 0.02 0.02 u 0.25 0.12 -0.25 0.28 -0.69 0.02 0.51 -0.20 -0.08 0.01 0.03 0.02 0.02 0.01 0.00 M. ccdifornianus and M.gaUoprovincialis are shown in Table 2. The fist component explained 71% of the total variance and was also considered a size axis. The number of ribs had a low corre- lation with this axis. The second component explained 10% of the total variance. The number of ribs (rib) and the shell width (sw) were positively correlated with this component whereas the width of the adductor muscle scar (wbrs) was negatively correlated. Components 3 to 15 accounted for 19.4% of the total variance, but their eigen values were smaller than one and they are not explained further. The graphic presentation of the component scores shows a Figure 3. Principal component scores plots between PC2 vs. PCI for M. calif ornianiis. Phenotype A. open circles; Phenotype H, bold squares. clear difference between phenotypes of M. californianus and among these phenotypes and M. gaUoprovincialis (Fig. 5). DISCUSSION Figure 4 shows different shell characteristics among M. cali- fornianus specimens, and the PCA and LR support this visual perception confirming that there are two phenotypes in M. cali- fornianus. one with ribs and the other with a smooth shell, and Figs. 4 and 5 show a morphological differentiation between both phenotypes of M. californianus and M. galloprovincialis. The original description by Conrad (1837) for Mytilus califor- nianus was done from specimens collected by Thomas Nuttal in upper California. Conrad describes "shell ovate elongated, in- flated; anterior margin straight; posterior side emarginated; ribs not very numerous, slightly prominent broad, rounded; lines of growth very prominent"". This description agrees with phenotype A studied here, where the rib number goes from 4 to 14 and they are very prominent. In phenotype B. however, the ribs are not distin- guishable and the growth lines are very prominent. Intraspecific differences in shell sculpture on specimens from different habitats have been noted in several gastropod species from the genus Lil- torina (Struhsaker 1968. Johannesson et al. 199.3. Rush 1997). These differences have been related to the degree of wave expo- sure— extreme ribbed and with nodes forms live on dry raised benches, not generally subject to horizontal water swash; while extreme smooth forms predominate on low. moist benches subject to strong wave swash. It is probable that a similar relation occurs among M. californianus phenotypes and wave action or their po- sition along the intertidal zone. We are carrying out a field study to explore this. The presence of ribs has been conelated with shell strength; the ribbed mussel Geukensia emissa has a stronger shell than M. edulis. this strength was correlated with shell mass, shell curvature and valve thickness (Majewski 1995). This could also be 138 Caceres-Martinez et al. Figure 4. Mytiliis califoniianiis of different sizes showing the two phenotypes found in this study: ( Al with rihs and (B) no ribs. For comparison, Mytilus gallopruvincialis of similar sizes also were included in figure (C). Note that different phenotypes appeared since young specimens. TABLE 2. Eigenvalues, explained variance ( 7r ), cumulative explained variance I '''< ) and eigenvectors from the principal component analysis between Mytiliis califoniianiis and Mytilus galloprorincialis morphometric data from the Pacific coast of Baja California. Principal Components 10 II 12 13 14 Eigenvalue VarianceCvJ- ) Cum. var.(%l rib si sh sw a aanis pam Ihr wbrs pam-piTi pam-vm hp Im pl-vm 10.587 VCSSI 70.581 0.015 0.301 0.278 0.229 0.281 0.266 0.275 0.292 0.184 0.286 0.276 0.259 0.281 0.258 0.25.^ 1.502 10.013 80.594 0.621 -0.043 -0.205 0.^94 0.016 0.000 0.05 1 0.037 -0.476 0.007 -0.201 0.030 -0.169 0.253 022.^ 0.751 5.009 85.603 0.737 -0.010 0. 1 82 -0.339 -0.163 0.036 0.074 -0.083 0.428 0.091 0.190 -0.035 -0.094 -0.144 -0.101 0.459 3.063 88.665 0,001 -0.139 0.182 -0.291 0.220 -0.102 -0.334 -0.023 0.145 -0.326 -0.166 0.553 -0.223 0.325 0.274 0.3 I I 2.075 90.741 -0.044 -0.017 -0.268 0.279 -0.222 -0.114 -0.228 0.144 0.582 -0,061 -0.074 -0.373 -0.007 0.187 0437 0,277 1.844 92,584 -0,011 0,014 -0.142 0.269 0.073 -0.722 0.298 0.022 0.237 0.107 0.034 0.294 -0.128 0.074 -0,339 0.263 1 .755 94.139 -0.030 -0.118 -0.265 0.277 -0.169 0.592 0.048 0.030 0.287 -0,122 -0.197 0.336 -0. 1 .% 0.044 -0.432 0.216 0.173 1.442 1.155 95.781 96.937 Eigenvectors -0.077 -0.125 -0.074 0.040 0.028 0.046 0.454 -0.179 0.094 -0.002 -0.211 0.222 -0.138 -0.585 0.516 0.020 -O.IOI 0.I7I 0.000 0.588 0.087 0.317 -0.216 0.185 -0.170 -0.344 -0.453 -0.075 0.215 -0.147 0.143 0.956 97.893 0.215 0.169 0.016 0.126 0.294 -0.100 -0.264 0.459 0.05 1 -0.318 -0.258 0.031 0.341 -0477 -0.129 0.100 0.667 98.560 0.052 0.007 -0.034 -0.211 -0.383 -0.094 0.344 -0.098 -0.020 -0.257 -0.309 0.093 0.652 0.268 0.027 0.079 0.527 99.086 0.062 -0.004 -0.077 0.077 0.166 -0.010 -0.388 -0.480 0.102 0.580 -0.317 0. 1 35 0.329 -0.053 -0.018 0.074 0.496 99.582 0.086 -0.161 -0.156 0.273 0.198 0.019 -0.080 -0.467 0.02! -0.432 0.573 0.055 0.283 -0.062 0.037 0.045 0.299 99.882 -0.032 -0.209 0.764 0.467 -0.322 -0.065 -0.092 -Olio -0.004 -0.073 -0.109 -0.019 -0.016 -0.074 -0.040 0.018 0.118 100.000 0.013 -0.866 -0.047 -0.074 0. 1 1 3 -0.006 0.036 0.353 -0.016 0.222 0.098 -0.012 0.210 0.016 0.000 Phenotypes of the California Mussel 139 CM O Q. 2 0 -2 4 ■ ■ ^ TtTT ▼ t'V ▼ ' T -10 -5 0 5 PC1 10 15 Figure 5. Principal component scores plots between PC2 vs. PCI for M. californiaiiiis Phenotype A, open circles; Phenotype B, bold squares; and M. galloprmincialis bold triangles. the case for M. caUfoniiiiniis where the presence of ribs might indicate a stronger sheH. In accordance with Seed (1968), variations in the M. cJiilis shell form can be attributed to differences in age, habitat, growth rate, and density. Old mussels have heavier shells, down-turned divergent innboes, and varying degrees of incurvature of the ven- tral shell margin than the young ones do. In this study, small and large individuals showed similar morphometric characteristics; therefore, the age or size of these mussels (which grow in the same habitat) seems to have little influence on the variability of the studied morphological characters. In relation to the habitat. Seed (1968) comments that in areas free of predators (like the upper shore) old individuals are common, whereas in areas where the mussel turnover is rapid there is a predominance of young mussels. Also, the presence of predators can affect shell morphology. M. edulis has been found to ha\e a smaller shell lencth. heii;ht and width with larger posterior adductor muscle, thicker shell, and more meat per shell volume when a starfish was present (Reimer & Tedengren 1996). In the Baja California region. M. califor- niunus is the dominant species where there is high wave action, whereas M. gallopravincialis is the dominant species in protected bays with thinner shell and more meat than M. californianus (Harger 1970. Harger 1972). It has been observed that shore level has an influence on the morphology and physiology of M. gallo- provincialis in the Adriatic see (Dalla Via et al 1987). Low shore level mussels have higher and narrower shells and a higher dry weight ratio whereas high shore mussels have a higher o.xygen consumption rate. When cultivated Mytihis edulis was transplanted between two different locations there were some morphological differences that were considered to be due to genetic variation (Stirling & Okumus. 1994). The same characters found in parents of distinct ecotypes also occurred in progeny raised in the labora- tory thereby indicating that the phenotypic differences have a ge- netic basis (Struhsaker 1968). The presence of two phenotypes and similar morphometric characteristics of the shell in small and large M. californianus in all three locations indicates not only some similarity among environments but it also strongly suggests that the presence of ribs is genetically produced. To our knowledge. there is no record on hybridization between M. californianus and M. gallopravincialis. which could result in a heavy shell without ribs. Our morphological results showed a clear difference between both phenotypes of M. californianus and M. galloprovincialis, which may suggest that phenotype B of M. californianus, is not the result of hybridization with M. galloprovincialis. Further studies using genetic markers will help to discard whether there has been any degree of introgression between these two species due to hy- bridization, which has been found in other Mvtilus species (Geller et al 1994). ACKNOWLEDGMENTS The authors thank Antonio Figueras Jr., Antonio Figueras Montfort. Andy Beaumont; for encouraging us to finish this study, and Biologist R. Vasquez Yeomans from CICESE for his help with the sample analysis. This work was supported by projects numbers 623106 and 6231 13 of CICESE. LITERATURE CITED Beaanuiiil. A. R.. Seed R. & P. Garci'a-Martfnez. 1989. Electrophorelic and morphometric criteria for the identification of (he mussels Mytilits edulis and M. gcdloprovincialis. In: J. S. Ryland & P. A. Tyler, editors. Reproduction. Genetics, and Distribution of Marine Organisms. Den- mark: Olsen and Olsen. pp. 251-258 Conrad T. A. 1837. Description of new manne shells, from upper Califor- nia. Collected by Thomas Nuttall Esq. J. Acad. Nal. Sci. Phila. 7:227- 242 Dalla Via G. J.. U. Tappemer. & G. Bilterlich. 1987. Shore-level related morphological and physiological variations in the mussel Mytiliis gtd- loprinincialis (Lamarck. 1819) (Molusca Bivalvia) in the north Adri- atic Sea. Mond. Zool. Ilal. 21:293-305 Figueras A. & A. J. Figueras. 1983. Variabilidad ecomorfica del mejilkin silvestre y cultivado en Espafia (gen. Mytilii.s) y relacion con su posicion sistematica. Investigacion Pesquera Al:yi-li Geller J. B.. J. T. Carlton & D. A. Powers. 1994. PCR-based detection of m( DNA haplolypes of native and invading mussels on (he nor(heas(ern Pacific coast. Lati(udinal pauern of invasion. Mar. Biol. 117:243-249 Gosling. E. M. 1992. Systematics and geographic distribution of Mytiliis. In: E. Gosling, editor. The mussel Mytihis: ecology, physiology, ge- netics, and culture. A(ns(erdam: Elsevier Science Publishers B.V. pp. 1-20 Harger, J. R. 1970. Comparisons aniong grow(h charac(eris(ics of (wo spe- cies of sea mussel. Mytiliis edulis and Mytiliis califoniianiis. The Ve- liger 13:44-56 Harger. J. R. 1972. Compe(i(ive coexistence: maintenance of interacting associations of the sea mussel. Mytiliis edulis and Mytihis califor- nianus. The Veliger 14:387-410 Inoue, K.. S. Odo. T. Noda. S. Nakao. S. Takeyama. E. Yamaha. F. Ya- masaki & S. Harayama. 1995. A possible hybrid zone in the Mytihis edulis complex in Japan revealed by PCR inarkers. Mar. Biol. 128:91- 95 Johannesson, K., B. O. Johannesson & E. Rolan-Alvarez. 1993. Morpho- logical differentiation and genetic cohesiveness over a microenviron- mental gradient in the marine snail Littorina sa.\atihs. Evolution 47: 1770-1787 Koehn. R. K. 1991. The genetics and taxono(ny of species in the genus Mytihis. Aquaculture 94:125-145 Majewski, M. 1995. Comparahve mechanical shell strength of the blue mussel a Mylilus edulis and the ribbed mussel Ceukensia demissa. In: J. P. Grassle. A. Kelsey. E. Dates & P. V. Snelgrove. editors. 23rd 140 Caceres-Martinez et al. Benthic Ecology Meeting. New Brunswick, New Jersey. (Abstract only). McDonald, J. H. & R. K. Koehn. 1988, The mussels Mylilus gallopiovin- cialii and M. trossulus on the Pacific coast of North America. Mar. Biol. 99:111-118 McDonald. J. H., R. Seed & R. K. Koehn. 1991. Allozyme and morpho- metric characters of three species of Mylilus in the Northern and South- em hemispheres. Mm: Biol. 1 1 1:323-335 Ohresser, M., P. Borsa & C. Delsert. 1997. Intron-length polymorphistn at the atin gene locus mac-1: a genetic marker for population studies in the marine mussels Mytihis galloproviniiulis Lmk and M. ('(/»//,v L. Mol. Mar. Biotech. 6:123-130 Rawson, P. D. & T. J. Hilbish. 1995. Distribution of male and female mtDNA lineages in population of blue mussels, Mytilus trossulus and M. galloprovincialis. along the pacific coast of North America. Mar. Biol. 124:245-250 Reimer, O. & M. Tedengren. 1996. Phenotypical improvement of morpho- logical defenses in the mussel Mytilus ediilis induced by exposure to the predator Asterias rubens. Oikos 75:383-390 Rugh, S. N. 1997. Differences in shell morphology between the sibling species Liltorina scutulata and Litlorinu plena (Gastropoda; Piosobran- chia). The Veliger 40:350-357 Seed. R, 1968. Factors influencing shell shape in the mussel Mylilus edulis. J. mar. bid Ass. U.K. 48:561-584 Seed, R. 1992, Systematics evolution and distribution of mussels belonging to the genus Mylilus: An overview. Am. Malacol. Bull. 9:123-137 Soot-Ryen, T. A. 1955. A report on the family Mytilidae (pelecypoda). .Mlait Hancock Pacific 20:1-175 Sokal, R. R. & F. J. Rohlf. 1995. Biometry. 3rd ed. New York: W. Freeman and Company. 887 pp. Stiriing, H. P. & 1. Okumus. 1994. Growth, mortality, and shell morphol- ogy of cultivated mussel (Mytilus edulis) stocks cross-planted between two Scottish sea lochs. Mar. Biol. 119:115-124 Struhsaker, J. W. 1968, Selection mechanisms associated with intraspecific shell variation in Liltorina picia (Prosohranchia: Mesogastropoda). Evolution 22:459-480 Jiiiiival oj Shellfish Research. Vol. 22. No. 1. 141-140. 2()(U. ADJUSTMENTS OF LIMNOPERNA FORTUNEI (BIVALVIA: MYTILIDAE) AFTER TEN YEARS OF INVASION IN THE AMERICAS G. DARRIGRAN,' C. DAMBORENEA.' P. PENCHASZADEH," AND C. TARABORELLi' ^Division Zoologia Inveitehnulos. FCN v Miiseo, UNLP. Paseo del Bosque s/ir ( IWO) La Plata. CONICET Argentina: -Dep. C. Bioldgicas. FCEyN. USA. Cnulad Univcrsitaria. Pah II. Niii'ic:.. Piso 4°. Buenos Aires. MACN-CONICET. Argentina ABSTRACT Limnoperna fortunei (Dunker, 1857) or golden mussel invaded South America through the Ri'o de la Plata estuary in 1991. Ten years later, the golden mussel lives in the main rivers of the Plata Basin. The gonadal cycle and the population settlement in a temperate climate are discussed in this article. This basic knowledge is needed to assist industries that may suffer the effects of macrofouling and also increment the ability to predict potential invasions of other countries. The study of population density and reproductive cycle was performed in Ri'o de la Plata estuary. Argentina. The temporal variation of population density from data of settlement and age structure collected between 1991 and 2001 is presented. The reproductive cycle between August 1998 and March 2000 was analyzed. Through the analysis of oocyte percentages four gonad spawning events were observed. The spawning events appear regulated by temperature changes. After the initial increase in population density following the invasion, there was a decrease. The population appeared stabilized at one third of the initial peak. KEY WORDS: in\asion. Limnoperna foriimei. freshwater, bivalve, reproductive cycle. Neotropical Region INTRODICTION Limnoperna fortunei (Dunker 1857). or golden mussel, is a freshwater invasive bivalve, from the southeast of Asia. It invaded South America in 1991. through the Rio de la Plata estuary. This represents the first record of L. fortunei for the American conti- nent. Ten years later, the golden mussel lives in the main rivers of one of the most itnportant Basins of the Neotropical Region (Bonetto 1994). the Plata Basin (the Rio de la Plata, and the Uru- guay. Parana, and Paraguay rivers). Since 1999. this species in- vaded the Guaiba Basin in the south of Brazil (Mansur et al. 1999). The golden mussel spreads 240 km/year, upstream along the Plata Basin. (Darrigran & Ezcurra de Drago 2000). The golden mussel attaches to every available hard substrate. This lifestyle (epifaunal) is atypical in local freshwater bivalves. The attachment capability and the great adaptability and reproduc- tive capacity of these mussels make this species very effective invaders (Darrigran 2000). The mussels impact on the natural en- vironment (displacement of native species — Darrigran et al. 1998b, Darrigran et al. 2000 — or change of native fish diet — Penchaszadeh et al. 2000) as well as on human activities (macro fouling in fresh water (Darrigran 2000. Darrigran & Ezcurra de Drago 2000). Detailed infomiation about the life cycle of this harnitiil inva- sive species provides a basis for the development and application 40 30 20 10 liC Au Oc IDe Fe Ap Ju Au Oc I3e Fe 1998 1999 2000 Figure 1. Monthly variation of mean air tenipiTature (line) and water temperature (bars) during sampling period in Bagliardi Htach. Rio de la Plata. HVithout data. of control strategies. The impact caused by this species in human activities (plugging of water intake for industrial cooling, power generation, and potable water) resembles what happened in the north hemisphere with the zebra mussel Dreissena polynwrplia (Pallas. 1771 ). The study of reproductive cycle, age structure and temporal density variation, is essential to generate sustainable techniques for golden tnussel prevention and control. Details of the reproductive cycle, and the population settlement in temperate climate are discussed in this article. This type of knowledge is not only essential to assist biologists and ecologists in the industries which tnay suffer frotn this new economic- environtnental problem in the Neotropical Region, but it is also necessary for predicting potential invasions of other countries in the north hemisphere such as USA (Ricciardi 1998) and southern Europe. TABLE 1. Date and number of specimens histologically processed per sample. Date Size range (mm) Males Females 2.V08/98 27 0.6-2.5 17 10 25/09/98 30 0.6-2.6 23 17 30/10/98 29 0.4-2.5 18 11 27/1 1/98 17 0.5-2.6 14 13 23/02/99 14 0.5-2.9 13 11 19/04/99 20 0.8-2.2 7 13 15/05/99 24 0.7-2.2 14 10 30/06/99 29 0.7-1.9 13 16 26/07/99 25 0.7-2.1 10 15 27/08/99 28 0.5-1.8 19 9 21/10/99 32 0.6-2.1 22 10 27/11/99 34 0.5-1.7 23 11 16/12/99 31 0.5-1.7 14 17 26/01/00 27 0.6-2.1 16 11 22/02/00 35 0.7-2.1 25 10 12/03/00 29 0.6-2.2 18 11 Total 431 266 195 141 142 Darrigran et al. DENSITY (ind/m- 200,000- 150,000- 100,000 50,000H a r^^ rh r+i I I I I I I I I I I I Oct Oct March Oct March Oct March Oct Oct Oct Oct Oct 1991 1992 1993 1993 1994 1994 1995 1996 1997 1998 1999 2000 Oct 2001 Figure 2. Temporal variation of mean density (bars) and standard deviation (lines) oX Limnuperna fortumi in Ba^liardi Beach, Rio de la Plata. • 4-5 ind/m". *\Vithout data. MATERIAL AND METHODS To study the golden mussel population density and reproduc- tive cycle, samples were collected along the rocky banks of Ba- gliardi Beach (34°55'S: 57°49'W). Rio de la Plata estuary. Ar- locality has a temperate regimen ranging from approximately 1 I °C to 3I°C (Fig. I). The physicochemical features of the Rio de la Plata may be found in Darrigran (1999). The density data were obtained partly from Darrigran. et al. (1998b) and through sam- pling carried out ad-hoc (October 1998 and October 2001) in gentina. South America. — where the mussel was found for the first Balgliardi Beach. Samples of mussels were collected for density time in 1991 (Pastorino et al. 1993). The water temperature in this analysis from the fringes with macrobenthos from a rectangular 20 15 10 5 0 October 1992 n = 481 III... March 1995 n= 1059 ■.■iiMMlii >N 15 u c OJ in J cr Ol 5 .1 October 1 993 n = 677 I.. I.ll October 1998 n = 289 I 20 15 10 5 0 October 1 994 n = 631 llllll... October 2001 n = 289 Illllllllll.llil.. length (mm) Figure 3. Size frequency (%) ot Limnoperna fortunei in Bagliardi Beacli. Rio de la Plata. L. FORTUNEi Ten Years After the Invasion 143 area, variable in size, according to Darrigran et al. ( 1998b). For the age structure analysis, the niaxinium shell length was measured and the length frequency distribution was made at 1 mm class intervals (see Fig. 3 later). The dates of sampling for reproductive cycle analysis, per- formed at low tides, may be observed in Table 1. The maximum shell length of the 431 collected individuals was taken. The ma- terial was fixed in Bouin solution and the histologic processing was performed according to Darrigran et al. (1999). Approximately 25 oocytes with conspicuous nucleolus, both free in the follicular lumen and attached to the follicle wall, for each gonad were measured. The percentage of males with sper- matozoids and the percentage of follicular occupation on the mantle were calculated for each sample. The latter was calculated using magnification (x200) in three different sections of the mantle, (upper, middle, and lower) through the visual estimation of field. The lysis periods were detemiined by microscopical analysis. RESULTS The temporal variation of population density found on the rockv litoral zone of Baszliardi Beach between 1991 and 2001 is given in Figure 2. From 1991 to 1995, the density increase was remarkable (from four to five individuals/nr to over 100.000 ind/m~). The population density then decreases and stabilizes at approximately 40,000 ind/nr. In Figure 3 it is shown that since 1 994 the population has had an age structure where most size class intervals are represented. The female and male tollicles grow in the mantle and in the visceral mass. During this study 0.25% hermaphrodite specimen, with female, male, and mixed follicles were recorded. The gonad growth is characterized by growing follicles. In this stage the follicles are small and there exists an abundant connec- tive tissue between them. A more developed stage shows young oocytes on the wall, many stalked oocytes (Fig. 4A) and abundant spermatogoniums in the males (Fig. 4D). In a later stage the fol- licles are bigger and the follicular lumen contains abundant oo- cytes half-grown and also almost fully grown oocytes (60-80 p.). When fully mature, the female and male follicles reach the maxi- mum size. Male follicles are packed with spermatozoa (Fig. 4E) and females" follicles with fully-grown (80-100 p.) oocytes. When the gonads are spent and partially spent, the follicles contain large spaces. Partially spent gonads retain genital products. Figure 4. Female and male tollicles in different development stages. (A) Female follicle partiallj gro«n with voiing oocytes on the wall and many stalked oocytes, scale bar = 1(10 fi. (B) .Spawned female follicles with abundant yellow bodies (arrows!, scale bar = 5(1 p. (C) Female follicles partly spawned, scale bar = 100 p. (Dl Developing male follicles, scale bar = 50 fi. (E) Fully developed male follicles, scale bar = 100 \i. (Fl Male follicles partly spawned, scale bar = 50 jj. 144 Darrigran et al. IL 23/08/98 )(= 57 06 DS = 57 06 n = 224: N = 10 Ij. 23/02/99 X = 75 22 DS = 21 14 = 262, N=11 il 26/07/99 X = 45 19 DS = 45 19 n = 267, N lll^ 16/12/99 X = 43 02 DS = 17 93 n = 353, N=17 llx. 25/09/98 x = 56 91 DS = 20 27 n = 400. N= 17 III] L_ Liilijili. 19/04/99 X = 60.57 n = 225, N=13 27/08/99 X = 38 99 DS = 14 95 n= 177, N=9 I L. 26/01/00 X = 47 65 OS = 16 £ n = 292; N=ll 31/10/98 X= 51.67 DS = 5167 n = 300. N = 1 1 u 15/05/99 X=46 52 DS = 46 52 n= 170. N=10 21/10/99 X = 51 92 DS= 17 91 n = 248; N 22/02/00 X = 49 94 n = 225. N=10 j1 iIL LJ. 30/06/99 X = 44 87 DS = 21 72 n = 352. N=16 Jul 110 1» 130 0 10 K Ti oocyte diameter (um; 12/03/00 X = 55 03 DS = 22 83 n = 320. N=11 K X) 40 so K 70 so so ICO 110 120 130 Figure 5. Frequency I in percentage) of oocyte sizes (ji) in different samplings, x, mean oocyte size; DS, standard deviation; n, number of oocytes; N, number of females. In males spennatozolds and spermatocites are observed (Fig. 4F|. Partly developed oocytes, oogonies, and young oocytes are re- tained on the female follicle walls (Fig. 4C). Oocitary lysis phe- nomena (Fig. 4B). with yellow bodies are evident for a short time after spawning is completed. The body length at which the follicle, either female or male, development is completed, varies seasonally. The smallest shell length at which follicles differentiate is 5.5 mm. for both males and females (Fig. 5). During this study (August 1998 to March 2000). oocyte growth was always recorded. Froin May 1999 until August 1999, the oocytes smaller than 20 p. were 309f of the total oocytes examined. The change in frequency of oocytes <20 p, and >60 p, indicates two reproductive peaks each year. The first peak occurs at the end A B oocyte (%) S. JJrlrl J rlbll.rhftrllJ De I Fe 1999 * * ¥ males (%) De I Fe 2000 n = 266 Figure 6. Temporal variation. (.\) Percentage of oocytes bigger than 60 fi (full bars) and smaller 20 yt (empty bars). The arrows indicate moments of gamete liberation. (B) Percentage of males with sperm. ■ Without data, n, number of male individuals. L. FOHTUNEi Ten Years After the Invasion 145 90 80 70 60 50 40 30 occupation of the mantle (%) n = 431 1 M Au Oc De Fe Ap Ju Au Oc De 1998 1999 2000 Finiirt' 7. Temporal variation of mantlt occupation. Female follicles (full barsi and males (empt> bars), n, total number of considered males and females. 'Without data. of winter or beginning of spring (August to September of 1998. October to November of 1999) and the second peak is recorded during the summer (February of 1999. March of 2000). During these periods in the female follicles the oocytes bigger than 60 |ji. dominate, while smaller oocytes are scarce (<20%). During the period of study gonad recuperation were observed (October 1998 and May to June of 1999). Through the analysis of oocyte per- centages present in the gonad, four spawning events were observed (Fig. 6A): ( i ) From September to October 1998. (2) February 1999 to May 1999. It is the most important for its duration and magnitude. (3) in July to August 1999, the least important. (4) between October and December 1999, Figure 6B shows the percentage of males with sperm through- out the period considered. The pattern agrees in general with that observed for females. The spawning pattern mentioned is similar to the follicular occupation of the mantle (Fig. 7). The percentage of occupation decreases during the spawning periods and stays low during the recuperation period (June. July, and August 1999). Lysis phenomena were observed in several samples (Fig. 8). They are more important during May to August 1999, and coincide with recuperating follicles or in partial evacuation. DISCUSSION The bivalve sexual processes are generally related to ambient temperature (Lubet 1983). The results presented here for a popu- lation of L. fiirliiiu'i. as well as those observed in the first study (Darrigran et al. 1999), those performed for a Hong Kong popu- lation (Morton, 1982), and the analysis of larvae density in the Ri'o de la Plata (Cataldo & Boltovskoy 2000) show the strong relation- ship between ambient water temperature and the reproductive cycle. The spawning events are regulated by changes in tempera- ture, and increases and decreases of temperature rule the gameto- genesis in this species. During the initial study (Darrigran et al. 1999). we found that oocytes were always present in the mussels even during the resting period. Periods of scarce proliferation were recorded from Decem- ber 1993 to May 1994. This study was performed a short time after the first record of L. forlimei in the Americas (Pastorino et al. 1993). The analysis of reproductive biology at that time differen- tiated numerous spawning events (five were recorded), many of them of low magnitude. Between September 1992 and January 1993 (the first period), two spawnings of reduced intensity were recorded and between February 1993 and November 1994 (the second period) three spawnings were recorded (two of these of higher magnitude). During the first period, the oocytes bigger than 60 |xni and those smaller than 20 |jLm are always present and their proportion is similar (about 309H. the spawnings are low in mag- nitude but the proportion of oocytes bigger than 60 \vm is always larger than 20%. During the second period, the spawnings are more intense and result in a diminution of the bigger oocytes proportion (by 10%). In contrast to the first period, the oocytes bigger than 60 (xm reach more than 60% (Darrigran et al. 1999). The population analyzed here shows a predictable reproductive pattern. Only two major spawnings are observed throughout the year, one when summer temperatures are recorded and the other with spring temperatures. A small winter spawning is also ob- served. This pattern, after 10 years of settlement in America, is similar to that described by Morton (1982) for the population of Hong Kong where the spawnings take place between May to June and November to December. The pattern shown during the first study [only after a year of settlement in the location considered (Darrigran et al. 1999)] could be due to the recent invasion. Morton (1982) describes short spawnings for a month in spring and a month in autumn. In this study in South America, mainly in autumn, the evacuation continues from April 1999 to May 1999. The presence of larvae in the Ri'o de la Plata, between August and April (Cataldo & Boltovskoy 2000). also indicates that the spawn- ing periods are longer than those described by Morton (1982). Similar to what was found in the first study of the golden mussel reproductive cycle (Darrigran et al. 1998a) 0.25% of the population was hermaphrodite. According to the variation of population density, this species, at the beginning of the invasion in temperate climate, presents a noticeable increase of density. Then, it decreases its density to a third part and stabilizes. At the same time, it presents an age structure with most class intervals represented. These facts would indicate a stable settlement of the population to the en\ ironment. % Ll * * Au Oc De Ap Ju Au Oc De I Fe Figure 8. Percentage of females with follicles where lysis phenomenon occur. *Withoul data. n. number of considered females. 146 Darrigran et al. The initial increase recorded in a temperate climate could also be observed in a subtropical climate. Despite the preliminary stud- ies of this species invasion in the south of Brazil, subtropical climate (Mansur et al. 1999). the golden mussel presents an in- crease in its population density similar to that observed in this study. Two years after its first record (Mansur et al. 2001 a. Mansur et al. 2001b). the maximum density is 62.100 ind/m". The golden mussel, like other invasive species, is opportunistic. This fact makes it difficult to relate the reproductive pattern with environmental variables and to determine the different facts that might be modified in the reproductive cycle. L. forliinei. for its great adaptability and reproductive capacity, increases its distribu- tion permanently by occupying environments of particular fea- tures. ACKNOWLEDGMENTS The authors thank Renata Claudi for her comments on a draft version of the manuscript. This work was partly financed by grants BID 1201 OC/AR PICT 01-03453 from the Agenda Nacional de Promocion Cientffica y Tecnologica, Argentina; Facultad Ciencias Naturales y Museo, Universidad Nacional de La Plata (UNLP) and Fundacion Antorchas. LITERATURE CITED Bonetto. A. A. 1994. Austral rivers of South America. In: R. Margalef. editor. Limnology Now: a paradigm of planetary problems. Amslerdan: Elsevier Science, pp. 425-472. Cataldo, D. H. & D. Boltovskoy. 2000. Yearly reproductive activity of Umnoperna fortuiu'i (Bivalvia) as inferred from the occurrence of its larvae in the plankton of the lower Parana river and the Rio de la PUiia estuary (Argentina). Aqiialic Ecology 34:307-317. Darrigran, G. 1999. Longitudinal distribution of molluscan communities in the Rio de la Plata estuary as indicators of environmental conditions. Malacological Review- (Suppl.) 8:1-12. Darrigran, G. 2000. Inva,sive Freshwater Bivalves of the Neotropical Re- gion. Dreissena 11:7-13. Darrigran, G. & I. Ezcurra de Drago. 2000, Invasion of Limnopenm for- timei (Dunker, IS.'i7) (Bivalvia: Mytilidae) in America. Nautilus 2:69- 74. Danigran, G., M. C. Damborenea & P. Penchaszadeh. 1998a. A case of hermaphroditism in the freshwater invading bivalve Limnoperna for- timei (Dunker. 1857) (Mytilidae) from Rio de la Plata. .Argentina. Iberus 16:99-104. Darrigran, G., S. M. Martin, B. Gullo & L. Armendariz. 1998b. Macroin- vertebrates associated to Limnoperna fortunei (Dunker. 1857) (Bi- valvia, Mytilidae). Ri'o de La Plata, Argentina. Hxdrobiologia 367: 223-230. Darrigran, G., P. Penchaszadeh & M. C. Damborenea. 1999. The life cycle oi Umnoperna forlunei (Dunker, 1857) (Bivalvia:Mytilidae) from a neotropical temperate locality. J. Shellfish Res. 18:361-365. Darrigran, G.. P. Penchaszadeh & M. C. Damborenea. 2000. An invasion tale: Limnoperna fortunei (Dunker, 1857) (Mytilidae) in the neotropics. International Aquatic Nuisance Species and Zebra-Mussels Confer- ence. 10:219-224. Lubet. P. 1983. Experimental studies on the action of temperature on (he reproductive activity of the mussel (Myliliis eilnlis L. Mollusca, Lamel- lihranchia). J. Mollusc. Studies (Suppl.) 12A: I(J0-I05. Mansur, M. C. D., L. M. Zanirichinitti & C. Pinheiro dos Santos. 1999. Limnoperna fortunei (Dunker, 1857), Molusco Bivalve invasor, na ba- cia do Guafba, Rio Grande do Sul, Brasil. Biociencius 7:147-150. Mansur, M. C. C. Santos, G. Darrigran, G. Heydrich, C. Quevedo & L. Iranco. 2001a. Preferencias e densidades do mexilhao dourado Lim- noperna fortunei (Dunker, 1857), em diferentes subtratos da bacia do Guai'ba, Rio Grande do Sul. Brasil. RESUMOS V Congresso de Eco- logia do Brasil: 246. Mansur, M. C. D., C. Pinheiro dos Santos, G. Darrigran. 1. Heydrich. C. Barbosa Quevedo & L. Bernades Iranco. 2001b. Densidade e cresci- mento populacional do mexilhao dourado Limnoperna fortunei (Dunker, 1857), na bacia do Guaiba e novos registros na Laguna dos Patos, Rio Grande do Sul. Brasil. RESUMOS XVII Encontro Brasileiro de Malacologia: 61. Morton, B. 1982. The reproductive cycle in Limnoperna fortunei (Dunker, 1857) (Bivalvia: Mytilidae) fouling Hong Kong's raw water supply system. Oceanologia el Limnologia Sinica 13:312-324. Pastorino. G.. G. Darrigran, S. M. Martin & L. Lunaschi. 1993. Limno- perna fortunei (Dunker, 1857) (Mytilidae), nuevo bivalvo invasor en aguas del Ri'o de la Plata. Neotropica 39:34. Penchaszadeh, P., G. Darrigran. C. Angulo, A. Averbuj, N. Brignoccoli, M. Brogger, A. Dogliotti & N. Pirez. 2000. Predation on the invasive freshwater mussel Limnoperna fortunei (Dunker, 1857) (Mytilidae) by the fish Leporinus obtusidns Valenciennes, 1846 (Anostomidae) in the Rio de la Plata, Argentina. J. Shellfish Res. 19:229-231. Ricciardi. A. 1998. Global range expansion of the Asian mus.sel Limno- perna fortunei (Mytilidae): Another fouling threat to freshwater sys- tems. Bu'fouling 13:97-106. J<:nniiil ofSlwllfish Research. Vol. 22. Nci. 1. 147-lh4. 2(KI,V QUANTITATIVE EVALUATION OF THE DIET AND FEEDING BEHAVIOR OF THE CARNIVOROUS GASTROPOD, CONCHOLEPAS CONCHOLEPAS (BRUGUIERE, 1789) (MURICIDAE) IN SUBTIDAL HABITATS IN THE SOUTHEASTERN PACIFIC UPWELLING SYSTEM WOLFGANG B. STOTZ,* SERGIO A. GONZALEZ, LUIS CAILLAUX, AND JAIME ABURTO Universidad Catolica del Norte, Facultud de Ciencias del Mar, DepariaiueiUo de Biolo^iia Marina, Casilla 117. Coquiinhd. Chile ABSTRACT Landings of Concholepas coinholepas. a carnivorous gastropod and valuable fishery resource, appear disproporlion- ately high compared with herbivores or suspension feeding mussels. The species has been previously described as feeding on a great variety of prey, the most important being barnacles, mussels, and tunicates. To quantitatively evaluate published information on the diet of C. comluilepas. an analysis of the stomach contents of 92.'i individuals was performed, representing a wide size-range, broad geographical distribution (29''30'S to 32°08'S), and different community types (variety of potential prey choices). The diet was based principally on suspension feeders, such as barnacles iBaUiiui.s Uicvis and juveniles of .Aii.\tii)nu'.i;iihcilanus fi.siikicii.s) 175%) and the ascidian Pyiira chilensis (16%). An additional sampling, in which abundance of prey in the habitat and microhabitats occupied by the gastropod was determined, showed that the gastropod positively selects these prey species, the ascidian being the most preferred. The rest of the diet was made up of Calyptraea Irochifoniii.s and mytilid bivalves. According to the literature, intertidal individuals of this species only feed at night. To confirm this behavior for subtidal populations, two 24-h samplings (analyzing digestive tract contents) were performed at a single location. No distinct circadian cycle of feeding for subtidal populations was found, most animals feeding most of the time. This, together with the characteristics of diet, made mainly by suspension feeders, which transfer energy from primary productivity in the water column which varies along the coast, to benthic carnivores, help to explain the high productivity of the gastropod and its variability along the coast of Chile. A'£l' WORDS: feeding beha\ ior, circadian rhythm, selectivity, carnivorous gastropod, Chile, subtidal, upwelling system INTRODUCTION The muricid gastropod Concholepas concholepas (Bruguiere 1789) ("Chilean abalone") is distributed from 12°S to 55°S along the Peruvian and Chilean coasts and is an important predator oc- cupying rocky shores (Castilla 1981. Castilla &. Paine 1987). It is a valuable product in artesanal fisheries (Castilla & Jerez 1986) along its entire distribution. In Chile, the highest landings ranged between 6,369t and 25,()()Ot between 1978 and 1988 whereas the fishery was unregulated; the maximum value was recorded in 1980 (SERNAP 1999). In region IV (between 29' 30'S and 32°08'S, 320 km coast) in the period between 1985 and 2000 landings fluctuated between 258 and 2,2 19t for this carnivorous gastropod species. In the same period and along the same stretch of coast the herbivo- rous gastropods FissurcUa spp. (eight different species that are fished) and Tegula aira (Lesson), which share the habitat with C concholepas. together registered landings between 695 and 1525t. The aim of this work was to investigate what kind of food sustains the comparatively important production of this high trophic level carnivore, the ecological position to which C concholepas is usu- ally assigned, Stotz (1997) has shown that within management areas the abundance of C. concholepas is related to the amount of food, the species overexploiting its food source when not fished, and then migration to other areas. Thus, the knowledge of diet and feeding behavior is also of importance in developing a manage- ment strategy of the species within management areas. According to published literature, C. concholepas has been observed feeding on a variety of prey, the most often mentioned being barnacles, mussels, and tunicates (Viviani 1975, Castilla & Cancino 1979, Castilla & Guisado 1979, Castilla et al. 1979. DuBois et al. 1980, Castilla 1981, Guisado & Castilla I98.\ Sotn- *Corresponding author. E-mail: wstotz@socompa.cecun.ucii,cl mer 1991. Sommer & .Stotz 1991 ). Bui quantitative feeding infor- mation is scarce; the number of published observations for indi- viduals feeding in their natural subtidal habitats was less than 96, observed at two localities (Castilla et al. 1979, Guisado & Castilla 1983, DuBois et al. 1980, Sommer 1991). These did not represent the entire spectrum of subtidal communities in which the gastro- pod lives. There are also qualitative observations (Viviani 1975, Castilla et al. 1979, Castilla 1981 ) that increase the data regarding the prey diversity of C. concholepas but do not allow evaluation of the relative dietary importance of the different prey species of this gastropod. The published quantitative information on food types con- sumed by C concholepas was obtained by feeding behavior ob- servations (Castilla et al. 1979). DuBois et al. (1980) stated "an individual is feeding when one observes an unusual extension of the foot over a potential prey species or when the individual shows movements to remove a prey." This includes lifting individuals to check for empty shells, direct observations of ingestion of prey, empty spaces on the substrate in front of the mouth or of the "shell teeth", which the species has on the anterior border of the shell, proboscis introduced into the prey, or prey held by the propodiuni and directed to the mouth (Castilla ct al. 1979). This method gath- ers information on the specific prey being consumed at the mo- ment of observation. Thus, those prey species that are more diffi- cult to consume and for which the process of ingestion lasts longer will have a higher probability of being observed. Also, in order not to disturb animals and thus record observations of natural feeding behavior, observations have been limited to individuals found on open surfaces. Feeding by individuals found in crevices or on the undersides of boulders, including most juveniles and medium- sized individuals of C. concholepas (Castilla & Cancino 1979, Guisado & Ca.stilla 1983, Sommer 1991, Stotz & Lancellotti 1993) cannot be easily observed. Thus, observations of C. concholepas on open surfaces will focus only its feeding on prey abundant on 147 148 Stotz et al. such places and food composition described using this method may not necessarily reflect the relative importance of the different prey species in the diet of C. concholepas. In contrast, the analysis of digestive tract contents provides a quantitative measure of food consumption over a certain time in- terval, representing the range of prey species and their relative importance in the diet of the predator. Only in case digestion rates for different prey species differ greatly, some bias may occur. This is the first work in which feeding of C. concholepas has been studied through the analysis of the contents of the digestive tract. According to published information. C. concholepas feeds only at night (Castilla & Guisado 1979. Castilla & Cancino 1979. Castilla et al. 1979. Guisado & Castilla 1983). However, this has been conckided mainly from laboratory experiments mostly using individuals collected in the intertidal zone. Only DuBois et al. (1980) have made observations in the subtidal. recording the feed- ing activity of 96 individuals of this species. Intertidal gastropods search out and consume food mainly at night to avoid desiccation (Underwood 1979, Branch 1981, Hawk- ins & Hartnoll 198.^. Lowell 1984). Subtidal populations of C. concholepas, not exposed to this stress, may feed mainlv at night for other reasons: ( I ) to avoid visual predators active during da> - time (Castilla & Cancino 1979) and/or (2) to capture prey that respond to visual stimuli and may be able to escape predation by C. concholepas during the day. Visual predators, which are known to include C. concholepas in their diet, such as the sea-otter Lmrafelina (Molina) (Castilla & Bahamondes 1979), the sea lion Otariaflavescens (Shaw) (Aguayo & Maturana 1973) and the fishes Pimelometopon macidatus (Perez) and Sicyases sanguineus Miiller & Troschel (Viviani 1975). do not figure prominently in the monality of this gastropod species. L. felina has been suggested to be highly specialized on fish and Crustacea as prey (Sielfeld 1990); O. flavescens does not appear to prey on gastropods firmly attached to substrates, as is the case for C. concholepas (George-Nascimento et al. 1983); and the fish species prey mainly on juveniles of C. concholepas which, according to our observations, are hidden in crevices in the sub- tidal. Prey selection is an unlikely factor promoting night time feeding, as the main prey of C. concholepas are sessile species, such as the barnacles Auslromegabalanus psittacus (Molina). Balanus laevis Bruguiere, and Jehlius cirratus (Darwin); the tuni- cate Pyura chilensis (Molina); the mitilid Peruniytilus puipuratus (Lamarck); and the hemisessile gastropod Calyptraea trochifonins (Boml (Castilla & Guisado 1979. Castilla et al. 1979. DuBois et al. 1980, Guisado & Castilla 1983, Castilla & Durdn 1985, Moreno et al. 1986, Sommer 1991. Sommer & Stotz 1991). Therefore, there appears to be no strong argument that subtidal populations of C. concholepas feed exclusively at night. Nevertheless, this needs to be investigated, which is one aim of this work. This work reports food composition and feeding behavior (cir- cadian feeding rhythm and food selection) for C. concholepas based on the analysis of the food content in the digestive tract. A greater variety of habitats than in previous studies were sampled, including open surfaces, crevices, the undersides of boulders, hold- fasts of the subtidal kelp Lessonia trabeculata (Villouta & San- telices), and under the canopy of this algae along an extensive stretch of coast from 29°30'S to 32°08'S (ca. 320 km). On one site the sampling and analysis of the digestive tract contents of a large number of individuals collected over a 24-h cycle was conducted. For some of the individuals sampled along the coast and in dif- ferent communities, the abundance of potential prey in the envi- ronment is quantified to establish to what degree the food in the gut represents the availability of prey. This allows us to study whether there is some kind of preference for some prey species. MATERIALS AND METHODS Study Sites Individuals of C concholepas were collected at several sites along the ca. 320 km of coast of the Coquimbo Region, between Pichidangui (32°08'S) and Punta Choros (29°30'S) (Fig. 1 ). The sites were chosen considering accessibility and being representa- tive of different coast and community types. A qualitative de- scription of subtidal communities of each sampling site is provided in Table I . Quantitative data of communities in which the gastro- pod was sampled are provided in Tables 3 and 4. For the 24-h sampling the site at Punta Lagunillas (30°05'S; 7|-26"W). located ca. 15 km south of Coquimbo, was chosen. It is a rocky point forming the northern border of Bahia Guanaqueros (Fig. I ). .-M- though it is an exposed coast, it has an irregular configuration that creates sheltered ponds that allow for safe diving through the surf and at night. The substrate is formed by different sized boulders that are covered by a dense kelp forest formed by small and bushy (many blades, short stipes) individuals of Lessonia trabeculata. It corresponds to community type I (Table I). Quantitative data for the community at this site are given in Table 3. Larger individuals of C. concholepas are found mostly within the kelp forest, w hereas smaller individuals are mainly hidden in crevices or on the under- sides of boulders. PACIFIC 29 " S OCEAN A ELTEMBLADOR Punla Choros \ TOTORAULLO ^^S^ NORTE \ P LINT A LAGUNILLAS J 30^ s PUERTO X.'S ALDEA ^ Coquimb o — DEVACA --^^^BahlaTongoy SAN y] LORENZO 1 3,o s PUERTO 1 OSCURO >\ HUENTELAUQEN \ ISLA ^ I HUEVO ^^^ / LASTINICUNAS \ T Vilos 32 s TOTORAULLO ^V SUR ""^f 7 Bah aPichidan^ ui Figure I. Location of the study sites along the coast in the region of Coquimbo (region IV). Diet and Feeding Behavior of C. concholepas 149 TABLK 1. Siibtidal fommunitifs »herf ('. coiuhiilepus was collected a general description ol' each community is given. Type Communities Localities I Kelp hed nf Lcssonia traheciiUita El Tenihlador, Punla Lagimillas. Puma Lengua de Vaca, San Loren/o. Caleta Las Conchas. Totoralillo Sur (isle and bay) II Barren gniund Toralillo Norte (rock). Puerto Oscuro III Barnacles and seaweeds Toioralillo Norte (isle) IV Colonies of Pxiini chilen.\ii Puerto Aldca General description of the subtidal community types Type tcimmunity General Description Kelp bed of Lessonia traheculata Barren ground Barnacles and seaweeds IV Colonies of Pxiira chileusis Community characterized by the kelp Lessonia traheculata. Under the canopy, dense patches of barnacles (e.g.. Balaims laevis) and to a lesser extent the ascidian Pyiiru chilensis are found. In crevices and on the underside of boulders are observed aggregations of the gastropod Calyptraea trocliifdiinis. sponges and small patches of barnacles. Community characterized by an high cover of calcareaus crustose algae and high densities of the black urchin Tetrapygiis niger. In crevices and on the underside of boulders are observed aggregations of Pyiira chilensis, of C. trochyfonnis and patches of barnacles. Community dominated by extensive patches of barnacles, specially by Austmmegabalaniis pssittacus, which can be covered by a dense mat of the red algae Gelidiiim chllense. Also aggregations of the ascidian Pyura chilensis may be present in crevices. Community formed mainly by aggregations of the ascidian Pyiira chilensis. which covers most of the surface. The ascidians could be partly covered by the algae Glgartina chamissoi. On the underside of boulders aggregations of Calvpiniea trochiformis can be observed. Sampling of C. concholepas Along the Coast to Describe Diet Individtials were collected by Hookah di\'ing from the intertidal down to a maximum depth of 25 m. At each site two divers collected all C cimcholepa.t that they were able to find within approximately 1 h of diving, which allows the inspection of an area of about 200-500 m". Individuals of all sizes were collected and the searches included the undersides of boulders. Table 2 summa- rizes the number and size range of individuals collected at each site of the samplings undertaken between January 1994 and December 1995. Experiments for the Identification of Prey and Food Retention Time in the Gut The identification of each prey item was aided by a simple experiment in which known prey were offered to individual C. concholepas. Three groups of 10 adult individuals (70-110 mm peristomal length) were collected at Punta Lagunillas and main- tained in tanks with running seawater. Each group was offered one of the most important prey items described in the literature (Som- mer & Stotz 1991): the barnacles Aiistromegahulanus psittacits and Bdhiiuis laevis. the gastropod Calyptraea trochiformis. and the TABLE 2. C. concholepas: Number of individuals collected in the field, number of entire digestive tracts analyzed in the laboratory, size range, number of individuals with food in their tracts, and number of individuals with recognizable prey in their digestive tracts are given. Sample Sample Individuals Recognizable Size Size Size \>itb Food Prey Field Labor. Range Localities (N") (N") (mm) No. % No. Vf Playa EI Teniblador 76 74 24-122 54 73.0 47 87 Totoralillo Norte (rock) 21 21 37-93 13 61.9 10 76.9 Totoralillo None (isle) 9 8 15-122 4 50.0 4 1 00.0 Punta Lagunillas (August) 166 166 2I-I2I 122 73.5 110 90.2 Punta Lagunillas (January) 282 260 7-125 235 90.4 225 95.7 Puerto Aldea 13 13 102-129 13 1 00.0 11 84.6 Punta Lengua de Vaca 52 45 51-116 33 73.3 22 66.7 San Lorenzo 188 158 16-126 105 66.5 93 88.6 Puerto Oscuro 7 7 59-100 5 71.4 5 lOO.O Isla Huevos 54 54 24-131 52 96.3 52 1 00.0 Totoralillo Sur (isle) 95 72 69^7 65 90.3 62 90.4 Totoralillo Sur (bay) 51 47 26-125 40 85.1 39 97.5 Total ini4 925 7-131 741 80.1 680 91.8 150 Stotz et al. ascidian Pyiira chilensis. Individuals were maintained continu- ously with food, sampling after the initial 48 h, and then daily, two individuals. Sample animals were dissected and their stomach and gut contents exaiuined. The physical characteristics of each prey item after ingestion by C. conclwlepas were recorded and then used as a reference in the analysis of stomach and gut contents from individuals sampled in nature. To measure the time the food is held in the digestive tract, a field experiment was performed at Punta Lagunillas on October 25-26, 1995. Therefore, all the individuals collected during a 30-min period at 1800 h and again at 0600 h of the next day were maintained in a mesh bag in the water in the study site, without food. Every 2 h, six individuals of this mesh bag were sampled and sacrificed, fixinc the visceral mass in 10% saline formalin. In the laboratory, the proportion of individuals with food in the stomach or gut in each sample was determined. Samplings to Compare Diet with the Food A railable in the Environment At seven sites (El Temblador. Punta Lagunillas. Punta Lengua de Vaca. Huentelauquen, Isla Huevos. Tinicunas. Totoralillo sur) (Fig. 1), between January 1996 and March 1997. samplings were repeated, but this time recording also abundance of prey in the environment. For each C. conclwlepas individual collected, the density and percent cover of species present on the spot, was recorded. A 0.25-m" quadrant with 100 regularly distributed points STOMACH Pyura chilensis INDETERMINATE 89,6 CIRRIPEDIA Q 3 Calyptrsea ''■^ ' trvctiiformis INTESTINE 83 9 CIRRIPEDIA INDETERMINATE 73 8,3 ^"^ chilensis TOTAL Pyura chilensis 15,88 INDETERMINATE Calyptraea trochifonvis o,65 74 67 CIRRIPEDIA 0,05 MYTILIOAE m CIRRIPEDIA MYTILIDAE ^v8i Pyura chilensis INDETERMINATE Calyptraea trochiformis Figure 2. Dietary composition of Conclwlepas conclwlepas. Diet and Feeding Behavior of C. concholepas 151 was used. The quadrant was loL-ated with its center on the spot were the C. conclioU-pas indi\ idual was captured. For four of these seven sites (Isla Huevo. Punta Lengua de Vaca. Punta Lagunillas. and El Temblador) (Fig. 1). a general quantitative description of communities present on the site was done. A 50-ni long and 2-m wide transect was placed parallel to the coastline. For less frequent species their abundance in the entire transect area (100 m") was counted, whereas for smaller, more frequent species five 0.25-ni" quadrants, distributed regularly along the transect, were used. To quantify the laminarian algae Lcssduia inilicculata. the transect was divided into 2.^1 areas of 2 x 2 m. estimating percent cover within each of these areas. Within these same areas the percent cover of each substrate type was estimated in those cases in which the bottom was a mixture of sand and rocks. This estimate was used to correct abundance and per- cent cover estimates of species, in order that they refer only to rocky bottom. 24-b Sampling al Punta iMgiiiiillas The 24-h sampling was accomplished twice: on October 24 and 2.S. 1994 and August .S and 6. 1996. Dives took place at 1700. STOMACH > u c (D <1> 00 i fe ^ EES i m 60 1 x^-' :|; 40 i 5 - CN CM i ^ ^ 1 1 Z 2 c o (A Surl Sur2 Total o o o O illo illo i- - - m n to uj 2 2 -^ as 5 2 2 a o £ ^— INTESTINE > u c 0) 3 a u. 100 . 80 1 60 40 m iiizza^ m TOTAL 100 m CIRRIPEDIA MYTILIDAE Pyura chilensis Calyptraea trochlformis INDETERMINATE Figure 3. General dietary composition of Cnnchnlepas concholepas from each sampling site. 152 Stotz et al. 2100, 0100, 0500, 0900, and 1300 h. On each dive, two divers sampled the subtidal at depths between 4 and 10 m, collecting each C. concholepas they were able to tlnd within a half-hour dive. Searches were concentrated beneath the canopies of L. trabeciilata and included the undersides of boulders. At night searches were conducted using underwater flashlights. Diving was conducted us- ing a compressor on the beach that provided air to the divers through a hose (Hooka diving). In the 1996 sampling, the indi- viduals collected by each diver were considered as replicate samples. Processing of Samples All samples of C. concholepas were processed immediately after collection. Peristomal length of individuals were measured with calipers and grouped into se\ en si/e classes from <30 mm to >130 mm (see Figs. 4 and 5). Each specimen was taken out of the shell and the visceral mass dissected and fixed in 10% saline formalin. Visceral masses of all individuals from each size class were stored together in a single container and transported to the laboratory. In the laboratory, the digestive tract of each individual was dissected; the contents emptied separately for stomach and gut in two Petri dishes, diluted with tap water, and spread on the bottom of the dish. The relative abundance of each prey item was recorded for each individual using a dissecting microscope. Therefore the dish was put over a point matrix, recording the food item over each point, and calculating its proportion to all the points covered by the sample. Also the presence of prey species, which were present, but not registered over any point, were annotated. For C. concholepas from the 24-h sampling a measure of full- ness was recorded. Fullness and digestion level was determined Using the following scale: Fullness: Full: contents occupy ca. 100% of the volume of the stomach or gut. Medium: contents occupy around 50% of the \ olume of the stom- ach or gut. Presence: contents occupy around 10% of the volume of the stom- ach or gut. Empty: no contents registered. Digestion level: Some digestion: entire structures are observed, such as pieces of cirri, 2ills, muscles, etc. STOMACH > o z LU a UJ 100 80 - 60 40 20 ^^^ ^ m INTESTINE > o z tu r> o lU a: u. 100 80 60 40 20 0 i^*c/j m CIRRIPEDIA MYTILIDAE ^vvi Pyura chilensis INDETERMINATE Calyptraea trochiforwis Figure 4. Dietary composition of Concholepas concholepas in different size classes (length of peristomal opening). Diet and Feeding Behavior of C. concholepas 153 STOMACH INTESTINE > o z lU o ai u. >- o z lU a Ul a: u. 100) sa so 4a 20^ 100 80i 6o^ 4a 2a I 0 + ^S3^^^SSS 80i I 2(y 10Q 80, 601 4a 2a EL TEMBLADOR ^? LAGUNILLAS O z LU a UJ (t LL o z UJ o UJ 100| 801 ■^1 61 4a 2a 10Q ! so] 40* 2a 0 ^^ 100| 8a 60 4a 20 '' 0 Wi 100 80 60 40 20 LAS CONCHAS ^^ TOTORALILLO SUR T- CO SIZE CLASSES (Cm) CIRRIPEDIA Pyura chilensis INDETERMINATE Figure 5. Dietary composition of Concholepas concholepas in different size classes (lengtli of peristomal opening) Iroin lour sampling localities. mate significance levels, using the following relations: Species A Other spp. Total Medium: structures could still be identified, but already with some one degree of freedom (Sokal & Rolf 1969, Pearre 1982) to esti- digestion. Total digestion: soft parts are completely digested, only pieces of shells or hard skeletons can be identified. Prey Selection Analysis To determine the degree of selection of prey by C. cdiuhdlepas an index proposed by Pearre (1982) was used. This allows the estimation of the selection index C. but also using a x' tc'st with In the diet A, «./ \ + e. = c In the environment .4,, fi„ .4, + s„ = D ■\, + .4,, = ,4 «,, + «,, = H ■\ + ■'>„ + Bj + B„ = N 154 Stotz et al. 10 12 18:30 Starvation period (hours) Figure 6. Percentage of Individuals v\ith contents In the stomach and Intestine during the starvation periods beginning In the morning (A) and In the afternoon (B). Where: Aj = Proportion of species A in the stomach /4,, = Proportion of species A in the environment Bj = Proportion of the rest of species in the stomach fi„= Proportion of the rest of species in the environment The index "C" is obtained from the followina relation: Where: N X' N (A,rB,,-A„- BJ-- A- B- CD) The index C varies between -1 and +1. A significant positive value indicates that the prey species was preferred and rejected with a significant negatise value. Values around zero means that the prey species is consumed in the same proportion it appears in the environment. For estimation of the index only those species found in the diet of C. concluilepus where considered. For the cal- culations, the density of invertebrates present in the quadrant was transformed into percent cover to have all the values on the same scale. For this, the area occupied by an average indi- vidual was estimated, calculating its proportion within the 2.?00 cm" of the sampled area. This proportion was multiplied by the number of sampled individuals, thus obtaining their percent cover. Once this proportions where estimated, a correction for poten- Degree of Fullness n Empty ^ Presence B Medium ■ Full Digestion Level D Empty ^ Total B Medium ■ Some 0 2 4 6 8 1012 1416 0 2 4 6 8 10 12 14 16 STARVATION PERIOD Figure 7. Prey digestion level (first column: A, C) and degree of fullness (second column: B, D) of stomach and intestine during the starvation periods beginning In the morning (first line: A, B) and in the afternoon (second line: C, D). Diet and Feeding Behavior of C. concholepas 155 MORNING SAMPLE Cirripedia (principally Balanus laevis) Indeterminate Totally digested AFTERNOON SAMPLE Mollusca Pyura chilensis Calyptraea trochiformis Cirripedia (principally Balanus laevis) Mollusca Indeterminate Totally digested Pyura chilensis Figure 8. Prey composition of Concholepas concholepas in the starva- tion experiment at Punta Lagunillas. tial prey species was done. Therefore, the percent cover values for algae and empty space was eliminated, calculating a new propor- tion considering that potential prey species cover 100% of the substrate. For these analyses, only the content of the stomach was used because this represents the most recently ingested food, most prob- ably from the sampled spot. Also, empty or destroyed stomachs were not considered. RESULTS Diet Of the 1.014 individuals of C. concholepas collected at nine sites (Table 2) visceral masses of 925 individuals were examined. Of these, only 741 individuals (SO.I^r), covering a size range from 7-131 mm peristomal length, had food in their digestive tracts (Table 2). Only 8.2% of the digestive tracts had contents that could not be identified because the process of digestion was already too ad- vanced (Table 2). About 98% of the individuals examined fed on one prey type. Only 18 individuals (2%) had more than one prey item in the digestive tract. The most important prey items were barnacles, representing 89.6% of the stomach contents, and 83.9% of intestinal contents (Fig. 21. The second most important prey item, the ascidian P. chilensis. represented 5.47r and 8.3% of the stomach and gut con- tents, respectively. The remainder of the prey was Calyplraea trochiformis, mitilids. and unidentified materials. Differences be- tween stomach and intestine were produced by more advanced digestion in the latter. That favored recognition of the ascidian in the intestine because its remains were recognized mainly by color, which was not affected by digestion. C. trochiformis was not found in the intestine. But these different digestion rates of the various prey did not change the general dominance of barnacles in the diet. The dietary importance of barnacles was most pronounced at Caleta Las Conchas, where they represented the only prey. In contrast, at Puerto Aldea, where C. concholepas was introduced by fishermen, barnacles were entirely replaced by P. chilensis (Fig. 3). With only two exceptions (Puerto Aldea and Lengua de Vaca). in all sites the barnacles were the predominant prey (Fig. 3). even though the basic community structure varied (Table I ). Prey composition did not differ among the different size groups within the pooled sample, where barnacles were always the dom- inant prey item (Fig. 4). The same analysis made at selected sam- pling sites, also showed in general, with only two exceptions (El Temblador 9-1 I cm; Totoralillo Sur 5-7 cm) (Fig. 5) that the barnacle was the predominant prey. Although in all cases the smallest and the biggest indi\iduals only fed on barnacles, interme- diate-sized individuals showed a slightly more varied diet (Fig. 5) Identification of Prey and Food Retention Time The feeding experiments with known prey items allowed gen- eral descriptions of the prey after ingestion by the gastropod. Skel- etal plates, cirri, and eggs were observed in the stomach and gut when C. concholepas fed on barnacles. When the ascidian Pxuru chilensis was the prey, an orange or red mass sometimes contain- ing syphons was observed. In the case of Calyptraea trochiformis. while-colored muscular tissue and egg capsules could be recog- nized. Comparison of these characteristics with those observed in the digestive contents of individuals collected in the field allowed the identification of most prey items. Regarding food retention, the percentage of individuals with content in the digestive tract is highest (83.3%) in the morning (0630 h) and in the evening (1830 h) when just sampled. As the starvation period increases, the proportion of individuals with con- tent in the digestive tract fluctuates, decreasing after 12 h of star- vation (Fig. 6). The decrease is more evident and regular for the stomach, not so much for the intestine. The stomach appears com- pletely empty after 16 h of starvation. Accordingly, the percentage of full stomachs or those with the content showing some digestion decreases as the starvation period increases (Fig. 7). Nevertheless, the tendency is not that clear, close to the end of the experiment appearing again individuals with full stomach or intestine, and showing just some digestion (Fig. 7). This suggests that some contamination of the experiment may have occurred. The problem probably stems on the fact that the shells of the individuals put together in the mesh bag were not cleaned. Thus the barnacles, which normally are attached to the shell, might have been con- sumed by some of the experimental indi\ iduals. Considering this possible contamination, the experiment suggests that the retention time in the stomach is around 6 h. whereas in the intestine the food seems to be retained up to 16 h. The prey species the experimental individuals had ingested were the same as described above for the individuals sampled along the coast (Fig. 8). Prey Selection by C. concholepas The most important prey species are not the most abundant species in the habitat (Table 3). Barnacles appear in small patches. 156 Stotz et al. TABLE 3. Abundance of macroalgae and invertebrates (percent co>er and density, mean and standard deviation) in the rocky subtidal in which Concholepas concholepas was collected at lour sites. Temblador Lagunillas Lengua de Vaca Isla Huevo Percent cover (%) Algae Rhodophyta Mesophytlwn sp. Corallina officinalis Gelidium chilense Calcareus algal crusts Phaeophyta Glossophora kiinthii Lessonia irtibccnUilii Porifera annellida Phnii^inalopoma sp. Roiiunuiiellii piisudata Spionidae Crustacea BaUmus laevis Httluiut\ flosciitii\ Austromei>ubalaiuis psitlacus Bryozoa Bugula sp. Briozoa indeterminated Hemichordata Pyura chilensis Free space Density (ind.m"') Mollusca Nassarius gayii Crassilabnim crassilnhrwn Tegula sp. Mitrellii iinifasiiura Crepiilula sp. Tegula Iridentala Calyptnwa Iroclnformis Density (ind. 1 00m"-) Cnidaria Anemonia alicemartiinie Phymactis clematis Phymanlhea pluvia Mollusca Concholepas concholepas Fissurella cosrara Fissurella ciimingii Crustacea Paroxanthiis barbiger Taliepus denumis Homalaspis plana Rhynchncinetes typiis Echinoderniata Aelionidiwn chilensis (Holoduiroidea) Meyenaster gelalinosus Stichaster strialus Heliaster helianihus Tetrapygiis niger 19.8 ±25.07 4.6 ± 10.29 5.0+ 11.18 2.4 ±2.51 68.0 ±28.72 29.8+ 17.04 0.2 ±0.45 5.6 ± 12.52 0.2 ± 1.79 0.8 ± 1.79 9.8 ± 10.43 21.2 ± l,V81 15.2 ±25.52 1.6±2.19 0.8 ± 1.79 20 361 16 2 45.6 ± 27.57 0.4 ± 0.55 4.0 ± 6.42 10.0± 11.16 70.0 ± 15.55 2.2 ±4.92 3.6 ± 5.68 1.6 ±2.30 10.6 ± 10.67 0.4 ± 0.8 1.2 ± 1.64 20.8 ± 23.86 0.6 ± 1 .34 1.6 ±3.58 1.0 ± 1.00 0.4 ± 0.89 95 2 55 1 3 17 1 5 57.0 ± 19.46 4.6 ±4.67 6.2 ± 8.90 1.2± 1.79 60.8 ±21.78 0.2 ± 0.45 6.6 ± 7.47 3.0 ±6.7 1 6.0 ± 7.04 15.2 ± 15.32 20.8 ± 29.04 4.8 ± 10.73 0.8 ± 1.79 1.6 ±2.19 0.8 ± 1.79 1 25 23 6 1 1 5 1 584 49.8 ± 23.22 0.4 + 0.55 10.0 ± 20.20 !3.8± 13.18 49.2 ±28. 12 1.0 ± 1.73 17.4± 13.92 0.4 ± 0.89 7.2 ± 16.10 124.8 ±265.85 164.0 ±257.74 125.6 ±265.38 26.4 ± 36.40 26 2 1 mostly associated to the area immediately around the holdfast of Lessonia Irabeciilata. where fronds do not wipe the rock. Pyura chilensis is mostly restricted to crevices. Percent cover of both prey species together lluctuates between 10 and 20% cover. But C. concholepas within the habitat selects microhahitats in which his prey species, mainly barnacles, are more abundant. In those mi- crohahitats percent cover of barnacles may increase up to almost 80% (Table 4). The polychaeta Phragmatopoma sp.. which con- Diet and Feeding Behavior of C. concholepas 157 TABLE 4. Proportion (%) of potential prtj in the different microhuhitats In Hhlcli Concholepas loiichohpas was captured on seven study sites. lA-nj^ua de Totoralillo Las El Temblador \ aca Huentelauquen Isla Huevo Sur Tinicunas Laguniiias Main prey species Pxura chilensis 14.34 6.16 1.01 Cirripedia 24.75 8.52 21.14 68.04 74.63 Phragmatopoma sp. Other potential prey Porifera annellida 45.66 8.76 43.74 6.88 8.13 27.68 2.52 4.88 14.63 Polvchaeta indeterniined 10.16 56.91 Romunclu'lla pusUilaui 3.68 6.47 Mollusca Calyplniea InKJiifonnis 0.29 1.75 Fix.surella spp 0.81 0.19 0.49 Timiciii elegans 0.31 Brachiodomes granulaia 0.22 2.26 Crassilahnim crassiUihnim 0.22 0.10 0.56 Tegiila spp Naisariiis gayii 0.51 0.41 0.41 5.37 Bryozo Brvozoa L59 8.42 9.76 Cnidaria Hydriv.oa indeterminate Echinodermata Tetnipygus niger Hemichordata 4.41 3.25 1.96 9.82 38.34 1.75 6.88 5.58 0.67 1.38 5.90 0.46 0.34 3.66 0.67 0.92 0.40 0.55 34.09 6.64 structs tubes of sediment attached to the rock surface, in some areas gets very important, covering together with the barnacles most part of the space in some sites (Table 4). The digestive tracts of C. concholepas from the sampled sites contained mainly barnacles and P. chilensis. Although barnacles are the most abundant prey species in the environment. P. chilensis was only rarely found, mostly in very low abundance. Only in one site the ascidian was important in the environment (El Temblador, Table 4). Barnacles appear in four of the seven sites as being positively selected (Table 5. Fig. 9). In the remaining three sites barnacles are consumed proportionally to their abundance in the environment. P. chilensis was present only in four of the seven sites (Table 4), being always positively selected (Table 5 1. On one site (Las Tinicunas) P. chilensis did not appear registered in the environment (its proportion less than 1%), but was in the digestive tract of the gastropod. When the data from all the sites are grouped and analyzed together, it is shown that only P. chilensis is posi- tively selected, the rest of preys being consumed proportionally to their abundance in the environment (Fig. 9H). Circadian Feeding Rhythm A total of 275 individuals were collected, representing a size range between 29 to 120 mm of peristomal length in the first 24-h sampling period. For the second period 88 and 84 individuals were sampled by each diver, representing a size range between 2(1 to 1 19 mm of peristomal length (Table 6). Numbers collected during individual sampling hours varied from 13 individuals at 2100 h to 71 individuals at 1300 h in the first sampling period and seven individuals at 2100 h to 28 individuals at 1700 h for the second sampling period (Table 6). As some of the samples were destroyed during the transport to the laboratory, the analysis is based on 254 individuals for the first sampling period, and on 66 and 81 indi- viduals respectively for the two replicate samples of the second sampling period. TABLE 5. Selection index C and x" for main prey species of Concholepas concholepas on seven sites. Lengua de El Temblador Vaca Huentelauquen Isla Huevo Totoralillo Sur Las Ti nicunas Lagu C nillas C X' C X^ C X- C X^ C x' C x' X^ Pxura chilensis 0.18* 6.64 0.47* 43.89 0.26* 13.39 0.34* 20.47 0.28* 16.22 Cirripedia 0.19* 6.95 0.05 0.45 0.80* 128.54 a 10 2.15 -0.(39 1.53 -0.43* 32.35 0.23* 10.37 Phragmatopoma sp. -0.39* 30.42 -0.53* .55.98 -0.63* 79.55 -0.32* 20.33 -0.16* 5.00 -0.09 1.76 Other species 0.03 0.21 0.01 0.0 1 -0.34 23.30 -0.13 3.23 0.14* 4.19 0.25* 11.11 -0.40* 3 1 .59 Values with * show significant positive or negative selection. 158 Stotz et al. TABLE 6. Date and time of 24-h samplings, number of individuals collected in the field, number of entire digestive tracts analyzed in the laboratory, and inditiduals with food in their digestive tracts (number and percentage). Sample Individuals Indi viduals Size Field Analyzed in the with Food Date Time (No.l Lab (No.) (No.) (%l 24 OCT 1994 17:00 44 42 31 73.8 21:00 13 13 11 84.6 01:00 46 40 31 77.5 05:00 44 44 37 84.1 09:00 57 52 41 78.8 13:00 71 63 45 71.4 Total 275 254 196 77.2 5 AUG 1995 17:00 28 19 15 78.9 (Replicate 1) 2 1 :00 7 5 5 100 01:00 S 7 6 85.7 05:00 15 7 6 85.7 09:00 15 15 14 93.3 13:00 15 13 11 84.6 Total 8S 66 57 86.4 5 AUG 1995 17:00 21 21 21 100 (Replicale 2) 21:00 7 7 7 100 01:00 12 12 9 75.0 05:00 16 13 12 92.3 09:00 15 15 13 86.7 13:00 13 13 10 76.9 Total 84 81 72 88.9 Considering all the individuals analyzed for the entire 24-h sampling, the individuals with food in their digestive tract (stom- ach and/or gut), represent 77.2% for the first sampling period and 86.4% and 88.9%. respectively, for the two replicate samples for the second sampling period (Table 6. Fig. 10). During the different sampling hours the proportion of individuals with food in their digestive tract for all sampling hours represented at least 71.4%. Although no clear pattern appears, in all sampling periods, the highest values were always registered at the late afternoon and early morning, thus suggesting that feeding intensity increases during the afternoon and in the second half of the night, or at dawn and dusk. Nevertheless, no statistical difference was detected be- tween day (individuals sampled at 0900. 1300. and 1700 h) and night (individuals sampled at 2100. 0100. 0500 h). as well as between the different replicate samples (sampling in October 94. and each diver in August 96) (3 x 3 G test, x" = 1 1.714: df = 7: P > 0. 1 ). Neither statistical difference was detected between dif- ferent hours (Contingency Table 6*3*2; x" = 32.7304; df = 27; P > 0.05). At the different sampling hours different degrees of fullness were observed (Fig. 10). Although no clear pattern can be identi- fied, the stomach shows a slight tendency of greater fullness in the afternoon or late afternoon hours, decreasing during night, with the same tendency repeating during the early morning hours. For the intestine it is observed, that as the stomach empties, the intestine increases in fullness (Fig. 10). Thus, again the data suggest that intake of new prey tends to increases at dawn and dusk. In all sampled individuals during both 24-h samplings, bar- nacles appear as the main prey species, with proportions ranging from 41.9% to 75.2%. with a mean value of 57.9% (Fig. 11). The second most important prey was Pyiini chilensis, which comprised 17.7% to 40.3% of the digestive tract contents. The remaining individuals had other preys of minor importance, such as Ciilrp- tniea trochifonnis (Fig. 11). The food composition also did not vary greatly with sampling time, and barnacles were always the dominant prey item. DISCUSSION Concholepas concholepas fed almost exclusively on barnacles and the ascidian Pyiini cliileiisis. The similarity in diet composi- tion among individuals from different localities and among differ- ent size classes, suggests that this is a general characteristic for subtidal populations of this species. These data support corresponding literature data (Castilla et al. 1979. DuBois et al. 1980. Sommer 1991 ), but show quantitatively, that barnacles were usually the most consumed prey in the differ- ent community or microhabitat types where C. concholepas was found. The smaller individuals of C. concholepas live on the un- dersides of boulders or in crevices (Stotz 1997. Guisado & Castilla 1983. Sommer 1991). where potential prey is probably different from that present on the rock surfaces where larger individuals live. Nevertheless, all size groups had consumed very similar food types. This suggests a strong feeding preference for barnacles, which nevertheless seems not always supported by the analysis with the selection index. With the pooled data. P. chilensis appears as the most preferred prey species. However, the preference is better shown by the fact that the gastropod is always found in microhabitats in which the barnacles predominate. And within such microhabitat the index is not any more able to show a pref- erence. Considering all the prey species described, the preference extends in general to suspension feeders. A similar behavior has been described for Acanthina lugubris angelica, the diet of which was restricted exclusively to sessile suspension feeders (Vermeij et al. 1994). The diet based on suspension feeders seems to be a general pattern for benthic predators, such as diverse gastropods and seastars (Table 7). The most common barnacles in subtidal communities are Baki- nus laevis and Aiistromegabalantis psituiciis. Individuals of the latter species are mostly small individuals with 0.5-1 cm basal diameter, while the species is able to growth to sizes of ca. 5-cm basal diameter. But in the I'egion. barnacles of such big size are seldom observed. Feeding based on barnacles that are small, sessile, and form a uniform cover on the substrate makes C. concholepas conceptually resemble a grazer. The feeding of C. concholepas is similar to the "grazing" of hydroid colonies by nudibranchs. or even to grazing gastropods, for example, the keyhole limpets Fisurella spp. (Moreno & Jaramillo 1983, Moreno et al. 1984. Godoy & Moreno 1989). This observation applies to many gastropods and starfishes (Table 7). It is a well-described characteristic for intertidal whelks (Dayton 1971. Paine 1966. Menge & Sutherland 1987). habitat in which the sessile suspension feeders are the main space occupiers, but less known for species living in the subtidal. where a wider variety of potential prey species may be expected. In fact. C. concholepas makes use of a wider variety of prey in such habitats, including mobile predators as crabs and even fishes (personal ob- servations), but quantitatively only the suspension feeders are im- portant. The feeding behavior of C. concholepas, not showing a clear circadian rhythm, differs from what has been published previously Diet and Feeding Behavior of C. concholepas 159 .2 Q 1U0 N=18 A >. o 50 ** * r-1 n S "- 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. LITERATURE CITED Acuna. E.. J. Moraga & E. Uribe. 1989. La zona dc Coqulniho: iin sistema nerftico de alta productivldad. Revista Paciticu Sur (Niiinero especial): 14.'i-153. Aguayo. A. & R. Maturana. 1973. Presencia del lobii niarino comiin {Otiiria flmesceiis) en el literal chileno. Biologia Pesquera. Chile 6: 45-75. Bakun. A. & C. S. Nelson. 1991. The seasonal cycle of wind-stress curl in sobtropical eastern boundary current regions. / Pliys. Oceanogr. 21:1815- 18,34. Bayne. B, L. & C. Scullar. 1978. Rates of feeding by Thais (Niici'lhil lupiUus (L.). / Exper. Mcirme Biol. Ecol. 32:1 13-132. Blackmore. G. 2000. Field evidence of metal transfer from invertebrate prey to an intertidal predator, Thais clavigera (Gastropoda: Muricidae). Esniurine Coastal Shelf Sci. 51:127-139. Branch. G. M. 1981. The biology of Umpets: physical factors, energy flow and ecological interactions. Oceanogr. Marine Biol. Annu. Rev. 19: 235-280. Brown, K. M. & W. B. Stickle. 2002. Physical constraints on the foraging ecology of a predatory snail. Marine Freshwater Behav. Physiol. 35: 157-166. Castilla. J. C. 1981 . Perspectivas de investigacion en estructura y dinamica de comunidades intermareales rocosas de Chile central. II. Depreda- dores de alto nivel trdfico. Medio Ambienle 5:190-215. Castilla, J. C. & J. Cancino. 1979. Principales depredadores de Conchole- pas concholepas (Mollusca: Gastropoda: Muricidae) y observaciones preliniinares sobre mecani.smos conductuales de escape y defensa. Bio- logia Pesquera. Chile 12:115-123. Castilla. J. C. & 1. Bahamondes. 1979. Observaciones conductuales y eco- logicas en Intra f el ina (Camivora: Mustelidae) en las zonas Central y Centro-Norte de Chile. Arch. Biol. Med. Exper. 12:119-132. Castilla, J. C. & L. R. Duran. 1985. Human exclusion from the rocky intertidal zone of central Chile: the effects on Concholepas conchole- pas (Gastropoda). Oikos 45:391-399. Castilla, J. C. & Ch. Guisado. 1979. Conducta de alimentacion nocturna de Coticholepas concholepas {MoWwica: Gastropoda: Miricidae). Biologia Pesquera. Chile 12:125-130. Castilla. J. C. & Jerez. G. 1986. Artisanal fishery and development of a date base for managing the loco Concholepas concholepas resource in Chile. Canadian Special Publication of Fishery and Aquatic Science 92:133-139. Castilla, J. C. & Paine, R.. T. 1987. Predation and community organization on Eastern Pacific, temperate zone, rocky intertidal coast. Revista Cliil- ena de Historia Natural 60:131-151. Castilla. J. C. Ch. Guisado & J. Cancino. 1979. Aspectos ecologicos y conductuales relacionados con la alimentacion de Concholepas con- cholepas (Mollusca: Gastropoda: Muricidae). Biologia Pesquera. Chile 12:99-114. Dayton, P. K. 1971. Competition, disturbance, and community organiza- tion: the provision and subsequent utilization of space in a rocky in- tertidal community. Ecol. Monogr. 41:351-389. Dayton, P. K., J. Rosenthal, L. Mahen & T. Antezana. 1977. Populatitm structure and foraging biology of the predaceous Chilean asteroid Mey- enaster geluimoMis and the escape hiology of its prey. Mar. Biol. 39:361-370. Dietl. G. 2000. Successful and unsuccessful predation of the gastropod Niicella lapillus (Muricidae) on the mussel Mytilus edidis from Maine. VV//;??;- 43:319-329. Dubois. R., J. C. Castilla & R. Cacciolatto. 1980. Sublittoral observations of behaviour in the Chilean 'Loco' Concholepas concholepas (Mol- lusca: Gastropoda: Muricidae). V'('//.?£'/- 23:83-92. Duggins. D. 1983. Starfish predation and the creation of mosaic patterns in a kelp-dominated community. Ecology 64:1610-1619. Fonseca, T. R. & M. Farias. 1987. Estudio del proceso de surgencia en la costa chilena utilizando percepcion remota. Invest. Pesquera 34:33^6. Gaymer, C. P., J. H. Himmelman & L. E. Johnson. 2001. Distribution and feeding ecology of the seastars Leptasterias polaris and Aslerias vul- garis in the northern Gulf of St. Lawrence. Canada. J. Marine Biol. Assoc. t/A' 81:827-843. George-Nacimento. M.. R. Bustamante & C. Gyarzun. 1985. Feeding ecol- ogy of the South American sea lion: Otaria flavescens: food contents and food selectivity. Marine Ecol. Prog. Series 21:135-143. Godoy. C. & C. Moreno. 1989. Indirect effects of human exclusion from the rocky intertidal in southern Chile: a case a cross-linkage between herbivores. Oikos 54:101-106. Gossehn. L. A. & F.-S. Chia. 1996. Feeding habits of newly hatched juveniles of an intertidal predatory gastropod, Nncella emarginata (De- shayes). / Exp. Marine Biol. Ecol. 176:1-13. Grahame. J. 1987. Plankton and fisheries. London: Edward Arnold Pub- lishers, 140 pp. Guisado, Ch.. & Castilla, J. C. 1983. Aspects of the ecology and growth of an intertidal juvenile population of Concholepas concholepas (Mol- lusca: gastropoda: Muricidae) at Las Cruces. Chile. Mar. Biol. 78:99- 103. Hawkins. S. J. & R. G. Hartnoll. 1983. Grazing of the intertidal algae by marine invertebrates. Oceanogr. Marine Biol. Aniui. Rev. 21:195-282. Himmelman, J. 1991. Diving observations of subtidal communities in the northern Gulf of St. Lawrence. In: J.-C. Therriault, editor. The Gulf of St. Lawrence: small ocean or big estuary'.' Canadian Special Publication of Fisheries and Aquatic Sciences. Fisheries and Oceans. Canada pp. 319-332. Himmelman. J. & Y. Lavergne. 1985. Organization of rocky subtidal com- munities in the St. Lawrence estuary. Naturaliste Canadiene 1 12:143- 1.54. Himmelman. J. & C. Dutil. 1991. Distribution, population structure and feeding of subtidal seastars in the northern Gulf of St. Lawrence. Ma- rine Ecol. Prog. Series 76:61-72. Hughes, R.. M. Burrows & S. Rogers. 1992. Ontogenic changes in foraging behaviour of the dog whelk Nncella lapillus (L). J. Exper, Marine Biol. Ecol. 155:199-212. Keough, M. & A. Butler. 1979. The role of asteroid predator in the orga- nization of a sessile community on pier piling. Mar. Biol. 51:167-177. 164 Stotz et al. Koh-Sian Tan. 2000. Feeding habits of Chicoreiis copticimis (Neogas- tropoda Muricidae) in a Singapure Mangrove. Menfit, Italy: 3° Work- shop Inlernacionale di Malacologia. Lowell. R, B. 1984. Desiccation of intertidal limpets: effects of shell size, fit to substratum, and shape. / Exper. Marine Biol. Ecol. 77:197-207. Menge. B. 1974. Competition for food between two intertidal starfish species and its effect on body size and feeding. Ecology 53:635-644. Menge, B. 1979. Coexistence between the seastars Asterias vulgaris and A. forbesi in a heterogeneous en\ironnient: A non equilibrium explana- tion. Oecologia 41:245-272. Menge, B, 1992a. Community regulation: under what conditions are bot- tom-up factors important on rocky shores? Ecology 73:755-765. Menge. B. 1992b. Effect of feeding on the environment: Asteroidea. In: M. A. Jangoux & J. Lawrence, editors. Echinoderm nutrition. Roterdam, Netherlands. A Balkema, pp. 521-551. Menge. B. & J. Sutherland. 1976. Species diversity gradients: synthesis of the roles of predation, competition, and temporal heterogeneity. Am. Naturalist 110:351-369. Menge, B. & J. Sutherland. 1987. Community regulation: variation in disturbance, competition, and predation in relation to environmental stress and recruitment. Am. Naturalist 130:730-757. Moreno, C. A. & E. Jaramillo. 1983. The role of grazers in the zonation of intertidal macroalga of the Chilean coast. Oikos 41:73-76. Moreno, C. A., J. P. Sutherland & H. F. Jara. 1984. Man apredator in the intertidal zone of southern Chile. Oikos 42:155-160, Moreno, C. A.. K. M. Lunecke & M. L Lepez. 1986. The response of an intertidal Concholepas concholepas (Gastropoda) population to protec- tion from man in southern Chile and the effects on benthic sessile assemblages. Oikos 46:359-364. Paine, R. T. 1966. Food web complexity and species diversity. Am. Natu- ralist 100:65-75. Pearre, S. 1982. Estiinating prey preference by predators: Uses of various indices, and a proposal of another based on x2. Can. J. Fish Aquatic Sci. 39:914-923. Perez, E. P. & W. B. Stotz. 1992. Comparaciones nitiltiples de parametros gravimetricos entre poblaciones submareales de Concholepas con- cholepas (Bruguiere, 1789) en el Norte de Chile. Revista de Biologi'a Marina. Valparaiso 27:175-186. Pino. C, Oliva, D. & Castilla. J. C. 1993. Ritmos de actividad en Fissurcllu crassa Lamarck 1822 y F. latimarginata Sowerby 1835: Efectos del ciclo de marea, fotoperiodicidad y estacionalidad. Xlll Jornadas de Ciencias del Mar. Vina del Mar, Chile, 26-28 de Mayo. Powers, S. & J. Kittinger. 2002. Hydrodynamic mediation of predator-prey interactions: differential patterns of prey susceptibility and predator success explained by variation in water flow. J. Exp. Marine Biol. Ecol. 273:171-187. Raymont. J. E. G. 1980. Plankton and productivity in the oceans. Vol. 1. Phytoplankton. Oxford. UK: Pergamon Press Ltd.. 489 pp. Rilov, G., A. Gasith & Y. Benayahu. 2002. Effect of an exotic prey on the feeding pattern of a predatory snail. Marine Environ. Res. 54:85-98. Saier, B. 2001. Direct and indirect effects of seastars Asterias rubens on mussel beds {.Mylilus edulis) in the Wadden sea. J. Sea Res. 46:29^2. SERNAP. (Servicio Nacional de Pesca). 1999. Anuario estadi'stico de pesca. Chile: Ministerio de Economia. Fomento y Reconstruccion. Sielfield, W. 1990. Dieta del chungungo iLutra felina (Molina. 1782)) (Mustelidae. Camivora) en Chile austral. Investigaciones Cientificas y Tecnologicas. Serie Ciencias del Mar 1:23-29. Sokal. R. S. & F. J. Rohlf. 1969. Biometry. San Francisco: W. H. Freeman and Company. 776 pp. Sommer. H.-J. 1991. Zur Okologie ausgewahlter Benthosarten im chilenis- chen Felslitoral: Wiederbesiedlungsversuche mit Concholepas con- cholepas. Diplomarbeit aus dent Zoologischen Instilul der Christhian- Albrechts-Univeristat zu Kiel. Kiel. BRD. 81 p. Sommer. H.-J. & W. Stotz. 1991. ,,Puede el loco Concholepas coiuholepas (Bruguiere. 1789) vivir en fondos blanqueados? XI Jornadas de Cien- cias del Mar. Viiia del Mar (Chile). 27-29 de Mayo. Stotz, W. 1997. Las areas de manejo en la ley de pesca y acuicultura: primeras experiencias y evaluacion de la utilidad de esta herramienta para el recurso loco. Estiidios Oceanologicos 16:67-86. Stotz, W. & Perez, E. 1992. Crecimiento y productividad del loco Conc- holepas concholepas (Bruguiere. 1789) como estimador de capacidad de carga en areas de manejo. Invest. Pesqueras 37:13-22. Stotz, W. & D. Lancellotti. 1993. Asentamiento del loco Coiuholepas concholepas (Bruguiere. 1789) en la zona intermareal: Excepcion o regla. XIII Jornadas de Ciencias del Mar. Vit'ia del Mar (Chile). 26 al 28 de Mayo Thomas. A.C.. F. Huang. P.T. Strubb & James, C. 1994. Comparison of the seasonal and interannual variability of phytoplankton pigment concen- trations in the Peru and California Current systems. J. Geophysical Res. 99:7355-7370. Tokeshi. M.. C. Estrella & C. Paredes. 1989. Feeding ecology of a size structured predator population, the South American sun star Heliaster heliamhus. Marine Biol. 100:495-505. Underwood. A. J. 1979. The ecology of intertidal gastropods. Adv. Marine Biol. 16:111-210. Vasquez. J. 1993. Abundance, distributional patterns and diets of main herbivorous and carnivorous species associated to Lessonia traheculata kelp beds in northern Chile. Facultad de Ciencias del Mar. Universidad Catolica del Norte. Cocpnmho Chile Serie Ocasional 2:213-229. Vasquez. J. & J. Castilla. 1984. Some aspects of the biology and trophic range of Cosmasleria lurida (Asteroidea. Asteriinae) in belts of Mac- rocystis pyrifera at Puerto Toro. Cliile Medio Ambiente 1:47-5 1 . Vermeij. G. J.. H. A. Lescinsky, E. Zipser & H. E. Vermeij. 1994. Diet and mode of feeding of the muricid gastropod Acanthinucella liigubris angelica in the northern Gulf of California. Veliger 37:214-215. Viviani. C. A. 1975. Las comunidades marinas litorales en el Norte Grande de Chile. Iquique. Chile: Publicacidn ocasional Laboratorio de Ecologi'a Marina. 196 pp. Whittaker. R. H. 1975. Communities and Ecosystems (second edition). New York: Macmillan Publishing Co.. Inc.. 387 pp. .loiiriHil ,.f Shellfish Research. Vol. 22. No. I. I6.';-I64, 200.^. FEEDING AND GROWTH IN THE KEYHOLE LIMPET, FISSURELLA FICTA (GMELIN, 17911 D. A. LOPEZ.* M. L. GONZALEZ. AND M. C. PEREZ Ltihoiaiono de Cultivos Marinas. Dcpariumeiiio dc Aciiiculliira. Univcisidad de Los Liigos, Casilla 933, Osorno. Chile ABSTRACT The feeding habits and growth relationships ol the keyhole limpet ("lapa") hissiirelhi pieki were analyzed in the field and under laboratory conditions. This species is of significant commercial value and considerable ecological importance in southern Chile. F. picta is not strictly a herbivore, although it prefers algae; the quantity of vegetable items con.sumed compared with animal items did not vary seasonally. The items most commonly found in the stomach of F. picta were the algae Ulva sp, Polysophonia sp and Gelidhim .tp. The abundance pattern of the principal items did not vary seasonally. However, there was greater diversity in the summer. The relative abundance of items in the diet was closely associated with their relative abundance in the environment. Under laboratory conditions, adults showed a higher consumption rate for the alga Gracilaiia chileii.\is (artificial diet) than for Ulva sp (natural diet). The preferred alga is not usually found in the natural habitat of F. picta and has a lower caloric value than that of Ulva sp. C. chilensis proved to be the best source of energy available for growth in juveniles. Keyhole limpets feeding on the chlorophyte alga Ulva sp show a negative energy balance. Specimens maintained in suspended systems and fed with the artificial diet (G. chilensis) reached the average commercial size of 5.^ mm in -3 y; the average survival rate was 90"/?. The results suggest that keyhole limpets prefer food with a high energetic scope for growth, although in field conditions they consume food with a lower energetic content but high in abundance. Factors such as morphology or palatability of food are more important than caloric value or presence in the natural habitats of keyhole limpets. This information is important for the culture of the keyhole limpet. KEY WORDS: feeding, scope for growth, keyhole limpet. Fi.'.siirella picia INTRODUCTION Keyhole limpets ("lapas") of the genus Fissurella are grazing molluscs that consume a wide variety of macroalgae in the inter- tidal zone (Branch 1981, Hawkins & Hartnoll 1983). Previous studies indicate that they also ingest other types of food, such as crustaceans, small molluscs, coralline algae, ostracods, and sponges, although they remain preferentially herbivores (Ward 1966. Bretos 1978, Santelices & Coirea 1985, Osorio et al. 1988). Among Chilean species of lapas, Fissurella crassa is classified as a generalist herbivore, which prefers to consume foliacious algae, such as Ulva sp.. Emeromorpha sp and Porphyni sp (Bretos 1978, Santelices et al. 1986). Data available on F. maxima, based on studies of its stomach contents, indicate that this species is euriphycophagous (Osorio et al. 1988). Experimental field studies on F. picta suggest that this species is a nocturnal herbivore, which migrates during the night to the middle intertidal zone (Jara & Moreno 1984. Moreno et al. 1984), to feed on the algae Iridaea horycma and Ulva rigida. F. picta. has an important commercial value, and over- harvesting has resulted in the depletion of natural stocks in south- ern Chile (Bretos 1978. Bretos 1988). In addition, human exploi- tation of other species has, indirectly, had a negative effect on keyhole limpet recruitment (Lopez et al. 1999). This species also has ecological importance given that it can modify the spatial and temporal distribution patterns of intertidal macroalgae (Mi)reno et al. 1 984). Knowledge of the diet and dietary preferences of F. picta is necessary to evaluate its growth rate in artificial cultures and to interpret the ecological role of the population under field condi- tions. Published literature suggests that the interaction between quan- tity and quality of food with factors such as pH. temperature and salinity, influences growth in mobile marine invertebrates (Newell 1979. Frantzis & Gremare 1992). The effect of type of food in- gested on growth can be determined by measuring the increase in *Conesponding author. Fax: -1-56-6-420-5271; E-mail: dIopezCfl'ulagos.cl weight or size of the animals, or in terms of energy through scope for growth, established by evaluating the components of the energy balance (Paine 1971, Bayne & Newell 1983. Gonzalez et al 1990, Gonzalez et al. 1993, Thompson & MacDonald 1991, Navarro & Torrijos 1994, NavaiTO & Torrijos 199.S). The aims of this study are to determine the feeding habits of the keyhole limpet, F. picta (Gmelin) in the field and under laboratory conditions and to es- tablish the relationship between feeding and growth. MATERIALS AND METHODS Stomach Conteiil in the Wild The feeding habits of keyhole limpets were observed in the intertidal and subtidal zone of Metri Bay (4r36'S, 72°42'W), in southern Chile. The stomach contents of 40 F. picta specimens (between 32.9 and 64.8 mm total length) were analyzed per season. Specimens collected at high tide were immediately injected with formalin dissolved in seawater to stop digestion. The stomach contents were analyzed over a 100-point grid (81 mm~). Thus, it was possible to determine ( I ) the relative frequency of vegetable and animal items; (2) the relative frequency of empty and full stomachs; and (3) the quantity and frequency of each item in the diet. A reference collection of all fronds of alga species present in different habitats and at different periods of the year was estab- lished to facilitate the identification of alga species consumed by lapas. Analysis was carried out under a dissecting scope. The relative abundance of sessile species present in the study area was verified during each season, based on coverage, using a 100-point grid 0.0625 m~ along ten linear transects of 15-18 m in the inter- tidal zone (Bumham et al. 1980). The statistical comparison between vegetable and aniinal con- tent in keyhole limpets was carried out by the x" test. The differ- ences in dietary preference and energy consumed and lost in ani- mals feeding on Ulva sp. and G. chilensis were analyzed with a r-tesl. Using correlation analysis, the relative abundance of algae in the diet was associated with the food supply of algae available in the environment. 165 166 Lopez et al. "Scope for growth" with Natural and Artificial Diets Juveniles of F. picta (length between 25.0-32.3 mm) were collected from the rocky intertidal zone in Metri Bay. The animals were separated into two groups and acclimated in the laboratory at 10°C ± 1°C for 20 days. During the experimental phase, each group was fed ad libitum with Ulva sp (Chlorophyla) or C. chil- ensis (Rodophyta). All the parameters of energy balance were standardized as joules per day per gram of shell-free dry weight (J • d~' • gdw~'). (using 1 cal = 4.18 J) (Lucas & Beninger 1985). Animal dry weight was obtained using the regression equation for length ver- sus dry weight, calculated for 150 keyhole limpets with lengths between 20.0-36.0 mm. The experimental procedures for the two groups were as fol- lows: To evaluate the effect of natural and artificial diets on ingestion rate, 40 F. picta specimens of 42.2 ± 9.5 mm total length, collected in the middle and lower rocky intertidal zone of Metri Bay, were transferred to aquaria for an acclimation period of 13 days at 15°C ± 1°C. The specimens were permanently submerged and the water was changed every 5-7 days. The ingestion rate of two types of macroalgae was compared: Ulva sp, which is the most frequent item found in the habitat of F. picta (natural diet) and G. chileiisis. a rhodophycean species of alga, not present in the keyhole limpet's natural habitat (artificial diet). G. chilensis is the principal species used in artificial culture with an average annual production of 821 19.5 ton y~' (Semap 1998). The two alga species have distinct forms: Ulva sp is foliaceus and G. chilensis is ramified. Each alga species was offered ad libititm to two groups of twenty animals of similar sizes kept in 1-L individual aquaria. The ingestion rate was measured gravimetrically, at 7-day intervals. An aquarium containing only alga samples was used as a control. The inges- tion rate was obtained by comparing differences in alga weight at the beginning and end of the experiment, expressed in grams of dry weight of algae consumed per individual per day (gdw • ind"' • d"'). Measurement of alga consumption was adjusted ac- cording to percentage weight variation of algae in the controls. No animal items were used as food because F. picta feed principally on algae and an important fraction of animal items in its diet are epiphytic organisms. The caloric contents of the Ulva sp and G. chilensis used in the experiments was measured with a Parr bomb calorimeter. Energy consumed (C) was determined using the ca- loric value of the algae. The energy loss due to metabolism (R) was measured in 39 animals as the standard oxygen consumption in a 145-mL hermetic flask using a WTW-530 oxygenometer (0.01 mg 0,/l accuracy). For conversion into energy, the Thompson and Bayne ( 1974) oxi- caloric value of 1 niL O, = 19.95 J was used. The excretion rate of ammonia (U) was determined in 40 in- dividual keyhole limpets measuring the concentration of ammonia accumulated over a period of 15 min in 200 ml aquaria, using the Solorzano method (Solorzano 1969). Conversion into energy units was carried out using the Elliot and Davison ( 1975) constant of 1 mg NH4* = 24.85 J. The energy loss through feces (F) was measured in 15 keyhole limpets that were placed individually in 1-L aquaria containing filtered seawater (mesh size: 1 |j,m) that was changed daily and with a constant supply of air. The feces were collected every 1 2 h according to methods described by Navarro and Thompson (1996). rinsed with isotonic solution of ammonium formate, kept in containers, and dried in a Memmert 500 furnace at 75°C until a constant weight was reached. The caloric value of the feces was determined in a Parr adiabatic bomb calorimeter. Energy loss through mucus (M) was evaluated by filtering water through 120-|jLm mesh. The energy values of scope for growth were calculated accord- ing to the following equation, using above average calculated val- ues: P = C-(F-^R + U-fM) where P = scope for growth; C = energy from food consumed: F = fecal energy loss; R = metabolic energy loss; U = energy loss due to excretion and M = mucus. Determination of Absorption Efficiency Absorption efficiency was calculated using the Conover equa- tion (Conover 1966): AE = (F-E) (I -E)x F' 100 where AE = absorption efficiency (9<-); F = ash-free dry weight food/total dry weight food and E = ash-free dry weight feces/total dry weight feces. To determine the algal and fecal organic matter content, algae and feces were carefully rinsed with distilled water and then dried in a Memmert 500 furnace at 75°C. until constant weight was reached. The samples were then incinerated in a muffle furnace at 450°C for 4 h. The organic matter was obtained by establishing the difference between the constant weight and the weight of the ash of each sample after incineration. The results of all the above determinations were then compared (differences between animals fed with a diet of G. chilensis or Ulva sp), using one-way ANOVA after logarithmic transformation (Sokal & Rohlf 1979). Dietary Preference — Natural and Artificial Diets The same quantity of Ulva sp and C. chilensis (volume and weight) was supplied simultaneously to a group of 20 individuals of 46.7 ± 9.5 mm total length. The amount of algae consumed by each specimen was determined daily, based on the biomass varia- tions, with an electronic balance (±0,01g accuracy). A control was also set up. Growth of Keyhole Limpets in Suspended Systems Feeding on an Artificial Diet The direct effects of the artificial diet on keyhole limpets' growth and mortality were determined in ailificial cultures. This study was carried out over 12 months in Metri Bay. At this location, average water temperature varies between 9.6°C (winter) and I8.2"C (summer); salinity fluctuated between 28%f and 32%t during the study period. Two hundred and forty specimens of F. picia collected from the intertidal zone were placed in trays ("lintemas") that were sus- pended from a raft. Specimens were fed ad libitum with the red alga G. chilensis. Four size categories were used. Initial average size and the standard deviations of keyhole limpets placed in ex- perimental growth systems (n = 20 per group) were; group 1 : 25.9 + 1.3 mm; group 2: 31.9 ± 1.9 mm; group 3: 37.8 ± 0.7 mm and group 4; 45.0 ± 0.9 mm. The experiments were replicated three times. Total weight and maximun length were measured monthly. Feeding and Growth in Fissurella picta 167 n Without gasinc content ■ With gasdic content 100 80 60 40 20 0 lL Ax s w Sp Figure without (Sp). SEASON 1. Relative seasonal frequencj of Fissiirella picta with and gastric content. Summer (Si; Autumn (A); Winter (W); Spring RESULTS D = Vegetable items 80 n J 1 = Animals iteins Aimjr^ ? «° « 40 01 3 T « 20 . 0 i >< o 80 60 40 20 Sloinaih Contents in the Wild WINTHR The relative frequency of F. picta specimens with empty stom- achs was less in autumn and winter than in summer and spring (Fig. 1). The percentage of vegetable items was always signifi- cantly higher than the animal items {P < 0.05), with no variation between different periods of the year (Fig. 2). The most frequent items present in F. picta stomachs were the algae Ulva sp, Pohsiplioitia sp, and Gelidiuin sp, especially during autumn. The main animal items were cirripedes and juvenile bi- valves (Fig. 3). There was a positive correlation between the rela- tive abundance of food items present in the stomachs throughout the year and the relative abundance of these items in the environ- ment (r = 0.891; n = 65: P < 0.05). Scope for Growth The diets used in scope for growth measurement had different energy values. The energy content of Ulva sp ( 1 3,990.5 J • gdw^' ) was higher than that of G. chilensis ( 1 1.101 2 J • gdw"' ). The type of food intluenced the energy balance and the scope for growth. The scope for growth was highest when F. picta consumed G. chilensis (Table 1 ). The negative energy balance in specimens fed with Ulva sp was due to energy loss (Table 1 . 1 The amount of energy consumed by F. /nrfcv juveniles did not vary significantly in animals fed with G. chilensis and those fed with Ulva sp (t = 100 5- 80 5" 60 c s « e ll 20 0 D Vegetable items ■ Animals items S w Sp SEASON Figure 2. Relative seasonal frequency of vegetahle and animal items in gastric content o( Fissiirella picta. Summer (S): Autumn (A): Winter (W); Spring (Sp). >< u ou - 60- SPRING 40 20 0 T n 1. 80 £ 60 >t u S 40 20 4 U SlMJBi a Ex Ch Jb Items Figure 3. Seasonal frequency (average ± standard deviation) of food items in the stomachs of Fissurella picta. Ulva sp (U); Chondrus sp (Ch); Gelidiuin sp ((i); I'olysiphonia sp (P); Fnteroinorpha sp (E); Cir- ripeds (C); juvenile hivalves (Jb); Sodilittorina araucana (I.). 0.098; df = 28; P< 0.005) (Table 1.). The quality of food affected the metabolic losses in F. picta (Fig. 4A). Oxygen consumption was significantly higher in animals fed with Ulva_sp than in those fed with G. chilensis (t = 5.48: df = 37; F < 0.001 ). The energy loss due to excretion was significantly higher in animals fed with G. chilensis, 23.0 J • d"' • gdw~', than in those fed with Ulva sp, 5.4 J • d"' • gdw-' (t = 8.10; df = 13; P < 0.001). The fecal energy loss was also affected by the quality of food (Fig. 4B). Specimens fed Ulva sp had significantly higher fecal energy los.ses than those fed G. chilensis (ts = 6.56; df = 13; P < 0.001 ), Since no mucus was found in the aquaria, and given that this value would only represent IVc of the energy ingested in herbivorous molluscs (Paine 1971 ). energy loss through mucus (M) was not considered. 168 Lopez et al. TABLE 1. Energy ingested, energy loss and scope for growth in Fissiirella piciii juveniles fed with L'lva sp (natural dietl or Gracilaria chitensis (artificial diet) in joule/day/gram dry weight of soft parts. Food Parameter Ulva sp Gracilaria chileiisis Range of energy ingested (J ■ d"' gdw"') Total energy loss (J • d~' ■ gdw" Average scope for growth (J -d"' -gdw-') 605.3-1.504.3 740-1.920.3 803 ±238.6 4(.)y ± 140.2 -10.4 390.6 Absorption Efficiency Absorption efficiency was highest in specimens fed Ulva sp. 83.4%, and lowest in those fed G. cliilensis. 74,6% (x" = 0.49; df = 1; 0.05). Dietary Preference — Natural and Artificial Diets In specimens of F. piclci. consumption rates of C. chilensis (artificial diet) were higher than those of Ulva sp (natural diet) (t = 76.12; df = 27; P < 0.001 ) and they also presented a greater preference for C. chilensis than for Ulva sp (t = 19.89; df = 28; P<0.00\). Growth of Keyhole Limpets in Suspended Systems The alga G. cliilensis proved to be suitable food for growth and survival in keyhole limpets. The annual average survival rate was 90'7(- under these experimental conditions. The growth rates of the animals varied according to size. Using these data it was cal- culated that F. picta reached 26.0 mm in about 14 mo. Thus, the average commercial size of .53 mm would be achieved in approxi- mately 3 y (Table 2). DISCUSSION The results obtained indicate that F. picta is preferentially a herbivore, as has been described for other species of this genus. (Osorio et al. 1988. Santelices et al. 19861. However, it also con- sumes animal items. Similarly, the high consumption of foliaceus species such as Ulva sp (Jara & Moreno 1984) was also confirmed. This can be associated with the food supply available in the envi- TABLE 2. Growth in four groups in = 20) of Fissurella picta in suspended cultures, feeding on Gracilaria chilensis (artit'icial diet). Initial Length Final Length Time Group ( mm 1 (mml (Month) 1 25.9 ±1.31 38.6 ± 4,4 12 48.2 ±0.1 2! 2 31.98+ 1.97 46.3 ± 5.7 12 3 37.86 ± 0.76 50.0 ± 5.3 12 4 45.03 ± 0.95 54.6 ± 1.2 X 55.3 ± 2.3 14 er 21:268- 273. Bretos. M. 1988. Pesqueria de lapas en Chile. Medio ,\mlnen!e 9:7-12. Burnham. K. P.. D. R. Anderson & J. L. Laake. 1980. Estimation of density from line transect sampling of biological population. Wildlife Mono- graphs 72:10-202. Buschmann. A. H. 1991. Algal communities of a wave protected intertidal rocky shore in Southern Chile. In: U. Seeliger, editor. Algal commu- nities in Latin America Florida. New York: Academic Press pp, 91- 103. Conover, R. J. 1966. Assimilation of organic matter by zooplankton. Lim- nol. Oceanogr. 1 1 :338-354. Elliot. J. M. & W. Davison. 1975. Energy equivalents of oxygen consump- tion in animal energetics. Oecologia 19:195-201. Frantzis. A. & A. Gremare. 1992. Ingestion, absorption and growth rates of Paracentrotus lividus (Echinodermata: Echinoidea) fed with different macrophytes. Mar. Ecol. Prog. Series 95:169-183. Gonzalez. M. L., M. C. Perez. D. A. Lopez & M. S. Buitano. 1990. Efecto de la temperatura en la disponibilidad de energia para crecimiento de Coneholepas eoncholepas (Bruguiere). Rev. Biol. Mar. 25:71-81. Gonzalez. M. L.. D. A. Lopez. M. C. Perez & J. M. Castro. 2002. Effect of temperature on the scope for growth in juvenile scallops .Argopecien piirpuraliis (Lamark. 1819). Aqiiaculture Intemalional. Hay. M. E. & W. Fenical. 1992. Chemical mediation of .seaweed-herbivore interactions in Plant-animal interactions in the Marine Benthos. In: D. M. John. S. J. Hawkins & J. H. Price, editors. Systematics Association Special Volume No 46. Oxford: Claredon Press, pp. 319-338. Hawkins. S. J. & R. G. Hartnoll. 1983. Grazing of intertidal algae by marine invertebrates. Oceanogr. Mar. Biol. Ann. Rev. 21:195-282. Jara. F. H. & C. A. Moreno. 1984. Herbivore and structure in the mid- littoral rocky community: a case in Southern Chile. Ecology 65:28-38. Lopez. D. A.. M. L. Gonzalez, J. M. Uribe. R. 1. Martinez & P. A. Vergara. 1999. Effect of cirripeds on the recruitment of the keyhole limpet Fissurella picta (Gmelin). Ciencias Marinas 25:75-90. Lowe. E. F. & J. M. Lawrence. 1976. Absorption efficiency of Lyiechinu.s variegatus (Lamarck) (Echinodermata: Echinoidea) for selected marine plants. ./. E.xp. Mar Biol. Ecol. 21:223-234. Lucas. A. & A. G. Beninger. 1985. The use of physiological condition indices in marine bivalve aquaculture. Aquaculture 44: 1 87-200. Moreno. C. A.. J. P. Sutherland & H. F. Jara. 1984. Man as predator in the intertidal zone of Southern Chile. Oikos 42:155-160. Navarro. J.M. & R.A. Torrijos. 1994. Seasonal variation in oxygen uptake and ammonia excretion in the predatory gastropod Coneholepas con- eholepas (Bruguiere. 1789). Compar. Biochem. Physiol. 108:39-46. Navarro. J. M. & R. A. Torrijos. 1995. Fisiologia energetica de Conehole- pas coneholepas (Bruguiere. 1789) (Gastropoda: Muricidae) en la bahfa de Yaldad. sur de Chile. Rev. Chilena de Historia Natural 68: 61-77. Newell. R. C. 1979. Biology ol intertidal animals. Mar. Ecol. Suney Ltd. Fawerham. Kent. 757 pp. Osorio. C. M. E. Ramirez & J. Salgado. 1988. Gastric contents of Fis- surella nucxima (Mollusca: Archaeogastropoda) at Los Vilos. Chile. Veliger 30:347-350. Paine. R. T. 1971. Energy flow in natural population of the herbivorous gastropod Tegula funebralis. Limnol. Oceanogr. 16:86-98. Santelices. B. & J. Correa. 1985. Differential survival of macroalgae to digestion by intertidal herbivore molluscs. J. E.xp. Biol. Ecol. 88:183- 191. Santelices. B.. J. Vasquez & I. Meneses. 1986. Patrones de distribucion y dietas en un gremio de moluscos y herbivoros en habitats intermareales expuestos de Chile Central. Publicaciones Periodicas. P. Universidad Catolica de Chile. Monogr. Biol. 4:147-171. Servicio Nacional de Pesca. (Semap) 1998. Anuario Estadi'stico de Pesca.. Valparaiso. Chile. 240 pp. Sokal. R. & J. Rohlf 1979. Biometria. San Francisco: Freeman & Com- pany. Solorzano. L. 1969. Determination of ammonia in waters by the phenol- hypochlonte methods. Limnol. Oceanogr. 14:799-801. Thompson. R. J. & B. L. Bayne. 1974. Some relationships between growth metabolism and food in the mussel Myiihis edidis. Mar. Biol. 27:317- 326. Thompson. R. J. & B. A. MacDonald. 1991. Physiological integrations and energy partitioning. In: S. E. Shumway, editor. Scallops: biology, ecol- ogy and aquaculture. Developments in aquaculture and fisheries sci- ence. 21. Amsterdam: Elsevier, pp. 347-378. Tugwell. S. & G. M. Branch. 1992. Effect of herbivore gut surfactants on kelp polyphenol defense. Ecology. 73:205-215. Ward. J. 1966. Feeding, digestion, and histology of the digestive tract in the keyhole limpet Fissurella harbadensis Gmelin. Bull. Mar. Sci. 16:668- 683. 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 Bennett. R. Jr.. N. Thanassi & H. I. Nakada. 1971. Hepatopancreas gly- cosidases of the abalone (Hcilioris rufescens). Comp. Biochem. Physiol. 406:807-811. Coote, T. A. 1998. The protein, energy and lysine requirements of greenlip abalone [Huliinis laevigata). Ph.D. Dissertation. University of Tasma- nia. .Australia. 1 18 pp. Ousel. G.. H. Kluge. K. Glaser, O. Simon, G. Harmann. J. v. Lengerken & H. Jeroch. 1997. An investigation into the variability of extract viscos- ity of wheat — relationship with the content of non-starch- polysaccharide fractions and metaholisable energy for broiler chickens. Arch. Anim. Niitr. 50:121-135. Elyakova, L. A.. N. M. Shevchenko & S. M. Avaeva. 1981. A comparative study of carbohydrase activities in marine invertebrates. Comp. Bio- chem. Physiol. 69B:905-90S. Fleming. A. E. 1995. Growth, intake, feed conversion efficiency and chemosensory preference of the Australian abalone. Haliolis rubra. .■Kqttaciilnire. 132:297-311. Hardy, R. E. 1997. Understanding and using apparent digestibility coeffi- cients in fish nutrition. Aquacultitre Mag. May/June:84-85. 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- ricult. Chem. 7:328-331. Ragan. M. A. & K. W. Glombitza. 1986. Phlorotannins. brown algal polyphenols. Prog. Phycol. Re.s. 4:129-241. Reed. J. D. 1995. Nutritional toxicology of tannins and related polyphenols in forage legumes. / Anim. Sci. 73:1516-1528. Royal Australian Chemical Institute. 1999. Cereal Chemistry Division- Official Methods. Dumas (combustion) total nitrogen determination. Method No: 02-03. SAS Institute Inc. 1988. SAS/STAT® Users guide. Release 6.03 Edition. Cary. NC: SAS Institute Inc.. 1028 pp. Schweiger. R. G. 1978. Carbohydrate Sulfates. A symposium sponsored by the ACS Division of Carbohydrate Chemistry at the 174"' Meeting of the American Chemical Society. Chicago. Illinois. August 30-31, 1977. ACS Symposium Series 77. pp. 214-243. Shepherd. S. A. 1975. Distribution, habitat and feeding habits of abalone. Aiisi. Fisheries 34:12-15. Shepherd. S. A. & P. D. Steinberg. 1992. Food preferences of three Aus- tralian abalone species with a review of the algal food of abalone. In: S. A. Shepherd. M. J. Tegner & S. A. Guzman del Proo. editors. Aba- lone of the world. Biology, fisheries and culture. Oxford: Blackwell Scientific Publications, pp. 169-181. Steinberg. P. D. 1989. Biogeographical variation in brown algal polyphe- nolics and other secondary metabolites, comparison between temperate Australasia and North America. Oecologia. 78:373-382. Stewart. W. D. P. 1974. Algal physiology and biochemistry. In: W. D. P. Stewart, editors. Oxford: Blackwell Scientific Publications, Osney Mead. 989 pp. Wee. K. L.. G. B. Maguire & S. Hindrum. 1994. Methodology for digest- ibility studies with abalone 2. Comparison of markers in an artificial diet for blacklip abalone (Haliolis rubra) and greenlip abalone (//. laevigala). In: L. M. Chou. A. D. Munro. T. J. Lam, L. K. K. Cheong, J. K. Ding, K. K. Hooi, H. W. Khoo. V. P. E. Phang, K. F Shim & C. H. Tan. editors. Proceedings of the Third Asian Fisheries Forum. Singapore. 26-30'" October. 1992. pp. 152-155. JoiiiiKil ,>f Shellfish Research. Vol. 22. No. I. I77-IS4. 2()()_V revip:vv of techniques to prevent introduction of zebra mussels (dreissena polymorpha) during native mussel (unionoidea) conservation activities W. GREGORY COPE,'* TERESA J. NEWTON," AND CATHERINE M. GATENBY' ^ North Carolina State University, Department of Environmental and Molecular Toxicology, Box 7633. Raleigh. North Carolina 27695; ^United States Geological Siiney. Upper Midwest Environmental Sciences Center, 2630 Fanta Reed Road, La Crosse, Wisconsin 54603: Academy of Natural Sciences, Patrick Center for Environmental Research. I'-MM) Ben Franklin Parkway. Philadelphia. Pennsylvania 19103 ABSTRACT Because ot the declines in diversity and abundance of native freshwater mussels (superl'amily Unionoidea). and the potential decimation of populations of native mussels resulting from the rapid spread of the exotic zebra mussel Dreissena polymorpha. management options to eliminate or reduce the threat of the zebra mussel are needed. Relocating native mussels to refugia (artificial and natural) has been proposed to mitigate the threat of zebra inussels to native species. Relocation of native inussels to refugia such as t~ish hatchery facilities or natural habitats within their historic range, which are unlikely to be infested by zebra mussels, necessitates that protocols be developed to prevent the inadvertent introduction of zebra mussels. Several recent studies have developed such protocols, and have assessed their effectiveness on the health and survival of native mussels during subsequent relocation to various refugia. The purpose of this project is to synthesize and evaluate the current protocols and to develop a set of procedures that resource managers and researchers should consider before conducting conservation activities in zebra mussel infested waters. We found that the existmg protocols have many common points of concern, such as facility modification and suitability, zebra mussel risk assessment and management procedures, and health and disease management procedures. These conservation protocols may have broad appli- cability to other situations and locations. A summary and evaluation of the mformation in these main areas, along with recommended guidelines, are presented in this article. A'£)' WORDS: relocation, Unionidae, Dieissenu polymorphu. conservation, refugia INTRODUCTION Native freshwater mussels of the families Martiariliferiihw and Unionidae (supeifamily Unionoidea) are one of the most rapidly declining fauna! groups in North America. About 67% of the nearly 300 native species found in North America are considered vulnerable to extinction or already extinct (Bogan 1993, Williams et al, 1993). The decline of native mussel populations in Noilh America has occurred steadily since the mid 1 800s and has been attributed to overharvest, construction of dams and impoundments, sedimentation, navigation, pollution, and habitat degradation (Fuller 1974, Bogan 1993, Naimo 199?, Brim Box & Mossa 1999, Vaughn & Taylor 1999). An additional recent threat to the native fauna has come from the introduction of the zebra tnussel Dreis- sena piilyiiiorpha. This species colonizes native mussels and im- pedes their movement, reduces the ability to feed and eliminate wastes, and coinpetes for food and space ( Mackie 1 99 1 , .Schloesser et al. 1996. Strayer 1999). Because of the declines in diversity and abundance of native mussels and the rapid and severe impacts of zebra inussels on native mussels (Gillis & Mackie 1994. Nalepa et al. 1996). a national strategy for the conservation of native freshwater mussels was developed to provide a framework for preventing further population declines and species extinction (National Native Mus- sel Conservation Committee 1998). This document identified a number of conservation needs and outlined goals, strategies, and tasks to address these needs. Listed among these was the recom- mendation to develop management options for eliminating or re- ducing the threat of zebra mussels to native mussels. These options included relocating native mussels to artificial and natural refugia. Although tiiany mussel relocations have had poor success (e.g.. *Corresponding author. E-mail: greg_cope@ncsu.edu Cope & Waller 1995), recent studies conducted with improved techniques, experimental design, and monitoring programs, have been successful (Dunn et al, 2000, Cope et al, 2003). Thus, with the increased likelihood of successful relocation efforts, and the continued range expansion and adverse effects of zebra mussels on native tnussel populations, any relocation done to conserve native mussels necessitates that protocols be developed to prevent the inadvertent introduction of zebra mussels. Several recent studies have developed protocols to ensure that zebra mussels would not be inadveHently introduced during native mussel conservation activities and have assessed the health and survival of native mussels during subsequent relocation (Patterson et al. 1997, Patterson et al, 1999. Gatenby et al, 2000, Nichols et al. 2000. Hallac & Marsden 2001. Newton et al, 2001), The pur- pose of this project was to synthesize and evaluate the current protocols and to develop a set of procedures that resource manag- ers and researchers should consider before conducting native tnus- sel conservation activities in zebra mussel infested waters. RESULTS AND DISCUSSION Almost all of the recent native mussel salvage and relocation projects have used some type of quarantine to prevent the inciden- tal introduction of zebra mussels. The exceptions are those studies intended to remove zebra mussels from fouled native mussels and replace them back to their original location (e.g., Schloesser 1996, Hallac & Marsden 2000), By necessity, most of the quarantine protocols have been location and facility specific. For example, Gatenby et al. (2000) reviewed procedures for relocating native mussels from the Ohio River. Likewise, Newton et al, (2001) developed a specific set of procedures for relocating native mus- sels from the Mississippi River to artificial ponds and to fish hatchery facilities. However, these and other protocols developed for specific studies have many common points of concern, such as 177 178 Cope et al. TABLE 1. Summary of collection and quarantine-related conditions and procedures, and recommended guidelines for preventing introduction of zebra mussels during native mussel conservation activities. Condition or Procedure Reference Gatenbv et al. (2000) Newton et al. (20011 Recommended Guidelines Collection setting Time of collection July. September. October \W5 May 1W5 Species of native mussels No. of native mussels Native mussels analyzed for disease and pathogens before relocation Air temperature (X) Water temperature (°C) Mechanism for removing zebra mussels from native mussels Method for holding scrubbed native mussels at collection site Emersion time (min) during collection and processing Transportation to quarantine facility Quarantine facility Type Mussel density (no./ni") Water source Water temperature ("O Dissolved oxygen (mg/L) pH Potas.sium (mg/L) Alkalinity (mg CaCOj/L) Hardness (mg CaCO,/L) Total ammonia nitrogen (mg/L) Unionized ammonia (|ji.g/L) Total residual chlorine (p.g/L) Nutrition/feeding Amhiema plicata, Quadnila ptistulosa, ElUptio crassidens. Pleurobema cordatum. Obliquaria reftexu, Ponmnhis ulanis 27(» No 20-28 Hand scrubbed vMth plastic- bristled brushes Mesh bacs in river* 20 Between moist burlap in coolers with ice (no direct contact of mussels and ice) Above-ground tanks, l-t-500 L Well water LS0-2.'S() 2-28 6-14 7.2-8..'i 1.6 90 90 £1.0 2-66 ■1 X 10'' cells/mL three times per week in quarantine; relocation ponds were fertilized with a nitrogen;phosphorous (N:P) ratio of 10:1 (1.0 mg/L N. 0. 1 mg/L P) with NH^NO, and NaHPOj salts Early spring, before zebra mussel spawning begins (water temperatures <15°C) or mid to late fall when natives have greater energy reserves and juvenile zebra mussels are visible (>2-5 mm shell length) Amblema plicata, Fusconaia flava, Leptodea fragilis, Obliquaria reflexa. Quadrula qiiailnihi 768 Yes 6-18 11-14 Hand scrubbed with plastic- bristled brushes under x2 magnification Hatchery truck with aerated well water Between moist burlap in coolers with ice (no direct contact of mussels and ice) Pond (0.04 ha), mussels held in 8-2720 L mesh baas If possible Early spring or late fall temperatures; minimize differences between air and water temperature Early spring or late fall temperatures; minimize differences between air and water temperature Hand scrub with plastic-bristled brushes under magnification Hold in zebra mussel-free water after scrubbing Keep to minimum, but <20 Between moist burlap in coolers with ice in plastic bags for transport durations <12 h; no direct contact of mussels and ice bags -19-159 Keep to minimum, hut <1.50 water Well water 1.1-27 <28 6-20 >6 7.8-10.6 6.5-9.0 2.6 <4 110-160 >15 180-200 >50 0.03-0.2 <1.0 2-20 .3 g/m" of 10:10:10 N:P:K fertilizer added to quarantine pond 2 weeks prior to adding unionids; relocanon ponds were not fertilized <25 <17 X 10' cells/mL or 4.0 mg dry wt./L twice daily or 2.0-5.0 x lO"* cells/mL or 1.9 mg dry wt./L on a continuous basis (Gatenby 2000, 2002); suitable algal species include Neochloris oleoabimdans. Bracteacoccus grandis. and Pliaeodactylum tricormHum continued on next page Preventing Zebra Mussel Introduction 179 TABLE 1. continued Reference Condition or Procedure Gatenbv et al. (2(H)0) Newton et al. (2001) Recommended Guidelines Da\s in quarantine Minimum of 30. but up In 120; re-inspected under 4x niagnirieation Disinfection of equipment and Chlorine solution of 25 mg/L supplies Monitoring Temperature, dissolved oxygen, and pH All other water quality variables Disease and inortalit\ Dessication for up to 4 d Twice daily Daily to weekly Not specified 35; re-inspected under 2x magnification Not specified Daily Daily to weekly Not specified Minimum of 30; re-inspect under magnification Chlorine solution of 25-250 mg/L, depending on type of material; dessication in warm dry air for 3-5 d At least daily Daily to weekly At least weeklv ' All native mussels were rinsed with a high pressure hose before being placed into the quarantine facility. facility modification and suitability, zebra mussel risk assessment and management procedures, and native mussel health and disease management procedures, that may have broad applicability to other situations and locations. Facility-Specific Concerns and Procedures The availability of aquatic facilities for long-term captive care of freshwater mussels is limited. Thus, most of the salvage and quarantine facilities have involved the short-term use of state and US Government owned fish hatchery ponds and raceways or simi- lar research aquaculture facilities (Dunn & Layzer 1997. Pinder et al. 1999. Gatenbv 2000. Newton et al. 2001). The main facility concerns have focused on the type of rearing or holding system (e.g.. ponds, raceways, or above-ground tanks capable of housing hundreds to thousands of mussels), the facility's proximity to the source of relocated mussels (to reduce transportation time and handling stress), on-site water quality for maintenance of mussel health, and production of an algal-based food supply. The objec- tives of any given conservation project will likely dictate the type of facility or holding system used and any modifications that may be required. Nonetheless, whether used for short-term quarantine or for long-term captive care, all facilities should be able to pro- vide space for isolation and quarantine, water quality characteris- tics to meet requirements for shell growth and metabolic processes, and food quantity and quality to support growth and reproduction (Table 1). Specific isolation and containment modifications are probably necessary at most facilities to control and contain source water inflow and potentially contaminated outflow. For example, the outflow of water from quarantine units may need to be passed through filtration or disinfectant treatments to remove or kill po- tential zebra mussels before the water is discharged through nor- mal routes. Containment procedures commonly used at facilities conducting zebra mussel research have included filtration of out- flow water through small mesh bags ( 100 (xm or smaller), chlorine treatment tanks (230 mg/L for I h). and sand filtration units (J. J. Rach, U.S. Geological Survey. Upper Midwest Environmental Sci- ences Center. La Crosse, WI, pers. com.). Additional facility pre- cautions may include the capping of all exterior drains to prevent the release of potentially contaminated water from the affected areas and the development of a flood risk assessment, if the facility is within a designated floodplain. The type of facility selected, however, may influence the rela- tive success of the conservation project. Success could depend on its use only as a short-term quarantine facility for subsequent re- location to a natural or artificial system, or its use for long-term captive care. For example. Newton et al. (2001) relocated five species of native mussels (1,392 mussels total) from the Upper Mississippi River to a fish hatchery pond after 35 d of quarantine in an artificial pond (81% of mussels survived during quarantine). Mussel survival in the hatchery pond averaged SO^c after 1 y. but only 35% 3 y after relocation. Of the mussels in a handling-control treatment that were placed back into the Mississippi River after quarantine, survival was 80% after 1 y and 75% after 3.3 y. The authors attributed the differences in survival between the hatchery pond and riverine relocated mussels to inadequate nutritional re- sources in the pond. This study illustrates the potential utility of natural or managed refugia over artificial refugia for long-term conservation (Nichols et al. 2000. Cope et al. 2003). Gatenby (2000) observed similar decreases in survival of six large river species relocated to pond refugia after a 30-d quarantine in above- ground tanks. Mean survival of native mussels during quarantine was 97%. Mean survival after 1 y in the ponds ranged between 82 and 93%. depending on species. Despite an abundance of a suit- able algal food supply and adequate water quality conditions in the ponds, however, the survival of relocated mussels decreased to 44%- after 2 y and to 5% after 3 y. Gatenby (2000) attributed the mortality to high water temperatures in July and August during years 2 and 3 of that study. Large river species of mussels relo- cated (with no quarantine period) to fish hatchery raceways with flowing water and sediment also showed high survival (95%) after 1 y (Dunn & Layzer 1997). but their long-term (3-5 y) success in this type of system is unknown. The relocation of native mussels after quarantine to natural refugia or raceway systems supplied by natural river water will likely have greater success for long-term preservation of the mus- sels than retention in artificial pond refugia for two key reasons: water temperature and food quality. These two cotnponents are critical to the livelihood of any aquatic organism. Rapid fluctua- tions in temperature, unnaturally high temperatures, and inad- equate food supplies are known to cause stress in aquatic organ- 180 Cope et al. isms, and can lead to mortality (Bayiie et al. 1973). Thus, tem- peratute. food quality, and food quantity will also be key components to the success of native mussel captive care programs. Zebra Mussel Risk Assessment and Management Procedures Because the threat of zebra mussels to native mussels has been the primary causal factor for initiating most mussel conservation activities, special precautions have been necessarily incorporated into the collection and handling protocols where native mussels are relocated. These precautions taken during collection, transport, processing, and quarantine of native mussels are of utmost impor- tance. Only the careful collection and handling of native mussels from zebra mussel-infested waters will ensure that hatchery fish, native mussels, and other aquatic species in the ecosystem are protected from the incidental introduction of zebra mussels. In situations where there is unceitainty in the co-existence of zebra mussel populations in the watershed, the most prudent and conservative approach is to treat all native mussels as if they originated from zebra mussel-infested waters. A review of zebra mussel range distribution and population dynamics in the particu- lar river basin is also warranted. Particular items of interest in- clude, the nearest known reproducing population of zebra mussels to the native mussel collectiiin site, the relative density and poten- tial spawning periods of zebra mussels at that site, and the likeli- hood of an undetected presence at the native mussel collection site (e.g.. lack of an active monitoring program). The optimum time for collection of native mussels for a given conservation project is largely unknown. Conservation projects, however, should strive to select periods that reduce the stress associated with handling as much as possible. Potential criteria include choosing a period that coincides with the absence of zebra inussel larvae in the water column, minimizes the temperature differential between air and water, and does not inteiTupt the re- productive cycle for most of the species being relocated. Zebra mussel contamination can be minimized by collecting native mus- sels during early spring or late fall periods when zebra mussel larvae are likely not present in the water column (e.g., water tem- peratures <15' C. Mackie 1991 ) or when the settled juveniles are of a sufficient size to be easily seen (e.g.. 2-^ mm in shell length), respectively. Freshwater mussels are categorized as either long- term (bradytictic) or shon-term (tachytictic) brooders. Long-term brooders, like many species of lampsilines and anodontines. be- come gravid in late summer, retain the developing glochidia in the gill marsupia throughout winter, and spawn in early spring (Mc- Mahon & Bogan 2001 ). In contrast, short-term brooders, like many species of amblemines. become gravid in early spring and spawn in late summer (McMahon & Bogan 2001). Newton et al. (2001) collected native mussels in early spring when water temperatures ranged between 1 1 and 14°C. a period before zebra mussel spawning, which generally occurs when water temperatures reach 15 to 17°C (between May and June), in north- em temperate regions of the United States and Canada (Mackie 1991 ). The collection of native mussels in early spring also has an added potential benefit of reduced energetic stresses associated with handling because of the cooler water temperatures (Jokela 1996, Newton et al. 2001). For example, glycogen concentrations in Amhleina plicata were highest between May and July and dropped precipitously thereafter — a pattern that closely paralleled reproduction in this short-term brooder (Monroe & Newton 2001 ). Similarly, Jokela et al. (1993) observed that glycogen concentra- tions decreased substantially between July and October in An- (uldiita pisciinilis. a long-term brooder. Ftirthennore. Jokela ( 1996) suggested that transplanting females before fertilization or during the early development of the brood had no detectable effect on reproductive output. Data on energetic reserves in marine bivalves contradict the recently reported data in freshwater bivalves. In the marine envi- ronment, it has been suggested that mussels collected in fall may be able to better withstand handling stress because of their higher energy reserves and because their metabolism is slowed by the cooler water temperatures (Bayne et al. 1973). For example, by mid to late fall, the marine species Mytihis edulis and M. trossulus had accumulated abundant carbohydrate energy reserves (Hawkins & Bayne 1985. Kreeger 1993, Kreeger et al. 1995). The differ- ences between marine and freshwater species may be caused by differing reproductive strategies. Results from a recent study with native freshwater mussels, however, suggest that some species of native mussels may build up their energy reserves in fall (Gatenby 2002). Obviously, this is an area where additional research is needed. When native mussels are collected from multiple sites in a watershed with a known or suspected gradient in zebra mussel density, working from the least infested site to the most infested site will reduce potential zebra mussel contamination of boats and other equipment. Optimally, boats used to collect or deploy native mussels in zebra mussel infested areas should be cleaned (before and after) by a high-pressure hot-water wash and diver wet suits, supplies, and equipment (e.g.. ropes, buckets, etc.) used in the study should be disinfected with a mild solution of chlorine bleach (25 mg/L) or air dried (3-5 d) before use (Gatenby et al. 2000). If the quarantine or relocation facility is also an operational fish hatchery or aquaculture center, precautionary measures to protect endemic wild species and cultured fish species should be consid- ered. Before entrance into the facility, a subsample of native mus- sels should be obtained from the collection site and submitted to a United States Fish and Wildlife Service. National Fish Health Center (Newton et al. 2001 ) or similar laboratory, to assess poten- tial disease and pathogen presence (see section later on native mussel health and disease management procedures). After screening for diseases and pathogens, collection of native mussels should proceed with procedures to minimize contamina- tion from adult and larval zebra mussels. These include scrubbing individual native mussels with plastic bristled brushes, visual in- spection of all exterior surfaces of the shell with magnifying lenses, and holding cleaned natives in zebra mussel-free water (Table 1 ). Care should be taken during scrubbing and inspection to avoid overlooking small zebra mussels that may be attached in crevices, in areas of shell erosion (native mussels with severely eroded or damaged valves should be discarded), or along the hinge line (Gatenby et al. 2000, Newton et al. 2001). Only personnel experienced in mussel biology should conduct the inspections to ensure accuracy and efficiency of these procedures. During collection and processing of native mussels, emersion (exposure to air) and thermal stress should be kept to a minimum. Recent studies have shown that handling mussels over a range of emersion air temperatures ( 15-35°C) and emersion durations ( 15- 60 min) did not acutely impair survival, behavior, or biochemical composition (Bartsch et al. 2000, Greseth et al. 2003). A minimal emersion time (<20 min). however, is generally recommended from recent efforts (Table 1 ). Moreover, water temperature and Preventing Zebra Mussel Introduction 181 dissolved oxygen concentrations in the holding \esscls during col- lection should be measured frequently (at least once per hour) and maintained at or near (±2 'O the ambient stream conditions at the time of collection with non-chlorinated ice and external aeration, if possible (Gatenby et al. 2000). Depending on the proximity of the native mussel collection site to the quarantine facility (a transport time generally <12 h). mus- sels should be transported in coolers covered with moist burlap and kept cool (within ±2°C of the water collection temperature, if possible) w ith ice in plastic bags without direct contact of ice bags and mussels (Gatenby et al. 2000. Newton et al. 2001. Cope et al. 200.^). This method is advantageous over the use of water-filled, aerated tanks (Chen et al. 2001) because of the reduced need for costly and cumbersome trucks and equipment and of miniinizing potential problems associated with maintaining stable dissolved oxygen concentrations in water during transport. At the quarantine facility, native mussels have generally been held for a minimum of 30-35 d (Gatenby et al. 2000, Newton et al. 200 1 ) to allow any small or previously undetected zebra mussels to become visually apparent on re-inspection. The 30-35 d quaran- tine period is based on reported zebra mussel growth rates of 0.06-0.15 mm/d (Mackie 1991. Martel 1995. Chase & Bailey 1999), which would allow a newly settled zebra mussel to reach a visible shell length of about 2-5 mm during quarantine. During this time, basic water quality measurements (e.g., temperature, dissolved oxygen, and pH) should be taken at least daily. Other water chemistry variables such as alkalinity, hardness, potassium, total ainnionia nitrogen (TAN), and unionized ammonia should be measured at least weekly to ensure that water quality conditions for minimum life requirements are met (Table 1 ). In addition, mussels in quarantine should be monitored at least weekly for disease (see section below on native mussel health and disease management procedures) and mortality. Isolation of native mussels from other aquatic species, their contact water, nets, or other equipment at the quarantine facility is necessary to protect organismal health and the physical facility. These concerns can largely be addressed by applying standard best practices for maintaining fish health. Disinfection of equipment and supplies for native mussel quarantine should be guided by National Fish Health Policy and Procedures, Part 713, sections FWI and FW 3 (USFWS 1995): chlorine (200-250 mg/L for 1 h), .sodium or potassium salts (saturated solutions) or other chemical treatments (e.g., benzalkonium chloride at 100 mg/L for 3 h) and desiccation (3-5 d) have been successfully used or recommended (Reid et al. 1993, Waller et al. 1996. Gatenby et al. 2()()()). After the minimum quarantine period (30-35 d). individual mussels are thoroughly re-inspected by hand with magnifying lenses to evaluate the presence of zebra mussels. If zebra mussels are not found, the mussels are deemed zebra mussel-free and can be relocated elsewhere (e.g.. to natural or artificial systems or to other facilities for long-term captive care). Because no zebra mus- sels were found after quarantine in the study of Newton et al. (2001). the mussels were subsequently relocated to fish hatchery ponds. In contrast. Gatenby et al. (2000) found zebra mussels on initial re-inspection and consequently held native mussels in quar- antine for additional 30 d intervals each time zebra mussels were found, up to a total of 120 d. Because of declines in mussel health and condition over time during quarantine (Patterson et al. 1997. Newton et al. 2001). Gatenby et al. (2000) recommended re- inspection of mussels at 7 d intervals after the initial 30 d period when zebra mussels are found, and to hold them onlv for 30 additional days after the last zebra mussel is found, to shorten the overall quarantine time. However, the added stress of handling native mussels more frequently must be weighed against the prob- ability of earlier detection of zebra mussels. Additionally, native mussels could be treated with chemical disinfectants. Certainly, the benefit of this type of treatment must be weighed against the risk of added stress and reduced fitness in the native mussels, but a study by Waller and Fisher (1998) found that limited application of specific chemicals (e.g., 20,000 mg NaCl/L for 6 h) may be feasible for certain tolerant native species. They cautioned, however, that chemical disinfectants cannot guar- antee the elimination of all zebra mussels from native mussel shells and stated that pre-treatnient or multiple treatment (e.g., once per week) of native mussels and their holding tanks may be most valuable for reducing the time held in quarantine. Many fish hatchery and aquaculture facilities may already be using various chemical treatments (Waller et al. 1996. Edwards et al. 2000. Edwards et al. 2002) or hazard analysis protocols such as the Aquatic Nuisance Species-Hazard Analysis Critical Control Point (ANS-HACCP) approach (Gunderson & Kinnunen 2001) to pre- vent the spread of zebra mussels and other aquatic nuisance spe- cies during their activities, which may be adapted to the collection, transport, and quarantine of native mussels. ,\ative Mussel Health and Disease Management Procedures Although liltle is known about the diseases of native freshwater mussels, recent studies have shown the potential for pathogen transmission among native mussels and fish (Starliper et al. 1998, Starliper & Morrison 2000). The primary concern for fish hatchery or aquaculture facilities that contain native mussels is the potential for transmission of disea.se and pathogens between host mussels and hatchery fish. Transmissions from hatchery fish to mussels and from mussel to mussel are also important vectors to control for maintaining mussel health. Therefore, a pathogen and disease monitoring plan for native mussels, similar to that commonly used for hatchery-reared fish, should be considered. Hatchery personnel are routinely trained in fish health protocols and record keeping: these procedures could easily be adapted for monitoring mussel health. The United States Government standards and protocols currently exist for a disease control and classification system for coldwater fish (salmonid) pathogens — similar guidelines for warmwater fish or native mussels do not exist (USFWS 1995). Revisions to the United States Fish and Wildlife Service, Fish Health Policies and Procedures are currently underway to include warmwater fish and other aquatic organisms (Richard Nelson, United States Fish and Wildlife Service, La Crosse Fish Health Center, Onalaska, Wl, pers. com.). Until those changes are imple- mented, however, native mussels may only be screened in the near term for reportable coldwater pathogens and diseases. On a posi- tive note, a recent study evaluating the effect of depuration on the transmission of the bacterial fish pathogen Aeromonas salmoni- cicla (the causative agent offish furunculosis) between the unionid Anihic'ina plicata and two strains of Arctic char Scilveliniis alpinus found that the minimum 3()-d quarantine of native mussels recom- mended for preventing the spread of zebra mussels was sufficient for depuration of the fish pathogen and eliminating transmission of the disease (Starliper 2001 ). Therefore, when adequate safeguards and standard best practices for fish health are used in combination with a 30-d quarantine, disease and pathogen transmission risks should be minimal. Native mussels held in quarantine should be 182 Cope et al. screened before being placed in tlie quarantine facility and moni- tored monthly throughout the duration of their captive care to document disease and pathogen incidence and history. More re- search and policy development is needed in this area to ensure protection of fish and native mussels. Maintaining the physiologic condition of native mussels during quarantine is difficult because diet and nutritional requirements are poorly understood. Although the specific time course for changes in biochemical indices of mussels caused by quarantine is un- known, recent studies have shown that substantial decreases in glycogen concentrations occur in as little as 7-35 d after quaran- tine. For example. Patterson et al. (1997) found that glycogen concentrations in mantle tissue in Amhieinii plicata and Quadriila pustidosii dropped significantly after 7 d in quarantine and by day 30. concentrations had declined to only 15-31% of that measured in wild-caught specimens. Likewise, glycogen concentrations in foot tissue of A. plicata decreased 44% from 279 ± 191 mg/g dry weight at day 0 to 178 ± 105 nig/g dry weight after 35 days in quarantine (Newton et al. 2001 1. Based on the poor physiologic condition of native mussels after quarantine shown by previous studies, it is critical to provide the best source of nutrition during quarantine. Previous studies have relied on an algal-based diet, either produced //; situ by stimulating algal growth with fertilizers in ponds or cultured indoors on site and added directly to mussel holding tanks (Gatenby et al. 1997, Patterson et al. 1997. 1999, Gatenby 2000, Gatenby et al. 2000, Newton et al. 2001 ). A number of algae have been tested as food for juvenile and adult mussels (Gatenby et al. 1997, Gatenby 2000, Beck 2001). Recent biochemical analysis of three algae (Neochlo- ris pleoahmulans. Bnuteacticciis gnmdis. and Phacodactyliiiu lii- ainuttuiii) indicate that these could be nutritionally suitable for maintaining freshwater mussels in captivity (Gatenby et al. 2002). If mussels are to be quarantined or relocated to ponds, the follow- ing should be kept in mind: ( 1 ) standard commercial pond fertil- izers should not be used to stimulate growth of algae; (2) the potassium levels in commercial fertilizers are toxic to freshwater mussels (Imlayl973); (3) the nitrogeniphosphorous ratio (N:P) of the standard 10:10:10 nitrogen:phosphorous:potassiuni (N:P:K) fertilizer will not promote suitable algae for mussels that typically require an N:P ratio of 10:1 (McCombie 1953); and (4) an unsuit- able, or indigestible filamentous blue-green algal bloom will result when 10: 10: 10 N:P:K is used. Therefore, we recommend using the fertilizers indicated in Table I, following Gatenby et al. (2000). Although feeding requirements for native mussels will likely de- pend on the species involved, temperature conditions, and meta- bolic activity, Gatenby et al. (2000) recommended that native mus- sels be fed 1 x 10'' cells/niL or 4.0 mg dry weight/L twice daily (Table 1 ). This was a conservatively high recommendation based on initial feeding studies and assimilation efficiencies. This con- centration resulted in the greatest assimilation of organic carbon, but a significant amount of this ration went unused by the animals (Gatenby 2000). More recent data indicate that a diet ration of 2.0-5.0 X lO'* cells/niL or 1.9 mg dry weight/L per feeding cham- ber should maintain mussel condition during summer growth pe- riods (Gatenby 2002). Particle concentrations should be monitored and not allowed to drop below 60% of this recommended ration. Feeding frequency will depend on the species and total biomass being held in captivity (Gatenby 2002). Thus, monitoring the par- ticle concentration on a daily basis is necessary. Initially, particle concentration may need to be monitored two to three times daily until the manager is familiar with the particle depletion rate or clearance rate of the native mussels held in captivity. CONCLUSIONS AND RECOMMENDATIONS Native freshwater mussels should only be relocated from ex- isting areas as a la.st resort (Cosgrove & Hastie 2001 ). Other op- tions to relocation and salvage, such as periodic cleaning of zebra mussels from native mussels and replacement (Hallac & Marsden 2000, Hallac & Marsden 2001 ), and the use of natural or managed refugia (Nichols et al. 2000), should be considered as first alter- natives where practical. For example, Hallac & Marsden (2000, 2001 ) suggested that periodic cleaning and replacement might be a viable option for conservation of native mussels, especially in areas where food is not limiting and where collection and cleaning are logistically feasible. If, however, freshwater mussel relocations are required to conserve localized populations from zebra mussels or other catastrophic events, the concerns and procedures de- scribed in this article should provide general guidance for devel- oping plans to prevent the incidental introduction of zebra mussels during these activities and for maintaining the health of the native refugees while under captive care. In addition, procedures for ensuring long-term viability of na- tive mussel populations need to be considered throughout the plan- ning and Implementation process. For example, similarities in wa- ter quality, substratum characteristics, food, and necessary fish hosts among the systems are critical elements in a native mussel relocation strategy. Additional ecological and evolutionary con- cerns, such as retention of genetic diversity of the mussel popula- tions, need to be carefully considered before relocating native mussels to natural refugia, especially if the mussels are to be relocated between river basins or between sub-basins of the same river system (Villella et al. 1998, Storfer 1999). Because of costs and limited availability of facilities for quar- antine and captive care of native mussels, the United States Fish and Wildlife Service and its resource conservation and manage- ment partners may wish to designate several facilities within re- gions of the United States that can accept, hold, and screen mussels for disease and pathogens. These facilities may include state or national fish hatcheries, research or aquaculture centers, and fish health centers. To our knowledge, this synthesis represents the "state-of-the- science"" for minimizing the incidental introduction of zebra mus- sels during native mussel conservation activities and for ensuring their short-term and long-term health and viability. Readers of this article should be cautioned that the information presented is only recommended guidelines and that future improvements to proce- dures will be made through research and policy development. ACKNOWLEDGMENTS This project was funded by the United Stales Fish and Wildlife Service, through a contract with the Freshwater Mollusk Conser- vation Society. Linda Drees and Tina Proctor provided valuable insight on the relevance of the project to resource managers. Steve Ahlstedt, Arthur Bogan, Heidi Dunn, Jerry Fairis. Doug Jensen, Patricia Morrison, Pam Thiel, and Kurt Weike provided informa- tion critical to preparation of the document. The authors thank Robert Anderson, Heidi Dunn, Richard Neves, Jeixine Nichols. Tom Watters. and Kurt WeIke for reviewing a draft of the docu- ment. Preventing Zebra Mussel Introduction 183 LITERATURE CITED Barlsch. M. R. D. L. Waller. W. G. Cope & S. Giitreuter. 2000. Emersion and thermal tolerances of three species ofunionid nuisscK: survival and behavioral effects. / Shellfish Res. 19:233-:4(). Bayne. B. L., R. J. Thompson & J. Widdows. 1973. Some effects of temperature and food on the rate of oxygen consumption by Mylilus ediilis. In: W. Wieser. editor. Effects of temperature in ectothermic organisms. Berlin: Springer-Verlag. pp. lSl-193. Beck, K. M. 2001. Development of an algal diet for rearing juvenile fresh- water mussels (MSc thesis). Blacksburg: Virginia Polytechnic Institute and State University. 58 pp. Bogan, A. E. 1993. Freshwater bivalve extinctions (Mollusca: Unionoidal: a search for causes. Am. Zool. 33:599-609. Brim Box. J. & J. Mossa. 1999. Sediment, land use, and freshwater mus- sels: prospects and problems. J. N. Ant. Benrhol. Soc. 18:99-1 17. Chase. M. E. & R. C. Bailey. 1999. The ecology of the zebra mussel {Dieissena polymorpha) in the lower Great Lakes of North America: I. Population dynamics and growth. / Great Lukes Res. 25:107-121. Chen, L. Y.. A. G. Heath & R. Neves. 2001. An evaluation of air and water transport of freshwater mussels (Bivalvia: Unionidae). .4m, Malacoi Bull 16:147-154. Cope. W. G. & D. L. Waller. 1995. Evaluation of freshwater mussel relocation as a conservation and management strategy. Reg. Rivers Res. Manage. 11:147-155. Cope. W. G., M. C. Hove. D. L. Waller, D. J. Hornbach. M. R. Bartsch. L. A. Cunningham. H. L. Dunn & A. R. Kapuscinski. 2003. Evaluation of relocation of unionid mussels to in situ refugia. ./. Molluscan Stud. 69:27-34. Cosgrove. P. J. & L. C. Hastie. 2001. Conservation of threatened fresh- water pearl mussel populations: river management, mussel transloca- tion and conflict resolution. Biol. Cons. 99:183-190. Dunn. C. S. & J. B. Layzer. 1997. Evaluation of various holding facilities for maintaining freshwater mussels in captivity. In: K. S. Cummings, A, C. Buchanan, C. A. Mayer & T. J. Naimo, editors. Conservation and management of freshwater mussels II: initiatives for the future. Pro- ceedings of a UMRCC Symposium. 16-18 October 1995. St. Louis. Missouri. Upper Mississippi River Conservation Committee, Rock Is- land, Illinois, pp. 205-213. Dunn. H, L.. B. E. Sietman & D. E. Kelner. 2000. Evaluation of recent Unionid (Bivalvia) relocations and suggestions for future relocations and reintroductions. In: R. A. Tankersley. D. I. Warmolts. G. T. Wat- ters. B. J. Armitage. P. D. Johnson & R. S. Butler, editors. Freshwater Mollusk Symposia Proceedings. Columbus. OH: Ohio Biological Sur- vey, pp. 169-183. Edwards. W. J.. L. Babcock-Jackson & D. A. Culver. 2000. Prevention of the spread of zebra mussels during fish hatchery and aquaculture ac- tivities. N. Am. J. Aquaculture. 62:229-236. Edwards, W. J., L. Babcock-Jackson & D. A. Culver. 2002. Field testing of protocols to prevent the spread of zebra mussels Dreissena poly- morpha during fish hatchery and aquaculture activities. N. Am. J. Aqua- culture. 64:220-223. Fuller. S. L. H. 1974. Clams and mussels (Mollusca: Bivalvia). In: C. W. Hart. Jr. & S. L. H. Fuller, editors. Pollution Ecology of Freshwater Invertebrates. New York: Academic Press, pp. 215-273. Gatenby, C. M. 2002. Seasonal changes in the biochemical and physiologi- cal condition of native freshwater mussels (final report). Cincinnati: Mussel Mitigation Trust Fund. 26 pp. Gatenby, C. M. 2000. A study of holding conditions, feed ration, and algal foods for the captive care of freshwater mussels (PhD dissertation). Blacksburg: Virginia Polytechnic Institute and State University. 237 pp. Gatenhy. C. M., B. C. Parker & R. J. Neves. 1997. Growth and survival of juvenile rainbow mussel, Villosa iris (Lea 1829) (Bivalvia: Unionidae) reared on algal diets and sediment. Am. Malacoi. Bull. 14:57-66. Gatenby, C, M., D. M. Orcutt, D. A. Kreeger. B. C. Parker. V. A. Jones & R. J. Neves. 2002. Biochemical composition of three algal species proposed as food for capli\'e for freshwater mussels. J. Appl. Phycol. 15:1-11. Gatenby. C. M., P. A. Morrison. R. J. Neves & B. C. Parker. 2000. A protocol for the salvage and quarantine of unionid mussels from zebra mussel-infested waters. In: R. A. Tankersley. D. 1. Warmolts. G. T. Watters, B, J. Armitage, P. D. Johnson & R. S. Butler, editors. Fresh- water Mollusk Symposia Proceedings, Ohio Biological Survey. Colum- bus. OH: pp. 9-18. Gillis. P. L. & G. L. Mackie. 1994. Impact of the zebra mussel. Dreissena polymorpha, on populations of Unionidae (Bivalvia) in Lake St. Clair. Can. J. Zool. 72:1260-1271. Greseth. S. L.. W. G. Cope. R. G., Rada, D. L. Waller & M. R. Bartsch. 2003. Biochemical composition of three species of unionid mussels after emersion. / Molluscan Stud. 69:101-106. Gunderson. J. L. & R. E. Kinnunen. 2001. Aquatic nuisance species- hazard analysis and critical control point training curriculum. Minne- sota Sea Grant Publication No. MNSG-Fl 1 and Michigan Sea Grant Publication No. MSG-00-400. 78 pp. Hallac. D. E. & J. E. Marsden. 2000. Differences in tolerance to and recovery from zebra mussel [Dreissena polymorpha) fouling by Ellip- tio complanala and Lampsilis radiata. Can. J. Zool. 78:161-166. Hallac, D. E. & J. E. Marsden. 2001 . Comparison of conservation strategies for unionids threatened by zebra mussels (Dreissena polymorpha): pe- riodic cleaning vs quarantine and translocation. / N. Am. Benthol. Soc. 20:200-210. Hawkins. A. J. S. & B. L. Bayne. 1985. Seasonal variation in the relative utilization of carbon and nitrogen by the mussel Mytilus edulis budgets, conversion efficiencies and maintenance requirements. Mar. Ecol. Prog. Ser. 25:181-188. Imlay, M. J. 1973. Effects of potassium on survival and distribution of freshwater mussels. Malacologia 12:97-1 13. Jokela. J. 1996. Within-season reproductive and somatic energy allocation in a freshwater clam. .Anodonia piscinalis. Oecologia 105:167-174. Jokela. J., L. Uotila & J. Taskinen. 1993. Effect of castrating trematode parasite Rhipidocotyle fennica on energy allocation of freshwater clam Anodoma piscinalis. Fund. Ecol. 7:332-338. Kreeger, D. A. 1993. Seasonal patterns in utilization of dietary protein by the mussel Mytilus trossulus. Mar. Ecol. Prog. Ser. 95:215-232. Kreeger, D. A.. A. J. S. Hawkins. B. L. Bayne & D. M. Lowe. 1995. Seasonal variation in the relative utilization of dietary protein for en- ergy and biosynthesis by the mussel Mytilus edulis. Mar. Ecol. Prog. Ser. 126:177-184. Mackie. G. L. 1991. Biology of the exotic zebra mussel. Dreissena poly- morpha, in relation to native bivalves and its potential impact in Lake St. Clair. Hydrobiologia 219:251-268. Marlel. A. 1995. Deinography and growth of the exotic zebra mussel [Dreissena polymtnpha) in the Rideaii River. Ccui. J. Zool. 73:2244— 2250. McCombie. A. M. 1953. Factors influencing the growth of phytoplankton. J. Fish. Res. Board Can. 10:253-282. McMahon. R. F. & A. E. Bogan. 2001. Mollusca: Bivalvia. In: J. H. Thorpe & A. P. Covich. editors. Ecology and classification of North American freshwater invertebrates. New York: Academic Press, pp. 331—129. Monroe. E. M. & T. J. Newton. 2001. Seasonal variation in physiological condition of Amblema plicata in the Upper Mississippi River. J. Shell- fish Res. 20:1167-1171. Naimo. T. J. 1995. A review of the effects of heavy metals on freshwater mussels. Ecotoxicol, 4:341-362. Nalepa. T. F.. D. J. Hartson. G. W, Gostenik. D. L. Fanslow & G. A. Lang. 1996. Changes in the freshwater mussel community of Lake St. Clair: from unionidae to Dreissena polymorpha in eight years. J. Great Lakes R«. 22:354-369. National Nafive Mussel Conservation Committee. 1998. National strategy for the conservation of native freshwater mussels. J. Shellfish Res. :7:I4I9-1428, 184 Cope et al. Newton, T. J.. E. M. Monroe. R. Kenyon, S. Gutreuler, K. I. Welke & P. A. Thiel. 2001. Evaluation of relocation of unionid mussels into arti- ficial ponds. J. N. Am. Benlhol. Soc. 20:468-485. Nichols. S. J., M. G. Black & J. D. Allen. 2000. U.se of on-site refugia to protect unionid populations from zebra mussel-induced mortality. In: R. A. Tankersley. D. 1. Warmolts, G. T. Watters. B. J. Arniitage. P. D. Johnson & R. S. Butler, editors. Freshwater Mollusk Symposia Pro- ceedings. Columbus. OH: Ohio Biological Survey, pp. 67-75. Patterson. M. A., B. C. Parker & R. J. Neves. 1997. Effects of quarantine times on glycogen levels of native freshwater mussels (Bivalvia: Unionidae) previously infested with zebra mussels. Am. Malacol. Bull. 14:75-79. Patterson. M. A.. B. C. Parker & R. J. Neves. 1999. Glycogen concentra- tion in the mantle tissue of freshwater mussels (Bivalvia: Unionidae) during starvation and controlled feeding. Am. Mulucol. Bull. 15:47-50. Pinder. M. J.. M. A. McGregor. C. D. Slickley & J. J. Ferraro. 1999. The use of state fish hatchery for the cultivation of freshwater mussels. In: P. D. Johnson, R. S. Butler & G. Benz, editors. Musseling in on Biodi- versity. Proceedings of a Freshwater Mollusk Conservation Society Symposium, 17-19 March 1999. Chattanooga, TN. 36 pp. Reid. D. F.. J. Bidwell. J. Carlton. L. Johnson. E. Marsden & S. J. Nichols. 1993. Zebra-mussel specific contaminant protocols. National Oceanic and Atmospheric Administration. Contribution No. 890. 28 pp. Schloesser. D. W. 1996. Mitigation of unionid mortality caused by zebra mussel infestation: cleaning of unionids. N. .Am. J. Fish. Manage. 16: 942-946. Schloesser, D. W.. T. F. Nalepa & G. L. Mackie. 1996. Zebra mussel infestation of unionid bivalves (Unionidae) in North America. Am. Zool. 36:300-310. Slarliper. C. E. 2001. The effect of depuration on transmission of Aero- monas salmonicida between the freshwater bivalve .Amhiciiui plicala and Arctic char. J. Aqual. Animal Health. 13:56-62. Starliper, C. E. & P. Morrison. 2000. Bacterial pathogen contagion studies among freshwater bivalves and salmonid fishes. J. Shellfish Res. 19: 251-258. Starliper, C. E.. R. Villella. P. Monison & J. Mathias. 1998. Studies on the bacterial flora of native fieshwater bivalves from the Ohio River. Biomed. Let. 58:85-95. Storfer. A. 1999. Gene flow and endangered species translocations: a topic revisited. Biol. Cons. 87:173-180. Strayer. D. L. 1999. Effects of alien species on freshwater mollusks in North America. J. N. Am. Benthol. Soc. 18:74-98. USFWS. 1995. United States Fish and Wildlife Service. Service Manual (700 Series), Part 713, Fish Health. Washington. DC. Vaughn. C. C. & C. M. Taylor. 1999. Impoundments and the decline of freshwater mussels: a case study of an extinction gradient. Cons. Biol. 13:912-920. Villella. R. F.. T. L. King & C. E. Starliper. 1998. Ecological and evolu- tionary concents in freshwater bivalve relocation programs. J. Shellfish Res. 17:1407-1413. Waller. D. L. & S. W. Fisher. 1998. Evaluation of several chemical dis- infectants for removing zebra mussels from unionid mussels. Proi;. Fish-Ctdt. 60:307-310. Waller, D. L.. S. W. Fisher & H. Dabrowska. 1996. Prevention of zebra mussel infestation and dispersal during aquaculture operations. Prog. Fish Cidt. 58:77-84. Williams. J. D.. M. L. Wan-en, Jr.. K. S. Cummings, J. L. Hanis & R. J. Neves. 1993. Conservation status of freshwater mussels of the United States and Canada. Fisheries 18:6-22. 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. « , ^i**3!F»e*7 » ♦ ^' . 0.' 1 -^4:^iil^ R^ fly JU % b • * .; • • /## t ▼ ^ » 4 • ■ 0 0 ^ $ 3 N '!_■«• • ►4 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- 192 Marshall et al. 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 0 68 ±22 225 ±118 124+56 1,828+622 1,522 ±565 0 56 ±22 80 ±38 8.0 ±7.5 57 ±28 350±136 38 ±33 291 ±130 649 ±272 94 ±35 2.148 ±617 1.758 ±635 78+58 366±138 412±189 ANNUAL - 2,610 ±1,136 89 ±40 98 ±45 1,158 ±328 45 ±16 138 ±59 326 ±116 1,334 ±362 286 ±85 Figure 3. Average abundance (# clams/m-) of CorbUiila fliiminea by size class (graph) and total (table. ±95% CI) at CV river sites. retained on the 1.0-nim sieve averaged 2.0 mm in shell length: on the 2.0-mm sieve. 4. 1 mm; on the 3.4-mm sieve, 6.7 mm; on the 6.3-mm sieve, 14.1 mm; on the 12.5-mm sieve, 19.3 mm; and on the liJ-mm sieve, 31.1 mm. Individual clam growth was monitored monthly in 1993 and 1994 using shell length measurements to the nearest 0.1 mm. In the river near the CY plant intakes, marked clams maintained in lan- tern nets were used for monitoring growth. In the CY discharge canal. 12 clams collected randomly from lantern nets were mea- sured monthly to assess growth. Clam fecundity was determined monthly using techniques of Aldridge and McMahon ( 1978). Several hundred adult clams (>8.0 mm in shell length) were collected from the river in May/June of 1991 through 1994 and held in lantern nets placed at two locations, one in the river near CY plant intakes, the other in the CY dis- charge canal. Clams were collected monthly from river nets until winter, when no live clams remained in lantern nets. In the CY discharge canal, all clams were dead by June (when water tem- peratures at this site exceeded 37°C). In this study, data for fecun- dity in the discharge canal were collected from November 1 992 to July 1993, and June and July fecundity data were at ambient river temperature due to a power plant shut down. Twelve clams were ANNUAL 11,482 ±4,416 616,.±227 555±253 subsampled monthly from each net. In the laboratory, each clam Figure 4. Average abundance (# clams/m") oi Corbiciila fluminea by was held under static conditions at 20 "C for 24 h in a lOO-ml size class (graph) and total (table. ±95% CI) at MS river sites. Month Year 1992 1993 758 +688 10,413 ±7,233 23,275 ±5,494 43 ±36 878+451 928 ±339 1994 i'38"±68 786 ±577 533 ±295 196 Morgan et al. beaker filled with filtered Connecticut River water. The number of juveniles released during this period, determined with a lOx dis- secting microscope, was recorded as an index of spawning activity. Additional fecundity assessments were made by dissecting these clams and noting the presence of brood. Maturity of gametes was assessed by removing egg and sperm cells from the gonadal tissues and examining the cells under a compound microscope (400x), Statistical analyses were performed using SAS version 8 soft- ware (SAS Inc., Cary. NC). Abundance data in figures are pre- sented using arithmetic means and non-transformed data. Statisti- cal comparisons of abundance data were always carried out after log transformation. The relationships between winter clam survival (detlned as the ratio of May clam abundance to November clam abundance from the previous year, expressed as a percentage) and temperature or river tlow indices were assessed using the rank- order Spearman correlation. Growth and reproduction data were not transformed prior to statistical testing. RESULTS Abundance Corhiciila abundance exhibited high intra- and inter-annual variability. Year to year abundance fluctuations were considerable at all ambient temperature river sites (Figs. 3, 4. 5; note different vertical scales). At CY. mean annual clam abundance in 1992, 1995, and 1999 (range 1.158-2,610 clams/nr) was significantly higher (P < 0.05) than in all other years (range 45-326; Fig. 3). At MS, mean annual abundance in 1992 (11.482 clams/m") was sig- nificantly higher (f < 0.05) than in 1993 or 1994 (616 and 555 clams/nr, respectively. Fig. 4). At SM, mean annual abundance was low, with 82 clams/nr in 1993 and 67 clams/nr in 1994 (Fig. 5). Of ambient temperature river sites, seasonal abundance at CY Month Year 1993 1994 5 8 11 112 ±30 52 ±21 0 114 ±103 88+75 ANNUAL 82 ±27 67 ±43 Figure 5. .Average abundance (# clanis/ni") of Corhiciila ftuminea by size class (graph) and total (table. ±95'7f CD at S.M, over a 10-year period was significantly higher (P < 0.05) in No- vember than in May or August. November abundance at CY ranged from 80 clams/nr in 1996 to 5,209 clams/m" in 1992. By contrast, over the 3 years surveyed at MS ( 1992-1994) and 2 years surveyed at SM ( 1993 and 1994), abundance was not significantly different (P > 0.05) between August and November samples. No- vember abundance at MS in 1992 (23,275 clams/m~) was the highest observed during the study. Lowest November abundance occurred at SM in 1993 (52 clams/nr). At all sites, clam abun- dance in May was significantly (P < 0.05) lower than that in either August or November. Of thermally infiuenced sites, seasonal clam abundance in the CY discharge canal had significant differences (P < 0.05) among the three sampling periods (Fig. 6). May abundance ranged from 0-92 clams/m". August abundance ranged from 0-12.174 clams/ m". November abundance ranged from 24 to 880 clams/m". At the MS discharge. August and November abundance estimates were not significantly different (P > 0.05). ranging from a low of 322 clams/nr in November 1993 to a high of 7.100 clams/m" in No- vember 1992 (Fig. 7). As with river sites. May abundance at both CY and MS discharge sites was significantly lower (P < 0.05) than that in .August and November. Annual abundance was variable at the CY discharge site. A pooled f-test of total abundance during operational (1991-1996) vs. post-operational years (1997-2000) indicated that clam abun- dance increased significantly (P = 0.007) during post-operational years. This increase was the result of higher abundance of larger size class clams (7-14 mm and 19-31 mm) following power plant shutdown. At the MS discharge site, total clain abundance was significantly higher (P < 0.05) in 1992 (3.322 clams/nr) than in 1993 and 1994 (496 and 549 clams/nr. respectively: Fig. 7). Clam abundance was not significantly different (P > 0.05) between the river and discharge sites at MS, except for the largest clams (31 mm size-class), which were most abundant at the MS discharge site. In fact, the largest clam measured during the entire study (37.6 mm) was collected at MS in August 1992. Winler Snnival Declines in clam abundance from November of one year to May of the next were used to determine winter survival; values at CY ranged from 0% in 1994 and 1996 to 55% in 1995 (Fig. 3). The effects of winter water temperatures and peak river fiows on clam winter survival were examined using Spearman-ranked correlation (Table I ). The severity of winter water temperatures, as indicated by the number of days with average water temperature <2°C, was not significantly correlated (r^, = -0.65, P = 0.081) with clam winter survival. The number of days, however, sl°C was nega- ti\ely correlated (r., = -0.73. P = 0.040) with winter survival, and average December through April water temperature was positively correlated (r.,= +0.87. P = 0.004). Highest average monthly flow in the Connecticut River typically occurs in April (Fig. 8). Ac- cordingly, the number of days each year exceeding 1.700 mVs in April was used as an index of spring freshet severity. This index was negatively correlated with winter clam survival (r.. = -0.91. P = 0.002). Data from 1993 were omitted from this analysis because a single storm in March caused total mortality of clams at our sampling sites. Growth Corbicida growth rates under ambient river conditions exhib- ited seasonal cycles, and growth of marked clams was size- E m O c; u c ro ■D c < CORBlCfLA IN THE LOWER CONNECTICUT RlVER 12,096 197 95 11 5 8 96 11 5 8 97 11 5 8 r- 98 11 5 8 00 Month Year 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 5 8 11 34 ±58 178±181 2 ±5.0 12, 174 ±30.269 96 ±121 32 ±22 0 42 ±26 0 60 ±54 880 ±1254 24 ±29 38 ±50 90 ±187 8.0 ±5.1 48 ±75 44 ±70 92 ±62 6 ±15 24 ±6.3 20 ±22 62 ±83 212 ±227 76 ±37 243 ±172 210 ±73 4±10 30 ±35 268 ±181 ANNUAL - 4,091 ±8,454 25 ±14 313 ±397 51 ±53 33+28 41 ±27 98+78 173 ±63 101 ±83 Figure 6. .Average abundance (# liams/m'l of Cnrhiciiki Jhiiniiiea b> size class (graph) and total (table, ±95'7f CI) at C\ discharge. dependent (Figs. 9 and 10). In 1993, clams with an initial shell length of -14.5 mm had a higher growth rate (0.54 mm/wk) from June to October than those starling at -17.5 mm (0.41 mm/wk). and -21.7 mrt) (0.35 mm/wki. A similar size-dependent relation- ship was also observed in the 1994 study; clams with an initial length of -12 mm grew fastest from June lo October (0.51 mm/ wk), followed by -20 mm (0.32 mm/wk) and -30 mm (0.14 mm/ wk) clams. Growth rates were significantly different [P < 0.05) among the three size classes through August. In September through December, however, mean monthly growth rates for all size classes were generally low and not significantly different from each other. Clam growth rates in the CY discharge canal from November 1992 to February 1993 were £0.18 mm/wk. when water tempera- tures were 13-19°C, I0-12°C above ambient river temperatures (Table 2). As these clams were not marked, negative growth rates could occur as a result of mortality of large individuals. Growth rates were as high as 0.27 mm/wk from March to May when water temperatures ranged from I3-27°C. Maximum growth rates at this site occurred during June (0.38 mm/wk) and July (0.33 mm/wk), when canal temperatures were similar to those at ambient river conditions because of a power plant outage. All clams died after the power plant restarted and discharge water temperatures ex- ceeded 37"C (July). ReprodiictiDii Microscopic examination of gametic tissues of clams held un- der ambient river and CY discharge conditions show that eggs and sperm were continually present as long as clams were alive (Fig. 1 1 ). For clams held at ambient river temperatures, the presence of embryos and veligers in the demibranchs (brooding) and the active release of juveniles occurred primarily over a 4-month period (June to September). By October, only one clam out of 48 exam- ined was still spawning. The maximum number of juveniles re- leased per clam per day typically occurred in August across all 4 years in which reproduction was monitored (2.862 juveniles/clam/ day; Fig. 12). This pattern of juvenile release allowed maximum recruitment to occur just after the period of maximum river water temperature (July, with a 4-year average of 27.5°C). The number of juveniles released per adult in August was positively correlated with the size of the clam (r,= 0.77; P < 0.01; Fig. 13). The reproductiv e cycle of Corbicuta in the CY discharge canal was seasonally shifted (Fig. II). Brooding and releasing of juve- niles first occurred in November 1992 when discharge tempera- tures averaged I8.3°C, and ceased from December through Feb- ruary when temperatures averaged 35°C in 4 days. By Au- gust 18. 1993 all clams held in the CY discharge were dead. DISCUSSION Corbicula fluminea was first documented in the Connecticut River in May 1990 (Morgan et al. 1992). the first report of this nonindigenous clam in New England waters. Before this discov- ery. Coiiiicula was not expected to colonize the Connecticut River because water temperatures routinely fall below 2^C for prolonged periods. It is commonly accepted among researchers that the lower lethal temperature limit for Corbicula is ~2°C (Homing & Keup 1964. Bickel 1966. Mattice & Dye 1976. Rodgers et al. 1979. Cherry et al. 1980). Corbicula abundance varied seasonally as well as annually, but 3500 3000 2500-1 to 5 2000' I 1500- 1 1000' < 500- 95 96 Year Figure 8. Connecticut River daily flow rates (mVs) at the Thompson- ville, CT gaging station in April from 1991 to 2000. clearly peaked in 1992. Survival of clams from one year to the next is positively coirelated with the average December to April water temperatures and negatively correlated with the number of days the river water temperature was below I °C and the number of days that river flows exceeded 1700 mVs in April. For example, no clams were observed in May at our Connecticut Yankee sampling sites following the two coldest winters (1993-1994 and 1995- 1996). when river water temperatures dropped below 2^C for 12- 15 weeks and the highest winter survival occurred in 1995 when daily average river flow in April never exceeded 1700 m /s. Low survival at Connecticut Yankee and Middletown Station during the winter of 1992-1993. when water temperature did not drop below 2°C, was attributed to winter storm Joshua (March 13. 1993). This storm produced low water levels ( 1-2' below normal) and left shoal areas, specifically our sampling areas, exposed to air temperatures as low as -8°C. freezing sediment and clams (NUSCO 1994). Higher winter survival at Middletown Station sites, when com- pared with those around Connecticut Yankee, was attributed to the influence of the Middletown Station thermal dischaige. River wa- ter temperatures seldom dropped below 2°C in the Middletown Station discharge mixing zone (NUSCO 1994). Other over- wintering populations likely exist in the river in refugia provided by other industrial thermal discharges or in areas of the river receiving regular influxes of groundwater that maintains a tem- perature of 9.0 ± 2°C (R. Lewis. State of Connecticut Geologist; pers. comm.). Graney et al. ( 1980) and Kreiser and Mitton ( 1995) suggest that warm water refugia such as these were assisting the Asiatic clam in expanding its geographical range northward. Clam densities in the Connecticut Yankee discharge canal were TABLE 1. Spearman correlations coefficient ( rj for percentage winter survival of Cnrhicula fluminea at CY \ersus indices of winter temperatures and Connecticut Rixerflow. Variable r^ Prob >lrl n" Mean Std Error Min. Max. Percentage Survival'' _ _ 8 12.7% 6.23% 0% 54.9% Ave. Winter Temp.*^ -1-0.87 0.004 8 2.93 0.37 1.32 4.86 No. Days S1°C -0.73 0.040 8 54.9 8.75 17 93 No. Days s2°C -0.65 0.081 8 70.6 8.12 28 103 Flow a 1 700 ni'/s'' -0.9 1 0.002 8 6.4 1.54 0 13 ' 1993 data were omitted because of the mortality caused by the March storm Josliua (see text). '% Survival = (May abundance/prior November abundance) x 100, ' Average Winter Temperature = the annual December to April mean daily Connecticut River temperature at CY. ' Number of days in April when the Connecticut River flow equaled or exceeded 1700 m'/s. CORBICULA IN THE LOWtR CONNECTICUT RlVER 199 Jun 1.1 - 1.0- ^ 0.9- -0.8- ^ 0.7- Ld 0.6- < 0.5- ^ 0.4- |0.3. g 0.2. 0.1 - 1 <- -^^ V ^^-. "^^ 0.0- Jul Aug Sep Nov Dec Figure 9. Corhiciila Jhiminca growth rates (mni/«k) in 1993 for marked clams within initial size classes based on shell length. Vertical bars represent two standard deviations around the mean growth rates for three individuals in each size class. most \ariable. Large numbers of small (2 mm) clams that appar- ently survived passage through the power plant cooling water sys- tem characterized transient populations in the canal. A permanent population, however, was not established during power plant op- eration because summer water temperatures often exceeded 37°C, the upper lethal temperature limit for Corbicula in our study. Mc- Mahon and Williams (!986b) reported similar findings for Cor- bicula living in the themial discharge of the Handley Power Sta- tion in Texas. Following Connecticut Yankee closing in 1996, size range of clams collected in the discharge canal has increased with shell lengths now ranging from 2-19 mm. These results indicate not only that clams are successfully over-wintering in the canal under ambient river temperatures, but also surviving for >1 year. The canal essentially has become a cove where circulation is de- pendent on semidiurnal tidal exchange, and not \ ulnerable to high spring freshet water Hows. Clam abundance in the Middletown Station discharge area also fluctuated, but was consistently higher than abundance at CY dis- charge during the same period. Similar to the CY discharge, the population near the MS discharge was dominated by clams 2 mm in size. In contrast, however, to the CY discharge, clams of all size clas.ses, including those in the 31 mm class, were regularly col- lected at the MS discharge. The presence of larger size clams suggests that this area provided a more stable refugium. The 37- mm clam collected at this site in 1992. along with growth rates observed during our study, suggests that Corbicula has been I n 4- 1— 5 o 0.3- (r o 0.2- 0.1 • 0.0- Aug Sep Figure 10. Corbicula fluminca growth rates (mm/wkl in 1994 for marked clams within initial size classes based on shell length. Vertical bars represent two standard deviations around the mean growth rates for two to five individuals in each size class. 200 Morgan et al. TABLE 2. Corbicula fluininea growth in the C\ discharge canal from November 1992 to July 1993. Date Growth Weeli Average Length (mm) SE Minimum Length (mm) Maximum Length (mm) Growth Rate (mm/wk) 1 1/10/92 0 20.20 12 0.69 15.3 1 2/22/92 6 20.04 12 0.38 18.1 01/26/93 11 20.97 12 0.59 17.7 02/23/93 IS 20.89 12 0.48 18.6 03/23/93 19 21.96 12 0.43 19.2 04/29/93 24 23.04 12 0.42 20.6 05/18/93 27 22.57 12 0.31 20.4 06/24/93 32 24.57 11 0.40 21.5 07/22/93 36 25.89 12 0.39 24.2 24,0 21.5 26.5 23.8 24.4 24.9 24.1 26.1 27.6 -0.026 0.185 -0.019 0.267 0.205 -0.172 0.378 0.330 .Ian i Feb Mar Apr May -lun .Aug Sep A fnhicn River Conditions WINTER MORTALITY Uts Sp.rn, Brooding Releasiim Ju\cniles i i i 1 1 i Cooling Water Discharge Conditions 1 1 i Fees SUMMER MORTALITY Spcnn Brood inj^ Rclcasmc.luvcnilc.s Figure 11. Summarization of the 1991 to 1994 annual reproductive cycle of Corbicula Jhiiniiiea under ambient Connecticut River condi- tion,s and the thermallv elevated conditions of the CV cooling water discharge. present in the river since 1988. Winter water temperatures were moderated by the Middletown Station thermal discharge, and sum- mer thermal stress was reduced because of rapid dilution of dis- charge waters with ambient river water. In addition, the MS ther- mal discharge flow was only ~\59r that of CY. Ciiibicida growth in the Connecticut River under ambient wa- ter temperatures is consistent with reports by other researchers in North American (Morton 1977. Britton et al. 1979. Eng 1979. Mattice 1979, McMahon 1983. Welch & Joy 1984, Joy 1983. Matlice & Wright 1986. McMahon & Williams 1986b. Doherty et al. 1990, French & Schloesser 1991). and was primarily influenced by water temperature. Growth began in May when water tempera- tures rose above IO°C and continued until December when water temperatures dropped below this threshold. Other researchers re- ported 9-15°C to be the lower temperature threshold for growth of Corbicula fluininea in their studies (Hall 1984. Mattice & Wright 1986, McMahon & Williams 1986a, French & Schloesser 1991). o o Q 40 30 5 o ;;^ 20 LU > 3 O 10- . >^. ■ ^ / p \ N. - 1 ^ . / 1 / I \ \ \ / 1 \ \ , / 1 \ \ ■ 9 / \ \ \ ■ 1 1 I \ ■ 1 1 I 1 ^ \ * / ■ \ « - A 1 \ . y / \ \ . / \ ^ ■ ■' 1 V > - =^ , 1 y . . , ^> ^ 9 ^ 30 25 2 < 20 3 15 73 4 5 B 7 MONTHS OF THE YEAR 10 11 12 10 Figure 12. Corhiciila fluininea fecundity from 1991-1994. (- -) and water temperature (- - : - -) for clams held in ambient temperature Connecticut River water COHHICULA IN THE LoWhR CONNECTICUT RlVER 201 o o >- < < _J O •\ > 3 O d 75 50 25- n =47 r=0.77 p<0.0001 .^<'-€> 100- .■■0^-^^' 75; a .^<^>>" ,„-'- 50- o o , Q' ^^^ , '' o .'.^ ,-' o yh- ^^^'' ° : ^^ '' rx.^ -■d' o ■ '--°'c ■^^-' ol T 1 1— ro^ 1 1 °o 1 1 1 1 1 1 1 1 1 1 — 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 LENGTH OF CLAM (mm) Figure 13. Linear regression with 95 '7r CI on mean predicted \aliies for the number of Juveniles released per day in relation to shell length (mm) of the spawning Corbktila jlumiiwa during the peak spawning month of August in the mainstem Connecticut River. MONTHS OF THE YEAR Figure 14. Corhiciila Jhiminea fecundity (-0-) and water temperature (- - 0 - -) for clams held in the discharge canal at CV from November 1992 to August 1993. 202 Morgan et al. Highest growth rates occurred in July and August, when river water temperatures pealced (25-30°C). and growth rates were sig- nificantly higher for the smaller clam sizes. The upper temperature tolerance of Corhicithi determined in this study is within ranges reported by other researchers in labo- ratory and field experiments (Mattice & Dye 1976. Dreier 1977. Mattice 1979. Cairns & Cherry 1983. McMahon & Williams 1986a). Corhuula growth in the CY thermal discharge canal was initiated in November 1992 when water temperatures dropped to <35°C. Growth continued until August 1993. when water tempera- tures were >37°C and clams died. Seasonal water temperatures also control reproductive cycles of the Connecticut River Corbiciila population. The presence of eggs and sperm was continuous in the Connecticut River population of this species as long as water temperatures supported its survival. Brooding and releasing of juveniles occurred when water tempera- tures were between 17-28^C. typically from June to October. Spawning temperatures of 14— 27°C were reported by other re- searchers in North America (Eng 1979. Mattice 1979. Hall 1984. Cherry et al. 1986. Foe & Knight 1986; McMahon & Williams 1986a: Doherty et al. 1987; Rajagopal et al. 2000). A single annual spawning peak for the Corhicithi population in the Connecticut River occurred in August. Others reported two Corbiciila spawning peaks, one in spring and one in fall (Heinsohn 1958. Aldrige & McMahon 1978. Eng 1979. McMahon 1983. Foe & Knight 1986. McMahon & Williams 1986a). Several others have reported a single spawning peak (Bickel 1966. Homback 1992, Mouthon 2001). The presence of a single reproductive peak in the Connecticut River population may be related to longer pe- riods of cold-water conditions, more severe spring Hooding, and the quantity and quality of available food. The altered thermal regimen within the CY discharge canal shifted the period of reproduction from the ambient river period of June through September to November and March through May when water temperatures in the canal ranged between 16-30'C. Spawning during July and August 1993 occurred because the power plant was off-line and the discharge water temperatures were not elevated. These results demonstrate that thermal dis- charges can alter the reproduction cycle of Corbiciila. Aldridge and McMahon (1978) and Dreier and Tranquilli (1981) reported that Corbicula fliiminea spawning activities stopped at tempera- tures of 30-34°C. most likely due to thermal stress. Graney et al. ( 1980) speculated that elevated temperatures in thermal discharges may e.xtend the spawning season into the winter. In conclusion, this study showed that the Connecticut River has supported a fluctuating Corbiciila population for at least 10 years. Cold water temperatures (<2°C) for several weeks, and high water flow in the spring caused high mortality of clams in the river during the winter and early spring. Growth and reproduc- tion for Corbiciila in the Connecticut River peaked in July and August when river temperatures ranged between 24-30°C and only one spawning peak occurred each year. The key to Corbicii- la's unexpected success in establishing a population in the Connecticut River is its ability to colonize refugia from cold win- ter water temperatures and spring freshet flows that cause high clam mortality. Following the closing of (he CY power plant. Corbiciila continued to populate the CY river sites establish- ing a more mature population in the discharge canal. Based on our observations of Corbiciila in the Connecticut River, we ex- pect that this species will continue to successfully colonize other rivers and lakes in New England, where similar winter refugia exist. LITERATURE CITED Aldridge. D. W. & R. F. McMahon. 1978. Growth, fecundity, and hioen- ergetics in a natural population of freshwater clam. Corhiciilii inanilen- sis Philippi. from North Central Texas. J. Molt. Studies. 44:49-70. Bickel. D. 1966. Ecology of Corbicula manileiuis in the Ohio River at Louisville Kentucky. Sterkiana 23:19-24. Boyd. W. A. 1976. Hydrology. In: D. Merriman & L. M. Thorpe, editors. The Connecticut River ecological study, the impact of a nuclear power plant. Bethesda. MD: Amer. Fish. Soc. Monogr. No. 1. pp. 25-34. Britton. J. C. D. R. Coldiron, L. P. Evans. Jr.. C. Golightly. K. D. O'Kane & J. R. TenEyck. 1979. Reevaluation of the growth pattern in Cor- bicula fluminea (Miiller). In: J. C. Britton. editor. Proceedings. First International Corbicula Symposium. October 13-15, 1977. Fort Worth. Texas: Texas Christian University, pp. 177-192. Burch. J. Q. 1944. Checklist of West North American marine mollusca from San Diego. California to Alaska. Minutes of the Coiuhological Club of Southern Califoniia 38:11-18. Cairns. J.. Jr. & D. S. Cherry. 1983. A site-specitlc field and laboratory evaluation of fish and Asiatic clam population responses to coal-fired power plant discharges. Water Sci. Tech. 15:10-37. Chen, L. 1990. Aquaculture in Taiwan. Cambridge MA: Fishing News Books. Cherry. D. S.. J. H. Rodgers. Jr.. R. L. Graney & J. Cairns, Jr. 1980. Dynamics and control of the Asiatic clam in the New River. Virginia. Bull. Va. Water Resour. Res. Cent. 123:1-72. Cherry. D. S.. R. L. Roy. R. A. Lechleitner. P. A. Dunhardt. G. T. Peters & J. Cairns. Jr. 1986. Corbicula fouling and control measures at the CELCO Plant. Virginia. In: J. C. Britton & R. S. Prezant. editors. Second International Corbicula Symposium, June 21-24. 1983. Hatties- burg, Mississippi. Amer Malacol. Bull. Special Edition No. 2. pp. 69-81. Counts. C. L.. lii. 1981. Corbicula jhnninea (Bivalvia: Corhiculidea) in British Columbia. Nautilus 95:12-13. Doherty. F. G.. D, S. Cherry & J. Cairns, Jr. 1987. Spawning Periodicity of the Asiatic Clam Corbicula fluminea in the New River. Virginia. .Am. Midi. Nat. 117:71-82. Doherty. F. G., D. S. Cherry & J. Cairns. Jr. 1990. Multiseasonal tissue growth trends in Corbicula fluminea (Bivalvia: Corbiculidae) from the New River. Virginia. Nautilus. 104:10-15. Dreier. H, 1977. Study of Corbicula in Lake Sangchris. In: The Annual Report for Fiscal Year 1976, Lake Sangchris Project. Sect. 7. Urbana: Illinois Natural History Survey, pp. 1-52. Dreier, H. & J. A. Tranquilli, 1981. Reproduction, growth, distribution and abundance of Corbicula in an Illinois cooling lake. In: R. W. Lawrimore & J. A. Tranquilli, editors. The Lake Sangchris Study: case history of an Illinois cooling lake. Bulletin Illinois Natural History Survey, No. 32. pp. 378-393. Eng, L. L. 1979. Population dynamics of the Asiatic clam, Corbicula fluminea (Miiller). in the concrete-lined Delta-Mendota Canal of Cen- tral California In: J, C. Britton, editor. Proceedings. First International Corbicula Symposium. October 13-15. 1977. Fort Worth, Texas: Texas Christian University, pp. 39-68. Foe, C. & A. Knight. 1986. A thermal energy budget for juvenile Corbicula fluminea. Amer. Malacol. Bull. 2:143-150. Foehrenbach, J, & D. Raeihle. 1984. A further range extension of the Asiatic clam. NY Fish and Game J. 31:224-225. French, J. R. P. II & D. W. Schloesser. 1991, Growth and over-winter survival of the Asiatic clam, Corbicula fluminea. in the St. Clair River. Michigan. Hydrobiologia 219:165-170. Gardner, J. A., Jr.. W. R. Woodall. Jr.. A, A. Staats, Jr. & J. F. Napoli. CORBICVLA IN THE LOWER CONNECTICUT RiVER 203 1976. The invasion of the Asiatic clam (Corbicula munilensis Philippi) in the Altanuiha River. Georgia. Naiilihis 90:117-125. Goss. L. B. & C. Cain. Jr. 1975. Power plant condenser and service water system fouling by Corbicula. the .Asiatic clam. Presented at the Bio- fouling Workshop held at John Hopkins University, Baltimore. Mary- land, June 16-19, 1975. Graney, R. L., D. S. Cherry, J. H. Rodgers, Jr. & J. Cairns. Jr. 1980. The influence of thermal discharges and substrate composition on the popu- lation structure and distribution of the Asiatic clam, Corbicula flu- minea. in the New River. Virginia. Nautilus 94:130-135. Hall, J. J. 1984. Production of immature Corbicula fluminca. in Lake Norman. North Carolina. Nautilus 98:153-157. Heinsohn. G. E. 1958. Life history and ecology of the freshwater clam, Corbicula fluniinea. MSc Thesis, Santa Barbara. CA: L'niversity of California. Homback, D. 1992. Life history traits of a riverine population of the Asian clam Corbicula fluminca. .Am. Midi. Nat. 127:248-2.S7. Homing. W. B. & L. Keup. 1964. Decline of the Asiatic clam in the Ohio River. Nautilus 78:29-30. Ingram, W. M. 1959, Asiatic clams as potential pests in California water supplies. / Amer. Wateniorks Assoc. 51:363-370. Isom, B. G. 1986. Historical review of the Asiatic clam {Corbicula) inva- sion and biofouling of waters and industries in the Americas, June 21-24, 1983, Hattiesburg, Mississippi. Amer. Malacol. Bull. Special Edition No. 2. pp. 1-5. Joy. J. E. 1985. A 40-week study on growth of the Asian clam. Corbicula fluminca (Miiller). in the Kanawha Ri\er. West Virginia. Nautilus 99: 110-116, Kreiser, B, R, & J. B. Mitton. 1995. The evolution of cold tolerance in Corbicula flinninea. (Bivalvia: Corbiculidae). Nautilus 109:111-112. Magdych. W, P, 1981. An efficient, inexpensive elutriator design for sepa- rating benthos from sediment samples. Hycirobiologia 85:157-159. Mattice, J. S. 1979. Interactions of Corbicula sp. with power plants. In: J.C. Britton, editor. Proceedings. First International Corbicula Symposium. October 13-15, 1977. Fort Worth, Te.xas: Texas Christian University, pp. 119-138, Mattice. J, S. & L. L. Dye. 1976. Thermal tolerance of the adult Asiatic clam. Pages 130-135 In: G. W. Esch & R. W. McFarlane. editors. Thermal Ecology II. Conf. 750425. Nat. Tech. Info. Serv.. Springfield, VA. Mattice. J. S. & L. L. Wright. 1986. Aspects of growth of Corbicula fluminca. June 21-24. 1983. Hattiesburg, Mississippi. Amer. Malacol. Bull. Special Edition No. 2. pp. 167-178. McMahon. R. F. 1977. Shell size-frequency distributions of Corbicula manilensis from a clam-fouled steam condenser. Nautilus 91:54-59. McMahon, R. F. 1983. Ecology of an invasive pest bivalve. Corbicula. Pages 505-561 in W,D, Russell-Hunter (ed.). The Mollusca. Vol. 6. Ecology. Academic Press, Orlando, FL. McMahon, R. F. 2002. Evolutionary and physiological adaptations of aquatic invasive animals: /■ selection versus resistance. Can. J. Fish. .Aquat. Sci. 59:1235-1244. McMahon, R. F. & C. J. Williams. 1986a. A reassessment of growth rate, life span, life cycles and population dynamics in a natural population and field caged individuals of Corbicula fluminca iMiiller) {Bi\alvia: Corbiculacea), June 21-24, 1983, Hattiesburg. Mississippi. Amer. Ma- lacol. Bull. Special Edition No, 2, pp. 151-166. McMahon, R. F. & C. J. Williams. 1986b. Growth, life cycle, upper ther- mal limit and downstream colonization rates in a natural population of the freshwater bivalve mollusc, Corbicula fluminca (Miiller) receiving thermal effiuent, June 21-24. 1983. Hattiesburg. Mississippi. Amer. Malacol. Bull. Special Edition No. 2. pp. 231-239. Merriman. D. & L. M. Thorpe. 1976. The Connecticut River ecological study, the impact of a nuclear power plant. Amer. Fish. Soc. Mono- graph No. 1. Bethesda. MD. Morgan. D. E.. M. Keser. J. F. Foertch & J. M. Vozarik. 1992. The Asiatic clam. Corbicula fluminca. extends its North American distribution to the Connecticut River [abstract only]. In: W. J. Blogoslawski, editor. In: Aquaculture and environmental stewardship: Milford Shellfish Bi- ology Seminar 1991. Environmental management, pp. 521-529. Morton, B. 1977. The population dynamics of Corbicula fluminca (Bi- valvia: Corbiculacea) in Plover Cove Reservoir. Hong Kong. J. Zool. 181:21^2. Mouthon, J. 2001. Life cycle and population dynamics of the Asian clam Corbicula fluminea (Bivalvia: Corbiculidae) in the Saone River at Lyon (France). Hydrobiologia 452:109-1 19. NSI. (Nautical Software, Inc). 1995. Tides & Currents for Windows, Ver- sion 2.0. nauticalswts'aol.com. Beaverton, Oregon. NUSCO (Northeast Utilities Service Company). 1994. 1993 annual report on Corbicula studies at Connecticut Yankee. As submitted to the Con- necticut Department of Environmental Protection on January 27. 1994. NUSCO Letter No. D07282. 19 pp. Page. T. L.. D. A. Neitzel. M. A, Simmons & P. F. Hayes. 1986. Biofouling of power plant service systems by Corbicula. June 21-24. 1983. Hat- tiesburg. Mississippi. Amer. Malacol. Bull. Special Edition No. 2. pp. 41-45. Prokopovich, N. P. 1969. Deposition of clastic sediments by clams. Sedi- men. Petrol. 39:891-901. Rajagopal, S.. G. van der Velde & A. bij de Vaate. 2000. Reproductive biology of the clams Corbicida flumimdis and Corbicula fluminca in the river Rhine. Arch. Hydrobiol. 149:403^20. Rodgers, J. H., Jr.. D. S. Cherry. K. L. Dickson & J, Cairns, Jr. 1979. Invasion population dynamics and elemental accumulation of Cor- bicula fluminea in the New River at Glen Lyn, Virginia. In: J. C, Britton. editor. Proceedings. First International Corbicula Symposium. October 13-15. 1977. Fort Worth. Texas: Texas Christian University, pp. 99-110. Rozsa. R. 2001. Hydrology of the lower Connecticut River. In: G. D. Dreyer & M. Caplis. editors. Living resources and habitats of the low er Connecticut River. December 2001. New London. CT: The Connecti- cut College Arboretum Bull. No. 37. pp. 18-22. Sinclair. R. M. 1964. Clam pests in Tennessee water supplies. J. ,Am. Water Works Assoc. 56:592-599. USGS. (United States Geological Survey). 2002. Water resources of the United States, surface-water data for Connecticut, August 15. 2002. loi>ical Laboratory. University of Maryland. Center for Environmental Science. P.O. Box .^(S. Solomons. Maryland 206HS: ^Department of Fisheries and Oceans. Canada. Gulf Fisheries Center. P.O. Box 5030. Moncton. N.B. EIC 9B6: "'Marine Bioloi;ic Laboratory. 7 MBL Street. Woods Hole. Massachusetts 0254.1 .ABSTRACT The thraustochytrid protist quahog parasite unknown (QPX) has caused mass mortalities of hard clams (Mercenaria nicneiuiria) in Atlantic Canada and Massachusetts. It typically secretes copious mucus in vivo and in vitro. M. mercenaria plasma contains naturally-occurring agents that modulate growth of QPX cultures. This activity was shown by exposing washed, mucus-free QPX (wQPX) to filter-sterilized M. mercenaria plasma. Low plasma protein concentrations (<10 |xg/ml) in the medium tended to stimulate QPX growth; higher concentrations (10-50 (jLg/ml) produced dose-dependent inhibition. If wQPX were incubated for various times before exposure to an inhibitory concentration of M. mercenaria plasma, a time-dependent protection from the plasma was observed; total protection was seen after -24 h preincubation. This effect was probably a result of the re-establishment of the mucoid coats around the wQPX during preincubation. These data suggest th;it ihe mucoid secretion of QPX may represent an important virulence factor. KEY WORDS: quahog parasite unknown (QPX). Mercenaria mercenaria. virulence factors, clam diseases INTRODUCTION Whyte et al. (1994) described a protistan parasite that caused high mortalities in a hard clam (Mercenaria mercenaria) hatchery on Prince Edward Island, Canada; the causative agent was named quahog parasite unknown (QPX). This organism was similar or identical to the clam pathogen first observed by Drinnan and Hen- derson ( 1963) in New Brunswick, Canada. Subsequently, QPX has been cited as the cause of mass mortalities of M. mercenaria in Massachusetts (Smolowitz et al. 1998) and has been reported in several Virginia coastal embayments (Ragone Calvo et al. 1998). Molecular phylogeny studies based on sequencing of I8S riboso- mal RNA suggest that QPX is a member of the phylum Labyrin- thulomycota (Maas et al. 1999. Ragan et al. 2000). in the thraus- tochytrid phylogenetic group (Stokes et al. 2002). A medium developed by Kleinschuster et al. (1998) has per- mitted in vitro cultivation of QP,\. In culture, thalli were shown to grow and mature into sporangia containing numerous vegetative endospores. The endospores were released on rupture of the spo- rangia and in turn matured to form thalli. and the stages of the vegetative life cycle were repeated. Whyte et al. ( 1994) and Klein- schuster et al. ( 1998) reported conversion of endospores to motile zoospores in sterile seawater. Later studies (Brothers et al. 2000). however, were unable to replicate these findings. The vegetative life stages of QPX have been observed in the tissues of infected M. mercenaria. In many instances, the QPX cells were seen in histo- logic sections to be enclosed by a translucent space; this was initially attributed to lysis of host tissue by enzymes secreted by the parasite (Whyte et al. 1994). Subsequently. Smolowitz et al. ( 1998) determined that in live animals, the space is occupied by a muco-fibrillar substance produced by the parasites; and that this substance is removed by histologic processing. It was suggested in that study that phagocytosis of the parasite in the clams' tissues is inhibited by the mucofibrillar secretions of the parasite. The disease caused by the Canadian strain (CA QPX) as de- scribed by Whyte et al. ( 1994) is similar to that described for the Massachusetts strain (MA QPX) by Smolowitz et al. (1998). MA QPX. however, primarily infected the mantle and gill and some- times produced nodules; CA QPX infections were more commonly seen in the connective tissue of the foot and were rarely associated with nodules. Areas of infection by CA QPX and MA QPX trig- gered inflammatory responses involving extensive infiltration of adjacent host tissues by hemocytes. with some evidence of phago- cytosis and/or encapsulation of the parasites. Inflammatory foci caused by MA QPX sometimes contained phagocytic multinucle- ated giant cells similar to those produced /;) vitro by Anderson (1987). Apparently QPX infection elicits a vigorous cellular re- sponse, but this activity is insufficient to control the disease. Hu- moral QPX modulatory agents in M. mercenaria plasma are de- scribed for the first time in this article, and Ihe role of QPX mucoid secretions in protection from them. MATERIALS AND METHODS *Corresponduig author. Tel.: -^ 1-4 10-326-7247; Fax; +1-410-326-7210; E-mail; andersonts'cbl. umces.edu QPX These studies were carried out using MA QPX obtained from Dr. R. Smolowitz, Marine Biologic Laboratory, Woods Hole, MA. They were propagated in the medium of Kleinschuster et al. (1998). The initial seeding density was 10"'/ml and the cultures were maintained at 23°C and were har\ested at 7 d ( 168 h) while still in exponential growth phase. The QPX cells were enveloped by a heavy mass of mucoid secretion, which was routinely washed off the cells by dilution with a saline solution. lO (25 ppt. Instant Ocean®, Aquarium Systems Inc.; Mentor, OH), followed by re- peated centrifugations (300 x g, 10 min, 21-0, x3). Washed QPX (wQPX) were >90'7f viable by the trypan blue exclusion assay (Hanks & Wallace 1958) and almost immediately resumed mucus secretion. The numbers of QPX cells in particular cultures and cell numbers required for subsequent experiments were quantified spectrophotometrically using a standard curve of the numbers of 205 206 Anderson et al. wQPX (as determined in a Ineniacytometer) as a function of tlieir absorbance at 560 nm. C9G Another thraustochytrid. C9G. closely related to QPX (Ander- son et al., in press) was isolated from gill tissues of Canadian M. meirenaria and provided by Mr. G. S. MacCallum and Dr. S. McGladdery, Gulf Fisheries Center. Moncton. Canada. Like QPX. C9G was maintained in the medium of Kleinschuster et al. ( 1998) at 25°C and subcultured at 7 d. M. mercenaria Plasma M. mercemiria. collected from the Ware River. VA by a com- mercial supplier; were maintained with recirculating water (25 ppt. 10. 1 1°C). Hemolymph samples were withdrawn by syringe from an adductor muscle hemolymph sinus and held on ice in polypro- pylene tubes. The hemocytes were centrifuged out of suspension (300 X g. 10 min. 4°C). The pooled supernatant (plasma) was sterilized by filtration (0.2 |j.m pore size), and assayed for protein content (BCA kit. Pierce Co.. Rockville. IL). Individual plasma samples from three to four hard clams were pooled and were frozen (-20°C) in aliquots. The frozen samples were used soon because the QPX-modulatory activity declined after -2 mo in stor- age. In one series of experiments, plasma was heat-treated by exposure to 65°C for 10 min. the plasma was cooled to room temperature (~25°C) before use. Immediate Exposure of Thraustochylrids to Plasma QPX cells from 7d cultures were washed, as described above, and resuspended (2.5 x lUVml) in 25 ppt lO. Plasma protein con- centration was standardized (usually to 0.2 mg/ml ) by dilution with 10 and serial dilutions prepared. Replicate culture flasks for each protein concentration tested were prepared with experimental (1.9 ml Kleinschuster" s minimal essential medium (KMEM), 0.1 ml QPX suspension, and 0.5 nil plasma dilution), control (1.9 ml KMEM. 0. 1 ml QPX suspension, and 0.5 ml lO). and the necessary blanks. After 7 d incubation at 24°C. the contents of each flask were removed, and the QPX washed thoroughly and quantified, as described previously. In related experiments. QPX or C9G were incubated for 2 h in lO containing plasma, washed, and resus- pended in KMEM. Percent inhibition was determined using the following formula: % inhibition = 1 experimental value control value X 100 Delayed Exposure to Plasma In the delayed exposure experiments. wQPX were permitted to incubate in KMEM for various time intervals <24 h before expo- sure to 40 jjLg/ml M. mercenaria plasma proteins. The QPX cells resumed typical secretory activities during these pre -exposure pe- riods, as seen by microscopic examination. This plasma protein concentration was selected because it had been shown in previous immediate exposure experiments to inhibit -95% of the growth of QPX cultures. Viability Assays QPX viability tests were carried out using viability/cytotoxicity kit #1 (Molecular Probes, Eugene, OR). The test is based on the differential permeability of live and dead cells to a pair of fluo- rescent stains. Cell populations exposed simultaneously to both dyes become differentially stained: live cells are stained green and dead cells appear red. This assay was used to check wQPX viabil- ity after exposure to 10 or M. mercenaria plasma. RESULTS Effects of M. mercenaria Plasma on Washed QPX At the lower plasma concentrations tested, inhibition was low and variable, with some pools actually stimulating growth (Fig. 1 ). However, at plasma protein concentrations s 10-50 jxg/ml, a dose- dependent inhibition was consistently recorded (-100% inhibition was seen at >50 (j.g/ml). The inhibitory EC^,, was calculated to be -19 p-g/nil. When this procedure was carried out with heat-treated (65"C. 10 min) plasma, the stimulatory effects of the lower con- centrations were not evident (Fig. 2). The inhibitory EC^,, for heated plasma was -32 (xg/ml; therefore, this heat treatment only partially inactivated (-40%) the growth inhibitory factor(s). The inhibitory effects of M, mercenaria plasma were exerted in a short period. When wQPX were exposed to 40 p.g/ml plasma for 2 h, washed free of plasma and cultured for 7 d in plasma-free medium, the resultant QPX cell numbers were 80.7 ± 13.3% (n = 3) reduced as compared with untreated controls. A similar degree of inhibition (94.3 ± 5.\%. n = 4) was seen when 40 |j,g/ml plasma was left in the medium for the entire duration of the assay. No significant difference was found between these means by way of a 2-tailed, unpaired Mest. The inhibition produced by 2-h ex- posure of wQPX to 40 p,g/ml plasma protein did not result from QPX-cidal activity. Plasma-treated and untreated wQPX were similar (treated: 94.0 + 1.7%r, n = 3; and untreated: 94.0 ± 3.0%-, // = 3 viable). A degree of specificity for M. mercenaria plasma is also indicted because exposure of wQPX to 40 |xg/ml produced >90%i inhibition, whereas under the same conditions. C9G was minimally inhibited (Fig. 3). Reactions of M. mercenaria plasma with mucus-enveloped QPX The typical response obtained by exposing wQPX immediately to M. mercenaria plasma (Fig. 1) was not seen after comparable 100 75 50 25 0 -25 -50 -75 -100 c o 10 20 30 40 — I 1 50 60 Protein Cone. (Mg/ml) Figure 1. QPX-modulatory activity of M. mercenaria plasma ex- pressed as percent inhibition of cultures after 7 d incubation. Final plasma protein concentration in tlie medium is indicated. Linear re- gression ly = lll..^[log xl - n.M: r- = 0.7497) of log-transformed concentrations was used to calculate tlie inhibitory EC;,, = 18.99 (ig/ml. QPX Mucoid Secretions 207 C o !c c 100 n 75 50 25 0 -25 -50 H -75 ■100 0 10 20 30 40 50 60 Protein Cone, (pg/ml) Figure 2. QPX-modulatory activity of heat-treated (65'C. 10 niin) M. inerccnaria plasma expressed as in Figure I. Linear regression (\ = 48.22|log \| - 22.71; r" = 0.67151 of log-transfornied concentrations was used to calculate the inhibitory V.C=,„ = 32.20 Mg/ml. exposure of vvQPX that was incubated for 24 h before the addition of plasma (Fig. 4). The lowest dose tested (3.75 |jLg/ml) apparently produced some inhibition, whereas all other doses (:£60 jjig/ml) seemed to stimulate the QPX cultures. The apparent inhibition produced by the lowest concentration tested was not significantly different from zero (P > 0.05. one sample Mest. 2-tailed). The higher concentrations tested were all stimulatory. (P > 0.05. one sample ;-test. 2-tailed). wQPX cells were either immediately ex- posed to a highly inhibitory plasma concentration (40 |jig/ml) or allowed to incubate in plasma-free medium for 2-24 h before exposure; in these delayed exposure experiments, a time- dependent linear decrease in growth inhibition was ob.served (Fig. 5 1. Unlike QPX. C9G cells in culture secreted no mucoid material visible in preparations examined under the microscope. Preincu- bation of washed CQG cells for 24 h before exposure to 40 p.g/ml plasma had no significant protective effect as compared with cells immediately exposed. 100 c o c 75- 50- 25 C9G QPX Figure 3. The effects of 40 pg/nil M. merciiiaria plasma proteins on growth of 7 d cultures of QPX and C9G, a closely related thraus- tochytrid also isolated from hard clams. The protists were exposed to the plasma for 2 h. washed, and cultivated (7 d) in KMEM. Mean percent inhibition and standard deviations are indicated. o luu- • • n • u t • • 100 • • • • • • • • • • 200 r- • r 0 10 20 30 40 50 60 Protein Cone, (pg/ml) Figure 4. (Irowth of wQPX preincubated lor 24 h in plasma-free me- dium before exposure to 40 (ig/ml M. menenaria plasma. The QPX cells continuously secreted mucoid material during the preincubation period. DISCUSSION When wQPX cells were introduced into media containing vari- ous concentrations of M. meixenahu plasma, their subsequent growth was altered according to plasma concentration. This may be seen in Figure 1 where 7 d QPX culture growth was often stimulated in the presence of low plasma levels but consistently suppressed at >I0 jjig/ml. These effects could be explained by the presence of two QPX-modulatory agents in the plasma. Stimula- tion at low protein levels might be caused by a factor with high QPX-affinity and low to moderate activity. The effect of this stimulator would be lost at higher protein levels if a low QPX- affinity. higher activity inhibitor were present. The presence of two growth modulators was also suggested by the differences in ther- mal sensitivity (Fig. 2). Heat treatment of 65°C for 10 min seemed to eliminate all stimulatory activity: however, the inhibitory effects persisted with somewhat reduced activity. The growth modulating activity of M. menenaria plasma takes place rapidly after inter- action with wQPX. If wQPX was exposed to an inhibitory con- centration of plasma (40 |jLg/ml) for 2 h. and then washed free of plasma proteins before growing the culture in plasma-free me- dium, culture growth was inhibited to about the same extent be- cause it would have been if the cells had been continuously ex- 100 -25 -50 8 12 16 20 24 28 Time (hrs) Figure 5. Effects of length of wQPX preincubation before exposure to 40 ng/ml M. menenaria plasma. Mean percent inhibition and standard deviations are indicated. 208 Anderson et al. posed to 40 ^Lg/ml plasma. These experiments could not establish whether the inhibitory effects produced by M. mercenaria plasma on the cell density of 7 d QPX cultures were caused by growth inhibition or by cidal activity. Direct killing was ruled out by the fact that 40 |xg/ml exposed, (potentially highly inhibited) wQPX and unexposed wQPX were -95% viable. Figure 3 presents evidence that the QPX-inhibilory plasma fac- tor shows target specificity; C9G growth was hardly affected by 40 p.g/ml. Sequence analysis of C9G placed it in the thraustochytrid phylogenetic group as a sister taxon to Thnnistocliytiium pachy- denniim. and these sequences were grouped with QPX with a parsimony jackknife support value of 100 (Anderson el al. in press). Clearly. QPX sensitivity to low (<40 |xg/ml) plasma con- centrations exceeds that of C9G; however, C9G growth was in- hibited (-60%) by exposure to >180 |Jig/ml plasma (Anderson et al. in press). Because the pathogenicity of C9G for M. Dierceinirici has yet to be established, it is not known whether inhibition dif- ferences caused by clam plasma between QPX and C9G reflect differences in pathogenicity. Incubation of wQPX in plasma-free medium allowed the cells to resume mucus secretion. The cells underwent minimal division for the first 48 h in culture, then proceeded lo grow w ith a doubling time of -3 d (QPX growth curve not shown). The wQPX cells were suspended in a loose gelatinous mass by 24 h. This mucoid secretion often infiltrated the entire culture medium by 7 d in culture. When the cells were permitted to develop their mucoid covering for 24 h before the addition of plasma (Fig. 4). concen- tration of -7-60 p-g/ml failed to inhibit QPX growth in 7 d cul- tures. Unexpectedly, the lowest concentration tested (3.75 jjig/ml) seemed to have inhibitory activity, but the mean of these experi- mental values were not significantly different from zero. These data suggested that the mucus material might protect QPX from A/. iiicrceiuiria humoral defense mechanisms such as antimicrobial factors. This hypothesis was supported by the results of the de- layed exposure experiments, where protection from growth inhi- bition was dependent on the time of incubation before exposure to 40 |j.g/ml plasma protein (Fig. 5). Because QPX cells in clam tissues are typically enveloped by mucus, a role of this secretion as a virulence factor seems likely. This is supported by a recent report that clams injected with wQPX did not develop infections or dis- ease (Smolowitz et al. 2001). ACKNOWLKDGMENTS This study was supported by Maryland Sea Grant, NOAA, grant number NA06RG010I. This is Contribution No. 3642 of the University of Maryland Center for En\ ironmental Science, Chesa- peake Biological Laboratory. LITERATURE CITED Anderson. R. S.. B. S. Kruus, S. E. McGladdery, K. S. Reece & N. A. Stokes. 2003. A thraustochytrid protist isolated from Meirciuiria mer- cenaria: molecular characterization and host defense responses. Fish. Shellfish Immunol, (in press). Anderson, R. S. 1987. Polykaryon formalion by Meiccmirin iiicrceiiariu hemocytes. Biol. Bull. l72:236-:45. Brothers. C, E. Marks & R. Smolowitz. 2000. Conditions affecting the growth and zoosporulation of the prollstan parasite QP.X in culture. Biol. Bull. 199:200-201. Drinnun. R. E. & E. B. Henderson. 1963. 1962 mortalities and a possible disease organism in Neguac quahaugs. Annual Rept. No. Bl I. Biologi- cal Station. St. Andrews, New Brunswick. Hanks, J. H. & J. H. Wallace. 195S. Determination of cell viability. Proc. Soc. E.\p. Biol. Med. 98:188-192. Kleinschuster, S. J.. R. Smolowitz & J. Parent. 1998. /;; vino life-cycle and propogation of quahaug parasite unknown. J. Shellfish Res. 17:75-78. Maas, P. A. Y., S. J. Kleinschuster, M. J. Dykstra, R. Smolowitz & J. Parent. 1999. Molecular characterization of QPX (quahog parasite un- known), a pathogen of Mercenaria mercenaria. J. Shellfish Res. 18: 561-567. Ragan. M. A., G. S. MacCallum, C. A. Muiphy, J. J. Cannone. R. R. Gutell & S. E. McGladdery. 2000. Protistan parasite QPX of hard-shell clam Mercenaria mercenaria is a member of Lahyrinthulomycota. Dis. .Aqual. Org. 42:185-190. Ragone Calvo. L. M.. J. G. Walker & E. M. Burreson. 1998. Prexalence and distribution of QPX. Quahog Parasite Unknown, in hard clams Mercenaria mercenaria in Virginia, USA. Dis, Aquat. Org. 33:209- 219. Smolowitz. R.. D. Leavitt. B. Lancaster. E. Marks. R. Hanselmann & C. Brothers. 2001. Laboratory based transmission studies of quahog para- site unknown (QPX) in Mercenaria mercenaria. J. Shellfish Res. 20: 555. Smolowitz, R., D. Leavitt & F. Perkins. 1998. Observations of a protistan disease similar to QPX in Mercenaria mercenaria (hard clams) from the coast of Massachusetts. J. Inverlehr. Pathol. 71:9-25. Stokes. N. A.. L. M. Ragone Calvo. K. S. Reece & E. M. Bun-eson. 2002. Molecular diagnostics, field validation, and phylogenetic analysis of Quahog Parasite Unknown (QPX), a pathogen of the hard clam, Mer- cenaria mercenaria. Dis. Aquat. Org. 52:233-247. Whyte, S. K.. R. J. Cawthom & S. E. McGladdery. 1994. QPX (quahaug parasite X), a pathogen of northern quahaug Mercenaria mercenaria from the Gulf of St. Lawrence. Canada. Dis. Aquat. Org. 19:129-136. J.Hinuil ofSlwllJhh Re.scanh. Vol. 22. No. 1. 209-212, 200.^. A PORTABLE AND PRACTICAL METHOD TO MONITOR BIVALVE FEEDING ACTIVITY IN THE FIELD USING TIME-LAPSE VIDEO TECHNOLOGY BRl'CE A. MACDONALD* AND LISA M. NODVVELL Dcpcinniciit of Biology. Centre for Coiisral Stiullcs and Aquaciihnrc. University of New Briins\vu± Saint John. P. O. Box 5050 Saint Jolin. New Brnnswicl<. Canada. E2L 4L5 ABSTRACT We developed a simple iiielhod to measure leeding activity of Mylilii.s filiilis using a canicorder placed inside an underwater housing, a plastic frame for holding mussels and time lapse videography. Exhalant siphon area, indicative of feeding activity, was monitored in laboratory mussels exposed to filtered seawater and various concentrations of microalgae, including Pavlova lulheri or TetraseUnis suecica. Exhalant siphon area increased as algal concentration increased from zero to -25-30 x 10' cells ml"', hut declined again at higher concentrations. Advantages of this method include portability and relatively low cost, high resolution of data over shon and long temporal scales, potentially large sample sizes, and minimum logistics required for deployment in a variety of different environments. Once relationships between exhalant siphon area and other indicators of feeding such as filtration rate have been established, this method could greatly miprove our understanding of bivalve feeding in situ and how they respond in dynamic natural conditions. KEY WORDS: Mvrilus etliilis. bivalve feeding, time-lapse recording, exhalant siphon area, particle concentration INTRODUCTION There have been numerous studies on measuring feeding ac- tivity in a variety of suspension-feeding bivalves over the last several decades. There has recently been much discussion and debate on whether or not bivalves have the capability of physi- ological regulation or are pumping at full capacity all the time (Jorgensen 1996, Bayne 1998, Hawkins et al. 2001 ). This includes numerous comments on the proper interpretation of the published literature and diverse opinions on the reliability of some of the methods used (Cranford 2001. Riisgard 2001. Widdows 2001), One such method considered to have good potential for assess- ing feeding activity remotely with little interference by the ob- server and minimal disturbance to the bivalve is the estimation of valve gape and siphon area in mussels (Newell et al. 2001 ). Posi- tive relationships have been reported between pumping rates of mussels, valve gape and the exhalant siphon area (Jorgensen I960. Riisgard & Randlov 1981. Famme et al. 1986. j0rgensen et al. 1988. Jorgensen 1990) and between exhalant siphon area and mus- sel filtration rates (Newell et al. 2001). Filtration rates of mussels have been shown to be linked to particle concentration with low levels observed for filtered water but increasing with natural levels of seston before decreasing again at higher seston loads (Foster-Smith 197.5. Winter 197.^. Bayne 1993). Riisgard and Randl0v ( 1981 ) found comparable reductions in filtration rates and valve gape of blue mussels at densities of Plmeodactylum trieonmutwn lower than 1.500 cells ml" and higher than .W.OOO cells ml"'. Newell et al. (2001) found a similar apparent threshold for the filtration response to particle concen- tration to occur at 2.000-6,000 particles ml"' in a Hume environ- ment. Dolmer (2000a, 2000b) observed that high algal concentra- tions may lead to decreases in valve gape as well as estimates of filtration in the field. There is ample evidence to suggest that exhalant siphon area is a useful indicator of feeding activity in mussels and it is responsive to variations in the concentration of suspended particles. The pur- pose of this study was to develop a ponable and reliable method to *Corresponding author. E-mail: bmacdon@unbsj.ca; Fax: +1-5U6-648-581 1. remotely estimate exhalant siphon area for numerous undisturbed mussels simultaneously. It would be particularly advantageous if the method could be deployed to the field where mussel response could be continuously evaluated while natural seston and flow conditions are monitored. The combination of time lapse capabili- ties and high resolution image of a digital camcorder, a portable underwater housing, a plastic frame for holding mussels, and readily available image analysis software provides an effective tool for studying mussel feeding activity. Exhalant siphon area was monitored in this study in mussels exposed to various concentra- tions of cultured microalgae in the laboratory en\ ironment. MATERIALS AND METHODS Mussels [Mytilus edidis Linnaeus 1758) were collected from an inlet in the Pasamoquoddy Bay. New Brunswick and transported to University of New Brunswick in Saint John. New Brunswick, Canada. Mussels were acclimated to laboratory conditions for a minimum of 2 d and a maximum of 7 d. Experiments were per- formed in a 530 I (244 cm long, 66 cm wide, and 33 cm deep) tank with well mixed recirculating seawater. flowing approximately 5-10 cm s"'. Experiments were performed in full room light and temperature and salinity were maintained at I2°C and 35-36'^f. respectively. Water was prc-filtered in the tank with three inline filters of 20. 5. and I p.m. Mussels were exposed to filtered sea- water and cultured microalgae ranging in initial concentration from 5.000-85.000 cells ml"' while siphon area was monitored over periods of hours using time-lapse videography. Mussels were exposed to experimental conditions for 30-60 min prior to mea- surements to ensure feeding activity had resumed. With a few exceptions experiments for each series of mussels typically ran tor 2_t h to ensure a good time series of measurements and a detect- able change in particle concentration. Algal concentration was measured using an electronic particle counter (Coulter Multisizer II) with a 100 p-m tube orifice diameter. Algal diets provided in experiments were one of Pavlova lutlieri (Provasoli-Guillard CCMP1325) or TetraseUnis suecica (Provasoli-Guillard CCMP904) or a mussel spat formula of Nanocliloropsis ocidata. Chaetoceros-B, and Phaeodaelyltim iricorniaum (Innovative Aquaculture Products Ltd.). 209 210 MacDonald and Nodwell At least one day prior to the experiments Velcro was attached to the mussel shell using cyanoacrylate cement and. after drying, mussels were attached to individual plastic posts also covered in velero. The posts containing the mussels were secured to a plastic plate and attached to a frame connected near the lens of a video recording device (Fig. 1 A). The number of mussels observed (usu- ally 9-12 adults) in the video frame depended on the size of the mussels and the efficiency of arranging mussels to adequately view the external siphon. A Sony Mini DV (model DCR-TRV900) three ccd camcorder was enclosed in an Amphibico 900 underwa- ter housing and set to an interval recording mode of 2 s every 30 s over the entire period of each experiment to capture siphon activity. Multiple images from the mini DV tapes were collected using the photo feature of the camcorder and stored on memory cards before being transferred to a personal computer (Fig. IB). Varia- tion in siphon area was estimated for individual mussels using the program Image J (NIH public domain Java image processing pro- gram— URL: http://rsb.info.nih.gov/ij). Siphon area was calibrated using a 1 cm mark on the mussel posts. The inherent variation in measuring exhalant siphon area was 2.4-3.8%. To standardize individual responses for different sizes of mussels to different algal concentrations, exhalant siphon area data were converted to per- cent of maximum values observed for each mussel. RESULTS There was a consistent decline in algae over time in all the experiments, indicating removal of microalgae by the mussels in the course of the experiments (Fig. 2). Exhalant siphons were opened, confirming feeding activity by the mussels. The fitted lines for the uptake rates of algae typically had r" values exceeding 0.90-0.95 in all examples. The percent maximum exhalant siphon area in individual mus- sels exposed to filtered seawater (no algae) was consistently lower than the siphon areas reported for the same mussels exposed to microalgae (Fig. 3A). A similar trend of greater exhalant siphon area was akso observed for groups of mussels exposed to different concentrations of microalgae compared to those held in filtered seawater (Fig. 3B). Note that mus.sel exhalant siphon area was still approximately 20-309f of the maximum when exposed to filtered seawater. The percent maximum exhalant siphon area in mussels in- creased with increasing particle concentrations to a maximum of near 90-95% at concentrations approaching 25-30.000 cells mP' (Fig. 4) Further exposure to concentrations above 30.000 cells ml"' resulted in a decline in percent maximum exhalant siphon area. DISCUSSION By modifying an underwater housing and combining it with a high resolution camcorder capable of time-lapse videography we have developed a simple and relatively inexpensive method to remotely study bivalve feeding behavior. There have been other devices developed to remotely monitor bivalve activity but, for various reasons, they have not been readily adopted by scientists working on bivalves. This includes The Musselmonitor* devel- oped as a biological early warning system containing sensors to record shell opening and closing while mussels are exposed to various pollutants (Baldwin & Kramer 1994). Manuel and Lob- siger ( 1999) de\eloped the MarineCanary'^' as a biomonitoring tool using an underwater camera and a time-lapse system to assess the marine environment through changes in bivalves" valve gape and mantle activity. Using this new method we have established a positive relation- ship between exhalant siphon area and the concentration of cul- tured microalgae, also observed by Newell et al. (2001) in their study. Feeding activity is this study was confirmed by the con- tinuous decline in the concentration of microalgae in the experi- y = -976.76x + 6671 R' = 0.9487 Figure I. {\). .\n adjustable plastic frame attached to the front of an underwater video housing containing a high resolution camcorder with time-lapse capabilities. Mussels are secured with \ elcro to move- able posts inserted into a plate positioned in front of the video lens. (B) A tvpicai black and while photo made from a video frame captured from the mini DV tape. Exhalant siphons are clearly visible for several mussels simultaneously. Elapsed Time (h) Figure 2. An example of variation in declining algal concentration, attributable to mussel feeding, during a typical medium — low concen- tration experiment. Time-Lapse Video Technique to Estimate Mussel Feeding w < e o a R 0) Mussel #2 Mussel #3 Mussel #5 Mussel #8 100 ■ No Algae =1 10-20000 cells ml B 20-30000 cells ml n 30-45000 cells ml"' * i Figure 3. (A) Variation in individual mean percent maximum exhal- ant siphon area of representative mussels held in filtered sea« ater and exposed to microalgae in different algal concentrations (5-45.000 cells nil"'). (B) Mean response for groups of mussels exposed to filtered seawater and three different experimental concentrations of microal- gae (5—15.000 cells ml"'). Values are means ± 1 SE. mental tanks (Fig. 2). The shape of the line when fitted to semi-log transformed data (i.e.. rate of clearance) was comparable to the reduction observed by Riisgard (1991) when Myliliis edutis was grazing on Rhodomonas baltica. The positive relationship between particle concentration and exhalant siphon area was apparent until concentrations reached 25.000-30.000 cells ml"' and exhalant si- phon area appeared to decrease with further increases in concen- tration (Fig. 3B). Clausen and Riisgard (1996) also observed that mussels partly closed their valves and reduced the opening of the exhalant siphon at high algal concentrations but they found this reduction to occur at around 13-24.000 cells ml"'. Note that we did observe some moderately high values for siphon area for mus- sels at the highest algal concentrations. This may have been an artefact of the experimental design where a group of starved mus- sels were exposed initially to \ery high concentrations of microal- gae. There are several advantages to the time-lapse videography method for the observation of feeding activity in bivalves. This includes its size. cost, portability and readily available components including public domain software. A variety of underwater hous- ings are available today for most commercial camcorders capable of using time-lapse technology. Because of the small size of the housing, they can be placed unattended in a wide variety of habi- tats for extended periods of time — up to 10-15 hours with the new 3 < It (/) fli TO LU 100 90 SO 70 60 50 40 30 20 10 z i i s 10000 30000 .50000 70000 Algal concentration (cells ml'^) Figure 4. Variation in percent maximum exhalant siphon area of mus- sels exposed to different concentrations of microalgae. The closed dia- mond represents an experiment where 8 mussels were subjected to algal concentrations from no algae to 45.000 cells ml"'; the open circle, 13 mussels subjected to algal concentrations of 0-85.000 cells ml"': the open triangle, 8 mussels subjected to algal concentrations of 0-17,000 cells ml', \alues are means ± 1 SE. generation of long-life batteries. Short-term bivalve feeding re- sponses will be estimated more accurately //; sUu by monitoring their activity continuously and unintenaipted rather than relying on measurements at regular intervals or convenient points in time. It is not necessary, as with more traditional methods to measure feeding activity, to confine the bivalve in any kind of experiment chamber, which may facilitate measuring the change in particle concentration over tune but exposes the bivalve to unrealistic flow conditions. Harrington et al. (2002) have successfully used this method to compare feeding activity in mussels held near salmon cages to mussels held in adjacent reference sites. We have ob- served between 8 and 12 mussels simultaneously, an obvious ad- vantage for sampling rate and statistical power o\er methods that observe a single bivalve at a time. However, there exists a trade-off between the number of mussels that can be observed and the resolution of the siphon area for individuals obtained from the video tape. Filtration rate by mussels is a function of pumping rate, particle concentration and filtration efficiency, such that control over pumping rate is viewed as a major factor contributing to energy acquisition by bivalves. As any one of these factors changes, there may be an uncoupling between exhalant siphon area and filtration rate. In order for this method, or any other method that measures exhalant siphon area, to be used to estimate a rate of feeding the variation in relationships between exhalant siphon area and filtra- tion or pumping rate must be established in future studies. We are proposing that this method, using a camcorder in an underwater housing, a plastic frame for holding mussels and time lapse videography, is a practical and potentially useful tool to address many questions on how bivalves respond, in real time, to changes in a naturally dynamic environment. ACKNOWLEDGMENTS This research has been supported in part by funds from AquaNet. the Network of Centres of Excellence for Aquaculture. Financial !i: MacDonald and Nodwell support was also provided through a NSERC research grant held by for constructing the mussel posts and frame and for technical assistance B. A. MacDonald. The authors would like to thank Wayne Armstrong and Kelly Barrington for assistance in conducting the experiments. LITERATURE CITED Baldwin. 1. G. & K. J. M. Kramer. 1994. Biological Early Waining Sys- tems (BEWS). In: K. J. M. Kramer, editor. Biomonitoring of Coastal Waters and Estuaries. Boca Raton: CRC Press, pp. 1-28. BaiTingtom. K. A.. B. A. MacDonald & S. Robinson. 2002. Assessing the feeding behaviour of blue mussels (Mytilus ediili.s). living within an Atlantic salmon (Salmo salarl aquaculture site, with time-lapse videog- raphy. Aquaculture Association Of Canada Meeting. Charlottetown. PEI, September 18 to 22. 2002. Bayne, B. L. 1993. Feeding physiology of bivalves: time-dependence and compensation for changes in food availability. In: R. F. Dame, editor. Bivalve Filter Feeders in E.stuarine and Coastal Ecosystem Processes. Berlin: Springer-Verlag. pp. 1-24. Bayne. B. L. 1998. The physiology of suspension feeding bivalve mol- luscs: an introduction to the Plymouth "TROPHEE" workshop. J. Ex/i. Mar. Biol. Ecol. 219:1-19. Clausen, I. & H. U. Riisgard. 1996. Growth, filtration, and respiration in the mussel Mytihis editlis: no regulation of the filter-pump to nutri- tional needs. Mar. Ecol. Prog. Sen 141:37-45. Cranford. P. J. 2001. Evaluating the "reliability" of filtration rate measure- ments in bivalves. Mar. Ecol. Prog. Ser. 215:303-305. Dolmer. P. 2000a. Algal concentration profiles above mussel beds. ./. Sea Res. 43:1LVII9. Dolmer, P. 2000b. Feeding activity of mussels Mylilii.s edulis related to near-bed currents and phytoplankton biomass. J. Sea Res. 44:221-231. Faninie, P.. H. U. RiisgSrd & C. B. J0rgensen. 1986. On direct measure- ments of pumping rates in the mussel Myliliis edulis. Mar. Biol. 92: 323-327. Foster-Smith, R. L. 1975. The effect of concentration of the suspension on the filtration rates and pseudofecal production for Mytilus edulis L., Cerasloderma edule L. and Venerupis pulla.stra (Montagu). J. E.xp. Mar. Biol. Ecol. 17:1-22. Hawkins. A. J. S.. J. G. Fang. P. L. Pascoe, X. L. Zhang & M. Y. Zhu. 2001. Modelling short-term responsive adjustments in particle clear- ance rale among bivalve suspension-feeders: separate unimodal effects of seslon volume and composition in the scallop Chlmiiys farreri. J. E.xp. Mar. Biol. Ecol. 262:61-73. Jorgensen, C. B. 1960. Efficiency of particle retention and rate of water transport in undisturbed lamellibranchs. J. Cons. Cons. Int. E.xplor. Mer. 26:95-116. Jorgensen. C. B. 1990. Bivalve filter feeding: hydrodynamics, bioenerget- ics. physiology and ecology. Fredensborg. Denmark: Olsen and Olsen. Jorgensen. C. B. 1996. Bivalve filter feeding revisited. Mar. Ecol. Prog. Ser 142:287-302. Jorgensen, C. B., P. S. Laren. F. Mohlenberg & H. U. Riisgard. 1988. The mussel pump: properties and modelling. Mar. Ecol. Prog. Ser. 45:205- 216. Manuel. J. L. & U. Lobsiger. 1994. The MarineCanary'^': Using time-lapse observation of mussel behaviour to assess the marine environment. Bull. Aquacul. Assoc. Canada. 99:23-30. NeweU. C. R., D.J. Wildish & B. A. MacDonald. 2001. The effects of velocity and seston concentration on the exhalant siphon area, valve gape and filtration rate of the mussel Myriliis edulis. J. Exp. Mar. Biol. Ecol. 262:91-111. Riisgard. H. U. 1991 . Filtration rate and growth in the blue mussel. Mytilus edulis Linneaus. 1758: dependence on algal concentration. J. Shellfish Res. 10:29-35. Riisgard, H. U. 2001. On measurement of filtration rates in bivalves — the stony road to reliable data: review and interpretation. Mar. Ecol. Prog. Ser. 211:275-291. Riisgard, H. U. & A. Randlov. 1981. Energy budgets, growth and filtration rates in Mytilus edulis at different algal concentrations. Mar. Biol. 61:227-234. Widdows, J. 2001. Bivalve clearance rates: inaccurate measurements or inaccurate reviews and misrepresentation? Mar. Ecol. Prog. Ser. 221: 303-305. Winter, J. E. 1973. The filtration rate of Mytilus edulis and its dependence on algal concentration, measured by continuous automatic recording apparatus. Mar. Biol. 22:317-328. Jounmi of Shcllfisl, ficscanli. Vol. 22. No. I. 2I.V223. 2(J03. PARALYTIC SHELLFISH TOXINS IN PUGET SOUND, WASHINGTON STATE VERA L. TRAINER.'* BICH-THUY L. EBERHART.' JOHN C. WEKELL.' NICOLAUS G. ADAMS,' LINDA HANSON," FRANK COX," AND JUDY DOWELL" Mariiie Biotoxins Prngniin. Emiroiimcutid Consenaiion Division. Northwest Fisheries Science Center. National Marine Fisheries Service. National Oceanic and Atmospheric Administration. 2725 Montlake Boulevard East. Seattle. Washington 9H1 12 and ~\Vasliini>ti>n State Department of Health, Food Safety and Shellfish Programs. 7171 Cleanwater Lane. Olympia. Washington 98504 ABSTRACT The first illnesses and only deaths in Washington State resulting from paralytic shellfish poisoning were documented in the 1940s, resulting in the establishment of one of the longest monitonng programs for paralytic shelltlsh toxins in commercial and recreational shellfish in the United States. An analysis of the Washington Department of Health's monitoring data for the Puget Sound area has allowed us to examine temporal changes in shellfish toxin levels and geographical distribution of shellfish harvesting closures. The values of toxins in shellfish were normalized to control for variable levels of toxin accumulation in different shellfish species by dividing individual values by the yearly average for a given species. These normalized values increased significantly over the past five decades, indicating that the ob.served increase in paralytic shellfish toxin levels in Puget Sound shellfish was not caused by the shift in species monitored. A geospatial map of the first shellfish closures or paralytic shellfish-poisoning event in each Puget Sound basin suggests that over time, toxigenic .Mexainlriiim cells have been transported from northern to southern Puget Sound. Shallow sills that restrict the exchange of water between adjacent basins have hindered the transport of toxic dinofiagellates. especially because these cells generally do not prosper in mixing conditions that are characteristically found at sills. Large-scale events, such as the bloom that occurred in the Whidhey and Central basins in 1978. may have been induced by global climate changes or shifts, such as the Pacific Decadal Oscillation. Although greater numbers of closures have been observed over time in basins of Puget Sound, closures as a percentage of total samples analyzed have decreased or remained constant in all basins, indicating that the Washington Department of Health has established an effective monitoring program to protect public health while allowing for maximum harvest potential. KEY WORDS: paralytic shellfish poisoning, saxitoxin, Puget Sound INTRODUCTION Background Paralytic shellfish poisoning (PSP) is an acute illness in hu- mans caused by eating bivalve shellfish (e.g.. mussels and clams) that have ingested dinoflagellates that produce neurotoxic com- pounds. The dinofiagellate. Alexandriiim catenella (Whedon and Kofoid) Balech, previously described as belonging to the genus Conyaidax Whedon and Kofoid or Protogonyaulax Taylor, has been identified as the primary causative organism on the west coast of North America, but recent evidence indicates that at least five known species oi Alexandrium can produce PSP toxins (PSTs) in Northwest waters (Homer et al. 1997). These dinofiagellates occur either as single cells or as chains of cells. Their two flagella enable them to vertically migrate to the surface during the day and to depth at night, giving them advantages over nontlagellated phy- toplankton. Generally, dinofiagellates thrive in stratified water be- cause of their motility and ability to move to nutrient-rich areas within the water column. When conditions for growth become less favorable, A. catenella cells form resting cysts that settle to the sediments, where they await the return of fa\orable growth con- ditions (Anderson 1980). Historically, PSP has been known in the Pacific Northwest and Alaska for centuries. Records of PSP events date back as early as June 15, 1793 (Vancouver 1798), when a member of Captain George Vancouver's exploration team died after eating contami- nated mussels harvested in the uncharted coastline of what is now known as British Columbia. In 1799. 100 Russian hunters died after consuming toxic mussels near Sitka. Alaska (Halstead 1965). The first recorded outbreak of PSP on the eastern shore of Van- *Corresponding author. E-mail: Vera. L.Trainer(S'noaa. gov couver Island. Canada, in October 1957 caused serious illness in a number of people (Waldichuk 1958) and resulted in a mandatory monitoring program for PSTs in Washington State. The PSTs include saxitoxin and at least 12 structurally related chemical compounds (see. for example. Baden I9 in the data is caused by a disproportionate increase in sample size over time in certain basins relative to other basins. During recent decades, more reports of PSP illness, especially in south Puget 216 Trainer et al. TABLE 2. ShellHsh collected by the Washington State Department of Health (1957-1999). Mytiltis ediilis Saxidomus gigaiUeus Protothaca staminea Crassostrea gigas Other* Basin Total (%) C^f) (%» {%) (%) North 10.175 IS 19 11 31 21 Northwest 5.%! 12 22 38 13 15 Whidbey 3.696 55 29 7 1 8 Central 13.673 25 25 26 8 16 South 5.644 43 4 4 33 16 * Other species (not all shellfish) include: Ccuiccr iDUiiistcy. Chmuc sp., Clilamys nihida. ClinociirJiiim nultallii. Cnissadoma giganlca. Cnissasireu sikumea. Ensis americwms. Fusirriton oregoneiisis. Haliotis kamlSLluitkana. Mcicomii nasuhi. Macoimi sectu. Modiolus modiolus. M\a aienaria. Mvtihis californiaiuis. Mytihis galloprovincialis. Ostrea edulis, Ostiea hirida. Panopea abruplu. Purastichopus califomicus, Patinopcclen caurinus. Polinices lewisii. Tapes philippiiuiriini. Tn'siis niilhillii. Sound, have required an increase in PST testing. The different sampling intensity as well as the shift in shellfish species collected over time has necessitated data reduction for the purpose of trend analysis. Because we examined the data for trends in PST activity, only samples having quantifiable levels (S32 ixg STXeq/100 g) of PST by mouse bioassay were included. All the quantifiable PST data for San Juan Island shellfish are shown in Figure 2A. San Juan Island was chosen because one of the longest historical records in Puget Sound is available from this site. Data were simplified by showing only the highest annual level of PST (Fig. 2B). Averages per decade (Fig. 2C) of those maximum annual levels were cal- culated in all shellfish from the San Juan area from the 1950s to the 1990s. Finally, data were normali/ed to control for different rates of uptake and depuration of PSTs in all shellfish tested by dividing individual PST values by the average for that species. The maxi- mum normalized PST values were determined for each year then averaged for the decade (Fig. 2D). When the nomialized maxima per decade for the 1950s through 1970s were compared with the past two decades ( 1980s and 1990s), the more recent two decades were significantly higher (/-test. P < 0.001 ). The rise in PST values over the past several decades is clearly seen in Figures 2C and D. PST ill Basins of Puget Sound A series of environmental factors such as the presence of bounding sills, river input, and unique bathymetry were used to divide Puget Sound into distinct basins (Strickland 1983; Fig. 3). Sites that show typical PST le\els within a given basin were se- lected for this study upon recommendation by WDOH. Because central and south Hood Canal shellfish have remained essentially free of PSTs. this arm of water west of the Central basin was not included as part of this analysis. A summary of averages by decade of maximum PSTs in all defined basins in Puget Sound showed increasing magnitude of toxins in all shellfish monitored at all sites with the exception of Whidbey and Central basins (Fig. 3). In the North basin. Samish Bay had relatively low levels of PSTs during the past three decades, whereas San Juan Island and Georgia Strait had more intense toxic events with the average by decade of an- nual maximum levels increasing from the 1970s to the 1990s. In the Northwest basin we observed obvious increases in levels of PSTs in both Sequim and Discovery bays over several decades. In the Whidbey basin, PST levels remained relatively low, except for an anomalously high level of toxin (30,360 jxg STXeq/100 g) in 1978 at Holmes Harbor. Levels of this magnitude had never before (and have not yet again) been observed in Washington State. Rec- ord levels of shellfish toxin measured from Whidbey Island south to central Puget Sound in 1978 were responsible for the anomalous peaks seen in Holines Harbor and Agate Pass in the 1970s (Fig. 3). In the Central basin. Quartermaster and Kilisut harbors, as well as 7000 6000 5000 4000 3000 - 2000 1000 0 ii^jkiiiiiiiLliLii I ''58 1^63 1968 1973 1978 1983 1988 1993 1998 1958 1963 1968 1973 1978 1983 1988 1993 1998 Figure 2. PST levels (^g STXeq/100 g) in all shellflsh from San Juan Island, collected from Jan 1958 to Nov 1999. All data (A), maximum annual PST levels (B), and average per decade of annual maximum PST levels (C», and normalized average per decade of annual maxi- mum PSTs (U) are shown. Paralytic Shkllfish Toxins in Puget Sound 217 North K:Wm Wliidlx'x Basin ■ <;i'iir};ia Strait (.^) D Saiiiish Bay (4) . . ^ Sanjuaii Maud (? a % 500 ■ • 50s 60s 71)s «0s 90s tfl Northwest Basin ■ DKuiM'O lla< III □ Scquim Hay l2l 50s t)Ob 70s 80s W: 47 N 123 W 122 W ■ Iliilme', II^irlK>rl6l ^ 1 501) • SlVimCi.yc- I7l -a KMMI ' J 50(1 • ^ „. i . 1 ■ s(K 60s 70s SOs lOs Central Basin 2000 ■ \Bati- l'assi»i □ Kilisulllarlxin'll ^ 1500+ E3 yuartt-niLislvr HarlwnlO) S 1000 • • SOs 60s 70s SOs 90s South Basin ICarrlnlcl llli 3 Cast InU't |12| SOs 60s 70s mis i)Os Figure 3. A^ erages per decade of maxinium PSTs (pg STXeq/100 g) in each Puget Sound basin. The locations of representative sites in each basin are numbered. Agate Pass, showed clear increases in average of annual niaximinii levels over the past two decaiJes. In the South basin. PST levels have recently reached record highs. Carr Inlet had its first shellfish harvesting closures in 1988. although monitoring had been done at this site since 1957. Before 1988. PSTs had only occasionally been measured in the South basin but at levels below regulatory limit. Nearby Case Inlet had its first closure in 1991. Since the 1991 event, this area has experienced more frequent toxic events and higher levels of PSTs. reaching a maximum of 1.^.769 fxg STXeq/ 100 g in blue mussels in 2000. Frequency of PST Closures The frequency of PST closures over time in each Puget Sound basin is shown in Table 3. Although the number of samples col- lected over time has increased, closures as a percentage of total TABLE 3. Number of closures in Puget Sound basins, also as a percentage of total samples analyzed during each decade. Northwest North VVhidbey Central South Decade Closures % Closures % Closures 9( Closures % Closures % 1950s 32 25 1 2 0 0 0 0 0 0 1960s 195 45 2 1 0 0 0 0 ND* ND 1970s 227 27 260 20 165 39 109 18 (1 0 1980s 610 34 827 22 119 7 912 23 238 22 1990s 387 14 486 10 31 1 1088 12 998 22 *ND = No data 218 Trainer et al. samples analyzed in each basin were variable. However, in general a decrease in percentage of closures in each basin during the 1990s relative to previous decades was evident, except in the South basin, where 22% of the samples analyzed resulted in closures in both the 1980s and 1990s. Seasonal Duration of Closures The greatest number of closures during each decade occuired from July through No\ ember with 81'^ of all closures occurring during these months in the 1950s. 69% in the 1960s, 63% in the 1970s, 65% in the 1980, and 73% in the 1990s (Table 4). Spread of PSTs The historical record of PSP events causing illness and death in humans and initial shellfish closures in the different regions of Puget Sound is shown in Figure 4. The death of three people and illness of two others after their consumption of mussels and butter clams from the beach in Sekiu in 1942 was the first evidence of high levels of PSTs in Washington State. The death of three mem- bers of the Ucluelet Tribe after eating mussels containing PSTs on the west coast of Vancouver Island, British Columbia, Canada, was recorded three days prior to the mortalities in Sekiu (L. Han- son, pers. comm.), indicating that this event was probably wide- spread in the Pacific Northwest. From 1942 to 1957, Washington State monitoring was sporadic and was actually temporarily stopped in 1946 because of blanket closures that were in effect at this time (Lilja 1978). Monitoring for PSTs in Washington became formalized in 1957 after a large outbreak of PSTs occurred in British Columbia, Canada (Waldichuk 1958). During this year, the first shellfish closure occurred in Sequim Bay when a level of 162 jxg STXeq/100 g was measured in butter clams. The first shellfish closure in the San Juan Islands occurred in 1958 when a level of 122 |j.g STXeq/lOO g was measured in butter clams. In the early 1970s, when WDOH monitoring efforts increased, shellfish con- taining PSTs were found further east in Lummi Bay (Fig. 4) when 465 (ig STXeq/lOO g was measured in Pacific oyster in 1973. In 1978, anomalously high PST levels (up to 30,360 (jig STXeq/lOO g) caused the first shellfish closures in both Whidbey and Central Puget Sound basins. Over a period of several weeks, the contami- nation spread southward in Puget Sound to an area between Seattle and Tacoma in south-central Pucet Sound. In 1987. levels of PSTs 49.0 N 47.0 N 125,0 W 124.0 VV 22.0 \V First Shellfish Flarvesting Closures and PSP Event in Each Region Record ^'c.ir Rccn.)n Location ot first closure and/or PSP event Five cases of PSP in Sekiu. three deaths Sequim Bay/Discovery Bay San Juan Islands Lummi Bay Whidbey Basin/Central Basin. 9 cases of PSP Northern Hood Canal Carr Inlet, I case of PSP Case Inlet Totten and Eld Inlets Figure 4. First recorded PSP events and shellflsh harvesting closures in each Puget Sound basin. Locations of each event are numbered on the map of Puget Sound, in northern Hood Canal were measured above the closure limit for the first time since WDOH sampling began (234 (xg STXeq/lOO g in Pacific oyster). The first closures of shellfish harvesting in south Puget Sound in 1988 were due to PST levels up to 10,982 |xg STXeq/lOO g in Carr Inlet. One person was hospitalized after ingesting oysters from Minter Bay, Carr Inlet in September 1988 (F. Cox, pers. comm.). In 1991, the first incidence of shellfish 1 1942 NW 1 1957 NW 3 1958 N 4 1973 N 5 1978 C 6 1987 C 7 1988 S 8 1991 S 9 1997 sw TABLE 4. Number of monthly closures, also as a percentage of total closures during each decade. 1950s 1960s 1970s 1980s 1990s Month Closures % Closures % Closures % Closures % Closures % Januarv 1 ■^ 10 5 40 5 80 3 82 4 February 0 0 11 5 30 4 49 2 66 3 March 0 0 8 4 35 5 97 4 53 1 April 4 9 14 7 65 8 173 7 60 3 May 2 4 1 0 33 4 155 6 70 3 June 2 4 2 1 42 5 238 9 147 7 July 3 7 24 12 101 13 442 17 353 16 Ausiist 13 28 20 10 109 14 449 18 337 15 September 5 11 27 13 98 13 408 16 321 14 October 6 13 41 20 115 15 260 10 393 18 November 10 ■>■> 29 14 65 8 III 4 204 9 December 0 0 I.S 7 37 5 94 4 150 7 Paralytic Shellfish Toxins in Puget Sound 219 closures occurred in Case Inlet, with levels of 779 ixg STXeq/100 g in blue mussels. In the fall of 1997. PST levels up to 6799 (j.g STXeq/lOO g were measured in Eld and Totten inlets, causing the first shellfish closures in these small southv\'estern finger inlets of south Puget Sound. Pre\ious routine monitoring, necessitated by ihe presence of commercial shellfish operations at these sites, de- tected only low levels of PSTs that were below the regulatory limit of SO (xg STXeq/lOO g (Saunders et al. 1982. Determan 2000). For example, the first measurement of PST in Carr Inlet was in 1981 at a level of 51 |xg STXeq/lOO g in blue mussels. When the highest annual PST levels exceeded 80 ixg/lOO g e\en once at a particular monitoring site during a given decade, that site was shown to have a closure during that decade (Fig. 5). Although samples were tested in several areas throughout Puget Sound, in the 1950s and 1960s the only areas with shellfish clo- sures were in the Northwest and North basins. In the 1970s, the number of sampling sites increased substantially, and closures were seen in central Puget Sound. During the 1980s, the first closures were seen in the eastern inlets of the South basin; shellfish closures occurred throughout much of south Puget Sound in the 1990s. An increase in the number of monitoring sites sampled over the decades is evident. Data from the 1970s indicated the high number of closures in 1978 in the Whidbey basin, however by the 1990s, few closures were observed here. The actual numbers of samples tested for toxins and closures in each basin as a percent of the total closures in all of Puget Sound are shown in Table 5. The greatest number of closures occurred in the Northwest basin in the 1950s (977f of all closures) and 1960s (999J- of all closures), in the North basin in the 1970s (34% of all closures), in the Central basin in the 1980s (34% of all closures), and in the Central (36% of all 1950s 1960s 49.0 N 48.5 N - 48.0 N 47.5 N 47.0 N 123.5 W 123.0 W 122.5 W 122.0 W 1970s 1980s 1990s Figure 5. Closures because of PST in shellflsh at all Puget Sound monitoring sites for each decade. Symbols represent maximum values for each cittade sIhih n as open circles (below 80 (ig STXeq/lOO g) or solid circles (greater than or equal to 80 jig STXeq/lOO g). Data for the 19S0s include only 1957-1959. 220 Trainer et al. TABLE 5. Number of samples analyzed for PSP toxins in each basin and closures in each basin as a percentage to total closures in Pugct Sound. Northwest North Widbey Central South Total Total Closures as a % of Total Decade Number C* ) Number (^n Number ['^r) Number (1) Number C?-) Measurements Closures Measurements 1950s 130 97 53 3 1 0 13 0 4 (1 201 33 16 1960s 433 99 202 1 1 0 17 0 ND" ND 653 197 30 1970s 841 30 1302 34 423 Tl 607 14 24 0 3197 761 24 1980s 1793 23 3759 31 1704 4 3966 34 1080 9 12302 2706 22 1990s 2764 13 4859 16 1566 1 9070 36 4536 33 22795 2990 13 Highest level* (dale) 3074(9/17/901 5968 (7/27/99) 30360(9/28/78) 4822(7/11/90) 10982(10/22/98) * Highest level = (ig STXeq lOOg" * ND = no dala. . all in blue mussels. closures) anti South basins (339f of all closures) in the 1990s. Whereas the highest percentage of total closures occurred in north- ern Puget SouncJ in the 1950s, the greatest percentage has nwie recently occurreiJ in the central and south Puget Sound regions. Closures as a percent of total measurements made have decreased since the 1960s. DISCUSSION There is speculation that harmful algal bloom events are in- creasing in intensity, frequency, duration, and geographical loca- tion; however, the long-term monitoring data needed to support these ideas are often insufficient for trend analysis. Because of documented illnesses and deaths due to PSP beginning in the 1940s. Washington State has one of the longest monitoring histo- ries for PSTs in the United States with the State of Maine ha\ ing the next oldest monitoring program, established in 1958 (Shum- way et al. 1988). Data collected in Washington State are compi- lations of PST measurements at shellfish harvest sites designated by the WDOH to have the greatest risk for human exposure to PSP. Although the location and frequency of monitoring at these sites have changed substantially over the years, we were able to use tlic data to establish trends for Puget Sound shellfish closures due to PSTs. Spread of PSTs into Central and Southern Panel Sound Until the last decade, the only Puget Sound basins with no measured PSTs were southern Hood Canal and the southernmost inlets of Puget Sound (Rensel 1993, Determan 2000). Since the 1980s, the frequency of PST detection has increased in southern basins of Puget Sound, an area that contains the region's most productive shellfish-growing beaches. Shallov\ sills that restrict the exchange of water between adjacent basins (Strickland 1983; see also Fig. I ) have likely hindered the movement of toxic dinotlagel- lates, especially because these cells generally do not prosper in mixing conditions that are characteristically found at sills. AUw- aiidriiim cells thrive in stratified environments, presumably due to the supply of nutrients, trace minerals, and natural humic sub- stances that may serve as growth stimulants at the density interface (see. for example, Anderson 1997). Therefore, sills, which are found at several sites in Puget Sound (Fig. I ), have likely delayed the spread of Alexandriiim cells to the South basin. A geospatial map showing the first accounts of shellfish clo- sures or PSP in each region of Puget Sound (Fig. 4) suggests that over time, toxigenic Alexamiriwn cells, cysts or both have made a slow progression from northern Puget Sound to the south. The numbers of cysts and cells likely have increased over the decades in the areas near sills, eventually reaching a critical mass that enabled their survival during transport over these natural barriers. Conditions for Alexandriuin cell growth are ideal in south Puget Sound because of the many shallow, poorly flushed bays and inlets where thermally-caused stratification occurs during summer months, allowing ideal grow th conditions for dinollagellate cells to persist for weeks (Rensel 1993). However, the initial population of Alexwulnum cells or cysts probably entered south Puget Sound only in recent years. The first detectable PSTs in south Puget Sound were noted in Carr Inlet (57 |j.g STXeq/100 g in blue mussels) in 1981. Some anecdotal evidence from the epidemio- logic record also supports the gradual spread of toxigenic Alexan- driuin cells into south Puget Sound. Of the nine people who be- came ill after eating mussels from Carr Inlet in the suinmer 2000, one woman who was sick during that event pre\ iously had eaten shellfish from the same beach in Carr Inlet for more than 50 y with no PSP symptoms (Cox 2000). A possible pathway of cells into Puget Sound was through the Strait of Juan de Fuca. into the Northwest basin and western San Juan Islands, then past the sill to the south of the San Juan Islands and Rosario Strait into Bellingham Bay. From the Northwest ba- sin, cells may have been transported southward to the Whidbey basin, past the sill at Admiralty Inlet to central Puget Sound, and also past the sill at the entrance to Hood Canal to northern Hood Canal. Finally, from the Central basin, cells spread into south Puget Sound past the sills at Tacoma Narrows and the Nisqually (Fig. 1 ). The hydrographic separation of the ea.stem and western inlets of south Puget Sound (Ebbesmeyer et al. 1998) can explain the temporal lag in the first documented shellfish harvesting clo- sures in Case Inlet in 1991 compared with Totten and Eld inlets in 1997 (Fig. 4). The Initial Population of .\lexandrium in Washington State The first recorded PSP event in Washington State, at Sekiu in 1942 (Fig. 4), coincided with three deaths on the western coast of Vancouver Island, Canada. The next PSP episode in British Co- lumbia was in the inland waters of the Strait of Georgia in 1961 when 61 people fell ill (Taylor & Homer 1994). It is possible that the source of the "seed"" population of toxigenic A. catenella cells in Washington State originated from the inland or coastal waters of Canada. Indeed, the first documented PSP event in all of North America dates back to 1793, when four members of Captain PARAL^■TIC' Shellfish Toxins in Puget Sound 221 George Vaneoiiver's erew beeame sick and one died of PSP during exploration of present day British Columbia (Quayle 1969). Unlike its neighbor to the north. Washington State had no recorded ill- nesses or deaths of humans with descriptions of PSP symptoms before 1942. Alexainlriiim calenella is the chief source of PSP off the west coast of British Columbia and eastern Vancouver Island (Taylor & Harrison. 2002) and evidence suggests that the earliest recorded PSP outbreaks were at least partially because of blooms of this dinotlagellate species (Quayle 1969). Because prevailing winds and currents are from the north during the summer months (Mickey 1989), when growth conditions for Alexandrium are op- limal. and because the inlet to Puget Sound is at the north end of this tjord. a north to south transport would support the natural dispersal of algal cells from Canada. The routes of toxigenic cell dispersal in the Pacific Northwest could be defined in the future by a study of population genetics of A. catenella isolates from both British Columbia and Washington State. Incriasid I'ST Levels Because of increases in aquaculture activity as well as the measurement of PSTs in new areas of Puget Sound, the number of samples taken annually for PST testing has increased steadily from 1988 to the present time (Table 5). However, increased sampling frequency has not resulted in a higher percentage of closures dur- ing the latter decades (Table 3). The majority of closures during each decade was in July through November; a shift to more clo- sures in earlier or later months has not been observed in recent years. In addition, no correlation between the highest toxin levels and total number of samples collected annually was observed (Table 5), suggesting that apparent increases in PST intensity are not due to increased sampling. Because mussels can accumulate higher levels of PSTs, the shift of reliance on oyster and clam samples in the inonitoring program in the 1960s to mussel samples in the I99()s (Table 1) may account for some of the observed increase in toxin intensity. However, the normalized maximum \alues of PSTs in all shellfish have also increased over the past fi\e decades (Fig. 2D), showing a statistically significant increase during the 1980s and 1990s compared with the three previous decades, suppoiling the fact that the increase in PST levels in Puget Sound shellfish was not due to the change in shellfish spe- cies monitored over the years, PST Inlensily Versus Human Population Growth Over the last four decades, modern human development has extensively altered the shoreline habitats of Puget Sound (see the Department of Ecology, Water Quality Monitoring web page, http://www.ecy.wa.gov/programs/eap/mar_wat.html ). A compari- son of maximum PST averages per decade and population esti- mates (of all counties bordering Puget Sound) over the last 40 y shows a high level of correlation (r = 0.987; Fig. 6). Although statistical correlation does not establish a causal link, it does sug- gest that some factor(s) associated with population growth may influence the magnitude of PSTs at any given site. Increased nu- trients to our coastal environment may provide more favorable growth conditions for Alexandrium cells that populate a given basin. It has been speculated that the lack of nitiogen in surface and subsurface waters of Puget Sound has been a major factor limiting the further spread of PSTs into bays and inlets otherwise suitable for A. calenella (Rensel 1993). Land clearing, logging, aerial forest fertilizing by timber companies, direct sewage out- 5(M)() 4(M)() 3000 2000 , 1(M)0 1,0 1.5 2,0 3,0 4,0 Population (Millions) Figure 6. Maximum PST average per decade versus population esti- mates. Census data for counties Ijordering Puget Sound were obtained from the rollowing site: http://H«\v, census.gov/population/cencounts/ «aiy009().txt falls, agricultural runoff, and e\'en aquaculture operations have increased the amounts of nutrients, including nitrogen, that are supplied to the coastal ecosystems of Puget Sound (Howarth 2001). Inlets and fjords with low flushing rates that adjoin urban- ized shorelines have the greatest sensitivity to nutrient addition (Mackay & Hairison 1997). The increased levels of PSTs in the .semi-enclosed bays of south Puget Sound in recent years may, at least partially, be explained by increased eutrophication and gen- erally poor circulation. Indeed, south Puget Sound is described by the Washington State Department of Ecology as one of the areas most susceptible to impacts of eutrophication (Cusimano 2002). Because the depth of south Puget Sound inlets is much shallower and flushing time is slower, nutrient inputs to surface waters pro- vide ideal growth conditions for A. catenella. Natural Events Although the intensity of PSTs in shellfish has increased with time (Fig. 2), toxic events do not occur in each basin in every year. For example, shellfish closures have occurred in northern Hood Canal only in 1991. 1996. and 1997-1999. What sets those years apart from all other years'? Environmental conditions such as water temperature, mixed layer depth, sunlight, and nutrients all work together to increase the chance of a toxic event in a particular basin and in any given year (Rensel 1993. Nishitani et al. 1988). In addition to microscale, basin-specific environmental factors that result in a periodicity of Alexandrium blooms, large-scale occur- rences, such as the bloom that occurred in the Whidbey and Cen- tral basins in 1978, may have been motivated by global climatic events or shifts. In 1977. a large shift to a positive Pacific Decadal Oscillation occurred, with a resulting ecological response to the environmental changes. This period was marked by an enhance- ment of overall productivity that appeared to be closely related to changes in upper ocean mixed-layer depths and temperatures (Mantua et al. 1997). Indeed, an exceptionally deep surface layer of warm water was believed to have exacerbated the 1978 Whid- bey basin bloom (Erickson & Nishitani 1985), Toxin levels of that magnitude have not been measured since that year in Whidbey basin, giving credence to the possibility that some unique, large- scale environmental factors influenced the occurrence of this event. The linkage of harmful algal bloom magnitude and fre- quency to climatic regime shifts has been suggested in recent 222 Trainer et al. studies (Epstein et al. 1998, Hayes et al. 2001). The specific co- variance of levels of PSP toxins in shellfish with strong El Nino/ Southern Oscillation events (Erickson & Nishitani 1985) and other environmental parameters such as the condition of oysters in Wil- lapa Bay (Ebbesmeyer et al. 1995) has been suggested. Effective Monitoring Although greater numbers of closures have been observed over time in many of the basins of Puget Sound, the percentage of closures relative to the total sites monitored in a given basin has decreased in all but south Puget Sound (Table 3). Although PSP toxins pose a serious threat to commercial and recreational shell- fishing operations, the large number of sites monitored by WDOH allows the agency to pinpoint areas within a basin that are safe for harvest. This rigorous monitoring has resulted in a greater propor- tion of open than closed sites for shellfishing in the Puget Sound region where the risk for PSP is extreme. Increased HAB events and interest in commercial shellfish operations in all regions of Puget Sound and wide-scale, year-round recreational harvest op- poilunities will likely result in a mandate for the WDOH to sustain its rigorous sampling efforts. In the future, improved monitoring methods (e.g.. molecular probes for cells and rapid analytical as- says for toxins) will be essential for cost-effective and timely management of the fishery in Puget Sound. CONCLUSIONS The following conclusions can be obtained from our study. 1 ) There has been a significant increase in the magnitude of PSTs in Puget Sound shellfish with time. 2) The geographical scope of shellfish closures caused by high levels of PSTs in Puget Sound has increased over the past four decades. The first recorded shell- fish closures in the Northwest basin in the 1950s, the Central basin in the 1970s, and the South basin in the 1980s are likely due to the spread of A. calenella cysts and/or cells from north to south. 3) Shellfish closures in south Puget Sound may have been delayed until recent years by the physical blockage of cell movement by sills to the north. Hydrographic blockage may also explain the delayed appearance of PSTs in the southwestern finger inlets of south Puget Sound. 4) Increased shellfish closures caused by PSTs over the past few decades are not just the result of greater numbers of samples collected over time. 5 ) Global climate changes, such as the Pacific Decadal Oscillation and increased eutrophication in nearshore areas, are possible explanations for the increased mag- nitude of PSTs in shellfish today. ACKNOWLEDGMENTS Thanks to the numerous volunteers who have collected shell- fish samples for WDOH over the years. The authors thank Rita Homer and Tim Determan for their constructive comments on an earlier version of this manuscript. Funding for database construc- tion and analysis was provided by the National Ocean Service, NOAA, through the Environmental Services Data and Information (ESDIM) program, project 0I-4I4F, "Access to Pacific Region Harmful Algal Bloom (PACHAB) Data." The authors thank Michelle Tomlinson for assistance with the database. LITERATURE CITED Anderson, D. M. 1997. Bloom dynamics of toxic Alexandiium species in the northea.stern U.S. Limnoi Oceanogr. 42:1009-1022. Anderson, D. M. 1980. Effects of temperature conditioning on develop- ment and germination of Gonyuulax tamarensis (Dinophyceae) hy- pozygotes. J. Phycol. 16:166-172. Associauon of Official Analytical Chemists. 1990. Paralytic shellfish poi- son biological method. In: W. Horowitz, editor. Official methods of analysis. Association of Official .^^alytical Chemists. Inc., Washing- ton. DC. pp. 881-882 Baden, D. G. 1983. Marine food-borne dinotlagellate toxins. In G. H. Bourne & J. F. Danielli. editors. International Review of Cytology. Academic Press, New York, Vol. 82. pp. 99-150. Bricelj. V. M. & S. E. Shumway. 1998. Paralytic shellfish toxins in bivalve molluscs: occurrence, transfer kinetics, and biotransformation. Reviews Fish. Sci. 6:315-383. Burns, R. 1985. The shape and form of Puget Sound. Washington Sea Grant Program. Seattle: UW Press, 100 pp. Cox, F. 2000. Preliminary report of paralytic shellfish poisoning outbreaks. Slate of Washington Department of Health. Office of Food Safety and Shellfish Programs. Sept. 20. 2000. Cusimano. R. F. 2002. South Pugel Sound Model Nutrient Study (SPASM). Washington State Department of Ecology, http:// www.ecy.wa.gov/programs/eap/spasm/spasm_descrip.html. Determan, T. 2000. Temporal and spatial distribution of paralytic shclHish poisoning (PSP) in Puget Sound 1999: A report for the Puget Sound ambient monitoring program. Olympia, WA: Office of Shellfish Pro- grams, Washington State Department of Health, 10 pp. Ebbesmeyer, C. C, R. J. Stewart & S. Anderson. 1998. Circulation in southern Puget Sound finger inlets. Olympia, WA: Proc. 1998 Puget Sound Research Conf., Puget Sound Water Quality Action Team. Ebbesmeyer. C. C, R. Chiang. A. Copping, G. M. Erickson, R. A. Homer. W. J. Ingraham, Jr. & L. Nishitani. 1995. Decadal covariations of Sequim Bay paralytic shellfish poisoning (PSP) with selected Pacific Northwest environmental parameters. Seatde: Puget Sound Research '95, Washington Sea Grant, pp. 415-421. Epstein, P., B. Sherman, E. Spanger-Siegfried, A. Langston. S. Prasad & B. Mckav. 1998. Marine ecosystems: emerging diseases as indicators of change. Boston: Harvard Medical School, 85 pp. Erickson, G. & L. Nishitani. 1985. The possible relationship of El Nino/ southern oscillation events to interannual variation in Gonyaulax popu- lations as shown by records of shellfish toxicity. In W. S. Wooster & D. L. Fluhany. editors. SeaUle: El Nino North, Washington Sea Grant Program, pp. 283-289. Halstead. B. W. 1965. Poisonous and Venomous Marine Animals of the World. Volume One - Invertebrates. U.S. Washington, DC: Govern- ment Printing Office, 663 pp. Hayes. M. L., J. Bonaventura, T. P. Mitchell, J. M. Prospero, E. A. Shinn. F. Van Dolah & R. T. Barber. 2001. How are climate and marine biological outbreaks functionally linked? Hydrobiologia 460:213-220. Hickey. B. M. 1989. Patterns and processes of circulation over the Wash- ington continental shelf and slope. In: M. R. Landry & B. M. Hickey, editors. Coastal Oceanography of Washington and Oregon. New York: Elsevier, pp. 41-1 15. Horner, R. A.. D. L. Garrison & F. G. Plumley. 1997. Harmful algal blooms and red tide problems on the U.S. west coast. Linmal. Ocean- ogr. 42:1076-1088. Howarth, R. B. 2001. Intertemporal social choice and climate stabilization. Im. J. Environ. Polliii. 15:386-105. Lilja, J. 1978. Shellfish Control Program, Food and Housing Section. Health Services Division. Department of Social and Health Services, 15 pp. Mackay. D. L. & P. J. Harrison. 1997. Nitrogenous nitrogen sources and sinks in the Juan de Fuca Slrail'Strait of Georgi;i/Puget Sound estuarine system: assessing the potential for eutrophication, Estiiar. Coast. Shell- fish Sci. 44:1-21. Paralytic Shllli isn Toxins in Puget Sound 223 Manlua. N. J.. S. R. Hare. Y. Zhang. J. M. Wallace & R. C. Francis. 1997. A Pacific interdecadal climate oscillalion with impacts on salmon pro- duction. Bull. Am. Meleorol. Soc. 78:1 069- lOSO. Nishilani. L. 1990. Suggestions for the Washington PSP monitoring pro- gram and PSP research. Olympia, WA: Prepared lor DOH Office of Shellfish Programs, Olympia. 12 pp. Nishitani. L.. G. M. Erickson & K. Chew. 1988. PSP research: implications for Puget Sound. In: Proceedings of the First International Meeting on Puget Sound Research (vol. 2), Puget Sound Water Quality Action Team. Seattle, WA. pp. 392-399. Nishitani, L, & K. K. Chew. 1988. PSP toxins in the Pacific coast states: monitoring programs and effects on bi\al\c industries. ./. Sl>ellfi.sh Res. 7:653-669. Quayle. D. B. 1969. Paralytic shellfish poisoning in British CoUimhia, Bull. Fish. Res. Bd. Can. 168:1-68. Rensel, J. 1993. Factors controlling paralytic shellfish poisoning (PSP) in Puget Sound, Washington. J. Shellfish Res. 12:371-376. Saunders. S. T., T. Sample & R. Matsuda. 1982. Paralytic shellfish poi- soning: its history, processes and impacts as applicable to Puget Sound. Seattle: Municipality of Metropolitan Seattle, Water Pollution Control Department. Shumway. S. E.. S. Sherman-Caswell & J. W. Hurst. 1988. Paralytic shell- fish poisoning in Maine: Monitoring a Monster. / Shellfish Res 7:643- 652. Strickland. R. M. 1983. The fertile fjord. Seattle: University of Washington Press, 145 pp. Taylor. F. J. R. & P. J. Harrison. 2002. Harmful marine algal blooms in western Canada. In F. J. R. Taylor & V. L. Trainer, editors. Harmful algal blooms in the PICES countries of the North Pacific. Sidney: North Pacific Marine Science Organization, pp. 77-88. Taylor, F, J. R. & R. Horner. 1994. Red tides and other problems with harmful algal blooms in Pacific Northwest coastal waters. In: R. C. H. Wilson, R. J. Beamish, F. Aitkens & J. Bell, editors. Review of the Marine Environment and Biota of Strait of Georgia, Puget Sound and Juan de Fuca Strait. Can. Teeh. Rep. Fish. .Aqiiat. Sci.. No. 1948. pp. 175-186. Vancouver, G. 1 798. A voyage of discovery to the North Pacific Ocean and round the world. Seaman poisoned by mussels. In G. G. Robinson & J. Robinson, editors. Vol. 2, Fourth book, chapter 2. Patemo.ster-Row and J. Edwards. London, pp. 260-287. Waldichuk. M. 1958. Shellfish to.xicity and the weather in the Strait of Georgia during 1957. Fish. Res. Bd. Canada. Prog. Rep. Pacific Coast Stations 112:10-14. Wekell. J. C. D. L. Gauglit/. H. J. Barnett. C. L. Hatfield. D. Simons & D. Ayres. 1994. Occurrences of domoic acid in Washington State razor clams (Siliqua patiila) during 1991-1993. Nat. Toxins 2:197-205. JiHiniul ofShclllhh Ri'scanh. Vol. 22. No. I. 225-233. 2(103. FEEDING SOUTHERN ROCK LOBSTER, JASVS EDWARDSII HUTTON, 1875. PHYLLOSOMATA IN CULTURE: RECENT PROGRESS WITH LIPID-ENRICHED A^reMM MATTHEW M. NELSON,'* BRADLEY J. CREAR,-t PETER D. NICHOLS,' AND DAYID A. RITZ' ^Department of Zoology, University of Tasiuania. GPO Box 252-05, Hobart, TAS 7001. An.stralia: ~Marine Re.searcli Laboratories, Tasmanian Aijiiaciibure and Fisheries Institute, University of Ta.smania, Taroona, TAS 7053. Australia: and 'CSIRO Marine Research, GPO Box 1538, Hobart, TAS 7001, Australia ABSTRACT Jasus fchvanlsii phylloMnna larvae were successfully grown in static culture with antihiotics Ironi newly hatched to stage V with high survival. Feeding phyllosomata on Artemki salitia Linnaeus, 1758. enriched with ( 1 ) a triacylglycerol (TAG)-rich Al DHA Selco-Chaeroceros inuelieri Lemmermann. 1898. nutrient source or (2) a formulated ethyl ester (EE)-rich nutrient source was compared with the more novel approach of using a formulated mussel powder-polar lipid diet attached to mesh. Individuals showed an increase to stage V in dry mass (0.1-1.5 mg) and total length (2.1-6.1 mm). Survival o( Anemia-'iti phyllosomata was high (92-98'5'f from stages II-III; 497? mean total survival). Animals fed the mussel powder-polar lipid diet had low molt success, although the presence of faecal trails confirmed they were consuming the diet. Total lipid remained generally constant in .4(Vfm(V;-fed phyllo- somata from newly hatched to stage V ( 155 mg g ' dry mass): this was notably higher than observed for previous feeding trials. The major lipid class in all phyllosomata samples was polar lipid, followed by sterol, with TAG as a minor component only, and EE not detected. The main fatty acids were 18;l(n-9)c. 18:2(n-6), 16:0. eicosapentaenoic acid [20:5(n-3l]. 18:0. 18:l(n-7lc. arachidonic acid (20:4(n-6)]. and docosahe.xaenoic acid |DHA; 22:6(n-3)]. Levels of the essential polyunsaturated fatty acids (PUPA), namely, arachi- donic acid, eicosapentaenoic acid and. in particular. DHA. decreased, on both a relative and absolute basis, from newly hatched to stage V. although phyllosomata fed the EE-rich enriched Artemia diet showed higher essential PUFA content together with oil content. This experiment further \alidates that lipids and fatty acids are important nutritional component in rock lobster larvae and that feeding phyllosomata with lipid-enriched Artemia maintains excellent growth and survival in early stages. Strategies will be needed, however, to either overcome the issue of low DHA. in particular, delivered by Arieinia (because of retroconversion), or to supply DHA by alternate means at later stages. KEY WORDS: Arleiuiu. enrichment, fatty acids, Ja.su.s I'dwanlsii, lipids, lobster, phyllosoma INTRODUCTION Rock lobster in Australasia has recently attracted the interest of a number of research institutions for its potential as a valuable aquaculture species. The fishery for southern rock lobster, Jasus edwardsii Hutton, 1875, boasts a value of over A$2()0 million in Australia (Punt & Kennedy 1997) and NZ$100 million in New Zealand (Breen & Kendrick 1997). As wild fishing pressure esca- lates (Booth & Phillips 1994). future exploitation of the rock lob- ster marketplace will logically be realized through aquaculture (Phlegeret al. 2001). As an aquaculture species, rock lobster possesses the allure of potentially high financial reward. Equally great is the challenge for research scientists because the larval phase, including metamor- phosis from phyllosoma to puerulus, is extensive (Phillips & Sas- try 1980. McWilliam & Phillips 1997), currently requiring close to a year in culture (Tong et al. 2000). To conquer this challenge, several vital aspects of culture of rock lobster phyllosomata can be identified as follows: (1) exploration of feeding capabilities of phyllosomata (Johnston & Ritar 2001, Nelson et al. 2002a) to determine appropriate format of feed presentation; (2) determina- tion of nutritional requirements to focus further the feed format; (3) a suitable aquarium design (Kittaka & Booth 2000. Ritar 2001 ) to optimize exposure of animals to the food source while mini- mizing microbial loading (Igarashi et al. 1990, Diggles et al. 2000). This study examines the second aspect (noted above), nutrition, and in particular the requirements for lipids. To focus this aspect. *Corresponding author. E-mail: mmnelsonC^utas. edu.au tCurrent address: Geraldton Fishermen's Co-operative. P.O. Box ' aldton. WA 6531. Australia. features of lipid nutrition under examination include: (1 ) total lipid content, the mg g ' of the lipid provided in the diet and that incorporated into larvae; (2) the lipid classes, examination of the delivery, and incorporation of types of lipids, such as triacylglyc- erol (TAG), polar lipid (PL) and ethyl ester (EE); and (3) the profile of fatty acids (FA), which are components of lipid classes. Building on the studies of lipids and FA in wild phyllosomata (Phlegeret al. 2001) and potential prey items (Nichols et al. 2001 ). we have examined enrichment of Arlcniici with essential polyun- saturated fatty acids (PUFA) (Phleger et al. 2001, Nelson et al. 2002b, Smith et al. 2002) and feeding of these TAG-enriched Artemia to phyllosomata (Nelson et al. 2003). The evidence amassed to date from these studies indicates that wild phylloso- mata largely obtain, and therefore may require, lipid in a PL form rather than in a TAG form. However, Artemia store their lipid enrichment as TAG (McEvoy et al. 1996, Sorgeloos et al. 1998, Harel et al. 1999). With this in mind and because phyllosomata do consume static food items (e.g., mussel pieces) (Kittaka 1997b, Matsuda & Yamakawa 2000, Nelson et al. 2002a). the present study was performed to provide phyllosomata a diet presented at a feed station (i.e., formulated diet attached to aquaria), a format cunently receiving attention (Cox & Johnston 2003), A compari- son was made for feed-station fed larvae to animals fed Anemia, enriched with either a TAG-rich product or with a novel docosa- hexaenoic acid (DHA)-rich EE product, by examining the effects on J. edwardsii phyllosomata survival, growth and lipid composi- tion. METHODS Artemia Enricliiiieiit 3, Ger- Decapsulated Anemia cysts (INVE, Great Salt Lake Prime Gold) were hatched at 28 ± IC in .50-L white fiberirlass cones in 225 226 Nelson et al. 0.2-|jim filtered brackish water (27 ± 1 g leg"'), with vigorous aeration and a 150 W light suspended 0.5 m above the water. After 24 h, Arteinia nauplii were removed from the hatching cones. rinsed in freshwater for 2 min and transfened into 1000-L tanks of filtered seawater (0.2 (xm. 34 ± 1 g kg"', 27 ± \°C). Anemia were fed twice daily with a rice pollard-soy flour-wheat flour brine shrimp diet (Eyre Peninsula Aquafeeds. South Australia) at a rate to maintain a Secchi depth of 25-30 cm. The environmental pa- rameters remained stable for the duration of the on-growing pe- riod; salinity (35.7 ± 0.2 g kg"' ), pH (8.3 ± 0.0), dissolved oxygen (7-7.2 mg L"'). and temperature (26.9 ± O.TC). After 5 days, 80.000 /4rte/)?/n with a total length of 1.5 ± 0.2 mm were removed from the on-growing container, rinsed in freshwater for 2 min and transferred to the 50-L white fiberglass cones containing 10 L of filtered seawater to achieve a density of 4 niL"'. Anemia were enriched for 24 h with 0.6 g L"' of three nutrient sources (i.e.. Aireniia enrichment diets): (DAI DHA Selco (INVE Group, Belgium). (2) The microalga Chaetocems nuielleri Lemmermann 1898. (3) Ethyl ester-mussel: a mixture of New Zealand Greenshell mussel (Pema canaliculus Gmelin. 1791 ) powder (NIWA Research. Auckland. New Zealand)-DHA (66'7f) EE oil (CSIRO Marine Research. Hobart. Australia)-AA (39%) TAG marine oil (Sun-TGA40S, Suntory Limited, Osaka, Japan )-Greenshell mussel polar lipid (NIWA Research, Auckland, New Zealand) (56:30:10:4 by mass). C. muelleri were cultured and the cell density was measured daily as described in Wilkinson (2000). The nonalgal enrichment diets were prepared daily by homogenizing ingredients suspended in seawater. Experimental Aquaria Three-hundred phyllosomata aquarium"' were grown from newly-hatched to stage V in 3-L plastic static aquaria on three diet treatments; each treatment was conducted in triplicate. The diet treatments consisted of: (1) Anemia enriched with Al DHA Selco and Anemia en- riched with C. muelleri (1:2 v/v). (2) Anemia enriched with the Ethyl ester-mussel nutrient source (as described above). (3) Mussel powder-polar lipid feed station diet [Greenshell mussel powder-Greenshell mussel polar lipid-lyprinol (from Greenshell mussel) (NIWA Research, Auckland, New Zealand)-sodium alginate (81:10:5:4 by mass)] af- fixed to 8 X 17 cm meshes (bird netting) with 10% CaCK solution. The water in aquaria was changed daily. After recording any molts/mortalities, the contents of each aquarium were poured through a l.000-|xm screen, retaining the phyllosomata while the uneaten feed and debris went to waste. The aquaria were cleaned, refilled with seawater, and larvae were washed back in. Phylloso- mata were provided with new diets once daily in the afternoon. Anemia were fed to phyllosomata at a rate of 3 Anemia niL"'. Oxytetracycline was added to the water at 20 mg L"' daily. After each molt, all animals were counted and 10 phyllosomata aquarium"' were measured for total length, carapace length and carapace width utilizing a dissecting microscope, digital camera and Scion Image Beta 4.0.2 software (Scion Corporation, Freder- ick, MD). Lipid Extraction Anemia and phyllosomata samples were filtered through 4.7- cm Whatman glass filters (GF/F) and rinsed with 0.5 M ammo- nium formate. Sample numbers of phyllosoma taken for lipid analyses were as follows: 400 newly hatched (sampled at start before distribution of larvae to aquaria); from each aquarium 50 stage II. 35 stage III, 25 stage IV, and 15 stage V; all midstage. Samples were lyophilized to determine dry mass and quantitatively extracted overnight using a modified Bligh and Dyer (1959) one- phase methanol:chloroforni:water extraction (2:1:0.8 v/v/v). The phases were separated by the addition of chloroform: water (final solvent ratio, 1:1:0.9 v/v/v methanol:chloroform:water). The total solvent extract was concentrated using rotary evaporation at 40°C. Lipid Classes An aliquot of the total solvent extract was analyzed using an latroscan MK V THIO thin-layer chromatography-flame- ioni/ation detector (TLC-FID) analyzer (Tokyo. Japan) to quantify indi\idual lipid classes (Volknian & Nichols 1991 ). Samples were applied in duplicate to silica gel SIII chromarods (5-|xm particle size) using 1-p.L micropipettes. Chromarods were developed in a glass tank lined with pre-extracted filter paper. The primary sol- vent system used for the lipid separation was hexane:diethyl ether: acetic acid (60:17:0.1), a mobile phase resolving nonpolar com- pounds such as wax ester (WE). TAG, free fatty acids (FFA) and sterols (ST). A second nonpolar solvent system of hexane:diethyl ether (96:4) was also used to resolve hydrocarbons, WE, TAG. and diacylglyceryl ether (DAGE). After development, the chromarods were oven dried and analyzed immediately to minimize absorption of atmospheric contaminants. The FID was calibrated for each compound class (phosphatidylcholine, cholesterol, cholesteryl ole- ate, oleic acid, squalene, TAG [derived from fish oil[, WE [derived from orange roughy oil], and DAGE [derived from shark liver oil]; 0.1-10 |jig range). Peaks were quantified on an IBM-compatible computer using DAPA Scientific software (Kalamunda, Western Australia). TLC-FID results are generally reproducible to ±5-10% of individual class abundances (Volkman & Nichols 1991). Fatty Acids An aliquot of the total lipid was /)i:7«,s-methylated to produce fatty acid methyl esters (FAME) using methanol:chloroform:conc. hydrochloric acid (10:1:1, 80°C, 2 h). FAME were extracted into hexane:chloroform (4:1, 3 x 1.5 ml) and treated with /V.O-bis- (trimethylsilyl)-trifluoroacetamide (BSTFA 50 p.L. 70'-C, over- night) to convert ST and alcohols to their corresponding TMSi ethers. Gas chromatographic (GO analyses were performed with a Hewlett Packard 5890A GC (Avondale, PA) equipped with an HP-5 cross-linked methyl silicone fused silica capillary column (50 m X 0.32 mm i.d.). an FID, a split/splilless injector, and an HP 7673A auto sampler. Helium was the cartier gas. After addition of methyl nonodecanoate and methyl tricosanoate internal injection standards, samples were injected in splitless mode at an oven temperature of 50''C. After 1 min, the oven temperature was raised to 150°C at 3()X min ', then to 250'C at 2°C min"', and finally to 300°C at 5°C min '. Peaks were quantified with Waters Mil- lennium software (Milford, MA). Individual components were identified using mass spectral data and by comparing retention time data with those obtained for authentic and laboratory stan- dards. GC results are subject to an error of ±5% of individual Feeding Soui-hbrn Rock Lobster in Culture 227 cnmpiment area. GC-mass speL-trometric (GC-MS) analyses were pertoniied on a Fiiinijjan Thermoquest GCQ GC-mass spectrom- eter (Austin. TX) fitted with an on-column injector. The GC was fitted with a capillar)' column siinilar to that described above. RESULTS Miiri>liiiiiiilncs The increase in total length and mass in phyllcsomata was similar between the Iwo Artemia diet treatments, with a good fit of exponential trend lines observed (R' > 0.999; Fig. 1 ). Phylloso- mata fed the A 1 DHA Selco-C mitellch-Artcmia diet treatment showed a greater increase in total length (2.1 to 6.1 mm) and mass per individual (0.1 to 1.5 ing dry mass) from stages 1 to V than did larvae fed ethyl ester-mussel-enriched Arteinia (total length: 5.9 mm; mass per individual: 1.2 mg dry mass; Fig. 1). Percentage survival was >68% between each stage and was highest from stages II-IIl (92-98'^f-: Table 1 ). Total survival to stage V was high for animals fed Anemia enriched with either the Al DHA Selco- C. miwllcii (57%) or ethyl ester-mussel (42%) nutrient sources. There were no differences in intermolt period for Artemia-itd phyllosomata among treatments. Intermolt periods were 9. 11, 12, TABI.K 1. Intermoll period (days) and pfrci'nlayt survival of phyllosomata fed different diets. Diet Al Mussel Selco-C. Ethyl Powder-Polar muellerf' Ester-Mussel" Lipid' Survival'' I-II 81.8 ±4.8 76.2 ± 15.9 - II-III 97.5 ± 2.2 92.3 ± 3.5 93.5 + 7.2 III-IV 87.3 ± 6.0 86.0 ± 6.7 5 1 .3 ± 3.6 IV-V 81.1 ± 14.5 68.3 ± 6.8 - Total 56.5 ± 12.6 41.5+ 11.2 - Intermolt period I-II 9 9 - II-III II 11 10 III-IV 12 12 - IV-V 1.^ 15 - " Presented as mean " Enriched Anemia. ■^ Feed station. ± SD: It = 3 L5 - 1 - 0.5 » Al DHA Selco-C. muelleri O Ethyl ester-mussel = 0.0884e" y = 0.I101e" R^ = 0.9998 St II Total Length (mm) Fijjure I. Dry mass as a function of total length of y. edwardsii phyllosomata from stages I to V on two diet treatments of Artemia enriched with either A I DHA Selco-C muelleri or ethyl ester-mussel nutrient sources. Presented as mean ± SD: filled with exponential trend lines. 228 Nelson et al. and 13-15 days to commencement of molt for stages 1-11. 11-111. III-IV. and IV-V, respectively. Phyllosomata fed the Mussel powder-polar lipid diet failed to molt to stage II on the feed station diet alone. Two animals re- mained alive at stage I for 30 days, at which time they were put on the Ethyl ester-miissel-enriched Anemia diet. At day 41 they suc- cessfully molted to stage 11. and were sampled at day 36. After sampling stage II animals at day 15. phyllosomata fed A I DHA Selco-C. »i»e//cn'-enriched Artemiu were divided and half were put on the Mussel powder-polar lipid diet. After 10 days, these animals molted to stage 111. were sampled at day 30, but failed to molt to stage IV. After sampling stage IV animals at day 37. phyllosomata fed ethyl ester-mussel-enriched Anemia were di- vided and half were put on the Mussel powder-polar lipid diet. They did not molt to stage V. but were sampled concurrently with Anemia-fed phyllosomata at day 56. Lipid Content and Classes The two nutrient sources were lipid-rich with Al DHA Seico higher than EE-mussel (960 and 410 mg g"' dry mass, respec- tively; Table 2). Al DHA SeIco was dominated by TAG (88%) and ethyl ester-mussel by EE (55%), with TAG the second most abundant lipid class (28%). Lipid content of Anemia enriched with A I DHA Selco-C. muellcri and ethyl ester-mussel was identical (250 mg g ' dry mass). TAG was the major lipid class (46-51% of total lipid), followed by PL (37-40%). ST (5-6%), FFA (4-10%), diacylglycerol (DG; 0.7-1.9%). and WE (0.1-0.3%) were minor components. In Anemia-fed phyllosomata. although lipid per individual gen- erally increased from newly hatched to stage V (8 to 180 |a.g), the absolute lipid content remained generally constant in Anemia-fed phyllosomata from newly hatched to stage V (Table 2). Total lipid was 155 mg g"' dry mass in newly hatched phyllosoma and in- creased slightly from stage 1 to stage II ( 207 and 1 73 mg g ' for A I DHA Selco-C. miielleri and ethyl ester-mussel Anemia-fed phyl- losomata, respectively) and to stage IV ( 157 and 176 mg g"' ). By stage V. total lipid decreased to the starting (newly hatched) value in ethyl ester-mussel Anemia-fed phyllosomata ( 156 mg g ' ) and was slightly lower in animals fed Al DHA Selco-C. miielleri- enriched Anemia ( 128 mg g ' ). PL comprised the major lipid class in all phyllosoma samples (73-87% of total lipid), followed by ST (4-8%; mainly cholesterol), FFA (2-8%). DG (2-i%), and WE (0-0.5%). Minor TAG was detected (0-0.4%). Stage IV phyllosomata fed the Mussel powder-polar lipid diet were similar to those fed either A/7e)?!(a diet, with 156 mg g"' dry mass of lipid and PL the dominant lipid class (83%; Table 2). ST were comparatively higher (10%). Compared with other phyllo- soma, PL (73%) and DG (1%) were lower in stage 111 Mussel powder-polar lipid feed station-fed animals, with a proportionate TABLE 2. Percentage lipid class composition of nutrient sources, enriched Artemiu. feed station, and phyllosomata. Free Wax Ethyl Fatty Ester Ester Triacylglycerol Acid Diacylglycerol Sterol Lipid Lipid as Mass nigg"' Indiv' Polar Drv Dry Lipid Mass Mass (pg) Nutrient souces Al DHA SeIco Ethyl ester-mussel Anemia Al DHA Selco-C. muelleii Ethyl ester-mussel Feed station Mussel powder-polar lipid Phyllosomata Newly hatched Al DHA Selco-C. inuelletT^ II IV V Elhyl ester-mussef II IV V Mussel powder-polar lipid*' III IV 0.0 + 0.0 0.7 ±0.0 .S?.! ±."1.8 U. 1 ± 0.0 0.3 ± 0.2 2.S±0.1 l.7±0.1 0.3 ± 0.0 0.3 ± 0. 1 O.-'S ± 0.3 0..'i±().l 2.7 ±0.2 1.0 ±0.3 88.2 ±0.8 1.6 ±0.1 - 0.8 ±0.0 9.5 ± 0.9 958.5 ±18.7 27.9±0.7 5.4±1.6 0.1 ±0.0 1.4±0.4 9.3±3.1 411.9±44.2 45.9±().4 10.0±0.2 l.y±().3 .';.6±0.0 .m5±1.0 2.';9.7±12.8 50.5 + 0.2 3.5 ±0.5 0.7 ±0.1 5.2 ±0.0 39.7 + (19 2-'i4.2 + 0.3 12.5±0.3 26.2±0.1 0.4±().l 3.5±0.3 54.6±0.9 191.0±11.4 0.2±0.1 9.9±0.1 - 9.9±0.1 78.3±0.1 l.M.6±9.4 8.0+1.1 5.5 ±5.0 1.5 ±0.4 4.3 ±1.7 88.7 ± 6.8 207.5 ±19.6 0.2 ±0.1 4.7 + 0.6 3.9 + 0.7 6.9 ± 0.2 84.1 ± 1.4 157.2 ±13.9 0.3 ±0.1 2.1+0.2 3.0 ±0.3 7.1 ±0.6 87.2 + 0.9 127.8 + 9..S 8.1 ±3.4 1.5 ±0.0 5.5 ±0.4 84.8 ± 3.7 173.0 ±17.5 0.2 ±0.1 4.9 + 0.8 3.0 + 0.4 6.3 ± 0.4 85.1 ±1.1 175.6+18.4 0.4±0.1 2.3±0.5 3.2±().2 7.5 ± 0.4 86.1 ±1.3 l.S6.(l±19.3 1.1 ±0.1 9.2 ±1.2 0.8 ±0.3 12.8 ±0.8 73.4 ±0.9 .S4.2 ± 7.5 0.5±0.3 2.7 + 0.3 2.2±0.9 10.1 ±1.9 83.4±2.5 155.5 + 86.4 72.7 + 6.1 LS6.0±8.0 188.9 ±25.2 61.3 ± 13.3 137.3 ±8.3 180.0± 13.3 46.7 ± 10.8 133.3 ±57.0 Presented as mean ± SD; /( = 3; (-), below detection. " Enriched Anemia. '' Feed station (molted from staae 11 lo 111 unlv). Feeding Southern Rock Lobster in Culture 229 iiiL-rease in ST (L^'/M and WE {i9c). Lipid content was a third that of other Artciiiia-t'eiS samples (54 mg g"' dry mass). Fatly Acids The FA ill the two nutrient sources differed markedl) (Table 3). hi Al [)HA Selco. dominant FA in decreasing order of propor- tional abundance of total FA were: palmitic acid ( 1 6:0; 1 79r ). EPA (159^). oleic acid [18:l(n-9)c; 14%]. palmitoleic 1 16:l(n-7)c. 97r], DHA (8%). myristic acid (14:0; 7%) and linoleic acid |18:2(n-6): 5'/f]. The ethyl ester-mussel nutrient source was doniinated by PUFA (75%). with major FA as DHA (37%). AA (13%). EPA (12%) and 16:0 (7%). The major FAs in enriched Anemia were as follows: I S: 1 ( n-9 )c (32-36%). l8:2(n-6) (23-27%). 16:0 (9-1 I7f). m-vaccenic acid |18:l(n-7)c: 4%|. stearic acid (18:0: 4%). and 16:l(n-7)c (2-4%: Table 3). Anemia enriched with ethyl ester-mussel had higher essential PUFA (3% AA. 6% EPA. 7% DHA) than those enriched with Al DHA Selco-C. miielleri (1% AA. 2% EPA, 1% DHA). The Mussel powder-polar lipid diet was dominated by 16:0 ( 19%). EPA ( 14%). and DHA (14% ). with A A at 3% of total FA. Com- pared with Anemia, levels in the Mussel powder-polar lipid diet of 18:0 fatty aldehyde (6%), 20:l(n-9)c (4%) and minor C„ PUFA (3%) were elevated, and levels of 18:l(n-9)c (3%) and 18:2(n-6) (2%) were lower. In ,4)7<7/;/(/-fed phyllosomata, the major FA were similar to those found m the enriched Anemia and in decreasing order of abundance were: 18:l(n-9)c (23-27% of total FA). 18:2(n-6) ( 17- 22%). 16:0 (9-11%). 18:0 (7-9%). EPA (7-11%). 18:l(n-7)c (4- 6%). DHA (4-6%). and AA (2-5%%: Table 4). These phylloso- mata experienced a decrease in essential PUFA, on both a relative (Table 4) and absolute basis (Fig. 2). from newly hatched to stage V. In phyllosomata fed ethyl ester-mussel-enriched y4rfe/»(rt, there was a concurrent drop in levels of AA (5^%. 4 to 3 mg g"' dry mass). EPA (21-9%. 14 to 6 mg g" ' ). and DHA ( 14-5%. 9 to 3 mg g''). with a similar, although more pronounced, decrease in ani- mals fed Al DHA Selco-C. mnelleri-enrkhed Anemia (AA: 3%, 2 mg g ' dry mass; EPA: 8%. 5 mg g"'; DHA: 4%, 2 mg g~'). Conversely, levels increased in 18:l(n-9)c (8 to 23-24%) and 18: 2(n-6) (1-18%). The FA profile of animals fed the Mussel pow- der-polar lipid diet closely reflected the diet, being dominated by 16:0 (12-15%). EPA (8-14%). DHA (6-9%). and AA (5-6%). Compared with Anemiu-fcd phyllosomata. levels in the Mussel powder-polar lipid-fed animals of 18:l(n-9)c (9-18%) and 18:2(n- 6) (5-13%) were lower, while in stages 111 and IV Mussel powder- polar lipid-fed animals, levels of 20:2(n-6) (2-3%) and 22:l(n-9) (3^%) were higher. DISCUSSION A major feature of previous Australian feeding trials with southern rock lobster phyllosoma has been comparatively poor survival. This trial, however, represents a turning point in Austra- lian rock lobster phyllosomata nutritional research, with greater than 80% survival of Anemia-fed phyllosomata through each stage from newly hatched to stage V. We believe that a primary differ- TABLE 3. Percentage fatty acid composition of nutrient sources, enriched Artemia. and feed station. Nutrient Sources Artemia Feed Station Al DHA Selco Ethyl Ester-Mussel Al DHA Selco-C. muelleri Ethyl Ester-Mussel Mussel Powder-Polar Lipid 14:0 6.9 ±0.2 0.7 ±0.1 1.3 ±0.1 0.5 ± 0.0 3.7 ± 0.3 16:l(n-7lc 8.8 ± 1.0 0.8 ±0.1 3.9 ±0.1 2.0 ± 0.0 5.3 ± 0.4 16:0 16.6 + 0.0 7.2 ±0.8 11.0 ±0.2 9. 1 ± 0. 1 18.5 ± 1.2 lS:4(n-3) 2.7 ±0.0 1.5 ±0.0 0.2 ± 0.0 0.3 ±0.0 2.1 ±0.1 lS:2(n-6) 5.1 ±0.2 3.6 ± 0.3 27.2 ± 0.3 22.9 ± 0.3 1.9 ±0.1 lS:l(n-9)c/18:3(n-3) 14.1 ±0.8 3.5 + 0.7 35.8 ± 0.2 31.8 ±0.6 2.6 ±0.1 18:l(n-7)c 3.0 ±0.1 0.5 ± 0.0 4.1 ±0.0 3.7 ±0.1 2.6 ±0.1 18:0 3.5 ± 0.2 3.4 ± 0.3 4.2 ± 0.0 4.4 ±0.1 5.1 ±0.0 18:1 Falde 0.1 ±0.0 - - 0.7 ±0.8 6.4 ±0.1 20:4(n-6) 0,9 ±0.0 1.^.2 ±0,6 0.5 ± 0.0 3.4 ±0,0 2.7 ±0.1 20:-'i(n-3) 14.9 ±0.6 12.3 ±0.3 2.4 ± 0.3 6.3 ± 0.0 13.9 ±0.4 2():4(n-3) 0.1 ±0.0 0.1 ±0.0 0.9 + 0.0 1 ,4 ± 0. 1 2.1 ±0.1 20:l(n-9)c 0.4 ± 0.4 0.4 ± 0.5 0.0 ± 0.0 0.8 ±0.1 3.6 ± 0.2 22:6(n-3) 7.9 ± 0.3 37.1 ±0.7 0.8 ±0.1 7.0 ±0.6 13.5 ±0.9 C,, PUFA - 0.7 ±0.0 - - 2.9 ±0.3 Ottier 14.8 15.0 7.6 5.7 13.0 Sum SFA 31.8 + 0.2 15.9 ±0.4 18.9 ±0.3 16.5 ±0.1 31.6± 1.6 Sum MUFA 30.7 ±1.5 8.5 ± 1.2 47.0 ± 0.6 40.8 + 0.1 18.1 ±0.3 Sum PUFA 37.4 ±1.3 74.7 ± 0.2 33.7 + 0.6 42.4 ± 0.5 43.1 + 1.8 Sum (n-3l 28.3 ± 1.0 52.9 ±0.9 3.7 ± 0.3 14.0 ±0.7 32.8 ± 1.5 Sum 01-6) 7.0 ±0.1 20.6 ± 1.0 28.2 ± 0.5 27.5 + 0.5 6.5 ± 0. 1 Ratio (n-3)/(n-6l 4.1 2.6 0.1 0.5 5.0 Ratio EPA/AA 15.8 0.9 5.2 1.9 5.1 Ratio DHA/EPA 0.5 3.0 0.3 1.1 1.0 Presented as mean ± SD: n = 3; (-). helow detection. AA, arachidonic acid; EPA. eicosapentaenoic acid; DHA, docosahexaenoic acid; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA. polyunsaturated fatty acids; Other includes components pre.sent at <2%: il5:0. al5:0. 15:0. il6:0. C|„ PUFA, I6:l(n-9)c, I6:l(n-7)t/16;2. 16:l(n-5)c. 16:0 Falde (fatty aldehyde), 16:1 Falde, 117:0, al7:0, 17:1. 17:0, 18;3(n-6), il8:0, 18:l(n-7)t, I8;l(n-5)c, 18:0 Falde, il9:0. 19:1. 20;3(n-6), 20;2(n-6), 20:l(n-ll)c. 20:l(n-9)c. 20:l(n-7)c, 20:0. C,, PUFA. 21:0. 22:5(n-6). 22;4(n-6). 22:5(n-3). 22:l(n-lll. 22:lin-7), 22:0. 24:1. 24:0, 230 Nelson et al. TABLE 4. Percentage fatty acid composition of phyllosomata from feeding trial. Diet Al DHA Selco-C n iielleri" Kthyl Ester-Mussel" Mussel Power-Polar Lipid'' Newly Hatched II IV V II IV V 11 III IV 16;l(n-7)c 4.1 +0.2 2.7 ±0.1 1 .8 + 0.0 3.1 ±0.2 1.6 ± 0.0 0.9 + 0.0 1.6 ±0.2 2,1 2.1 ±0.4 0.9 + 0.0 16:0 12.2 ±0.4 10.7 ±0.5 10.0 ±0.0 11.3 ±0.1 10.4 ±0.0 9.0 ± 0.2 10.1 ±0.3 15,4 15.1 ±2,2 12.2± 1.0 17:0 1 .6 ± 0.0 0.8 ±0.0 0.8 + 0.0 0.9 ± 0.0 0.8 + 0,0 0.6 ± 0.0 0.7 ±0.1 4.6 1,3 + 0.2 0.9 ± 0. 1 18:2(n-6) 0.7 + 0.0 17.4 + 0.4 21.7 + 0.2 18.3 ±0.3 17.1 ±0.1 20. 1 + 0.0 18.3 ±0.3 9.7 5.4 ± 0.8 13.0 ±2.4 18:l(n-9)c/18:3(n-3) 8.1 ±0.2 26.5 ± 0.4 27.4 ±0.2 23.5 ± 0.3 24.8 ± 0.3 24.9 ± 0. 1 22.7 ±0.5 14.5 9.4 ± 1.8 17.6 ±3.4 18:l(n-7)c 4.9 ±0.1 5.9 ±0.1 4.7 + 0.0 5.8 ±0.1 5.6 ±0,1 4.0 ± 0.0 4.5 ±0.1 3.9 4.1+1 .0 3.9 ± 0.5 18:0 8.3 ±0.0 7.4 ±0.1 7.2 ±0.1 8.5 + 0.2 8.3+0.1 7.5 ±0.1 8.7 ±0.1 13.3 10.9+ 1.2 1 1 .6 ± 1 .0 20:4(n-6) 5.1+0.1 2.4 + 0.1 2.0 ±0.0 2.6 ± 0. 1 4.7 ±0.2 4.3 ±0.1 4.3 ± 0. 1 6.1 5.2 ± 0.8 5.4 ± 0.3 20:5(n-3) 21.0±0.2 10.1 ±0.4 6.9 ±0.1 8.2 ±0.2 10.8±0.1 8.8 ±0.3 8.6 ±0.2 10.2 14.1 ±2.4 8.7 ± 0.5 20:2(n-6) 1.7 ±0.0 0.0 ± 0.0 - 1.5 ± 1.3 0.0 ± 0.0 0.8 ± 1.5 1.8 ± 1.6 - 2.6 ±0.2 2.1 + 1.8 20:Un-ll)c 0.3 ± 0.0 1 .7 ± 0. 1 2.3 ± 0.0 0.8 ± 1.3 1 .8 ± 0.0 1.7 ± 1.5 0.8 ± 1.4 - - 1 . 1 ± 1 .9 22;6(n-3) 13.5 + 0.3 5.3 ± 0.3 4.0 ±0.1 3.5 + 0.1 5.4 ±0.4 6.3 ± 0.2 4.9 ± 0.2 5.6 8.6 ± 1.5 5.9 ± 0.2 22:l(n-9) 0.8 ± 0. 1 0.1 ±0.0 1.6 ±0.2 1.8 ±0.3 0. 1 ± 0.0 1.7 ±0.2 1.9 ±0.1 0.5 3.9 ± 2.6 3.2+ 1.8 Other 17.5 9.0 9.7 10.3 8.6 9.2 10.8 14.0 17.1 13.4 Sum SFA 27.6 + 0.3 22.2 ± 0.3 21.1 ±0.2 24.6 ± 0.4 22.8 + 0.1 20.1 ±0.3 23.7 ± 0.9 35.6 .34.8 ± 5.0 29.9 ± 3.4 Sum MUFA 23.8 ± 0.3 40.2 ± 0.4 40.8 ±0.1 38.0 ± 1.0 37.0 ± 0.4 35.8 ± 1.4 34.4 ± 2.0 24.8 23. 1 ± 0.7 29.8 ± 2.0 Sum PUPA 44.4 ± 0.7 37.3 ± 0.7 37.9 ±0.2 35.7 ± 0.3 39.8 ± 0.6 43.0 ± 0.4 39.8 ±0.8 39.6 37.6 ±4.8 37.5 ± 1.6 Sum (n-3) 36.6 + 0.5 16.5 + 0.8 11.8 + 0.2 12.4 ±0.1 16.9 + 0.5 15.9 + 0.5 14.3 ±0.3 18.3 24.0 ± 4.0 15.6 ±0.3 Sum (n-6) 8.5 ±0.1 20.4 ± 0.3 25.4 ± 0.3 24.1 + 1.4 22.5 ±0.1 27.2+ 1.6 26.8 ± 1.9 21.3 15.2 ± 1.3 23.2 ± 2.6 Ratio (n-3)/(n-6) 4.3 0.8 0.5 0.5 0.8 0.6 0.5 0.9 1.6 0.7 Ratio EPA/AA 4.1 4.3 3.5 3.1 2.3 2.0 2.0 1.7 2.7 1.6 Ratio DHA/EPA 0.6 0.5 0.6 0.4 0.5 0.7 0.6 0.6 0.6 0.7 Presented as mean ± SD. /) = 3. " Enriched Artemia. ^ Feed station (molted from stage II to III only). '/! = 1. (-). below detection. AA. arachidonic acid; EPA. eicosapentaenoic acid: DHA. docosahexaenoic acid: SFA. saturated fatty acids: MLTA. monounsaturated fatty acids: PUFA, polyunsaturated fatty acids: (-), below detection: Other includes components present at <2'7c: 14:0, iI5:0, al5:0. 15:0. il6:0. C,,, PUFA. 16:l(n-9)c. 16:l(n-7)t/16:2, 16:l(n-5)c. 16:0 Falde (fatty aldehyde) il7:0, al7:0. 17:1. 17:0. 18:3(n-6). 18:4(n-3). il8:0, I8:l(n-7)t. 18:l(n-5)c. 18:0 Falde. i 19:0. 19:1. 20:3(n-6l. 20:4(n-3). 20:l(n-lllc. 20:l(n-7)c. C,, PUFA. 2 22:4(n-6). 22:5(n-3). 22:5(n-6), 22:l(n-n). 22:l(n-7). 24:1. 24:0. ence between this and the majority of previous trials has been the daily use of antibiotics in static culture. Although static culture and antibiotics are less appropriate for medium to large-scale culture of phyllosomata (Ritar 2001 ). they have been used in raising phyllo- somata to pueruli (Matsuda &. Yamakawa 2000). Additionally, the growth results from this trial, although similar to a previous trial, had much tighter standard deviations (Nelson et al. 2003). This suggests that larvae from this trial had more similar environmental parameters resulting from both aquarium design and use of anti- biotics. The successful use of antibiotics in static culture in this experiment highlights the fact that because the vital aspects of phyllosomata culture (i.e.. feeding capabilities, nutritional require- ments, aquariutn design and microbial loading) are intrinsically linked, advances cannot be readily made sequentially, and should ideally be performed concurrently. In previous trials, it has been difficult to test the effectiveness of feeding phyllosomata on Ar- temia. including using different enrichments, when experiments may be confounded by the adverse effects of microbial loading and aquarium design. The higher survival and good growth in this trial sugge.st that enriched Artemia may be adequate for early stage phyllosomata. The dominance of TAG in enriched Artemia illustrates the propensity for readily incorporating TAG from nutrient sources, as well as metabolizing EE to TAG for assimilation into their tissues. Similar results were observed when providing Artemia with a high-PL diet (Nelson, unpublished). These lipid class results are comparable to previous trials using 5-day old Artemia (Nelson et al. 2002b, Smith el al. 2002, Nelson et al. 2003). A distinction between this trial and an earlier trial (Nelson et al. 2003) is the detection of TAG in phyllosomata, albeit at low amounts and the higher relative proportion of DG. Although the difference is small, the presence of these short-term energy storage molecules is consistent with unproved larval health. In a prior feeding trial, total lipid content dropped markedly in phyllosoma to below 100 mg g"' by stage IV, and was also accompanied by poorer survival (Nelson et al. 2003). This result contributed to the hypothesis that phyllosoma, like puerulus (Jeffs et al. 2001 ), may be better served by use of PL, rather than TAG (Nichols et al. 2001, Nelson et al. 2003). Animals in the present trial did not experience the same marked decrease in lipid content. This finding may be the result of a number of reasons. First, if lipid is critical to survival, a drop in total lipid is associated with the poorer survival in previous trials. Maintenance of lipid at above 100 mg g ' in the present trial may therefore be linked with good survival. Second, because animals had high survival, but still did not have total lipid equal to wild phyllosomata (250 mg g ' lipid dry tnass Feeding Southern Rock Lobster in Culture 231 New hatch IV IV Al DHA Selco-C muellen Ethyl estei -mussel Diet & Stage Figure 2. Content (mg g') uf the essential long chain-polyunsaturated FA arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosa- hexaenoic acid (DHA) in / edwardsii phyllosoniata from stages I to V on two diet treatments o( Artemia enriched with either Al DHA Selco-C. miwlleri or cth>l ester-mussel nutrient sources. Presented as mean ± SD. at stage V) (Phleger et al. 2001). the class of lipid provided (i.e.. TAG in feeding trial versus largely PL in wild) was less effective. Thirdly, aquarium design and microbial loading can affect metabo- lism of lipids in larvae. For example, in previous trials, conducted in flow-through aquaria without antibiotics, and the present trial, conducted in static aquaria with antibiotics, Artemia that were similarly enriched with DHA Selco-C. nnielleri were fed to phyl- losomata. Animals from the present trial had 189 mg g"' lipid dry mass al stage V. while animals in the previous trial had 50 mg g ' lipid dry mass at stage V (Nelson et al. 2003). The animals in the previous trial either did not store lipid, or used more lipid as energy, while under the strain of microbes and/or swimming. Al- though Artemia supported excellent survival for larval stages I-V in the present trial, the Artemia diet may still not sufficiently condition phyllosomata for later stages; Artemia may not be pro- viding adequate total lipid for growth and high survival, especially if the aspects of aquarium design and microbial loading are not addressed. The current emphasis in phyllosoma culture in Australia is the use of Artemia for feeding stages I-V. This concept stems from developments with other aquaculture species, such as marine fin- fish, where it has been impossible to grow them during the early part of their life cycle without using live, motile feed (Olsen 1997. Castell et al. 1998). With rock lobster, complete rearing of phyl- losoma to puerulus was achieved by feeding on Artemia. fish larvae and/or iriussel tissue (Kittaka 1997b. a. Kittaka & Abrun- hosa 1997. Matsuda & Yamakawa 2000). Mussel gonad has been identified as the key to this success (Kittaka 1997b), used exclu- sively after the third instar (Kittaka 1997a). In culture, phylloso- mata have been observed ingesting inanimate food particles, such as lobster, prawn and mussel pieces at late stages (Thomas, un- published). Early stage animals have likewise been observed con- suming pieces of mussel, jellyfish and other inanimate foods (Mitchell 1971. Nelson et al. 2002a, Cox & Johnston 2003). Phyl- losomata in the present study were no exception. The larvae were observed consuming the Mussel powder-polar lipid feed station diet, a diet with which we attempted to build on the success of using mussel gonad. Additional evidence of feeding was the pres- ence of faecal trails, and molting, considering that phyllosomata do not molt when not feeding (Abrunhosa & Kittaka 1997). Never- theless, since phyllosomata fed the Mussel powder-polar lipid diet failed to molt properly beyond more than one stage, there is per- haps a necessary component either not present in sufficient amounts, or lost by leaching, in the feed station diet that contrib- utes to molting. Therefore, the use of Artemia up to the third instar (Kittaka 1997a) remains valuable for phyllosomata. However, to improve conditioning of larvae, the potential use of co-feeding of Artemia (Dhert et al. 1999), along with a PL source, should be examined, particularly for later stage animals. Of note is the decrease in essential PUFA from newly hatched to stage V phyllosomata. On a relative basis, Artemia-fed phyllo- somata and wild-caught animals at stage V had similar levels of 232 Nelson et al. AA (trial. ?,-i'7r: wild. 2-3%) and EPA (trial. 8-9%; wild, 7-9%), with markedly lower DHA in cultured animals (trial, 4-5%: wild. 16-17%) (Phleger et al. 2001). Results from a previous feeding trial are similar for relative levels of these FA (3-6% AA: 8-9% EPA; 2^% DHA) (Nelson et al. 2003). However, because the larval lipid remained above 189 nig g ' lipid dry mass at stage V. on an absolute basis this trial represents a marked improvement for incorporation of essential PUFA. The fact that the amount of total lipid remained the same to stage V. but there was a drop in the level of essential PUFA. in particular DHA. highlights the impor- tance of these FA. Higher absolute concentrations of these FA may be associated with enhanced survival and growth in this feeding trial compared with previous trials (Nelson et al. 2003, Hart et al., unpublished). Total lipid and levels of essential PUFA in phyllo- somata fed ethyl ester-mussel-enriched Anemia were higher than in larvae fed A I DHA Selco-C. nuielleri-emiched Artemia. Be- cause there was no direct association of enhanced FA profiles with survival and growth for phyllosomata from the two Artemia diet treatments, and the majority of lipid provided to phyllosomata through enriched Anemia was TAG. we propose that the impro\ed survival and growth in this trial may result from the presence of lipid in the diet (as described above) in combination with better health. Furthermore, we suggest it is likely that the PUFA profiles will have a more significant effect if provided in a PL form. These results also support the suggestion that co-feeding ai Anemia and a PL source should be trialed to improve larval condition. In conclusion, the use of antibiotics and static culture has en- abled a clearer picture of the effects of nutrition on larval health. Our experiment demonstrated that lipid-enriched Anemia support excellent growth and survival in early stages of phyllosomata. and we are now better placed to take nutrition of phyllosonia forward. The results suggest that the class of lipid provided \\\i Anemia may not adequately condition larvae, nor supply sufficient quantities of the essential PUFA. in particular DHA. for later stages. Thus, for successful culture of phyllosomata. the development of a formu- lated diet, which can provide the nutritional requirements in the right form to enhance long term conditioning of the larvae, is likely to be vital, particulariy for later stage larvae. ACKNOWLEDGMENTS We are extremely grateful to B. D. Mooney. G. G. Smith. A. J. Ritar. and C. W. Thomas for their invaluable expertise and assis- tance during the experiment. The Greenshell mussel products (powder, polar lipid and lyprinol) were kindly provided by Dr. A. G. Jeffs. NIWA Research. Auckland. New Zealand. D. Hold- sworth and B. D. Mooney managed the CSIRO GC-MS and GC facility. M. M. Nelson gratefully acknowledges a University of Tasmania Thomas A. Crawford Memorial Scholarship. This work was supported in part by the FRDC RLEAS Subprogram (2000/ 214) and FRDC project 1999/331. LITERATURE CITED Ahrunhosa. F. A. & J. Kittaka. 1997. The morphological development of juvenile western rock lobster Panuliius cygnus George. 1962 (Deca- poda. Palinuridae) reared in the laboratory. Bull. Mar. Sci. 61:81-96. Bligh, E. G. & W. J. Dyer. 1959. A rapid method of total lipid extfaction and purification. Can. J. Biochem. Pliysiol. 37:911-917. Booth. J. D. & B. F. Phillips. 1994. Early life history of spiny lobster. CrusUiceana 66:271-294. Breen. P. A. & T. H. Kendrick. 1997. .A fisheries management success story; the Gisbome. New Zealand, fishery for red rock lobsters Uasiis edwardsii). Marine Freshwater Res. 48:1 103-1 1 10. Castell. J. D.. L. D. Boston. D. A. Nanton, T. Blair. D. Douglas. J. Batt. C. Lanteigne. J. Castell & R. Henry. 1998. Factors affecting nutritional value of live feed organisms for use in larval marine t1sh culture in Atlantic Canada. Larval Culture, pp. 61-86. Cox. S. L. & D. J. Johnston. 2003. Feeding biology of spiny lobster larvae and implications for culture. Rev. Fisli. Sei. (In press). Dhert. P., M. G. Felix. K. Van Ryckeghem. I. Geurden. F. Thysen. E. Lebegue. P. Lavens & P. Sorgeloos. 1999. Cofeeding of phospholipids to turbot Scoplnlniliniis iiui.\inuis L. larvae as a tool to reduce live food consumption. .Acjiia. Niitr. 5:237-245. Diggles, B. K., G. A. Moss. J. Carson & C. D. Anderson. 2000. Luminous vibriosis in rock lobster Voiui verreau.xi (Decapoda: Palinuridae) phyl- losoma larvae associated with infection by Vibrio lianeyi. Dis. Aqnul. Org. 43:127-137. Harel, M., S. Ozkizilcik. L. E. P. Behrens & A. R. Place. 1999. Enhanced absorption of docosahexaenoic acid (DHA. 22:6n-3) in Artemia nauplii using a dietary combinalion of DHA-rich phospholipids and DHA- sodium salts. Comp. Bioeltem. Pliysiol. B 124:169-176. Igarashi. M. A., J. Kittaka & E. Kawahara. 1990. Phyllosonia culture with inoculation of marine bacteria. Bull. Jpn. Sac. Sei. Fi.'ih. 56:1781-1786. Jeffs. A.. P. D. Nichols & M. P. Bruce. 2001. Lipid reserves used by pueruli of the spiny lobster 7h.vh,v edwardsii in crossing the continental shelf of New Zealand. Comp. Biochem. Physiol. A 129:305-311. Johnston. D. J. & A. Ritar. 2(X)I. Mouthpart and foregul ontogeny in phyllosonia larvae of the spiny lobster Jasus edwardsii (Decapoda: Palinuridae). Marine Freshwater Res. 52:1375-1386. Kittaka. J. 1997a. Application of ecosystem culture method for complete development of phyllosomas of spiny lobster. Aipiaeiiliure 155:319- 331. Kittaka. J. 1997b. Culture of larval spiny lob.sters: a review of work done in northern Japan. Marine Freshwater Res. 48:923-930. Kittaka. J. & F. A. Ahrunhosa. 1997. Characteristics of palinurids (Deca- poda; Crustacea) in larval culture. Hydrohiologia 358:305-311. Kittaka. J. & J. D. Booth. 2000. Prospectus for aquaculture. In: B. F. Phillips & J. Kittaka. editors. Spiny lobster management. Oxford: Fish- ing News Books, pp. 465^73. Matsuda, H. & T. Yamakawa. 2000. The complete development and mor- phological changes of larval Panuhms longipes (Decapoda. Palinu- ridae) under laboratory conditions. Fish. Sei. 66:278-293. McEvoy. L. A.. J. C. Navarro. F. Hontoria. F. Amat & J. R. Sargent. 1996. Two novel Anemia enrichment diets conlaining polar lipid. .Aquacul- ture 144:339-352. McWilliam. P. S. & B. F. Phillips. 1997. Metamorphosis of the final phyllosonia and secondary lecithotrophy in the puerulus of Panulirus evgnus George: a review. Marine Freshwater Res. 48:783-790. Mitchell. J. R. 1971. Food preferences, feeding mechanisms, and related behavior in phyllosonia larvae of the California spiny lobster. Panuli- rus inlerruptus (Randall). M.S. San Diego State College. San Diego. California. USA. Nelson. M. M.. S. L. Cox & D. A. Ritz. 2002a. Function of mouthpans in feeding behavior of phyllosonia larvae of the packhorsc lobster. Jasus verreau.xi (Decapoda: Palinuridae). J. Cruslae. Biol. 22:595-600. Nelson, M. M., B. J. Crear, P. D. Nichols & D. A. Ritz. 2003. Growth and lipid composition of southern rock lobster Uasus edwardsii) phyllo- soma fed enriched Artemia. Aqua. Nutr. (hi press). Nelson, M. M.. B. D. Mooney. P. D. Nichols, C. F. Phleger, G. G. Smith. P. R, Hart & A. J. Ritar. 2002b. The effect of diet on the biochemical Feeding Southern R(jck Lobster in Culture 233 composition of juvenile Anemia: Potential formulations for rock lob- ster aquaculture. 7. World AquacuU. Soc. 33:146-137, Nichols. P., B. Mooney & A. Jeffs. 2001. The lipid, tally acid and sterol composition of potential prey items of the southern rock lobster Jasiis eclwtinhii: an aid to identification of food-chain interactions. Report 2001-CMR/MP2. CSIRO. Hoban. Tasmania. .Australia. Olsen. Y. 1997. Larval-rearing technology of marine species in Norway. Hyilmhiologici 358:27-36. Phillips. B. F. & A. N. Sastry. 1980. Larval ecology. In: J. S. Cobb & B. F. Phillips, editors. The biology and management of lobsters. New York: Academic Press, pp. 1 1-57. Phleger. C. F.. M. M. Nelson. B. D. Mooney. P. D. Nichols, A. J. Ritar. G. G. Smith. P. R. Hart & A. G. Jeffs. 2001 . Lipids and nutrition of the .southern rock lobster, Jcistis edwanl.ui. Ironi hatch lo pucrulus. hhinnc Freshwiiwr Res. 52:1475-1486. Punt. A. E. & R. B. Kennedy. 1997. Population modeling of Tasmanian rock lobster. Jiisus edwardsii, resources. Mariiw Frcxhwalcr Res. 48: 967-980. Ritar. A. J. 2001 , The experimental culture of phyllosoma lar\ ae of south- em rock lobster Uusus edwardsii) in a flow-through system. Ai/iiticKlr. Eng. 24:149-156. Smith. G. G.. A. J. Ritar. C. F. Phleger. M. M. Nelson. B. D. Mooney. P. D. Nichols & P. R. Hart. 2002. Changes in gut content and composition of juvenile Arteinia after oil enrichruent and during starvation. Ai/ikiciiI- tiire 208:137-158. Sorgeloos, P.. P. Coutleau. P. Dhert. G. Merchie & P. Lavens. 1998. Use of brine shrimp. Artemia spp., in larval crustacean nutrition: a review. Rev. Fisli. Sci. 6:55-68. Tong. L. J.. G. A. Moss. M. P. Paewai & T. D. Pickering. 2000. Effect of temperature and feeding rate on the growth and survival of early and mid-stage phyllosomas of the spiny lobster .hisiis edwardsii. Marine Freslmaler Re.'.: 5 1 :235-24 1 , Volkman. J. K. & P. D. Nichols. 1991. Application of thin layer chroma- tography-tlame ionization detection to the analysis of lipids and pol- lutants in marine environment samples. J. Plan. Chront. 4:19-26. Wilkinson. B. 2000. Microalgae production at the Marine Research Labo- ratories, Taroona. Tasmania. Australia: Tasmanian Aquaculture and Fisheries Institute. Hobart. Journal of Shellfish Ken'cinh. Vol. 22, No. I, 235-239, 2003. THE RELATIONSHIP BETWEEN HEMOLYMPH CHEMISTRY AND MOULT INCREMENT FOR THE SOUTHERN ROCK LOBSTER, JASUS EDWARDSII HUTTON R. J. B. MUSGROVE'* AND P. J. BABIDGE" ^SARDl Aquatic Sciences. P.O. Box 120. Henley Beach. GPO Box 397. .Adelaide. SA 5001. Aiisrralia SA 5044. Australia and -SARDl Biochemistry. ABSTRACT Growlh data are essential to rock lobster fisheries stock assessment. At present, predictions of growth tor a given year are based on data from previous years with the accuracy of the estimates being unknown until measures of growth are obtained in the year in question. This article tests the hypothesis that premoult hemolymph lipid concentration is a predictor of moult increment for the southern rock lobster. Jasus edwardsii. in the laboratory. The study was undertaken to develop a nonlethal means of moult increment prediction, which could then be used in the field. Premoult carapace length had no effect on percent moult increment (P > 0.05) in the laboratory. Both phospholipid and triglyceride were significantly correlated with percent moult increment. Phospholipid showed the highest coefficient at r^ = 0.66. Our data suggest that hemolymph phospholipid level has the potential to predict moult increment. However, the hemolymph lipid/moult increment data were gathered over a short time period and within a relatively controlled en\ironment. Further field studies are essential to better understand the relationship between hemolymph lipid level and moult increment in w ild populations of this species. KEY WORDS: Jasus ednardsii. lipid, moult increment, moulting, growth INTRODUCTION Growth data are essential to rock lobster fisheries stock assess- ment. At present, predictions of growth for a given year are based on data from previous years with the accuracy of estimates being unknown until measures of actual growth are obtained for the year in question. The shedding of all hard parts at ecdysis complicates measure- ments of growth in rock lobsters and other crustaceans. No struc- tures are retained (sensu fish otoliths) from which age at size, and therefore growth information, may be gathered. During recent work in South Africa, Cockcroft (1997) suggested that growth may be estimated from hepatopancrealic lipid level having found a significant relationship between moult increment and percent hepatopancreatic lipid during premoult in Jasus kdandii. The find- ing of this relationship was a significant advance, although it was still necessary to kill the animal to gather the data, a step that tnight be avoided by isolation of an equally useful hemolymph compo- nent. During a recent study. Musgrove (2001) found that hemolymph protein, in combination with hemolymph pigment level and moult stage, was useful in distinguishing between lob- sters at high and low growth sites within the South Australian fishery. He was able to show that grouping serum protein data by pigment stage with reference to the major pigment, astaxanthin. allowed the differentiation of lobsters at the beginning and those at the end of intermoult. Given the correlation between serum protein and 9^ dry weight, differences in lobster condition between high and low growth sites could be examined more thoroughly using this method. Hemolymph protein has been used successfully in other studies as a measure of condition (Leavitt & Bayer 1977. Musgrove 2001 ) but has not been shown to be useful in predicting moult increment. Given Cockcroft's work, premoult hemolymph lipid appeared to be the most likely to show a predictive relation- ship with moult increment. If hemolymph lipid could be used in place of total hepatopancreas lipid to predict moult increment, the necessity to kill the lobster would be avoided and multiple samples may be taken over time from the same individual. *Corresponding author. E-mail: musgrove.richard@saugov.sa.gov.au Phospholipids are the major circulating lipid and triglycerides the major storage lipid in crustaceans. Both are found in the hemolyinph and hepatopancreas (Chang & O'Connor 1983). The hepatopancreatic lipid component of Jasus lalandii is largely tri- glycerides (neutral lipids) with phospholipids (polar) of less im- portance (<14<7f) Cockcroft (1997). In the hepatopancreas, in- gested neutral lipids are cleaved to mono or diglycerides, which are then converted to phospholipids. These are expelled into the hemolymph and transported to various tissues, either for use as membrane components or conversion to triglycerides and storage (Chang & O'Connor 1983). The hepatopancreas of rock lobsters increases in size and lipid content through the moult cycle, reaching a maximum just before ecdysis (Musgrove 2000b, Cockcrof, 1997) it may also be ex- pected that other chemical compounds would show similar pat- terns. Thus, as the hepatopancreas reached maximum storage dur- ing late premoult (Musgrove 2001, Mercaldo-AUen 1991). so hemolymph lipid would reach maximum concentration. Further- more, as Cockcroft ( 1997) found that hepatopancreas lipid was an indicator of moult increment in the field, so may hemolymph lipid be, as phospholipid would be used for both the cell membranes of the expanding hepatopancreas and, after conversion to triglyceride, as the main lipid store. This article tests the hypothesis that premoult hemolymph lipid concentration is a predictor of moult increment for the southern rock lobster. Jasus edwardsii. in the laboratory and examines the relationship between hemolymph and hepatopancreatic lipid con- tent and tissue weight. The study was undertaken to develop a non-lethal means of moult increment prediction, which could then be used in the field. MATERIALS AND METHODS iMhoratory Experiment I: Relalinnship Betneen Moull liieremeiil and Premoult Hemolymph Level Forty lobsters (mean CL: 89.88 ± 0.60 mm, mean weight 364.6 ± 6.54g) were individually housed in 30-L plastic tanks in a flow- through system (0.4 L/h/tank) for 185 days. Each tank was inde- pendently supplied with air and water of a constant temperature 235 236 MUSGROVE AND BaBIDGE (18°C, which was similar to the average summer temperature in the area ot capture). Day length was set at 12 h and the lights covered with red cellophane to minimize disturbance. Lobsters were fed ad libinim daily on a mixed diet of artificial pellets (four pellets/feed. Geddes et al. 2000) and cockles (four cockles/feed. Donax deltoicles) in a rotation. Daily consumption was assessed by eye from day 52 and categorized as 0. <257r. 25-50%. and >507f. Excess food was removed and tanks cleaned each mornmg. taking care to minimize disturbance to the lobsters. Hemolymph samples (0.5 mL) were taken fortnightly from each lobster by pericardial puncture for analysis of hemolymph serum. Once the pigment stage of each hemolymph sample had been noted (Musgrove 2001 ) it was snap-frozen (-196°C) for later analysis. Pigment stage refers to the color of the hemolymph. which changes from light blue through beige to deep orange during the moult cycle, the beige becoming visible during intermoult (Musgrove 2001). If the lobster was immediately premoult. samples were taken before and after ecdysis. Pleopod samples were also taken periodically to track moult stage by examination of setal development (MusgroNC 2000). Laboratory Experiment 2: Relationship Between Moult Increment and Premoult Hemolymph Level in a Less-Controlled Environment Seven premoult lobsters were selected from animals that had been kept in an outside tank for several months with other species (echinoderms. other decapods) and fed two to three times a week on blue mussels (Mytilus sp.). The tanks were at ambient tempera- ture (about 16°C) and contained abundant limestone rocks, Mac- rocystis sp.. Ulva sp. and other aquatic macrophytes. The selected lobsters were measured (range. 65.3 to 103.8 mm CL). moult staged (after Musgrove 2000) and placed in plastic cages within the aquaria. They were fed mussels ad libitum 3 to 4 times a week. Pleopods were taken regularly to keep track of the moult stage and. during late premoult (Stage D,) a 0.2-mL hemolymph sample was taken, pigment staged, then snap frozen. Once each lobster had hardened (i.e.. at intermoult) it was re-measured. The data were then compared with those collected from laboratory experiment 1. For experiments 1 and 2. blood was taken during the afternoon to standardize postprandial effects on hemolymph lipid (sensii Dall 1981). Lobsters were fed after extraction was completed. In both cases, lobsters were not observed to feed during daylight. Field Study: Relationship Between Tissue Lipid and Hemolymph Lipid Level One hundred and thirty nine rock lobsters were collected from the wild fishery as described by Musgrove (2001) and hemolymph samples taken as described above within 3 h of capture, the pig- ment stage noted and the sample snap-frozen (-196"C) for later serum lipid analysis. A pleopod was also taken for moult stage determination by examination of setal development (Musgrove 2000). The lobsters were then frozen (-30°C) and retained for dissection and tissue analysis. Within two weeks of collection, lobsters were rapidly thawed and the abdominal tissue and hepatopancreas removed, weighed then dried to constant weight (60°C. 72 h). The tissue was then allowed to cool to room temperature in a desiccator over silica gel. reweighed (to nearest 0.1 mg) and dry weight and percent dry weiaht calculated. Hemolymph Serum Analysis All whole hemolymph samples from the laboratory study and a random selection of samples from the field collection (n = 139) were analyzed for triglyceride and phospholipid. The clotted hemolymph was thawed then broken up gently with a glass stirring rod and the sample centrifuged (Hettich EBA12 centrifuge, 15 min. 17.280g) to extract the serum. Serum aliquots were analyzed on a Cobas Mira Autoanalyser for triglyceride and phospholipid using commercially produced test kits (Roche). To test for phos- pholipid the triglyceride kit (Roche. No. 07 3679 1 ) was modified as follows. 250 units phospholipase C (Sigma No. P4014) were added to a 30-mL bottle of triglyceride reagent. The modified reagent was then incubated with the serum sample for 15 min at 37 "C (cf 6 min for ti-iglyceride) to convert the serum phospholipids to diglycerides, which were then converted to glycerol by the lipase in the kit. The incubation time was chosen by incubating a lecithin solution (2 mM) to give a result equivalent to 2 mM triglyceride. Accuracy was maintained for all tests using commer- cially available quality controls (Nycomed Farmer). Data Analysis If data were normally distributed or could be normalized analy- ses were performed using analysis of covariance (ANCOVA) or analysis of variance (ANOVA) with the GLM module (General Linear Models) on SPSS. If data could not be normalized, the Kruskel Wallis nonparametric ANOVA or the Wilcoxon Rank Sign were used. In all cases significance was accepted at P = 0.05. RESULTS iMhoratory Experiments I and 2 Percent Moult Increiiient, Tank Placement, and Feeding Regimen Premoult CL had no effect on % moult increment (P > 0.05, ANCOVA). and there was considerable overlap between the ranges of 9c moult increment recorded in the outside tanks (2.4 to 8.0% of premoult CL. n = 1) and those inside (0.8 to 5.2%. n = 9). The slopes of the percent moult increment; lipid regressions were the same for inside and outside tanks (P > 0.05. ANCOVA). For this reason, data from inside and outside tanks were pooled for further analyses. Hemolymph Serum Lipid and Moidt Increment Both lipid tractions were significantly correlated with percent moult increment (Table 1). Phospholipid showed the highest co- efficient at r = 0.66. Both phospholipid and triglyceride showed a progressive increase with pigment stage (Fig. 1 ) until PS3.0 to 4.0 then declined to PS4.5. TABLE 1. Relationship between percent moult increment and haeniolyniph lipid (Mmol 'l for phospholipid, triglyceride, and TP (triglyceride + phospholipid). Parameter P Phospholipid Triglyceride TP 0.0715 0.1140 -0.0786 2.072 2.248 1.765 0.66 0.40.1 0.641 .10.062 <0.001 II . 1 2,1 0.005 27.781 <0.001 The regression model is Log % moult increment = allog (lipid)]^. n 16. Hemolymph Chemistry and Moult Increment in Lobsters 237 35 30 25 o E 20 1.5 1.0 05 0.0 ■ ' ^^ E \ E i \ "5 T 5 T i !_i T " '\ 0.5 1 1.5 4.5 2 25 3 3.5 Pigment Stage Figure 1. Laboratory: mean hemolymph serum triglyceride (mmol/I.) ±SE, phospholipid Immol/L) ±SE, and triglyceride plus phospholipid (TPl (mmol/I, I ±SF, vs pigment stage. Closed dianKmd, TP; closed square, triglyceride; closed triangle, phospholipid. 1.8 1.6 1.4 1.2 1 0.8 06 0.4 0.2 0 0.5 1 1.5 3.5 4.5 2 25 3 Pigment stage Figure 3. Field: mean hemolymph serum triglyceride (mmol/l,( ±SE. phospholipid Immol/lj +SK. and triglyceride plus phospholipid (TPl (mmol/L) ±SE vs pigment stage. Closed diamond. TP: closed square, triglyceride: closed triangle, phospholipid. Feeding Rales Feetiing rate increased 4-fol(i after the moult in those lobsters for which there was data (Fig. 2). There were no se.xual differences in feeding rate (P > 0.03). Field Study Hemolymph Serum Lipid The field serum lipid data showed a progressive increase in lipid content with pigment stage (Fig. 3) in a similar fashion to that found in the laboratory, although in this case the peak occurred at PS4. Hemolymph Lipid and Hepatopancreas Weight Hemolymph lipid increased with hepatopancreas dry weight on both a total weight and a percentage basis (Fig. 4a and b) up to PS4. However, although hemolymph lipid was significantly cor- related with tissue weight during intermoull (Table 2). the rela- tionship declined after PS2-2.5. ImI'ihiiIoiv Fxperimeiits and Field Study Comparisons Moult Stage and Pigment Stage The relationship between moult stage and pigment stage was similar in the laboratory and the field (Fig. 3). In the following 30 25 20 15 10 05 00 fVt* 987654321 Weeks before moult 2 3 4 5 6 7 Weeks after moult Figure 2. Mean feeding rates (±SE| for 10 weeks before and after ecdysis. Four daily consumption categories (0, <25, 25-50, >50'!'r ) were used and assigned numbers from I to 4 (h = 18). analysis, comparisons are made between pigment stage-specific laboratory and field hemolymph lipids. Before this was done, analysis was undertaken to check that the same pigment stages had similar distributions of moult stages in the laboratory and the field. To facilitate the analysis each moult stage was assigned a number (1-11). The laboratory distribution of moult stages within each pigment stage was similar to that in the field (Mann-Whitney U. Zar 1984). The only significant difference was in PS 4 (U = 37.3. P = 0.014, mean moult stage|.,b„„,„ry = 9.46 ± 0.3Q. mean moult stage,-,^,j = 8.13 ± 0.249). otherwise P > 0.212. Hemolymph Serum Lipid Pigment stage-specific total lipid of laboratory animals was greater than that in the field until PS3.3 (Mann-Whitney U. P < 0.03: Fig. 6). The patterns in the relative importance of the two lipid fractions were also different. In the field, the proportion of phospholipid increased until PS 2.5 (Fig. 7) then fell until PS 4.5. in contrast to the laboratory where the peak was reached during PS 1 . Both laboratory and field showed the same trends after PS2.3. DISCUSSION The key result to come out of this study is the potential use of hemolymph lipid in the prediction of percent moult increment. Although further field studies are needed to be sure of the result, this outcome is potentially very useful because hemolymph lipid measurement does not require killing the lobster. Questions remain as to whether higher growth sites, showing higher serum protein content v\ould also ha\e higher moult increments. In this regard, significant differences were reported in mean serum protein level between sites by Musgrove (2001). The differences occurred mainly during intermoult, which is the period when hemolymph lipid is significantly correlated with both serum protein and hepatopancreas percentage dry weight. This may suggest a rela- tively higher degree of lipid accumulation at those sites, pointing to a higher moult increment. Dall (1981) suggested that the prin- cipal function of digestive gland lipid in Nephrops noii'egicus was 238 MUSGROVE AND BaBIDGE a) TABLE 2. 25 1 2' y 1 ). :V^ ■^ TP (mmol/l) -*■ bi J ■^ ^A r ^\ 1 — i ► 1 i 05 w 7^ ' I / i^ 1 ^ \ H \ 1 1.5 2 2.5 3 3.5 Pigment Stage 4.5 o E E P- 1 s \ 39 ss In ; -37 *•* - 35 1 •^ >. 33 ■D \ C r3l 0) o 6) a. Pigment Stage Figure 4. Comparison of (a) hepatopancreatic mean dry weight (g, ±SE). (b) mean percent dry weiglil {±SE). and triglyceride plus phos- pholipid (TPl \s pigment stage. Mean dry weight (g) standardized for carapace length using GLM analysis of SPSS. Data are displayed for a 97.9-mm CL lobster. Closed diamond, TP; closed square, weight (g). in the moulting process so one might expect that lipid accumula- tion in Jasiis edwordsii would be similarly focused. Cockcroft ( 1997) also found a significant relationship between moult increment and hepatopancreas lipid level, for Jasus lalaiulii. He reported that moult increment was positively related to peak "/flipid values occurring during late premoult in the hepatopan- creas, similar to the present study, where the significant relation- ship was between heiiiolymph lipid (|j.mol/Ll and '/<- moult incre- ment. Furthermore, he suggested a "window" period of reserve accumulation, essential for growth. This occurs from intermoult to early premoult. especially the former, as suggested for/ edwardsii by the relative increase in feeding rate after ecdysis. The period of reserve accumulation (PRA) would probably lead up to a "reserve saturation point" as suggested by Anger (1987) for crustacean larvae. Cockcroft found that lobsters starved during PRA, then feed during premoult. showed severely reduced growth rates and 10 Field data regression statistics for pigment stage-specific percent dry 9.5 weight and total dry weight versus total lipid (T + Pl. 9 Percent or 8.5 S £ Total Weight PS r F P n 8 Percent 1 0.722 17.33 <0.()01 25 7.5 1 l-.S 1 0.887 0.599 118.29 29.93 <0.001 <0.001 17 24 7 r^ 2.5 0.685 41.39 <0.()01 21 6.5 o 3 0.-U8 S.IO ,017 12 >3 0.067 1.93 0.176 29 6 Total Weight 1 0.532 24.96 <0.001 25 5.5 1.5 0.767 49.50 <0.001 17 2 0.389 13.37 0.001 24 5 2.5 0.282 7.47 0.013 21 3 0.000 2.9"*" 0.987 12 >3 0.078 2.29 0,142 29 Regression model is Lipid = a Weight'^ except for percent dry weight at pigment stage 1. where the best fit was given by the cubic model (lipid = a -I- Pi weight -I- p, weight^ + p, weight') even shrinkage. Those starved prior to moulting but led during PRA moulted with similar growth increments to those of control lobsters, which were fed throughout. Therefore, it is this PRA that is critical to future growth, influencing both moult increment and intermoult period (Cockcroft, 1997). The question is, why should the percent moult increment be correlated with the lipid level in the hemolymph at PS4.3, when it was not related to the hepatopancreas percent dry tissue at that stage? At PS4.5 about 95% of lobsters were beyond D,'". The rigorous investigation of this question is outside the framework of this study but it may be that the apparent decoupling of the rela- tionship between hepatopancreas weight and hemolymph lipid at the later pigment stages is due to a mobilization of lipid reserves from the hepatopancreas to the hemolymph in preparation for the energetic demands of ecdysis. The correlation may arise because the higher the level of stored lipid in the hepatopancreas, the creater the reserve that may be mobilized in readiness for ecdysis. D3 D, D,'" D,- 0/ Do C3 C2 B/C, A 0.0 1.0 1.5 3.5 4.0 4.5 5.0 2.0 2.5 3.0 Pigment Stage Figure 5. Mean moult stage (±SF) within each pigment stage for labo- ratory experiments (pooled, n = 40) and field study (n = 135). There were no lobsters at PS3.5 in the laboratory sample. Closed diamond, laboratory: closed square, field. Hemol\mph Chemistry and Moult Increment in Lobsters 239 2,5 ? 15 E E 05 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 Pigment stage Figure 6. Mean triglyceride and phospholipid (TP: nimol/I>) ±SE: field vs laboratory data by pigment stage. Significant differences (Mann-Whitney U testi between laboratory and field data are indi- cated by asterisks {**P < 0.001 1. Closed square, laboratory TF; closed diamond, field TP. The importaiK-e of phospholipid in the relationship fits in with Chang and O'Connor's (1983) contention that phospholipid is the main circulating lipid in crustaceans. Bligh and Scott (1966) re- ported that 65'7f of the total lipid in the hemolymph of the lobster, Hdiiiaiiis (iniericciiuis. was phospholipid, with the remainder al- most equally divided between triglycerides and sterols. The latter has a primarily structural role (Fraser, 1989). Free fatty acids com- prised only about 2.4% of the total lipid. O'Connor and Gilbert ( 1969) reported similar results for the land crabs. Gecarcinns lat- eralis and Canliosoma giianhmiu. Finally, the relative levels of hemolymph protein and lipid in the laboratory and the field suggest that the rate of accumulation differs, particularly in the early stages of the moult cycle. One would assume that these differences occur because captive lobsters did not have to hunt for food, more nutrients being directed to muscle accumulation and lipid storage earlier in the moult cycle. Our data suggest that hemolymph phospholipid level has the 0.7 065 0.6 Q. H •B 0 55 a. o 0.5 ,c a. o 045 s: Q. 0.4 0.35 0.3 ' - 0 0.5 1 1.5 2 2,5 3 3.5 4 4.5 5 Pigment Stage Figure 7. Hemolymph scrum phospholipid/! triglyceride plus phospho- lipid, nimol/L( ±SE: field vs laboratory data by pigment stage. .Signifi- cant differences (Mann-Whitney I' test) between laboratory and field data are indicated by asterisks (**/" < O.OUl). Closed square, labora- tory TP; closed diamond, field TP. potential to predict moult increment. However, the hemolymph lipid/moult increment data were gathered over a short time period and within a relatively controlled environment. Further field stud- ies are essential to better understand the relationship between hemolymph lipid level and moult increment in wild populations of this species. ACKNOWLEDGMENTS The authors would like to thank the South Australian Rock Lobster Industry for their support, for supplying the lobsters for this study, and for allowing us the use of their facilities for the initial data collection. We wiuild also like to thank Dr Stephen Mayfield and Dr Jason Tanner for critically reviewing the manu- script. Financial support for this study came from Fisheries Re- search and Development Corporation (Project 96/160). LITERATURE CITED Anger, K. 1987. The D„ threshold: a critical point in the larval developnienl of decapod cru.staceans. J. Exp. Biol. Ecol. 108:15-30. Bligh, E. G. & M. A. ScoU. 1966. Blood lipids of the lobster. Hoimuus americcmus. J. Fi.sh. Res. Bd. Can. 23:1629-1631. Chang, E. S. & J. D. O'Connor. 1983. Metabolism and transport of car- bohydrates and lipids. In: L. H. Mantel, editor. The biology of Crusta- cea. Vol 5: Internal anatomy and physiological regulation. New York: Academic Press, pp. 263-287. Cockcroft. A. C. 1997. Biochemical condition as a growth predictor in male west-coast rock lobster {Jasiis lalandii). Mar. Fresh. Res. 48:845- 856. Dall. W. 1981. Lipid Absorption and utilisation in the Norwegian lobster. Nephrops non'ei>iciis (L.). J. E.xp. Mar. Biol. Eeol. 50:33— 15. Fraser. A. J. 1989. Triacylglycerol content as a condition index for fish, bivalve and crustacean larvae. Can. J. Fish. Aqiial. Sci. 46:1868-1873. Geddes. M. C. S. R. Bryars. C. M. Jones. B. J. Crear, P. R Hart. C. Thomas & L. Linton. 2000. Determination of the optimal environmental and system requirements for juvenile and adult rock lobster holding and grow-out. Fisheries Research and Development Corporation Report 98/305: 141 pp. Leavitt D. F & R. C. Bayer. 1977. A refractometric method of determining serum protein concentrations in the American lobster. Aquaciilture 12: 169-171. Mercaldo-Allen. R. 1991. Changes in the blood chemistry of the American lobster, Homarus amerieanus. H. Milne Edwards. 1837. over the moult cycle. J. Shellfish Res. 10:147-156. Musgrove. R. J. B. 2000a. Moult staging in the southern rock lobster y 100 mm CW molt on the order of months (Miiliken and Williams 1984). Shedding of the exoskeleton (i.e.. ecdysis) occurs when 241 242 Chaves and Eggleston crabs secrete a new exoskeleton within the old one. The old exo- skeleton then cracks along suture-lines and the crab exits the old shell with a sofl-shell that is larger than the old one. Four or five hours after molting, the soft shell gradually hardens. Crabs in the soft-shell industry are collected shortly after molting and before the shell hardens. When crabs begin to secrete their new shell, a white-line becomes visible inside the cuticle of the crab's last appendage or swimmeret. This white line indicates that the crab will molt within two weeks. As molting time nears. the indicator line gradually changes color: a pink line peeler will molt within 1 wk. and a red-line peeler will molt within 3 days (Oesterling 1984). North Carolina 's Soft-Shell Blue Crab Industry Soft crab landings in North Carolina ha\e made up l.69r of the total blue crab landings for the past 8 y (-6,166.160 lbs), but the value of this fishery has averaged 6.6% of the total during that same time and increased to nearly 10% during 2001 (-$3,336,990; North Carolina Division of Marine Fisheries 2002). The increase in value of the soft crab fishery in North Carolina may be attrib- utable to drastic declines in the blue crab population and in hard crab catch (Eggleston et al. 2002). and increasing local, regional, and worldwide demand (Oesterling 199.'i). In North Carolina, peeler crabs are trapped as by-catch in the hard crab fishery using hard crab pots or targeted directly using peeler pots. Hard crab pots are constructed of 3.8-cni wire mesh, fitted with at least two escape rings of 5.9-cm inside diameter (North Carolina Division of Marine Fisheries 2002). and are baited with dead fish. Peeler pots are constructed of 2.54-cm mesh and are not fitted with escape rings because there is no size limit on peeler crabs (North Carolina Division of Marine Fisheries 2002). Peeler pots are either unbaited or are baited with a mature male crab whose urine may attract prepubertal female peeler crabs (Ryan 1966). Prepubertal female peeler crabs are attracted to male blue crab urine because they are only able to copulate during a brief period of 2-3 h after ecdysis. Two types of shedding systems, open and closed re-circulating, are used primarily in North Carolina. In open systems, water is pumped into shedding tanks from a nearby source such as a creek or bay. and drains back into the same water source. In closed systems, tanks are either tilled with well water and aquarium salt added, or water is trucked in from the nearest suitable source. Water drains into a biologic filter tank and is continuously pumped back into shedding tanks. Nitrogen fixing bacteria in filter tanks reduce the toxicity of ammonia in the water by reducing it to nitrite and then to nitrate (Wheaton 1977). Management and Operational hsues The rapid growth of the soft-shell blue crab industry in North Carolina and elsewhere has outpaced the generation of certain information to address key management and operational issues concerning this fishery. Input on key management and operational issues concerning the soft-shell crab industry in North Carolina were provided through direct communication with the North Caro- lina Division of Marine Fisheries (NC DMF) and through a series of public workshops that sought input from commercial crabbers as a part of the North Carolina Fisheries Resource Grant Program. administered through North Carolina Sea Grant. Specific manage- ment questions are described below. 1. Do white-line peelers in the soft-shell blue crab industry suffer relatively high mortality rates caused by long holding periods (e.g., held for weeks) compared with red-line peeler stages (e.g.. held for days)'? (Henry and McKenna 1998). 2. Are overall mortality rates of crabs in shedding operations relatively high, and does crab mortality vary with crab size? (S. McKenna. NC DMF, personal communication). Cur- rently, there is no size limit on peeler crabs in North Caro- lina. Specific questions raised by soft-shell crab shedders in NC during public workshops are described below. 1. Do peelers purchased by shedders suffer higher mortality than those they caught? 2. Is peeler crab mortality caused by low dissolved oxygen and high temperatures? 3. Is peeler crab mortality higher in closed than open systems? 4. Is peeler crab mortality elevated for crabs captured in hard crab pots as opposed to peeler pots' 5. Does peeler mortality increase with the crab density in hold- ing tanks? 6. Is peeler crab mortality and time-to-molt higher in males than females? Is male peeler crab mortality disproportion- ately high in the presence of female peelers? Such untested questions are the basis of at least one current regulation in North Carolina. For example, the NC DMF prohibits harvest of white-line peelers after June I each fishing season be- cause of assumed high mortality during summer months. More- over, much of the hypothesis testing in the present study was driven by the collective observafions of commercial crab shedders. The overall objectives of this study were to address the man- agement and operational questions raised above by quantifying: 1 ) mortality rates of white-line versus red-line peelers; 2) size- specific mortality rates of crabs in shedding systems; 3) mortality rates of peelers as a function of crab source (purchased or self- caught), system type (closed versus open), and gear (hard crab pot vs. peeler pot); 4) the relationship, if any, between peeler mortality and water quality parameters such as dissolved oxygen, tempera- ture, salinity, and nutrients; 5) the effects of crab sex on peeler mortality rates; 6) the effects of female crab presence or absence and molt stage on time-to-molt and survival of male crabs; and 7) the effects of crab density on time-to-molt and survival of male crabs. This information should lead to improvements in shedding technology, better fishery management, and improved profits. METHODS AND MATERIALS Study Locations Data were collected in collaboration with 1 1 different commer- cial crab shedders throughout coastal North Carolina (Fig. I ) from May until October 2001. Study locations were selected to represent a broad spectrum of water quality while simultaneously providing replicated closed and open systems and replicated use of purchased versus self-caught crabs (Chaves 2002). Both large-scale .seafood producers and small backyard operations were represented in this study. Seven locations used closed systems, three used open sys- tems, and one location used both an open and a closed system (Chaves 2002). Crab Collection Crabs were captured by commercial fishermen using hard crab and peeler crab pots. After capture, peeler crabs were either stored Blue Crab Mortality in North Carolina 243 Chaves^ Egglesion. Moruliiy of sofl crabs \ Elizabeth City ■, 1^ Albemarl Vv ar/e SountT ^ ** ^^ w • 4^ Pamlico Sound Cape Hatteras :»i^W t N Wilmington Cape Lookout A a an tic Ocean Cape Fear Figure 1. Map of North Carolina showing study locations (stars). in wooden baskets on the deck of a boat or were placed in coolers on ice. Once landed at the dock, crabs were placed in nearby shedding tanks or trucked to shedding operations up to 200 km away. At each of the 1 1 crab shedding study locations, premolt crabs were sexed and their carapace width measured (mm CW). Crabs were separated according to peeler stage (red-line vs. white- line) and then equally distributed among four experimental tanks measuring 1 .2 m wide x 2.4 m long and 20 cm deep. The crab sizes used ranged from 5-17.2 cm CW. A total of 49 experiments were conducted. A single experiment could last for -6 days or -21 days for red-line or white-line peelers, respectively, to allow crabs enough time to molt. Some shedders conducted experiments in either closed or open systems, using only purchased or self-caught crabs. Other shedders would conduct simultaneous experiments with red-line and white-line peelers that were self-caught. Others might sw itch systems and crab source from one month to the next. For example, a shedder might conduct an experiment with only purchased crabs in a closed system in one month, followed by an experiment using only self-caught crabs in an open system the next month. Each experiment at a specific location was treated as a single independent replicate, since a new grouping of crabs from varying sources was placed in the shedding tanks at the initiation of each experiment, and the experimental methods were standard- ized across locations. In the following Methods and Results .sec- tions, the objectives of the study are described within the topics of operational (system, crab source, gear, water quality) and biologic (crab molt stage, sex, density, size) considerations. Operational Cuiisideratiom Kffiits ot \\ ater Quality on Crab Mortality To quantity the effects of water quality on crab mortality, the following water quality parameters were measured daily at -0800 h; dissolved oxygen (DO) (mg/L), temperature (°C), salinity (parts per thousand; ppt), pH. and concentrations of nitrite (mg/L), nitrate (mg/L). and ammonia (mg/L). A weighted mean calculated from the number of crabs that died in each experimental tank per day divided by the number of crabs in the tank on that day was used. Thus, the response variable in all cases dealing with crab moilality was a weighted percent mortality/day. Tanks with red-line crabs were monitored for 6 days and tanks with white-line crabs were monitored for up to 21 days. If all crabs in a tank shed or died before the 6- or 2 1 -day period, the experiment was terminated. The experimental unit was each tank, and four leplicate tanks were used at each of the 1 1 sites. Statistical analyses used a multiple regression model with crab source (purchased vs. self-caught) as the independent variable and water quality parameters as independent continuous variables. In this case, crab source was highly significant (see below), which confounded mortality associated with the source of crabs and wa- ter quality parameters. Thus, in subsequent statistical analyses, the data were first divided into separate categories of self-caught ver- sus purchased crabs. In assessing the effects of water quality on crab mortality, we then used a backward, stepwise multiple regres- sion model. Alpha to enter and remove factors from the model was 0.10. A Levene's Median test assessed constant variance among the responses and a Kolmogorov-Sminiov test tested for normal- ity. In cases where the data failed to meet the assumptions, the data were transformed using ArcSine or log 10 transformations, which were successful in all cases. Effects of Shedding System, Crab Source. Gear Type, and Crab Density on Crab Mortality The mean daily crab peicent moitality in closed versus open systems, between self-caught versus purchased crabs, and between crabs caught in hard crab pots versus peeler pots was compared with three separate one sample / tests. The relationship between mean daily crab density and mean daily crab proportional mortal- ity pooled across 23 experiments using self-caught crabs and 26 experiments using purchased crabs was examined with a linear least squares regression model. Biologic Considerations Effects of Crab Sex, Size, and Peeler Stage on Crab Mortality. To quantify the effects of crab sex and peeler stage on crab percent daily mortality, we compared the mortality of male versus female crabs, and white-line peelers versus red-line peelers using an analysis of covariance (ANCOVA) model with crab sex and peeler stage as factors and crab size as a covariate. The data were noi'iiially distributed and variances were homogeneous. Effects of Female Crab Presence on Mortality and Time-to-Molt in Male Crabs The effects of female crab presence and their molt stage on the time-to-molt and moilality of male crabs were examined in a closed shedding system in Swan Quarter, NC. Twenty tanks, mea- suring 1.2 m wide x 2.4 m long and 20 cm high were filled with estuarine water from Albemarle Sound to a height of 15 cm. Once the tanks were filled, the pump was turned off and water was not allowed to circulate between tanks, thereby preventing any poten- tial pheromone contamination across tanks. Tanks were aerated by aquarium air pumps. Crabs were purchased from several fishermen and randomly assigned to one of the following three treatments: 1 ) one red-line male per tank (control |; 2) one red-line male and one intermolt female per tank; and 3) one red-line male and one red- line female per tank. All crabs were visually examined houriy to record the time that thev molted or died. When a red-line male crab 244 Chaves and Eggleston molted or died, the trial was stopped. Each male red-line peeler was an experimental unit and each treatment was replicated seven to nine times. We tested whether there was a treatment effect on a male crab's time-to-molt and percent daily mortality with a one- way analysis of variance (ANOVA). The data were log- transformed to meet assumptions of normality and homogeneity of variance. Effects of Increasing Male Crab Density in the Presence of a Red-Line Female on Mortality and Time-to-Molt of Male Crabs The effect of increasing male crab density on percent daily mortality and time-to-molt in male crabs was also tested at Swan Quarter, North Carolina. Four treatments were randomly inter- spersed among tanks: 1 ) one red-line male per tank [control]; 2) one red-line male and one red-line female per tank: 3) one red-line male, one red-line female, and one intermolt male per tank; and 4) one red-line male, one red-line female, and three intermolt males per tank. Each male red-line peeler was an e.xperimental unit and the response variables were time-to-molt and percent daily mor- tality. Each treatment was replicated five to se\en times. Effects of Crab Sex on Time-to-Molt Time-to-molt of male versus female crabs in the absence of other crabs was quantified in separate experiments at Swan Quar- ter. North Carolina. This experiment was conducted to determine if males simply took longer to shed than females regardless of any other factors such as presence of females or increasing crab den- sity. Each tank contained a single male or female red-line crab. The response variable was time-to-molt in hours. Each crab was an experimental unit and each treatment was replicated 10 times. The LIFETEST procedure in SAS was used to compare the distribution of male's time-to-molt in the presence and absence of red-line females and other male crabs. The data was right censored (experiments ended before a response could be observed) due to the early termination of several trials when male crabs died before molting. The censored data points can not be left out of the analy- sis because crabs that take longer to molt are also more likely to die. The LIFETEST uses both censored and uncensored times to molt when comparing distributions of times to molt for various treatments. An uncensored data point is an actual observation of the time-to-molt. but the time-to-molt for censored data points is a calculation based on the distribution of times to molt among non- censored data points. Chi-Square tests were used to detect differ- ences in mortality between treatments, and ANOVA was used to detect differences in the time-to-molt between male and female crabs. RESULTS Operational Considerations Effects of Water Qualit> on Crab Mortality Water quality was somewhat poorer in closed than open recir- culating systems (Table 1). For example. DO was lowest (2.9 nig/L) and nitrates highest (77.4 mg/L) in closed systems. Never- theless, most of the water quality values were well within tolerance limits of blue crabs (Manthe et al. 1983). The percent daily mor- tality of self-caught and purchased peeler crabs did not vary sig- nificantly with any of the water quality parameters recorded (mul- tiple regression; self-caught: all P > 0.08. purchased: all P > 0.16). Effects of Crab Source, Shedding System. Gear Type, and Crab Density on Crab Mortahty Mortality of peeler crabs was significantly higher for purchased than self-caught crabs (f test; t = -2.22, df = L.'iO, P = 0.03; Fig. 2). Shedding system type (i.e.. open vs. closed) did not signifi- cantly affect the mortality of self-caught it test; t = 1.23. df = 1,48, P = 0.22) or purchased crabs (r test, t = 0.32. df = 1.44, P = 0.15). For self-caught crabs, there was no difference in crab mortality between crabs caught by peeler pots or those caught by hard crab pots (peeler pots: mean daily percent mortality = 2%, SE = 0.007, /; = 16; hard crab pots; mean daily percent mortality = 3%, SE = 0.006, /I = 8; r = 0.54, df= 1,22, P = 0.60). We were unable to test the effects of gear type on mortality of pur- chased crabs because all purchased crabs came from hard crab pots. Surprisingly, the percent daily mortality of red-line male peel- ers decreased with increasing density of peelers held in shedding tanks for both self-caught and purchased crabs (Fig. 3). The de- clining trend in percent daily peeler mortality with density was significant for self-caught crabs (linear least-squares regression: F = 14.27. df = 1.17. P<0.01; Fig. 3A). and marginally significant for purchased crabs {F = 4.05. df = 1.19. P = 0.06; Fig. 3B). Biological Considerations Effects of Crab Sex, Size, and Peeler Stage on Crab Mortality Mortality rates of self-caught crabs were unaffected by crab sex and the covariate of crab size (two-way ANCOVA; sex: F = 3.06. TABLE L Means and ranges of water quality parameters measured. DO (mg/L) Temp ( C» Sal ippt) Nitrite Nitrate .\nimonia pH Closed systems Mean 5,9 25.4 20.1 0.2 21.7 0.5 7.4 Low value 3,0 17.4 3.7 0 0 0.1 7.1 High value 8.2 30.8 39.8 0.5 77.4 3.2 8.0 Open systems Mean 6.7 26.4 17.3 0.1 3.6 0.3 7.4 Low value 3.6 19.5 5.0 0.0 0.0 0.2 6.9 High value 9.7 31.8 35.9 0.2 8.3 0.5 7.8 Nitrate, nitrite and ammonia are presented as mg/L. Blue Crab Mortality in North Carolina 245 0.20 1 0.15 •^ 0.10 o E 0.05 0.00 (A) Self-caught self-caught purchased Crab source Figure 2. Mean daily proportional mortality (+ SE) of self-caught (A' = 24) and purchased (A' = 27l peeler crahs. .Asterisk denotes sig- nillcant difference. See text for details of statistical test. df = 1.20. P = 0.10: size; F = 3.03, df = 1.20. P = 0.10); however, self-caught white-line peelers experienced significantly higher mortality rates than self-caught red-line peelers (molt-stage: F = 3.4, df = 1.20, P = 0.03; Fig. 4A). Mortality rates of purchased crabs were not affected by crab size (one-way ANCOVA; F = 0.02. df = 1.44, P = 0.88): however, purchased male peelers experienced significantly higher mortality rates than purchased female peelers (F = 10.04, df = 1.44, P < 0.01: Fig. 4B). It was not possible to determine the effect of crab stage on purchased crabs because no white-line peelers were purchased. Effects of Female Crab Presence on Mortality and Time-to-Molt in Male Crabs There was no significant difference in time-to-molt between male red-line peelers held alone, held with intermolt females, or held with red-line females (ANOVA; F = 0.13: df = 3.23; P = 0.718; Fig. 5A). There was also no significant difference in mor- tality between males that were held alone, held with intennolt females, or held with red-line females (Chi-square test: x""" = 4.14. df = 1,44, P = 0.13). Effects of Increasing Male Crab Density in the Presence of a Red-I.ine Female on .Mortality and lime-to-Molt of Male Crabs Time-to-molt in male peelers varied significantly according to whether a red-line female and intermolt male were also present (ANOVA: F = 13.06; df = 2.10: P < 0.01). In this case, time- to-molt was significantly shorter among males held with one red- line female and one green male compared with the control group of a single red-line male held alone (Ryan's Q-multiple compari- son test: Fig. 5B). There was no significant difference in daily percent mortality of male peelers in the presence or absence of red-line female and intermolt male crabs (Chi-square test; x" = 3.06, df = 3.50, P = 0.38). Effects of Crab Sex on Time-to-Molt The average time-to-molt for male crabs was significantly shorter than for female crabs (ANOVA; F = 14.21, df = 1,19. P <().01. Fig. 5C). 0.4 n • y=0.27-0.002' R^=0.44 p<0.0001 ►x 0.3 • \^^ ro 0.2 • ^\ o \^^ E 0.1 0.0 - • • ^^ •• • 0 20 40 60 80 100 120 mean density (crabs/tank) (B) Purchased y=0.21-0.0007*x R'=0.14 p=0.0594 140 0.00 50 100 150 200 mean density (crabs/tank) 250 Figure 3. Relationship between mean daily proportional mortality of (.\) self-caught (,V= 18) versus (15) purchased (A" = 20) peeler crabs and mean density per tank. DISCUSSION The soft-shell blue crab industry is one of the fastest growing fisheries in North Carolina. In this study, we collaborated with a team of commercial crab shedders across 1 1 different locations spanning the entire North Carolina coast to address key manage- ment and operational questions intended to better manage the blue crab resource, improve shedding technology, and increase profits. The key findings were as follows; 1 ) significantly higher mortality of white-line than red-line peelers; 2) daily mortality rates of 10- 30% per tank (primarily poorly handled red-line peelers) in shed- ding systems, but no effect of crab size on mortality rates: 3) no relationship between moilality of peelers and water quality param- eters, such as DO, tempeiature, salinity, and nitrates: 4) signifi- cantly higher mortality of peelers purchased by crab shedders than peelers caught by the shedders: 5) no significant difference in peeler mortality between closed and open systems, or between those crabs captured by hard crab pots or peeler pots: 6) decreasing peeler mortality with increasing density of peelers in holding tanks; 7) significantly higher mortality rates for male than female 246 Chaves and Eggleston (A) Self-caught 0.20 n 0.15 (A) ^ 0.10 o E 0.05 0.00 white-line red-line Molt stage (B) Purchased U.4 0.3 J * ^ ^ 0.2 O T E 0.1 males females Crab sex Figure 4. Mean daily proportional niorlality (+ SE) of (A) self-caught white-line (h = 15) versus (B) red-line peelers (A' = 37), and (B) pur- chased males (A' = 21) versus females (// = 25). Asterisk denotes sig- nificant difference. See text for results of statistical tests. peelers, and significantly lower time-to-molt for males than fe- males; 8) no significant increase in male peeler mortality or time- to-molt in the presence of red-line females; and 9) a significant decrease in a male red-line peeler's time-to-molt in the presence of a red-line female and inlermolt male. Blue Crab Management Issues White-Line Peelers Mortality of self-caught white-line peelers was significantly higher than self-caught red-line peelers. We were unable to assess the effect of molt stage on purchased peelers because commercial shedders do not shed white-line peelers for fear of high mortality rates. White-line peelers in this study probably experienced higher mortality rates than red-line peelers because of the relatively long periods of time required for them to molt, in which they are more likely to suffer from accumulated stress, as compared with red-line peelers. Most crabbers will feed white-line peelers if they will eat. 100 ~ 80 e 3 o S 60 40 o E o 0) E ■^ 20 20 T T control 1 green female 1 red-line female (B) 120 100 O 80 I 60 I o % 40 E * * T control 1 red 1 red 1 red female female female 1 green 3 green male males (C) 120 1 -. 100 (A o 80 o 60 o Z 40 E 20 0 * male female Crab sex and molt stage Figure 5. Mean 1+ SE) time-to-molt for male red-line peelers as a function of crab sex and molt stage. Green = intermolt crabs. N = 7-9. Asterisk denotes significant differences between treatments. See text for results of statistical tests. Blue Crab Mortality in North Carolina 247 Depending upon temperature and where a crab is in the moh cycle, white-hne peelers cease to feed somewhere between 10-21 days hetore moltinL' (pers. obs.). The peeler crab fishery in NC is regu- lated on the assumption that male white-line peeler mortality is very high during summer months, and that to keep them would be a wasteful practice. Our results support this management practice; however, it is important to note that the mortality of self-caught w hile-line peelers was similar to that of purchased red-line peelers (compare Fig. 2 and 4A), highlighting the importance of crab source as a key determinant of peeler mortality. Oesterling (1984) suggested that it is not economical to keep white-line peelers for more than 10 days, and various coastal Sea Grant extension agents have urged crabbers that harvest peelers to take better care of them after capture. OMTiill and Size-Specific Crab Mortality Rates. Mortality rates of red-line peelers placed in shedding tanks in this study averaged \59( per day. This daily mortality rate is ex- tremely high when compounded over the typical 5-day duration of shedding, and is dramatically higher than natural mortal it\ rates. For example, the annual mortality rate for sub-adult and adult blue crabs is 50% (Eggleston 1998). For a hypothetical, yet realistic example of how mortality in shedding systems is compounded over time, assume that 100 crabs are placed in a shedding tank on Day 1. On Day 2, -15% of the crabs would have died and 507r would have molted successfully and been sold. This would leave 35 crabs on Day 2. Assuming these same daily percentages of death and successful molting, we would have five dead crabs and 17 crabs nu)lting successfully on Day 3, a total of two dead crabs and seven molting successfully on Day 4, and one dead crab and two molting successfully on Day 5, after which there would be one crab left. Thus, out of a starting population of 100 peelers in the shedding table, an average of 23 (23% ) would die iiver 5 days. The cumulative mortality for white-line peelers would likely be even higher since they are generally held two to four times longer than red-line crabs before they molt. Depending upon method of harvest and crab source, a high mortality of peelers could be expected mimediately after crabs are stocked, with a large decrease in mor- tality as stronger crabs shed. We did not detect any effect of crab size on crab mortality rates, contrary to popular belief that male peeler crab moilality increases with crab size, especially when males become very large. There may not have been enough contrast in our data to reveal a positive relationship between crab size and mortality since male peelers >16 cm CW were rarely observed. The belief among crabbers that male peelers experience high mortality is so prevalent that many have given large male soft-shell crabs the nickname "miracle crabs." Operational Considerations Source of Peelers Crab source was always the single greatest source of variation in crab mortality rates, with highest inortality in purchased peelers. Variability in mortality because of crab source is likely caused by different handling methods used by crabbers who shed their own peelers versus crabbers who sell peelers. Crabbers who shed their own peelers put forth great effort to ensure the survival of their peeler crabs, such as carrying a cooler with ice and wet burlap bags in their boats, which keep crabs cool and moist. Crabbers who sell peelers usually place peeler crabs in a wooden basket on the deck of their boat, unprotected from bright sun. wind, and extreme heat. Purchased peeler crabs may also experience significantly higher mortality rates than self caught peelers because they are more likely to travel greater distances from the point of capture to shed- ding systems. Long travel times may cause crabs to dehydrate, but also increase the likelihood that crabs will experience large changes in salinity from the point of capture to shedding tanks. Although blue crabs are euryhaline organisms that can survive in a broad range of salinities from 0 ppt to over 40 ppt, sudden large changes in salinity (i.e.. > 10 ppt in less than 24 h) may exceed a crab's ability to osmoregulate the tissues in its body, causing mor- tality (Engel and Thayer 1998). Although we were unable to record changes in salinity from the point of capture to a given shedding system, these salinity changes may be another source of stress, in addition to poor handling, leading to significantly higher mortality rates of purchased than self-caught peelers and is worthy of further research. If large changes in salinity prove to be an important source of stress for peelers, then shedders capable of regulating salinity in their systems could alter the salinity to accommodate the peelers they buy. A crabber who sells poorly treated peelers is unaffected by high mortality rates that may occur in shedding systems because market demand for peeler crabs ensures that they will receive top dollar for these peelers, despite their relatively poor treatment. One of the most obvious ways to reduce crab mortality in North Carolina's soft-shell crab industry is for shedders to capture their own crabs rather than rely on purchasing peelers. The majority of all peeler crabs in North Carolina shedding systems, however, are purchased crabs (Tony Roughlon, Seafood dealer and commercial fisherman, Columbia, NC. personal communication), and the data in this study indicate that purchased peeler crab mortality is 1 1% greater than self-caught crabs. The 11% difference in mortality rates of self-caught and purchased peelers could easily equate to a financial loss of over $776,044 per year for crab shedders alone (see Appendix for calculations). The relatively high mortality of purchased crabs could probably be reduced greatly by taking better care of peelers on the boat, such as using coolers or wet burlap sacks to keep crabs cool, inoist, and out of the sun and wind, and reducing the travel times between initial harvest, and placement in shedding systems. If the mortality rate of purchased crabs were reduced to that of self-caught crabs, the soft crab industry would increase in value by 11% ($776,044) without any increases in harvest size. Effects of Crab Density on Crab Mortality Mortality of both self-caught and purchased crabs decreased as crab density increased, in contrast to expectations. It is possible that crabs abandon aggressive behavior that causes mortality once density is increased to a certain level, as evidenced by some spe- cies of fish that abandon territorial behavior if density surpasses a certain threshold (Dr. Jon Shenker, Fl. Inst. Tech., personal com- munication). Many shedders feel that peelers can be stocked at extremely high densities (>250 crabs/tank) without any hannful effects as long as good aeration is maintained in the tanks and all crabs placed in the tank are red-line peelers. Failure to carefully examine the molt stage of each crab placed in tanks allows the 248 Chaves and Eggleston accidental entry of both intermolt and white-line peelers, which are known to cannibalize red-line peelers and soft crabs. Isolating crabs from each other may be a highly effective method of reducing crab mortality. One of the shedders in this study reduced mortality in his system by placing 100 plastic mesh cylinders in each tank to isolate crabs from each other. The cyl- inders were originally designed to eliminate cannibalism, but the shedder felt that the reduction in mortality was much greater than what would have been caused by cannibalism alone. Crab mortal- ity at the study location where the cylinders were used was con- sistently the lowest in the entire state during our study. Eliminating physical interaction between crabs may greatly reduce crab stress. and therefore reduce mortality. Although the use of mesh cylinders in shedding tanks also reduced the density of crabs that could be held in a shedding table, the lack of a decline in mortality with decreasing crab density in this study suggests that creating physi- cal barriers between crabs by using mesh cylinders explains the reduction in mortality of peelers. Most shedders that we spoke with were reluctant to try isolating crabs in tanks because they felt that cylinders would greatly reduce the capacity of shedding tanks during large runs of peeler crabs when tanks are stocked at den- sities of 200-300 crabs. These peeler runs only occur two or three times each year, however, and it is not likely that peeler supply would exceed the capacity of 100 crabs per tank during the rest of the shedding season. Biologic Considerations Effects of Crab Sex Crab sex had a significant effect on the mortality of purchased crabs but not on the mortality of self-caught crabs. Commercial crab shedders that purchased crabs report that they always ob- served higher male mortality than female mortality, whereas shed- ders that caught their own peelers reported that they never ob- served higher male mortality than female mortality. Several crab- bers have suggested that high male mortality associated with purchased crabs is the result of aggressive encounters that males experience in hard crab pots. We could not statistically test for a significant interaction between crab sex. crab source, and gear type on crab mortality because nearly all purchased crabs were caught in hard crab pots, and nearly all self-caught crabs were caught in peeler pots. Nevertheless, relatively high male crab mortality did coincide with the use of hard crab pots and not with peeler pots. Female peeler crab mortality may not be affected by the use of hard crab pots because female peeler crabs that enter hard crab pots are usually cradled with a pre-copulatory embrace by a male crab immediately after entry into a crab pot (personal observation i. The male crab protects the female from other crabs and attempts to mate with her (Dell Newman, commercial fisherman. Swan Quar- ter. NC. personal communication). Alternatively, when a male peeler crab enters a hard crab pot. he is not protected from ag- gressive encounters with intermolt crabs, and may experience in- juries or sub-lethal stress that will not become manifest until he is placed in a shedding system and dies. In a peeler crab pot. inter- molt crabs are rarely present, so males and females do not encoun- ter aggressive intermolt crabs. Whether peeler crabs face sub-lethal aggressive encounters with aggressive intermolt crabs in hard crab pots is unknown, but further research in this area may explain the higher mortality of purchased male than female peelers. Effect of Female Crab Presence and Increasing Male Density on Mortality and Tinie-to-Molt in Male Crabs Male red-line peelers held with a relatively low density of intemiolt male crabs experienced signit~icantly shorter times to molt than control crabs, but time-to-molt did not differ signifi- cantly between male red-line peelers held v\ith a relatively high density of intennolt males and the control treatment of no conspe- cifics. This result was contrary to the expectation that increasing male density would lead to longer times-to-molt. The biologic explanation for the decreasing time-to-molt of male peelers with a low density of intermolt male crabs is unclear and warrants further experimentation. Male red-line peelers experienced significantly shorter times to molt than female red-line peelers, contrary to our expectation that time-to-molt would be equal among males and females or that males might experience longer times to molt than females. The findings in this study differ from the opinions of many crab shed- ders that report male red-line peeler crabs in shedding tanks take longer on average to molt than female red-line peeler crabs. Fur- ther investigation might reveal the extent to which intermolt peri- ods differ between male and female blue crabs. In this study, male peeler crabs showed no ability to regulate their time-to-molt in response to different situations (i.e.. the presence of red-line fe- male, different levels of conspecific density). CONCLUSIONS Our study revealed sources of mortality in the North Carolina soft-shell blue crab industry that fisherman are capable of elimi- nating. The survival of self-caught peeler crabs is significantly higher than for purchased crabs. Implementing best management practices in the soft crab industry could encourage crabbers to take better care of peeler crabs by always placing them in a cooler on ice immediately after capture or underneath wet burlap sacks. The benefits of best management practices will likely include a reduc- tion in the mortality rate of peeler crabs in shedding systems, increased financial profits for crabbers who sell peelers that are now more likely to survive in shedding systems, and improved profits of shedding system operators who purchase peeler. It is important to reduce mortality in North Carolina's soft-shell blue crab industry because 1 ) soft-crab landings are increasing rapidly and becoming a larger component of overall landings; 2) approxi- mately 23% crabs placed in shedding systems die; and 3) there is an urgent need to conserve the blue crab spawning stock given the recent 80% decline and a highly significant stock-recruitment re- lationship for the blue crab in NC (Eggleston et al. 2002). ACKNOWLEDGMENTS We are extremely grateful to the blue crab shedding operators that participated in this project: Bob Austin, Murray and Kristina Bridges. Russel and Gerry Howell. Connie and Luke Ingraham, Santa Klotz and Jim Messina. Pam Mason. Dell Newman. Willy and Jake Phillips. Scott and Patti Rader. Tony Roughton and Vir- ginia Phelps, and Phillip Smith. We also thank Dr. David Dickey, Dr. Joe Hightower. Dr. Steve Rebach. Sean McKenna, David Nadeau. Geoff Bell. Eric Johnson. Todd Kellison. and Ashton Drew for scientific and editorial input. The authors thank the NC Sea Grant/Blue Crab Research Program (grant Ol-FEG-03) for funding this project, and Bob Hines. Dr. Steve Rebach. Marc Turano for their enthusiastic administration of this project. Blue Crab Mortality in North Carolina 249 LITERATURE CITED Arv. R. D.. Jr. & M. A. Poirrier 1989. Acute Tiwicity ot Nitrile In the Blue Crab iCcillinecle.s sa/'idiis). Progr. Fish Culiurisl 51:69-72. Chaves, J. C. 2002. Biological and operational factors causing mortality in North Carolina's soft shell blue crab industry. MS Thesis, North Caro- lina State University, Department of Marine. Earth & Atmospheric Sciences. Raleigh, NC 27695-8208 USA. 32 pp. Das, T. & W. B. Stickle. 1993. Sensitivity of crabs CaUmecU-s sapiihis and C similis. and the gastropod Siicmwnini luiemasumui to hypo.xia and anoxia. Mar. Ecol. Prog. Ser 98:263-274. Eggleston, D. B. 1998. Population dynamics of the blue crab in North Carolina: statistical analyses of fisheries survey data. Final Report for Contract M-6053 to the NC Department of Environmental Health and Natural Resources, Division of Marine Fisheries, 66 pp. Eggleston, D. B.. J. Hightower & E. Johnson 2002 Population dynamics of the blue crab Calliiwctes sapidus in North Carolina, Fishery Resource Grant report 99FEG-10 and 00-FEG-l I. 22 pp. Engel, D. W. & G. W. Thayer. 1998. Effects of habitat alteralion on blue crabs. / Sliellfr'.h Res. 17:579-585. Henry. L. T. & S. McKenna. 1998. Status and management of the blue crab fishery in North Carolina. / Shellfish Res. 17:465-468. Lakshmi. G. J.. C. M. Trigg. H. M. Perry & A. Venkataramiah. 1984. The effects of ammonia accumulation on blue crab shedding success, Mis- sissippi-Alabama Sea-Grant Consortium, Project R/RD-2, Ocean Springs, Mississippi. Manthe, D. P., R. F. Malone & H. M. Perry. 1983. Water quality tJuctua- tions in response to variable loading in a commercial, closed shedding facility for blue crabs. J. Shellfish Res. 3:175-182. Milliken. M. R. & A. B. Williams. 1984. Synopsis of biological data on the blue crab. Callinectes sapidus Rathbun. NOAA Technical Report Na- tional Marine Fisheries 1. FAO Fisheries Synopsis Number 138. North Carolina Division of Marine Fisheries 2002. Web-site (http:// www.ncfisheries.net) Oesterling. M. 1984. Manual for handling and shedding blue crabs {Calli- iieeles sapidus>. Virginia Institute of Marine Science, College of Wil- liam and Mary, Special Report 271, Gloucester Point, Virginia. Oesterling. M. 1995. The soft crab lishery. Virginia Marine Resource Bulletin 27:13-14. Ryan. E. P. 1966. Pheromonc: evidence in a decapod crustacean. Science 151:340-_341. Weis. J. S.. A. Cristini & K. Ranga Rao. 1992. Effects of pollutants on molting and regeneration in Crustacea. Am. Zool. 32:495-500. Wheaton, F. W. 1977, Aquacultural engineering. Wiley-Interscience. New York. APPENDIX Estimation of Annual Financial Loss for North Carolina's Soft-Shell Bine Crab Industry I sing Purchased Peeler Crabs Assume 2.565.434 purchased peelers are placed in shedders each year in North Carolina. Assume each dead crab represents a loss of $2.75 (purchase price = $0,75; lost revenue = $2.00). If one assumes a mortality rate of 16% (this study), then 410.469 dead crabs die at a cost of -$1,128,790 per year. Jouniiil of Shellfish Research. Vol. 22, No. I, 251-254, 2003. SEX-SPECIFIC RESPONSE TO DISTURBANCE IN A FIDDLER CRAB PABLO D. RIBEIRO.'"^* CAROLINA G. LUCHETTI," AND OSCAR O. IRIBARNE" ^UniversidaJ Nacional dc Mar del Plata. CC 573 Corrco Central. B7600WAG Mar del Plata. Argentina: and ^Universidad de Btieno.s Aires. Argentina CONICET. Argentina ABSTRACT Fiddler crabs are organism with an extreme sexual dimorphism. Male crabs have an enlarged claw used for sexual display and combat but not for feeding, which place them in foraging disadvantage when are compared with females. Given that avoiding disturbance (e.g., predators or human activity), courting, and feeding are incompatible behaviors, males should have different time budget to balance all the activities. In this study we experimentally evaluated the hypothesis that males of the Southwestern Atlantic fiddler crab Uca unif;i(arensis have a sex-specific response to disturbance. We performed an experiment where we applied an artificial disturbance (created by addition of flags). During a tidal cycle we found that males were more affected by disturbance than females. During the ebb tide, more males than females remained into their burrows because of the artificial disturbance. After disturbance (i.e.. when flags were removed) the male-to-female sex ratio on the surface increased in disturbed plots. However, once disturbance was interrupted the male-to-female sex ratio on previously disturbed plots differed from the observed in control plots, being smaller during the ebb tide and larger dunng the flood tide. The latter might indicate that male crabs increase their foraging effort to compensate the time they loss for feeding as consequence of disturbance. Disturbance also affected the proportion of courting males, but when disturbance was removed courtship returned to initial values of activity, which indicates that the cost of stop courting may be higher than cost of stop feeding. However, after 27 days of experimental disturbance comparison of body condition (dry weight in relation to their carapace width) showed no effect of disturbance, suggesting that males were able to compensate the decrease in feeding time. KEY WORDS: Uca unigiiayensis. fiddler crabs, disturbance, sex-specific response INTRODUCTION Fiddler crabs are interesting animals for studying the effect of sexual dimorphism on their behavior. Male tiddler crabs show an enlarged claw used for courtship displays (Crane 1975. Christy & Salmon 1984); its size gives them an advantage in combats, bur- row acquisitions (see Hyatt & Salmon 1979). and probably in mate acquisition. However, given that the enlarged claw is not used for feeding, male feeding efficiency is lower, which leads to different foraging strategies in males and females (Vaiiela et al. 1974. Weissburg 1993). Furthermore, the color of the claw makes males more conspicuous and visually detectable than females (Crane 197.5), and its size hinders escape from predators (Iribame and Martinez 1999). Thus, if the visual detectability by predators is related to predation rate (Utne-Palm 2000) male fiddler crabs may have higher predation insk than females because of their conspicu- ousness and their longer time they spend on the surface (although some field studies show the opposite situation; e.g.. Bildstein et al. 1989. P. Ribeiro. unpublished data). Fiddler crabs are intertidal organisms with surface activity (feeding or courtship) only during low tide remaining inside their burrows during high tide (Crane 1975. Wolfrath 1993). During the courtship season both sexes feed mostly during the first hours after the tide ebbs and then, males court (by waving their large chelae) until the tide start to fiood when they go back to feed (Crane 1975. Wolfrath 1993). However, males usually keep feeding longer than females before sheltering into their burrows while tide is flooding (Wolfrath 1993). This difference may be the product of lower feeding efficiency and/or higher energetic investment by males during waving. When a disturbance occurs (i.e.. predators, such as shorebirds. human activity) most tiddler crabs shelter into their burrows as a generalized antipredatory response (Frix et al. 1991. Iribame & Martinez 1999). However, the trade off between feeding, mating. *Corresponding author. E-mail: pdribeirCsmdp.edu.ar and survival may not be the same for males and females. There- fore, we expect the response to disturbance to be sex specific. For example, given the visual conspicuity of the enlarged claw . males should show an earlier response than females to disturbances or potential predators. However, given that males need to feed longer (Vaiiela et al. 1974), receding early inside buiTOWs may involve a higher cost in terms of food acquisition and loss of mating oppor- tunities. In this work, we experimentally evaluate the hypothesis that fiddler crabs show a sex-specific response to disturbance by study- ing the SW Atlantic fiddler crab Uca unignayen.sis (Nobili 1901 ). We predict the following responses to disturbance: ( I ) more males than females will shelter into their buiTows during ebb tide, given that the time available for feeding is still long and they may be. for their conspicuousness. at a higher risk of predation than females; (2) more females than males will shelter into their burrows during flood tide because time available for feeding is now short; (3) there will be an overall reduction in the time allocated to courtship during the whole tidal cycle; and (4) if males do not reduce the time allocated to courtship, their body condition will be affected. MATERIALS AND METHODS The study was conducted at the Mar Chiquita coastal lagoon (Argentina. 37°32' to 37''45"S and 57''19' to 57°26'W) from Feb- ruary 4 to March 3. 2000 (Austral summer). Uca uruguayensis occurs in the upper levels of the tidal flats, adjacent to the border of extensive marshes dominated by the cordgrass Spariiiia densi- flora (Spivak et al. 1991 ). We marked 16 plots (each 2 m long and 6 m width) parallel to the shoreline with four 50-citi height (30- mm diameter) iron stakes. Plots were arranged at the same tidal level and were separated from each other by 2 x 2 m areas. Eight plots were subjected to disturbance (disturbed) and the remaining eight plots were kept as controls (control). Treatments were sys- tematically assigned. Disturbance was applied by means of thirty flags consisting in iron stakes (30 cm high, homogeneously dis- 251 252 RiBEIRO ET AL. persed) with black and red nylon stripes (30 cm long. 2 cm width) added on their tips. Nylon stripes were easily waved by the wind and when approached to a crab induced it to shelter into its burrow. Control plots were without these nylon stripes but we walked on them to keep the same effect of setting up the nylon stripes as in the treatment plots. In all cases observation of crab behavior (focal census) were conducted using a 10 x 50 binoculars. 8 m from the plots and 5 min after the setting or extraction of the nylon stripes, to allow crabs reinitiate their activities after disturbance caused by the experimental setup. To assure that crabs were entering inside their burrows in re- sponse to disturbance, we quantified in 12 plots the density of crabs on the surface. In each plot we sampled one transect of 2-m long by 0.2-m wide counting the number of crabs. Then, in six of these plots we placed the nylon flags and quantified the number of crabs again following the same procedure. Finally, the nylon stripes were extracted and crabs were quantified again. Data was square root transformed to comply with the assumptions and two- factor repeated measures ANOVA (Neter et al. 1991 ) was used to evaluate the density of crabs on the surface in relation to treatment (disturbed-control) and the disturbance state (before-during-after; as the repeated measures factor). To evaluate the effect of disturbance on the sex ratio (males to females) and on the proportion of courting males we carried out an experiment encompassing a complete diurnal tidal cycle. The ex- periment began 4 h before low tide and finished 4 h after low tide. In disturbed plots, we applied two intervals of disturbance (from 4 to 2 h before low tide and from 0 to 2 h after low tide: thereafter during disturbance) and two intervals where disturbance was re- moved (from 2 to 0 h before low tide and from 2 to 4 h after low tide: thereafter after disturbance). To measure the sex ratio and the proportion of courting males we performed focal censuses. For this we started by randomly taking a male crab and then we succes- sively located the most near crab, which was sexed (a simple task because of the sexual dimorphism). This procedure was system- atically performed to reach a minimum quantification of 20 male crabs. For males we noted if they were feeding or courting (de- noted by the waving movement of the enlarged claw ). These ob- servations were performed for both periods of disturbance and for both periods where the disturbance was removed. Given that 4 hours since low tide represent the moment where crabs unplug (when ebbing) or plug their burrows (when flooding), the 4 h of disturbance affected the 50'/r of the available surface time. To fit parametric assumptions (Neter et al. 1991 ) the sex ratio was trans- formed to the square root of data and the proportion of courting males was transformed with the arc-sin of the square root of data. A three-factor repeated measures ANOVA (Neter et al. 1991 ) was used to evaluate the effect of treatment (disturbed-control), tidal state (ebb-tlood: repeated measures factor) and disturbance state (during-after: repeated measures factor) on 1) sex ratio on the surface and 2) the proportion of couiling males. We conducted a 27-day experiment to evaluate the effect o\' disturbance on the body condition of crabs. For this, during the diurnal tidal cycle of all days of this period we applied two inter- vals of disturbance and two intervals where the disturbance was removed (similarly as was explained before). After 27 days, 10 adult males and 10 adult females (carapace width larger than 9 mm) were sampled from each plots (a total of 80 crabs of each sex), measured (maximum carapace width, precision 0.02 mm), and then dried at 70°C for 48 h and weighed (precision 0.001 g). Carapace width was log transformed to fit linearity of model (Neter et al. 1991). Differences in dry weight between treatments (disturbed-control) in relation to carapace width were e\aluated with ANCOVA (Neter et al. 1991 ). Given the allometric growth of the enlarged claw of males, regression equations of dry weight in relation to carapace width of males and females are not parallel (f i,.ii4= 59.0.\ MS,,,,,, = 0.1534. P < 0,01 ). thus we made the analysis for each sex separately. RESULTS There was an interactive effect of treatment and disttirhance state on the density of crabs (f, .„ = 10.13. MS^.„^.^.^ = 12.14, P < 0.001 ). The density of crabs on the surface before disturbance was applied did not differed between the plots to be disturbed and the plots to be maintained as controls (disturbed plots: .v = 20.83, SE = 3.71: control plots: x = 19.58, SE = 5.35). However, the density of crabs on the surface in disturbed plots was lower than in control plots during the disturbance (disturbed plots: .v = 1.25, SE = 0.51: control plots: .v = 18.33, SE = 2.26). Once disturbance was interrupted, crab density on the surface returned to initial values (disturbed plots: .v = 16.67, SE = 2.55: control plots: .v = 23.75. SE = 1.93). An interactive effect between Treatment. Disturbance State and Tidal State affected the male to female ratio on the surface (F, u = 9.32,M5„„„ = 0.2535, /*< 0.01; Fig. 1 A). During disturbance the male to female sex ratio was higher in control plots than in disturbed ones. Nevertheless, the male to female sex ratio in- creased in disturbed plots after disturbance. During the ebb tide. 10 a a Z LU t- a: <> => X LU LU I CO h- DURING AFTER u J EBB FLOOD Figure I, F.ffects of disturbance on the behavior of the fiddler crab Uca uriiguayinsis. (\) Male-lo-feniaie sex ratio on the surface and (B) proportion of courting males. Limits of boxes represent the (1.75 and 0.25 percentiles, lines represent the 0.01 and 0.99 percentiles, and the line inside boxes is the median. Different lowercase letters indicate differences from multiple comparisons for three factors interaction. Different numbers indicate differences from multiple comparisons for the Treatment X Disturbance State interaction. Significance is at P < 0.05. C = control plots, D = disturbed plots, EBB = ebb tide. FLOOD = flood tide, DURING = during disturbance, and .AFTER = after disturbance. Sex-Specific Response in a Fiddler Crab 253 the increase in the male to female ratio after disturbance was not large enough to surpass the \ alues observed at control plots. Dur- ing the flooding tide, however, the male to female ratio observed after disturbance exceeded the value observed at control plots. The proportion of courting males was higher during the ebb tide than during the flood tide iF, ,^ = 25.19. M5^.„,,, = 0.70Q4. P < 0.001 : Fig. 1 B ). However, it was lower during the disturbance at disturbed plots than either after the disturbance or at control plots (interaction effect between Treatment and Disturbance state f , ,4 = 22.18. MS^,,^.^., = 0.5884. />< 0.001; Fig. IB). There were not sianificant interactions between Treatment and Tidal State ^F, 0.06. MS, 0.001 6. P > 0.8) nor between Tidal State and Disturbance state (f ,.,4 = 0.02. M5,ff,„ = 0.0002. P > 0.9) nor between Treatment. Tidal State and Disturbance state (f j ,4 = 2.36. MS^.„,^, = 0.0370. P > 0.1) on the proportion of courting males. There were no effect of disturbance on the dry weight of both males (F, 0.43. MS,, 0.0019. P > 0.5; Disturbed plots. Slope = 2.20. SE = 0.04. Elevation = 2.03. SE = 0.04; Control plots. Slope = 2.48. SE = 0.03. Elevation = 2.34. SE = 0.04) and females (F,;„ = 2.60. MS,,,,,, = 0.0020. P> 0.1; Disturbed plots. Slope = 0.91. SE = 0.01. Elevation = 0.80. SE = 0.01; Control plots. Slope = 0.99. SE = 0.01. Elevation = 0.88. SE = 0.01). DISCUSSION Artificial disturbances are useful for the study of behaxioral responses of organisms by simulating natural environmental con- ditions (e.g.. Bell 2001. Sloman et al. 2001). Responses to distur- bances are helpful for understanding how organisms face critical trade offs under changes in their envii-onment. Our disturbance e.vperinients show that the fiddler crab Uca iiniguayensis has a sex-specific response. Disturbance decreased the male to female sex ratio on the surface, indicating that more males shelter into iheir burrows in response to disturbance. However, during the ebb tide and after the disturbance the male-to-female sex ratio in dis- turbed plots was lower than in control plots. This pattern was the opposite to the observed during the flood tide, which suggest that the proclivity of crabs to shelter and stay inside their burrows may depend on the time available for feeding before the tide flood their habitat. Given that males need to feed for longer periods as a consequence of the sexual dimorphism ( Valiela et al. 1974. Weiss- burg 1992). the cost of stop to feed may be higher for them when the remaining feeding time is short. Our study encompassed the effect of disturbance at a teinporal scale of days in relation to the body weight and at a temporal scale of hours in relation to the behavioral avoiding response. Other works analyzed the effect at a lower, practically immediate, tem- poral scale where they look at the direct effect of the disturbance in the avoidance response of crabs. Frix et al. (1991). found that both male and feinale fiddler crabs Uca piigiUitor and U. pugiui.y shelter into their burrows at similar rates when simulated predators approach them indicating, in fact, that both sexes may perceive a similar risk of predation. However, females descend further into their burrows than males. This pattern could be expected it the female is the most preyed sex. as is recognized to happen in the Frix et al. ( 1991 ) study case (see also Bildstein et al. 1989). For the case of Uca iiniguayensis. we did not investigated if feinales and males shelter at similar rates, but instead, we know that during the disturbance males spend less time on the surface. The dispropor- tionate effect on males that we have observed may be expected from a high predation rate on male crabs. However, there are not evidences of high shorebird predation in our study site (Bogazzi et al. 2001): but in nearby population of U. unigiuiyensis (Sambo- rombon Bay; 36°22'S. 56°45'W) predation by migratory shore- birds is intense (Iribarne and Martinez 1999). Nevertheless, the occurrence and nature of sex-specific predation pressure is likely to be dependent on the predatory species present at the locale and their abundance because some predators prefer females whereas other prefers males (Iribarne & Martinez 1999). In any case, it was observed that the overall effect of predation is not male-biased (Ribeiro et al. unpublished data). Thus, the male-biased response to disturbance in this species is not related with the extant sex- specific predation pressure. This response, therefore, might have evolved under other selective forces than extant predation pres- sure. This scenario can occur with a higher relative abundance of shorebirds that specialize on males, such as the Ruddy Turnstone Aremina imcipres (Iribarne & Martinez 1999). Disturbance also decreased the proportion of courting males, which after disturbance returned to values similar to those ob- served in controls. This is contrary to the expectation that males may increase their foraging effort if they loss the opportunities to do it by evading the disturbance (or potential predators). This response suggests that courtship is risky when disturbance is in action, given that the male waving display may enhance their vulnerability to predator (P. Ribeiro. unpublished data) However, despite males lose a larger proportion of time avail- able for feeding than females, their body condition was not af- fected as consequence of disturbance. This might be because dis- turbance was not so severe or the experimental period was not long enough. Alternatively this result may indicate that crabs were able to successfully compensate in some way for the time they loss as consequence of disturbance, which is potentially available for feeding. Given that males are less efficient foragers than females (Valiela et al. 1974. Weissburg 1992) the mechanism solving this trade off should incorporate changes in their foraging effort and changes in the mechanisms of food delivery and extraction (Weiss- burg 1993). The fact that the proportion of males increased after disturbance and that it was higher in disturbed plots than in control ones during the flooding tide strongly suggest that males are in- creasing their foraging effort after disturbance. ACKNOWLEDGMENTS This project was partially supported by grants from the Uni- versidad Nacional de Mar del Plata. IFS-Sweden (A2501-2F). Fundacion Antorchas (Argentina AO 13672). National Geograph- ic Explorafion Grants (#6487-99). CONICET (PIP2851). and ANPCyT (#1-7213). all granted to O. I.). P. D. Ribeiro is sup- ported by a scholarship from CONICET. This work is part of the doctoral thesis of the first author. Bell. A. M. 2001. Effects of an endocrine disrupter on courtship and aggressive behaviour of male ihree-spined stickleback. GiisrcmsifKs aculealus. Anim. Behav. 62:775-780. LITERATURE CITED Bildstein. K. L.. S. G. McDowell & I. L. Brisbin. 1989. Consequences of sexual dimorphism in sand fiddler crab. Uca pugilator. Differential vulnerability to avian predation. Anim. Behav. 37:133-139. 254 RiBEIRO ET AL. Bogazzi. E.. O. Iribame. R. Guerrero & E. Spivak. 2001. Wind Pattern may explain the southern limit of distribution of a southwestern Atlantic fiddler crab. J. Shellfish Res. 20:353-360. Christy, J. H. & M. Salmon. 1984. Ecology and evolution of mating sys- tems of fiddler crabs (genus Ucci). Biol. Rev. 59:483-500. Crane. J. 1975. Fiddler crab of the world, Ocypodidae: genus Uca. Prince- ton. NJ: Princeton University Press, 736 pp. Frix. M. S., M. E. Hostetler & K. L. Bildstein. 1991. Intra- and interspecies differences responses of Atlantic sand (Uca pugilator) and Atlantic marsh ((/. pugna.x) fiddle crabs to simulated avian predators. / Crusi. Biol. 11:523-529 Hyatt. G. W. & M. Salmon. 1979. Comparative statistical and information analysis of combat in the fiddler crabs. Uca pugilator and U. [nigiw.x. Behaviour 9S:\-2?>. Iribame, O. O. & M. M. Martinez. 1999. Predatlon on the southwestern Atlantic fiddler crab {Uca uruguayensis) by migratory shorebirds [Plii- vialis dominica. P. squatarola. Arenaria imerpres and Nwnenius phaeopus). Estuaries 22:47-54. Neter. J.. W. Wasserman & M. H. Kutner. 1991. Applied linear statistical models. Regression, analysis of variance, and experimental designs. Irwin. IL: Homewood. 1181 pp. Sloman, K. A., A. C. Taylor. N. B. Metcalfe & K. M. Gilmour. 2001. Effects of an environmental disturbance on the social behaviour and physiological function of brown trout. Aiiim. Behav. 61:325-333. Spivak. E. D.. M. A. Gavio & C. E. Navarro. 1991. Life history and structure of the world southernmost Uca population: Uca uruguayensis (Crustacea. Brachyura) in Mar Chiquita Lagoon (Argentina). Bull. Mar. Sci. 48:679-688. Utne-Palm, A. C. 2000. Prey visibility, activity, size and catchability's (evasiveness) influence on Gobiusculus flavescens prey choice. Sarsia 85:157-165. Valiela. 1., D. F. Babiec, W. Atherton, S. Seitzinger & C. Krebs. 1974. Some consequences of sexual dimorphism: Feeding in male and female fiddler crab. Uca pugna.x (Smith). Biol. Bull. 147:652-660. Weissburg. M. 1992. Functional analysis of fiddler crab foraging: sex specific mechanism and constraints in Uca pugna.v (Smith). J. Exp. Mar. Biol. Ecol. 156:105-124. Weissburg. M. 1993. Sex and the single forager: gender-specific energy maximization strategies in tiddler crabs. Ecology 74:279-291. Wolfrath. B. 1993. Observations on the beha\'iour of the European fiddler crab Uca langeri. Mar. Ecol. Prog. Ser. 100:1 11-118. Journal ,yf Shfllfish Research. Vol. 22. No. 1, 255-262. 2003. GEOGRAPHICAL EXPANSION OF A NONINDIGENOUS CRAB, CARCINUS MAENAS (L.), ALONG THE NOVA SCOTIAN SHORE INTO THE SOUTHEASTERN GULF OF ST. LAWRENCE, CANADA DOMINIQUE AUDET,' DEREK S. DAVIS," GILLES MIRON,'* MIKIO MORIYASU,^ KHADRA BENHALIMA,' AND ROBERT CAMPBELL' ' Universite de Monclon. Depurteiucnt tie biologie. Monctun. Nuitvemi-Bnmswick El A 3E9 Canada; 'Nova Scotia Museum of Natural History. 1747 Summer Street, Halifax Nova Scotia B3H 3A6 Canada; Department of Fisheries and Oceans Gulf Rei^ion. Science Branch P.O. Box 5030 Moncton. New Brun.'iwick EIC 9B6 Canada ABSTRACT The European green crab. Cuniiui.s maenus. was first observed in the western Atlantic in the I9th century (from New Jersey to Massachusetts, USA). A northward expansion along the coast of New England has been observed in the first half of the second century. The green crab was ob.served in Canadian waters in Passamoquoddy Bay in 1951. The species has gradually invaded the Bay of Fundy in the 1950s, and the Atlantic coast of Nova Scotia from the 1960s to the mid 1990s, and reached the southern Gulf of St. Lawrence in the mid 1990s. Further westward expansion in the southern Gulf of St. Lawrence has been confirmed along the eastern coast of Prince Edward Island in 1997 and more recently in the Northumberland Strait at the border between Nova Scotia and New Brunswick. KEY WORDS: Carciiiii.', iiiacnas. green crab, geographical expansion, nonindigenous crab, northwestern Atlantic, southern Gulf of St. Lawrence INTRODUCTION Accidental and voluntary introduction of species has occuired as a result of expanded human settlement and international trade. Over the past 200 years, the invasions were mainly due to shipping activities. Various species of invertebrates with free-swimming larvae were accidentally introduced into many coastal areas when ships using ballast water appeared around 1880 (Carlton 1985). Rui/ et al. (2000) suggested that about 298 species of invertebrates and algae have been introduced in marine and estuarine regions in North Ainerica. Crustaceans and mollusks constitute ca 50% of the intruders. The green crab. Carciiius niaenas (Linnaeus. 1758), is a good example of a species that is now well established in estuarine habitats around the world. Carciniis inaeiias was originally distributed along the eastern Atlantic coast, from Norway to Mauritania including southern Ice- land (Broekhuysen 1936. Crothers 1968, Grosholz & Ruiz 1996). This species was recorded on the northeastern American coast in 1817 (Say 1817). Sporadic introductions in Brazil, Hawaii and Panama Bay were also recorded in the second half of the 19th century (Smith 1880). Australian occurrences were first docu- mented about a hundred years ago in Port Phillip Bay. Victoria (Thresher 1997). The crab has since expanded its distribution from South Australia to New South Wales in the late 1970s (Zeidler 1978) and on the east coast of Tasmania in 199."^ (Gardner et al. 1994). The green crab was first recorded in South Africa near Cape Town in 1983 and is now well established (LeRoux et al. 1990). The species also colonized the San Francisco Bay area (California. USA) in 1989 to 1990 (Cohen et al. 1995). The present green crab distribution on the eastern Pacific coast lies between Morro Bay (South California. USA) (Grosholz et al. 2000) and Esperanza Inlet on the west coast of Vancouver Island (British Columbia, Canada) (Glen Jamieson, pers. comm.). This rapid and irregular expansion, which occurred from 1997 to 1999. could be related to 'Corresponding author. E-mail: mirongfe'unioncton.ca an El Niiio event during the saine period (Behrens Yamada & Hunt 2000). According to these investigators, the green crab's range expansion is limited off the northwestern American coasts since then because of a declining recruitment. On the northeast American coast, the green crab was first docu- mented in New York and New Jersey in 1817 and slowly migrated northward towards New England where it was reported in Casco Bay (Maine. USA) in the early 1900s (Rathburn 1905). Through the following 50 years, the species has colonized various estuarine habitats along the coast of Maine up to the Bay of Fundy in Canada (Scattergood 1952, Glude 1955. MacPhail et al. 1955). The green crab is a voracious predator of a wide range of invertebrates (Elner 1981) with preferences for bivalve species (Ropes 1968) (e.g.. American oysters [Cras.sostrea virginica], soft-shell clams |A/v(( arenciria]. blue mussels [Myliliis eihilis] and northern quahogs \Mercenaria mercenaria]). Aquaculture stake- holders in the southern Gulf of St. Lawrence (SGSL) expressed serious concerns about a potential threat to cultured and wild shell- fish populations in the Canadian maritime provinces. The purpose of this paper is to document the northeast expan- sion of the green crab in eastern Canadian waters, from the Pas- sainaquoddy Bay area in New Brunswick (NB) along the shores of Nova Scotia (NS) to the SGSL. The possible effects on the shell- fish aquaculture industry are also discussed. MATERIALS AND METHODS Musium .\rclnvcs and liiWrvietts Unpublished museum records were examined from the Nova Scotia Museum of Natural History (NSMNH) (Halifax. NS). the Atlantic Reference Centre (ARC) (St. Andrews, NB) and the Ca- nadian Museum of Nature (CMN) (Ottawa. Ontario) to complete the history of occurrence of C. macna.', along NS and NB coasts. Interviews were carried out among twelve eel fishermen and four fishery officers in the fall of 1998. Eel fishermen were chosen because they were fishing in potential green crab habitats, and fishery officers for their frequent contacts with various fishermen. 255 256 AUDET ET AL. Interviews were held in northern NS and western Cape Breton Island (CBI) to obtain information on the year and location of the first green crab occurrence in commercial catches. Survey Annual observations on the presence and absence of green crabs were made during the summer period (June to September) from 1997 to 2001. Forty-six stations (estuary and river systems) were chosen at an interval of 30-50 km along (1) the coast be- tween the southwestern region of Bras d'Or Lakes and the tip of CBI: (2) between the western coast of CBI and Shippagan along the NB coast; and (3) around Prince Edward Island (PEI). (Table 1 and see Fig. 2). A frozen mackerel was placed in a modified pearl lantern net (30 x 30 cm with two openings) and immersed to the bottom at each observation site for a duration of 15-30 min to determine the green crab presence. RESULTS AND DISCUSSION Northward Expansion of C. maenas Along the .Xew England Coast, USA Rafmesque (1817). as stated in Fowler (1912). reported the presence of the green crab off the coasts of Long Island. New York and New Jersey in 181 7. and Say (1817) confirmed the presence of the green crab in estuarine habitats off the Atlantic coast of the United States in 1817. Smith (1880) stated that the range of C. maenas was limited in northwestern Atlantic waters in 1871 and 1872. At the time, the crab seemed to be found in great numbers and well established in Great Egg harbor (New Jersey), on the southern coast of Long Island (New York), in Long Island Sound (Connecticut), and in Vineyard Sound. Buzzards Bay and Proxince- town (Massachusetts) (Fig. 1). Rathburn ( 1905) reported that the crab reached Maine's Casco Bay area (Eagle Harbor. Haipswell and New Meadows River) in 1905. Green crab observations, how- ever, in Maine at that time were scarce and the species was not considered a regular member of the community before 1935 (Scat- tergood 1952). According to Scattergood ( 1952), the northern limit of its distribution was near Winter Harbor (Maine) from 1939 to 1942 (Fig. 1 ). Dow and Wallace ( 1952) reported that the presence of green crabs at Lakeman's Harbor on Spruce Island near Jonesport was observed in 1919 by a lobster fisher. There were no further reports until 1948 and by 1951, green crabs were abun- dant in Jonesport and also found in Lubec in Passamaquoddy Bay (Fig. I). Expansion of C. maenas /;/ the Bay of Fundy A specimen of C. maeiuis was discovered in 1 95 I m the estuary of the Digdeguash Ri\er (Fig. 1) in Passamaquoddy Bay near Oven Head (NB) (Scattergood 1952. MacPhail 1953). Five green crabs were also collected the same year at the mouth of Magagua- da\ic Ri\er. near St. George (ARC unpublished records). A small number of crabs were found a year later in the western Bay of Fundy. Crabs were observed, however, in great numbers in the entire Passamaquoddy Bay in Pocologan Harbor and in the Le- preau Basin (NB) by spring of 1953 (MacPhail et al. 19551. They then spread eastward in the Bay of Fundy (Welch 1968) where it was reported in Sandy Cove on the northern shore of St. Marys Bay (NS) and at the mouth of the Pereau River in the Minas Basin (NS). both in 1953 (MacPhail et al. 1955) (Fig. 1). By 1958. green crab populations were established in Minas Basin (Bousfield & Leim 1960. NSMNH unpublished records). The eastward range expansion in NS was confirmed with the presence of one crab in Wedgeport in 1954 (MacPhail et al. 1955). A survey made from Cape Fourchu to Three Fathom Harbor (between Lawrencetown TABLE 1. Confirmed sighting of green crab iCareinus maenas) in the southern Gulf of St. Lawrence and adjacent area during the summer period (June to September) between 1997 and 2001. The station number corresponds to the numbers in Figure 2. St. Site 97 98 99 00 01 St. Site 97 98 99 00 01 1 NB Shippagan 3 Tracadie } Escuminac 4 Richibuctou 5 Pointe-du-Chene 6 Murray Comer 7 Baie Verte 8 NS Pugwash 9 Sand Point 10 Caribou River II Meri2oniish 12 Malignant Cove 13 Bayfield 14 Strait of Canso 15 Mabou 16 Inverness 17 Maraaree Harbor 18 Petit Etans: H) Pleasant Bav 20 South Harbor :i Ingonish Tl Indian Brook 23 Baddeck - - - 24 Wagmalcook + + + + - - - 25 Dundee + + + + - - - 26 PEI Cap Traverse - - - - - - - 27 Clyde River - - - - - - - 28 Vernon Bridge - - + + - - - 29 Belle River - - - - - - - 30 Murray Harbor - + + + - - - 31 St. Mary's Bay - + + + - - - 32 Cardigan River + - + + + + + 33 Bay Fortune - + + + + + + 34 East Lake - - + + + + + 35 Naufrage - - + + + + + 36 St. Peters - - - - + + + 37 Winter Bay - - - - + + + 38 North Ruslico - - - - + + + 39 French River - - - - + + + 40 Indian River - - - - + + + 41 Bideford River - - - - + + + 42 Cascunipec Bay - - - - - - - 43 Anglo Tignish - - - - + + + 44 Miminegash - - - - + + + 45 Baie Egmont - - - - + + + 46 Summerside - - - - Green Crab Expansion in Eastern Canada 257 Figure 1. Historical records of sighting contlrmcd sighting. of Carciiiiix moeiuis from the eastern I SA to the eastern Canadian coasts. Date indicates the earliest and Petpeswick Inlet) hy Bousfield (1958) in 1956. revealed that C. maenas was present only in the Cape Fouichu and West Puh- nico areas in few numbers (CMN unpublished records). According to the NSMNH records, the presence of the green crab was con- firmed in Westport on Brier Island in I960. MacPhail et al. (1955) reported a low catch rate of green crabs (i.e.. an average of two crabs a day) at the mouth of the Sissiboo River in St. Marys Bay in the mid 1950s. From the first green crab sighting in the Bay of Fundy, the species expanded its range more than 400 km in 2 years. The crab density increased significantly from 1952 to 195.^ in Passamaquoddy Bay (MacPhail et al. 1955). FiirOwr Xorlhward Expansion Along the Eastern Coast of A'oia Scotia The spread of the green crab around the southwestern end of NS began at the latest in 1954 to 1956. The presence of the crab was reported in Lockeport on the southeastern coast of NS in 1960 (Anonymous 1961 ). Green crabs were considered hy fishermen to be abundant startnig from 1964 in the La Have Islands area only 4 years after their arrival on the southeastern coast of NS (ARC unpublished records). They were collected from Peggys Cove to Prospect Bay from 1964 to 1966. respectively (NSMNH unpub- lished records). After reaching Peggys Cove in the mid 196()s. the abundance of green crabs decreased considerably and the rate of expansion further north seemed to have diminished possibly due to the influence of the cold Nova Scotian coastal current (Davis & Browne 1996). During a survey made in Passamaquoddy Bay in 1954. about 300 crabs were caught per baited trap with a 24-h soak time. In 1958, the catch rate was recorded at 53 crabs per trap per day. It then dropped to 7.5 crabs in 1960 (Anonymous 1961). This de- crease of crab abundance in the Bay of Fundy seems to coincide with a general cooling period, which was reported from 1953 to 1962 (Lauzier & Hull 1962). A significant diminution of crab abundance was also observed in Trenton (Maine, USA) where catches decreased from 27 1 crabs per trap per day in 1953 to a total absence in 1958 to 1965 (Welch 1968). Welch (1968) suggested that this rapid decrease in crab abundance might be caused by severe winter conditions along the New England coast between the late 1950s and mid 1960s. Similarly, on the other side of the Atlantic, winter was particularly cold in 1962 to 1963 around the British Isles and large adult crabs did not survive the cold weather, resulting in a major drop in density (Clay 1967). However, juve- niles and smaller adult crabs survived and repopulated the British Isles. The recently established population in the Bay of Fundy was probably smaller than the one from the British Isles. Genetic varia- tions may explain both populations" response to a cold environ- ment. For instance, the Canadian green crabs may be less cold environment-adapted. It then took a longer period of time for the Maritime green crab populations to adapt to new environmental conditions through the cold period. 258 AUDET ET AL. Collections and records of intertidal animals made by NSMNH (unpublished) in Halifax Harbor and at Lawrencetown in 1965 to 1966 did not include C. imwiuis. No record of the species was made during an extensive study in Petpeswick Inlet near Halifax Harbor in 1971 (Davis 1972. NSMNH unpublished records) or in the St. Marys River estuary in 1973 (Davis 1976). No surveys were conducted during the period when the north- ern limit of the green crab distribution progressed toward the Canso area, CBI and the northern coast of NS. The green crab sampling program carried out by the NSMNH on the eastern shore of NS since the late 1970s was rather sporadic. No direct study has been conducted on C inaenas in this area until today. Green crabs were collected in Marie Jo.seph in 1982 and in Tor Bay (NS) in 1983, the species being most likely established at these localities before those dates. It was not observed in Guysborough Harbor in 1983. Green crab probably entered Chedabucto Bay around 1985, which potentially provided access to the Northumberland Strait through the Strait of Canso, and to the Bras d"Or Lakes through St. Peter's Canal. Anecdotal information suggested the presence of this species in the Bras d"Or Lakes before 1995 (Kara Paul & John M. Tremblay, pers. comm.). The species is widely distributed in the main lake since 2(J00. Expansion of C maenas /row Cape Breton Island Toward the Southern Gulf of St. Lawrence The westward expansion of this species within the last 20 years was rapid (Fig. 1). This species, considering that it was not re- ported frequently in northeastern CBI, may have invaded the SGSL through the Strait of Canso in the early 1990s. Squires ( 1990) misinterpretation of Bousfield and Laubitz's ( 1972) records led him to conclude that the species was present in Northumber- land Strait in 1 960. This result was due to the duplication of station number series (S-series) used for studies in the SGSL in 1960 and southwestern NS in 1963. Bousfiels and Laubitz ( 1972), however, did not record C. maenas in the Northumberland Strait during their studies. Eel fishermen interviewed from the western side of CBI caught green crabs in their nets for the first time in 1998. One fisherman from Margaree Harbor mentioned that he has been col- lecting green crabs since either 1994 or 1995. He latter stated that the abundance had increased in 1998. The earliest green crab re- port concerning St. Georges Bay was from an eel fisherman in Pomquet in 1997. The occurrence of green crabs in eel nets is directly related to the fishing effort during the eel fishing season. Most fishermen from Caribou up to Port Hastings have not en- countered crabs within the years preceding the survey. In this area, the fishing effort increased when fyke nets were first used in 1993. As a decreasing trend in eel density and fishing effort was ob- served in 1995 (Chaput et al. 1997, Paulin 1997), the chance of encountering green crab might also have decreased after 1995. Crabs were observed in great numbers in Antigonish (St. Georges Bay) in 1999 (Jim Williams, pers. comm.). A qualitative survey carried out along the coast of the SGSL (from NB to CBI and around PEI) from 1997 to 2001 (Table 1, Fig. 2) revealed that the green crab was present in estuaries along the northeastern shore of CBI and in the Bras d"Or Lakes in 1997. The survey also confirmed that C. maenas was present in Malig- nant Cove (NS) from at least 1997, which was the mo.st advanced expansion in the SGSL at the time. In 2000, the abundance pos- sibly became greater (the catching method used was greatly influ- enced by the abundance) and the distribution reached the eastern opening of the Northumberland Strait. The western limit of the green crab gradually moved from Merigotnish in 1998 to Caribou River in 1999, indicating that the crab has been moving westward along the coast of NS. Shellfish aquaculturists started to express their concerns regarding green crabs off the northern coast of NS when crabs were reported near Sand Point in Tatamagouche Bay (J. Mark Hanson & Andrea Locke, pers. comm.) and in Wallace Gulf of St. Lawrence ^4f Magdalen Q"^ Island Chedabucto Bay Bras d'OrLakes Figure 2. Sampling location for the survey ol the occurrence of Carcinus maenas conducted in the southern Gulf of St. Law rence and adjacent area during summer (June to September) between 1997 and 2001. Station number corresponds to the sampling sites described in Table 1. Green Crab Expansion in Eastern Canada 259 Bay (Marc Ouellette. pers. comm.) in 2000 and 2001 . respectively. The crab has recently (June 2002) been collected near Port Elgin in Bale Verte (NB) (J. Mark Hanson & Andrea Locke, pers. comm.). Green crabs were present in Tor Bay in 1983 and probably invaded Chedabiicto Bay around 1985. They then spread into St. Peters Bay to possibly reach the Bras d'Or Lakes before 1995 (D. Davis, unpublished). A lobster fisherman reported the presence of the species in Port Hastings, along the Strait of Canso in the early 1990s (John M. Tremblay. unpublished). It is difficult however, to trace the pathway of the species" expansion around CBL as little information was collected in the late 1980s and early 1990s. The species did not .seem to reach the SGSL through the Strait of Canso first because it was reported in St. Georges Bay only in 1997. The presence of C. inaenas was rather first observed in the SGSL in 1994. on the western coast of CBI. Still, there is no evidence of in\asion pathway into the western CBL Invasion of new habitats may be due to natural larval transport and migratory patterns, but may also be the result of transfer with other species (e.g., oysters, blue mussels, scallops [Placopecten nuii^ellwiicus]. American eels \Angiiilla msrnita]. and American lobsters) from already invaded regions. Roff et al. ( 1984) studied brachyuran larvae off the Scotian Shelf in 1977 to 1978 and re- ported that zoeae and megalopae of C. maenas were common, but restricted off the coast of southwestern NS. A blue mussel grower from Whitehead. 600 km northeastward (Fig. 1). collected green crabs in mid to late 1970s (John M. Tremblay. unpublished). This report is the only case of a simultaneous occurrence of the species at such distant locations throughout the northern geographic inva- sion history of this species in the western Atlantic. A low research effort on this species at that time may be the reason why we observe punctual invasions (i.e., not being observed in the White- head area for a long period after 1970). There is no reason to presume however, that the northeastward invasion of green crab along the NS coast is continuous and initiated by a single source from a southwestern area. The invasion of green crab could be the result of multiple invasions as suggested by Geller et al. (1997) for C. aestiiarU in Japan and in South Africa. Further comparative studies have to be carried out on the genetic characteristics of the species along the coast of NS. Invasion of Prince Edward Island The geographic distribution of green crabs in PEI was limited to the Cardigan River system, in the suinmer of 1997 (Table 1 . Fig. 2). In 1998, crabs were reported from Fortune Bay to Murray Harbor along the eastern coast of PEI. Our survey, held from 1997 to 2001, indicated that the geographical expansion from 1999 to present did not exceed Naufrage and Vernon Bridge on the north and south shores, respectively. Intensive surveys conducted by the PEI Department of Fisheries. Aquaculture and Environment (PEIDFAE). however, showed that C. maenas was mainly re- stricted to the southeastern coast in 1999. the distribution including North Lake on the north shore and Gascoigne Cove on the south shore. In 2000 crabs were detected in the Charlottetown Harbor area, and in 2001 the western limit of the distribution moved toward Victoria on the south shore and Savage Harbor on the northern shore. Ovigerous females were observed in samples col- lected in eastern PEI in the summer of 1999 (Gillis et al. 2000). This observation suggests that this species is locally self- reproductive. There have been isolated reports of green crabs in the blue mussel and American oyster culture sites in Cascumpec and Malpeque Bays in the northwestern part of PEI in 2000 (Neil J. MacNair. pers. comm.). We consider that these crabs might have been accidentally introduced around the Island by way of aqua- culture activities, as no further report on the presence of green crab in this area was made since then. According to an investigation carried out in 1998 and 1999 by the PEIDFAE. the green crab likely arrived as a result of natural larval transport from NS (Gillis et al. 2000). There is no factual data, however, to support the arrival of this species in eastern PEI by larval transportation. If true, the megalopal settlement would have occurred as early as the mid 1990s, shortly after the known introduction of the crab on the western coast of CBI. Zoea larvae can travel with cuirents in the open sea. Larvae from CBI could be the source that fed the southeastern shores of PEI. Oceanographic conditions between PEI and CBI appear to support this hypothesis. Lauzier 1965 and Koutitonsky & Bugden (1991) showed that a gyre is induced by wind and internal wave activity at the mouth of St. Georges Bay. Currently, the green crab is observed from Pleas- ant Bay on the northwest coast of CBI down to Baie Verte at the NS-NB border and from Savage Harbor to Victoria, PEI. The gradual westward progression of green crabs is taking place at a similar rate on both sides of the Northumberland Strait. Potential Expansion in the Gnlf nf St. iMwrence The coastal habitats of PEI are rich in estuaries and are sur- rounded by the warm summer waters of the Magdalen Shallows and the Northumberland Strait. The environmental characteristics in the SGSL are ideal for a rapid and effective proliferation of the green crab. Warmer coastal temperatures in the summer and shorter winters would allow the species to grow faster and to expand their habitats as observed in the last 10 years in NS. Lauzier and Hull ( 1 962 ) showed that the Bay of Fundy area was under a general warming period in the 1940s and 1950s (the mean water temperature increased by 1.8°C from 1940 to 1953). Ac- cording to Pocklington et al. (1994). the warmest years were ob- served from 1951 to 1953. This was followed by a cooler period from 1953 to the mid 1960s and 1970s. The sea surface tempera- ture followed a siinilar trend. Temperatures were above normal from 1930 to 1960 and reached a maximum in the late 1950s. These investigators suggest, however, that there was a general cooling period from the 1 960s to present. In fact, water tempera- tures between 1981 and 1990 in eastern Canada seem to be near the long-term average and significantly colder than recorded dur- ing the warm conditions of the 1950s (Pocklington et al. 1994). The green crab may have invaded the Bay of Fundy during the warming period and this species has now reached the SGSL. Good seasonal environmental conditions in this area may contribute to a northwestern geographical expansion in the SGSL. Cohen et al. ( 1995). for instance, predicted that C. maenas could establish itself from California to Alaska, considering the wide range of tempera- tures and salinities the species can tolerate in the Pacific Ocean. At this point, the abundance of green crabs in PEI is lower than what is observed on the northern coast of NS. A mean catch rate of 10 ± 5 crabs per trap with a 24-h soak time was recorded in Basin Head between 2000 and 2002 on the eastern coast of PEI (Audet et al. in prep.). The same fishing gear captured hundreds of crabs in a few hours in Antigonish (NS) (Jim Williams, pers. comm.). As the green crab appears to be well established on the western coasts of CBI through the last decade, an increase in abundance and a westward expansion of the species are expected in PEI and NB in the near future. A close monitoring program is 260 AUDET ET AL. needed to follow the progression of green crabs on a possible northwestward expansion from the edge of the Northumberland Strait to the Chaleur Bay. where coastal temperatures (Savoie & Lanteigne 2002) are fa\orable to the species. Potential Impacts on the Aquaculture Industry The green crab is an omnivorous species. Its diet includes polychaetes, crustaceans, mollusks, and green algae (Crother 1968. Ropes 1968). Juvenile crabs are considered, among all, as green algae grazers, using the sea lettuce ( Ulva lactuca) beds as a refuge. Large adult males prey on various species including commercially exploited molluskan species (e.g., blue mussels and dogwhelks (Nucella lapilhis). flat oysters (Ostrea ediilis). Pacific oysters iCrassostrea gigas). the soft-shell clams and the northern quahog) (Glude 1955, Kaiser et al. 1993. Feare 1970. Marin et al. 1973, Mascaro & Seed 2000, Walton & Walton 20011. Naylor (1962) and Miron et al. (2002) observed that the feeding activity of the green crab varied considerably depending on water temperatures. They suggested that green crabs would cause certain damage to molluskan species during the summer period. Case studies from NS (MacPhail et al. 1955. Ropes 1968) and Maine (Smith 1954. Glude 1955) demonstrated a high vulnerability of molluskan spe- cies to green crab predation. Blue mussel and American oyster aquaculture are the most lucrative industries on PEI (Boghen 1995). Natural populations of soft-shell clams are currently heavily exploited and a trial production of northern quahogs is underway in PEI (Brown et al. 1995). The industry in the SGSL has an increasing interest in the cultivation of native shellfish. The rapid expansion of the green crab population in the same area may threaten the shellfish aquaculture industries. Some protective mea- sures could be used, such as fencing aquaculture sites to prevent intrusion of the green crab as practiced in Norway to protect scal- lops (Pecteii Diaxiiniis) against the brown crab (Cancer pagunis) predation (Strand et al. 1999). Physiological Adaptation and Limitation The green crab has a high reproductive potential (e.g., 200,000 eggs per female) (Broekhuysen 1936). They are also known to be tolerant to extreme environmental conditions (Broekhuysen 1936, Wheatly 1981, Abello et al. 1997). The green crab population established itself quickly in the North Pacific (Jamieson et al. 1998) by colonizing the intertidal habitat (0.7-1.4 m above mean lower low water) in sheltered areas. Green crab inhabits depths down to 10 m in the SGSL (Gillis et al. 2000). This is probably due to their physiological tolerance to low water temperature condi- tions during the winter period. Preliminary results obtained by Audet et al. (in prep.) revealed that key biological events (e.g.. molting, mating, and egg bearing) occur later in the SGSL com- pared with similar events occurring in the southern Atlantic (Ber- rill 1982). Temperatures are warmer during summer periods, reaching 26 C in lagoons on the eastern coast of PEI and -2°C during the winter season. Water temperature remains <10°C for at least 8 months of the year. Although the embryonic stages are vulnerable to fluctuating water temperatures and salinities (Naga- raj 1993, Anger et al. 1998), the species possibly adapted to a naiTow breeding time frame during the warmer months. Zoeal larvae, which prefer high salinities, probably migrate offshore dur- ing ebb tides and re-invade the estuarine habitats as euryhaline megalopae (Queiroga 1998). Nagaraj ( 1993) reported that the four planktonic stages of C. niaenas developed successfully in tempera- tures ranging from 10"C-25°C and salinities from 20 to 35'3f. This may be the reason why the green crab has successfully established itself in the Bay of Fundy and off the southeastern coast of NS during the last 50 years despite low mean surface water tempera- tures (12°C-14°C) (Harding et al. 1983). A threat to the ecological equilibrium is also possible. Long term effects are still difficult to identify at the moment, but may have great consequences. Carcinus maenos. with its high fecun- dity, high capability to tolerate a wide range of environmental conditions, and omnivorous feeding behavior, appears as an ex- cellent invader and can certainly displace endemic species. La- goons and estuaries around PEI that have been colonized by green crabs are also used by various commercial crustacean species such as the American lobster. Competition for space and food may be foreseen (Moody & Steneck 1993). The American lobsters, rock crabs {Cancer irroratus). and various mud crabs (Ritluopanopeiis harrisii and Drspanopeiis sayi) represent potential species that might have to compete with the green crab in the SGSL. On the North American Pacific coast, inter-specific competition forced juvenile Dungeness crab (Cancer magister) to emigrate from their natural oyster shell habitat. Green crabs also seem to be able to dominate equal size Dungeness crabs during altercations (Mc- Donald et al. 2001 ). It is therefore important to closely monitor the ecology of non-indigenous species, as their ecological effects are not well known (e.g., alteration of food webs, displacement of other resident crustacean species). ACKNOWLEDGMENTS The authors thank all eel fishermen and fishery officers from NS. NB and PEI (Canada) for the collection of valuable informa- tion on green crab sighting and Mr. Neil J. MacNair and his team (PEIDFAE, Charlottetown, PEI) for providing us with up to date information regarding the PEI green crab distribution. Special thanks are directed to Mrs. Kara Paul (Eskasoni Wildlife Com- mission, Eskasoni, NS, Canada) and Leslie E. Pezzack (Nova Scotia Museum of Natural History, Halifax Canada) and Drs. Glen Jamieson (DFO, Pacific Biologic Station, Nanainio, BC, Canada), Andrea Locke and J. Mark Hanson (DFO Gulf Fisheries Centre, Moncton, NB. Canada). John M. Tremblay (DFO Bedford Institute of Oceanography, Bedford. NS. Canada). Jim Williams (St. Fran- cis Xavier. Biology Department, Antigonish, NS, Canada), Hubert J. Squires (Paradise, NEED, Canada), and members of the Atlantic Reference Centre (St. Andrews, NB) and Canadian Museum of Nature (Ottawa. Ont.) for providing us valuable unpublished in- formation on the occurrence of the green crab in eastern Canada. We also thank Drs. J. M. Hanson and A. Locke (DFO Gulf Fish- eries Centre, Moncton, NB, Canada) who patiently reviewed the manuscript. LITERATURE CITED Abello. P., A. Aagaard. C. G. Warman & M. H. Depledge. 1997. Spatial variability in the population structure of the shore crab Carcinus nuie- nas (Crustacea: Brachyura) in a shallow-water tidal t]ord. Mar. Ecol. Proa. Sen 147:97-103. Anger. K., E. Spivak & T. Luppi. \9W. Effects of reduced salinities on development and bioenergetics of early larval shore crab. Carcinus nmenas. J. Exp. Mar Biol. Ecol. 220:287-304. Anonymous. 1961. Annual Report of the Fisheries Research Board of Canada 1960 to 1961. St. Andrews, N.B. Ottawa: Biological Station, pp. 47-70. Green Crab Expansion in Eastern Canada 261 Behrens Yamada. S. & C. Hunt. 2000. The arrival and spread of the European green crab. Carcimis maeiuts. in the Pacific Northwest. Dreissena 1 1:1-7. Berrill. M. 1982. The life cycle of the green crab Caiciniis macnas at the northern end of its range. / Cnisi. Biol. 2:31-39. Boglien. A. D. 1995. Introduction: The state of aquaculture in Atlantic Canada. In: A. D. Boghen. editor. Cold-water aquaculture in Atlantic Canada. Moncton: The Canadian Institute for Research on Regional Development, pp. 1-33. Bousfield, E. L. 1958. Littoral marine arthropods and mollusks collected in western Nova Scotia. 1956. Pwc. Nova Scoria liisl. Sci. 24:303-325. Bousfield. E. L. & A. H. Leim. 1960. The fauna of Minas Basin and Minas Channel. Nal. Mus. Can. Bull. 166:1-30. Bousfield, E. L. & D. R. Laubitz. 1972. Station lists and new distributional records of littoral marine invertebrates of the Canadian Atlantic and New England regions. Biol. Oceanogr. Publ (No. 5), Can. N. Mus. Nat. Sci. 51 pp. Broekhuysen. G. L. 1936. On development, growth and distribution of Caicinickw inaenas (L.). Archs. Neer. Zool. 2:257-399. Brown. J.. M. Helm & J. Moir. 1995. New-candidate species for aquacul- ture. In: A. D. Boghen. editor. Cold-water aquaculture in Atlantic Canada. Moncton: The Canadian Institute for Research on Regional Development, pp. 341-362. Carlton. J. T. 1985. Transoceanic and interoceanic dispersal of coastal marine organisms: the biology of ballast water. Oceanogr. Mar. Biol. Annu. Rev. 23:313-371. Chaput. C. A. Locke & D. Cairns. 1997. Status of American eel {Angiiilla roslrata) from the southern Gulf of St. Lawrence. In: R.H. Peterson, editor. The American eel in eastern Canada: stock status and manage- ment strategies. Proceedings of eel workshop, January 13-14, 1997, Quebec city. Qc. Can. Tech. Rep. Fish. Aquat. Sci. No. 2196. pp. 69-93. Clay, E. 1967. Literature survey of the common fauna of estuaries. 16. Carcimis maenas L. Imperial Chemical Industries Limited. Brixham Laboratory. PVM45/A/916. 8 pp. Cohen. A. N., J. T. Cariton & M. C. Fountain. 1995. Introduction, dispersal and potential impacts of the green crab Carcimis maenas in San Fran- cisco Bay. California. Mar. Biol. 122:225-237. Crothers. J. H. 1968. The biology of the shore crab Carcimis macnas (L.). The life of the adult crab. Field Stud. 2:579-614. Davis. D. S. 1972. The Atlantic Shore. Environmental Studies Series. Halifax : Nova Scotia Museum. 41 pp. Davis. D. S. 1976. The Estuary of St. Mary's River. Nova Scotia 1973. Curatonal Report. No. 34. Halifax : Nova Scotia Museum. 23 pp. Davis, D. S. & S. Browne. 1996. Ocean Currents. In: D. S. Davis & S. Browne, editors. Natural history of Nova Scotia. Vol, 1 . Halifax: Nim- bus Publishing and the Nova Scotia Museum. 517 pp. Dow. R. L. & D. E. Wallace. 1952. II. Observations on green crabs (C. inaenas) in Maine. Maine Dept. Sea Shore Fish. Fish. Circ. 8:1 1-15. Elner, R. W. 1981. Diet of green crab Carcinus maenas (L.) from Port Heben. Soulhweslern Nova Scolia. J. Shellfish Res. 1:89-94. Feare, C. J. 1970. Aspects of the ecology of an exposed shore population of dogwhelks Nucella lapillus (L.). Oecologia 5:1-8. Fowler. H. W. 1912. The Crustacea of New Jersey. In: Annual Report of the New Jersey State Museum for 1911. Trenton. NJ: MacCrcllish & Quigley. pp. 409-412. Gardner, N. C. S. Kwa & .A. Paturusi. 1994. First recording of the Euro- pean shore crab, Carcinus maenas in Tasmania. The Tasmanian Natu- rali.s! 116:26-28. Geller. J. B., E. D. Walton, E. D. Grosholz & G. M. Ruiz. 1997. Cryptic invasions of the crab Carcinus detected by molecular phylogeography. Mol. Ecol. 6:901-906. Gillis. D. J.. J. N. MacPherson & T. T. Rattray. 2000, The status of green crab {Carcinus maenas) in Prince Edward Island in 1999. Prince Ed- ward Island Department of Fisheries and Tourism., Tech, Rep. No. 225. 309 pp. Glude, J. B. 1955, The effects of temperature and predators on abundance of the soft-shell clam. Mxa arenaria. in New England. Trans. .Am. Fish. Soc. 84:13-26. Grosholz. D.. G. M. Ruiz & C. A. Dean. 1996. Predicting the impact of introduced marine species: Lessons from the multiple invasions of the European green crab Carcinus maenas. Biol. Cons. 78:59-66 E. Grosholz. E. D.. G. M. Ruiz. C. A. Dean, K, A. Shiriey, J. L. Maron & P. G. Connors. 2000. The Impacts of a nonindigenous marine predator in a California Bay. Ecolo.i;x 81:1206-1224. Harding. G. C. K. F. Drinkwater & W. P. Vass. 1983. Factors influencing the size of American lobster (Homarus americanus) stocks along the coast of Nova Scotia, Gulf of St. Lawrence, and Gulf of Maine: a new synthesis. Can. J. Fish. Aquat. Sci. 40:168-184. Jamieson, G. S., E. D. Grosholz, D. A. Armstrong & R, W. Elner. 1998. Potential ecological implications from the introduction of the European green crab. Carcinus maenas (Linneaus). to British Columbia. Canada, and Washington. USA. J. Nat. Hist. 32:1587-1598. Kaiser. M. J., R. N. Hughes & R. N. Gibson. 1993. Factors affecting diet selection in the shore crab. Carcinus maenas (L.I. ,\nim. Behav. 45: 83-92. Koutitonsky. V, G, & G. L. Bugden. 1991. The physical oceanography of the Gulf of St. Lawrence: a review with emphasis on the synoptic vanability of the motion. In: J.-C. Therriault. editor. The Gulf of St. Lawrence: small ocean or big estuary? Can. Spec. Publ. Fish. Aquat. Sci. 113. pp. 57-90. Lauzier, L. M. 1965. Drift bottle observations in Northumberiand Strait, Gulf of St. Lawrence. / Fish. Res. Board Can. 22:353-368. Lauzier. L. M. & J. H. Hull, 1962, Sea temperatures along the Canadian Atlantic coast 1958-1960. Fish. Res. Bd. Can. Prog. Rep. Atl. Coast Sta. 73:11-15, LeRoux, P. J., G. M. Branch & M. A. P. Joska. 1990. On the distribution, diet and possible impact of the invasive European shore crab Carcinus maenas (L.) along the South African coast. S. Afr. J. Mar. Sci. 9:85-93. MacPhail. J. S. 1953. .Abundance and distribution of the green crab. A clam predator. In: A,W.H. Needier, director. Repon of the Atlantic Biologi- cal Station for 1953. Fish. Res. Board Can. Appendix no. 22. pp. 33-34. MacPhail. J. S.. E. I. Lord & L. M. Dickie. 1955. The green crab— A new clam enemy. Fish. Res. Board Can. Prog. Rep. Atl. Coast Sta. 63:3-12. Marin. J.. R. Bellail & D. Latrouite. 1973. Predation de I'huitre plate, Ostrea edulis. par le crabe enrage. Carcinus maenas. Comite des crus- taces. coquillages et benthos. Cons, Intern. Explor, Mer. CM 1973/K: 1 1 . 6 pp. Ma,scar6. M. & R. Seed. 2000, Foraging behavior of Carcinus maenas (L.I: species-selective predation among four bivalve prey, J. Shellfish Res. 19:293-300. McDonald. P. S., C. J. Gregory & D, A. Armstrong. 2001 . The competitive and predatory impacts of the nonindigenous crab Carcinus maenas (L.) on early benthic phase Dungeness crab Cancer magister Dana. J. E.xp. Mar. Biol. Ecol. 258:39-54. Miron, G.. T. Landry & N. G. MacNair. 2002. Predation potential by various epibenthic organisms on commercial bivalve species in Prince Edward Island: preliminary results. Can. Tech. Rep. Fish, Aquat, Sci. No. 2392. 33 pp. Moody. K. E. & R. S. Steneck. 1993, Mechanisms of predation among large decapod crustaceans of the Gulf of Maine Coast: functional vs. phylogenetic patterns. / E.xp. Mar. Biol. Ecol. 168:111-124. Nagaraj, M. 1993. Combined effects of temperature and salinity on the zoeal development of the green crab, Carcinus maenas (Linnaeus, 1758) (Decapoda: Portunidae). Sclent. Mar. 57:1-8. Naylor, E. 1962. Seasonal changes in a population of Carcinus maenas (L. 1 in the littoral zone. J. Anim. Ecol. 31:601-609. Paulin. L. 1997. Eel fishery in the Gulf Fisheries Sector. Maritimes Region. In: R. H. Peterson, editor. The American eel in eastern Canada: stock status and management strategies. Proceedings of eel workshop, Janu- ary 13-14, 1997, Quebec city, Qc. Can. Tech, Rep. Fish, Aquat. Sci. No. 2196. pp. 25-33. Pocklington, R.. R. Morgan & K. Drinkwater. 1994. Why we should not expect "greenhouse warming" to be a significant factor in the Canadian 262 AUDET ET AL. coastal zone in the near future. In: P. G. Wells & P. J. Ricketts, editors. Costal Zone Canada 1994. Cooperation in the Coastal Zone: Confer- ence Proceedings. Vol.: 4. Costal Zone Canada Association. Bedford institute of oceanography. Dartmouth, N.S.. Canada. Queiroga. H. 1998. Vertical migration and selective tidal stream transport in the niegalopa of the crab Carcinits maenas. Hydrobiol. 375:137-149. Rathbum. M. J. 1905. Fauna of New England. Occas. Papers Boston Soc. N. Hisr. 7. 117 pp. Roff. J. C. L. P. Fanning & A. B. Stasko. 1984. Larval crab (Decapoda: Brachyura) zoeas and megalopas of the Scotian Shelf Can. Tech. Rep. Fish. Aquat. Sc. No. 1264. 22 pp. Ropes. J. W. 1968. The feeding habits of the green crab. Cuicimis maenas (L.). Fish. Bull. 67:183-203. Ruiz, G. M., P. W. Fofonoff. J. T. Carlton. M. J. Wonham & A. H. Mines. 2000. Invasion of coastal marine communities in North America: Ap- parent patterns, processes, and biases. Anmi. Rev. Ecol. Sysr. 31:481- 531. Savoie, F. & M. Lanteigne. 2002. Coastal temperature monitoring prograin for 2001 : southern Gulf of St. Lawrence. Can. Data Rep. Fish. Aquat. Sc. No. 1091. 69 pp. Say, T. 1817. An account of the Crustacea of the United States. J. Acad. Nat. Sci. Phila.. I. pp. 53-63. Scattergood. L. W. 1952. The distribution of the green crab. CarciniJes maenas (L.) in the Northwestern Atlantic. Maine Dept. Sea and Shore Fish. Ore 8:2-10. Smith. S. I. 1880. Crustaceans of the Atlantic coast. The stalk-eyed Crus- taceans of the Atlantic Coast of North America north of Cape Cod. Trans. Conn. Acad. Sci. 5:27-138. Smith, O. R. 1954. Fencing in flats may sa\e clams from green crab. Maine Coast Fish. 8:27-138. Squires, H. J. 1990. Decapod Crustacea of the Atlantic coast of Canada. Can. Bull. Fish. Aquat. Sci. 221. 532 pp. Strand. 0., G. A. Haugum. E. Hansen & A. Monkan. 1999. Fencing scal- lops on the seabed to prevent intrusion of the brown crab Cancer pagiinis. In: Book of abstracts. 12th International pectinid workshop May 5-11, 1999. Bergen Norway. 58-59 pp. Thresher, R. E. 1997. Proceedings the first international workshop on the demography, impacts and management of introduced populations of the European crab, Carcinus maenas. CSIRO CRIMP. Technical Report, 11, Hobar. Tasmania, Australia: Centre for Research on Introduced Marine Pest. 101 pp. Walton. W. C. & W. C. Walton. 2001. Problems, predators, and perception: management of quahog (hard clam), Mercenaria mercenaria. stock enhancement programs in southern New England. ./. Sliellfish Res. 20:127-134. Welch. W. R. 1968. Changes in abundance of green crab, Carcinus maenas (L.). in relation to recent temperature changes. Fish. Bull. 67:337-345. Wheatly, M. G. 1981. The Provision of oxygen to developing eggs by female shore crabs [Carcinus maenas). J. Mar Biol. ,Ass. UK. 61:1 17- 128. Zeidler. W. 1978. Note on the occurrence of the European shore crab C. maenas (Linn., 1758) in Australia. S. Aust. Nat. 53:11-12. Joiinuil of Shellfish Ri-scurch. Vol. 22. No. I. 26,V2(i7. 200.^. MINIMUM ENVIRONMENTAL POTASSIUM FOR SURVIVAL OF PACIFIC WHITE SHRIMP LITOPENAEUS VANNAMEI (BOONE) IN FRESHWATER WILLIAM J. MCGRAW* AND JOHN SCARPAt Harbor Branch Oceaiiogruphic Instinuion. Inc. Acjiuiciilturc Division 5600 US 1 North, Fori Pierce, Florida 34Q46 ABSTRACT The effect of three essential osiiioregiilalory ions (Mg"*, K*. and SOj"") on the short-term survival of Pacific white .shrimp Litopemieus vunnamei in freshwater (200 ppm for L. vannamei survival (Scarpa, unpublished data). Chloride and Na* have been determined by Chen and Chen (1996) to be the major ions contributing (88.4%) to heniolymph osmolality in marine shrimp. The addition of Ca"* to freshwater is thought to be necessary for the survival of shrimp because this ion is needed to form the exoskeleton. which is shed repeatedly during molting (Villalon 1991. Wyban & Sweeny 1991 ). Shrimp exuvia is composed mainly of CaCO, (99% of the inorganic portion; Richards 1951). L. vannamei does not possess internal Ca"* reserves like some freshwater crustaceans (McWhin- nie 1962). Therefore Ca"* must be continually absorbed from the environmental medium (Robertson 1953. Greenaway 1983). In the present study. Mg"*. SO4"". and K* ions were examined for their effect on survival of postlarval L. vannamei in "artificial" freshwater (i.e.. distilled water with sodium, chloride, calcium and carbonate). Magnesium and SO4"" were not found to have a criti- cal effect on short-term survival. Magnesium and Ca"* have been linked to membrane integrity (Douglas & Home 1997) and Mg"* concentrations in heniolymph have been conelated with crusta- cean activity (Mcfarland & Lee 1963). Sulfate is the third most prominent ion in seawater. but it has been shown to be nearly undetectable in shriinp heniolymph at low salinities (Dall & Smith 1981). Ti"eatnient solutions without |ii)tassium in the present study had lower survivals compared with solutions with K*. Potassium was shown in all three experiments to be a significant factor contrib- uting to the short-term survival of L. vannamei. The addition of 1 ppm of potassium doubled survivals over treatment waters with only Na*. Ca"*. and Mg-*. Compared with the other essential ions, K* is a minor constitu- ent in brackish and fresh water (Home 1969). but this ion plays a major role in metabolism of invertebrates (Schmidt-Nielsen 1990). Potassium was suggested by Robertson (1953) to be important in the maintenance of neroniuscular efficiency in decapods, whereas other authors have discussed the iinponance of K* in cmstacean metabolism (Gross 1958. Bursey & Lane 1971. Dall & Smith 1981. Schmidt-Nielsen 1990). Enzyme activity is directly depen- dent on K* concentration, which is maintained within narrow lim- its in the heniolymph of penaeids despite changing environmental salinity (Gross 1958. Bursey & Lane 1971. Dall & Smith 1981). In the marine environment, K* was constantly regulated in the heniolymph of P. dnnraiiim as the salinity of the extemal medium TABLE 3. Mean (±SE, n = }) 24- and 48-h survival CXr 1 of PL-28 /,. vannamei at different K* concentrations (ppm). Solution Na* Ca-* Mg=* K* ci- Total Ions 24-11 % Survi 1 290 54 53 0 626 1023 47(14.5)" 2 290 54 53 1 627 1025 83(11.5)" 3 290 54 53 10 635 1(342 90 (5.8)" 4 290 54 53 25 649 1071 93 (3.3)" 5 290 54 53 50 694 1141 93 (3.3)" Control 181 44 31 10 280 546 80(14.1) 48-h ^, Survival 37(3.33)" 80(6.7)" 77 (8.8)" 77 (3.3)" 90(10.0)" 60(5.0) Total ion value for control does not include trace elements. Survival values followed by a different superscript are significant at the /•' < 0.05 level. 266 McGraw and Scarpa changed (Bursey & Lane 1971 ). Potassium concentrations of 9-10 meq/L in the hemolymph were maintained between salinities of 7 to 35 ppt. whereas CI" and Na* concentrations were similar to that of the surrounding medium. Four Australian shrimp species stud- ied by Dall and Smith ( 1981 ) showed hemolymph K* concentra- tions were maintained between 5 and 1 5 meq/L over a range of 1 0 to 30 ppt salinity, with a trend of K* accumulation with increasing salinity. Potassium ions in P. monodon hemolyinph were strongly regulated during changing environmental salinity (Lin et al. 2000). Shrimp transferred from 45 to 1 5 ppt showed K* levels reached a steady state after 4 h. Euryhaline penaeids sampled from Gulf Coast waters showed higher K* concentrations in muscle tissue compared with stenohaline species taken from the same area (Mc- Farland & Lee 1963). The ability of K* to be stringently regulated in the hemolymph can be partly explained by the regulation process of this ion. Gross (1958) stated that hemolymph Na* and K* concentrations were maintained in an intertidal crab {Pachygrapsus crassipe.s) via in- tracellular pools as well as active uptake under hypotonic condi- tions. Changes in the Na* and K* concentrations between the hemolymph and surrounding medium were 84 and 68%. respec- tively, of the total hemolymph ion change while the additional 16 and 32% were assumed to come from internal salt pools. Hemolymph K* concentrations were maintained within narrower limits than Na* concentrations, despite the change in ion concen- tration of the surrounding medium. These salt pools were thought to be an ecological adaptation to buffer the ionic change between incoming and outgoing tides in an estuarine environment (Gross 1958). Gilles and Pequeux ( 1983) made a similar determination. A large decrease in the intracellular K* concentration of crustaceans appears to occur immediately following the application of hypo- osmotic conditions. The decrease in intracellular K* concentration being inversely proportional to the extracellular K* concentration, with lower extracellular K* concentrations producing a greater release of K* from isolated cells. The increase in pH between the first, second and third experi- ments of the present study did not appear to increase survival of shrimp. Optimum pH values of 6.6-8.5 for L. vannamei have been reported by Tsai (1990) and the pH values for all experiments listed here were within that range (within 0.1 pH value). Pillai and Diwan (1999) did not find any correlation of pH (7.04-7.84) with ion concentrations in the hemolymph of the shrimp Metapeiuieiis inonoceros taken from a tropical estuary over an 18-nio period. Wickens ( 19841 observed good growth and survival of P. monodon at pH values ranging from 6.7 to 7.9. Although the present study used PLs of slightly different ages for each experiment (PL- 18 to -28), it is unlikely that osmoregu- latory ability differed between these age groups. McGraw et al. (2002) found that PL- 10 L. vannamei had significantly lower sur- vival than PL- 15 and -20 when subjected to various acclimation rates, however, survival of PL- 15 and -20 age groups were not different from each other. This is probably caused by the full development of gills and osmoregulatory capacity of postlarval L. vannamei. which occurs at approximately PL- 12 (Lucu 1990. Pe- queux 1995, Van Wyk et al. 1999). Similar results have been ob- served for other penaeid species postlarvae (Olin & Fast 1992, Tsuzuki et al.. 2000). It is also unlikely that major environmental ion deficiencies may be compensated through dietary supplementation and. there- fore, feed supplementation during the trial would have had little impact. Dietary calcium supplementation for catfish cultured in calcium-free water had little effect on body calcium levels or spi- nal deformities (Scarpa & Gatlin 1993). The present study establishes the importance of potassium to L vannamei survival in freshwater solutions. Potassium addition to ponds as potash (K,0) has been recommended for pond fertiliza- tion (Boyd 1990) and ion supplementation (Boyd 2002). Potash has been used as a source to increase potassium concentrations in ponds for growing shrimp (Teichert-Coddington. Green Prairie Aquafarm, personal communication, 2000); however, the eco- nomical feasibility of this practice for shrimp culture in inland freshwater locations has yet to be determined. Because of this, decisions regarding potential inland sources of saline well water for growing L. vannamei should focus in part on the presence and concentration of potassium in water sources. ACKNOWLEDGMENTS We thank the Harbor Branch Institution post doctorate fellow- ship program for providing funds for this research. Special thanks to the HBOI library personnel for providing many invaluable li- brary searches and interlibrary loan documents. Gratitude is also expressed to those who have critically reviewed this manuscript. This is HBOI contribution 1495. LITERATURE CITED Boyd. C. E. 2002. Ions in low salinity shrimp aquaculture. Aquaculture America 2002 Book of Abstracts. World Aquaculture Society. Baton Rouge. LA: Louisiana State University, p. 31. Boyd. C. E. )990. Water Quality in Ponds for Aquaculture. Auburn Uni- versity. AL: Alabama Agricultural Experiment Station. 482 pp. Bursey, C. R. & C. E. Lane. 1971. Osmoregulation in the pink shrimp Penneus diioraritm Burkenroad. Coiiip. Biocii. Pliysiol. 39A:483— 193. Chen. J.-C. & C.-T. Chen. 1996. Changes of osmotic and electrolyte con- centrations in the haemolymph of Penaeiis japonicus exposed to ajii- bient ammonia. Comp. Bincli. Physiol. 114C:35-38. Dall. W. & D. M. Smith. 1981. Ionic regulation of four species of penaeid prawn. / Exp. Mar. Biol. Ecol. 55:214-232. Douglas. W. S. & M. T. Home. 1947. The interactive effects of essential ions and salinity on the survival of Mysidopsi.s hiMa in 96-H acute toxicity tests of effluents discharged lo marine and estuarine receiving waters. Em: To.x. Chem. 16:1996-2001. Ednotf. M. 2001. Fish Farming News, Vol 9. pp. 45-55. Feth. J. H. 1970. Saline groundwater resources of the contermmoiis United States. Water Resources Res. 6:1454-1457. Gilles. R. & A. Pequeux. 1983. Interactions of chemical and osmotic regulation with the environment. In: F. J. Vemberg & W. B. Vemherg. editors. The Biology of Crustacea. Vol 8: Environmental adaptations. New York: Academic Press, pp. 109-177. Greenaway. P. 1983. Uptake of calcium at the postmolt stage by the marine crabs Callinectes sapidus and Carcimis maenus. Comp. Biocli. Phy.sioi. 75A:I81-184. Gross. W. J. 1958. Potassium and sodium regulation in an intertidal crab. Biol. Bull. 114:334-347. Harvey, D. J. 2002. Aquaculture Outlook, http://www.usda. mannlib. comell.edu/reports/erssor/livestock/Idpaqs/200 1/Idpaqs 1 4.pdf. Hopkins. S.. P. A. Sandifer. C. L. Browdy & J. D. Holloway. 1996. Com- parison of exchange and no-exchange water management strategies for the intensive pond culture of marine shrimp. / Sliellflsli Res. 15:441- 445, Pacihc White Shrimp Survival 267 Home. R. A. 1969. Marine Chemistry. The Structure of Water and the Chemistry ot the Hydrosphere. Neu York: John Wiley and Sons Ine.. .'568 pp. Laramore. S.. C. R. Laraniore & J. Scarpa. 2(101. Effect of low salinity on growth and survival of postlarvae and juvenile Liloiieiuieiis vannanwi. J. World Aqua. Soc. 32:385-392. Lentner. M. & T. Bishop. 1993. Experimental Design and Analysis. Blacksburg. VA: Valley Book Company. 585 pp. Lin. S.-C, C.-H. Liou & J.-H. Cheng. 2000. The role of antennal glands in ion and body volume regulation of cannulated Penaeus monodon reared in various salinity conditions. Conip. Binch. Physiol. 127A:121- 129, Liicu, C. 199(1. Ionic regulatory mechanisms in crustacean gill cpithelia. Camp. Blochem. Physiol. 97A:297-306. McFarland, W. N. & B. D. Lee. 1963. Osmotic and ionic concentrations of penaeidean shrimps of the Texas coast. Bidl. Mar. Sci. ddf Caribbean 13:391-417. McGraw W. J. & J. Scarpa. 2002. Determining ion concentrations for Lilopenaeus vannumei culture in freshwater. Gtohul .Aiiiituuliitre .Ad- vocate 5:36-38. McGraw. W. J., D. A. Davis. D. Teichert-Coddington & D. B. Rouse. 2002. Acclimation o{ Litopenaeus vaimamei postlarvae to low salinity: Influence of age, salinity endpoint and rate of salinity reduction. J. World Aqua. Soc. 33:78-84. McWhinnie. M. A. 1962. Gastrolith growth and calcium shifts in the fresh- water crayfish Orconecres virilis. Coinp. Bioch. Physiol. 7:1-14. Ogle. J.T.. K. Beaugez & J. M. Lotz. 1992. Effects of salinity on survival and growth of postlarval Penaeus vannaniei. Gulf Res. Reports 8:415- 421. Olin. P. G. & A. W. Fast. 1992. Penaeid PL harvest, transport, acclimation and stocking. In: A.W. Fast and J. L. Lester, editors. Marine shrimp culture: principles and practices. Amsterdam: Elsevier, pp. 301-320. Pequeux. A. 1995. Osmotic regulation in crustaceans. J. Crust. Biol. 15: 1-60. Pillai. B. R. & A. D. Diwan. 1999. Ionic regulation pattern in the shrimp, Metapenaeus inonoceros from a tropical estuarine environment. J. Aqua. Tropics 14:237-246. Richards. A. G. 1951. The Integument of Arthropods. Minneapolis: L'ni- versity of Minnesota Press, 411 pp. Robertson, J. D. 1953. Further studies on ionic regulation in marine inver- tebrates. J. E.xp. Biol. 30:277-296. Samocha, T. M.. A. L. Lawrence & D. Poser. 1998. Salinity effect on growth and survival of juvenile Penaeus vannamei in a semi-closed recirculating system. Israeli J. Aquaculture-Bamidgeh 50:55-59. Samocha. T. M., L. Hamper, C. R. Emberson, A. D. Davis. D. Mcintosh, A. L. Lawrence & P. M. Van Wyk. 2002. Review of some recent developments in sustainable shrimp farming practices in Texas. Ari- zona and Florida. / Appl. Aqua. 12:1^2. Saoud, I.. D. A. Davis. D. Teichert-Coddington & D. B. Rouse. 2002. Salinity tolerance of Pacific white shrimp. In: Aquaculture America 2002 Book of Abstracts. World Aquaculture Society. Baton Rouge. LA: Louisiana State University, p. 295. Scarpa, J. & D. M. Gatlin, III. 1993. Responses of channel catfish Ucta- lurus punctatus) swim-up fry to dietary calcium in soft and hard water. Comp. Biochem. Physiol. IO6A:803-808. Scarpa. J. & D. E. Vaughan. 1998. Culture of marine shrimp, Pennaeiis vaimamei. in freshwater. Aquaculture 98. Book of Abstracts. World Aquaculture Society. Baton Rouge. LA: Louisiana State Universitv. p. 473. Schmidt-Nielsen. K. 1990. Animal Physiology: Adaptation and Environ- ment. 4th ed. New York: Cainbridge University Press, pp. 304-313. Tsai. C. K. 1990. Water quality management. In: D. M. Akiama. editor. Proc South East Asia Shrimp Management Workshop. Philippines: Soybean Association, pp. 56-63. Tsuzuki, M. v., R. O. Cavalli & A. Bianchini. 2000. The effects of tem- perature, age and acclimation to salinity on the survival of Farfante- penaeus paulensis postlarvae. J. World Aqua. Soc. 31:459—468. Van Wyk. P., M. Davis-Hodgkins. R. Laramore. K. Main. J. Moutain & J. Scarpa. 1999. Farming marine shrimp in recirculating freshwater pro- duction systems: a practical manual. FDACS Contract #4520. Talla- hassee, PL: Florida Department of Agriculture Consumer Services. Villalon, J. 1991. Practical manual for semi-intensive commercial produc- tion of marine shrimp. Galveston, TX: Texas A&M University, 104 pp. Wickens, J. F. 1984. The effect of reduced pH on carapace calcium, stron- tium and magnesium levels in rapidly growing prawns {Penaeus mon- odon Fabricius). Aquaculture 41:49-60. Wyban, J. A. & J. N. Sweeny. 1991. Intensive shrimp production technol- ogy. The Oceanic Institute Shrimp Manual. Honolulu. HI: The Oceanic Institute, 158 pp. Joiirmil oj Shdlfhh Research. Vol. 21, No. 1, 269-279, 2003. PHYSIOLOCxICAL AND GENETIC VARIATIONS IN DOMESTICATED AND WILD POPULATIONS OF LITOPENAEUS VANNAMEI FED WITH DIFFERENT CARBOHYDRATE LEVELS LETICIA ARENA.' GERARD CUZON." CRISTINA PASCHAL,' GABRIELA GAXIOLA.' CLAUD SOYEZ,- ALAIN VAN WORMHOUDT.' AND CARLOS ROSAS'* ^Lahoratorio cle Biologi'a Marina Expcriincntiil. Apclo. Post 69. Cd. del Carmen. Camp.. Mexico: 'Centre Oceanologic/tic dii Pacifiqiie. BP 7004. Taravao. Taliiti, French Polynesia: and Station du Biologic Marine du Mti.seum National d'Histoire Naturelle el du College de France. BP 225. 29900. Concarneau. France ABSTRACT The relationship between polymorphism of a-amylase and physiologic and hiochemical hehaMur of /.. vniiiuiiiici was used to determine whether artificial selection based on body weight and body size affect the adaptation ability of shrimp to use dietary carbohydrates as a source of energy. Shrimp fitness was addressed by measurement of energy balance using growth (P), oxygen consumption (R). and ammonia excretion (U) of juveniles from wild. 7th, and 25th generations of cultured shrimp. Hemolymph glucose, digestive gland glycogen, amylase activity, and amylase polymorphism was also evaluated in the three shrimp populations. Heterozygosity, amylase activity, and starch metabolism were affected by artificial selection of L vaiinamei. Shrimp from a 25th- cultured generation had less heterozygosity and physiologic alteration than did wild shrimp. Shrimp from a 7th-generation cultured shnmp population showed an intermediate state of genetic and physiologic alteration. Although a statistical comparisons cannot be made between the three studied populations, it is evident that there is a reduction in amylase activity related to shrimp domestication, with high values in wild shrimp (between 24 to 39 lU mg"' protein), intermediate in 7th-generation cultured shrimp (between 16 to 25 lU mg'' protein), and low in 25th-generation cultured shrimp (between 3.6 to 15.8 lU mg"' protein). A reduction in the frequency of alleles of amylase genes possibly related to domestication of shrimp was also demonstrated. It appears that the reduction of allele frequency of ainylase genes affected the adaptative ability of shrimp to use dietary carbohydrates as a source of energy and molecules and caused farmed populations to be protein dependent. Results of energy balance studies indicate that there are differences in production efficiency (P/AS) between populations: a reduction in P/AS as a function of generations of farmed shrimp suggests that efficiency with which shrimp transform energy into biomass is reduced with artificial selection. A'£l' WORDS: Liiupciiaciis vuimamei. physiology, genetics, populations, domestication, bioenergetics. blood parameters INTRODUCTION The Pacific white shrimp L. vuwuiiiici (Boone) is the most important shrimp species cultivated in the Antericas and the sec- ond in word production (Ben/ie 2000). More than 90% of the shrimp cultivated in 1998 on the American continent were L. van- luiinei (132 000 t; Rosenberry 1998). For that reason, shrimp farm- ers are establishing selective breeding programs for L. vannamei throughout the natural range of the species, as well as the US Atlantic coast and Bra/il (Sunden & Davis 1991. Paiva-Rocha 2001. Garci'a-Calleja 2000). These programs are motivated in pail by the serious disease problems caused by uncontrolled farmed population movements (Wyban et al. 1993. Bedier et al. 1998) and are focused to obtain better profitability through the selection of body weight or body size for optimal harvest. Although a better growth rate has been observed in breeding programs with L. van- namei. the impact of reported reduction of genetic diversity (Sunden & Davis 1991 ) on the general physiology of shrimp is not known (Benzie 1998). In a recent study Xu et al. (2001 1 showed a reduction in genetic diversity in cultured P. numodon compared with wild populations. That genetic differentiation pattern among populations was related to the prevalence of IHHNV viral disease in the same populations, indicating that the change in genetic diversity of shrimp could change the disease susceptibility of cul- tured or wild shrimp, affecting their fitness. Assimilation (As) is the key characteristic of living organisms because it is a direct index of the energy allocated to body weight or cametes or to maintain homeostasis. According to Lucas ( 1993). *Corresponding author. E-mail: cr\'@hp.fciencias.unam.mx As = P -I- R, where P is the energy allocated to production of biomass or gametes and R is the metabolizable energy. Although the fitness of a population has reproductive consequences, in a practical sense many researchers have been using the energy bal- ance on juvenile forms to determine how the environmental fluc- tuations or types of food affect the energy allocation in Crustacea trying to predict the environmental or nutritional consequences in energy pailitioning (Mayzaud & Conover 1988. Stickle et al. 1989, Du-Preez et al. 1992. Koshio et al. 1992. Hopkins et al. 1993, Rosas et al. 1993, Rosas et al. 1995, Guerin & Stickle 1997, Rosas et al. 1998, Rosas et al. 2001). The energy derived from food depends on mechanisms of transformation of dietary components that, in turn, depends on the ability of organisms to hydrolyze. absorb, and assimilate those dietary nutrients (Ceccaldi. 1998). In a series of recent articles, we have demonstrated that energy allocation derived from dietary car- bohydrates (CHO) has been found to be a limiting factor in L. stylirostris. L. vannamei. and L. seliferus (Rosas et al. 2000a, Rosas et al. 2000b. Rosas et al. 2001 ). In these works, we reported that glucose uptake in metabolism was limited because of satura- tion of a-amylase when shrimp are fed with diets above 33% CHO. At the same time, the digestive gland was saturated with glycogen in shrimp fed with diets >33% CHO, affecting nutrient absorption and in consequence growth rate and biomass produc- tion. Shrimp fed without dietary CHO can produce their own CHO using the gluconeogenic pathway, demonstrating that shrimp pro- tein metabolism is well adapted to produce its own metabolic energy despite energy lost through ammonia excretion. Shrimp a-amylase is one of the best-studied polymorphic di- gestive enzymes in shrimp. Two allelic forms were measured in Aselhis aquaticus, four isoforms in Palaemonetes varian.'i, seven 269 270 Arena et al. isoforms in P. elegans three isoforms in P. serratiis and L. van- nainei. and three in Farfaiuept'iiaeiis nolialis. in L. schmitti. and in L. setifems (Lomholt & Christensen 1970. Christensen & Lomholt 1972. Van Womihoudt 1983. Van Wormhoudt & Favrel 1988. Diaz et al. 1995, Le Moullac et al. 1996, Ball et al. 1998. Arena 1999. Garci'a-Machado et al. 2001). This enzyme can be induced or repres.sed by dietary CHO. protein levels, or by circadian. annual, or moult cycles (van Wormhoudt 1974. van Wormhoudt 1977). Van Wormhoudt et al. (1980) reported a reduction in amylase activity in Palaemon serratiis as a function of the increase in dietary glucides. Rosas et al. (2000a) showed an increase in a-amylase of L. stylirostris as a function of an increase in dietary CHO levels. Lovett and Felder (1990) stated that a significant increase in amylase activity of L. setifems postlarvae might be a response to low levels of CHO in the postlarval diet. Le Moullac et al. (1996) reported a reduction of amylase activity in L. van- nainei when the amount of this protein increa.sed in diets, showing that a-amylase gene expression could be repressed by casein, re- flecting the control that diet has on activity of amylase isoforms. In the present research, a relation between polymorphism of a-amy- lase and physiologic and biochemical behavior of L. vannamei was used to study whether artificial selection based on body weight and body size affected the ability of shrimp to use dietary CHO as a source of energy. Shrimp fitness was addressed through measure- ment of energy balance using growth, oxygen consumption, and ammonia excretion of juveniles from wild, 7th, and 25th genera- tions of cultured shrimps. Hemolymph glucose, digestive gland glycogen, amylase activity, and amylase polymorphism was also evaluated in the three shrimp populations. MATERIAL AND METHODS The study was divided between two experiments. The first was conducted in Mexico where comparisons were made wild and 7th-generation specimens of L. vaniuiniei. The second experi- ment was conducted at the French Marine Research Institute (IFREMER) Tahiti facilities with 25th-generation specimens of L. vannamei. Both experiments were conducted under the same basic conditions and with the same experimental diets. Experimeiilal Conditions For experiment I , live wild L. vannamei {n = 200; 0.8 ± 0. 1 g wet weight) were collected from Huizache and Cainianero Lagoon on the Pacific Coast of Mexico. Shrimp were transported by plane in plastic bags with cool sea water {30%c salinity; 20°C) to the Experimental Marine Biology Laboratory of National Autono- mous University of Mexico in Cd. del Carmen. Campeche. Mexico. Shrimp were acclimated to laboratory conditions for 2 wk before any experimental procedure was initiated. During this pe- riod shrimp were maintained in a circular external pond (20 nr) with aerated (O^ > 5.0 mg/L) natural seawater (32%c; 29 ± 2°C). During acclimation, shrimp were fed twice each day on a com- mercial shrimp diet containing 45% protein (Api Aba camariin ultra, Malta Clayton SA"' ). At the same time, a sample of 7th- generation cultured shrimp in = 200; 0.03 ± 0.03 g live weight) from a farm located in Sisal. Yucatan, was transported to the laboratory in cool sea water (35%c salinity, 24°C) and acclimated under identical conditions to those described above. After 2 wk of acclimation, a sample of each population was removed and distributed in 90-1 plastic tanks. For experiment one, shrimp were reared for 55 to 58 days in a flow-through sea water system (32%r salinity) at a density of 10 shrimp per tank. For experiment 2. we used 1600 postlarvae (0.009 ± 0.001 wet weight) of 25th-generation L vannamei obtained in the IFREMER hatch- ery facilities. In IFREMER shrimp were reared in 800-L tanks for 36 days in a flow-through sea water system (36%o salinity) at a density of 100 shrimp per tank. In both experiments shrimp were fed three times a day (0800, 1400, and 2000 h), uneaten food particles were removed twice a day (0730 and 1700 h) and water quality variables were main- tained as temperature 28 ± 1°C, dissolved oxygen >5.0 mg/L, and pH >8.2 ± 0.3. In both locations the photoperiod was set at 12h/12h. Samples of digestive gland for biochemical and genetic analysis from experiment 1 were stored at -80'"C and then freeze- dried until analysis. Digestive glands from 25th-generation culti- vated shrimp were freeze-dried at the IFREMER facilities in Tahiti before analvsis. Diets L. in shrimp was higher in shrimp fed with low dietary CHO than that in shrimp fed with high dietary CHO (P < 0.05). No differences were observed between wild shrimp fed with high or low dietary CHO levels (Table I; P > 0.05). Survival was not affected by dietary CHO in either of the shrimp populations. A mean value of 69% survival was obtained in all treatments (Table 1). Experiment 2 L. iY((i;!(»)/c/ juveniles from Tahiti population (25th generation) were not affected bv dietary CHO (Table 2; P > 0.05). TABLE 2. Dailj growth coefficient for juveniles of L vannamei from 25th-cultured generation: Experiment 2. HCHO LCHO Initial weight. 0 0.009 ±0.001'' 0.009 ±0.001" Final weight. 0 0.72 ± 0.04 1.02 ±0.05 Survival, % 85 ± 5 88 ±6 Time, days 36 36 DGC, 9c 1.91 ±0.7'' 2.21 ±0.78" N 8 S Different letter means statistical differences, P < 0.05. Values are mean ± SE. HCHO. hiah dietary carbohydrates; LCHO. low dietary carbohydrates. Ph^ .sioLOGic, Genetic Variations in L. vannamei 273 45- A 1 40- 1 2 35 c 'o) 30 5 25 1 20- 1 15- Amylase CJl o Wild HCHO Wild LCHO 7th HCHO 7th LCHO Population origin 18 B b '|> 12 - 5 10- activity, O) C» a Amylase ! 1 25th HCHO 25th LCHO Dietary CHO level Figure 1. Amylase ac'ti>it> h> wild and 7th-f;eneration cultured /,. vannamei (A) and 25th-generatlon cultured L. vannamei (Bl. Mean ± SE. Different letter means statistical differences at f < 0.05 level. Experiment 2 A significantly high glucose hemolymph level was measured in 25th-generation shrimp fed with high CHO ( 1 mg/niL) that was 2.6 times the value in shrimp fed with low CHO (0.39 mg/mL) (Fig. 2B; P < 0.05). Digestive Gland Glycogen Experiment 1 Digestive gland glycogen concentration was affected by dietary CHO and the origin of shrimp (Fig. 3A). In wild and 7th-generation cultured shrimp, a high glycogen concentration was measured in shrimp fed with low dietary CHO (P < 0.05). Experiment 2 In 25th-generation cultured shrimp, the high dietary glycogen level was measured in shrimp fed with high dietary CHO (2.0 mg/g) that was 30% higher than that in shrimp fed with low dietary CHO (1.4 mg/g: P < 0.05; Fig. 3B). Pattern of Allozyme Variation An eight-band pattern was observed in the electrophoretic analysis of amylase. These patterns were classified into two sys- tems; system 1 with alleles a, b, and c and system two with five alleles: a, b. c. d. and e (Fig. 4). Both systems were polyinorphic (Table 3). System 1 was inore conservative than system 2. In such a system, alleles a. b. and d were rare with an allelic frequency <0.05. A reduction in H in system 2 was observed into domesti- cated populations, with high values in wild shrimp (H = 0.29) and low values in 25lh-generation cultured shrimp populations (H = 0.08). reflecting a high percentage of homozygosity. Amylase loci from wild and 7th-generation cultured shrimp were in equilibrium. Locus from the 25th-generation of cultured shrimp showed sig- nificant deviation from Hardy-Weinberg proportions (heterozygo- sis deficit: P < 0.05) (Table 4). Energy Balance Experiment 1 Oxygen consumption of 12-h fasting shrimp was affected by dietary CHO in both wild and cultured populations (Table 5). The 1,20 1 A 1 00 - 1 0 80 - cn ^ 0 60 o ^0 40 b O 1 0,20 - 0 00 - a b S b f WHCHO WLCHO 7 HCHO 7 LCHO Population Origin and Dietary CHO level cn E o o O 1 z - B b 1 - 0 8 - 0 6 - a 0 4 - ¥ 0.2 - 0 ■ i 1 HCHO LCHO Dietary CHO. level Figure 2. Glucose hemolymph level of wild and 7th-generation cul- tured L. vannamei (A) and 25th-generation cultured shrimp (B). Mean ± SE. Different letter means statistical differences at f < 0.05 level. 274 Arena et al. 6 T o) 4 E c 0) O) o o 2 3 - 1 -- T '^ i : b F a ^ ! \ 1 WLCHO WHCHO 7LCH0 Dietary CHO level 7HCH0 TABLE 3. Genetic diversity {Hj in wild and cultured populations of L. vannamei. Population System I He System 2 He Wild 7th generation 25th generation 0.66 0.51 0.51 0.29 0.27 COS weight). Ammonia excretion increased after feeding, reaching a ma.ximum value between 0.5 to 3 h after feeding depending on shrimp group (Table 6). The highest postprandial amtnonia excre- tion value was recorded in 7th-generation shrimp fed with low dietary CHO and the lowest in wild shrimp fed with high dietary O) 4 E £=" 3 O) O O LCHO HCHO Dietary CHO level Figure 3. Digestive gland glycogen of u ild and 7th-generation cultured L. vannamei {.\) and 25th-generation cultured shrimp iB). Mean ± SE. Different letter means statistical differences at P < 0.05 level. highest oxygen consumption was measured in 7th-generation cul- tured shrimp (0.63 mg 0-,/h/g wet weight) fed with high dietary CHO (P < 0.05). The lowest oxygen consumptio// value was in wild shrimp fed with low dietary CHO (0.19 mg 0,/li/g wet weigh; P < 0.05). The oxygen consumption rate increased after feeding in each treatment (Table 5). Oxygen consumption of shrimp during feeding followed either of two patterns: one for wild shrimp fed with low dietary CHO and the other for the remaining shrimp groups. During feeding, oxygen consumption of wild shrimp fed with low dietary CHO was significantly lower than in w ild shrimp fed high dietary CHO shrimp and 7th-generation shrimp fed with high or low dietary CHO. In each, oxygen consumption increased rapidly after feeding and decreased afterwards until reaching levels similar to those at the start of experiment. The time required to achieve oxygen consumption peak was higher in 7th-generation shrimp fed with high dietary CHO (2 h; than in all remaining shrimp groups (0.5 to 1 h). Ammonia Excretion Ammonia excretion in fasting wild shrimp (mean value of 0.06 mg N-NH^/h/g wet weight) was significantly lower than in 7th- generation shrimp (mean value of 0.15 mg N-NHj/h/g wet System 1 0.6 >. 05 u o 04 £ 03 < 0 1 0 7th generation cultured shrimp DHCHO BLCHO System 1 a b System 2 25th generation cultured shrimp DHCHO SLCHO b c System 1 b c System 2 Figure 4. .Allele frequencies of wild, 7th-, and 25th-generation cul- tured I., vannamei fed with high dietary CHO (HCHO) and low dietary CHO (IXHOl. Physiologic, Genetic Variations in L. vannamei 275 TABLE 4. Allelic fri'i|ui'ncies comparison among different populations of/., ramitimci from wild (Mexico), 7th-generation cultivated shrimp and 25th-!;eneratiun cultivated shrimp. Wild 7th Generation 25th Generation Population SI S2 SI S2 SI S2 Wild 7th generation 25th t>eneralion NS NS NS. without significant statistical difference. *Statistical differences aX P < 0.05 level. CHO. Intermediate values were recorded in tlie remaining shrimp groups ( P < 0.05 ). The respiratory energy (/^y,,,.,,) varied between populations and was affected by dietary CHO (Table 7). Of the /?Toiai. 1'7'7<- was wasted in /J^,,, in wild shrimp fed with low dietary CHO in com- parison with the .^.4-4'^ waste as R.^y,, in the remaining shrimp groups (Table 7). R„^, was observed between S.^-y7Vr of /^x,,,,,, with the lowest value in wild shrimp fed with low dietary CHO. There were statistical differences between C/Totai between popula- tions and between treatments in 7th-generation cultured shrimp (Table 7; P<0.05). The percentage of Lfj^,,.^, that was t/^„„, varied between shrimp populations with the lowest value in wild shrimp fed with low dietary CHO (37%) and the highest (82%) in 7th-generation shrimp fed with low dietary CHO. The energy wasted after feeding (t/pp) was higher in wild shrimp fed with low dietary CHO (6.^% of t/y,„^|) than that in 7th-gene]ation cultured shrimp fed with the same diet (18% of U-y^,^,). Absorbed energy {Ah = P + R + U) showed differences between shrimp groups and was affected by distary CHO with high values in wild shrimp fed with high dietary CHO (S24J"' g"' WW day"') and low values in 7th-generation cultured shrimp fed with same diet (598 J"' g"' ww day"': Table 7). Ut-„,^| varied between 5-1 1% of Ab with low values in wild shrimp fed with high dietary CHO and high values in 7th- generation cultured shrimp fed with high and low dietary CHO (11% and 10% ). Between 89 and 95% of Ab was assimilated. The energy assimilated {AS) was the result of adding R to P. The .4.v value was affected differently in each shrimp population. In wild shriinp the highest value was observed in shrimp fed with high dietary CHO whereas in 7th-generation cultured shrimp the high- est value was observed in shrimp fed with low dietary CHO (Table 7). Respiratory efficiency (R/AS) was lower in wild than in 7th- generation cultured shrimp and was affected by dietary CHO in each shrimp group (Table 6). Inversely, growth efficiency (P/AS) was higher in wild than in 7th-generation shrimp and highest in shrimp fed with low dietary CHO in both shrimp groups. Experiment 2 Oxygen Consumption No difference was measured in 12-h fasting oxygen consump- tion values between treatments (mean value of 0.23 mg O^/h/g wet weight; Table S: P > 0.05). A similar ma.ximum oxygen consump- Uon value was observed in both dietary shrimp groups (0.32 mg Oi/h/g wet weight). The time to reach the peak was different between treatments with 1 h for shrimp fed with high dietary CHO and 2 h for shrimp fed with low dietary CHO (Table 8). Ammonia Excretion In 25th-generation shrimp, 12-h fasting shrimp had similar val- ues of ammonia excretion between treatments (mean value of 0,022 mg N-NH,/li/g wet weight; P > 0.05; Table 9). After feed- ing, the ammonia excretion increased. The time to reach the peak was similar in both treatments with high values in shrimp fed with high dietary CHO (0.040 mg N-NH,/h/g wet weight) and low values in shrimp fed with low dietary CHO (0.035 mg N-NH,/h/g wet weight; P < 0.05). Dietary CHO affected /^y^,,^,, (Table 10). Shrimp fed v\ith high dietary CHO had the higher proportion of energy from /?t„,„, that was channeled to /^^^,^, (96%) and at the same time the lower proportion of ^x„,.,i that was used in R^h, (4%). In contrast the higher proportion of energy of f/^,,, ,, that was lost as f/^..^, was in TABLE 5. Oxygen consumption (mg ();/li/g«vv) of L. vannamei after 12-h fasting (time = 0) and at lime increments after feeding: Experiment 1. Time After Feeding, h Wild HCHO ECHO 7th Generation HCHO ECHO 0 0.5 1 2 3 4 0.44 ± 0.09-' 0.59 ±0.11" 0.61 ±0.11" 0.58 ± 0.07" 0.52 ± 0.08" 0.46 ± 0.06" 0.19±0.(1V' 0.44 ± 0.04" 0.46 + 0.07" 0.28 ± 0.05" 0.39 ± 0.04" 0.22 ± 0.03-' 0.65 ± 0.06" 0.61 ±0.09" 0.65 ± 0.09" 0.72 ± 0.04" 0.48 ± 0.05" 0.35 ± 0.04- 0.54 ± 0.05^ 0.57 + 0.08" 0.70 ± 0.09" 0.59 ±0.11" 0.59 ± 0.06" 0.61 ±0.14" Different letter means statistical diflerences. P < 0.05. Values are mean ± SE. HCHO, high dietary carbohydrates; LCHO, low dietary carbohydrates. 276 Arena et al. TABLE 6. Ammonia excretion (mg N-NH/li/gww) of L vannamei after 12-h fasting (time = (l( and at time increments after feeding: Experiment 1. Time After Feeding, h Wild HCHO ECHO 7th Generation HCHO ECHO 0 0.5 1 2 3 4 0.067 ± 0.002' 0.18 ±0.004" 0.12 ±0.003'' 0.19 + 0.003'' 0.08 ± 0.003° 0.11 ±0.003" 0.05 + 0.008' 0.15 ±0.02" 0.19 ±0.03"'^^ 0.21 ±0.03*^^ 0.22 ± 0.06" 0.07 ± 0.009-' 0.15 ±0.02" 0.15 ±0.05" 0.16 ±0.007" 0.16 ±0.03" 0.25 ± 0.03"^ 0.06 ± 0.0 r 0.14 ±0.02" 0.34 ± 0.03'' 0.35 ± 0.04" 0.24 ± 0.03" 0.33 ± 0.05'-' 0.14 ±0.04" Different letter means statistical differences, P < 0.05. Values are mean ± SE. HCHO, high dietary carbohydrates; ECHO, low dietary carbohydrates. shrimp fed with low dietary CHO (87'7r ) in comparison to 15% lost in C/f„„, in shrimp ted with high dietary CHO. Inversely the pro- portion of t/xotai 'hat was lost as U^p was higher in shrimp fed with high dietary CHO (25%) than in shrimp fed with low dietary CHO {M^c: Table 10). In both treatments QSVr of energy absorbed {Ab) was assimilated [AS). Dietary CHO affected AS and growth and respiratory efficiencies. Shrimp fed with low dietary CHO showed the higher AS and growth efficiency (72%) compared with shrimp fed with high dietary CHO (61%; Table 10). DISCUSSION In L. vannamei shrimp from the 2,5th-cultured generation ex- hibited less heterozygosity than did wild shrimp. From results obtained, the 7th-generation cultured shriinp showed an interme- diate genetic and physiologic alteration. Although results demon- strate significant genetic differentiation among cultured and wild populations when based upon only an amylase allozyme marker, we acknowledge the necessity to confirm such differences at the mtDNA level through sequence variation of the amylase gene as recommended by Xu et al. (2001) and Garcia-Machado et al. (2001). A more detailed study involving molecular biology and genetic alterations by domestication of L. vannamei is in process. As a consequence of selection in cultured populations, carbohy- drate metabolism routes (hydrolysis, absorption, and synthesis) in shrimp fed with different dietary CHO was affected. A different enzyme activity-dietary CHO relation was observed depending on population characteristics; wild shrimp amylase activity was in- duced by high dietary CHO whereas low dietary CHO induced a high amylase activity in cultured shrimp. If reduction of heterozy- gosis means a reduction in amylase genes, then amylase activity induction was a compensatory response to obtain the highest pos- sible glucose from the diet, increasing enzyme synthesis when shrimp are fed with low dietary CHO. On the contrary, in wild shrimp an excess of dietary CHO induced amylase activity because those shrimp have all the isoforms of the atnylase enzyme to respond directly to the dietary starch. If atnylase production in domesticated shrimp is efficient enough to process dietary CHO, it can be analyzed in a general context. Although a statistical com- parison cannot be done among the three studied populations, it is evident there is a reduction in amylase activity as a function of dotnestication. with high values in wild shrimp (between 24 to 39 lU mg"' protein), intermediate in 7th-generation cultured shrimp (between 16 to 25 lU mg~' protein), and low in 25th-generation cultured shrimp (between 3.6 to 15.8 lU mg"' protein; Fig. 1). Such reduction indicates that the reduction of allel frequency of amylase genes affected the adaptative ability of shrimp to use TABLE 7. Energy balance in juveniles of L. vannamei: Experiment 1. Wild HCHO ECHO 7th Generation HCHO ECHO 167.3 ± 18.8 162.1 ±26.7 6.0 ± 0.8 6.8 ±0.8 173.3 168.9 46.1 ±6.1 60.3 ± 8.6 18.5 ±2.4 12.9 ± 1.6 64.6 73.2 359.9 ± 46 500.9 ± 65 597.8 743.0 533.3 669.8 89.2 90.1 32.5 25.2 67.5 74.8 /?„„. J/day/gww S^Hi- J/day/gww /?To>ai- J/day/gww (/„,, J/day/gww f PPNE. J/day/gww ^Tniai- J/day/gww p. J/day/gww Absorption (Ah) J/day/gww Assimilation (As) J/day/gww Ef assimilation. As/AI^ Respiratory efficiency, '7r R/As Production efficiency, % P/A.s 147.1 ± 1.9 5.2 ±0.8 152.3 24.7 ± 0.73 13.8 ±1.9 38.5 633.4 ± 70 842.2 785.7 95.3 19.4 80.6 57.1 ± 10.7 11.6± 1.1 68.7 18.5 ±2.70 31.4 ±4.7 49.9 614.6 ±73 732.6 683.3 93.3 10.1 89.9 Mean ± SE. HCHO, high dietary carbohydrates; ECHO, low dietary carbohydrates. Physiologic, Genetic Variations in L vannamei 277 TABLE 8. Oxygen consumption (mg 0,/h/g\v\M of/,, ycninamei (2Sth generation) 12 h lasting (time = 0( and at time increments after feeding: Experiment 2. Time .\fter Feeding, h HCHO I.CHO 0.24 ± 0.02' 0.31 ±0.02*" 0.29 ± 0.02" 0.28 ± 0.02" 0.26 + 0.02"" 0.25 ± 0.02" 0.27 ± 0.02" 0.21 ±0,01' 0.31 ±0.02" 0.33 ± 0.02" 0.27 ± 0.02'-' 0.27 ± 0.02'' 0.28 ± 0.02" 0.25 ± 0.02" Dit't'erent letter means statistical differences. P < 0.05. Values are mean ± SE. HCHO. liicli dietary carbohydrates; LCHO. low dietary carbohydrates. dietary CHO as a source of energy and molecules, which could cause farmed populations to be protein dependent. Juveniles of Uloiwiiaeiis vaimainei can synthesize their own glucose from protein through a gluconeogenic pathway (Rosas et al. 2001). Shrimp fed with low dietary CHO had digestive gland glycogen levels that were higher than when fed with high dietary CHO because the enzymatic system is induced to synthesize CHO from protein (Cuzon et al. 2001 ). In the present study, an increase in digestive gland glycogen was measured in wild and 7th- generation shrimp fed with low dietary CHO indicating that an induction mechatiism is working. In contrast, in the 2,5th- generation farmed shrimp, that mechanism appears to be working in the opposite direction, producuig more digestive gland glycogen in shrimp fed with high dietary CHO than in shrimp fed with low dietary CHO. If Amylase genes are repressed after 25th genera- tions of selection then a high probability exists that other genes could be repressed also, producing changes and reducing the glu- coneogenic route in shrimp. This indicates that artificial selection of shrimp favored more than size and harvest weight, as it also favored protein metabolism bv acting on shrimp digestive capacity. The use of high levels of animal protein in shrimp feeds in all phases of shrimp culture, from larvae to broodstock (including Anemia, krill, Cyclops, high qual- < 100 90 80 70 60 50 40 30 20 10 0 HCHO-W LCHG-W HCHO-7 LCHO-7 HCHO-25 LCHO-25 Diet and group shrimp Figure 5. Growth eniciency (P/AS,%) of wild (W) 7lh (7)-. and 25th (25)-generation cultured L. vannamei fed with different carbohydrates levels. HCHO. high dietary carbohydrates; ECHO, low dietary car- bohydrates. ity fish meal, and squid) is responsible for activation and repres- sion of genes. For amylase, Le Moullac et al. (1996) reported a reduction of enzyme activity in L. vannamei after an increase in dietary protein, which was related to a regulating role of amino acids on amylase expression. They observed a disappearance of one amylase mRNA associated with a high protein level suggest- ing that a regulation of amino acids would take place at the tran- scriptional level. Because, in selected shrimp, protein metabolism was favored and growth rate depended on dietary protein (An- drews et al. 1972). one can explain why 7th and 25th-generation farmed shrimp possess a higher growth rate than wild shrimp (Tables 1 and 2). There are several costs that are necessary to take into account with the breeding programs that only take into account the size of shrimp at harvest, which is also related to growth efficiency. From results on energy balance, there are differences in production ef- TABLE 10. Energy balance of L. vannamei (25th generation): Experiment 2. TABLE 9. excretion (mg N-NH,/h/gHH ) of L vannamei (25th Dietary CHO Ammonia HCHO LCHO generation) 12-h fasting (time = 0) and at time feeding: Experiment 2. /e„„. J/day/gww 72.07 ± 6.0" 54.1 ±2.6" Ri,n,. J/day/gww /?j^„ji, J/day/gww U,^,. J/day/gww t/ppNE, J/day/gww (/j„,^i, J/day/gww 3.0 ± 0.85" 75.1 7.4 ±0.7" 2.5 + 0.3" 10.3 ±1.7" Time .\fter Feeding, h HCHO LCHO 64.4 8.9 ±0.2" 1.4-^0.1" 0 0.020 ± 0.002" 0.024 ± ().()004" 9.84 10.2 1 0.030 ± 0.002" 0.028 ±0.001" P. J/day/gww 96.78 ± 14" 137.6 ±20.6" 2 0.040 ± 0.002' 0.035 ± 0.00 r Absorption (AIj). J/day/gww 181.7 212.2 3 0.030 ±0.001" 0.029 ±0.001" Assimilation (As). J/day/gww 171.8 202.0 4 0.037 ± 0.002"-^ 0.026 ±0.001" Ef assimilation As/Ah 94.6 95.2 5 0.029 ±0.001" 0.030 ± 0.0009" Respiratory efficiency % R/As 43,7 31.9 6 0.03 ±0.001" 0.027 ± 0.0008" Production efficiency. 9c P/As 56.3 68.1 Different letter means statistical differences. P < 0.05. Different letter means statistical differences. P < 0.05. Values are mean ± SE. Values are mean ± SE. HCHO, high dietary carbohydrates; LCHO. low dietary carbohydrates. HCHO, high dietary carbohydrates; LCHO, low dietary carbohydrates. 278 Arena et al. ficiency between populations (Fig. 5); a reduction of the P/AS ratio depending on the generations of farmed selected shrimp indicate that efficiency with which shrimp transform energy into biomass is reduced with artificial selection. That situation has several impli- cations on coastal ecology. When selected shrimp are lost by pond break caused by floods or hurricanes they could be liberated to surroundings environment. If those shrimp are from a breeding program based on size only, they shrimp could growth faster and consume more protein than wild shrimp, wasting energy due to its reduced assimilation efficiency and wasting other nutrients offered by the natural environment in the form of CHO and in conse- quence changing the relation between nutrients and consumers. In this same sense a reduction in PIAS ratio could have implications on the shrimp industry if is considered that a reduction in produc- tion efficiency could means the use of foods with more and more fish meal to satisfy the protein requirement of shrimp provoking that the shrimp industry to compete with other industry that use fish meal to produce meat for human consumption. On the other hand, selection shrimp programs could have rel- evance for the health of farmed shrimp. Recently. Xu et al. (2001) showed that there is a relation between genetic diversity and IHHNV sensitivity of P. moiwdon from Philippines. Although such relation is not understanding at all it could means that at the same time that shrimp are selected for size some other genes related with virus tolerance could be selected as well, provoking a segregation of the genes involved in virus resistance. If such con- cepts are applied to L. vannamei from breeding programs we could help to develop an industry based on rapid growth, low efficiency and vulnerable shrimp. It will necessary change looking for an shrimp based in the conception of breeding program that try to select shrimp that have wider adaptative ability to respond de- mands including all that are related to feed composition, produc- tivity, and sustainability (Fenucci et al. 1982, Boureau et al. 2000). and biosecurity (Xu et al. 2001 ). ACKNOWLEDGMENTS Thanks to Ellis Glazier for editing this English-language te.xt. The authors thank the ECOS Mexico-France program for its sup- port to researcher exchanges during this study. Special thanks are given to Adriana Paredes. Ariadna Sanchez. Manuel Valenzuela. Gabriel Taboada. and Gabriela Palomino for help during the ex- periments. The present study was partially financed by CONACYT through proyect .^1 137B to Carlos Rosas. Special thanks are given to Industrias Pecis for its support. LITERATURE CITED Andrews, J. W., L. V. Sick & G. J. Baptist. 1972. The innuence of dietary proteins and energy levels on growth and survival of penaeid shrimp. Aquaculture 1:341-347. Arena, L. 1999. Caracterizacion de poblaciones de camaron bianco Lito- penaeus vannamei y L. setiferus: estudio morfometrico y molecular. Facultad de Ciencias. UNAM, 1-81 pp. Ball, A. O., S. Leonard & R. W. Chapman. 1998. Characterization of (GT) sub(nl microsatellites from nLitive while shrimp [Penaeus setiferus). Mol. Ecol. 7:1251-1253. Bedier. E., J. C. Cochard, G. Le Moullac & J. Patrols. 1998. Selective breeding and pathology in penaeid shrimp culture: The genetic ap- proach to pathogen resistance. J. World Aquacuh. Soc. 29:46-51. Benzie. J. A. H. 1998. Penaeid genetics and biotechnology. Aquaculmre 164:23-47. Benzie, J. 2000. Population genetic slriiclure in Penaeid prawns. Aqiiacult. Res. 31:95-119. Brito. R., C. Rosas, M. E. Chimal & G. Gaxiola. 2001. Effect of different diets on growth and digestive enzyme activity in Litopenaeus vannamei (Boone. 1931) early postlarvae. Aquacuh. Res. 32:257-266. Bureau, D. P., P. A. Azevedo, M. Tapia-Salazar & G. Cuzon. 2000. Pattern and cost of growth and nutrient deposition in fish and shrimp: potential implications and applications. In: E. Cruz-Suarez, D. Ricque-Marie, M. Tapia-Salazar & R. Civera-Cerecedo, editors. Avances de Nutricion Acuicola, Meniorias del V Simposium Internacional de Nutricion Acuicola, Vol. V. Merida Yucatan, Mexico: CINVESTAV-Merida, pp. 25-49. Ceccaldi, H. J. 1998. A synopsis of morphology and physiology of diges- tive system of some crustacean species studied in France. Rev. Fish. Sci. 6:13-19. Cho, C. Y. 1992. Feeding systems for rainbow trout and other salmonids with reference to current estimates of energy and protein requirements. AquaciiUure 100:107-123. Chrislensen. B. & B. Lomholt. 1972. Amylase heterogeneity in Paluemon- etes varians (L.) (Crustacea: Decapoda). Ophelia 10:63-65. Davis, B. 1964. Disc electrophoresis. Part 11, Ann. NY. Acad. Sci. 121:404- 421. Diaz, R., M. E. Marquez, G. Bspinosa & V. Berovides. 1995. Morphologi- cal and electrophoretic studies of three species of cultured penaeid shrimps. Rev. Invest. Mar. 16:83-88. Drach, P. & C. Tchernigovtzeff 1967. Sur le methode de determination des stades d'inlermue el son application generale aux crustaces. Biol. Mar. 8:595-610. Du-Preez, H. H.. H.-Y. Chen & C. S. Hsieh. 1992. Apparent specific dynamic action of food in the grass shrimp Penaeus monodon Fabri- cius. Camp. Biochem. Physiol. 103A:173-178. Fenucci, J., A. Casal, A. Lawrence & Z. Zoula. 1982. The assimilation of protein and carbohydrate from prepared diets by the shrimp, Penaeus slyliroslris. J. World. Maricult. Soc. 13:13-1-145. Garci'a-Calleja, I. E. 2000. CENIACUA a la vanguardia en desarroUo de genetica en camaron de cultivo. Panorama Acuicola 5:14—17. Garcfa-Machado, E., A. Robainas, L. G. Espinosa, M. Oliva, J. Paes, N. Verdecia & M. Monnerot. 2001. Allozyme and mitochondrial DNA variation in Cuba population of shrimp Faifantepenaeus notialis (Crus- tacea Decapoda). Mar. Biol. 138:701-707. Guerm, J. L. & W. B. Stickle. 1997. Effect of salinity on survival and bioenergetics of juvenile lesser blue crabs, Callinecles similis. Mar. Biol. 129:63-69. Hopkins, J. S., R. D. Hamilton, P. A. Sandifer, C. L. Browdy & A. D. Stokes. 1993. Effect of water exchange rate on production, water qual- ity, effluent characteristics and nitrogen budgets of intensive shrimp ponds. / World. Maricult. Soc. 24:304-320. Koshio, S., J. D. Caslell & R. K. O'Dor, 1992. The effect of ditferent dietary energy levels in crab-protein diets on digestibility, oxygen con- sumption and ammonia excretion of bilaterally-ablated and intact ju- venile lobster, Homurus americanus. Aquaculture 108:285-297. Le Moullac, G., B. Klein, D. Sellos & A. Van Wormhoudt. 1996. Adap- tation of trypsin, chymotrypsin and a-amylase to casein level and pro- tein source in Penaeus vannamei (Crsutacea Decapoda). J. Exp. Mar. Biol. Ecol. 208:107-125. Lomholt, B. & B. Chrislensen. 1970. Amylase polymorphism in the water- bug Asellus aqualicus. revealed by cellular acetate electrophoresis. W^'/-«//m.s 64:177-180. Lovelt, D. L. & D. L. Felder. 1990. Ontogenetic changes in the digestive activity of larval and postlarval while shrimp Penaeus setiferus (Crus- tacea. Decapoda, Penaeidae). Biol. Bull. 178:144-159. Lucas, A. 1993. Bioenergetique des animaux aquatiques. Paris: Masson. Mayzaud, P. & R. J. Conover. 1988. 0:N atomic ratio as a tool to describe zooplanklon metabolism. Mar. Ecol. Prog. Ser. 45:289-302. Physiologic, Genetic Variations in L. vannamei 279 P;iiva-Ruclia. I. 2001. El cultivo de camariin marino en Brasil. I'tiiKiiaiiiu Aciiicola 6:40-44. Rosas. C, C. Vanegas. I. Tabares & J. Ramirez. 1993. Energy balance of Callinecles rathbimae. Contreras 1930 in floaling cages in a tropical coa.stal lagoon. J World Aquaciih. Soc. 24:71-79. Rosas. C. A. Bolongaro-Crvenna. A. Sanchez. G. Ga.xiola. L. Soto & E. Escobar. 1995. Role of the digestive gland in the energetic metabolism of Peiweiis seliferiLs. Biol. Bull. 189:168-174. Rosas. C. E. Martinez. G. Gaxiola. E. Diaz. R. Brito & L. Soto. 1998. Effect of dissolved oxygen on the energy balance and survival of Pe- luiciis selifenis juveniles. Mar. Ecol. Prog. Ser. 174:67-75. Rosas. C. G. Cuzon. L. Arena. L. Arena. P. Leniaire. C. Soyez & A. Van Wormhoudt. 2000a. Influence of dietary carbohydrate on the metabo- lism of juvenile Litopemieiis st\liri>siris. ./. E\p. Mor. Biol. Ecol. 249: 181-198. Rosas. C. G. Cuzon. G. Gaxiola. E. Taboada. C. Pascual. R. Bnto. M. E. Chinial & A. VanWormhoudt. 2000b. El metabolismo de los carbo- hidratos de Litopeiuieiis setiferus. L vannamei y L. stylirostris. In: E. Cruz-Suarez. D. Ricque-Marie. M. Tapia-Salazar, R. Civera-Cerecedo. editors. Avances de Nutricion Acm'cola, Memorias del V Siniposium Intemacional de Nutricion Acui'cola. Vol V. Merida. Yucatan. Mexico: CINVESTAV-Merida, pp. 25-19. Rosas. C. G. Cuzon. G. Gaxiola, C. Pascual. G. Taboada. L. Arena & A. Van Wormhoudt. 2002. An energetic and conceptual model of the physiological role of dietary carbohydrates and salinity on Litopi-naeiis vannamei juveniles. / Exp. Mar. Biol. Ecol. 268:47-67. Rosenberry, R. 1998. World shrimp farming. Shrimp Neivx Int. 1 1:31 1. Stickle, W. B.. M. A. Kapper, L. L. Liu. E. Gnaiger & S. C. Wang. 1989. Metabolic adaptations of several species of crustaceans and molluscs to hypoxia: tolerance and microcalorimetric studies. Biol. Bull. 177:303- 312. Sunden. S. L. F. & S. K. Davis. 1991. Evaluation of genetic variation in a domestic population of Penaeus vannamei (Boone): a comparison with three natural populations. Aquaculture 97:131-142. Van Wormhoudt. A. 1974. Variations of the level of the digestive enzymes during the intermolt cycle of Palaemon serralus: influence of the sea- son and effect of the eyesialk ablation. Comp. Biochem. Physiol. 49A; 707-715. Van Wormhoudt. A. 1977. Activites enzymatiques digestives chez Palae- mon serratus: variations annuelles de lacrophase des rythmes circadi- ens. Biochem. System. Ecol. 5:301-307. Van Wormhoudt, A. 1983. Immunoquantitative variations of amylase dur- ing molt cycle at different seasons on Palaemon serratus. Mar. Biol. 74:127-132. Van Wormhoudt, A., J. H. Ccccaldi & B. J. Martin. 1980. Adaptation de la teneur en enzymes digestives de Fhepatopancreas de Palaemon ser- ralus (Crustacea Decapoda) a la composition daliments experimentaux. Acjuacutlure 21:63-78. Van Wormhoudt, A. & P. Favrel. 1988. Electrophoretic characterization of Palaemon elegans (Crustacea. Decapoda) a-amylase system: study of amylase polymorphism during the intermolt cycle. Comp. Biochem. Physiol. 89B;201-207. Wright. S. 1965. The interpretation of population structure by F-statistlcs with special regards to system of mating. Evolution 19:395^20. Wyban. J.. J. S. Swingle, J. N. Sweeney & G. D. Pruder. 1993. Specific pathogen free Penaeus vannamei. Worhl Aquacult. 24:39—45. .\u, Z., J. H. Primavera, L. D. de la Peiia, P. Pettit, J. Belak & A. Alcivar- Warren. 2001. Genetic diversity of wild and cultured black tiger shrimp [Penaeus minuulon) in the Philippines using microsatellites. Aquacul- ture 199:13-40. Zar, J. H. 1974. Bioslalistical analysis. Englewood Cliffs. NJ: Prentice Hall. Joiunul of SlwUthli Research. Vol. 22. No. 1. 2S1-2S4, 2I)().\ EFFECT OF TEMPERATURE ON POST-PRANDIAL METABOLISM OF BROWN SHRIMP FARFANTEPENAEUS CALIFORNIENSIS LUCIA OCAMPO,'* CARLOS ROSAS,' AND HUMBERTO VILLARREAL' 'Ccntro de liivcsrigacioncs Bioldi^ica.s del Noroeste (CIBNOR) P.O. Bo.\ I2H. La Paz. B.C.S. 23000. Mc.xico and 'Gnipo de Muhcultura. Lah. Ecoflsioloi^fa. Faciiltad de Cleiuia.s. UN AM. P.O. Box 69, Ciiidad del Carmen, Canipeelie 24140. Mexico ABSTRACT The effect of three temperatures 09. 23. and 27°C) on the postprandial metaboHsm (apparent heat increment) of iu\cnile larfantepenaeus californiensis was evaluated. The unfed metabolic rate and post-prandial metabolic rates were determined with an intermittent-tlow respirometer during 5 h. A peak in oxygen consumption was found 2 h after feeding at 19 and 27"C whereas at 23°C the peak was found after I h. The unfed metabolic rate at 23°C was not different from that at 27°C . The maximum metabolic rates of fed animals were 2.1, 1.6, and 1.7 times that of unfed animals in order of increasing exposure. The highest apparent heat increment was found at 27°C. Energy loss varied from 4.11 to 11.43 J. Calculated Q,„ thermal coefficients indicate metabolic overcompensation for temperature changes between 19 and 27°C, and between 19 and 23"C, except at the maxmium metabolic rate. In contrast. Q|,,s for temperature changes between 23 and 27°C indicate compensation. KF.Y WORDS: energy loss. Faiiaiuepemieus califoruicnsis. oxygen consumption, postprandial metabolism, temperature INTRODUCTION Rtihner ( 1902) defined the heat increment resulting from bio- chemical reactions to ingestion of a meal as specific dynamic effect. Since then, various terms, such as specific dynamic action, heat of nutrient metabolism, thermogenic action, calorigenic effect of food, postprandial respiration, and heat increment, have been used widely to represent energy losses associated with feeding in ectotherms (Johling & Davies 1980. Beamish & Trippel 1990). The physiologic basis of this increased heat production includes postabsorptive processes related to ingestion, particularly of pro- tein-rich food, the metabolic work required for formation of ex- cretory nitrogen products, and the synthesis in the tissues of pro- teins and fats from the newly absorbed food derived substrates like amino and fatty acids. The energies required for grasping, chew- ing, and swallowing food are technically distinct from the heat increment but are difficult to separate experimentally (Beamish & Trippel 1990). The apparent heat increment (AHI) is the energy required for the mechanical processes of feeding and the ingestion and digestion of food (Hewitt & Irving 1990). In homeotherms. heat increment has multiple influences, including tiine spent in eating, muscular work, secretion of saliva, fermentation heat, transport of the absorbed nutrients, hormonal effects, and pharma- cokigical effects of food constituents, and is related to the enthalpy change associated with the generation of ATP (Blaxter 1989). In fish, there is ample evidence that AHI is influenced by meal size and feeding frequency, temperature (Bret 1976); size of the ani- mal (Beamish 1974); quantity, quality, and proportions of the di- etary energy components (Smith et al. 1978); and the nutritional status (Hart 1980). Despite the amount of information published on AHI. experimental techniques have varied greatly among the stud- ies, and observations of the effects of temperature on AHI have not been consistent. In addition, there is little information on AHI in Penaeids (Hewitt & Irving 1990. DuPreez et al. 1992. Rosas et al. 1996). DuPreez et al. ( 1992) reported that the AHI for P. immodon *Corresponding author. Tel +6i; E-mail; locampo@cibnor.mx -125-36-33; Fax: -1-612-125-36-2.5; Fabricus ranged from 2-17% when fed commercial pellets and from 2.4 to 19.5% when fed shrimp flesh. Rosas et al. (1996) reported that the highest AHIs were found for P. duoranim Burkenroad and P. notialis Perez Farfante feeding on a 65% diet, whereas the lowest were found for P. setifeiiis Linnaeus and P. schmitti Burkenroad fed a 40% protein diet. The authors con- cluded that AHI varied with diet protein content for all these species. Brown shrimp Faifantepenaeus californiensis Holmes is cur- rently being evaluated as a cold-tolerant species with potential for aquaculture at our center. Studies of nutritional and metabolic aspects that are influenced directly by factors such as temperature are important to better understand the physiology of this species. This study presents information about the effect of temperature on the AHI of juvenile F. californiensis. Some physiologic responses and possible mechanisms of adaptation are discussed. MATERIALS AND METHODS Juvenile F. californiensis from the Centro de Investigaciones Biologicas del Noroeste experimental shrimp farm were selected randomly, fed a commercial diet containing 35% crude protein (RANGEN®) with filtered seawater at a salinity of 37 ppt. A photoperiod of 12-hL:12-hD was maintained throughout the study. Shrimp were acclimated ( 1' C/day) at three different temperatures (19, 23, and 27°C) for a period of 5 days. After a 24-h starvation period. 12 animals of each temperature treatment were placed indi\ idually in an intermittent flow respirometer system similar to the one described by Villaireal (1989) 2 h before commencing the test to minimize the effect of handling and previously calibrated at each experimental temperature. The fasting metabolic rate was determined for 2 to 3 h thereafter. Next, shrimp were allowed to feed on commercial pellets for 1 h. Uneaten food was siphoned out completely and collected, and water was replaced completely. Oxygen intake was recorded hourly for 5 h after ingestion of the meal with an oxygen electrode (Yellow Spring Instruments, Model 58). Water was replaced completely after each record to prevent accumulation of ammonia. At the end of the experiment, shrimp were weighed on a digital balance after blotting. Data were cor- 281 282 OCAMPO ET AL. ■c O 00 o S S £^ o u oi 0.9 r 0.8 - 19°C 23°C ■27°C 0.7 0.6 - 0.5 - = 0.4 - 0.3 ■- 0.2 0.1 Time after feeding (h) Figure 1. Respiratory metabolism (mgOi/g shrimp/h) of juvenile Farfantepenaeus califontiensis after feeding on a 35% crude protein diet (RANGEN®) at different temperatures (°C|. Unfed respiration is sliown as the respiration at time 0. n = 12 shrimp/temperature. *Signifieant differences. reeled for oxygen consiiniption with u control respirometer with no shrimp. AHI (J) at each temperature wa.s calculated as: AHI = (maximun postprandial rate - unfed rate)(20.06) over the period studied (Rosas et al. 1996, Lucas 1993). Differ- ences between treatments were defined by one-way ANOVA and the Tukey multiple range test. RESULTS Respiration (mg O^/g shrimp/h). as a function of time, is shown in Figure 1 . Time 0 was defined as the end of the 24-h starvation period. The highest unfed metabolic rate occurred at 27°C, but it was not significantly different from that at 23°C {P > 0.05). The lowest unfed metabolic rate was at 19°C and represented approxi- mately 55% of the value at 23 and 27''C. A tendency to increase metabolic rates at all temperatures after feeding was observed, but this increase was signitlcant only at 19°C. At 19 and 27°C, the highest rate was reached after 2 h. At 23°C. the maximum was observed after 1 h, and was sustained over 2 h. The highest overall increase in metabolic rate after feed- ing of 677c occurred at 27°C. whereas at 23 and 19 C. the meta- bolic rate increased 59% and 110%, respectively (Table I). The time after commencement of feeding until the appearance of the first feces varied from 30 to 60 min at 27 and 23T, whereas at 19°C the time was approximately 90 min. AHls are shown in Table 1. The highest AHI was at 27"C and the lowest AHI was at 23°C. When AHI was expressed as energy lost, values varied from 4.1 1 to 11.43 J. These values were cor- rected for the time needed to reach the peak and represent the metabolic efficiency of heat loss. The highest value was at 27°C and the lowest was at 23°C. Qii, coefficients were calculated for temperature increments between 19 and 23°C, and 19 and 27 ^C, and are shown in Table 2. A Qui value of 2 indicates a doubling of the metabolic rate with an increase in temperature of 10°C. Q,,, for 23-27 for the unfed period showed adaptation, whereas Q^s for 19-23 and 19-27 showed overcompensation. Q„, for 23-27 for the feeding period showed compensation for almost the entire trial except during the second hour. Qm for 19-23 showed overcompensation, except for the second hour when there was adaptation. Little compensation was observed between 19 and 27°C in this experiment. TABLE L Mean effect of temperature on unfed and postprandial metabolism (mg02/g shrimp/h). apparent heat increment (AHL J), and energy lost (J) in ju>enile Farfantepenaeus calif orniensis. Temp. Unfed Rate (°C) (mgO,/g Shrimp/h 1 ± SD Maximum Postprandial Rate Increase AHI Time to Reach Energy Lost (mgO,/g Shrimp/h) ± SD (%) (J) Peak (h) (J) 19 ().I42±0.()68-' 23 0.349 ± 0.066'' 27 0.428 + 0.091'' 0.403 ± 0.063" 0.554 ± 0.077" 0.713 ±0.142' no 4.2.^ "> 39 4.11 1 67 5.72 2 S.46 4.11 1 1 .43 N = \2 shrimp/temperature. Entries with the same letter are not statistically different (P > 0.05) Post-Prandial Metabolism of F. cauforniensis 283 TABLE 2. Calculated Q,,, values for unfed and postprandial metabolic rales in juvenile Farfanlepenaeus californiensis. Time After Q,„ Q,„ Q,„ Feeding (hi (19-2.Vt) ( 19-27 C) (23-27 C) 0 4.49 2.7.^ 1.6(1 1 6.02 2.61 1.L1 2 L95 2.04 2.14 3 3.02 L67 0.92 4 5.19 2.54 1.24 5 3.5 2.09 1.2.'; Values for Q,,, were calculated using the formula Q,,, = iRJR,) exp"""-""'. where ft, ;ind /?, are the metaholic rates at temperatures r, and /,, respec- tively. DISCUSSION DuPreez et al. (1992) conciucJed that the magnitude and dura- tion of oxygen consumption peaks could be influenced by diges- tion rate, environmental temperature, and activity of the animal. In our study, a peal^ was seen 2 h after feeding at 19 and 27°C. and decreased thereafter, showing that oxygen consumption of F. cali- forniensis was affected only by food in the experimental device used. Perhaps the amount of food consumed or the digestion rate were responsible for sustaining maxiinum post-prandial rate over 2 h at 23°C. The induction in oxygen consumption for F. cuUfomiensis was approximately 43% higher at 19°C than at 27°C (Table 1). Fur- thermore, the maximum metabolic rate at 19°C equaled the pre- feeding rate at 23'C. Juveniles at I9"C appeared lethargic, and v\e observed a lower ingestion rate at this temperature. Although pre- liminary trials indicated that 5 h was adequate for complete diges- tion of food, we noticed that the digestive tract of some shrimp at \TC still contained food at the end of the experiment. The slower appearance of feces showed that more time is needed to finish digestion at 19°C. and could be related to a decrease in the appetite and the general movement of shrimp. DuPreez et al. (1992) found two peaks for P. monodon. 30 min and 6 h after feeding. Perhaps we would have found a second peak if the trial had continued past 5 h at 19 C, when the shrimp reinitiated digestion. Villarreal and Ocampo (1993) concluded that F. californiensis postlarvae and juveniles exposed to a temperature range from 19 to 3rC could adjust their normal metabolic rate with gradual, short- term temperature modifications. Similar results were obtained in this study, in which the unfed metabolic rate at 23°C was close to that at 27"C. It seems that organisms at 23°C have a mechanism for metabolic compensation or adaptation to temperature varia- tions at this stage of life (Q,„ for 23-27 = 1.66). This equivalence in metabolic rate could be explained as a modification in enzyme kinetics. Ocampo and Ezquerra (2002) found that the effect of temperature on total //; vivo protease activity of F. cciliforniensis at 23°C was 58% higher than that at 21°C, for shrimp that had been acclimated for 50 days to the temperatures, and suggested that different digestive protease enzymes arise as an adaptation mecha- nism to temperature and dissolved oxygen variations. In general, homeostatic regulation of enzymatic catalysis in animals can be accomplished in two ways: by modifying enzyme concentration, or by modifying catalytic efficiency (Hochachka & Somero 1973). When enzymatic concentration is increased, the rate of reaction increases. At 23°C. juveniles might increase their reaction rate as a quantitative strategy for temperature compensa- tion (see QioS). However, this quantitative strategy might be less efficient during cold adaptation since less time is required for changing enzyme concentration via synthesis of new protein. This metabolic reduction enhances the resistance of F. cuUfomiensis to low temperature stress. The process seems to involve controlled decreases in metabolism and organelle function, coupled with si- multaneous controlled stabilization of macromolecule and or- ganelle structures (Hochachka 1990). DuPreez et al. ( 1986) calculated AHl as the increase in oxygen consumption over the time until oxygen consumption decreased to the prefeeding level. In our study, AHl was expressed as the dif- ference in oxygen consumption between unfed and fed animals (Rosas et al. 1996). AHl is best expressed as percent metaboli/able energy (Blaxter 1989). Taylor et al. (1987) stated that most ani- mals displayed maximum metabolic rates that were 5 to 20 times the normal rates. However, some experiments did not take into account stress caused by handling, and the need for a "resting" period in the chamber before initiating the trial. Rosas et al. (1996) reported the maximum metabolic rate of fed P. schmitli was 2.6 to 3.6 times that of unfed. In our study, maximum metabolic rates found were 2.1. 1.6. and 1.7 times that of unfed animals, with increasing temperature. We emphasize that the heat increment AHl corresponds to the production of ATP (maintenance heat increment) and tissue energy deposition (production heat increment). Cho and Kaushik (1990) estimated the heat increment of feeding for a maintenance ration is approximately one third of the total heat increment, and the rest is used for productive gain. In general, AMI might range from 1 1- 24% of digestible energy (6-19% of gross energy intake: Beamish & MacMahon 1988), and is a more or less constant fraction of dietary energy (Brody 1964). Further research is needed to relate protein intake with AHl in F. calif imiensis. However, the results of this experiment show that F. californiensis juveniles present a metabolic strategy to digest food efficiently at 23°C, leaving more time to consume food and saving heat energy loss. This strategy is not related to their optimum aquacullure temperature, but is related to their physiologic optimum, which would indeed be a good tem- perature to maintain the animals. ACKNOWLEDGMENTS Luci'a Ocampo was a student-fellow of CONACYT, Mexico. Thanks to Jean-Charles Guillaume for observations and sugges- tions and the CIBNOR editing staff. Beamish, F. W. H. 1974. Apparent specific dynamic action of largemouth bass. Micropterits salmoides. J. Fish. Res. Board Can. 31:1763-1769. Beamish, P. W. H. & P. D. MacMahon. 1988. Apparent heat increment and feeding strategy in walleye. Slizosledion viireum vitreiim. Aquacuhure. 68:73-82. LITERATURE CITED Beamish. F. W. H. & E. A. Trippel. 1990. Heat increment: a static or dynamic dimension in bioenergetic models'? T. Am. Fish Soc. 1 19:649- 661. Blaxter. K. 1989. Energy metabolism in animals and man. Cambridge. UK: Cambridge University Press. 284 OCAMPO ET AL. Brett. J. R. 1976. Feeding metabolic rates of young sockeye salmon. On- corhynhus nerka. in relation to ration level and temperature. Fish. Mar. Sen: Res. Dew Tech. Rep. 675:43. Brody. S. 1964. Bioenergetics and growth, with special reference to the efficiency complex in domestic animals. New York: Hafner Publish- ing Co. Cho. C. Y. & S. J. Kaushik. 1990. Nutrition energetics in fish: energy and protein utilization in rainbow trout iSnImo gciirdiieri). World Rev. Nun: Diet. 61:1,^2-172. DuPreez. H. H.. A. McLachlan & J. F. K. Marais. 1986. Oxygen consump- tion of a shallow water teleost, the spotted gunter, Pomadasys com- mersonni (Lacepede, 1802). Comp. Biochem. Phys. A. 84:61-70. DuPreez, H. H., H. Y. Chen & C. S. Hsieh. 1992. Apparent specific dynamic action of food in the grass shrimp, Penueus monodon Fabri- cus. Comp. Biochem. Phys. A. 10.3:173-178. Hart. R. C. 1980. Oxygen consumption in Caridina nilotico (Decapoda Atyidae) in relation to temperature and size. Freshwtiter Biol. 10:215- 222 Hewitt. D. R. &. M. G, Irving. 1990. Oxygen consumption and ammonia excretion of the brown tiger prawn Penaeus esciilentiis fed diets of varying protein content. Comp. Biochem. Phys. A. 96:373-378. Hochachka. P. W. & G. N. Somero. 1973. Biochemical adaptation. Cam- bridge. MA: Princeton University Press. Jobling. M. & P. S. Davies. 1 980. Effects of feeding on metabolic rate, and the specific dynamic action in plaice. Pleuronectes pUnessa. L. J. Fish Biol. 16:629-638. Lucas, A. 1993. Bioenergetique des animaux aquafiques. Paris: Masson. Ocampo, L. & J. M. Ezquerra. 2002. Digestive protease acrivity in juvenile Faifantepenaeiis califoniiensis as a function of dissolved oxygen and temperature. Aquae Res. 33:1073-1080. Rosas. C. A. Sanchez. E. Diaz. L. A. Soto. G. Gaxiola & R. Brito. 1996. Effect of dietary protein level on apparent heat increment and post- prandial nitrogen excretion of P. setiferus. P. schmitli. P. diioranim. and P. notialis postlarvae. J. World ,\qiiaciill. Soe. 27:92-102. Rubner. M. 1902, Die Gesetze des Energieverbrauchs bei del Emahrung. Leipzig and Vienna: Deuticke. Smith, R. R., G, L. Rumsey & M. L. Scott. 1978. Heat increment associ- ated with dietary protein, fat carbohydrate and complete diets in salmo- nids: comparative energefic efficiency. J. Nutr. 108:1025-1032. Taylor. C. R.. R. H. Karas, E. R. Weibel & H. Hopeler. 1987. Adaptative variation in the mammalian respiratory system in relation to energetic demand. II. Reaching the limits to oxygen flow. Resp. Physiol. 69:7-26. Villarreal, H. 1989. Feeding, growth and energetics of the freshwater cray- fish Chera.x leiuiinumus (Smith) with special emphasis on its potential for commercial culture. Ph.D. Thesis. University of Queensland. Aus- tralia. Villarreal. H. & L. Ocampo. 1993. Effect of size and temperature on the oxygen consumption of the brown shrimp Penaeus cidifornieiisis (Holmes, 1900). Comp. Biochem. Phys. A. 106:197-101. Jininwl of Shellfish Research. Vol. 22, No. 1. 285. 2003. ABSTRACTS OF TECHNICAL PAPERS Presented at The 23rd Annual MILFORD AQUACULTURE SEMINAR Milford. Connecticut February 24-26, 2003 285 Milt'ord Aqiiaciilture SL-inmar. Milford. Coiiiiecticul Abstracts. February 2003 287 CONTENTS Walter J. Bloguslawski Overview. 23rd Milfiird Aquaculture Seminar 289 Kathleen Becker and Kim Tetrault Photo doLiinienlation as a vital element in community based shellfish restoration programs 289 David Berry Insuring your aquaeulture erop 289 Don Bishop Economies, marketing and how they relate to growers husbandry methods 289 Diane Broiisseau, Sara Brady, and Allison Schaffer Preliminary investigations of shelter competition among the Asian shore crab and native mud crabs 290 Susan Biinsick Governing offshore aquaeulture: Progress and challenges 290 Joe Buttncr and Dale Leavitt Augmenting the lobster catch: Oyster aquaeulture in modified lobster traps 290 Lisa Calvo. Eugene Burreson, Susan Ford, John Kraeuter, Dale Leavitt. and Roxanna Smolowilz Variation in QPX susceptibility w ith host genetic origin 291 Julie Coininsky, Maureen Mikos, and Katie Sicona The potential of heat shock treatment for imprcned salinity tolerance of Siiliitd India 291 Todd Corayer Deep water, loiigline shellfish farming in NaiTagansett Bay 291 Barry A. Costa-Pierce The Rhode Island Aquaeulture Initiative 292 Yvonne Coursey, Nina Ahmad, Barbara McGee, Nancy Steimel, and Mary Kimble Embryonic blood cell formation in Liiiiulus polyphciuiis ( horseshoe crab) 292 Peter DeSanctis and Kim Tetrault Preliminary lindnigs on the effect of manipulating photoperiod on gonadal index of the bay scallop (Argopecten irrailiaiis iiiadians ) 292 Mark Dixon and Gary Wikfors Rotifer production on microalgal diets: Defining parameters for optimal production 293 Gef Flimlin, Michael Celestino, John Kraeuter, Robert Macaluso, and Michael Kennish Raritan Bav hard clam fishery management: Getting the data to make decisions 293 Tessa Getchis, Cori Rose, John Volk. Peter Francis, Robin Bray, Mark Johnson, and R. Michael Payton Aquaeulture policy in Connecticut — Constructing a permitting roadniap for stakeholders 293 Jack Grundstrom. Bonnie McAneney, Scott Weston, Mark Fregeau, and Joe Buttner Community efforts to restore local clam Hats 294 Edward Jaskolski, Michael Rice, and Karin Taninii Growth of Rhode Island quahogs. Meirenaria incrcenaria, in experimental upwellers as a part of the North Cape Oil Spill Restoration Project 294 Richard Karney and Enid Sichel In search of labor saving culture strategies for the bay scallop. Argopecten irradians irradians 295 Dale Leavitt. Brad Morse, Scott Soares, and Keith Wilda There is something fishy about that cranberry bog! 295 Clyde MacKenzie. Jr. The spread of sea lettuce in estuaries of North America and Europe and its potential effects on shellfish culture 295 Christopher Martin, Dean Perry, David Nelson, Robin Katersky, Stephen Metzler, Fu-Lin Chu, and Eric Lund Ciyptlu'cddiiiiiiiii cohnii. heterotrophic marine dinotlagellate: Is it a good alternate source of essential fatty acids for first-feeding larval finfish? 296 Paul Mangle Urban aquaeulture in Connecticut 296 Mary Morgan, Kathleen Becker. Marion Maino, and Kim Tetrault The first 1 8 months of a community-based shellfish restoration project for eastern Long Island. NY 296 Jessica Miische and David Bengtson Effects of weaning strategies on growth and survi\al of ju\enile summer flounder. Paraliclitliys dciiiatiis 297 288 Abstracts. February 2003 Milford Aquaculture Seminar. Milford. Connecticut David Nelson, Dean Perry, and Edward Baker Natural spawning of blacis sea bass. Centroprislis striata, at the NMFS Milford Laboratory and the UMASS Dartmouth Laboratory w ith observations on spawning beha\ ior 297 David Nelson, Dean Perry, Robin Katersky, and Stephen Metzler Grow th of juvenile black sea bass, Centropristis striata, in a recirculating seawater system 298 Christopher Parkins The potential of polychlorinated biphenyls contamination of aquaculture products through feed 298 Dean Perry, David Nelson, Robin Katersky, Mark Dixon, and Stephen Metzler Effects of high levels of ammonia. pH, and salinity in algal feeds on the mass production of rotifers 299 Cori Rose, Peter Francis, Robin Bray, and Tessa Getchis Evaluation factors for aquaculture gear applications 299 Anthony Rossomando. Ryan Kilmartin, John Roy, and Richard Cooper A comparison of mortahtv m the American lobster. Hoinaiiis americanus. using two methods of tagging 300 Otto Schmid, Armand DeLuca, and Kim Tetrault It takes a communitv to build a hatchery 300 Laurie Stafford, Jessica Miische, and David Bengtson Effects of container size on growth and metamorphosis of larval summer flounder. Paralichtliys deiuatus 300 Sheila Stiles, Joseph Choronianski, and Dorothy Jeffress Genetic strategies for culture and stock enhanceinent of bivalves 301 Amandine Surier and Richard Karney Oyster triploidy trials on Martha's Vineyard 301 John Wadsworth, Tessa Getchis, and Nancy Balcom Razor clam, Ensis directits, growth rates in Niantic River, Connecticut 302 Bill Walton The long and winding road; Towards sustainable fisheries management and meaningful shellfish restoration ( Wellfleet. MA ) 302 Scott Weston, Bonnie McAneney, Mark Fregeau. and Joe Biittner Mo\ ing tow ards cominerciali/ation of softshell clam culture on Massachusetts' Northshore 302 James Widman, Jr. and David Veilleiix Demand feeding of bav scallops. Artiopecteii irradians irradiaiis using an automated control system 302 Gary Wikfors, Barry Smith, Shannon Meseck, Mark Dixon, and Jennifer Alix A decision tree for designing a process to produce microalgal feeds for aquacultured animals 303 William Wilcox and David Grunden Initial in\ estigation of an annual Proioccmntm bloom in Lagoon Pond, Martha's Vineyard 303 Lawrence Williams, Tessa Getchis, and hike Sunila An update on blue mussel culture in Long Island Sound 304 Milford Aqiuicultuie Seminar, Miltord, Connecticut Abslrach. February 2003 289 OVERVIEW. 23rd MILFORD AQUACULTURE SEMINAR. Walter ,|. Blogoslawski, United States Department of Commerce. National Oceanic & Atmospheric Administration, National Marine Fisheries Service. Northeast Fisheries Science Center, Miltord Laboratory. 212 Rogers Ave.. Milford. CT 06460. There were 162 registrants for the 2.^rd Milford Aquaculture Seminar, a gathering of industry, research, and academic interests. By blending both the theoretical and practical aspects of aquacul- ture. the meeting permitted attendees an exchange of technology in aquaculture methods outside their own expertise and provided a forum where the latest innovations were mtroduced and discussed. Forty-two formal papers and posters were presented by attend- ees from eleven US states, the District of Columbia and Canada. Meeting attendees represented three vocational aquaculture high schools. !.■? universities, five marine labs, and se\eral state and federal institutions involved in shellfish and finfish aquaculture. A highlight of the meeting was a set of papers reviewing the aqua- culture research activities at the NMFS Milford lab in algae and tlsh culture, fish feeds, scallop culture, and the role of genetics in culture and enhancement of aquacultured products. Other papers co\ered crop insurance, fish fanning in cranben'y bogs and how pol- lutants can bioaccumulate in culture feeds. Mr. Tim Keeney. NOAA Deputy Assistant Secretary for Oceans and Atmosphere, descnbed NOAA's position on aquaculture during a luncheon address. The Seminar has de\eloped a tradition of offering the latest information available in the field in an informal atmosphere. This has succeeded in promoting a free exchange among all with an interest in the success and future of aquaculture. This Seminar continued that approach which allowed all attendees to enjoy and learn from the formal presentations and afforded informal oppor- tunities to di.scuss the latest developments pertinent to this impor- tant expanding field. At this year's seminar thirty-three separate aquaculture com- panies met in an evening session for their annual industry group meeting of the East Coast Shellfish Growers Association. The Association's goals are to promote and protect shellfish members" needs in state and regional contexts and involve all stakeholders in the task of enhancing the shellfish aquaculture industry. In addi- tion, federal and state agencies involved in regulation of offshore aquaculture described the new permitting system and how it might affect the indu.stry's development. The meeting was sponsored by the National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford, CT. Abstract printing was courtesy of the U. S. Depart- ment of Agriculture, Northeastern Regional Aquaculture Center, N. Dartmouth, MA. PHOTO DOCUMENTATION AS A VITAL ELEMENT IN COMMUNITY BASED SHELLFISH RESTORATION PRO- GRA.MS. Kathleen Kmet Becker, and Kim Tetrault. Cornell Cooperative Extension of Suffolk County Marine Program, Marine Environmental Learning Center, Southold. NY 11971. Community nnoUenient in local programs dedicated to vari- ous aspects of shellfish restoration has grown dramatically in re- cent years. Documentation is increasingly important and expected and can be used as a powerful tool to benefit any program. The Special Projects in Aquaculture Training (SPAT) program has. from its onset in January of 2001, compiled an extensive library of photographic images as one pail of its documentation process. The photo documentation provides an ongoing chronology of the program's projects and growth. It is being used to document scientific data collection and community involvement in restora- tion and stewardship activities. It is useful in the grant application and subsequent reporting process. As a visual aid and information sharing tool, it is being used to educate and to communicate to a broader public through posters, marketing and Power Point® pre- sentations. Photographic recognition of individual volunteer participation in restoration activities highlights the grass roots efforts and helps illustrate the social dynamics of a program. ® The use of trade names is to identify products and does not imply endorsement by the National Marine Fisheries Service. INSURING YOUR AQUACULTURE CROP. David Berry, Hartford Company, 2625 S. 158th Plaza, Omaha, NE 68130. As an aquaculturist, you face various inherent financial risks. Among them are the loss of the initial investment in the crop, loss of the investment in any growing facilities, and loss of income associated with the finished crop. This session will help you better understand these risks, and ways to minimize them. It will explain how insurance can diminish your financial losses, and some of the other functions it performs. An overview of some risks for which insurance can be purchased will be given, such as power failure, disease, and windstorm. Attendees will also be given an outline of the underwriting and claims process involved with an insurance policy, and a brief review of the Federal Clam Insurance Program. ECONOMICS, MARKETING AND HOW THEY RELATE TO GROWERS HUSBANDRY METHODS. Don Bisliop, Bishop Aquatic Technologies Inc., Fukui North America, P.O. Box 669. IIO-B Bonnechere Road. Eganville, Ontario, Canada KOJ IT. The current Shellfish production in the United States and Canada has a wholesale trade of approximately 243 million USS. There is a substantial amount of imported shellfish that when added to this further creates a serious economic sector of the seafood industry. It is estimated that with an increased supply of safe, quality, branded product that the market place could be in excess of 325 million USS over the next decade. Consumer taste and consumption patterns are in constant change in our brand conscious society, the understanding of this and the relationship to social class structure and the buying habits 290 Abstracts. February 2003 Milford Aquuculture Seminar, Milford. Connecticut present evidence and opportunity for the shellfisli industry to grow very profitably. To address and take advantage of these factors, shellfish grow- ers have to deliver what the customer wants and not just what the shellfish grower can supply. Technology and strategies have been developed from the larval stage though husbandry practices to point of sale marketing that will attract and develop new and repeat customers. The challenge the industry will face will be the supply of a "Safe, Quality, Branded product" that can be sold at a premium price; this means that farm yield and efficiency is an equally im- portant part of the equation. The information presented will allow growers and industry spe- cialists in attendance to learn what is available in production tech- nology and marketing initiatives as well as the direction that they may take to develop a more profitable shellfish business for them- selves or their specific regional area now and in the future. PRELIMINARY INVESTIGATIONS OF SHELTER COM- PETITION AMONG THE ASIAN SHORE CRAB AND NA- TIVE MUD CRABS. Diane J. Brousseau. Sara Brady, and Allison Schaffer, Biology Department, Fairfield University, Fair- field. CT 06824. This study examined the potential impact of the recently intro- duced Asian shore crab, Hemigrapsus sanguineus, on shelter uti- lization by two native species of mud crabs. Euiypaiwpeus de- pressus and Paiu)peus herbstii. using laboratory experiments and field sampling at two sites in western Long Island Sound (Black Rock Harbor, BRH: Milford Harbor, MH). Abundance and distri- bution patterns of these species differed at the two sites. Similar numbers of mud and Asian crabs were found under rocks at BRH, but Asian crabs outnumbered mud crabs 15:1 at MH. Asian crabs were most abundant at mid-tide level, whereas 90% of the mud crabs occurred low in the intertidal. This is likely due to the low tolerance for desiccation exhibited by xanthid crabs (Grant & Mc- Donald 1979). At low tidal elevation, where most of the overlap occurred, between-site differences in under-rock microhabitat uti- lization were present. Only mud crabs were found beneath 75% of the rocks sampled at BRH. but at MH, mud crab species alone were found under only 5% of the rocks. Relative crab densities likely affect competitive outcomes and ultimately space utilization patterns. Results of shelter competition experiments conducted in the laboratory did not support the hypothesis that H. sanguineus affects shelter utilization by native mud crabs. The percentage of mud crabs occupying shell shelters remained unchanged when Asian crabs were present, but the percentage of Asian crabs oc- cupying shell shelters decreased relative to controls in trials where mud crabs were present. These findings suggest that E. depressus and P. Iicrhstii may affect patterns of habitat use by H. sanguineus. especially in the lower intertidal, where these species occur to- gether. However, direct experimental manipulations in the field coupled with long-term monitoring are needed to fullv understand the role of competitive interactions in determining the local dis- tribution of these species. GOVERNING OFFSHORE AOLIACULTURE: PROGRESS AND CHALLENGES. Susan M. Bunsick, Marine Policy Con- sultant. 3 1 14 Wisconsin Ave., NW, #702, Washington, DC 20016. Six key components of a governing framework for offshore aquaculture are identified, and used as benchmarks in assessing progress toward the development of offshore aquaculture policy for the U.S. Exclusive Economic Zone. From an aquaculturist's perspective, the most important components of a governing system for offshore aquaculture are mechanisms for ( 1 ) granting a range of rights to the aquaculturist and (2) protecting those rights. From the broader perspective of a national government, there is a need for mechanisms that (3) protect the rights of other legitimate users of public waters and (4) consider a range of other important na- tional interests and policy priorities. There is also a need to (51 develop administrative systems that are fair, effective, and effi- cient. This may include a requirement that (6) the aquaculturist provide some form of compensatii)n in exchange for the right to locate and operate an aquaculture operation in public waters. Fed- eral agencies, the research community, and others have begun to address the development of a governing framework for offshore aquaculture in the United States. While these initiatives have re- sulted in some progress, challenges remain. AUGMENTING THE LOBSTER CATCH: OYSTER AQUA- CULTURE IN MODIFIED LOBSTER TRAPS. Joe Buttner, Northeastern Massachusetts Aquaculture Center and Department of Biology. Salem State College, Salem, MA 01970; Dale Leavitt, Roger Williams University, One Olde Ferry Rd., Bristol, RI 02809. Traps used by commercial fishers to capture the American lobster {Hoinarus americanus) are constructed and fished in ways that approximate technologies commonly employed to culture the eastern oyster (Ciassostrea virginica). By modifying traditional lobster traps to incorporate trays for oysters it was hypothesized that oysters would survive, grow, and augment the income of lobstermen while promoting acceptance of aquaculture among commercial fishers, local cominunities, and regulatory agencies. To explore the biological feasibility and practical integration of oyster aquaculture in modified lobster traps a 2-y. cooperative study involving commercial lobstermen, regulatory agencies, and research/extension personnel was initiated in 2001. Ten lobstermen, six from Massachusetts" Northshore and four from Massachusetts" Southshore/Cape Cod/Islands were identi- fied, trained, and provided with modified traps and oysters from an approved source. Modified traps were fished adjacent to or in the same line as unmodified traps between May/June and October/ Milford AquacultLire Seminar, Milt'ord, ConneLticut Absuacls. February 2003 November in 2001 and 2002. Lohstermen recorded information (date, deptli fished, capture rale, and handling elTorl) in standard- ized journals. Results indicated that oysters in modified lobster traps sur- vived, grew, and can be managed without excessive handling or interfering with lobster removal. Lobster capture rales varied widely and it is unclear whether the modified lobster traps fish differently from traditional traps. Oyster growth was temperature dependent and integration of oyster culture in lobster traps seems most appropriate on the Southshore, Cape Cod, and the Islands. It is likely that the tech- nology can be transferred to other areas and applied to other bi- valves, providing supplemental income to lobsterfishers while nur- turing acceptance of aquaculture and perpetuating an important New England tradition, commercial fishing. VARIATION IN QPX SUSCEPTIBILITY WITH HOST GE- NETIC ORIGIN. Lisa M. Ragone Calvo. and Eugene M. Bur- reson, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point. VA 23062; Susan E. Ford and John N. Kraeuter, Haskin Shellfish Research Laboratory, Rutgers Uni- versity, 6959 Miller Ave.. Port Norris, NJ 08349; Dale F. Leavitt, Roger Williams University. One Olde Ferry Rd.. Bristol, RI 02809; and Roxanna Snioiouitz, Marine Biological Laboratory, Woods Hole, MA 02543. In recent years epizootics of quahog parasite unknown (QPX), a protistan pathogen of hard clams, Merceiuiria mercenaria. have occurred in Massachusetts, New York, New Jersey, and Virginia. Although it has been found in wild hard clam populations, this parasite has most seriously affected cultured hard clams suggesting *^at aquaculture practices may promote or predispo.se clams to the disease. In this investigation we examined the influence of host genetic origin and geographic location on QPX disease suscepti- bility. Five clam strains, originating from Massachusetts, New Jersey, Virginia, South Carolina, and Florida were produced at a single hatchery and evaluated during a 3-year period for growth, survival, and QPX susceptibility at three QPX endemic sites (Mas- sachusetts, New Jersey, and Virginia). Severe winter-associated clam losses occurred at the Massachusetts site precluding comple- tion of the study at that location. At the Virginia site, mortality at the termination of the experiment was 79% in Florida clams and 52% in South Carolina clams, as compared to 369f in Virginia. 33% in Massachusetts, and 209c in New Jersey clams. Differences between stocks were significant with mortality in the Florida and South Carolina clams being significantly higher than in the north- em clams. QPX prevalence in the South Carolina and Florida stocks ranged from 19-21% and 27-29% respectively in the sec- ond and third year of the study, while QPX prevalence in the Virginia, New Jersey, and Massachusetts slocks was 10% or less. Mortality was significantly correlated with QPX prevalence during the second and third years of the investigation. A similar trend was observed at the New Jersey site. Mortality al the termination of the experiment was estimated to be respec- tively 53%. 40%, 20%. 6%, and 4% in the Florida, South Carolina, Virginia, Massachusetts, and New Jersey clam stocks respectively. QPX was first detected in the clams 14 mo after planting. At 17 and 22 mo after planting. pre\alences ranged from 13-18% in the Florida stock. 20-38% in the South Carolina stock. 0-18% in the Virginia clams, and 0-5% in the New Jersey and Massachusetts clam stocks. These results suggest that genotype-environment in- teractions are important determinants of QPX disease. As such, hard clam culturisis should consider the geographic origin of clam seed an important component of their QPX disease avoidance/ management strategies. THE POTENTIAL OF HEAT SHOCK TREATMENT FOR IMPROVED SALINITY TOLERANCE OF SALMO TRUTTA. Julie Cominslvy. Maureen Mikes, and Katie Sicona. Bridgeport Regional Vocational Aquaculture School. 60 Saint Stephens Road, Bridgeport, CT 06605. Heat shock treatment has been applied to cross protection stud- ies, including salinity, ammonia, and nitrogen compounds. Brown trout. Sciliiio iniiici. were selected for this study to determine the potential of heat shock treatment for improved tolerance of salinity stresses. The heat shock was conducted in 10-gallon freshwater tanks for 10 minutes. Visual observations were conducted at 30- sec intervals. These visual observations included swim patterns and orientation, mucus excretion, respiratory motion rate, and scale loss. The fish were then removed from the heat shock treat- ment tanks and transported to the post shock recovery tank system. The post shock recovery tank system consisted of four 10-gallon tanks in a cold-water bath. The heat shock treatment range was 23-29°C set at 2°C intervals. As the shock temperatures increased, negative behavior patterns were observed, with mortalities occur- ring at 29°C. Based on these observations 27°C was determined the optimal temperature to perform the heat shock treatment in the salinity applications of 5 ppt to 20 ppt at 5 ppt intervals. Noticeable disparities between the control set and the heat shock set were not realized until the 20 ppt concentration was conducted. At 20 ppt the heat shock data showed a 100% survival rate over 96 h of salinity exposure, while, the control set showed a 70% survival rate over 96 h of salinity exposure. DEEP WATER, LONGLINE SHELLFISH FARMING IN NARRAGANSETT BAY. Todd Corayer, Salt Water Farms LLC, 30 George St., Wakefield, RI 02879. Salt Water Farms is developing a multi-species aquaculture business specifically sited to make use of an underutilized water column, and excellent environmental factors in an effort to estab- lish responsible, large-scale shellfish aquaculture in waters with many historical users. 292 Abstracts. February 2003 Milford Aquaculture Seminar, Milford. Connecticut Anchored with two different custom mooring configurations for this dynamic, open water site and serviced by our 36' vessel, the New Hope, we have deployed both vertical cage assays for Crassostrea vir}>inica culture and blue mussel drop-socking, in conjunction with spat collectors. Approximately 300,000 oysters, at an average size of 25 mm were confined in cages at densities that reflected market size spatial requirements. Mytilus edidis spat collectors, both svnthetic and recycled pot waip. were set to iden- tify spawning patterns and preferences. Blue mussels were also set into socking and have been examined throughout their grow-out. In cooperation with the University of Rhode Island, seasonal grab samples are being conducted, to determine any effects the farm may have on the benthic environment. Despite the usual learning curve, we experienced sufficient growth rates to enable a reasonable percentage of oysters to reach market size by the end of the growing season. Positioned mid- water, the design is an effort to establish a prototype large-scale farm that can operate successfully in the midst of other recreational and commercial users. Our main goal is to build a farm business where both animal and gear stocking densities have been thor- oughly tested and analyzed to have minimum environmental and social impact, while operating profitably. THE RHODE ISLAND AQUACULTURE INITIATIVE. Barry A. Co.sta-Pierce, Rhode Island Sea Grant College Program, Graduate School of Oceanography. University of Rhode Island, Narragansett. RI 02882. In an attempt to ele\ ate Rhode Island from last place among the 50 states in aquaculture production. Senator Jack Reed obtained 1.5 million USS for developing aquaculture in the Ocean State. The Rhode Island Aquaculture Initiative (RIAI) is a unique col- laboration that unites federal and state interests as well as aca- demic, regulatory, and industry resources. Funding from the National Oceanic and Atmospheric Admin- istration was awarded to the Rhode Island Coastal Resources Man- agement Council (CRMCl, the states lead regulatory agency for aquaculture. CRMC, in turn, enlisted the Rhode Island Sea Grant College Program to administer the project. In 2002, the RIAI di- rected 600,000 USS of that money toward aquaculture research and development in the state through a series of multi-year re- search grants and 1-y "mini-grants", awarding funding for projects that seek to improve the health and longevity of farmed shellfish, educate students and coinmunities about aquaculture, address con- cerns about aquaculture"s effects on the environment, help re- searchers and aquaculturists access aquaculture data, and reduce conflicts between aquaculturists and traditional capture fishermen. Funding for new capacity-building projects and industry-relevant aquaculture research has been made to help jump-start a new era ot aquaculture development in Rhode Island — a place where every- one says our collective challenges are among the greatest any- where— and help Rhode Island become a world-class aquaculture research and development center. EMBRYONIC BLOOD CELL FORMATION IN LIMULUS POLYPHEMUS (HORSESHOE CRAB). Yvonne Coursey, Nina Ahmad, Barbara McGee, Nancy Steimel, and Mary Kimble, Department of Biology. University of South Florida, 4202 E. Fowler Ave., SCA 1 10. Tampa. FL 33620. Invertebrates produce blood cells, but unlike vertebrates where blood cell production (hemopoiesis) takes place primarily in the bone maiTow, hemopoietic sites in invertebrates vary from species to species. The blood cells (amebocytes) of Limulus polyphemus Linnaeus are among the most widely studied of any invertebrate. Despite having received an enormous amount of attention the site(s) of blood cell forination in Limulus have remained elusive. The primary goals of this research were to determine where horse- shoe crabs (Limulus polyphemus) produce their blood cells, and when during embryogenesis blood cell production begins. To distinguish Limulus amebocytes froin other tissue, a poly- clonal antibody was raised against purified coagulogen protein, the major protein found in the amebocyte granules. The anti- coagulogen antibody allowed the identification of maturing em- bryonic blood cells from all other embryonic cells. Blood cell production begins in the developing embryo at stage 18, approxi- mately half way through embryonic development. Embryonic blood cells are located in body cavities. Blood cells mature in circulation, as seen by the increase in granulation of blood cells comparing stage 1 8 to stage 20 embryos. The presence of coagu- logen in the granules was confirmed using the anti-coagulogen antibody. PRELIMINARY FINDINGS ON THE EFFECT OF MA- NIPULATING PHOTOPERIOD ON GONADAL INDEX OF THE BAY SCALLOP (ARGOPECTEN IRRADIANS IRRADl- ANS). Peter N. DeSanctis, and Kim Tetrault, Cornell Coopera- tive Extension of Suffolk County-Marine Program. Marine Envi- ronmental Learning Center, Southold, NY 11971, Using gonadal index as a measure of fecundity, a preliminary experiment was performed in an attempt to demonstrate the effect of light on the reproductive capacity and rate of conditioning of the bay scallop. Populations of scallops were exposed to a regime of ambient light, continuous light, or continuous dark. All other vari- ables, such as water temperature and feed were held constant for the three test populations. In this initial and abbreviated study, it was observed that differences in gonadal index varied with pho- toperiod. It was found that scallops subjected to continuous light showed in a higher gonadal index throughout the test period as compared to the other treatments. Subsequent investigations will address fecundity, egg size and quality and minimizing condition- tiine to spawning. Milford AquacLillure Seminar, MiU'ord, Coniieclicut Absnaas. February 2003 293 ROTIFER PRODUCTION ON MICROALGAL DIETS: DE- FINING PARAMETERS FOR OPTIMAL PRODUCTION. Mark S. Dixon and Gary H. VVikfors, USDOC. NCAA. National Marine Fisheries Ser\ ice. Northeast Fisheries Science Center. Mil- ford Laboratory. Milford. CT 06460. Production of live feeds can be a bottleneck in finfish aquacul- lure. Producing sufficient numbers of rotifers on live microalyac can be an especially problematic step in this process. A balance of producing large volumes of suitable quality algae, maintaining appropriate growth paraineters for rotifers, and timing is required for success. Rotifer production at the Milford National Marine Fisheries Service Lab has had \aried levels of success and failure during fintlsh aquaculture prtijects. These e.xperiences. and a re- view of literature, suggest that a narrow set of parameters in both microalgae culture and rotifer culture must be met to assure con- sistent live feed production. Previous work at the Milford lab identified the Tetraselmis strain. PLY429. of microalgae as the best food for rotifer production of ten algae tested. It was also found that maintaining algal densities of six million cells per mil- liliter yielded the highest conversion efficiency of algal biomass to rotifer biomass. This study focuses on identifying the specific param- eters required to re;ir rotifers successfully on a moderate scale. Six. 30-liter, round-bottom drain vessels were used to test a single parameter at two different levels (each level in triplicate) at a time. Parameters tested included: ammonia level, light level, algal cell density, and mixing method. Rotifers were stocked at 50/ml to begin each trial. Total rotifers produced, percent of roti- fers with eggs, and various water/culture quality parameters were measured during each trial. Maintaining ammonia levels below 1 ppm in the algae and rotifer cultures was essential to rotifer growth. Illuminating the "green" rotifer cultures to levels of 1000 microeinsteins/square meter/second PAR at the surface led to higher rotifer production and reduced ammonia levels compared to room light alone. Maintaining algae densities at a constant high level (2-3 million cells/ml) produced more rotifers than letting rotifers graze down the algae population. Adequate bubbling to keep dissolved oxygen levels over 5 mg/1 throughout the "green water" culture was also essential to rotifer production. When all tested parameters were optimized, and with full 30-liter vessels, it was possible to consistently produce 500 rotifers/ml (15 million per tank) from 50/ml in 5-7 days. RARITAN BAY HARD CLAM FISHERY MANAGEMENT: (JETTING THE DATA TO MAKE DECISIONS. Gef Flinilin. Rutgers Cooperati\e Extension. 1623 Whitesville Rd.. Tt)ms River. NJ 08755: Michael Celestino, NJ DEP Bureau of Shell- fisheries, Nacote Creek Research Station. P.O. Box 418. Route 9. Milepost 51. Port Republic. NJ 08241 : .John N. Kraeuter, Haskin Shellfish Research Laboratory. Rutgers L!ni\ersity. 6959 Miller Ave.. Port Norris. NJ 08349: Robert J. Macaluso, Brookdale Communitv Colleize. Sandv Hook Field Station. Building 53. Sandy Hook. Highlands. NJ 07732: and Michael Kennish. Rut- gers Institute of Marine and Coastal Sciences. Cook College. 71 Dudley Rd.. New Brunswick. NJ 08901. The hard clam fishery in the Raritan and Sandy Hook Bays was a \iable entity until a hepatitis outbreak in the early 1960s closed the fishery. In 1983. a state-sponsored relay program and private depuration allowed the fishery to re-open in Monmouth County. At this time there are about 200 full and part-time clammers working those areas. They supply two depuration plants or move their catch to relay beds in appixned water in Ocean County for a 30-days purging process. They harvest about 35-40 million clams a year that have a dockside value of over 5 million USS. Some clammers can gross over $100,000 in this operation. Although a stock assessment was done by the State in 1983 a lack of funds prevented more surveys until 2000 when the New Jersey Department of Fish and Wildlife Bureau of Shellfisheries surveyed the same area again. At the same time. Rutgers Haskin Shellfish Lab. Rutgers Institute of Marine and Coastal Sciences, and Rutgers Cooperative Extension along with Brookdale Com- munity College conducted studies to determine the age and growth of the shellfish and a natural mortality study. Since the fishery appears to be very lucrative, there is increased interest by others to open depuration plants. The information from these three studies can allow the industry and the state to work better together to manage the harvest pressure and the participation in the area. Fortunately, the data show that the stocks are at higher levels than when harvest restarted in 1983. possibly allowing for further ex- ploitation. AQUACULTURE POLICY IN CONNECTICUT- CONSTRUCTING A PERMITTING ROADMAP FOR STAKEHOLDERS. Tessa S. Getchis. Connecticut Sea Grant. University of Connecticut. 1080 Shennecossett Road. Groton. CT 06340: Cori M. Rose, United States Army Corps of Engineers. New England District. 696 Virginia Road. Concord. MA 01742: John Volk. Connecticut Department of Agriculture. Bureau of Aquaculture. P.O. Box 97. Milford. CT 06460; Peter Francis and Robin Bray, Connecticut Department of Environmental Protec- tion. Office of Long Island Sound Programs, 79 Elm Street. Hart- ford. CT 06106: Mark Johnson, Connecticut Department of En- vironmental Protection. Fisheries Division. P.O. Box 719. 333 Ferry Road, Old Lyme, CT 06371; R. Michael Payton, Connecti- cut Department of Environmental Protection, Boating Division, P.O. Box 280, 333 Fen^ Road. Old Lyme. CT 06371. The permitting system for marine-based aquaculture in the State of Connecticut has had a complete overhaul in the past 2 y. As floating and submerged shellfish structures (longlines. cages, bags, racks, etc.) have been shown to be an efficient and produc- tive method for growing shellfish, their use has grown dramati- cally. The implementation of these types of gear has raised a number of permitting issues concerning; navigation, boater safety, aesthetics, environmental effects, liabilitv. etc. 294 Abstracts. February 2003 Milford Aquaculture Seminar, Milford, Connecticut A new aquaculture permitting policy was set up in Connecticut in October of 2001. The Connecticut Department of Agriculture. Bureau of Aquaculture (DA/BA) has collaborated with the United States Army Corps of Engineers (US ACE) and the Connecticut Department of Environmental Protection (DEP) to develop the Connecticut General Programmatic Permit for Aquaculture. The extensive new permitting process requires review of the above listed issues and others by a number of state (DA/BA. DEP). federal (USACE. National Marine Fisheries Service. United States Fish & Wildlife Service, United States Environmental Protection Agency), and in some cases, local officials. The Connecticut Sea Grant Extension Program (SGEP) has sponsored a workshop series on aquaculture policy and the per- mitting process. SGEP's partners include USACE, DA/BA, CT DEP, and municipal shellfish and harbor management commis- sions that aid in workshop development. The series includes work- shops specialized for various stakeholders including growers, policy-makers, extension services, researchers, educators, and the general public. The intent of the.se workshops is to provide stake- holders with information on Connecticut's aquaculture permitting process from local, federal and state perspectives, and to address the questions or concerns of these stakeholders. The goal of this workshop series is to facilitate communication and information transfer among stakeholders in the aquaculture permitting process. A list of objectives or ""needs" was developed at the first planning meeting. The immediate needs from the policy makers' standpoint were: (1) To develop a roadmap for aquaculture permittmg in Con- necticut. (2) To develop an online ""Guide to Aquaculture in Connecticut." (3) To develop a new strategic plan for aquaculture in Con- necticut. COMMUNITY EFFORTS TO RESTORE LOCAL CLAM FLATS. Jack Grundstrom. Shellfish Constable. Rowley. MA 01969; Bonnie McAneney, Scott Weston, Mark Fregeau, and Joe Buttner, Northeastern Massachusetts Aquaculture Center and Department of Biology, Salem State College. Salem. MA 01970. Since May 1999. officials and volunteers (primarily shellfish- ers) have released or redistributed millions of wild-caught and hatchery-reared softshell clams (Mv(( areiiaria) onto approved tidal flats in Rowley, Massachusetts. Initially, 6 capture nets (35' X 8' nets with a 1/4" x 1/8" mesh) were installed on flats in the Rowley River. Only two nets successfully collected wild clam seed. In 2000, 20 capture nets were set and all nets retained seed; some nets collected thousands of clams per square foot. Most clams caught in 2000 were distributed among local flats. -200,000 were transferred to the Northeastern Massachusetts Aquaculture Center's (NEMAC) Cat Cove Marine Laboratory and over- wintered. Concun-ently, the same number was held using spat bags in the Rowley River. These clams were seeded in the spring of 2001 and covered with predator exclusion netting (35' x 14' or 50' X 14' with a 1/4" x 1/4" mesh). In 2001, over 60 capture nets were deployed and all collected softshell clam seed with maximum den- sity reaching a few hundred per square foot. High densities were reduced by replacing the capture nets (35' x 8' ) with larger preda- tor exclusion nets (50' x 14'). Between 1999 to 2001 natural recruitment yielded large numbers of clam seed; however, in 2002 almost no seed was col- lected under capture nets in Rowley (and nearby towns such as Gloucester and Ipswich). Poor recruitment was partially mitigated by hatchery production. The town of Rowley received over 800,000 hatchery-reared clams from NEMAC. Clams were cul- tured in a Floating Upwelling System (FLUPSY). In the fall, clams were planted and covered by predator exclusion nets, to be har- vested when they attain market size. To restore and maintain healthy clam flats requires broad community support that includes monitoring and record keeping, facilitating wild recruitment, pos- sibly a hatchery, creative networking, and a lot of work! GROWTH OF RHODE ISLAND QUAHOGS. MERCE- NARIA MERCENARIA. IN EXPERIMENTAL UPWELLERS AS A PART OF THE NORTH CAPE OIL SPILL RESTO- RATION PROJECT. Edward Jaskolski and Michael A. Rice, Department of Fisheries, Aniinal and Veterinary Science, Univer- sity of Rhode Island, Kingston, Rhode Island 02881; Karin Tamnii, Department of Environmental Management Coastal Fish- eries Laboratory. 1231 Succotash Rd. Wakefield. RI 02879. The growth of northern quahogs, Mercenaiia mercenaiia. spawned from native Rhode Island non-notata broodstock was evaluated in experiment upwellers for the purpose of evaluating seed production methods for shellfish restoration projects. Down- wellers were constructed to accommodate 1.2 million small (400 fxm to 1 mm) hatchery-reared seed. Seed were moved to upwellers once they reached an average valve length of -2 mm. Upwellers were purchased from commercial sources and deployed at two sites. The primary location for the study was the Rhode Island Department of Environmental Management Coastal Fisheries Lab, Jerusalem, Rhode Island, vsith a secondary location at Roger Wil- liams University, Bristol, Rhode Island. Growth of the quahog seed was determined as a function of location, stocking density, le\'els of biofouling. and water tlow through the upweller silos. The quahog seed reached a maximum size of 13 mm at the end of the 20()2-growing season. To minimize overwintering and preda- tion loss the quahogs were overwintered in benthic cages. The seed will be field planted in designated shellfish restoration sites in the 2003 season when they reach an average valve length of 20 mm. This is publication number 3972 of the College of the En\ironment and Life Sciences, University of Rhode Island. Milford Aquacultuie Seminar, Milford. Coiiiicclicut Abstracts, February 2003 295 IN SEARCH OF LABOR SAVING CULTURE STRATE- GIES FOR THE BAY SCALLOP. ARGOPECTEN IRRADl- ANS IRRADIANS. Richard C. Karney, Marthas Vineyard Shellfish Group. Inc.. P. O. Box 1352. Oak Bluffs. MA 02557; Enid K. Sichel. Woods Hole Oceanographie Institution. Woods Hole. MA 02543. Farming the bay scallop. Argopecteii inadians inadians. is a labor-intensive proposition, primarily due to biofouling control on the netting of culture structures. Attempts to field culture small, early juveniles (2 mm) requires the use of small-mesh nettings { 1.5 mm) that require almost daily brushings to maintain adequate wa- ter flow to support survival and growth. Larger mesh nettings used to grow older scallops require less frequent cleaning, however, the number of cages required increases dramatically as the scallops grow. Several culture strategies including, reduced densities, cage- less culture methods using artificial eelgrass, biodegradable burlap nurseries, and adhesives were investigated as possible means of avoiding the labor costs associated with net cleaning. Juvenile scallops were cultured in spat bag nurseries at four densities (-3,000. 5,000. 7,000. and 11,000/bag) to determine if simply lowering the density could reduce the requirement for fre- quent bag brushing. Although growth correlated inversely with density, growth at even the lowest density was poor. "C-weed®". an artificial polyethylene (HOPE) eelgrass at- tached to a weighted aerated pipe, was investigated for its potential as both a spat substrate for setting scallops and a cageless field nursery system. Seed scallops that had set on the C-weed® grew well in the field but the initial set on the artificial eelgrass in the hatchery was poor. The use of biodegradable burlap to set and field-culture juvenile scallops remains a superior method. Twenty commercially available adhesives were tested for pos- sible application in a cageless culture methodology that involves attaching juvenile scallops to polyethylene netting with the adhe- sives. Several promising adhesives have been identified for further investigation. ® The use of trade names is to identify products and does not imply endorsement by the National Marine Fisheries Service. THERE IS SOMETHING FISHY ABOUT THAT CRAN- BERRY BOG! Dale Leavitt. Roger Williams University. One Olde Ferry Rd.. Bristol, Rl ()2S09; Brad Morse, DoubleM Cran- berry Company, 980 Walnut Plain Rd., Rochester, MA 02770; Scott Soares, Mass Department Food & Agriculture. 251 Cause- way St., Boston, MA 021 14; Keitli Wilda. Western Mass Center for Sustainable Aquaculture, University of Massachusetts. Am- herst. MA 01002. Over-production and limited market development ha\e de- creased the farmgate value of cranberries to the point where it is not covering production costs. Cranberry farming in southern New England is an economically important industry that also controls vast amounts of undeveloped land in an area that is rapidly ap- proaching build-out. It is imperative to develop alternate crops to permit cranberry farmers to stay in business thereby protecting an important sector of the local economy and protecting the land. We ha\e been involved during the past 2 y in modifying a new fish farming technology, the partitioned aquaculture system (PAS), to allow its use within a cranberry bog system while not changing the overall physical structure of the cranberry bog. A demonstration bog/PAS fish farm has been operating for part of one stimmer growing largemouth bass, yellow perch, and brown bullheads. Al- though a full grow-out season has not been reali/.ed. preliminary growth data suggest that the bog/PAS fish farm has potential to allow the cranberry farmer to produce an alternate crop within the bog system. While some water quality parameters were not com- pletely controlled (i.e., pH and dissolved oxygen) we were able to grow fish at a rate comparable to other fish farms. At this time we are planning to further develop the farm next summer to enhance phytoplankton production, the means of removing soluble nitrogen from the fish waste, by way of better control of water quality. In this presentation, we plan to introduce the concept of the parti- tioned aquaculture system and demonstrate its application w ithin a cranberrv bog. THE SPREAD OF SEA LETTUCE IN ESTUARIES OF NORTH AMERICA AND EUROPE AND ITS POTENTIAL EFFECTS ON SHELLFISH CULTURE Clyde L. Mackenzie. Jr., USDOC. NCAA. Northeast Fisheries Science Center. James J. Howard Marine Sciences Laboratory. 74 Magruder Road. High- lands. NJ 07732. In recent decades, the distribution of sea lettuce, Ulva sp.. has spread due to increasing loads of nutrients in estuaries in North America and Europe. The sea lettuce covers vast areas of shallow fiats in some years. A 2000 study in New Jersey and a 2001 study in Italy show that sea lettuce has a detrimental effect on macro- fauna. In New Jersey, small invertebrates were 2% as abundant on the surface of sea lettuce, U. lactuca, sheets and 25% as abundant under the sheets as they were on unvegetated sand bottoms nearby. In the Venice Lagoon in Italy the presence of sea lettuce U. rigida substantially changed the species composition of macrofauna and lowered their density from what it was 30 y earlier. The results suggest that the presence of sea lettuce substantially decreases abundance of small invertebrates and changes their species com- position. Sea lettuce crowds out eelgrass. Zostera marina, softshell clams, Mya arenaria. and forces northern quahogs. Mercenariu mercenaria. to emerge from the bottom. Aquaculturists who grow softshell clams and quahogs should remove sea lettuce from their planted beds. This can be done with a haul .seine: a 50- or 100-foot minnow seine is suitable. Removal needs to be done twice a sum- mer, initially about the first of June and again in late July or eariy August. Controlling sea lettuce also improves the condition of the overall ecosvstem in estuaries. 296 Abstracls. Febniary 2003 Milford Aquaculture Seminar. Milfoid, Connecticut CRYPTHECODINWM COHNII, HETEROTROPHIC MA- RINE DINOFLAGELLATE: IS IT A GOOD ALTERNATE SOURCE OF ESSENTIAL FATTY ACIDS FOR FIRST- FEEDING LARVAL FINFISH? Christopher Marthi. Dean Perry, David Nelson, and Robin Katersliy, USDOC. NCAA. National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford. CT 06460; Stephen Metzler. End to End. 415 Port Centre Parkway. Portsmouth. VA 2.^704; Fu-Lin Chu and Eric Lund, Virginia Institute of Marine Science, College of William and Mary, Gloucester Point. VA 23062. Traditionally, fish culturists have turned to autotrophic microal- gae for enrichment of larval prey (i.e.. rotifers and brine shrimp nauplii). For this puipose. algal strains have been selected for their essential fatty acid composition. Two long-chain polyunsaturated fatty acids (PUFAs) have received special attention since they have been shown to improve growth and survival of larval fish. These are docosahexaenoic acid (DHA. 22:6n-3) and eicosapen- taenoic acid (EPA. 20;5n-3). Their role in the normal development of the brain and visual system of young fish is of particular inter- est. The amount of DHA and EPA varies widely among microal- gae with some strains containing more of one than of the other. Consequently, it is usually necessary to enrich with a mixture of two or more different algal strains in order to achieve the desired fatty acid level in larval prey. Our objective is to demonstrate that a commonly occun'ing heterotrophic dinotlagellate. Crxpthccod- liiitiiii cohiiii Biechler. could serve as a source of DHA in this application. C. cohnii is a small, colorless dinotlagellate commonly found in association with decaying macroalgae (especially Fuais spp.) in the littoral /.one of temperate and tropical waters. It is readily isolated from its fa\ored substrate and cultured on seawater en- riched with simple sugars and yeast extract. Having a short gen- eration time (8-10 hrs). cultures attaining a density of ca.lO'' cells/ mL may be achieved in 3—1 days. It grows best in the absence of light and. while it is not fussy, seems to produce more fatty acids at 30'C than at lower temperatures. C. cohnii is an excellent source of DHA. Purification of DHA from this organism has led to com- mercial production of enrichment products for use specifically in aquaculture. We chose American Type Culture Collection strain 30772 \Ciyptliecodinium cohnii Biechler] for this work based on its re- ported fatty acid composition. We demonstrated the ease with which it could be grown in axenic batch culture on simple media. We showed that live cells of this heterotrophic dinotlagellate are acceptable to both rotifers and brine shrimp. Moreover, we docu- mented the transfer of PUFAs from C. cohnii to larval fish via enriched rotifers. Finally, we confirmed that larval tautog fed twelve days on rotifers enriched with live cells of C. colinii accu- mulated DHA and EPA in the near-optimum ratio 3.31 ± 0.02. In contrast, larvae fed rotifers enriched with a mixture of Isoclirvsis sp. and Tetraselmis sp. accumulated DHA and EPA in the ratio 1.42 ±0.07. URBAN AQUACULTURE IN CONNECTICUT. Paul D. Maugle, Mohegan Aquaculture LLC. .'i Crow Hill Rd.. Uncas- ville. CT 06382. Aquaculture in Connecticut has for the last 130 years tradition- ally harvested native-set shellfish from the bottom. Connecticut's oysters are among the most valued oysters reared in the United States. Knowing that bottom harvesting of native-set shellfish is not inherently sustainable in Eastern Connecticut waters. Mohegan Aquaculture LLC has chosen to have, at its core, a shellfish hatch- ery, coupled with mid-water and surface rearing of shellfish. The hatchery will be based on the systems installed by the Garbo Lobster Company in the village of Stonington. Connecticut. This hatchery when complete is projected to have the productive capa- bility of more than 200 million shellfish seed each year. This would make it one of the most productive on the east coast of the United States. The company's goal is to become a leading North American aquaculture producer of premium marine shellfish. With that goal in mind Mohegan Aquaculture will look to control the entire pro- duction process from culturing the microalgae that form the live feed for the larval shellfish to packing the finished product. This ensures that the company can deliver a superior quality eating experience to both ethnic and white tablecloth markets. When completed, the Stonington facility will house several profit and cost centers including a commercial scale shellfish hatchery, a wet storage facility, a commercial scale nursery, up- wellers. support space for near-shore longline and tray operations, and mooring facilities for shellfish har\est and long-line tender boats. The company has adapted several proven aquaculture tech- nologies to create its own proprietary production systems. Mohegan Aquaculture's production model utilizes a three spe- cies portfolio approach — bottom seeding of quahog clams, scallop, and oyster production in various types of floating midwater and corral structures. The enterprise will also strive to augment its production capabilities by working w ith third party contract grow- ing partners. This hybrid production approach allows the enterprise to focus on its core business competencies including production techniques, management expertise, and shellfish-value adding while utilizing available outside production capacity. THE FIRST 18 MONTHS OF A COMMUNITY-BASED SHELLFISH RESTORATION PROJECT FOR EASTERN LONG ISLAND, NY. Marv F. Morgan. Kathleen K. Becker, Marion Maino, and Kim Tetrault, Cornell Cooperative Exten- sion of Suffolk County Marine Program, Marine Environmental Learning Center. Southold. NY 1 1971. Cornell Cooperative Extension of Suffolk County. New York is in the second year of an expansion of its Marine Program to include a community-based shellfish restoration model to foster Milford AqLiaCLiltiire Seiniii;ir. Miltord. CoiuieL-licut Ahsiniciy FebmuiA 2003 297 stewardship of the Peconic Estuary. Special Projects in Aquacul- ture Training (SPAT) is based on the understanding that enhance- ment of shellfish beds contribute greatly to the health iif estuarine ecosystems, and that local communities can play a significant role in stewardship and restoration. Bay scallops. Aii;opeclen iinulians inacliuns. hard clams. Mercenaria inerccnaiia iiolata. and Eastern oysters. Cnissostrea virginica are commercially, recreationally, ecologically, and historically important species to the Peconic Es- tuary, which currently supports only ]'/c of its historic stocks. Nationally published data ha\e indicated that hands-on oppor- tunities in the environment help people become good caretakers of the environment. From the beginning, it has been a goal of the project to involve community members in a long-Iemi effort both to restore locally important marine resources and to develop a stewardship ethic. While capturing the interest and dedication of community members is a labor-intensive, year-round undertaking, the project has motivated many members of our Long Island com- munity. As of August 2002. 209 marine shellfish gardens are being maintained by individuals or families totaling 284 individuals. The community is involved in varying degrees in everything from tend- ing their own aquaculture gardens, attending monthly seminars, building and operating a community hatchery, developing an all- volunteer creek water quality testing team, public education efforts such as speaking at local civic groups, donating materials and supplies, and to creating and selling a cookbook to raise funds for the continuance of the project. EFFECTS OF WEANING STRATEGIES ON GROWTH AND SURVIVAL OF JUVENILE SUMMER FLOUNDER. PARALICHTHYS DENTATUS. Jessica Musche and David A. Benglson, Department of Fisheries. Animal and Veterinary Sci- ence. University of Rhode Island, Kingston, RI 02881. The transition from live feed to formulated diets, which aqua- culturists call weaning, is a difficult period in the hatchery rearing of some marine fish. Elevated mortality during this period is often due to cannibalism as some fish adapt to the new diet and grow while others do not adapt. Certain strategies have been developed with other species to optimize the weaning process. We conducted four experiments on the effects of weaning strategies on the growth and survival of newly metamorphosed juvenile summer flounder. Parol ichlhys dentalus. The goal of these experiments was to reduce growth variability and increase survival. Each experiment consisted of three treatment groups and one control group, with three replicates (75-L aquaria) each. The con- trol group in each experiment consisted of fish that were fed live brine shrimp nauplii throughout the course of the experiment. In each experiment, the actual weaning period lasted for two weeks, followed by several weeks of feeding with the new diet. All aquaria were on a fiow-through system, receiving Narragansetl Bay water at 20°C. In experiment I. we attempted to determine the optimum age at which to wean the fish. Fish were weaned onto a dry commercial pellet at 2, 4, or 6 wk post-metamorphosis. Fish that were weaned at 6 wk post-metamorphosis had the smallest mean lengths, but they had the least variability in growth and the highest sur\i\al. We. therefore, conducted each of the remaining experiments be- ginning at six weeks post-metamorphosis for that group of fish. In experiment 11, we studied the timing of weaning diet pre- sentation. Fish were given either a dry diet in the morning and brine shrimp nauplii in the evening, both dry diet and brine shrimp simultaneously in the morning and e\ening, or a dry diet and brine shrimp on alternate days. There were no significant differences in sur\ i\al or growth among the treatments; however, the fish given brine shrimp and dry diet simultaneously had the lowest variability in growth. In experiment 111, we explored the use of intermediate weaning diets. Fish were weaned directly to a dry pellet, weaned to frozen adult brine shrimp and then a dry pellet, or weaned to a semi-moist pellet and then a dry pellet. While there were no significant dif- ferences in growth between the fish fed dry pellet only and those fed frozen brine shrimp, the fish fed the semi-moist diet had a significantly lower growth rate. The fish fed frozen brine shrimp had the lowest variability in growth of the treatments. There were no significant differences in survival among treatments. In experiment IV, we attempted to use already-weaned fish to teach unweaned fish to accept a pelleted diet. Aquaria in each treatment were provided no already-weaned fish, one already- weaned fish, or five already-weaned fish. Clear barriers that al- lowed water to flow through were placed in the tanks to separate already weaned from unweaned fish. At the end of the experiment there were no significant differences in survival or growth among treatments, and very little difference in growth variability. In each experiment, the control groups had the highest survival. The con- trol groups also had the lowest variability in size, with the excep- tion of the first experiment in which those fish weaned at 6 weeks post-metamorphosis had the lowest variability in size. NATURAL SPAWNING OF BLACK SEA BASS. CENTRO- PRISTIS STRIATA, AT THE NMFS MILFORD LABORA- TORY AND THE UMASS DARTMOUTH LABORATORY WITH OBSERVATIONS ON SPAWNING BEHAVIOR David A. Nelson and Dean Perry. USDOC, NCAA, National Marine Fisheries Service, Northeast Fisheries Science Center, Mil- ford Laboratory, Milford, CT 06460; Edward Baker. 1.^6 Beech- wood Hill Tr, Exeter, RI 02882. The black sea bass, Cenlriiphstis striata, is an important sport and commercial fishery along the United States Atlantic coast. Black sea bass are managed under the Magnuson-Stevens Fishery Conservation and Management Act and by the Atlantic States Marine Fisheries Commission. Because the black sea bass is a temperate reef species and is unavailable to bottom trawlers, cap- ture is limited to anglers and pot fisheries. The demand for black 298 Abstracls. February 2003 Milford Aquuculture Seminar. Milt'ord. Connecticut sea bass exceeds supply and the hiyh market \alue has prompted researchers to evaluate its potential for commercial aquaculture. Reproductively, black sea bass are protogynous hermaphro- dites, developing first as females and later, at 3—4 y of age. trans- forming into males. Early attempts at spawning black sea bass centered around artificial spawning, collecting adult black sea bass in spawning condition and hand-stripping both males and females. Later attempts focused on inducing ovulation by intramuscular injection of two hormones; human chorionic gonadotropin or luteinizing hormone releasing hormone analog (LHRHa) and hand stripping. Milford Laboratory and UMASS Dailmouth Laboratory, have used photothermal manipulation to induce spawning. Black sea bass were placed in tanks of ambient, flowing seawater (10"C). The day/night cycle was controlled by a timer that turned fluores- cent lighting on and off. Lighting was adjusted every three days to simulate the day/night cycle that was occurring in nature until 15 h of light and 9 h of darkness was reached. When ambient temperature reached 1 8-20°C and the day /night cycle was 1 5 h of light and 9 h of darkness the black sea bass spawned. Fish were allowed to spawn in the tanks and embryos were collected on a 500- |xm screen or in a 800- ixm net. Fish were spawned under these conditions from mid-April to the middle of July at the Dartmouth Laboratory and from the end of May until the beginning of July at the Milford Laboratory. Percent viable embryos ranged from 0% (first eggs produced) to lOOVr (in the middle of the spawning season). We have also made observations on the spawning behavior of black sea bass in the course of our conditioning procedures. One dominant male (alpha) appeared to control spawning. The domi- nant male segregated other males and females in the tank (10 females to 5 males at Milford and 9 females to 5 males at UMASS). This one male prevented other males from mingling with the females. When spawning occurred a female would swim up to the alpha male and present herself. Both fish would move to a separate portion of the tank where the female would release eggs and the male would release milt. When spawning was complete the female returned to the other females and the alpha male positioned himself between the females and the other males. GROWTH OF JUVENILE BLACK SEA BASS, CENTRO- PRISTIS STRIATA, IN A RECIRCULATING SEAWATER SYSTEM David A. Nelson, Dean M. Perry, and Robin Kater- sky, USDOC. NOAA. National Marine Fisheries Service, North- east Fisheries Science Center, Milford Laboratory, Milford, CT 06460; Stephen Metzler, End to End Technical Services Inc., Suite 102, 415 Port Centre Parkway, Portsmouth, VA 23704. The black sea bass. Ceniropristis striata, is currently being investigated as a potential aquaculture species. Work to date has focused on spawning and larval development, conditions for cul- ture of larvae, stocking densities of larvae and juveniles, feeding trials of juveniles and sub-adults, cannibalism in juxeniles, habitat preferences in juveniles, and the effect of water velocity on the juveniles position and moxement. Many of the studies on juveniles have been conducted with wild-caught black sea bass. Although black sea bass show great potential for aquaculture. studies have not demonstrated the time required to produce a market-size fish. Our goal is to grow black sea bass from larvae to market size adults (454-680 grams) in 24 months. Black sea bass are spawned naturally by photothermal manipulation. Embryos are collected on a 5()()-(j.m stainless steel screen. Viable embryos float and are separated from dead embryos in an Imhoff cone. The embryos are placed in 1,140 L cone-bottom tanks filled to 1,100 L with 20°C seawater. These tanks are part of a closed, recirculating seawater system with a biofilter and U.V. light. Embryos are allowed to hatch (48 h) and grow into juveniles in these tanks. Fish remain in this system for 3-4 mo and are culled by size before being trans- ferred to two 1,067 L half-round tanks ( 120.5 cm diameter x 60.2 cm depth x 180.7 cm length). Filtered seawater ( IO-|xm) is recir- culated in these tanks with 10% water replacement/day. These tanks have biofilters and U.V. lights associated with them. Flow rate is 113.6-151.4 L/min. Temperature in these tanks is main- tained at 20 ± 1°C. Fish are weighed and measured on the day of transfer and once every two weeks thereafter. After 477 days in this recirculating seawater system, fish have grown from an initial mean length of 91 mm and a mean weight of 15.6 g to mean lengths of 232.3 mm and 197.7 mm and mean weights of 242.2 g and 177.9 g in the two tanks. Juvenile black sea bass that were produced from fish spawned in 2002 have grown from mean lengths of 80 and 106.9 mm at transfer to 1 17.2 and 146.5 mm in 83 days. Mean weights have increased from 10.9 and 23.6 g to 32.8 and 65.5 g in 83 days. Fish spawned in 2001 had specific growth rates of 0.499r and 0.56% per day. Black sea bass spawned in 2002 had specific growth rates of 0.6% and 0.8% per day. THE POTENTIAL OF POLYCHLORINATED BIPHENYLS CONTAMINATION OF AQUACULTURE PRODUCTS THROUGH FEED. Christopher Parkins. Bridgeport Regional Vocational Aquaculture School, 60 Saint Stephens Road, Bridge- port, CT 06605. Polychlorinated Biphenyls (PCBs) are a group of industrial organochlorine chemicals that are a major environmental concern. They are used commercially because they are chemically inert liquids, have low vapor pressures, are inexpensive to produce and are excellent electrical insulators. Due to the fact that PCBs are inert chemicals and soluble in fatty tissues. PCBs undergo bio- magnification. Most aquaculture products rely on commercially processed feeds. These feeds are based on wild-stock fishmeal, which may be contaminated with PCBs found in the natural environment. Through the consumption of these aquaculture products PCBs pose numerous health risks to humans. These include birth defects. Milford Aquatulture Seminar. Milford. Connecticut ALmraas. February 2003 299 carcinogenic potential and negative impacts to the immune system. The feed types, which are being tested, include Zeigler Trout Feed®. Silver Cup Floating and Sinking Trout Feed® and Hartz Turtle Feed®. The PCBs are extracted from the feed samples using a microwave extraction system following EPA method 3546. A temperature programmable gas chromatograph with a dry electro- lytic conductivity detector (DELCD) was used following EPA method 8082 to determine the qualitative level of Aroclor® 1260 in the samples. Two trials were performed which showed the ab- sence of Aroclor® 1260 in all tested samples. ® The use of trade names is to identify products and does not imply endorsement by the National Marine Fisheries Service. EFFECTS OF HIGH LEVELS OF AMMONIA. PH, AND SALINITY IN ALGAL FEEDS ON THE MASS PRODUC- TION OF ROTIFERS Dean M. Perry. David A. Nelson. Robin Katersky. and Mark Dixon, USDOC, NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center. Milford Laboratory, Milford, CT. 06460; Stephen Metzler. End to End Technical Services Inc., Suite 102, 415 Port Centre Parkway, Portsmouth. VA 23704. The rotifer, Bmclnonus pikatilis. has been widely used as a live food for feeding the larval stage of marine fishes. Successful aquaculture of marine fish requires adequate and reliable produc- tion of high-quality, nutritious rotifers. One method of culturing rotifers is to feed them microalgal diets that promote rapid growth and reproduction. The rotifers used in our aquaculture studies of the tautog and black sea bass were fed the algal strain Tel rase I mis sp. (PLY 429). This alga not only promotes rapid reproduction of the rotifers, but also contains the n-3 and n-6 polyunsaturated fatty acids that have been shown to promote growth and survival in larval marine fish. Tetraselinis was cultured under semi- continuous conditions in three large, open rectangular fiberglass tanks that received constant lluorescent lighting. These tanks were maintained between 2()0-3()(l L. Rotifers were fed Tetraselinis from two of the three tanks on a rotating basis. Initially, for about 1 week, rotifers showed an increase from 4 to 16 million. After that time, the rotifer population declined to five million and remained at that level for 2 wk. During that time, and for the next 3 mo. sporadic measurements of ammonia, salinity, and pH were taken in each of the three algal tanks. High levels of unionized ammonia (>1 mg/1). and abrupt changes in salinity (±5 ppt) and pH (±1 pH unit) in the algal tanks coincided with decreases in the rotifer population. Those measurements indicated that either individual fluctuations in salinity. pH and ammonia, or a combination of two or more of these factors adversely affected rotifer production. We conclude that changes in salinity, pH. and ammonia levels, as well as increased numbers of bacteria and ciliates in algal cultures can be counterproductive to maintaining high rotifer populations. It is recommended that algal tanks be inonitored daily during high ro- tifer production times for salinity, pH, and ammonia levels. Also. large open algal tanks should be monitored on a regular schedule for bacteria {Vibrio) and ciliates. Some alternatives to using live algae include concentrated algal pastes, baker's yeast and com- mercial products. EVALUATION FACTORS FOR AQUACULTURE GEAR APPLICATIONS. Cori Rose, Senior Project Manager, United States Army Corps of Engineers, New England District. Regula- tory Division. 696 Virginia Rd., Concord. MA 01742: Peter Fran- cis and Robin Bray, Connecticut Department of Environmental Protection. Office of Long Island Sound Programs. 79 Elm Street. Hartford. CT 06106; Tessa S. Getchis, Connecticut Sea Grant. University of Connecticut. 1080 Shennecossett Road, Groton, CT 06340. In response to the expansion of aquaculture activities and uti- lization of developing rearing techniques, there is an increasing need for review and evaluation of aquaculture proposals to ensure adequate protection of the environment, wild populations and their habitat, and the compatibility of such enterprises with existing users of the public resource. Regulatory agencies (federal, state, and local) are mandated to review applications for foreseeable future impacts, which a grower may not consider or be aware of. It is the charge of such agencies to achieve a balance between sometimes competing interests while ensuring appropriate regula- tion of the industry with due regard to the environment and its many users. For example, it is some or all of these agencies' responsibility to ensure that granting of a permit, lease or other authorization will not adversely impact marine resources or pose unacceptable disease, ecological, health, safety, or welfare risks to persons, the environment, or aquatic resources. In addition, agency determinations must also ensure that an authorized activity does not conflict with or negatively impact any recreational, commer- cial or other use of the proposed project area, or adversely impact the \ alue or use of private property in and around the area. The charge to an applicant proposing an aquaculture project, especially for a project that entails innovative technologies not currently used in a geographical area or for the culture of non- indigenous stock, is to provide enough information for regulators to make a reasoned decision. However, this can be a daunting task and the various parties' differing expectations regarding the amount and type of information needed may result in costly delays or protracted regulatory reviews. The purpose of this talk is to impart the type of information that should be submitted along with an application for aquaculture in Connecticut in order to facilitate the state/federal joint regulatory review process; and also to dis- cuss regional guidance that currently exists to aid aquaculture ap- plicants, convey expectations of the standard level, and provide the quality of information that may be solicited from regulatory agen- cies when seeking authorization of aquaculture projects. 300 Absiracts. February 2003 Milford AqiKicLilture Seminar. Milford. Connectieut A COMPARISON OF MORTALITY IN THE AMERICAN LOBSTER, HOMARUS AMERICANUS. USING TWO METHODS OF TAGGING. Anthony Rossomando. Ryan Kil- martin, and John Roy. The Sound Sehool. 60 South Water St., New Haven. CT 06519: Richard Cooper, UCONN. 1084 Shen- necossett Road. Groton. CT 06340. The American lobster. Hoinanis anieiicaiuis. has been the sub- ject of tagging studies for the past several decades. The benthic life cycle of the lobster and the ease with which they are trapped make them a species that lends itself readily to recapture studies. Popu- lation declines in southern New England during the past decade have made investigations into the recruitment methods of this spe- cies a priority for several studies. The means by which the species propagate makes the female lobster the preferred sex for many studies. A large percentage of the female animals that survive to maturity will bear eggs annually. The impact of tagging female lobsters in Southern New England, where the population is declin- ing, warrants the investigation of the stress caused by the tagging procedure. Outcomes from catch and release studies that depend on the capture of tagged animals to produce data are influenced by re- capture percentages. While many factors influence the success of the recapture rate, mortalities that result from the capture, tagging, and subsequent release of aquatic animals adversely affect all study outcomes. Investigators and scientific researchers have used many methods of marking animals that have been taken in this type of study. Students from the Sound School Regional Aquacul- ture Center conducted a study to compare the effects of tagging adult female lobsters with both Floy tags and Back tags. The results from this study indicate that mortalities associated with the stresses caused by tagging increased in tagged specimens. Mortalities of 19.1% and 14.3% were recorded in Back and Floy tagged lobsters respectively while the lobsters held as controls had mortalities of 9.59r. The students at the Sound School have had first hand experience with the dramatic declines in the lobster populations in western Long Island Sound during the late 1990s. We belie\e that it is becoming increasingly important to monitor accurately the existing stocks of lobsters at all levels of the fishery. However, it has become increasingly apparent through our studies that tagging efforts, which employ either the Floy or Back tag to study Hoiiuinis (iinericniuis. may be inflicting substantial mortali- ties among the sampled portion of the population. IT TAKES A COMMUNITY TO BUILD A HATCHERY. Otto Schmid, Arniand DeLuca, and Kim Tetrault, Cornell Co- operative Extension of Suffolk County Marine Program. Marine Environmental Learning Center. Southold. NY 11971. The program Special Projects in Aqiiaculture Training (SPAT) at Cornell Cooperative Extension, in Southold. New York, has just completed its second year of operation, having attracted over 200 families volunteering over 1 1 .000 h. Construction of a community hatchery began in the fall of 2001 and was made operational in the spring of 2002. SPAT members supplied all of the labor necessary to do the carpentry, plumbing and electrical work. This became a valuable learning experience for 12 core workers, augmented by numerous additional SPAT members on an "as available" basis. Utilizing many recycled materials, donations of supplies and equipment and volunteer labor, the cost of the hatchery was mini- mized. During the 2002 winter layover following its initial grow- ing season, the layout and exterior were revised and improvements made to make the hatchery more efficient, in addition to adding a maintenance annex for tools and equipment. The hatchery at this time houses six 400 L larval rearing conicals along with the nec- essary aquaculture equipment needed to produce approximately 6-9 million larvae per spawn. Selected species of bivalves are spawned in the hatchery and the larvae are raised through metamorphosis, at which time they are moved to downwellers in the Marine Center nursery. All coni- cals are maintained three times weekly at which time the equip- ment is cleaned and the larvae are culled and restocked to a desired density. Larvae are fed a mixed diet of algae produced at the Marine Center. Larvae are set using a variety of techniques. The community hatchery is the product of the diverse talents of many individuals. It serves as an invaluable tool for practicing the concepts learned during the training initiatives of the program in a hands-on and productive manner. As with many of the components of the SPAT program, the hatchery is a work in progress and is unique in many ways. The individuality and commitment of the SPAT members have allowed the hatchet^ to perform effectively in its first year of operation and is anticipated to greatly increase production in the 2003-growing season. The emphasis will be placed on the production of bay scallops (Argopecten irradians irniJIaiis) with a target goal of 10-1.5 million post-set. EFFECTS OF CONTAINER SIZE ON GROWTH AND METAMORPHOSIS OF LARVAL SUMMER FLOUNDER. PARALICHTHYS DENTATUS. Laurie Stafford, Jessica Miische, and David A. Bengtson. Department of Fisheries. Ani- mal and Veterinary Science. University of Rhode Island. Kingston. Rl 02881. Commercial aquaculture of the summer flounder, Paralichthys dentatus. began in the 1990s. Although research on optimum con- ditions and methods to rear the larvae has been conducted for years, many factors remain to be studied. Growth rates of summer flounder begin to vary greatly in the late larval period. Because metamorphosis is size-dependent, the fastest growing fish settle first and often cannibalize their slower-growing siblings who settle later. Many variables may affect growth and metamorphosis. We examined effects of container size by conducting an experiment of 49 days duration in which larvae were raised from age 12 days (after hatch) through metamorphosis. Fish were obtained from GreatBay Aquaculture in Portsmouth, NH and were the result of Milford Aquaculture Seminar. Milford. CoihiclUlui Ab<,lraci\. February 2003 301 their first pureh Fl male x Fl female crosses. The experiment consisted of three treatments: 2-L, 20-L, and 150-L containers with four replicates of each treatment and stocking densities in all con- tainers of 10 fish/L. Three specific variables were examined: sur- vival, growth (as measured by total length), and the rate of meta- morphosis (as measured by settlement times of the fish; settled fish were removed from each container every 3 days). Although there were no significant differences in survival among the three treat- ments, container size did affect growth and metamorphosis. The length of the fish in the 20-L containers was significantly greater than in the other two treatments until shortly before metamorpho- sis, after which the fish in the 1 50-L containers surpas.sed the other treatments (.^NOVA. P < 0.05). Analysis of the distributions of settlement over time indicated that fish in the 20-L containers metamorphosed earlier than fish in the 150-L containers (Kolmo- gorov-Smimov test, P < 0.U5). but metamorphosis of fish in the 2-L containers was not significantly different from that of fish in either 20-L or 150-L aquaria. Because commercial aquaculture of summer flounder larvae is conducted in volumes of 1.000 L or greater, our results may have more significance for the research community than for the industry. Nevertheless, container size can affect summer flounder larval growth and metamorphosis. GENETIC STRATEGIES FOR CULTURE AND STOCK ENHANCEMENT OF BIVALVES. Sheila Stiles. Joseph Choronianski, and Dorothy Jeffress, USDOC, NCAA, National Marine Fisheries Service. Northeast Fisheries Science Center. Mil- ford Laboratory. Milford. CT 06460. Genetic selection, which exploits the heritable component of sanation through breeding, has enhanced significantly the effi- ciency of livestock and crop production in agriculture. Aquacul- tured species lag behind, but ha\e provided some successes. How- ever, inadvertent selection and. therefore, narrowing of the gene pool can occur with standard hatchery practices, such as spawning small numbers of broodstock and screening or culling larvae and juveniles for size. This inbreeding effect can be minimized by developing appropriate strategies such as introducing new brood- stock to increase genetic diversity. In addition, in some hatcheries, there may be certain characteristics that are desired to improve or increase production. Selecting animals for growth, disease resis- tance or shell color could increase the frequency of these traits. For example, some scallops have obvious shell markings, such as stripes, which could be used in stock identification as nomta clams are used in the clam industry, and oysters with disease resistance. Selective breeding studies are underway employing scallops with striped shells as markers for stock enhancement, as one major limitation to previously conducted stock enhancement programs has been a lack of identification of stocks. The objective of this project is to investigate the feasibility of producing, through se- lective breeding, increased numbers of bay scallops with distinc- tive phenotypes for field identification. These naturally-occurring scallops with distinctively \isible markers at low frequencies of 1-5% are being developed to determine the reproductive success or genetic contributions of transplanted populations to stock en- hancement efforts possibly in sanctuaries. Preliminary laboratory results indicate a positive response to selective breeding with an increased frequency of at least 50% of scallops with striped shells, and favorable growth and survival. Other components of a breeding program, which could include marker-assisted selection (MAS) and quantitative trait loci (QTLs). should consider the following points: the status of the population, the goals to be attained (i.e.. harvesting, stock resto- ration or enhancement), facility and personnel needs, mating schemes, genetic monitoring (for genetic diversity), and periodic assessments. Genetic monitoring methods include cytogenetics, allozyme, mt and nuclear DNA analyses, and PCR technology. Alternative biotechnological and supplemental approaches to breeding encompass technology of gene transfer and chromosome engineering, such as induced polyploidy (triploidy and tetra- ploidy). Genetic applications additionally could involve DNA- based probes and assays to detect disease agents as in MSX and Deniio studies with oysters, as well as the generation of molecular tags to identify stocks of shellfish. All of these diverse aspects are applications of genetics to aquaculture and fisheries management that should be considered in strategies to maximize production. OYSTER TRIPLOIDY TRIALS ON MARTHA'S VINE- YARD. Amandine Surier and Richard C. Karney, Martha's Vineyard Shellfish Group. P. O. Box 1552, Oak Bluffs, MA 02557. Triploidy is the condition of possessing three times the haploid number of chromosomes in the cell nucleus. Because triploid bi- valves are sterile, their meat quality remains constant throughout the year and the energy usually used for reproduction is diverted towards somatic growth and disease resistance. Because of those unique qualities, their production has attracted worldwide attention since the early 1980s. At this time triploidy has been successfully applied to the economic benefit of the Pacific oyster industry on the west coast of the US and also in France. Under funding from the Sailors" Snug Harbor foundation of Boston, triploidy was induced in the American oyster Crassostrea viriiinica in an attempt to locally produce triploid strains of oysters for the growers of the island of Martha's Vineyard. Triploidy was induced with a low risk chemical. 6-DMAP that has been shown to be slightly less efficient than Cylochalasin B but much safer to handle and is water-soluble. The success of induction was mea- sured by flow cytometry at the Virginia Institute of Marine Sci- ence. By the third attempt, a 12-min treatment at a concentration of 400 (iM yielded 949r triploidy. After 9 days of development, differential mortality led to a percentage triploidy of over 96% in that same batch. Although we were successful in producing the triploid oysters. 302 Abstracts. February 2003 Millord Aquaculture Seminar, Milford, Connecticut the late production date coincided with deteriorating water quality prevalent later in the summer. Due to a toxic algae bloom iPio- roceiitriiin sp.), high bacteria {Pseiichvtioiuis sp.) and a minor oil spill, only a couple of thousand triploid and diploid control sur- vived and their growth was altered by the exposure. On October 2nd the surviving animals were transferred to one of the growers" high flow, tidal upweller nurseries. However the oysters did not grow and only time will tell if they were hardy enough to survive overwintering. RAZOR CLAM, ENSIS DIRECTUS, GROWTH RATES IN NIANTIC RIVER, CONNECTICUT. John Wadsworth, Nian tic Bay Shellfish, LLC. 15 First Street. Waterford, CT 06385, USA; Tessa S. Gefchis and Nancy Balcom. Connecticut Sea Grant, University of Connecticut. 1080 Shennecossetl Road. Groton, CT 06340. In 2001. Niantic Bay Shellfish. LLC partnered with the North- eastern Regional Aquaculture Center as part of a regional project to develop growout culture methods for the razor clam. £;?,v/,v directus. Approximately 10.000 seed (20 mm) were distributed evenly (one clam per 6.45 square cm) into felt-lined wire cages (0.6m:length x 0.6 nrwidth x 0.3 m:height) and were filled to a height of 15 cm of sediment. The cages were set and buoyed on leased ground in the Niantic Ri\er in Waterford. Connecticut. MLW was 0.6-0.9 m (site-dependent) with a tidal height of 0.85 m. Monthly inventories to determine clam density and growth (length and width to ± 0.01 mm) were performed beginning in September 2001. The clams increased in length from 18.84 ± 2.22 mm to 74.25 ± 6.54 mm in the first 2 y of the project. Grow-out trials have continued with limited success, as surviving clams are slow growing and have been increasingly susceptible to predation by green crabs. THE LONG AND WINDING ROAD: TOWARDS SUSTAIN- ABLE FISHERIES MANAGEMENT AND MEANINGFUL SHELLFISH RESTORATION (WELLFLEET, MA). Bill Walton. Wellfleet Shellfish Department. 300 Main Street. Well- tleet. MA 02667. Over the last year, the Town of Wellfleet (Cape Cod. MA. USA) has begun the long and often contentious process of devel- oping a long-term shellfish management plan. Here I describe the evolution of this plan from a traditional fisheries management approach (e.g.. gear limitations, increased fees, etc.) to a commu- nity-driven document that relies on input from the shellfishing community while promoting sustainability. Topics will include spawnmg sanctuaries, cultching. predator control, disease manage- ment, and monitoring efforts. In addition. I will review several steps we have taken toward increasing the efficiency of local shell- fish lestoration efforts. MOVING TOWARDS COMMERCIALIZATION OF SOFT- SHELL CLAM CULTURE ON MASSACHUSETTS" NORTHSHORE. Scott Weston. Bonnie McAneney. Mark Fregeau, and Joe Buttner, Northeastern Massachusetts Aquacul- ture Center and Department of Biology. Salem State College, Sa- lem, MA 01970. To support community-initiated enhancement and aquaculture efforts on Massachusetts' Northshore that target the softshell clam (A/vo areiuiria). the Northeastern Massachusetts Aquaculture Cen- ter (NEMAC) produced nearly 2 million juvenile clams in 2002. Beyond serving as a regional hatchery and nursery, NEMAC ex- panded outreach efforts that include technical assistance, educa- tional activities and networking with shellfishers and regulators. Survival of clams spawned by NEMAC personnel in 2002, to 2.0 mm, exceeded 80%. Resultant juvenile clams were distributed in July to Massachusett"s sites: 650,000 (ave. In. = 2.5 mm) to Rowley and 170,000 (ave. In. = 3.5 mm) to Martha's Vineyard. Another 220,000 clams (ave. In. = 14.0 mm) over-wintered in Smith Pool at NEMAC's Cat Cove Marine Laboratory (CCML) were also transferred to Rowley. About 800.000 juvenile clams were retained in spat bags placed in protective plastic cages and floated in Smith Pool. By the end of the growing season, clams had grown to 6.0 mm (ave. In.) with survival rates approaching 95%. Clams are being over-wintered in submerged cages for release onto approved flats in the spring/early summer 2003. To facilitate and expand clam production, a dual use Dock/ Floating Upwelling System (FLUPSY) was acquired by the Town of Rowley, through NEMAC's small grants program. Clams (650.000) were cultured in the FLUPSY as a cooperative effort involving Rowley shellfishers (maintenance and coordination), the Boy Scouts (maintenance and data collection) and NEMAC (tech- nical support, environmental monitoring and supplies). Sur\iving clams were released onto the Rowley tidal tlats and covered with predator exclusion netting (6.4 mm mesh). NEMAC also advanced private seed collection and grow-out projects in Ipswich and Gloucester. Massachusetts by providing materials and training. It is anticipated that clams spawned at the CCML will attain market size in 2003. the beginning of a sustainable, shellfish aquaculture industry on Massachusetts" Northshore. DEMAND FEEDING OF BAY SCALLOPS, ARGOPECTEN IRRADIANS IRRADIANS USING AN AUTOMATED CON- TROL SYSTEM. James C. Widman Jr. and David J. Veilleux. USDOC. NOAA. National Marine Fisheries Service. Northeast Fisheries Science Center. Milford Laboratory. Milford. CT 06460. We have developed a system that allows juvenile bay scallops. Aifiopecten imidiatis irradians. to be exposed to near-constant concentrations of phytoplankton, even as scallops consume it. Chlorophyll-a fluorescence levels are used to monitor phytoplank- ton cell concentration in the juvenile scallop culture system. Sea- water from the scallop culture is continuously pumped through a Milford Aquaciilture Seniiiiai. Milford. Connecticul Ahsumis. February 2003 303 WET® labs submersible nmiriuiieler using a Poiidinaster® mag- netic drive pump. The fluorometer outputs an analog signal (volt- age) proportional to the fluorescence. The analog signal is mea- sured by an ADAC® model 5516 DMA data acquisition board installed in a personal computer. An algorithm reads the voltage/ fluorescence and switches a relay on or off depending on the value. When the fluorescence drops below a preset value, the relay turns on and starts adding phytoplankton to the scallop culture with a peristaltic pump. On reaching the desired fluorescence (algal cell concentration), the algorithm switches the relay off which in turn stops the addition of algae to the culture system. By continuously monitoring the fluorescence level of the culture water, the algal cell concentration can be maintained and scallops are fed on demand. Scallops with an initial mean shell height of 7.2 mm grew to a mean shell height of 18.4 mm in 78 days using our prototype system. This growth was achieved while testing the mechanics and logic of the system. Additional monitoring systems are being built so we can analyze how algal cell concentration affects scallop growth. Our goal is to maximize scallop growth while minimizing phytoplankton consumption. This system would be amenable to feeding oysters, clams, mussels, brine shrimp, rotifers, and other phytoplankton grazers. ® The use of trade names is to identify products and does not miply endorsement by the National Marine Fisheries Service. A DECISION TREE FOR DESIGNING A PROCESS TO PRODUCE MICROALGAL FEEDS FOR AQUACUL- TURED ANIMALS. Gary H. Wikfors. Barry C. Smith, Shan- non L. Meseck, Mark S. Dixon, and Jennifer H. Alix, USDOC, NOAA, National Marine Fisheries Service, Northeast Fisheries Science Center, Milford Laboratory. Milford, CT 06460. Research, commercial, and publicly-funded mariculture facili- ties generally have a need to produce microalgal cultures to feed molluscan broodstock. larvae, and post-set, or to rear zooplankton as live feed for larval finfish or crustaceans. Too often, facilities and procedures for microalgal feed production are based on inap- propriate, previously-existing examples, leading to production pro- cesses that are under-scaled, expensive to operate, and undepend- able. A production process based on quantitatixc and qualitative needs would be preferable; a decision tree seems to be a useful tool for designing a microalgal feed-production process in a new aqua- culture operation or improving an existing facility. Main considerations for process-design decisions are defined by the nutritional and water-quality needs of the animals being fed — What? How Much? And How Often'.' Choices of "What" microalgae to grow will, to some extent, constrain the shapes and configuration of containers, and culture management options (i.e.. batch or some form of continuous or semi-conlinuous culture man- agement). In some applications, especially very intensive, recircu- lating systems, microbiological, and chemical aspects of water- quality become critical in defining feed-culture quality. Once ac- ceptable qualitative food requirements arc identified, then the quantitative characteristics of the process — "How Much and How Often" — must be addres.sed. Daily harvest volumes can be calcu- lated by dividing algal biomass quantities required to feed animals by estimated (conservatively!) biomass densities per unit volume of algal culture using the selected culture management. Options to replicate small production units many times or to monitor and manage several large cultures intensively can be considered in the context of dependability of the process. Rationale for choices made in Milford Laboratory microalgal feed-production processes — and surprises and problems encountered during operation of these pro- cesses— will be discussed. INITIAL INVESTIGATION OF AN ANNUAL PROROCEN- TRUM BLOOM IN LAGOON POND, MARTHA'S VINE- YARD. William M. Wilcox, Marthas Vineyard Commission, PO Box 1447, Oak Blutfs, MA 02557, USA; David W. Grunden, Oak Bluffs Shellfish Department, PO Box 1327, Oak Bluffs, MA 02557. For 5 out of the last 6 years blooms of Prorocemniin have been observed in Lagoon Pond on Martha's Vineyard. This salt-water embayment supports various uses including recreational boating and shellfishing. It has two hatcheries located on its shores: the Martha's Vineyard Shellfish Group's shellfish hatchery and the Massachusetts Division of Marine Fisheries Lobster Hatchery. This project is the first to investigate the possible causes of this almost annual Proroccntnini bloom. This report summarizes the lah and field data collected from five sample locations in Lagoon Pond during the period from mid-May through mid-September 2002. The data reported include vertical profiles of temperature and dissolved oxygen; dissolved and particulate nutrient analyses from both surface and deep sample sites; and chlorophyll, bacterial and phytoplankton analy- ses, and transparency. July and August rainfall was 5.5 inches less than the historic average for these two months. In addition, water table levels throughout the outwash plain set new records for monthly low stands as measured since 1991 (Wilcox 2003). This produced a lower than usual amount of fresh water input from rainfall, ground- water, runoff and fresh water surface inflow. As these sources are major contributors of nitrogen, there was less input of this nutrient to the system. During the course of study, the Lagoon maintained good water column transparency v\ith Secchi depth averaging 3 to 3.4 meters and never falling below 2.1 meters. The dissolved oxygen satura- tion was typically above 80 percent in the surface water but fell briefly to a low of 12 percent at 5 to 7 meters depth at one station. The pond is always limited by the availability of nitrogen but cycles between times when silica is and is not limiting to the growth of phytoplankton. In July, the Shellfish Hatchery quahogs became heavily fouled with vorticella, hydrozoans and bryozoans and showed symptoms 304 Abslmcts. February 2003 Milt'ord Aquaculture Seminar. Millord. Connecticut of lack of food or poor quality food. There were some indications of mild bacterial infection hut the usual die-off did not materialize in July as it has typically in previous years. A groundwater survey was conducted and. at the seeps sampled, nitrogen was 20 to 100 times more concentrated than in the pond. Silica was about an order of magnitude greater but onho-phosphate was roughly equal to the in-pond concentration. Groundwater is clearly a source of nitrogen and silica to the sys- tem. The Upper Lagoon Pond discharges through a herring ladder at the Madeiras run. This freshwater pond experienced a severe algae bloom with chlorophyll a concentrations rising from 8.2 micro- grams per liter in mid-May to over 50 by mid-June. A second bloom began in mid-August, peaked at 143.-5 txgfL on August 19 and continued through the last sample round on September 12. The data collected hint at a complex cycle of phytoplankton populations, grazers including jellyfish, water quality, and shell- fish survival at the MVSG Hatchery. The jellyfish were numerous this year and. it is suspected, their feeding on copepods and other grazing organisms freed up primary phytoplankton growth, which is influenced by both the availability of nitrogen and silica. Low levels of nitrogen in the system probably subdued the phytoplank- ton bloom that, as a result, was not as excessive as perhaps pre- vious vears. AN UPDATE ON BLUE MUSSEL CULTURE IN LONG IS- LAND SOUND. Lawrence Williams, Jessie D.. Inc.. 68 Anchor- age Drive, Milford, CT 06460; Tessa S. Getchis, Connecticut Sea Grant, University of Connecticut, 1080 Shennecossett Road, Groton, CT 06340-6048; Inke Sunila, Connecticut Department of Agriculture, Bureau of Aquaculture. P.O. Box 96, Milford, CT 06460. As fisherman and commercial shellfish harvesters continue to struggle with fisheries disasters in Long Island Sound including disease, drought, storms, invasive species, etc., they are able to depend less and less on traditional fisheries. Maritime industry members have partnered with state and federal agencies to inves- tigate the possibility of new/alternative species for culture in Long Island Sound. In 2001. a pilot project was initiated to investigate long line culture of blue mussels {Myliliis ediiUs) in Long Island Sound. A natural set of seed mussels was collected on the long lines in the area of Charles Island off the coast of Milford, Connecticut, USA. Newly set mussels were observed on the long lines in the spring, summer, and fall of 2001 and 2002. The mussels reached market size of approximately 2.5 inches in less than 10 months. An investigation into the feasibility of a commercial-scale op- eration involving blue mussel culture in Long Island Sound has been proposed. Jininml of Shellfish Research. Vol. 22, No. 1, 305. 2003. ABSTRACTS OF TECHNICAL PAPERS Presented at The 95th Annual Meeting NATIONAL SHELLFISHERIES ASSOCIATION New Orleans. Louisiana April 13-17. 2003 305 National Shellfisheries Association, New Orleans. Louisiana Abstracts. 20U3 Annual Meeting, April 13-17, 2003 307 CONTENTS George R. Abbe. Candace A. Morrell, Carol B. McColloiigh and Christopher F. Diingan Environmental effects on Pcrklnsus muriiiiis infection rates, growth and survival among dermo-disease-free juvenile oysters in the Patuxent River, Maryland during drought conditions 317 Charles Adams, Effte Philippakos, Alan Hodges, David Mulkey. Dorothy Comer and Leslie Stunner Economic impact of the cultured hard clam industry in Florida 317 Standish A. Allen Jr., A. J. Erskine, Elizabeth Walker, Ronald Zebal and Gregory A. DeBrosse Production of tetraploid Suminoe oysters. C. unnkcnsis 317 Yvonne C. Allen, Charles A. Wilson. Harry Roberts, John Siipan and Ralph Pausina Ground truthing hydroacoustic data with commercial ovster dredging 317 L. S. Andrews, B. Posadas, D. Barrage and Michael Jahncke Oyster irradiation: Pathogenic Vibrio response and consiuiier difference testing 318 Linda S. Andrews and Susan De Blanc Response of Vibrio vubujicits and V. parahafiuulyticiis 03:K6 318 William S. Arnold Population collapse, depensation effects, and the time-scale of recovery of hard clam (Mercenaria spp.) fisheries 318 William S. Arnold, Sarah L. Walters, Sarah C. Peters, Theresa M. Bert and Jon S. Fajans Influence of congeneric aquaculture on hard clam [Mercenaria spp. ) population genetic structure 318 Corinne Audemard, Lisa M. Ragone Calvo, Kimberly S. Reece, Eugene M. Burreson and Kennedy T. Paynter lit situ determination of Perkinsits marinus transmission dynamics 319 Jean-Christophe Avarre. Yannick Gueguen, Evelyne Bachere and Jean-Michel Escoubas Functional genomics: A powerful approach to study the immune response of the Pacific oyster Crassostrea gigas 319 Patrick D. Banks Biological assessment of storm effects on the Louisiana public oyster resource: Tropical Storm Isidore and Hurricane Lili 319 Carta D. Beals and Shirley Baker Clearance rates and feeding selectivity of Crassostrea virginica and Mercenaria mercenaria; implications of increased eutrophication in the Suwannee River Estuary 319 Donald L. Bishop Engineering and economics as related to Oysters Grown in the Gulf of Mexico 320 Karine Bouilly. Helen McCombie, Alexandra Leitdo and Sylvie Lapegue Persistence of atrazine impact on aneuploidy in the Pacit~ic oyster, Crassostrea gigas 320 Daniel Bourque, Thomas Landry, Jeff Davidson and Neil McNair Impact of an invasive tunicate in Atlantic Canada: Recruitment and competition 320 V. Monica Bricelj, John Kraeuter, Eric N. Powell, John M. Klinck, Eileen E. Hofniann. Ray Grizzle and Stuart Buckner A simulation model of the population growth of hard clams {Mercenaria mercenaria). III. Effects of brown tide 320 Kenneth M. Brown. Gary Peterson, Mike McDonough, Patrick Banks and Brian Lezina Deterrents to black drum predation on oyster leases 321 Nicole T. Brun, V. Monica Bricelj, Emmanuel E. Egbosimba. Thomas H. MacRae and Neil W. Ross Stress responses in scallops and hard clams to heat and cold shock 321 Eugene M. Burreson. Kimberly S. Reece, Karen L. Hudson and Christopher F. Dungan Perkinsns chesapeaki and Perkinsus amirensi are the same species 321 David Bushek, Donnia Richardson, Yvonne Boho, Loren Coen and Jennifer Cardinal Evaluating shell quaiantine duration to limit the transfer of Perkinsus marinns when planting oyster cultch 321 Kevin R. Calci High hydrostatic pressure inactiv alion of viruses 322 Lisa M. Ragone Calvo, Gene M. Burreson, Susan E. Ford. John N. Kraeuter, Dale F. Leavitt and Roxanna Smolowitz Host genetic origin an important determinant of QPX disease 322 Mark D. Camara and Standish K. Allen Jr. Experimental evaluation of crosses within and among five commercial strains of hard clams, Mercenaria mercenaria. across a salinity gradient in Virginia waters 322 308 Ab.stnicts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana Ruth H. Carmichael, Andrea C. Shriver, Erica T. Weiss and Ivan \ aliela Growth of quahogs (Mercenaria mcnenaria) and softshell elams (Mya arenaria) in response to eutrophie-driven changes in food supply and habitat 323 Ryan B. Carnegie. Mark D. Caniara. Lisa M. Ragone Calvo, Kimberly S. Reece and Patrick M. Gaffney Development of a single nucleotide polymorphism (SNP) marker set for the hard clam. Merct'iuiriu mciccuaiia 323 Robert M. Cerrato, Amy E. Streck and Darcy J. Lonsdale Trophic interaction between hard clams and natural assemblages of plankton 323 Maria del Refugio Castaneda Chavez, Erasino Orrantia B., Violeta Pardio Sedas and Fabiola Lango Reynoso Presence of pathogenic bacteria in the lagoon systems La Mancha and Alvarado Veracruz. Mexico in water and oyster ( Cnissoslrea virgiiiica) 323 Daniel P. Cheney. Andrew D. Suhrbier. Aimee E. Christy, Hector S. Beltran. Jonathan P. Davis. Kenneth M. Brooks and Frank J. Smith Mussel gi-owth and food utilization in relation to water column conditions on raft systems in Puget Sound, Washington 324 Marnita M. Chintala and Karin A. Tamnii Assessing the effect of habitat alteration on shellfish populations 324 Mary C. Christman. Cynthia J. Giffen. Jon H. Volstad and Lynn W. Fegley Design and implementation of a survey of commercial blue crab effort in the Maryland portion of the Chesapeake Bay 324 Fu-Lin E. Chu and Jean-Francois Samain An integrated approach to bivalve domestication: introductory remarks 324 Loren D. Coen and Majbritt Bolton-Warberg Evaluating the impacts of harvesting practices, boat wakes and associated shoreline erosion on intertidal creek habitats in the southeastern US: Managers and restoration programs take note 325 David W. Cook History of post-harvest treatment to reduce Vibrio spp. in shellfish 325 Hua Dan Freshwater pearl culture and production in China 325 Richard L. Darden and Brian R. Kreiser Population genetics of the blue crab ( Callinectes xapidtis) in the Gulf of Mexico 325 Patricia M. da Silva. Antonio Villalha and Jose Fuentes Growth and mortalitv of different Osiica cdiilis stocks cultured in the Ri'a De Arousa (Galicia. NW Spain) 326 Patricia M. da Silva, Antonio Villalba. Maria J. Carballal and Jose Fuentes Differences in disease susceptibility among Ostrea edidis stocks cultured in Galicia (NW Spain) 326 Joth Davis and Dennis Hedgecock Crossbreeding in pacific oysters 326 Lewis E. Deacon The effect of algal toxins on the isolated ventricle of the clam. Mercenaria incrccnavia 326 Lionel Degremont, Pierre Boudry. Patrick Soletchnick, Edouard Bedier. Michel Ropert. Arnaud Huvet, Jeanne Moal and Jean Francois Samain Genetic basis of summer mortality in juvenile cupped oysters 327 Maryse Delaporte. Philippe Soudant. Jeanne Moal and Christophe Lambert Impact of environmental and nutritive conditions on defense mechanisms of oysters during an annual cycle 327 Leonard DiMichele. Stephan Towers and Donald Shepherd Mucin secretions and nacre deposition in the formation of pearls 327 Angela K. Dukeman. Norman J. Blake and William S. Arnold Reproduction in tfame scallops. Liiiui scahra scal'iv (born 1 778). from the lower Florida Keys 327 Christopher F. Dungan. Kimberly S. Reece and Karen L. Hudson In vitro propagation of Peil500C for 10 min. Processing at this T/T did not adversely affect the sensory qualities consumers expect in raw half-shell oysters. POPULATION COLLAPSE, DEPENSATION EFFECTS, AND THE TIME-SCALE OF RECOVERY OF HARD CLAM {MERCENARIA SPP.) FISHERIES. William S. Arnold*, Florida Fish & Wildlife Conservation Commission Marine Re- search Institute 100 8th Avenue SE St. Petersburg, FL 33701. The commercial fishery for naturally occuixing hard clams has a brief but eventful history in Florida waters. The first known fishery, initiated in the early I900"s on the southwest coast of the state, constituted one of the largest hard clam fisheries on record. The population that supported that fishery collapsed in the 1940s ■ and has never recovered. Smaller fisheries developed in the early I980"s and the early I990's in the Indian River on the Florida east coast. Those fisheries also collap.sed, apparently in response to freshwater inputs, and similarly have not recovered. The observed lack of recovery of any of those populations may result from depensation effects at low population density. Recovery may be protracted even with intervention. An alternative management ap- proach, taking into account the vagaries of hard clam recruitment and population survival, is proposed for consideration. INFLUENCE OF CONGENERIC AQUACULTURE ON HARD CLAM {MERCENARIA SPP.) POPULATION GE- NETIC STRUCTURE. William S. Arnold*, Sarah L. Walters, Sarah C. Peters, Theresa M. Bert, Jon S. Fajans, Rorida Fish & Wildlife Conservation Commission Marine Research Institute 100 Sth Avenue SE St. Petersburg FL 33701. An aquaculture-based hard clam industry is developing on the west coast of Florida. There, the species Mercenaria campechien- sis predominates in the natural clam population whereas M. mer- cenaria is the predominant species utilized by the industry. The species hybridize extensively, and this study was conducted to measure the genetic impact of M. mercenaria aquaculture on the natural population of M. campechiensis near Cedar Key, Florida. Clams (N = 257) were analyzed for genetic composition, age, and the presence and stage of gonadal neoplasia. Results indicate that the genetic composition of the clam population has changed since the 1993 advent of aquaculture. Mercenaria mercenaria were non- existent prior to the initiation of aquaculture but increased in abun- dance post-aquaculture, as did hybrid clams. There was no differ- ence in abundance of M. campechiensis pre- vs. post-aquaculture. All taxa exhibited a high incidence (> S(Wc) of gonadal neoplasia, but it is not clear if this high incidence results from the introduc- tion of aquaculture or if neoplasia predates that introduction. These results indicate that Mercetiaria culture can influence naturally occurring congeneric populations in the vicinity of the culture operation, although the long-term implications of that influence remain to be seen. National Shellfisheries Association, New Orleans. Louisiana Ahstracrs. 2003 Annual Meeting, April 13-17, 2003 319 IN SITU DETERMINATION OF PERKINSUS MARINUS TRANSMISSION DYNAMICS. Corinne Audeniard*. Lisa M. Ragone Calvo. Kimberly S. Reece, Eugene M. Burreson. Kennedy T. Paynter, Virginia Institute of Marine Science, The College of William and Mary. Gloucester Point, Virginia 23062. Dermo disease, caused by the protozoan parasite Perkinsus marinus, is currently the most widespread and lethal infectious disease of the oyster, Crassostrea virginica. During the last de- cade, it has spread into low salinity areas raising que.stions about parasite transmission dynamic. Our objective is to determine the transmission dynamics of P. marinus in low to moderate salinity areas. The functional relationship between disease-related mortal- ity, ambient parasite abundance, and infection acquisition by naive oysters was examined in three Chesapeake Bay tributaries-the Ma- gothy. Patuxeiit. and James Rivers. From June through October 2002. water samples were collected weekly and parasite cell num- bers were quantified using real-time PCR. Concurrently, on a monthly basis, naive .sentinel oysters were deployed and monitored for P. marinus acquisition and local oysters were monitored for mortality and infection levels. The three studied rivers showed very different P. marinus abundance: the high salinity site in the James river showed up to 3000 cells/1, whereas the Patuxent site showed less than 20 cells/1 during the whole study and. the Mag- othy showed no parasite detection. These abundances and P. mari- nus incidence in sentinel oysters were significantly correlated with mortality of local oyster population and with salinity. arrays were designed with cDNAs encoding proteins involved in physiological functions such as immunity, wound healing, cell proliferation or cell motility, in order to assess the effect of envi- ronmental stresses on oyster health. BIOLOGICAL ASSESSMENT OF STORM EFFECTS ON THE LOUISIANA PUBLIC OYSTER RESOURCE: TROPI- CAL STORM ISIDORE AND HURRICANE LILI. Patrick D. Banks. P.O. Box 98000 Baton Rouge. LA 70898. Effects of Tropical Storm Isidore and Hurricane Lili on Loui- siana's public oyster resource were determined using a combina- tion of square-meter and dredge sampling. Pre and post storm samples were statistically analyzed for differences in percent mor- tality and density of oysters (Crassostrea virginica). Results were di.scussed in relation to environmental parameters such as salinity, precipitation, and storm surge. Although percent mortality of oys- ters in square meter samples significantly increa.sed on some pub- lic oyster grounds following the storms, it was generally < 40%. Oyster density data from square meter samples yielded mixed results and dredge samples indicated a slight increase in percent mortality of oysters in the Lake Pontchartrain basin following the storms. Negative effects of Tropical Storm Isidore and Hurricane Lili on the public oyster resource exhibited large spatial variation (likely due. in part, to extensive spatial variation of Louisiana's oyster habitat) with significant effects only occurring on some of the public grounds sampled. FUNCTIONAL GENOMICS: A POWERFUL APPROACH TO STUDY THE IMMUNE RESPONSE OF THE PACIFIC O'S STER CRASSOSTREA GIGAS. Jean-Christophe Avarre, Yannick Gueguen, Evelyne Bachere and Jean-Michel Es- coubas*. Defense and Resistance in Marin hnertebrates (DRIM) UMR5098 (IFREMER. CNRS, UMII) Universite de Montpellier 112 place Eugene Bataillon. CC80. 3409.S Montpellier. FRANCE Most of knowledge on oyster innate iminunity is based on biological activities, and molecular features of immune effectors remain largely unknown. To progress in oyster immune gene char- acterization we generated expressed sequence tags (ESTs) from a hemocyte cDNA library built from Crassostrea gigas subjected to bacterial challenge. A total of 1 142 randomly selected clones were single-pass sequenced. According to sequence similarities, a pu- tative function could be assigned to 54V(r of the clones (for more details, visit the database web site http://www.ifremer.fr/ GigasBase). Among them. 20 genes potentially involved in immu- nity were identified. To investigate the expression pattern of these genes. cDNA arrays were developed. Oysters were experimentally injected with several Vilvio strains isolated from moribund ani- mals during mortality outbreaks, and gene expression was com- pared with unchallenged animals. First results showed that some of these genes were over-expressed after bacterial challenge suggest- ing their involvement in defense mechanisms. Likewise. cDNA CLEARANCE RATES AND FEEDING SELECTIVITY OF CRASSOSTREA VIRGINICA AND MERCENARIA MERCE- NARIA; IMPLICATIONS OF INCREASED EUTROPHICA- TION IN THE SUWANNEE RIVER ESTUARY. Carla D. Beals* and Shirley Baker. Department of Fisheries and Aquatic Sciences. University of Florida. 7922 NW 71st Street. Gainesville. FL 326.'i3. The objective of this study is to examine the potential effects of increased eutrophication of the Suwannee River Estuary on the feeding biology of clams and oysters. My hypotheses are that 1 1 the presence or absence of particular phytoplankton species will affect bivalve clearance rates; and 2) bloom concentrations of some phytoplankton species will reduce the particle selection and clearance rates of bivalves. Oysters and clams collected from the Estuary will be subjected to two concentrations of plankton (av- erage and Suwannee bloom densities) and four types of plankton assemblages: 1 ) natural phytoplankton. 2) monospecific cultures of phytoplankton (not included in selectivity experiments). 3) labo- ratory-manipulated phytoplankton assemblages, and 4) phy- toplankton and niicro-zooplankton combinations. Changes in clearance rate or particle selection ability will have implications for the future productivity of clams and oysters in the Suwannee River Estuary. 320 Abstracts, 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana ENGINEERING AND ECONOMICS AS RELATED TO OYSTERS GROWN IN THE GULF OF MEXICO. Donald L. Bishop. Bishop Aquatic Technologies Inc., P.O. Box 669. 1 lO-B Bonnechere St. Eganville. Ontario. Canada. KOJ 1X0 / Engineer- ing and Economics as Related to Oysters Grown in the Gulf of Mexico. Globally the demand for a quality, safe, consistently available shellfish continues to outpace production. To deliver to the current consumers as well as to yet to be developed markets will take the focused involvement of biological, engineering and business plan- ning aspects to further develop the industry. Currently there is little correlation between the individuals that specialize in these areas, yet solutions have been implemented and proven by a small mi- nority of shellfish producers that understand how to match together these dynamics. Husbandry technologies have been developed that allow for the control of shell shape, appearance, size, meat yield and even shelf life pre harvest. Unfortunately many within the scientific community are unfamiliar with this area. These tech- nologies also input control to bio fouling, and production manage- ment further enhancing yield and profitability. Economics related to new technologies with return on invested capital per acre per year are significant. In the past economic models have been cre- ated for the oyster industry based on past input, output and cost of operation numbers. New husbandry technology and processes change this processes significantly, a discussion relating these to- gether with physical and biological aspects will be co\ered. within and between generations and also support the genetic basis previously found for this phenomenon. IMPACT OF AN INVASIVE TUNICATE IN ATLANTIC CANADA: RECRUITMENT AND COMPETITION. Daniel Bourque*, Thomas Landry DEO. P.O. Box 5030. Moncton. New Brunswick, EIC 9B6. Jeff Davidson. University of Prince Edward Island, 550 University Avenue. Charlottetown. Prince Edward Is- land, CIA 4P3; Neil McNair. PEI Department of FAE. P.O. Box 2000, Charlottetown, Prince Edward Island CIA 7N8. The presence of the club tunicate. Sr\ela clava, was recently noted in Eastern Prince Edward Island (PEI), Canada. This tuni- cate presents a significant fouling problem for the blue mussel {Mytibis ediilis) farms. S. clava has had a negative impact on mussel culture, attaching in high densities to mussel socks and equipment, competing for food resources and fouling equipment. This tunicate is spreading rapidly in the waters of PEI and seems to be mainly from anthropological mode as opposed to natural mode. Recruitment, abundance and growth of S. clava were stud- ied on a temporal scale. The impact of this new fouling organism was investigated by evaluating its competition for food in relation to the mussels. The eradication of this invasive tunicate from PEI waters is considered impractical and therefore the development of farm management strategies is considered as the only economi- callv viable solution. PERSISTENCE OF ATRAZINE IMPACT ON ANEUP- LOIDV IN THE PACIFIC OYSTER. CRASSOSTREA GIGAS. Karine Bouilly*. Helen McCombie. Alexandra Leitao, and Sylvie Lapegue. IFREMER. Laboratoire de Genetique et Patholo- gic. Avenue de Mus de Loup. 17390 La Tremblade. France. Aneuploidy is the alteration of the normal diploid chromosome number. In the Pacific oyster. Crassostrea gigas. hypodiploid aiieiiploid cells have regularly been reported as has a negative correlation between this phenomenon and growth and evidence for a genetic basis. We previously demonstrated a positive relationship between a pollutant, atrazine. and aneuploidy in Crassostrea gigas adults and juveniles. To e\aluate the persistence of this impact, the present study focused on a sample of the same juveniles exposed to different atra/Jne treatments (0.01 nig/1 which represents a peak value found m a polluted environment and 0.1 mg/1) for three and a half months and evaluated them for aneuploidy after another two and a half months in non polluted conditions. Their aneuploidy level remained significantly different between the treatments ap- plied. In addition, our study examined the offspring of the same adult population previously treated and found that these offspring exhibited significantly higher aneuploidy levels when the parents had been exposed to atrazine. These results demonstrate the per- sistence of the atrazine impact on Pacific oyster aneuploidy in time A SIMULATION MODEL OF THE POPULATION GROWTH OF HARD CLAMS [MERCENARIA MERCE- NARIA). III. EFFECTS OF BROWN TIDE. V. M. Bricelj*, J. N. Kraeuter, E. N. Powell, J. M. Klinck, E. E. Hofmann, R. Grizzle, and S. Buckner. Institute for Marine Biosciences Na- tional Research Council Canada 1411 Oxford Street Halifax. NS B3H 3Z1. Brown tides oi Aureococcus anophagejferens have occurred in Great South Bay, NY, since the mid-1980's. Peak concentrations usually occurs in June or July and have been attributed a role in the decline of hard clam populations. Using a physiologically-based model, simulations were run to examine the effect on clam popu- lation growth by a) timing of blooms, b) Aureococcus concentra- tion (105 to 2x106 cells ml-I). c) bloom duration, and d) food supply. Brown tide effects were incorporated into the model by assuming dose-dependent feeding inhibition of juveniles/adults. A brown tide-induced, larval mortality function was generated based on laboratory results obtained with bay scallop larvae. Sensitivity of the model output lo variation in larval mortality was assessed. Effects of a time-dependent, juvenile mortality function, based on published data for 2 mm clams which experienced high moralities after prolonged (4-wk) exposure to high brown tide levels (4x105 cells ml-1) were also tested. Preliminary results of a modeled brown tide effects on individual scope for growth and egg produc- National Shellfisheries Association. New Orleans. Louisiana Abslracls. 2003 Annual Meeting. April 13-17. 2003 321 tion o\er a clam's lifespan for inUiNiduals varying in initial body size and genotype will be presented. DETERRENTS TO BLACK DRUM PREDATION ON OYS- TER LEASES Kenneth M. Brown*, (iary Ptttrson, Mike Mc- Donough, Patrick Banks, and Brian Lezina. Department of Bio- logical .Sciences. Louisiana State University. Baton Rouge. Loui- siana 70803. Preliminary experiments indicated large black drum were ef- fective predators, and small oysters were preferred, but that salin- ity did not affect feeding. Further laboratory experiments indicated scent of dead black drum (as hypothesized by lease holders) did not lower fish feeding. Field experiments in Barataria Bay in 2 seasons indicated scent reduced feeding by 10-20 %. but only at one site in one season. We conclude scent is not effective under most conditions. Mortalities to all predators ranged from 60 % to 90 % within the four weeks on leases. The importance of black drum and southern oyster drills varied among sites, as did temporal patterns of mortality. Fish caused 74 % of mortality, and oy.ster drills 23 %. and fish and oyster drill predation were inversely related. Laboratory experiments with sound deterrents indicate "drumming" by male black drum in the frequency range of 40-60 Hz does not deter predation. Sounds in the range of 10-30 Hz may deter fish, but are impractical because they require considerable power to broadcast over leases. We hope in future work to deter- mine whether limited gill netting or trot line fishing can decrease oyster mortality to black drum without impacting other fish popu- lations through by-catch. STRESS RESPONSES IN SCALLOPS AND HARD CLAMS TO HEAT AND COLD SHOCK. Nicole T. Brun*' \ V. Monica Bricelj", Emmanuel E. Egbosiniba", Thomas H. Mac- Rae', and Neil W. Ross". 'Biology Department. Dalhousie Uni- versity, Halifax NS B3J IZl, "National Research Council. Institute for Marine Biosciences. Halifax NS B3H 3Z1. In response to various stressors, such as temperature, organisms increase production of stress proteins (SPs). Heat shock SP re- sponses have been studied in mussels, but limited information is available for other bivalves. Cold shock Stress Protein Response (SPR) has not been previously investigated. Sea scallops Pla- copecten magellaiiictis. a deeper water species, and the estuarine bay scallop Argopecten irradians inadians differ in temperature tolerance: the former is susceptible to high temperatures, whereas the latter may be more vulnerable to low temperatures. Juvenile hard clams Meneiiana mercenaha suffer heavy mortalities during over wintering in Atlantic Canada and the mid-Atlantic US. The SPR to acute heat shock, determined by SP-70. was ciimpared in the two scallop species (10°C increase for 3h). No differences in SP-70 expression were observed in sea scallops, except at 2 Id. when levels were significantly lower than initial, control levels. In contrast. SP-70 in bay scallops increased significantly during and following heat shock, attaining a maximum by I2h, and exceeded control levels after 8d-recovery. The SPR of bay scallops and hard clams to acute cold shock (17°C decrease for 3h) was examined to determine if this stressor also modulates SP-70. The latter in- creased significantly in both bivalves, with levels still increasing after 8d and 24h respectively. The same samples are being ana- lyzed using SP-40. Characterization of the SPR to acute tempera- ture shock may have application in acquired thermo tolerance of bivalves transferred from hatchery to field growout sites. PERKINSUS CHESAPEAKI AND PERKINSUS ANDREWSI ARE THE SAME SPECIES. Eugene M. Burreson*. Kimberly S. Reece, Karen L. Hudson. Christopher F. Dungan. Virginia Institute of Marine Science Gloucester Point. VA 23062. Perkinsiis chesapeaki was described from the soft-shell clam. M\a arenaha. and Perkinsus andrewsi was described from the Baltic macoma. Macoma halthica. both from Chesapeake Bay. Sequence analysis of the internal transcribed spacer region (ITS), the large subunit ribosomal RNA gene and actin genes from clonal Perkinsus cultures derived from both hosts revealed that the two species are synonymous. Multiple DNA clones of each region were sequenced from each clonal isolate. Phylogenetic analyses based on all three sequences placed isolates derived from the two different hosts into a monophyletic group. Polymorphisms were detected at each locus and sequence variation was observed within clonal isolates at the multi-copy genes. ITS sequences from each isolate were found in each of two monophyletic sister clades. One clade included the GenBank deposited ITS sequence for Perkinsus chesapeaki. and the sister clade included P. andrewsi ITS se- quences. These results suggest variation observed among ITS se- quences of these isolates is representative of polymorphisms within a single parasite species from two different hosts. GenBank deposited P. chesapeaki and P. andrewsi ITS sequences represent sequence variants from a single Perkinsus species. The name P. cliesapeaki has priority under Article 23 of the International Code of Zoological Nomenclature. EVALUATING SHELL QUARANTINE DURATION TO LIMIT THE TRANSFER OF PERKINSUS MARINUS WHEN PLANTING OYSTER CULTCH David Bushek*. Donnia Richardson, Yvonne Bobo, Loren Coen and Jennifer Cardinal. Baruch Marine Field Lab. University of South Carolina. PO Box 1630. Georgetown. SC 29442. Freshly shucked oyster shell can carry harmful organisms such as predators, non-natives or pathogens in remaining tissues. Thus, planting fresh oyster cultch may spread harmful organisms. De- composing Perkinsus marinus infected oyster tissue is a major source of infective stages of P. nuirinus. Therefore, we examined 322 Absinicls. 2003 Annual Meeting, April 13-17. 2003 National Shellfisheries Association, New Orleans, Louisiana changes in P. iiuiiiiuis abundance in tissues of shucked and whole Gulf Coast oysters deployed in replicate shell piles between March and July 2002 in Charleston, SC. Parasite abundance was deter- mined by RFTM body burden assay, and parasite enlargement in RFTM used to indicate viability. After 31 days, only 13% of shucked oysters contained any tissue and total parasite abundance had declined to 0.05%. No tissues remained in subsequent samples. Tissues decayed much slower in whole oysters, but para- site abundance still declined rapidly with just 1% remaining after only 31 days. After 1 15 days, only two whole oysters contained any observable tissue and total parasite abundance was a mere 0.005% of the original number. The impact of climate and shell pile configuration should be more closely evaluated, but simply quarantining oyster shell for one month or more on land can dra- matically reduce the abundance of P. marinus. minimizing the potential for transmission. HIGH HYDROSTATIC PRESSURE INACTIVATION OF VIRUSES. Kevin R. Caici*, US FDA PO Box 158 Dauphin Island, AL 36528. Viruses have been epidemically linked to the majority of the illnesses associated with consumption of raw shellfish. The ma- jority of the implicated shellfish were traced back to growing areas in approved status which were thought to have become contami- nated by illegal overboard discharges or failures of proximal wastewater treatment facilities. High hydrostatic pressure (HHP) processing is in use commercially to reduce Vibrio sp. in shellstock oysters. Investigations are underway, to determine if HHP might serve as a post-harvest treatment process to improve the safety of oysters as related to viruses. Viruses under study include hepatitis A virus and SM-17, a surrogate for Norwalk-Like virus. Oysters (Crassostrea virginica) accumulated virus in a flow through sea- water system. Shucked meats were packaged in plastic pouches before subjecting to HHP processing. Results show HAV to be the more pressure resistant requiring pressures > 400 MPa to achieve a 3 log 10 reduction in 1 min. A similar reduction could be achieved with SM-17 in 1 min at 275 MPa. The wide range of pressures required to inactivate different viruses may make it dif- ficult to select a pressure, that will be effective in destroying all viral contaminates in oysters without damaging the quality of the ovsters. HOST GENETIC ORIGIN AN IMPORTANT DETERMI- NANT OF QPX DISEASE Lisa M. Ragone Calvo*. Gene M. Burreson, Susan E. Ford, John N. Kraeuter. Dale F. Leavitt, Roxanna Smolowitz. Virginia Institute of Marine Science. Col- lege of William and Mary, Gloucester Point, VA 23062. Epizootics of QPX (Quahog Parasite Unknown) a protistan pathogen of hard clams, Mercenaria mercenaria have occurred in maritime Canada and Massachusetts, New York, New Jersey, and Virginia, USA. Although it has been found in wild hard clam populations, the parasite has most seriously affected cultured hard clams, suggesting that aquaculture practices may promote or pre- dispose clams to the disease. In this investigation we examined the influence of host genetic origin and geographic location on QPX su.sceptibility. Five clam strains, originating from Massachusetts, New Jersey, Virginia, South Carolina, and Florida were produced at a single hatchery and evaluated for growth, survival, and QPX susceptibility at three QPX endemic sites (Massachusetts, New Jersey and Virginia). Severe winter-associated clam losses oc- curred at the Massachusetts site precluding completion of the study at that location. At both the New Jersey and Virginia sites the South Carolina and Florida clam stocks exhibited significantly higher QPX prevalences and lower survival than the New Jersey and Massachusetts clam stocks: while clams from Virginia had QPX prevalences and survival rates that were intermediate to the more '" northern" and " southern" clam stocks. These results sug- gest that genotype-environment interactions are important deter- minants of QPX disease. EXPERIMENTAL EVALUATION OF CROSSES WITHIN AND AMONG FIVE COMMERCIAL STRAINS OF HARD CLAMS, MERCENARIA MERCENARIA, ACROSS A SA- LINITY GRADIENT IN VIRGINIA WATERS. Marli D. Ca- niara*, Standish K. .Yllen Jr. Aquaculture Genetics and Breeding Technology Center Virginia Institute of Marine Science PO Box 1346 Gloucester Point, VA 23062. Cultured Mercenaria mercenaria are a multi-million dollar in- dustry in Virginia. Grow-out sites vary from ocean salinity outside to mid-salinity estuarine sites inside the Chesapeake Bay. Pres- ently, the industry uses essentially undomesticated genetic stocks, and we know very little about the suitability of stocks to varying environmental conditions. We evaluated the genetic influence of hard clam strain selection on growth along a salinity gradient in Virginia as well as the potential for enhancing production by out- crossing available strains. We first created all fifteen possible com- binations within and among five brood stock strains in the hatch- ery. We subsequently raised the juveniles in common conditions until they reached approximately 10 mm, at which point we split the groups for planting at five sites encompassing the range of salinities at which clams are grown. We measured them and com- pared the growth of these groups in the hatchery, nursery, and field, estimated the correlations among the performance measures between life stages, compared the performance of within- and among-strain crosses, and assessed site-specifity. We discuss the results and their implications for strain selection, hatchery spawn- ing procedures, and future efforts in selective breeding for superior hard clam strains. Niitional Shelirisheries Association, New Orleans, Louisiana Ahsrracts. 2003 Annual Meeting. April 13-17, 2003 323 GROWTH OF QUAHOGS (MERCENARIA MERCENARIA) AND SOFTSHELL CLAMS (M)A ARENARIA) IN RE- SPONSE TO ELTROPHIC-DRIVEN CHANGES IN FOOD SUPPLY AND HABITAT Ruth H. Carnikhael*. Andrea C. Shriver. Erica T. Weiss, and Ivan \'aliela. Boston University Marine Program, Marine Biological Laboratoiy. 7 MBL Street, Woods Hole, MA 02543. In recent years increased urbanization has increased nitrogen loads to coastal estuaries, prompting eutrophication and changing estuarine features. Increased N loads increase phytoplankton and microphytobenthos concentrations, result in accumulation of or- ganic matter from detritus of algae, reduce sediment and water column oxygen content, and may change sediment composition. These changes likely affect growth and survival of commercially important bivalves like quahogs and soft-shell clams. To determine how eutrophication-related changes affect these bivalves, we trans- planted ju\eniles into estuaries of different land-deri\ed N loads, measured changes in sediment and water column properties, and recorded growth and survival of bivalves. We used N stable iso- topes to link responses of bivalves to their food supply and land- derived sources of N for management. We found growth rales of quahogs and soft-shell clams increased as land-derived N loads increased their food supply. Water column food sources had a greater effect on growth than sediment sources, and low salinity and high particulate organic matter may have limited growth in some areas despite increased food supply. N stable isotope analysis linked these growth responses to land-derived N primarily from wastewater sources. DEVELOPMENT OF A SINGLE NUCLEOTIDE POLY- MORPHISM (SNP» MARKER SET FOR THE HARD CLAM, MERCENARIA MERCENARIA. Ryan B. Carnegie*. Mark D. Camara, Lisa M. Ragone Calvo, Kimberly S. Reece. and Patrick M. Gaffney. Virginia Institute of Marine Science P.O. Box 1346 Gloucester Point. VA 23062. In aquaculture. molecular genetic markers can be used to e\ alu- ate the diversity of wild shellfish stocks to be introduced into hatchery breeding programs, to control pedigrees in hatchery lines, and to track the performance of outplanted seed. While progress has been made in developing molecular markers for Crassttstrea spp.. the hard clam Meirenaria inercenaria. an extremely valuable commercial species in eastern North America, has received rela- tively little attention. Our objective was to develop a set of single nucleotide polymorphism markers (SNPs) for M. inercenaria. Hemocyte and mantle complementary DNA (cDNA) libraries were created in plasmid vectors and then sequenced. Screening for SNPs is being done using a panel of clams encompassing the genetic diversity of VIMS hatchery stocks and reflecting the wide geographic distribution of M. mercenaria. SNPs demonstrating Mendelian inheritance will be immediately useful for evaluating the relative performance of clams produced from Massachusetts, New Jersey, Virginia, and South Carolina broodstock that are now deployed at two QPX-enzootic and three QPX-free sites in Vir- ginia. The markers will also be useful for characterizing wild M. mercenaria germplasm diversity, and may begin to reveal allelic variation underpinning the variable susceptibility of East Coast clams to QPX. TROPHIC INTERACTION BETWEEN HARD CLAMS AND NATURAL ASSEMBLAGES OF PLANKTON Robert M. Cerrato*. Amy E. Streck. and Darcy J. Lonsdale. Marine Sciences Research Center Stony Brook University Stony Brook, NY 11794-3000. To examine whether intensive grazing by hard clams or cope- pods shifts the composition of the plankton community toward species of different nutritional quality, we conducted experiments in 400-liter tanks at three locations in Great South Bay, NY. Treat- ments were created by varying adult clam and copepod abun- dances. After a 2-week acclimation period, several juvenile (2 mm) clams were added to each tank and allowed to grow for 4 weeks. In one location, where growth under ambient conditions was high, juvenile growth declined by 57% in the treatment with high adult clam grazing, suggesting that juveniles and adults were competing. In the other two locations, where growth under ambi- ent conditions was moderate to poor, juvenile growth improved by 60 to 200% in treatments with high adult clam grazing. Plankton composition was altered in the high adult copepod treatments, but no effect on juvenile hard clam growth was observed. Examination of clearance and assimilation rates of naive clams exposed to treat- ment water indicated that observed increases in juvenile clam growth were related to food quality rather than quantity. Our re- sults suggest that intense grazing by hard clams can have a positive effect on the nutritional value of the plankton. PRESENCE OF PATHOGENIC BACTERIA IN THE LA- GOON SYSTEMS LA MANCHA AND ALVARADO VER- ACRUZ, MEXICO IN WATER AND OYSTER (CRASSOS- TREA VIRGINICA ). Maria del Refugio Castafieda Chavez*, Erasmo Orrantia B., Violeta Pardio Sedas, Fabiola Lango Rey- noso. Carr. Veracruz-Cordoba Km 12 C.P. 94290. Boca del. Ver. Mexico. Mexico maintains the 6th place in world-wide oyster produc- tion, contributing the Gulf of Mexico with 76% of the total vol- ume. In this coastal area, 30 and 36 sampling stations were estab- Ikshed in the coastal lagoons of Alvarado. and La Mancha. Water and oyster samples were taken during one annual cycle, and mi- crobiological analysis were performed to determine according to the Mexican Official Norm NOM-031-SSA 1-1993. Three stocks of pathogenic vibrios were isolated from water samples of Lagoon of Aharado. Vilvio alginolyticus. V. cliolerae (IN.ABA) and V. choierae No-OI. besides Salmonellas and total coliforms. The V. 324 Abstnicls. 2003 Annual Meeting. April 13-17, 2003 National Shellfisheries Association. New Orleans, Louisiana cholerae serotype INABA was reported in Alvarado during the months of July. August and September. The V. alginolyticus was reported in January. V. cholerae No-01 was reported in the La Mancha during the rainy season exclusively. Analysis for V. chol- erae no-01 from oyster samples of the Alvarado is not significantly different to those reported from the oyster banks of La Mancha. It was concluded that fecal discharges is the main cause of pollution representing a health problem that must be considered due to the possibility of survival of microorganisms when oysters are raw consumed and not subjected to depuration. efforts ha\'e been conducted to restore many of the altered areas back to their original habitat value. What is not always clear is how to define the value of a habitat to a particular species of interest. This information is important to assess the impacts of habitat al- teration on species that utilize those areas. The impacts of these types of alterations to the critical life support functions of shellfish populations will be reviewed. A characterization of the habitat conditions that support the survival and continued viability of shellfish populations is needed to properly assess habitat alteration and e\aluate the success of restoration efforts. MUSSEL GROWTH AND FOOD UTILIZATION IN RELA- TION TO WATER COLUMN CONDITIONS ON RAFT SYSTEMS IN PUGET SOUND. WASHINGTON Daniel P. Cheney*. Andrew D. Suhrbier, .\iniee E. Christy. Hector S. Beltran. Jonathan P. Davis, Kenneth M. Brooks, and Frank ,|. Smith. 120 State Ave. NE #142 Olympia. WA 98501. Suspended mussel and oyster culture on the U.S. west coast is predicted to increase significantly in coming years. Description of the changes associated with the culture of these crops is essential for the siting and evaluation of new culture facilities and in im- proving yield and production of existing facilities. This research had three general objectives: 1 ) to assess at large-scale farm sites, mussel growth and yield against a suite of measured physical, chemical and biological variables; 2 ) to compare the same suite of variables with measurements of mussel feeding and biodeposit production; and 3) to utilize available nutrient and yield models to estimate potential mussel carrying capacity in the farming area. During a two year period (2001-03). multiple observations were made of water currents, water chemistry, phytoplankton. mussel growth, seston removal and absorption, fouling, and fish utilization at commercial mussel raft culture sites in Totten Inlet and Penn Cove. Washington. Although parameters, such as water currents and phytoplankton abundance varied markedly inside and outside the raft units and under different tidal regimes, these effects were localized and did not correlate with mussel growth. This research is supported by a Sea Grant National Marine Aquaculture Initiative arant. ASSESSING THE EFFECT OF HABITAT ALTERATION ON SHELLFISH POPULATIONS. Marnita M. Chintala*. U.S. EPA. NHEERL. Atlantic Ecology Division, Narragansett. Rl 02882; and Karin A. Tanimi. NOAA/R.l. Dept. of Environmental Management. Narragansett, Rl 02882. Habitat provides a variety of life support functions for many species, such as providing shelter, substrate, food, and nursery areas. Habitat alteration is one of the most important causes of declines in ecological resources in North America, and habitats essential to the well being of shellfish species are rapidly being affected by many land-use activities. As a result, many restoration DESIGN AND IMPLEMENTATION OF A SURVEY OF COMMERCIAL BLUE CRAB EFFORT IN THE MARY- LAND PORTION OF THE CHESAPEAKE BAY Mary C. Christman,* Cynthia J. Giffen. Department of Animal and .Avian Sciences. University of Maryland. College Park. MD 20742; Jon H. Volstad. Versar Inc.. 9200 Rumsey Rd., Columbia. MD 21045; and Lynn W. Fegley. MD DNR. Tawes State Office Building. 580 Taylor Ave.. Annapolis. MD 21401. The Maryland Department of Natural Resources (MD DNR. requires estimates of the fishing effort expended by commercial crab fisheries in the Chesapeake Bay. We designed a three-prong approach to obtaining instantaneous estimates of effort in the bay. We collected field data on the commercial pot and trotline crab fisheries, and telephone surveys for supplementary information. Sampling included -160 stratified random transects for pots each month to obtain estimates of spatially explicit pot densities. Survey stations were modified transects; planar boards were used to de- lineate the width of each transect. The trotline surveys were per- fonned using both aerial flyovers and roving intercept surveys to quantify the mean number of lines per boat and mean trotline length. We describe methods for merging this information in ways that can be used to estimate effort for similar fisheries. AN INTEGRATED APPROACH TO BIVALVE DOMESTI- CATION: INTRODUCTORY REMARKS. Fu-Lin E. Chu*, Virginia Institute of Marine Science. College of William and Mary. Gloucester Point. VA 23062; Jean-Francois Saniain*. If- remer Centre de Brest, BP 70, 29820, Plouzane, France. Environmental and disease stresses are worldwide problems and have caused severe mortality in many cultivated and feral bivalve populations. For years, scientists in France and US have devoted time and effort in an attempt to improve the yields via multi-disciplinary research. To coordinate activities of researchers from various scientific disciplines in US and France, a US-France Workshop on "Domestication of bivalve molluscan shellfish" was held in La Tremblade, France, 2002. Via the meeting several short- term US-France collaborative projects have been developed. To accelerate information and technology exchange, ideas for future technical workshops have been established. Currently a five-year National Shellfisheries Association. New Orleans. Louisiana Abxnucrs. 2003 Annual Meeting. April 13-17. 2003 325 multi-disciplinary research project on Crassostreci gifias summer mortality is being conducted in France. Six disciplines are con- tributing together to test the hypothesis of a complex interaction between oyster, environment and opportunistic pathogens. The study focuses on mortality dynamics in the field and determines the relative role of different putative factors in contributing to the mortality. EVALUATING THE IMPACTS OF HARVESTING PRAC- TICES, BOAT WAKES AND ASSOCIATED SHORELINE EROSION ON INTERTIDAL CREEK HABITATS IN THE SOUTHEASTERN U.S.: MANAGERS AND RESTORATION PROGRAMS TAKE NOTE. Loren D. Coen* and Majbritt Bolton-WarbiTji, Marine Resources Research Institute. SCDNR. 217 Fon Johnson Rd.. Charleston. SC 29412 and Graduate Marine Biology Program. Grice Marine Lab. College of Charleston. 20,'i Fort Johnson Rd.. Charleston. SC. 29412. In areas where oysters are intertidal and fringe marsh-lined creeks, they can act as shoreline "stabilizers". Recent work (FL. SC. and NC) suggests that harvesting and boating, in addition to natural phenomena, can significantly impact natural intertidal habitats and restoration/enhancement efforts. We assessed oyster populations prior to applied treatments, evaluating the direct im- pacts of four common harvesting practices on oyster population recovery at 12 sites, paired with controls. Concurrently, recruit- ment, survival, and growth were also examined annually and popu- lations reassessed -3 years later to evaluate "recovery". Simulated boat wake experiments used shell treatments (with and without mesh) to evaluate impacts of wakes on restoration efforts. Results are discussed and current larger-scale study designs applying our findings are summarized. Four study sites were established in 1999 to measure shoreline erosion. Over 2?-38mo. rates ranged from ~0-23cm/month; overall bank losses were from 69-1.54 cm. In 2001, we expanded sampling at nine additional sites using our SCORE program. Erosion rates (4-16mo.) ranged from ~2-8cm/ month, with overall losses from 13- 104cm. These and other results suggest that anthropogenic impacts may be having much greater impacts on critical intertidal habitats than previously perceived. HISTORY OF POST-HARVEST TREATMENT TO RE- DUCE VIBRIO SP. IN SHELLFISH David W. Cook, Food & Drug Administration Gulf Coast Seafood Lab P.O. Box 158 Dau- phin Island, AL 36528. Vibrio vidnificiis was first recognized as the cause of primary septicemia in humans and its relationships to shellfish consump- tion established in the early 1970's. K vulnificus is a naturally occurring pathogen and it densities in shelltlsh at harvest are re- lated to growing water temperature. To control illnesses caused by this bacterial species, several approaches including time- teniperaliire controls, harvest restrictions and consumer education have been initiated. Research into post-harvest processing mitiga- tion strategies to reduce Vibrio numbers without destroying the raw characteristic of the shellfish was undertaken. In 1996, the first commercial post-harvest treatment process, a mild heat treatment, was recognized as capable of reducing V. vidnificus in shellfish to a non-detectable level. Two other processes, freezing and high- hydrostatic pressure processing, were validated in 2002 by com- mercial processors. Other processes under study are depuration, relaying of shellfish to waters free of V. vidnificus and irradiation. Post-harvest processes for reducing V. parahaemolyticus in shell- fish to non-detectable levels are also being validated. FRESHWATER PEARL CULTURE AND PRODUCTION IN CHINA. Hua Dan*. Freshwater Fisheries Research Center (FFRC). Chinese Academy of Fisheries Sciences. Wuxi City 214081. Jiangsu Province. CHINA Lustrous pearls have been called the queen of jewels, biil the occurrence of quality pearls in wild mussels is rare. The technolo- gies of freshwater pearl culture were developed in China some 2,000 years ago. However, commercial pearl culture dates back only to the late 1960s. Gradual changes in technology and in the type of mussel used {Hxriopsis cuiningii). resulted in the produc- tion of greater quantities of larger and more lustrous round, near- round, and baroque cultured pearls of various colors. Today, there is a great demand for cultured freshwater pearls, and China pro- duces 95*7^ of those pearls sold in the world market. China pro- duces an estimated 800 to lOOO metric tons of freshwater cultured pearls annually, of which roughly 400 to 500 metric tons are exported to different continents and countries worldwide. Pearls 8 mm and larger represent a large percentage of those exported. This presentation will re\ iew the techniques of freshwater pearl culture in China, to include principles of pearl formation, mussel operation procedures, and mussel culture post-implantation. POPULATION GENETICS OF THE BLUE CRAB (CALLI- NECTES SAPIDUS) IN THE GULF OF MEXICO. Richard L. Darden* and Brian R. Kreiser, Department of Biological Sci- ences. Universitv of Southern Mississippi. Hatliesburg. MS 39406. Gene flow among populations of the blue crab {Callinectes sapidus) is determined by larval dispersal and adult crab move- ments. Assessment of population genetic structure allows infer- ences about historic and contemporary patterns of gene flow. A total of 1.920 crabs were collected from 26 locations around the Gulf of Mexico coast between Naples. Florida and Brownsville. Texas during 2001-02. A 650-base pair portion of the mitochon- drial cytochrome oxidase I (COI) gene was amplified and se- quenced for individuals from each location. Preliminary results seem to indicate that Gulf of Mexico blue crab populations are not 326 Abslnicls. 2003 Annual Meeting, April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana genetically homogeneous. We will place these results into the between cumulative mortality and burden of pathological condi- context of blue crab life history as well as prevailing theories tions was significant, concerning blue crab dispersal and migration. GROWTH AND MORTALITY OF DIFFERENT OSTREA EDULIS STOCKS CULTURED IN THE RIA DE AROUSA (GALICIA, NVV SPAIN). Patricia M. da Silva*. Antonio Vil- lalba. and Jose Fuentes. Centre de Investigacions Marinas. Aptdo. 13. 36620 Viianova de Arousa, Spain. Nowadays. Bonamiosis is the most important constraint for the Galician oyster industry. The development of a disease-resistant stock by a selective breeding program seems a promising measure. Oysters harvested from four genetically different populations were used as broodstock to obtain 5 families from each stock in a hatchery. Two of these stocks were obtained from two B. ostreae- free areas in Ireland and Greece, and the other two from Coroso and Ortigueira. two Galician areas where the parasite is present. Spat of every family is being cultured in the Ria de Arousa since Sept 2001. Growth and mortality data for one-year culture period are analyzed in this presentation. Results show significant differ- ences in growth and mortality, both among stocks and families. On average, Galician and Greek stocks perform better (faster growth and lower mortality) than the Irish one. However, the importance of the differences detected among families in both variables di- minishes the relevance of those among stocks. CROSSBREEDING IN PACIFIC OYSTERS. Joth Davi.s*. Taylor Shellfish Farms, Quilcene, WA 98376; Dennis Hedgecock. Bodega Marine Laboratory. Bodega Bay, CA. Intraspecific hybrid lines of Pacific oysters {Crassastrea giaas) were made in 2001 by crossing inbred oysters in a full factorial mating design at the Taylor Shellfish Farms bivalve breeding fa- cility in Quilcene, WA. Two cohorts of hybrid oysters were reared from inbred lines produced by the Molluscan Broodstock Program. Families generated from individual pair-matings were reared and set using standard techniques. Seed from individual families was reared in the field in a 2 month replicated experiment to test for differences in yield among hybrid families. Oysters were subse- quenlly redeployed in replicate cages for a 1 2-month yield trial. Final yield measurements (total count and biomass) made in Au- gust 2002 demonstrated a positive correlation between yield at the seed stage and yield in harvest-ready oysters. Inbred lines and hybrid combinations that generated superior yield at both the seed and harvest stages were identified. Stock improvement via cross- breeding emphasizes yield testing at the seed stage to help predict tlnal yield in oyster production, and offers some advantages over the cost and effort associated with traditional selection and breed- ing programs. DIFFERENCES IN DISEASE SUSCEPTIBILITY AMONG OSTREA EDULIS STOCKS CULTURED IN GALICIA (NW SPAIN). Patricia M. da Silva*. Antonio Villalba, Maria J. Car- ballal. and Jose Fuentes. Centro de Investigacions Marinas. Aptdo. 13, 36620 Viianova de Arousa, Spain. Bonamiosis is the bottleneck for Galician oyster industry. A program to develop a Bonamia ostreae resistant strain is being performed. Oysters from different populations were selected as broodstock: Ireland and Greece bonamiosis-free areas, and two Galician areas. Ortigueira (bonamiosis is epizootic), and Coroso (low bonamiosis pressure). Five families per stock were trans- ferred to a raft in the Rfa de Arousa on September 2001 . Mortality is estimated monthly and samples of each family are taken and historically processed. The most prevalent pathological conditions detected until October 2002 were intranuclear inclusions, suggest- ing viral infection, and disseminated neoplasia. RLO in digestive epithelia and Haplosporidium-like plasmodia were rare. Haemocytic infiltration, granulocytomas and necrosis were also observed. B. ostreae was detected in September and October 2002 with very low prevalence, although increment is expected in the second year. Significant differences in the burden of pathologic conditions were detected anions stocks and families. Correlation THE EFFECT OF ALGAL TOXINS ON THE ISOLATED VENTRICLE OF THE HARD CLAM, MERCENARIA MER- CENARIA. Lewis E. Deaton. Biology Department. University of Louisiana at Lafayette. Lafayette. LA 70504. While many species of algae have been associated with mass mortalities of shellfish, relatively little is known about the specific effects of algal toxins on the organ systems of mollusks. Isolated ventricles in aerated seawater were exposed to varying concentra- tions of saxitoxin, brevetoxin 2 and brevetoxin 9. Saxitoxin had no effect, even at a concentration of Ix 10-6 M. Brevetoxin 2 caused a prolonged negative inotropy in the ventricles; the threshold is about 1 X 10-9M and the effect is dose-dependent. Brevetoxin 9 (I X 10-9 M) caused a decrease in the amplitude and increase in the diastolic tone; these effects were transitory. Higher doses (10-8. 10-7 M) of brevetoxin 9 did not increase the inhibitory effect. The hearts of bivalves are myogenic, and are not affected by the neural Na-i- channel blocker, tetrodotoxin. The lack of any effect of sax- itoxin is therefore unexceptional. Brevetoxins open Na-i- channels; whether this is the mechanism of their inhibition of the Mercenaria ventricle will require further study. National Slielltisheries Association. New Orleans. Louisiana Ahstnict.s. 2003 Annual Meeting. April LVI7. 2003 327 GENETIC BASIS OF SUMMER MORTALITY IN JUVE- NILE CUPPED OYSTERS. Lionel Degremont*, Pierre Boudry and Patrick Soletchnick. LGP-LCPC. F- 1 7390 La Trem- hlade; Edouard Bedier. LCB. F-.'i647() La Trinite; Michel Rop- ert. LCN. F- 1 4520 Port-en-Bessin; Arnaud Huvet, .Jeanne Moal and Jean Francois Samain, LPL F-2y2S0 Plouzane. The French project "Merest", coordinated by IFREMER, aims to understand the causes of the simimer mortalities in Crassostrea gii^tis. In 2001, three sets of families were bred following a nested half-sib mating design. 1 7 halt-sib families (HSF) were obtained in this first generation (Gl) and reared in 3 sites. Significant differ- ences in survival were observed among HSF. and some HSF showed high levels of mortality in all sites, clearly indicating a genetic basis for survival. In 2002. a second generation (02). including divergent selection and inbred lines, was constituted. Monitoring of survival and growth of G2s were the same as in 2001. Significant differences in survival were found between the offspring of the "high" and "low" selected groups and between inbred lines. The high realized heritability for survival indicates that selective breeding programs could efficiently improve sur- vival of juvenile oysters. MUCIN SECRETIONS AND NACRE DEPOSITION IN THE FORMATION OF PEARLS. Leonard DiMichele* and Stephan Towers. Department of Wildlife and Fisheries Sciences. Texas A&M University, College Station, TX 77843; Donald Shepherd. Professional Pathology Laboratories. Ltd, P.O. Box 326. Tow. TX 78672 Cultured pearls originate within a pearl-sac formed by the in- sertion of a nucleus and graft tissue into a surgically created pouch. Within the pouch, the host animal initiates a classical wound heal- ing response and then nacre-secreting cells from the graft prolif- erate, lining the lumen of the pouch. Maturing pearl-sac epithelia from a freshwater mussel (LInionidae: Cyrloiniias tampicacnsis) were examined. Mucopolysaccharide secretions gradually in- creased after 30 days of development. By day 43. all mucins were actively secreted by host epithelia. Although the pearl-sacs were morphologically mature, there was no evidence of calcium secre- tion. However, natural pearl-sacs in the same mussels exhibited calcium secretions. The various proteins and calcium secretions formed an aragonite - protein laminate (nacre). Using atomic force microscopy and acid extractions, we characterized natural pearls and shell nacre of Cyrtomiias tampicoensis. Our results were simi- lar to those reported from several salt water species and were consistent with evidence from Asian freshwater mussels. IMPACT OF ENVIRONMENTAL AND NUTRITIVE CON- DITIONS ON DEFENCE MECHANISMS OF OYSTERS DURING AN ANNUAL CYCLE. Maryse Delaporte*. Philippe Soudant. Jeanne Moal and Christophe Lambert, Maryse De- laporte Laboratoire de Physiologic des Invertebres Centre Ifremer de Brest, BP 70 29280 Plou/ane (France). In the frame of MOREST project, a common biological mate- rial, resulting of a mixture of different families produced in ex- perimental hatchery, was reared in two different environmental fields: Normandy and Charente. Concomitantly, a pool was con- ditioned at the Ifremer Argenton hatchery with three different al- gae levels: 4%, S'/r and 12% of algal dry weight per oyster dry weight. During the experiments, five immune parameters were studied in parallel with survival rate and reproductive status (stages and intensity). Site location, seasonal variations and experimental diet level clearly intluenced oyster immune parameters. Hemocyte counts were higher for oysters reared in Normandy than those reared in Charente and ni hatchery. Granulocyte percentage was drastically reduced in hatchery conditions compared to in situ conditions. Moreover, hemocyte activities were also affected by the in situ conditions and dietary treatments in relation to reproductive cycle and mortality events. In example, in vitro haemocyte adhesion capacities were more affected by pathogenic Vibrio when oysters suffered mortality. REPRODUCTION IN FLAME SCALLOPS, LIMA SCABRA SCABRA (BORN 1778), FROM THE LOWER FLORIDA KEYS. Angela K. Dukeman*. 100 8th Avenue SE St. Petersburg, FL 33701. Norman J. Blake and William S. Arnold. Sex ratio, gonadal characteristics, and the reproductive cycle of the flame scallop. Lima scabra scabra (Bom 1778), collected from Boca Chica Key. FL were investigated over a 21-month period from January 1998 through September 1999. Gametogenic cycles were examined using qualitative and quantitative methods and the results were analyzed within the context of environmental varia- tion. Gamete development was initiated in winter and coincided with cooler water temperature and moderate food concentration. Maximum gamete ripeness and size occurred in late summer, when water temperatures were at maximum values (33 C), and food quantities were increasing (>0.2 ug/l). Both quantitative and quali- tative results indicated a clearly defined spawning event that oc- curred in autumn in association with decreased female gonad size, increased presence of partially spawned, spent, and early gameto- genic gonads, rapidly decreased water temperature (~7 degrees), and maximum measured chlorophyll-a concentrations (1 ug/I). Less defined periods of spawning activity occurred in February and June but could not be related to specific changes in environ- mental conditions. The presence of ripe and partially spawned flame scallops and adequate chlorophyll-a concentrations through- out the year suggests a continuous spawning reproductive strategy, common in tropical marine invertebrates. 328 Abslnicts. 2003 Aiiiuial Meeting. April 13-17, 2003 National Shellt'isheries Association, New Orleans, Louisiana IN VITRO PROPAGATION OF PERKINSUS SP. PARA- SITES FROM JAPANESE MANILA CLAMS. RUDITAPES PHILIPPINARUM. Christopher F. Dungan*, Maryland DNR. Cooperative Oxford Laboratory. Oxford. MD 21654; Kimberly S. Reece. and Karen L. Hudson. Virginia Institute of Marine Sci- ence. Gloucester Point. VA 23062 Perkinsus sp. is destructive parasites of Manila clams. Rudi- tapes philippinarum from Korea. Japan, and Spain, but parasite isolates are not reported from this host. Gills of Japanese Manila clams collected in Gokasho Bay, Mie prefecture were infected by Perkinsus sp. parasites at 97% prevalence and moderate infection intensities. Parasite cells in gill and gonad tissue samples were enlarged for 48h at 28C in Ray's fluid thioglycollate medium: then inoculated into DME: Ham's F-12 Perkinsus sp. culture medium. Enlarged parasite cells zoosporulated to produce hundreds of mo- tile zoospores, which subsequently gave rise to schizogonic in vitro cell lines that zoosporulated intermittently at low frequency. Four Perkinsus sp. isolates were propagated, cryopreserved, and cloned. In vitro cell morphologies and cell cycles of these isolates differed from those reported for other Perkinsus sp.. and DNA sequences suggest that at least one of our isolates is genetically distinct from described Perkinsus species. REPRODUCTIVE STRATEGY: VARIABILITY OF RE- PRODUCTIVE PATTERN IN TWO POPULATIONS GE- NETICALLY DETERMINED OF CR.ASSOSTREA GIGAS. Martha F^nriquez-Diaz*. Stephane Pouvreau, Caroline Fabi- oux, Yvette Le Coguic. Jean Claude Cochard, Marcel Le Pen- nec; UMR PE2M. IFREMER; BP70. 29280 Plouzane. France. In the literature, the reproductive cycle of C. giiicis has been well described and is generally characterized in three steps: ( 1 ) energy storage: (2) gamete development and (3) spawning. But the genetic intra-variability of this cycle has been scarcely investigated in C. gigas. During the French MOREST program, a genetic se- lection based on the survival criteria allowed to obtain a resistant stock (named "' R") and a susceptible stock (named " S"). The gametogenic activity of these two stocks was characterized in field (South Brittany. France) on the basis of quantitative histology of the gonad (gonad volume, number and egg size) and by the ex- pression of the vasa gene, specific marker of the germinal cell. Results showed that the reproductive strategy, especially the re- production effort and the spawning intensity, was strongly differ- ent between the two groups and these results suggest that a genetic triggering mechanism might exist for the onset and flexibility of gametogenesis. THE ROLE OF HEAT SHOCK PROTEINS IN TOLER- ANCE TO PARASITIC STRESS IN THE EASTERN OYS- TER. CRASSOSTREA VIRGINICA. Vincent G. Encomio*; Fu- Lin E. Chu. Virginia Institute of Marine Science. College of Wil- liam and Mary. Gloucester Point, VA 23062. Thermal stress could affect disease resistance mechanisms by depressing immune defense and physiological fitness . We are investigating the relationship between heat tolerance and P. mari- nus resistance among Dermo "resistant" and "susceptible" oyster stocks and the role of heat shock proteins (hsps) in protection of oysters from thermal and disease stress. Results revealed that Chesapeake stocks had higher thermal tolerance than Louisiana stocks. Levels of hsp 70 did not vary between these two stocks and only increased slightly as water temperatures increased. No con- sistent differences in thermal tolerance were found among Chesa- peake resistant and susceptible stocks, and a resistant hatchery strain. Exposure of oysters to a sublethal heat shock improved their survivorship when subsequently exposed to a lethal temperature. We are presently examining how induced thermo tolerance and hsps mediate interactions between parasitic and thermal stress in uninfected and P. marinus challenged oysters This study is sup- ported by ODRP. Sea Grant. NOAA (Award*: 1 14926-GL100I4. Project* VA-OD-01-05). HISTOLOGICAL EXAMINATION OF GAMETOGENESIS IN GENETIC TRIPLOID CRASSOSTREA ARIAKENSIS IN CHESAPEAKE BAY A.J. Erskine* and Standish K. Allen, Jr., College of William and Mary. Virginia Institute of Marine Sci- ence. P.O. Box 1346, Gloucester Point. VA 23062. Combating the loss of the oyster resource in Chesapeake Bay has been ongoing for decades. Recently, focus has turned to the non-native Suminoe oyster, Crassostrea ariakeusis and the possi- bility of its introduction as reproducing diploid or a triploid for aquaculture only. In field tests, triploid C. ariakensis has exhibited high survival, growth, and disease tolerance in Chesapeake Bay. As reported for several other shellfish species, triploidy often re- sults in abnormal or arrested gametogenesis. Documenting the extent of gamete development in triploid C. ariakensis is an im- portant biological variable addressing the risk associated with non- native introduction. Nine diploid females and one tetraploid male were used as parents for this triploid spawn. These genetic triploids were deployed at six sites along Chesapeake Bay ranging from low salinity (~13%c) to high salinity (-35%r). Diploid native controls were sampled at each site to track the "normal" cycle of gameto- genesis. Paraffin histology of triploids revealed abnormal gamete production typical of triploid. However, a few sites produced un- usually mature ova and spermatozoa for triploids. Samples late in the season indicated spawning had occurred in both diploid and triploid males and females. National Sliellfisheries Association. New Orleans. Louisiana Ahstimls. 2(103 Annual Meeting, April 1.V17, 2003 329 EFFECTS OF INBREEDING ON PERFORMANCE TRAITS IN PACIFIC OYSTERS (CRASSOSIREA GIGAS). Ford Evans*, Sean Matson, John Brake, and Chris Langdon. Hat- field Marine Science Center, Oregon State University. Newport. OR, 97365. Understanding the elTects of iiihrccding ls critical lo the long- term viability of shellfish breeding programs, hibreeding depres- sion in shellfish is well documented among the offspring of self fed individuals and full-sib crosses. This study was conducted to determine if crossing more distantly related parents would result in measurable inbreeding depression of performance traits in aduh raised in a commercial inter-tidal growing environment. Families were created with average estimated inbreeding coefficients (F) of 0. 1/16, and 1/5. Average family yield, individual growth rate, and survival were recorded after the first and second growing seasons. After two growing seasons, significant inbreeding depression of yield and individual growth rate was observed in families with F= 1/16 and F= 1/5. Significant depression of survival at harvest was observed only in families with F= 1/5. These results empha- size the importance of maintaining pedigree records in breeding programs to help avoid the dcleteriiius effects of inbreeding de- pression, even among crosses of distantly related parents. TRACKING THE SPREAD OF AN INVASIVE MUSSEL (MYTILIDAE: PHRNA VIRWIS) IN FLORIDA Jonathan S. Fajans*, Patrick Baker. Department of Fisheries and Aquatic Sciences, University of Florida, 7922 NW 7r' ST, Gainesville, PL 32653 The green mussel Feiiia virulis was introduced to Tampa Bay, Florida in 1998. Since April 2002. we have been conducting sur- veys to chart the population growth of the mussel and monitor its spread. Three sites within the Bay were chosen as representatives of estuarine. introdtiction epicenter, and oceanic environments. Monthly collections were made for population density estimates. Densities within the Bay have reached 4033. 3675. and 41 17 per square meter, respectively. Coastal sites throughout Florida were visited annually to determine presence or absence of P. viriclis. As of January 2003 the range of pt)pulations has been extended to Fort Myers Beach to the south of Tampa Bay and Indian Rocks Beach to the north. Additionally, a new population has been found south of St. Augustine extending to Ponce Inlet on Florida's east coast, and several specimens have been reported from Pensacola in the panhandle. OYSTER VASA-LIKE GENE: A SPECIFIC MARKER OF THE GERM CELL LINEAGE IN CRASSOSTREA GIGAS. Caroline Fabioux*. .Arnaud Huvet. Frederic LeRoux, Marcel LePennec, Jean-Claude Cochard. UMR PE2M. Itiemer, BP70 29280 Plou/ane, France. Identification of physiological mechanisms implied in repro- duction of Crassiistrea i>igii.s is essential to improve control re- production in hatchery. Origin and developmental pattern of first germ cells in oyster are steel unclear underlying the need of mark- ers for gametogenesis initiation. The vasa gene, isolated from sev- eral organisms such as Drosophila, Caenorhabditis. Xenopus or Zebrafish are specifically expressed in germ cells and are essential for gonad differentiation. We isolated and characterized an homo- logue of the vasa gene in C. .i;/,?ai by RT-PCR. The spatio- temporal expression pattern of vasa gene was established by In Situ Hybridization or real-time PCR. Results showed that vasa is only expressed in germ cells and not in somatic cells. Moreover, vasa appeared differentially expressed during gametogenesis: from high expression in oogonia and spermatogonia to zero in gametes. Oyster Vasa-like gene appeared to be a relevant marker of germ cells for further studies such as the analysis of environmental effect on the kinetic of gametogenesis and reproductive effort of C. gigas. MANIPULATION OF ENVIRONMENTAL PARAMETERS FOR OUT-OF-SEASON EGG AND LARVAL PRODUC- TION IN BLUE CRAB BROODSTOCK {CALLINECTES SAPWUS). Andrea Findiesen*, Oded Zniora. Moti Harel, and Yonathan Zohar, Center of Marine Biotechnology, University of Maryland Biotechnology Institute. Baltimore. MD; Alicia Young- Williams and An.son H. Hines. Smithsonian Environmental Re- search Center, Edgewater. MD. Blue crab production techniques are being developed at the Center of Marine Biotechnology (COMB) to evaluate the possi- bility of restocking the diminished Chesapeake Bay blue crab population. Mature mated females were introduced into 2 m3 tanks with phase-shifted environmental conditions. By manipulating photoperiod, temperature and salinity, we have successfully in- duced females to ovulate, produce egg masses (sponges) and pro- vide viable larvae all year-round. We also have been able to pro- duce up to four successive sponges per female. Sponge production seems to be affected by a combination of photoperiod and tem- perature: long photoperiod ( 14 hours light: 10 hours dark) and high temperature (23oC) generated the most sponges. Our data indicates that high temperatures, though optimal for sponge production, in- crease susceptibility to disease when exposed over long durations of time. Sand is necessary for sponges lo adhere properly to a female's abdomen. There doesn't seem to be any difference be- tween inaintaining the females at 25 or 30 ppt. Future work may include hormonal manipulation of broodstock to provide more predictable ovulation and larval production. 330 Absrnicis. 2003 Annual Meeting, April 13-17, 2003 National Shellfisheries Association. New Orleans, Louisiana MANAGING AND MONITORING INTERTIDAL OYSTER REEFS WITH REMOTE SENSING IN COASTAL SOUTH CAROLINA. Mark Finkbeiner*. Bill Stevenson. Bill Ander- son. Mike Yianopolous, Loren Coen. Ginger Martin, and Karen Cullen. NCAA Coastal Services Center 2234 South Hob- son Ave, Charleston. SC 29405. Intertidal oyster reefs are a keystone species in South Caroli- na's estuaries and a major commercial and recreational resource. The South Carolina Dept. of Natural Resources (SCDNR) is re- sponsible for conserving oyster reefs and regulating their harvest. The current oyster reef database for South Carolina was developed by field assessment in the 1980s and an update is needed to assess resource status and trends across the coastal zone. Coastal devel- opment and associated waterway usage are suspected of altering the extent and density of the state's oyster resources. The NCAA Coastal Services Center is working with SCDNR to develop meth- ods for using high-resolution remote sensing data to assess inter- tidal oyster reefs along the South Carolina coast. The objective of the project is to provide SCDNR with a new methodology for assessing intertidal oyster resources. The project examined digital and analog aerial photography in two pilot areas located in Charleston and Beaufort Counties. A variety of image processing and photogrammetric methods were evaluated includ- ing manual delineation, spectral clustering, and digital texture analysis. The.se methods focused on determining the perimeter and spatial characteristics of oyster reefs. Results of this study will support future efforts to update the entire state database. IS COPPER REQUIRED FOR EASTERN OYSTER SET- TING AND METAMORPHOSIS? William S. Fisher, US. En vironmental Protection Agency National Health and Environmen- tal effects Research Laboratory Gulf Ecology Division Gulf Breeze, FL 32561. Recent field research with eastern oysters demonstrated higher defense activities, including hemocyte numbers, locomotion and bactericidal ability, associated with locations exhibiting relatively high chemical contamination. Copper and zinc, found in high con- centrations in tissues of oysters collected from these sites, are known to accumulate almost exclusively in amebocytes. These data have led to a re-evaluation of potential roles for copper and zinc in oyster physiology. A role for copper in setting and meta- morphosis of oysters was previously proposed by Herbert F. Prytherch (1934), who found that larval oysters would not set or metamorphose without 0.05 to 0.6 mg L- 1 copper for at least short durations in the surrounding water. High concentrations were not toxic for these short durations, and setting was stimulated within minutes of copper addition. Salinity altered the amount of time required for larvae to fix to the substrate but was not ultimately critical to setting. Consequently, oyster setting near river mouths may be due to incoming copper rather than the variable salinity to which it is sometimes attributed. If true, our understanding of oyster distributions and larval setting success would be greatly altered. Yet. by all appearances, these observations have never been validated. COMPARISON OF PACIFIC OYSTER (CRASSOSTREA GI- GAS) REARING RESULTS (SURVIVAL. GROWTH. QUAL- ITY) IN FRENCH FARMING AREAS, AFTER A 10-YEARS MONITORING (1993-2002) BY THE IFREMER/REMORA NETWORK. Pierre-Gildas Fleury*. Erwan Le Ber. Serge Claude, Florence Cornette, Florence d'Aniico. Patrice Guil- pain. Hubert Palvadeau. Stephane Robert, Patrick Le Gall, Michel Ropert. Charlotte Simonne. Catherine Vercelli. IFREMER. F-56470 La Trinite-sur-mer. France. Since 1993 the network IFREMER /REMORA has carried out annual standard monitorings of survival, growth and quality crite- ria of the Pacific oyster ^Crassostrea gigas) among the main French farming areas. The network provides data series for each site, mean values (references) and allows multifactorial analysis of oyster rearing results. It must be pointed out that no general cor- relation was found between growth and mortality. A large range of results was exhibited both between years and between sites. How- ever, unusual mortalities, annual variations of growth, or increas- ing infestation by the worm Polydora could be focused and quan- tified. Moreover, local trends may be of interest for collective oyster management. At last. REMORA data may support various types of studies, such on oyster quality, biological indicators for coastal waters or explanatory models of the oyster-farming eco- systems. EVALUATION OF RARITAN AND SANDY HOOK BAY HARD CLAM, MERCENARIA MERCENARIA. STOCKS FOR FISHERY MANAGEMENT. George E. Flimlin, Jr.*, Michael Celestino, John N. Kraeuter, Robert J. Macaluso, Michael Kennish. Rutgers Cooperative extension 1623 Whites- ville Rd. Toms River. NJ 08755. The hard clam fishery in the Raritan and Sandy Hook Bays took a twenty year hiatus started by a hepatitis outbreak in the early 1960's. The use of a clam relay and depuration allowed clammers to re-enter the fishery in 1983. Since then the fishery has grown steadily to about 200 full and part-time participants sup- plying clams to two depuration plants with others relaying their catch to approved beds in another county for purging. A stock assessment was done the State in 1 983 with no further evaluation until 2000 when the Bureau of Shellfisheries covered the same area again. Simultaneously, studies were done examining the age and growth of the shellfish as well as a natural mortality study. Armed with this information, the industry and the state can better work together to manage the harvest pressure and the par- ticipation in the area. Analysis of the data indicates that the stocks are at higher levels than when harvest restarted in 1983, possibly allowing for further exploitation. National Shellt'ishenes Association. New Orleans, Louisiana Abstracts. 2003 Annual Meeting. April 13-17. 2003 331 POTENTIAL PATHOGENS ASSOCIATED WITH ABNOR- MAL MORTALITIES. Celine Garcia,* Isabelle Arzul. Francl< Bcrthe. Bruno Chollet, .Jean-Pierre Joly, Nolwenn Kerdudou, Laurence Miossec. Maeva Robert and Jean-Louis Nicolas. Ge- netic-Pathology and Aquaculture Shellfish Research Laboratory. IFREMER 17300 La Tremblade. France. In France abnormal mortalities of mollusks affect many species of bivalves. They occur mainly in summer and concern all the French coastline. For Crassostrea gigas. they affect all life"s stages but more particularly spat. A pathology monitoring net- work. REPAMO. was created at the beginning of the nineties in France in order to answer European requirements as regards mol- lusk pathology. REPAMO observes whether there is abnormal mortality and keeps track of the health situation of mollusk stocks including the presence of pathogens notifiable to the European L'nion and OIE. When mortalities occur, the network REPAMO. samples the populations and performs different types of analysis (histology. bacteriology, viral detection) in order to detect potential patho- gens. In France, different agents have been sometimes associated with abnormal mortalities of bivalves such as herpes-virus in Cnis- sostreci gigas. Bacterial agents can be also involved. Indeed hemolymph of moribund oysters from open sea and from hatchery are often invaded by one Vibrio species belonging to V. spleiuUdus group or V. aestiiarianus. These observations suggest that Vilirio could induce or aggravate mortality in oysters weakened by envi- ronmental or physiological (maturation) factors. SEASONAL VARIATION IN THE PHYSIOLOGICAL STA- TUS OF THREE SPECIES OF MUSSELS IN THE ALLE- GHENY RIVER, PA. Catherine M. Gatenby*, The Academy of Natural Sciences 1900 Benjamin Franklin Parkway Philadelphia. PA 19103; Danielle A. Kreeger, Deborah Raksany, and Rich- ard J. Neves. A necessary precursor to identifying suitable feeding regimes for maintaining endangered freshwater inussels in captivity is de- fining their nutritional requirements. Similarly, a better under- standing of their physiological status and use of food resources is needed to assess their role in natural systems and develop man- agement plans that protect existing populations from further de- cline. We quantified the seasonal and interspecific variation in condition index and tissue biochemistry of representative unionid families from a large bed in the Allegheny River. Condition [jeaked in July and was similar between November and May for all species. Protein content peaked in November and May for EUiptio (Ulatata and Lasiiiigoini costaia (>40'7r). but did not differ season- ally in Actinoiiaias ligaineiitinu (>307r). Lipid content was high in November and May for A. ligamentina and E. dilatata ( >29'7c ). but peaked in July in L. costata. (23%). Carbohydrate content was similar among species and times. The overall physiological status and specific demands for protein and lipid varied considerably among seasons and species. Hence, the formulation of diets for maintaining captive mussels should target these changing de- mands. As well, efforts to assess the ecological importance of mussels should anticipate variation in physiological rate functions. CHARACTERIZATION OF VIBRIO ISOLATED FROM PA- CIFIC OYSTERS* SPAT SUFFERING FROM SUMMER MORTALITY OUTBREAKS Melanie Gay*, Laboratoire de Genetique et Pathologic IFREMER 1 7390 La Tremblade France. Guenaelle Lancelot, Bruno Chollet, Tristan Renault, Nathalie Cochennec. Franck Berthe, Christophe Lambert, Gwenaelle Choquet, Christine Paillard, Manolo Gouy, Frederique Le Roux, Philippe Goulletquer. The pathogens related to summer mortality outbreaks are a heipes virus and two bacterial strains one belonging to Vibrio splemlidiis biovar II and the other to Vibrio splendidus spp. How- ever, the feature pathogen/opportunist of these strains is still un- known. Several strains belonging to the genus Vibrio have been identified as pathogen for different mollusk species. In the context of the French program Morest. experiments of cohabitation have been used to demonstrate the potential presence of a transmissible infectious agent in batches of oysters suffering from summer mortality outbreaks. More than one hundred Vibrio strains have been isolated from these experiments. These strains have been phenotypically and genotypically characterized. Their virulence has been evaluated by infection trials. Two Vibrio lentus strains have been selected. The mortality rate induced by them injected together is always higher than the mor- tality rate induced by each strain injected individually. A histo- logical examination of injected animals showed damaged hemocytes and muscle. However, bacteria have only been ob- served in the tissue surrounding the muscle and in the kidney. We have shown that physiological and genetic factors had an effect on the sensitivity of Crassostrea gigas to the experimental model of bacterial infections. RESTORATION OF BAY SCALLOPS IN HIGHLY MODI- FIED AND RELATIVELY PRISTINE HABITATS ON THE WEST COAST OF FLORIDA, USA. Stephen P. Geiger* and William S. Arnold. Florida Fish and Wildlife Conservation Com- mission Manne Research Institute 100 8th Avenue S.E. St. Peters- burg. FL 33701 USA. The density of scallops in many populations within Florida has declined greatly while other populations have remained healthy enough to allow recreational harvest. We have been attempting to restore four of the populations that experienced declines. Two of these populations exist in coastal areas with expansive seagrass meadows and low i?iipact from de\elopment. Two populations exist in embayments which have been modified by anthropogenic impacts such as hardened shorelines, filled wetlands, channeliza- tion, and construction of causeways. In one coastal population. 332 Abstmcts. 2003 Annual Meeting. April 13-17, 2003 National Shellfisheries Association. New Orleans. Louisiana adult density has returned to har\estable levels. Good management practices, natural variability, and restoration efforts may have all played a role. The density in the second coastal community has also increased but not to harvestable levels. Neither population in embayments has recovered despite restoration efforts. Evidence from surveys of adult scallops and recruitment of spat in these four populations as well as three additional populations where no res- toration efforts occurred suggest that habitat alteration may am- plify negative variations in the population. One example is the rate of recovery from declines related to harmful algal blooms. Con- tinued de\elopment in northwest Florida may exacerbate the popu- lation declines, especially if those regions serve as a source for recruits in other areas. CIS to examine the effects of culture density and location on seston depletion in Tracadie Bay, an important site in the PEI mussel industry. Models have been constructed at several levels including box models of the estuary, and fully coupled physical- biological models set up on a detailed hydrodynamic grid. In the latter case, maps of seston depletion and biodeposition are gener- ated as a function of culture density and distribution. Model results are integrated as data layers in the GIS, and calculations are made w ithin grid cells using spatially explicit conditions to predict mus- sel growth and bioenergetics. A comprehensive field program in- cluding moorings with current meters, particle sensors, sediment traps, and surveys with a towed vehicle was used to provide boundary conditions as well as groundtruthing of model results. FLOW CYTOMETRY AS A TOOL TO QUANTIFY OYS- TER PHAGOCYTOSIS. RESPIRATORY BURST AND AP- OPTOSIS. Michael Goedken*. and Sylvain De Guise. Depart- ment of Pathobiology and Veterinary Science. University of Con- necticut. 61 N Eagleville Road. U-89. Storrs. CT 06269. The parasites Perkiiisus iiuiriiiiis and Haplosporidium nelsoiii have generated losses in the hundreds of millions of dollars. The relationship between parasites and oyster defense mechanisms is unclear. A better understanding of the iinmunopathologic associa- tion may reduce these economic losses. Defense mechanisms of the Eastern Oyster {CrassDstii'a virginica) were quantified at the single cell le\el utilizing flow cytometry. Phagocytosis was mea- sured using fluorescent beads. Respiratory burst activity was quan- tified as the increase in dichlorofluorescein-associated fluores- cence upon stimulation. Apoptosis was evaluated with TUNEL assay. Three sub-populations of heniocytes (granulocytes. h>ali- nocytes and intermediate cells) were identified with unique func- tional characteristics. Granulocytes were most acti\e at phagocy- tosis and peroxide production while hyalinocytes were relatively inactive. TUNEL assay application allowed quantification of hemocyte apoptosis. which was more frequent in dividing cells. Flow cytometry can rapidly, accurately and directly quantify the morphology and function of a large number of individual cells, and will lead to a better understanding of the bivalve immune system and susceptibility to disease. INTEGRATION OF MODELING AND GIS IN STUDIES OF CARRYING CAPACITY FOR BIVALVE AQUACULTURE Jon Grant. Marie .Archambault *, Cedric Bacher. and Peter Cranford. Dpt Oceanograph> Dalhousie University Halifax. NS B3H4J1 Canada. Estimation of carrying capacity for bivalve culture is important in predicting the effect of the environment on culture yield, as well as the effect of culture on the environment. Areas of Prince Ed- ward Island (Canadian Maritimes) appear saturated with respect to mussel farms, and there is a requirement for estimation of culture density relative to sustainability for growth rates and ecosystem health. We have combined field studies, biophysical modeling, and MAPPING AND CHARACTERIZING EASTERN OYSTER iCRASSOSTREA VIRGl.MCA) REEFS USING UNDERWA- TER VIDEOGRAPHY AND QUADRAT SAMPLING Jenni- fer Greene*. Ray Grizzle and Jamie Adams. Jackson Estuarine Laboratory University of New Hampshire iS.'i Adams Point Rd. Durham. NH 03824. This project attempts to develop an economical technique to map oyster ( Cra.v.vo.sfrra virginica) reef boundaries as well as char- acterize the general health of oyster populations using videography and quadrant sampling. In New Hampshire, oyster monitoring by resource managers has been impeded by lack of an effective meth- odology for determining distribution and abundance. Videography was conducted in Great Bay, NH by systematically imaging mul- tiple sampling cells in a grid covering two study reefs. In each cell. a 5-10 s digital video image was recorded (0.25 m2 area) with location determined by DGPS. A representative still image was selected for each cell and combined into a photomontage overiaid onto a geo-referenced base map using ArcView GIS. Quadrat samples (0.25 m2) were collected from 8-10 of the imaged areas on each reef and all live oysters were counted, measured and returned to the reef. Initial results suggest that systematic videog- raph\ can accurately delimit reef boundaries, yield quantitative data on shell densities, and provide information on reef characteristics and structure. Additional reefs will be sampled in 2003 using a com- bination of continuous video transects with peri(xiic camera drops in an attempt to pro\ ide finer scale determination of reef boundaries. THE EFFECTS OF BACKGROUND CONCENTRATIONS OF THE BROWN TIDE ALGA AUREOCOCCUS ANOPH- AGEFFERENS ON GROWTH AND FEEDING IN THE BI- VALVE MERCENARIA MERCENARIA. Dianne I. Green- field*. Darcy J. Lonsdale. Robert M. Cerrato, and Glenn R. Lopez. Marine Sciences Research Center. Stony Brook University. Stony Brook NY 11794-5000. This study examined the extent to which background levels, defined as concentrations too low for toxicity to inhibit feeding, of Aureococciis anopliagefferens (brown tide) influenced the growth National Shellfisheries Associalioii. New Orleans. Louisiana Abstracts. 2003 Annual Meeting. April 13-17. 2003 333 and feeding physiology of hard clanis. Mcnenaria iiicireiuiria. in the laboratory compared to other phytoplankton common to Long Island. NY. waters. Juvenile clams were fed either unialgal cul- tures or diets mixed with background levels of brown tide. Ab- sorption efficiency (AE) was determined using the 14C:51Cr dual- tracer method and growth was determined by biomass change. Results showed that unialgal diets resulting in the highest AE. specifically Isocliiysis galhana and Thalassiosira pseudonana. re- sulted in rapid M. mercenaria growth. A unialgal diet of Nitzschiu closteriiim resulted in a comparatively low AE and loss in clam biomass. Diets mixed with brown tide resulted in a significantly lower AE than the corresponding unialgal diet for all phytoplank- ton species except N. closteriiim. Additionally, mixed diets re- sulted in slightly less clam growth than unialgal diets. This sug- gests that when brown tide occurs in the field at background levels, clams may suffer subtle, chronic effects. Moreover, the responses of M. mercenaria to each diet have implications for understanding how phytoplankton community composition influences bixalve growth in the field. A SIMULATION MODEL OF THE GROWTH OF HARD CLAMS {MERCENARIA MERCENARIA), IV. EFFECTS OF CLIMATE CHANGE. Raymond E. Grizzle*, Eileen E. Hof- mann. .|ohn M. Klincli. Eric N. Powell. John N. Kraeuter, V. Monica Bricelj. and Stuart C. Buekner. Jackson Estuarine Labo- ratory University of New Hampshire 83 Adams Point Rd. Durham. NH 03824. A phvsiologically-based model that simulates indi\idual growth of the hard clam. Mercenaria mercenaria. in response to changes in environmental conditions has been developed. We are applying this base model to esaluate the effects of possible climate change scenarios. Thus far. our climate change modeling has fo- cused on water temperature and the timing of spring and fall phy- toplankton blooms because these are major factors that control growth of hard clams. Actual water temperature data sets from Great South Bay. NY as well as sites in Chesapeake Bay and North Inlet. SC were used to simulate the long-term warming trend pre- dicted by all major climate change inodels. Each data set was used in combination with different spring and fall phytoplankton bloom scenarios. When bloom times were held constant, long-term warm- ing resulted in increased growth and the predicted rates matched published values for clams from each area from which water tem- perature data were used. The timing of blooms had a dramatic effect on growth, suggesting that year-to-year variations may be more important than overall temperature trends as climate change ensues. Details on the modeling results will be presented and dis- cussed. STATUS OF BLUE CRAB POPULATIONS IN LOUSIANA BASED ON FISHERY INDEPENDENT DATA COLLEC- TIONS (1967-2002) WITH OBSERVATIONS ON RELA- TIVE ABUNDANCE IN OTHER GULF STATES. Vincent Guillory*. Louisiana Department of Wildlife and Fisheries. P.O. Box 1 89. Bourg, Louisiana 70343; Harriet Perry, Center for Fish- eries Research and Development. Gulf Coast Research Laboratory, College of Marine Sciences, the LIniversity of Southern Missis- sippi. P.O. Box 7000. Ocean Springs. Mississippi 39566-7000; and tlie Blue Crab Technical Taskforce, Gulf States Marine Fish- eries Commission. The 33-year ( 1967-2000) database from the Louisiana Depart- ment of Wildlife and Fisheries bottomfish/shrimp monitoring pro- gram is the most extensive and continuous fishery independent blue crab data set in the Gulf of Mexico. Long term and recent trends in recruit (<4() mm CW), juvenile (40-99 mm CW). sub- legal (100-124 mm CW). and legal 0123 mm CW) blue crabs were examined, as well as overall catch per unit of effort sub-legal crabs did not significantly change over the long term, although overall CPLIE for these size groups showed a significant increase from 1967-1989 with a significant decrease in recent years ( 1990- 2002). Catch per unit of effort of recruits significantly increased over the long term with a downward trend noted in cuirent data. Trends in relative abundance are discussed in relation to habitat changes in northern Gulf of Mexico estuaries and to biological factors such as predation. BREEDING AND EVALUATION OF EASTERN OYSTER STRAINS SELECTED FOR MSX, DERMO AND JOD RE- SISTANCE. Ximing Guo*, Susan Ford, and Gregory De- Bros.se. Haskin Shellfish Research Laboratory. Rutgers Uni\er- sity. 6939 Miller Avenue. Port Norris. NJ 08349; Roxanna Smolowitz. Marine Biological Laboratory. 7 MBL Street. Woods Hole. MA 02543; Inka Sunila. Bureau of Aquaculture and Labo- ratory. Milford. CT ()6460. Rutgers University has been breeding oysters for disease- resistance since the early 1960s and produced strains showing strong resistance to MSX and some resistance to Dermo. Breeding at the F.M. Flower Oyster Company has produced a strain (FMF) showing superior growth and JOD-resistance. We undertook a project to evaluate the Rutgers NEH strain, the FMF strain and their hybrids (HYB) along with a global susceptible control (ME) and local controls that were normally cultured at each of the four deplovment sites. Oysters were produced in June 2000. deployed in Jul\ 2000 and evaluated for 27 months. Dermo exposure was heavy at most sites, while MSX and JOD infections were low or absent. At Cape Shore (NJ) where infection was the heaviest. NEH and HYB had the lowest cumulative mortality. 43.5% and 43.6% respectively, compared with 82.3% for FMF. 99.4% for ME and 81.1% for the local control (Delaware Bay wild). In growth. HYB was the same as FMF and faster than NEH. while ME and the local 334 Absmicls, 2003 Annual Meeting, April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana controls grew the slowest. The hybrid offered the highest yield by surviving as well as the NEH strain and growing as fast as the FMF strain. MARKETING IMPLICATIONS OF CONSUMER ATTI- TUDES TOWARD OYSTERS Terrill R. Hanson*, Lisa O. House. Benedict C. Posadas. Dept. of Agricultural Economics P.O. Box 5187 Mississippi State. MS 39762. As US consumption of oysters declined during the 1990's an understanding of why consumers purchase and consume oysters is important to marketing oysters effectively and reversing this trend. In 2000-2001. a survey was administered to U.S. residents on the topic of seafood consumption. Results and findings of this survey are useful for sellers to use in targeting consumers likely to in- crease their oyster consumption and for processors to use in de- signing programs likely to improve food safety considerations. Reasons for eating oysters included enjoyment of the flavor {SOVc of consumers) and addition of variety to their diet (37%). Oyster consumer's identified price (38%), product safety (29%), and lack of availability of fresh product (20%) as the main reasons for not consuming oysters more often. Forty-three percent of oys- ter consumers and 54% of those concerned about product safety indicated their consumption of oysters would increase if depura- tion methods were used to 'guarantee' oyster safety. Sixty-one percent stated a willingness to pay of $0.34/oyster over the raw- oyster price for a 'guaranteed' safe product. This option may be profitable if depuration costs do not exceed the increases in con- sumer's willing to pay. HOW MANY LARVAE STAY AT HOME? MEASURING PATTERNS OF LOCAL OYSTER RECRUITMENT USING MOLECULAR MARKERS. Mattliew Hare*. D. Merritt and K. Paynter. Biology Department University of Maryland College Park. MD 20742; S.K. Allen. ,Ir.. E.M. Burreson. M.D. Camara. Ryan Carnegie. M. Lucixenbach and K.S. Reece. Recruitment enhancement is one of the primary objectives of oyster restoration in the Chesapeake Bay. Given the tremendous spatial and temporal variability in recruitment patterns that have been documented in Chesapeake Bay oysters, correlations between broodstock plantings and spat set provide only a partial and po- tentially misleading index of recruitment strength from enhance- ment efforts. We are using the unique genetic signature of disease- tolerant selected strains to measure the geographic scale of recruit- ment provided by restored reefs. With the highly variable DNA polymorphisms that we have developed, every newly settled oyster is "tagged" with a genotype that links it to its parents and its source population. In cooperation with several agencies and organizations we planted CROSBreed selected strains in the Great Wicomico River in 'Virginia and the Little Choptank River in Maryland dur- ing the summer of 2002. Genetic markers provide better than 95% accuracy for assigning indi\ idual oysters to their source, selected strain or natural broodstock. CROSBreed oysters planted in 'Vir- ginia averaged 6 cm. large enough to potentially breed during 2002. We will present analyses of 2002 Virginia spat indicating the proportion of recruitment derived from planted oysters versus natural broodstock. SUITABILITY OF OYSTER CLUSTERS AS HABITAT FOR REEF-RESIDENT FISHES AND DECAPOD CRUSTA- CEANS IN THE CALOOSAHATCHEE ESTUARY Leslie H. Haynes.* Arielle Poulos. Lacey K. Smith. Aswani K. Volety and S. Gregory Tolley. 1227 S.W. 25th St. Cape Coral. FL 33914. The habitat suitability of oyster clusters for reef-resident fishes and decapod crustaceans was examined in the Caloosahatchee Es- tuary. Lift nets representing three habitat treatments were deployed during three seasonally wet and three seasonally dry months on an oyster reef located in the lower estuary. Nets contained either no oyster shell (control), dead, articulated clusters, or live oyster clus- ters. Based on the results of Hester-Dendy sampling conducted at the same site, nets were deployed for a period of 1 month to ensure full recruitment. Analysis of variance indicated that articulated oyster shell (dead or living clusters) exhibited greater species rich- ness, biomass. and dominance than did the controls. Furthermore, organism abundance was higher in living oyster clusters compared to dead, articulated clusters; both treatments with oyster shell ex- hibited significantly greater abundances than the control. In addi- tion, biomass of all treatments was significantly greater during the dry season than during the wet season. The results of this study suggest that the habitat value of oyster clusters to reef-resident fishes and decapod crustaceans lies primarily in the three- dimensional structure created; dead, articulated oyster shell exhib- ited levels of as.sociated biomass and species richness similar to that of clusters containing living oysters. ALGAL FOOD QUANTITY AND QUALITY AFFECT IM- MUNE FUNCTION IN OYSTERS STRESSED BY HIGH TEMPERATURE. Helene Hegaret*. Gary Wilvfors. NCAA Fisheries. Milford. CT. USA. Philippe Soudant. LEMAR, lUEM- UBO, Plouzane. France, and Jean-Fran^-ois Saniain. Laboratoire de Physiologic des Invertebres. IFREMER. Brest. France. Oyster seed from a hatchery must resist environmental stresses when planted in the sea. We conducted an experiment to analyze the influence of nutrition on oyster. Crassostrea virigiiiica's. im- mune capability. Cultured microalgal diets were varied factorially in quantity ( 10 and 50% dw/dw microalgae/oyster soft tissue per day) and quality (Skeletonema. Tetraselmis, and a 50/50 mix), with unfed controls. Oysters were fed five weeks at 20°C and then temperature-stressed for one week at 28°C. Before and after heat stress, we used flow-cytometry and multivariate statistics to ana- lyze the following hemocyte functions: viability, aggregation. National Shell! ishcries Association. New Orleans, Louisiana Ahsiracly 2003 Annual Meeting, Apiil 1.^-17, 200.^ 335 phagocytosis, and respiratory burst. Discriminant Analysis showed significant effects of food quantity and quality on hemocyte func- tion. Principal Components Analysis revealed the main effects of heat stress to be increased respiratory burst and decreased phago- cytosis; this decoupling of the two steps in pathogen defense was more severe in starved or poorly-fed oysters. ASSESSING FEASIBILITY OF STOCK ENHANCEMENT FOR CHESAPEAKE BLUE CRABS (CALLINECTES SAPI- DUS). Anson H. Hines*. Jana L.D. Davis, Alicia Young- Williams. Smithsonian Environmental Research Center, Edgcwa- ter. Maryland 2 1 037, USA; Yonathan Zohar. Oded Zmora, Uni- versity of Maryland Biotechnology histitute, Baltimore. Maryland 21202, USA. In overexploited. recruitment-limited fisheries, enhancement with hatchery-produced juveniles, coupled with traditional man- agement techniques and habitat restoration, may be required for effective stock management. Enhancement, used most frequently for finfish stocks, has rarely been attempted with crustaceans. The Chesapeake blue crab stock exhibits key characteristics as an ap- propriate candidate for enhancement: SIVr decline in biomass over the past decade, recruitment limitation, and extensive habitat with reduced juvenile mortality and densities below carrying capacity. The goals of this work were ( I ) to determine the enhancement potential of blue crab subpopulations by releasing hatchery-reared crabs (25.000 juveniles <23mmCW) on spatial scales of 10-15 ha, and (2) to identify factors intluencing survivorship of hatchery crabs in the wild. In four separate cohorts (3,700-9,500 juveniles) that were sampled over S-16 weeks, released tagged hatchery crabs successfully enhanced local subpopulations, growing rapidly and surviving to contribute to the spawning stock. Hatchery and wild crabs were similar in most respects, but differed initially in burial rate, carapace morphology, and susceptibility to predation. How- ever, differences disappeared within days in the field, suggesting ways to improve success of future released crabs. These initial results contribute to determining whether enhancement on a larger scale is possible. A SIMULATION MODEL OF THE POPULATION GROWTH OF HARD CLAMS (MERCENARIA MERCE- NARIA). I. MODEL DEVELOPMENT AND IMPLEMENTA- TION. Eileen E. Hofmann*, ,|ohn M. Klinck, Eric N. Powell, John Kraeuter, Monica Bricelj. Ray Grizzle, Stuart Buckner. CCPO, Crittenton Hall Old Dominion University Norfolk, VA 23529, A physiologically-based model that simulates the population growth of hard clams, Mercenaria incrcenaria. in response to tem- perature, salinity and food supply has been developed and applied in Great South Bay. The model structure model allows indepen- dent simulation of shell and tissue growth, which permits calcu- lation of animal conditi()n as a diagnostic. Also, length and age are independently tracked, thereby allowing specification of size- frequency and age-frequency distributions to describe population structure, and more importantly to define age dependent, as well as size-dependent, processes. The model structure includes a genetic component that permits simulation of a range of genotypes, which are combined into cohorts to construct a population. The simulated hard clam growth obtained using environmental conditions char- acteristic of Great South Bay match weight and length values that are ob.served for populations in this region. The extension of the simulations of individual clams to cohort and populations scales shows the imponance of assimilation rate and the apportionment between reproductive and somatic growth in determining inter- cohort variability and overall long-term population characteristics. The results of these simulations as well as those that examine model sensitivity to assumptions made for key model processes and parameteri/ations will be presented. COMPARISON ALONG THE NEW ENGLAND COAST OF EPIDEMIC SHELL DISEASE IN THE AMERICAN LOB- STER, HOMARUS AMERICANVS. Andrea C. Hsu*, Boston University Marine Program Marine Biological Laboratory Woods Hole. MA 02543; Roxanna M. Smolowitz, Marine Resources Center Marine Biological Laboratory Woods Hole. MA 02543: Andrei Y. Chistoserdov. Department of Biology University of Louisiana at Lafayette Lafayette. LA 70504; and Hemant M. Chikarmane. Marine Resources Center Marine Biological Labo- ratory Woods Hole, MA 02543. During the last six years, shell disease has been found at high levels in wild lobsters along the New England coast. This study utilizes a combination of scanning electron microscopy (SEM). denaturing gradient gel electrophoresis (DGGE). and histological analysis to describe and define bacterial cells on the infected cara- pace of wild-caught lobsters. Diseased lobsters used in this study were collected starting from Eastern Long Island Sound. New York, up toward Cape Cod Bay. Massachusetts, with control ani- mals from Maine. SEM analysis revealed and statistical tests verified five sepa- rate morphological types of bacteria present in shell lesions. Halo- like holes surrounding all bacterial types suggest boring as their causative mechanism for degrading the lobster carapace. Prelimi- nary DGGE data indicated up to fourteen independent phylotypes of bacteria were present in lobster lesions. At least two of them were found in all diseased lobsters used in this studv. Histopath- ologically. the carapace matrix was usually absent or lattice-like cuticular remnants were found attached to underlying less de- graded cuticle. Bacteria were the predominate organisms found at the leading edge of erosions. Combined results from SEM, DGGE, and histological analyses present evidence that an assemblage of bacteria may be the cause of New England epidemic shell disease. 336 Absinicls. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana TENNESSEE'S PEARL CULTURE INDUSTRY. Don Hubbs. Mussel Program Coordinator. Tennessee Wildlife Resources Agency. P.O.B. 70. Camden. TN 38320. The Tennessee Wildlife Resources Agency (TWRA) regulates freshwater pearl culture in Tennessee. Administrative rules, proc- lamations and contracts are employed to regulate the industry, and protect and manage its utilization of the native mussel resource. Although experiments in pearl culturing began in the 1960's, gov- erning regulations were not developed until 1988. A panel com- posed of TWRA fisheries personnel and industry representatives drafted the first regulations. The washboard, Megalonaias iien-o.sa (Rafinesque, 1820). is the primary freshwater mussel species used by the pearl culture industry. Because washboards command the highest price in the commercial shell market, and legal-sized in- dividuals can be scarce, industry experts convinced the TWRA to permit the use of sub-legal sized washboards for economic rea- sons. Contracts, seasons, and quotas were established to control the harvest of wild washboard mussels for the pearl culture industry. Permission for use of sub-legal sized washboards for pearl culture proved unpopular with many commercial shell harvesters and wholesale shell dealers. EVIDENCE OF A COLD SHOCK RESPONSE IN VIBRIO VULNIFICUS, A HUMAN PATHOGEN TRANSMITTED VIA RAW EASTERN OYSTERS, CRASSOSTREA VIR- GINICA, FROM THE GULF OF MEXICO. Kristi L. Huels*, 203 Swingle Hall Auburn University. Auburn. AL 36049-5419. Yolanda J. Brady, Mary A. Delaney, Joel A. Bader. This study examined the response of Vibrio vulnificus to incu- bation at 13 and 4o C. It focused on changes in protein expression using one and two dimensional gel electrophoresis. Although dif- ferent proteins were expressed following cooler temperature ex- posure no major cold shock protein was identified. As hypoth- esized, longer incubation times at l3o C resulted in increased variations. Proteins expressed at the cooler temperature were only transiently expressed, classical of stress responses. These prelimi- nary results suggest there is a cold shock response active in V. vulnificus that requires further investigation in order to properly evaluate and alter the general management practices for collection and processing of the Eastern Oyster, Crassostrea viri^inica. from the Gulf of Mexico. PREVALENCE AND ABUNDANCE OF PERKINSUS MARI- NUS AND PERKINSUS CHESAPEAhl/ANDREWSl IN CHESAPEAKE BAY OYSTER BEDS Karen L. Hudson*, Kimberly S. Reece, Christopher F. Dungan, and Rosalee M. Hamilton. Virginia Institute of Marine .Science. Gloucester Point. VA 23062. Three described species oi Perkinsus have been reported in the Chesapeake Bay region of the United States. Perkinsus inarinus is a well known pathogen of the eastern oyster. Crassostrea vir- ginica. Perkinsus chesapeaki and Perkinsus undrewsi are more recently described species from the soft-shell clam. Mya arenaria and the Baltic clam. Maconia Ixdthicu. respectively. Recent mo- lecular studies, however, suggest that these two species are syn- onymous (Dungan et al. 2002). In 2001. Coss et al. reported P. aruirewsi infections in oysters. The routine test used to diagnose P. niariinis infections from oysters. Ray's fluid thioglycollate me- dium (RFTM) however, is not species-specific. The objective of this study was to survey oyster beds in the Chesapeake Buy area located adjacent to a variety of clam species in order to assess prevalence and abundance of Perkinsus species in oyster and clam hosts. Prevalence was assessed by standard PCR using two spe- cies-specific PCR primers: one P. nnn-inns-^pecifii: and the other P. chesapeaki I andrewsi- specific. Abundance was accomplished using quantitative PCR using the same species-specific primers. Two species-specitlc in situ hybridization probes were developed and tested. Results of the assay development and en\ironmental screening will be presented. A FISHERY-ORIENTED MODEL OF MARYLAND OYS- TER POPULATIONS Stephen J. Jordan* and Jessica Vani- sko. USEPA. Gulf Ecology Division 1 Sabine Island Drive Gulf Breeze, PL 32561. We used time-series data to calibrate a model of oyster popu- lation dynamics for Maryland's Chesapeake Bay. Model param- eters were fishing mortality, natural mortality, recruitment, and carrying capacity. We calibrated for the Maryland bay as a whole and separately for 3 salinity zones. Simulations indicated that a long-term declining trend in the Maryland bay-wide stock of har- vestable oysters could be reversed by controlling fishing mortality and enhancing recruitment. For example, an exponential increase in stock size was predicted by simulating a 40% reduction in fishing mortality; initial losses to the fishery were more than com- pensated by large gains after a few years. In the low salinity zone, where the harvestable stock has been maintained largely by relay- ing seed oysters, recruitment rates are too low to support a sig- nificant population increase, but stocks in the medium and high salinity zones appear to have potential for recovery within 10-20 years. The model is sensitive to mortality and recruitment rates, but not to carrying capacity, which is much larger than current stock sizes. Measures of uncertainty for model predictions include (1) confidence limits for mean predicted trends, and (2) percent- ages of iterative simulations that satisfy specified criteria. NutiDiial Shellfisheries Assoeialion. New Orleans. Louisiana Ahslmcts. 2(103 Annual Meeting. April 13-17. 2003 337 DEVELOPMENT OF BIOMARKERS FOR PERKINSUS MARINUS RESISTANCE IN THE EASTERN OYSTER (CRASSOSTREA VIRGIMCA). Stephen L. Kaattari and Christopher Earnhart. Department ol Environmental and Aquatic Animal Health. Virginia Institute of Marine Science, Col- lege of William and Mary. Gloucester Ponit. VA 23062. The development of biomarkers for the determination of Per- kin.'iiis mariiuis resistance in the eastern oyster would be of great utility to the oyster industry and would also serve as an important tool in the study of disease pathogenesis. To achieve such a goal we have capitalized on the observation of the ability o't P. inaiinus cells to respond in a specific manner to extracts of susceptible oyster tissue. Generally co-incubation of P. inaniuis with host tissue extracts can elicit a variety of effects including altered cel- lular differentiation, protease expression, growth rates, infectivity. and parasite lethality. The application of this analysis to stock assessment and deployment decisions, as well as their use in the selection of future oyster broodstock could provide a needed com- petitive edge to the American oyster industry. Further, investiga- tion in this arena should yield useful models for the analysis of the developmental process of oyster protozoan parasites. EVALUATION OF COMMERCIAL POST HARVEST TREATMENTS FOR CONTROL OF VIBRIO VULNIFICUS IN OYSTERS. Marilyn B. Kilgen*. Department of Biological Sciences, Nicholls State University, Thibodaux, LA 70310. Post harvest treatments of freezing, low dose ionizing irradia- tion, and hydrostatic high pressure (HHP) were commercially tested in collaboration with oyster industry members from the gulf and east coasts. Six vinegar-based oyster marinades were also developed in collaboration with the NSU Chef John Folse Culinary Instiiiiie. All reduced Vihric vKliiificits levels from 240,000 MPN/g to non-detectable levels (<3MPN/g) after 24 hours of marinating at 35F. and one received the highest sensory score from 1,116 tasters (80%) at the Louisiana Boat Show. Commercial cryogenic (liquid C02) freezing of half-shell oysters reduced V. viilnificii.s levels from 460,0(10 MPN/g to 0.74 MPN/g by 6 weeks post freezing. Commercial ionizing irradiation with Co60 reduced levels of Vihiid Yubuficus in live shellstock oysters from 46().0()() MPN/g to <0.3 MPN/g at 1.0 kilogray (KGy). In recent studies, live oysters were treated with hydrostatic high pressure for the first time. In commercial applications, 35,000 psi for 3 minutes at 70F was determined to be most economically feasible and was validated to reduce V. vulnificus from > 100.000 to <3 MPN/g. It was also initially discovered in these studies that oyster adductor muscle protems were denatured at the shell attachment resulting in me- chanical shucking of the oyster. THE BLUE CRAB FISHERY OF THE HUDSON RIVER ES- TUARY. Gregg Kenney*. Andrew Kahnle, Kathy Hattala, and Steven H. Jury. 21 South Putt Corners Road New Paltz, NY 12561. Despite its economic and recreational importance, there has been relatively little systematic inquiry into blue crab {Callinectes sapidus) abundance, distribution, and habitat utilization in the Hudson River Estuary. Blue crab abundance is generally consid- ered to have increased in this system as indicated by expanding recreational and commercial fisheries. The New York State De- partment of Environmental Conservation has implemented a pro- gram to investigate the extent of the commercial fishery and sea- sonal movements of blue crabs in the estuary. The commercial fishery catch was monitored during the 2000. 2001 and 2002 crab- bing seasons and fishery independent sampling was conducted weekly throughout the 2002 season. Catch per unit effort was found to fluctuate temporally and spatially in a manner similar to that found in other temperate estuaries. The relationship between blue crab abundance and changes in temperature and salinity is presently being analyzed. This project will provide data that can be used to monitor changes in relative abundance and distribution of blue crabs in the Hudson River to fulfill the goal of maintaining a sustainable fishery. POLINICES PULCHELLUS: THE JAMES DEAN OF GAS- TROPODS: LIVING FAST, DYING YOUNG Peter Kingsley- Smith*. VIMS P.O. Box 1346 Gloucester Point. VA 23062. The gastropod. Poliniccs puklwllus. is patchily distributed on subtidal muddy sand in Red Wharf Bay. Wales, LIK. Competent pediveligers metamorphosed in response to sediment collected from the adult habitat, such that the adult distribution may be explained by preferential larval settlement. Polinices pulcheltus densities were significantly higher in summer than in winter, which it is proposed arose from mating aggregations. Small indi- viduals (4-5 mm) were present throughout the year indicating an extended period of low-level recruitment, which was reflected in the year-round production of egg collars in the laboratory. Larger females had higher fecundities than smaller females, however, smaller females continued to lay egg collars later in the year. Small females (4-5.9 mm) grew rapidly during the warm, summer months (April to August), attained sexual maturity at 8-9.9 mm and began laying egg collars in mid-Septeinber. The relationship between shell length and statolith diameter was determined for newly hatched larvae through to fully-grown adults ( 16 mm). Es- timates of mean shell length at the formation of the first and second prominent rings supported the conclusion that prominent statolith rings are formed annually. Polinices putchellus reaches its maximum si/c in 2-3 years. 338 Abstracis. 2003 Annual Meeting. April 13-17, 2003 National Shellfisheries Association. New Orleans. Louisiana OBSERVATIONS ON THE UNUSUAL ABUNDANCE OF TROPICAL CALLINECTES SPECIES IN THE SOUTH AT- LANTIC BIGHT IN FALL 2002. AND REMARKS ON THE NON-INDIGENOUS CHARYBDIS HELLERII. David M. Knott*, Elizabeth L. Wenner. and Susan L. Thornton, South- eastern Regional Taxonomic Center at the Marine Resources Re- search Institute, South Carolina Department of Natural Resources. Charleston. SC 29412. Tropical species of Callinecles typically appear in the South Atlantic Bight only occasionally, and then usually only in isolated occurrences. In fall 2002. however, commercial fishermen near Charleston reported the capture of mature C. exasperatus and C. bocourti in abundances great enough to warrant inquiry about their identity and the legality of selling them. Although quantitative estimates are not available (landings reports do not include species composition), we believe that these species were fairly common and widespread in the vicinity, beginning in mid-October. A single specimen of C. lan'atus was also collected by SCDNR staff in mid-November, at about the same time that the last C. bocourti was seen. The more recent collection of moribund C. exasperatus on December 10. after water temperatures had dropped below 1 l°C, indicates that these species may be unable to survive typical winter condition in the area. Possible explanations for this unusual event will be discussed. Additional remarks will address the inva- sive portunid Charybdis hellerii in the SAB. and the original report of its occurrence in the western Atlantic will be updated. A SIMULATION MODEL OF POPULATION GROWTH OF HARD CLAMS (MERCENARIA MERCENARIA). II. EF- FECTS OF FISHING. Kraeuter*, Haskin Shellfish Research Laboratory Rutgers University 6959 Miller Axenue Port Norris. NJ 08349; Powell. Hofmann, Klinck. Grizzle, Bricelj and Buck- ner. A physiologically-based model that simulates hard clam, Mer- cenaria mercenaria. growth in response to environmental condi- tions of temperature, salinity and food supply has been developed. We are applying this base model to evaluate the effects of various fishing scenarios on Great South Bay. New York clam popula- tions. Comparison of fishery-independent samples with landings suggests the population was heavily over fished in the late 1970 until at least the mid I980's. Base simulations illustrate the effect of changing salinity and food environments. The spawner/recruit relationship is based on limited data so the effects of variation in this parameter have been evaluated. Fishing simulations evaluate the effects of proportional fishing (all marketable sizes of clams are removed in proportion to their abundance) at various percent- ages of removal. In addition, exclusive removal of various per- centages of commercial size categories; littleneck, topneck. cher- rystone or chowder is explored. Finally, population recovery rates are evaluated under scenarios of a total fishing ban or limited percentage removal. In general, simulations indicate recovery time is on the order of a decade or more, and fishing proportionally or on littlenecks at greater than 25 to 35% of adult standing stock will decrease fishing yields and clam populations. GENOMIC SIGNATURE TAGS: A NOVEL METHOD FOR GENOMIC PROFILING WITH APPLICABILITY TO SHELLFISHERIES RESEARCH. Maureen K. Krause*. Dept. of Biology. Hofstra University. Hempstead. NY 11549; John J. Dunn. Daniel van der Leiie, Sean McCorkle. Biology Dept.. Brookhaven National Laboratory. Upton. NY 1 1973. Genomic Signature Tags (GSTs) are the products of a new high-throughput, direct sequence-based approach for characteriz- ing genomes that does not rely on a priori knowledge of the genome. Our technique produces large numbers of positionally defined tag sequences that can. in principle, provide limited rep- resentation of all the DNA molecules in a sample. A GST analysis of the 4.7 Mb Yersinia pestis EV766 genome validates that the GST technique provides a route to obtaining numerous sequence tags that can be used to identify the DNA source. Additionally, our data show that the presence or absence of particular tags can be used to characterize intraspecific genetic variability. One exciting extension of GST analysis, ribosomal GSTs. shows tremendous potential for analyzing microbial communities, including those that may be associated with shellfish disease. Overall knowledge of microbial cotnmunities associated with diseased versus non- diseased shellfish remains poor due to constraints of culturability and microscopy. Ribosomal GST profiles have the potential to not only determine what microbial species are present, but their rela- tive abundance, as well. The power of our approach is that it is both qualitative and quantitative, and can directly provide se- quence information without electrophoretic isolation of amplicons. SPATIAL AND TEMPORAL VARIATION IN OYSTER FIT- NESS IN SAN ANTONIO BAY, TEXAS, 1998-2002. D. Kree- ger*. R. Thomas, H. Hertler. and D. Raksany. Patrick Center for Environmental Research. Academy of Natural Sciences. 1 900 Ben Franklin Parkway. Philadelphia. PA 19103. Adult oysters {Crassostrea virginica) were sampled eleven times between October 1998 and May 2002 from four locations in San Antonio Bay to quantify spatial and temporal variability in body size, condition index, and tissue biochemical composition. All measures of oyster fitness varied spatially, seasonally and among years. Seasonal differences were consistent with expected norms for healthy adults that undergo an annual reproductive cycle (fall/winter conditioning: winter/spring gametogenesis and spawn- ing). This pattern was not observed every year at every location, however, and spatial and inter-annual variability interacted strongly. Spatial variation was greatest along an axis extending from the upper to lower bay, and inter-annual differences were greatest at upper bay locations. Seaward oysters were consistently Natioiuil Shellfisheries Association. New Orleans. Louisiana Absimcis. 2003 Annual Meeting. April 13-17. 2003 339 fit. hut ovster fitness in the upper hay varied widels hetvseen drv and wet years. Follov\ing major floods, upper bay oysters had a smaller size (or were inorhid) and demonstrated a subdued sea- sonal conditioning cycle compared to seaward oysters: whereas, in drier years upper bay oysters were largest and attained the highest condition of all locations. The stable oyster beds in the lower bay appear to serve as critical broodstock that provide larvae to re- plenish upper bay stocks follov\ing major disturbance events. RECONSTRUCTING THE GROWTH OF HARD CLAMS. MERCENARIA MERCENARIA, UNDER BROWN TIDE CONDITIONS. Cathy A. Laetz* and Robert C. Cerrato. 4.^01 Greenwood Ave N Apt # 102 Seattle WA 98103. Hard clams have been an important resource in Great South Bay. New York for decades despite severe population declines. One suspected cause of declines in recent years is brown tides, or blooms of the phytoplankton Aureococcus anophagefferens which have been found to cause a slowing or cessation of feeding activity in various shellfish. Growth in hard clams planted in Great South Bay for one year during a brown tide bloom v\ as similar to growth in clams measured in years prior to brown tides. Similarly, ar- chived shells from the Town of [slip's annual shellfish surveys showed comparable growth rates between brown tide and pre- brown tide years. Rapid shell growth was observed in the spring and fall, whereas no growth occurred when water temperatures fell below 6"C. Although there was no relationship between brown tide concentration and clam growth, a strong relationship was ob- served with temperature, which accounted for65'A of the variation in shell growth rate. In contrast to other shellfish, brown tide does not appear to have as great a negative effect on the growth of hard clams in Great South Bay. possibly due to acclimation, growth compensation, or population selection over time. "bad"). Their immunological status was compared after four months in the three sites. Another comparison was performed after 13. 15. and 17 months but only in one site. Concomitantly, im- munological parameters of triploTdes and diploide oysters were followed during summer in Charentes. Significant differences were measured between good and bad families but were less marked during the year 2. Triploi'des and diploides presented clear differences. To discuss the possible genetic transmission of im- mune parameters, status of 8 divergent families from crossed good or bad families was studied. OPTIMIZATION OF SPERM CRYOPRESERVATION FOR THE PACIFIC OYSTER CRASSOSTREA GIGAS: EVALUA- TION OF COOLING RATE Paul Lang* and Chris Langdon, Coastal Oregon Marine E.xperiment Station. Hatfield Marine Sci- ence Center. Oregon State University. Newport. Oregon. 97365. Sperm suspension with a concentration of 109^ dimethyl sulf- oxide was prepared from calcium-free Hanks' balanced salt solu- tion (-800 mOsmol/kg) and sperm of five Pacific oysters (Cras- sostrea giges). Plastic straws (2.5-mL) were filled with 2 mL suspension, placed in a chamber previously cooled to either -30 °C or -70 °C. and plunged into liquid nitrogen (-196 °C) when inter- nal straw temperature fell within ~2 C of the chamber temperature (7 min at -30°C. 4 min at -70"Cl. Straws were thawed in a water bath (70 "C) for 30 sec. Eggs of females (n = 5) were fertilized using thawed or fresh (control) sperm at equal sperm-to-egg ratios (20:1 ). and incubated in lO-mL tubes. Fertilization (the percentage of eggs to have reached the 4-cell stage) was 22% ± 9% for eggs fertilized with sperm cooled to -30°C. 51'7r ± SVc for -70 °C. and 57 ± 49c for fresh sperm. Larval development (the percentage of initially fertilized eggs to have reached D-stage) was 29% ± 10% for eggs fertilized with sperm cooled to -30°C. 62% + 13% for -70"C. and 72% ± 5% for fresh sperm. IMMUNOLOGICAL STATUS OF SELECTED CRASSOS- TREA GIGAS FAMILIES AND DESCENDANTS, REARED IN DIFFERENT ENVIRONMENTAL CONDITIONS. Chris- tophe Lambert*. Laboratoire des sciences de fenvironnement marin (LEMAR) Institut Universitaire Europeen de la Mer (lUEM) Universite de Bretagne Occidentale (UBO) Place Coper- nic. technopole Brest Iroise 29280 Plouzane. FRANCE: PhHippe Soudant. Gwenaelle Choquet, Christine Paillard. Stephane Frouel. Lionel Degremont. Maryse Delaporte. Jeanne .Moal. Pierre Boudry. Patrick Soletchnick. Michel Ropert. F^douard Bedier, Tristan Renault. Beatrice Gagnieres Arnaud Huvet and Jean-Francois Saniain. Defense mechanisms variability in Crassostrea gigas is sus- pected to result from genetic factors. In the French program MO- REST, bi-parental families, obtained from a nested half-sib cross- ing design, were reared four months in three culture sites. Six families were selected on their survival performance ("good" and FAMILY-BASED SELECTION IMPROVES YIELDS OF PACIFIC OYSTERS. CRASSOSTREA GIGAS. Chris Lang- don*. Sean Matson, John Brake. F'ord Evans. Coastal Oregon Marine Experiment Station and Dept. Fisheries and Wildlife. Or- egon State University. Newport. Oregon 97365. The Molluscan Broodstock Program (MBP) was established in 1 995 to improve yields of Pacific oysters on the West coast, U.S., through family-based selection. Parental families (PI ) in three co- horts of about 60 families each were selected based on superior live weight and meat yields at harvest. Live weight yields of prog- eny (Fl) from crossing PI selected families were significantly greater than those of non-selected control families in four out of seven trials (ANOVA, p<0.001). resulting in an average gain of 9.5% after one generation of selection. ANOVA indicated a sig- nificant (P<0.01) genotype by environment interaction effect on yields for families planted at both inter-tidal and sub-tidal sites. 340 Abstracts. 2003 Annual Meeting. April 13-17, 2003 National Shcllt'isheries Association, New Orleans. Louisiana Nonetheless, it was possible to identify four to six "generalist" families that were among the top ten families at both sites. Further evaluation is needed to determine if the best strategy to improve oyster yields will be to select "generalist" families or whether it will be more effective to develop site-specific lines instead. M. inerceiniriii abundance differed and was nonadditive. Sediment and faunal effects in shell hash were not different, although there was some indication the sediment effect may be greater. In sand and large shell pieces, alternative prey availability may be more important for small M. ineirenaiia survival than physical refuge from predation. AN EVALUATION OF SEA SCALLOP CLOSED AREA BOUNDARIES IN THE MID-ATLANTIC J. David Lange. Jr., William D. DuPaul*. and David B. Rudders. VIMS PC Bo.\ 1346 Gloucester Point, VA 23062-1346. A formal area management strategy for the U.S. sea scallop fishery is being developed under Amendment 10; Sea Scallop Fishery Management Plan. Area closures impacting the sea scallop fishery occurred in 1994 on Georges Bank to protect groundfish resources. Also, in 1998, area closures in the mid-Atlantic (Hudson Canyon and Virginia Beach) were enacted to protect concentra- tions of pre-recruit scallops. This study determined if scallop abun- dance was reflective of closed area boundaries designated by co- ordinates on navigational charts. Data was collected among two post-closure stock abundance surveys. A total of 329 standard survey tows were conducted both inside and outside the closed areas. Survey data were evaluated and results indicate that the use of electronic vessel monitoring systems to track fishing activity can be an effective tool in the enforcement of area management strategies. The effective bound- ary is described as the location at which the scallop population differed as a result of an absence of fishing mortality due to the protection provided by the area closures. THE ROLE SUBSTRATE CHARACTERISTICS HAVE IN ALTERING THE BEHAVIOR, GROWTH AND SURVIVAL OF JUVENILE (POST-SETTLEMENT) MERCENARIA MERCENARIA. Amy A. Larson*, and Robert M. Cerrato, De- partment of Biology San Diego State University 5300 Campanile Drive San Diego, California 92182-4614. Indirect effects can be the primary structuring mechanism in soft-sediment communities, but can be overlooked in experiments that do not test for effects at appropriate levels of habitat com- plexity. Interactions between physical factors (azoic sediments) and biotic factors (faunal communities from the different sub- strates) on growth and predation of small Mercenaria mercenaiia were tested in different habitat types: sand, shell hash, large pieces of shell and a control with no substrate. In the sand, competition between small M. mercenaria and infauna reduced growth of M. mercenaria. Shell hash and the associated fauna had no effect on growth. On large pieces of shell, both competition and effects of the substrate were important, and the combined effect of the two was additive, resulting in the slowest growth rate overall. Preda- tion rates were approximately equivalent in the different habitat types, but the relative importance of physical and biotic factors on ONE MAN'S DREAM: AMERICAN CULTURED PEARLS GIna Latendresse*, American Pearl Company. 807 Watts Lane #B. Nashville, TN 37209. The late John Latendresse was the visionary behind pearl cul- ture in the United States. His forty-five year journey from local fisherman, entrepreneur, shell exporter, pearl importer and finally to the originator of the American cultured pearl walks us through the many facts of his life and details of his business success and failures. His venture into pearl culture started with a challenge from a Japanese colleague. Later he would be known as an evan- gelist for pearl culture in the United States, To accompany this presentation will be a display of exceptional American natural pearls and jewelry designs with American cultured pearls, to in- clude coin. bar. triangle, loaf, cabochon, teardrop .ind round. ZOOPLANKTON INGESTION BY BIVALVES— MORE FOOD FOR THOUGHT. Clare Lehane* and John Davenport. Dept. of Zoology & Animal Ecology, University College Cork, Lee Makings. Prospect Row. Cork. Ireland. Bi\al\es have generally been thought of as herbivorous, gain- ing nutrition from phytoplankton. However, since the 19th century researchers ha\e reported finding zooplankton species in the stom- achs and excreta of bivalves. A study was carried out to determine if four species of bivalves, namely blue mussels, common cockles, queen scallops and horse mussels could ingest zooplankton. Though a series of suspended cage experiments and sampling bi- valves from their natural habitats, it was determined that all four species ingested zooplankton representative of that found in the water column on the days of experiment. A second experiment dealt with determining if a fabricated bed of blue mussels could deplete zooplankton in overflowing water. It was found that zoo- plankton numbers were lowest in the middle of the bed. signifying that mussels may have the ability to affect zooplankton popula- tions to some degree. SPECIES-SPECIFIC VARIATION IN THERMAL TOLER- ANCE DURING LARVAL DEVELOPMENT IN BLUE MUS- SELS. MYTILUS SPP. Susan J. Limbeck*, and Paul D. Raw- son, School of Marine Sciences, University of Maine. Orono. ME 04469. Two species of blue mussel, Mytihis eclidis and Mytilits tros- siihis. are sympatric throughout much of the Canadian Maritime Provinces and into the Gulf of Maine. While the distribution of A/. National Shellfisheries Association. New Orleans. Louisiana Ahslrmls. 2003 Annual Meeting. April 13-17. 2003 341 eihilis extends south to the Mid-Atlantic, that of M. trasuilKs ends abruptly in the Gull of Maine. We hypothesized that species- specific variation in larval thermal tolerance influences differences in distribution. Previously, we found that M. lro\siihi\ experienced higher mortality than M. cJiilis when larvae were held at tempera- tures above 1 5 C throughout development. Our current experiment examines whether species-specific sensitivity to elevated tempera- tures is dependent upon larval age. Growth and mortality were monitored for larvae exposed to three experimental temperatures at three time points during development. Preliminary analysis sug- gests that M. trosMiliis larvae experience higher mortality at 1 S^C and 22°C but the effect is dependent on larval age. The importance of these findings with respect to patterns of larval dispersal and coastal water temperatures in the Gulf of Maine will be discussed. VARIATIONS IN THE SIZE STRUCTURE OF LOBSTER (HOMARUS AMEKICANUS) POPULATIONS WITHIN THE OFFSHORE FISHERY Susan A. Little \ Winsor H. Watson. HI. and Rudman Hall. Dept of Zoology University of New Hampshire Durham. NH 03824. The offshore lobster fishery is cunently managed as one unit (Area 3), although it extends from New Jersey to Maine. To de- termine if there were differences in the size structure of lobster populations within Area 3. we examined 36,815 lobsters from three regions: North: Georges Bank and offshore Gulf of Maine; Middle; offshore Massachusetts to south of Cape Cod. and; South: offshore Rhode Island to New Jersey. Each region included mid- shelf areas (30-40 fathoms) out to continental shelf canyons (120- 150 fathoms). In the North 2% of the catch was sub-legal, com- pared to 409r in the Middle and 29Vc in the South. This pattern was reflected in the average size in each region: North = 110mm CL (carapace length); Middle = 89mm and; South = 91 mm. There were also more lobsters > 100mm CL in the North (37%) than in the Middle (4%) and South (3%). and. conversely, more lobsters <65mm CL in the Middle (3%) and South (5%). than in the North (5 cm were common in the region and had begun to colonize the tubes of Riftia pachyptila. concomitant with declining concentrations of H2S in the venting diffuse flow fluids. Over the next 5-6 years, the abun- dance of mussels increased dramatically until most of the existing communities previously dominated (in biomass) by tubeworms were now dominated by extensive populations of mussels. SUSPENSION-FEEDING BIVALVES. MARINE AGGRE- GATES AND THE ACCESSIBILITY OF SMALL PAR- TICLES. M. Maille Lyons*, J. Evan Ward, Department of Ma- rine Sciences. University of Connecticut. Groton CT 06340. Marine aggregates (i.e., marine snow, organic aggregates. floes, and detritus) are common in coastal waters where large populations of bivalves dominate benthic communities. Suspen- sion-feeding bivalves actively pump seawater through their pallial cavities and extract particles for food. Retention efficiencies of small, freely suspended, particles (~1 p,m) are generally low. Small particles are often concentrated within aggregates. The retention efficiency of the larger, amorphous aggregates (>10 |j,m) should be 100%. However, the percentage of aggregates ingested, compared to the percent rejected as pseudofeces, is not known. The focus of this study is to determine the fate of the particles embedded within aggregates. Determining the ingestion rate of aggregates is an important step in assessing the role aggregates play in trophic interactions between water column microbiota and benthic bi- valves. To test the hypothesis that the presence of marine aggre- gates increases the accessibility of small, otherwise poorly re- tained, particles, experiments were designed using artificial aggre- gates generated on a rolling table. Fluorescent beads ( I |xm and 10 |xm) were incorporated into aggregates in order to track the fate of embedded particles. The percentage of beads in the bivalves gut. evaluated by direct counts, was compared to controls (fluorescent beads not incorporated into aggregates). Preliminary results indi- cate species specific differences, increasing the accessibility of small particles for the sea scallop (Placopecten inagellanicus), but showing no significant increase in accessibility for the marsh mus- sel (Geukensia demissa). SHELLFISH RESTORATION: IT'S NOT JUST BIOLOGY THAT MATTERS. Sandra L. Macfarlane. Coastal Resource Specialists P O Box 1 164 Orleans. MA 02653. Shellfish restoration projects have been practiced in most coastal states for years. But as stocks decline, water quality de- grades and population pressures increase throughout the coastal zone, restoration projects have become more urgent in many sec- tors. While biological factors such as predator/prey relationships and physical factors such as current and sedimentation are impor- tant for the success of a restoration program, other less tangible factors can be equally important. Increasingly, the success of a project may depend on a holistic approach of embayment man- agement that often includes land use issues, topics such as storm- water runoff, nutrient loading, proliferation of docks or erosion control structures and other human use impacts, issues that may not considered when planning a restoration effort. As coastal area population increases, land use and human marine use issues may have greater influence on the success of the restoration effort than traditional biological and physical factors. And yet. as land uses continue to degrade water quality, shellfish restoration projects are being considered as a counter measure, using the natural filtering capacity of shellfish to boost water quality. This paper discusses these issues as challenges to shellfish restoration efforts. EVIDENCE FOR NATURAL SELECTION FOR RESIS- TANCE TO PSP TOXINS IN EARLY LIFE HISTORY STAGES OF THE SOFTSHELL CLAM. MYA ARENARIA. S. MacQuarrie*. V. Monica Bricelj. 1411 Oxford St. Halifax. Nova Scotia. Canada. B3H 3Z1. Our prior research has demonstrated that sensitivity to paralytic shellfish poisoning (PSP) toxins, measured by behavioral and physiological indices, varies significantly among Mya arenaria populations with differing toxin exposure histories. Populations from PSP-affected areas are predominantly resistant whereas naive populations are dominated by sensitive individuals. An extensive survey of M. arenaria populations supports this correlation over a wide geographical range. This study identifies the life history .stages susceptible to selective pressures and demonstrates the po- tential for strong selection. Effects of toxin exposure were deter- mined for larvae and spat from a population previously character- ized as sensitive. Veliger larvae exposed to bloom levels of a highly toxic Alexandrium tamarense strain (PR18b) in a mixture with non-toxic algae showed no increased mortality relative to controls. However, spat (3.5mm) exposed to a monospecific sus- pension of PR 1 8b suffered 95% mortality after 1-week exposure, resulting in a population dominated by resistant clams. Video ob- servations suggest that anoxia of the pallial cavity may be respon- sible for mortalities. Ingestion of toxic cells is necessary to induce mortality and a single bloom of sufficient toxicity is capable of selecting for resistance at these stages. Results will be discussed in context of ecological relevance and fitness consequences. National Shellt'isheries Association, New Orleans, Louisiana Abstiuas. 2003 Annual Meeting. April 13-17. 2003 343 OPTIMIZING OYSTER PRODUCTIVITY IN CARAQUET BAY: COORDINATING RESTORATION AND AQUACUL- TURE. J. F. Mallet , and T. Landry. DFO. P.O. Box 5030. Moncton NB. EIC 9B6. Caraquet Bay represents the most northern location with a sus- tainable oyster (Crassostrea virginica) population. In recent years, a decrease in the productivity of oysters from the natural beds has generated interest in restoration projects. To determine the benefits of restoration activities, information on the distribution, abundance and population structure of oysters was collected in 1999. Over 60% of the oysters found were pre-recruits (35-75mm). These oysters were mainly found in the northern portion of the bed, which is locally renowned for its "stunted oysters". In 2001, "stunted" oysters along with control oysters were transfened to four stations and monitored for growth. Results to date show that growth oysters are associated with site and bottom conditions. In 2002, samples of "stunted" oysters were placed at three stations in various holding situations to evaluate the effects of vertical posi- tioning in the water column and tidal position. The results from this experiment revealed that oyster productivity varies in relation to their geographical location. They will provide key information for the oyster fishers and aquaculturists to develop management strateaies. ROSEIMARINA CRASSOSTREAE (GEN. NOV.. SP. NOV.) ASSOCIATED WITH JOD-SIGNS IN THE ABSENCE OF SIGNIFICANT MORTALITIES, AND FIRST ISOLATION FROM A NEW YORK EPIZOOTIC Aaron P. Maloy*. 5735 Hitchner Hall University of Maine Orono ME. 04469: and Katherine J. Boettcher. The alpha-proleobacterium Roseiiiiariiio crassostreae (gen. nov., sp. nov.) has. to date, been isolated exclusively from episodes of juvenile oyster disease (JOD) in .Maine. With few exceptions, isolates have been recovered from animals that probably would have died from the disease. Mortalities have been reproduced by experimental exposure to the bacterium, although without typical JOD-signs (e.g. conchiolin). Herein we describe induction of con- chiolin in oysters challenged with R. crassostreae. Further, we report a 907f correlation between conchiolin and colonization by Roseimarina in a natural (but unusual) occurtence of JOD. The affected animals were from Maine's Damariscotta River where cumulative mortalities were < 5% in 2002 (down from 50% in 2001 ). Thus, these bacteria were isolated in the absence of signifi- cant mortalities. In fact, most of the 9% of animals with conchiolin appeared otherwise healthy. Finally, we document the first isola- tion of R. crassostreae from JOD outside of Maine. Analyses of the 16S-23S rDNA internal transcribed spacer region revealed that isolates from a 2002 New York epizootic were the same genotype (GTl) as those from Maine epizootics in 1997 and 1998. For unknown reasons, a slightly different genotype (GT2) appeared in Maine in 2000. and thereafter replaced GTl as the etiological acent here. FINDING THE WHEAT IN THE CHAFF— OYSTER LAR- VAL FEEDING IN TURBID. LOW SALINITY CONDI- TIONS. Roger Mann and Peter Kingsley-Sniith. VIMS P.O. Box 1346 Gloucester Point, VA 23062. Oyster restoration efforts in the Middle Atlantic States focus on a combination of benthic habitat refurbishment and brood stock supplementation, predominantly in low salinity sanctuaries from endemic disease. Central to this approach is the assumption that efforts increase local recruitment, yet we are ignorant of the in- fluence of low salinity, elevated turbidity, and limited food avail- ability on the survival and growth of oyster larvae. We suggest that in high turbidity regions available food is essentially diluted by indigestible inorganic material, and larvae may be food limited despite an apparently adequate absolute concentration of food be- cause the relative food concentration is low. If this is the case then watershed management practices emphasizing nutrient reduction policies in excess of concomitant sediment load reduction may serve to reduce larval survival in receiving water bodies, and com- promise restoration efforts. We offer numerical estimates of the impact of elevated turbidities on oyster restoration through de- creased lar\al survival. We then investigate larval feeding behav- ior, as a proxy for overall larval viability, under both controlled salinity-turbidity conditions in the laboratory, and along a salinity- turbidity cline in the York-Mattaponi river systems of the Chesa- peake Bay. CHARACTERIZATION OF SUMMER MORTALITIES OF CRASSOSTREA GIGAS OYSTER IN RELATION TO PHYSIOLOGICAL PARAMETERS. M. Mathieu*. B. Dubois, K. Costil. C. Heude. A. Huvet, K. Kellner, S. Pouvreau. Physi- ologic Ecophysiologie des MoUusques Marins IFREMER Univer- site de Caen. 14032 Caen Cedex, France. Pacific oysters are characterized by high fecundity, and follow a seasonal breeding pattern beginning in autumn with gamete maturation in spring and early summer. Summer mortalities occur during spawning period, but according to the environmental con- ditions gametes are spaw ned or reabsorbed. In Normandy, which is the northern more oyster cultivation area in France, spawning is often partial or absent. Mortalities were observed in both situations but more often when gamete release is delayed. The implication of hemocytes in gamete resorption as in storage tissue restoration was observed. The level of fecundity varies with trophic conditions. Storage material is accumulated in specific cells mainly in autumn and winter, and then mobilized to support gametogenesis. Resorp- tion of gametes contributes to storage tissue development. Incor- poration of metabolites in storage cells is performed by diffusion through cell membrane and by two transport systems sensitive to 344 Abstnicts. 2003 Aniuuil Meeting. April 13-17. 2003 National Shellfisheries Association. New Orieans. Louisiana internal regulation. The storage tissue follows a seasonal cycle regulated by internal factors, with reversion of its metabolism in summer. PERKINSUS MARINUS INFECTION RATES IN SPECIFIC- PATHOGEN-FREE JUVENILE OYSTERS PLANTED IN THE PATUXENT RIVER, MARYLAND. Carol B. MeCol- lough* and Christopher F. Dungan. Sarbanes Cooperative Ox- ford Laboratory. Oxford. MD 2l6.'i4; George R. Abbe and Can- dace A. Morrell. Academy of Natural Sciences Estuarine Re- search Center, St. Leonard, MD 20685. Specific-pathogen-free (SPF) oysters were set and reared in artificial seawater and transferred to four sites in the Patuxent River along a salinity gradient. Three sites were adjacent to natural oyster bars and one, Sandgates, was remote from existing oyster populations. Samples of 30 oysters were assayed at 2 and 4 weeks post-deployment for infection by P. mariuus using an enhanced RFTM whole body burden technique. Assays continued at 4-week intervals. Deployments were made in May and September 2002. From the May deployment. SPF oysters placed at Sandgates, re- mote from existing populations, acquired infections by day 27 (13%' prevalence), as did juveniles deployed at TC (7%) and HP (3%). At all sites oysters acquired infections within 62 days, with prevalences of 10%, 63%, 43%, and 37%^ (TC - HP). By 91days post-deployment all sites, with the exception of TC, had infection prevalences greater than 90%, and these elevated prevalences con- tinued through 127 days. At 9 1 days TC prevalence remained low at 10%, but by 127 days it also increased, to 53%. In October prevalences declined at TC. GA. and SG (154 days), and all re- mained above zero into mid-November. SPF juveniles deployed in late September acquired P. mariuus infections by 27 days at all sites, however prevalences were low and declined at 55 days, with infections detected then only at HP. IS MERCENARIA MERCENARIA A HOST FOR PERKIN- SUS SPECIES? Ayana McCoy*, Shirley Baker, Ruth Francis- Floyd, and Anita Wright, University of Florida Department of Fisheries and Aquatic Science 7922 NW 71st St Gainesville. Fl 32653. Perkinsus marimts is an endoparasitic protistan that infects the Eastern oyster. Crassostrea virginica. This parasite has caused mass mortalities of oysters along the Atlantic and Gulf coasts. The commercially important Mercenaria mercenaria is cultured in ar- eas naturally populated by C virginica. Whether the hard clam, M. mercenaria, is susceptible to Perkinsus infection or serves as an intermediate host has not been well studied. Therefore, the objec- tives for this study were ( 1 ) to examine the diversity of Perkinsus species associated with A7. mcrccnarici and C. virginica in the environment, and (2) to experimentally test the susceptibility of hard clams to P. marinus and P. andrewsi infections. M. merce- naria and C. virginica were collected from the Cedar Key area on the Gulf Coast of Florida. Both species-specific PCR assays and standard Ray's Fluid Thioglycollate Media assays were u.sed to identify associated parasites and determine levels of infection. Laboratory studies are in progress to determine virulence of Per- kinsus species in M. mercenaria. This project should help to pro- \ ide an indication of virulence potential of Perkinsus species for the hard clams on Florida's Gulf Coast and the possible threat of these parasites to the rapidly growing aquaculture industry in the region. RECOMMENDATIONS TO OYSTER HARVESTERS ON REMOVING HOOKED MUSSELS, ISCHADIUAf RECUR- VUM. Earl J. Melancon. Jr.*. Biology Department, Nicholls State University. Thibodaux, La 70310; Dale Diaz, Mississippi Department of Marine Resources. Biloxi. Miss. 39530; Badiollah Asrabadi. Math Department, Nicholls State University. Our results indicate that high salinity as a physiological factor to kill mussels is of minimal value to removal success. Predation, often by the blue crab, is the driving force on the removal of mussels from oysters. In addition, the physical process of harvest- ing with a dredge, using water cannons to move oysters on deck and then again to plant overboard can resulted in as much as a 33-38% direct mussel mortality; in turn, the dead and dying mus- sels attract predators. Planting to down-bay (high salinity) habitats will remove mussels within a short period of time if predators are present; perhaps in as little as week in summer water temperatures (25-30°C). Use of water cannon to move oysters on deck sup- presses temperatures within the pile and allows relaying during summer months without harming oysters. The spray from the hose, the dripping from the stacked oysters and evaporative cooling all work together to keep air temperatures well below the heat toler- ance of mussels and oysters. Observations also suggest that culti- vation by breaking up oyster clusters may reduce mussel fouling in mid-bay and down-bay sites, but not necessarily at up-bay (low- salinity) sites. A COMPARISON OF CRYOGENIC FREEZING TECH- NIQUES AND THEIR USEFULNESS IN REDUCTION OF VIBRIO VULNIFICUS IN RETAIL OYSTERS D. Mestey and G.E. Rodrick*, University of Florida Dept. Food Science and Human Nutrition, Gainesville, Fl. 3261 1. Freezing the oysters and storing them at freezing temperatures suppress the number of recoverable V. vulnificus from the oyster meat. There are various methods that can be used to achieve a frozen product. In this study the effectiveness of carbon dioxide and nitrogen was analyzed. A comparison of freezing whole oys- ters versus half shell oysters with this two freezing methods was also studied. The oysters were processed using the commercial practices at each of the three seafood-processing plants. The samples were analyzed using the guidelines provided by the FDA Bacterial Ana- National Shellt'isheries Association. New Orleans, Louisiana Absrracls. 2003 Annual Meeting. April 13-17. 2003 345 lytical Manual. An initial sample of fresh unfrozen oysters was analyzed to determine the initial \'ihriii viiliiifkus load followed by analysis of frozen samples at 1.7. 14 and 21 days after storage at -10°C. The study demonstrates that there is lower number of recover- able V. viilnifkiis when C02 is used for freezing than when nitro- gen is used, but the overall decrease in V. vulnificus load in the fresh to frozen product is by 200,000 organisms per gram of oyster meat. There are few organisms recovered by 21 days regardless of the freezina method. USING MOLECULAR GENETIC TECHNIQUES TO AS- SESS OYSTER RESTORATION PROGRAMS AND PRO- JECTS. Coren A. Milbury* and Patrick M. Gaffney. College of Marine Studies. 700 Pilottown Road. Lewes. De 19958. Restoration efforts are becoming essential in managing many of our ecological resources. Equally important are the assessment and monitoring of restoration programs. Recent advances in ge- netic techniques allow for the use of high-throughput and cost effective methods in restoration assessment and monitoring. We have used molecular methods to assess a restoration project by the Maryland Oyster Recovery Partnership and the University of Maryland. Four million Louisiana oysters were planted in the Choptank Ri\er. Maryland. Crassostrea virginica exhibits region- ally diagnostic mitochondrial haplotypes. which provide a means to genetically differentiate Gulf Coast oysters from native oysters. Detection of newly recruited spat possessing the Gulf Coast hap- lotype in the Choptank River confirms the survival and propaga- tion of the outplanted oysters and the contribution of new progeny. A high-throughput, synthesis-by-sequencing technique (Pyrose- quencing) was used to determine the mitochondrial hap- lotypes of spat collected in the Choptank River. Of 4,566 spat analyzed. 94.2% possessed the North Atlantic haplotype. 5.39^ had the South Atlantic haplotype. and 0.1% possessed the Gulf Coast haplotype. The results demonstrate the contribution of the out- planted Louisiana oysters to the resident Choptank River popula- tion, and show that effective monitoring of stock enhancement projects can be achieved with high-throughput molecular genotyp- ing techniques. CREATING SALT MARSHES TO ENHANCE PRODUC- TION OF FISHERY SPECIES Thomas J. Minello* and Lawrence P. Rozas, National Marine Fisheries Service. Southeast Fisheries Science Center, Galveston Laboratory, 4700 Avenue U. Galveston. TX 77551. U.S.A. Salt marshes in the northern Gulf of Mexico are valuable nurs- ery habitats for fishery species such as penaeid shrimps and blue crabs. Extensive marsh loss has led to numerous restoration pro- jects in the region, but little design information has been available for optimizing fishery productivity from these created wetlands. We have sampled the small-scale spatial distributions of shrimps and blue crabs in natural and created marsh systems and developed models to 1) estimate populations of these fishery species in marshes of different land- water configurations and 2) simulate population changes in created marshes with different land-water patterns. The amount of vegetation-water interface or edge in salt marshes is an important characteristic that can determine the fish- ery value of these habitats. Marsh creation projects that maximize edge are likely to be most productive for commercially important decapod crustaceans. Terracing and the formation of small marsh islands are two restoration techniques that produce a great amount of marsh edge and should provide productive habitats for penaeid shrimps and blue crabs. GENETIC VARIABILITY IN REPRODUCTION AND SIM- MER MORTALITY IN CRASSOSTREA GIGAS. Jeanne Moal*. Edouard Bedier, Pierre Gildas Fleury, Ainie Langlade, ■^'vette LeCoguic. Lionel Degreniont. Pierre Boudry. Jean Rene Le Coz, Stephane Pouvreau. .Martha Enriquez-Diaz, Christophe Lambert. Philippe Soudant, Jean Francois Sa- main. Ifremer. centre de Brest BP 70 29280 Plouzane. France. Bi-parental families were produced in hatchery and tested in the field in 2001. Two sets of 5 families were constituted, selected on their high (R) and low (S) survival. These two sets were reared in Brittany from March to November 2002. Samplings were per- formed twice a month to obtain data on biometry, survival, repro- ductive cycle, biochemical composition, adenylate energy charge, hemolymph parameters (ions and defense system) and muscle strength. "R" and ■"S" oysters exhibited different reproductive effort and spawning strategy. "R" oysters allocated less energy in gonad than "S" ones and presented a complete spawning at the end of August contrary to the "S" which spawns partially. Mortality started in July when the seawater temperature reached 19"C and affected mainly "S" oysters. Concentrations of Na and CI ions in hemolymph were different for "S" and "R" from May to June. A bacterial increase in hemolymph (R and S) was observed during the same period. The adenylate energy charge was more lowered for "R" than for "S" oysters, just before the spawning event How- ever, other physiological and immunological parameters were similar between the two sets during the phases of maturation and mortality but discriminated groups after the spawning peak. PRELIMINARY PATHOLOGICAL INVESTIGATION OF THE WHITE ABALONE. HALIOTIS SORENSENI. James Moore*. Thea Robbins. Carolyn Friedman. Neal Hooker. Thomas McCormick. Melissa Neunian. Bodega Marine Labo- ratory P. O. Box 247 Bodega Bay CA 94923 USA. Populations of white abalone HalUnis sorenseni. deep water inhabitants, were severely exploited in the 1970s following serial depletion of several other species found in shallower water. This 346 Abslracls. 2003 Annual Meeting, April 13-17. 2003 National Shellt'isheries Association. New Orleans, Louisiana species appears to be nearing extinction and in 2000 became the first marine invertebrate to be listed under the federal Endangered Species Act. Acquiring health information is critical for planning recovery of this species. White abalone broodstock were collected in 1999-2000 prior to federal listing. Deaths of eleven of these animals appeared to be related to collection injuries or water qual- ity problems rather than infectious disease. The etiological agent of withering syndrome (WS-RLP, withering syndrome associated Rickettsiales-like prokaryote), was not detected in any of the dead animals by histology or PCR. Juveniles produced from broodstock were held at 12. 15 and ISC and were exposed to the WS-RLP. Marked losses of and pedal atrophy in animals with severe WS- RLP infections demonstrated that white abalone are susceptible to withering syndrome. As in other abalone species, cool water pro- vided some refuge from WS-RLP pathogenicity. No other signifi- cant pathogens were observed. The susceptibility of white abalone to WS must be considered in the formulation of recovery plans. UTILIZATION OF POST-HARVEST TREATMENT AS A STRATEGY FOR REDUCING VIBRIO VULNIFICUS ILL- NESSES. Ken B. Moore. ISSC 209-2 Dawson Road Columbia SC 29223. Illnesses and deaths associated with the consumption of raw molluscan shellfish continue to be a significant public health con- cern for the Interstate Shellfish Sanitation Conference (ISSC). In 1995, the ISSC highlighted three main approaches for reducing V. vulnificus-Telated illnesses and deaths involving high-risk consum- ers. These included education of "high-risk" groups to avoid raw shellfish, more rapid post-harvest refrigeration of shellfish to pre- vent increases in numbers of the pathogen, and encouraging and promoting shellfish post-harvest treatments to reduce Vibrio vKlnificits to non-detectable levels. The role of post-harvest treat- ment as a strategy to control Vibrio vulnificus has become more significant since the passage of the mandatory Vibrio vubtificus Illness Reduction Plan by the ISSC Voting Delegates in 2000. The establishment of collective illness reduction goals for core states has created a proactive approach for addressing Vibrio vubiificus- related illnesses and deaths. The ISSC remains committed to Vibrio vulnificus illness reduction and is continuing efforts to iden- tify additional effective strateaies. CHARACTERIZATION OF NATURAL KILLER CELL- LIKE ACTIVITY IN THE EASTERN OYSTER, CRASSOS- TREA VIRGINICA. Brenda M. Morsey* and Sylvain De Guise, Department of Pathobiology and Veterinary Science, University of Connecticut, 61 N Eagleville Road, U-89. Storrs, CT 06269, USA. Natural killer (NK) cells are an important part of the innate immune system of mammals. However, little is known about NK- like cell activity in the Eastern Oyster, Crassostrea virginica. NK- like cell activity of oyster hemolymph cells was measured by a flow cytometric assay in which oyster hemocytes were incubated with DiO-labeled K-562 target cells, and propidium iodide to label dead target cells. For every individual oyster tested, higher effec- tor-to-target cell ratios resulted in higher levels of target cell death. Moreover, NK-like activity of individual oysters was further en- hanced by recombinant human interleukin-2. Enhancement of NK- like cell activity by interleukin-2 was more pronounced in pooled oyster hemolymph compared to individual oyster hemolymph samples. Our data demonstrate for the first time the presence of NK-like cell activity in a marine invertebrate. This activity can be enhanced by physiologically relevant concentrations of mamma- lian interleukin-2 which further suggest that some structural and functional homologues of the mammalian innate immune func- tions are conserved in invertebrates such as the oyster. The im- portance of oyster NK-like activity in protection against disease and pathogen control will be assessed. FOOD AVAILABILITY IN A MUSSEL RAFT Jessica Munro* and Carter Newell. Great Eastern Mussel Farms, P.O. Box 141. Tenants Harbor, Maine. 04860. Current speed, phytoplankton concentration, detritus concen- tration, mussel biomass and mussel density are important deter- mining factors in the growth rate of raft cultivated mussels. Peri- odic measurements of flow and food with depth inside and outside mussel rafts are used to determine seasonal and site specific food availability and mussel raft consumption. Field data is collected with Seabird CTD and current meter casts, water sampling, and weighing mussel lines with a crane scale. Seasonal stratification causes vertical variation of food availability to mussel rafts in Maine waters. The depletion of available food is a function of the biomass of a mussel raft and mussel raft hydrodynamics. COMPARING TWO MYA ARENARIA POPULATIONS AS POTENTIAL CANDIDATES FOR SEEDING OPERA- TIONS. Bruno Myrand*, Station Technologique Maricole des Iles-de-la-Madeleine, Cap-aux-Meules, Canada, GOB I BO, Rejean Tremblay, Societe de Developpement de ITndustrie Maricole. Gaspe, Canada, G5X 1T5; Lise Chevarie. Societe de Developpe- ment de ITndustrie Maricole, Cap-aux-Meules, Canada, GOB IBO; Fabrice Pernet, Universite du Quebec a Rimouski-Centre Aqua- cole Marin de Grande-Riviere, Grande-Riviere, Quebec, GOC IVO; and Diego Mantovani, Institut des biomateriaux du Quebec. Universite Laval, GIK 7P4. It is important to identify a source of clams for seeding in lles-de-la-Madeleine. Two populations were examined: Havre- aux-Basques (HB) and Dune-du-Nord (DN). No neoplasia were found. Both populations belong to the same stock and have a low multilocus heterozygosity. Growth was better at DN site for both populations and better for the DN clams at both sites. The HB clams had a very limited growth. These results will be interpreted according to scope for growth measurements. The fragility of the National Shcllfislieries Association. New Orleans. Louisiana Abstracts. 2003 Annual Meeting, April 13-17, 2003 347 shell was higher tor HB elanis. Therefore, the HB clams appear unsuitable tor seedine. PROPAGATION OF FRESHWATER MUSSELS FOR FRESHWATER PEARL PRODUCTION. Richard J. Neves*. Jess W. Jones, William F. Henley, and Rachel A. Main. Fresh- water Mollusk Conservation Center, Virginia Cooperative Fish and Wildlife Research L'nit, Virginia Tech. Blacksburg, VA 24061. The commercial harvest of mussel species suitable for pearl production could provide an incentive to replace wild-caught adults with laboratory-reared juveniles to sustain populations. The Freshwater Mollusk Conservation Center at Virginia Tech was the first facility in the LInited States to begin an annual propagation and release program focused on endangered mussel species. Initial research to identify host fishes, develop production and culture methods, and test culture technology required nearly 10 years of experimentation. Endangered juvenile mussels were released first in 1997. and subsequent annual releases total nearly 370.000 ju- veniles of 10 species. A new facility dedicated to propagation was completed in 2002, with capacity to address commercially har- vested species, as well as those under federal protection. Should the harvest of particular species such as those with colored nacres increase, then culture techniques are now available to replace har- vested specimens with progeny produced from the parental popu- lation. AN EXPERT SYSTEM FOR THE OPTIMIZATION OF SHELLFISH RAFT CULTURE. Carter Newell* and John Ri- chardson. Great Eastern Mussel Farms P.O. Box 141 Tenants Harbor. Maine. An expert system combining computer-based methodologies for determining tidally driven flows, wave heights, flow through shellfish raft systems, and consumption of food by the shellfish with specially designed data collection techniques is being used to improve shellfish production on mussel rafts in Maine. Elements of the expert system are being incorporated into a single computer that operates in a "point and click" manner. A large scale flow model develops tidal flow boundary conditions for the three di- mensional computational fluid dynamics (CFD) raft model, and predicts wave conditions relative to mooring specifications and site risk assessment. The detailed CFD raft model predicts cunent speed and chl a consumption relative to ambient flow speed and direction, shellfish biomass, and density distribution. Field data collection involves flow profiles, wave gauges, CTD casts, sedi- ment traps and feeding chambers. Mussel biomass on culture ropes is monitored using a crane scale. Optimization of production cycles on shellfish rafts involves careful consideration of raft hy- drodynamics, seasonal changes in food availability, and stocking densities. LINKING HARD CLAM {MERCENARIA MERCENARIA) REPRODUCTION TO PHYTOPLANKTON COMMUNITY STRUCTURE: II. PHYTOPLANKTON COMMUNITY STRICTURE AND FOOD COMPOSITION Roger I.E. New- ell*. Horn Point Laboratory UMCES Cambridge. MD 21613. Christopher Gobler. and Stephen T. Tettelbach. Hard clam. Mercenaria mercenar/a. recruitment has declined in some southern bays of Long Island, NY and we hypothesized that this was associated with changes in the phytoplankton com- munity structure and overall patterns of primary production. We collected hard clams over an annual cycle for analysis of repro- ductive condition from five south shore bays of Long Island. Con- currently, ambient water was filtered for analyses of organic car- bon and nitrogen, total and size-fractionated chlorophyll, and mi- croscopic counts for the harmful brown tide picoplankter. Aiireococciis anophagejferens. We found appreciable differences in seston composition that related to the observed differences in hard clam reproductive and tissue condition. Bay Shore and Patchogue had the highest total Chl a levels and organic carbon nitrogen and carbon of any bay. Paradoxically, clams from this location had the lowest condition index and reproductive effort. The size fractionated Chl a data, however, showed that the high levels of organic material at these two locations was mainly con- tributed by cells < 2 \i.m which are too small to be efficiently retained by adult hard clams and hence have no nutritional value. In addition, both Bay Shore and Patchogue had brown tide blooms at cell concentrations that inhibit adult hard clam feeding. We conciude that changes in the floristic composition of the phy- toplankton community in at least some of the Long Island south shore bays is translating into appreciable differences in hard clam condition and ultimately into reducing tcital reproductive effort. COMMERCIAL IMPLEMENTATION OF HIGH PRES- SURE PROCESSING (HPP) FOR PACIFIC OYSTERS. David H. Nisbet*, Nisbet Oyster Co., P.O. Box 338 Bay Center, WA 78527. High Pressure Processing (HPP) was first used commercially on Pacific oysters, Crassostrea gigas by Nisbet Oyster Co.. Inc. a cultivator, processor and packer of Pacific oysters on Willapa Bay in Washington State. Initial pilot scale experimentation was cen- tered on the oyster shucking protocol for pressure and dwell time regimes. Physical material flow proved a major obstacle to resolve in the feed and outfeed of the equipment. .\n engineering study was commissioned to determine real-time throughput capabilities of commercially available HHP equipment. Building design, prod- uct flow and ergonomics were also researched as the company expanded its processing facility. When the commercial high- pressure equipment installation was completed, studies were un- dertaken in collaboration with the Oregon State University Sea- food Laboratory and Seafood Consumer Center. Extended sensory analysis and Vibrio control studies were considered most impor- 348 Ahsnacts. 2003 Aiinuul Meeting. April 13-17, 2003 National Sliellfisheries Association, New Orleans, Louisiana tant, as well as the development of other possible \alue added product candidates. The commercial considerations tor high pres- sure processing included specific end product related studies and building design features including product flow, throughput analy- sis, ergonomics, equipment maintenance, and cleanup. Addition- ally, the physical size of Pacific oysters relative to available hy- drostatic chamber size capabilities constitutes special consider- ations for HPP commercial installations. OPTIMAL PLANTING CONDITIONS FOR MAXIMUM REPRODUCTIVE OUTPUT OF CAGE-PLANTED SCAL- LOPS. ARGOPECTEN IRRADIANS. IN ANCLOTE. FLORIDA. Melanie L. Parker*. William S. Arnold and Dan C. Marelli, Florida Marine Research Institute 100 Eighth A\enue SE St. Petersburg. FL 33701. As part of an ongoing effort to restore bay scallop populations on the west coast of Florida, we compared the growth, survivor- ship and gonadal development of bay scallops planted in cages at \arious densities and planting conditions in the Anclote estuary. To test density effects, scallops were planted in 0.6-in L x 0.6-m W cages, constructed from 12.7-mm-mesh. within a seagrass bed. Densities of 50, 150 and 300 scallops per cage were tested in triplicate. Growth, survivorship, and gonadal development were monitored every six weeks between July 1999 and July 2000. Planting at 150 scallops per cage resulted in the most live scallops available for fall spawning. To test the effect of planting condition. 50 scallops per cage were planted in triplicate in each of four treatment combinations including within and outside a seagrass bed and either directly on the substrate or raised 20 cm above the substrate. Growth and survivorship were monitored every si.x weeks between September 1999 and April 2000. Results indicate that growth and survivorship were significantly lower in the cages planted directly on the substrate within the seagrass bed. but no significant difference was detected among the remaining treat- ments. WATER LOSSES. SEASONAL MASS LOADING. AND BEST MANAGEMENT PRACTICES FOR CRAWFISH PONDS. Landon D. Parr*, Robert P. Romaire. and W. Ray McClain. Louisiana State University AgCenter. Aquaculture Re- search Station. 2410 Ben Hur Road. Baton Rouge. Louisiana 70820. Some crawfish (Procamhanis clarkii and P. zoiuingiili(s) ponds discharge into impaired water bodies in Louisiana. The objectives of this research were to develop water discharge models, determine seasonal mass loading of solids and nutrients, assess effluent qual- ity during final drawdown (May through June), and identify best manageinent practices for crawfish ponds. Average crawfish pond water loss during a production cycle was 228 cm and was parti- tioned among evapotranspiration (68%). precipitation overflow (13%). final drawdown (13%). and infiltration (6%). Modeling indicated that 15-cm of water storage capacity reduced precipita- tion oserflow by 28% in high precipitation years. 61% in average precipitation years, and 100% in low precipitation years. Predicted mass loading was greatest in the winter (precipitation overflow) and late spring through early summer (final drawdown). During final drawdown, total suspended solids (TSS) were high in the first 5% and last 20% of water discharged. During final drawdown, deep vegetated ditches provided the best TSS reduction compared to narrow , shallow, non-vegetated ditches. Slow draining from the water surface and avoiding drainage of the final 20% of the pond \olume are recommended best management practices. The final 20% of the pond volume can be treated in deep vegetated ditches, setthng basins, or constructed wetlands. EFFECTS OF KARENIA BREVIS ON SHELLFISH: DOES STRAIN MATTER? Susan E. Pate.* Jeffrey J. Springer, and JoAnn M. Burkholder. Center for Applied Aquatic Ecology, North Carolina State University, Raleigh, NC 27606; Sandra E. Shuniway, Department of Marine Sciences, University of Con- necticut, Groton, CT 06340, Red tides are found in the Gulf of Mexico and the coast of Florida and consist primarily of the toxic dinoflagellate, Karenia hrevis (Davis). Previous studies show lipid-soluble polyether tox- ins (bre\'etoxins, PbTx) can accumulate by .several species of shell- fish exposed to A', hrevis. Bloom characteristics, shellfish grazing rates, and biotransfoniiati\'e processes influence shellfish toxin le\'els. Little is known regarding interactions between shellfish and varying strains of K. hrevis. Experiments were conducted involving three bivalve species (Argopeeteii irradians. Crassostreu yiri>iniea, Mereenaria inerce- naria). The three K. hrevis strains represent low, moderate, and high levels of brevetoxin production and were introduced at cel- lular concentratiims during a bloom event. Behavioral response and grazing rates were determined for each species versus each K. hrevis strain. In addition, we microscopically examined fecal ma- terial to determine whether cells remained intact and viable after passage through the digestive tract. Preliminary results indicate that some cells pass through the shellfish digestive tract intact. ASSESSMENT OF THE EPIZOOTIOLOGY OF PERKIN- SUS SPP. ON THE ATLANTIC COAST OF USA USING GENUS-. SPECIES-, AND STRAIN-SPECIFIC MOLECU- LAR PROBES. Wolf T. Pecher*. Jose A. F. Robledo. Eric J. Schott. and Gerardo R. Vasta. Center of Marine Biotechnology 701 East Pratt Street Baltimore. MD 21202. P. marinus represents a major cause of mortality of the eastern oyster {Cnissostrea viriiiiiica) along the Gulf of Mexico and At- lantic coasts of the USA. Based the fluid thioglycolate medium (FTM) assay. Perkinsiis infections attributed to P. marinus have been reported as far north as Maine but although infection preva- National Shellfisheries Association. New Orleans. Louisiana Ahsinicls. 2003 Annual Meeting. April 1.^-17. 2003 349 lence in Northeast regions may be high, it may not correlate w ith oyster mortality. In addition to the influence of environmental factors, the presence of other PcrkiiisLi.s species/strains that evhibit reduced pathogenicity for C. viri;ii}iLa may explain the.se observa- tions. Two recently described species. P. clwsupeaki and P. tm- drewsi that test positive by the FTM assay, can also be present in clams and oysters, but their \irulence remains unknown. Thus, the accurate pre\ alenee assessment of Perkinsiis v/7/j is needed for the detailed understanding of epizootic events. To discriminate be- tween P. iiniriiins. P. aiidrewsi and other Perkinsiis species our laboratory has developed species-specific PCR-based assays. We are applying these molecular probes to investigate the epizootiol- ogy of Perkinsiis species and strains in oysters, hard clams, and other shellfish along the East Coast (from ME to VA). [Supported by ODRP. NOAA award NAO6RG01U1-5. through the MD Sea Grant College, to GRV). ECOLOGICAL EFFECTS OF FISHING: BIOLOGICAL, PHYSICAL, AND SOCIOLOGICAL IMPACTS OF DER- ELICT AND ABANDONED CRAB TRAPS IN MISSISSIPPI. Harriet Perry*. Kirsten Larsen, Center for Fisheries Research and Development. Gulf Coast Research Laboratory. College of Marine Sciences. The University of Southern Mississippi. P.O. Cox 7000. Ocean Springs. Mississippi 39566-7000; Bill Richard- son and Traci Floyd. Mississippi Department of Marine Re- sources. 1141 Bayview Avenue. Suite !01. Biloxi. Mississippi 39530. The wire crab trap dramatically changed the Gulf of Mexico Blue crab (Cailinectes sapidiis Rathbun) fishery. Crab traps were introduced in Louisiana and Texas as early as 1948 and by the mid-1950s were widely accepted throughout the Gulf. While adop- tion of the crab trap had a positive impact on fishing efficiency and harvest, proliferation of traps has resulted in an increase in the problems associated with lost or discarded traps. Derelict traps contribute to the mortality of blue crabs and other bycatch, exac- erbate user group conflicts, create visual pollution, and may cause damage to sensitive habitats. Derelict traps result form abandon- ment of fishable traps by fishermen and the inadvertent loss of actively fished traps from: I ) weather/hydrological factors, 2) de- terioration of buoys, lines, or knots, 3) negligence in assembling and maintaining gear, 4) use of plastic jugs/bottles as floats, 5) clipping of float lines by vessel propellers, and 6) intentional cut- ting of buoy lines by vandals. Conservative estimates of trap loss for the Gulf of Mexico approach 250.000 traps per year. Hundreds of traps litter coastal waters in eastern and western Mississippi Sound. Concern over the magnitude of the problem and the po- tential impacts to the blue crab resource prompted Mississippi to develop a program to remove these traps form near shore waters. THE REGISTRY OF TUMORS IN LOWER ANIMALS: A RESOURCE FOR BIVALVE CULTURE HEALTH STUD- IES. Esther C. Peters*. Tetra Tech, Inc., Fairfax. VA 22030; Marilyn J. Wolfe and Jeffrey C. Wolf. Experimental Pathology Laboratories, Inc., Herndon, VA 20172-0474. Neoplastic diseases have been recognized in several orders of bivalves. Of particular concern for culture efforts are hemopoietic neoplasms of mussels and soft-shell clams and gonadal neoplasms of quahogs. The etiologies of these diseases are unknown but studies suggest that factors which could be manipulated in culture, such as diet, genetics (hybridization or breeding for disease resis- tance and faster growth rate to market size), and environmental conditions (water quality, crowding) could influence the develop- ment of these and other cellular proliferative disorders. The Reg- istry of Tumors in Lower Animals (RTLA) has been moved to Experimental Pathology Laboratories. Inc.. under contract to the National Cancer Institute, and will continue to provide a global resource for investigators interested in bivalve diseases. The col- lection of contributed specimens and reprints will be expanded and Internet access to a searchable and illustrated database provided. The RTLA welcomes visitors (by appointment) and will offer diagnosis of bivalve diseases contributed for archi\ ing and training in comparative histopathology. USING CREATED OYSTER REEFS AS A SUSTAINABLE COASTAL PROTECTION AND RESTORATION TOOL. Bryan Piazza*, John Plunket, John Supan and Megan La Peyre. U.S.G.S. Louisiana Fish and Wildlife Cooperative Re- search Unit. School of Renewable Natural Resources. Louisiana State University Agricultural Center, Baton Rouge, LA 70803. Protection and restoration of coastal shorelines remains a pri- ority worldwide. This study tested the viability of creating sus- tainable oyster reefs for use as a coastal protection and restoration tool in Caillou (Sister) Lake. Louisiana. Six oyster shell reefs (approximately 25 m x 2 m x 0.75 m) were created along the shoreline during June 2002 in two areas representing typical low and high-energy environments. Reefs were located approximately 3-5 m from shore (60 - 90 cm deep). Marsh vegetation was dominated by Spartina allerniflora. Jiincus roemerianus. and Dis- tichlis spicata. The value of reefs for protecting shorelines was determined by tracking shoreline position and adjacent marsh health (vegetation biomass, redox, sediment accretion) at paired cultched and non-cultched sites. Reef sustainability was deter- mined by measuring recruitment and survival of oyster spat. Fish- eries value of the reef was quantified by sampling nekton. Recruit- ment and survival of oyster spat increased throughout the spring and summer. Fish community usage of cultched and non-cultched sites was similar and dominated by Anchoa mitchilli. Shoreline retreat appears to be slightly higher in high energy, non-cultched sites. Minimal movement and reworking of shell through two tropical storm events showed that reefs were stable. 350 Abstracts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana BLUE CRAB {CALLINECTES SAPIDUS) GENETIC STRUCTURE AND DIVERSITY. Allen R. Place*. Colin R. Steven, and Xiaojun Feng. Center of Marine Biotechnology Suite 236 701 E. Pratt Street Baltimore. MD 21202. A responsible approach to marine stock enhancement requires that potential negative impacts upon the gene pools of wild popu- lations be mitigated through the use of genetically sound breeding and release protocols. Studies over the past decade of patterns of genetic variation and divergence in a variety of pelagic marine organisms have demonstrated that high dispersal potential at any of several life-history stages does not necessarily indicate high levels of actual gene flow and uniformity in population structure. Three published studies describing the population genetics of Calliiiectes sapidiis all indicate substantial gene flow, with values sufficiently high to infer panmixia between all blue crab popula- tions from New York to Texas. Despite this high level of gene flow, two striking patterns of temporal and geographic differen- tiation occurred: genetic patchiness and clinal variation. These studies were done with protein polymorphisms (allozymes) which are less diagnostic of population substructure than the more vari- able genetic markers found in mitochondrial and nuclear DNA. To help distinguish hatchery-raised crabs from wild cohorts we have characterized the genetic variability in both the mitochondrial ge- nome and nuclear genomes of Calliiiectes sapidus. The implica- tions of these findings to the overall genetic structure of Calli- nectes sapidus will be addressed. A COMPARISON OF FINFISH ASSEMBLAGES ON SUB- TIDAL OYSTER SHELL (CULTCHED OYSTER LEASE) AND MUD BOTTOM IN BARATARIA BAY. LOUISIANA John Plunket*. Megan La Peyre. U.S.G.S. Louisiana Fish and Wildlife Cooperative Research Linit. School of Renewable Natural Resources. Louisiana State University. Baton Rouge. LA 70803. Recent research suggests that oyster reefs provide unique three- dimensional habitat for many tlsh species. Along the northern shore of the Gulf of Mexico, oyster shell bottoms are predomi- nantly flat, sublidal and cultched. providing a very different habi- tat. In this study, we compared finfish assemblages and gut con- tents at subtidal oyster shell (cultched oyster lease) and mud bot- tiims in Barataria Bay. Louisiana. Three mud and three shell sites were sampled from October 2001 to October 2002. using gill nets with mesh ranging from 25.4-63.5 cm. and 60 x 50 cm substrate trays. Data from the gill nets were used to compare fish assem- blages, and to document diets through gut content analysis. Data from the substrate trays were used to document benthic fish and invertebrate communities associated with the subtidal cultched oyster shell habitat. Finfish abundance was greater at shell (N = 223) versus mud (N= 170) bottoms, with higher numbers of sciaenid fishes over shell. Substrate trays collected a variety of benthic fish and invertebrates, primarily naked gobies (Gohiosoma base), skilletfish (Gobiesox stnimosis). toadfish (Opsaiuis beta) and xanthid crabs. These results support the contention that shell bottoms support unique communities of tlsh. as compared to mud bottom habitats. FIBER DIGESTION IN THE BLUE CRAB. CALLINECTES SAPIDUS. Allen R. Place*. Andrea Findiesen. and Nilli Zmora. Center of Marine Biotechnology 701 East Pratt St. Baltimore. MD 21202. A wide range of digestive enzymes have been reported in Crus- tacea indicative of the diverse dietary preferences of the different species. Two of the most important carbon containing compounds in the blue crab diet are chitin (an unbranched homopolymer of b 1-4 linked N-acetyl-D-glucosamine residues, NAG) and cellulose (an unbranched homopolymer of b 1-4 -D-glucose residues. Glc). The traditionally held view of chitin and cellulose digestion in higher animals and invertebrates has been that gut microbes confer the ability to degrade these two polymers. However, recently the genes for chitinase and cellulase have been detected in the ge- nomes of Crustacea. Accordingly, using degenerate primers de- signed from aligned sequences of chitinases and cellulases, we have begun screening a heptopancreas cDNA library of the blue crab. Currently, we have isolated a 479 bp fragment that is highly homologous to the vertebrate and insect chitinase and just starting to probe for crab cellulases. Given that these two polymers are the two most abundant and renewable energy resource on earth, ef- fective utilization of these fibers especially in diets for aquaculture rearing will be an important key to improving production and feed conversion efficiency in the future. A COMPARISON OF NEKTON USAGE OF MUD BOT- TOM, CREATED LIMESTONE. SHELL. AND NATURAL SHELL REEF HABITATS IN TERREBONNE BAY. LOUI- SIANA. John Plunket*. Gary Peterson. Bryan Piazza and Megan La Peyre. U.S.G.S. Louisiana Cooperative Fish and Wild- life Research Unit. School of Renewable Natural Resources, Loui- siana State University Agricultural Center, Baton Rouge, LA 70803. Restoration of coastal environments increasingly involves habi- tat creation for fisheries species. The creation of artificial reefs is based on the assumption that estuarine hard-bottom habitats sup- port more diverse, complex communities than soft bottom habitats. In Louisiana, the creation of artificial reefs has recently become a focus of activity among recreational fisherman and coastal man- agers. In 2002, we compared finfish abundance on a natural shell reef, a created clam shell reef, a created limestone rubble reef, and a mud bottom site in lower Lake Pelto, Louisiana. The four sites were sampled over one year using 200' experimental gill nets, an 8' otter trawl and fish traps. On average, species diversity was two times higher on natural and created reefs (N= 15), as compared to mud bottom (N = 7). The created limestone and natural reef con- sistently supported the more diverse, as well as the more even (Pielou's J) communities throughout the year. Sorenson's commu- National Shellfisheries Association. New Orleans. Louisiana Abstracts. 2003 .-Xnnual Meeting. April 13-17. 2003 3.SI nity similarity index indicates large dissimilarities between the created reefs and the mud bottom (S<0.23). and more similar com- munities between both created and natural reefs (S>0.5). The two artificial reefs support communities of greater diversity and even- ness than mud bottom habitat, and are comparable to natural reefs in diversity, but vary in species composition. CONSUMER PREFERENCES AND ATTITUDES TOWARD IRRADIATED OYSTERS. Benedict C. Posadas* and Linda S. Andrews. Mississippi State University. Coastal Research and Ex- tension Center 2710 Beach Blvd. Ste. 1-E. Biloxi, MS 39531. Consumer attitudes and preferences toward raw oysters in gen- eral, and irradiated oysters, in particular, were evaluated from results of consumer surveys conducted through personal and tele- phone interviews. Seventy five interviews were conducted at the MSU-Coastal Aquaculture Unit Open House in Gulfport. Missis- sippi on December 6. 2001. Another survey was conducted at the MSU-Coastal Research and Extension Center booth among 140 participants of the 2002 International Boston Seafood Show in Boston. Massachusetts on March 12-14. 2002. Telephone inter- views with a simple random sample of adults living in the Balti- more and Houston MSAs in households w ith telepht)nes were done by the Survey Research Unit (Social Science Research Center at Mississippi State University) in June of 2002. Households were selected using random digit dialing procedures. Of the eligible respondents contacted in the Baltimore Metropolitan Statistical Area (MSA). 610 completed the interview and S5 refused to par- ticipate. Of the eligible respondents contacted in the Houston MSA. 606 completed the interview and 67 refused to participate. FORM AND FUNCTION IN OYSTER REEFS: INFLUENCE OF REEF MORPHOLOGY ON HABITAT FUNCTION AND OYSTER SURVIVAL Martin H. Posey*, Troy D. Alphln, Heather D. Harwell and Thomas J. Molesky. Center for Marine Science. UNC-Wilmington 5600 Marvin K. Moss Lane Wilming- ton. N.C. 28409. With the decline in natural oyster's reefs there is increasing interest in restoration of reef habitat for fishery and ecosystem functions. Oyster reefs provide important structural habitat and have significant ecosystem impacts. However, the function of oys- ter reefs varies with reef morphology, especially venical complex- ity that may affect 3-diniensional characteristics of the reef sur- face, edge convolution that may affect encounter surfaces for in- tertidal reefs and reef fragmentation. We have begun a multi-year study examining the influence of vertical complexity, edge con- volution and fragmentation on faunal use. ecosystem function, and oyster settlement and survival on intertidal created oyster reefs in southeastern North Carolina. Reefs have been established with blocked high and low vertical coinplexity and circular versus con- voluted edge as well as small and larce frasment reefs. We are assessing sediment nutrient fluxes, benthic microalgae. infauna. epifauna. and nekton use of these reefs through a variety of sam- pling approaches to examine community responses to variations in landscape factors. Reefs were established in 2002 and initial re- sults indicate strong effects of vertical complexity and fragmenta- tion and weaker effects for edge characteristics. Efforts to restore oyster reefs should consider the potential influence of reef design on ultimate habitat function. REPRODUCTION, BIOENERGETIC AND SUMMER MORTALITY OF CRASSOSTREA GIGAS: EXPERIMEN- TAL .APPROACH. Stephane Pouvreau*. Martha Enriquez- Diaz, Pierrick Le Souchu, .Jean Paul Connan, Bertrand Le Roy, Christian Mingant. .Jeanne Moal, Maryse Delaporte, Jean Rene Le Coz, and Jean Francois Saniain, *UMR PE2M WVPhysiologie et Ecophysiologie des Mollusques MarinsWV". Sta- tion Experimentale dWVArgenton. 1 1 presqu\\\"ile du vivier. 29840 Argenton (FRANCE). As a part of the French MOREST program, we examine ex- perimentally, in 2002. the relationships between food level, repro- ductive processes, bio-energetic status and mortality on 3 batches of the same hatchery oyster population, produced in 2001 . Each lot underwent a same annual temperature cycle (from 8 to 20 "C). a same food composition (4 algae), but a different food level: low (-30 cell.jjLl-1 ). medium (-60 cell.|jil-l) and high (-100 cell.|jLl-l). Each month, several parameters were followed: (I) somatic growth, storage, gametogenesis using quantitative histology: (2) clearance rate, absorption efficiency, oxygen consumption and scope for growth (SFG). (3) biochemical composition. Results demonstrated that oysters under high food availability showed an accelerated gametogenesis and the highest reproductive effort. At the maximum of gametogenesis development (i.e. July), these oys- ters exhibited also the highest oxygen consumption and conse- quently the lowest SFG values. E.xperimental infection (by Vibrio lentils as infectious agent) confirmed this relative weakness in relation with the reproductive effort. As a conclusion, it appears that food level that controls the reproductive effort can generate a bioenergetic imbalance at high trophic conditions. Thus, summer mortalities in eutrophic areas could be partly explained by these processes. A COMPARISION AND FEASIBILITY STUDY OF TWO DIFFERENT BIOMONITORING SYSTEMS USING THE BLUE MUSSEL, MYTILUS EDULIS. AND THE AMERICAN LOBSTER, HOMARUS AMERICANVS. Heidi Pye*, Winsor H. Watson III, Christopher Rillahan, Rachel Hamilton, and Jennifer Wishinski. 46 College Rd Zoology Department-UNH Durham. NH 03820. The advantages of biomonitoring in accordance with traditional techniques include: 1. behavioral and physiological responses are more sensitive indicators of contaminant-induced stress. 2. While 352 Abstracts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana traditional instrumentation measures specific substances, organ- isms integrate all stressors to provide an indicator of overall water quality. 3. and if utilizing keystone species the information will help assess impact of the contamination at population and com- munity levels. To effectively use a bioindicator it is necessary to characterize its response and sensitivity (detection threshold) to contaminants. Our goal was to compare the response and sensi- tivities of the American lobster. Homanis americanus and the blue mussel. Mytihis echilis. to four different heavy metals (CuCl. CrC13. PbCI2, CdC12) common in the Great Bay Estuary. In gen- eral, detection levels were lower for mussels (O.Sppm CuCl. 30ppm CdC12) than lobsters (Ippm CuCl. 50ppm CrC13. > 50ppm PbC12. CdC12). Clear responsiveness was limited to CuCl which occurred close to lethal levels (for H. americanus Ippm response. 2ppni LD50). Given these results we would rec- ommend using mussels, due to their higher sensitivity and ease of use. The only drawback is that mussels are sensitive to a variety of other environmental perturbations that can make responses to heavy metals difficult to elucidate. LARVAL ECOLOGY: MOLECULAR TOOLS FOR THE BLACK BOX? Paul D. Rawson. School of Marine Sciences 5751 Murray Hall. University of Maine Orono, ME 04469-5751. Many marine invertebrates, including ecologically and com- mercially valuable shellfish, have biphasic life histories with a relatively long-lived and highly dispersive larval stage. Ecologists have recognized the role that larval supply and settlement play in population and community dynamics while geneticists have fo- cused on the impact that larval dispersal has on the distribution of genetic variation. Dispersal and settlement, in turn, are dependent on the local abundance of larvae, which can be extremely variable in space and time. Traditional methods for identifying and enu- merating larvae can be time consuining. and because of the mor- phological similarity between larvae of many species, requires specialized training. Thus, our understanding of the links between planktonic processes that generate larval patchiness and larval settlement can perhaps be represented by a black box. Molecular methodologies, in particular PCR-based methodologies, provide tools for peering into this black box by allowing the rapid, and perhaps quantitative, analysis of larval abundance. We will discuss the development of some of these methodologies, the advantages and pitfalls associated with them, as well as providing examples of their application from work currently being conducted in our lab. STATUS OF PERKINSUS MARINVS IN GALVESTON BAY. TEXAS: RESULTS OF THE DERMOWATCH PROGRAM Sammy M. Ray.* Department of Marine Biology. Texas A&M University at Galveston. Galveston. TX 77553; Thomas M. So- nlat. Department of Biology. Nicholls State University. DermoWatch is a web site (www.blueblee.com/dermo). a monitoring program and an online community for the management of the oyster parasite, Pcrkinsus inariniis. The web site contains an embedded model, which calculates a time to critical level of dis- ease from an initial weighted incidence of disease and water tem- perature and salinity. Six public reefs and three private leases in Galveston Bay have been sampled monthly since December 1998. The web site displays the most recent data from each site on the home page and archives all data, such that an historical record is maintained. Historical data show high levels of disease during the drought years of 1999 and 2000. With the cessation of the drought in 2001 and heavy rains associated with tropical storm Allison in June of 2001, disease levels throughout the Bay have been de- pressed. SEASONAL AND TEMPORAL VARIABILHTY IN CONDI- TION INDEX AND TISSUE BIOCHEMISTRY OF ELLIP- TIO COMPLANATA. Deborah Raksany*. Catherine M. Gatenby and Danielle A. Kreeger. The Academy of Natural Sci- ences 1900 Ben Franklin Pkwy Philadelphia. PA 19103: Due to diminishing biodiversity and habitat, it is imperative that we better understand the biology and the ecological function- ing of our existing freshwater mussel populations. Temporal vari- ability in the condition and physiological status of marine shellfish has been well studied, but there remains a dearth of knowledge with respect to these trends in freshwater mussels. Our goal was to quantify variability in physiological condition of Elliptic) coinpla- iiata. a common freshwater mussel in the Atlantic drainage. Con- dition index and proximate tissue biochemistry (protein, lipid, and carbohydrate) were monitored in adults collected from a healthy population over a three-year period. Both parameters varied sea- sonally and among years for similar seasons. For example, condi- tion index peaked in August of 2000. but reached its peak in October of the following year. These results reflect the reproduc- tive and seasonal conditioning processes of these animals, which may be responsive to environmental cues. By understanding tem- poral shifts in the physiological status of these animals in nature, we will be better equipped to gauge their functional roles in fresh- water ecosystems and formulate appropriate diets to sustain them in captivity. NUCLEIC ACID-BASED AQUATIC PATHOGEN MO- LECULAR DIAGNOSTICS FOR DETECTION, RE- SEARCH AND ENVIRONMENTAL MONITORING Kim- berly S. Reece. Virginia Institute of Marine Science. Gloucester Point. VA 23062. Advances in molecular genetic technology have facilitated progress on many fronts of aquatic disease research including pathogen identification, detection, and studies examining transmis- sion dynamics, epizootiology. virulence mechanisms and host/ parasite/environment interactions. Probes for in situ hybridizations and primers for use in PCR are now available for many pathogens found in the aquatic environment. These nucleic acid-based mo- National Shelltisheries Associatiiin. New Orleans, Louisiana Ahsimay 2003 Annual Meeting. April 13-17. 2003 353 leciilardeteetion methods can improve sensitivity and efficiency of disease diagnoses and detection of organisms in environmental samples, especially where it is difficult and/or time-consuming to isolate and identify pathogens. Rapid and accurate molecular de- tection assays have been developed to facilitate both field moni- toring programs and studies to examine the effects of various environmental parameters on growth and distribution of patho- gens. Studies that employ different molecular detection techniques will be presented including those where real-time PCR assays are being used to quantify the number of pathogen cells in water samples for environmental monitoring programs and disease trans- mission studies. In situ hybridization assays ha\'e been developed for confirming the identity of parasites in host tissues and for detecting pathogenic organisms in the gut contents of bivalves that, because of their filter-feeding behavior, are natural integrators of the water column. VALIDATION OF POST-HARVEST PROCESSING OF VI- BRO PARAHEMOLYTICUS IN OYSTERS: SPEED BUMPS ON THE ROAD FROM THE RESEARCH LAB TO THE PROCESSING PLANT. P. VV. Reno*. V-C. Su. M. Morrissey, and D. Nisbet. HMSC 2030 S. Marine Science Dr. Newport, OR 97365-52%. Small scale laboratory experiments were canied out to deter- mine the efficacy of high pressure processing in inactivating Vibrio paralu'inolyticus (VP), particularly serotype 03;K6, with in Pacific oysteis. Oysters were held at HMSC isolation facility with u\ -irradiated, sand-filtered seawater that had no detectible VP in the incoming water or in oysters. The oysters were exposed \ ia bath for 3 h in static seawater with between 103 and 106 cfu/mL of VP. Bacterial counts per gram of oyster meat approximated the VP count per niL water. Bacterial counts remained stable in oys- ters for at least 10 h at IOC. The results of these tests indicated >l05/g reduction in colony counts was achieved at 3IOmP;i/2min in a 1.5-L pressure unit. Transfer of the technology from the small scale (1.5 L capacity) research laboratory to a pressure unit oper- ating under commercial processing conditions was undertaken to validate the process to accede to anticipated FDA requirements. Using a commercial pressure unit of 42 L volume, a series of time/pressure combinations are currently under way to determine the efficiency of killing under commercial conditions on oysters exposed by the techniques used in the research laboratory. The process is still ongoing, but results appear promising. COMPUTATIONAL FLOW MODELING OF AQUACUL- TURE SYSTEMS. John Richardson*, Alden Research Labora- tory, 30 Shrewsbury St., Holden. MA 01520-1843; Carter Newell. Great Eastern Mussel Farms, P.O. Box 141. Tenants Harbor. Maine 04860. The successful design of floating raft-culture systems requires knowledge of how water circulates through the raft-culture struc- tures. In this research advanced Computational Fluid Dynamics (CFD) Techniques were used to model fiow through ratl-culture systems used to grow oysters and Blue Mussels. The basic mod- eling techniques are general, and they can also be used to model the flow through other types of aquaculture systems (marine or teiTestrial). The analysis techniques used for this study are capable of accurately simulating the 3-dimensional flow of water through raft-culture structures located in areas with complex bathymetries. The analysis scheme can. additionally, be used to simulate the transport of nutrients and wastes through the floating rafts. CHARACTERIZATION OF THE CRASSOSTREA VIR- GINICA SLCllA GENE (FORMERLY NRAMP). Jose A. F. Robledo* and Gerardo R. Vasta, COMB, UMBI. University of Maryland. Baltimore. MD 21202, USA. Pt'ikinsits imiriniis has been associated to extensive damage to oyster populations, with catastrophic consequences for shellfish- eries. Although, selective breeding approaches for development of disease-resistant oyster stocks are promising, the identification of genes that are directly linked to disease-resistance/susceptibility represents an attractive alternative. The Slclla (former Nramp: natural resistance-associated macrophage protein) is a divalent cat- ion transporter, demonstrated to be a determinant of resistance/ susceptibility to intracellular pathogens. Most parasites have de- veloped efficient mechanisms for iron acquisition from their hosts. Reciprocally, most hosts have developed mechanisms to prevent pathogens from acquiring iron. Iron sequestration from the patho- gen is also a non-specific host response to infection (nutritional immunity), and SIcl la is a critical component in this response. We have already characterized the P. inariinis SIcl 1 (PmSlcl la) and obtain partial sequence of the C. viriiiuica Slclla (CvSlclla). Sequence information was used for screening a C virginica ge- nomic library resulting in several clones" positives for CvSIcl la. The characterization of CvSlclla gene in both host and parasite will provide insight into their competition for iron, and yield in- formation on the mechanisms underlying disease susceptibility (Grant No NA06RG010I-5 from ODRP. NOAA, through the Maryland Sea Grant College to GRVj. PERKINSUS MARINUS CELLULAR BIOLOGY USING EX- PRESSION SEQUENCE TAGS (EST). Jose A. F. Robledo*. Eric J. Schott. and Gerardo R. Vasta COMB, UMBI, University of Maryland, Baltimore, MD 21202, USA. During the last five years virtually all fields of biology have benefited from the tremendous volume of information generated by genomic approaches. Embedded within genomic sequence data is information needed for identifying targets for drug development and for dissecting the biological aspects that may constitute the basis for infectivity and pathogenicity. Perkinsus marimis has been associated with mass mortalities of the eastern oyster, Crassoslrea viriiinica. for more than 50 years and although substantial progress 354 Abstracts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana in understanding the disease has been accomplished, effective pre- vention or treatment methods are still lacking. Previously, we pre- sented a dataset consisting of 300 ESTs generated from two P. monniis cDNA libraries constructed using P. inariniis propagated in standard culture medium and in medium supplemented with C. virginica serum. We now present the analysis of a more extensive EST dataset corresponding to both libraries, focusing on those P. mahniis genes or metabolic pathways that may be unique to this parasite, or which have been targeted for intervention in other parasite species. Based on our increased knowledge of P. inariiiiis genomics/biology. possible strategies to enhance anti-parasite re- sponses in the oyster will be discussed [ODRP. NOAA award NA06RG0101-5. through the MD Sea Grant College, to GRV]. DEVELOPMENT OF AN INDIVIDUAL. ENERGY- BALANCE BASED. GROWTH MODEL FOR THE MA- NILA CLAM (RUDITAPES PHILIPPINARUM). J. Five Sainte Marie*, S. E. Ford, E. Hofmann. F. Jean. J. Klinck. C. Paillard. E. Powell. LEMAR. Univ. Bretagne Occidentale Inst. Univ. Europeen de la Mer. Place Copernic F-29280 PLOUZANE. To study Brown Ring Disease in the Manila clam. Ritditapes pliiUppiiiaiiim. caused by the bacterial pathogen Vibrio tapetis. an environment-host-pathogen interaction model is being developed. As a base upon which to build a population model, an individual growth model, which does not include the pathogen, was first developed. The aim of the present study was to calibrate, to vali- date and to do a sensitivity analysis on this model. The model simulates the length and weight increase of an average individual under forcing of two environmental variables: temperature and food. Model simulations approximate the scope for growth and spawning events observed in nature. On the other hand, the simu- lations showed that chlorophyll a concentrations are not an ad- equate substitute for food availability for this infaunal bivalve. Although additional data are needed to develop a relationship be- tween growth and food availability in the field, .sensitivity analysis showed that this model is responsive to the parameters that deter- mine scope for growth. sure to superoxide and hydrogen peroxide (H202). but not hypo- chlorite. These findings are consistent with two observations: ( I ) Viable trophozoites are able to destroy hydrogen peroxide in vitro; (2) extracts of P. imiriiuis contain abundant iron-type superoxide dismutase (FeSOD) activity, as well as ascorbate dependent per- oxidase (APX) activity. We previously described the cloning and characterization of two P. imiriiiKs FeSODs that have the potential to convert superoxide to H202 in vivo. Recombinant PmSODl and PmSOD2 proteins have been crystallized for structural analy- ses, and used to raise specific antisera for immunolocalizations. The APX activity appears to be a 35 kD protein. Continuing analy- sis of P. marinus SOD and APX functions will be presented. The unique characteristics of the P. marinus antioxidant system may provide the basis for disease prevention or therapy strategies | Sup- ported by ODRP. NOAA award NA06RG0I0I-5. through the MD Sea Grant College, to GRV]. CORRELATION OF FLAT PEARL STUDIES WITH PEARL SAC FORMATION IN A FRESHWATER MUSSEL (CYRTONAIAS TAMPICOENSIS). Donald Shepherd*. Profes- sional Pathology Laboratories. Ltd. P.O. Box 326, Tow. TX 78672. Flat pearl studies can illustrate the process of biominerali/atiiin of molluscan shell, by placement of a flat material, between the mantle and inner shell of the mussel. Protein and calcium carbon- ate crystals can be evaluated by specific stains and light and po- larization microscopy. The initial stage is secretion of a protein layer of glycoproteins on the insert as the nucleating protein sheet. After several days, secretion of calcium carbonate crystals begins trom the epithelial cells of the mantle. These crystals are calcite. which form rhomboid crystals by 15 to 17 days. A .second crystal forms on the calcite crystals; it is an isoform of calcium carbonate - aragonite. The switch from calcite to aragonite is accomplished by a change in acid protein secretion (Lustrin A). The aragonite forms small bricks as in a wall to form the mother-of-pearl nacre. Photos of natural pearls from Tampico pearly mussels will be presented to illustrate natural pearl formation. THE ANTIOXIDANT PATHWAY OF PERKINSUS MARI- NUS: FUNCTIONAL ANALYSIS AND LOCALIZATION OF TWO IRON SUPEROXIDE DISMUTASES. Eric J. Schott*. Jose A. F. Robledo. Wolf T. Pecher. Florence Okafor. and Gerardo R. Vasta, Center of Marine Biology 701 E. Pratt St Baltimore. MD 21202. The economic and environmental impacts of Pcrkinsiis mari- nus epizootics make imperative the understanding of this parasite's virulence mechanisms. It has been proposed that viable P. marinus trophozoites rapidly suppress or detoxify reactive the oxygen burst characteristic of oyster hemocytes. We now report that cultured P. marinus trophozoites are remarkably insensitive to transient expo- CHARACTERISATION OF SUMMER MORTALITIES OF C.GIGAS OYSTER IN FRANCE IN RELATION TO ENVI- RONMENTAL PARAMETERS. P. Soletchnik. M. Ropert. A. Huvet. J. Moal. L. Dcgremont. E. Bedier, J.F. Bouget. B. Dubois, JL. Martin. M. Enriquez Diaz. N. P'aury. O. Le Moine, T. Renault. B. (lagnaire and J.F. Samain. Ifremer 17390 La Tremblade, France. Field characterization of summer mortality was performed in France in the frame of the Morest project. Natural and hatchery spat were compared between three oyster production areas in France. Regardless of the natural or hatchery origin, oysters died during the reproduction period after temperature reaches 19°C. National Shelltishcries Association, New Orleans, Louisiana Absiivcrs. 2003 Annual Meeting, April 13-17. 2003 355 Thus, in southern ureas, temperature accelerated gametogenesis of small spat ( 10mm) as well as adults, and mortality appeared for the two age classes. In contrast, sexual maturation proceeded more slowly in northern where spat mortality was lower compared to 18 months old oysters. Hov\e\er, critical gametogenesis and tempera- ture were not sufficient to induce mortalities, as observed in e.x- aniples w ith stable environment. Alternatively sediment proximity m addition to oyster manipulations increased mortality during spring and summer, suggesting that some additional environmental stresses were necessary to reproduce the phenomena. These inter- action processes will be detailed in the other Morest contributions. course of recent human history - a decadal time scale. Analysis of long term trends in oyster settlement periodicity since 1960 in three major sub estuaries (James, Piankatank and Great Wicomico Rivers) of the Chesapeake Bay show marked changes in this pe- riodicity within the 40 year time frame with the 50th percentile of cumulative recruitment occun'ing between day 194 and 250 of the year depending on year and location. Significant coherence in interannual variation is observed across a wide range of sites. These are discussed in relation to pre- and post-disease (both MSX and Pfrkinsus) events, periods characterized by high and low river flow, varying harvest pressure, and trends arguably associated with alobal warming. A COMPARISON OF TWO OYSTER {CRASSOSTREA VIR- GINICA) STOCKS TO DETERMINE SUITABILnV FOR USE IN OYSTER REEF RESTORATION IN VIRGINIA Laurie Carroll Sorabella* and Mark W. Luckenbach, Virginia Institute of Marine Science P.O. Box 1346 Gloucester Point. VA 23062. Restoration efforts for eastern oysters (Crassostrea virginica) in Virginia have focused on constructing sanctuary reefs that are intended to serve as spawner sanctuaries. Frequently, these reefs are stocked with hatchery-produced oysters to enhance regional recruitment rates. An important unresolved issue is the suitability of specific oyster stocks to achieve maximal reproductive output on sanctuary reefs. The efficacy of using stocks selected for aqua- culture verses wild stocks for oyster reef restoration is not well established. We compared the performance of two hatchery-reared oyster stocks, the CROSBreed selected stock and a wild-caught oyster stock (Lynnhaven), after deployment onto reefs in the Lafayette River (Chesapeake Bay). Performance was evaluated based on growth, survival, female fecundity, sex ratio, disease status and cumulative egg production. Results indicate that repro- ductive performance of the two stocks varied depending on which disease predominated. Where MSX disease pressure was high, the CROSBreed stock outperformed the Lynnhaven stock for cumu- lative egg production; where dermo disease pressure was high, the Lynnhaven stock outperformed the CROSBreed stock. This work suggests that to maximi/'e reproductive output, broodstocks used in reef restoration should be selected based on knowledge of disease pressure in the region. DECADAL SCALE CHANGES IN SEASONAL PATTERNS OF OYSTER RECRUITMENT IN THE VIRGINIA SUB ES- TUARIES OF THE CHESAPEAKE BAY. Melissa South- worlh* and Roger Mann. Virginia Institute of Marine Science P.O. Box 1346 Gloucester Point, VA 23062. Reproductive periodicity of sessile estuarine invertebrates re- flects local seasonality of both environmental (temperature, salin- ity) and biological (food) parameters. Estuaries are ephemeral fea- tures in geological time, but considered somewhat constant in the FIRST REPORTED OCCURRENCE OF MSX IN CANADA. Mary F. Stephenson*, Sharon E. McGladdery, Michelle Mail- let and Anne Veniot. Gulf Fisheries Centre. Department of Fish- eries and Oceans. P.O. Box 5030. Moncton. New Brunswick. Canada EIC 9B6: Gary Meyer, Pacific Biological Station. De- partment of Fisheries and Oceans, 3190 Hammond Bay Road, Nanaimo. British Columbia. Canada V9T 6N7. The first reported occurrence of MS.X (HupUispdiidian iiclsdni) in American oysters (Cnissostrea virf>iiiic(i) was observed on the Atlantic Coast of Canada in October 2002 associated with mor- talities of >80% in adult oysters from St. Patrick's Channel. Bras d"Or Lakes, Nova Scotia. Histological examination revealed the plasmodial stage of MSX with confirmation using DNA probes received from the Office International des Epizooties (OIE) Ref- erence Laboratory for Haplosporidiosis at the Virginia Institute for Marine Science. In collaboration with the Provinces. Industry, and First Nations, an extensive disease survey was conducted from October to December 2002 while affected areas were closed to the harvest of oysters. Heavy infections, adult oysters with plasmodia or spores, were contained within Bras d'or Lakes while light back- ground levels were described from other areas. Stakeholders con- tinue to work collaboratively on the development of MSX control strategies within Atlantic Canada. A QUANTITATIVE. REAL-TIME PCR ASSAY TO DE- TECT THE PARASITIC DINOFLAGELLATE HEMATOD- INWM SP. IN BLUE CRABS, CALINECTES SAPIDUS. Colin R. Steven*, Kristen Hunter-Cevera, Allen R. Place, Mike Sheppard, and Dick Lee, Center of Marine Biotechnology Suite 236 Baltimore, MD 21202. Hematodinium sp. is a parasitic dinotlagellate that infects and kills several species of commercially valuable crustaceans, includ- ing the blue crab. This dinoflagellate is found in several different morphologies in the hemolymph and tissues of blue crabs. Hema- todinium infections in the Chesapeake Bay show strong salinity and temperature dependencies during their seasonal fluctuations. We present our work towards the development of an ultra sensi- 356 Ahstnicts. 2003 Annual Meeting. April 13-17. 2003 National Sliellfisheries Association. New Orleans, Louisiana tive. real-time, fluorescence-based. PCR assay for the detection and quantification of Hciuntddiniiiiii infection. This assay builds on a previously developed PCR-based diagnostic that relies on specific oligonucleotide primers designed against a section of the Hematodiniiim 18S rRNA gene (AF421184). Our quantitative, real-time assay incorporates a fluorescently-labeled. gene-specific probe as well as two gene-specific primers which allow us to accurately detect approximately 1.4 Heniatiicliniiiin cells/ml hemolymph. This new diagnostic tool will allow investigators to quickly and easily monitor the extent and severity of Heniatod- iiuiim infections in blue crabs, and ensure that infected crabs are not released from hatcheries. THE MITOCHONDRIAL GENOME OF THE BLUE CRAB, CALLINECTES SAPIDUS. Colin R. Steven*. Xiaojun Feng, Allen R. Place, and Jeffrey L. Boore. Center of Marine Biotech- nology Suite 236 701 E. Pratt Street Baltimore. MD 21202. In animals. mtDNA is generally a small (15-20 kB) genome containing 37 genes that is maternally inherited. There is generally a single large non-coding region which, for a few animals, is known to contain controlling elements for replication and tran- scription. Animal mtDNA displays extensive intraspecific poly- morphism {often in the non-coding cimtrol region) and often evolves faster than typical single-copy nuclear DNA. Most mtDNA variants involve nucleotide substitutions or small length changes; gene order is highly stable over short evolutionary time. No published studies using blue crab mitochondrial polymor- phisms exist and the only crustacean mitochondrial genome de- posited in GENBaiik is that for Artemia. Recently the DOE Joint Genome Institute has begun a Mitochinidrial genomics program. We ha\e initiated a collaborative project to sequence the entire Callinectes sapidits mitochondrial genome which w ill allow us to find variable regions for distinguishing the mothers of hatchery derived juveniles from those in the wild. Depending of the vari- ability observed, these same markers would assist in defining the genetic substructure of blue crab in the Chesapeake Bay. Investigators use microsatellites to distinguish genetic subpopula- tions as well as individuals at the genetic level with a very high degree of certainty. We have isolated approximately two dozen dinucleotide and tetranucleotide microsatellite loci, and are in the process of screening these loci to determine their usefulness. Once validated, these microsatellite loci will be used to examine the genetic structure of the Chesapeake Bay blue crab fishery, and to determine the impact that restocking efforts would have on the natural fishery. SETTLEMENT, SURVIVAL. AND PREDATION OF RED KING CRABS ON NATURAL AND ARTIFICIAL SUB- STRATA. Bradley G. Stevens*. NMFS/NOAA Kodiak Fisheries Research Center 301 Research Ct.. Kodiak. AK 99615; and Kathy Swiney. In tests with structurally complex live substrata, postlarxal (glaucothoe) and juvenile red king crabs ParaUthodcs ccinitsclniti- iiis prefen'ed hydroids and algae, over sand or worm colonies. Survival to stage CI was highest for controls, least on sand, and intermediate on other substrata. Predators (larger crab) caused in- creased mortality of glaucothoe, but neither shelter presence or type, or predator size had any effect. Survival of juvenile crabs was significantly decreased by shelter absence, predator presence, and predator size. Density of juvenile crabs on shelters was higher than that o( glaucothoe. and increased in the presence of larger preda- tors, whereas that of glaucothoe did not. Despite active selection for complex substrata by settling glaucothoe. significant predation occurs there, and behavior of glaucothoe is not compensatory. In contrast. juNcnile crabs modify their behavior to achieve higher densities in sheltered habitats, which dampens the effect of preda- tion. These survisal strategies have probably evolved to compen- sate for the much greater risk of predation in open habitats. Bio- genic oases are important to settling larvae, and should be pro- tected from disturbance by fishing activities. Knowledge of settlement behavior is essential prior to considering the potential of king crabs for stock enhancement or aquaculture. DEVELOPMENT OF MICROSATELLITE MARKERS IN THE BLUE CRAB, CALLINECTES SAPIDUS. Colin R. Steven, Johnathan Wilkes, Allen R. Place. Jessica Hill, and Brian Masters. Center of Marine Biotechnology Suite 236 701 E. Pratt Street Baltimore. MD 21202. Current tagging methods for blue crabs, which include, fluo- rescent elastomers and coded wire tags can be expensive, labor- intensive and/or relatively short-lived. We have initiated the iden- tification and characterization of genetic markers, or microsatel- lites, to augment current tagging methods. Microsatellites. or simple sequence repeats (SSRs), are tandemly repeated units of two to six nucleotides, located randomly throughout the genome of all organisms. The high variability among these loci has become a powerful and popular tool for ecology and population genetics. USE OF LOG PILING STRUCTURES AS ARTIFICIAL HABITATS FOR RED KING CRABS PARALITHODES CAMTSCHATICUS. Bradley G. Stevens*. NMFS/NOAA Ko- diak Fisheries Research Center 301 Research Ct.; J. Eric Munk, and Peter A. Cummiskey. Juvenile king crabs use wooden dock pilings as habitats. We studied whether pilings could be used to mitigate for natural habi- tat lost during construction of a breakwater. Scuba divers counted organisms on six piling structures and adjacent seatloor areas at quarterly intervals. Site, sea.son, and their interaction had signifi- cant effects on abundance. Abundance of juvenile (age 0 tol-i-) king crabs increased steadily from July 1997 through March, then declined in June I99S. Crab abundance was significantly higher on pilings than on the adjacent substratum, and at more exposed sites National Shclltisheries Associatiim. New Orleans. Louisiana Abslmcls. 2003 Annual Meeting. April I. VI 7. 200.^ 357 than at sheltered sites. Red king erabs were assoeiated with the presenee of green urehins. deeorator crabs, leather stars, and sculpins. Each site could be discriminated by their unique com- munity of inhabitants. Why juvenile king crabs are attracted to pilings is unknown. Pilings are inefficient habitats that are not structurally complex, do not persist in the environment, and may not be the best structure for habitat enhancement. For these rea- sons, and because there is no evidence that RKC are habitat- limited, we do not recommend the use of pilings as artificial habi- tats to mitigate for the loss of natural habitat. SUSTAINABLE COMMUNITY DEVELOPMENT VIA AN INSHORE MOLLUSCAN AQUACULTURE PARK: A CON- CEPT FOR THE GULF OF MEXICO John E. Supan \ La Sea Grant College Program. LSU. Baton Rouge. LA 70803. Industrial parks are areas permitted and/or zoned for the op- eration of prescribed businesses without the need for individual permitting. Such community programming is commonly used in the economic development of inner cities and rural areas across the nation. This same concept can be applied to coastal waters delin- eated and permitted for certain farming activities for economic development of coastal regions. The concept of state aquaculture parks was proposed in March \9H9 by the National Research Council's Committee on Assess- ment of Technology and Opportunities for Marine Aquaculture in the U.S as a means of fostering entrepreneurship through technol- ogy transfer and commerciali/.ation. A well-planned and adminis- tered aquaculture park can circumvent user conflicts, navigation, security, and liability issues that may otherwise hinder such use of coastal waters. A public entity could be the authority that selects the site, obtains public input, necessary permits. Coast Guard ap- proval, and administers park operations, such as leasing areas to farmers, providing security. The Gulf region's semitropical climate provides ideal condi- tions for sea farming. Oyster genetics research has created superior stocks, which can be coupled with technically ad\anced grow-out methods in a park setting to achieve their full economic potential. HISTORY OF THE DEVELOPMENT, COMMERCIALIZA- TION AND SUCCESSFUL MARKETING OF THE FIRST HACCP-BASED P0ST-HARVF:ST PROCESS FOR THE RV- MEDIATION OF VIBRIO SP. IN RAW OYSTERS— THE AMERIPIREPROCESS". .lohn Tesvich* and Patrick Fahey. AmeriPure Processing Company. Inc.. Franklin. LA. The development of a hot water/cold shock treatment to reme- diate Vibrio sp. In raw oysters without removing the oyster from its natural shell was initiated in response to growing public health concerns and marketplace reaction to raw oyster-related Vibrio sp. Illnesses. This led to the patenting and commercialization of the first raw. yet dead (as a result of the PHT) in-shell oyster product, as well as the first HACCP-based PHT process to remediate Vibrio sp. Ill raw oysters. The marketing of a value-added raw oyster with reduced risk of infection and excellent shelf life opened markets previously closed to raw oysters, particularly from the Gulf of Mexico. It akso paved the way for other HACCP-based PHT pro- cesses and has sparked considerable interest among other oyster processors to license the AmeriPure Process*. The process is de- pendable, simple and economical. It is also adaptable to large and small operations with equipment that is easy to maintain and avail- able from numerous manufacturers/fabricators. SELECTION OF APPROPRIATE HABITATS/SITES FOR BAY SCALLOP RESTORATION. Stephen T. Tettelbach*. Christopher V. Smith, Peter Wentzel. Natural Science Division Southampton College of Long Island University. Southampton, NY 11968. Strategies for restoration of bay scallop, Argopectcn irradums irrculiciiis. stocks include collection of setting larvae in spat bags, direct seeding of juveniles or adults on the bottom ("free- planting"), or placement of broodstock in protective enclosures. Aggregations of the latter type of enclosures are often refen'ed to as spawner sanctuaries. Larval collection is often attempted adja- cent to spawner sanctuaries or. when data are available on tidal circulation patterns, in other areas where larvae are likely to be entrained. In Long Island. New York waters, we have evaluated potential sites for free-planting of scallops on the basis of several criteria, including: historical scallop productivity, anticipated lar- val dispersion, predator abundance, bottom characteristics (includ- ing sediment type and presence of SAV's). degree of exposure to prevailing NW winter winds (which can cause stranding of scal- lops on adjacent beaches), and the potential for scallop burial (in winter) by shifting sediments. Placement of net enclosures has been based on most of the above criteria, but additional factors of particular importance include water depth, potential hazard to navigation, and suitability for securing appropriate permits. The choice of appropriate strategies and habitats/sites must be consid- ered simultaneouslv. LINKING HARD CLAM (MERCENARIA MERCENARIA) REPRODUCTION TO PHYTOPLANKTON COMMUNITY STRUCTURE: I. CLAM GROWTH AND REPRODUCTIVE CYCLF^S. Stephen T. Tettelbach*, Natural Science Division, Southampton College of Long Island, Roger I.E. Newell, Chris- topher Gobler. Hard clam. Merccnariu mcrccnaria. populations and fisheries have declined dramatically in the south shore bays of Long Island, New York since the mid- 1 970s. We hypothesized that this decline in recruitment was associated with variation in either the timing of gametogenic development or synchronicity of reproduction w ithin the population due to changes in cnerall patterns of primary pro- duction. Quantitative histological techniques were used to assess the reproductive cycles of adult (>4() mm) female hard clams, from 358 Abstracts. 2003 Aniuuil Meeting, April 13-17. 2003 National Sliellfisheries Association, New Orleans. Louisiana October 2000 - October 2001. at tlve sites in south shore bays of Long Island. For comparison, we also sampled tv\ o sites in Raritan Bay, New Jersey where regular hard clam recruitment supports a large fishery. Timing of peak reproduction was nearly identical at the 5 south shore bay sites and was 1 to 2 weeks later in Raritan Bay. There were appreciable differences in reproductive effort between locations, with female clams from Bayshore and Patchogue showing the lowest and the two Raritan sites having the highest Gamete Volume fraction. Clam growth and condition in- dex differed even more dramatically between sites, with poorest growth and condition also being exhibited at the Bayshore and Patchogue sites. University Southampton, NY 11968. INFLUENCE OF FRESHWATER INPUT ON THE HABI- TAT VALUE OF OYSTER REEFS IN THREE SOUTH- WEST FLORIDA ESTUARIES S. Gregory Tolley*, Aswani K. Volety, Mike Savarese and James T. Winstead, Florida Gulf Coast University, 10501 FGCU Blvd S, Fort Myers. FL 33965. In order to examine the influence of freshwater input on the habitat value of oyster reefs, a spatiotemporal comparison of reef- resident fishes and decapod crustaceans was conducted during three seasonally dry and three seasonally wet months in three Southwest Florida estuaries: the Caloosahatchee and Estero rivers, and the Faka-Union Canal. Lift nets containing 5 liters of oyster clusters were deployed monthly at three sites along the salinity gradient of each system. Salinities within each system varied both spatially and seasonally, with mean salinities being significantly higher downstream and significantly lower during wet months. Analysis of variance also indicated significant spatial and seasonal differences in the community metrics examined. Overall results suggested that abundance, biomass, and species richness of reef- resident organisms increased downstream where salinities were higher. Diversity (H") and richness were also greatest downstream in the Caloosahatchee. but diversity in the Faka-Union was highest upstream. In general, both biomass and diversity exhibited a sig- nificant positive correlation with salinity. Our results suggest that freshwater input (salinity) plays a significant role in structuring oyster-reef communities in southwest Florida estuaries. These re- sults can be used to inform water management practices as well as efforts at oyster-reef restoration. HISTOLOGICAL EVALUATION OF EARLY PEARL-SAC DEVELOPMENT IN THE TAMPICO PEARLY MUSSEL {CYRTONAIAS TAMPICOENSIS). Stephan Towers*, and Le- onard DiMichele. Department of Wildlife and Fisheries Sciences, Texas A&M University. College Station. TX 77843: and Donald Shepherd, Professional Pathology Laboratories, Ltd. P.O. Box 326. Tow, TX 78672. Pearl-sac development in the Tampico pearlymussel was evalu- ated histologically. Hemocytes massed at the wound entrance, sealing it off and staunching blood loss. Hemocytes also lined the incision track. Mucopolysaccharides formed an extracellular ma- trix important in wound healing, restructuring of blood sinuses, and development of a basal membrane. Epithelial cells originating from the graft began to proliferate onto the newly formed basal membrane. The peari-sac was formed, and tall columnar cells be- gan active secretions by day 30. Our results indicate that pearl-sac development is remarkably consistent across taxa and among im- plantation sites. The primary role of the host is seemingly to seal the wound, reconstruct blood sinuses, and provide a basal mem- brane. The role of the donor tissue is to provide epithelial seed cells. Both epithelia (lateral and medial) of the graft may prolif- erate, but only those from the lateral surface of the mantle appear to be involved with pearl formation. MODELING INDIVIDUAL EASTERN OYSTER {CRASSOS- TREA VIRGINICA) GROWTH IN THE MARYLAND POR- TION OF THE CHESAPEAKE BAY. Jessica Vanisko*. Co operative Oxford Laboratory. MDDNR. Oxford. MD 21654: and Thomas Miller. Chesapeake Biological Laboratory. UMCES, So- lomons MD 20688. Eastern oyster populations have declined dramatically in the Chesapeake Bay during the last century. A clear and quantitative description of oyster population dynamics is essential for the implementation of effective restoration efforts. Growth remains an important, but poorly understood component of these dynamics providing the link between spat (young-of-year oysters) and the reproductive and fishable stocks. Catch-at-length data collected at 55 sites from fishery-independent surveys were used in a length- based analysis of growth through modal decomposition, allow ing the mean growth of individuals within a cohort to be followed through time over a maximum of 6 years. Initial sizes of spat were highly variable both temporally and spatially (mean = 24.03. CV = 32.81%). Maximum and minimum observed growth were 1.02-46.22 mm/yr. Growth rates declined with age class. Growth rates were also highly variable among sites due to site-specific differences such as salinity. These data were used to develop re- gion-specific age-length relationships for oysters. EVALUATION HACCP IN THE OYSTER ACTIVITY IN THE LAGOON SYSTEM ALVARADO, VERACRUZ: MEXICO. Itzel G. Villa*. Fabiola L. Reynoso. and Ma. del Refugio C. Chavez, km 1 2 carr. Veracruz-cordoba. boca del no. Veracruz. Mexico cp 94290. In the last 30 years the oyster production in the national envi- ronment has not been stable, presenting this in the State of Ver- acruz a descending behavior, diminishing of 40,569.4 t obtained in 1988 to 9,653.8 t for 1994, that which demonstrates that the oyster veracruzana's activity faces limitations, caused by the over exploi- tation, the contamination, the accumulation of sludge in the coastal lagoons, the climatological interferences, the ecological changes National Shellfisheries Association. New Orleans, Louisiana Absiimls. 2003 Annual Meeting, April 13-17, 2003 359 and the sanitary problems: in some cases these alterations ha\e caused serious problems of public health and even the exhaustion of the banks of oyster. Considering that the concept HACCP in- volves all the potential dangers of security of the foods (biological, chemical and physical), either that they happen in natural form, for en\'ironmental changes or that was generated by failure in the production process. The present project carried out a diagnose of the lagoon system of Alvarado using the HACCP v\ith the aim to propose a handling plan for the exploitation of the American oyster [Crassosirea virginiai). and this way to guarantee its sanitary quality as food for human consumption and to fulfill the regula- tions sanitary to product exportation. REMOTE SENSING TO MAP AND ASSESS INTERTIDAL SHELLFISH RESOURCES IN THE SOUTHEASTERN USA. Jeffrey S. Vincent. USC Dept. of Geography; Dwayne E. Porter, use Baruch Inst, and School of Public Health. Loren Coen, SCDNR Marine Resource Research Inst.; Dave Bushek*, USC Baruch Inst.; and Steve Schill. GeoMetrics. Inc., Baruch Marine Field Laboratory /USC. PO Box 1630. Georgetown, SC 29442. Oyster resources in the southeastern USA are predominantU intertidal. Water clarity and tidal stage limit the use of passive remote sensing systems while shallow water limits the ability of sonar to accurately map beds and reefs. Oysters can be observed directly during low tide exposure, but inaccessibility and other problems make mapping these intertidal oyster resources difficult and tedious with questionable accuracy. Currently, maps are pro- duced via a lengthy process of ground surveys and manual inter- pretation of aerial photographs, both of which are time-consuming and prone to human error. This project is developing a library of hyperspectral imagery to identify spectral end members of shell- fish from in situ and remotely sensed (HyMAP) imagery. Prelimi- nary results indicate separation in hyperspectral characteristics of oyster resources compared to surrounding habitats. Furthermore. HyMAP spectral end members show reasonable separation and similarity with in-situspectral end members. We will use these spectral characteristics to classify and map the distribution and condition of intertidal shellfish resources. If successful, we will develop an automated mapping technique in a GIS environment that can be used by resource managers to obtain more timely information on the changing condition of oyster resources and better direct enhancement/restoration efforts. HISTORY OF THE COMMERCIAL APPLICATION OF HYDROSTATIC HIGH PRESSURE PROCESSING TO MOLLUSCAN SHELLFISH Mike Voisin. P.O. Box 3916 Houma, La. 70361-3916. The history of the commercial application of Hydrostatic High Pressure to molluscan shellfish will be discussed by the CEO of the firm that developed the application. The challenges and op- portunities during the development of this revolutionary process will be discussed. The process reduces certain {Vibrio) bacteria to non-detectable levels and shows potential to inactivate viruses in shellfish, at the same time the shellfish's inuscle releases from the shell creating an easily processed product with reduced labor cost and increased yields. ESTABLISHING MINIMUM FLOWS AND LEVELS OF FRESHWATER IN THE CALOOSAHATCHEE RIVER, FLORIDA. USING RESPONSES OF OYSTERS. Aswani K. Volety*. S. Gregory Tolley and James T. Winstead, Florida Gulf Coast University, 10501 FGCU Blvd, Fort Myers, FL 33965. Alterations in freshwater intlow resulting from watershed de- velopment and water management practices have impacted salinity and water quality and led to declines in oyster populations within southwest Florida estuaries. In the Caloosahatchee Estuary, Florida watershed management is typified by large freshwater re- leases during wet summer months and little or no releases during dry winter months. Effects of watershed management on oysters were investigated to provide guidelines for establishing minimum How s and levels of freshwater in the Caloosahatchee Estuary, Re- productive patterns, Pfrkiijsiis marinus disease, spat recruitment, and juvenile oyster growth, were investigated. Oysters in the Ca- loosahatchee Estuary spawn continuously from April-October. Up- stream, sub-tidal locations exhibited good spat recruitment, low disease intensity, and higher juvenile growth rates compared to downstream, intertidal sites. High freshwater flows during summer flush out oyster larvae and spat from areas with suitable cultch and/or reduce salinities to unfavorable levels for spat settlement and survival. Limited freshwater releases during winter coupled with decreased releases in summer will result in suitable condi- tions for survival and enhancement of oyster reefs. Water quality targets that should sustain, enhance and restore oyster reefs have been both identified and communicated to water resource managers. DECLINING INTERTIDAL OYSTER REEFS IN FLORIDA: DIRECT AND INDIRECT IMPACTS OF BOAT WAKES Linda Walters*. Paul Sacks. Lisa Wall. Jeffrey Grevert, Daniel Lejeune, Samantha Fischer, and Andrew Simpson, Department of Biology University of Central Florida Orlando, FL 32816. Numerous intertidal reefs of the eastern oyster Crassostrea viifiinica have dramatically declined over the past 50 years along the east coast of central Florida. Many reefs are significantly smaller than in the past and have large dead margins on their seaward edges. It is hypothesized that these differences are due to increased recreational boating activity. To better understand the impact of boating on intertidal oyster reefs, we have begun to run replicated field trials in Mosquito Lagoon that include a motorboat passing a reef at one of three speeds (5, 10, 20 mph), one of three distances from shore (15, 30, 45 ml and one of two propeller 360 Ahstnicts. 2003 Annual Meetmg. April 13-17. 2003 National Sliellfisheries Association. New Orleans. Louisiana angles (45 and 90 degrees). On shore, observers have recorded dislodgment of shells, flow rates, w ake height. v\ ind speed, propa- gation time, and turbidity. With the present configuration, all 3 variables had a significant impact on the oyster reef. CHROMOSOMAL MAPPING OF RIBOSOMAL RNA GENES AND TELOMERIC REPEATS IN ZHIKONG AND BAY SCALLOPS. Yongping Wang,*' - and Ximing Guo.' 'Haskin Shellfish Research Laboratory, Rutgers University. 6959 Miller Avenue. Port Norris. NJ 08349. USA: -Experimental Ma- rine Biology Laboratory. Institute of Oceanology CAS. Qingdao. Shandong 266071. PRC. Chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes, and the \ertebrate telomeric repeat (TTAGGG)n were studied in two scallop species, zhikong scallop Clikimys faneri and bay scallop Argopecten irradians. using fluo- rescence in situ hybridization (FISH). Probes were made by PCR amplification, labeled with digoexigenin-1 1-dUTP and detected with fluorescein-tagged anti-digoxigenin antibodies. In zhikong scallop, the major and minor genes were mapped to two different regions of Cliromosome 5. The major rRNA genes were located at the telomeric region of the short arm. while the 5S rRNA gene w as located at an interstitial site on the long arm. In bay scallop, the major rRNA genes had two loci one on Chromosome 4 and the other on Chromosome 8. both at telomeric regions of the short amis. The 5S rRNA was found at an interstitial site of an acro- centric chromosome (Chromosome 10). In both species, the ver- tebrate telomeric repeat hybridized to telomeres of all chromo- somes, and no interstitial sites were observed. The finding of major differences in the distribution of the rRNA genes between the tv\ o species suggests that chromosomal rearrangements may have played an important role in the esolution of scallops. PRODUCTION OF TRANSPARENT EXOPOLYMER PAR- TICLES (TEP) BY BIVALVES J. Evan Ward . Kari B. Hei- nonen, Michael P. McKee, Bridget A. Holohan. Department of Marine Sciences. University of Connecticut. Groton. CT 06340; Bruce .\. MacDonald. Department of Biology. University of New Brunsw ick. Saint John. N.B.. Canada, E2L 4L5. In the marine environment, dissolved polysaccharide-rich or- ganic matter coalesces to form transparent exopolymer particles (TEP). In turn. TEP has substantial impact on the flocculation of phytoplankton and other particles into aggregates (marine snow) which increase deposition of organic matter to the benthos. Pre- vious studies have demonstrated that exudates and lysates from phytoplankton and bacteria contribute to the production of TEP. Little is known, however, about other sources of TEP precursors, especially in near-shore environments. The purpose of this study was to investigate production of TEP by several species of bi- \al\es iMytiliis cJiilis. Argopecten irradians. Crassostrea vir- ginica ). In laboratory studies, several individuals of one bivalve species were isolated in static or recirculating seawater chambers and al- lowed to feed for up to 9 hr. In the field, groups of oysters were isolated in flow-through, benthic chambers and allowed to feed for 1 to 2 hr. Water samples were taken periodically and analyzed for TEP. dissolved organic carbon (DOC), and bacterial numbers. TEP cimcentration was determined using an Alcian Blue staining tech- nique and quantified using a spectrophotometer. Results indicated that TEP concentration in chambers with actively feeding bivalves w as significantly higher than in control chambers without bivalves. No significant differences in bacterial numbers were found be- tween control and experimental chambers suggesting that the ef- fects of bacteria were similar in all treatments. Mixed results were obtained for DOC concentration. Our results indicate that bivalves do produce TEP. probably during feeding when large volumes of water pass o\'er mucus-coated feeding structures. We suggest that bivalves may be an important source of TEP in near-shore waters. ESTIMATING THE IMPACT OF BAY SCALLOP RESTO- RATION EFFORTS USING GENETIC DATA. Ami E. Wil- bur. Biological Sciences/CMS University of North Carolina- Wilmington 5600 Mar\in K. Moss Lane Wilmington. NC 28409. Shellfish populations in many areas are being augmented with hatchery-produced animals in an effort to counteract the effects of overfishing, habitat degradation and disease. While such efforts have the immediate effect of increasing local abundance, it is the expectation that the restorations will have a more dramatic effect on subsequent generations. Until recently, it has been difficult to evaluate the contribution made by such restorations because the offspring of hatchery-produced animals are not readily distinguish- able from wild conspeciflcs. The constraints of hatchery method- ologies, however, prevent the production of stocks that mimic natural populations with respect to genetic variation. These inevi- table genetic differences between hatchery-produced and wild stocks can be used to differentiate individuals in the cohort fol- lowing restoration. Recent efforts to assess the contribution of hatchery-produced bay scallops based on sequence analysis of mi- tochondrial DNA markers serve as a field test of this approach. Assessment of restoration efforts in Florida provided no genetic evidence of a contribution from the hatchery stock despite sub- stantial increases in abundance following the restoration. In con- trast, a substantial contribution from hatchery-produced scallops deployed in Chincoleague Bay was suggested by mtDNA analysis, indicating that the restoration effort was in part responsible for the increase in abundance. National Shellfisheries Association. New Orleans. Louisiana Ahsinicls. 2003 Annual Meetnig. April 13-17. 2003 361 COMPARATIVE SPERMATOZOON ULTRASTRUCTURE OF ARCIDAE BIVALVES ARCA OLIVACEA AND SCAPHARCA BROUGHTONI. VVan-Xi Vang*, School of Lite Sciences, Zhejiang University, Hangzhou 310012, China; Jun- Quan Zhu. Department of Marine and Fisheries, Ningbo Univer- sity. Ningbo 31521 I.China. The uitrastructure of mature spermato/oon of two Arcidae fam- ily species Ana olivticea and Scapliana briiughtoni was com- pared using transmission electron microscopy for the first time. The mature spermatozoon of both species consists of a head which is composed of a cone-shaped acrosome and a round nucleus and a tail region. Spermatoaoon of both species has a round solid nucleus, which exhibits a triangular posterior invagination, hous- ing the centriolar complex and proximal portion of the axoneme. The acrosome of Scapliana bidiiglitoni is fat while that of Area olivacea is very thin. In Scapliana hroKi^htoiii. the subacrosomal space contains an axial rod and a basal plate, while in Ana oli- vacea. no such structures were obserxed. Within the middle piece, the spermatozoon of Scapliana hnmf>hhmi has five spherical mi- tochondria, and in contrast, only four mitochondria were observed in Area olivacea. Both species has long whip-like end portion, which is composed of an axoneme w ith the typical 9+2 structure. MICROSCOPIC OBSERVATION OF TEGUMENT AND CEMENT GLAND DISTRIBUTION OF FEMALE PLEO- POD IN CHINESE MITTEN CRAB. ERIOCHEIR SINENSIS. Wan-Xi Vang*, College of Life Science. Zhejiang University, Hangzhou 310012. China; Antonina dos Santos, Inst. Nac. In\. Agraria e das Pescas IPIMAR. A\. de Brasilia, s/n 1449-006 Lis- boa, Portugal; Luis Narciso and Ricardu Calado. Laboratorio Maritimo da Guia-Faculdade de Ciencias da Universidade de Lis- boa. Estrada do Guincho, 2750-642 Cascais, Portugal; Hong Zhou, Jian-Ping Lu and Nai-Cheng Jiang. College of Life Sci- ence. Zhejiang University. Hang/hou 310012. China; Xue-Ping Ving. Department of Biological and En\ ironmental Science. Wen- zhou Normal College. Wenzhou 325027. China. Eriocheir .sineii.sis is a vitally important economic species of China. In recent years, its production falling down partially be- cause of egg-loss during larval aquaculture. To reveal possible causes of egg loss, we primarily studied the pleopod tegument structure and its cement gland distribution. The pleopod tegument consists of exoskeleton (subdivided into epicuticle. exocuticle and endocuticle) and epithelial cell layer, while the cement glands lie closely to the epithelial cells, with fine gland tubules come across the exoskeleton. We primarily consider that cement glands in the pleopod function in the egg attachment in Eriocheir sinensis. IMMUNOLOGICAL STUDIES ON THE ORIGIN OF THE LAMELLAR COMPLEX (LCX) DURING THE SPERMIO- GENESIS OF MACROBRACHIUM NIPPONENSE (DE HAAN). Wan-Xi Vang. School of Life Science. Zhejiang Uni- versity. Hang/hou 310(112. China. Lamellar complex (LCX) is a transient organelle, which is believed to be deri\ed from Golgi apparatus and lysosome during spermiogenesis of caridean shrimp Macrohrachiuni nipponensc (de Haan). Conventional electron microscopical evidence shows that, in the round spermatid, no LCX observed surrounds the nucleus while saccules of Golgi apparatus begin to separate and move to the nucleus along with the condensation of cytoplasm. Typical LCX can be seen when nucleus of spemiatid begins the sickle-shaping process, and it locates on the convex side of the nucleus. One important feature is that lysosomes merge into the Golgi saccules while the saccules open a cut or cuts. Most part of the LCX conies from Golgi apparatus. To prove this, we use GM130 monoclonal antibody to localize the Golgi apparatus. Im- munofluorescence data show that GMI30 exists mostly in the LCX. and immunocytochemistry results show that gold particles (representing GM130) distribute mainly on the LCX. All these evidence support that the idea that LCX originates mostly from Golgi apparatus. INTERTIDAL OVSTER RESTORATION ALONG AN ERODING SHORELINE: AN ASSESSMENT OF SUB- STRATE TVPES FOR STABILIZATION AND PROPAGA- TION. Guy M. Vianopoulos. and William D. Anderson*, Ma- rine Resources Division. South Carolina Department of Natural Resources, Charleston. South Carolina 29422. Gulf coast Crassostrea virginica shell. South Carolina inter- tidal oyster shell, whelk shell {Biisycon spp.) and intertidal seed oysters were established as cultch material along an eroding inter- tidal shoreline ( 1.83m mean tidal range) to compare the efficacy of substrate types for propagation to three-dimensional oyster popu- lations. Four treatment areas were asses.sed for matrix accumula- tion, growth and recruitment over a three-year period. Shell treat- ments were covered with polypropylene netting (Cinlotlex *) mesh size of 3. 1 75cm x 3.8 1 cm to provide stabilization. Recycled South Carolina intertidal oyster shell and whelk shell demonstrated the best matrix propagation, with whelk shell accumulating the most spat. Gulf coast shell recruited lov\er numbers, but grew larger spat. Transplanted intertidal seed oysters suffered mortali- ties during the three-year study but continued to recruit significant numbers of spat. 362 Abstracts. 2003 Annual Meeting. April 13-17. 2003 National Shellfisheries Association. New Orleans. Louisiana THE MORPHOLOGY AND ULTRASTRUCTURE OF SPERMATOZOON OF THE GASTROPOD BULLACTA EX- ARATA. Xue-Ping Ying. Department of Biological and Environ- mental Science. Wenzhou Normal College. Wenzhou 325027. China; Wan-Xi Yang*. College of Life Science. Zhejiang Uni- versity. Hangzhou 310012. China. The morphology and ultrastructure of spermatozoon of mud snail Biillacta exafcita are first described. It is composed of a head in which a simple cap-shaped acrosomal complex and a elongated nucleus are included and a tail containing middle piece, principle piece and end piece. The nucleus is cylindrical, tapering gradually towards the anterior tip. A posterior nuclear fossa is observed clearly. In the Middle piece, there is a ring consisting of 5 occa- sionally 6 mitochondria, which closely contacted the posterior por- tion of the head. The proximal centriole lies in posterior nucleus fossa and the distal one is in the center of the mitochondrial ring. The principal piece with 9-1-2 structure consists of axoneme and lateral fins. The end piece is short with relatively simple structure. FINE STRUCTURAL ANALYSIS OF SPERMATOZOON OF THE BIVALVE BARBATIA VIRESCENS AND ITS EVO- LUTIONARY CHARACTERISTICS. Jun-Quan Zhu. Depart ment of Marine and Fisheries, Ningbo University. Ningbo 315211. China; Wan-Xi Yang*. School of Life Sciences. Zhejiang Uni- versity. Hang/hou 310012. China. The ultrastructure of mature spermatozoon of Baihatia vire- sceiis was observed using transmission electron microscopy and its evolutionary significance was analyzed. The mature spermatozoon consists of a head and a tail. The head is composed of an apical, umbrella-shaped acrosome and cylindrical nucleus. In the longi- tudinal sections, striations can be seen clearly, which come across outer acrosomal membrane. The nipple-shaped subacrosomal space contains small granules. The nucleus has a Ll-shaped anterior invagination and an inserted V-shaped posterior one. The nucleus is highly condensed. The tail of the spermatozoon includes a middle piece sunounded by five or occasionally six spherical mi- tochondria and a long whip-like end piece with an axoneme with the typical 9-h2 structure. A phylogenetic path can be traced by comparative study of sperm ultrastructure in the Family Anidae. The spermatozoon of B. viresceiis has a very important role in the reproductive evolution of the Family Arcidae. POPULATION GENETIC STRUCTURE OF THE SUMI- NOE OYSTER AS INFERRED FROM RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) AND MICROSATELLITE MARKERS. Qian Zhang*. Karen L. Hudson, Standish K. Allen Jr. and Kimberly S. Reece. Virginia Institute of Marine Science. College of William and Mary. Glouc- ester Point. VA 23062. The Suminoe oyster. Crassostrea ariakeitsis. is being evaluated and considered as a non-endemic aquaculture species for Chesa- peake Bay. To date, published reports on the taxonomic status and genetic characterization of this species have focused on inter- specific relationships within the genus Crassostrea. and little is known about the population genetic structure of C ariakensis in its native range. In this study, we used restriction fragment length polymorphism (RFLP) markers based on the mitochondrial cyto- chrome oxidase I (COll gene and the first internal transcribed spacer (ITS- 1 ) region of the nuclear ribosomal RNA gene region to examine the genetic variation within and among five geographi- cally separated samples of C. ariakensis and hatchery stocks. RFLP data using nuclear and mitochondrial loci showed that the samples shared common haplotypes. but significant frequency dif- ferences were observed between the samples in the northern group (Northern China and Japan) and southern group (Southern China) indicative of population structure (P= 0.000). These results sup- port previous phylogenetic analy.ses based on ITS 1 and CGI DNA sequences of 5-10 individuals from each sample. Microsatellite markers are currently being employed to further examine the popu- lation structure and to determine whether a bottleneck effect has occurred in hatchery stocks. CHARACTERIZATION OF KEY CDNAS OF THE ENDO- CRINE AXES REGULATING REPRODUCTION AND MOLTING IN THE BLUE CRAB. CALLINECTES SAPIDUS. Nilli Zmora* and John M. Trant. 701 E. Pratt St. Baltimore. MD 21202. For the first time in brachyurans. a number of cDNAs encoding key hormones, enzymes and receptors of the reproductive/molting endocrine axes were isolated from the blue crab. Using 5' and 3" RACE. O-methyltransferase. the major regulatory enzyme for me- thylfamesoate (MF) production, was isolated from the mandibular organ. The deduced amino acid (AA) sequences are 74% identical to the Metapeiiaeits ensis enzyme. The enzyme that activates ec- dysone, 20-hydroxylase (CYP4), was isolated from the Y-organ and is 59% identical to the Cherax quadricarinatiis enzyme at the AA level. The ecdysone receptor (ECR) and vitellogenin (Vg) cDNAs were isolated from ovary. The AA sequence of ECR shares -96'7(- identity with the Fiddler crab and many insect ECRs. A 2 kb fragment of the 5' -terminus of a putative Vg transcript was iso- lated, however there is a low degree of homology when compared to crayfish and penaeid Vg sequences. Our attempts to isolate the mandibular-organ-inhibiting-hormone (MOIH) from the X-organ were unsuccessful. The above cDNAs, together with the published molt inhibiting hormone (MIH) sequence, will be used to develop the molecular assays for the investigation of the endocrine regu- lation of reproduction, molting and growth. Natiiinal ShcllCisheries Association. New Orleans. Louisiana AhstniLls. 2003 Annual Meeting, April 13-17. 2003 363 HATCHERY MASS PRODLCTION OF BLUE CRAB iCALUNECTES SAPIDUS) JUVENH^ES Yonathan Zohar*. Oded Zmora, Andrea Findiesen, Emily Lipnian. John Stubble- field. Anson H. Hines and Jana L.D. Davis. Center of Marine Biotechnology, University of Maryland Biotechnology Institute 701 E. Pratt St. Baltimore. Maryland 21202. USA. Responding to the rapidly declining abundance and harvests of the blue crab in the Chesapeake Bay. a multidisciplinary re- search program was established to study the blue crab basic biol- ogy, develop hatchery technologies for its mass production and examine the feasibility of its stock enhancement. This presenta- tion will address the hatchery work. Exposing wild-caught, mated blue crab females to phase-shifted photo-thermal conditions re- sulted in out-of-season hatchinc of millions of zoeae I. Larval rearing to the zoea 8/megalopa stage was conducted at densities of 40-1 10 individuals per liter based on a diet comprised of microal- gae. rotifers and Anemia naiipUi. Zoeae 8/megalopae were produced in an average 22 days, and survival rates of 41.5%. Maximal survival was 74%. Secondary growth of zoeae 8/mega- lopae to 20 mm juvenile crabs was conducted at lower densities of 2-40 individuals per liter. To reduce cannibalism, ample shelter structure was introduced and the crabs were graded by size. Diet was comprised of adult Artemia, shredded squid and artificial pel- lets. In large-scale conditions, 20 mm juvenile crabs were pro- duced in 64 days at a survival rate of 46%. During summer/spring 2002. we produced 40,000 juvenile crabs, of which 25,000 were individually tagged and experimentally released to the Chesapeake Bay. THE NATIONAL SHELLFISHERIES ASSOCIATION The National Shellfisheries Association (NSA) is an international organization of scientists, manage- ment officials and members of industry that is deeply concerned and dedicated to the formulation of ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man- agement of shellfish resources. The Association has a membership of more than 1000 from all parts of the USA. Canada and 18 other nations: the Association strongly encourages graduate students" mem- bership and participation. 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E-mail: sandra.shumway@uconn.edu or sandrashumway@hotmail.com Membership information may be obtained from the Editor or the Treasurer using the form in the Journal. Institutional subscribers should send requests to: Journal of Shellfish Re- search. P.O. Box 465. Hanover, PA 17331. Meegan E. Vandepeer and Robert J. Van Barneveld A comparison of the digestive capacity of blacklip {Haliotis nihni) eind grecnlip (Haliotis laevii^aia) abalone 171 W. Gregory Cope, Teresa J. Newton and Catherine M. Gatenby Review of techniques to prevent introduction of /cbru mussels {Drcissciin jxilxnidiplui) during native mussel (Unionoidea) conservation activities 177 W. L. Marshall, S. M. Bower and G. R. Meyer A comparison of the parasite and symbiont fauna of cohabiting native if'ioiolluuci .siaiiiincii) and introduced ( Venerupis philippinarum and Nuttalia obscurata) clams in British Columbia 1 85 D. E. Morgan, M. Keser, J. T. Swenarton and J. F. Foertch Population dynamics of the Asiatic clam. Corhicula flmninea iMiiller) in the lower Connecticut River: Establishing a foothold in New England 193 Robert S. Anderson, Brenda S. Kraus, Sharon McGladdery and Roxanna Smolowitz QPX. a pathogen of quahogs (hard clams), employs mucoid secretions lo resist host antimicrobial agents 205 Bruce A. Macdonald and Lisa M. Nodwell A portable and practical method to monitor bi\alve feeding activity in the field using time-lapse video technology 209 Vera L. Trainer, Bich-Thuy I^ Eberhart, John C. W'ekell. Nicolaiis G. Adams. Linda Hanson, Frank Cox and Judy Dowell Paralytic shellfish toxins in Puget .Sound, Washington state 213 Matthew M. Nelson, Bradley J. Crear, Peter D. Nichols and David A. Ritz Feeding southern rock lobster, Jasus edwardsii Hutton. 1875. phyllo.somata in culture: Recent progress with lipid-enriched Arlcniia 225 R. J. B. Musgrove and P. J. Babidge The relationship between haemolymph chemistry and moult increment for the southern rock lobster. Jasu.s cdwurdsii Hutton 235 Juan C. Chaves and David B. Eggleston Blue crab mortality in the North Carolina soft-shell industry: Biological and operational effects 241 Pablo D. Ribeiro, Carolina G. Luchetti and Oscar O. Iribarne Sex-specific response to disburbance in a fiddler crab 251 Dominique Audet, Derek S. Davis, Gilles Miron, Mikio Moriyasu, Khadra Benhalima and Robert Campbell Geographical expansion of a nonindigenous crab. Caiciniis maoias (L.). along the Nova Scotian shore into the soulheastem Gulf of St. Lawrence. Canada 255 William J. McGraw and John Scarpa Minimum environmental potassium for survival of Pacific white shrimp Litopmuwiis vwwamei (Boone) in freshwater 263 Leticia Arena, Gerard Cuzon, Cristina Pascual, Gabriela Gaxiola, Claud Soyez, Alain van Wormhoudt and Carlos Rosas Physiological and genetic variations in domesticated and wild populations of Litopenaeus vannamci fed with different carbohydrate levels 269 Lucia Ocampo, Carlos Rosas and Humberto Villarreal Effect of temperature on post-prandial metabolism of brown shrimp Faifaiuepenaeus ralifonuensis 281 Abstracts of technical papers presented at the 23rd Annual Milford Aquaculture Seminar. Mi"'ird, Connecticut, February 24-26. 2003 285 Abstracts of technical papers presented a( the 95th Annual Meeting of the National Shellfisheries Association. New Orleans. Louisiana, April 13-17. 2003 305 COVER PHOTO: Mussels. Mylilus cdtdis. Photo: S. E. Shumway. The Journal of Shellfish Research Is indexed In the following: Science Citation Index*. Sci Search®. Research Alert®. Current Contents*/Agriculture. Biology and Environmental Sciences. Biological Abstracts. Chemical Abstracts. Nutrition Abstracts. Current Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography, Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts. JOURNAL OF SHELLFISH RESEARCH Vol. 22, No. 1 June 2003 CONTENTS Mingfang Zhou and Standish K. Allen, Jr. A review ot published work on Crassoslrea ariakensis 1 Jonathan H. Grabowski, Sean P. Powers, Charles H. Peterson, Monica J. Powers and David P. Green Consumer ratings of non-native [Crassostrea gigas and Crassosueti iiriakcnsis) vs. native (Crassostrea virginica) oysters 21 Ziniu I'm, Xiaoyu Kong, Liusuo Zhang, Ximing Guo and Jianhai Xiang Taxonomic status of four Crassostrea oysters from China as infened from mitochondrial DNA sequences 31 John N. Kraeuter, Susan Ford and Walter Canzonier Increased biomass yield from Delaware Bay oysters (Crassostrea virginica) by alternation of planting season 39 Lisa House, Terrill R. Hanson and S. Sureshwaran U.S. consumers: Examining the decision to consume oysters and the decision of how frequently to consume oysters 51 John N. Kraeuter, Michael J. Kennish, Joseph Dobarro, Stephen R. Fegley and G. E. Flimlin, Jr. Rehabilitation of the northern quahog (hard clam) [Merceiiaria iiieneiuiria) habitats by shelling — 1 1 years in Bamegat Bay. New Jersey 61 Jorge L. Gutierrez and Oscar O. Iribarne Spatial variation in the body mass of the stout razor clam, Tagelus plelwiiis: Does the density of burrowing crabs, Cluismagnalliiis graniilata. matter? 69 William R. Congleton, Jr., Bryan R. Pearce. Matthew R. Parker and Robert C. Causey Mariculture siting — Tidal currents and growth of Mva arcnaria 75 A. Campbell and M. D. Ming Maturity and growth of the Pacific geoduck clam, Panopea ahriipla. in southern British Columbia, Canada 85 M. A. Delaney, Y. J. Brady, S. D. Worley and K. L Huels The effectiveness of N-halamine disinfectant compounds on Perkinsus marinus. a parasite of the Eastern oyster Crassostrea virginica 91 A. Louro, J. P. De la Roche, M. J. Campos and G. Roman Hatchery rearing of the black scallop, Chlaniw varia (L.) 95 Lorelei A. Grecian, G. Jay Parsons, Patrick Dabinett and Cyr Couturier Effect of deployment date and environmental conditions on growth rate and retrieval of hatchery-reared sea scallops, Placopecten nuigellanicus (Gmelin, 1791 ), al a sea-based nursery 101 Seifu Seyoum, Theresa M. Bert, Ami Wilbur, William S. Arnold and Charles Crawford Development, evaluation, and application of a mitochondrial DNA genetic tag for the bay scallop. Argopecten irraJians Ill A. P. Maloy, B. J. Barber and P. D. Rawson Gametogenesis in a sympatric population of blue mussels, Mytilus edulis and Mytilus trossidus, from Cobscook Bay (USA) 119 F. M. Suplicy, J. F. Schmitt, N. A. Moltschaniwskyj and J. F. Ferreira Modeling of tllter-feeding behavior in the brown mussel, Perna perna (L.), exposed to natural variations of seston availability in Santa Catarina, Brazil 125 Jorge Cdceres-Marti'nez, Miguel A. Del Rio-Portilla, Sergio Curiel-Ramirez Gutierrez and Ignacio Mendez Gomez Humardn Phenotypes of the California mussel, Mxtihis californiainis. Conrad (1837) 135 G. Darrigran, C. Damborenea, P. Penchaszadeh and C. Taraborelli Adjustments of Limnoperna fortunei (Bivalvia: Mytilidae) after ten years of invasion in the Americas 141 Wolfgang B. Stotz, Sergio A. Gonzalez, Luis Caillaux and Jaime Aburto Quantitative evaluation of the diet and feeding behavior of the carnivorous gastropod, Concliolepas concholepas (Bruguiere, 1789) (Muricidae) in subtidal habitats in the southeastern Pacific upwelling system 147 D. A. Lopez, M. L. Gonzalez and M. C. Perez Feeding and growth in the keyhole limpet, Fissurella picla (Gmelin, 1791) 165 CONTENTS CONTINUED ON INSIDE BACK COVER JOURNAL OF SHELLFISH RESEARCH VOLUME 22, NUMBER 2 SEPTEMBER 2003 The Journal of Shellfish Research (formerly Proceedings of the National Shellfisheries Association) is the official publication of the National Shellfisheries Association Editor Sandra E. Shumway Department of Marine Sciences University of Connecticut Groton, CT 06340 Standish K. Allen. Jr. (2004) Aquaculture Genetics and Breeding Technology Center Virginia Institute of Marine Science College of William and Mary P.O. Box 1346 Gloucester Point, Virginia 23062 Shirley Baker (2004) University of Florida Department of Fisheries and Aquatic Sciences 7922 NW 7r' Street Gainesville, Florida 32653-3071 Bruce Barber (2005) School of Marine Science University of Maine 5735 Hitchner Hall Orono, Maine 04469 Brian Beal (2004) University of Maine 9 O'Brien Avenue Machias, Maine 04654 Neil Bourne (2003) Fisheries and Oceans Pacific Biological Stadon Nanaimo, British Columbia Canada V9T 6N7 Andrew R. Brand (2003) University of Liverpool Port Erin Marine Laboratory Port Erin, Isle of Man IM9 6JA United Kingdom Eugene Burreson (2003) Virginia Institute of Marine Science P.O. Box 1346 Rt. 1 208 Create Road College of William and Mary Gloucester Point, Virginia 23062 Wnrme Biological Laboratory ' Woodi Hole Oceanographic Institution Library OCT 2 7 2003 Woo'Js I >oifc, Ma 543 EDITORIAL BOARD Peter Cook (2004) Austral Marine Services Lot 34 Rocky Crossing Road Warrenup Albany, W.A. 6330. Australia Simon Cragg (2004) Institute of Marine Sciences University of Portsmouth Ferry Road Portsmouth P04 9LY United Kingdom Leroy Creswell (2003) University of Florida/Sea Grant 8400 Picos Road, Suite 101 Fort Pierce, Florida 34945-3045 Lou D'Abramo (2004) Mississippi State University Department of Wildlife and Fisheries Box 9690 Mississippi State, Mississippi 39762 Christopher V. Davis (2004) Pemaquid Oyster Company, Inc. P.O. Box 302 1957 Friendship Road Waldoboro, Maine 04572 Ralph Elston (2003) Aqua Technics/Pacific Shellfish Institute 455 West Bell Street Sequim, Washington 98382 Susan E. Ford (2004) Rutgers University Haskin Shellfish Research Laboratory 6959 Miller Avenue Port Norris, New Jersey 08349 Raymond Grizzle (2003) Jackson Estuarine Laboratory Durham, New Hampshire 03824 Karolyn Mueller Hansen (2004) 1524 Barley Circle Knoxville, Tennessee 37922 Journal of Shellfish Research Volume 22, Number 2 ISSN: 0730-8000 September 2003 www.shellfish.org/pubs/jsr.htm Mark Luckenbach (2003) Virginia Institute of Marine Science Eastern Shore Lab P.O. Box 350 Wachapreague, Virginia 23480 Bruce MacDonald (2004) Department of Biology University of New Brunswick Saint John, New Brunswick Canada E2L 4L5 Roger Mann (2004) Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Islay D. Marsden (2004) Department of Zoology Canterbury University Christchurch, New Zealand Jay Parsons (2005) Memorial University Marine Institute Box 4920 St. John's, Newfoundland Canada AlC 5R3 Tom Soniat (2004) Biology Department Nicholls State University Thibodaux, Louisiana 70310 J. Evan Ward (2004) Department of Marine Sciences University of Connecticut 1080 Shennecossett Road Groton, Connecticut 06340-6097 Gary Wikfors (2004) NOAA/NMFS Rogers Avenue Milford, Connecticut 06460 Joiirihil oj Slifllfish Research, Vol. 22, No. 2, 365-375, 2003. BIOCHEMICAL INDICATOR OF SEA SCALLOP {PLACOPECTEN MAGELLANICUS) QUALITY BASED ON LIPID CLASS COMPOSITION. PART I: BROODSTOCK CONDITIONING AND YOUNG LARVAL PERFORMANCE FABRICE PERNET,' * REJEAN TREMBLAY." AND EDWIN BOURGET" ^GIROQ. Pavilion Vachon. Uuiversite Laval. Cite Universitaire, Quebec, Qc. Canada. GIK 7P4; 'Univer.site du Quebec a Rinwuski — Centre .Aqiuicole Marin. 6 Rue du Pare. Centre Aqiuteole Marin MAPAQ. 6 Rue du Pare CP. 340. Grande-Riviere. Qc, Canada. GOC IVO: and ''Vice-reetorat a la Recherche. Pavilion Central, Univer.site de Sherhrooke. Sherbrooke, Qc. Canada. .1 IR 2R1 ABSTRACT The aim of this study was to test the validity of a lipid based indicator of larval quality of sea scallop Placopeclen ma,uell(iiiici(S. Objectives were 2-fold: ( 1 ) to determine the link between lipid class content and reproductive state of adults in the field and in the laboratory and (2) to follow lipid class content, growth, and survival during embryonic and early larval development. Adult scallops were periodically sampled during gametogenesis for lipid class and histological analysis of the gonads in the field at two locations and in the laboratory after feeding three different diets. Females were induced to spawn and lipid class content, larval growth, and survival of five batches of eggs were followed for 8 days after fertilization. Site, diet, and time had significant effects on lipid class composition of male and female gonads and gametogenesis of females. Triacyglycerol accumulation during vitellogenesis was characteristic of female gonads and explained respectively 56.4% and 71.3%' of the variability in maturity and egg size. When spawning was induced, no major effect of location or diet on lipid composition of gonad and subsequent eggs was detected. Nevertheless, the mean number of eggs produced by females increased with atresia level in gonad, suggesting that egg quantity was incompatible with egg quality. Lipid class composition during embryogenesis and young larval development showed a high demand for triacyglycerol. KEY WORDS: broodstock nutrition, gametogenesis. hatching, larval growth, lipids, scallop, Placopeclen INTRODUCTION The expansion of aquuculttire has increased the demand for juveniles of a wide range of bivalve species. As a consequence, hatcheries need to produce large quantity of eggs and larvae of good quality. Larval production has to be considered in two phases: broodstock conditioning and larval rearing. Under northern temperate conditions, young larvae of most bivalves rely on en- dogenous sources of energy during embryogenesis before the tran- sition to exogenous sources. The diet provided to adults can affect the biochemical composition of their gonads and their resulting eggs and larvae. For instance, the essential fatty acid composition of the microalgae fed to adult scallop Peeten maximiis was re- flected in the composition of gonads, eggs and larvae until 5 days after fertilization (Delaunay et al. 1992, Samain et al, 1992). Then, maturity and hatching success of eggs from adult P. maximus were improved with a diet based on Isoehrysis sp. rich in essential fatty acids (Soudant et al. 1996a, Soudant et al. 1996b). Given the above, the performance of young larvae is directly linked to ma- ternal nutrition. Studies focusing on gametogenesis and early larval develop- ment highlight the primordial role of lipids and the relative im- portance of triacylglycerol (TAG), For example, gametogenesis in the scallop Argopecten purpuratus was associated with an increase in the ovary lipid content (Barber & Blake 1981 ). The sea scallop Placopeclen inai;ellaiucii.s stores large quantities of TAG in the gonads before spawning (Napolitano & Acknian 1992). Then, lipid reserves accumulated in the eggs of the scallop Crassadoma gi- gantea cover 47,6% of energetic needs during embryogenesis (Whyte et al. 1991). TAG were preferentially cataboli/.ed during egg development of the clam Mercenaria inercenaria and the oys- *Corresponding author. National Research Council 1411 O.xford Street Halifax, Nova Scotia, Canada, B3H 3Z1. Telephone: 902-426-8289; Fax: (902) 426-9413; E-mail: fabrice.pemet@nrc.ca ter Crassostrea virginica (Gallager et al. 1986). Finally, the mass of lipid in the eggs is correlated with the hatching success of P. maximus larvae (Dorange 1989, Devauchelle & Mingant 1991). The reproductive cycle of the sea scallop, like other marine bivalves, includes five distinct periods: vegetative, cytoplasmic growth, vitellogenesis. spawning and. finally, resorption of non- released gametes (see Barber & Blake 1991, Eckman 1996), Spawning is synchronous among individuals at a particular site, and most populations display a single annual spawning period extending over 1 or 2 mo between July and October, depending on latitude. In other scallop species, site-specific variation in game- togenic cycles has been observed (e.g.. Bricelj et al. 1987). Histological preparation of gonadal tissue provides the means to assigned numerical values to the developmental stages. For instance, mean egg diameter is indicative of the stage of the ga- metogenic cycle. Eggs gradually increase in size during gameto- genesis, reaching a maxitnum size prior to spawning and decrease sharply after spawning as mature eggs are released (Barber & Blake 1981, Barber et al. 1988, Paulet & Boucher 1991). Histology allows quantification of the fraction of the gonad occupied by developing, mature, and resorbing (atresic) gametes (Beninger 1987, MacDonald & Bourne 1987). The aim of this .study was to test the validity of lipid class composition of gonad, egg, and larvae as a predictor of quality of sea scallop P. magellanicus. In the present article, "quality" refers to a set of physiological variables (lipid composition) that could explain the variability of reproductive state of adult (maturity, atresia and egg size) and larval performance (survival and growth). For example, the mass of lipid in bivalve larvae is a good indicator of quality because it has been positively correlated with growth and survival (Gallager et al. 1986). To our knowledge, gonad quality has never been assessed using lipid composition. We de- signed our study with two objectives in mind: 1 ) to examine the link between lipid class variation in the gonads and reproductive state of adults, in the field and in the laboratory- and 2) to examine 365 366 Fernet et al. the link between lipid class composition of gonads and eggs with performance during early development (number of eggs released, growth, and survival), MATERIAL AND METHODS Animal Maintenance This study was conducted at the experimental hatchery of Min- istere de 1' Agriculture, des Pecheries et de TAlimentation du Quebec at Grande-Riviere (Gaspe coast, Quebec, Canada). Male and female adult scallops of comparable size (110 cm ± 10 cm) were harvested by SCUBA diving at the end of May 2001 at two sites on Gaspe coast: Perce (Si) at a depth of 30 m and Pointe Saint-Pierre (S2) at a depth of 20 m (48 °30'N; 65 °15'0). Labo- ratory held animals were maintained in a flow-through sea water system (28 ppt) and fed continuously with living microalgae. The latter were produced semicontinuously in the f/2 nutrient mixture (Guillard 1975). Temperature was maintained at 8 °C from the beginning of the experiment until July 9th and temperature fluc- tuated between 10 and 13 "C until spawning. Photoperiod was set at a constant cycle 16:8 (light/dark). Spawning was induced by thermal rise to 16 °C and mechanical shock using air-lift systems. Fertilization was made with a mixture of sperm at ca. 10 sperm to each oocyte. The fertilized eggs were left undisturbed in 1000 L Xactic® tanks (one tank per treatment) at 12-14 °C. Swimming embryos were then siphoned 24 h later into another tank, where they were maintained in suspension with a light bubbling. Four days after fertilization, as D-veligers emerged, water was poured through a 20-|xm pore mesh, and the tank was cleaned. Larvae were reared until day 8 at a density of 1.5 larvae per niL. Experimental Design Adult Conditioning and Field Sampling Adult scallops collected in the bay of Perce were fed from the beginning of June until mid-July 2001 with three artificial diets June V July (Fig. 1 ). The daily dry mass of algal ration was adjusted to 4% of scallop dry mass for each adult diet. Based on preliminary measurements, mean dry mass was assumed to be ca. 25 pg celP' for Isochiysis sp., Chaetoceros inuelleri, and Pavlova lutheri and 150 pg cell"' for Skeletonema costatum. Adult diet A was a mix of hochi-ysis sp. (clone T-iso), P. lutheri. S. costatum (40/40/20 cells), adult diet B consisted of the standard mix of Isochiysis sp., P. lutheri. S. costatum. and C. muelleri (25/25/25/25 cells) and, finally, adult diet C was made of Isochiysis sp. and C. muelleri (25/75 cells). The adult scallops harvested at Pointe Saint-Pierre were conditioned from mid-July to mid-August with the standard diet B (treatment S2B, Fig. 1 ). Microalgae were harvested every 3 to 4 days for lipid class (n = 15) and fatty acid (« = 5) analyses during the entire feeding period. Adults maintained in the labora- tory on the three artificial diets were periodically sampled in trip- licate and their gonads analyzed from the beginning of the experi- ment (early June, t^) during vitellogenesis (early July, t,) until spawning (mid-July, t;). Adults living in the wild at the two sites were sampled at t,,, t,. and t, but also later during the reproductive cycle (mid-August, t, and mid-September, ly. Fig. 1 ). Three pieces of ca. 100 mg of gonad were collected to perform lipid class (both sex) and histological analyses (females only). Spawning Induction and Larval Rearing Females from SI -fed diets A, B, C, and females from S2 were induced to spawn in separate buckets in mid-July and females from treatment S2B were induced to spawn in mid-August (Fig. 1). Wild animals were induced to spawn right after arrival in the laboratory. Number of spawning females varied from two to five per treatment. Eggs of each female were counted and sampled separately for lipid class analysis and size measurement. Egg fer- tilization was conducted with a mixture of spermatozoa from three to five males per treatment. Individual spawnings were pooled in one tank per treatment for fecundation and embryonic develop- Aug. h Sept. LAB 3 diets FIELD 2 sites SI- 82- FIELEH-LAB S2B 1 site+l diet 0 ■D t Gametogenesis D Spawning Larval rearing i Sampling period f Larval sampling (day) : 0 4 Figure 1. Experimental design. \ Biochemical Indicator of Sea Scallop Broodstock Quality 367 ment fno treatment replication). Each group of 4-day-old larvae was separated among three tanks of ca. 200 to 400 L and fed different diets: a) /.mchiysis sp. and Pavlova lutheri (50/50 cells). b) Isochnsis sp. and Chaetoceros imielleri (50/50 cells), and c) Isochnsis sp. with C. muelteri grown under silicate deprivation to enhance TAG accumulation (50/50 cells). Larvae were sampled for lipid analysis, density and growth measurements on day 4 and 8 after fertilization. Laboratory Analysis Sample Collection Samples of 10 mL microalgal culture, 10,000 eggs, and 5000 larvae were filtered on prebaked GF/C filters at 450 °C and stored in 1 mL of dichloromelhane in amber glass vials with Teflon liner caps under nitrogen at -20 C until lipid extraction. Gonad samples for lipid analysis (ca. 100 mg) were directly stored in dichloromelhane. Gonad samples for histologic analyses were stored at room temperature in Helly's fixative. Finally, samples of eggs and larvae used for size measurements were stored in 10'7f formaldehyde. Lipid Extraction Lipids were extracted after a 4-day to 1-mo storage period. Microalgae. egg, and larvae samples were first sonicated three times in 1.5 mL of CHXL-MeOH (2:1; v/v) in an ice bath to remove the organisms from the filter. Gonads were ground in 6 mL of CH,CK-MeOH (2:1: v/v). KCl (0.88'7f) was added to the pre- vious solution to obtain CHXL-MeOH-KCl (2: 1 :0.6; v/v/v; Folch et al. 1957). The homogenates were mixed and centrifuged at 4000 rpm for 2 min to obtain a biphasic system. The lipid fraction (lower phase) was removed and transferred to a clean tube. The solvent was evaporated under a nitrogen fiow and lipids suspended in 0.05, 0.1. or 1 niL CH-,CU for eggs and larvae, microalgae, or gonads, respectively. Lipid extracts of microalgae were fractionated in two aliquots to analyze lipid classes and fatty acid. Manipulations were carried out on ice and under nitrogen whenever possible. Lipid Class Composition Lipids (0.5% to 10% of total extraction depending on sample tissue) were spotted onto the S-lII Chromarods (latron Laborato- ries Inc., Tokyo, Japan) using a Hamilton syringe. Four different solvent systems were used to obtain three chromatograms per rod according to Parrish (1987). This method separates aliphatic hy- drocarbons (HCs), ketones (KETs), TAGs, free fatty acids (FFAs), free fatty alcohol (ALCs), free sterols (STs), diglycerides (DCs), acetone mobile polar lipids (AMPLs), and phospholipids (PL). Between each development, Chromarods were scanned by the flame ionization detection system of the analyser latroscan Mark-V (latron Laboratories Inc., Tokyo, Japan). Lipid classes were identified and quantified with the use of standard calibration curves obtained for each lipid class. The load applied to the rod ranged from 0.05 to 5.9 |xg. Within each set of rods, one was used for the lipid standard and another one for extraction blank. Fatty Acid Composition Lipid extract of microalgae was analyzed by gas chromatogra- phy. Fatty acid methyl ester (FAME) were prepared from about 0.2 mg of the total lipids following the method of the American Oil Chemists" Society using BF,/CH,OH (12%; AOCS, 1989). FAME were suspended in 40 (xL of hexane, and a 2-p,L aliquot was injected with a 1:37 split in a Perkin Elmer Sigma 300 capillary chromatograph, equipped with a Supelco Omegawax™ 320 fused- silica capillary column 30 m x 0.32 mm x 0.25 ixm ID. The following chromatographic conditions were used: 190 °C for 20 min, followed by an increase of 4 °C min"' to 210 °C for 25 min, followed by an increase of 5 °C min"' to 240 °C for 5 min. Helium was the carrier gas at a flow rate of 2 mL min"'. The gas chro- matograph was equipped with flame ionization detectors and the integrator software Varian Star Chromatography Workstation 5.51. FAME were identified by their retention times compared with standard (Supelco 37 component FAME Mix, Menhaden Fish Oil and PUFA-3, Supelco Bellefonte, PA) and quantified with tricosanoic acid (c23:0) as an internal standard. Notation used in fatty acid identification is L:BnX where L is the chain length, B is the number of double bonds and nX is the position of the double bond closest to the terminal methyl group. Histology Female gonads were dissected and stored in Helly's fixative. After rinsing, the samples were dehydrated through an ascending alcohol series, cleaned in toluene and embedded in paraffin. Speci- mens were sectioned (6 |j.m m thickness) and stained with Harris hematein and eosin. Examination of gonad sections was made using a compound microscope at a magnification of 40x with an image capture kit CoolSNAP-Pro cf Digital Kit™ 4.1. Percentage area of gonads occupied by mature and atresic eggs and size dis- tribution of eggs for each sampling date were measured with Im- age-pro plus® 4. 1 .0 package software. Eggs were considered ma- ture when stalked or ripe. Eggs with a much deformed appearance (jigsaw-puzzle shapes) were considered as atresic (Dorange 1989). Three counts were made for each tissue section and one tissue section was examined per individual. Growth Measurements Shell size was calculated as the average of the length (anterior- posterior distance) and height (dorsal-ventral distance) of larvae. Larvae were measured using a compound microscope (magnifica- tion of 40x) with an image capture kit CoolSNAP-Pro cf Disital Kit"! 4.1. Data Analysis Diet composition, in terms of lipid class and fatty acids, was submitted to one-way multiple analysis of variance (MANOVA). Fatty acids were grouped as saturated (SEA), monounsaturated (MUFA), and polyunsaturated fatty acids (PUFA). Among PUFA, 20:5n3 (eicosapentaenoic acid. EPA), 22:6n3 (docosahexaenoic acid, DMA), total n3 and n6 and finally n3-n6 ratios were distin- guished. As the /; value was smaller than the number of fatty acids (independent variables), there were not enough degrees of freedom to apply MANOVA without data grouping. Lipid class composition and histology of gonads depending on treatment (diets A, B, and C and sites SI, S2) and time (from early June, t„ to mid-July, t,), were investigated using two-way MANOVA by sex. The total sum of squares was partitioned be- cause of the asymmetric experimental design (Underwood 1997). At t(„ there were only two treatments (SI, in which A, B, and C were confounded and S2) whereas at t, and t^, there were five distinct treatments. Consequently, contrasts were carried out be- tween control and experimental treatments (t^, vs. t,) and then a 368 Pernet et al. two-way MANOVA was run among experimental treatments at t, and t,. Site-specific effects on the independent variables (lipid class composition and histology of gonads) from t, to tj were investigated using a two-way MANOVA by sex. It was not pos- sible to perform an overall analysis due to the fact that animals in the laboratory were not sampled from t^ to tj. then leading to an asymmetric design (Fig. 1 ). One-way MANOVA was used to investigate treatment effects on lipid class content and histological data of ovaries prior to spawning. Each individual was considered as an experimental unit (n = 3 per treatment). One-way MANOVA was used to investigate treatment effects on lipid class content, size and quantity of eggs produced by adults. Each batch of eggs produced per female was considered as an experimental unit (n = 2 to 5 per treatment). The number of replicates among adult feeding regimes differed. Age effects on lipid class content and growth from early em- bryogenesis until day 8 were analyzed using one-way MANOVA. As previously mentioned, individual spawnings were pooled in one tank for fecundation and embryonic development. The pooled eggs and larvae from each treatment were considered as experi- mental units {II = 5). Values of lipid class content and size of the pooled eggs used for fecundation were obtained by weighing the contribution of each female to the total number of eggs in the group (individual fecundity). This is a means to assess the initial composition of the pooled eggs, allowing their inclusion in the data analysis. Finally, lipid class composition, quality, size and survival of 8-day-old larvae were subjected to one-way MANOVA to deter- mine larval diet effects (/; = 3). When overall differences were detected. Least-square means multiple comparison tests (LSMean. SAS Institute Inc. 1999- 2000. Gary, NO were used to determine which means were sig- nificantly different. Probability levels were divided by the number of degrees of freedom of the tested factor (Bonferroni correction ). Homoscedasticity was tested using Levene's test and was con- firmed by graphical examination of the residuals (Sherrer 1984). A stepwise multiple regression was used to examine the rela- tionships between histological data as response variable [% area of gonads occupied by both mature and atresic eggs and egg diam- eter) and lipid class content as explanatory variable (/! = 49). Another model was used to assess the relation between spawning performance as response variable (number and size of eggs at release and hatching success) and gonad histological data (egg maturity, atresia and size) and lipid composition as explanatory variable. Finally, a stepwise multiple regression was also used to examine the relationship between larval performance as response variable (growth and survival) and egg lipid composition as ex- planatory variable (n = 5). A significant threshold of 0.05 was adopted for all statistical tests. All statistical analyses were carried out using SAS 8.01 (SAS institute Inc. 1999-2000). RESULTS Lipid Class and Fatty Acid Composition of Diet Diets A. B. and C fed to giant scallop contained respectively 88.31. 74.67, and 129.88 mg of lipid per g of algal dry mass {P < 0.001). Lipid class composition of diets A and B differed from diet C particularly in TAG {P < 0.001, Fig. 2). TAG content of diet C was 100 and 10 times higher than that of diets A and B respectively. Diet C contained significantly higher level of MUFA O < O 5 u. J P u. ^ Lipid class Figure 2. Mass of each lipid class (±SD, n = 14) expressed In mg per g dry mass in diet A (D). B (D ), and C (■) (For compositions of diet, see text). Lipid classes detected were wax estser (WE), ketone (KET), triacylglycerol (T.\G). free fatty acid (FF.\), fatty alcohol (.\LC). cholesterol (ST), acetone mobile polar lipid (.\MPL), and phospho- lipid (PL). and PUFA than diets A and B. and differences of PUFA were mainly attributed to n6 fatty acid (Table I ). Values of 20:5n3 and 22:6n3 did not differ among diets (P = 0.061 and P = 0.082, respectively). Gonad Lipid Class Content and Maturation General Pattern We examined the influence of three diets in the laboratory and two sites in the field on ovarian lipids and histology from vitello- genesis until the resorption period. Based on the percentage of mature eggs and egg diameter measurements, the first major spawning probably occurred at the beginning of July (t, ) and lasted until the beginning of August (t,. Fig. 3). Egg size increased sig- nificantly from t(, to t| and reached a maximum in mid-July (t,). Thereafter, egg maturity and size gradually decreased in gonads until end of September (tj). Adults reared in the laboratory dis- played partial spawning at t, and were induced to spawn at t, (July II. 12, and 15 for diets B, A. and C, respectively). Total lipid level (TL) of female gonad varied from 6-16% dry mass during the experiinental period (Fig. 4f). The highest level of TL was observed at the end of vitellogenesis (t, ), followed imme- diately by a sharp decrease during spawning (t, to t,, P = 0.008, Fig. 4f). TL remained low until the end of the experiment. In male gonad, TL increased from tg to t, (Fig. 41). The gonad lipid fraction varied between 4.5 to 7.5% dry mass. This suggests that, during gametogenesis. lipids were highly solicited in females but only moderately in males. TAG explained 84% of the variability of TL in female gonads, whereas PL explained 65% of the variability of TL in male gonad during the course of experiment. Time, Treatment, and Site-Specific Effects on Ovaries Mature eggs in the female gonads varied in time depending on the diet. There was a highly significant time effect from t,, (end of May) to t| (beginning July, P = 0.003) and a significant interac- tion of treatment and time [from t, to t, (mid-July)J (P < 0.001, Biochemical Indicator of Sea Scallop Broodstock Quality 369 TABLE 1. Fatty acid mass (nig g ' dry mass) and % (relative to the sum of fatty acid mass) in adult diets A ilsochrysis sp., Pavlova liilheri, and Skeletonema costaliim 40/40/20), B [Isochrysis sp., P. lutheri. S. coslatiim, and Chaeloceros muelleri 25/25/25/25), and C. ilsochrysis sp. and C. muelleri 25/15). ii = S. Diet A Diet B Diet C Fatty Acid Mass % Mass % Mass % 12:0 14:0 14:ln5 l.'5:0 16:0 I6:ln7 16:2n4 16:3n4 18:0 lS:ln9 IS:ln7 18:2n6 18:3n6 I8:3n3 I8:4n3 20:0 20:ln9 21:0 20:4n6 20:4n3 20:5n3 22:0 22:ln9 21:5n3 22:5n3 24:0 22:6n3 0.05 5.63 0.19 0.16 3.22 3.22 0.91 3.68 0.24 2.30 0.73 3.13 0.23 1.67 2.66 0.02 0.00 0.00 0.21 0.01 6.80 0.06 0.00 0.00 0.00 0.00 3.99 0.12 14.40 0.49 0.40 8.22 8.24 2.34 9.40 0.62 5.89 1.85 8.00 0.58 4.27 6.80 0.06 0.00 0.00 0.54 0.02 17.40 0.15 0.00 0.00 0.00 0.00 10.20 0.09 4.66 0.18 0.17 3.10 5.21 1.14 4.56 0.31 1 .55 0.59 2.02 0.26 1.03 1.75 0.00 0.00 0.00 0.46 0.01 7.13 0.07 0.00 0.00 0.02 0.00 2.86 0.23 12..54 0.48 0.47 HM 14.01 3.(J6 12.27 0.84 4.17 1.60 5.43 (171 2.77 4.70 0.00 0.00 0.00 1.23 0.02 19.18 0.18 ().()() 0.00 0.06 0.00 7.70 0.05 10.80 0.38 0.44 8.01 15.36 2.21 7.40 0.67 3.11 1.39 4.03 0.8! 2.01 3.05 0.08 0.07 0.00 1.73 0.02 12.65 0.07 0.02 0.00 0.05 0.01 4.27 0.06 13.73 0.48 0.56 10.18 19.52 2.80 9.41 0.86 3.95 1.76 5.13 1.03 2.56 3.88 0.10 0.08 0.00 2.19 0.03 16.07 0.09 0.02 0.00 0.06 0.02 5.43 ISFA IMUFA IPUFA Xn3 Vn6 In3/In6 9.38 6.44 23.29 15.13 3.57 4.91 23.98 16.47 59.55 38.68 9.12 8.40 7.53 21.24 12.80 2.74 5.27 22.61 20.26 57.14 34.43 7.37 20.14 20.31 38.22 22.05 6.56 ^.^3 25.60 25.82 48.58 28.03 8.35 Fig. 3). At t|. animals fed diet C showed a higher maturity than those fed diets A and B. Scallops in the field showed maturity levels between those fed diets A and B. At t,, the maturity of field scallops and scallops fed diet B dropped to values observed at t,, the maturity of those fed diet C remained high whereas that of scallops fed diet A increased significantly to reach the le\el of those fed diet B at t,. Thus, scallops maintained in the laboratory initiated vitellogenesis faster than those in the field, and those fed diet C matured more quickly and completely than those fed diets A and B. Thus, scallops fed diet B and adults maintained in the field started to spawn at t,. earlier than adults fed diets A or C with lower maturity levels. Levels of atresia in female gonads showed significant time and treatment effects. In fact, percentage area of gonads occupied by atresic eggs increased from t^, to t, {P = 0.019) and decreased markedly between t, and t, (P = 0.023), except for scallops from Pointe Saint-Pierre (S2). The lowest level of atresia was observed in scallops fed diet C. There was no site effect on maturity and atresia levels in female gonads. Scallops 3 (0 E O) LU E ■Jo O) tu - — • — -S1 — ■ — -S2 s? 30- - - -a- ' -A Q - - -A - -B (A 9 20 - a-A---r . - O- - -C L. ,-5 / \ ■--, ■ - * - -S2B < 10 J • ' ' J ^' V L T ' ' y^ .^ A\. n J ^'-l^^^^ ^--^^^-^ i^~ May 9 June 8 July 8 Aug 7 Sept 6 Oct 6 Figure 3. Female giant scallop maturity, egg size (mean diameter), and atresia as a function of time and treatment (±SD, n = 3). Broodstock in the field (normal line) at Perce (SI, #), Pointe Saint-Pierre (S2. ■), and in the laboratory (dashed line) fed with diet A (D), B (open triangle), and C (O) and Pointe Saint-Pierre-fed diet B since mid-July (S2B, ■). from Perce (SI) had smaller eggs than those from Pointe Saint- Pierre (S2; Fig. 3). In ovaries, TAG levels sharply increased at the end of vitello- genesis. between t,, to t, (P = 0.005). and dropped sharply during the spawning period, from t, to t, {P = 0.016. Fig. 4a). TAG levels gradually decreased from t, to tj {P < 0.001 ). Levels of TAG were highly variable depending on treatment and time. From t, to t,, scallops from Perce (SI), and those fed diets A and C had significantly higher TAG content than those fed diet B. scallops from Pointe Saint-Pierre (S2) exhibiting intermediate levels. Fi- nally, ovarian TAG levels were influenced by location since there were significant interactions of site x time (P = 0.03). In fact. 370 Pernet et al. Ovary (A (fl 19 E ■D a May 9 June 8 July 8 Aug 7 Sept 6 Oct 6 May 9 June 8 July 8 Aug 7 Sept 6 Oct 6 — S1 -82 Time B S2B Figure 4. Mass of each lipid class expressed in mg per 100 mg dry mass ( % ) in ovary (left) and testis (right) as a function of time and treatment (±SD, n = 3). Broodstock in the field (normal line) at Perce (SI, #1, Pointe Saint-Pierre (S2. ■). and in the laboratory (dashed line) fed with diet A (D), B (open triangle), and C (C) and Pointe Saint-Pierre-fed diet B since mid-July (S2B. ■). Lipid classes detected were TAG, FFA, ST, AMPL, and PL. TL were obtained by summation of each lipid class. scallops from Pointe Saint-Pierre (S2) did not exhibit a significant gonadal TAG increment as observed in scallops from Perce (SI ) or those reared in the laboratory. TAG maxima occurred at different times depending on site (t, for SI and t, for S2). FFAs were a minor lipid class in female gonads accounting for ca. 6% of total lipid (Fig. 4b). In fact, there was no accumulation or depletion of FFAs from tg to t, but FFAs markedly increased from t, to ti in all the treatments. Thereafter, FFA levels gradually decreased toward initial values until t4 except for scallops from S 1 . ST content decreased from t„ to t, {P = 0.003), reaching the lowest value at t, (Fig. 4c). Values of ST measured at t, and tj were intermediate between t, and ty, suggesting a slow recovery after spawning. ST were affected by treatment in such a way that scallops fed diet B exhibited the highest level, those fed diets A and C and from Perce (SI) were intermediate and scallops from Pointe Saint-Pierre (S2) were the lowest. Moreover, a site effect was detected on ST because levels in scallop gonads from 81 were lower than those from 82 (P = 0.012). Biochemical Indicator of Sea Scallop Broodstock Quality 371 AMPL. mainly glycolipids. pigments and remaining neutral lipids, gradually increased during the period of study (P < O.OOl. Fig. 4d|. There was no significant effect of treatment but a sig- nificant interaction of time x site (P = 0.008). PL content of gonads gradually decreased during the study as a function of treat- ment and site (Fig. 4e). Particularly, scallops from Pointe Saint- Pierre (S2) showed a pronounced drop from t, to t, (P = 0.014). TL decreased throughout the spawning period, trom t, to tj (Fig. 4f). Time, Treatment, and Site-Specific Effects on Testes Lipid class composition of male gonad consisted mainly of structural lipids, such as ST and PL (ca. 88% of TL). TAG were consistently low (Fig. 4g). FFA were a minor lipid class in testes and accounted for ca. 2% of total lipids (Fig. 4h). There was a significant interaction of time and site on FFA content ( P < 0.00 1 ). but no clear pattern emerged. ST content increased from t„ to t, (P = 0.028) but subsequently was not affected by time, diet, or site (Fig. 4i). AMPL gradually increased all over the period of study with no significant effect of diet or site (Fig. 4j). The AMPL pattern observed in testes was similar to that in ovaries (student f-test, P = 0.487). PL increased before spawning from to to t, (P < 0.001 ) and decreased markedly from t, to t^ (Fig. 4k). There was no significant difference in PL in the male and female gonads (student t test, P = 0.198) except that PL content in testes in- creased from t(, to t,. Finally, TL content in testes increased from t|, to t, (P < 0.001 ) and gradually decreased until t4 as for PL (Fig. 41). TL was also infiuenced by site since values in scallops from Perce (SI) were lower than those from Pointe Saint-Pierre (S2, P = 0.041). Lipid Class Composition and Histological Analyses A stepwise multiple regression was conducted to examine the relationship between lipid class and female maturity (Table 2). The model, including TAG and FFA. explained 61.7% of the variabil- ity of maturity. Based on this model, increasing ovarian TAG increases maturity level. TAG" only explained 16.7% of the vari- ability of maturity, but it indicates that TAG values above a certain threshold are linked with lower maturity. TAG alone explained 56.4% of the variability of maturity. Variability of atresia in fe- male gonads was weakly linked with ST (r^ = 0.12). The model suggests that atresia increases as ST decreases. Finally, egg size was related to TAG and ST. The model suggests that egg size increases with TAG until a threshold (quadratic effect) and also increases as ST decreases. However, the influence of ST (partial r = 0.03) was negligible compared with that of TAG (partial r- = 0.68). Lipid Class Content, Growth, and Survival During Embryonic and Early Larval Development Females fed diets A. B. C. and originating from Pointe Saint- Pierre (S2) were induced to spawn in mid-July (t,) and those from S2B in mid- August (t,). Lipid composition and maturity of gonads collected prior to spawning differed markedly in relation to treat- ment. Significant effects were detected in FFA (P < 0.001). ST (j)=0.Q\5). and PL (P = 0.035). Individual fed diet C showed the highest maturity, followed by those fed diet A and finally those from S2. S2B and B (P < 0.001). It seemed that broodstock con- ditioning just after the first major spawning (S2B) maintained but did not increase maturity (Fig. 3). TABLE 2. Stepwise multiple regression analysis using lipid class composition of female gonads as explanatory variables and maturity, atresia, and size of eggs in gonad as response variables, n = 49. Maturity Regression Equation \ = 4.4.1 + 7.89 X X FFA, r- = 0.617, f TAG - 0.47 X < 0.000 TAG- - 8.04 Regression Step No. Source of Variance Partial r^ P Value 1 2 TAG TAG^ FFA 0.396 0.167 0.052 <.000 <.000 0.015 Atresia Regression Equation Y = 18..39 - 27.08 X ST, r= = 0.1 20. P = 0.014 Regression Step No. Source of Variance Partial r' P Value 1 ST 0.120 0.014 Egg Size Regression Equation Y = 31.70 + 4.87 X xST, r- = 0.713, f TAG - 0.. < 0.000 19 X TAG' - 10.34 Regression Step No. Source of Variance Partial r P Value 1 2 3 TAG TAG' ST 0.473 0.210 0.030 <.000 <.000 0.034 There was no effect of the treatment on lipid composition of eggs (except in FFA. P< 0.001, Fig. 5) and on the number of eggs released (Table 3. P= 0.366). In contrast, egg size varied according to the treatment (P = 0.004) and was correlated with size of 8-day-old larvae (r = 0.82, P = 0.035). Eggs released by fe- O) 6 O) 507f larvae had devel- oped visible eyes). Larvae were allowed to settle until day 40. Swimming larvae were harvested at day 4. 8. 12. 20. 28. 32. 36. and 40 after fertilization for growth, survival, and lipid class analy- sis whereas settled larvae were sampled at the end of the experi- ment (day 40). Behavior of individual pediveliger larvae on day 36 and 40 for each diet treatment and replicate culture was recorded on videotape using an endoscopic camera (see below). Endoscopic recorded behavior were categorized as follows: ( 1) larvae actively exploring or (2) not moving, remaining attached to the screen, (3) actively swimming, or (4) passive in the water column. Based on these observations, a time budget (relative time spent by each larvae exhibiting a specific behavior) was determined. Exploration distance and exploration rate (distance. time"') were measured for each larvae as well. The complete experiment was repeated from mid-August to September, without the behavioral aspects. However, the second experiment was conducted without replicates because of the poor Laboratory Analysis Shell size was average of length (anterior-posterior distance) and height (dorsal-ventral distance) of at least 30 larvae. Larvae were measured using a compound microscope at a magnification of 40x with image capture kit CoolSNAP-Pro cf Digital Kit ' ^' 4. 1 . Lipid analysis was conducted as previously described (Pernet et al. 2003). Briefly, 5000 veliger larvae or 500 pediveligers were filtered on prebaked GF/C filters and stored in dichloromethane at -20°C until lipid extraction. Samples were sonicated and lipids were extracted according to Folch et al. (1957). Then, lipids were spotted onto the S-III Chromarods (latron Laboratories Inc., To- kyo, Japan) and separated according to Parrish (1987). Lipid classes were quantified with the analyser latroscan Mark-V (latron Laboratories Inc., Tokyo, Japan). Fatty acid composition of mi- croalgae was analyzed by gas chromatography. Fatty acid methyl ester (FAME) were prepared following the method of the Ameri- can Oil Chemists' Society (AOCS 1989) and injected in a Perkin Elmer Sigma 300 capillary chromatograph, equipped with a Su- pelco Omegawax^''' 320 fused-silica capillary column. FAMEs o> E in (0 rs EXP I 130 70 10 i aab aaa aaa aoi J EXP aaa aaa ^ aaa ^ i aab 1 I J aaa M Q. 2 -1 Q. ID h- lU < Lipid class < O -J < CO a. < Figure 1. Mass of each lipid class (±SD, n = 9 and h = 6 for experiments I and II, respectively ) expressed in mg per g dry mass in diet A {Isochrysis sp. + Pavlova lutheri. □), B (Isochrysis sp. + Chaetoceros muelleri. D ). and C (Isochrysis sp. + Chaetoceros muelleri with silicate deprivation, ■) during experiment I and 11. Lipid classes detected were wax ester (WE), ketone (KET), triacylglycerol (TAG), free fatty acid (FFA), fatty alcohol (ALC), cholesterol (ST), acetone mobile polar lipid (.\.MPL), and phospholipid (PL). Biochemical Indicator of Sea Scallop Larvae 379 were identified by their retention times compared with standard (Supelco 37 component FAME Mix. Menhaden Fish Oil and PUFA-3, Supelco, Bellefonte, PA| and quanlilied with tricosanoic acid (c23:0) as an internal standard. The methodology used to examine e.\pk)ratory behavior of competent larvae was previously described (Walters et al. 1999). The equipment used was an Olympus K 17- 1 8-90 endoscope (1.7 mm diameter. 186 mm length) attached to a video camera fixed to a micromanipulator arm, allowing two-dimensional movement. Video output was sent to a video 8 mm integrated to a monitor (Optiscan lUS-l ). An Olympus 250 W high-intensity xenon light source (model ILV-2) provided cold light to the extremity of the endoscope. Behavioral observations were performed on swimming larvae sampled in settling tank during water renewal on day 36 and 40 for diets B and C. These larvae were carefully transferred to circular tank type "Plankton Kreisel." A two-dimensional (23 x 16.5 cm) polypropylene collector (200-(xm pore mesh) was placed perpendicular to the tlow (ca. 1 cm s '). Observation of larval behavior in each trial lasted until the larvae had stopped movement for 5 min or was out of the field covered by the camera (5.5 x 3.5 cm). Between each recording, collector was shaken and rinsed to detach larvae and new larvae were injected into the system. Be- havioral observations lasted for average 6.3 min, at least 1 min to a maximum of 33 min. Data Analysis The lipid class (n = 15) and fatty acid compositions (/i = 5) of the diets were compared by one-way multiple analysis of vari- ance (MANOVA). Fatty acid were grouped in saturated (SFA), monounsaturated (MUFA). and polyunsaturated fatty acids (PUFAs). Among PUFAs, 20:5n3 (eicosapentaenoic acid, EPA), 22:6n3 (docosahexaenoic acid, DHA), total n3 and n6 and finally n3-n6 ratio were distinguished (see Pemet et al. 2003). Shell size, lipid class composition, and quality of swimming larvae depending on diet and age were investigated by two sepa- rate two-way MANOVA. Groups of larvae fed diet A were lost at day 28, leading to an unbalanced design. Then, the first analyzes included data from day 4 to 28 whereas the second analyses in- cluded data from day 28 to 40. Where differences were detected. Least Square Mean multiple comparison tests (LSMean) were used to determine which means were significantly different with prob- ability levels divided by the number of degrees of freedom of the tested factor (Bonferroni correction). Shell size, lipid class com- position, and quality of larvae depending on diet, age and behavior were investigated by one-way MANOVA. Treatments consisted of different combinations of factors: diets B or C at days 28 and 40 and. at day 40, larvae settled or swimming. Contrasts were per- formed to verify a posteriori particular effect. Exploration rate and exploration distance of larvae depending on diet and age were submitted to two-way analysis of variance. Homoscedasticity was tested by running Levene's test and was confirmed by graphical examination of the residuals (Sherrer 1984). Finally, to compare survivorship according to diet from day 4 to 28, Life Test procedures were used. When differences were detected, the x" comparison tests were applied to determine which treatments differed significantly. A significant threshold of 0.05 was adopted for all statistical tests. All statistical analyses were run in SAS 8.01 (SAS Institute Inc., 1999-2000, Gary, NG). RESULTS Lipid Class and Fatly Acid Composition of Ixinal Diet As expected, lipid class content differed among larval diets (P = 0.0221 ). TAG levels were highly different among diets (Fig. 1 ). In experiment I. diet C had higher TAG content than diets A (/> < 0.0001 ) and B (P < 0.0001 ) and diets A and B also differed {P = 0.0333). In fact, diets A. B and C contained 2.68, 8.92 and 65.11 mg.g"' algal dry mass of TAG which represent 2.32, 8.31, and 31.19% of lipid class composition respectively. Free fatty acids (FFA), cholesterol (ST), and total lipids (TL) levels were also different among diets (Fig. 1 ). During the course of experi- ment II, microalgae lipid class composition revealed nearly the same differences of TAG and total lipid content among diets. However. FFA and ST contents were the same whereas ketone levels were different (P = 0.0001 ). In short, the lipid composition of the diatom Chaetocerus mitelleri was altered by varying the silicate level in the culture medium. The TAG content of cells TABLE L Fatty acid mass (mg g"' dry mass) and % (relative to the sum of fatty acid mass) in larval diet X {Isochrysis sp. + favlora lutheri), B ihochrysis sp. + Chaetocerus mitelleri), and C {Isochrysis sp. + Chaetoceros muelleri with silicate deprivation) during experiment I and II (H =5). Fatty Acid A B C Mass % Mass % Mass % 12:0 0,07 0.09 0.07 0.08 0.20 0.09 14:0 13.29 17.24 12.01 14.80 28.80 13.16 14:ln5 0.58 0.75 0.52 0.64 0.53 0.24 15:0 0.23 0..10 0.40 0.49 1.79 0.82 16:0 7.56 9.81 9.15 11.27 53.73 24.55 16:1 117 6.06 7.87 11.18 13.77 48.37 22.10 16:2n4 0.86 1.12 1.28 1.57 2.46 1.13 16:3n4 0.42 0.55 4.44 5.46 4.42 2.02 18:0 0.24 0.32 0.98 1.21 3.67 1.68 18:ln9 4.55 5.91 4.32 5.32 6.32 2.89 18:ln7 1.20 1.56 1.38 1.70 1.77 0.81 18:2n6 3.50 4.,S4 4.76 5.87 5.45 2.49 18:3n6 0.72 0.94 0.81 0.99 6.55 2.99 18:3n3 4.33 5.61 3.44 4.24 3.48 1.59 18:4n3 1 1 .56 15.00 8.33 10.26 13.48 6.16 20:0 0.02 0.02 0.09 0.11 0.09 0.04 20:ln9 0.23 0..W 0.29 0.36 0.09 0.04 21:0 0.02 0.02 0.00 0.00 0.56 0.25 20:4n6 0.28 0.36 0.90 1.11 7.58 3.46 20:4n3 0.07 0.09 1.43 1.76 0.65 0.30 20:5n3 1043 13.53 8.26 10.17 19.47 8.89 22:0 0.06 0.08 0.16 0.19 0.38 0.17 22:ln9 0.04 0.06 0.07 0.09 0.06 0.03 21:5n3 0.00 0.00 0.00 0.00 0.00 0.00 22:5n3 0.02 0.02 0.03 0.03 0.27 0.12 24:0 0.02 0.03 0.02 0.02 0.00 0.00 22:6ii3 10.71 13.89 6.88 8.47 8.68 3.97 SSFA 21.51 27.90 22.87 28.17 89.21 40.76 IMUFA 12.68 16.45 17.77 21.88 57.14 26.11 IPUFA 42.90 55.65 40.55 49.95 72.49 33.13 In3 -17.11 48.14 28.37 34.94 46.03 21.03 In6 4.51 5.84 6.47 7.97 19.58 8.95 In3/In6 8.24 5.18 2.42 380 Pernet et al. 300 ♦ 4 8 12 16 20 24 28 32 36 40 4 8 12 16 20 24 28 32 36 40 Day Figure 2. Growth of larvae fed diet A (■), B ( ♦ ), and C (•) depending on larval stage and experiment 1 1 or III. Distinction between settled (open symbol) and plantonic (filled symbol) larvae was made at day 40 (±SD. ;i = 2 and n = 1 for experiments I and II. respectively). cultured in the silicate deprived medium was higher than those in the complete medium. Fatty acid analyses performed of diets A, B, and C during experiment I and II revealed that diets A and B did not differ (Table 1 ). However, silicate deprivation enhanced fatty acid accu- mulation in diet C with no distinction among SFAs. MUFAs, or PUFAs. Relative content of PUFAs from diets A and B (ca. 53%) differed from that of diet C (33%). As a consequence, diet C may be qualified as a high TAG-rich SFA and MUFA diet. The three diets had all essential fatty acid requirements, each of them con- taining eicosapentaenoic (EPA. 20:.'in3) and docosahe.xaenoic acid (DHA. 22:6n3). Diet C was the richest in total n3 and 20:5n3 whereas diet A exhibited the highest level of 22:6n3. DHA-EPA ratios of diets A. B and C were respectively 1.03. 0.83 and 0.45. Larval Growth and Sunival Diet and time interacted in their effects upon larval growth (P = 0.032). The difference of larval size among diets first ap- peared when larvae had reached competency at day 28 (Fig. 2). In fact, larvae fed diet C exhibited a higher mean size (220.09 |j.m) than larvae fed diet B (204.56 (xm. P = 0.0011) and diet A (200.74 p.m. P < 0.0001). There was no size difference between larvae fed diets A and B (P = 0.3960). Consequently, diet C increased larval growth by ca. 20% compared with diets A and B. However, these results were not maintained at day 40 because larvae fed diet B reached the same size as those fed diet C (re- spectively 257.16 and 248.96 |xm. P = 0.339). Furthermore, settled larvae were larger than swimming ones (253.9 vs. 23 1 jjim. P = 0.004). Larvae in experiment II showed the same tendency as in experiment I. but dietary effects seemed more pronounced (Fig. 2). In fact, mean size of larvae fed diets A, B and C at day 28 were respectively 199.29. 210.65 and 230.90 jj-m and discrepancies among size related to diets were maintained after settlement. Larvae fed diet A suffered high mortality on day 28 in both experiments (Fig. 3). In experiment I. survivorship patterns were similar between larvae fed diets B and C but larvae fed diet B seemed to have a better settlement success than those fed diet C at day 40. In experiment II. larvae fed diet C seemed to promote higher survival than those reared on diets B and A at competency. 100 > 3 (0 EXP o n r- 4 8 12 16 20 24 28 32 36 40 4 8 12 16 20 24 28 32 36 40 Day Figure 3. Survival of larvae fed diet A (■). B ( ♦ ), and C (•) depending on larval stage and experiment (I or II). The distinction between settled (open symbol) and planktonic (filled symbol) larvae was made at day 40 (±SD, h = 2 and ;i = 1 for experiments I and II, respectively). Biochemical Indicator of Sea Scallop Larvae 381 and a higher settlement success than larvae fed diet B trespectively 4.6% vs. 2.7%. Fig. 3). Lipid Class Composition During Larval Cycle From D-Veligers to Competency Age and diet interacted in their effects up(in lipid class com- position of sea scallop larvae [P = 0.0001 ). particularly. TAG, ST. PL and. as a consequence, total lipid TL (Figs. 4 and 5). FFA and acetone mobile polar lipids (AMPL) were accumulated during lar- val development without any diet effect. From the beginning of the experiment to day 20. TAG content of larvae was constant with no difference attributable to diet. TAG content of 28 days old larvae fed diets B and C rose by factors of 15 and 70 respectively, while it remained constant for larvae fed diet A (Figs. 4 and 5). Consequently, the high TAG level in diet C was correlated with a better accumulation of TAG in larvae prior to metamorphosis compared with larvae fed diet A (P < 0.0001) and B (P = 0.012). Moreover, diet B allowed a greater accumu- lation of TAG than diet A (P = 0.0007). The two structural lipids. .ST and PL followed the same trend (Fig. 4 and 5). From the beginning of larval development until day 20. ST and PL were gently rising, independently of diet. After day 20. these lipids were accumulated at different rates according to diet. PL and ST contents of larvae fed diets B and C showed a marked rise, whereas they remained low for larvae fed diet A. 25 20 -I 15 10 -I AMPL 0) t c (0 (0 flj 60 50 40 30 20 10 0 PL -T ! r- 4 3 -I 2 1 ST — , 1 1 1 1 — ~T 1 1 1— 4 8 12 16 20 24 28 32 36 40 8 12 16 20 24 28 32 36 40 Day Figure 4. Lipid class profile of larvae of experiment I fed diet A (■), B ( ^ ), and C (#) depending on larval stage. Distinction between settled (open symbol! and planktonic (filled syniboll larvae was made at day 40. Lipid classes detected were TAG. FFA, ST, AMPL, and PL. TL was obtained by summation of eacb lipid class (±SU, n = 2). 382 Pernet et al. 60 50 40 30 20 10 5.0 2.5 0.0 AMPL ^ 12 0) ra t y (9 C 6 (0 (A 3 $ 1 1 r- o - ST •- 2 /" o / o 1 - ^.^ 0 - , --t--^^^'^-^ 4 8 12 16 20 24 28 32 36 40 Day Figure 5. Lipid class profile of larvae of experiment II fed dies A (■), B ( ♦ ), and C (•) depending on larval stage (no replication). Distinction between settled (open symbol) and planktonic (filled symbol) larvae was made at day 40. lipid classes detected were TAG. FFA, ST, AMPL, and PL. TL was obtained by summation of each lipid class. FFA displayed distinct patterns compared with the other lipid class. Despite a significant time effect on FFA content. FFA were not accumulated during larval development in experiment I (Fig. 4). In fact, the level of FFA in young larvae at day 8 was the same as that of pre-competent larvae at day 28 (P = 0.16). Average FFA content was 9.01%. High levels of FFA in sample may be attributed to the presence of moribund larvae. The presence of large amounts of FFA in animal tissues (more than 10%) is usually an indication of lipid degradation and decreases in the amounts of TAG and PL. Finally, AMPL content of larvae increased gradually from day 4 to competency without any measurable effect of the feeding regimen (P = 0.543, Figs. 4 and 5). AMPL consist principally of pigments and may reflect ingestion of microalgae. From Competency to Settlement In experiment 1. TL level in larvae fed with diet B increased significantly from day 28 to 40 (P = 0.041). This effect was mainly attributable to the augmentation of PL level during this period (P = 0.009, Fig. 4). This pattern was not observed in larvae fed diet C. Levels of TAG in larvae fed diet C were higher than those fed diet B (P = 0.003). Settled larvae had higher TAG and TL content than planktimic ones (P = 0.018 and P = 0.015 Biochemical Indicator of Sea Scallop Larvae 383 respectively). However, data of experiment II showed inverse re- sult since settled larvae ted diet C had a lower TAG and TL contents than planktonic larvae (Fig. 5). Larval Quality ST and PL were highly correlated with larval size, confirming the adequacy of these structural lipids as weighting factors for the size dependency of TAG levels (Fig. 6). Thus, TAG-ST and TAG- PL ratios were used as indicators of larval quality. From D-Veligers to Competency Age and diet interacted in their effects upon TAG-ST and TAG-PL ratios (P < 0.001, Fig. 7). Between day 4 and day 20. there was no difference in either TAG-ST or TAG-PL ratios. At day 20, larvae fed diet C showed higher ratios of TAG-ST and TAG-PL than those fed diets A and B. From day 20 to day 28, just before settlement, a sharp rise of both ratiiis was observed for larvae fed diets B and C in both experiments. High TAG level in diet C suggests higher larval quality prior to metamorphosis com- pared with diets A and B. Correlation analysis showed a significant positive relation be- tween size of 28-day-old larvae and their TAG-ST and TAG-PL ratios at day 20 (Table 2). There was also a positive relation between survival of 28-day-old larvae and TAG-ST and TAG-PL ratios at day 8. The strength of this relationship gradually de- creased with age of larvae (Table 2). From Competency to Settlement TAG-ST and TAG-PL ratios of planktonic larvae did not vary significantly during settlement [P = 0.258 and P = 0.359 respec- tively. Fig. 8). Planktonic larvae fed diet B showed lower levels of TAG-ST and TAG-PL ratios (P = 0.001 and P < 0.001, respec- tively). The level of ratios of settled larvae remained constant com- pared with those observed at competency (Fig. 7). However, there was a significant effect of diet on TAG-ST and TAG-PL ratios of settled larvae. Larvae fed diet C maintained higher TAG-ST and TAG-PL ratios than larvae fed diet B. Settled and swimming lar- vae at day 40 had the same ratio values, despite different TAG levels. A negative correlation between larval quality (TAG-ST ratio) of 28-day-old individuals and settlement rate at day 40 was ap- parent (Fig. 9). The higher the TAG-ST ratio, the lower was the settlement rate. However, this relation was not significant for TAG-PL ratio. Larval Behavior During Seltlement Larval behavior time budgets (relative time spent by each lar- vae exhibiting a particular behavior) at day 36 showed no evident effect of feeding regimen or larval quality. Larvae fed diets B (low TAG-ST ratio) and C (high TAG-ST ratio) spent most of their time swimming in the water column. Active exploration of the collector consisted in ca. \6'7c of observation time whereas immobility on (0 t (A JS O a (A (A r^ = 0.88 EXP II PL ST 100 150 200 250 300 Larval size (|im) Figure 6. Relation between structural lipid a.s cholesterol (STI and phospholipids (PI, I, and larval size in experiments I and II (/; = 39 and n = 19, respectively). 384 40 n 0) 5 0 O a: 3.0 2.5 2.0 1.5 1.0 0.5 0.0 TAG/PL -1 1 1 1 1 1 1 r- 4 8 12 16 20 24 28 32 36 40 Day Figure 7. Triacvlglycerol (TAG)-cholesterol (ST) or TAG-phospholipid (PI-1 ratios of larvae-fed diet A (■), B (♦), and C (•! depending on larval stage in experiment I and II. Distinction between settled (open symbol! and planktonic (filled symbol) larvae was made at day 40 (±SD, H = 2 and n = 1 for experiments 1 and II, respectively). the screen accounted for ca. 34% of observation time of each larvae (Fig. 10). However, larvae fed diet B or C exhibited differ- ent time budgets at the end of the experiment (day 40). In fact, larvae fed diet C showed active exploration of the collector for 9'7c of the observation time whereas larvae fed diet B exhibited little exploration behavior {<2'7f ). The relative time spent immobile on the screen by larvae was higher at day 40 compared with day 36, and. at day 40, it was higher for larvae fed diet B than those fed diet C. Exploration rate of 40 day old larvae seemed to be higher than observed for 36-day-old larvae fed diet B (P < 0.05, Fig. 1 lA). Finally, exploration distance was similar for larvae fed both diets (Fig. IIB). TABLE 2. Matrix of Pearson correlation coefficients between size or survival of 12-, 20-, and 28-day-old larvae with TAG-ST and TAG-PL ratios of 8-. 12-. and 20-dav-old larvae. ■Age TAGAST TAG/PL Variable (d) 8 12 2(1 8 12 20 Shell Mze 12 -0.03 1 0.327 (jjini) 20 -0.070 -0.061 -0.023 -0.062 28 0.519 0.667 0.739 0.070 -0.056 0.734 Survival 12 0.289 -(J.0I5 (%r 20 0.449 0.427 0.4.59 0.210 28 0.706 0.543 0.313 0.760 0.476 0.309 " Based upon initial number of 4-day-old larvae. Data of experiment I and II were pooled {n = 9 larval cultures Significant probabilities are in bold {P < 0.05). DISCUSSION Lipid Composition of Larvae During the first 20 days, lipid reserves of the larvae of the sea scallop Phicopecten magelkmicus remained low (Figs. 4 and 5). A similar pattern for total lipids occurs in Japanese scallop Pati- nopecten yessoensis (Whyte et al. 1987). From late embryogenesis to 20 days, energy from food intake might be insufficient to sustain sitiiultaneously larval growth and lipid accumulation. In contrast, a continuous increase of TAG from day 3 is normal in developing larvae of the great scallop Pecten maximus and suggests that food was efficiently assimilated (Delaunay et al. 1992). Thus, the ob- served pattern in sea scallop larvae might reflect a lag in the metabolic and digestive processes of food assimilation and storage. From day 20 to 28, TAG and structural lipids accumulated as the larvae reached pre-metamorphic condition. During this period, dietary sources of energy were directed toward growth, develop- > Biochemical Indicator of Sea Scallop Larvae EXP I 385 TAG/PL 28 32 36 40 28 32 36 40 Day Figure 8. Triacjlglyccrol (TAG)-cholesterol (ST) and TAG-phospholipid (PL) ratios of competent planlvtonic larvae fed diet B (0) and C (O) depending on age in experiments I and II (±SD. ;i = 2 and h = I for experiments I and II, respectively). nient iif primary gill filaments and foot, as well as storage to meet the energy demand for metamorphosis (Whyte et al.. 1987). Finally, during settlement, from day 28 to 40, lipid levels re- mained stable. These results contrast with the low lipid levels following metamorphosis of oysters (Holland and Spencer, 1973; Labarta et al., 1999), scallops (Whyte et al. 1992) and barnacles (Lucas et al. 1979). Three reasons might be evoked to explain this pattern. Firstly, a low level of lipid reserves following metamor- phosis of marine invertebrates is still debated. For example, lipids provided 59.3'Jf of energy needs during metamorphosis of Ostreci echilis (Holland and Spencer. 1973) whereas another study showed that metamorphosis of the same species was fuelled mainly by 50 0 r^0.66,p =0.049 £^ 40 J ♦ ** c • oi \^ E 30 - ^N^ a> \s^ S N. 20 - 10 - ♦ X,^^ r^=0.30,p =0.259 10 20 30 40 0 1 TAG/ST TAG/PL Figure 9. Triacylglycerol (TAO-cholesterol (ST) and TAG-pholpholipid (PL) ratios of larvae fed diet B ( ♦ ) and C (•) at competency (day 28) as a function of ttie settlement success. Settlement success is tile percentage of settled larvae at day 40 based of the number of competent larvae at day 28. Data of experiments I and II were pooled (h = 6 larval culture). 386 Pernet et al. Diets DietC Day 36 TAG/ST= 9.69 TAG/PL= 0.79 N=2S 36% 16% 44% TAG/ST= 16.67 TAG/PL=1.63 N=19 32% 15% 47% Day 40 TAG/ST= 8.04 T.AG/PL=0,54 N=8 TAG/ST= 23.43 TAG/PL= 1.55 N=27 53%l 11% 83% 34% Figure 10. Time budgets for sea scallop larvae according to diet and time. Recorded behavior were larvae actively crawling (D) or remain- ing fixed on the screen <■) swimming (■ ) or passive in the water column {[J )■ Each pie chart represents the proportion of observation time spent by each larva exhibiting a specific behavior. (n:2 replicate culture) proteins, lipids accounting for only 16.8% (Rodriguez et al. 1990). Secondly, low TAG levels have been observed in newly settled larvae (Holland & Spencer 1973). In our experiments, larvae may not have been as newly settled as necessary to observe a TAG depletion. Lipid analysis were performed on sample of ca. 5000 larvae containing settled larvae of different ages. Thus, some of them may have started recovering energy reserves and increasing the mean lipid level of the cohort. For instance, post-metamorphic larvae of O. ediilis had recovered pre-metamorphic neutral lipid mass 4 to 11 days after settlement (Holland & Spencer 1973). Finally, sea scallop larvae may continue to feed on microalgae during settlement and counterbalance the lipid utilization. In fact, the oyster Crassostrea rirginica has the ability to feed during settlement and metamorphosis (Baker & Mann 1994). The feeding hypothesis agrees with the maintenance of dietary differences in TAG levels during settlement. Effects of Dietary Triglyceride Enrichment The diet enriched in TAG promoted higher larval growth (Fig. 2). Chaetoceros muelleri grown under silicate limited conditions leads to the highest growth rate of juvenile oysters at low feeding rations whereas at higher feeding rations, the silicate limited cul- ture was a poorer diet than the control culture (Enright et al. 1986). The high level of calorie-rich SFAs and MUFAs in the silicate- limited diet was evoked by the authors to explain higher growth rates of the oysters at the lowest feeding ration. At the higher feeding ration, the fatty acid composition, and particularly the relatively low content of 22:6n3 became a limiting factor and might explain the lower growth rate of oyster larvae fed with the silicate limited cells. In our experiments, high growth rates were obtained with a diet including silicate limited culture of C. muelleri (diet C) probably because SFA and MUFA were 4 times higher and PUFA were also twice higher than in diets A and B. Abnormally high mortality was observed for larvae fed diet A in both experiments and was correlated with extremely low TAG and low total lipid levels in diets and larvae. This agrees with previous studies showing a positive relation between larval lipid content and survival (Gallager et al. 1986, Delaunay et al. 1992, Ouellet & Taggart 1992). It seems that low lipid or TAG level in the feeding regimen might have deleterious effect on survival, whereas there was no difference in survival for the other regimes. TAG levels in competent larvae were correlated with TAG levels in diet (Figs. 4 and 5). A possible explanation is that the metabolic cost involved in TAG synthesis and storage could be lower when feeding upon microalgae rich in SFA and MUFA. TAG, or more generally, total lipid rather than with other bio- chemical sources such as protein or carbohydrate. However, con- clusions based on these observations must be tempered by the possibility of effects as a result of variations of other unmeasured variables such as digestibility or palatability due to diatom cell wall properties or biochemical compounds such as amino-acids and vitamins (see Robert & Trintignac 1997. for review). In short, we cannot argue a direct causal relationship between diet and larval lipid composition. Liinal Quality We used two indicators to assess larval quality: TAG-ST and TAG-PL ratios. During larval development prior to competency, both ratios have led to the same results and conclusions. As PL have an energetic role during early larval development or starva- tion period (Tocher et al. 1985. Fraser el al. 1988, Fraser 1989, Delaunay et al. 1992) the use of the more conservative TAG-ST ratio should be more appropriate to estimate larval quality. How- ever, PL depletion was not observed in our study of sea scallop larvae, as reported for larvae of Patinopecten yessoeiisis (Whyte 1987). This allows us to use both indicators without distinction. Based on these indicators, a positive relation between size of 28-day larvae and larval quality at day 20 was found (Table 2). It has previously been reported that poor growth was related to a low TAG-organic matter (OM) ratio in D-larvae. while higher levels of ratio were not necessarily reflected by growth rate (Delaunay et al. 1992). Consequently, it seemed that ST was a better denominator than OM. This might be due to the fact that OM values include both structural and storage molecules, thus decreasing accuracy of a storage-structure ratio. A positive relationship between number of competent larvae produced and 8 d larval quality indices was found (Table 2). This is in accordance with results of previous studies. Indeed, risks of mortality of the shrimp Pandalus borealis were well correlated with the condition indices of the larval group as measured by the proportion of larvae exhibiting a TAG-wet mass ratio <0.2 (Ouel- let & Taggart 1992). Moreover, high mortality of larvae of scallop P. maxiiHHs could be related to an abnormally low initial TAG-OM ratio (Delaunay et al. 1992). Our study suggests that survival at competency was partly explained by the efficiency of recovery of lipids after embryogenesis and. as discussed by Delaunay et al. (1992), reflects the difficulties of the transition to the D-veliger stage. Finally, a negative correlation between the quality (TAG-ST ratio) of competent larvae (28-day-old) and settlement success (at 40 days) was found (Fig. 9). The better the quality, the lower the settlement success. In the barnacle Semibalanus balanoides, it ap- pears that cyprids of high physiological condition, as measured by Biochemical Indicator of Sea Scallop Larvae 387 B36 C36 B40 C40 J^ B36 C36 840 C40 Figure 11. Exploration rate (Al and distance (B) as a I'unction of diet (B or C) and age of larvae (36 or 40 days old). Exploration rate is the distance traveled by exploring larvae (h varied from 2 to 14 as a function of treatment combination) per minute. Exploration distance is the average value of the distance traveled by all the observed larvae (;i varied from 8 to 28). Vertical bars represent standard errors between replicate cultures. a TAG-ST ratio, settle in the best quality habitats compared with those in low condition (Miron et al. 1999). In fact, TAG-ST ratio of cypiids was highest at low intertidal level, the preferred attach- ment location site. This suggests that larvae fed with rich TAG diet C and having high TAG-ST ratio might delay metamorphosis to encounter better quality settlement sites, whereas larvae fed with poor TAG diet B and having low TAG-ST ratio would not have enough reserves to make to a meticulous selection of settling site and therefore would settle more rapidly. According to this sce- nario, larvae in good condition would delay metamorphosis until reaching a critical physiologic threshold where settlement would become urgent. In support of this hypothesis, behavioral observa- tions showed that larvae fed diet C (high TAG-ST) spent 12-15% of observation time exploring the substratum whatever the age, whereas larvae fed diet B (low TAG-ST) did not explore the sub- stratum at day 40 (Fig. 10). Thus, it seemed that larvae fed diet B lost selectivity with age as observed with the barnacle Balaniis amphitrilc (Rittschof et al. 1984. Pechenik et al. 199.^. Qian & Pechenik 1998. Miron et al. 1999). Moreover, in our study, larvae spent 15% to 47% of observation time swimming in the water column. Such swimming activity in competent larvae has previ- ously been reported for the bryozoan Bugula ncritiua (25% to 45% of observation time), which was attributed to the stillness of the water (Walters et al. 1999). Similarly, substrates became attractive to the cyprids of Balamis amphitrite in flow, whereas no explora- tion was observed in individuals from the same cohort placed in still water (Miron et al. 2000). Larvae of other invertebrate species also seem to avoid settlina in low flow conditions (Mullineaux & Butman 1991. Pawlik et al. 1991). For example, larvae of the polychaete Phragmatopoma califoniica tumbled along the bottom in the presence of fast flow, whereas they were swimming in the water column in slow flow (Pawlik et al. 1991). Thus, the low settlement success observed in larvae of high quality might be the effect of delayed metamorphosis because of inappropriate hydro- dynamic conditions in our experiment. If this interpretation was correct, larval quality need to be considered jointly with hydrody- namics to fully understand the decision process of settling larvae. ACKNOWLEDGMENTS The authors thank E.-J. Arsenault and S. Bourget for their assistance in the culture of larvae and microalgae and all the staff of Centre Aquacole Marin de Grande-Riviere of Ministere de FAgriculture des Peches et de TAlimentation du Quebec for help in hatchery and laboratory. Thanks are also addressed to E. Dem- ers from Centre de Transformation des Produits Aquatiques (CTPA) for teaching GC analyses and fatty acid identification. Funding for this research has been provided by CORPAQ (Conseil des Recherches en Peche et en Agro-alimentaire du Quebec). MAPAQ and GIROQ (Groupe Interuniversitaire de Recherches Oceanographiques du Quebec). We are grateful to Dr. L. Fortier from Universite Laval, who kindly let us use the latroscan. and Dr. B. A. MacDonald from University of New Brunswick for his en- doscope camera. Thanks are also addressed to G. Daigle, Depar- tement de mathematique et statistique. Universite Laval, Quebec for validating the statistical analysis, and V. Moreau. M. Cusson and L. Lapointe for their constructive and critical discussions. REFERENCES AOCS. 1989. American Oil Chemist" Society Official method Ce lb-89. In: Official methods and recommended practices of the American Oil Chemist" Society. Champaign, IL. Baker. S. M. & R. Mann. 1994. Feeding ability during seulemeni and metamorphosis in the oyster Crassostrea virginica (Gmelin. 1 74! ) and the effects of hypoxia on post-settlement ingestion rates. / £.v/i. Mar. Biol. Ecol. 181:239-253. Bourne, N.. C. A. Hodgson & J. N. C. Whyte. 1989. A manual for scallop culture in British Columbia. Can. Tech. Report Fish. Aqiial. Sei. 1694: 216. Coombs. J. P.. P. J. Halicki. O. Holm-Hansen & B. E. Volcani. 1967. Studies on the biochemistry and fine structure of silica shell formation in diatoms. 11. Changes in concentration of nucleosidetriphosphates in silicon starvation synchrony of Noviciilii pelliciilo.m (Breb.) Hilse. E.xp. Cell. Res. 47:315-328. Culliney. J. L. 1974. Larval development of the giant scallop Placopecten magellanicus (Gmelin). Bioll. Bull. 147:321-332. Delaunay. P.. Y. Marty. J. Moal & J.-F. Samain. 1992. Growth and lipid class composition of Pecten nia.ximiis (L.) larvae grown under hatchery conditions. J. E.xp Mar. Biol. Ecol. 163:209-219. Delaunay. F.. Y. Marty. J. Moal & J.-F. Samain. 1993. The effect of monospecific algal diets on growth and fatty acid composition of Pecten ma.ximus (L.) larvae. J. Exp. Mar. Biol. Ecol. 173:163-179. Enright. C. G. F. Newkirk. J. S. Craiaie & J. D. Castell. 1986. Growth of 388 Pernet et al. juvenile Ostrea ediilis L. fed Chaeloceros gracilis Schutt of varied chemical composition. / Exp. Mar. Biol. Ecol. 96:15-26. Folch. J.. M. Lees & G. H. Sloane-Stanlez. 1957. A simple method for the isolation and purification of total lipids from animal tissues. / Biol. Chem. 226:497-509. Eraser. A. J.. J. C. Gamble & J. R. Sargent. 198S. Changes in lipid content, lipid class composition and fatty acid composition of developing eggs and unfed larvae of cod [Gadii.s morlnia). Mar. Biol. 99:307-314. Fraser, A. J. 1989. Triacylglycerol content as a condition index for fish, bivalve and crustacean larvae. J. Fish. Res. Board Can. 46:1868-1873. Gallager. S. M.. R. Mann & G. C. Sasaki. 1986. Lipid as an index of growth and viability in three species of bivalve larvae. Aquacidturf 56:81-103. Guillard. R. R. L. 1975. Culture of phytoplankton for feeding marine invertebrates. In: W. L. Smith, editor. Culture of marine invertebrate animals. New York: Plenum Press, pp. 29-60. Hakanson. J. L. 1989. Condition of larval anchovy (Engraulis mordax) in the southern California bight, as measured through lipid analysis. Mar. Biol. 102:153-159. Hakanson, J. L., Coombs. S. H. and Re, P. 1994. Lipid and elemental composition of sprat (Sprattiis spruuus) larvae at mixed and stratified sites in the German Bight of the North Sea. 51:147-154. Holland. D. L. & B. E. Spencer. 1973. Biochemical changes in fed and starved oysters. Ostrea edidis L. during larval development, metamor- phosis and early spat growth. J. Mar. Biol. Assoc. U.K. 53:287-298. Holland. D. L. 1978. Lipid reserves and energy metabolism in the larvae of benthic marine invertebrates. In: D. C. Malin, editor. Biochemical and biophysical perspectives in marine biology. London: Academic Press pp. 85-123. Labarta, U.. M. J. Femandez-Reiriz & A. Rerez-Camacho. 1999. Energy, biochemical substrates and growth in the larval development, meta- morphosis and postlarvae of Ostrea edulis (L.). J. Exp. Mar. Biol. Ecol. 218:22.5-242. Lochmann, E.. L. Maillet Gary. T. Frank Kenneth & T. Taggart Chnsto- pher. 1995. Lipid class composition as a measure of nutritional condi- tion in individual larval Atlantic cod (Gadus morluia). J. Fish. Res. Board Can. 52:1294-1306. Lombardi. A. T. & P. J. Wangersky. 1991. Influence of phosphorus and silicon on lipid class production by the marine diatom Chaetoceros gracilis grown in turbidostat cage cultures. Mar. Ecol. Prog. Ser. 11: i9-ll. Lucas, M. I., G. Walker. D. L. Holland & D. J. Crisp. 1979. An energy budget for the free-swimming and metamorphosing larvae of Balaniis balunoides (Crustacea: Cirripedia). Mar. Biol. 55:221-229. Miron, G.. B. Boudreau & E. Bourget. 1999. Intertidal barnacle distribu- tion: a case study using multiple working hypotheses. Mar. Ecol. Prog. Ser. 189:205-219. Miron. G.. L. Walters. R. Tremblay & E. Bourget. 2000. Physiological condition and barnacle larval behavior: a preliminary look at the rela- tionship between TAG/DNA ratio and larval substratum exploration in Balaniis aniphitrite. Mar. Ecol. Prog. Ser. 198:303-310. Mullineaux. L. S. & C. A. Butman. 1991. Initial contact, exploration and attachment of barnacle {Balaniis amphitrite) cyprids settling in flow. Mar. Biol. 110:93-103. Ouellet, P. & C. T. Taggart. 1992. Lipid condition and survival in shrimp (Pandalus borealis) larvae. / Fish. Res. Board Can. 49:369-378. Ouellet. P.. T. Taggart Christopher & T. Frank Kenneth. 1995. Early growth, lipid composition, and survival expectations of shriinp Pan- dalus borealis larvae in the northern Gulf of St. Lawrence. Mar. Ecol. Prog. Ser. 126:163-175. Parrish. C. C. 1987. Separation of aquatic lipid classes by chromarod thin-layer chromatography with ineasurement by latroscan flame ion- ization detection. /. Fish. Res. Board Can. 44:722-731. Pawlik. J. R.. C. A. Butman & V. R. Starczak. 1991. Hydrodynamic fa- cilitation of gregarious settlement of a reef-building tube worm. 5a- ence 251:421^24. Pechemk. J. A.. D. Rittschof & A. R. Schmidt. 1993. Influence of delayed metamorphosis on survival and growth of juvenile barnacles Balaniis aniphitrite. Mar. Biol. 115:287-294. Pernet. F.. R. Tremblay & E. Bourget. 2003. Biochemical indicator of giant scallop iPlacopecten magellaniciis) quality based on lipid class com- positions. Part I: broodstock conditioning and young larvae perfor- mance. J. Shellfish Res. 22. Qian. P. Y. & J. A. Pechenik. 1998. Effects of larval starvation and delayed metainorphosis on juvenile survival and growth of the tube-dwelling polychaete Hydroides elegans (Haswell). / Exp. Mar. Biol. Ecol. 227: 169-185. Rittschof, D., E. S. Branscomb & J. D. Costlow. 1984. Settlement and behavior in relation to flow and surface in larval barnacles, Balaniis aniphitrite Darwin. J. Exp. Mar. Biol. Ecol. 82:131-146. Robert, R. & P. Trintignac. 1997. Microalgues et nutrition larvaire en ecloserie de mollusques. Haliotis 26:1-13. Rodriguez, J. L.. F. J. Sedano, L. O. Garcia-Martin, A. Perez-Camacho & J. L. Sanchez. 1990. Energy metabolism of newly settled Ostrea edulis spat during metamorphosis. Mar. Biol. 106:109-1 1 1. Sherrer. B. 1984. Biostatistique. Montreal: Gaetan Morin, 467 pp. Soudant, P., Y. Marty. J. Moal & J.-F. Samain. 1996. Fatty acids and egg quality in great scallop. Aquacullure Int. 4:191-200. Tocher. D. R., A. J. Fraser. J. R. Sargent & J. C. Gamble. 1985. Lipid class composition during embryonic and early larval development in Atlantic herring {Cliipea harengus). Lipids 20:84-89. Walters. L.. G. Miron & E. Bourget. 1999. Endoscopic observations of invertebrate larval substratum exploration and settlement. Mar. Ecol. Prog. Ser. 182:95-108. Webb, K. L. & F.-L. E. Chu. 1983. Phytoplancton as food source for bivalve larvae. In: G. D. Pruder. ed. Proceedings of the Second Inter- national Conference on Aquaculture Nutrition: Biochemical and Physi- ological Approaches to Shellfish Nutrition. Louisiana State University. Baton Rouge. LA, pp. 272-291. Whyte. J. N. C. 1987. Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aipiacultiire 60:231-242. Whyte. J. N. C. N. Bourne & C. A. Hodgson. 1987. Assessment of bio- chemical composition and energy reserves in larvae of the scallop Patinopecten yessoensis. J. Exp. Mar. Biol. Ecol. 113:113-124. Whyte. J. N. C. N. Bourne & C. A. Hodgson. 1989. Influence of algal diets on biochemical composition and energy reserves in Patinopecten yes- soensis (Jay) larvae. Ac/iiaciiltiire 78:333-348. Whyte. J. N. C. N. Bourne & N. G. Ginther. 1991. Depletion of nutrient reserves during embryogenesis in the scallop Patinopecten yessoensis (Jay). J. Exp. Mar. Biol. Ecol. 149:67-80. Whyte, J. N. C, N. Bourne, N. G. Ginther & C. A. Hodgson. 1992. Compositional changes in the larva to juvenile development of the scallop Crassadoma gigantea (Gray). J. Exp. Mar. Biol. Ecol. 163:13- 29. Jounud oj Shellfish Research. Vol. 22, No. 2. 389-.W1. 2()().V A RAPID TEST FOR THE DETERMINATION OF THE SPAWNING STATUS OF THE BAY SCALLOP, ARGOPECTEN IRRADIANS (LAMARCK, 1819) STEPHEN L. ESTABROOKS* Nantucket Marine Laboratory, 0 Eastern Street. Nantucket. Massachusetts 02554 ABSTRACT The bay scallop. Argopecten iiraduiiis irnidians (Lamarck. 1819). is a generally semelparous. commercially imponant marine bivalve found along the shores of the Northeast Atlantic from Cape Cod, Massachusetts to New Jersey. It can be found in areas of varying conditions, including current flow, nutrient levels, salinities, siltation levels, all factors that can affect its size when it becomes legally harvestable. A harvestable scallop is defined as having a visible growth ring signifying that it has completed its reproductive cycle. However, there are areas on the island of Nantucket, MA. that produce scallops that lack this classic growth ring, giving rise to disagreements between scallop fishermen and regulatory agencies concerning the legality of harvesting them or returning them to the water. A rapid. 10-min test has been developed to quickly determine whether scallops in a particular area have spawned. It was determined that bay scallops, at least those found in Nantucket waters, retain mature spermatozoa in their gonads throughout the scallop harvest season, which in Massachusetts, runs from October through March of the next year. Detection of their presence could be useful in determining their spawning status. A small piece of male gonad is removed, homogenized bnetly. and stained to detect the presence of these residual spermatozoa. It is hoped that the implementation of this rapid test will help to settle some of these local disputes, which should help ensure the protection of seed scallops. KEY WORDS: scallops, Argopecten. spawning, seed INTRODUCTION The bay scallop, Argopecten irradians irradians (Lainarck. 1819), found in shallow bays along the Northeastern United States coast from Cape Cod to New Jersey, is a hermaphroditic, generally semelparous bivalve that is sexually mature at the age of I yr (Belding 1910). The reproductive period inay last from mid-.lune into September in the waters surrounding Cape Cod. depending on local conditions, after which time scallops may be harvested be- cause most will not survive to complete a second reproductive season (Belding 1910, Marshall 1960, Taylor & Capuzzo 1983. Tettelbach et al. 1999). The waters surrounding the island of Nantucket. Massachu- setts, have yielded steadily declining scallop harvests from a high of 117,000 bushels in 1980 to a low of 6.800 bushels in 1998 (Curley 2002). It has been long recognized that seed scallops must be protected because they are the primary source of the next year's harvest (Belding 1910). Scallops resulting from late spawning tend to be much smaller the next year, although many may catch up in size with scallops spawned earlier in the year, again, depending on local conditions (Auster & Stewart 1984). However, many of these late-spawned scallops may lack the distinctive growth ring, generating confusion among fishermen and regulatory agencies as to whether these are seed scallops and should not be harvested (MacFarland 1991). Massachusetts laws governing the taking of mature bay scal- lops require the presence of a ""well defined raised annual growth ring" (MGL.cl30,s.70). However, in some areas of Nantucket, varying conditions, such as water temperature, food supply, cur- rent flow, and heavy siltation. among others, can lead to scallops lacking this distinctive ring. In addition, several investigators have documented late spawning events that give rise to smaller scallops with very small growth rings very near the hinge line (McFarland 1991, Tettelbach et al. 1999, Tettelbach et al. 2001). Scallops *Correspondence. Tel.: 1-843-546-4047; E-mail; estabrooks(a'sccc.tv spawned near the beginning of the spawning season, generally beginning around the middle of June when water temperatures reach 15°-16°C in Nantucket waters, in an area of good current flow and sufficient food, can often yield seed scallops that are as large or larger than the average adult by the time harvest season arrives (Kelley & Ceely 1980). During one very productive year, 1990, in Pleasant Bay on Cape Cod, MacFariand (1991), found that if scallops were harvested based on size alone, as was recom- mended by local fishermen, 66'7f of those harvested would have been large seed. In 1999. one area. Madaket Harbor, was closed to scalloping because of the presence of a large number of seed, and the fol- lowing year saw the initiation of much stricter enforcement of taking only scallops with the distinctive growth ring. This has led to controversy because Madaket Harbor has generally poorer growing conditions and often yields scallops without the classic annual ring. Fishermen point to a fine line, usually within a cen- timeter of the hinge, as the annual growth ring, giving rise to the local term "nub" scallop, i.e., a scallop that has spawned (1-t- yr) but that demonstrates no normal growth ring. Others state that this is a first-year scallop that has not spawned (O-i- yr) and there- fore should be returned to the water. Heretofore, confirmation has relied on preserving scallops in formalin and sending the speci- mens to a laboratory to have histologic slides prepared and read, with the results often obtained weeks later. To help eliminate this confusion, a rapid yet definitive test was developed to aid local regulatory agencies in differentiating seed scallops from those that have spawned. This test is based on the observation by the author over several years that bay scallops, at least those found in Nan- tucket waters, retain residual mature sperm in their gonads into March and April of the following year, whereas residual eggs are generally resorbed quickly, most likely because of their high energy content and the scallop's need to store energy for the up- coming winter. The purpose of this investigation was 2-fold: to determine whether this sperm retention was a sporadic event or was generally found throughout the scallop population and sec- ondarily, to develop a rapid test to detect the presence of the residual sperm. 389 390 ESTABROOKS METHODS Fifty bay scallops that displayed the classic annual growth ring (1+ yr) were collected each month from Nantucket Harbor from October 1998 through March 1999. Scallops were obtained from three sources, SCUBA diving, from commercial scallopers. and from upwellers and lantern nets inaintained at Nantucket Marine Labora- tory. Because the purpose of this study was to see whether scallops that had spawned retained sperm throughout the harvest season, only those scallops that had clearly spawned were used. In addition to the presence of the growth ring, the gonad was large and flaccid, the upper shell was generally encrusted with flora and fauna, and the bottom valve was distinctly of a greater curvature than the upper shell. Also, 25 known seed scallops (0+ yr) that had been obtained in July and August of 1998 from sets onto onion bags and subsequently maintained in upwellers and lantern nets were also tested each month. Residual sperm in bay scallops were examined by clipping a small piece (1-2 mm" is sufficient) from any part of the male gonad (see Fig. 1) and placed in a disposable 1.5-mL plastic coni- cal tube containing approximately 0.5 mL of \0'7c formalin (this amount is not critical). The tissue was ground for 15-30 sec using a pellet pestle, and a drop of this mixture was placed onto a glass slide and spread. Once air-dried, the slide was dipped in methanol to fix the tissue, and stained for 10-30 sec in a Safranin: Wright-Giemza: Water (1:2:10) stain. The slide was rinsed briefly in running tap water, air-dried and read at lOOOx magnification under oil immersion. Results can be hastened by drying the slide at each step on a slide warmer or hot plate. This procedure also lends itself to sampling by needle biopsy if sacrificing of the scallop needs to be avoided (Schneider et al. 1997). RESULTS Sperm with characteristic bullet-shaped heads (S) were stained grayish-blue with Wright-Giem/a, and the tails (T) were stained i Figure L Anatomy of the bay scallop, Argopecten irradians (L.) seen with the upper or left valve removed: male gonad (MG); female gonad (FCl; adductor muscle (.AMI; gill (CL); digestive gland (DG); heart (HTl; mantle (MT). • -. u. i 4 Figure 2. Residual sperm ( KKlOx) seen in a representative bay scallop harvested in February 2001 confirming that it has previously spawned and is legally harvestable. Sperm heads (S); tails (Tl; hemocytes (H). pink by the safranin (Fig. 2). These can be readily distinguished from the larger nucleated hemocytes (H) that tend to become more pervasive in the gonad as the season progresses, ostensibly phago- cytizing the remaining sperm. Late in the scallop season, i.e., February and March, some scallops may demonstrate mostly tails (Fig. 2). Results for 100% of the seed scallops (O-t- yr) tested negative for the presence of spermatozoa. In the post spawning scallops ( 1-1- yr), of 300 individuals tested from October through March, 241 displayed residual sperm, 55 demonstrated only tails, and the remaining 4 tested negative (these were from the scallops collected in March). DISCUSSION Because one of the primary concerns in maintaining a semelpa- rous and commercially important species, such as Argopecten irradians irradians. is ensuring the survival of the seed scallops, it is important that they not be taken before their contributing to the following year's population. Traditional methods of detemiining whether bay scallops have spawned, in addition to the presence of a distinct annual growth ring, include a larger gonad, grayish in color or with a whitish line or band as compared with a much smaller gonad, shiny black in color, as seen in reproductively immature scallops. Also, a dis- tinctly curved lower or right shell as compared with the upper shell and a generally rougher appearance due to a greater longevity in the water with a concomitant accumulation of flora and fauna upon its upper shell help to separate the older scallop from its younger cohort (Belding 1910). However, these differences are often subjective and can lead to controversy between fisherman and local regulatory agencies. In addition, the spawning season in Massachusetts waters, as de- scribed by Belding (1910) and Sastry (1963). generally has run from the middle of June through mid-August but now seems to be Spawning Status of the Bay Scallop 391 extended to include September (Kelley & Sisson 1981. McFarland 19<:)1). Tettelbach (1999) found similar results for some scallop populations in New York state. In Nantucket, these late-spawned or nub scallops lack the dis- tinctive growth ring seen in earlier-spawned scallops. These may spawn the next year, but in one experiment. 807f of these nub scallops held in cages survived yet an additional year {2+ yr) and 50% of those spawned (Conant. K. Assistant Town Biologist. Nan- tucket. MA, personal communication, 2002). However, many fish- ermen see a line, usually falling within 10 mm of the hinge, as the growth ring, and harvest these as adults. McFarland ( 1991 ) found that 9% of the scallop population had growth rings between 4 and 8 mm from the hinge line, of which 50% spawned the next year and the remaining 50% spawned the following year. It remains unclear as to the significance of the contribution of this small portion of the population, but it inay play a role in the persistence of some scallop populations (Tettelbach et al. 1999). Although the significance of this secondary spawning remains unclear, it is ab- solutely clear as to the significance of taking scallops that have yet to spawn. It is hoped that the development of a rapid yet simple test to determine v\'hether scallops in an local area have spawned or not will be useful in reassuring both regulatory agencies and scallop fishermen that only adult animals are being harvested. ACKNOWLEDGMENTS This research was supported by grants from the PADI Foun- dation. The Nantucket Land Council, and the Nantucket .Shellfish and Harbor Advisorv Board. LITERATURE CITED Auster, P. J. & L. L. Stewart. 1984. Compensatory growth in the bay scallop. Argopeclen irradians (L.). J. Northwest Atlantic Fish. Sci. 5:103-104. Belding. D. L. 1910. A repon upon the scallop fishery of Massachusetts. Boston: The Commonwealth of Massachusetts. 150 pp. Curley. T. 2002. Shellfish Catch Reports. Nantucket. MA: Nantucket An- nual Town Report. Shellfish and Marme Department. Kelley. K. & M. Ceely. 1980. Studies of bay scallops, Argopecleu irradi- ans. on Nantucket 1979-1980 season. Nantucket. MA: Shellfish and Marine Department, pp. 16-34. Kelley. K. M. & J. D. Sisson. (1981). Seed sizes and their use in deter- mining spawning and setting times of bay scallops on Nantucket. In: K.M. Kelley. editor. The Nantucket Bay scallop fishery: the resource and its management. Nintucket. MA: Shellfish and Manne Depart- ment, pp. 43—19. MacFarlane, S. L. 1991. Managing scallops .4/;i;()/)fcrc)i irradians irradi- ans (LamiU'ck) in Pleasant Bay. Massachusetts; large is not always legal. In: S. E. Shumway and A. P. Sandifer. editors. IntT Compen- dium of Scallop Biology and Culture. World Aquaculture Soc. 264- 272. Marshall. N. I960, Studies on the Niantic River. Connecticut with special reference to the bay scallop. Aequipecten irradians. Limnol. Oceanogr. 5:86-105. Massachusetts General Laws. Chapter 1 30. Section 70. Sastry, A. N. 1963. Reproduction of the bay scallop, Aequipecten irradians Lamarck. Influence of temperature on maturation and spawning. Biol. Bull. 125:146-153. Schneider. P.. R. Smolowitz, C. Smith. J. Degiorgis & M. McCafferty. 1997. Comparison of three techniques for evaluating seasonal game- togenesis in Spisula solidissima. Biol. Ball. 193:233-234. Taylor. R. E. & J. M. Capuzzo. 1983. The reproductive cycle of the bay scallop. Argopecten irradians irradians (Lamarck), in a small coastal embayment on Cape Cod. Massachusetts. Estuaries 6:431-435. Tettelbach. S. T. C. F. Smith. R. Smolowitz. K. Tetrault & S. Dumais. 1999. Evidence for fall spawning of Northern Bay scallops Argopecten irradians irradians (Lamarck, 1819) in New York. / Shellfish Res. 18:47-58. Tettelbach, S. T., P. Wenczel & S. W. T. Hughes. 2001. Size variabihty of Juvenile (O-t- Yr) Bay Scallops Argopecten irradians irradians (Lamarck, 1819) at Eight Sites in Eastern Long Island. New York. The Veliger 44(4):389-397. Jomnal of Shellthh Research. Vol. 22, No. 2. 393--W9, 2003. OPTIMIZATION OF SETTLEMENT OF LARVAL ARGOPECTEN PURPURATUS USING NATURAL DIATOM BIOFILMS RUBEN AVENDANO-HERRERA,' CARLOS RIQUELMES,'* FERNANDO SILVA,' MIGUEL AVENDANOD,- AND RUTE IRGANG' ' Liihoratorio de Ecologia Mkiohiami. Departameuto de Acuicidtuni, Uuiversidad de Antofagasta Cusilla 170, Antofagasta; 'Departameuto de Aciiicidtiira. Uuiversidad de Antofagasta, Casilla 170, Antofagasta ABSTRACT Larval settlement is a critleul stage in the artificial production of Argopeclcii iniipurauis. The study investigated the feasibility of improving post-larval settlement of this .species using a substrate (cultchi that was pre-treated with a biofilm of native diatoms. Four species of diatoms were isolated from the surface of collectors that had high numbers of juvenile scallops (spat). These four species were able to attach themselves and grow on a polystyrene substrate. Scallop post-larval settlement was evaluated experimentally in two ways: (I) laboratory experiments in lO-L buckets; and (2) under natural condition by in situ experiments at the Marine Reserve "La Rinconada" (Antofagasta. Chile). Effects of biofilm treatments were examined using collectors that were coated with diatoms and collectors handled using normal culture methods (new netlon held in filtered seawater that did not have a biofilm). Results of the laboratory experiments showed a higher percentage oi A. purimmhis post-larval settlement on collectors coated with Fwgiliaropsis pseudomma compared with control collectors (P > 0.03). Results comparing biofilms of the diatoms F. pseudonaiia and Navicida venela showed higher settlement on collectors pretreated with N. venehi ( 1.136 ± 172 spat per collector "' ). Statistical analysis showed that the addition of diatom biofilms enhanced spatfall and always produced larger settlement compared with untreated collectors. These results indicate that addition of cultured diatom biofilms improves scallop larval settlement. KEY WORDS: Argopecten purpuratus. diatoms, biofilms, post-larval settlement INTRODUCTION The northern Chilean scallop, Argopecten piirpiiraliis (Lama- rck 1819), is the most important commercial bivalve species in Chile. Production in 1999 was 20.668 I. valued at $13 million (US) and the industry provided 3.600 direct jobs (Lozano 2000). The aquaculture production, however, is not sufficient to satisfy the international demand for this species. A major reason is the large variation in natural seed production (Navarro et al. 1991, Disalvo 1991. Avila et al. 1994. Riquelme et al. 1995. Avendano et al. 2001) that supplies about 30% of the annual Chilean production (Farias et al. 1998). A major problem with seed supply occurs at metamorphosis when larvae settle on a substrate (Keough & Downes 1982). Fol- lowing attachment larvae undergo considerable morphologic and physiologic changes as they metamorphose from a pelagic to a benthie existence (lUanes 1990). There are generally a large num- ber of larval mortalities (Tremblay 1988. Bourne et al. 1989. Castagna 1975). Ambrose et al. (1992) reported that there was little information concerning factors that influence scallop larval settlement and this led to numerous studies on the subject in the mid 1990s. A goal of these investigations was to increase settle- ment by improving substrates for settling larvae including color of the substrate, size, monofilament density and composition of the collector (Miron et al. 1995. Pouliot et al. 1995. Pearce & Bourget 1996). Further, the mechanism by which scallop larvae detect and settle on a particular substrate is still not understood (Harvey et al. 1997). Many studies showed that biologic, chemical and physical factors could induce larval settlement of marine invertebrates (Weiner et al. 1989; Bonar et al. 1986. Christensen 1989, Maki et al. 1990, Chevolot et al. 1991). Many of these studies showed that bacterial films were important for triggering larval settlement (Meadows & Campbell 1972, Kirchman et al. 1982. Weiner et al. Corresponding author. E-mail; criquelmeCs'uantof.cl 1989, Maki et al. 1990, Pearce & Bourget 1996). Bacterial com- munities were found associated with other microorganisms such as diatoms forming a multi-specific biofilm that was firmly attached to a substrate. These multi-specific biofilms emitted several types of signals, including; ( 1 ) peptic (Zimmer-Faust & Tamburri 1994) or associated fatty acids (Pawlik 1986) and (2) polysaccharides and glycoproteins (structure of a biolfilm) (Hadtleld 1986) that would stimulate marine invertebrate larvae to settle (Pawlik 1992. Keough & Raimondi 1995). Harvey et al. (1995). using electron microscopy showed that biofilms were not only composed of bacteria but microalgae and detritus as well. The various organisms may have different effects on settlement of different species of scallops. Benthie diatoms that colonize substrates might not only be a source of nutrition for more advanced post-larval stages of marine invertebrates (Takami et al. 1997) but also may be necessary for the settlement of mollusc larvae, as shown in abalone culture (Seki 1980, Hahn 1989). The purpose of this study was to isolate native diatoms from scallop collectors that had high levels of settled spat and evaluate the feasibility of improving post-larval scallop settlement by using of biofilms composed of specific diatom species. Results of labo- ratory and in situ field experimental work are reported here. MATERIALS AND METHODS The study was perfomied in three stages; ( I ) isolation of native diatom species: (2) laboratory experiments undertaken at the hatchery of the Facultad de Recursos del Mar de la Uuiversidad de Antofagasta (FAREMAR, Faculty of Marine Resources at Anto- fagasta University); and (3) in situ field experiments undertaken in "San Jorge" bay at the Marine Reserve "La Rinconada" (27°03'24"S-70°5r30"W). Isolation of Diatoms Diatoms isolation was undertaken at the Cultivos Marinos In- ternacionales hatchery in "Inglesa" bay, Chile (27°03'24"S- 393 394 Avendano-Herrera et al. 70°5r30"W). Netlon collectors with high levels of scallop-spat settlement were selected (more than 2.500 spat per collector"'). Seventy pieces of netlon mesh were cut into 100 cm" sections. They were washed several times with a marine saline solution (SSM) (Austin 1988), and placed in Schott bottles with 50 ml of seawater filtered to 0.2 |jim. Diatoms were removed from the mesh with an Ultrasonic Homogenizer (Cole-Parmer) for 60 sec. The resulting solution was diluted in test tubes with 9 mL of F/2 com- mercial Fritz Chemica Inc. supplemented with sodium metasilicate (F/2M, Guillard & Ryther 1962). These tubes were incubated for 7 d at 20 ± rC. photoperiod 12:12 and a light intensity of 100 (jimol m"~s~'. Dominant diatom species were isolated using the microfishing technique described by Hoshaw and Rosowski ( 1979). Four species were identified after the method of Rodriguez (1998) as: Navicula veneta (Kutzing), Navicida ciyptocephala (Hustedt). Navicula menisciihis (Schuman) and Fragilariopsis psciicloiuma {}i-ds]e). The species were then purified by exposure to a wide range of antibiotics (Hoshaw & Rosowski 1979). Adherence of Diatoms to Polystyrene Substrates Adherence assays to attach diatoms to the experimental sub- strate were performed using the method of Gawne et al. (1998). Polystyrene petri dishes with a diameter of 3 cm were filled with 5 mL of filtered seawater (0.2 |xm) autoclaved for 15 min at 12rC and inoculated with either species of N. veneta (Nv). N. crylo- cephala (Nc), N. menisculus (Nm), and F. pseudonana (Fp) at a concentration of 5 x 10' cells x ml"' (3.5 x 10*^ cells x cm"-) in the pre-stationary phase. Three replicates of each culture were incubated in a controlled environment room at 20 ± 1 °C and a photoperiod of 12:12 for 48 h. Each petri dish was washed 5 times with 0.2 (im filtered seawater to insure that only those diatoms adhering to the bottom of the dishes were retained, non-adhering diatoms were thus eliminated. Diatom adherence at incubation times of 1,6. 12, 24, and 48 h were recorded by direct count with an inverted microscope Olympus 1X50 at a magnification of x 1 00 (Guillard 1973) The percentage of diatom adherence was calcu- lated by comparing the concentration of inoculated diatoms with those observed on the bottom of the petri dish. Growth of Diatoms on Polystyrene Substrates Polystyrene petri dishes with a diameter of 3 cm were filled with 4 mL of F/2M solution autoclaved for 15 min at 121°C. Each dish was inoculated with one of the four species of diatoms from the pre-stationary phase at a concentration of 5 x 10"* cells x ml"' (2.8 X 10^ cells X cnr). To assess the growth of diatoms, microal- gal counts were performed every 48 h for a period of 144 h under identical conditions to those in the adherence experiments. Stage U-Laboratory Experiments on Settlement of Post-Larval Scallops The effect of native diatoms on the settlement of A. purpwatits post-larvae, was evaluated by conforming settlement among coated with biofilm of the four species of diatoms. (a) Determination of post-larvae settlement substrate pre- treated with diatoms (according to the criteria of diatom adherence on substrate). Bioassays were performed in buckets containing 10 L of 1 |j.m filtered seawater and no aeration. Each bucket was inoculated with strains of diatoms in the stationary phase (Fox 1983) at a concen- tration of 5 X 10' cells X ml"'. After inoculation of the diatoms, a piece of netlon collector was placed in each bucket (length x width = 30 X 60 cm ) and incubated for 48 h. A set of collectors that were placed and kept in 10 |j.m-filtered seawater was pre-treated ac- cording to procedures done by commercial companies (natural). The control was new netlon that did not have a biofilm (Ct s/b). At the end of the incubation period, "eyed" scallop larvae (>220 p.m) were added to each bucket at a density of 1 larva x mP', and maintained for a 7-day-period. During this time, the water in each bucket was changed daily, larvae were filtered on a 120 |j.m screen, washed on 205 (jlhi screens, and returned to their respective buckets. Larvae were fed daily with a mixed diet of 7,500 cells X ml"' of Chaetoceros calcitrans and 10.000 cells x ml"' of C gracilis. After seven days the netlon collectors were removed, cleaned with horsehair brush and the spat collected on a 205-|jLm screen. The number of attached spat was determined using an Olympus BH2 stereoscopic microscope. Results were expressed as "percent settlement" calculated by comparing the number of attached spat on collectors to the number of "eyed" larvae added to each bucket (Avendaiio-Herrera et al. 2002). Settlement i Number of attached post-larvae x 1 00% I X lO"* "eyed" larvae (b) Determination of post-larvae settlement on substrate pre- treated with diatom (according to the criteria of diatom growth on substrate). Bioassays to assess diatom growth were performed in buckets containing 10 L of 1 p,m filtered seawater using a constant 24-h photoperiod with a light intensity of 50 ixmol m"" s"' and aeration. Buckets were inoculated with diatoms at concentrations similar to the polystyrene substrate growth experiments. To stimulate growth during the incubation period, treatments and controls were en- riched with the addition of F/2M. Netlon spat collectors measuring 30 X 60 cm that are typically used by commercial companies were placed in each bucket and incubated for a 96 h period. The bioassays with larvae were carried out as previously de- scribed. Stage Itl-in Situ Field Experiments of A. Purpuratus Attachment to Collectors Treated with Diatoms When the effect of the four diatom species on settlement of scallop larvae was known from the laboratory experiments, strains of F. pseudonana and N. veneta were selected for further testing in the natural environment. Buckets with 20 L of l-|j.m filtered sea- water were inoculated with diatoms in the stationary phase at a concentration of 5 x lO"* cells x ml"' and incubated with aeration and constant 24 h photoperiods at a light intensity of 50-|jimol m' s"'. A biofilm was established on one set of collectors using the method commonly used by commercial companies (Natural) and as control was used new the control used new netlon without biofilm (Ct s/b). Treatment and control buckets were enriched with the addition of F/2M and incubated for 10 days after inoculation of the diatom six netlon spat collector (30 x 60 cm) and placed in each bucket. A collector from each experiment was sampled to determine the density of diatoms attached to the surface of the collectors at the end of the incubation period. Three pieces of netlon were cut into 25 cnr pieces, washed repeatedly with SSM and placed in 50-ml Schott bottles, and the diatoms attached to pieces of netlon were removed using an Ultrasonics Homogenizer for 60 sec. The number of diatoms attached to the monofilaments of each piece of Settlement of Larval A. pukpuratus and Diatom Biofilms 395 netlon was determined by direct counting using a Neubauer cham- ber and an Olympus BH-2 microscope. Results were extrapolated for the complete area of the collectors (1.800 cnr). The five remaining collectors from each treatment and control were placed in 1 x 1 mm "onion" bags, labeled, and placed in the ocean at a depth of 16 m at the Marine Reserve Area for 38 days (January \5 to February 22. 2002). Prior to placing the collectors in the water, plankton-sampling method was used to assess A. piiipiiiatus spatfall. Water temperature was recorded to evaluate larval and spat growth during the 38-d period (17°C ± T'C). After 38 days the collectors were removed from the ocean following the method of Wallace (1982) and taken to the Labora- torio de Ecologia Microbiana de la Universidad de Antofagasta (Microbial Ecology Laboratory of the University of Antofagasta) to assess spatfall. The effect of diatom biofilms on settlement of /\. piirpiiraliis was determined by counting the juveniles (spat) that were firmly attached to the monofilament of the treated collectors. Results were not affected by those spat that fell off collectors during transport because the interest was on spat that were firmly attached to the collectors. Each collector was removed from the onion bag. washed with circulating water for 5 min. and the at- tached material collected was deposited on a 205-|xm-mesh screen. To avoid loss of spat, each collector was cleaned with a horsehair brush and the spat were preserved in (70%) ethanol for counting with a stereoscopic Olympus microscope. Statistkal Analysis The growth rate of diatoms was calculated using Guillard's equation (Stein 1979), which describes mean microalgal duplica- tion velocity: K = [3.322/(t- -t')] X (log N-/N') Where K is the mean microalgal duplication velocity of the microalgal biomass. N' is the cellular density of the beginning of the experiment, N" is the cellular density at the end of the experi- ment, t' is the time at the beginning of the experiment and t" the time at the end. Results were tested by ANOVA to compare growth rates and maximum density values (Sokal & Rohlf 1980). To evaluate the effect of diatoms on settlement of larvae in laboratory experiments, the results were tested by ANOVA with the statistical significance criteria (alfa = 0.05) and Multiple LSD Comparison Test (Sokal & Rohlf 1980). The influence of selected diatoms on settlement of larvae in the "in situ" field experiments was realized counting the collector naturally pre-lreated (Natural) as one treatment. Results were submitted to the Dunnet test com- paring results from the various treatments to those of the control (Zar 1984). RESULTS Isolalion of Diatoms Four species of diatoms were isolated from the microflora that was attached to the surface of scallop collectors. Only four species could be purified to an axenic condition using a mixture of che- motherapeutics and these were; Navicula veneta (Kutzing). Na- vicula ciyptocephala (Hustedt), Navicula menisculus (Schumann), and Fragilariopsis pseudonaim (Hasle). Adherence and Growth of Diatoms on Polystyrene Substrates N. veneta rapidly colonized plastic substrates without the ad- dition of nutrients and 100% adherence was observed 48 h after inoculation (Fig. 1). A similar situation was observed with N. ci-\ptocepliala and N. menisculus. Two growth patterns were observed for the four diatom species when F/2M was added to the cultures, an accelerated growth for N. veneta and N. menisculus and slower growth for N. cryptocephala and F. pseiitlonana. Figure 2 illustrates that the four species were in the exponential phase of growth after 96 h of culture and a maximum cell production was observed after 144 h. When dupli- cation velocity was compared, the rate for rapid growing species was (K = 1.57 ± 1 duplication x days"') and the slow growing species was (K = 1.38 ± 1 duplications x day"'), the difference was significant (P < 0.05). Stage II-Effect of Diatoms on Attachment of Scallop Larvae in Laboratory Studies Results of experimental laboratory studies showed a higher percentage of post-larvae attached to collectors incubated with diatoms for 96 h in 1 (xm filtered seawater enriched with the addition of F/2M (criteria of diatom growth on substrate) com- pared with collectors incubated with diatoms for 48 h in 1 p,m filtered seawater (criteria of diatom adherence on substrate) (Fig. 3). Collectors treated with F. pseudonana had a larger number of spat attached to the collectors. The breakdown was 2.567 ± 205 and 7,727 ± 107 post larvae x collector"' under the criteria of diatom adherence and diatom growth on substrate, respectively. Collectors without biofilms had lower numbers of spat on them. Statistical analysis of settlement of larvae between collectors with and without diatoms films after 48 h incubation showed a signifi- cant difference between treatments with different diatom species and the control that had no film {P < 0.05). There was no statistical difference between settlement on the control and cultch that had been treated with 10 p. in filtered seawater. The only significant difference in settlement on cultch treated with different species of diatoms was cultch treated with F. pseu- donana (P < 0.05), incubated with diatoms for 96 h in I (xm filtered seawater enriched with the addition of F/2M, and where settlement reached 77.27% of the total available post-larvae. Un- der laboratory conditions, collectors treated with 10 |xm filtered seawater showed a significant increase in the number of settled larvae (33.84 ± 7.13%). This number was close to the average of those treated with the five species of diatoms (31.91 ± 2.21%). 125 1 too - r 75 50 - Figure 1. Percent attachment of the axenic diatoms Navicula veneta Navicula (Nv), cryptocephala (Nc). Navicula menisculus (Nm), and Fragilariopsis pseudonana (Fp) incubated for 48 h on polystyrene sub- strate,s. Vertical lines show standard deviation. 396 Avendano-Herrera et al. 240 1 .-/!i ■^E _«_ Nv -»-Nc -*-Nm -»«-Fp >; ■fo " __...z:.... o 2 '^° 1 g ^ Z- ^ ■55 -^ S 60 o o U ^^ 0 ^ 0 48 Time (hours) 95 144 Figure 2. Growth curves of the axenic diatoms Navicula veneta (Nv), Navicula cryptocephala (Nc), Navicula menisciiliis (Nm) and Fragilari- opsis pseudonana (Fp) grown with the addition of culture medium marine phytoplaniiton F/2M. \ ertical lines show standard deviation Stage Ul-in Situ Field Experiments of Settlement of A. Purpuratus Larvae on Collectors Treated with Diatoms The best results for stimulating settlement in scallop lar\ae were found with the diatoms F. pseiuloiuma and N. veneta and these species were used in field experiments. After 10 days of incubation the concentration of these two diatoms on cultch was 73 ± 3.5 X lO'* and 47 ± 3.7 x 10'* cells x cm"", respectively. Plankton sampling showed a concentration of 8,197 larvae x m""* with a mean size of 181.4 ixm in water column in the Reserve. Recorded seawater temperatures during the 38 days of the experiment in the area of "Rinconada" did not show drastic changes. Results of settlement on collectors after 38 days in the natural environment are shown in Figure 4. Collectors treated with the diatom N. veneta had a higher number of spat (1,156 ± 172 spat x collector"') compared with the controls and other treatments. Sta- tistical analysis showed that spat settlement on collectors with diatom biofilms was always higher than controls (P < 0.05). DISCUSSION The presence of diatoms in the microflora of biofilms on sub- strates is a natural phenomenon and formation of such a micro- environment on a clean surface is inevitable (Cooksey & Wiggles- worth-Cooksey 1995). Harvey et al. (1955) showed that the sec- ondary surface colonizers after bacteria were a diverse species of 100 1 80 60 40 20 ■JJJri Cts/b natural Nv Nc Treatments Nm Fp D Adherence ■ Growth Figure 3. Percentage of A. purpuratus spat that settled on collectors pre-treated with native diatoms according to diatom adherence crite- rion (White rectangle = 48 hi and diatom growth criterion (Black rectangle = 96 h). Vertical lines show standard deviation. cts^ Treatments Figure 4. Number of A. purpuratus spat on collectors coated with different diatoms species in field experiments. Vertical lines show stan- dard deviation. benlhic diatoms and these have been traditionally used as a settle- ment surface for abalone (Seki 1980, Hahn 1989). In this study, the diatoms N. veneta and N. menisculus adhered better and grew faster on the plastic substrate compared with iV. cryptocephala and F. pseudonana. The diatoms N. veneta and N. menisculus are probably more adapted to adherence and formation of a primary biofilm on such substrate compared with the other two species (Characklis & Bryers 1990). Numerous investigators have stated (hat adherence and development of a biofilm are associated with the physical and chemical properties of the substrate (Wiggles- worth-Cooksey & Cooksey 1992. Callow & Fletcher 1994). Struc- ture of the diatoms has an important role in facilitating adherence as well as the production of extracellular polymers that interact with the substrate and may affect diatom adherence positively or negatively (Wetherbee et al. 1998). Results of larval settlement in laboratory experiments showed variable rates of settlement for the four species of diatoms. The presence of spat was always greater when diatoms were present compared with clean substrates. Studies of the biology and culture of marine invertebrates indicate that before settling on a substrate, the larvae require biofilm capable of emitting to the environment chemical signals that stimulate their settlement (Kavouras & Maki 2000.). Studies of the effect of microbial biofilms on attachment of pectinid larvae have shown variable results. In laboratory experi- ments, Hodgson and Bourne (1988) showed higher attachment of Chlamxs hastata on surfaces that had biofilms compared with surfaces without biofilms and Parsons et al. (1993) reported similar results for Placopecten magellanicus. In this study, the higher percentage of spat attached to collectors that were exposed to 10 |jLm filtered seawater under controlled growth conditions (3,384 spat X collector"'), may have resulted from stimulation by live organisms in the biofilm. Microbenthic components are considered to be among the principal diatom components. Cyanoficeas epi- benthic and their associated bacteria (Meadows & Anderson 1968) in the presence of nutrients would increase their density, coloniz- ing a higher percentage of the substrate that are used for adherence, favoring the settlement of scallop larvae. Because they colonize the substrate and cause higher spatfall. characteristics of biofilm growth or production of some unidentified substance may cause this higher spatfall. The composition of natural diatom biofilms and associated microfiora that colonized the netlon are unknown but they could be variable and produce changes in composition and Settlement of Larval A. purfuratus and Diatom Biofilms 397 structure of the community that could produce irregular settlement (Suzuki et al. 1987). Butman et al. ( 1988) suggested that stimulation of invertebrate larval settlement is commonly enhanced by substances that enrich the substrate. In metamorphosis of scallops, it has been shown that if no stimulation is present then no settlement will occur thereby suggesting that specific stimuli may be necessary for different species (Padilla 1979). There were significantly more spat on collectors incubated for 96 h with F. pseudonana compared with collectors with N. veneta, N. cnptocei'hala and N. inenisciiliis that may indicate a selectivity of scallop spat for a specific species of diatoms. Studies of the ingestion of A. piirpiiratus larvae exposed to probiotic bacteria (II, 77 y C33) showed larvae selected two of the strains (Riquelme et al. 2000). Reasons for selection of biofilm surfaces for settlement are unknown but various theories exist. Bourne and Hodgson ( 1991 ) proposed that selection was due to differences in nutrition that occurred during transition among the ciliate velum of the planktonic phase and the filamentous gill of the young benthonic. Bivalve larvae may respond to colonized substrates of biofilms that serve as a bridge between planktonic feeding and tillering feeding, by using the foot for pedal feeding. Observations made under microscopy have allowed visualizing the gradual detach- ment caused by the movement of the food and the ingestion (Un- published MS). Studies of settlement of abalone larvae showed that the success of settlement and density of juveniles on cultch depended on diatom species (Daume et al. 1999). Initial studies of attachment of five species of diatoms on polyethylene showed minor colonization of the F. pseudonana strain after 48 h of in- cubation but none by the other four species (Unpublished MS). Probably the majority of spat in the F. pseudonana treatment settled because of the formation of a primary biotllm that was favored by the incorporation of nutrients and also physical char- acteristics of the substrate surface (Characklis & Marshall 1990). The diatoms are not only a nutrient source for marine invertebrate larval stage, but they also have the capacity to liberate chemical stimuli or extracellular component into the environment (Welher- bee et al. 1998). This extracellular component could be absorbed by pectinid larvae, improving the larval survival in the substrate (Pearce & Bourget 1996, Kavouras & Maki 2000). It is also pos- sible that the gradual biofilm detachment could be used as food (Unpublished MS). Some investigators report that the microbial biofilm gradually detached from the substrate, and the detachment of cells from the biofilms is a nature process in the biofilm devel- opment (Stoodley et al. 2001). This detachment phenomenon shows that the gradual cell detachment from the biofilm could be used as food for the pectinid larvae. Studies on settlement of abalone larvae to biotllms with 18 species of diatoms showed better attachment of the larvae to biofilm with high density of diatom (Kawamura & Kikuchi 1992). Increase in density of colo- nizing diatoms on the substrate, static conditions in the experi- ment, and relative confinement of larvae probably facilitated de- tection of diatom biofilms by larvae. Results of adherence of diatoms F. pseudonana and N, veneta in field experiments showed similar values (lO*" cells x cm""). Collectors incubated with N. veneta had the majority of spat and showed that pre-conditioning the surfaces with a diatom biofilm is a preferred substrate for scallop larvae. Pearce and Bourget (1996) proposed that larvae of the sea scallop. Pkicopecten magellanUus. were able to choo.se between different substrates for settlement, favoring monofilaments with a biofilm. Harvey et al. (1997) found a significant effect of natural biofilms on bivalve settlement (66%) and of Pecten magellanicus (35%) compared with cultch without a biofilm. Hence there is a preference of settlement substrates by some pectinid larvae, one criterion being determined by nutrition (Bourne & Hodgson 1991 ). The stimulus for settlement can be due to various factors including pectinid species, diatom composition, and density of the biotllm. Diatoms may be effective because of a particular microcosm with extracellular material that enhances settlement. Further, the constant supply of artificial substrates with specific and adecuated biofilms is the key to produce higher growth and survival (Hahn 1989, Takami et al. 1997). In conclusion, diatom biofilms enhanced settlement of A. pur- pwatus larvae in laboratory and field experiments, spatfall reach- ing higher values than on collectors without biofilms or using traditional biofilms. This suggests that native diatom biofilms may be used to increase production of spat of other bivalves, including northern Chilean scallop Argopecten purpuratus. ACKNOWLEDGMENTS The authors thank Professor Ismael Kong for the revision and commentary. Dr. Neil Bourne for the invaluable critical reading and improvement to the manuscript. Professor Marcela Cantillanez for her collaboration during the "in situ" stage, and Professor Luis Rodriguez for the identification of native diatoms. This study was financed by the project FONDEF N° DOOI1I68. LITERATURE CITED Ambrose, W. G., C. H. Peterson. H. C. Summerson & J. Lin. 1992. Ex- perimental tests of factors affecting recruitment of bay scallop (Ar- gopecten irradians) to spat collectors. Aquaculture 108:67-86. Austin, B. 1988. Marine Microbiology. London: Cambridge University Press. 221 pp. Avendaiio, R., C. Riquelme, R. Escribano & N. Reyes. 2001. Sobreviven- cia y crecimiento de post-larvas de Argopecten purpuratus (Lamarck. 1819) en Bahia Inglesa. Chile.- efectos del origen. distribucion en la bahi'a y bactenoflora larval. Rev. Cliil. Hist. Nat. 74:669-679. Avendaiio-Herrera. R.. C. Riquelme & F. Silva. 2002. Utilizacion de bio- peli'culas bacterianas en el asentamiento de larvas de de Argopecten purpuratus (Lamarck, 1819) en un hatchery comercial. Rev. Biol. Mar. Ocean. 37:35^1. Avila, M., M. Seguel, H. Plaza, E. Bustos & R. Otaiza. 1994. Estado de situacion y perspectivas de la Acuicultura en Chile: Informe CORFO- IFOP 94/1. 170 pp. Bonar, B., R. Weiner & R. Colwell. 1986. Microbial invertebrate interac- tions and potential for biotecnology. Microly. Ecol. 12:101-110. Bourne, N., C. A. Hodgson & I. N. Whyte. 1989. A manual for scallop culture in British Columbia. Can. Tech. Rep. Fish. Aquat. Sci. 19:64, 215 p. Bourne, N. & C. A. Hodgson. 1991. Development of a viable nursery system for scallop culture. In: S. E Shumway & P. A. Sandifer. editors. An International Compendium of Scallop Biology and Culture. World Aquaculture Workshops. No. 1. Baton Rouge: The Worid Aquaculture Society, pp. 273-280. Butman, C. A., J. P. Grassle & C. M. Webb. 1988. Substrate choices made by marine larvae settling in still water and in a flume How. Nature 333:771-773. Callow, M. & R. Fletcher. 1994. The influence of low surface energy materials on bioadhesion. A review. Int. Biodeterior. Biodegr. 34:333- 348. 398 Avendano-Herrera et al. Castagna, M. 1975. Culture of the bay scallop Argopecten irradiims. in Virginia. Mar. Fish. Rev. 37:19-24. Characklis, W. G. & J. D. Bryers. 1990. Biofilms. In: W. G. Characklis & K. C. Marshall, editors. Biofilms in Wastewater Treatment. New York: John Wiley & Sons, Inc. pp. 671-696. Characklis. W. G. & K. C. Marshall. 1990. Biofilms. In: W. G. Characklis & K. C. Marshall, editors: Biofilms in Wastewater Treatment. New York: John Wiley & Sons, Inc. pp. 3-16. Chevolot, L., J, C. Cochard & J. C. Yvin. 1991. Chemical induction of larval metamorphosis of Pecten maximus with a note on the nature of naturally occurring triggering substances. Mar. Ecol Prog. Ser. 74:83- 89. Christensen, B. 1989. The role of extracellular polysaccharides in biofilms. Bioteaiology 10:181-202. Cooksey K. E. & B Wigglesworth-Cooksey. 1995. Adhesion of bacteria and diatoms to surfaces in the sea: a review. Aqua!. Microb. Ecol. 9: 87-96. Daume, S., W. Brand-Gardner & J. Woelkeriing. 1999. Preferential settle- ment of abalone larvae diatom films v/s non-geniculate coralline red algae. Aquaculture 174:243-254. Dillon, P, S„ J. Maki & R. Mitchell. 1989. Adhesion of Enteromorpha swarms to microbial films. Microb. Ecol. 17:39-47. Disalvo, L. 1991. Vibriosis y problemas del cullivo de ostion {Argopeclen purpuratus). En: IV Congreso Latinoamericano de Ciencias del Mar. 30 Septiembre a 4 Octubre. 1 72 pp. Farias, A., I. Uriarte & J. C. Castilla. 1998. A biochemical study of the larval and postlarval stages of the Chilean scallop .Argopecten purpu- ratus. Aquaculture 166:37—17. Fox, J. 1983. Intensive algal culture techniques. In: J. Mcvey, editor. Hand- book of mariculture. Volume 1 . Crustaceans Aquaculture. Boca Raton. Florida: CRC Press, pp. 15-31. Gawne, B., Y. Wang. K. Hoagland & M. Gretz. 1998. Role of bactena and bacterial expolimer in the attachment Achnanthes Umgipes (Bacillari- ophyceae). Biofouling 13:137-156. Guillard, R. & J. Ryther. 1962. Studies of marine planktonic diatoms. Cyclotella nana. (Hustedt) and Detonula confenruea (Cleve). Can. J. Microb. 8:229-237. Guillard, R. 1973. Division rates. In: J. R. Stein, editor. Handbook of Physiological Methods, Culture and Growth Measurement. Cambridge, Massachusetts: Cambridge University Press, pp. 289-312. Hadfield, M. G. 1986. Settlement and recruitment of manne invertebrates: a perspective and some proposals. Bull. Mar. Sci. 39:418-425. Hahn, K. O. 1989. Induction of settlement in competent abalone larvae. In: K. O. Hahn, editor. Handbook of Culture of Abalone and Other Marine Gastropods. Boca Raton, Florida: CRC Press, Inc. pp.lOI-112. Harvey, M„ G. Miron & E. Bourget. 1995. Resettlement of Iceland scallop (Chlamys islandica) spat on dead hydroids: response to chemical cues from the protein-chitinous perisarc and associated microbial film. / Shellfish. Res. 14:383-388. Harvey, M., E. Bourget & N. Gagne. 1997. Spat settlement of the giant scallop, Placopecten magellanicus (Gmelin 1791), and other bivalve species on artificial filamentous collector coated with chitinous mate- rial. Aquaculture 148:277-298. Hodgson, C. A, & N. Bourne. 1988. Effect of temperature on larval de- velopment of the spiny scallop, Chlamys hastata Sowerby, with a note on metamorphosis. J. Shellfish Res. 7:349-366. Hoshaw, R. W & J. R. Rosowski. 1979. Method for microscopic algae. In: J. R. Stein, editor. Handbook of Physiological Methods, Culture and Growth Measurement. Cambridge, Massachusetts: Cambridge Univer- sity Press, pp. 53-67. Illanes. J. 1990. Cultivos de moluscos en America Latina. Mems. 11 Re- union Grupo Trabajo Tecnologico. Ancud. Nov. 1989. Bogota DE Colombia, pp. 230, Kavouras, J. H. & J. S. Maki. 2000. Biofilm effects on the attachment behavior of Zebra Mussels. Big Sky, Montana: ASM Workshop — Biofilm July 2000. pp. 16-20. Kawamura, T. & H. Kikuchi. 1992. Effects of benthic diatoms on settle- ment and metamorphosis of abalone larvae. Suisanzoshokii 40:403- 409. Keough. M. J. & B. J. Downes. 1982. Recruitment of marine invertebrates: the role of active larval choices and early mortality. Oecologia 54:348- 352. Keough, M. J. & P. T. Raimondi. 1995. Responses of settling invertebrate larvae to bioorganic films: effects of different types of films. J. E.xp. Mar. Biol. Ecol. 207:59-78. Kirchman, D.. S. Graham, D. Reish & R. Mitchell. 1982. Bacteria induce settlement and metamorphosis of Janua (Dexiospira) hrasiliensis Grube (Polychaeta: Spirorbidae). J. Exp. Mar. Biol. Ecol. 56:153-163. Lozano, M. 2000. Compendio y directorio de la acuicultura y la Pesca de Chile. Aquanoticias Intemacional. Technopress S.A. pp. 355. Maki, J. D., O. Rittschof. U. Samuelsoon, A. Szewzky, B. Yule, S. Kjelle- berg, J. D. Costlow & R. Mitchell. 1990. Effect of marine bacteria and their exopolymers on the attachment of barnacle cypris larvae. Bull. Mar Sci. 46:499-511. Meadows, P. S. & J. G. Anderson. 1968. Microorganism attached to marine sand grains. / Mar Biol. Ass. UK. 48:101-115. Meadows, P. S. & J. 1. Campbell. 1972. Habitat selection by aquatic invertebrates. Adv. Mar Biol. 10:271-282. Miron, G„ P. Pelletier & E. Bourget. 1995. Optimizing the design of giant scallop (Placopecten magellanicus) spat collector: Flume experiments. Mar Biol. 123:285-291. Navarro, R., L. Sturia, O. Cordero & M. Avendaiio. 1991. Fisheries and aquaculture: Chile. In: Shumway, S. E. (ed.). Scallops: Biology, Ecol- ogy and Aquaculture. Amsterdam: Elsevier Science Publishers. pp.1001- 1015. Padilla, M, 1979. DesarroUo larval del ostion Chlamys (Argopecten) pur- purata (Lamarck 1819) en condiciones de laboratorio (Mollusca, Pele- cypoda). Cienc. Tec. del Mar. CONA 4:41-52. Parsons, G. J., M. J. Dadswell & J. C. Roff. 1993. Influence of biofilm on settlement of sea scallop Placopecten magellanicus (Gmelin, 1791 ), in Passamaquoddy Bay, New Brunswick, Canada. J. Shellfish. Res. 12: 279-283. Pawlik, J. R. 1986. Chemical induction of larval settlement and metamor- phosis in the reef-building tube worm Phragmatopoma calijornica (Polychaeta: Sabellariidae). Mar. Biol. 91:59-68. Pawlik, J. 1992. Chemical ecology of the benthic marine invertebrates, Oceanog. Mar. Biol. Annu. Rev. 30:273-335. Pearce.C. M. & E. Bourget. 1996. Settlementof larvae of the giant scallop, Placopecten magellanicus (Gmelin). on various artificial and natural substrata under hatchery-type conditions. Aquaculture 141:201-221. Pouliot, F., E. Bourget & M. Frechette. 1995. Optimizing the design of giant scallop (Placopecten magellanicus) spat collectors: field experi- ments. Mar. Biol. 123:277-284. Riquelme, C, G. Hayashida, A. Toranzo, J. Vilches & P. Chavez. 1995. Pathogenicity studies of a Vibrio anguillarum- related (VAR) sU'ain causing an epizootic in Argopecten purpuratus larvae culture in Chile. Dis. Aqua. Org. 22:135-141. Riquelme, C, R. Araya & R. Escribano. 2000. Selective incorporation of bacteria by Argopecten purpuratus larvae: implications for the use of probiotics in culturing systems of the Chilean scallop. Aquaculture 181:25-36. Rodriguez, L. 1998. Guia del Curso Fitoplancton Marino. Universidad de Antofagasta. pp.61. Seki, T. 1980. An Advanced biological engineering system for abalone seed production. International Symposium on Coastal Pacific Marine Life. Bellingham: Western Washington University, pp. 45-54. Sokal, R. & J. Rohlf. 1980. Introduccion a la bioestadistica. De. Reverte S.A. Barcelona, pp. 362. Stein, J. 1979. Handbook of Psychological Methods, Culture and Growth Measurement. Cambridge: Cambridge University Press, pp. 446. Stoodley, P., S. Wilson, L. Hall-Stoodley, J. Boyle, H. Lappin-Scott & J. Costerton. 2001. Growth and detachment of cell cluster from mature mixed-species biofilms. App. Envir. Microbiol. 67:5608-5613. Suzuki, H., T. lonka & Y. Aruga. 1987. Changes of algal community on Settlement of Larval A. pukfuratus and Diatom Biofilms 399 the plastic plates used for rearing the ahalone Haliotis discus hanncii. Nip. Siiis. Gakkaishi 53:2163-2176. Takami, H.. Kawamura. T. & Y. Yamashita. 1947. Survival and growth rates of post-larval abalone Haliotis discus hamuli fed conspecific trail mucus and/or benlhic diatom Cocconeis sculellum var. Parva. Ac/ua- culture 152:129-138. Takami. H.. T. Kawamura & Y. Yamashita. 1997. Contribution of diatoms as food sources for postlarval abalone Haliotis discus hannai on a crustose coralline alga. MoUuscan Res. 18:143-151. Tremblay. M. J. 1988. A summary of proceedings of the Halifax sea scallop workshop, August 13 and 14, 1987. Can. Tech. Rep. Fish. Aquat. Sci. No. 1605. 12 pp. Wallace, J. C. 1982. The culture of Iceland scallop. Chlamxs islandica (O. F. Muller). 1. Spat collection and growth during first year. Aqtiacidture 26:311-320. Weiner. R., M. Walch, M. P. Labare, D. Bonar & R. Colwell. 1989. Effect of biofilms of the marine bacterium Alterotnonas colwelliuna (LST) on set of the oysters Crassostrea gigas (Thunberg, 1793) and C. Virginica (Gmelin. 1791). J. Shellfish Res. 8:117-123. Wetherbee, R., J. Lind, J. Burke & R. Quatrano. 1998. The first Kiss: establishment and control of initial adhesion by rapids diatoms. / Pineal 34:9-15. Wigglesworth-Cook.sey. B. & K. Cooksey. 1992. Can diatoms sense sur- faces? State of Our Knowledge. Biofouliiig 5:227-238. Zar, J. H. 1984. Biostatistical analysis. 2nd ed. Prentice-Hall. Englewood Cliffs, New Jersey. 718 pp. Zimmer-Faust, R. K. & M. N. Tamburri. 1994. Chemical identity and ecological implications of waterbome, larval settlement cue. Liitmol. Otfwiogr 39:1075-1087. Jounml of Shellfish Research. Vol. 22. No. 2. 40 1 -+02, 2003. ADHESIVES TO ATTACH JUVENILE BAY SCALLOPS TO PLASTIC NETTING IN AQUACULTURE ENID K. SICHEL AND RICHARD C. KARNEY The Woods Hole Oceanographic Institution. Woods Hole, Massachusclts 02543; The Martha 's Vineyard Shellfish Group. Inc.. Oak Bluff's. Mas.sachiisctts 02557 ABSTRACT Fanning Ihe hay scallop. Argopeclen irradians inadiims. is a labor-intensive effort, primarily due to biofouling control on the netting of culture cages. We tested commercially available adhesives for possible application in a cageless scallop aquaculture methodology: attaching juvenile hay scallops via adhesives to polyethylene netting. The new culture method holds promise to minimize the culture structure surface area subject to biofoulmg and to facilitate harvesting. We present results for five adhesives. KEY WORDS: hay scallop, Aif^opecteii imulians. aquaculture INTRODUCTION Traditionally, bay scallops have been reared in floating cages or lantern nets in the United States. Biofotiling of cage netting and a subsequent decrease in water flow and food availability is a major obstacle for growers of filter feeding shellfish. Physical removal of fouling organisms by brushing and power washing represents a labor-intensive expense. Cageless culture methods, the topic of this article ehminate Ihe labor and expense of cleaning fouled netting. Methods used to attach seed shellfish to floating structures include, piercing the "ears" of scallops to attach a line or using adhesives to bond the shell to netting. A student group has tested adhesives for tagging marine mammals (private communication). Ear hanging experiments with bay scallops -10 mm diameter at the Martha's Vineyard Shellfish Group (MVSGl cii. 1990 were performed by piercing the ears with a Dremel® tool and stringing them on monofilament line. Although the shell surfaces were heav- ily covered with fouling organisms, the scallops grew at remark- able rates. Over the course of the growing season, the drilled ear failed to grow and eventually the weight of the growing scallops caused the ears to break. In an attempt to replace ear hanging with an alternative cage- less method, we report our recent study to find suitable adhesives and specialty cements to affix juvenile scallops to hanging struc- tures in the water column. The ideal adhesive must be strong, adhere to damp surfaces, set up quickly, cure under water and not break down in seawater. Further, it must not injure the shellfish, interfere with their growth, or leave any toxic residue in the tissue. To be useful in aquaculture, the adhesive must be cost-effective both in the cost of the labor to affix the shellfish to netting and the cost of the adhesive. METHODS We attached bay scallops to high-density polyethylene netting (ADPl Enterprises, Inc. Durethene BOP-2L, mesh size 2.25-inch by 2.2.'>-inch). The animals were quickly blotted dry and the ad- hesive was applied in air. After a cure time of about 15 min, the scallop was immersed in seawater, either in a tank or off a dock at the MVSG facility. The adhesives are listed in Table I . Testing Tests of adhesives on live scallops were "pass or fail" tests. If the adhesive held the .scallop to the netting, it was graded pass. If the adhesive failed to hold and the scallop dropped off, the test was graded fail. We discovered that it was important to engulf the polyethylene netting in adhesive to form a good bond. Shells were about 3 cm in height. Scallop shells were cleaned free of algal fouling. Live animals were scrubbed with a brush in buckets of seawater to remove algae, tubeworms, barnacles, and other fouling. Shells were blotted dry with paper towels. In addition to removal of biofouling, some tests were peiformed by touching each shell top with anhydrous ethanol or blowing dry with compressed air. No significant im- provement in results was noted with these drying techniques, prob- ably because of the high ambient humidity. When bonding shells to netting, it is easiest to place the animal on top of the netting with the adhesive sandwiched between the animal and the netting. How- ever, the animal "drools", which keeps the adhesive wet, and "claps", which disturbs the bond as it is setting up. We tried both configurations (shells under netting and shells on top of netting) and found no significant differences in bonding. Our results are shown in Table 2. The last column (#bonds) is the number of animals bonded to netting at the beginning of the study. The time that the animals were out of water is noted in the second column. In three cases, anhydrous alcohol flowed past the shell edge and the adhesive bonds were intact but several animals died. The number of bonds to empty shells (dead animals) is noted in parentheses. TABLE 1. List of adhesives Adhesive Manufacturer Adhesive Type Ceramicrete Bone & dental cement .SI 458 PSI-326 (Smart Glue) Fastcure epoxy 0.51 135-08107 Prism 454 Dr. Arun Wagh, Argonne Nat. Lab. Stoelting co. Polymeric systems Inc 3M Company Loctite Co. Ceramic cement +5% phosphoric acid Cement/methacrylic acid ester/amine Two-pan epoxy Two-part epoxy Single component 401 402 SiCHEL AND KARNEY TABLE 2. Results on bay scallops. Adhesive Air Time Life Test Conditions Results # Bonds PSI-326 (Smart Glue) 15 niin Seawater; 19 wk 3 out of 12 intact (2 dead) 12 PSI-326 (Smart Glue) 15 min Seawater: 14 wk 5 out of 24 intact 24 PSI-326 (Smart Glue) 15 min Alcohol dry: seawater; 15 wk 4 out of 12 intact (3 dead) 12 Fastcure epoxy 051 135-08107 15 min Seawater; 4 mo 7 out of 25 intact 25 Fastcure epoxy 051 135-08107 15 min Seawater; 14 wk 8 out of 24 intact 24 Fastcure epoxy 051 135-08107 15 min Alcohol dry; seawater. 14 wk 4 out of 12 intact (1 dead) 12 Stoelting bone cement 51458 20 min Seawater; 17 wk 0 out of 4 intact 4 Stoelting bone cement 51458 20 min Seawater; 16 wk 6 out of 12 intact 12 Stoelting bone cement 51458 15 min Alcohol dry; seawater; 14 wk 9 out of 12 intact (5 dead) 12 Ceramicrete 20 min Seawater; 20 wk 2 out of 5 intact (1 dead) 5 Ceramicrete 30 min Seawater; 19 wk 6 out of 9 intact 9 Prism 454 15 min Seawater, 5 wk 25 out of 25 intact (1 dead) 25 CONCLUSIONS The most promising adhesives are Fastcure epoxy 051135- 08107 (3M Company) and Ceramicrete (developed by Dr. Arun Wagh, Argonne National Lab.). The Ceramicrete powder was mixed with phosphoric acid (diluted to 5% by weight in water) to speed the setting time. Dr. Arun Wagh has recommended another additive (magnesium oxide) to further speed setting. Initial tests of "quick set" Ceramicrete with magnesium oxide additive were dis- appointing. Stoelting bone cement also proved to be a good adhe- sive for this application but may be too expensive; additionally, it sets up too fast to use with large numbers of animals at a time. Initial tests of Prism 454 (Henkel Loclite, Rocky Hill, CT) were promising but the cost of this one-part adhesive is high. Equally important for all adhesives are tests for toxicity, which remain to be done. Proper curing of epoxies requires that the ambient tem- perature be sufficiently high for the thermal energy to support molecular motion so that the chemical reaction of resin and hard- ener can go to completion. A good rule of thumb is that the reaction should occur at temperatures above the glass transition temperature, T„. (The glass transition temperature is the tempera- ture at which a polymer changes from a glassy to a rubbeiy state. Above Tg, portions of the polymer molecules are mobile.) There- fore, application of adhesives in winter poses additional chal- lenges. Resins that are liquid at 0 °C and materials with T^, near 0°C would be useful for cold weather curing. ACKNOWLEDGMENTS This project was supported in part by NSF grant DUE-0I0I632 and by the Southeastern Massachusetts Aquaculture Center (SEMAC). We benefited from many helpful suggestions from Dr. A. Pocius, 3M Company. Polymeric Systems, Inc. and 3M Com- pany generously provided free samples of adhesives. Assistance was provided by student technician, Ann Bodio. LITERATURE CITED A high school student research project, "Upward Bound" in Ohio in 2002, evaluated adhesives to attach tags to whales. Marine Quest 1492, University of Akron, Goodyear Polymer Center, Akron, OH. Gary Harp was the graduate student advisor (private communica- tion). Hamada, T., N. Yamashita, T. Watanabe & S. Natsume. 2001. Drilling position of the ear affects growth and mortality of scallop {Palino- pecten yessoensis. Jay) in ear-hanging culture. Aquaculture 193: 249-256. Harold Hudson, J. 1972. Marking scallops with quick-setting cement. Proc. Nm. Shellfish Assoc. 62:59-61. Lemarie, D. P., D. R. Smith, R. F. Villella & D. A. Weller . 1996. Evalu- ation of tag types and adhesives for marking freshwater mussels. (Ab- stract only). J. Shellfish Res. 15:528. Mallet, A. 2000. Reports on oysters attached with masonry cement to lines for aquaculture. Presentation at the Northeast Aquaculture Conference and Expo in Portland, Maine. December 7-9. Wagh, A. 2002. Perfect Patch? Adrian Cho (ed). Science 295:619. Joiinuil of Shellfish Research. Vol. 22, No. 2, 403-4U8, 200.^. EVIDENCE FOR THE INVOLVEMENT OF CYCLIC AMP IN THE METAMORPHOSIS OF BAY SCALLOP, ARGOPECTEN IRRADIANS (LAMARCK) LARVAE TAO ZHANG, HONGSHENG YANG, HUAYONG QUE,* GUOFAN ZHANG, SHILIN LIU, YICHAO HE, AND FUSUI ZHANG Institute of Ocecmology, Chinese Acadciity of Sciences. 7 Ncinhai Road Qint^duo. Shandong 266071. China ABSTRACT The putative involvemenl of cyclic AMP (cAMPi in the metamorphosis of bay scallop /I /xo/w/ch irradians larvae has been investigated on three integrated aspects. First, we conducted experiments on response of competent larvae to selective inhibitors of phosphodiesterase (PDE), theophylline, and caffeine, which presumably lead to elevated concentration of intracellular cAMP by preventing the degradation of cAMP to 5'-AMP. Second, the endogenous levels of cAMP were determined during larval development. Third, monitoring the variation of cAMP content in larvae when exposed to neuroactive compounds tested (i.-DOPA and epinephrine) and to elevated concentrations of potassium ion. was carried out to examine the possible role of cAMP as a second messenger in metamorphic pathway stimulating artificially. Consistent results have been obtained in all the three experiments. The two putative PDE inhibitors that were tested stimulated metamorphosis in A. irradians larvae significantly above control level in a dosage-dependent manner. The inductive effects did not vary significantly with exposure time. At the optimum concentration of 1.0 mM. percent metamorphosis increased by 33% and 36.0 1'/r when subjected to theophylline and caffeine respectively. The endogenous level of cAMP varied dramatically over larval development. In particular, significant increase in cAMP content from 2129 pmoL/(mg protein! for eyed larvae (Day 13 post-fenilization, PF) to 15,195 pmol/(mg protein) for spats (Day 17 PF) occurred dunng the metamorphic process. This finding indicates that metamorphic pathway involves cAMP in appreciable quantities. Furthermore, the endogenous cAMP content increased significantly in competent larvae exposed to excess potassium ion, epinephrine, or l-DOPA, suggesting cAMP plays an important role in metamorphic signal transduction pathway triggered by the three chemical cues. Evidences presented here show that cAMP becomes involved in the metamorphic pathway oi A. irradians larvae. KEY WORDS: cAMP, metamorphosis, Argopecten irradians. catecholamines, L-DOPA, PDE inhibitors INTRODUCTION Larval metamorphosis is a crucial process in the development of most murine invertebrates. Evidence indicates that this process is triggered by specific endogenous and exogenous chemical cues (Burke 1983, Baloun & Morse 1984, Coon & Bonar 1986, Yool et al. 1986, Bonar et al. 1990, Inestrosa et al. 1993a, Leise & Had- field 2000. Pires et al. 2000, Zhang et al. 2002a, Zhang et al. 2002b). Recent evidence suggests that neurotransmitters (norepi- nephrine, dopamine, and 5-hydro.\ytryptamine) play an important role in regulating metamorphosis of mollusk larvae (Coon & Bo- nar 1986. Pires et al. 2000, Zhang et al. 2002a). The cAMP/PKA (protein kinase A) pathway is one of the most important signal transduction pathways involved in the neurotransmitter regulation. Previous studies revealed or inferred that the cAMP, as an important mediator of cellular metabolism and cell-to-cell signal- ing, was possibly involved in the metamorphosis of certain species of marine invertebrates, such as the polychaete Pluigmatopoma califoniica (Jensen & Morse 1990), the barnacle Balanus amphi- trite amphitrite (Clare et al. 1995), the red abalone Haliotis rufe- scens (Baxter & Morse 1987). It remains unclear as to whether cAMP is involved in the metamorphosis of some marine inverte- brate species such as of Crassostrea gigas (Coon & Bonar 1987. Bonar et al. 1990, Coon et al. 1990) and Hydroides elegans (Holm et al. 1998). The signal transduction pathway involving cAMP, however, is incompletely understood, in the majority of previous studies, because there is no direct proof of variation in endogenous larval cAMP level during the metamorphic process. It is still unknown whether cAMP is involved in the metamor- phosis of the bay xaXXo^ Argopecten irradians (Lamarck). In this study, we investigated the potential role of cAMP and sought the *Corresponding author. E-mail; hqueCs'ms.qdio.ac.cn direct evidence on the involvement of cAMP in the metamorphosis of A. irradians larvae. We designed three experiments to test the putative involvement of cAMP in the metamorphosis of A. irra- dians. The first experiment investigates larval response to phos- phodiesterase (PDE) inhibitors. PDE is known to function in stimulating the hydrolysis of cAMP to 5'-AMP. Response of lar- vae exposed to PDE inhibitors (e.g., theophylline and caffeine), which is assumed to increase endogenous cAMP level, would provide proof revealing function relationship between cAMP and the metamorphic pathway. The second experiment is designed to manifest potential role of cAMP in the natural metamorphic pro- cess by monitoring endogenous levels of cAMP over larval devel- opment. The third experiment is designed to elucidate the possible relationship between the inductive activities of the commonly adopted exogenous chemical cues and cAMP. Results from the three experiments are expected to provide fuller understanding of the signal transduction pathway that involves cAMP in marine mollusks. MATERIALS AND METHODS Collection of Larvae Larvae of the bay scallop. Argopecten irradians (T^amarck). were obtained from Xujia Maidao Hatchery, Institute of Oceanol- ogy Chinese Academy of Sciences. Larvae collected with Nitex screen were cultured with the methods as described by Zhang et al. (1986, 1991). Test of Chemical Cues All experiments were conducted in 6-well plastic tissue culture plates using l-p-m filtered natural seawater at 23°C, 32 ppt. The selective chemical cues, including the two PDE inhibitors (the- ophylline and caffeine), l-DOPA, epinephrine (Fluka), KCI, pre- 403 404 Zhang et al. pared as 10 stock solutions in distilled water prior to experiments, were kept under 4°C. All chemicals were purchased from Sigma Company unless denoted. For experiments, the stock solutions of chemicals tested were allowed to be equilibrated to the desired temperature and then were diluted to the appropriate concentration with seawater containing A. inadiaiis larvae. Approximately 50-100 larvae in 10 niL of filtered seawater were placed in each well of the plastic tissue culture plates. Seawater in controls was diluted with distilled water to match that in experimental groups. Test solutions of PDE in- hibitors were applied at concentrations of lO""*. 10"~. 10"', 1, and 10 mM in seawater. Exposure time of PDE inhibitors varied from 1 h to 24 h. For the assessment of endogenous cAMP, competent larvae were exposed to l-DOPA or epinephrine at a concentration of 10 |xM for 8 h, or to 13.42 mM KCl for 24 h. On completion of the treatment, larvae were rinsed and re- placed in fresh filtered seawater to be ready for other procedures. Larvae were cultured for an additional 72 h before they were fixed with iodine and observed under a dissecting microscope to deter- mine the percentage of larvae that had metamorphosed and the mortality rate. Metamorphosed larvae were verified by the com- plete formation of dissoconch, the newly grown adult shell. Three replicates were conducted for each experiment with 50-100 larvae per replicate using different batches of larvae. Analysis of Endogenous cAMP Content Samples of different developmental stages of A. irradians lar- vae for cAMP assay were taken as follows: D-stage larvae (Day 3 post-fertilization, PF), umbo-stage larvae (Day 7 PF), 10% eyed larvae (Day 10 PF), 100% eyed larvae (Day 12 PF). 100% eyed larvae (Day 13 PF) and spats (Day 17 PF). Larvae following exposure to elevated concentration of K*. l-DOPA, or epinephrine were also sampled for cAMP assay. For the measurement of cAMP content -100-200 larvae were used in each sample. The extraction of cAMP was carried out by homogenizing in 5% trichloroacetic acid and centrifuging at 3,000 rpm for 30 min. The supernatant was washed with saturation ether to remove trichloroacetic acid and then dried on 70-75°C water bath. The residue was redissolved in TE buffer for cAMP assay as described by Oilman (1970). The P-E 240 Elementary Analyzer (Perkin Elmer, USA) was used for protein assay. The amount of larvae was counted prior to the measurement of cAMP content in larvae. The cAMP content is finally expressed as follows, with its unit of pmol cAMPAmg protein): Content of cAMP = (cAMP content per larva)/(protein content per larva) Where the protein content per larva was calculated using the following: Protein con- tent per larva = (absolute content of nitrogen x 6.25)/(larval amount). Data Analysis Percentage of response of larvae to chemical cues was com- pared by two-way analysis of variance (ANOVA). All analyses were conducted usmg Microsoft Excel program. RESULTS Influence of Theophylline on Larval Metamorphosis and Mortality Theophylline exhibited high and consistent inductive activity on the metamorphosis of A. irradians larvae. The percentage of metamorphosed larvae increased by over 14% at concentrations of 0.001-10 mM for 1-24 h of exposure compared to controls. The- ophylline induced larvae to metamorphose in a concentration de- pendent manner (P < 0. 1 ). Increased concentration of theophylline led to an increase in percentage of larvae that had metamorphosed. At theophylline concentration of 1.0 niM, there is an average maximum increment of 33% over control levels. The mean per- centage of larvae metamorphosing increased by 23.15% and 21.97% in response to 0.1 mM and 10 mM theophylline respec- tively (Table 1 ). On the other hand, the effect of exposure duration on the metamorphosis of A. irradians larvae was not significant (P > 0. 1 ). The average metamorphosis increment varied from 1 9.07% to 26.1% for the exposure duration of 1-24 h at various concen- trations of theophylline (Table 1 ). Theophylline treatment at concentrations lower than 1 niM for brief periods of time did not cause obvious mortality to the larvae of A. irradians. In 1 1 of 20 cases, larvae in theophylline-treated groups showed higher survival rates than that in the control groups (Table 2). Lethal effect emerged, however, when high concentra- tions of theophylline or prolonged exposure time were applied. The larval mortality increa.sed by 24.69 ± 3.56% when treated with 10 mM theophylline for 16 h compared with that of the control group. It seemed that prolonged exposure time had more impact on larval survival, as suggested by the increase in larvae mortality, by 17.64 ± 3.56%, 15.48 ± 3.45%, and 39.23 ± 4.36% for 24 h exposure at the concentration of 0.1, 1.0, and 10 mM, respectively. Overall, exposure to theophylline had no significant effect on the TABLE L Effects of theophylline concentration and exposure time on metamorphosis of A. irradians. Increment of Percent Metamorphosed 1 Larvae Above Controls Level 1 (mM) 1 h 8h 16 h 24 h Average 0.001 22.43 ± 3.45 12.97 ±2.56 17.50 ±4.23 20.91 ±2.56 18.45 0.010 18.74 ±2.76 17.29 ±1.29 8.40 ±1.73 13.84 ±2.10 14.57 0.100 20.79 ± 2.25 28.64 ± 3.65 23.94 ± 4.72 19.23 ± 2.89 23.15 1.000 21.33 ±4.67 39.49 ± 6.45 33.37 ± 4.30 37.81 ±2.57 33.00 10.00 29.54 + 5.12 32.13 ±2.64 12.16±1.26 14.06 ± 1.14 21.97 Average 22.57 26.10 19.07 21.17 Competent larvae were exposed to theophylline as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater that had been diluted to match thai in experimenlal group correspondingly. Three replicates were made with 50-100 larvae per replicate. Metamorphosis was defined as complete formation of dissoconch. Data are expressed as mean percentage and standard deviation. Cyclic AMP in Metamorphosis of the Bay Scallop 405 TABLE 2. Effects of theophylline concentration and exposure time on mortality of A. irradians. Concentration (mM) Increment of Larval Mortality Relative to Controls Level 1 h 8 h 16 h 24 h Average 0.0(11 2.73 ± 0.45 -2.84 ± 0.23 -2.61 ±0.78 -8.03 ± 0.89 -2.69 0.010 10.96 ± 2.32 -7.53 ±1.23 0.26 ± 0.09 0.05 ± 0.02 0.94 0.100 -4.62 ± 0.98 -21.16 + 2.45 -8.99 + 2.11 17.64 ±3.56 -4.28 1 .000 -13.65 + 2.34 -4.00 ±1.34 -0.91 ±0.08 15.48 ±3,45 -0.77 10.00 -4.26 ± 0.89 3.56 ± 0.78 24.69 ± 3.56 39.23 ±4.36 15.81 Average -1.77 -6.39 2.49 12.87 Competent larvae were exposed to theophylline as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate. Data are expressed as mean percentage and standard deviation. mortality of A. irradians larvae in coinparison with larvae in con- trols (P> 0.1). Influence of Caffeine on Larval Metamorphosis and Mortality Caffeine like theophylline stimulated the inetainorphosis of A. irradians larvae remarkably. The metamorphosis increment aver- aged over 10% for the treatment of caffeine at varying concentra- tions for 1-24 h. The action of caffeine on the metamorphosis was dose-dependent. Concentration of caffeine had significant effect on the metamorphosis induction (P < 0. 1). Optimum inducing of metamorphosis was achieved at a concentration of 1 .0 niM caf- feine, with mean percentage of metamorphosed larvae increasing by 36.01% among the four exposure durations (Table 3). The next effective concentration for caffeine was 10 mM. averaging incre- ment of 26.43% metamorphosis. Caffeine exposure time does not appear to significantly influence the percentage of metatnorphosed larvae. Among various durations of exposure time, metamorphosis increased by 19.65-22.02% above the control levels (Table 3). The correlation between treatment duration and efficiency of larval metamorphosis inducing was not significant (P > 0. 1 ). Statistic analysis showed that the caffeine treatment had sig- nificant effect on larvae mortality of A. irradians (P < 0.01). In particular, larvae mortality occurred more than control levels when the exposure time was prolonged to 24 h or when caffeine con- centration reached 10 mM. Increment of larvae mortality rose up to 21.29 ± 0.95% when larvae were treated with 10 mM caffeine for 24 h compared to the control groups. Exposure of A. irradians larvae to caffeine below 24 h or 10 mM, however, resulted in the increase in larval survival (Table 4). Variation of Endogenous Levels of c AMP During Larval Development The endogenous cAMP level increased over the time-course of the development in A. irradians larvae (Fig. I). The larval cAMP content underwent a gradual increase from 10% eyed larvae to 100% eyed larvae and eventually a sharp climb in the cAMP level following the completion of metamorphosis. The content of cAMP increased by 6.1 times from eyed larvae (100%, Day 13 PF) to spats (Day 17 PF). This result showed that cAMP played an active role in the metamorphic pathway that naturally occurs in A. irra- dians. Variation of Endogenous cAMP Level Following Excess K* Treatment Larvae of A. irradians underwent an increase in the cAMP level significantly above the control groups" level when exposed to excess potassium ion (Fig. 2). The content of cAMP in larvae treated with 13.42 mM KCI for 24 h was elevated to 7.8 times higher than that in the control groups, that was, from 2129 pmol/ (mg protein) to 18.656 pmol/(mg protein). Variation of Endogenous cAMP Level Following Treatment of Epinephrine or 1.-DOPA The content of cAMP in A. irradians larvae increased follow- ing the treatment of IO-|jlM epinephrine or l-DOPA for 8 h (Fig. TABLE 3. Effects of caffeine concentration and exposure time on metamorphosis of A. irradians. Concentration ImMl Increment of Percent Metamorphosed Larvae Above Controls Level 1 1 h 8h 16 h 24 h Average 0.001 12.55 ± 1.34 21.52 ±2.45 16.00 ±3.13 12.46 ±2.56 15.63 0.010 7.78 + 0.99 8.39 ± 1.12 8.40 ±1.45 24.98 ± 3.56 12.39 0.100 19.10±2.12 12.08 ± 1.56 9.42 ±1.68 13.70 ±1.98 13.58 1.000 23.92 ±2.53 35.73 ± 2.97 46.43 + 3.76 37.94 ± 3.98 36.01 10.00 34.89 ±4. 13 32.37 ± 2.38 20.00 ± 1.99 18.44 ±2.01 26.43 Average 19.65 22.02 20.05 21.50 Competent larvae were exposed to caffeine as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate. Metamorphosis was defined as complete formation of dissoconch. Data are expressed as mean percentage and standard deviation. 406 Zhang et al. TABLE 4. Effects of caffeine concentration and exposure time on mortality of A. irradians Concentration (mM) Increment of Larval Mortality Relative to Controls Level 1 h 8h 16 h 24 h Average 0.001 -4.50 + 0.14 -5.60 ±0.14 -6.43 ± 0.45 5.53 ± 0.97 -2.75 0.010 -8.60 ±0.87 -9.47 ± 0.87 -6.60 ± 0.76 6.07 ± 0.74 -4.65 0.100 -4.98 ± 0.57 -2.88 + 0.30 -9.29 ± 0.89 5.10 + 0.78 -3.01 1.000 -4.07 ± 0.49 -6.59 ±0.71 -5.73 ±0.76 9.75 ± 0.89 -1.66 10,00 -3.49 ± 0.37 5.64 ±0.45 1.07 ±0.02 21.29 ±0.95 6.13 Average -5.13 -3.78 -5.40 9.55 Competent larvae were exposed to caffeine as indicated and then allowed for recovery for 72 h. Larvae in control group were kept in filtered seawater that had been diluted to match that in experimental group correspondingly. Three replicates were made with 50-100 larvae per replicate. Data are expressed as mean and standard deviation. 3). Larval cAMP content in treated groups increased by 1.5 and 10.7 titnes than that ot the control groups, (i.e., from 4007 pmol/ [mg protein] in the control groups to 9882 pniol/[nig protein] and 46,824 pniol/[mg protein]) for epinephrine and l-DOPA treatment groups respectively. DISCUSSION It is generally believed that the pathway taking cAMP as sec- ond messenger is an important signal-transduction pathway in in- vertebrate tissues. Involvement of cAMP in larval metamorphosis varies with the species of marine invertebrates and there is no evidence of involvement of cAMP in metamorphosis of Pacific oysters Crassostrea gigas. The alpha- 1 adrenergic receptor served as the receptor of norepinephrine to regulate metamorphosis of C. gigas larvae (i.e., norepinephrine was through intracellular mes- sengers DG [diglyceride] and IP, [1,4,5-trisphosphoinositidel) not cAMP, to regulate metamorphosis of C. gigas larvae (Coon & Bonar 1987, Bonar et al. 1990, Coon et al. 1990). One report revealed that cAMP level in the gastropod Concholepas conchole- pas larvae reduced by 20 times during metamorphosis (Inestrosa et al. 1993b). An earlier report, dealing with the metamorphosis of this species, revealed that the degree of larval internal protein phosphorylation increased during the metamorphosis process 18000 16000 • ■i 14000 2 ^12000 E 10000 r 8000 - < 6000 4000 2000 0 15195 1)96 1402 1770 m. 2129 3 7 10 12 13 17 Developing days (PF) Figure 1. The variation of endogenous level of cAMP in A. irradians larvae during consecutive different developing stages as follows: D- stage larvae [Day 3 post-fertilization, (PF)|, umho-stage larvae (Day 7 PR), 10% eyed larvae (Day 17 PF). Data on the top of the bars rep- resents cAMP content. Data are averages of three duplicates with standard deviation indicated as vertical bars. (Campos et al. 1991 ). Therefore, Inestrosa et al. (1993b) concluded that the phosphorylation of protein following metamoiphosis had no relation with PKA but presumably was triggered by other kinds of kinase, including PKC. The investigators, however, did not clarify whether or not cAMP is involved in the metamorphosis of C. coiuholepas larvae. On the other hand, several studies revealed that cAMP is in- volved in the process of settlement and metamorphosis in certain invertebrate species (Jensen & Morse 1990, Clare et al. 1995). It has been shown that cAMP is involved in the morphogenetic path- way in the larvae of the red abalone. Haliotis rufescens (Baxter & Morse 1987). Cholera toxin has been found to be effective in inducing metamorphosis in Cassiopea andromeda larvae, whereas db-cAMP is not effective in initiating the settlement and metainor- phosis of the same species (FitI et al. 1987). Furthermore, endog- enous cAMP level in C. andromeda larvae did not undergo sharp variation as did in mammal species. Based on earlier observation, the authors concluded that cAMP is involved in the metamorpho- sis, but not in initiating the settlement and metamorphosis process. The drug induction method has been used for the research in signal-transduction pathway in the process of marine invertebrate larvae settlement and metamorphosis, i.e., the signal transduction pathway could be inferred from the response of larvae to the spe- cific drug that induces larval settlement and metamorphosis. This method is simple and practical and much progress has been made through this method. Because of the complexity of the biochemical reaction involved in the metamorphic process, there is still linii- 25000 r m. Figure 2, The variation of cAMP content in A. irradians larvae ex- posed to elevated K*, Larvae were treated with 13.42 mM KCl for 24 h. Data on the top of the bars represent cAMP content. Data are averages of three duplicates with standard deviation indicated as vertical bars. Cyclic AMP in Metamorphosis of the Bay Scallop 407 60000 50000 g^40000 ail E o 30000 £ S 20000 < 10000 9882 m Control Epinephrine L-DOPA Figure 3. The variation of cAMP content in A. irradians larvae ex- posed to epinephrine and 1,-DOPA. Larvae were treated with 10 jjM of the testing neuroactive drug for 8 h. Data on the top of the bars represent cAMP content. Data are averages of three duplicates with standard deviation indicted as vertical bars. tation in using this method because of deviation to some extent. Therefore, we combined both drug induction method and direct assay of endogenous level of cAMP in larvae for veiification of involvement of cAMP in the process of metamorphosis in A. ir- radians. As for drug induction, theophylline and caffeine, which could affect the intracellular level of cAMP. were used to test the mechanism of A. irradians metamorphosis. Results presented here show that both theophylline and caffeine are effective in promot- ing metamorphosis in A. irradians larvae. On the other hand. cAMP level is found to vary different larval developmental stages, especially with significant increase from eyed larvae to spats. Fur- thermore, the internal cAMP level in larvae increases significantly following exposure to excess K*. epinephrine or l-DOPA that are known as the common inductive agents for settlement and meta- morphosis in larval marine invertebrates. All these results suggest that cAMP is involved in the metamorphosis of A. irradians lar- vae. The drastic increase of cAMP, however, occurs after meta- morphosis not before. Therefore, we believed that the process of metamorphosis of A. irradians larvae was not triggered by cAMP. although cAMP is involved in this process. The triggering process might be through other pathways. Of particular interest is that mortality of metamorphosing lar- vae exposed to either theophylline or caffeine in most of the cases was much lower than that in controls. The increase in larval sur- vival was possibly due to the metamorphosis promotion of com- petent larvae in treated groups, which shortens the time elapsed in the metamorphic process. We have found that the delay of meta- morphosis resulted in increasing loss of competent larvae in A. irradians. This finding indicates that theophylline or caffeine is potentially useful for promoting yield of metamorphosed spats in A. irradians. which is essential for the efficiency of seed produc- tion in commercial hatcheries, and their use as exogenous meta- morphosis inducers by hatcheries engaging in seed production of bay scallops in China will result in promising and cost efficient commerciali/ation of bay scallop aquacullure. Ill the settlement and metamorphosis model of C. gigas, L- DOPA, as the precursor of neurotransmitter, is absorbed by the larvae and transformed into dopamine, which initiates the settle- ment of C. gigas larvae followed by the secretion of neurotrans- mitters, such as norepinephrine. This process causes metamorpho- sis in the larvae through the a_, adrenergic receptor (Bonar et al. 1990. Coon et al. 1990). In this study, the intracellular level of cAMP increased significantly following exposure of larvae to l- DOPA and epinephrine, which suggests that P-adrenergic receptor is involved in the metamorphosis of A. irradians larvae (i.e., epi- nephrine regulates the metamorphosis of A. irradians through 3-adrenergic receptor). It seems that the mechanism of A. irradi- ans metamorphosed was obviously different from that of C. gigas. In this study, endogenous level of cAMP increased with expo- sure of A. irradians larvae to excess potassium ion. It is generally believed that K* induces larval metamorphosis through directly depolarizing excitable cells involved in the larval perception of inductive stimuli (Yool et al. 1986. Baloun & Morse 1984). It, however, remained unknown as to how depolarization causes lar- val metamorphosis. Based on our results, we propose that as a result of cell membrane depolarization resulting from excess po- tassium ion exposure, nerve impulse occurs and then increases intracellular level of c AMP through certain pathways, which trig- gers phosphorylation of PKA. and eventually regulates metamor- phosis of larvae of bay scallops. In summary, this study shows that the second messenger cAMP is involved in the regulation of metamorphosis in A. irradians larvae. Since cAMP functions by activating PKA, it means that PKA is possibly involved in the metamorphosis of the bay scallop larvae. However, further proof of PKA involvement in metamor- phosis of this species has to be found in future studies. ACKNOWLEDGMENT We thank Mr. Jianghu Ma at Maidao Hatchery for providing larvae for experiments and to numerous scholars at lOCAS who extended their help to this study. This study was supported by China Natural Science Foundation Grant No. ,^9970.'i88 and No. 30200214. LITERATURE CITED Baloun, A. J. & D. E. Morse. 1984. Ionic control of settlement and meta- morphosis in larvae Haliotis rufescens (Gastropoda). Biol. Bull. 167: 124-138. Baxter. G. & D. E. Morse. 1987. G protein and ducylglycerol regulate metamorphosis of planktonic moliuscan larvae. Proc. Nail. Acad. Set. USA. 84:1867-1870. Bonar. D. B.. S. L. Coon. M. Walch. R. M. Weiner & W. Fin. 1990. Control of oyster settlement and metamorphosis by endogenous and exogenous chemical cues. Bull. Mar. Sci. 46:48-1-498. Burke, R. D. 1983. Neural control of metamorphosis In Deiulra.ster e.xcen- tricus. Biol. Bull. 167:176-188. Campos, E. O., M. Gonzalez & N. C. Inestrosa. 1991 . Biochemistry of the metamorphosis in Coiichnleivis cunchoh'pas. Arch. Biol. Med. Exp. 24:R-I95. Clare. A. S., R. F. Thomas & D. Rittschof. 199.'). Evidence for the involve- ment of cyclic AMP in the pheromonal modulation of barnacle settle- ment. J. Exp. Biol. 198:655-664. Coon. S. L. & D. B. Bonar. 1987. Pharmacological evidence that alpha-I adrenoreceptors mediate metamorphosis of the Pacific oyster Crusso- slrea giga.t. Neurosci. 23:1 169-1 174. Coon. S. L. & D. B. Bonar. 1986. Norepinephrine and dopamine content of larvae and spat of the Pacific oyster, Crassostrea gigas. Biol. Bull. 171:632-6.W. Coon, S. L.. W, K. Fill & D. B. Bonar. 1990. Competence and delay of 408 Zhang et al. metamoq^hosis in the Pacific oyster Crassostrea gigas. Mar. Biol. 106: 379-387. Fitt, W. K.. D. K. Hofman, M. Wollc & M. Raliat. 1987. Requirement of exogenous inducers for metamorphosis of a.xenic larvae and buds ot Cassiopea andromedu (Cnidaria: Scyphozoa). Mar. Biol. 94:415^22. Oilman, A. G. 1970. A protein binding assay for adenosine 3 '.5 '-cyclic monophosphate. Proc. Natl Acad. Sci. USA 67:305-312. Holm. E. R., B. T. Nedved. E. Carpizo-Ituarte & M. G. Hadfield. 1998. Metamorphic-signal transduction in Hydroides elegans (Polychaeta: Serpulidae) is not mediated by a G protein. Biol. Bull. 195:21-29. Inestrosa. N. C, M. Gonzalez & E. O. Campos. 1993a. Metamorphosis of Concholepas concholepas (Bruguiere 1789) induced by excess potas- sium. J. Shellfish Res. 12:337-341. Inestrosa, N. C., M. Gonzalez & E. O. Campos. 1993b. Molecular changes induced by metamorphosis in larvae of prosobranch Concholepas con- cholepas Bruguiere (Mollusca: Gastropoda: Muricidae). i. E.\p. Biol. Ecol. 168:205-215. Jensen, R. A. & D. E. Morse. 1990. Chemically induced metamorphosis of polychaete larvae in both the laboratory and ocean environment. / Chemical Ecol. 16:911-930. Leise. E. M. & M. G. Hadfield. 2000. An inducer of molluscan metamor- phosis transforms activity patterns in a larval nervous system. Biol. Bull. 199:241-250. Pires. A., R. P. Croll & M. G. Hadfield. 2000. Catecholamines modulate metamorphosis in the opisthobranch gastropod Phestilla sihogae. Biol. Bull. 198:319-331. Yool, A. J., S. M. Grau. M. G. Hadfield, R. A. Jensen, D. A. Markell & D. E, Morse. 1986. Excess potassium induces larval metamorphosis in four marine invertebrate species. Biol. Bull. 170:255-266. Zhang, F., Y. He, X. Liu, J. Ma, S. Li & L. Qi. 1986. A report on the introduction, spat-rearing and experimental culture of bay scallop, Ar- gopecten imidians Lamarck. Ocean. Limn. Sinica 17:367-374. Zhang, F.. Y. He, X. Liu, J. Ma, S. Li & L. Qi. 1991. Introduction, spat-rearing and experimental culture of bay scallop, Argopecten irra- dians Lamarck. Chin. J. Oceanol. Limnol. 9:123-131. Zhang, T., H. Que, H. Yang, S. Liu, Y. He & F. Zhang. 2002a. The changes of neurotransmitter content during metamorphosis of bay .scallop Ar- gopecten irradians larvae. Ocean. Limn. Sinica 3:239-244. Zhang. T.. H. Que. H. Yang, S. Liu. Y, He & F. Zhang. 2002b. Induction of metamorphosis of different day-old bay scallop Argopecten irradi- ans by chemical cues. J. Fishery Sci. China 9:228-233. Journal of Shellfish Rf.morh. Vol. 22. No. 2, 409-414. 2003. DEPURATION CONDITIONS FOR GREAT SCALLOPS {PECTEN MAXIMUS) WILLIAM J. DORR,* JENNIFER FARTHING, AND IAN LAING Centre for Eiiviriiiiineii! Fisheries and Aqiuieiilture Seieuee. Weymouth Laboratory. Barraek Road. Weymouth. Dorset. United Kingdom ABSTRACT Trials were undertaken to deternilne appropriate conditions for depurating hand-gathered great scallops {Ptrteii maxi- mu.f). Scallops were contaminated with Escherichia coli to levels consistent with those requiring depuration hy relaying in sewage impacted waters for a minimum of 2 weeks. These scallops were then purified for 42-48 h in both laboratory and small-scale commercial depuration systems under varying conditions. Levels of E. coli were monitored before and after depuration to assess the effect of temperature, salinity, shellfish-loading arrangements, and the use of artificial seawater on the depuration process. Self-righting trials were used to assess the amount of stress imposed on the scallops caused by transport, handling, and the depuration procedures. Results to date demonstrate that the use of artificial seawater cannot be recommended. During depuration, natural seawater should be maintained at a salinity ^SO'^r and at a temperature >10°C. Our results demonstrate that scallops could be depurated in a double layer within trays at a nominal density of 250 scallops m"" with a shellfish-to-water ratio of 1:12 (kg:L). KEY WORDS: great scallops, depuration conditions, purification INTRODUCTION Sewage-containinated bivalve mollu.scan shellfish can present a significant health risk if consumed raw or lightly cooked (Rippey 1994. Cliver 1997). To minimize these health risks, most countries operate legislative controls on the harvesting and placing on the market of live bivalve shellfish (Lees 2000). Such controls gener- ally rely on the use of Escherichia coli as an indicator of fecal pollution in these shellfish. European Community (EC) Directive 91/492 (Anon 1991) stipulates such controls for the EC and re- quires classification of shellfish harvested areas depending on the degree of fecal pollution, as judged from monitoring for E. coli contamination of bivalve tlesh. This classification determines whether bivalve shellfish can be sold direct for consumption or must be treated before sale. There are four classification categories (Table I ). Bivalves from category B areas require short-term self- purification in tanks of clean seawater by a process termed depu- ration (Richards 1988). All bivalves sold for consumption whether treated or not must comply with an end-product standard of <230 E. coli 100 g^'. There is increasing interest in farming great scallops (Pecten maximus) in Europe (Chataigner 1996, Dao et al. 1998) and sev- eral studies have examined the environmental requirements for cultivation of this species (e.g., Brynjelsen & Strand 1996, Fleury et al. 1996, Chauvaud et al. 1998, Laing 2000, 2002). However, scallops held in inshore areas have been shown to be as capable of accumulating equal amounts of sewage-derived micro-organism as other commercially cultivated bivalve shellfish (Silk 2000). The availability of pristine (category A) waters for scallop cultivation is limited in the United Kingdom. Most (64%) of the 249 recog- nized shellfish-harvesting areas in England and Wales are cur- rently classified as category B. About 69% of over 120 Scottish shellfish sites are category B for all or part (seasonal classification) of the year. At least two of the three present scallop farms in Northern Ireland are likely to hold a B classification (Heath & Pyke 2002). The market for scallops is predominantly for a live product. Where bivalves are sold as live product the treatment process most commonly used is depuration, which represents a major control point during the production of bivalve molluscs *Corresponding author. E-mail: w.j.doreCScefas. co.uk world wide (Richards 1998). Depuration has not been applied to scallops landed in the United Kingdom because they are tradition- ally considered to be fished in offshore locations deemed to be microbiologically secure and so are exempt from classification requirements (Anon 1991 ). To realize the full aquaculture potential of great scallops in the United Kingdom, there is a need to apply successful treatment processes that will remove microbiological contaminants. Depuration is likely to be the preferred option. Depuration relies on bivalves continuing filter-feeding activity when placed in tanks of clean seawater and purging themselves of sewage contatninants. To ensure this is achieved, suitable condi- tions must be met. Criteria for some of these conditions are com- mon to all species, such as adequate water quality, shellfish con- dition, and system design. However, some conditions, such as temperature, salinity, and loading arrangements, vary depending on the species depurated. These conditions have been carefully determined in the United Kingdom for a variety of bivalve mol- luscan species, including oysters (Ostrea edulis, Crassostrea gi- gas), mussels (Mytilus edulis). cockles (Cardium edule), and clams (Ensis spp., Mercenaria inercenaria. Tapes philippinarwn. T. de- ciissatus. and Spisula .solida). Some preliminary investigations have been conducted to determine the effect of a number of con- ditions for scallop depuration (Heath & Pyke 2002). However, it was determined that further work would be required to define these and other conditions more closely before regulatory authorities could sanction the use of depuration as a treatment process for scallops. This study investigated the effect of temperature, salinity, and emersion time before depuration and shellfish-loading arrange- ments on scallop purification, principally using E. coli elimination as a measure of depuration efficiency. The aim of the study was to produce sufficient information that would allow minimum depu- ration criteria for scallops to be determined. MATERIALS AND METHODS Experimental Animals and Environmental Contamination Market-size scallops were obtained from a commercial culti- vation site and were distributed into lantern nets at field sites that were impacted by sewage contamination. The nets were filled with six to seven scallops in each of the 12 compartments and sus- pended from floating pontoons with the top of the net at least 1 m 409 410 DORE ET AL. TABLE 1. Criteria for classifying bivalve molluscan slielirish harvesting areas (EU Shellfish Hygiene Directive 91/492/EEC). Classification E. coli Category 100 g-' Flesh Comment A Less than 230 Suitable for consumption. Can be marketed. B Less than 4,600 Depuration needed (or relaying in category A area or cooking by an approved method) C Less than 46.000 Relaying (minimum of 2 mo) m category A or B area needed (or cooking by an approved method) Prohibited Above 46,000 Cannot be taken for placing on the market from the seawater surface. The nets were deployed for at least 2 weeks to allow microbiological contamination of the scallops. Two field sites were used throughout the study and both had previously been identified as areas where the scallops would reliably accu- mulate E. coli to a level at which they would require depuration. After contamination, scallops were collected from the field site as required for depuration experiments. They were transported in groups of 60-70 animals in 40-L rectangular plastic bins covered with a dampened hessian sack to maintain a high level of humidity. For all experiments scallops were transported to the laboratory in less than 3 h. Depuration Tanks Experiments were conducted in two types of depuration tanks both using UV sterilization. Laboratory scale systems had dimen- sions of 1050 mm (length) by 300 mm (width) by 450 mm (depth) with a working volume of 200 L. Seawater was recirculated lengthways through the tank at a rate of 400 L h~' and sterilized by irradiation in a 15 W UV sterilizer (type l5/3p: UVAQ Ltd., Sud- bury, UK). Temperature was inaintained by placing the whole tank in a controlled temperature room. Dissolved oxygen levels were maintained by the use of a spray bar for recirculated water. Shell- fish were depurated in plastic mesh baskets (no. 41042; Sommer Alibert [UK] Ltd.. Droitwich. UK) raised off the base of the tank to avoid recontamination by voided fecal material. Standard design small-scale commercial systems (SFIA 1995) had dimensions 1 140 mm (length) x 950 mm (width) x 600 mm (depth) with a working volume of 550 L. Seawater was recircu- lated through the tank at a rate of 900 1 h"' and sterilized by irradiation in a 15 W UV sterilizer (type 15/3p; UVAQ Ltd.. Sud- bury. UK). Scallops were loaded into six mesh baskets (no. 41042; Sommer Alibert [UK] Ltd.. Droitwich. UK) stacked three high in two columns. Temperature was maintained by the use of an aquarium heater (Tronic 100 watt; Hagen [UK] Ltd.. Castleford. UK) or chiller units (model RA680; Teco Ltd.. Ravenna. Italy). Dissolved oxygen levels were maintained by the use of a spray bar for recirculated water. Baskets in the bottom layer were raised off the base of the tank by 50 mm to avoid recontamination by voided fecal material. Depuration Natural or artificial seawater was circulated through the depu- ration system and UV irradiated for at least 24 h before each experiment. Artificial seawater was made using a standard salt mix widely used in the UK for shellfish depuration from a commercial supplier (Seainix; Peacocks Ltd. Glasgow. UK). Contaminated scallops were thoroughly washed and damaged or gaping shellfish discarded. Prior to depuration an initial sample of 20 or 30 scallops was removed and analyzed as duplicate or triplicate samples of ten animals. Scallops were loaded into mesh baskets with cupped shell down generally in a single layer except in loading configuration experiments. For all experiments, except a trial investigating the effect of length of emersion, depuration commenced within 4 h of shellfish collection. After an initial trial using what was believed to be optimal conditions, trial parameters were changed to investigate the effect of artificial seawater. salinity levels, temperature, emer- sion time before depuration, and loading arrangements. Details of the parameters investigated are discussed further in the relevant part of the results section. A control treatment where the parameter under investigation was not varied was included for each experi- ment. All depuration experiments were run for between 42 and 48 h, after which time duplicate samples of 10 scallops for each treat- ment were removed for E. coli analysis. Levels of dissolved oxy- gen, temperature, ammonia, and pH were recorded periodically throughout the depuration period. E. coli Analysis Scallops were thoroughly washed and scrubbed under running potable water. Dead and open scallops not responding to percus- sion were discarded. Ten scallops were aseptically opened using a flame-sterilized shucking knife to sever the adductor muscle and meats and intravalvular fluid removed. These were diluted and homogenized as described previously for oysters (Dore & Lees 1995). Diluted homogenates were assayed for E. coli using a standard most-probable number (MPN) method used for shellfish analysis (Donovan et al.l998). Briefly, this is a five-tube, three-dilution procedure involving inoculation of tubes containing minerals modified glutamate broth (CM607; Oxoid Basingstoke UK) fol- lowed by incubation at 37''C for up to 48 h. Tubes displaying acid production were confirmed as containing E. coli by subculture on to Tryptone Bile Glucuronide Agar and incubation at 44°C for 24 h. After incubation, the number of tubes that were positive for (3-glucuronidase activity after subculture was recorded. The MPN was then calculated by reference to standard tables (Donovan et al. 1998). The nominal limit of sensitivity for the assay is 20 MPN 100 g~'. All results are expressed as an average for the duplicate samples. Self-Righting Experiments Self-righting experiments were performed on some surplus scallops as a simple assessment of pre- and postdepuration stress levels in the animals (Minchin et al. 2000). For these trials. 10-20 scallops were placed upside down (flat shell down) in 25 cm depth of sea water in a 3()0-L rectangular tank supplied with a continuous flow of aerated unfiltered sea water at ambient temperature and salinity (>30%f ). The number of scallops self-righting after 1 h was recorded and the result compared with that for control ani- mals. Repeated observations were made with the same scallops every 3-5 days for up to 15 days or until at least 50% of the scallops in both control and treatment groups righted within 1 h. Mortality of the scallops in the tanks was recorded. After each Depuration Conditions for Great Scallops (Pecten maximus) 411 experiment the scallops that self-righted were marked with a small spot of permanent ink and all scallops were returned to the normal (cupped side down) position until the next observation in the ex- periment. Scallops collected from the cultivation site and delivered di- rectly to the laboratory provided the control for predepuration self-righting trials to ensure that the results from the depuration experiments were not compromised by stress caused by the effects of holding, transporting, and handling of the animals during the contamination phase. Control scallops for assessing the effect of depuration (in the control depuration treatment) were taken from surplus animals collected from the field site. Comparisons were also made of post- depuration scallops from individual treatments compared with scallops from the control depuration treatment. RESULTS Initial Depuration Experiment An initial experiment was conducted under what was expected be acceptable conditions for scallop depuration based on require- ments for oysters. Conditions for the experiment were salinity levels of 36%o, temperature of 15°C ± 1°C, with a scallop to water ratio of approximately 1:50 (1:1 ratio being equivalent to 1 kg to I L of seawater). Dissolved oxygen levels were maintained above 90% saturation throughout the experiment. E. coli levels of 805 MPN 100 g"' (consistent with a category B classification) were reduced to nondetectable levels indicating that it was possible to purify category B scallops under these conditions. Further inves- tigations varied one parameter at a time. Artificial Seawater and the Effect of Salinity Concentration Experiments were conducted to investigate the effect of salin- ity. Initial trials used fresh tap water to make artificial seawater from standard salt mixes. However these trials produced high mor- tality rates and poor levels of E. coli elimination were observed (Table 2). These results apparently indicate that decreasing salinity causes an increase in mortality. However the fact that 20% mortality occurred in the control treatment (35'^f) compared with no mor- talities in the initial trial using natural seawater at a siinilar salinity described above, indicates that salinity alone was not responsible for the mortalities. A further trial comparing artificial seawater made up to a final concentration of 30%t with natural seawater diluted with freshwa- ter to also give a final concentration of 30%f demonstrated 100% mortality in the tank using artificial seawater compared with TABLE 2. Percentage mortality of scallops during depuration under varying salinity ranges. Trial Date Salinitv (%) Mortality (%) 14/2/01 27/2/01 14/2/01 27/2/01 25 30 35 35 100 55 20 20 no mortality using diluted natural seawater. E. coli levels in shell- fish in the natural seawater tank were reduced from 265 MPN 100 g"' to 20 MPN 100 g"' indicating successful depuration. To determine whether the problem associated with using arti- ficial seawater was because of the salt mix used or the fresh water in which it was diluted, an experiment comparing artificial seawa- ter (30 '^(f ) prepared by adding potable water and using freshwater that had been treated by passing through an activated charcoal filter. Scallops depurated in seawater made up in untreated water had a 20% mortality level compared with no mortalities in scallops depurated in filtered water. E. coli reductions in the scallops during depuration also differed; initial levels of 2.300 MPN 100 g~' were reduced to <20 MPN 100 g"' in the treated water tank compared with 300 MPN 100 g"' in the untreated water tank. Chlorine and ammonia levels recorded during these experiment were low in both tanks (<0.06 mg mL~' chlorine and <0.02 mg mL"' for ammonia). It therefore appears that there was some unknown con- stituent in the untreated water salt solution that was causing the mortality in scallops, which was removed by treatment with acti- vated carbon filtration. Further salinity trials were performed using natural seawater that was diluted in fresh water treated by passing through an ac- tivated carbon filter. Other parameters during these experiments were maintained at optimal conditions. Dissolved oxygen levels were maintained above 80% saturation and temperature at 15°C ± 1°C. Scallop to water ratios were maintained at approximately 1:50. Results are shown in Table 3. A salinity concentration of 28%c or higher appeared to allow successful elimination of E. coli although caution should be used in interpreting some of this data given the relatively low initial E. coli levels observed in some of the trials. In all further experiments investigating the effect of other physiologic parameters, full .saline natural seawater (range 35 to 38%c) was used. Temperature Trials Results from the experiments to investigate the effect of tem- perature on depuration efficiency are shown in Table 4. Depuration at 10. 16. and 20" C was shown to be effective at reducing £. coli to end product levels (<230 E. coli MPN 100 g"' ) even from levels consistent with a category C classification (>4600 E. coli MPN 100 g"'). In contrast a minimal reduction (10%) was observed when depuration was carried out at 7°C. TABLE 3. E. coli levels in scallops before and after depuration under varying salinity ranges. E. coli MPN 100 g-' Post Percent Trial Date Salinity (%) Predepuration Depuration Reduction 13/3/0 1 25 465 210 55.2 24/4/01 28 330 <20 >94 13/3/01 30 465 <20 >96 24/4/01 30 330 <20 >94 2/5/01 30 2.300 <20 >99 Artificial seawater was made using standard salt water mixes dissolved In potable standard water. All values are averages of duplicate samples. Artificial seawater was made using standard salt water mixes dissolved in water treated with an activated charcoal filter. 412 DORE ET AL. TABLE 4. E. coli levels in scallops before and after depuration under varying temperature ranges. Temperature E. coli MPN 100 g-' Post Percent Trial Date CCl Predepuration Depuration Reduction 31/7/01 10 2200 <20 >99.1 31/7/01 16 2200 <20 >99.1 21/8/01 10 9750 220 95.5 21/8/01 20 9750 <20 >99.8 4/9/01 7 600(1 5400 10 All values are averages of duplicate samples. Emersion Time Before Depuration One trial was performed to assess the effect of the length of time scallops were emersed before depuration had on the treatment process. Scallops were held out of water at 15°C ± TC for a total of 6. 10. and 22 h before being placed in depuration tanks at 14°C ± 1°C for 42 h. Initial E. coli levels of 1200 MPN 100 g"' were reduced to 30. 30. and 145 MPN 100 g'' for 6, 10. and 20 h emersion treatment respectively. All scallops were successfully reduced from a category B level to end product standard, although it appears that 20 h emersion may have a detrimental effect on the efficiency of depuration compared with a lO-h immersion period. Loading Arrangements An initial trial was conducted with 60 scallops loaded in two layers, cup side down, into one basket in a laboratory scale depu- ration tank under optimal conditions. The scallops moved substan- tially and several scallops escaped from the basket and on to the base of the tank amongst fecal strands that had settled there. Scal- lops did not escape from the basket in the control tank where only 20 animals were placed in one basket. After depuration, samples were taken randomly from the top and bottom layers of the treat- ment basket. Although reductions of E. coli were similar between the control and treatment (96% and 98%. respectively), scallops that had escaped the basket and were sitting on the base of the tank were not tested. Further trials placed mesh nets over the baskets so that the scallops could not escape. A space was left between the scallops and the net so that it did not impinge on the ability of the scallops to open and filter. A trial was conducted to confirm that scallops could be depu- rated in two layers. Sixty scallops were loaded into one basket in two layers and 20 scallops into another basket in the same system to act as a control. Initial E. coli levels of 3500 MPN 100 g~' were reduced to 30 and 20 MPN 100 g"' in the top and bottom layer of the treatment basket, respectively. E. coli levels in the control were reduced to 30 MPN 100 g"'. Three trials were conducted in the commercial scale depuration system fully loaded with scallops on the basis of a double layer of 50-60 scallops in each of six baskets. This gave a scallop to water ratio of about 1:12. Trials were conducted at 1 5 ± 1 °C and salinity of 36%f'. Control tanks containing just 20 scallops were also used (scallop to water ratio in excess of 1 :50). In all cases E. coli levels were reduced to below 230 MPN 100 g"' (Table 5). Dissolved oxygen decreased in the treatment tanks in all three trials, but remamed above 70% saturation at all times. Total ammonia in the three treatment tanks increased to a level between 2.5 and 5 mg L~' during the three trials compared with maximum levels 0.5 mg L"' in the control tank but did not appear to have a detrimental effect. Self-Righting Experiments The percentage number of scallops self-righting in the control groups was variable between experiments, from 40-80%, but was generally consistent for each batch of scallops for every repeated observation within experiments. It was often the same (marked) animals that righted on each occasion. It was shown that holding scallops at the field site and trans- porting them to and from the site did not apparently impose any stress. In eight righting trials these scallops performed similariy to control scallops delivered directly to the laboratory from the cul- tivation site. The mean percentage righting responses, for the first observations only, were 64.4% for control scallops and 55.6% for treatment scallops. A paired r-test showed that the difference was not significant (t = 1.08. P = 0.314. for 7 df). The self-righting response of scallops following depuration in the control treatment was similar to that for scallops from the same batch collected from the field site at the same time but not depu- rated. Mean righting response was 63.8% and 60.8% respectively (paired t = 0.515. P = 0.634. for 4 df). Scallops from the artificial seawater treatment, which was found to be not suitable for depuration, did not show any self- righting above 10% over the 14 days for which this experiment was continued, by which time there was 40% mortality. With the salinity experiments, scallops depurated at 25%o took 20 days to recover to a level of self-righting response of only 28.6%, although there was no mortality in this group. Scallops in the 289ft treatment showed no difference to the control scallops immediately following depuration at this salinity, with the same number of scallops self-righting in both groups. At low temperatures, scallops from the 7°C depuration treat- ment did not show any recovery above 10-20% self-righting over 15 days, by which time there was 70% mortality. In three obser- vations, between 50-70% of the scallops depurated at 10°C self- righted in 1 h. a similar result to those from the control treatment. It was also shown that scallops from an ambient temperature of 18°C that were held at 10°C for 42 h in a simulated depuration experiment showed no sign of stress as measured by these experi- ments. A similar number of control (untreated) scallops and scal- lops from this treatment self-righted. In four separate self-righting trials using scallops from the high density (double layer) depuration experiments, including the three carried out in the commercial scale systems, there were no differ- ences in righting response between high and low stocking densities in the depuration tanks. The mean righting responses, for the first observations only, were 64.8% (control, 20 scallops in tray) and 61.1% (double layer, 50-60 scallops in tray; paired t = 0.502. P = 0.65. for 3 df). DISCUSSION The predepuration self-righting experiments showed that the conditions used for holding and handling the experimental ani- mals, including transportation to and from the field site and the laboratory, did not cause stress to the scallops. Most of the other infomiation available on transporting scallops is in respect of mov- Depuration Conditions for Great Scallops (Pecten maximus) 413 TABLE 5. E. coli levels before and after depuration in scallops taken from various positions throughout small-scale commercial depuration systems. Sample Position E. coli MPN 100 g- Predepuration Post Depuration % Reduction 99.8 99.7 98.2 99.2 99.6 88.1 92.4 97.9 92.4 92.4 97.9 97.9 97.9 95.7 97.9 97.9 98.9 97.8 98.9 97.8 97.8 4/12/01 Control Top basket top layer Top basket bottom layer Bottom basket top layer Bottom basket bottom layer 17/12/01 Control Left top basket top layer Left top basket bottom layer Right top basket top layer Right basket bottom layer Left mid. basket top layer Left mid. basket bottom layer Right mid. basket lop layer Right mid. basket bottom layer Left bottom tray Right bottom tray 26/2/02 Control Left top basket Left bottom basket Right top basket Right bottom basket 9,100 925 10,000 20 30 165 20 40 115 70 20 70 70 20 20 20 40 20 20 110 220 110 220 220 Scallops were loaded in plastic mesh trays in double layers at a density of approximately 50-60 scallops per tray. ing juveniles from a hatchery or collector site to a cultivation site. This has shown that periods of up to 12 h out of water have no observable effect, provided the scallops are maintained in a humid atmosphere (Maguire et al. 1999. Christophersen 2000. Minchin et al. 2000). One of the current depuration experiments showed that the process could be run effectively with scallops that had been immersed for this amount of time. Longer periods of emersion may compromise the ability of the scallops to depurate, and there was some indication of this from the results of this study. Initial trials had indicated that it was possible to successfully depurate category B level scallops using standard procedures used in the United Kingdom without any detrimental effects on the product quality. Subsequently this study concentrated on finding the minimum acceptable requirements for parameters, such as tem- perature, salinity, etc., when depurating scallops. The use of artificial seawater during depuration is common practice for a wide range of species. However, results during this study indicated that the use of artificial seawater is unacceptable under the conditions applied here. This finding does not concur with previous work (SFIA 1996). which demonstrated that it was possible to use artificial seawater in tanks for degritting scallops without any reduction of scallop activity or increased mortality. However, no details of what was used to dilute the artificial salt mix were given for that study. The scallop depuration trials con- ducted by Heath and Pyke {2001 ) used natural seawater only. The results obtained here indicated that an unidentified constituent of the tap water used to make the artificial seawater was responsible for the mortalities observed. Artificial seawater has been used on numerous occasions at the CEFAS Weymouth laboratory to un- dertake depuration trials with other shellfish species without any effect on shellfish quality, and this result is unique to these scallop trials. Because it was not possible to identify the constituent re- sponsible for the problem, it remains unclear whether this situation is unique to the water used at this laboratory or would be a wide- spread problem if used in the field. However, until further work is done to investigate the use of artificial seawater during scallop depuration the use of artificial seawater in this role cannot be recommended. Salinity was found to have a critical effect on efficiency of depuration and levels of 25%o had a detrimental effect on the rate of E. coli clearance. The next lowest concentration of salinity that was investigated was 28%t. and E. coli levels were successfully eliminated at this salinity. However only one trial was conducted at this salinity, and the initial level of E. coli in this trial was only 330 MPN 100 g"'. It is questionable whether this can be consid- ered a suitable challenge to test this condition. Given this it is recommended that in the absence of further work a minimum salinity concentration of 30%c for scallop depuration should be maintained during depuration. This is a relatively high salinity compared with minimum concentrations set for other species. This is not surprising given that scallops are an open seawater species that will normally be exposed to full-salinity seawater. It should be noted that the consistent availability of natural seawater at a sa- linity in excess of 30%c might present a constraint in some com- mercial settings. This requirement should be carefully considered by operators at the outset of any plans to depurate scallops. Temperature was also found to have a significant effect on E. coli reduction. Minimal reductions were observed at 7°C, whereas E. coli levels were successfully depurated at 10°C. These results do not agree with those from previous studies (Heath & Pyke 414 DORE ET AL. 2001), which concluded that temperatures as low as 6.6°C could effectively reduce levels of E. coli. They are, however, in agree- ment with the results of McNamara (SFIA 1996), who recom- mended a temperature range of 10-1 8°C for scallop degritting based on measurements of shellfish activity. In the absence of any further work, a minimum temperature of IO°C is recommended for use during scallop depuration. No experiments were conducted to define upper temperature limits for depuration. This is because higher temperatures do not usually compromi.se the depuration process, although they may affect product quality. In the one ex- periment (Table 4) in these trials in which scallops were depurated at 20°C the scallops depurated effectively, but there was some post depuration mortality. Heath and Pike (2001) recommended an up- per temperature limit of 16°C for depuration and McNamara (SFIA 1996) a limit of I8°C for degritting. Although scallops depurated to below the end product standard in all treatments during the single experiment investigating emer- sion time, there was some evidence that animals immersed for 22 h reduced E. coli levels less successfully compared with those immersed for 10 h. However this single result must be considered inconclusive, although it does concur with previous work (SFIA 1996). That study concluded that scallop activity was reduced during degritting in scallops that were immersed for 24 h before processing. A general conclusion from various studies on the effect of emersion time on viability of great scallops is that they should not be kept out of water for longer than 12 h (Maguire et al. 1999, Christophersen 2000. Minchin et al. 2000). It was possible to depurate successfully in a double layer with nominal capacity of 230 scallops m'~. Scallops loaded at this density showed a considerable tendency to move and, if left un- confined, escaped from the basket and deposit themselves on the base of the tank. This is considered unacceptable during depuration as much of the fecal material excreted by the scallops during the depuration cycle will settle on the base of the tank. Movement of the scallops in this area will resuspend this material, which may be reingested and recontaminate the scallops with microbiological organisms present in the sediment. During this trial, plastic mesh was placed over the baskets to prevent the scallops escaping. It is critical for the depuration process that scallops should be contained within the basket. Any procedure for doing this must not interfere with the ability of the scallops to open and filter. The lowest scallop to shellfish water ratio investigated in this trial was 1:12 because this was the maximum that could be achieved using the double layer arrangement. This is a higher ratio than may be found in some of the high-intensity systems that may be used to depurate other species. These may have shellfish-to-water ratios of as low as 1 :3 when fully loaded. However it is considered unlikely that high-density depuration of scallops is likely to be required in the near future. Given this and results from density trials conducted elsewhere (Heath & Pyke 2001 ), it is recommended that scallop to water ratios should not fall below 1:12. In general, results from the post depuration self-righting (stress) experiments gave good agreement with the results from the depu- ration experiments. That is, scallops from conditions that sup- ported effective depuration showed no difference in stress to con- trol scallops, whereas scallops from conditions in which they did not depurate showed high levels of stress, sometimes accompanied by subsequent high mortality. Also, the lower temperature and salinity limits, below which the scallops will not depurate effec- tively, are similar to the lower limits for optiinuni growth perfor- mance of great scallops (Laing 2000. 2002). ACKNOWLEDGMENTS This research was funded by the UK Department for Environ- ment, Fisheries and Rural Affairs. LITERATURE CITED Anon. 1991. Council directive of 15 July 1991 laying down the health conditions for the production and placing on the market of live bivalve molluscs (91/492/EEC). Offic. J. Eur. Comm. 268:1-14. Brynjelsen, E. & O. Strand. 1996. Test production of king scallop in intermediate culture — 1995—1996. Fisken Havet Havforsknini^sinsntut- rer 18:34. Chataigner, E. P. J. 1996. L'aquaculture de la coqullle Saint-Jacques (Pecten maxiniKs): un complement pour la peche. Toulouse France. Ecole Natl. Veterinaire de Toulouse, (France), 117 pp. Chauvaud, L., G. Thouzeau & Y. M. Paulet. 1998. Effects of environmen- tal factors on the daily growth rate of Peclen mav/'/HHi juveniles in the Bay of Brest (France). / Exp. Mar. Biol. Ecol. 227:83-1 1 1. Christophersen, G. 2000. Effects of air emersion on survival and growth of hatchery reared great scallop spat. Aquaciilt. Int. 8:159-168. Cliver, D. O. 1997. Virus transmission via food. Food Teclviol. 51:71-78. Dao. J. C. N. Lake, M. Nonnan & S. Tilseth. 1998. Scallop seabed cul- tivation in Europe. In: K. G. Barthel, H. Barth, M. Bohle-Carhonell. C. Fragakis, E. Lipiatou & P. Martin, editors. Third European Marine Science and Technology Conference, Lisbon, 23-27 May 1998, Vol 5: Fisheries and Aquaculture AIR: 1990-94. Selected projects from the research programme for Agriculture and Agro Industry including Fish- eries, Donovan. T. J., S. Gallacher, N. J. Andrews, M. Greenwood, J. Graham, J. E. Russel, D. Roberts & R. Lee. 1998. Modification of the standard method used in the United Kingdom for counting Eschericliiu coli in live bivalve molluscs. Com. D/.s. anil Public Health 1:188-196. Dore, W. J. & D. N. Lees. 1995. Behavior of Escherichia coli and male- specific bacteriophage in environmentally contaminated bivalve mol- luscs before and after depuration. Appl. Environ. Microbiol. 61:2830- 2834. Heath, P. & M. Pyke. 2002. King Scallop (Pecten maximus) Depuration Tnals. J. Shellfish Res. 20:117-120. Fleury, P. G., C. Mingant & A. Castillo. 1996. A preliminary study of the behaviour and vitality of reseeded juvenile Great Scallops, of three sizes in three seasons. Aquaciilt. Int. 4:325-337. Laing, I. 2000. Effect of temperature and ration on growth and condition of king scallop [Pecten tna.\'innis) spat. .Aquaculture 183:325-334. Laing, 1. 2002. Effect of salinity on growth and survival of king scallops (Pecten maximus). Aquaculture 205:171-181. Lees, D. N. 2000. Viruses and bivalve shellfish. Int. J. Food Microbiol. 59:81-116. Maguire, J. A., D. Cashmore & G. M. Bumell. 1999. The effect of trans- portation on the juvenile scallop Peclen maximus (L.) Aquacult. Res. 30:325-333. Minchin. D., G. Haugum, H. Skjaeggestad & O. Strand. 2000. Effect of air exposure on scallop behavior, and the implications for subsequent sur- vival in culture. Aquacult. Int. 8:169-182. Richards, G. P. 1988. Microbial purification of shellfish a review of depu- ration and relaying. J. Food. Prot. 51:218-251. Rippey, S. R. 1994. Infectious diseases associated with molluscan shellfish consumption. Clin. Microbiol. Rev. 7:419. SFIA. 1996. Degritting of King Scallops. Seafish report No. SR468. Hull. UK: Seafish Industry Authority. Silk. R. 2000. Might scallops need depuration? Shellfish New.'i 10:7-8. Joiirmil of Shellfish Research, Vol. 22, No. I, 41S-42I, 2003. CIRCADIAN METABOLIC RATE AND SHORT-TERM RESPONSE OF JUVENILE GREEN ABALONE (HALIOTIS FULGENS PHILIPPI) TO THREE ANESTHETICS OSCAR CHACON,' MARIA TERESA VIANA,' ANA FARIAS," CARLOS VAZQUEZ,' AND ZAUL GARCIA-ESQUIVEL' * ^Instititto de Investigaciones Oceanologicas. Universidad Autonoma de Baja Ccdifomia. Apdo. Postal 453, 22 800 Enseiiada. B.C. Mexico: 'Instituto de Aciiicultura, Universidad Austral de Chile, Campus Puerto Montt. Puerto Monti. Chile: and ^Universidad Autonoma de Mexico, Facultad de Ciencias Veterinarias. Ciudad Universitaria. Mexico. DF ABSTRACT Time-course experiments were performed on juvenile green abalone {Hiilmlis fiili;fii.^\ to assess the degree of stress caused by the anesthetics magnesium sulfate (MS), benzocaine (BZ). and phenoxyethanol (PE). Metabolic rate (VO,) of abalone was reduced by 65. 35. and 18% during short-term (10 or 20 mini exposure to MS. BZ. and PE. respectively. Abalones significantly increased their VO. above control values (1.5-fold) after removal of PE from metabolic chambers, whereas those treated with MS or BZ recovered their VO, to preanesthesia values. Visual criteria of recovery generally coincided with those of metabolic measurements (i.e., 80% of abalone regained "normal" activity after 35 min postanesthesia), yet metabolic measurements showed that "fast" recovering abalone treated with PE maintained high VO, values during 3-h postanesthesia. Abalone treated and nontreated with anesthetics exhibited a circadian metabolic rhythm, with 20-35% higher rates observed during dark than light hours. Despite the short-term metabolic alterations with MS. BZ. and PE, the present study suggests that all three anesthetics may be safely used in abalone. However, detailed evaluations are still needed to assess the effect of anesthesia on other physiological variables. The results obtained in this study highlight the importance of physiological evaluations when different chemical substances are used in aquatic invertebrates. KEY WORDS: abalone, anesthetics, circadian rhythm, metabolism, Haliotis fidgens INTRODUCTION Current culture methods for abalone involve vaiious steps in which organisms need to be dislodged from their rearing substra- tum for the purpose of size grading, adjusting densities, tagging, and/or transfening from indoor to outdoor culture facilities (Hahn 1979, Juefeng & Shiuan 1996). Because of the natural ability of abalone to strongly adhere on most surfaces, forced removal not only results in excess mucus production with the consequent en- ergy losses (Peck et al. 1987, Davies & Williams 1995, McBride et al. 2001 ), but it can also result in injuries in the .soft tissues that may eventually result in death (White et al. 1996). It is thought that the lack of clotting mechanisms in abalone facilitate prolonged bleeding and/or the appearance of bacterial infections in wounded tissues (Armstrong et al. 1971 ), thus increasing the probabilities of death (Juefeng & Shiuan 1996). Farms have produced several solutions to remove abalone from the substrate without producing injuries. All of them are aimed to relax the soft tissues or decrease the degree of awareness in aba- lone and include thermal shock and desiccation (Hahn 1989) in addition to the use of anesthetic substances, such as CO,, urethane, chloral hydrate, barbitol, diethyl carbonate, benzocaine, ethyl al- cohol, propylene phenoxytol, potassium chloride, procaine hydro- chloride, MS-22, pentobarbital, magnesium sulfate, and phenoxy- ethanol (Hahn 1989. Juefeng & Shiuan 1996, White et al. 1996, Aquilina & Roberts 2000). The last three substances have been reported as effective and nonlethal anesthetics for abalone because organisms usually recover within the first few hours of application (Hahn 1989, White et al. 1996. Aquilina & Roberts 2000). Excess magnesium sulfate interfere with neuromuscular trans- mission signals in mammals because magnesium ions block the *Con-esponding author. Tel. -1-52-646-174-4601; Fax -I-52-646- 1 74-5303; E-mail; sgarcia(a'uabc.mx release of the neurotransmitter acetylcholine from motor nerve endings, by competitively binding to N-methyl-d-aspartate (NMDA). a glutamic acid receptor (Iwatsu et al. 2002). The overall effect of this blockade is a sedative effect of the neuromuscular system, followed by muscle paralysis, respiratory depression, coma, and death (Swain & Kaplan-Machlis, 1999). It is thought that phenoxyethanol also binds competitively to NMDA receptors (Mushoff et al. 1999) and causes depression of the central nervous system and hypoxia when delivered in excess (American Veteri- nary Medical Association 2001 ). The mechanism of action of local anesthetics, such as benzocaine (BZ), is a blockade of the voltage- activated sodium channel at the neuronal cell membrane, which prevents the generation and conduction of the nerve impulse (Cat- terall & Mackie 1996). Excess BZ in mammals may result in prolonged sedation, cardiac arrhythmias, respiratory depression, tremors, and death (Catterall & Mackie 1996). The effectiveness of anesthetics in marine molluscs has been largely evaluated on the basis of visual observations, such as the degree of gaping respon.se after tactile stimuli in bivalves (Culloty & Mulcahy 1992, Heasman et al. 1995, Mills et al. 1997), degree of muscle relaxation, and coloration in squids (Garcia-Franco 1992), and degree of adhesion, muscle relaxation, and mortality in abalone (Hahn 1989, White el al. 1996, Aquilina & Roberts 2000). In most cases, short-, medium-, or long-term effects of anesthetics have not been studied in detail, even though the magnitude of stress during and shortly after the application of anesthetics is well documented with visual observations. Therefore, detailed under- standing of the effect of anesthetics on abalone is still needed at the physiologic level, especially because they represent a potential tool for research and management. Several physiological parameters have been reported to in- crease at night in abalone, including motor (Donovan & Carefoot 1998), feeding activities (Barkai & Griffiths 1987), and metabolic rate (Uki & Kikuchi 1975). The latter is known as a highly sen- 415 416 Chacon et al. sitive parameter in molluscs because it readily changes in response to stress factors, such as temperature (Newell 1973, Paul & Paul 1998). pH (HaiTis et al. 1999), nitrite (Harris et al. 1997) and ammonia concentrations (Harris et al. 1998), and starvation (Gar- cia-Esquivel et al. 2002). Respiration is controlled by the central nervous system, and therefore it is not surprising that metabolic rate of abalone is affected by substances such as magnesium sul- fate (Edwards et al. 2000). In the present study, time-course mea- surements of metabolic rate were performed in juvenile green aba- lone (Haliotis fulgens) in the presence and absence of anesthetics to assess the magnitude and duration of metabolic stress produced by magnesium sulfate, phenoxyethanol and benzocaine. METHODS Experimental Conditions One and a half year-old juvenile abalone (Haliotis fulgens) with shell lengths ranging from 25 to 35 mm, originally obtained from BC Abalone farm in Erendira B.C., Mexico, and maintained at the laboratory facilities at the University of Baja California, were used for the different experiments. Abalone was kept in a shallow water tray (180 x 90 x 20 cm, length x width x height) under flow- through (ca. 300 mL min"'), aerated seawater conditions. Seawater temperature was maintained at 23 ± 1°C with a digitally controlled heater (CLEPCO. 1000 watts) located in a reservoir. Inert food was offered at night on a regimen of 12 h per day, with a diet (Table 1 ) made in the laboratory as recommended by Viana et al. (1996). Light intensity was kept at ca. 2 x 10"*^ p-E/s/cm" with several layers of a plastic mesh (70% shed) placed around the system. A photoperiod of 12:12 (Light: Dark) was maintained throughout the study. Experimental Design Experiments were of two types: (1) Time series to identify circadian changes in the metabolism of H. fulgens. and (2) Time series to assess the short-term (3 h) and medium-term (2 days) effects of magnesium sulfate (MS), phenoxyethanol (PE), and BZ on the metabolic rate. Circadian Changes Twenty-four abalone were randomly selected from the main- tenance tray and distributed among eight respiration chambers (three organisms per chamber) with a volume of 1.8 L each. Four additional chambers were also used as controls (without organ- isms). Chambers were maintained with open flow and without food during 24 h for the abalone to acclimate to the system. Feces and any remaining particles were siphoned out from each chamber before beginning the first measurement of oxygen consumption. Respiration was measured using closed-cell respirometry. Incuba- tions of ca. 1- to 1 .5-h duration were conducted every hour during a total period of 48 h. At the end of this period, the live weight and total length of the experimental abalone was recorded. Anesthetics Two experiments were carried out with using the anesthetics magnesium sulfate (MS. Sigma M-75()6) at a final concentration of 4% w/v (Hahn 1989); phenoxyethanol (PE, Sigma P-1 126) at 0.1% v/v (Edwards et al. 2000) and benzocaine (BZ, Sigma E-1501) at 0.01% v/v (Hahn 1989). The latter was dissolved in 95% ethanol (10% w/v) before use. All final solutions were prepared in 5-p,m filtered seawater just before application. TABLE L Percent composition (dry weight basis) of the balanced diet used offered to juvenile green abalone Haliotis fulgens. Ingredients (g/100 g diet) Balanced Diet Fish meal* Corn starch Kelp meal'' Corn tlour'' Gelatin (50 blooms) Soybean meal' Cellulose'' Modified starch*^ Mineral mixture' Vitamin mixture^ Fish silage'' Stay-C Choline chloride Sodium benzoate BHT' Composition (%) Protein Ash Nitrogen-free extract 30.00 14.66 10.00 10.00 10.00 8.00 5.00 5.00 4.00 1.50 1.40 0.20 0.10 O.iO 0.04 30.8 ± 0.7 12.6 ±0.1 6.3 ±0.1 50.3 ± 04 * 64% protein. '' Made from Macrocyslis pyrifera. '' Corn flour (Maseca). " 39Vf protein, 2V7c lipid. '' «-cellulose (Alphacel). ■■■ Modified com starch (Clearjel®). ' ICN vitamin diet fortification. * ICN salt mixture #5 Brigges. '' Acid fish silage from tuna viscera. ' Ascorbyl polyphosphate (kindly donated by Roche). ' Butylated hydroxy toluene. In the first experiment, a step-wise approach was used for quantifying the short-term response of juvenile abalone exposed to anesthetics. Metabolic rate was measured before, during, and after the application of anesthetics. Each anesthetic was evaluated on different days using four replicate chambers per anesthetic (three organisms per chamber), three control chambers (abalone without anesthetic), and three reagent controls (seawater with anesthetics, but no abalone). Experimental organisms were transferred to the chambers 24 h before the treatment, as described in the previous section. At the end of this period, chambers were cleaned and respiration rate of abalone was measured during 1-1.5 h. The water was completely renewed ( 100% oxygen saturation) and an- esthetics were added directly into the incubation chambers, while recordings of oxygen consumption continued. Abalones were in contact with anesthetics for a fixed period of 10 min (BZ and PE) or 20 min (MS). These treatment periods were based on prelimi- nary visual observations of the organism's response to these an- esthetics. Chambers were flushed with fresh seawater (ca. 6 vol- ume changes) after the exposure period to eliminate anesthetics. It was assumed that anesthetics got rid off the chambers during flush- ing, as the reagent control and experimental chambers regained a constant oxygen baseline afterwards. Incubations continued every 1 or 1.5 h during the following 3 h to measure the metabolic response postanesthesia on the same organisms. CiRCADiAN Rhythm and Anesthetics in H. fulgens 417 The second experiment consisted of a time series of 48 li v\'ith simultaneous measurement of VO, in abalone treated with all three anesthetics (MS, PE, BZ) to assess the duration of metabolic stress. Experimental abalone were removed from the maintenance tray with the help of a spatula and transferred to plastic buckets con- taining I L of seawater. When all organisms had adhered to the walls, all three anesthetics (MS, PE, BZ) were added separately into the buckets and the anesthetized organisms were transferred to metabolic chambers (3 abalone per chamber), where respiration measurements began 3h later. Incubations lasted between 1 .3 and 2 h, with a measurement frequency of ca. 8h, and a total elapsed time of 47 h. At the end of each trial, oxygen consumption rate was measured in the same experimental chambers without abalone, to correct for the oxygen consumed by sources other than abalone (electrodes, microorganisms). A total of three replicates per treat- ment (anesthetics) and two control replicates (abalone without an- esthetics) were used for this trial. Measurements Metabolic Rate Oxygen consumption by H. fulgens was recorded every 30 sec with two computer-controlled polarographic oxygen sensors (Strathkelvin Instruments Ltd.. Ireland). Each oxygen meter had six channels, such that 12 chambers could be monitored simulta- neously. Aerated seawater was used for calibration to 100%, and sodium sulfite was used for 0% calibration. A magnetic stir bar ( lO-mm diameter x 8-mm length) was used for mixing the water in each incubation chamber under a perforated acrylic sieve (4-mm mesh) to prevent a direct contact between the stir bar and organ- isms. After incubations all abalone from the chambers were blot dried with a piece of cloth, measured with digital calipers (MAX- CAL, ± 0.03 mm) on their longest dimension, and weighed in a portable scale (AND SV-200, ± O.Olg). Oxygen consumption rate (metabolic rate, VO,l was estimated from the corrected slope of the oxygen evolution curve (abalone minus non-abalone cham- bers), after transforming the VcO-, saturation to |j.mol of dissolved O, in seawater. from known values of oxygen solubility (Green and Cairitt. 1967). The following equation was used for calculat- ing metabolic rate: VO,, = (Cs*m*60)/(100%* Wwt) (I) where VO,^. = metabolic rate of the experimental organism (|j.L O, /g/hT Cs = total amount of O, in the incubation chamber at 100% saturation (jjiL O,). m = slope of the O^ evolution curve (9'rO-,/min) 60 = factor used to transform from minutes to hours Wwt = live weight (g) of organisms in the incubation chamber. Visual Assessment of Anesthesia and Recovery Direct observations of abalone behavior during and after ap- plication of each anesthetic were conducted on 36 organisms dis- tributed in 12 buckets (three organisms per bucket). The time taken from the application of anesthetics to the moment an abalone fell off the walls of the bucket was considered as the period needed for induction to anesthesia. Similarly, the time taken for an anesthe- tized abalone to regain an upright position (ventral side firmly attached to the container's walls) was considered a visual criterion for recovery postanesthesia (White et al. 1996). Mortality was evalu- ated on anesthetized organisms after 2 or 4 weeks postanesthesia. Statistics In all cases, time-dependent changes of metabolic rate were statistically tested using an analysis of variance (ANOVA) with repeated measures. When significant differences were found, least squares pre-planed comparisons of means were used to identify specific differences. These tests were carried out using a general linear model procedure (GLMi included in the statistical package SAS. version 6.08 (SAS, 1998). RESULTS Short-Term Effect of Anesthetics Juvenile abalone treated with all three anesthetics exhibited time-dependent differences in their metabolic rate (VO,), as this was significantly reduced (Table 2) by 65% (MS), 35% (BZ), and 18% (PE) of initial values during the exposure period (Fig. la-c). TABI^E 2. Results of repeated analysis of variance for comparison of short-term (5 h) and long-term |48 h) changes of metabolic rate of juvenile abalone, Haliotis fulgens, after exposure to three anesthetics (Anest). Source of Variation Mean Squares F DF MS PE BZ MS PE BZ Short term Replicates 3 28.0 60.9 46.9 0.9 NS 3.8* 1.0 NS Between Subjects (Anest) 1 390.5 1137.8 30.7 12.8** 70.3** 0.7 NS Within Subjects (lime) 3 824.5 431.3 234.5 27.0** 26.6** 5.1** Time x Anest 3 842.0 439.6 378.3 27.6** 27.1** 8.2** Error 17 30.5 16.2 46.2 Long term Replicates 2 51.8 3.1 NS Between Subjects (Anest) 3 141.2 8.4** Withm Subjects (time) 6 115.22 68.8** Time x Anesth 18 46.9 2.8** Error 47 16.7 Short-term trials were conducted independently for each anesthetic and its control whereas the long-term trial was conducted simultaneously for the anesthetics magnesium sulfate (MS), phenoxyethanol (PE). henzocaine (BZ) and a control without anesthetics (C). * P < 0.05; **P < 0.01 ; NS = not significant at P > 0.05. 418 Chacon et al. D) CM o 80 - (b) r<^-^^^ 3. S 60 ■ -< ^J^^^-^ (T o ^i o 3 40 J u Elapsed Time (h) Figure 1. Short-term changes of metabolic rate in juvenile green aba- lone {Haliotis fiilgens) before, during, and after exposure to magne- sium sulfate (a), phcoxyethanol (bt, and benzocaine (cl. Arrows indi- cate the moment when anesthetics were added i and removed T from the respiration chambers. Abalone from the MS and BZ treatments re-established their VO, after flushing away the anesthetic from the respiration chambers, and remained similar to control values thereafter (P > 0.05). In contrast, abalone treated with PE significantly increased their VO. by a factor of 1 .5 above control values (Table 2) and remained high for the ne.xt 2 h posttreatment. with a trend to decrease thereafter (Fig. lb). Oxygen consumption of reagent control chambers (an- esthetics, without abalone) also increased in the presence of these chemicals. It accounted for lO^r (MS). 13% (PE), and 54.3% (BZ) of the total O. consumed by experimental abalone during the ex- posure period, yet no significant O, consumption was observed in the reagent chambers (P > 0.05) after anesthetics were flushed away (data not shown). Diel Changes of Metabolic Rale A circadian rhythm was observed in juvenile abalone in the absence of anesthetics (Fig. 2a). with significantly higher meta- bolic rate observed during dark than light hours (P < 0.01 ). Meta- bolic rate (VO-,) decreased and remained relatively constant (42- 32 jjiL OMg Wwt) during the period of 10;00 to 16:00 h (light conditions), and significantly increased (P < 0.01) by ca. 20% during the period of 22:00 to 4:00 h (dark conditions). The tran- sition period (light switched on or off. at 8:00 and 20:00 h. re- spectively) was characterized by rapid changes of metabolic rate, such that abalone exhibited most of the time a dark- or a light- adapted metabolic rate (Fig. 2a). The circadian VO, pattern was maintained throughout the 48-h measuring period, even though the absolute values showed a trend to decrease in the second day of the trial (P < 0.05). Abalone exposed to anesthetics exhibited signifi- cant anesthetic and time effects (Table 2), and the same circadian rhythm identified above. VOj values obtained at 8:00 or 20:00 h (Fig. 2b) also corresponded to the transition period. Higher values (65 to 70 [jLLO,/h/g Wwt) were observed during dark hours and lower values during daylight hours (Fig. 2b). Time vs anesthetic interaction was also significant (Table 2). PE-treated abalone ex- hibited significantly higher VO, values than control organisms (P < 0.05) during the first measurement (3 h postanesthesia), yet these differences were not statistically significant thereafter (P > 0.05). Visual Criteria Based on motor activity, it was observed that SM acted slowly and asynchronously on juvenile abalone. These organisms showed a complete relaxation of the mantle and became narcotized some- DAY NIGHT DAY NIGHT I 3. B o 75 60 45 30 90 (a) 75 - 60 45 30 16 24 32 40 48 Elapsed Time (h) Figure 2. Two-day recordings of metabolic rate exhibited by juvenile green abalone (Haliolis fulgens) without anesthetic treatment (a) and after a lO-min exposure (b) to the anesthetics magnesium sulfate (MS), phenoxyethanol (PK), and benzocaine (BZ). A control treatment (aba- lone, no anesthetics) is also shown. Time 0 = 10:00 AM. CiRCADiAN Rhythm and Anesthetics in H. fuluens 419 TABLE 3. Visual assessment of minimal (min) and maximal (max) exposure period (minutes) necessary to anesthetize juvenile abalone. Haliotis Julgeiis, by magnesium sulfate (MS), phenoxyethanol (PE) and benzocaine (BZ). MS PE BZ ID Min Max Min Max Min Max 1 2.4 19.0 2.3 4.2 4.5 4.5 2 3.2 5.1 1.1 4.2 3.7 6.5 3 2.3 25.0 1.9 2.7 2.7 4.4 4 2.7 18.6 2.3 5.7 2.6 4.2 5 4.0 20.0 2.3 4.2 3.0 9.6 6 2.1 23.0 2.1 5.6 2.5 4.4 7 2.4 34.0 2.8 3.0 2.4 9.2 8 4.0 19.0 3.2 4.3 4.5 4.8 9 1.5 23.0 2.6 3.2 2.9 2.9 10 14.0 23.0 2.4 3.1 6.5 7.1 11 12.0 26.0 3.0 4.7 2.4 3.1 12 2.4 18.0 2.6 3.3 3.7 6.1 Mean+ SE 4.4+ 1.2 21.1 + 1.9 2.4 + 0.2 4.0 + 0.3 3.4 + 0.3 5.6 + 0.6 Data based on evaluations of 12 independent chambers per each anesthetic (15 abalone/chamber). time between 2 and 21 min after exposure. In contrast, organisms expose(i to PE anii BZ became generally narcotized within the first 2 to .^ min (Table 3) and were characterized by their rigidity. Recovery postanesthesia varied among anesthetics. About 80% of abalone regained their normal upright position after 18 min (PE) 25 min (BZ) and 35 min (MS) post-anesthesia, and nearly 100% had been recovered after 1 h in all treatments (Fig. 3). DISCUSSION Short-Term Effect of Anesthetics The induction/recovery periods visually determined for H. fiil- gens in this study were similar to those reported for other mollus- can species, including the abalone H. gigantea and H. midcie (Hahn 1989, White et al. 1996), the scallop Pecten fumatus (Heasman et al. 1995) and the pearl oysters Pinaata albina and P. margu- ritifeni (Norton et al. 1996). The heterogeneous anesthetizing ef- fects of MS contrasted with the homogeneous, rapid action of PE and BZ assessed visually. Similar observations have been previ- ously reported for H. midae (Hahn 1989, White et al. 1996) and may be related to the degree of access of these anesthetics to the site of action. It is known that topical anesthetics like BZ readily and locally interact with any nerve cell receptor (American Vet- erinary Medical Association 2001). whereas MS affects the smooth muscle or the central nervous system of vertebrates (Swain & Kaplan-Machlis 1999) by blocking the release of the neurotrans- mitter acetylcholine. The relaxation symptoms of abalone tissues observed in this and other studies (White et al. 1996) are consistent with the symptoms described for the neuromuscular system of humans (i.e.. Swain & Kaplan-Machlis 1999) and other vertebrates (American Veterinary Medical Association 2001). To our knowledge, this is the first study that documents in detail a time-course response of metabolism in marine inverte- brates exposed to anesthetics, including the observation of rapid depression of the respiratory system and concomitant recovery following the elimination of anesthetics from the chambers (Fig. 1). White et al. ( 1996) recorded an inhibitory response of the tarsal muscle of H. midae when exposed to MS. PE, and procaine, but their study was more focused at demonstrating the effectiveness of these substances as anesthetics, rather than documenting time- dependent physiologic effects on abalone. The short-term (i.e., <3 h postanesthesia) metabolic response of abalone was only partially coincidental with visual criteria of recovery. In this regard, the VOt exhibited by H. fulgens immediately after MS and BZ were flushed away from the incubation chambers were similar to the controls, whereas organisms treated with PE maintained a high VO2 even after 3 h postanesthesia (Fig. lb). Conversely, visual observations suggested that organisms exposed to PE and BZ re- covered faster and more uniformly than those exposed to MS (Fig. I ). Although no other physiological variables were measured in this study, it has been reported that the trout OncDihyiulinx iiiykiss experienced an increase in blood pressure after being exposed to PE (Fredricks et al. 1993). Therefore, the actual physiologic state of abalone (this study) was more likely reflected in the metabolic response curve, as this variable is highly sensitive to exogenous 0) > o o (1> a. 0) > 3 B 3 o . -^::jC^Z-^^-<^- '~Z..--- PE BZ ■ MS ^-^ ,.•■■ n = 36 75 / '' ■' 50 ■ / /J 25 ■J ! t n - }y Time Post-anesthesia (min) Figure 3. Cumulative percent recovery from anesthesia of juvenile abaicme, Haliotis fulgens), based on visual criteria. Magnesium sulfate (MS), phenoxyethanol (PE), and benzocaine (BZ). 420 Chacon et al. and endogenous perturbations. The reason for the post-anesthesia increase of VO, in the presence of PE is not known and need further and detailed studies in abalone. Diel Changes The consistent circadian rhythm exhibited by control and an- esthetic-treated H. fidgens suggests that these chemicals did not significantly affect the functioning of the central nervous system after a few hours of application. Furthermore, the observed rhythm is consistent with previous reports of night-accelerated metabolism in the Japanese abalone H. discus hannai (Uki & Kikuchi 197.'i). Other physiologic variables, such as food intake (Barkai & Grif- fiths 1987) and motor activity (Donovan & Carefoot 1998), typi- cally increased at night in abalone. Circadian physiologic rhythms have also been observed in other molluscan species. For example, Watanabe ( 198.^) reported that the repair and growth of shell in the bivalves P. murtensii and Helisoma duryi was highest under con- tinuous darkness. Taken together, these results suggest that the endogenous clock of these species may be cued by light. It has been demonstrated that changes in light intensity/photoperiod are among the major environmental cues responsible for the activation of clock genes in organisms ranging from fruit flies to mammals (Schibler & Lavery 1999). In addition, it has been shown that the ocular circadian rhythm of the marine snail Bulla i;inildiana is a complex process regulated at the level of transcription, translation, and phosphorylation and involves the presence of a cyclin- dependent protein kinase, whose activity coincides with a circa- dian clock (Krucher et al. 1997). Earlier reports suggest that the hypothesis of a light-controlled circadian rhythm may still be con- troversial haliotids. In this regard. Jan et al. (1981) found that Haliotis diversicolor supertexta exhibited a circadian metabolic rhythm when exposed to a natural diurnal cycle, but not under continuous light. Accordingly. Peck el al. (1987) did not find significant differences in the metabolic rate of H. iLdtercidata be- tween day and night (12:12 L:D photoperiod), and suggested that such behavior resulted from an excess in the available food. The contradictory results found among haliotids may either suggest that there are species-specific differences in the response to envi- ronmental cues and/or there are subtle methodological differences that may explain the observed results. In this regard, it was shown that the gastropod A. californica exhibited a circadian feeding rhythm under conditions of 12:12 L:D photoperiod, with shorter feeding response associated with light hours. Such a pattern con- tinued when organisms were shifted to a 0:24 (L:D) photoperiod (Kohn 1983). but the whole experimental period lasted only three days, and therefore no conclusive results could be drawn as to the role of light on cueing the observed rhythm. Therefore, detailed and long-term experiments are needed to test whether the circadian rhythm observed in this and other studies can be generalized in haliotids. Despite these controversies, the results of the present study may have implications for growth/production protocols in H. fidgens because the total energy drain (i.e., respiration) of H. fid- gens is highest at night. The trend of decreasing metabolic rate observed during the second day of measurement (Fig. 2a) was likely due to a higher amount of food remaining in the abalone's gut (i.e., SDA component of VO.) in the first day of measurement. It has been shown that a complete gut evacuation can take between 18 h to 7 days in abalone (Wee et al. 1992, Maguire et al. 1993, Britz et al. 1996, Mai et al. 1998). Overall, the results of this study highlight the importance of physiologic evaluations when different chemical substances are used in aquatic invertebrates. The combined visual and metabolic evaluations confirmed that all three anesthetics might be poten- tially used for handling abalone, since all of them effectively in- duced anesthesia, rapid post-anesthesia recovery and no mortality. Nevertheless, careful evaluations are still needed to assess the long-term effects of anesthesia on other physiologic variables such as growth, food intake and activity of abalone. In this regard, Edwards et al. (2000) found that the abalone H. laevigata and H. rubra exhibited a significantly lower growth rate than control or- ganisms after 6 weeks of exposure to PE and BZ. ACKNOWLEDGMENTS Financial support was partially obtained through a grant CONACYT (G281I9B) awarded to MTV and a grant (SINVE 002-DE) awarded to ZGE by Gobiemodel Estado de, Baja, Cali- fornia. The authors thank Marco A. Gonzalez, Roberto Escobar, and Laura Gomez for their valuable help during most of the trials. Thanks to two anonymous reviewers who helped to improve the article. LITERATURE CITED American Veterinary Medical Association. 2001. Report of the AVMA Panel on Euthanasia. JAVMA 218:669-696. Aquilina, B. & R. Roberts. 2000. A method for inducing muscle relaxation in the abalone, Haliotis iris. Aquacultiire I90:403^0S. Armstrong, D. A., J. L. Armstrong. S. M. Krassner & G. B. Pauley. 1971. Experimental wound repair in the black abalone. Haliotis crachemdii. J. /nvertetv. Pathol. 17:216-227. Barkai. R. & C. Griffiths. 1987. Consumption, absorption efficiency, res- piration and excretion in the South African abalone Haliotis midae. S. Afr. J. Mar. Sci. 5:523-529. Britz, P., T. Hecht & J. Knauer. 1996. Gastric evacuation time and diges- tive enzyme activity in abalone Haliotis midae fed a formulated diel. S. Afr. J. Mar Sci. 17:297-303. Catterall, W. & K. Mackie. 1996. Local anesthetics. In: J. G. Hardman, L. E. Limbird, P. B. Molinoff R. W. Ruddon & A. G. Gilman, editors. Goodman and Gilman's the pharmaceutical basis of therapeutics. 9lh ed. New York: MacGraw-Hill, pp. 331-347. Culloty. S. & M. Mulcahy. 1992. An evaluation of anesthetics for Ostrea edulis (L.). Aquaculture 107:249-252. Davies. M. S. & G. A. Williams. 1995. Pedal mucus of a tropical limpet. Cellaiia grata (Gould): energetics, production and fate. J. Exp. Mar. Biol. Ecol. 186:77-87. Donovan, D. & T. Carefoot. 1998. Effect of activity on energy allocation in the northern abalone Haliotis kamtschatkana (Jonas). / Shellfish Re.s. 17:729-736. Edwards, S., C. Burke, S. Hindrum & D. Johns. 2000. Recovery and growth effects of anesthetic and mechanical removal on greenlip (Hali- otis laevigata) and halcklip [Haliotis rubra) abalone. 4"' International Abalone Symposium. University of Cape Town. South Africa, pp. 16. Fredriclcs, K.. W. Gingerich & D. Fater. 1993. Comparative cardiovascular effects of four fishery anesthetics in spinally transected rainbow trout. Oiicorhynclms mykiss. Comp. Biochem. Physiol. C. 104:477-483. Garcia-Esquivel, Z., V. M. Bricelj & H. Felbeck. 2002. Metabolic depres- sion and whole-body response to starvation by Crassostrea gigas post- limae. Comp. Biochem. Physiol. A. 133:63-77. Garcia-Franco, M. 1992. Anesthetics for the squid Sepioteiithis sepioidea (Mollusca: Cephalopoda). Comp. Biochem. Physiol. C. 103:121-123. CiRCADiAN Rhythm and Anesthetics in H. fulgens 421 Green, E. & D. Carritt. 1967. New tables for oxygen saturation of seawater. J. Mar. Res. 25:140-147. Hahn, K. 1989. Handbook of culture of abalone and other marine gastro- pods. Boca Raton, FL: CRC Press, pp. 1 13-134. Harris, J.. G. Maguire, S. Edwards & S. Hindrum. 1997. Effect of nitrite on growth and oxygen consumption for juvenile greenlip abalone. Haliotis laevigata Donovan. J. Shellfish Res. 16:395-401. Harris, J.. G. Maguire, S. Edwards & S. Hindrum. 1998. Effect of ammonia on the growth rate and oxygen consumption of juvenile greenlip aba- lone. Haliotis Uievigata Donovan. .Aquacuhure 160:259-272. Harris. J., G. Maguire. S. Edwards & S. Hindrum. 1999. Effect of pH on growth rate, oxygen consumption rate, and histopathology of gill and kidney tissue for juvenile greenlip abalone, Haliotis laevigata Donovan and blacklip abalone, Haliotis rubra Leach. / Shellfish Res. 18:61 1- 619. Heasman, M., W. O'Connor & A. Frazer. 1995. Induction of anesthesia in the commercial scallop, Pecteit fumatiis Reeve. Aiiiiuculliire 131:231- 238, Iwatsu, O., T. Ushida, T. Toshikazu, N. B. Lawand ct H. Yamamoto. 2002. Peripheral administration of magnesium sulfate and ketamine hydro- chloride produces hypesthesia to mechanical stimuli in humans. / Health Set. 48:69-72. Jan, R.-Q., K.-T. Shao& K.-H. Chang. 1981. A study of diurnal periodicity in oxygen consumption of the small abalone {Haliotis Jiversieolor supertexta Lishke). B. I. Zool. Acad. Sinica 20:1-8. Juefeng, S. & K. Shiuan. 1996. Seed production and grow-out culture techniques for abalone Haliotis discus hatmai Ino. Chinese Academy of Sciences, Qingdao, China. 22 pp. Krucher, N. A., L. Meijer & M. H. Roberts. 1997. The cyclin-dependent kinase (cdk) inhibitors, olomoucine and roscovitine, alter the expres- sion of a molluscan circadian pacemaker. Cell. Mol. Neurohiol. 17: 495-507. Maguire. G., K. Wee & S. Hindrum. 1993. Digestibility studies. The "ins" and "outs" of abalone guts. Austasia Aquae. 7:42-49. Mai, K., G. He & W. Xu. 1998. Studies on postprandial changes of diges- tive status and free amino acids in the viscera of Haliotis discus hamuli Ino, J. Shellfish Res. 17:717-722. McBride, S. C, E. Rotem. D. Ben-Ezra & M. Shpigel. 2001. Seasonal energetics of Haliotis fidgens (Philippi) and Haliotis tuherculata (L.). / Shellfi.'ih Res. 20:659-665. Mills, D.. A. Tlili & J. Norton. 1997. Large-scale anesthesia of the silver- lip pearl oyster, Pinetada ma.xiina Jameson. J. Shellfish Res. 16:573- 574. Mushoff, U., M. Madeja, N. Binding, U. Witting & E. Speckmann. 1999. Effects of 2-phenoxyethanol on N-methyl-d-aspartate (NMDA) recep- tor-mediated ion currents. Arch. Toxicol. 73:55-59. Newell, R. 1973. Factors affecting the respiration of intertidal inverte- brates. Am. Zool. 13:513-528. Norton. J.. M. Dashorst, T. Lansky & R. Mayer. 1996. An evaluation of some relaxants lor use with pearl oysters. Aquacuhure 144:39-52. Paul, A. & J. Paul. 1998. Respiration rate and tolerance of pinto abalone Haliotis kamtschatkana. J. Shellfish Res. 17:743-745. Peck, L., M. Culley & M. Helm. 1987. A laboratory energy budget for the ormer Haliotis tuherculata L. / Exp. Mar. Biol. Ecol. 106:103-123. Schibler. U. & D. J. Lavery. 1999. Circadian timing in animals. In: U. E. A. Russo, D. J. Cove, L, G. Edgar, R. Jaenisch & F. Salami, editors. Development, Genetics. Epigenetics and Environmental Regulation. Berlin: Spnnger-Verlag, pp. 487-505. Uki, N. & S. Kikuchi. 1975. Oxygen consumption of the abalone. Haliotis discus hamuli in relation to body size and temperature. Bull. Tohoku Reg. Fish. Res. Lab. 35:75. Swain. R. & B. Kaplan-Machlis. 1999. Magnesium for the next millen- nium. Southern Med ,/. 92:1040-1047. Viana M.T., L.M. Lopez. Z. Garcia- Esquivel & E. Mendez. 1996. The use of silage from fish and abalone viscera as an ingredient in abalone feed. Aqiiacidture 140:87-98. Watanabe, N. 1983. Shell repair. In: K. M. Wilbur & A. S. M. Saleuddin, editors. The Mollusca, vol. 4. New York: Academic Press, pp, 289- 316. Wee, K.. G. Maguire & S. Hindrum. 1992. Methodology for digestibility studies with abalone 1. Preliminary studies on feeding and defecatory behavior of black abalone. Haliotis rubra, fed natural and artificial diets. In: Allan. G.L. and Dall, W. (Eds.). Proceedings of Aquaculmre Nutrition Workshop, NSW Fisheries, Brackish Water Fish Culture Res. Station. Salamander. Australia, pp. 192-196. White, H.. T. Hecht & B. Porgieter. 1996. The effect of four anesthetics on Haliotis midae and their suitability for application in commercial aba- lone culture. Aquacuhure 140:145-151. Journal of Shellfish Research, Vol. 22, No. 2, 423-+30, 2003. LABORATORY HYBRIDIZATION OF THE MUSSELS, MYTILUS TROSSULUS AND M. GALLOPROVINCIALIS: LARVAL GROWTH, SURVIVAL AND EARLY DEVELOPMENT SEAN E. MATSON,'* JONATHAN P. DAVIS," AND KENNETH K. CHEW' ^Oregon State Universit}: Hatfield Marine Science Center. 2030 SE. Marine Science Dr.. Newport. Oregon 97365: 'Taylor Resources, Inc.. Quilcene. Washington 98376: and ^University of Washington School of Fisheries and Aquatic Sciences. Seattle. Washington 98 J 95 ABSTRACT Expenmenis were performed to determine whether hybrid larvae of Mytihis trossiilus (Baltic mussel) and Mytilus galloprovincialis (Mediterranean mussel) could be produced in a shellfish hatchery environment and whether early development, survival, or growth differences existed between the two species and their reciprocal hybrids at full and reduced salinity. Hybrids of these two species are uncommon in Puget Sound. Washington and on the northern west coast of North America. Broodstock were screened morphologically and positively identified at two nuclear DNA loci using polymerase chain reaction and restriction fragment length polymorphism techniques. Hybrid larvae were produced in both reciprocal combinations, and were successfully reared through metamorphosis. There was no apparent hybrid vigor because hybrids did not grow consistently larger (or survive better) than the parental crosses, nor did one reciprocal cross grow consistently larger than the other. Both reciprocal hybrid crosses and the parental cross, M. rro.ssuliis. grew faster than the other parental cross, M. galloprovincialis. at low salinity (20 ppt). These results concur with the two species' physiologic and ecological characteristics. Mytilus trossiilus grows well in areas of low and variable salinity (much of Puget Sound) and M. galloprovincialis grows well in areas of stable, full salinity, and recruits pooriy in Puget Sound. Hybrids showed generally lower fertilization rates and slower early development than parental crosses, although they were sufficient to produce larval cultures and postlarvae. The successful fertilization, growth, and survival of these hybrids suggests that some factor other than genetic incompatibility is likely responsible for the rarity of these hybrids in Puget Sound. One such factor could be the limited overlap of the spawning periods of the two species in this region. A differentia! species growth-response to salinity was observed in this study. KEY WORDS: hybrid, mussel, Mytihis irossuliis. galloprovincia INTRODUCTION Two species of mainne mussels found in Puget Sound, Wash- ington are Mytilus trossiilus (Baltic mussel) and M. galloprovin- cialis (Mediterranean mussel). The two species occur both sym- patrically and allopatrically in Puget Sound. Both species possess distinct ecological and physiologic characteristics (Johnson 1978, Kautsky 1987, Brooks 1991,Margus 1991. Sarver & Loudenslager 1991, Sarver & Foltz 1993, Geller et al. 1994, Hoffman & Somero 1995, 1996). Mytilus tros.ndus thrives in areas of widely fluctuat- ing salinity, as is the character of much of Puget Sound, whereas M. iialloprovincialis occurs in bays with more stable, high salinity (Brooks 1991). These two species hybridize naturally in isolated populations in Puget Sound (Brooks 1991, Suchanek et al. 1996). Their hybrids also occur in other bays in Washington, Oregon, and California. Although these two species often co-occur throughout their distri- bution within Puget Sound, the overall frequency of hybrids has remained very low (Brooks 1991, Suchanek et al. 1996). One reason this laboratory attempt at hybridizing these two species was performed was to help elucidate what sort of barrier to hybridiza- tion may be responsible for this lack of hybrids in the wild. Bar- riers to hybridization can be rooted in genetic incompatibility or physiologic and ecological differences, such as disease, salinity tolerance, or the tiining of spawning events. Both mussel species are commercially important within the United States and throughout the world. Their physiologic and ecological differences pose challenges to tho.se who culture them. Mytilus trossulus suffers high mortalities before the end of its second year of life becau,se of high summer water temperatures and the disease hemic neoplasia (Brooks 1991). Up to 75% of aM. trossiilus mussel crop in Penn Cove, Puget Sound often dies before *Corresponding author. E-mail: sean.matsonCa'oregon.state.edu lis it is old enough to be harvested (Brooks 1991). Mytilus gallopro- vincialis typically grows to a larger size than M. trossulus and is resistant to hernic neoplasia (Brooks 1991). Mytilus galloprovin- cialis has been observed suffering significant mortalities when salinities have dropped to 20 parts per thousand (ppt), and 100% mortality below 10 ppt (Kautsky 1987, Margus 1991). Low salin- ity conditions have repeatedly coincided with substantial mortali- ties of M. galloprovincialis in Holmes Harbor. Puget Sound. Washington (Kurt Johnson. Taylor Resources, personal commu- nication), eventually leading to the closure of the mussel farm there. The financial implications of the aforementioned mortalities are severe enough to wanant examining biologic alternatives that might ameliorate lost farm revenues and even closures as a result of salinity- and disease-related crop loss. One such alternative worth examining is hybridization of the two mussel species. Myti- lus trossulus and M. galloprovincialis possess characteristics that if expressed in a hybrid (variable salinity tolerance of M. trossulus and the disease resistance of M. galloprovincialis) might result in increased mussel production for industry. Mytilus galloprovincia- lis X M. edulis hybrids carry some of M. galloprovincialis' disease resistance to a trematode parasite (Coustau 1991). Sturgeon hy- brids have been found to be more resistant to thermal and salinity shock than either parental species (Chikhachev 1979). Both inter- specific and intraspecific hybrid vigor have been documented in bivalve mollusks (Loosanoff 1954. Hedgecock et al. 1996. Bayne et al. 1999) and it could occur in hybrids of these two Mytilus species. Hybrid vigor is defined here as an increase in growth or survival of hybrid crosses over pure-species crosses. This investigation was performed to determine whether hybrid larvae of two locally occurring species of marine mussels (A/. trossulus and M. galloprovincialis) could be produced in a shell- fish hatchery environment, and whether survival and growth dif- ferences existed between the two species and their reciprocal hy- brids at full and reduced salinity. This was the necessary first phase of evaluating the culture potential of hybrid mussels. 423 424 Matson et al. METHODS Broodstock Broodstock mussels were collected from areas known to have essentially monospecific populations. After preliminary Brood- stock selection was made based on morphology, molecular meth- ods were used to positively identify all of the Broodstock in this study. Penn Cove was chosen for collection of M. trossuhis based on Brooks 1991. 1996 and 1997. Only mussels several years old that tit the typical morphology for its species were collected. Hy- brids often have a shell morphology intermediate to that of the parent species (Lubet 1984). Large mussels (1.5 inches or longer) of either species are easier to tell apart than very young ones, so only larger mussels were collected. These two species have different morphologies (Brooks 1991. McDonald & Koehn 1991). The valves of Mytilus trossuhis are typically narrow and long. The ventral shell margin is usually concave or straight. The anterior end (the umbo) is gradually bent; sometimes referred to as "beaked." The periostracum of M. tros- sulus is typically thin and rubs off near the umbo. In sagittal section, the ventral shell margin is straight. M. galloprovincialis has a very broad valve. The ventral margin is often convex. The umbo appears sharply hooked. M. galloprovincialis ' periostracum is typically thick and black. In sagittal section, the ventral shell margin is rolled inward at the joining of the two valves. Mussels that fit these criteria were chosen for Broodstock. Those that ap- peared intermediate to these morphotypes were not collected to avoid hybrid Broodstock. Molecular Identification Broodstock mussels were identified using two different types of nuclear DNA markers (Heath et al. 1995. Rawson et al. 1996). These diagnostic molecular markers enabled positive discrimina- tion between the three members and hybrids of the Mytilus com- plex. Both of these nDNA markers were based on the polymerase chain reaction and one used restriction fragment length polymor- phism analysis. The first marker is based on the Glu gene, which encodes the mussel polyphenolic adhesive protein (Rawson et al. 1996). That protein is key in mussel attachment to the substrate. The Glu-5' marker enables differentiation between all three spe- cies in the Mytilus complex; Mytilus trossuhis. M. galloprovincia- lis. and M. ediilis. In this study, tissue samples were digested using CTAB isola- tion buffer (IB) and proteinase k ( 10 mg/niL). CTAB was used to remove mucopolysaccharides in the bivalve tissue that could co- extract with the DNA and negatively affect later polymerase chain reaction (PCR). The CTAB IB (2% w/v CTAB, 1.4 M NaCl, 0.2% w/v 2-mercaptoethanol, 20 mM EDTA, 100 mM Tris/HCl, pH 7.5) was preheated at 50''C. Mantle edge tissue was chopped up with a razor blade and put into 1 .5-inL polypropylene centrifuge tubes with 10 (J.L of proteinase k and an equal volume of CTAB IB. The mixture was incubated at 55"C for 3 h in a Rossi agitating incu- bator, vortexed for 10 s each, and then held at 50°C in a water bath overnight. In the morning, DNA was extracted from the tissue digestion with an equal volume of 24:1 chloroform:isoaniyl alco- hol mixture. The mixture was centrifuged at I 1,500 g in a mi- crofuge for 10 min. It was necessary to repeat the extraction two or three times to get a clear supernatant. Two volumes of 100% ethanol were added and the mixture was held in a -20°C freezer over night to allow precipitation of the DNA. The next day, the extraction was centrifuged for 30 min at 1 1 ,500 g. The alcohol was removed and the pellet was rinsed with 0.5 mL of 70% ethanol. The pellets were dried in a centrifugal evaporator and then dis- solved in 100 p.L of TE ( 10 mM Tris/HCl, 1 mM EDTA, pH 7.6). A "Gene Quant" spectrophotometer (Pharmacia) was used to quantify DNA stock solutions. The stock solutions were diluted to make a 100 ng/jji.L working solution for use in PCR and were stored in refrigerator at 4°C. The stock solutions were frozen at -20°C for long term storage. The sequences of the primers used for Glu-5' (Rawson et al. 1996) in this study were: 5'-GTAGGAACAAAGCATGAACCA- 3' (forward) and 5'-GGGGGGATAAGTTTTCTTAGG-3' (re- verse) The PCR recipe of Rawson et al. (1996) and their thermal cycler protocol were adapted. The end concentrations of chemicals in the PCR were 0.8x TBE buffer (20x TBE buffer solution: 121 g/L Tris base, 61.7 g/L boric acid, 7.44 g/L Na2EDTA*2H20), 0.32 dNTPs, 1.5 mM MgCU, 4 |j.M forward primer, 4 |jiM reverse primer, 4 ng/p-L of DNA template, and 0.04 U/|xL of Taq DNA polymerase. The total reaction volume was 12.5 p.L, and samples were amplified in a Techne thermal cycler using a hot-start pro- tocol. The thermal cycler protocol used for this marker was one cycle of 94° for 3 min and then 24 cycles of 94° for 20 sec, 53° for 20 sec, and 72° for 45 sec. After PCR. the products were size- fractionated on 3% agarose TBE gels and stained with SYBR green (Molecular Probes) for approximately 1 h. They were visu- alized using a Molecular Dynamics 575 Fluorlmager. The banding pattern observed for Glu-5' in M. galloprovincialis was one band of 300 base pairs (bp) and one 500 bp band or just one 300 bp band. One 240 bp band was observed for M. trossuhis. The second DNA species marker used was also PCR-based but was followed by restriction fragment length polymorphism analy- sis (Heath et al. 1995). This codominant marker was based on internal transcribed spacer (ITS) regions between the 18S and 28S nuclear rDNA coding regions. Heath et al. (19951 showed that it worked very well in distinguishing M. trossuhis from M. gallo- provincialis or Mytilus edulis. This marker cannot distinguish be- tween M. galloprovincialis and M. edulis. but because M. edulis is not yet known to occur in any of the Broodstock collection sites, or anywhere else in Puget Sound. It was reasonable to use this marker in conjunction with the Glu-5' marker and morphologic screening. The sequences of the primers used (Heath et al. 1995) in this study for the ITS marker were 5'-GTTTCCGTAGGT- GAACCTG-3' (forward) and S'-CTCGTCTGATCTGAGGTCG- 3' (reverse). The Heath et al. (1995) PCR recipe and thermal cycler protocol were both optimized for the facility where the work was performed. The end concentrations of chemicals in the PCR were Ix buffer, 0.8 mM dNTPs, 1.5 mM MgCU, 0.3 p,M forward primer, 0.3 p.M reverse primer, 0.5 ng/(xL of DNA template, and 0.05 U/|xL of Taq DNA polymerase. The thermal cycler protocol used was 94° for 3 min and then 30 cycles of 94° for 20 sec, 50° for 20 sec, and 72° for 45 sec. The total reaction volume was I6p,l and a Techne thermal cycler was used. The PCR was hot-started. After PCR. 1 jjiL of each product was electrophoresed on a 1.5% agarose gel to check for amplification success. The products were then cut with Hluil restriction endonuclease overnight. Conditions for one digestion reaction was 0.04 p,L Hhal enzyme, 1.0 p-L lOx NEB #4 buffer (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, 1 mM DTT, pH 7.9 @ 25°C), 0.1 \x.L of lOOx bovine serum albumin, and 3.5 p.L of sterile double distilled water, and 5 |jiL of template DNA in TE (100 ng/p-L). The frag- Laboratory Hybridization of M. trossulus and M. galloprovincialis 425 ments were separated on a S'/r agarose gel. stained with SYBR green for approximately 1 h and visualized using a Fluorlmager. Heath et a), reported that in M. ciliilis and M. i^iilloprovincialis. the 1250-bp PCR product was cut into two 45()-bp fragments and two 180-bp fragments. In Mytilus trossulus. the 1250-bp product was cut into two 280-bp fragments, two 1 80-bp fragments, and a few fragments smaller than 100 bp. In this study, the PCR product was closer to 1050 bp long in both species. The two species mark- ers, Glu-5' and ITS both worked well at distinguishing M. trossu- lus from M. galloprovincialis and from hybrids. All Broodstock individuals were positively identified using the Glu-5' marker. All but three males and two females were identified at the ITS locus. Those few were most likely not identifiable because of sample degradation. Both banding patterns were seen when equal portions of DNA from each species were mixed together and amplified (with either marker). This shows that one species' DNA was not preferentially amplified over the other's. Hybrid mussels would show the banding patterns of both of their parents (Rawson et al. 1994). The use of two loci increased the power of detection of hybrids. Larval Rearing Mussels were spawned and reared in 12-L plastic bags at the Taylor Shellfish Hatchery on Dabob Bay. Washington. It was nec- essary to condition M. trossulus Broodstock for a few weeks be- fore spawning was attempted. M. trossulus normally spawn in March through May in Puget Sound (Johnson 1978). The mussels were held in tanks at ambient Dabob Bay temperature (10 to I2°C) and were fed large amounts of algae to encourage the necessary development of the gonad. Brenko and Calabrese (1969) found that food was the primary controlling factor for gonad develop- ment in Baltic mussels, and that rise in water temperature was the triggering factor for spawning in the natural environment. It was not necessary to condition the fully ripe M. galloprovincialis Broodstock. as they were already in spawning condition. They could not be held in water overnight because they spawned out in the holding tank before morning when this was attempted. M. galloprovincialis are typically in spawning condition December through March in Puget Sound. Up to 100 mussels of each type were used in spawning attempts for the first experiment to ensure that enough mussels actually spawned to make the crosses. Only approximately one fourth of tho.se mussels that were induced, spawned enough gametes to produce a culture. Approximately .^00 mussels of each species were induced in the spawning attempts that led to the second experiment. The mussels were induced to spawn by agitation, followed by heat shock (Loosanoff & Davis 196.^). The mussels were taken from ambient IT or 14°C water, shaken in buckets for approximately two minutes, and then placed into spawning trays with flowing seawater. The water temperature in the trays was changed from as high as 24° to as low as 11 °C repeatedly. Dense, live algal food was also added occasionally for periods of about 20 min to encourage spawning. Once a mussel began spawning, it was immediately removed from the tray, its sex was identified, the mussel's interior and exterior was rinsed with seawater, it was placed in a separate clean dish, and was allowed to spawn further. After that, its interior and exterior and its dish were rinsed a second time. Then it was al- lowed to spawn the gametes that would be used in the crosses. When all of the individuals had spawned, the mussels were re- moved from the dishes and the gametes were screened. All screens were cleaned and soaked with hot fresh water between batches of gametes. Spawning mussels, each in its own dish, were kept sepa- rated in different areas on different tables by both sex and by species. Every batch of eggs and sperm were carefully examined under the microscope for contamination by other gametes. A batch of eggs or sperm was only used in a cross if it was observed to have zero signs of development in it, and any contaminated ga- metes were discarded. Fertilization was confirmed in each culture under the microscope. After fertilization, the embryos were screened and rinsed to remove excess sperm. All screens were cleaned and soaked with hot fresh water between batches of em- bryos. Two samples of one ml each were taken after fertilization for early development analyses and later growth measurements. Sixty-four different mussels were used in all, to produce the 32 pair matings used in the experiment. Eight individuals of each sex were used to establish each cross (16 parents for each cross). Each replicate represented one single-pair mating. No replicates shared either a sire or dam. The four crosses made were M. trossulus x trossulus (TT). galloprovincialis sperm x trossulus egg (GT). tros- sulus sperm x galloprovincialis egg (TG). and galloprovincialis x galloprovincialis (GG). The low-salinity treatment was 20 ppt and the high salinity was 30 ppt. The embryos were then placed in 32 separate 12-L culture bags, with four replicate bags per cross by salinity treatment. (See Fig. I for a graphical description of the experimental design.) The culture bags were hung in a water bath with a thermostat-controlled immersion heater and circulating pumps. Culture temperatures were maintained at 18°C. The cul- tures were covered with shade cloth to prevent algal growth. The larval density and the algal density of each culture were both standardized (regulariy made equal between cultures). Larval density was equalized twice per week (at each water change) to prevent density-dependent growth or survival. This was performed by counting the larvae in each culture, and then decreasing the water volume in all bags until they had the same larval density as the culture with the highest survival (10 larvae/ml initially, de- creasing to 3 larvae/mL by day 14). The algal density was equal- ized once per day, by counting the algae in each culture, and then feeding a different amount to each culture to maintain the desired algal density (20,000 cells/mL initially, gradually increased to 80.000 cells/mL at pediveliger). Larvae were initially fed 20.000 cells/mL of naked flagellates Mytilus trossulus speim X Mytilus trossulus egg (TT) Higli salinity Low salinity QQQQ QQQQ Mytilus trossLilus speim x Mytilus gaUopimincialis egg (TG) High salinity Low salinity SQQQ 0QQQ Mytilus galloprovincialis speim X Mytilus galloprmincialis egg (GG) High salinity Low salinity QQ00 QQQQ MytHus galloprovincialis sperm X Mytilus trossulus egg (GT) High salinity Low salinity QQQQ QQQQ Figure 1. Two-factor experimental design used in examining survival and growth in larvae of M. trossulus, M. galloprovincialis. and their reciprocal hybrids at high and low salinities (.'2 ppt and 2(1 ppl. re- spectively). The i2 larval cultures were produced by 32 separate-pair matings. Eight cultures were used in each of four crosses, and four cultures were used lor each salinity level within each cross. For ex- ample, the M. trossulus sperm by M. galloprovincialis egg cross in- cluded four cultures at high salinity and four at low salinity. 426 Matson et al. (Isochrysis sp. Tahitian isolate). Algal cell concentration was de- termined using a Coulter Counter model ZBI. On the day follow- ing fertilization, the cultures were fed a mixture of flagellates and diatoms {.Tahitian Isochnsis. Chaetoceros calcitrans. Thalassio- sira pseudonana [University of Washington 3H clone], and Ske- letonema [species unidentified]). A mixture of two algal species supported faster growth than one alone, according to Bayne (1965), when he fed Isochiysis galhana and Monochiysis liitheri together. The amount of food given increased incrementally to a maximum of 80,000 cells/mL at the pediveliger stage. The density of larvae and algae was kept equal between cultures to reduce the possible influence on larval growth rate due to crowding. Sampling for survival was done twice per week at each water change. Each culture was condensed to 100 mL and one count was taken. The variability between counts was kept below 5% (tested beforehand) by condensing the culture and using a paddle stirrer. Cultures were resuspended in one liter between counting and bag refilling. Bags were cleaned with bleach, sodium thiosulfate. and rinsed with hot water at each water change. Bag water volumes were then adjusted to equalize larval density and larvae were resuspended in their bags. Two samples of 1 mL each were taken after fertilization for early development analyses and later growth measurements. Esti- mates of the proportions of larvae at each developmental stage present in the cultures were made from those samples as well. A total of 200 larvae were counted from each sample, and the number of larvae at each developmental stage was noted. Fifty larvae were chosen randomly and the distance from umbo to lip (shell length) of each was measured in microns using a compound light micro- scope and ocular micrometer. Figure 2. Early development of hybrid and pure specie.s Mytilus lar- vae. Larvae of the TT cross developed significantly faster than those of the GT hybrid cross iP = 0.007). TT. M. trossulus; GT. M. gallopro- vincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. gal- loprovincialis egg; GG, M. galloprovincialis. Bars represent the mean transformed proportion of the larvae that were at or beyond the blastula stage after 12 h at 16 C at a salinity of 30 ppt. growth in the low salinity treatment than in the high salinity treat- ment, while the GG cross did not. It had a slightly higher mean growth in the high salinity treatment than in the low salinity treat- ment. Survival RESULTS Early Development Early development was measured as the proportion of embryos that had developed to the blastula stage or beyond, at 12 h post- fertilization. This proportion was arcsine transformed to conform to the normality and homogeneity of variance assumptions of the analysis of covariance. It was also adjusted for egg density by using egg density as a covariate. The regressions for the covariate were significant for developmental success (proportion of blastu- las; P < 0.0001 ). The slopes of the lines for the different crosses were equal for developmental success (NS cross by egg density interaction). Cross (P = 0.045) was a significant factor. The mean development of the TT cross was significantly higher than that of the TG cross" mean (P = 0.007. Fig. 2). No other differences were significant. Growth At day 3. the mean length of the GG cross was significantly higher than those GT and TT crosses (P = 0.026 and < 0.001. respectively). The TG cross's mean length was significantly higher than the TT cross's mean length also (P = 0.001. Fig. 3). These results are similar to an earlier experiment performed with the same crosses (Matson 2000). The low salinity treatment was ap- plied at day 3. No significant differences exi.sted in growth be- tween crosses or salinities froiu day 3 to 7. Salinity was a highly significant factor affecting growth be- tween day 3 and 14 (P < 0.001, Fig. 4, Table 1). Cross was not a significant factor from day 3 to 14 (P = 0.256). The three crosses with a M. trossulus component (TT. GT, and TG) had higher mean Cross was a significant factor {P = 0.011) affecting day 3 survival. The TT cross's mean survival was significantly higher than those of the GT, TG, and GG cross's mean survival (P = 0.042. 0.026. and 0.020 respectively. Fig. 5). No significant dif- ferences existed in survival between crosses or salinities from day 3 to 7. or from day 3 to 14, though there was an interesting pattern in the means. Each hybrid cross survived most like its sire (TG cross survived better at low salinity, GT cross survived better at high salinity). 1 UJO 1 "5 ■1? =3 sn - fin - T i It -tn - A .-.t. E^• c *7 3n - ■ '-I ?? n 1_J TT GT TG GG Cross Figure 3. Shell length of hybrid and pure species Mytilus larvae at day 3. At day 3. the mean length of the GG cross was significantly greater than those GT and TT crosses (P = 0.026 and <0.001, respectively). TT, M. trossulus; GT, M. galloprovincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis. Cultures were maintained at 18 C in 30 ppt seawater. Laboratory- Hybridization of M. trossulus and M. galloprovinciaus All GLov?(^2Dppti ■ Kigb(30ppt.i Figure 4. Change in shell length of hybrid and pure species Mytilus larvae from day 3 lo day 14 at two salinities (20 ppt and 30 ppt). Salinity was a significant factor affecting growth of the larvae (P < O.OOI ). Larvae of the TT. (JT, and TG crosses grew more than at low salinity than at high salinity. Cultures were maintained at 18°C. TT, M. trossulus; GT, M. galloprovincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis. Figure 5. Survival of hybrid and pure species Mytilus larvae at day 3. The mean survial of the TT cross was significantly higher than the mean survival of the GT, TG, and GG crosses {P = 0.042, 0.025, and 0.020, respectively). TT, M. trossulus: GT, M. galloprovincialis sperm x A/, trossulus egg; TG, M. trossulus sperm x A/, galloprovincialis egg; GG, A/, galloprovincialis. Cultures were maintained at 18°C in 30 ppt seawater. DISCUSSION Barriers to Hybridization Hybrid larvae were produced in both species-egg combinations and larvae were successfully reared through settlement. The suc- cessful fertilization, growth, and survival of these hybrids suggest that some factor other than genetic incompatibility is responsible for the rarity of these hybrids in Puget Sound. One such factor could be the limited overlap of the two species' spawning periods in Puget Sound. This would be an example of a partial temporal barrier to hybridization. Both M. trossulus and M. galloprovincia- lis. have one peak or mass-spawning time per year and one or more periods when a much smaller proportion of each species spawns (trickle-spawning). Mass spawning of M. galloprovincialis occurs in the late-winter through early spring in Totten Inlet (Dr. Jonathan Davis, personal communication). Brooks (1991) found ripe M. galloprovincialis during November and December of 1988 though 1990 in Puget Sound. M. trossulus that were examined at the same time had gonads that were still in the resting stage with little gamete formation. M. trossulus typically mass-spawn in March or April in Holmes Harbor in Puget Sound (Johnson 1978). These observations support M. galloprovincialis being primarily a win- ter-spawner and M. trossulus being a primarily spring-spawner in Puget Sound, and thus also support the theory of a partial temporal barrier to hybridization. The comparatively low abundance of M. galloprovincialis in the region (Suchanek et al. 1996) may interact with or exacerbate the effects of a temporal barrier. There would likely be fewer opportunities for hybrids to be formed if the two species spawning times are different and if one of the species was present in much lower numbers than the other. Mytilus gallopro- vincialis, in this case, occurs in Puget Sound at much lower abun- dance than M. trossulus (Suchanek et al. 1996, Brooks 1991), probably due to M. galloprovincialis' preference for high, stable TABLE 1. Two-factor analysis of variable table for growth (change in shell length from day 3 to 14) of the four crosses of Mytilus pure-species and hybrid larvae at high and low salinities. Dependent Variable LENGTH Difference in Microns Day 3 to 14 Type III Sum Mean Eta Noncent. Observed of Squares df Square F Sig. Squared Parameter Power Source corrected model 14626.461 7 2089.494 4.539 0.003 0.58 31.771 0.965 Intercept 284579.457 1 284579.5 618.1 0 0.964 618.145 1 Cross 1993.008 3 664.336 1.443 0.256 0.158 4.329 0.33 Salinity 8030.93 1 8030.93 17.44 0 0.431 17.444 0.979 Cross* salinity 3901.449 3 1300.483 2.825 0.061 0.269 8.474 0.6 Error 10588.661 23 460.377 Total 315391.818 31 Corrected total 25215.122 30 TT, M. trossulus: GT. M. galloprovincialis sperm x M. trossulus egg; TG, M. trossulus sperm x M. galloprovincialis egg; GG, M. galloprovincialis. Salinity was a significant factor affecting growth of the larvae (P < 0.001). Computed using alpha = 0.05 R squared = 0.580 (Adjusted R squared = 0.452) 428 Matson et al. salinity bays and M. trossiiliis' preference for low temperature, variable salinity bays (Margus 1991. Sarver & Foltz 1993, Hilbish 1994. Geller 1994. Hoffman & Somero 1995. 1996). There are many examples of barriers to hybridization among mollusk and echinoderm species, and some of these are temporal in nature. A temporal barrier to hybridization is believed re- sponsible for the lack of natural hybridization of the sea stars Leptasterias polaris and Asterias vulgaris (Hamel & Mercier 1994). The two sympatric species were shown to hybridize readily in the laboratory, although they do not in the wild, due to their distinct breeding seasons. Another example of a temporal barrier to hybridization is found in Montastraea corals. Szmant (1997) found that the coral, Montastraea faveolata consistently spawned I to 1.5 h before M. franksi and M. annularis in experi- ments. These species have very specific spawning periods that are both seasonal and closely related to lunar cycles. Szmant dis- cussed this as a potential temporal barrier to fertilization and hy- bridization in these species because there was no inherent pre- zygotic barrier to cross-fertilization among three species he stud- ied. Other examples of barriers to hybridization include genetic, ecological, geographic, and physiologic barriers. In the hard clam, Mercenaria mercenaria x M. campechiensis hybrids were shown to have an excessive susceptibility to gonadal neoplasia, relative to either parental species (Bert et al. 1993). This cellular disease acts as a barrier to hybridization by decreasing fitness of the hy- brids relative to the parental species. Genetic barriers to hybrid- ization have been shown in oyster species. Allen et al. (1993) attempted hybridization between Crassostrea gigas and C. vir- ginica, as well as between C. rivularis and C virginica. They found that larvae survived only 8 to 10 d and grew little; therefore, the hybrids were considered genetically inviable. Allen and Gaffney (1993) found that C. gigas and C. rivularis yielded viable hybrids when crossed in the laboratory. Crassostrea gigas and C. sikamea have also been hybridized, although they were only suc- cessful in one direction (Dr. Anja Robinson, pers. comm.). An unusual physiologic barrier to hybridization is seen in the interaction of the urchins, A. vulgaris and S. droebachiensis ga- metes. Although shown to be physiologically able to hybridize with L. polaris (which has a different spawning period), the eggs of A. vulgaris were observed to disable heterospecific sperm from S. droebachiensis (believed to have an overlapping spawning pe- riod) within 12 sec (Hamel & Mercier 1994). It is thought that a very diffusible substance involved in the phenomenon is secreted only from mature eggs that appears to disable sperm in the direct vicinity of an egg. Salinity, Growth, and Sunival A differential species response to salinity was observed in this study. From day three to 14, the TT, GT, and TG crosses all grew much faster at low salinity, and the GG cross grew slightly faster at high salinity (Fig. 3). These differences agree with the two species' physiologic and ecological characteristics (Kautsky 1987, Margus 1991, Hoffman & Somero 1995, 1996, Sarver & Foltz 1993, Geller etal. 1994, Johnson 1978, Brooks 1991), and are likely to be inherited genetic differences. Mytilus tros- sulus has been shown to be a low, variable salinity mussel and M. galloprovincialis has been shown to be a high, constant salinity mussel (Brenko et al. 1977, His 1989, Margus 1991, Sarver & Foltz 1993, Hilbish 1994, Hoffman & Somero 1996). These data suggest that the hybrids inherited the ability to grow well in low salinity water from their M trossulus pa- rent. Significant cross-dependent differences in survival existed only during the first week, where the TT cross survived better than the GT, TG, and GG crosses (Fig. 5). There were no sig- nificant between-cross differences in larval survival after the first week. These data are in agreement with a previous experiment by Matson (2000). Hybrid larvae of both reciprocal crosses survived through settlement and as juveniles. No significant survival differences existed between crosses or salinities from day 3 to 7, or from day 3 to 14, though there was an interesting pattern in the means by day 14. Each hybrid cross survived most like its sire (TG and TT crosses survived better at low salinity, GT and GG crosses survived better at high salinity). It may be worth examining this more closely, perhaps with greater replica- tion (more than 4x per cross at each salinity, more than /; = 32 total) to see if salinity tolerance/preference may be paternally in- herited. Hybrid Vigor Although significant cross-dependent differences were found in early growth, survival, and eariy development, most of them seem to have been due to factors other than the phenomenon of hybrid vigor. Hybrid vigor was defined here as an increase in fitness of the hybrids over either of the parental crosses, exhib- ited in either growth or survival. Hybrids were not consistently larger than parental crosses, nor did one reciprocal consistently grow faster than the other. These findings concur with a previous between-cross experiment (Matson 2000). The hybrid cross using M. galloprovincialis eggs generally grew larger than its recipro- cal, which used M. trossulus eggs, during the first week. This may have been because of maternally dependent conditioning ef- fects, or species-specific temperature effects that were also maternally dependent. Early cross-dependent larval growth was probably also influenced by maternal effects (Lannan et al. 1980). These maternal effects may have been the result of species- or population-dependent differences in egg nutrition (Bayne 1978), or differences in egg condition (Lannan et al. 1980), reflected by the different peak spawning times of each species in Puget Sound (Johnson 1978, Brooks 1991). When spawned, the M. galloprovincialis mussels were at the end of their spawning season, and the M. trossulus mussels were almost at their peak. This observation concurs with previous seasonal examinations of these two species gonadal condition (Johnson 1978, Brooks 1991). These results are similar to those of Beaumont et al. 1993 (in terms of lack of hybrid vigor), who hybridized M. galloprovincia- lis with M. edulis. Beaumont et al. (1993) found that after initially higher mortality, veliger larvae of both reciprocal hybrid crosses grew as fast (Trial Three) or significantly faster than (Trial One) M. galloprovincialis larvae in one trial, but not the other. Hybrid crosses didn't grow consistently faster than parental crosses. Lubet ( 1984), who created hybrid M. galloprovincialis x M. edulis mus- sels and examined their juveniles and adults in the field, concluded that those two species are closely related, and exhibit minimal barriers to hybridization as well as minimal fitness differences between hybrids and parentals. Laboratory Hybridization of M. tkossulus and M. gallofrovincialis 429 ACKNOWLEDGMENTS Thanks to Paul Bentzen, Ginger Arbrust. Patrick, and Pam Jensen from the University ot Washington (UW) Marine Molecu- lar Biotechnology Laboratory; thanks to Hal Beattie and Amily Caffe at the Washington Department of Fish and Wildlife. Pt. Whitney Shellfish Laboratory, and thanks to William Hershberger from UW. Special thanks to everyone at the Taylor Shellfish Hatchery. Quilcene. WA. This project was funded by the Victor and Tamara Loosanoff Endowed Fellowship and the Research and Scholarship Committee at the University of Washington School of Fisheries. LITERATURE CITED Allen. S. K. & P. M. Gaffney. 1993. Genetic confirmation of hybridization between Crassostrea gigas (Thunberg) and Crassostrea rivularis (Gould). Aquacuhure 113:291-300. Allen, S. K.. P. M. Gaffney. J. Scarpa & D. Bushek. 1993. Inviable hybrids of Crassostrea virginica (Gmelin) with C. rivularis (Gould) and C. gigas (Thunberg). Acjiiaciilnire 113:269-289. Bayne. B.L. 1965. Growth and the delay of metamorphosis of the larvae of Mytilus edulis (L.) Ophelia 2:1-47. Bayne. B. L.. P. A. Gabbot & J. Widdows. 1975. Some effects of stress in the adult on the eggs and larvae of MxuIks edulis L. J. Mar. Biol. Assoc. UK 55:675-689. Bayne. B. L., D. L. Holland. M. N. Moore, D. M. Lowe & J. Widdows. 1978. Further studies on the effects of stress in the adult on the eggs of Mytilus edulis. J. Mar. Biol. Assoc. (7A' 58:825-841. Bayne. B. L.. D. Hedgecock. D. McGoldrick & R. Rees. 1999. Feeding behavior and metabolic efficiency contribute to growth heterosis in Pacific oysters (Crassostrea gigas Thunberg). J. E.xp. Mar. Biol. Ecol. 233:115-130. Beaumont, A. R., A. K. M. Abdul-Matin & R. Seed. 1993. Early devel- opment, survival and growth in pure and hybrid larvae of Mytilus edulis and M. galloproviiicialis. J. Molluscan Studies 59:120-123. Bert. T. M.. D. M. Hesselmann. W. S. Arnold, W. S. Moore. H. Cruz- Lopez & D. C. Marelli. 1993. High frequency of gonadal neoplasia in a hard clam (Mercenaria spp.) hybrid zone. Mar. Biol. 1 17:97-104. Brenko. M. & A. Calabrese. 1969. The combined effects of salinity and temperature on larvae of the mussel Mytilus edulis. Mar. Biol. 4:224- 226. Brenko, M.. C. Claus & S. Bubic. 1977. Synergistic effects of lead, salinity and temperature on embryonic development of the mussel Mytilus gal- loproviiicialis. Mar. Biol. 44:109-115. Brooks. K. M. 1991. Epizootiology of hemic neoplasia in Mytilus trossulus in Washington State. Part 2. / Shellfish Re.s. 10:233. Brooks, K. M. 1991. The genetics and epizootiology of hemic neoplasia in Mytilus edulis. Doctoral dissertation. University of Washington. 283 pp. Brooks, K. M. 1996. September. 1996, Baseline electrophoretic survey of mussels, Mytilus edulis trossulus and Mytilus edulis galloprovincialis in Holmes Harbor, Washington. Technical report for Taylor United Inc.. p. 7. Brooks, K. M. 1997. 1997 .Annual electrophoretic survey of mussels. Myti- lus edulis trossulus and Mytilus edulis gallopro\incialis in Holmes Harbor. Washington. Technical report for Taylor United Inc., p. 8. Chikhachev, A. S.. V. A. Boryakin & V. V. Tyan'kin. 1979. Resistance of young sturgeon hybrids (Acipenseridae) to extreme conditions. / Ich- thyol. 19:155-158. Cobb, G. W. 1997. Design and analysis of experiments. New York: Springer-Verlag. pp. 674-677. Coustau. C. 1991. Differential susceptibility to a trematode parasite among genotypes of the Mytilus fi/H/is/galloprovincialis complex. Genetic Res. 57:207-212. Geller, J. B., J. T. Carlton & D. A. Powers. 1994, PCR-based detection of mtDNA haplotypes of native and invading mussels on the northeastern Pacific coast: latitudinal pattern of invasion. Mar. Biol. 1 19:243-249. Hamel, J. F. & A. Mercier. 1993. Occurrence of interspecific cross- fertilization among echinoderms and niollusks. Invertebrate Reprod. Devel. 26:221-228. Heath. D. B.. P. D. Rawson & T. J. Hilbish. 1995. PCR-based nuclear markers identify alien blue mussel (Mytilus sp.) genotypes on the west coast of Canada. Can. J. Fisheries Aquatic Sci. 52:2621-2627. Hedgecock. D., D. J. McGoldrick, D. T. Manahan. J. Vavra, N. Appelmans & B. L. Bayne. 1996. Quantitative and molecular genetic analyses of heterosis in bivalve molluscs. J. Exp. Mar Biol. Ecol. 203:49-59. His. E.. R. Robert & A. Dinet. 1989. Combined effects of temperature and salinity on fed and starved larvae of the Mediterranean mussel Mytilus galloprovincialis and the Japanese oyster Crassostrea gigas. Mar Biol. 100:455-463. Hilbish, T J.. B. L. Bayne & A. Day. 1994. Genetics of physiological differendation within the marine mussel genus Mytilus. Evolution. 48: 267-286. Hoffmann. G. E. & G. N. Somero. 1995. Evidence for protein damage at environmental temperatures: seasonal changes in levels of ubiquitin conjugates and hsp70 in the intertidal mussel Mytilus trossulus. J. Exp. Biol. 198:1.509-1518. Hoffmann. G. E. & G. N. Somero. 1996. Interspecific variation in thermal denaturation of proteins in the congeneric mussels Mytilus trossulus and M. galloprovincialis: evidence from the heat-shock response and protein ubiquitination. Mar. Biol. 126:65-75. Johnson, K. W. 1979. The Relationship Between Mytilus edulis Larvae in the Plankton and Settlement for Holmes Harbor, Washington. Master's Thesis. University of Washington, pp. 28-39. Kaulsky. N. 1987. Growth and size structure in a Baltic Mytilus edulis population. Mar. Biol. 68:117-133. Lannan, J. E. 1980. Broodstock management of Crassostrea gigas I. Ge- netic and environmental variation in survival in the larval rearing sys- tem. Aquacuhure 21:323-336. Lannan. J. E., A. Robinson & W. P. Breese. 1980. Broodstock management of Crassostrea gigas II. Broodstock conditioning to maximize larval survival. Aquacuhure 21: 337-345. Loosanoff, V. L. 1954. New advances in the study of bivalve larvae. Am. Sci. 42:607-624. Loosanoff, V. L. & H. C. Davis. 1963. Rearing of bivalve mollusks. Adv. Mar. Biol. 1:90-95. Lubet. P.. G. Prunus, M. Masson & D. Bucaillet. 1984. Reches experimen- tales sur I'hybridization de Mytilus edulis L. et M. galloprovincialis Lmk. (Mollusques Lamellibranches). Bull. Sociuetae Zool. France 109: 87-98. Mallet. A. L.. E. Zouros, K. E. Gartner-Kepkay. K. R. Freeman & L. M. Dickie. 1985. Larval viability and heterozygote deficiency in popula- tions of marine bivalves: evidence from pair matings of mussels. Mar. Biol. 87:165-172. Margus, D. 1991. Growth and survival of mussels (Mytilus galloprovin- cialis Lmk.) in an on-going trial in the Krka estuary, central Adriatic. Yugoslavia. Oebalia. XVII:209-220. Matson. S. E. 2000. Hybridization of the mussels Mytilus trossulus and M. galloprovincialis: Larval growth, survival, and early development. Master's Thesis. University of Washington. 112 pp. McDonald, J. H.. R. Seed & R. K. Koehn. 1991. Allozymes and morpho- metric characters of three species of Mytilus in the Northern and South- em Hemispheres. Mar. Biol. 111:323-333. Rawson, P. D, & T. J. Hilbish. 1991. Genotype-environment interaction for juvenile growth in the hard clam Mercenaria mercenaria (L.). Evolu- tion. 45:1924-1935. 430 Matson et al. Rawson. P. D., K. L. Joyner, K. Meetze & T. Hillbish. 1996. Evidence for intragenic recombination within a novel genetic marker that distin- guishes mussels in the Mytilus eduUs species complex. Heredity- 11: 599-607. Sarver, S. K.. and D. W. Foltz. 1993. Genetic population structure of a species' complex of blue mussels {Mytilus spp.) Mne. Biol. 1 17:105-1 12. Sarver. S. K. & E. J. Loudenslager. 1991. The genetics of California populations of the blue mussel: Further evidence for the existence of electrophoretically distinguishable species or subspecies. Biochem. Systematics Ecol. 19:183-188. Sokal. R. R. & F. J. Rohlf. 1997. Biometry, Third Edition. New York: Freeman, pp. 119-122. Suchanek, T. H., J. B. Geller. B. R. Kreiser & J. B. Mitton. 1997. Zoo- geographic distributions of the sibling species Mytilus galloprovincialis and M. trossulus (Bivalvia: Mytilidae) and their hybrids in the north Pacific. Biol. Bull. 193:187-194. Szmant. A. M., E. Weil, M. W. Miller & D. E. Colon. 1997. Hybridization within the species complex of the sleractinan coral Montastraea annu- laris. Mar. Biol. 129:561-572. Weiss, N. A. 1995. Introductory statistics. Fourth Edition. Reading, MA, p. 424. Zar, J. H. 1996. Biostatistical analysis. New Jersey; Prentice Hall, pp. 290-291, Jcninuil i>t Shellfish Research. Vol. 22. No. 2. 431-434, 2003. RIBOSOMAL RNA CHARACTERIZATION OF NON-TRANSCRIBED SPACER AND TWO INTERNAL TRANSCRIBED SPACERS WITH 5.8S RIBOSOMAL RNA OF PERKINSUS SP. FOUND IN UNDULATED SURF CLAMS (PAPHIA UNDULATA) FROM THAILAND SUFANNEE LEETHOCHAVALIT,'* E. SUCHART UFATHAM," KWANG-SIK CHOI,' FICHAN SAWANGWONG/ KASHANE CHALERMWAT/ MALEEYA KRUATRACHUE^ 'institute of Marine Science. Bunipha University. Bangsaen. Clwnhiiri 20131. Tliailcmd: 'Faculty of Science. Department of Biology. Burapha University. Bangsaen. Chonburi. 201 3 f Tliailanil: ^Faculty of Applied Marine Science, College of Ocean Science. Cheju National University. 1 Ara 1-Dong Jeju City Jeju-Do 690-76-56. S. Korea: ^Faculty of Science. Department of Aquatic Science. Burapha University. Bangsaen. Chonburi 20131. Thailand: ^Faculty of Science. Department of Biology. Mahidol University, Rama 6 road. Payathai. Bangkok. 10400. Thailand ABSTRACT The genetic divergence of Perkinsiis sp. loLind in the undulated surf clam i.Puphiu iimliilala) from the Gulf of Thailand and other known Perkiiisiis species was exammed using the non-transcribed spacer and two internal transcribed spacers with 5.S S rRNA gene. The sequences of non-transcnbed spacer (NTS) and internal transcribed spacer region (ITS) that includes the 5.8S rRNA gene flanked by ITS I and ITS2 (ITSI-5.8S-ITS2) were cloned and sequenced. The sequences were compared with those of Perkin.sus olseni from Australia. P. allimiiciis from Korea. P. marimis and P. aiidrewsi from the United States and P. qugwadi from Canada. The lengths of the obtained nucleotide sequences of NTS. ITS-1 5.85 rRNA and ITS-2 were 1,167, 183, 159, and 371 bp, respectively. The nucleotide sequences of NTS and ITS-5.8S rRNA of Thai Perkiiuus and P. olseni showed 98.69% and 99.85% identity, respectively. When compared with P. allanticus identities were 96.27 and 99.71%, in P. marinus 75.38 and 94.88% and in P. andrewsi 46.55 and 86.23%. The nucleotide sequences of ITS-5.8S rRNA between Thai Perkinsiis and P. chesapeaki showed an identity of 87.05%. This is the first report of the occurrence of Perkinsiis sp in the Gulf of Thailand. KEY WORDS: Perkinsiis sp.. Pupliia iindutata. nucleotide sequence, non-transcribed spacer, internal transcribed spacerl, internal transcribed spacer 2 INTRODUCTION The pathogenic protozoans. Perkinsiis spp. causes Perkinsosis disease in marine bivalves (Andrews 1988). According to Perkins (1976) and Levine (1978). they were classified as an apicom- plexan. However, recent molecular phylogenetic analyses by Sid- dall et al. (2001) and Recce et al. (1997) have placed these para- sites within the Dinoflagellata. Traditionally, diagnosis of Perkin- sus infection depends on the fluid thioglycollate medium (FTM) assay for identification and Choi's 2 M NaOH digestion technique on FTM cultivated tissues for quantification (Choi et al. 1989, Almeida et al. 1999). However, the FTM assay does not discrimi- nate different Perkinsiis species and has a potential to introduce misleading positive results between Perkinsiis and other di- noflagellate species (Almeida et al. 1999). A more precise appli- cation for detection, identification, and numeration of these para- sites is based on molecular characterization. The internal tran- scribed spacers (ITS), 5.8 S regions of the ribosomal RNA (rRNA) and non-transcribed spacer gene (NTS) can be used to discriminate among the Perkinsiis species because these regions are largely non-coding with high evolutionary rate, and have been used to identify Perkinsiis species isolated from different hosts and geo- graphical regions (Kotob et al. 1999. Robledo ct al. 1999, Robledo et al. 2000). These NTS and ITS regions have also been used to distinguish between strains and species of other protozoa (Cai et al. 1992, Goggin 1994, Cunningham 1997). We have identified Per- kinsiis in the undulated surf clam, Paphia lunliihila, a major com- mercial species from the Gulf of Thailand using FTM assay. In this study, we have characterized the Thai Perkinsiis ribosomal RNA, the nucleotide sequences of ITS-5.8S rRNA, and NTS and com- *Corresponding author. E-mail: sp02l7@yahoo.com; Fax: -1-66-38-391674 pared the sequences with rRNA sequences that have been reported for other known Perkinsiis species. MATERIALS AND METHODS Isolation of Prezoosporangia Live specimens of the undulated surf clam {Paphia iindidata) were obtained from food markets in Chonburi Province. Thailand. The infected gills of clams were cultured in fluid thioglycollate medium supplemented with streptomycin (500 |j.g/ml) and peni- cillin G potassium (500 unit/ml) at 27°C in the dark for 3 days. The tissue was then digested by trypsin (0.25% in sterilized seawater) at rootn temperature for .3-4 h. and the obtained prezoosporangia were then isolated by filtration through a silk net. The resulting pellets were finally washed 3 times using sterilized seawater and at each washing the pellets were cenlrifuged at x490,i? for 8 min. DNA Isolation Genomic DNA was extracted from prezoosporangia using a DNA trap kit, according to details provided by the manufacturer (Tissue Protocols for DNA isolation, DNA TEC, Thailand). PCR Amplification The complete region of ITS1-5.8S-ITS2 and NTS genes were amplified from genomic DNA using a forward primer of the small subunit (SSU) 5'AGGAAGGAGAAGTCGTAACAAGG 3' (Hamaguchi et al. 1998) and a reverse primer of the large subunit (LSU) 5'ACCCGCTGAATTTAAGCATA 3' (Goggin 1994). The NTS region was amplified by using a forward primer 5' AAGTC- CTTAGGGTGCTGCTGGCT 3' and reverse primer 5' CTACTG- GCAGGATCAACCAGGT 3' (Park et al. 2002). The polymera.se 431 432 Leethochavalit et al. chain reactions were performed in a final volume of 20 |xL reac- tion mixture containing 2 jxL of 10 x reaction buffer (50 mM KCL; 10 mM Tris-HCL, pH 8.3: 1.5 mM MgCK). 2 jxL of 1 mM de- oxyribonucleoside-5'-triphosphate (dNTPs)(Proniega Corp., Madison, WI), 1 (jlI of 5 |jlM upstream primer, and 1 (jlL of 5 jjlM downstream primer, 0.8 p.L of 5 units Taq DNA polymerase (Promega Corp.) and 4 |xL of 5 ng/fil of DNA template. The final volume was adjusted with sterilized distilled water to 20 (xL. The mixture was amplified in a PCR Thermal Cycler (GeneAmp®PCR system9700, Albertville, MN) for 35 cycles with initial denatur- ation at 94°C for 3 min. The process was followed by 35 cycles of denaturation at 94°C for 30 sec and annealing at 55°C for 30 sec. The primer extension was performed at 72°C for 1 min followed by a final extension at 72°C for 5 min. The PCR products were analyzed utilizing 1% agarose gel electrophoresis and visualized for DNA bands under UV light and photographed using digital photography (Delidow et al. 1993, Hamaguchi et al. 1998, Carne- gie et al. 2000). DNA Cloning and Sequencing The PCR products of the ITSI-5.8S rRNA- ITS2 and NTS fragments were excised from the agarose gel and purified by a cleaning reagent consisting of exonuclease I (Exo I) and shrimp alkaline phosphatase (SAP). The NTS and ITS-5.8S fragments were cloned into pGEM™-T Easy vector (Promega Corp.) and isolated. At least one clone of each NTS and ITS-5.8S fragment was sequenced following standard procedures in an automatic DNA sequencer, ABI PRISM model 377 (Dicker et al. 1993, Hamaguchi et al. 1998). The nucleotide sequences of NTS were analyzed for nucleotide similarities with P. olseni (GenBank ac- cession number AF466527), P. atlanticus (AF438150), P. marimts (AF497479), P. andrewsi (AF102171). The nucleotide sequences of ITS1-5.8S-ITS2 were analyzed for nucleotide similarities with P. mariniis (AF497479), P. andrewsi (AFI02171), P. atlanticus (AF473840), P. olseni (U07701), P. qiigwadi (AFI51528), and P. chesapeaki (AF09154I) by BLAST and CLUSTALW programs provided by GenBank and the European Bioinformatics Institute. RESULTS From this study, the sequences of the NTS. ITS-1, ITS-2, and 5.8S rRNA fragments PCR amplified from prezoosporangias of Thai Perkinsiis found in P. imdulata were 1 167,183, 371, and 159 bp in length, respectively. These sequences were submitted to GenBank and given an accession number (AF522321 ). The nucle- otide sequences of NTS from Thai Perkinsiis were compared with the completed sequences of P. olseni isolate P 01 (Murrell el al. unpublished data). P. atlanticus (Park et al. 2002), P. mahnus isolate TXsc (Robledo et al. 1999), and P. andrewsi (Coss et al. 2001). The sequence similarity between the NTS region of Thai Perkinsus and P. olseni. P. atlanticus. P. marimis. and P. andrewsi were 98.69%, 96.27%, 75,38%, and 46.55%, respectively (Table I ). To determine the ITS with 5.8S rRNA sequence similarities of the Thai Perkinsus. we compared the complete sequences of this species with completed sequences of P. marimts isolate TXsc (Robledo et al. 1999), P. andrewsi (Coss et al. 2001), P. atlanticus (Park et al., in press), P. olseni (Goggin 1994), P. qugwadi (Hervio et al.. unpubl. data), and P. chesapeaki (Kotob et al. 1999). The ITS-5.8S rRNA sequences of Thai Perkinsus was 94.887r similar to P. marinus isolate TXsc, 88.34% similar to P. andrewsi, 99.71% similar to P. atlanticus. 99.85% similar to P. olseni, 68.02% simi- lar to P. qugwadi, and 87.05% similar to P. chesapeaki (Table I ). The sequence of 5.8S rRNA of Thai Perkinsus showed 100% similarity to P. olseni, P. atlanticus, and P. marinus. DISCUSSION Several species of Perkinsus have been reported from different locations in the worid including Australia (Goggin 1994). China (Liang et al. 2001 ). Japan (Blackbourn et al. 1998, Hamaguchi et al. 1998, Choi et al. 2002). Korea (Park & Choi 2001), New Zealand (Goggin 1994), Portugal (Azevedo 1989) and USA (Mackin et al. 1950). There has been no report of Perkinsus sp. in any species of shellfish in Thailand and no species of shellfish in Thailand has been reported to exhibit symptoms of Perkinsosis diseases. However, additional research in this area may reveal otherwise. In our study, we targeted and analyzed the NTS. ITS-1, ITS-2, and 5.8S rRNA genes for species-specificity of Thai Perkinsus found in P. undulata. The results showed that the NTS region of Thai Perkinsus is slightly different from that of P. olseni (1.31%) and P. atlanticus (3.73%) but highly different to P. marimis (24.62%) and P. andrewsi (53.45%). As proposed by Coss et al. (2001 ). this implies that the NTS region of P. andrewsi is dramati- cally different in both length and sequence from those of P. mari- nus and P. atlanticus. Robledo et al. (1999) concluded that the NTS region can accumulate a high degree of sequence variability between closely related species. The sequence of 5.8S rRNA of Thai Perkinsus showed 100% similarity to P. olseni. P. atlanticus. TABLE 1. The length and sequence similarity ( % ) of non-transcribed spacer, internal transcribed spacerl, 5.8S ribosomal RNA and internal transcribed spacer2 of Thai and other Perkinsus species. Length NTS Length ITS-1 Length 5.8S rRNA Length ITS-2 Accession Organism (bp) (^similarity) (bp) (% similarity) (bp) (% similarity) (bp) (% similarity) No. Thai Perkinsus 1.167 100 183 lUO 159 100 371 100 AF522321 P. marinus KL-ig 75.38 197 85.27 161 100 372 93.27 AF497479 P. andrewsi 1,551 46.55 185 79.45 159 98.74 368 82.60 AF102171 P. atlanticus — — 183 99.45 159 100 371 99.73 AF473840 P. atlanticus 1.146 96.27 — — — — — — AF438150 P. olseni — — 183 99.45 159 100 371 100 U07701 P. olseni 1,153 98,69 — — — — — — AF466527 P. qugwadi — — 204 47.05 158 93.63 363 63.08 AF102171 P. chesapeaki — — 18S 87.76 159 96.22 379 82.45 AF091541 Characterization of Spacers in Thai Perkinsus 433 and P. marinus. As reported by Goggin (1994). the 5.8S rRNA sequence regions from P. olseni. P. atlanticiis. P. marinus. and unidentified Perkinsus (from/4, trapezia and C. pacificus) were ail identical. However, the 5.8S rRNA sequence of Thai Perkinsus differs at 2 positions when compared with P. andrewsi and 10 positions when compared with P. ijugwudi. Coss et al. (2001 ) reported that 5.8S rRNA of P. andrewsi differed from 5 isolates of Perkinsus spp. reported by Goggin (1994) in 2 positions but dif- fered from P. qujiwadi in 14 positions. Murrell et al. (2002) re- cently updated the phylogenetic position of the genus Perkinsus and considers P. olseni and P. aikinticus to be synonyms. Our results show high levels of sequence homology in 1TS-5.8S rRNA region among Thai Perkinsus. P. olseni. and P. allanticus. The nucleotide sequences of ITS-5.8S rRNA in Thai Perkinsus were highly similar to P. olseni (99.85%) and P. atlanticus (99.7 IVr). In this region. Thai Perkinsus differs from P. olseni at 1 position in ITS-1 but differs from P. atlanticus at 2 positions in both ITS-1 and ITS-2. Goggin (1994) found that P. olseni from Australia and P. atlanticus from Portugal had an identical se- quence for ITS I but differed in ITS-2 at .^ positions by substitution of one nucleotide and he suggested that these two species belong to a single species. From our study the Thai Perkinsus is most closely related to P. olseni and P. atlanticus. At the same time, the Thai Perkinsus showed genetic divergence at the ITS-5.8S rRNA region from P. marinus. P. andrewsi. and P. cjui^wadi. The nucle- otide sequence of Thai Perkinsus ITS-5.8S rRNA showed 94.88% homology to P. marinus. 86.23% homology to P. andrewsi, and 68.02% homology to P. qugwadi. At this fragment, the sequences of Thai Perkinsus versus those of P. marinus and Thai Perkinsus versus P. andrewsi were more different in ITS-1 (14.73% and 20.55%) than ITS-2 (6.73% and 17.40%). Goggin (1994) also reported that the sequences of ITS-1 and ITS-2 of P. marinus from American oysters differed significantly from P. olseni. P. atlanti- cus. and an unidentified Perkinsus from Anadara trapezia and Chama pacificus. Furthermore, he found that the variation among 4 isolates of Perkinsus and P. marinus was greater in the ITS- 1 (23%) than the ITS-2 (7-8%) region. Goggin (1994) concluded that 12% differences of nucleotide deletions were most common in the ITS-1. Our study shows that the sequences of lTS-1 and lTS-2 in Thai Perkinsus and P. qugwadi were substantially different. Coss et al. (2001) also found genetic divergence from ITS-1 and ITS-2 regions, between P. andrewsi and P. qugwadi and suggested that P. qugwadi is not closely related to the other Perkinsus species. In conclusion, molecular evidence of the ribosomal RNA from Perkinsus found in Paphia undulata from the Gulf of Thailand shows that it is distinctly different from P. marinus. P. andrewsi. and P. qugwadi. Although homology of NTS, ITS-1, 5.8S rRNA, and ITS-2 sequence in Thai Perkinsus with P. olseni and P. at- lanticus are high, the level of homology required to discriminate between species of Perkinsus have not been determined (Goggin 1994). Therefore, we do not specify a species-specific name for Perkinsus sp. found in Paphia undulata from the Gulf of Thailand at this point in time. ACKNOWLEDGMENTS The authors thank the Institute of Marine Science for use of facilities and laboratory space. Partial research funding was pro- vided by the Graduate Program in Biological Science, Graduate School and Faculty of Science, Burapha University. The Shellfish Aquaculture and Research Laboratory, Faculty of Applied Marine Science. College of Ocean Science. Cheju National University provided funds for travel and research in Korea. We also thank Dr Wansuk Senanan for reading and commenting on the manuscript. LITERATURE CITED Almeida, M., F. Berthe, A. Thebault & M. T. Dinis. 1999. Whole clam culture as a quantitative diagnostic procedure of Perkinsus atlanticus ( Apicomplexa, Perkinsea) in clams Riidiiapes decussatus. Aquaculture 177:325-332. Andrews. J. D. 1988. Epizootiology of the disease caused by the oyster pathogen Perkinsus marinus and its effects on the oyster industry. In: W. S. Fisher, editor. Disease processes in marine bivalve molluscs. American Fisheries Society Special Publication, pp. 257-264. Azevedo, C. 1989. Fine structure of Perkinsus atlanticus n. sp. (Apicom- plexa, Perkinsea) parasites of the clam Ruditapes decussatus from Por- tugal. J. Parasitol. 75:627-635. Blackboum, J.. S. M. Bower & G. M. Meyer. 1998. Perkinsus qugwadi sp. nov. (incertae sedis). a pathogenic protozoan parasite of Japanese scal- lops, Patinopecten yessoensis. cultured in British Columbia, Canada. Can. J. Zool. 76:942-953. Cai, J., M. D. Collins, V. Mcdonald & D. E. Thompson. 1992. PCR clonmg and nucleotide sequence determination of the 18S rRNA genes and internal transcribed spacer 1 of the protozoan parasites Cryptosporid- ium pan-um and Ciyplosporridium muris. Biochimica et Biopliysica Acta. 1131:317-320. Carnegie, R. B., B. J. Barber, S. C. Culloty. A. J. Figueras & D. L. Distel. 2000. Development of a PCR assay for detection of the oyster pathogen Bonamia ostreae and support for its inclusion in the Haplosporidia. Dis. Aquat. Org. 42:199-206. Choi, K.-S., K.-I. Park, K.-W. Lee & K. Matsuoka. 2002. Infection inten- sity, prevalence, and histopathology of Perkinsus sp. in the Manila clam. Ruditapes philippinarum. in Isahaya Bay. Japan. / Shellfish. Res. 21:119-125. Choi, K.-S.. E. A Wilson, D. H. Lewis, E. N. Powell & S. M. Ray. 1989. The energetic cost of Perkinsus marinus parasitism in oysters: quanti- fication of the thioglycollate method. J. Shellfish. Res. 8:125-131. Coss, C. A., A. F. Robledo, G. M. Ruiz & G. R. Vasta. 2001. Description of Perkinsus andrewsi n. sp. isolated from the Baltic clam (Macoma halthica) by characterization of the ribosomal RNA locus, and devel- opment of species-specitlc PCR-based diagnostic assay. J. Eukaryot Microbiol. 48:52-61. Cunningham, C. O. 1997. Species variation within the internal U'anscribed spacer (ITS) region of Gyrodactylus (Monogenea: Gyrodactylidae) ri- bosomal RNA genes. J. Parasitol. 83:215-219. Delidow, B. C, J. P. Lynch. J. J. Peluso & B. A. White. 1993. Polymerase chain reaction. In: B. A. White, editor. Methods in molecular biology: PCR protocol. New Jersey: Humana Press, pp. 1-40. Dicker. A. P.. M. Volkenandt & J. R. Bertino. 1993. Manual and automated direct sequencing of product generated by the polymerase chain reac- tion. In: B. A. White, editor. Methods in molecular biology: PCR protocol. New Jersey: Humana Press, pp. 143-152. Goggin. C. L. 1994. Variation in the internal transcribed spacers and 5.8S ribosomal DNA from five isolates of marine parasite Perkinsus (Pro- tista. Apicomplexa) based on small subunit ribosomal RNA. Mol. Bio- chem. Parasitol. 60:65-70. Hamaguchi. M.. N. Suzuki & H. Usuki. 1998. Perkinsus protozoan infec- tion in short-necked clam Tapes ( = Ruditapes) philippinarum in Japan. Fish Patho. 33:473^80. Kotob, S. I., S. M. McLaugWin, P. Van Burkum & M. Faisal. 1999. Discrimination between two Perkinsus spp. isolated from the softshell clam, Mya arenariu. by sequence analysis of two internal transcribed 434 Leethochavalit et al. spacer regions and the 5.8S ribosomal RNA gene. Parasitol. 1 19:363- 368. Levine. N. D. 1978. Perkinsus gen. n. and other new ta.xa in the protozoan phylum Apicomplexa. J. Parasitol. 64:549. Liang. Y.-B., X.-C. Zhang. L.-J. Wang. B. Yang, Y. Zhang & C.-L. Cai. 2001. Prevalence of Perkinsus sp. in the Manila clam, Rudihipes phit- ippiiuiniin. along the northern coa.st of the Yellow Sea in China. Oceanol. Limnol. Sinica. 32:502-511. Mackin. J. G., H. M. Owen & A. Collier. 1950. Preliminary note on the occurrence of a new protistan parasite, Dermocystidiiim marinum n. sp. in Crassostrea virginica (Gmelin). Science 1 1 1 :328-329. Murrell. A., S. N. Kleeman, S. C. Barker & R. J. G. Lester. 2002. Syn- onymy of Perkinsus olseni Lester & Davis, 1981 and Perkinsus atlan- licus Azevedo, 1989 and an update on the phylogenetic position of the genus Perkinsus. Bull. Eur Ass. Fish Pathol. 22:258-265. Park. K.-L & K.-S. Choi. 2001. Spatial distnbution of the protozoan para- site Perkinsus sp. found in the Manila clams Rudimpes philippinarum. in Korea. Aquaculture 203:9-22. Park, K.-L, Y.-M. Park, J. Lee & K.-S. Choi. 2002. Development of a PCR Assay for detection of the protozoan Parasite Perkinsus. Korean J. Em: Biol. 20:109-117. Perkins, F. O. 1976. Dermocysiidiunt marinum infection in oysters. Mar. Fish. Rev. 38:19-21. Reece, K. S., M. E. Siddall, E. M. Burreson & J. E. Graves. 1997. Phy- logenetic analysis of Perkinsus based on actin gene sequences. J. Para- sitol. 83:17-423. Robledo, A. F., A. C. Wright, A. March & G. R. Vasta. 1999. Nucleotide sequence variability in the nontranscribed spacer of the rRNA in the oyster parasite Perkinsus inarinus. J. Parasitol. 85:650—656. Robledo, A. F.. C. A. Coss & G. R. Vasta. 2000. Characterization of the ribosomal RNA locus of Perkinsus atlanticus and development of a polymerase chain reaction-based diagnostic assay. J. Parasitol. 86: 972-978. Siddall, M. E.. K. S. Reece, T. A. Nerad & E. M. Burresson. 2001. Mo- lecular determination of the phylogenetic position of a species in the genus Colpodella (Alveolata). Am. Mus. Novilates 3314:1-10. Journal of Shellfish Research, Vol. 22, No. 2, 435^M1, 2003. A STUDY OF GONADAL DEVELOPMENT IN RUDITAPES DECUSSATUS (L.) (MOLLUSC A, BIVALVIA), USING IMAGE ANALYSIS TECHNIQUES: INFLUENCE OF FOOD RATION AND ENERGY BALANCE M. DELGADO AND A. PEREZ CAMACHO* Instituto Espauol cle Occauografia. Miiellc de Animas. s/n. E- 15001 A Conma. Spain ABSTRACT This study evaluated the inlluence of food availability on sexual maturation ui RiiclinqH-x ilecussaUis (L.) in conditions of positive (daily rations of 0.10, 0.24, 0.42. and 0.96<;f). zero (O.OS'/f ration), and negative energy balance (0.025% ration). The percentages correspond to the organic weight of the phytoplankton supplied as a proportion of the live weight of the clams. The gonadal occupation index (GOI) and the percentage of ripe oocytes in the gonad, calculated using image analysis techniques, were taken as indicators of the degree of sexual maturity. Gonadal development in R. decussauis occurred under all food rations and energy balance conditions, even when the organic weight of the clams decreased during the period of sexual development. All conditions registered a gradual increase in GOI and the percentage of ripe oocytes throughout the experimental period. Maximum values for GOI varied between 30% and 40% in females and between 55% and 75% in males, according to the amount of food available. Similarly, mature sexual cells were observed under all experimental conditions, with maximum percentages in females of between 30% and 40%. The extent of gonadal development is directly related to the amount of food available, which in turn has a direct bearing on the rate of gonadal development, with smaller rations leading to a lower rate of increase in the gonadal occupation index and the percentage of ripe oocytes. KEY WORDS: food ax'ailability. gonadal development, image analysis. Rudilapes decitssams INTRODUCTION The majority of studies of the reproductive cycle of R. decus- satus in its natural habitat (Perez-Camacho I9S0. Beninger 1982, Shaffee & Daouidi 1993. Villalba et al. 1993). are based either on indirect indicators of gonadal development (condition index, go- nadosomatic index, flesh weight), the discharge of gametes, smear techniques (Berthou et al. 1980), or histologic studies of the gonad that describe the various stages of gametogenesis (Holland & Chew 1974). A more objective determination of the degree of maturity is provided by methods that measure the area occupied by sexual cells and the frequency distribution of oocyte sizes. As a result, it has been possible to produce more accurate inter- and intraspecies comparative analyses of several bivalve species (Navarro et al. 1989, Xie & Burnell 1994, Lai^elle et al. 1994, Rodri'guez- Moscoso & Amaiz 1998). However, the data on bivalve reproduc- tive histology provided by image analysis are more accurate and precise than that obtained by the traditional stereological method in which an ocular graticule is used (Lowe et al. 1982). Temperature is one of the main factors influencing the game- togenic cycle in bivalves (Sastry 1975, Mann 1979). It would appear to define both the starting point and the rate of gonadal development, whereas diet appears to have a direct effect on the duration of gametogenesis (Lubet 1980-1981). The above- mentioned studies, however, tend to support the involvement of several environmental parameters on sexual activity in bivalves. The reproductive phenomenon is studied in the natural habitat. making it difficult to separate the particular effect of one factor from those of the others. In fact, there are very few studies of the individual infiuence of each environmental variable on the repro- ductive process under controlled conditions (Sastry 1966. Gima- zane 1972, Bayne et al. 1975, 1978. Pipe 1985). The use of image- analysis techniques to determine the effects of a single environ- mental variable, in this case food availability, on gonadal development in R. decussanis is the main aim of this study. MATERIALS AND METHODS Breeding Stock *Corresponding author. E-mail: alejandro.perez@co.ieo.es The experiments were performed in two years running, using clams of two sizes. In the first experiment specimens of R. decus- sanis with a length of 20.8 ±0.15 mm (mean plus standard devia- tion) and a live weight of 1.60 ±0.31 g were used. In the second experiment, average clam length was 36 ± 0.19 mm and live weight 9.97 ± 1.53 g. Experimental Design and Conditions The experiments were performed in a flow-through system containing seawater filtered through a p.m cartridge and main- tained at a constant temperature (18°C) and salinity (33%o). As a consequence of the large number of individuals in each experi- ments (400 and 420) and long duration of the surveys (46 and 70 days), clams were maintained within large groups, in plastic tanks of 12 1. In this way, food concentration is more stable and equal for all clams at each experimental conditions are closer to natural ones. Food consisting in different rations of the microalga Isoch- rysis galbana was added to the circulating water on a continuous basis by means of a variable flow peristaltic pump. The different rations were obtained by maintaining food concentration constant and varying both the flow of water into the tanks and the number of clams per tank. Through-flow in the vessels was reduced after each sampling, to adjust it to the number of clams remaining. Experiment I The following daily food rations, with percentages correspond- ing to the organic weight (ask free dray weight) of food supplied as a proportion of the live weight of the clams, were assayed in this experiment: 0.24% (Al). 0.48% (A2), and 0.96% (A3). The initial number of specimens was 140 for ration, and the number of clams for tank 1 40. 70. and 35 for the rations A 1 , A2, and A3 respectively. The experimental period lasted 46 days, with samples being taken on days 12, 26, 35, and 46. On each occasion 10 specimens from each diet were used to determine soft tissue dry 435 436 Delgado and Perez Camacho weight, with a further 10 specimens used for histologic studies. Where necessary, the number of specimens per sample was in- creased to obtain a minimum of four specimens of each sex. Experiment 2 The rations used in this experiment were 0.025% (BI), 0.05% (B2). and 0.10% (83). The initial number of specimens was 200 for ration Bl, and 100 for rations 82 and 83. and the number of clams for tank 200. 100, and 50, for the rations Bl. B2. and 83, respectively. The experimental period lasted 70 days and samples were taken on days 25, 41, and 70, with 10 specimens being used to determine soft tissue dry weight and a further 10 specimens used for histologic studies. Where necessary, the number of specimens per sample was increased to obtain a minimum of four specimens of each sex. Soft Tissue Growth: Total, Somatic and Gonadal The anatomic features of the gonad in this species make it difficult to separate from the rest of the organism, so indirect methods are usually used to determine the changes that take place (Perez Camacho 1979). In our case, total clam flesh growth (FG) corresponds to the difference between initial and final dry weight (DW). DW was obtained by freeze-drying the total amount of soft tissue. When there was an increase in weight during the experimental period, gonadal growth (GG) was calculated from the difference between the DW of the initial sample (when gonadal development was nil, or very little) and that of the final sample (when the gonad was well developed). To discount any growth of the organism during the experimental period, initial DW was calculated for a standard clam of the same length as the mean length of the final sample, using the length-DW equation of the initial sample. So- matic growth (SG) was taken as the difference between the in- crease in total DW and sonadal srowth (FG-GG). stage of vitellogenesis, or ripe, when their maximum diameter exceeded 50 |jim (Vilela 1950). Males Colorimetrics was used to analyze images of the male clams, with each different part of the soft tissue being color-coded. This division of soft tissue corresponded to gametes (deep purple stain), muscle tissue and reserves (deep and pale pink stain), and empty zones (white). The area occupied by each color in the image being studied was measured, and the previously mentioned expression (GOD was calculated, the area occupied by gametes corresponding to that occupied by spermatozoids, spermatids, sperinatocytes and spermatogonia. Each specimen was assigned a mean value for GOI and a percentage of ripe oocytes present in the gonad, obtained from the nine images analyzed in each case. Statistical Methods Comparisons between the different rations for flesh dry weight, conditioning index, gonadal occupation index and oocyte diameter were established by analysis of variance (ANOVA) for a signifi- cance level of 95%, and by analysis of covariance (ANCOVA) to compare slopes of the regression lines of those equations having the greatest determination coefficient. Cochran's test was used to guarantee the homogeneity of the variances. When there was a direct relationship between the mean and the standard deviation, logarithmic Iransfomiation was used to homogenize the variances. Parameters expressed as percentages were modified, prior analysis using angular transformation (arcsineV%). Multiple comparisons between experimental conditions were performed with the mul- tiple rank test using the least significant difference (LSD) method. All the statistical analyses were performed with Statgraphics plus 3.0 software, according to the methods described by Snedecor and Cochran (1980) and Zar (1974). RESULTS Histology and Image Analysis A conventional histology protocol was followed. The soft tis- sues were fixed with Bouin's fixative, sealed in paraffin, and 4-|jLm slices were taken. Harris" hematoxylin and eosin stain was used (Bancroft and Stevens 1996). For each specimen, nine fields of vision of the gonad were chosen at random, corresponding to three different depths in the body of the clam. Microimage software (Olympus) was used to process and analyses the images obtained. Females Because sexual maturation in venerids is characterized by an increase in size of the gonadal follicles and their progressive oc- cupation by ripe gametes, which then separate from the follicle walls, it was decided to focus on the area of the gonad occupied by oocytes. The area of each of the oocytes visualized was obtained automatically (Microimage software). On average, measurements of more than 500 oocytes were obtained for each specimen. The gonadal occupation index was defined as follows: GOI: (area occupied by gametes/area of the field analyzed) x 100. Gametogenic development in females is also characterized by a considerable increase in oocyte size, and maximum diameters were therefore measured. Oocytes were considered to be in the final Total. Somatic, and Gonadal Growth The clams in experiment 1 were fed daily rations of 0.24, 0.42, and 0.96%. All three diets produced a positive energy balance, leading to a considerable increase in flesh dry weight (DW) that was directly proportional to the amount of food available (Fig. la). The total increase in DW, expressed as a percentage of initial DW, was 35.8% for ration Al, 48.9% for A2, and 80.4% for A3. The differences between the increases in DW recorded for each of these diets were statistically significant (ANOVA, P < 0.001 ; mul- tiple rank test (LSD), P < 0.05). In experiment 2, diet 83 (0.10%) produced a positive energy balance leading to an increase of 18.6% in DW over the initial value. For diet 82 (0.05%) DW stayed approximately constant during the experimental period, indicating a zero energy balance, as corresponds to a maintenance diet. Diet Bl (0.025%) led to a negative energy balance and a loss of 20% DW by the end of the experimental period (Fig. lb). The differences between the varia- tions in DW of clams fed with these diets were statistically sig- nificant (ANOVA, P < 0.05; multiple rank test (LSD), P < 0.05). Most of the energy acquired by the clams in positive energy balance conditions in our experiments was expended on gonadal development. Accordingly, as can be seen in Figures la and lb, gonadal growth accounts for 90% of the total increase in DW for the highest diets (experiment 1). and 98% for diet 83 in expert- Gonadal Development in R. decussatus 437 80 70 ei 60 £ SO % 40 o I. Ul o 20 10 0 P y Al a: Diets Aj □ GG B SG 100 80 60 'm 40 E 71) j= 0 S -20 £ -40 o -60 -80 -100 -120 W r ^^fl^K' \y / y^ i\ B2 Diets B3 □ GG B SG □ FG Figure I. Total flesh growth (FG), somatic growth (SG), and gonadal growth (GG) during the experimental period, (a) Experiment I (46 days): diets Al (0.24% ), A2 (0.48% ), and A3 (0.96% ). (b) Experiment 2 (70 days): diets Bl (0.025%), B2 (0.50%), and B3 (0.10%). ment 2. Gonadal development in diets Bl and B2 occurred at the expense of previously stored reserves, and cannot therefore be quantified by the same method. GOI GOI increased throughout the experimental period in both males and females for all diets. Although there was clear evidence of gonadal development in all cases, there were obvious differ- ences, attributable to the different rations. Statistical comparisons were based only on data from samples taken up to days 26 (ex- periment 1) and 41 (experiment 2). Partial spawning observed in the experimental tanks after these dates would have affected the interpretation of the data corresponding to later samples. Experiment I Females The two highest rations in experiment 1 (A3 and A2) both produced a rapid increase in the GOI to approximately 35'7r by day 12, after which it remained constant (Fig. 2a). The rate of increase for ration Al was slower, and although maximum GOI was simi- lar to those for diets A2 and A3 (Fig. 2al this did not occur until day 35. GOI was related to time by means of a potential equation (Table 1). A comparison of the slopes of these equations after applying logarithmic transformation reveals statistically signifi- cant differences between the lowest ration (Al ) and the two high- Females 50 - 40 . 5 30 - I i o o 20 10 0 b Males r 10 20 30 40 50 Conditioning period (days) o A3 ^ A2 A Al O X 15 10 20 30 40 50 Conditioning period (days) A3 A2 Al Figure 2. Gonadal occupation index (GOI) during experiment 1 with diets Al (0.24% l, A2 (0.48% ), and A3 (0.96% ). (al Females, (b) Males. Average data (± SD). est (A2 and A3). No significant differences were observed between the latter two rations (P > 0.05). The amount of food available did not lead to any significant difference in the maximum GOI for any of these diets (ANOVA. P > 0.05). Males The GOI was much higher in males than in females. Maximum values of between 60 and 75% were obtained, according to the amount of food available. This factor, together with the energy balance, has a more noticeable effect on variations in the male GOI: there is a constant increase throughout the experimental pe- riod, with the highest diets showing the greatest rate of increase (Fig. 2b). There was a marked decrease in the GOI of clams fed on ration A3 after day 26, once maximum GOI (75%) had been reached. This coincided with the partial spawning observed in the experimental tanks. The best fit between GOI and time (Fig. 2b) is given by a linear equation (v = a + bx). Comparison of pairs of regression lines (Table 1) shows significant differences between the slopes of these equations {P < 0.05). The ANOVA performed between the maxi- mum values of the GOI for each ration shows significant differ- ences (P < 0.05) between the lowest diet (A I) and the two highest (A2 and A3). No statistically significant differences were observed between the latter two rations. 438 Delgado and Perez Camacho TABLE 1. Parameters of the regression lines between the gonadal occupation index (%, y) and time (days, x). Diets Comparison of Slopes Females Males Females Males Al (1) 17. IS 0.57 0.74 0.0040 12 A1-A2 0.0100 A2(l) 18.65 0.65 0.75 0.0001 13 A1-A3 NS A3(1) 18.51 0.59 0.69 0.0005 13 A2-A3 0.0200 Al (2) 33.61 6.64 0.63 0.0036 11 A1-A2 0.0040 A2 (2) 33.10 11.15 0.70 0.0007 12 A1-A3 0.0020 A3 (2) 27.87 15.55 0.88 0.0000 11 A2-A3 0.1040 Bl (3) 6.45 18.63 0.85 0.0001 10 B1-B2 0.0010 B2(3) 6.78 26.26 0.91 0.0000 13 B1-B3 0.0001 B3(3) 5.69 31.59 0.97 0.0000 9 B2-B3 0.0500 Bl (1) 8.41 14.32 0.78 0.0001 13 B1-B2 NS B2(!) 9.38 13.25 0.80 0.0004 10 B1-B3 NS B3(l) 13.24 13.35 0.66 0.0001 16 B2-B3 NS (1) Potential model (y '"); (2) Linear model (y = a + bx); (3) Logarithmic model (y bliix). NS. not significant; /;. number of observations. Experiment 2 Females Ma.ximum GOI (39.81'7c) was reached after 41 days (Fig. 3a) for those clams fed on a ration that produced a positive energy balance (B3). Clams fed on ration B2. the maintenance ration, did not reach the same GOI until the end of the experimental period (day 71). Diet Bl, with a clearly negative energy balance, gave both a slower rate of increase in the GOI and a lower maximum value (35.5%). The differences in maximum GOI values between rations were not, however, statistically significant (ANOVA, P > 0.05). Although maximum gonadal occupation is similar for all the diets in this experiment, the rate of gonadal development is deter- mined by the amount of food available, and a comparison of the GOI time regression slopes (Table 2) shows statistically significant differences (ANCOVA, P < 0.05). Males Variations in the GOI of males in experiment 2 were similar for all rations, with maximum values of around 60% (Fig. 3b). Com- parisons between the slopes of pairs of regression lines (Table 2) show no significant differences between any of them (ANOVA. P > 0.05), and neither were there any significant differences be- tween the maximum values obtained for each ration (ANOVA, P > 0.05). Percentage of Ripe Oocytes Experiment 1 The percentage of ripe oocytes (i.e.. with diameters of over 50 (xm) in the clams in experiment I increased rapidly during the first two weeks of the experimental period to approximately 25%. This rate of increase then diminished, and by day 26 average values of 26.3, 32.3, and 34.8% were recorded for rations Al, A2. and A3, respectively. After this date partial spawning was observed in the tanks containing clams fed on the two highest rations (A2 and A3), this being reflected in a decrease in the percentage of ripe oocytes, followed by a subsequent recovery (Fig. 4a). Although the maximum percentage of ripe oocytes is similar for all three rations at close to 40% (ANOVA, P > 0.05), the rate of increase of this percentage is directly related to the amount of food available, and the increase of the slopes of the regression lines between the percentage of ripe oocytes and time coincides with an increase in food (Table 2). The corresponding ANCOVA shows significant differences between the slopes of rations Al and A3, at a 95% confidence level. No statisticallv significant differences a Females O o 20 40 60 Conditioning period (days) X B3 ^. 82 ^ Bl 20 40 60 Conditioning period (days) 80 B3 82 Bl Figure 3. Evolution of the gonadal occupation index (GOI) during experiment 2 with diets Bl (0.025% ), B2 (0.05% I, and B3 (0.10% ). (a) Females, (b) Males, .\verage data (±D). Gonadal Development in R. decussaws 439 TABLE 2. Parameters of the regression lines between the proportion of ripe oocytes {%, v) and time (days. \). Diets a b r P /I Al 9.92 10.29 0.71 0.0003 A2 10.39 12.96 0.70 0.0004 A3 8.72 13.6 0.76 0.0000 Bl -7.41 7.47 0.73 0.0320 B2 -11.66 12.87 0.87 0.0000 B3 -11.33 12.74 0.92 0.0001 9 Linear model (y = a + b.x). P. probability level; n. number of observations. periment 1 (Fig. 4b). The lower percentages recorded at this point for clams fed with ration B3 coincide with partial spawning ob- served in the experimental tanks. As in experiment 1, the increase in the percentage of ripe oocytes was directly related to the amount of food available, al- though the differences between maximum percentages of ripe oo- cytes for each ration were not statistically significant (ANOVA. P > 0.05). Accordingly, comparison of the slopes of the regression lines between the percentage of ripe oocytes and time (Table 2) shows statistically significant differences (ANCOVA. P < 0.05) between the lowest ration (Bl: 0.()25'7f ) and the two highest (82: 0.057f. B3: 0.10%). No statistically significant differences were observed between the latter two rations. were observed between the slopes of rations A2 and A3, or Al and A2. Experiment 2 If the amount of available food is lower, as in experiment 2. where daily rations of 0.025. 0.050. and 0.10% were used (rations B 1 . B2. and B3. respectively), the percentage of ripe oocytes in the gonad increases at a lower rate than for the higher diets in experi- ment 1 (daily rations of between 0.26% and 0.96%). Under these conditions, after 25 days the average percentages of oocytes with a diameter greater than 50 ixni were 12.8. 14.6. and 23.5%. for rations Bl. B2. and B3. respectively, and 70 days were needed to reach values similar to those of the third sample (day 25) in ex- a 60 50 fc 40 i 30 a 20^ 10 0 .^ b 60 50 1 -i- ,_, 40 ??; 1 4> 30 ^ 20 K in cd 0 J, ml 12 26 35 46 Conditioning period (days) BA3aA2oAl [t -10 1 25 41 70 Conditioning period (days) a B3 B 82 Q Bl Figure 4. Percentage of ripe oocytes during the experimental period. (ai Experiment 1: diets Al (0.24% ), A2 (0.48% ). and A3 (0.96% ). (bl Experiment 2: Bl (0.025%), B2 (0.05%), and B3 (0.10%). Average data (±SD). DISCUSSION Image analysis has been used by several authors to compare the reproductive cycles of different species, using methods based on the frequency of different sizes of oocyte or the proportion of gonadal tissue occupied by oocytes. For example. Laruelle et al. (1994) and Xie and Bumell (1994) detected differences in the extent and intensity of reproductive activity in species, such as R. decussatus and Ruditapes philippinarum (Adams and Reeve). The parameters used in the present study, i.e.. the percentage of ripe oocytes and the gonadal occupation index, would also seem to be good indicators of the degree of gonadal maturity in R. decussatus. although they give no indication of the total amount of gonadal tissue. Navarro et al. (1989) associated interannual differences in the reproductive cycle of Ceiastodenna edute (L.) with fluctuations in the nutrient storage cycle caused by variations in food availability. The amount of food available in the environment is a determining factor of the amount of energy incorporated by the animal, and must therefore affect processes such as somatic and reproductive growth, as our experiments clearly show. Accordingly, when the daily amount of available food (ex- pressed as a percentage of clam live weight) is equal or greater than 0.10%. as in rations 83. Al. A2. and A3, a positive energy balance ensues. In these situations, there is a corresponding in- crease in the amount of clam soft tissue, which under the tempera- ture conditions prevailing in our experiments, corresponds princi- pally to an increase in reproductive tissue. Ration B2 produces a zero energy balance, in which energy acquisition and expenditure by the organism were equal. When there is a negative balance, as in the case of ration B 1 . the energy obtained from food is insufficient to meet the energy demands of the organism, resulting in a considerable loss of body weight. Gonadal development took place in both situations, possibly as a result of the high temperature at which the experiments were per- fonned. but in this case at the expense of previously stored re- serves. Our results show that the amount of available food influences both the extent of gonadal development and the rate of gonadal maturation, with the higher rations producing a faster rate. Simi- lariy, Buchanan et al. (1998) detected differences in the gameto- genic development and the conditioning index of Crassostrea vir- ginica (Gmelin). which he associated with nutritional and tempera- ture differences between laboratory conditions and the natural medium that produce a faster rate of gonadal development in specimens conditioned at a higher temperature and optimal nutri- tional conditions. 440 Delgado and Perez Camacho There are noticeable differences in the GOI of males and fe- males, with maximum values ranging from 55-75% for the former and 35 and 40% for the latter (Figs. 2 and 3). Spontaneous release of gametes can occur when these values are reached. A similar phenomenon is observed regarding the proportion of ripe oocytes. with spawning taking place when percentages reach between 30 and 40% (Fig. 4). These are not total spawnings because the variations in DW. GOI, and the percentage of ripe oocytes are only moderate, and in the case of the last-mentioned parameter they are followed by a rapid recovery. In this respect our results coincide with the period of continued spawning described by Laruelle et al. (1994) and Rodri'guez-Moscoso (2000) for R. decussatus, characterized by partial but continued release of gametes once a certain level of gonadal occupation has been reached. This reproductive strategy regulates the continued and progressive process of follicular oc- cupation, which does not appear to be compensated by an adequate degree of reabsorption of gametes in this venerid. The spawning period starts earlier under favorable nutritional conditions, since the first partial spawnings correspond to the diets with the greatest abundance of food. These partial discharges of gametes may. on the other hand, be responsible for the reduced synchronization between specimens, and for the high degree of variation in the data from the final stages of the experiment. Toba et al. (1993) also describe a greater synchronization between specimens in the early stages of gonadal maturation in R. philippinaniin in Tokyo Bay. which decreases considerably in the later stages of maturity. Bayne (1975), however, in contrast with the findings of our study, discovered a certain increase in the rate of gametogenic development in Mytihts ediilis (L.) under conditions of nutritional stress during the initial stages of gametogenesis, although in this species this process is completed by the reabsorption of gametes. In a later study on the effects of thennal and nutritional stress on the eggs of M. edulis, Bayne et al. ( 1978) establish a relationship between decreases in the volumetric fraction of gametes and spawning periods when temperatures are high and food abundant. When food is scarce, these decreases correspond to reabsorption processes or a low level of gametogenesis. In our case, and has already been mentioned, decreases in GOI for the higher diets are associated with spontaneous spawnings, but we have seen no sig- nificant decreases associated with nutritional deficiency in either zero or negative energy balance situations. Based on the relationship between gonadal development and the accumulation and use of nutrients, species can be classified as being either conservative or opportunist (Bayne. 1976). In the former category, gametogenesis takes place at the expense of pre- viously accumulated reserves (Zandee et al. 1980, Bayne et al. 1982). In the latter, gametogenesis occurs when there is an abun- dance of food in the environment, and sexual maturation parallels the accumulation of nutrients. Our results show that the behavior of R. decussatus varies according to the amount of food available. When there is an abun- dance of food it adopts an opportunist behavior, developing the gonad at the expense of ingested food, but when food is scarce it behaves like a conservative species, with gametogenesis taking place at the expense of accumulated reserves. ACKNOWLEDGMENTS We are grateful to P. Espineira. G. Rico. H. Regueiro. C. Pena, and P. Mallo for their technical assistance. This study was financed by the project PGIDT - 99MAR60401. M. Delgado was supported by a research personnel training grant from the European Social Fund - Spanish Oceanographic Institute (1998-19991 and by a grant from the Consello Regulador do Me.xillon de Galicia (Board of Control of the Galician Mussel) (2000-2001 ) while working on this study. LITERATURE CITED Bancroft, J. D. & A. Stevens. 1996. Theory and practice of histological techniques. New York: Churchill Livingstone, pp. 765. Bayne, B. L. 1975. Reproduction in bivalve molluscs under environmental stress. In: Physiological ecology of estuarine organisms. Columbia. SC: University of South Carolina Press, pp. 259-277. Bayne, B. L. 1976. Aspects of reproduction in bivalve molluscs. In: M. Wiley, editor. Estuarine processes. Uses, stresses and adaptation to the estuary. New York: Academic Press, pp. 432-448. Bayne. B. L.. P. A. Gabbott & J. Widdows. 1975. Some effects of stress in the adult on the eggs and larvae o{ Myiiliis nlulis (L.). J. Mar. Biol, /t.s.s. U.K. 55:675-689. Bayne. B. L.. D. L. Holland, M. N. Moore. D. M. Lowe & J. Widdows. 1978. Further studies on the effects of stress in the adult on the eggs of Myliliis edulis. J. Exp. Mar. Biol. Ass. U.K. 58:825-841. ^ayne, B. L.. A. Bubel. P. C. Gabbott. D. R. Livingstone, D. M. Lowe & M. N. Moore. 1982. Glycogen utilisation and gametogenesis in Mytilus edulis. Mar. Biol. Utt. 3:89-105. Beninger. P. G. 1982. Etude biochemique comparee de deux populations de bivalves. PhD Thesis. University of Bretagne Occidentale, pp. 193. Berthou, P.. Y. Le Gall, P. Djabali & M. Yahiaoui. 1980. Biologie et peche de la praire Veinis verrucosa (mollusque veneridae) en Manche occi- dentale (Bretagne et Normandie): Reproduction et fecondite. Commu- nication CIEM, C. M., pp. 18. Buchanan. J. T.. G. S. Roppolo. J. E. Supan & T. R. Tiersch. 1998. Conditioning of eastern oysters in a closed, recirculating system. J. Shellfish Res. 17:1183-1189. Gimazane. J. P. 1972. Etude experimentale de faction de quelques facteurs extemes sur la reprise de I'activite genitale de la coque. Cerastoderma edule (L.). Mollusque Bivalve. C. R. Soc. Biol 166:587-589. Holland. D. A. & K. K. Chew. 1974. Reproductive cycle of the Manila clam ( Venerupis japonica) from Hood Canal. Washington. In: Proceed- ings of the National Shelltlsheries Association. 64:53-58. Laruelle. F.. J. Guillou & Y. M. Paulet. 1994. Reproductive pattern of the clams Ruditapes decussatus and Ruditapes pinlippinarum on intertidal flats in Bnttany. / Mar Biol. Ass. U.K. 172:69-96. Lowe. D. M.. M. M. Moore & B. L. Bayne. 1982. Aspects of gametoge- nesis in the marine mussel Mytilus edulis L. / Mar. Biol. Ass. U.K. 62:133-145. Lubet, P. 1980-1981. Influence des facteurs extemes sur la reproduction des lamellibranches. Oceanis 6:469^89. Mann. R. 1979. The effect of temperature on growth, physiology, and gametogenesis in the Manila clam Tapes philippinarum (Adams and Reeve. 1850). / £.v/j. Mar. Biol. Ecol. 38:121-133. Navarro, E.. J. I. P. Iglesias & A. Larranaga. 1989. Interannual variation in the reproductive cycle and biochemical composition of the cockle Cerastodenna edule from Mundaca estuary (Biscay. North Spain). Mar Biol 101:50.^511. Perez Camacho, A. 1979. Biologia de Venerupis pullastra (Montagu. 1983) and Venerupis decussata (Linne, 1767) (Mollusca. Bivalvia). con es- pecial referenda a los factores determinantes de la produccion. PhD Thesis. University of Santiago de Compostela. pp. 217. Perez Camacho, A. 1980. Biologia de Venerupis pullastra (Montagu, 1803) and Venerupis decussata (Linne, 1767) (Mollusca: Bivalvia) con Gonadal Development in R. decussatus 441 especial referenda a factores determinanles de la produccion. Bol. In.sl. Esp. Oceunogr 281:353-358. Pipe. R. K. 1985. Sea.sonal cycles and effects of slarvalion on egg devel- opment in Myriliis ediilis. Mar. Ecol. Prog. Ser 24:121-128. Rodriguez-Moscoso. E. 2000. Histofisiologi'a de la reproduccion de la almeja fina Ruditupes decussatus (Linne, 1758) en la n'a de Arosa (Poblacion natural y poblacion de cultivo). PhD Thesis. University of Santiago de Compostela. pp. 202. Rodriguez- Moscoso. E. & Arnaiz. R. 1998. Gameiogenesis and en- ergy storage in a population of the grooved carpet-shell clam. Tapes decussaius (Linne. 1787). in northwest Spain. Aquaculiure 162:125- 139. Sastry. A. N. 1966. Temperature effects in reproduction of the bay scallop Aequipecten irradians Lamarck. Biological Bulletin. Marine Biological Laboratory. Woods Hole. Mass. 130:118-134. Sastry. A. N. 1975. Physiology and ecology of reproduction in marine invertebrates. In: F. J. Vemberg. editor. Physiological ecology of es- tuarine organisms. Columbia. SC: University of South Carolina Press. pp. 279-299 Shaffee. M. S. & M. Daouidi. 1993. Gametogenesis and spawning in the carpet-shell clam. RudiUipes decussatus (L.) (Mollusca: Bivalvia) from the Atlantic coast of Morocco. Aquaculture Fisheries Management 22:203-216. Snedecor. G. W. & W. G. Cochran. 1980. Metodos estadisticos. Ci'a Ed. Continental. Buenos Aires. Toba. M.. Y. Natsume & H. Yamakawa. 1993. Reproductive cycles of Manila clam collected from Funabashi Waters. Tol,„ Cp and Lp) and ciwip,, {P^. C,, and Lp) are the contents of each component in the feces (F) and the diet (D), respectively. The absorption rates of the different biochemical components were obtained from the product of the ingestion rate of the biochemical component in question and its absoi-ption effi- ciency. Component absorption rates were transformed to energetic units using the following energy equivalents: 18.0 Kj (g protein)"'. 17.2 Kj (g carbohydrate)"', and 35.2 Kj (g lipid)"' (Beukema & de Bruin 1979). Statistical Analysis The differences observed in the different physiological param- eters between the experimental diets used and between the two species studied in this experiment were submitted to statistical analysis of variance ( ANOVA, P < 0.05; Zar 1 984). Angular trans- formation (arc sin V(AE/100)) was used to transform the results for absorption efficiency in order to guarantee standardisation of the data. The Bartlett test was used to check homogeneity of the vari- ances. In the case of non-homogenous variances, logarithmic or reciprocal transformation was used to transform the data, after which their homogeneity was once again checked. RESULTS Characteristics of the Diets Table 1 shows the characteristics of the diets used. The main components of the organic fraction in the natural diets were pro- teins and lipids, each accounting for approximately 40*. whereas the proportion of carbohydrates is much lower at 17.1%. In the carbohydrate-rich diet, however, the relative percentages of pro- teins and lipids are much lower, with values of 7.0 and 13.3%, respectively, the main component being carbohydrates, which ac- count for 79.7%. Both diets were assayed at concentrations similar to those ob- served in their natural environment (Navarro et al. 1991, Babarro et al. 2000). The ratio of the concentration of organic matter to total particulate matter was 0.73 for the natural diet and 0.82 for the carbohydrate-rich diet. Food concentrations, expressed as en- ergy equivalents, were similar for both diets, being 14.1 and 15.1 J L"' for the natural and carbohydrate-rich diets, respectively. Physiological Parameters Average clearance rates (CR). organic ingestion rates (//?„), organic absorption efficiencies {AEj and organic absorption rates (ARj together with their standard deviations for a specimen of I g flesh dry weight for each species of clam and for both diets are shown in Table 2. Organic ingestion rates of natural diet were significantly higher in V. piillastra than in R. clecussalits {P < 0.05, ANOVA test). When clams were fed on the carbohydrate-rich diet organic ingestion rates were significantly higher than those regis- tered for the natural diet. This increase in the ingestion rate was much more noticeable in R. clecussalus than in V. pullastni. thus leading to higher rates in R. ilecussatiis The absorption efficiencies of total organic material were simi- lar for both species fed on the natural diet (ANOVA; P > 0.05), with a value of close to 70%. However, when the clams were fed on the carbohydrate-rich diet, absorption efficiencies decreases in both species at around 37%. Thus, the increase in the proportion of carbohydrates in the diet leads to an increase in the ingestion rate, and this in turn supposes a decrease in the efficiency with which the ingested food is absorbed. The relation between the ingestion rate and the absorption efficiency is given by a model that fits the equation ,4£= a*L/?^ in which a = 6.37 (±0.575) and b = -0.404 (±0.095) (r = -0.9493, R- = 90.13%. P = 0.0507). The organic absorption rate (/1R„) behaves in a similar manner to the //?^, in natural-diet fed clams: the AR^^ was significantly higher (ANOVA. P < 0.05) in V. piillastra than in R. deciixsaliis. When the carbohydrate diet was used, organic absorption rate was three times greater than that for the natural diet in the case of R. deciissaliis. but only 50% higher in comparison with the natural diet in the case of V. piillastra. Absorption of Biochemical Components The difference in biochemical composition between the two diets determines the ingestion rates of each biochemical compo- nents of the diet. In V. piillastra, although the total ingestion rate of the carbohydrate-rich diet is three times greater than that of the natural diet, the quantity of protein ingested in the former is only half that in the latter (Table 2). The value of lipids ingested is similar in both diets in this species, whereas the quantity of car- bohydrates ingested is much greater in the carbohydrate-rich diet. In the case of R. deciissalns. however, the protein ingestion rate is the same for both diets whereas lipid ingestion doubles with the carbohydrate-rich diet, in which the quantity of carbohydrates in- gested increases considerably. Although total organic absorption efficiency is the same for both species when fed on the same diet, the efficiency with which proteins are absorbed by V. piillastra on the carbohydrate-rich diet is less than that of R. deciissalns. and this, together with the smaller amount of proteins ingested by V. piillastra, as described above. 446 Albentosa et al. o ■i £ 2 ■a V Si 13 U ■O u 4rf u 1- O' 3 1- C S o o T= " (S 4> ^ U "- 9 .J S T B3 < J2 t- ■■j ,u Q = ■S S. "5- Ij e , •*- a a c ■a. « .^ ^ la !>. Si ■*" ^ «tf s .a ■o •= c .s a u M 'C 5 i a -o vj >-■ ^ .= ^3 O « -P 3 h t" ■s s ss a < J ri — -T U-, X •T < +1 ri +1 +1 ^ +1 ^ Vi ^ vi -r r- ^ U-, u-j — r'-, O +1 *^ d +1 1^^ oo O «~. W~i ^ OC ^D •/". L 5 — U-) ^ O) u g +1 +1 "5 +1 •< -g ■^ - £■- ri *"' ON C +1 ON -^ "^^ w*-, ^ ON -:; +1 q +1 00 ,C' O 00 oo — ^ >c — Ov ri — ' ^ "~ r-' ON ■3 rr-, ^ ri I/-, — o — ^ +1 u-i « S o r^ 0\ w r*"! ri rt oo -_ r- O^ ^ r^; _ n O "" ■? +1 W-; +1 f~- OS ri d ri O U", ri ON ^C ■Ov ^ r^ o - -f +1 *f, +1 ^ ^: u-i r- n "^ rj d ^ ■H o X < 9- Si ^ -- a ^ -^ 3 •- OS S 4J ^ ~ < H "o gives us a protein absorption rate whicin is 60% lower than that observed for the natural diet. Lipid absorption efficiencies are simihir between the two species, and between diets for each spe- cies, at around 56%. thus producing the same difference in lipid absorption rates as have previously been described for ingestion rates, i.e., R. deciissarus doubles its lipid absorption rate, whereas that of V. piiUasira remains the same when the diet changes. Car- bohydrate absorption efficiencies decrease for both species with the carbohydrate-rich diet, but because carbohydrate ingestion rates are very high on this diet, the absorption rates for this com- ponent are nevertheless much higher than those observed for the natural diet. Whereas the highest absorption rate was given by proteins in both species when fed on the natural diet, the main component absorbed were carbohydrates when clams were fed on the carbo- hydrate-rich diet, due to the fact that they comprise the highest quantity of the organic matter ingested. In R. deciissuiiis. protein absorption rates maintain similar values (53.5 ixg ind~' h~ ) to those described for the natural diet (33.8 (jig ind"' h"'). in spite of the noticeable decrease of the protein content of the diet. V. pul- liisini. however, is unable to maintain the same level of protein absorption as with the natural diet (84.5 |xg ind"' h"' ). dropping to 35.3 p.g ind^' h"'. a lower rate than any of those obtained for R. deciissatiis on either of the diets. The increase in total ingestion for both species fed on the carbohydrate-rich diet leads to a consid- erable increase in the carbohydrate absorption rate when compared to that obtained with a natural diet, although this increase is much greater in R. Jecii.ssaius. with an AR^ of 244.4 as opposed to 178.2 |jig of carbohydrates absorbed in V. pullastra. With regard to lipid absorption, this is maintained at a similar level to the natural diet (A/?, . 66.4 (xg ind^' h"') by V. pullastra when fed on a carbohy- drate-rich diet (A/?L. 55.4 ^g ind"' h"': ANOVA. P = 0.0838) whereas in the case oi R. decussatus it doubles (75.0 p.g ind"' h~' ) its value with respect to the figure obtained for the natural diet (40.2 (jLg ind"' h"'). Absorption Levels Expressed in Units of Energy Figure la and b shows the absorption rates of the biochemical components of the diet e.xpressed in their energy equivalents. In both species the greatest amount of energy absorbed comes from lipids, when they are fed on a natural diet (54% of total energy absorbed), but from carbohydrates when they are fed on a carbo- hydrate-rich diet. On a natural diet, the energy absorbed by V. pullastra is 70% higher than that absorbed by R. decussatus, but on a carbohydrate-rich diet R. decussatus absorbs 40% more energy than V. pullastra. When fed on a carbohydrate-rich diet. R. decus- satus absorbs three times the energy than it does when fed on a natural diet and is thus able to maintain protein absorption, in energy terms, at similar levels for both types of diet. However, V. pullastra reduces the contribution of energy supplied from proteins to the total of absorbed energy by 50% when fed on a carbohy- drate-rich diet in comparison with a natural diet. The energy ab- sorbed from carbohydrates when both species were fed on a car- bohydrate-rich diet was greater in R. decussatus than in V. pullas- tra. In the case of lipids, the amount of energy deriving from this component in R. decussatus when fed on a carbohydrate-rich diet is almost double that obtained on a natural diet, whereas in V. pullastra this figure decreases. Absorption of Diet Biochemical Components in Clams 447 Natural Diet □ Proteins H Carbohydrates □ Lipids D Total R decussatus V- pullastra Carbohydrates-rich Diet □ Proteins H Carbohydrates □ Lipids □ Total I B R. decussatus V. pullastra Figure 1. Absorption rates (ARl of biochemical components, ex- pressed in energy equivalents, in two clam species, Ruditapes decussa- tus and Venerupis pullastra when fed on a natural diet (a) and a car- bohvdrate-rich diet (b). DISCUSSION Physiological Parameters The most important difference observed between the feeding physiology of the two species of clam when fed on a natural diet are caused by the ingestion rates. According to the results of our study, organic ingestion rates in V. pullastra are SOVr higher than in R. decussatus (Table 2; V. pullastra/R. decussatus index, Vp/Rd = 1 .50), which when taken together with the slightly higher food absorption efficiency in V. pullastra gives a total organic absorp- tion rate for this species that is almost 60% higher than that of R. decussatus {Vp/Rd = 1.59). This difference in energy absorbed is in consonance with the findings of other authors (Perez-Camacho 1980, Beiras et al. 1993, Albentosa et al. 1996. Laing et al. 1987, Laing & Child 1996). who in their studies note that both growth and food consumption rates in R. decussatus are lower than those observed in other venerids such as V. pullastra or Ruditapes phil- ippinarum. When the clams are fed on a carbohydrate-rich diet, important differences can also be observed in the feeding behavior of the two species, although of an opposite nature to those described for the natural diet. In these circumstances, the ingestion rate for R. de- cussatus when fed on a carbohydrate-rich diet is higher than that observed for V. pullastra. giving us in this case an index of Vp/Rd = 0.74. An increase in ingestion is observed in both species when fed on the carbohydrate-rich diet, this increase being of the order of 6 and 3 times greater in R. decussatus and V. pullastra. respec- tively. It is therefore true to say that the effect of the diet is the same in both species, i.e., an increase in ingestion when compared with the natural diet, although quantitatively much greater in the case of R. decussatus. If we bear in mind that the food concentra- tion, expressed as total particulate matter, is only 1 .4 times higher in the carbohydrate-rich diet than in the natural diet, these quan- titative differences would not account for the increase in ingestion observed. Furthermore, when expressed in terms of energy, the food content of both diets was similar (Table 1 ). Navarro et al. (2000) describe a feeding behavior similar to the one observed in our study, in Argopecien purpuratus. These authors describe an increase in ingestion of up to 6 times, just as is the case with R. decussatus in our study, when the microalgal diet is supplemented with carbohydrates obtained from potato starch. They also note a similar behavior when the diet is supplemented with lipids, but this time the ingestion rate increases by a factor of 8 in comparison with that obtained on a pure microalgal diet. They suggest the existence of chemical receptors on the gills or labial palps that are capable of detecting specific nutritional components of the diet and which would stimulate an increase in the clearance rate and hence the ingestion rate. The com flour starch used in the present study consists of particles of a much greater density than the microalgal cells, or if expressed in terms of unit volume, the organic content of com flour starch particles are some 4 times greater than that of microalgae cells (unpublished data). If we consider that bivalves are continuous filter-feeders, i.e.. their digestive system is continu- ously occupied by food, then we can assume that the digestive capacity of both species, expressed in terms of the amount of organic matter that can be contained inside the digestive tract, must be much greater when the clams are fed on a carbohydrate-rich diet than when fed on a natural diet, because of the above-mentioned difference in particle density between the two diets. Given the great similarity of food concentration at which both diets were assayed (0.6-0.8 mg POM L"'), the total occupation for an equal volume of the digestive system would be obtained by the existence of higher clearance rates for the carbohydrate-rich diet, which would account for the differences found between the ingestion rates for the two diets. Total organic matter absorption efficiency is reduced by half in both species when they are fed on a carbohydrate-rich diet, owing to the considerable increase in the ingestion rate. The relation between food ingestion rate and absorption efficiency has been much studied in bivalves (Foster-Smith 1975, Navarro & Winter 1982, Bayne & Newell 1983, Beiras et al. 1993, Albentosa et al. 1996. Ibarrola et al. 1998) with a similar behavior being described in all instances, i.e.. a decrease in absorption efficiency as the ingestion rate increases, principally because of the reduced transit time through the digestive tract and hence the reduced length of time during which food is exposed to the digestive enzymes. Na- varro et al. (2000) also describe a decrease in absorption efficiency when the microalgal diet is supplemented with either carbohy- drates or lipids, although to a lesser extent than that observed in the present study. Total organic matter absorption efficiency within each diet was the same for both species, so the differences observed between species in absorption rates ( Vp/Rd = 1 .59 for the natural diet and Vp/Rd = 0.72 for the carbohydrate-rich diet) reflect the differ- ences observed in the ingestion rate. Although there is a consid- erable decrease in the efficiency with which ingested food is ab- sorbed when the clams are fed on a carbohydrate-rich diet, the total organic matter absorption rates are higher, even more so in the case of/?, decussatus (Vp//?d = 0.72). The absorption of total organic matter was three times higher in the carbohydrate-rich diet than in 448 Albentosa et al. the natural diet for R. decussaius. whereas in the case of V. pul- lastra this increase was only 1.5 times greater. Absorption of Biochemical Components There are few references in the literature to the process of absorption of the various biochemical components of the diet in bivalves (Kreeger & Langdon 1994, Ibarrola et al 1996. 1998). particularly when the biochemical composition of the diets differs as much as it does in the present study. Ibarrola et al. (1998). in studies of specimens of Cenistoderma editle fed on diets consist- ing of microalgae and sediment in varying proportions (some of which are comparable with the natural diet assayed in our study), show that the most efficiently absorbed biochemical component in high quality diets (i.e.. diets with the highest proportion of organic matter) are carbohydrates, whereas in low quality diets lipids are the most efficiently absorbed component. The authors attribute this high rate of carbohydrate absorption in high quality diets to an increase in the activity of certain carbohydrases to be found in the digestive gland. Protein absorption efficiency, however, remains unaffected by the quality of the diet. In our study, on the other hand, the biochemical component that is most efficiently absorbed by both species is protein, regardless of diet. This discrepancy may be due to interspecies differences between enzyme production in the digestive systems of cockles and clams, or also to the different biochemical composition of the microalgae used in the two studies. The high protein content (63.9*7^) of the microalgal portion of the diets assayed by Ibarrola et al. (1998) when compared to the pro- tein content of the two diets used in the present study (40.5% for the natural diet and 7.0% for the carbohydrate-rich diet) may well account for the differences in protein absorption efficiency regis- tered between the two studies. The effect of diet on the feeding behavior of the two species in our study, i.e.. the increase in ingestion and the resulting increa.se in absorption, allows R. decussaius to compensate for the nutri- tional deficiencies of the carbohydrate-rich diet, whereas V. pul- lastni seems unable to compensate fully for these deficiencies because it does not maintain the same level of protein absorption as observed in the natural diet. This latter level of absorption can be taken to be sufficient for this species, because it is a reflection of the conditions found in its natural habitat. If we consider that protein absorption is of fundamental importance for all organisms, because proteins are the source of necessary essential amino acids in the biosynthetic routes in the metabolism, this leads us to sup- pose that V. pullastra has a lesser capacity to respond to diets with a high carbohydrate content than does R. decussatus, which may be an indication of the existence of different metabolic routes in the two species. Studies that have been performed by our research group (re- viewed by Labarta et al. 1997) in connection with feeding behav- ior, the biochemical composition of body tissues, and the nutri- tional requirements of the two species of venerids studied in the present work suggest that lipid demand is higher in V. pullastra than in R. decussatus. whereas carbohydrate demand is higher in the latter than in the former, provided that there is sufficient pro- tein in the diet. This may be related to the mechanism by which each species adapts to its specific habitat: R. decussatus. which characteristically inhabits the inter-tidal zone and is subject to periods of emmersion as a result of the tidal cycle, would possess an anaerobic metabolism in which carbohydrates are a more ap- propriate source of energy than lipids. V. pullastra, on the other hand, a species that is permanently submerged because of its sub- tidal habitat, would not have these same nutritional requirements, which are more appropriate to an anaerobic metabolism, and would instead find lipids to be a more relevant source of energy, since they are the appropriate fuel for the aerobic routes of the metabolism. In our experiment both species were exposed to a completely unbalanced diet that contained a very high proportion of carbohydrates at the expense of protein and lipids. Both species responded in a similar manner, in qualitative terms, showing a considerable increase in ingestion which allowed them to counter the low protein content (proteins being an essential component of the diet) of the unbalanced diet. In quantitative terms, however, there are major differences between the two species: R. decussatus is able to maintain protein absorption levels, and even manages to double the quantity of lipids absorbed, whereas V. pullastra is unable to keep protein absorption at the same level, registering a 60% decrease, and is barely able to maintain lipid absorption. These results would appear to reinforce the previously mentioned hypothesis regarding metabolic differences between the two spe- cies. ACKNOWLEDGMENTS Funding for this research was provided by Comision Intermin- isterial de Ciencia y Tecnologia. Spain, project MAR99-0240- C02. The authors thank P. Espineira from the Centro Ocean- ografico de A Corufia (lEO) for her helpful technical assistance in the physiological measurements and L. Nieto and B. Gonzalez from the Instituto de Investigaciones Marinas (CSIC) for their technical assistance in the biochemical assistance. This study was conducted in accordance with the legal and ethical standards of the countries involved. LITERATURE CITED Albentosa. M.. A. Perez-Camacho &. R. Beiras. 1996. The effect of food concentration on the scope for growth and growtli performance of Ruditupes decussatus (L.) seed reared in an open-tlow system. Aqua- culture Nutr. 2:213-220. Babarro. J. M. F.. M. J. Femandez-Reiriz & U. Labarta. 2000. Growth of seed mussel (Mxtihis galloprorincialis LMK): effects of environmental parameters and seed origin. / Shellfish Res. 19:187-193. Bayne. B. L. & R. C. Newell. 1983. Physiological energetics of marine molluscs. In: A. S. M. Saleuddin & K. M. Wilbur, (editors) The mol- lusca. vol 4. New York: Academic Press, pp. 407-515. Bayne. B. L.. A. J. S. Hawkins, E. Navarro & J. 1. P. Iglesias. 1989. Effects of seston concentration on feeding, digestion and growtli in the mussel Mytilus edulis. Mar. Ecol. Prog. Scr. 55:47-54. Bayne. B. L.. J. I. P. Iglesias. A. J. S. Hawkins. E. Navarro. M. Heral & J. M. Deslous-Paoli. 1993. Feeding behavior of the mussel. Mytilus edulis: responses to variations in quantity and organic content of the seston. J. Mar. Biol .Ass. U.K. 73:813-829. Beiras, R.. A. Perez-Camacho & M. Albentosa. 1993. Influence of food concentration on energy balance and growth performance of Venerupis pullastra seed reared in an open-flow system. Aquacullure 116:353- 365. Beukema. J. J. & W. de Bruin. 1979. Calorific values of the soft parts of the tellinid bivalve Macotna halthica (L.) as determined by two meth- ods. J. Exp. Mar. Biol. Ecol. 37:19-30. Bligh. E. J. & W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911-917. Absorption of Diet Biochemical Components in Clams 449 Cranford, P. J. 1995. Relationships between food quantity and quality and absoiption efficiency in sea scallops Pkuopecten mafielUmicus. J. E.xp. Mar. Biol. Ecol. 189:123-142. Conover, R. J. 1966. Assimilation of organic matter by zooplankton. Liin- nol. Oceanogr. 11:338-345. Delgado, M. 2002. Maduracion sexual en Ruditapes decussatus (L.): Im- plicaciones energeticas y bioquimicas. Ph. D. Thesis. Universidad de Santiago de Compostela, Spain, pp. 307. Femandez-Reiriz. M. J., A. Perez-Camacho. M. J. Ferreiro. J. Blanco. M. Planas. M.J. Campos & U. Labarta. 1989. Biomass production and vanation in the biochemical profile (total protein, carbohydrates. RN.'\. lipids and fatty acids) of seven species of marine microalgae. .■\iiiui- cullure 83:17-37, Foster-Smith. R. L. 1975. The effect of concentration of suspension and inert material on the assimilation of algae by three bivalves. J. Mar. Biol Assoc. U.K. 55:411-418. Griffiths, C. L. & J. A. King. 1979. Some relationships between size, food availability and energy balance in the ribbed mussel Aulacomya ater. Mar Biol. 51:141-149. Hawkins, A.J. & B. L. Bayne. 1985. Seasonal variation in the relative utilization of carbon and nitrogen by the mussel Myiihis ediilis: bud- gets, conversion efficiencies and maintenance requirements. Mar. Ecol. Prog. Ser. 25:181-188. Ibarrola, I.. J. I. P, Iglesias & E. Navarro. 1996. Differential absorption of biochemical components in the diet of the cockle Cerastodenna editle: enzymatic responses to variations in seston compositions. Can. J. Zool. 74:1887-1897. Ibarrola, 1.. E. Navarro & J. I. P. Iglesias. 1998. Short-term adaptation of digestive processes in the cockle Cerastodenna ednlc e.xposed to dif- ferent food quantity and quality. J. Comp. Physiol. B 168:32^0. Iglesias. J. 1. P.. M. B. Urrutia. E. Navarro. P. Alvarez-Joma. X. Larretxea. S. Bougrier. & M. Heral. 1996. Variability of feeding processes in the cockle Cerastodenna edule (L.) min response to changes in seston concentration and composition. J. Exp. Mar. Biol. Ecol. 197:121-143. Iglesias. J. I. P.. M. B, Urrutia. E. Navarro & I. Ibarrola. 1998. Measuring feeding and absorption in suspension-feeding bivalves: and appraisal of the biodeposition method. / Exp. Mar. Biol. Ecol. 219:71-86. Kreeger. D. A. & C. J. Langdon. 1993. Effects of dietary protein coment on growth of juvenile mussels. Mytiliis trossiilus (Gould. 1850). Biol. Bull. 185:123-139. Kreeger. D. A. & C. J. Langdon. 1994. Digestion and assimilation of pro- tein by Mvtiliis trossiilus (Bivalvia: MoUusca) fed mixed carbohydrate/ protein microcapsules. Mar. Biol. 118:479^88. Labarta, U., M. J. Femandez-Reiriz, M. Albentosa & A. Perez-Camacho. 1997. Evaluacion de dietas, vivas e inertes. para el cultivo de juveniles de bivalvos. In: Costa et al. (editors), Actas del VI Congreso Nacional de Acuicultura. Madrid: Ministerio de Agricultura Pesca y Alimentacion. pp. 1-10. Laing, I„ S. D. Utting & R. W. S. Kilada. 1987. Interactive effect of diet and temperature on the growth of juvenile clams. J. Exp. Mar. Biol. Ecol. 113:2.V38. Laing. I. & A. R. Child. 1996. Comparative tolerance of small juvenile palourdes {Tapes decussatus L.) and Manila clams (Tapes pliilippi- nanim Adams & Reeve) to lov\ temperature. J. Exp. Mar. Biol. Ecol. 195:267-285. Langdon, C. J. 1989. Preparation and evaluation of protein microcapsules for a marine suspension-feeder, the Pacific oyster Crassostrea gigas. Mar. Biol. 102:217-224. Lowry. O. H.. N. J. Rosebroug & A. L. Fair. 1951. Protein measurement with the pholin-phenol reagent. J. Biol. Chem. 193:265-275. Marsh. J. B. & D. B. Weinstein. 1966. Simple charring method for deter- mination of lipids. J. Lipid Res. 7:574-576. Navarro. E. J. I. P. Iglesias. A. Perez-Camacho, U. Labarta & R. Beiras. 1991. The physiological energetics of mussels (Mytilus galloprovin- cialis Lmk.) from different cultivation rafts in the Ria de Arosa (Gali- cia. NW Spain). Aquaculture 94:197-212. Navarro. J. M. & J. E. Winter. 1982. Ingestion rate, assimilation efficiency and energy balance in Mytilus chilensis in relation to body size and different algal concentrations. Mar. Biol. 67:255-266. Navarro, J. M. & R.J. Thompson. 1996. Physiological energetics of the horse mussel Modiolus modiolus in a cold ocean environment. Mar. Ecol. Prog. Ser. 138:135-148. Navarro, J. M., G. E, Leiva. G. Martinez & C. Aguilera. 2000. Interactive effects of diet and temperature on the scope for growth of the scallop Argopecten purpitratus during reproductive conditioning. J. Exp. Mar. Biol. Ecol. 247:67-83. Perez-Camacho, A. 1980. Biologia de Venerupis pullasira (Montagu. 1803) y Venerupis decussata (Linne. 1767) (MoUusca. Bivalvia). con especial referencia a los factores determinantes de la produccion. Bo- letin del Institiito Espanol de Oceanografia V(4). Perez-Camacho. A., A. Villalba, R. Beiras & U. Labarta. 1997. Absorption efficiency and condition of cultured mussel (Mytilus edulis gallopro- vincialis Linnaeus) of Galicia (NW Spain) infected by parasites Mar- teilia intestinalis Steuer. / Shellfish Res. 16:77-82. Strickland. J. D. & T. R. Parsons. 1968. A practical handbook of sea water analysis. Bull. Fish. Res. Board. Can. 167:173-174. Thompson. R. J. & B. L. Bayne. 1972. Active metabolism associated with feeding in the mussel Mytilus edulis L. / Exp. Mar. Biol. Ecol. 9:1 1 1- 124. Urrutia. M. B.. J. I. P. Iglesias. E. Naviirro & J. Prou. 1996. Feeding and absorption in Cerastodenna edule under environmental conditions in the bay of Marennes-Oleron (Western France). / Mar. Biol. Assoc. U.K. 76:431^50. Widdows. J. 1978. Combined effects of body size, food concentration and season on the physiology of Mytilus edulis. J. Mar. Biol. Assoc. U.K. 58:109-124. Zar. J. H. 1984. Biostatistical analysis. Englewood Cliffs. NJ: Prentice- Hall. Jounuil of Shellfish Ri'.minh. Vol. 22. No. 2, 451-464. 2003. THE PERSISTENCE OF NEW JERSEY'S OYSTER SEEDBEDS IN THE PRESENCE OF OYSTER DISEASE AND HARVEST: THE ROLE OF MANAGEMENT STEPHEN R. FEGLEY,'* SUSAN E. FORD," JOHN N. KRAEUTER," AND HAROLD H. HASKIN- t Comi?ii; School of Ocean Studies. Maine Maritime Academy. Castine. Maine 04420; and ~Haskin Shellfish Research Laboratory. Rutgers University. 6959 Miller Avenue. Port Norrls. New Jersey 08349 ABSTRACT New Jersey'^ Delaware Bay oyster fishery developed along a pathway common to many fisheries. Perennially large harvests led to depletion of the oyster resource, which led to increasing, but ineffective, harvest restrictions and cumbersome nianagemeni. In the 1950s, two events altered the management structure. In the beginning of the decade, a university researcher dedicated himself to having oystermen and the state regulatory agency use information from research and monitoring programs directly in their decision making. He achieved limited success until a previously unknown oyster disease, eventually called MSX, occurred that threatened to drive the oyster fishery to extinction. The presence of MSX led oyster harvesters to become dependent on the information provided by the university. In addition, the regulatory agency and its regulations had to be responsive to shon-tenn changes in the intensity and prevalence of disease. A tripartite management structure developed in which: I ) the oystermen. researchers, and state regulatory agency acted cooperatively and 2l flexible guidelines were developed that could respond to annual variation in oyster abundance and disease. Several aspects of this management arrangement could prove useful in other fisheries. KEY WORDS: oyster, management, fishery INTRODUCTION Over the past decade, an increasing sense of urgency to develop effective, nontraditional approaches to fisheries management has developed. Too frequently, government-directed management has had problems sustaining fisheries resources at hai^estable levels while providing economic and social stability for the fishery par- ticipants (McGoodwin 1990, Hannesson 1996). Alternative man- agement models that have been suggested include adaptive man- agement (Waiters 1986), ecosystem management (Schramm & Hubert 1996), and responsible management (FAO 1995). All al- ternative management models suggested to date involve greater participation by fishery participants in the management decision processes, a management structure generally referred to as co- management. Many observers of and participants in fisheries are wary of including the principal users of the resource: they doubt that those who would gain immediate benefit from using a re- source would sacrifice current profit for future sustainability (Jentoft et al. 1998). In contrast, Jentoft et al. ( 1998) have argued that there are numerous social and institutional elements that allow a more positive expectation of the outcome of co-management models. Co-management has developed in some fisheries without a de- liberate effort to develop a nontraditional management program (Jentoft & McCay 1995). Contingent needs can lead all partici- pants in a fishery to search for an operating environment to solve certain problems. Such is the case with New Jersey's Delaware Bay oyster fishery. A detailed examination of the ontogeny and structure of this particular fishery provides several benefits. First. it allows those who are considering developing co-management programs to learn from the successes and failures of those who have already incorporated co-management. Co-management pro- grams are emerging. For example, in the state of Maine, co- management has been legislated recently for the lobster fishery *Corresponding author. E-mail: sfegley@iTima.edu tDeceased. (Acheson et al. 2000). Other fisheries in the region are expected to follow the same path. Second, the contingent need that had to be solved in New Jersey's Delaware oyster fishery was the presence of diseases that affected the resource. Apparently several popula- tions of marine species have an increasing incidence of disease- induced mortality (Harvell et al. 1999). Managing in the presence of disease may be a more common feature of fisheries in the future. Accordingly, we present the following case study. Historically low abundances of the eastern oyster, Crassostrea virginica. presently occur throughout much of the middle Atlantic US coast. Many factors have contributed to the decline of the large oyster populations that existed in Chesapeake and Delaware Bays, including management that failed to prevent overharvesting (Ha- ven et al. 1978, Kennedy & Breisch 1983). A major factor con- tributing to recent declines of midcoast oyster populations and frustrating restoration efforts is the presence of one or more oyster diseases. Disease-induced mortalities have been so intense that in some areas oysters are rare and local oyster fisheries have become extinct (Bosch & Shabman 1989). In Delaware Bay, the principal oyster disease organism for most of the past four decades has been the MSX parasite Hap- lospohdium nelsoni. Since 1990, a southern oyster parasite, Per- kinsiis marinus, which causes Dermo disease, has invaded the Bay becoming the principal disease agent affecting oysters. Epizootics produced by both parasites have caused extensive oyster mortali- ties in Delaware Bay; however, large numbers of oysters persist. Continued high abundances of oysters in Delaware Bay have been possible because many natural oyster beds occur in a spatial refuge from disease in the upper regions of the Delaware estuary. Salini- ties in this area frequently fall below levels necessary to sustain MSX infections. The Dermo parasite survives in these reduced salinities but does not produce lethal infections. The natural beds have been a primary source of seed oysters for the industry since the mid 1800s. Until recently, direct marketing from the beds had been prohibited. All seed oysters had to be transplanted to private leases in the lower bay where their growth and meat quality would be greatly enhanced before marketing. With the advent of Denno 451 452 Fegley et al. disease, movement of oysters into the lower Bay became uneco- nomical, and limited direct marketing from the beds began (Ford 1997). Because the natural beds are located in the upper estuary, the seed resource would have survived the depredations of oyster dis- ease without human intervention. However, a management scheme that developed shortly before the 1957-1959 MSX outbreak sta- bilized postepizoolic yields from seed oysters. Oysters still had to be transplanted onto leased grounds where enhanced growth and fattening was now countered by higher disease pressure that in- creased mortality. After the outbreak of Dermo disease, an entirely new strategy had to be developed to sustain the industry in the face of a disease with very different characteristics. We believe that a description of New Jersey's management structure provides in- sights for those desiring an effective management structure for many fisheries. Below we describe the physical and biologic con- text of the seed oyster fishery in Delaware Bay. Next, we provide a brief history of the fishery and describe the development of the present management structure and how it functions. We then sum- marize important aspects of the role of oyster diseases and how the management scheme responded to challenges from the diseases. We conclude by highlighting the unique elements of the manage- ment structure that we feel led to its success. It is important to note that the authors were participants in many events described below and may be burdened with preconceptions as to the value and importance of different aspects of the management structure. How- ever, our direct and extensive knowledge of the inner workings of the management structure allows us to place events and circum- stances in a context that would not be available to an outsider. Physical Description of Delaware Bay Detailed descriptions of the physical and bathymetric charac- teristics of Delaware Bay are available elsewhere (Shuster 1939, Maurer & Watling 1973, Galperin & Mellor 1990a, 1990b). Dela- ware Bay is bounded on the north and east by New Jersey and on the south and west by Delaware (Fig. 1 ). The bay extends 75.2 km from its southeastern-facing mouth between Cape May and Cape Henlopen to the entrance of the Delaware River in its northwestern comer. The average depth is ca. 10 m with the greatest depths occurring near the central long axis of the bay. The eastern side of the bay has extensive tidal flats. The bottom consists largely of soft-substrates (sands and muds) with hard substrate limited to spatially discrete oyster beds and cobble aggregates. Delaware Bay experiences predominately semi-diurnal tides with a 1-1.25 m tidal range near its mouth. Around 12% of the annual freshwater input enters the bay from the Delaware and Schuylkill Rivers. Salinity near the mouth ranges from 30-3 1 ppt and decreases with distance in a roughly uniform fashion up the bay to 0-4 ppt near Wilimington, DE. Water temperatures range from -1.8 to 29.0°C annually. Oysters in Delaware Bay Historically, natural oyster beds existed throughout Delaware Bay (Ford 1997). Before the mid- 1800s, however, harvest prac- tices and the distribution of oyster predators (primarily oyster drills, Urosalpinx cinerea and Euplcuni caudata) eliminated beds in the lower bay. The geographic location of extant natural (seed- oyster) beds has remained fairly constant and predates the appear- ance of MSX in Delaware Bay (Engle 1953, Maurer et al. 1971 ). These oyster beds occur in several small rivers entering the bay NEW JERSEY DELAWARE Figure 1. Location of the seedbeds (shaded areas) and the planting (leased) grounds (areas inside the broken lines) in Delaware Bay. The double line extending down the center of the bay represents the ship- ping channel. It separates the New Jersey and Delaw are portions of the bay. .Abbreviations for the seedbeds are the same as in Table 1. The labels of five small beds, located inland of EIS and NWB-STR, are indicated by letters (a— NPT. b— HGS, c— HWN, d— VEX, and e— BDN ). DPW is the Deepw ater site. (Broadkill. Leipsic, Mispillion. Murderkill, and St. Jones Rivers in Delaware; Back, Cedar, and Nantuxent Creeks and Cohansey and Maurice Rivers in New Jersey) and in the bay itself between Egg Island Point in the south and Arnold's Point in the north (Fig. 1 ). Most of the beds are in the eastern or New Jersey half of the bay. Oyster beds vary in size from those that are a few m" in area ("lumps") to some that exceed 6 x 10'^ nr. The density of oysters per unit area is highly variable within and between beds. Salinity of the water immediately over the oyster beds varies with distance from the mouth of the bay (Engle 1953. Maurer & Watling 1973. Fegley et al. 1994). Over the lowermost beds (those closest to the mouth of the bay) bottom salinity typically ranges from 16.0-20.0 ppt. The uppermost beds generally experience a bottom salinity ranging from 7.0-15.0 ppt. Oysters experience reduced predation rates on all but the lowermost beds because the most abundant and effective oyster predators in Delaware Bay, the oyster drills, are inhibited by salinities less than 15 ppt (Engle 1953). In contrast to survival, oyster growth rates decline along the decreasing salinity gradient in Delaware Bay. Oysters transplanted from the lowermost beds have generally required a single growing season to reach marketable size whereas most oysters moved from the uppermost beds have needed to remain on the planting grounds at least 2 years before they could be landed. Adult oysters spawn throughout the summer with most repro- Management of New Jersey's Oyster Seedbeds 453 ductive activity occurring from mid June to mid July. The larvae remain in the water column trom 10 to 20 d. depending on water temperature. Oyster spat set over most of the bay. The densest sets generally occur in the eastern portion of the bay south of Egg Island Point, where no beds exist and where oysters rarely survive to adulthood because of high predation. disease, and winter ice mortalities (Engle 195,^, Ford & Haskin 1988). Oyster Fisheries in Delaware Bay Several detailed accounts of the history of Delaware Bay oyster fisheries exist (Miller 1962, Maurer et al. 1971, Ford 1997). The following description, based on these histories, concentrates on the New Jersey portion of the fishery. In colonial times, natural oyster beds occurred throughout the bay although then, as now, most beds were located in the eastern (New Jersey) portion. Oysters were harvested directly from the beds and most were taken directly by ship to markets in Philadel- phia. The concept of planting small "seed" oysters onto private leases for growth and fattening before taking them to market was introduced to Delaware Bay in the mid 1 800s. Leases were estab- lished in the lower bay because the market quality of oysters was better there and because the local, natural beds had been largely destroyed by that time. Transplanted oysters were usually left on these relatively high-salinity leased grounds for 1-3 y before they were marketed. The seed oysters came primarily from the extant natural "seed" beds in the upper bay and in the creeks where low salinity protected small-sized oysters from predation. These beds remained a "public" resource. The practice of planting oysters was codified independently by laws in the States of Delaware and New Jersey. Until recently, planting seed oysters was the principal means of producing oysters in Delaware Bay. As planting became more widespread, the oyster fishery became dominated by com- panies that owned large schooners and used dredges to harvest oysters; hand tongers oystering from small boats have remained a marginal component of the fishery since that time (Fig. 2). From 1900 to 1930. Delaware Bay oyster landings produced between one million and two million bushels annually (Ford 1997). After 1930 and until the mid 1950s, the productivity of the industry declined slightly and annual landings remained at or just below one million bushels (-40 million L, Fig. 3). Landings of this magnitude, although supplemented by planting of seed oysters collected from outside of the estuary (primarily Chesapeake Bay and Long Island Sound), removed tremendous numbers of oysters from the natural seeds. By the early 1900s, seedbeds near the planting grounds were reported to be out of production. Subse- quent harvest practices (e.g., failure to return oyster shell to the seedbeds and the introduction of engines into the sailing schooners used to dredge seed oysters) and physical-biologic interactions (e.g., persistent droughts that increased the range and abundance of oyster drills) led to further degradation of the seedbeds. Finally, several years of poor recruitment onto the seedbeds and some unexplained mortalities of adult oysters in the 1940s and 1950s left oyster abundances on the seedbeds at historical lows. Development of Oyster Seed Fishery Management Legislation enacted by the States of New Jersey and Delaware during the 19th century attempted to regulate oyster fisheries in both states (Ford 1997). The overall goal was to preserve the oyster resource. Specific laws introduced culling (returning oyster shells to the bottom), restricted taking oysters from public seedbeds to a > Q CO Z o BOATS IN NEW JERSEY'S DELAWARE BAY OYSTER FISHERY C T vessels > 5 gross metric tons ^^M vessels < 5 gross metric tons o vessels dredging seed beds IVISX Denno 1900 1910 1920 1930 1940 1950 YEAR 1960 1970 1980 1990 Figure 2. The number of vessels registered annually in New Jersey's Delaware Ba> fisher). Open bars represent vessels greater than 5 gross metric tons in size. Shaded bars represent vessels less than 5 gross metric tons in size. From 1958 to 1991, the number of vessels partici- pating in seedbed dredging each year is indicated by the diamonds. In the last 3 yr (1995 to 1997) the diamonds indicate the number of boats harvesting the seedbeds in the spring and fall for direct marketing of the oyster seed. The broken vertical lines indicate the first year the respective diseases were observed in the bay. No data were available in vears when the beds were closed. specific season, allowed the first private leasing of grounds, and created a variety of organizations to monitor and enforce the leg- islation. Enforcement was a perennial problem and. at the request of many oystermen. the State of New Jersey took control of both the public and private grounds in 1899 (the State of Delaware had done so in 1 873, just two years after private grounds were devel- oped there). The principal regulation affecting the New Jersey seedbeds limited the period for oyster dredging to May and June. During this period, known as bay season, licen.sed vessels were permitted to take as many oysters as they could dredge and carry from the seedbeds for transplanting onto private leased grounds. Beyond limiting the length of bay season, there were no attempts to restrict the numbers of oysters taken from the beds. Prior to the 1950s, the seedbeds were closed to harvest only once, in 1928, to protect a large set of spat (newly settled oysters up to one year of age; Nelson 1929). During this time, information on year-to-year changes in oyster abundance on the seedbeds was not gathered. Few data were available to provide a basis for decisions by man- agement. Management of New Jersey's oyster resource can be traced to 1888. In that year Julius Nelson, a member of Rutgers University's New Jersey Agricultural Experimental Station, convinced the school to create the Department of Oyster Culture. Julius Nelson, and later his son, Thurlow. became leaders in the field of oyster biology and established a tradition of using scientific methods to produce information useful to the oyster industry (Nelson 1913, 1928, 1947). In the early 1950s, when oyster abundances on the Delaware Bay natural seedbeds reached historical lows, the De- partment of Oyster Culture, then under the direction of Harold Haskin. began studying the factors limiting oyster abundance on the seedbeds and gathering data that would suggest management strategies to rehabilitate the beds. The collection of data on oyster life-history in Delaware Bay in a regular and consistent manner 454 Fegley et al. O LU _l O > to o 100 -I 80 60 40 -I 20 0 NJ Seed bed harvest Tf MSX Dermo Ml|l|l|l^ 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 80 ^ 60 NJ market landings from Delaware Bay in o 0 30 25 20 15 -, 10 1880 1890 1900 1910 1920 1930 1940 1950 1960 1970 1980 1990 Market value of Delaware Bay oysters ' I ' ' ' ' I ' ' ' ' I ' ' 1880 1890 1900 19101920 1930 1940 1950 1960 1970 1980 1990 YEAR Figure 3. Historical changes in seed bed harvest, market landings, and market value in New Jersey's Delaware Bay fishery. The broken ver- tical lines indicate the first year that the respective diseases were ob- served in the bay. The absence of bars prior to 1957 indicate that no data are available. The absence of bars after 1957 indicate 0 values for the respective measures. Harvests are mostly for direct marketing and not planting starting in 1996. has continued for 45 y and has provided the basis for what we believe has been an effective management scheme. At its inception the seedbed rehabihtation program consisted of two key elements: gathering quantitative data on oysters (Research Component) and advocating the use of these data in making man- agement decisions (Applied Component). The research component consisted of several studies conducted yearly including: ( 1 ) deter- mining the temporal and spatial abundance patterns of oyster lar- vae. (2) determining the temporal and spatial patterns of oyster spat settlement and fouling organisms (invertebrates that compete with spat for space) onto artificial collectors, (3) detecting annual changes in the abundances of spat, yearlings, and older oysters on the seedbeds, and (4) estimating the volume of seed oysters trans- planted from the beds. Much of the funding for monitoring in the early years derived from University sources, a condition that is uncommon in our experience. The applied component entailed a determined effort on the part of the Director of the Department of Oyster Culture (Haskin) to convince the state management agency and, more importantly, the oystermen of the need for additional restrictions on seed transplants and of the usefulness of scientifi- cally collected data in decision-making. The use of scientifically collected data is now an accepted element. Both components have had continued importance in the overall management of the re- source. Research Component Of the several studies in the research component, two have been consistently of greatest use to management of the resource: collecting dredge samples from seedbeds and estimating the amount of oyster seed transplanted during bay season. Since the onset of Dermo disease, data on infection levels and oyster mor- tality rates have also been used on a regular basis in making management decisions. For dredge sampling, several grids, each consisting of contigu- ous 275-m X 370-m rectangles (approximately 0.2 min of longi- tude by 0.2 min of latitude, respectively), were created for each of the 25 spatially largest seedbeds that had historically contributed the bulk of oyster production. Each year, generally between No- vember and March, approximately 10% of the grids were chosen from each bed using a stratified random sampling design. Samples were taken from the middle of each grid. In the grid an oyster dredge (with a 71-cm tooth bar and a bag capacity of -80 L) was towed on the bottom for one minute at constant boat speed (i.e. approximately constant effort) three separate times. Approxi- mately 13-14 L of the contents of each of the three hauls were retained, pooled, and returned to the laboratory as a single sample. First, the volumes of live oysters (adults, yearlings, and spat), cultch (oyster shell with no live oysters attached), and debris (sponges, algae, wood, etc.) of each sample were estimated. Then the following quantitative attributes were determined by direct examination: ( 1 ) the number of oysters older than I y, (2) the number of "yearlings"" (oysters that were about 1 y old), (3) the number of spat, (4) the number of ""boxes"" (articulated but empty oyster valves), (5) the number of "gapers"" (recently or nearly dead oysters that do not fully close their valves when handled), and (6) the number of dead spat and, if any distinctive drill or crab valve damage was apparent, the source of spat mortality. Estimates of seedbed yields were made by research crews every day that dredging occurred on the seedbeds from 1956 to 1991. How many and which boats dredged, which beds the boats dredged, and estiinates of the volume of oysters moved to the planting grounds at the end of the day were obtained by direct inspection. Estimating the volume of oysters harvested was done by noting the size of the pile on the deck and the position of the water line on the oyster boat. In several years research crew esti- mates were compared with estimates of seed oyster volume made by the boat captain and by direct measurements. Remote observer estimates were generally within W7c of the captain"s estimates and of direct measures. Estimates of the percent composition of commercial dredge samples were also made during bay season. On Thursday (usually) of each week of seed planting season uncalled 40 L samples of oysters and shell were taken directly from the decks of oyster boats. Boats were selected on the basis of which beds they dredged. The beds of interest were those that had experienced the greatest amount of dredging activity during the week or that had begun the week with relatively low percentage (by volume) of oysters. On shore a committee composed of industry members, managers, and laboratory personnel sorted the samples into oyster (live adults, yearlings, and spat) and shell (anything without an oyster attached) and estimated the relative volumes of the two portions. This information was then used to decide whether to Management of New Jersey's Oyster Seedbeds 455 close some of the beds or to end the seed tninsplant early. If the average percent of oysters by volume was less than 40*^ for a bed the committee gave serious consideration to closure. The 40% value was a "rule of thumb" benchmark that was never supported by statute or regulation. It was not supported by scientific evidence. When the seedbed rehabilitation program be- gan the approximate percent oyster on many beds was around 40'7f and many felt that it should not go lower. The industry members understood this measure (as opposed to more complex statistical indices) that required simple math and that they could derive on their own via examination of dredge hauls. Also, when percent oyster did drop much below 407c harvesting oysters became pro- hibitively expensive for boats using manual culling. Use of the 40% rule was flexible. Depending on other factors (abundance of oysters elsewhere, number of spat in the sample, perceived eco- nomic needs of the oystermen) a bed could be closed before the percent oyster measure reached 40% or at a considerably lower percentage (as low as 20% in a few cases). Applied Component A shellfish council, officially consisting of industry members appointed by the Governor, had long been in place to advise the state agency in charge of the seedbeds (the council also supervised the private leases approving transfers, vacancies, boat licenses, etc.). In the mid 1950s incorporating research results into the coun- cifs decision-making proved difficult. The concept of managing oyster beds with recently collected data was foreign to both the state agency (NJ Bureau of Shellfisheries) and the oystermen. However, the greatly depleted condition of the beds indicated that restrictions on seed transplants would be austere for some time to come. The patent threat to the fishery by the condition of the seedbeds and the persistent efforts of the Director of the Depart- ment of Oyster Culture advocating the utility of research results led to the development of a tripartite management scheme. An independent source of information. Rutgers University, was added, in an informal advisory role, to the shellfish council and state regulators (Fig. 4). This system remains in effect today. In late winter, several months prior to the beginning of bay season, data collected from the seedbeds by the university re- searchers are presented to the shellfish council and representatives of the state management agency. The primary concerns are the Shellfish Council * INDUSTRY INDEPENDENT RESEARCH AND MONITORING STATE MANAGEMENT AGENCY RESOURCE Oyster seed beds Figure 4. Diagram illustrating the relationships among the compo- nents of the New Jersey Delaware Bay oyster fishery. .Solid lines indi- cate formal informational pathways. Broken lines indicate informal informational pathways. The X represents the control of industry ac- cess to the seedbeds via state management. relative compositions of dredge samples taken from the seedbeds (percent oyster) and the seedbed spat abundances. An oral presen- tation of these data (usually supplemented with a written sum- mary) is made to the shellfish council members who use this information and, in some years, their own direct observations of the beds, to decide ( I ) whether there will be a bay season. (2) how long the season will be, and (3) whether any beds will be excluded from fishing. The council's recommendations are then submitted to the state management agency (specifically the Commissioner of New Jersey Department of Environmental Protection who directs the Bureau of Shellfisheries), where they have generally been ap- proved. Onset of MSX Disease In the spring of 1957, widespread mortalities of oysters planted the previous year occurred on the New Jersey leased grounds. Within two years the epizootic had killed over 90% of the oysters on the planted grounds and almost half of those on the seedbeds (Haskin et al. 1966). The causative agent, H. netsoni (popularly referred to as MSX), was identified in 1958 and has remained enzootic in the estuary (Ford 1997). Since 1957, dockside landings of oysters from Delaware Bay have remained well below a half a million bushels (-20 million L) of oysters annually (Fig. 3), al- though significant undeireporting of these landings may be occur- ring (Haskin & Ford 1983). Uninfected oysters residing in salinities greater than 15 ppt can become infected with H. nelsoni from June to early November. The disease progresses to a lethal stage within several weeks in susceptible oysters. Mortalities are delayed in Delaware Bay native stock; it has developed a degree of resistance to the disease (Ford & Haskin 1987). Some oyster deaths occur in late summer or fall of the first year of planting, but these are usually tolerably low (Ford & Haskin 1982). Mortalities are cumulative, however, and become unacceptably high if oysters are not marketed within a year. The large oyster mortalities produced by MSX on the planted grounds altered the practices of the Delaware Bay oyster fishery. First, importation of oyster seed from other regions ended. Second, only relatively large oyster seed could be transplanted from the seedbeds to the planting grounds because only a single growing season was likely to be available to growers. It was no longer possible for small oysters to survive in the lower bay for the two to three years necessary to reach market size. Planters could not stockpile oysters on their leases anymore. Third, oystermen con- centrated their planted oysters in a relatively small area of the bay less prone to disease. Leased bottom was made available that encroached onto the lower seedbeds in an attempt to provide less saline and less disease-ridden planting grounds. Fourth, oyster boats decreased operating costs by using automatic culling ma- chines instead of manual labor to separate oysters from cultch. Fifth, regulations were changed to permit marketing oysters earlier in the year. This allowed planters to land oysters as soon as they reached market size instead of waiting until 1 September as they had previously. Sixth, a limited fishery based on boat size was established in 1981 to prevent a large influx of participants during good times who had no commitment to preservation of the re- source. The onset of MSX disease initiated a long-term monitoring program that followed the spatial and temporal patterns of the disease in the bay and consequent oyster mortality (Ford and Haskin, 1982). Results garnered from this effort helped interpre- 456 Fegley et al. tation ot data acquired from the annual seedbed sampling program. At approximately one month intervals, oysters were dredged, using the same device described above, from the larger seedbeds and several locations on the planting grounds. The dredge samples were taken only from the most productive grids on the seedbeds. In contrast to the fall/winter seedbed sampling procedure, several successive dredge hauls were conducted until a bushel (-40.7 L) containing only live oysters, gapers, and bo.xes was obtained. All gapers and boxes were examined for evidence of shell damage that could be attributed to crabs, drills, or dredging. Gapers and boxes with undamaged valves were assigned to the nonpredation mor- tality category. The interiors of the boxes were further inspected to determine whether any fouling organisms had recruited onto the inner surfaces of the valves. Boxes with no fouling on the inner valve surfaces were considered "recently dead." Spatial and tem- poral variation in the rates of valve fouling were estimated by placing clean valves in the field at regular intervals and examining them at subsequent intervals for the presence of fouling organisms. Seasonal "fouling intervals" ranged from 2 to 3 wk in the summer and up to 10 wk in the winter (Ford & Haskin 1982). Estimation of the annual mortality from predation and nonpredation sources were made by accumulating mortalities determined over short in- tervals. MSX disease prevalence and intensity was determined via his- tologic procedures in live oysters and gapers collected during the mortality sampling. After sectioning and staining, the abundance and location of MSX parasites in the tissues were determined via microscopic examination. In local infections (nonlethal at the time of collection) the parasites occur only in the gills. In systemic infections parasites are distributed through all oyster tissues. Sys- temic infections are found in 90'7f of oysters that die of MSX disease (Ford & Haskin 1982). Onset of Derino disease Infections by the southern oyster parasite, P. mariniis, causative agent of Dermo disease, had been historically of little consequence in Delaware Bay. During the mid 1950s light infections were found in planted oysters after parasitized seed was imported from Virginia where the disease was endemic. Dermo infections became rare in the bay after importation of seed ended. The water tempera- tures in Delaware Bay were generally believed to be too cold for Dermo disease to persist (Ford & Haskin 1982) and sampling spe- cifically for Dermo disease ended in 1963. In the late summer of 1990, oyster mortalities that did not fit the pattern associated with MSX disease were documented in several locations in Delaware Bay (Ford 1996). The causative agent was quickly identified as P. inari- nus. Since 1990. Dermo infections have been persistent, wide- spread, and responsible for continuing oyster mortality in the bay. In contrast to the pattern of MSX distribution, Dermo infections have extended onto the seedbeds and caused substantial monalities of seed oysters. P. mariiuis is much more tolerant of low salinity than H. nelsoni. It survives on most of the seedbeds, even though it does not cause many lethal infections on the uppermost beds. Parasites proliferate rapidly in oysters transplanted to the planting grounds in spring, stimulated by both high temperature and high salinity. Under these conditions, transplanted oysters typically die before the fall market season. The consequences of a mortality pattern quite different from the delayed mortalities induced by MSX disease was forcefully demonstrated to the planters shortly after the onset of the Dermo epizootic. Planters were advised of the presence of Dermo disease in Delaware Bay immediately after it was identified in the summer of 1990. During the remainder of the summer and fall, the disease spread to all planting areas and to the lower seedbeds, but caused relatively little mortality and yields from planted oysters were the best in many seasons. The following year, the abundance of oysters on the seedbeds was the best since the early 1980s and nearly 300,000 bushels (1.2 x 10^ L) were moved to the planted grounds. A large majority of these oysters were already infected with P. manniis, which quickly proliferated. Despite advisories about relatively high infection levels by re- searchers (including warnings by oyster disease researchers from institutions other than Rutgers University expressed in a special public meeting), most planters, remembering the profitable results of the previous year, chose to leave their oysters on their leases rather than to harvest early. Mortalities, when they began, were severe and only about a quarter of the oysters survived to the fall market season. The MSX surveillance program was severely diminished after the mid 1980s because of funding limitations and an expressed hesi- tation by university administrators to commit to long-term monitoring programs. Tlie advent of Dermo disease, however, raised enough concern within the industry that limited monitoring was resumed. It centered primarily on disease diagnosis in oysters collected during the fall seedbed survey, which provided information on the spatial dis- tribution and intensity of Dermo disease on the natural beds at a time of peak prevalence and intensity (Ford & Tripp 1996). Because it is likely that oysters rarely, if ever, completely rid themselves of P. mariini.s. even under the low temperature and low salinity conditions that are unfavorable to the parasite (Ragone-Calvo & Burreson 1994, Ford et al. 1999). the fall sampling provided a good estimate of what percentage of oysters are infected on each bed. Subsequent sam- pling in the spring before bay season, provided additional infor- mation on infection intensity, which typically decreases over the winter in proportion to temperature and fresh-water influx. Infec- tion intensity in oysters likely to be transplanted provided a rough measure of whether infections would progress to the lethal stage relatively sooner or later after planting. The results from the Dermo disease surveillance program and from the earlier MSX program were presented to the shellfish council and to individual planters. In recent years, mailings to all lease holders describing the most recent levels of oyster mortality and disease prevalence were made. Delaware Bay Oyster Fishery Activity. 1953 to 1991 Seed dredging has occurred in most years since the first MSX epizootic (Fig. 5). Generally all of the beds were open, but oys- termen concentrated their efforts in just a few beds. The 1960s harvests were relatively small and came primarily from the upper- most beds. By the end of the 1 960s most oyster seed came from the beds in the middle of the seedbed region. Four beds, Cohansey, Shell Rock, Bennies, and New Beds, produced 68.2'7f of the oyster seed from 1958 to 1991. These are among the largest beds and perennially have relatively high abundances of moderately large oysters (Table I). As would be expected, samples collected for the weekly esti- mation of relative oyster volume during bay season were taken from where most of the harvest activity occurred. During the 1960s and early to mid 1980s the relative volumes of oysters in the samples were generally less than 40% (Table 2). Only a quarter of these samples ( 1 1 of 42 instances) was less than 30% and in only Management of New Jersey's Oyster Seedbeds 457 LU _l o > LU < C/5 LU O cr LU 0. ANNUAL MEAN PERCENT VOLUME OF OYSTERS 80 60 40 - 20 S n > ^ I X o o ■n !2 i ■o 0) ■¥ £ 0) "^ T- T I I" T II li T- I ill T T T T -r ^ ' III T 1955 A I960 1965 1970 MSX T 1975 YEAR T t: T (1) o — to ^ -D o: 00 CO T T CM (^ TT I ll 1980 1985 1990 Dermo 1995 Figure 5. Average (±1 SE) perteni volume of oysters in dredge samples for each year. The average is calculated across all beds; ;i ranges from 60 to 120 for any given year. The arrows indicate the first year the respective diseases were observed in the bay. The horizontal broken line indicates a percent volume of 40%. Text above each bar describes the length of the subsequent bay season. Abbreviations for seedbeds are the same as in Table 1. three instances were the proportions less than 209c. During the mid to late 1970s the relative oyster volume frequently exceeded 40%. but these data were never used to extend a seedbed harvest season beyond the length that had been agreed upon earlier in the year. Individual seedbeds were closed before the end of bay season only four times (Shell Rock Bed, 1961: Cohansey Bed, 1967; Shell Rock Bed. 1972; and Bennies Bed. 1974). Low percent oyster was the reason in half of these closures, while protection of spat led to the other early closures. The fishery benefited from very successful recruitment in 1972 although relative abundances of oysters on the beds were increas- ing before this year (Fegley et al. 19941. The large 1972 set pro- vided oysters until the early 1980s. The persistence t)f harvestable oyster seed for almost a decade after the 1972 set was aided by the management and harvest practices of the fishery participants. For instance, despite large abundance of oysters in 1974 the length of bay season was not extended to take immediate advantage of this bounty either that year or in any successive year. Within years, the efficiency of seed harvest (actual harvest/potential harvest x 100%) remained near 60% throughout the period (the potential harvest was based on estimates of the total abundance of oysters present on the seed bed large enough to be suitable for transplant). The observed efficiency was most likely a function of boat harvest limitations rather than conscious efforts of the harvesters. Oyster recruitment onto the seedbeds was relatively low in the years after 1972; another "large" set (only a third the si/e of the 1972 set) did not occur until 1 986. Restrained harvesting of the large 1 972 set by the fishery, combined with average or above-average annual Dela- ware River flow into the bay. remains the most likely explanation for the continued presence of oysters on the seedbeds into the late 1970s and early 1980s. In the mid 1980s seedbed harvests began to decline. During this time there were increased prevalences and intensities of MSX disease throughout the bay (Fig. 6); widespread mortality of oys- ters followed. This was the first time since the mid 1960s that the seedbeds exhibited such high levels of disease and predation. The mid 1980s were also the first time since the mid 1960s that the annual mean Delaware River flow remained below the long-term average for several successive years (Fegley et al. 1994). No seed dredging occurred for 3 yr (1987-1989). During this protracted closure of the fishery there were modest increases in the abun- dances of oysters on the beds and seed transplants began again in 1990. Unfortunately that was also the first year of a Dermo disease epizootic. The effects of Dermo di.sease upon the New Jersey oyster fish- ery have been substantial. Data provided by university researchers informed oystermen that most of the oysters they would plant were infected with the Dermo parasite and would not survive long after planting. Based on this monitoring information, the shellfish coun- cil voted to close the seedbeds in 1992. 1993. and 1994. By 1995. after 3 yr without a planting season, it was obvious that the tradi- tional transplant scheme would work no longer. In 1995. a new strategy was agreed upon and tried for the first time that allowed direct marketing from the seedbeds. Up to this time, all oysters removed from public seedbeds had to be trans- planted onto private grounds before they could be marketed. In the new scheme, which was developed by the Bureau of Shellfusheries and agreed to by the Shellfish Council, each licensed vessel re- 458 Fegley et al. TABLE 1. Some characteristics of the seed beds related to seed harvest. The area of the seed bed includes nonproductive bottom. Mean percent oyster is based on dredge samples taken in the random sampling program (1953-1991). Mean individual size is estimated by dividing the volume of a dredge sample consisting of oysters by the number of oysters present. The harvest data are the total volume of seed removed from each bed between 1958 and 1991. The five largest >alues in each category appear in boldface. The names of the beds, which are listed from those uppermost in the bay to those that are lowermost, are given below. Area % Oyster Indiv. Harvest Bed* (Hectare) Rank (±1 SD) Rank Size (niL) Rank L X 10' Rank RIS 162 13 73.6(13.5) 2 37 18 3,066 13 UAR 121 15 74.1(14.3) 1 51 16 787 17 ARN 232 9 70.7(18.4) 3 49 17 8.249 10 UMD 20 18 49.9(27.7) 13 63 15 1.S55 15 MID 374 7 64.9(17.2) 5 70 13 17,617 5 COH 545 3 62.2(17.5) 6 78 8 43,735 2 SHJ 454 4 66.5(18.5) 4 74 10 16,2.^1 6 SHR 404 5 61.7(20.6) 7 85 6 42,784 3 BNS 101 17 59.9 (22.3) 8 91 5 4.335 8 BEN 636 2 48.0(25.2) 14 98 4 40,941 4 NPT 212 11 53.0(22.8) 11 69 14 2.476 14 HGS 111 16 46.8(23.8) 15 76 9 11.270 7 NWB-STR 829 1 53.4(26.9) 10 85 7 62,496 1 HKN 202 12 55.0(19.4) 9 71 12 1,121 16 BDN 293 8 43.4 (25.9) 17 135 2 203 18 VEX 162 14 52.8(20.1) 12 74 11 4.051 11 EIS 394 6 43.6(25.4) 16 112 3 8.945 9 LDG TT) 10 33.0 (23.0) 18 146 1 3,479 12 * RIS, Round Island; UAR. Upper Arnold's; ARN. Arnold's; UMD, Upper Middle; MID. Middle; COH. Cohansey; SHJ, Ship John; SHR. Shell Rock; BNS, Bennies' Sand; BEN, Bennies; NPT. Nantu.xent Point; HGS, Hog Shoal; NWB, New; STR. Strawberry; HKN. Hawk's Nest; BDN, Beadon's; VEX. Vexton; EIS. Egg Island; LDG. Ledge. ceived a quota (of equal size, regardless of boat size). Vessel owners were required to buy a tag costing $1.25 per bushel for each bushel they expected to harvest up to their quota. A time period was set in which the quota was to be used. After this period the status of the resource, markets and other factors were evalu- ated, and another quota decision was made. In most cases the quota per boat was increased. This activity has generated a considerable amount of revenue (Table 3). Purchase of tags alone totaled $374,615 (through the fall of 1998). This money was deposited into an "Oyster Resource Development Account" that is used for shell planting and moving oysters from upper to mid bay beds. Direct marketing from public beds goes against a trend towards privatization, which is generally considered more efficient than public fisheries (Haven et al. 1978). Because direct marketing does not require maintaining leased private grounds nor capital invest- ment in moving oysters from the public to the private grounds there is a possibility that new participants could more easily enter the fishery. Yet, direct marketing from the New Jersey Beds has been the only obvious use of the resource under prevailing disease conditions. For instance, in 1991 and 1995 (the beds were closed from 1992 through 1994). a total of 390,000 bushels was taken from the seedbeds and transplanted to the leased grounds, but because of high subsequent mortality, only 63,000 bushels (2.6 x 10* L) were landed, producing a total return of $1,189,190. For each bushel removed from the seedbeds, direct marketing has re- turned nearly seven times more in dockside value compared with typical planting returns during periods of high Dermo disease (Table 4). The presence of Dermo disease has increased the reliance of oystermen and state officials on the results of university research and monitoring. In the past, information about MSX prevalence was of secondary importance to the shellfish council when they were deciding whether to have a bay season (MSX was generally uncommon on the seedbeds). In contrast, the high prevalences of Dermo disease in oysters on the seedbeds raised concerns about transplanting infected oysters, which could result in rapid prolif- eration of the disease and high oyster mortalities before they could be marketed. Data on Dermo disease prevalence has been the primary information leading to limited seed transplanting in the past few years. A 4-week bay season was agreed to in 1995, but the shellfish council closed the beds after two weeks. Shellfish council deliberations cover a range of issues when the council makes de- cisions on closures (Appendix I ). DISCUSSION Aspects of the Delaware Bay Management Structure Management of the Delaware Bay New Jersey oyster fishery has the elements common to many fishery management structures. It consists of a management agency, an industry, a means of data collection and evaluation, an industry council, and a set of statutes and regulations. The difference between this system and other fishery management structures is the way these entities relate to each other. Although these special relationships cannot, by them- selves, be credited with the continued persistence of harvestable oyster populations in Delaware Bay, we believe their implemen- tation has developed an atypical management program. There are at least six basic differences — some obvious, others subtle — between the Delaware Bay New Jersey management scheme and many others. First, as for several estuarine shellfish- Management of New Jersey's Oyster Seedbeds 459 TABLE 2. Weekly estimations of average percent oyster volume during seed bed harvest season. Values belovt' 40% are shaded. Bed designations are the same as in Table I. ND = no data. Year RIS ARN MID COH SH,I SHR BNS BEN OB NPT HGS NWB VEX LDG EIS AVG 1958 33.5 36.8 33.4 30.7 34 33.7 1%1 1962 1964 1966 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1990 ALL SEED BEDS CLOSED TO HARVEST (1959-1960) 35.6 40.3 37.1 24.1 32 29.8 31.7 30.3 ALL SEED BEDS CLOSED TO HARVEST (1963) 17.3 28.2 15.9 ALL SEED BEDS CLOSED TO HARVEST (1965) 74.3 38.6 33.5 23.7 ALL SEED BEDS CLOSED TO HARVEST (1967) 64.4 50.8 58.6 56 55.3 85.5 71 754 69.9 79 81.9 74 71 85.8 66.4 51.7 54.9 64 60.7 36.3 36 36.2 49.7 30.7 69.7 ALL SEED BEDS CLOSED TO HARVEST (1987-1989) 52.4 52 63 78.1 68.2 52.6 39.5 49.4 32 37.4 46.8 35 48. 7 45.5 47 55 45.5 36.6 28.3 32.2 63.5 58.7 52.2 59 54.8 30.8 19.2 25.9 23.6 34.3 30.9 20.4 43 ND ND ND 45.5 56.3 57.2 57.1 54.8 66.5 52.5 69.1 78 75.5 64 78.5 65 69.9 57 71 64.3 39 56.8 43.8 53.9 52.5 43.5 44.2 61.3 48.7 ND 31.3 37.3 38.6 44 35 37.1 31.8 27 43.7 31.8 35 42.3 25 30.9 52.2 eries, there is no formally written and passed management plan, nor has there ever been one. Only a number of very basic provi- sions are encoded in State statute and regulations. Second, the fishery has been closed to new entries since 1981. Third, all of the major participants are housed in close proximity to each other and have been for nearly a century (the Haskin Shellfish Research Laboratory is located at the home port of the New Jersey oyster fleet and the State Bureau of Shellfisheries has an office in the Laboratory building). Fourth, these three groups have worked, and continue to work, together closely in various combinations. Fifth, a tripartite relationship exists in which each entity has a specific role: the industry is represented by the Delaware Bay Shellfish Council, the State Bureau of Shellfisheries provides the adminis- trative support for the Shellfish Council and the Commissioner of the Department of Environmental Protection makes final decisions based on Council recommendations, and an independent group (in this case the Haskin Shellfish Research Laboratory) collects and provides data to the other two. Sixth, formal, informal, and per- sonal information exchange between all three parties takes place on a regular basis. The actual importance of these six differences in the development, evolution, and execution of the management strategy is not easily evaluated; however, the salient features of each are described below. I. The lack of a written plan provides flexibility. The process required to make changes can be adapted to the situation at hand and, with the exception of those portions that are en- coded in law, most issues are settled in Council meetings. All decisions are made openly (regularly scheduled shellfish council meetings are advertised in the paper, anyone may attend the meetings and express their opinions to the gath- ering, minutes are taken and distributed at the next meeting, newspaper journalists generally attend and publish articles on decisions within one to two days, and special unsched- uled meetings are held after all industry members have re- ceived notification by direct mailings.). The decision pro- cess however is not burdened by regulatory needs for formal hearings, published notices, comment periods, etc. If all three parties (industry. State, and the Laboratory) agree, even major changes can be accomplished relatively rapidly. The change to harvest practices brought about by Dermo disease provides an example of this flexibility. This disease caused such high losses in oysters that by 1995 it was ob- vious that the traditional movement of oysters from the seedbeds to the planted grounds in spring was neither com- mercially viable nor biologically desirable. Discussion be- gan in the fall Council meetings about harvesting directly from the seedbeds. This was in direct opposition to over 100 y of practice and the proposal generated a great deal of heated debate. In general, the older members of the fishery were opposed and the younger members thought that the new approach should be tried. At the end of March, after five to six meetings and an industry evaluation of the seed- 460 Fegley et al. XXXX X xxxxxxxxxxxxxxxxxx 1960 1965 1970 1975 1980 1985 1990 YEAR Figure 6. Percentages of live oysters infected with either MSX or Dermo disease on several seedbeds and one site located in the planting grounds (DPW. Deepwater). The absence of a symbol indicates no samples were taken from that location in that year. The sites are arrayed from the least saline (ARNi to the most saline (DPW). The abbreviations for the seedbeds are the same as in Table 1. beds in mid-March, general agreement was reached that a direct harvest should take place beginning in mid-April, with each boat limited to 1000 bushels instead of the normal unlimited transplantation. This change in statute was intro- duced into the New Jersey State legislature with three pro- visions: 1 ) a limited direct market program should be at- tempted, 2) if oysters were to be harvested from the beds, a per-bushel fee should be imposed with the proceeds used for bed rehabilitation, and 3) the harvest would have to be ac- tively managed to be successful; recommendations for open- ing and closing the season should be in the hands of the Council, with final authorization being given by the Com- missioner of the Department of Environmental Protection (Appendix 2). Once these aspects were "agreed upon", leg- islation was drafted, introduced into the NJ Legislature in May, passed and signed by the Governor by the first week in September. Fall direct harvest from the seedbeds began one week later. The closed nature of the fishery means that all participants are known and readily contacted for regular or special meet- ings. Mailings of informational bulletins are easily accom- plished. Contact is uncomplicated even though out-of-state interests control a significant part of the industry because they have local representatives who act in much the same fashion as other local fishery participants. The importance of all groups having significant on site rep- resentation cannot be overemphasized in fostering the flow of information and appreciation of differing outlooks. The close proximity permits daily contact among the parties, but more importantly, nurtures a sense of community. It allows each individual and group to become aware of the other's point of view and to understand their biases. This does not mean all groups agree on every issue, but it does allow interested participants to evaluate what is being said in a context broader than that of a formal meeting. Working together in various capacities is partly an out- growth of the close proximity of the different parties and adds to their overall ability to understand and communicate with each other. For instance, since 1989 the industry has donated a boat and captain for the Laboratory's annual sur- vey of the seedbeds. Without this donation continuation of the annual shellbed survey would have not occurred given the existing University resources during that time. The State often collects samples for the Laboratory, has collected samples of interest to the industry, and often allows industry members to sample the beds "out of season." Laboratory representatives regularly attend Shellfish Council meetings where they present results of ongoing projects or simply answer questions on issues of immediate interest. The tripartite scheme, with a party independent of the man- agement authority collecting basic data, holds in check the belief common to many fishermen that data obtained by manageiTient agencies are biased, or that the interpretation of those data is biased. In the current scheme, both the management agency and the industry are free to criticize data collection and/or evaluation in any way they see fit. This provides a check and balance, somewhat equivalent to "peer review" on the data collection and presentation pro- cess. In addition, a research organization can use funds from competitive funding sources to support research that does not have an immediate interest to management or the indus- try. However, these "pure" research projects can occasion- ally provide new information to the attention of the industry and the management agency that they would not have oth- erwise. The formal, informal, and personal relationships, as with the close physical proximity, allows communication and infor- mation exchange to take place on many different levels. What is said in private conversations is often not represen- tative of the positions presented in public meetings. This is because each group has personal views that may not be appropriate for expression in a formal meeting. For instance, the formal role of the researchers is to present the facts and to elucidate potential biologic risks. Their opinion on man- agement alternatives is frequently sought, and they may en- dorse certain options, but they generally refrain from advo- cating a specific action. These scientists may have views on whether the industry is making optimal economic use of the resource, but this would be not be expressed in a formal presentation of the data on the status of the resource. Simi- larly, an individual in the industry may think the resource is being exploited too heavily, but because of social relation- ships in a small community, not wish to express this view in Management of New Jersey's Oyster Seedbeds 461 TABLE 3. Direct murkt'liii); of oysters from Delaware Bay, New Jersey Seed Oyster Beds. Time Period Numher of Bushels Landed (vol. in L) Approximate \ alue of Bushels Landed Value of Tags Sold Spring 1996 (10 weeks) Fall 1996 (7 weeks) Spnng 1997 (10 weeks) Totals 17.828 (7..^ X 10') 42.570 ( 1.7 X 10'') 27.479(1.1 X 10") S7.S77(.^.6x lO*") $,^20,904 $89.^,970 $.'i77.0.'i9 $1,791,933 $22.28.5 $52,213 $34,349 $108,847 Time period includes the length of the dredging season. We present the harvest in the fishery's traditional bushels but we also convert those volumes into L. a formal meeting. The Industry iiieiiibers kuik to the Labo- ratory or the State to present this view in the formal context. Two obvious characteristics of management for the New Jersey seed oyster fishery has been the high degree of cooperation and mutual respect among the oystermen. State officials and university researchers. In formal Council discussions each entity generally honors each other's expertise and role. The relationship has been uneasy, particularly when the resource was scarce. In recent years, however, the restrictions on the fishery imposed by severe oyster disease have been important in maintaining a mutual dependency of the three parties. Scientific data have become recognized as being more significant than ever in the management process. Co- operation of all parties has been crucial in implementing and test- ing new practices. The persistence of disease and its potential to kill oysters has forced the industry to proceed cautiously and to husband the oyster resource thoughtfully. The industry may have acted in an equally prudent way in the absence of the existing management structure, although pre- 1950s fishery practices sug- gest otherwise. We believe that the interactive management struc- ture, described above, has fostered effective decisions about the use of the oyster resource in the presence of disease. Scientific Data: Formal Use Critical to the management structure has been the availability of current population data, collected in a consistent manner over a prolonged period. Although the data are clearly used, the manner of use has varied, depending on the status of the resource and the industry at the time. Below, we provide instances where the bio- logic data can be shown to have influenced Council decisions, others where more informal uses of the data are evident, and still others where the data were generally ignored. Prior to 1991 (when the Dermo disease epizootic became a decisive factor) the abundance of oysters and spat, and to a lesser degree MSX disease prevalence, were considered when decisions TABLE 4. Comparison of returns per bushel of oysters removed from the seedbeds by planting (in 1991 and 1995) and by direct marketing (1996-1997) during periods of high Dermo disease. Seedbed Oysters Fate Bushels (vol. in L) Average Return per Total Sales Bushel Leased grounds Direct marketing -m).(JOO ( 1 .6 X 10') 87,877 (3.6 X 10") $1,189,190 $1,791,933 $3.05 $20.39 We present the harvest in the fishery's traditional bushels but we also convert those volumes into L (vol. in L). were made about the length of seedbed season. A general, direct relationship of these measures and the resultant occurrence or length of the season is apparent (short or no season when percent oyster <40%, longer seasons when percent oyster >40%; Fig. 5). On specific occasions, the data clearly influenced decisions. In 1972 Bennies Bed was closed to dredging. At that time the relative abundance of oysters was over 40% and the proportion of oysters infected with MSX in the preceding two springs was low; how- ever, oysters were available on other beds and the opportunity to allow previous good sets on Bennies Bed to mature undisturbed by dredging was realized. The usefulness of this decision was never formally tested because in 1972 that bed and the remainder of the bay experienced another, even larger, recruitment event that proved to be an important source of oysters for years to come. Data use has been amply illustrated since 1991 when it was recognized that planting oysters infected with the Dermo parasite would likely result in unacceptably high losses of planted oysters and loss of shell from the seedbeds. This realization clo.sed the seed fishery for 3 consecutive years despite lost income to the fishery and the opposition by some industry members. The desire of these members to continue to plant as usual was muted because most participants in the fishery shared beliefs that restrained the degree of risk that the fishery as a whole would take. The shared beliefs included the following: I ) that the "disease problems" would eventually lessen (as they did with MSX). making preser- vation of the resource until that time an important and common goal; 2) that data gathered and presented by the "third-party" re- searchers were accurate and unbiased (although conclusions about the data were not always widely shared); and 3 ) that the experience of oystermen concerning when and where to plant, and when to harvest, were important in making decisions about the advisability of dredging seed oysters. Scientific Data: Informal Use There is no clear correlation in the long-term data between MSX prevalence and oyster mortality on pi'ivate leases. A major reason is because the total mortality on a particular ground is only partly a function of disease levels. It is also influenced by decisions of the lease holders who transplanted oysters. Oystermen fre- quently solicited information about MSX prevalence and intensity from the Laboratory. If MSX prevalence and intensity seemed to be increasing on the leased grounds, .some planters would equip extra boats to harvest oysters to insure they retrieved all market- able individuals before they died (L. Jeffries, pers. comm., 199.5). Not all lease owners availed themselves of the data or. if they did, acted on them. Oystermen were free to ignore the monitoring data and gamble that the disease would be less destructive than ex- pected. 462 Fegley et al. Little or No Data Use The "40% rule" was ignored on several occasions during the 1960s and late 1980s (2 and Fig. 5). The industry was still reeling from the financial losses caused by the initial MSX epizootic in the 1960s and was severely stressed again in the 1980s because of a drought that stimulated renewed MSX activity. Economic pres- sures clearly predominated over the biologic data; however, the data were not entirely ignored because the length of bay season was restricted to only 2 wk in most of these years. Economic and Financial Pressures Economic considerations continually threatened this manage- ment strategy. Oystemien had to maintain cash flow during pro- longed periods when oyster harvests were small or impossible; most responded by diversifying their activities. Boat owners who also owned shucking houses kept the houses active by shucking oysters from other locations (primarily Connecticut, but also from the Gulf Coast) or by processing surt' [Spisiila solidissima) and mahogany clams {Arctica iskmdka). Some oystermen moved boats into the Atlantic surf clam fishery or used them to harvest finfish. blue crabs (CalUuectes sapichis). whelks {Busycon spp.). or horseshoe crabs {Liimilus polyphemus) in Delaware Bay. Others diversified economically by direct marketing of multiple seafood products or managing marinas. Some of the older oystermen pos- sessed sufficient cash reserves to temporarily retire. Many younger participants left the industry; the on-again off- again nature of the fishery restricted their ability to reenter. The large costs of prepar- ing a boat to work in the fishery when economic return was so uncertain resulted in a de facto limited entry fishery prior to the establishment of a regulatory limited fishery. Only those who could risk substantial financial losses could continue to participate. CONCLUSION New Jersey's management of the Delaware Bay oyster seed fishery demonstrated an ability to respond relatively quickly to both threatening and promising changes in the dynamics of oyster populations and oyster mortality sources. Despite this flexibility and the fact that management is largely in the hands of the industry itself, the resource has been generally well conserved. In fact, the impact of seed dredging on the oyster population cannot be sta- tistically measured (Fegley et al. 1994). We suggest that the pri- mary reasons for the persistence of the resource include (1 ) the high degree of communication among the three parties involved in the management strategy. (2) the presence within the industry of a few individuals who took a long-term and relatively conservative management view and who were generally respected by others in the industry; and (3) the perception of a shared risk among industry members, which also constrained their activities. Not all aspects of the Delaware Bay management system may apply to other fisheries. For example, the fishery has relatively few participants who operate in a geographically constrained area. Part of the resource lies within an area where diseases and predators are absent or reduced by prevailing environmental conditions. Both of these conditions reduced the scale of management complexity in the present case. However, several characteristics of this fishery and its management structure could be exported to other locations. We argue they include the following: Human harvest activities on some parts of the resource need lo be limited. Long-term, reliable, third-party monitoring of the resource, disea.ses, and harvest ac- tivities should be integrated as a consistent part of the decision processes of the management structure. Continued personal con- tact through meetings, discussions and working together is essen- tial in transmitting information. Last and most importantly, the participants in the fishery should agree on the basic goals of the program and all must play a role in the management of the bed and its dependent fishery. Participating groups must agree on their respective formal roles, restrain themselves from "stepping be- yond"" their areas of expertise, and respect the role and viewpoints of the other participants. ACKNOWLEDGMENTS A large number of individuals contributed to the projects de- scribed in this paper. Three contributed more than the rest: Labo- ratory biologist, Donald Kunkle, and the two boat captains over the period from 1953 to 1990, William Richards and Clyde Phillips. In recent years, the supportive efforts of J. Dobarto and R. Reed of the New Jersey Bureau of Shellfisheries have been substantial. Financial support was received for much of this research from the State of New Jersey and from Public Law 88-309 funds. The authors thank Walt Canzonier for his comments and insight. This is New Jersey Agricultural Experiment Station Publication No. D-32403-1-03 and contribution no. 2003-19 from the Institute of Marine and Coastal Sciences, Rutgers University. APPENDICES /. An Example of Shellfish Council Deliberations Before the Advent of Large-Scale, Direct Marketing from the Seedbeds The following account describes deliberations by the Delaware Bay Shellfish Council during bay season of 1995. Oyster planters, representatives of the New Jersey State management agency, Rut- gers University personnel, and shellfish council members partici- pated in what was often a chaotic discussion. However, a consen- sus was reached. A cursory description is presented here to provide an example of the issues considered when making decisions and of how biologic information provided by the University was inte- grated with economic realities faced by planters. Bay season had begun on 10 April and was scheduled to last for a minimum of two weeks. A decision on the closing date was to be made near the end of the second week. On 20 April 1995, the shellfish council met to examine dredge samples that had been collected from the beds that day and to consider extending bay season. By that date approximately 3000 bu of oysters had been marketed directly from the beds at approximately $15-$ 17 per bushel. A little more than 20 boats harvested (seed for planting plus direct market) a total of about 100,000 bushels. Most of the harvest was from New, Bennies, and Bennies Sand Beds. Some harvest was from Ledge Bed. Sampling to determine percent oys- ter on the beds was conducted on 13 April and 20 April from Bennies Sand and New Beds, and from New Beds on 20 April. Mean percent oyster was high on both dates (Bennies Sand = 61% and New Beds = 63% on the 1 3th and New Beds = 62% on the 20"^). Although there was general agreement that plenty of oysters remained on all of the beds, several other concerns were discussed. First, prices for oysters marketed directly from the beds were low and only 3' oysters were acceptable. This meant that a good deal of costly on-board sorting was required to produce a marketable product. Second, as nearly all seed was infected with P. marinus, any oysters planted on the leased grounds would have to be mar- keted before July to avoid mortality. Third, the season had been Management of New Jersey's Oyster Seedbeds 463 good so far. Transplanting more oysters to the planting grounds would likely lead to decreases in profit because summer prices are usually low and the cost of moving oysters might not be recovered if subsequent mortality was high. Fourth, if oysters were not moved and they died on the seedbeds, at least the shells would remain as cultch. Fifth, if the beds remained open, everyone would keep fishing in spite of the economic risk. After listening to all these issues the shellfish council opted for a conservative strategy and decided to close the seedbeds for the season. //. An Example of Shellfish Couneil Deliberations After the Advent of Direct Marketing from the Seedbeds Direct marketing of oysters from the seedbeds has had mi.xed results. This process provided $4.3 million in revenues to the in- dustry for harvests in 1996. 1997. and the spnng of 1998, and allowed the industry to maintain a presence in the markets and maintain boats. Tag fees provided for an enhanced shelling effort. The down side to this form of landing was that the industry was restricted to the time period agreed to and could not stockpile oysters on the planted grounds to satisfy markets at other times. Because the oysters were harvested from lower salinity waters, the meat quality was not as good as in oysters from farther down bay and the price received for the product was not as high as it might have been. Chiefly because of these latter conditions, some indus- try members wished to plant oysters. The State achieved direct revenue ($1.25/bu) from oysters re- moved from the seedbeds for market, but would only receive pay- ment on planted oysters once they were landed. Thus in the former case the State (and directly the oyster industry accounts) received payment up front, while in the latter case the State took on the majority of the risk. If the oysters died on the planted grounds the resource would not be paid for. the shell would no longer be on the seedbeds, and no funds would have been generated to replace it. In 1997 the State and industry agreed to a spring direct harvest followed by an evaluation of the seedbeds to determine if a plant- ing season could be allowed in the summer of 1998. The chief reason for the planting would be to allow meat quality to improve during the late summer and fall. The chief worry was the level of the oyster disease Dernio. University researchers sampled for Dermo and reported to the council in an open meeting. Samples were removed in July from the five beds deemed by the industry to have the greatest probability of being harvested. The samples revealed that oysters on all beds were heavily infected with Dermo. The summer had been hot and dry and the forecast was for a continuation of these conditions. The discussion in the August 1998 council meeting was heated because some segments of the industry wished to move oysters anyhow, while others were reluctant to risk the resource. The latter group said that the resource would remain for later harvest if it was not moved. The group finally agreed to wait and monitor condi- tions further. Laboratory researchers took samples in August. Con- ditions had not improved and the Council deferred a seed move and decided to allow direct market harvest to begin (1500 bu/ license! beginning on 17 August. The council requested a Septem- ber sample of disease prevalence: it remained high. The council decided to have a 5-d transplant in an 8-d period beginning 7 October. To participate each boat would have to participate in a one day intermediate transplant (.5 and 6 October) in which oysters from up bay would be moved to an intermediate bed. Direct market harvest would cease when the transplant began. A meeting was scheduled for 1 October to make final adjustments to this plan. In October the direct market program allocation was increased by 1000 bu/license. otherwise the transplant program was to occur as decided earlier. As of the November council meeting the direct market program had landed approximately 73.000 bu: 10.000 bu were moved in the intermediate transplant and 58.800 bu were transplanted to the leases. Acheson. J. M., T. Stockwell & J. A. Wilson. 2000. Evolution of tlie Maine lobster co-management law. Marine Policy Rev. 9:52-63. Bosch. D. J. & L. A. Shabman. 1989. The decline of private sector oyster culture in Virginia: causes and remedial policies. Mur. Resource Econ. 6:227-243. Engle, J. B. 1953. Effect of Delaware River How on oysters in the natural seed beds of Delawaie Bay. Washington. DC: U.S. Fish and Wildlife Spec. Rep., 26 pp. FAO. 1995. Code of Conduct for Responsible Fisheries. COFI/95/2. Rome: Food and Agriculture Organization of the United Nations. 41 pp. Fegley, S. R.. S. E. Ford. J. N. Kraeuter & D. R. Jones. 1994. Relative effects of harvest pressure and disease mortality on the population dynamics of the eastern oyster {Crassostrea virginica) in Delaware Bay. NOAA Oyster Disease Res. Prog. Final Rep. Port Noms. NJ. 179 pp. Ford, S. E. 1996. Range extension by the oyster parasite Perkinsus marinus into the northeastern US: Response to climate change.' / Shellfish Res. 15:45-.56 Ford. S. E. 1997. History and present status of molluscan shellfisheries from Baraegat Bay to Delaware Bay. In: C. L. MacKenzie Jr.. V. G. Burrell Jr.. A. Rosenfield. & W. L. Hobart. editors. The history, present condition, and future of the molluscan fisheries of North and Central America and Europe. Vol. 1 . North America: U.S. Department of Com- merce, pp. 1 19-140. Ford, S. E. & H. H. Haskin. 1982. History and epizooliology of Huplospo- LITERATURE CITED ridiion nelson! (MSX). an oyster pathogen, in Delaware Bay. 1957- 1980. J. Invert. Pathol 40:1 18-141. Ford. S. E. & H. H. Haskin. 1987. Infection and mortality patterns in strains of oysters Crassostrea virginica selected for resistance to the parasite Haplosporidium nelsoni (MSX). / Parasitol 73:368-376. Ford. S. E. & H. H. Haskin. 1988. Management strategies for MSX (Hap- losporidium nelsoni) disease in eastern oysters. Am. Fish. Soc. Spec. Pub 18:249-256. Ford, S. E. & M. R. Tripp. 1996. Diseases and defense mechanisms. In: R. I. E. Newell. V. S Kennedy. A. F. Eble. editors. The Eastern Oyster Crassostrea virginica. College Park. MD: Maryland Sea Grant College, pp. 383^50. Ford, S. E., A. Schotthoefor & C. Spruck. 1999. In vivo dynamics of the microparasite Perkinsus marinus during progression and regression of infections in eastern oysters. J. Parasitol 85:273-282. Galperin. B. & G. L. Mellor. 1990a. A time-dependent, three-dimensional model of the Delaware Bay and River system. Part I : description of the model and tidal analysis. Esiuar. Coast. Shelf Sci 31:213-253. Galperin. B. & G. L. Mellor. 1990b. A time-dependent, three-dimensional model of the Delaware Bay and River system. Part 2: three-dimensional flow fields and residual circulation. Esiuar. Coast. Shelf Sci 31:255- 281. Haskin. H. H. & S. E. Ford. 1983. Quantitative effects of MSX disease {Haplosporidium nelsoni) on production of New Jersey oyster beds in Delaware Bay. USA. International Council for the Exploration of the Sea. CM 1983/Gen:7/Mini-Symposium. Goteborg. Sweden. Oct. 1983 464 Fegley et al. Haskin. H. H., L. A. Stauber & J. A. Mackin. 1966. Mmchiiua nelsoni sp. n. (Haplosporida, Haplosporiidae); causative agent of the Delaware Bay oyster epizootic. Science 153:1414—1416. Hannesson. R. 1996. Fisheries mismanagement; the case of the Atlantic cod. London: Fishing News Books. 160 pp. Harvell, C. D.. K. Kim. J. M. Burkholder. R. R. Colwell. P. R. Epstein. D. J. Grimes. E. E. Hofmann. E. K. Lipp. A. D. M. E. Osterhaus. R. M. Overstreet. J. W. Porter. G. W. Smith & G. R. Vasta. 1999. Emerging marine diseases — climate links and anthropogenic factors. Science 285: 1505-1510. Haven, D. S., W. J. Hargis & P. C. Kendall. 1978. The oyster industry of Virginia: its status, problems, and promise. Virginia Inst, of Marine Science. VIMS Special Papers Marine Sci. 4:1-1024. Jentoft. S.. B. J. McCay & S. C. Wilson. 1998. Social theory and fishenes co-management. Marine Policy 22:423^36. Jentoft, S. & B. J. McCay. 1998. User participation in fisheries manage- ment: lessons drawn from international experiences. Marine Policy 19:227-246. Kennedy, V. S. & L. L. Breisch. 1983. Sixteen decades of political man- agement of the oyster fishery in Maryland's Chesapeake Bay. /. En- viron. Manage 16:153-171. Maurer, D. & L. Watling. 1973. Studies on the oyster community in Dela- ware: the effects of the estuarine environment on the associated fauna. Int. Rev. Gesamplen. Hydrobiol 58:161-201. Maurer, D., L. Watling & R. Keck. 1971. The Delaware oyster industry: a reality? Trans. Am. Fish. Soc 1:100-111. McGoodwin, J. R. 1990. Crises in the World's Fishenes: People, Problems, and Policies. Stanford. CA: Stanford University Press. 235 pp. Miller. M. E. 1962. The Delaware oyster industry, past and present. Ph.D. dissertation. Boston University. Boston. MA. 329 pp. Nelson. J. 1913. Experiment in spat collecting. Fishing Gazette 30:515. Nelson, T. C. 1928. Relation of spawning of the oyster to temperature. £co/ogy 9:145-154. Nelson. T. C. 1929. Annual report for 1928 of the New Jersey Agncultural Experiment Station. New Brunswick, NJ, 105-112 pp. Nelson, T. C. 1947. Some contributions from the land in delermining conditions of life in the sea. Ecol. Monogr 17:337-346. Ragone-Calvo. L. & E. M. Burreson. 1994. Characterization of overwin- tering infections of Perkinsus marinus (Apicomplexa) in Chesapeake Bay oysters. J. Shellfish Res. 13:123-130. Schramm, H. L., Jr. & W. A. Hubert. 1996. Ecosystems management: implications for fisheries management. Fisheries 21:6-1 1. Shuster, C. N. 1959. A biological evaluation of the Delaware River estuary. Information Series, Publ No. 3. Wilmington. Delaware: Univ. of Dela- ware Marine Laboratories. 77 pp. Wallers. C. J. 1986. Adaptive Management of Renewable Resources. New York: MacMillan. 374 pp. Jotinuil ofShelljhh Rcscanh. Vol. 22. No. 2, 465-474, 20U3. INFLUENCE OF TIMING OF BROODSTOCK COLLECTION ON CONDITIONING, OOCYTE PRODUCTION, AND LARVAL REARING OF THE OYSTER, CRASSOSTREA GIGAS (THUNBERG), AT SIX PRODUCTION SITES IN FRANCE JORGE CHAVEZ-VILLALBA,'* JEAN BARRET." CHRISTIAN MINGANT,- JEAN- CLAUDE COCHARD,- AND MARCEL LE PENNEC' 'U.M.R. C.N.R.S. 6539. Institut Univcrsitaire Eitropeen de la Met: 29280 Plouzane. France: -IFREMER, Centre de Brest, Udnmitoire de Physiologie des Invertebres. BP 70. 29280 Plouzane. France ABSTRACT Ganietogenic development and response to conditioning procedures of six samples of oysters Crassoslrea gigas (Thunberg) collected in the Bassin d'Arcachon. each cultivated at a different production site along the Atlantic coast of France were compared simultaneously from December 1998 to July 1999. Oysters were conditioned with and without food (fed oysters and unfed oysters, respectively). Samples at northern production sites (Bale des Veys, Aber Benoit, and Baden) initiated gonadal development and spawning about one month earlier than those at southern production sites (Bouin. La Tremblade, and Arcachon). Three condi- tioning expenments (December 1998 to February 1999. February to April 1999. and April to June 1999) favored Bale des Veys and Aber Benoit oysters, because these resulted in higher body component indices and higher proportions of mature oocytes in the three conditionings that produced more gametes than the other samples in all expenments. Unfed oysters from Bale des Veys and Aber Benoit produced viable gametes and larvae in all the experiments. No significant difference was observed in larval culture (growth and mortality I among samples, of both fed and unfed animals. Differences in the timing of gametogenesis and response to conditioning among northern and southern samples seem adaptive and non-genetic in nature, since all oysters were collected from the same population in the bay at Arcachon. Nutrient recycling seems to have been an important regulating factor for gametogenesis in the northern samples. The occurrence of oysters in different locales having differences in the timing of gametogenesis and response to conditioning has implications for spat production in hatcheries. KEY WORDS: conditioning. Crassoslrea gigas. gametogenesis, larvae, oocytes INTRODUCTION The Pacific oyster Crassostrea gigas (Thunberg) was intro- duced to France to replace the Portuguese oyster C. angulata that was decimated by a virus in the 1970s (Heral 1989). The Pacific oyster exhibited high survival rates and adequate growth. Since 1982. spat importation from Japan and progenitors from British Columbia were no longer needed because the collection of juve- niles in French bays and lagoons became sufficient to sustain oyster cultures (Mann 1983). The oyster industry expanded exten- sively throughout this time and France became the fourth largest oyster producer in the world in 1994. At the present time, there is great inter-annual variability of spat setting at collection sites caused by collector overcrowding and hydroclimate variations (Robert & Gerard 1999). Moreover, the demand for spat increased continually over time, but hatcheries produced only 10% to 15% of the juveniles required by oyster farmers. Under these circum- stances, national management programs are directed at studying reproductive factors affecting this species under hatchery condi- tions to improve current spat production procedures. The culture of C. gigas in France is conducted in four stages; (I) spat collection; (2) intermediate culture; (3) culture; and (4) fattening (Cochard 1990). Juveniles (12 to 18 mo) are transferred from collection areas at Marennes-Oleron embayment and Arca- chon lagoon to production sites, where they continue development until reaching commercial size (24 to 36 mo later, depending on the region). In France, C. gigas reaches first sexual maturity in 12 *Corresponding author. Present address: Centro de Investigaciones Bi- ologicas del Noroeste, Guaymas Unit. (CIBNOR), A. P. 349. Guaymas, Sonera 85465. Mexico. E-mail: jechavez@cibnor.mx Fax: -1-52-622-221- 2238. to 18 mo (Soletchnik et al. 1997). This means that oysters can complete two reproduction cycles before the end of the harvest period at production sites. Since oysters are exposed to fluctuations in temperature, photoperiod. and quality and quantity of suspended fine particulate matter (seston) that affect their physiology and growth (Barille et al. 1994. Goulletquer et al. 1996). we would expect important geographic variations of gametogenesis along the French coast. Under laboratory conditions, many factors including those af- fecting gametogenesis and broodstock conditioning influence lar- val development in both early and late juvenile stages (Martinez et al. 2000). Le Pennec et al. (1998) pointed out that pectinid egg development and consequent larval production are extremely vari- able in hatcheries and that results are not reproducible from one year to the next. For C. gigas. Lannan et al. (1980) demonstrated that this variation is related to gonadal development of parental oysters and that this involved environmental and heritable com- ponents. Seasonal studies have shown that environmental factors, such as temperature and food availability, are closely related to reproductive performance in bivalves (Ruiz et al. 1992). For in- stance, the quantity of phytoplankton in temperate and high- latitude seas varies seasonally, producing cyclical changes in avail- ability of nutrients (Gabbott 197.'i. Abad et al. 1995). Regulatory substances with gonadotrophic action vary periodically, and they play important roles in spawning and in maturation of oocytes and adults (Deridovich & Reunova 1993). To obtain gametes in hatcheries in an optimum state of devel- opment, it is essential to know the gonadal stage of the parents at the time of conditioning, as well as the rate of gametogenesis during the conditioning intervals (Lannan et al. 1980). Moreover, during the conditioning of broodstock from different localities, seasonal variations in gonadal development must be identified 465 466 Chavez-Villalba et al. (Chavez-Villalba et al. 2002). The objective of this study is to discover the response to artificial conditioning procedures of oys- ters originating in Arcachon, but cultivated in six different geo- graphic regions of France. This was accomplished by simulta- neously comparing conditioning with and without food, oocyte production, and larval rearing during three consecutive periods of oyster cultivation from Bale des Veys (BV), Aber Benoit (AB). Baden (BA). Bouin (BO), La Tremblade (LT). and Arcachon (ARJ. MATERIAL AND METHODS Experimental Conditions At the end of November 1998, 150 oyster samples were taken from each of six production areas along the Atlantic coast of France, where they had been cultured in plastic bags on tables for almost two years. These oysters had been collected in the Bassin d' Arcachon, grown at Baden until they were 18 mo old. and dis- tributed in March 1 997 to the various culture sites. Two sites are situated along the English Channel: Bale des Veys (BV) in Nor- mandy and Aber Benoit (AB) in Brittany. Four are located along the Bay of Biscay on the Atlantic coast: Baden (BA), Bouin (BO). La Tremblade (LT) and Arcachon (AR) (Fig. 1). Specimens were obtained at the same time from all sites and transported immedi- ately to the IFREMER center at Brest, where they were placed in a tlow-through seawater system for one week. Temperatures in the tanks were maintained in close proximity to those found at the production sites at the time of collection; 10°C in December, 1 TC in February and 12°C in April. The procedure was conducted three times: December 98 (first conditioning), February 99 (second con- ditioning), and April 99 (third conditioning). Additionally, oysters were collected in June and July 99, but they were not conditioned because they were mature (Lango-Reynoso et al. 2000). After acclimation, each sample was divided into two groups for conditioning and transferred to seawater maturation tanks, where the temperature was increased 1°C per day until 19°C (heating period) and the photoperiod was adjusted to 16 h of daylight. Oysters were subjected to two conditions: with and without food. The fed groups had a diet used commonly for conditioning in experimental hatcheries: a mixture of two microalga species (10*^ cells of each species/day /animal) from monospecific cultures of Isochrysis aff. galbana Green (Clone T-iso: Tahitian Isochrysis) and Chaetoceros calcilrans Takano. The samples recovered in June were tested for histology only, and the samples collected in July were tested for histology and stripped immediately (see larval culture). Sampling Upon arrival at the laboratory, 20 oysters were chosen ran- domly from each sample for biometrical measurements. Weights of whole animals, empty shells, and soft tissue were determined to within ±1 mg using a digital balance. The soft tissues of 10 oysters were freeze-dried during 48 h, and dry weights were measured. The body component index of Walne and Mann (1975) was cal- culated: WMI-- DSTW* 1000 ' DSW where WMI is the Walne-Mann index, DSTW is the dry soft tissue weight in grams, and DSW is the dry shell weight in grams. Only fed oysters were considered for histologic study. Two samples of 10 oysters each were taken from each group during the conditioning experiments. The first sample was obtained before the heating period, and the second sample was taken at the end of the conditioning period. For individuals collected in June and July, the histologic samples were taken immediately after biometric measurements. Semi-quantitative Histologic Analysis Oysters used for histology were opened, and a section of ap- proximately 1 cm"* visceral mass was taken from above the peri- cardial area, and fixed in Bouin's solution for at least 48 h. Samples were dehydrated with a series of ethanol treatments of English Channel" "T^ Sale des^ys o\ » r— 1 . Normandy Aber BenoTt^^'^'*^ VO^S«^-^ _ ^ Brittany BadeiA^-n ATLANTIC Boul.j> OCEAN A ~ Bassin de Marennes-Oleron^ > La Tremblade ^ n Spat collection Bassin d'Arcachon 5 # Production sites 1 Figure L Location of the six Crassostrea gigas production sites studied. Spat collection and production sites are also marked. Influence of Timing of Broodstock Collection 467 increasing concentration, cleared in toluene, and embedded in par- affin following a standard procedure. Sections (5 (jini) were cut. mounted on glass slides, and stained with Groat's hematoxylin and eosin Y solution (Martoja & Martoja-Pierson 1967). The histology slides were examined under a microscope connected to a video camera to determin oocyte size and frequency, and gametogenic activity. Recorded images were processed by digital image analy- sis. Oocytes were measured and histology classified following the description by Lango-Reynoso et al. (2000). These operations were conducted on 100 randomly chosen oocytes per oyster, and measurements followed a standard bias reduction procedure for selecting measurement fields. Transects of gonad preparations were oriented to maximize coverage of the larger vertical or hori- zontal oocyte field axis. All oocytes with a well-defined germinal vesicle in a field were measured, and every oocyte measured was assigned to a reproductive stage based on diameter and histologic characteristics of the gonad (Table 1). Larval Rearing (larval yield estimation) Both fed and unfed oyster groups were considered for larval rearing. Following seven weeks of conditioning, oysters were opened and their sex was determined by observing a fresh smear sample from the gonad under a microscope. After this procedure, females and males were separated and gametes from both sexes were recovered using the scarification technique described by Allen and Bushek (1992). Gonads of all oysters were scarified by a light incision of the gonadal tegument. Oocytes were collected in beakers by rinsing the gonad with filtered seawater. Then the oocytes were passed through a 60-(a.m sieve to eliminate undesir- able material. Mature oocytes were retained in a 20-(xm sieve. These were rinsed several times and placed in 2 or 5-L beakers. To determine oocyte production, three 50-|jlL samples per group were examined, and counted under a profile projector. Males underwent the same procedure, but spermatozoa suspensions were examined under a microscope for mobility. Batches of spermatozoa of low mobility were discarded. A minimum of three batches was mixed together and diluted 10- to 20-ml P' for fertilization. Oocytes were fertilized in 3-L beakers, and checked for normal progress 0.5 to I h later (Robert & Gerard 1999). After fertilization, an equal number of embryos from all oysters of each group were pooled together and placed one group per tank in 150-L experimental tanks at concentration .^3 embryos per ml. After 48 h the tanks were emptied and the larvae recovered by sieving. Three 50-[xL larvae samples from each tank were taken for larval yield estimation: number of D larvae after 48 h of cul- ture/initial number of embryos. Standard methods were used during larval rearing. Tank sea- water temperature was maintained at 20°C throughout the experi- ment, and larval diet consisted of a mixture of three microalga species: 40% Chaetoceros pumilum. 40% l.soclirysis aff. gall'iiiui Green (Clone T-iso. Tahiti Isochiysix). and 20% Pavlova hiilicri. Feeding increased from an initial concentration of 80,000 cells/ml to a final concentration of 150,000 cells/ml. Seawater in the tanks was renewed every two days, and larvae were recovered by siev- ing. Larvae were measured during the second, ninth, and sixteenth days of culture by sampling I or 2 mL of seawater containing larvae from each experimental tank after sieving. Larval samples were placed on microplates and fixed with formaldehyde (5%). Two or three pictures of each sample were taken using a Scioncorp frame grabber and processed by image analysis for size evaluation (Scion Image for Windows). Larvae length was deemed equivalent to that of the major axis of the best-fitting ellipse. Data Analyses The oocyte proportion corresponding to each reproductive stage was calculated according to Lango-Reynoso et al. (2000). and arcsine transformed (Snedecor & Cochran 1972) for each oys- ter. The logarithms of oocyte production data were calculated. The transformed proportions and logarithms were compared using the Kruskal-Wallis test. A two-way ANOVA test was used to examine the effect of conditioning and sample on ( 1 ) early, growing, and mature oocyte categories; (2) the Walne-Mann index; (3) oocyte production; and (4) the D larval yield. A three-way ANOVA test was run to analyze the effect of conditioning, days of culture, and origin (sample) on larval culture. Statistics were analyzed at sig- nificance level a = 0.05. RESULTS Gametogenesis The results of oocyte evolution from December 98 until July 1999 are presented here without regard to conditioning experi- ments. The mean oocyte size for each sample is shown in Fig- ure 2. Oysters from the six samples had the same oocyte diameter distribution except the AB sample that had degenerating oocytes in December 1998. In this sample we detected that the proportion of oocytes in the early gametogenesis stage increased significantly (36.4-92.4%) at the same time as that of degenerating oocytes decreased significantly (44.8-0.0%) from December to February. TABLE L Reproductive scale for Crassostrea gigas proposed by Lango-Reyno.so et al. (2000). Each reproductive stage is based on an oocyte diameter (fim) interval. Cytological characteristics corresponding to each stage are included. Stage Interval (Mm) Histologic Description Early gametogenesis Growing Mature Degenerating 3.0-12.0 Follicles are elongated and often isolated in the abundant connective tissue, with walls consisting of primary oocytes of homogeneous size. 12.1-30.0 Start of oocyte growth. A large range in oocyte size at all gametogenic stages can be observed, including some free oocytes. Interfollicular connective tissue disappears. 30.1^1.0 Follicles of homogeneous size completely filled with mature oocytes with distinct nucleus. 41.1-60.0 Follicles containing degenerating oocytes, often elongated in shape, sometimes broken. Obvious redevelopment indicated by increased number of primary oocytes. 468 Chavez-Villalba et al. 50 40 30 20 10 BV a o AB I I I O 40 £ ra 30 0) 20 BA >» 10- o o O 50 40 30 20 10 — T- T I I I I I LT ^ I I I BO T I I I I I I 1 s AR T t 1 F=H=«= DJFMAMJJA DJFMAMJJA Time (1998 -1999) Figure 2. Evolution of oocyte diameters (mean ± SD) over time in Crassoslrea gif;as specimens collected at six production sites on the Atlantic coast of France: BV (Bale des Vevs), AB (Aber Benoit). BA (Baden), BO (Bouin), l,T (l.a Tremblade), and AR (Arcachnn). In the other samples, oocytes at this stage did not change signifi- cantly during the same period. In February, growing oocytes were observed only in the oysters from BV. AB. and BA (4.0. 8.0. and 3.0% respectively). In all samples, oocytes in growing and mature stages increased significantly from February to April, and from April to June, respectively. The BV. AB. and BA samples, which contained growing oocytes in February, were the same samples in which mature oocytes decreased significantly from June to July. The oocytes in the early gametogenesis and growing stages in- creased significantly in the BV (2.4-28% and 18.6^7.6% respec- tively) and AB (0.0-3.'^. 27r and 1.3^9.0% respectively) samples during the same period. In the other samples (BA. BO. LT and AR). no significant change was detected from June to July. Conditioning The results of the three conditioning procedures performed in this study are presented in Figure 3. In the BV sample, the pro- portion of growing oocytes increased significantly in the first (3.0- 18.8%) and second (3.7-30.7%) conditionings, and in the last ex- periment the proportion of oocytes in this category decreased sig- nificantly (57.0-7.0%). The proportion of mature oocytes increased significantly in this sample in all the experiments. By the end of the first conditioning, degenerating oocytes were no longer observed in the AB sample, and there was no significant change in growing oocytes, but the proportion of mature oocytes increased significantly in the three conditionings. For the BA and BO samples, the same pattern as for the AB sample was observed, except during the second conditioning experiment when the pro- portion of growing oocytes increased significantly (2.9-18.0% in BA. and 0.0-27.2% in BO). The proportion of growing oocytes also increased significantly in the second conditioning for the LT sample (0.0-37.2%), but that of mature oocytes increased signifi- cantly only in the second (0.0-41.5%) and third (0.0-89.7%) ex- periments. Finally, the proportion of AR sample growing oocytes increased significantly during the first (0.0-39.5%) and the second (0.0-21.8%) conditionings but the proportion of mature oocytes increased significantly only in the last experiment (7.0-88.2%). The two-way ANOVA test on early gametogenesis, growing, and mature oocytes showed that the effect of the sample was not significant in any conditioning experiment, but the effect of con- ditioning was evident on the proportion of early gametogenesis stage oocytes, which increased significantly in the first and the second conditionings. No significant conditioning effect was de- tected on oocytes at the growing stage during the three experi- ments. Finally, conditioning increased the proportion of mature oocytes significantly during the second experiment. Index Values of the Walne-Mann index (WMI) are presented in Fig- ure 4. The WMI increased significantly in BO (3rd experiment), LT (2nd and 3rd experiments) and AR (1st and 3rd experiments) by the end of conditioning. In the other samples no significant difference was detected between the start and end of conditioning. The two-way ANOVA test concerning the effect of condifioning showed that WMI values in the third experiment were significantly higher than those in the two previous conditionings, but these Influench of Timing of Broodstock Collection 469 120 80 120 >> 80 u C 40 0) 3 0 CT '20 0) ^ 80 First conditioning S E A^..^^^ Second conditioning S E .^^ -^^ A l/L-.^ k i/k l\ |/ Wo>^ Third conditioning S E .^^'^^^ BV --^^ AB BA .-J\ BO LT /^es=^ AR 40 $0 0 40 60 40 60 0 20 40 60 Oocyte diameter {\im) Figure 3. Temporal variation in oocyte diameter of specimens conditioned with food, from six sites of Crassostrea gigas production durin;- December 1998 to February 1999 (tlrst conditioning); February to April 1999 (second conditioning); and April to June 1999 (third conditioning). Each line represents one individual. S (start of conditioning). E (end of conditioning), BV (Bale des Veys), AB (Aber Benoit), BA (Baden), BO (Bouin). LT (La Tremblade), and AR (Arcachon). values were significantly lower than those of oysters collected in July. The WMI values of oysters from the AB sample were sig- nificantly higher than those from the other samples, except BV. Gamete Production The highest gamete production was observed in oysters col- lected in July except for those of BO, conditioned in April to June (Fig. 5). The two-way ANOVA test showed that there were sig- nificant conditioning and sample effects on gamete production. We observed that gamete production increased significantly from the first to the second experiment, and also from the second to the third conditioning. No difference between the last conditioning and that of oysters collected in July was observed. The AB sample pro- duced significantly more gametes than that of BO. We observed that only the unfed groups of AB and BV pro- duced gametes during the first conditioning, and that the quantity was significantly higher in AB oysters. In the second conditioning, four groups produced gametes, and are ranked by gamete quality as follows: AB, BV. BA. and BO. The AB and BV groups pro- duced significantly more oocytes than the BA and BO groups. In the third conditioning, two-way ANOVA revealed significant ef- fects of conditioning and sample on gamete production. All groups produced significantly more gametes except LT. which produced no oocytes during the three conditioning procedures. AB oysters produced significantly more gametes than any other group (Fig. 5). D Larval Yield The two-way ANOVA test showed no significant effect of conditioning or sample on D larval yield of either fed or unfed oysters. D larval yield for fed and unfed oysters during the three conditioning experiments, as well as that for animals collected in July are presented in Figure 5. During the first conditioning, the highest larval yield (80%) was observed for fed oysters from BV, while the lowest corresponded to those of AB and BA (28 and 22%. respectively). In the second conditioning, the BO group had the highest percentage (90%), while the lowest (51%) was ob- served for LT oysters. In the third conditioning, and for oysters collected in July, we found that larval yield was homogeneous (=60%) for all groups. We had technical problems with unfed oysters during the second experiment, so the D larval yield was not measured and consequently, no larvae were reared. Nevertheless, we observed no significant difference in larvae yields between fed and unfed oysters during the first and third conditionings. Larval Growth Three-way ANOVA demonstrated significant effects of condi- tioning and time on size of larvae produced by both fed and unfed oysters (Fig. 6). Larvae from fed oysters, were significantly larger in the third conditioning than in the tlrst or second conditionings. The first conditioning of unfed animals in groups BV and AB were larger also. There was no significant sample effect on larval size of fed or unfed animals. We compared the size of larvae from fed and unfed oysters, and those of oysters collected in July on the last day of culture (16th day), and found no significant difference. DISCUSSION The gametogenic development of Crassostrea gigas in this study is similar to that reported by Lango-Reynoso et al. (2000) for two populations in Brittany and one in Marennes-Oleron. We observed that the gametogenic cycle (December 1998 to July 470 Chavez-Villalba et al. 120 1 100 80- X 0) •a c 0) c 5 60- 40- 20 Start of conditioning I I 1 Irt 1 jA^ xu x IJ 120- 100- 80- 60 40 -f 20 0 End of conditioning 123N 123N 123N 123N 123N 123N BV AB BA BO LT AR Oyster samples Figure 4. Walne-Mann indices (WMI I of specimens conditioned « ith food, troni six sites of Crassostrea gigas production during December 1998 to February 1999 ( 1 1, February to April 1999 (2|. April to June 1999 (3). and July 1999 (M. BV (Bale des Veysl, AB ( Aber Benoit), BA (Baden), BO iBouinl. LT (La Tremblade) and AR (Arcachonl. An asterisk (*) represents a significant difference between WMIs at the start and end of conditionings in a Kruskal-Wallis test [P < 0.05). 1999) of all samples examined in this study followed the same pattern. Primary oocytes were evident from December to February. In the Aber Benoit sample, a large proportion of degenerating oocytes (457^) were detected in December, but not in February. Degenerating oocytes occur because the oysters at this site have partial spawnings from September to January, and gametes in the gonad are reabsorbed very slowly (Chavez-Villalba et al. 2001 ). Histologic observations show that only northern samples BV, AB, and BA had growing oocytes in February. Oocytes grew in all groups from February until maturity in June, and there were al- ways more than 75% mature oocytes. Histologic observations show that northern oysters spawned only partially between June and July, and that the proportions of mature oocytes were 25%. 18%. and 52%, respectively. Moreover, we detected primary and growing oocytes in the gonads of these samples during the same period, indicating the development of a new oocyte generation. In contrast, the proportion of mature oocytes of southern samples BO. LT. and AR continued above 80%. Lango-Reynoso et al. (2000) found that oysters at northern sites initiated gonad growth, achieved maximal gonad development, and began spawning about one month earlier than oysters from Marennes-Oleron. The results of this study and those of previous experiments in our laboratory (Chavez-Villalba 2001 ) were consistent with the observations of Lango-Reynoso et al. (2000). Differences between northern and southern oysters in the timing of gametogenesis during the condi- tioning experiments revealed that northern samples perfomied best in laboratory conditions. These oysters in the three conditionings presented higher Walne-Mann index values and higher propor- tions of mature oocytes and produced more oocytes than other samples in all experiments. Moreover, unfed BV and AB oysters produced viable gametes and larvae in all experiments. This dem- onstrates that differences between northern and southern sites in environmental influences regulate the initiation or completion of gametogenesis. Differences between populations in the term and extent of go- nad growth, apan from genetic differences, suggest the existence of environmental factors regulating gonad development (Barber et al. 1991). The differences found in this study should not be con- sidered genetic since all juvenile oysters were collected in the Bassin d'Arcachon. Thus, we believe that there is intraspecific variation in gametogenesis of C. gigas in France that is an adap- tation to different local environmental factors. Dinamani (1987) stated that the pacific oyster shows flexible reproductive behavior that includes changes in timing and length of gametogenesis de- pending on the environment in various regions of the world. It is known that water temperature is a principal environmental factor affecting gonad development in marine bivalves (Loosanoff & Davis 1963). Goulletquer and Heral (1997) pointed out that the temperate climate in France is affected by the Gulf Stream, with a geographic barrier around Brittany, limiting the distribution of marine species between the coldest regions in the north and warm- est in the south. The fact that oysters from northern locations Influence of Timing of Broodstock Collection 471 Gamete production x 10® D larval yield (%) 80 70 60 SO 40 30 20 10 0 80 70 60 50 40 30 20 10 0 80 70 80 50 40 30 20 10 0 80 70 60 50 40 30 20 10 0 First conditioning 100 ^ fl^ rl, gl- ^^ ^ -^ 1 1 a CL £D_ X 1 i w wo w wo BV AB w wo w wo BA BO Oyster samples Figure 5. Gamete production and D larval yield of specimens collected at the end of each conditioning with IW) and without (WO) food, at six sites of Crassostrea gigas production during December 1998 to February 1999 (first conditioning), February to April 1999 (second conditioning), April to June 1999 (third conditioning), and July 1999. BV (Baie des Veys), AB (Aber Benoit), BA (Baden), BO (Bouin), LT (La Tremblade), and AR (Arcachon). acclimated to lower temperatures than those from the south and began gonad growth earlier, eliminates temperature as the single regulator of gonad development in C. gigas. Goulletquer and Hera! (1997) indicated that another difference between northern and southern locations is variations in trophic conditions caused by tidal effects. Tidal cycles can vary markedly in the quality and amount of suspended particulate matter (Pastoureaud et al. 1996). We believe that differences detected in this study result from varia- tion in stored reserves that depend on food availability (Thompson et al. 1996). This view is supported by MacDonald and Thompson ( 1988). who reported site-specific variation in the gonad develop- ment of Placopecten inagellanicus. due to adaptation to local variations in environmental factors, most notably food availability. There is evidence that periods of reserve accumulation and gamete production are temporally separated in temperate species (Emmett et al. 1987, Thompson & MacDonald 1990). Berthelin et al. (2000) found that re.serves in C. gigas are constituted during the autumn and the winter, and that these reserves are used later in 472 Chavez-Villalba et al. N '55 (0 250 200 150 100 50 250 200 150 100 50 Fed oysters Unfed oysters Bale des Veys La Tremblade Arcachon 16 2 Time (days) 16 Figure 6. Change in larval size until day 16 of culture of specimens conditioned with and without food, from six sites of Crassoslrea gigas production during December 1998 to February 1999 (1). February to April 1999 (2), April to June 1999 (3), and July 1999 (N). gametogenesis. This suggests greater food accessibility that fa- vored nutrient accumulation in oysters from northern locations. Some considerations for assuming this are for example, that the region of Baie des Veys is a high carrying-capacity ecosystem (Goulletquer et al., 1996) and that the national production program of C. gigas in France gets the highest meat yield per year from the oysters of Aber Benoit (Goyard 1997). In contrast. Bouin oysters have poor growth rates and low biologic yields compared with the rest of French oyster production (Gerard, 1995). Heral et al. ( 1986) found evidence of biologic overload in the Marennes-Oleron basin (La Tremblade) produced by a huge oyster biomass (95,000 tons), and Pastoureaud et al. ( 1996) indicated low seston quality encoun- tered by oysters in this bay. Barber and Blake (1983) suggested that potential food supply for the scallop Argopeaen iiradians decreases with latitude and that metabolic rate increases with tem- perature. The metabolic rate in the Japanese oyster increases with temperature (Bougrier et al. 1995). Previous observations suggest that the metabolic rate of C. gigas increases with decreasing lati- Influence of Timing of Broodstock Collection 473 tude. bill there is less food that results in less energy for repro- duction. It was significant that the best laboratory performance coin- cided with partial spawning in nature. Ropert (1999) reported that ovsters in Baie des Veys have a partial spawning during their reproductive cycle, and Cha\ez-Villalba et al. (2001) found that oocytes left from the incomplete spawning of Aber Benoit oysters are slowly reabsorbed from September to January. This led us to think that apart from the advantage of ambient food at northern sites, it is possible that nutrient recycling from reabsorption of unreleased gametes within the gonad is a regulating factor in the timing of gametogenesis. Post-spawning reabsorption has been observed in C. gigas. in which gametes remaining after spawning are reabsorbed (Steele. 1998). Beninger and Le Pennec (1991) suggest that reabsorption of residual gametes leads to nutrient rec>cling in scallops. Le Pennec et al. (1991 1 found evidence for lipid catabolism during reabsorption of unreleased gametes in Pecten maximus and they suggested that the products of these catabolic activities could be stored as glycogen. This was con- firmed by several investigators, including Berthelin et al. (2000). and could indicate that nutrients in northern oysters could be re- cycled from residual gametes during the autumn-winter period and then used for gametogenesis. It would be interesting to compare the conditioning response of northern oysters maintained in natural conditions throughout the year with those returned to natural con- ditions in the autumn or winter after artificial spawn in July or August. We observed greater gamete production in fed oysters than unfed. Robinson ( 1992) found comparable results when comparing gamete production of C. gigas oysters maintained with and without food. However, in our study the D larva yield of animals condi- tioned with and without food was close, in particular for northern oysters. Moreover, we found that there is no difference in larval growth without considering broodstock culture conditions. It seems that these oysters maintain oocyte quality by reducing their number when there is not enough food. Gametogenic cycles in bivalves are strongly tied to glycogen stocking cycles and to ultimate synthesis, de novo, of lipids during spring \itellosenesis. which depends on stocked glycogen (Gab- bott 1975). Interruption of these cycles, due to artitlcial condition- ing at high temperature, might force oocyte development before sufficient glycogen has been accumulated for lipid synthesis. The consequence might be production of few gametes with low bio- chemical quality (Gallager & Mann 1986). Our observations sug- gest that the stocking reserve in unfed oysters allows production of fewer gametes of high quality. It seems that viability and survival of reared larvae are directly related to the initial quantity of lipids during gamete emission (Holland & Spencer 1973, Gallager & Mann 1986). Apparently, unfed BV and AB oysters maintain their lipid stock during conditioning, probably due to large glycogen reserves, which assures not only lipid synthesis but also gamete development. When comparing lar\al development in the three conditioning experiments, we observed that larval growth of fed and unfed oysters is significantly inferior during the first two experiments. Although Lannan et al. (1980) showed the importance of season in the timing of broodstock collection for artificial conditioning, they had no explanation concerning mechanisms that govern egg qual- itv and the variability of survival during larval rearing. However, Gallager and Mann (1986) noticed that growth and survival of Mercenaria mercenaria and C. virginica larvae were associated directly with the initiation and duration of conditioning. Berthelin (2000) found that glycogen stores in the gonads of C. gigas during autumn and the beginning of winter remained low in spring, while proteins and lipids increase significantly from March to April, coincident with the first phytoplankton blooms. Results of condi- tioning during December to February suggest that reserves used for larval growth in fed and unfed oysters were accumulated in autumn and winter, and reserve allocation during spring increased fecundity and larva growth but not necessarily D larval yields. Knowledge of the general condition of animals before exposure to experimental conditions is important to obtain gametes in the optimum state of development. This study shows that the stored reserves of northern oysters allow them to perform better during conditioning than southern oysters. The existence of oysters hav- ing distinct gametogenic development and therefore distinct re- sponses to conditioning has implications for oyster spat production in hatcheries. Broodstock from northern locations can be condi- tioned starting in December because they mature after six weeks of exposure at elevated temperatures (19°C). According to Chavez- Villalba et al. (2002) this occurs because 60% of oocytes in the gonad are mature after conditioning. The response of these oysters to artificial conditions can be maintained throughout gametogenic development, whereas the oysters from southern locations mature only after commencing conditioning in April. These conditioning experiments suggest that using oysters from northern locations in hatchery operations should result in substantially increased hatch- ery production. ACKNOWLEDGMENTS The authors thank CONACYT (Mexico) for a scholarship grant to Jorge Chavez-Villalba for PhD studies at Universite de Bretagne Occidentale, France. This work was supported by the project IFREMER/Contrat Uni\ersitaire LTBO, No. 98/2521426. Editing staff at CIBNOR reviewed and improved the English text. LITERATURE CITED Abad. M.. C. Ruiz, D. Martinez. G. Mosquera & J. Sanchez. 1995. Sea- sonal variations of lipid classes and fatty acids in flat oyster. Ostrea edulis, from San Cibran (Galicia, Spain). Camp. Biochem. Physiol. 110:109-118. Allen, S. K. & D. Bushek. 1992. Large-scale production of triploid oysters Crassostrea virginica (Gmelin) using "stripped" gametes. .Acjuaculuire 103:241-251. Barber, B. J. & N. J. Blake. 1983. Growth and reproduction of the bay scallop, Argopeaen irradiuns (Lamarck) at its southern distributional limit. J. Exp. Mar. Biol. Ecol. 66:247-256. Barber. B. J.. S. E. Ford & R. N. Wargo. 1991. Genetic variation in the timing of gonadal maturation and spawning of the eastern oyster, Cras- sostrea virginica (Gmelin). Biol. Bull. 181:21(3-221. Barille, L., S. Bougrier. P. Geairon & M. Robert. 1994. Alimentation experimentale de I'huitre Crassostrea gigas a I'aide de navicules bleues Haslea ostrearia (Simonsen) de differentes tallies. Oceanol. .\cta 17:201-210. Beninger, P. G. & M. Le Pennec. 1991. Reproductive System. In: S. E. Shumway. editor. Scallops. Biology. Ecology, and Aquaculture. Am- sterdam: Elsevier, pp. 133-223. 474 Chavez-Villalba et al. Berthelin, C. 2000. Etude du metabolisme du glycogene chez I'huitre creuse Crassostrea gigus. Impact sur la reproduction et les mortalites estivales. Doctoral Thesis. Caen. France: Universite de Caen Basse- Normandie. 156 pp. Berthelin. C. K. Kellner & M. Malhieu. 2000. Storage metabolism in the Pacific oyster (Crassoslrea gigas) in relation to summer mortalities and reproductive cycle (West coast of France). Coinp. Biochem. Physiol. 125:339-369. Bougrier, S.. P. Geairon. J. M. Deslous-Paoli. C. Bacher & G. Jontjuieres. 1995. Allometric relationships and effects of temperature on clearance and oxygen consumption of Crassostrea gigas (Thunberg). Aquacul- ture 134:143-154. Chavez-Villalba. J. 2001. Condilionnenient experimental de I'huitre Cras- sostrea gigas. Doctoral Thesis. Brest, France: Universite de Bretagne Occidentale. 186 pp. Chavez-Villalba, J., C. Mingant, J. C. Cochard & M. Le Pennec. 2001. Gametogenese chez I'huitre Crassostrea gigas de I'Aber Benoit (Bretagne. France), a la limite nord de son aire de reproduction. Hali- otis 30. pp. 1-12. Chavez-Villalba. J.. J. Pommier. J. Andriamiseza, S. Pouvreau. J. Barret. J. C. Cochard & M. Le Pennec. 2002. Broodstock conditioning of the oyster Crassostrea gigas: Origin and temperature effect. Aquaculture 214:115-130. Cochard. J. C. 1990. Presentaciiin del cultivo de moluscos en Francia. Actas del III Congreso Nacional de Acuicultura. pp. 941-955. Dendovich, I. I. & O. V. Reunova. 1993. Prostaglandins: Reproduction control in bivalve molluscs. Camp. Biochem. Physiol. 104:23-27. Dinamani, P. 1987. Gametogenic patterns in populations of Pacific oyster. Crassostrea gigas, in Northland, New Zealand. Aquaculture 64:65-76. Emmett. B., K. Thompson & D. Popham. 1987. The reproductive and energy storage cycles of two populations oi Mytihis edulis (Linne) from British Columbia. J. Shellfish Res. 6:29-36. Gabhott. P. A. 1975. Storage cycle in marine bivalve molluscs: A hypoth- esis concerning the relationship between glycogen metabolism and ga- metogenesis. Proceedings of the 9th European Marine Biology Sym- posium, pp. 191-21 1. Gallager, S. M. & R. Mann. 1986. Growth and survival of larvae oi Mer- cenaria mercenaria (L.) and Crassostrea virginica (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aquaculture 56: 105-121. Gerard, A. 1995. Genetique. aquaculture et pathologic (La Tremblade- Bouin). France: Rapport IFREMER (DRV/RA/GAP). 89 pp. Goulletquer. P.. J, P. Joly. A. Gerard. E. Le Gagneur. J. Moriceau. J. M. Peignon. S. Heurtebise & P. Phelipot. 1996. Performance of tnploid Pacific oysters Crassostrea gigas (Thunberg) reared in high carrying capacity ecosystem: survival, growth and proximate biochemical com- position. Haliotis 25:1-12. Goulletquer, P. & M. Heral. 1997. Marine molluscan production trends in France: From fisheries to aquaculture. In: US Dept. Comm., NOAA Tech. Rep. NMFS 129. pp. 137-164. Goyard. E. 1997. Reseau du suivi de la croissance de I'huitre creuse sur les cotes fran(jaises. REMORA-Resultats Nationaux. France: Rapport In- terne DRV-IFREMER. 28 pp. Heral, M., J. M. DesIous-Paoli & J. Prou. 1986. Dynamiques des produc- tions et des biomasses des huitres creuses cultivees (Crassostrea an- gulata et Crassostrea gigas) dans le bassin de Marennes-OIeron depuis un siecle. CIEM CM. 1986/F:41. Heral. M. 1989. Traditional oyster culture in France. In: G. Bamabe. editor. Aquaculture I. Paris: Lavosier. pp. 342-387. Holland. D. L. & B. E. Spencer. 1973. Biochemical changes in fed and starved oysters. Ostrea edulis L. during larval development, metamor- phosis and early spat growth. J. Mar. Biol. Assoc. UK. 53:287-298. Lango-Reynoso, F., J. Chavez-Villalba. J. C. Cochard & M. Le Pennec. 2000. Oocyte size, a means to evaluate the gametogenic development of the Pacific oyster. Crassostrea gigas (Thunberg). Aquaculture 190: 183-199. Lannan, J. E., A. Robinson & W. P. Breese. 1980. Broodstock management of Crassostrea gigas II. Broodstock conditioning to maximize larval survival. Aquaculture 21:337-345. Le Pennec. M., P. G. Beninger. G. Dorange & Y. M. Paulet. 1991. Trophic sources and pathways to the developing gametes of Pecten maximus (Bivalvia:Pectinidae). / Mar. Biol. Assoc. UK. 71:451^63. Le Pennec. M., R. Robert & M. Avendafio. 1998. The importance of gonadal development on larval production in pectinids. J. Shellfish Res. 17:97-101. Loosanoff. V. & H. C. Davis. 1963. Rearing of bivalve molluscs. Ai^v. Mar. Biol. 1:1-136. MacDonald. B. A. & R. J. Thompson. 1988. Intraspecific variation in growth and reproduction in latitudinally differentiated populations of the giant scallop Plucopecten magelhmicus (Gmelin). Bu>l. Bull. 175: 36-371. Mann. R. 1983. The role of introducing bivalve mollusks species in mari- culture. / World Mancul. Soc. 14:546-559. Martinez. G.. C. Aguilera & L. Mettifogo. 2000. Interactive effects of diet and temperature on reproductive conditioning of Argopecten pupuratus broodstock. Aquaculture 183:149-159. Martoja, R. & M. Martoja-Pearson. 1967. In: Mason et cie, editor. Initia- tion aux techniques de I'histologie animale. Paris. 1232 pp. Pastoureaud. A.. M. Heral. P. Prou, D. Razet & P. Russu. 1996. Particle selection in the oyster Crassostrea gigas (Thunberg) studied by pig- ment HPLC analysis under natural food conditions. Oceaiiol. Acta. 19:79-88. Robert. R. & A. Gerard. 1999. Bivalve hatchery technology: The current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France. Aquat. Living Resour. 12:121-130. Robinson, A. 1992. Dietary supplements for reproductive conditioning of Crassostrea gigas kumamoto (Thunberg). I. Effects on gonadal devel- opment, quality of ova and larvae through metamorphosis. J. Shellfish Res. 11:437-U1. Ropert, M. 1999. Etude des mortalites ostreicoles de I'hiver 199S/1999 en Bale des Veys. Rapport d'Etude. Convention Region/IFREMER (No. 079419). Ruiz. C. M. Abad. F. Sedano. L. Garcia-Martin & J. Sanchez-Lopez. 1992. Influence of seasonal environmental changes on the gamete pro- duction and biochemical composition of Crassostrea gigas (Thunberg) in suspended culture in El Grove. Galicia. Spain. J. Exp. Mar. Biol. Ecol. 155:249-262. Snedecor. G. W. & W. G. Cochran. 1972. Statistical Methods. Ames: Iowa State College Press. 593 pp. Soletchnik, P., D. Razet. P. Geairon, N. Faury & P. Goulletquer. 1997. Ecophysiologie de la maturation sexuelle et de la ponte de I'huitre creuse Cras.sostrea gigas: reponses metaboliques (respiration) et ali- mentaires (filtration, absorption) en fonction des differentes stades de maturation. Aquat. Living Resour. 10:177-185. Steele. S. 1998. The sexual cycle of the Pacific oyster Crasso.strea gigas in Southern Ireland. Doctoral Thesis. Ireland: University of Cork. 2 1 5 pp. Thompson. R. J. & B. A. MacDonald. 1990. The role of environmental conditions in the seasonal synthesis and utilization of biochemical en- ergy reserves in the giant scallop. Placopecten magellanicus. Can. J. Zooi 68:750-756. Thompson. R. J.. R. I. E. Newell. V. S. Kennedy & R. Mann. 1996. Reproductive processes and early development. In: V. S. Kennedy, R. I. E. Newell & A. F. Eble. editors. The Eastern Oyster Crassostrea virginica. Maryland: Maryland Sea Grant Book. pp. 335-370. Walne. P. R. & R. Mann. 1975. Growth and biochemical composition in Ostrea edulis and Crassostrea gigas. In: H. Barnes, editor. Ninth Eu- ropean Marine Biology Symposium. Scotland: Aberdeen University Press, pp. 587-607. Jounuil of Shellfish Research. Vol. 22, No. 2. 475-479, 2()().V APPEARANCE AND PATHOGENICITY OF OVARIAN PARASITE MARTEILIOIDES CHUNGMUENSIS IN THE FARMED PACIFIC OYSTERS, CRASSOSTREA GIGAS, IN KOREA MI SEON PARK,* CHANG-KEUN KANG, DONG-LIM CHOI. AND BO-YOUNG JEE National Fisheries Research & Development Institute. Sirani;-ri, Gijang-Guu. 619-902 Biisan, Republic of Korea ABSTRACT The ovarian parasite Marlcilioidcs chuui;mucnsis that ijilects the ovaries of Pacific oyster Crassostrea gigas ha.s increased in frequency in farmed oysters on the southern coast of Korean peninsula since the early 1990.S. The appearance and pathogenicity of the ovarian parasite in the farmed oyster in Jinhae Bay, Korea, were investigated in 1996 and 1997. Infection by M. chiingiiiucnsis was highest during spawning (from June to August) and gonadal regenerating .season of the oysters (from September to October), with prevalences ranging from 1,1. ,1 to 57.1% in 1996 and from 28.6 to 6l.59f in 1997, respectively. The surveyed oysters showed signs of recovery from the infection after October. Glycogen levels were considerably lower in M. chiingiiuieiisis-\nfected oysters those that of uninfected oysters. A rapid accumulation of glycogen was observed in uninfected oysters together with the gonadal regeneration after the summer spawning. By contrast, no increase in glycogen content was found in infected oysters until the end of the investigation. Lipid levels were slightly higher in the infected oysters than in the uninfected oysters. Serum protein concentrations were significantly lower in the infected oysters than in the uninfected oysters. Also, the increase of serum protein concentration after the summer spawning was apparent in the uninfected oysters but not in the infected oy.sters. These results indicate that the infections by M. chungimiensis may have an adverse impact on metabolic recovery after spawning of the oysters. A'£)' WORDS: Pacific oyster, Crassoslrea gigas. ovarian parasite, Marteilioides chungmiiensis INTRODUCTION The ovarian parasite Marreilloiiles chiingiiuien.^i.'^ is found in the ovaries of the Pacific oyster, Crassostrea ,?(,?fl.s. The parasite is a Protozoan belonging to the Phylum Ascetospora Sprague (Comps et al. 1986, Park & Chun 1989). It is found in the cyto- plasm of the ovum and measures 3-3,5 |jim in diameter (Comps et al. 1986). The effects of the ovarian parasite on the growth of oysters have been studied for the last 10 years. Park et al. ( 1999) reported that the parasite could induce ovary necrosis and prohibit normal growth of fertilized eggs. Oysters infected by this parasite show grossly visible ovary deformations, with lump-like hypertrophy that renders them unmarketable. Therefore, the parasite is consid- ered one of the most serious problems for oyster production in Korea, The parasite has been found in oysters from almost all oyster culture areas of Korea (Chun 1970, 1979. Park & Chun 1989, Park et al. 1999), although prevalence and intensity of in- fection vary with region and season. Infection levels along the southern coast have increased since 1990 and the oyster industry in this area is facing increasing production challenges as a result of poor seed collection. Studies of M. cliiiii^mia'nsis have concentrated on histopathol- ogy, infection dynamics, and transmission pathways, but little work on the effects of the parasite on the physiology of the oyster has been conducted. To better understand the effects of this ovar- ian parasite on oyster aquaculture, this study examined the rela- tionship between M. cluiiii>niuensis infection on the oysters and gonad regeneration and its related physiologic parameters, MATERIALS AND METHODS Collection of Oyster Broodstock Oysters were collected from the culturing sites around Chil- cheon Island in Jinhae Bay of Korea (Fig. I). Although many *Corresponding author. E-mail: parkms@nfrdi.re.kr oyster beds still operate in this bay, sulTicient seed for stocking purposes produced no longer in this formerly productive seed col- lection area since 1 990. For broodstock sampling, one oyster string was divided into three sections (upper, middle, and bottom), and 30 individuals were collected monthly from each section. Examination of M. chungmuensis Infection Levels Sampled broodstock were washed with clean seawater and shucked by hand. A 3-mm thick dorsoventral cross section through the anterior thiid of the soft tissues was fi,\ed in Davidson solution and processed for light microscopy. Paraffin-embedded sections (4 jxm) were stained with Harris' hematoxylin-eosin for microscopic examination. Because early stages of infection are difficult to de- tect by light microscope, additional tissue smears were made from ovarian tissues and stained with eosin-methylene blue (Fig, 2). Evaluation of Infection Levels and Oyster Condition Factors The oyster samples were divided into two groups, infected and uninfected, based on gross evidence of M, chungmuensis infec- tions, to evaluate infection effects on specific oyster condition parameters. To have an accurate gross indication of infection, oys- ters were collected from May to September when infections are most obvious to the naked eye (Fig. 3). Infection levels were divided into two levels by observation of slides that were smeared with reproductive tissues and stained with eosin-methylene blue (H group: heavy infection of >50'yf prevalence; M group: moderate infection of <50'7r prevalence). Of condition parameters, glycogen content (excluding ovarian tissues) was measured using the method of Whyte and Englar (1982); biochemical composition of the meat using the AOAC method (1990); and serum protein con- centrations using the Lowry method (1951). RESULTS M. chungmuensis Infection Levels Prevalences of infection from 1996 to 1997 ranged between 0.0 to 61.5% (Fig. 4), Infection levels of M, chungmuensis were high- 475 476 Park et al. K ORE A A ^^ (^ ■n\ .o. 128°10' 128° 30- Figure 1. Map showing sampling site in Jinhae Bay, Korea. 128°:50-E 34°55'N 34° 50- =J34°45' est in September 1996 (57.1%) and in August 1997 (61.5%). No infections were detected from January to April 1996. In 1997, the parasite was detected all the year round, with highest prevalences in August. Correlation Condition Factor to Levels of M. cliungmuensis Infection Glycogen Monthly mean glycogen levels ranged from 2.0 to 14.8%, from 2.1 to 16.0%, and from 4.0 to 20.2% in H group, M group, and uninfected oysters, respectively (Fig. 5). Glycogen levels in all the three groups were maxima in May and minima in August. Glyco- gen levels in the infected oysters were similar between H and M groups but consistently lower than in uninfected oysters (analysis of variance. P < 0.01 for all the sampling months. Table 1). The glycogen level increased abruptly in September when the gonadal tissues were regenerated (Fig. 5). However, recovery of glycogen level after summer spawning was observed in the infected oysters. Biochemical Analysis of Oyster Tissue Lipid levels fluctuated in the narrow ranges from 11.5 to 14.0%, from 10.0 to 13.0%. and from 5.0 to 9.0% in H group. "juT^IidFtMBWGW'^AOW '^^■■H Figure 3. The external view of an oyster with an advanced M. cliun- Figurc 2. I'hotdmicrograph of a smear preparation from a heavily gmHfn.vis infection associated with ovarian hypertrophy, rendering the infected ovary. Pa, parasite. Eosin-methylene blue (x200). meat 'iumpy" in appearance. Appearance and Pathogenicity of Ovarian Parasite M. chungmuensis All Figure 4. Monthly variations of the % prevalence of the ovarian paraste M. chungmuensis of the Pacific oyster, Crassostrea gigas. i ■ Heavy infection ■ Moderate infection nUninfection ^ i\ jH m Figure 5. Monthly variations of levels (% of dry tissue weight ± 1 SD) in the infected and uninfected oysters from May to September. M group, and uninfected oysters, respectively (Fig. 6). The lipid contents in the infected oysters were similar between H and M groups (paired /-test. P = 0.174) and showed slightly greater levels than those in the uninfected oysters (paired t test. P = 0.06 for both infected groups). Protein levels ranged from 56.4 to 62.09^, from 55.1 to 58.9% and from 53.2 to 55.8% in H group. M group, and the uninfected oysters, respectively. No apparent dif- ferences were found between infected and uninfected oyster (paired r-test, P = 0.111 between H and M groups, P = 0.562 between H-group and the uninfected oysters, and P = 0.673 be- tween M group and the uninfected oysters). Carbohydrate levels ranged from 4.2 to 17.0%>, from 9.1 to 20.1%. and from 8.7 to 25.6% in H group. M group, and the uninfected oysters, respec- tively. Because glycogen levels accounted for most of total car- bohydrate levels, temporal variations of carbohydrate levels par- alleled those of glycogen with maxima in May and minima in August (Fig. 6). Ash levels ranged from 1 1.5 to 21.1%. from 16.0 to 25.1%, and from 10.7 to 25.6 in H group, M group, and the uninfected oysters, respectively, with maxima in August and minima in May. No pronounced correlation to infection was found for ash content (paired /-test. P = 0.930. P = 0.384, and P = 0.085. respectively, for the same paired variables as the statistical treatment of protein content). Serum Protein Mean serum protein concentration ranged from 3.1 to 4.0 p,g/ (jlL, from 3.7 to 6.2 |jig/|jiL, and from 5.1 to 11.4 (jig/(jiL in H group, M group, and the uninfected oysters, respectively (Fig. 7). Serum protein concentrations in the infected oysters were signifi- cantly higher in H-group than in M-group (analysis of variance, P < 0.001 for each sampling month except September). Serum pro- tein concentrations were then significantly lower in infected oys- ters then in uninfected oysters (analysis of variance, P < 0.001 for all the sampling months. Serum protein concentration in unin- fected broodstock was highest in May and lowest in August, with an obvious increase after the summer spawning of the oysters. However, in H-group oysters, the low concentration of mean s 4.0 |jLg/fj.L remained constant from May to September. In M group oysters, the serum protein concentration was highest in May and lowest in August-September. Finally, no increase in the serum protein concentration after the summer spawning was observed in both groups of the infected oysters. TABLE 1. Results of ANOVA and Tukey post-hoc test (a = 0.05) for absolute values of tissue glycogen and serum protein in each sampling month. Uninfected Parameters Month H Group M Group Oysters P Tissue glycogen (% of dry tissue) May 14.8 ±2.5 = 16.0 ± 1.8 < 20.2 ± 2.6 <0.001 June 11.9 + 2.2 = 13.5 ±2.0 < 16.8 ±0.5 <0.001 July 3.8 ± 0.5 = 4.7 ±1.2 < 5.7 ± 0.9 <0.001 August 1.8 + 0.6 = 2.2 ± 0.5 < 4.0 ± 0.5 <0.001 September 2.0 + 0.7 = 2.1 ±0.7 < 6.8 ± 0.6 <0.001 Serum protein ((jLg/|j.L) May 4.0 ± 0.7 < 6.2 ±0.7 < 11.4 ±0.5 <0.001 June 3.5 ±0.4 < 6.2+1.3 < 8.3 ± 0.4 <0.001 July 3.7 ± 0.5 < 4.8 ± 0.3 < 7.2 ±0.5 <0.00l August 3.1 ±0.5 < 4.1 +0.5 < 5.1 ±0.8 <0.001 September 3.1 ±0.7 = 3.7 ± 0.7 < 6.5 ± 1.0 <0.001 Data represent mean ± 1 SD. 478 Park ET AL. Heavy infection 100% ■ ■ ■ ■ "■1 100% 80% 'II 1 80% s 60% 60% g m 40% llll 1 40% 20% 1 1 1 1 1 20% 0% ■ ■ ■ ■ JU 0% Moderate infection I ■ ■ ■ ■ Uninfection CO 1 00% 80% 60% 40% 20% 0% ■ Ash ■ Carbohydrates D Total Lipids ■ Proteins M J J A S Figure 6. Monthly variations in the biochemical composition of infected and uninfected oysters. DISCUSSION Park and Chun (1989) reported M. chungmuensis infection prevalences of 5.3-6.7% between 1986 and 1987 in the oyster growing area of Hansan-Geoje Bay in the southern coast of Korea. i ■ Heavy infection ■ Moderate infection D Uninfection i JL 1 Figure 7. Monthly variations of serum protein concentrations [\i?J\il^ ± 1 SD) in the infected and uninfected oysters from May to September. The parasite was detected from June to October. Park et al. ( 1999) reported prevalences of 15.0-18.6% in oysters from Goseong Bay and Geoje Bay in 1993, with infections detected from May to September. During a subsequent survey in 1997. the parasite was detected all the year round, with an average prevalence of 26.6%. Therefore, the prevalences of M. chimginiiensis have been in- creased annually during last decade in Korea. M. chungmuensis only infects oyster ovarian tissues, inducing necrosis of the ova and massive hypertrophy of the gonad. The parasite impedes development of the fertilized eggs (Matsuzato et al. 1997, Matsuzato & Masumura 1988, Park & Chun 1989). Park et al. (1999) compared infected eggs with uninfected eggs and concluded that the infected eggs did not undergo fertilization. And also uninfected eggs isolated from infected oysters could be fer- tilized but their growth pattern was abnormal. More than 80% of the fertilized eggs which were from infected oysters showed ab- normal shapes and died during the early umbo stage. To date, there is no clear evidence that the parasite M. chungmuensis induces mortality of the mature oyster. Glycogen reserves have been considered to be the main energy reserves both for the formation of gametes of marine bivalves, especially under conditions of nutrient stress and also for the main- tenance during nutritional stress (Beninger & Lucas 1984, Enco- mio & Chu 2000). It is accumulated in storage tissues of the digestive gland, gonad, and mantle (Berthelin et al. 2000). Glyco- gen reserved in the gonad and mantle is used for gamete matura- Appearance and Pathogenicity of Ovarian Parasite M. chungmuensis 479 tion. Lower content of glycogen in infected hroodstock indicates that the parasite may directly reproductive success. Serum protein increased after the summer spawning in the uninfected oysters, but decreased in infected oysters. Reduced serum protein as well as reduced glycogen content may exacerbate morbidity, but there is no clear evidence to date that this has a direct correlation to mor- tality of oyster hroodstock. Protein may not be used for gameto- genesis, but is considered to be an essential metabolic requirement (Berthelin et al. 2000). Increased serum protein after spawning is normal; however, this did not occur in infected oysters examined. Thus, as with tissue protein, the effects of infection may have a significant meta- bolic impact, whereby the drop in prevalence of infection in Oc- tober was caused the death of heavily infected oysters, rather than recovery from infections. The prolistan parasite Perkiiisus nuiiiinis has been responsible for high mortality of eastern oyster Crassostrea virginica in the United States. The physiologic effects of P. marinus infection are most apparent as a reduction in growth rate as well as reproductive capacity (Barber and Mann 1994, Paynter 1996, Dittman et al. 2001). The physiologic effects of M. chungmuensis infection on Crassostrea gigas may reduce reproductive capacity of oyster population in Korea. ACKNOWLEDGMENTS The authors thank Dr. Sharon E. McGladdery at Department of Fisheries and Oceans Canada, Gulf Fisheries Centre, Centre des Peches du Golfe DFO Headquarters, Moncton, Ottawa, Canada for her critical corimients and suggestions on the article. This work was supported by the Ministry of Maritime Affairs and Fisheries- Special Grants for Fisheries Research and Development Project in Korea. LITERATURE CITED AOAC. 1990. OtTicial Method.^ of Analysis of the Association of OtTicial Analytical Chemists. 15th Ed. Arlington. Virginia: Association of Of- ficial Analytical Chemists Inc. Barber, B. J. & R. Mann. 1994. Growth and mortality of eastern oyster, Crassostrea virginica (Gmelin, 1791 ). and Pacific oysters, Crassostrea gigas (Thunberg. 1793) under challenge from the parasite. Perkinsus marinus J. Shellfish Res. 13:109-114. Beninger, P. G. & A. Lucas. 1984. Seasonal variations in condition, re- productive activity and gross biochemical composition of two species of adult clam reared in a common habitat: Tapes ileciissatiis L.. (Jef- freys) and Tapes philiphiiiariim (Adams and Reeve). J. Exp. Mar. Biol. Ecol. 79:19-37. Berthelin. C. K. Kellner & M. Mathieu. 2000. Storage metabolism in Pacific oyster [Crassostrea gigas) in relation to summer monalities and reproductive cycle (west coast of France). Comp. Biochem. Physiol. 125:359-369. Chun, S.-K. 1970. Diseases of oyster. I. Pathological study. Bull. Korean Fish. Sac. 5:1-7. Chun. S.-K. 1979. Amoeba infection on oyster. Bull. Korean Fish. Soc. 12:281-285. Comps, M., M. S. Park & I. Desportes. 1986. Etude ultrastrurale de Mar- teilioides chungmuensis n.g., n. sp. parasite des ovocytes de I'huitres Crassostrea gigas) TH. Prolistologica 22:279-285. Dittman, D. E., S. E. Ford & D. K. Padilla. 2001. Effects of Perkinsus nmriiuis on reproduction and condition of eastern oyster, Crassostrea virginica. depending on timing. J. Shellfish Res. 20:1025-1034. Encomio, V. & F.-L. E. Chu. 2000. The effect of PCBs on glycogen reserves in the eastern oyster Crassostrea virginica. Mar. Environ. Res. 50:45-49. Lowry. O. H.. N. J. Rosebrough, A. L. Farr & R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem 193:265- 275. Matsuzato. T. & K. Masumura. 1988. Abnormal enlargement of the ovary of oyster. Crassostrea gigas (Thunberg) by an unidentified parasite. Inter. J. Aqua. Fish. Tech 9:3-7. Matsuzato, T., T. Hoshina, K. Y. Arakawa & K. Masumura. 1977. Studies on the so-called abnormal egg-mass of Japanese oyster, Crassostrea gigas (Thunberg). Distribution of the oyster collected in the coast of Hiroshima Pref., and parasite in the egg-cell. Bull. Hiroshima Fish. Exp. St. 8:9-25. Park. M. S. & S. K. Chun. 1989. Study on Marteilioides chungmuensis Comps et al., 1986 parasite of the Pacific oyster, Crassostrea gigas Thunberg. / Fish Pathol. 2:53-70. Park. M. S.. H. Y. Lyu & T. S. Lee. 1999. Investigation on the cause of bad natural seed collection of the Pacific oyster. Crassostrea gigas: rela- tionships between the conditions of mother shell and the viability of the released eggs and larvae based on the pathological and embryological survey. J. Korean Fish. Soc 32:62-67. Paynter, K. T. 1996. The effects of Perkinsus marinus infection on physi- ological processes in the eastern oyster. Crassostrea virginica. J. Shell- fish Res. 15:119-125. Whyte. J. N. C. & J. R. Englar. 1982. Seasonal variation in the chemical composition and condition indices of Pacific oyster, Crassostrea gigas. growing in trays and on the sea bed. Can. J. Fish. Aquat. Sci. 39: 1084— 1094. Joumcit ofShetlfish Research, Vol. 22, No. 1. 481-185. 2003. MOLECULAR PHYLOGENETICS OF FIVE CORBICULA SPECIES DETERMINED BY PARTIAL 28S RIBOSOMAL RNA GENE SEQUENCES GAB-MAN PARK'*AND EE-YUNG CHUNG" ^Dcpcirtmeii! o) Parasitology. Kwamhmg University College of Medicine. Gangnimg. Gangivon-Jo 210-701. Korea; 'Department of Marine Living Re.wurces. College of Ocean Science anil Technology, Knnsan National University. Knnsan 573-701. Korea ABSTRACT Partial 28S rihosomal RNA (rRNA) gene sequences of five species (C fliiminea. C. papyracea. and C leana from Korea. C. japonica from Japan, C. larifillifrii from China) in the genus Corbkuhi were investigated for their genetic divergence. Neighbor-joining analysis on the alignment of 412 base pairs of C. flmninea. C. largillerti. C. papyracea. C. leana and C. japonica (with Polymesoda maritima. P. caroliniana and Sphaerhtm comeiim chosen as an outgroup) provides a robust molecular phylogeny for the genus; (C. japonica, C. papyracea. C. largillieni. C. leana. C. fluminea. P. maritime. P. caroliniana. and S. corn results of this study provide potential use of 28S rRNA gene sequence for phylogenies in the family Corbiculidae. KEY WORDS: Corbiculoidea. Corbiciila spp.. 28S rRNA. phylogeny, China. Korea. Japan , The INTRODUCTION Corhicula is conservative, possessing few moiphologic char- acters useful for species discrimination and displaying a broad range of subtle variability, especially with respect to shell form and color. The genus Corbicula is present in freshwater, brackish wa- ter, and estuaries in southeastern Asia, Africa, the Indian subcon- tinent, the Pacific Islands, and South America, where it is an important component of benthic communities in both lentic and lotic environments (Leveque 1973, Britton & Morton 1979). In Korea, six species, C. fluminea, C. leana, C. fenouilliana, C. pa- pyracea. C. colorata and C. portentosa, are recognized based on shell form {Kwon et al. 1993). Corhicula species can be categorized as 3 major groups based on reproductive characters and ecology (Miyazaki 1936); the spe- cies belonging to Group 1 are monoecious, viviparous, and incu- batory. They have nonswimming planktonic veliger larvae and live in freshwater; the species belonging to group 2 are dioecious, oviparous, nonincubatory, and also live in freshwater regions; the species belonging to group 3 are dioecious and oviparous. They do not incubate the young, has free-swimming planktotrophic larvae and live in brackish waters. The phylogenic relationship among these three groups cannot fully be clarified with these taxonomic characters alone. Recently, the chromosome numbers and the de- grees of genetic differentiation from a Hmited number of species of the genus Corbicula have been investigated (Okaiiioto & Arimoto 1986, Lee & Kim 1997, Park et al. 2000). Within the tandemly repeated rRNA gene complex, coding sequences for small (18S) and large (5.8S + 2SS) subunit rRNA components are flanked by nontranscribed and internal transcribed spacer regions. As a result of functional constraints within the ribosome. coding regions are in general more conserved than the spacer regions (Raue et al. 1990, Mulvey et al. 1998). The 28S rRNA gene contains "conserved" core regions interspersed with more variable "expansion segments" or domains, designated D 1 to D18 (Raue et al. 1988). Hillis and Dixon (1991) reported that, if chosen carefully, many divergent domains in the gene coding for large subunit ribosomal RNA are useful for reconstructing recent events. Sequence data from the 28S rRNA gene have been suc- *Corresponding author: Tel: -1-82-33-649-7480; Fax: -1-82-33-641-1074; E-mail: gmpark@kwandong.ac.kr cessfuUy used for intergeneric resolution within the Corbiculoidea (Park & Ofoighil 2000). This study is base on analysis of se- quences from 5' end 28S rRNA gene five common Corbicula species. MATERIALS AND METHODS Sample Collection and DMA Extraction Five Corbicula species C. fluminea (Cheorwon. Gangwon Province); C. papyracea (Yeongwol, Gangwon Province); C. leana (Wanju, Chunbuk Province) from Korea, C. japonica (Iwaki, Fukushima Province) from Japan and C. largillieni (Tung-ting lake, Hunan Province) from China were analyzed and nucleotide sequences were applied to five specimens in each spe- cies (Fig. I). Polymesoda maritima (Corbiculidae, GBDB Acces- sion no. AF1310I0), P. caroliniana (Corbiculidae. GBDB AF131011) and Sphaerium cornium (Sphaeriidae), GBDB API 3 101 3) were also analyzed as an out group. Voucher speci- mens of the Corbicula species used in this study have been placed in the Department of Parasitology, Kwandong University College of Medicine. Korea. Genomic DNA was isolated from fresh tissues using DNeasy Tissue Kit (Qiagen #69504) following manufacturers instructions. The 28S gene regions were amplified by the polymerase chain reaction (PCR) from 20 to 40 ng of genomic DNA. For the 28S, primers used were forward 5'-GATTACCCGCTGAACTTAAG- CATAT-3' and 5'-GCTGCATTCACAAACACCCCGACTC-3' reverse and DIF and D6R were used (Park & Ofoighil 2000). PCR amplification was conducted over 40 cycles using the following conditions; 1 min at 95'"C. 1 min at 54°C, and 1 .5 min at 72°C with a final extension of 7 min at 72°C. The PCR products were purified A B C D E Figure 1. Shells of Corbicula species. A, Corbicula fluminea; B, C. leana; C, C. papyracea; D, C. japonica; E, C. largillierti. 481 482 Park and Chung 1. C. fluminea AACCAGGATTCCCCCAGTAACGGCGAGTGAAGCGGG-AAGAGCCCAGCACCGAATCTCCC 2. C. largillierti - 3 . C . papyra cea G • ■ • • 4 . C. japonica - . . . . 5. C. leana T G---- 6. P. maritima •••A TT ---A- 7. P. cariliniana •••A T -.... 8. S. corneum •••A TT - CT- 1. GGCCTGACGGGCGGCGAGAAATGTGGTGTATAGGCGGCCGATTGTTGCCGGGTCCGGCGCTCAA-GTCCTCCTGATCGTG 2 . CG A - 3. CG- 4. CG- 6 . ATG CC TT • 7 . - • • • ATG C CG • 8 . • A - • ATG ■ • • C - - • • AG C ■ • • AA GC - • AGTC •-G-T-C---G--A 1. GCCTTGCCCAGAGCGGGTGTCAGGCCCGT GGCGGCGCTGGAGACGGCGGCTTCGAGCCTCCTTGGAGTCGGGTTGTT 2. --- A 3. --- A 4. A 5. --- 6. T AT---T--T--- 7. T G---TG---T 8. ---A-A G-ATC- - • -CT-GAC-CGG- AA ■ 1 . TGGGAATGCAGCCCAAAGCGGGTGGTAAACTCCACCTAAGGCTAAATACTGGCACGAGTCCGATAGCGGACAAGTACCGT 2. A 3. 4. 5. 6. A by gel extraction (Qiagen Co.) and ligated into a T cloning vector (IPTG) and X-gal. DNA from positive recombinants was purified (Novagen Co.). Clones were generated by transforming Escheri- using the QIAprep spin plasmid kit (Qiagen Co.). DNA sequenc- chia coli NovaBlue competent cells provided in the T cloning ing was performed using the dideoxy chain termination method vector kit, according to the protocol of the supplier. The recom- and an automated DNA sequencer (Applied Biosystems, Model binant plasmid was screened using isoprophy-p-thiogalactoside 373A. Perkin Elmer). At least two clones were sequenced per Molecular Phylogenetics of the Genus Corbicula 483 1. AGGGAAAGTTGAAAAGAACTTTGAAGAGAGAGTTCAAGAGTACGTGAAACCGCATAGAGCCAAACGGGTGGATCCGCAG 2. G 3. 4. T 5. 6. T G 7. T G 8. A GT 1. 2. 3. 4. 5. 6. 7. AGTCGACCCGGGGAATTCAGCCCGGCGGGTGCC GA • • T GA- -C CAG- • ■ -G- • -T Figure 2. Aligned of 5' 28S rRNA gene sequences of Corbiculoidea. Dashes represent gaps in the alignment. isolate, and additional clones were sequenced as necessary to re- solve ambiguous sites. Sequences Analyses Nucleotide sequences were aligned using Clustal X (Thompson et al. 1997). Phylogenetic analyses were performed by a distance method, using Kimura 2-parameters distance, to obtain a neighbor- joining tree (Saitou & Nei 1987) and using the MEGA vl.OI program. (Kumar et al. 199.3). Gaps were considered as an addi- tional character state in pairwise comparisons. The statistical con- fidence of a particular cluster of sequences was evaluated by the bootstrap procedure (1000 replicates). RESULTS The alignment of the partial 28S rRNA gene sequences of C. fluminea. C. papyracea. C. leana. C.japonica, C. largillierti. Poly- mesoda maritima, P. caroliniana, and Sphaerium corneum is shown in Figure 2. Nucleotide sequence data reported in this study are available in the GenBank database under the accession num- bers; C. fluminea (AY052553), C. largillierti (AY052534). C. pa- pyracea (AY052555), C. japonica (AY052556) and C. leana (AY052557). The 28S sequence was 412 base pairs, which in- cluded gaps in length. Nucleotide sequence differences for the various pairs of Corbiculoidea are presented in Table 1 . For this gene segment, interspecies differences from recognized species within any species where clones from the different sequenced isolates (C. fluminea. C. papyracea. and C. leana). Interspecies variation within the genus Corbicula was detected at a low level of 0.73 to 1.70% (from 4-22 nucleotides). Among the genus, how- ever, Corbicula, Polymesoda, and Sphaerium exhibit more varia- TABLE 1. Nucleotide sequence differences between pairs of Corbicula taxa for 28S rRNA region. Species (Origin) 2 3 4 S 6 7 8 L Corbicula fluminea (Korea) 6 6 4 3 IT 18 56 1 C. largillierii (China) 5 4 7 18 16 57 3. C. papyracea (Korea) 4 6 Tl 20 57 4. C. japonica (Japan) 6 19 17 57 5. C. leana (Korea) 22 18 56 6. Pohmesoda maritima (USA) 12 62 7. P. caroliniana (USA) 56 8. Sphaerium corneum (Germany) 484 Park and Chung 79 1 Corticula papymcea *6 H— c japonica 87 791 '-"" 6Ji-C 79 C largilliaflj C tluminaa C leans 92 PoiymasoCa mantima -P carolimana Sphaenum comeum — Sphaeriidae Corblculidae Figure 3. Tree dipicting relationships among genus Corbicula inferred from 28S rRNA gene sequence data using P. maritina. P. caroliniana. and S. corneiim as an outgroup. A distance matrix was calculated using the Kimura-2-paramater model and the tree constructed using the Neighbor-Joining method. tions (1.70-15.1%, from 22-62 nucleotides). The distances be- tween the genus Polymesoda and Sphaerium and the various Cor- bicula species are significantly greater than some interspecies distances with the Corbicula genus. The phylogenetic tree shows relationships among the interspecies based on the 28S sequences (Fig. 3). Analyses using P. maritima. P. caroriniana and S. comeum as outgroups supported the monophyly of genus Corbicula. Also, in the neighbor-joining tree, monophyly was strongly supported for both the families Corblculidae and Sphaeri- idae. DISCUSSION In the family Corblculidae, there are three genera: Corbicula. Batissa and Polymesoda. Of these, only Corbicula has a signifi- cant number of freshwater and brackish-water species. The other genera are dominantly brackish-water clams and characteristically have reducing sediments in tropical mangrove swamps. There are marked ecologic and reproductive differences between interspe- cies of Corbiculoidea (Table 2). Geographic variation in physiol- ogy, sex determination, and reproduction are undefined. There are references in the literature to a single species (C fluminea) pos- sessing different sexual strategies (e.g., protandry, protogyny, separate sexes) in different parts of its range (Morton 1982). Re- production in these species must be by parthenogenesis, but mature sperm are found in the gonads. The earliest corbiculid (mid-Jurassic) and dreissenid (Eocene) fossils were clearly marine (Keen & Casey 1969, Nutall 1990) and all dreissenid and some corbiculid species retain an indirect mode of development involving broadcast spawning and a pelagic veliger larval stage (Morton 1985, Morton 1989, de Severeyn et al. 1994). A planktonic veliger larva is considered to be nonadaptive in riverine freshwater environments because it lives in colonies at upstream habitats (McMahon 1991). Some fresh- water corbicuid species have evolved parental care of young in association with a greatly reduced (C. fluminea ) (King et al. 1986) or completely absent (Neocorbicula limosa ) (Ituarte 1994) pelagic larval ontogeny. From the comparisons of the chromo- some numbers and karyotypes in three species, Okamoto and Arimoto (1986) assumed that the ancestral species of the hermaphroditic species including C. leana originated from the ancestral species of C. sandai that had originated from the an- cestral species of C. japonica. Lee and Kim (1997) reported that the genetic similarity coefficient of C. fluminea. C. leana, and C. colorata in freshwater was very closed (Rogers S <0.970). whereas C japonica in brackish-waters was genetically distant (S = 0.873) from them. In this study, despite widespread geo- graphic origins of the Corblculidae, their percentage sequence variation in the 28S rRNA was low, <5.3%. Phylogenetic tree calculated using neighbor-joining method is shown in Figure 3. Instead of a single monophyletic Corbicula lineage, C papyracea. C japonica. and C larf>illierti are members of a separate clade distinct from that shared by C. fluminea and C leana: (i.e., within the Corbiculinae there are two sister groups) both of which contain species currently assigned to Corbicula. Based on the 288 rRNA data, the genus Corbicula is indistinguishable by biologic habitat features; C. japonica live in brackish-water, while other species live in freshwater. Partial 28S rRNA gene sequences provide use- ful data for resolving phylogenies within the Corbicula species groups. TABLE 2. Habitats, reproduction, and chromosome numbers in 12 species of the superfamily Corbiculoidea. Habitats Reproduction Chromosomes Species 2n References Corbiculidae C. jhiminea Freshwater Hermaphrodite 54 Park et al., 2000 C. papyracea Freshwater Hermaphrodite 54 Park et al., 2000 C. leana Freshwater Hermaphrodite 54 Okamoto & Arimoto. 1986 C. colorata Freshwater Dioecious 38 Park et al., 2000 C. japonica Brackish-water Dioecious 38 Okamoto & Arimoto, 1986 C. sandai Freshwater Dioecious 36 Okamoto & Arimoto, 1986 Sphaeriidae Pisidium coreanum Freshwater Hermaphrodite 190 Park et al., 2002 P. ca.'^ertantnu Freshwater Hermaphrodite ca. 150, 180 ca. 190 Barsiene et al., 1996 Burch et al., 1998 Sphaerium comeum Freshwater Hermaphrodite 36 Keyl, 1956 S. occidenlale Freshwater Hermaphrodite ca. 209 Burch et al., 1998 S. striatinufii Freshwater Hermaphrodite ca. 68-98 ca. 152 Woods. 1931 Lee, 1999 Musculium secure-^ Freshwater Hermaphrodite ca. 247 Burch et al., 1998 Molecular Phylogenetics of the Genus Corbicul\ 485 LITERATURE CITED Barsiene. J., G. Tapia & D. Barsyte. 1996. Chromosomes of mollusks inhabiting some mountain spiings of eastern Spain. / Moll. Stud. 62: 339-54.^. Britton. J. C. & B. Morton. 1979. Corhiciila in North Aitierican: the evi- dence reviewed and evaluated. In: Proceedings of the First International Corbkulii Symposium, pp. 15-38. Burch, J. B., G. M. Park & E. Y, Chung. 1998. Michigan's polyploid clams. Michigan Academician 30:351-352. De Severeyn, Y. G.. H. J. Severeyn & J. J. Ewald. 1994. Early development of the estuarine mollusk Polymesoda solida (Philippi, 1846) (Bivalvia: Corbiculidae) in Lake Maracaibo. Venezuela. Am. Maluc. Bull. 1 1:51- 56. Hillis, D. M. & M. T. Dixon. 1991. Ribosomal DNA: molecular evolution and phylogenetic inference. Q. Rev. Biol. 66:411— 153. Ituarte, C. F. 1994. Corhiciila and Neocorhicula (Bivalvia: Corbiculidae! in the Parana. Uruguay, and Rio de la Plata Basins. Nautilus 107:129- 135. Keen. M. & H. Casey. 1969. Family Corbiculidae. In: R. C. Moore, editor. Treatise on Invertebrate Paleontology. Part N. Vol. 2, MoUusca 6, Bivalvia. Geol. Soc. Am. and Univ. Press, of Lawrence, Kansas, pp. 665-669. Keyl, H. G. 1956. Boobachtungen iiber die meiose der Muschel Sphacrium comeum. Chromosoma 8:12-17. King, C. A., C, J. Langdon & C. L. Counts, Jr. 1986. Spawning and early development of Corhiciila fluminea (Bivalvia: Corbiculidae) in labo- ratory culture. Am. Maine. Bull. 4:81-88. Kumar, S., K. Tamura & K. Nei. 1993. MEGA, Molecular Evolutionary Genetics Analysis, Version 1.01. Institute of Molecular Evolutionary Genetics. The Pennsylvania State University, University Park, PA. Kwon. O. K., G. M. Park & J. S. Lee. 1993. Coloured Shells of Korean. Seoul, Korea: Academy Publishing Company, pp. 1-445. Lee, J. S. & J. B. Kim. 1997. Systematic study on the genus Corhicula (Bivalvia; Corbiculidae) in Korea. Kor. J. Sysl. Zool. 13:233-246. Leveque. C. 1973. Dynamiquedes peuplements biologie et estimation de la production des mollulques benthiques du Larc Tchad. Cahiers O.R.S.T.O.M. Serie Hydrohiologie 7:117-147. McMahon. R. F. 1991. Mollusca: bivalvia. In: J. H. Thorp & A. P. Covich. editors. Ecology and Classification of North American freshwater in- vertebrates. New York: Academrc Press, pp. 315-399. Miya/.aki, K. 1936. On the development of bivalves belonging to the genus Corhicula. Bull. Jap. Soc. Sci. Fish. 5:249-254. Morton, B. S. 1982. Some aspects of the population structure and sexual strategy of Corhicula ci. fluminalis (Bivalvia: Corbiculacea) from the Pearl River, People's Republic of China / Moll. Stud. 48:1-23. Morton, B. 1985. The reproductive strategy of the mangrove bivalve Poly- mesoda (Geloina) erosa (Bivalvia: Corbiculoidea) in Hong Kong. Mai- acological Rev. 18:83-89. Morton, B. 1989. The functional morphology of the organs of the mantle cavity of Batissa viotacea (Lamerck. 1797) (Bivalvia: Cobiculacea). Am. Malacol. Bull. 7:73-79. Mulvey. M.. H. P. Liu & K. L. Kandl. 1998. Application of molecular genetic markers to conservation of freshwater bivalves. J. Shellfish Res. 17:1395-1405. Nutall, C. P. 1990. Review of the Caenozoic heterodont bivalve superfam- ily Dreissenacea. Palaeontology 33:707-737. Okamoto, A. & B. Arimoto. 1986. Chromosome of Corhicula japonica. C. saiidai and C. (Corhiculimi) leana (Bivalvia: Corbiculidae). Venus 45: 194-202. Park. J. K. & D. Ofoighil. 2000. Sphaeriid and Corbiculid clams represent separate Heterodont bivalve radiations into freshwater environments. Mol. Phylo. Evol 14:75-88. Park. G. M., Y. S. Yong, K. L. Im & E. Y. Chung. 2000. Karyotypes of three species of Corhicula (Bivalvia: Veneroida) in Korea. J. Shellfish Res. 19:979-982. Raue. H. A., J. Klootwijk & W. Musters. 1988. Evolutionary conservation of structure and function of high molecular weight rRNA. Pro. Biophy. Mol. Biol. 51:77-129. Raue, H. A., W. Musters, C. A. Rutgers. T. Van, Riet & R. J. Planata. 1990. rRNA: from structure to function. In; The ribosome; structure, function, and evolution. Washington, D. C; Amer. Soc. Microbiol, pp. 217-235. Saitou, N. & M. Nei. 1987. The neighbor-joining method; a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin & D. J. Hig- gins. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Re.'.. 25:4876-4882. Woods, F. H. 1931. History of the germ cells in Sphaerium striatnum (Lam.). J. Morpho. Physiol. 51:545-595. Joitmal ,\f Shellfish Research. Vol, 22. No. 2. 487-4').1. 200.^. DOMINANCE OF THE ASIATIC CLAM, CORBICULA FLUMINEA (MULLER), IN THE BENTHIC COMMUNITY OF A RESERVOIR ALEXANDER Y. KARATAYEV,'* LYUBOV E. BURLAKOVA,' THOMAS KESTERSON,' AND DIANNA K. PADILLA- ^ Department of Biology. Stephen F. Austin State University. P.O. Box 13003. SFA Station, Nacogdoches, Texas, 75962-3003 and 'Department of Ecology and Evolution. State University of New York at Stonv Brook, Stony Brook. New York 11794-5245 ABSTRACT Corbuula fluminea dominated the benthic community of Latce Nacogdoclies, East Te,\as, composing 97% of the total biomass of benthic invertebrates. C. fluminea appears to be restricted to the httoral zone. Lower depths have lower oxygen, especially during the stratified period, which may restrict the distribution of C. fluminea. C. fluminea was found only down to a depth of 4 m and had and extremely patchy distribution. The greatest density within a patch was found at 1 m depth (35.8 ± 13.8 m"-) and the greatest biomass withm a single patch was at 2 m ( 137.17 ± 69.21 g • m""). C. fluminea density differed significantly among substrate types. The maximum density (43 ± 14 xvC') was found in sediments with dead C. fluminea shells and course detritus, and the lowest density (3.6 ± 3.6 m"') was found in silt. The spatial distributions of C. fluminea and three species of unionids were similar both in depth and across substrates in the reservoir. We found no correlation between the densities of C fluminea and other benthic invertebrates. Finally, we contrasted the effect of C. fluminea on benthic coninuinities to what is known about the impacts of another invasive bivalve, the zebra mussel. KEY WORDS: Corhicula fluminea. benthic comnuinity. Hydrilla. invasive species INTRODUCTION Asiatic clams [Corbicula fluminea (Muller)] are native to Southeast Asia and have been successfully invading North Ameri- can water bodies since the beginning of the 20th century. They currently occur in 36 states in the United States and northern and central Mexico; however, they are not found in Canada (McMahon 1982, McMahon 1999, McMahon & Began 2001). C. fluminea invaded Texas in the 1960s and has now spread statewide (How- ells 1992). C. fluminea is a simultaneous hermaphrodite that is ovoviviparous. Fertilized eggs are brooded in the interlamellar spaces of the gills through the trochophore and veliger stage and released at the nonswimming pedi veliger stage (McMahon 1999). Because of a high reproductive potential (<68,000 pediveligers adult"' y"' ), C. fluminea can rapidly increase in population density within a short period of time (Aldridge & McMahon 1978, Mc- Mahon 1991, McMahon 1999, McMahon & Bogan 2001). C. flu- minea is infaunal, usually burrowing in soft sediments. Adults can grow to 50-70 mm in size and can live for 3—4 y (reviewed in McMahon 1999). One of the reasons for its success may be the ability of C. fluminea to feed both from the water column (using siphons; Cohen et al. 1984, Boltovskoy et al. 1995), and from the sediments (using the foot to pedal-feed; Reid et al. 1992, Haken- kamp et al. 2001) Carried into raw water systems on intake flows, C. fluminea nonswimming pediveligers and juveniles may settle in places with water currents below 1.2-1.5 m sec"' and form adult populations >20,000 m"- (McMahon 1999). The total damage caused by C. fluminea for US industries in 1986 was estimated at $1 billion (Isom 1986). C. fluminea can also play an iinportant role in at|uatic ecosystems as a benthic-pelagic coupler (Lauristen 1986, Haken- kamp & Palmer 1999). C. fluminea can reduce phytoplankton lev- els (Cohen et al. 1984), seston concentration (Leef et al. 1990), particulate phosphates (Greer & Zeibell 1972) and chlorophyll a levels (Beaver et al. 1991). Water clarification by clam filtering *Corresponding author. E-mail: akaratayev@sfasu.edu favors the growth of rooted macrophytes, shifting primary produc- tion from planktonic to benthic communities (Phelps 1994. Mc- Mahon 1999). As a consequence, C. flu)uinea is becoming a major component of benthic communities in freshwater environments across North America (McMahon 1983, Counts 1986, Poff et al. 1993. McMahon 1999). C. fluminea may also influence bottom fauna as a result of pedal-feeding via bioturbation of sediments or consuming benthic fauna directly (Hakenkamp & Palmer 1999, McMahon 1999, Hak- enkamp et al. 2001). Although there are some reports that the Asiatic clam can compete with native unionid bivalves (Kraemer 1979, Leef et al. 1990, Howells 1992), there are no data about the impact of this invasive bivalve on biodiversity and functioning of the macroinvertebrate community or productivity and food web interactions. Hakenkamp et al. (2001) found that an increasing abundance of C. fluminea was negatively associated with the abun- dance of benthic bacteria and flagellates but had no apparent effect on other benthic protists or meiofauna. This contrasts with studies of another invading bivalve, the zebra mussel, Dreissena polymor- plia (Pallas) (reviewed in Karatayev et al. 1997, Karatayev et al. 2002). We determined the abundance and distribution of C. fluminea along depth gradients and among substrate types and their role in the benthic community, especially possible impacts on native fauna including unionid bivalves. We also compared patterns of the distribution of C. fluminea and its impact on bottom inverte- brates with those found for zebra mussels. METHODS Study Area Studies were conducted at Lake Nacogdoches, a monomictic reservoir in East Texas (31' 37'N, 94"49'W), Lake Nacogdoches is the municipal water supply reservoir for the city of Nacogdoches, Texas. The dam-forming Lake Nacogdoches was completed in July 1976. The reservoir has a surface area of 8.94 km", maximum 487 488 Karatayev et al. storage capacity of 49.7 million nr. maximum depth of 13 m, and an average depth of 5.6 m. The upper shallow (<5 m depth) and more eutrophic part of the reservoir is situated north of an island in the lake and constitutes approximately 40% of the water body (Fig. 1). Bottom sediments in this shallow part are mainly silt and a mixture of silt and clay. The lower part of the reservoir is less eutrophic. deep ( up to 13m). and has a variety of substrates, including sand, gravel, clay, shells, course detritus, and silt, as well as various combinations of these. The drainage area of the reservoir is 231 km", and Loco Bayou is the primary tributary (Prater 1991). During December to March, there is a long period of homeothermy. In spring, summer, and fall the water column of the reservoir is stratified. A lack of mixing and high productivity in the reservoir cause complete oxygen depletion below the thermocline by late spring. As a result, the oxygen content at depths greater than 6 m never exceeds 1 mg L"' from May to August (Taylor 1980). In the early 1980s, Hydhlla verticillata (l.f.) Royal was acci- dentally introduced into the Lake Nacogdoches and by 1989 cov- ered approximately 45'7r of the reservoir (Prater 1991). H. verti- cillata spread mainly in the upper shallow part of the water body, where it completely covered the reservoir. In contrast, m the lower part less than 3% of the reservoir is covered with H. verticillata (Fig. 1). Sampling Protocol To determine the distribution of C. fluminea and its effect on the benthic community of Lake Nacogdoches, a total 96 bottom NACOGDOCHES RESERVOIR ELEVATION 279' (65 M) Figure 1. Location of transects sampled in Lake Nacogdoches. Shaded area represents the extent of the reservoir covered by Hydrilla verti- cillata. samples were taken in September (transect 1) and October (transects 2-6) 2001 (Fig. 1). For each transect, samples were collected from 1, 2. 3. 4. 6. and 8 m. except for transects 5 and 6. where samples were collected at depths of 1. 2, 3, and 4 m. These last two transects were situated at the upper shallow part of the reservoir with a maximum depth less than 5 m. In addition, the deep (profundal) part of the lake was sampled separately (6 and 10 m depth). Three or more replicate samples were taken at each depth with an Eknian grab (sampling area = 0.0233 m") and washed through a 550-(xm mesh. At each sampling point, water transparency, bottom temperature, pH, oxygen, and conductivity were recorded (Table 1). After sampling, all macroinveilebrates were transferred to containers with W7c neutral-buffered formalin and labeled. All macroinvertebrates were identified to the genus or species level, counted, and weighted to the nearest 0.0001 g after being blotted dry on absorbent paper (wet mass). For oligochaetes, only Braiuhiiira sowerbyi Beddard and Stylaria laciislris (Lin- naeus) were identified to species level. All C. fluminea and union- ids were cut open with a scalpel to remove water from the mantle cavity, measured, weighed (wet mass), and identified to species. The average mass of individual C. fluminea in a sample was cal- culated by dividing the total mass by the number of clams in the sample. Because several samples contained no C. fluminea (den- sity = 0), we used nonparametric Kruskal-Wallis test to analyze the data. When multiple statistical tests were conducted on the same data, we used a Bonferroni correction to determine the criti- cal alpha for significance. RESULTS Corbicula fluminea Distribution During our September sampling, the reservoir was still well stratified for temperature and oxygen to around 6 m depth (Table 1 ). In October, the lake was well mixed and both temperature and oxygen did not vary appreciably with depth. Oxygen content was low only at the deepest sampled site (10 m depth). Water pH and conductivity did not show sharp changes across the thermocline (Kruskal-Wallis test, P = 0.20). C. fluminea was found only in the lower part of Lake Nacog- doches (transects 1-4; Fig. 1). We did not find any live C. flu- minea, or even their dead shells, in the upper part of the reservoir, which was covered with H. verticillata (transects 5 and 6). We found a significant difference in some chemical parameters between regions of the lake with C. fluminea (transects 2-4, 1-4 m) and the area of the lake with H. verticillata. where we did not find clams (transects 5-6, 1-4 m). The pH was slightly higher in the upper region (7.96 ± 0.009. /; = 12) vs. 7.86 ± 0.011 [n = 1): Kruskal-Wallis test, P = 0.0005). Dissolved oxygen was slightly lower in area covered with H. verticillata (9.26 ± 0.06 (n = 7) vs. 9.76 ± 0.15 mg L-' (n = 12); Kruskal-Wallis test, P = 0.016), but this difference was only marginally significant (critical alpha with the Bonferroni Correction = 0.012). Conductivity was lower in the upper part of the reservoir (93.1 1 ± 0.28 (/( = 7) vs. 95.66 ± 0.53 m Siemens cm"' (n = 12); Kruskal-Wallis test, P = 0.002). Transect 1 was not included in these analyses as it was sampled 20 days earlier. The average (± SE) C fluminea density and biomass in the lower portion of the reservoir (transects 1^, depths 1-8 m) was 15.6 ± 5.3 m~' and 71.9 ± 18.8 g m"", respectively. There were no significant differences in density or biomass of C. fluminea be- tween the four transects (Kruskal-Wallis test, P > 0.44). in addi- Dominance of Corbicula in Benthos 489 TABLE 1. Oxygen concentration, temperature, conductivity and pH in Lake Nacogdoches. Depth (m) Parameter 1 2 3 4 6 8 10 Transect 1 Oxygen, mg • L"' 9,72(1) 9,.'i7 (1) 9,40(1) 9,26(1) 3,03(1) 0,45 ( 1 ) — Temperature. °C 27.8(1) 27,7 (1) 27,6(1) 27,6(1) 26,3(1) 24,3(1) — Conductivity. mSiemens cm-' 96,9 ( 1 ) 95,3(1) 94,3 ( 1 ) 94,2 (1) 97.7 (1) 116,7 (1) — pH 7,87(1) 7,90(1) 7,90(1) 7,87(1) 7,76(1) 7,60 ( 1 ) — Transects 2-4 Oxygen, nig ■ L ' 10,24 ±0,36 (3) 9,62 ±0,39 (3) 9,64 + 0,30(3) 9,52 ±0,25 (3) 7,83 ±0,40 (3) 6.25 ±2,15 (2) — Temperature, C 23,10 ±0,17 (3) 22,69±0..30(3) 22,83 ±0,12 (3) 22,82 ±0,12 (3) 22.45 ±0,05 (3) 22,30 ±0(2) — Conductivity. mSiemens cm-' 96,27 ± 1,77(3) 95,2 ±0,95 (3) 95,2 ±0,68 (3) 95,97 ± 1,06(3) 95,70 ±0,20 (3) 97,65 ±2.25 (2) — pH 7,87 ±0,03 (3) 7,86 ±0,02 (3) 7,86 ±0,01 (3) 7,84 ± 0,02 (3) 7,84 ±0,03 (3) 7,80 ±0,02 (2) — Transects 5-6 Oxygen, mg ■ L"' 9,30 ± 0,20 (2) 9,20 ±0(2) 9,33 ±0,12 (2) 9,15 (1) — — — Temperature. °C 22,30 ± 0,20 (2) 22,31 ±0,16(2) 22,41 ±0,27(2) 22,7(1) — — — Conductivity, mSiemens cm"' 92.60 + 0(2) 93,5 + 0.50(2) 93,55 ±0,75 (2) 92,50(1) — — — pH 7,96 ±0,03 (2) 7,95 ±0,04 (2) 7,96 ±0,005 (2) 7.97(1) — — — Profunda] Oxygen, mg ■ L"' — — — — 8,45 (1) — 0,85(1) Temperature, "C — — — — 23,4(1) — 21,3(1) Conductivity, mSiemens cm"' — — — — 93,6 ( 1 ) — 123,0(1) pH — — — — 7,14(1) — 7,80(1) Transect 1 was sampled in September; all other transects were sampled in October. Transects 5 and 6 were in an area of the lake covered with Hydrilla venicillala and no Corbicula flmninea. Transects 2-4 had C fliiminea and no H. verticiUata. In addition, we sampled two sites deep in the lake (profunda!. 6 and 10 m). Average values + standard errors of mean are given, sample sizes are in parentheses, — = no data. tion. density and wet mass did not differ significantly with depth down to 4 m (Kruskal-Wallis test: density: P = 0.29; bioniass; P = 0.40; Figs. 2 and 3). This lack of significance is probably caused by the high degree of patchiness (Index of Dispersion Test: I = 59.7; X- = I * (n - 1) = 59.7*(48 - I) = 2805. x',,025. 47 = 67.8, P < 0.001). Many samples had no C, fluminea, which resulted in an increase in the variance in observed means. The 1 150 I 100 50 12 12 12 fla^ I o I Live mussels ■i Dead shells Meant SE 12 m 12 3 4 5 6 Depth (m) Figure 2. Density of Hve and dead shells of Corbicula fluminea at different depths in Lake Nacogdoches, Averages, standard errors of mean, and sample sizes are given. mean density o'i C. fluminea was greatest at 1 m (35,8 ± 13,8 m""), and the maximum biomass was at 2 m (137,17 ± 69,21 g m""). The maximum density of C. fluminea in a single sample was 172 m"" (transect 4, I m) and maximum wet mass (soft body + shells) in a single sample was 770 g m"" (transect 2, 2 m). The average individual mass for C. fluminea (total sample mass/density) differed significantly with depth (ANOVA, Depth (m) Figure 3. Total wet mass (left axis) and average individual wet mass (total wet ma.ss divided by the number of clams in the sample, right axis) of Corbicula fluminea at different depths in Lake Nacogdoches. Averages, standard errors of mean, and sample sizes are given. 490 Karatayev et al. 16 .■& 8 12 12 rol Unionid Density H Unionid Wet Mass Mean ± SE 12 12 12 T 12 12 150 50 Depth (m) Figure 4. Density (left axis) and total net mass (right axis) of unionids at different depths in Lake Nacogdoches. Averages, standard errors of mean, and sample sizes are given. P < 0.029; Fig. 3). The smallest average individual mass (2.95 ± 1.02 g) was at 1 m. and the largest average individual mass (7.33 ± 0.86 g) was at 2 m. C. fluminea shells were found down to 6 m (Fig. 2). The dis- tribution of shells with depth was not uniform (Kruskal-Wallis test, P = 0.0002) and differed from the distribution of live C. fluminea (Kolmogorov-Smirnov test, P < 0.001). C. fluminea density differed significantly among substrate types (Kruskal-Wallis test, P = 0.04). Dead C. fluminea shells and course detritus had the highest density (43 ± 14 m"-). and the lowest density (3.6 ± 3.6 m~") was found in silt (Table 2). Dead C. fluminea shells were not found uniformly among substrate types. and were most abundant in clay with stones (272 ± 56 m""; Kruskal-Wallis test, P = 0.0008). Distribution of Benthic Animals We found 38 taxa (species, genera or higher taxa), including 17 chironomids (identified to the species or genus level). The average density of benthic animals, excluding bivalves, over the entire reservoir was 901 ± 91 m^~ with a biomass of 2.76 ± 0.29 g m"". The most abundant insect larvae were the chironomid Coelotanx- pus tricolor (Lowe) (185 ± 25 m"*, total number of samples = 96), Cliironomus sp. (69 ± 17 m""), and the phantom midge Clia- oborus punctipennis (Say) (137 ± 35 m""). Among oligochaetes, Brandnura sowerhyi Beddard was dominant (71 ± 12 m~"). Only a single species of amphipod, Hyallelu azteca (Saussure), was found (24 ± II m"-). Corhicula fluminea dominated the benthic biomass in the lit- toral zone from 1— t m in the lower part of the reservoir (transects 1—4) and was responsible for more than 97% of the total wet mass of the benthic community. At depths s 6 m. Cliironomus sp., C. punctipennis. and B. sonerbyi were responsible for 43%, 17%, and 26% of the total benthic biomass, respectively. In the upper region of the reservoir (transects 5 and 6) the average density (1165 ± 216 m"~) and average biomass (3.57 ± 0.54 g m~") of benthic animals were marginally higher (Kruskal- Wallis test, density: P = 0.073; biomass: P = 0.061) than in the lower part (excluding C. fluminea and unionids density 843 ± 104 m^", biomass 2.60 ± 0.35 g m""). However, because of the pres- ence of C. fluminea in the lower region of the reservoir, the total macrobenthos (including C. fluminea) biomass (74.5 ± 18.9 g m~") was 20 times greater than in the upper part. There were three species of unionids in the lake, Pyganodon grandis (Say), Ligumia subrostrata (Say), and Toxolasma texas- ensis (Lea). Two P. grandis. one L. subrostrata. and six T. texas- ensis were found on transects 2, 3, and 4 on depths of 1—4 m (Fig. 4). These unionids completely overlapped with the distribution of C. fluminea (Kolmogorov-Smirnov test, P > 0.10). For tran.sects 1—1. the maximum density and biomass of union- ids was in clay (17.9 ± 8.3 m"-, 97.5 ± 42.8 g • m"", total number of samples n = 12) and in course detritus with C. fluminea shells (10.8 ± 5.6 m"-, 98.4± 71.1 g ■ m'-.n = 12). The lowest density and biomass of unionids was in silt (3.6 ± 3.6 m"", 14.9 ± 14.9 g-m~", n = 12). There were no significant correlations between the C. fluminea density or the density of C. fluminea shells and any invertebrate taxon. DISCUSSION C. fluminea Distribution The exotic plant Hydrilla verlicillata covers approximately 45% of Lake Nacogdoches and is the dominant macrophyte spe- cies in this community (Prater 1991). Another exotic species. C. fluminea. dominated the benthic community of this reservoir. However, the spatial distribution of these two nuisance species did not overlap. During our study, neither live C. fluminea nor dead shells were found in the upper part of the reservoir, which is covered with H. verticillata. Prater ( 1991 ) sampled the benthos of Lake Nacogdoches monthly over 12 mo in 1989-1990 and never found C. fluminea in the H. verticillata region of the reservoir as well. Two factors may contribute to the absence of C. fluminea in the upper part of the reservoir. First, dense H. verticillata mats may deplete the oxygen in the water to levels below those critical for C. fluminea survival. We found a significant decrease in oxygen in TABLE 2. Abundance of live Corhicula fluminea and shells in various substrata in Lake Nacogdoches in 2U01, Substrate Type Density, Ind. m Biomass, g ■ m C. fluminea Shells m C. fluminea shells and course detritus Clay and stones Clay Sand Silt 43.0 ± 14.0(12) 19.1 ± 14.5(9) 25.1 ±8.3(12) 14.3 ± 14.3(3) 3.6 + 3.6(12) 162.68 ±56.01 (12) 95.36 ± 63.75 (9) 149.93 ±66.26 (12) 45.87 ±45.87 (3) 17.77 ± 17.77(12) 71.7 ±25.6 (12) 272.3 ±55.5 (9) 100.3 ±21.4 (12) 28.7 ±28.7 (3) 112.9 ±28.6 (24) The abundance of live C. fluminea was estimated from transects 1—4, from 1—4 m. The abundance of dead shells was estimated from transects 1-6 m. Average values ± standard errors of the mean are given. Sample size in parentheses. ■ and Dominance of Corbicula in Benthos 491 the portions of the lake covered with H. verlicillata. but this dif- ference was relatively small. Second, bottom substrates in the up- per part of the reservoir are predominantly silty clay, which may be unfavorable for C. fliiiiiiiieci. C. fluminea was found in all four transects in the lower part of Lake Nacogdoches at depths up to 4 m. and C. fluminea dead shells were found up to 6 m depth. Deeper in the reservoir. C. fluminea was probably limited by low oxygen, especially during the summer, when the water column is stratified and the oxygen content deeper than 6 m never exceeds 1 mg L"' (Taylor 1980). C. fluminea is known to be intolerant of even moderate hypoxia (Mc- Mahon 1991. McMahon & Bogan 2001 ). and low oxygen is con- sidered to be one of the main sources of mortality for C. fluminea (Sieckel 1986). Although live C. fluminea were most dense at 1 m depth and had highest total biomass at 2 m, their dead shells were most abundant at 3 and 4 m (Fig. 2). which suggested that the depth of maximum C. fluminea abundance may vary with time or that dead shells were transported to deeper water by water motion. C. fluminea density and biomass also varied among substrate types. Clams were most abundant in sediments formed by shells and course detritus and least abundant on silt. The mean population density and biomass of C. fluminea we found were very similar to those found 10 years earlier by Prater (1991). He found that clam density in 1989-1990 varied from 0 to 60 m"- (average 24.4 ± 5.5 m"'). This suggests that the population density of C. fluminea in Lake Nacogdoches is rather stable. C. fluminea can occur in dense aggregations, exceeding 2000 m'" (Gardner et al. 1976. Phelps 1992). These densities are much higher densities than those that have been recorded in Lake Nacogdoches. However, these higher densities were reported for a limited period of lime, shortly after initial invasion (Phelps 1994) or from a local spot in a water body (Eng 1979). For example, after the initial invasion in the Potomac River in 1977, C. fluminea reached a maximum density in 1986 (722 g m~" wet weight, including shell) but then sharply declined, and in 1992 was at 24.87^ of 1986 levels (Phelps 1994). In the sediment bars of the Delta-Mendota Canal, the maximum density of C. fluminea at one site was 1 3 1 ,200 m""; however, the average density was much smaller (Eng 1979). In another Texas lake. Lake Arlington, the mean density of C. fluminea in 1975 was very similar to the densities we found in Lake Nacogdoches (32.1 ± 16.5 nr-. Aldridge & McMahon 1978). Dominance in Benthos C. fluminea appears to dominate the benthic community of water bodies it invades (McMahon 1983, Counts 1986, McMahon 1991, Poff et al. 1993, McMahon 1999). We found that in littoral zone of Lake Nacogdoches C. fluminea comprises more than 97% of the total wet mass of the macrobenthic community. We found no correlations between C. fluminea density and biomass and other nonmolluscan invertebrates. Impact on I'nionids Whether C. fluminea and native bivalves compete is controver- sial (McMahon 1999, Strayer 1999). According to some authors, C. fluminea may out compete native unionids (Kraemer 1979, Belanger et al. 1985, Leef et al. 1990, Howells 1992). The com- petitive advantage of C. fluminea over native bivalves has been suggested because it has a much higher filtering rate than native species (Mattice 1979, Lauristen 1986). In addition, by being able to use both filter and pedal feeding, C. fluminea may have an advantage over native bivalves that are only able to filter feed (Hakenkamp & Palmer 1999). However, most of the evidence for the competitive impacts of C. fluminea on native bivalves is based on an analysis of their spatial distributions, and much of these data are anecdotal and qualitative rather than quantitative (Strayer 1999). According to many authors (reviewed in Strayer 1999), C. fluminea and native bivalves have nonoverlapping spatial distri- butions, implying that C. fluminea can out compete other bivalves. However, we found that in Lake Nacogdoches unionids and C. fluminea are both abundant and occupied the same areas. The depth distribution of C. fluminea and unionids was completely overlapping. In addition, both unionids and C. fluminea were abun- dant in the same type of substrate (course detritus with C. fluminea shells and clay). The lowest numbers and biomass of both C. fluminea and unionids were in silt. Several other authors have found that unionids and C. fluminea coexist (Clarke 1988. Beaver et al. 1991. Miller & Payne 1994). These data may suggest that the impact of C. fluminea on native unionids is not as strong as the impact of zebra mussels, which can cause mass mortality of unionids (reviewed in Karatayev et al. 1997). Impacts of Corbicula fluminea versus Dreissena polymorpha In Lake Nacogdoches C. fluminea was never found in areas over grown by H. verlecillata. In contrast, zebra mussels are often found at their highest densities on submerged macrophytes, which they use as sites for attachment (Lewandowski 1982, Lyakhnovich et al. 1994, Karatayev et al. 1998). During our study. C. fluminea was most abundant on shelly sediment. This sediment is also one of the best substrates for the zebra mussel (Lyakhnovich et al. 1994. Karatayev et al. 1998). Silt is the poorest substrate for both C. fluminea (Duarte & Diefenbach 1994) and D. polymorpha (Zha- din 1946, Draulans & Wouters 1988, Karatayev & Burlakova 1995) and often limits their distributions. Belanger et al. (1985) found in their field and laboratory studies that C. fluminea pre- ferred the following sediments in decreasing order: fine sand, or- ganically enriched fine sand, and coarse sand. C. fluminea, a bur- rowing animal, preferred tine sediments: however, the zebra mus- sel, which attaches to hard substrate, forms especially high densities on rocks (Lyakhnovich et al. 1994, Burlakova 1998). Low oxygen may be another important limiting factor for both C. fluminea (McMahon 1991, McMahon & Bogan 2001) and the zebra mussel (Mikheev 1961. Spiridonov 1972. Shkorbatov et al. 1994). Both C. fluminea (McMahon 1983, Counts 1986, McMahon 1991, Poff et al. 1993, McMahon 1999) and D. polymorpha (Sokolova et al. 1980, Karatayev et al. 1994) dominate benthic communities and are responsible for more than 95Vf of the bio- mass in lakes where they occur. C. fluminea live in soft sediment, crawl through sediment with a foot, and feed both as a filter feeder from the water column (Cohen et al. 1984, Boltovskoy et al. 1995), and from the sediments as a pedal feeder (Reid et al. 1992, Hak- enkamp et al. 2001) and thus may negatively impact burrowing detritivores (McMahon 1999). Zebra mussels, in contrast, can live only on the surface of the sediments, where they attach to hard substrates and each other with proleinacious byssal threads creat- ing complex three-dimensional structures (Karatayev et al. 2002). D. polymorpha constantly filter the water for both feeding and respiration. Filtered particles are either consumed or bound in mucus, preventing immediate re-suspension. This zebra mussel 492 Karatayev et al. activity builds a direct connection between the planktonic portion of water body and the benthos (benthic-pelagic coupling) and greatly enhances the rates of deposition of both organic and inor- ganic material on the bottom. D. polymorpha provide food and shelter for many benthic invertebrates, which have increased den- sity and biomass in zebra mussel beds. Simultaneously other spe- cies (mainly filter feeders) may decrease or disappear from the community (Karatayev & Burlakova 1992. Stewart et al. 1998. Stewart et al. 1999). This well-documented effect of zebra mussel on benthic communities contrasts with the unknown impact of C. fluminea on composition, structure and densities of native inver- tebrates. In a recent study Hakenkamp et al. (2001) found that when they experimentally increased C. fliiiiiiuea density in the field, there was no apparent impact on the abundance or taxonomic composition of the meiofauna. In some circumstances C. fluminea may compete with native bivalves for food or substrate. In contrast, the negative impact of D. pohmorpha on native unionids is more diverse. Besides re- source competition, zebra mussels also show direct interference competition through overgrowth of unionids. By attaching to unionids, zebra mussels can make it more difficult for them to burrow and move through the sediment. They can weight down their host unionid. resulting in burial in very soft sediments, can increase drag and the likelihood of dislodgement by water motion for species living near shore, prevent opening valves for respira- tion, feeding and reproduction, or preventing the closing valves (reviewed in Karatayev et al. 1997, Burlakova et al. 2000). Mass mortalities of unionids caused by D. polymorpha over- growth are most common during the initial stages of colonization, when mussel populations are growing rapidly. After initial peaks in zebra mussel abundance, D. polymorpha can coexist with unionid bivalves (Nichols & Amberg 1999, Buriakova et al. 2000). Similarly, we hypothesize that the strength of competition between C. fluminea and native bivalves may depend on various factors including unionid species, C. fluminea density, and time since C. fluminea invasion. ACKNOWLEDGMENTS We would like to thank Dmitry and Vadim Karatayev for as- sistance in the field and with sample processing. DKP acknowl- edges the support of the Distinguished Research Fellow program. Bodge Marine Laboratory, University of California. Davis (BML contribution 2184). LITERATURE CITED Aldridge, D. W. & R. F. McMahon. 1978. Growth, fecundity, and hioen- ergetics in a natural population of the Asiatic freshwater clam Cor- biciila manilensis Philippi, from North Central Texas. J. Moll. Studies 44:49-70. Beaver, J. R., T. L. Crisman & R. J. Brock. 1991. Grazing effects of an exotic bivalve (Corbicula fluminea) on hypereuthrophic lake water. Lake Reserv. Manage. 7:45-.'il. Belanger. S. E., J. L. Farris, D. S. Cherry & J. Cairns Jr. 1985. Sediment preference of the freshwater Asiatic clam Corbicula fluminea. Naulilus. 99:66-73. Boltovskoy, D.. I. Izaguirre & N. Correa. 1995. Feeding selectivity of Corbicula fluminea (Bivalvia) on natural phytoplankton. Hydrobiolo- gia 312:171-182. Burlakova, L. E. 1998. Ecology of Dreissena polymorpha (Pallas) and its role in the structure and function of aquatic ecosystems. Candidate Dissertation. Zoology Institute of the Academy of Science Republic Belarus. Minsk. Belarus. 168 p. (in Russian). Burlakova, L. E.. A. Y. Karatayev & D. K. Padilla. 2000. The impact of Dreissena polymorpha (Pallas) invasion on unionid bivalves. Int. Rev. Hydrobiol. 85:529-541. Clarke, A. N. 1988. Aspects of corbiculld-unionid sympatry in the United States. Malacology Data Net. 1:3-10. Cohen, R. R. H., P. Y. Dresler, E. J. P. Phillips & R. L. Cory. 1984. The effect of the Asiatic clam, Corbicula fluminea, on phytoplankton of the Potomac River. Maryland. Limnol. Oceanogr. 29:170-180. Counts. C. L.. III. 1986. The zoogeography and history of the invasion of the United States by Corbicula fluminea (Bivalvia: Corbiculidae). Am. Malacol. Bui Spec. Ed. 2:7-39. Draulans, D. & R. Wouters. 1988. Density, growth and calorific value of Dreissena polymorpha (Mollusca: Bivalvia) in a pond created by sand extraction, and its importance as food for fish. Aiuils. Soc. r. Zool. Belg. 118:51-60. Duarte, M. M. & C. O. Diefenbach. 1994. Microdistribution and abundance of freshwater mussels (Mollusca: Unionacea and Corbiculacea) in Su- zana Lake. Southern Brazil. Stud. Neotrop. Fauna Environ. 29:233- 250. Eng, L. L. 1979. Population dynamics of the Asiatic clam Corbicula flu- minea (Muller) in the concrete-lined Delta-Mendota Canal of Central California. In: J. C. Britton, editor. First International Corbicula Sym- posium. Fort Worth, TX: Texas Christian University Research Foun- dation, pp. 39-68. Gardner, J. A.. Jr., W. R. Woodall, A. A. Staas & J. F. Napoli. 1976. The invasion of the Asiatic bivalve Corbicula fluminea (Bivalvia). Hydro- biologia 127:105-115. Greer. D. E. & C. D. Zeibell. 1972. Biological removal of phosphates from water. / Water Poll. Control Fed 44:2342-2348. Hakenkamp, C. C. & M. A. Palmer. 1999. Introduced bivalves in fresh- water ecosystems: the impact of Corbicula on organic matter dynamics in a sandy stream. Oecologia 119:445-451. Hakenkamp. C. C, S. G. Ribblett. M. A. Palmer. C. M. Swan. J. W. Reid & M. R. Goodison. 2001. The impact of an introduced bivalve (Cor- bicula fluminea) on the benthos of a sandy stream. Freshwal. Biol. 46:491-501. Howells, R. G. 1992. Annotated list of introduced non-native fishes, mol- lusks, crustaceans, and aquatic plants in Texas waters. Management Data Series 78. Austin. TX: Texas Parks and Wildlife Department, Fisheries and Wildlife Division. Isoni, B. G. 1986. Historical review of Asiatic clam (Corbicula) invasion and biofouling of waters and industries in the Americas. Am. Malacol. Bui. Spec. Ed. 2:1-5. Karatayev, A. Y. & L. E. Burlakova. 1992. Changes in trophic structure of macrozoobenthos of an eutrophic lake, after invasion of Dreissena polymorpha. Biol. Vnutr Vod. Inf. Byull. 93:67-71. Karatayev, A. Y. & L. E. Burlakova. 1995. Present and further patterns in Dreissena population development in the Narochanskaya lakes system. Vestsi Akad. Navuk Belurusi. Ser Biyol. Navuk. 3:95-98. Karatayev, A. Y., L. E. Buriakova & D. K. Padilla. 1997. The effects of Dreissena polymorpha (Pallas) invasion on aquatic communities in Eastern Europe. / Shellfish Res. 16:187-203. Karatayev, A. Y., L. E. Buriakova & D. K. Padilla. 1998. Physical factors that limit the distribution and abundance of Dreissena polymorpha (Pall.). J. Shellfish Res. 17:1219-1235. Karatayev, A. Y., L. E. Buriakova & D. K. Padilla. 2002. Impacts of zebra mussels on aquatic communities and their role as ecosystem engineers. In: E. S. Leppiikoski et al., editors. Invasive Aquatic Species of Europe: Distributions, Impacts and Management. Monoraphiae Biologicae Se- ries. Dordrecht. The Netherlands: Kluwer Scientific Publishers, pp. 433-446, Dominance of Curbicula in Benthos 493 Karatayev, A. Y., V. P. Lyakhnovich, S. A. Afanasiev. L. E. Burlakova. V. P. Zakutsky, S. M. Lyakhov, M. P. Miroshnichenko. T. G. Moro/. M. Y. Nekrasova, I. A. Skalskaya, T. G. Kharchenku & A. A. Protasov. 1994. The place of species in ecosystem. In: J. I. Starobogatov. editor. Freshwater zebra mussel Dreissena potymorpha (Pall.) (Bivalvia, Dreissenidae). Systematics, ecology, practical meaning. (In Russian) Moscow: Nauka Press, pp. 180-195. Kraemer, L. R. 1979. Corbicula (Bivalvia: Sphaeriacea) vs. indigenous mussels (Bivalvia: Unionacea) in U.S. rivers: a hard case for interspe- cific competition? .4m. Zooi 19:1085-1096. Lauristen. D. D. 1986. Filter-feeding in Corbicida fliiiiuiifu and its effect on seston removal. J. N. Am. Beiuhoi Soc. 5:165-172. Leef, L. G., J. L. Burch & J. V. McArthur. 1990. Spatial distribution, seston removal, and potential competitive interactions of the bivalves Cor- bicula fliiminea and Elliplio complanala in a coastal plain stream. J. Freshwater Ecol. 24:409-416. Lewandowski, K. 1982. The role of early developmental stages in the dynamics of Dreissena potymorpha (Pall.) (Bivalvia) populations in lakes. 2. Settling of larvae and the dynamics of numbers of settled individuals. Ekol. Pol. 30:223-286. Lyakhnovich, V. P., A. Y. Karatayev. N. 1. Andreev, S. I. Andreeva. S. A. Afanasiev, A. K. Dyga, V. P. Zakutskiy. V. 1. Zolotareva. A. A. Lvova. M. Y. Nekrasova, V. F. Osadchikh, Y. V. Pligin, A. A. Protasov & G. M. Tischikov. 1994. Living conditions. In: J. 1. Starobogatov, editor. Freshwater zebra mussel Dreissena potymorpha (Pall.) (Bivalvia, Dreissenidae). Systematics, ecology, practical meaning. (In Russian) Moscow: Nauka Press, pp. 109-1 19. Mattice, J. S. 1979. Interaction of Corbictita with Power Plants. In: J. C. Britton, editor. First International Corbicuta Symposium. Fort Worth. TX: Texas Christian University Research Foundation, pp. 119-138. McMahon, R. F. 1982. The occurrence and spread of the introduced Asiatic freshwater clam, Corbicula fluminca (Muller), in North America: 1924-1982. Nautilus 96:134-141. McMahon, R. F. 1983. Ecology of an invasive pest bivalve, Corbicula. In: W. D. Russell-Hunter, editor. The mollusca. Vol. 6. Ecology. New York: Academic Press, pp. 505-561. McMahon, R. F. 1991. Mollusca: Bivalvia. In: J. H. Thorp & A. P. Covich, editors. Ecology and classification of North American freshwater in- vertebrates. San Diego: Academic Press, Inc., pp. 315-399. McMahon, R. F. 1999. Invasive characteristics of the freshwater bivalve, Corbicula fluminea. In: R. Claudi & J. H. Leach, editors. Nonindig- enous freshwater organisms: vectors, biology and impacts. Boca Raton. FL: Lewis Publishers, pp. 315-343. McMahon, R. F. & A. E. Bogan. 2001. Mollusca: Bivalvia. In: J. H. Thorp, & A. P. Covich, editors. Ecology and classification of North American freshwater invertebrates. San Diego: Academic Press, Inc., pp. 331- 430. Mikheev, V. P. 1961. Experiments on killing Dreissena with heated water. Biol. Vnutr Vod. Inf. Byult. 11:10-12. Miller, A. C. & B. S. Payne. 1994. Co-occurrence of native freshwater mussels (Unionidae) and the non-indigenous Corbicula fluminea at two stable shoals in the Ohio River. U.S.A. Malac. Rev. 27:87-97. Nichols, S. J. & J. Amberg. 1999. Co-existence of zebra mussels and freshwater unionids: population dynamics of LcptoJea fragilis in a coastal wetland infested with zebra mussels. Can. J. Zool. 77:423—432. Phelps, H. L. 1992. Possible development of the freshwater Asiatic clam. Corbicuta fluminea. as a new aquaculture product in the US. Aijuacul- ture '92. Oriando, FL, p. 186. Phelps, H. L. 1994. The Asiatic bivalve (Corbicula fluminea): invasion and system-level ecological change in the Potomac Riser estuary near Washington D.C. Estuaries. 17:614-621. Poff, N. L., M. A. Palmer. P. L. Angermeier, R. L. Vadas, C. C. Haken- kamp, A. Bely, P. Arensburger & A. P. Martin. 1993. Size structure in the metazoan community in a Piedmont stream. Oecologia 95:202- 209. Prater, M. A. 1991. A survey of the benthic macroinvertebrate community structure of the Lake Nacogdoches, a //v(in7/fl-infested East Texas reservoir. Master of Science Thesis, Stephen F. Austin State Univer- sity, Nacogdoches, Texas, 73 pp. Reid. R. G. B.. R. F. McMahon. D. O'Foighil & R. Finnigan. 1992. An- terior inhalant currents and pedal-feeding in bivalves. Vetiger. 35:93- 104. Shkorbatov, G. L., A. F. Karpevich & P. 1. Antonov. 1994. Ecological physiology. In: J. I. Starobogatov, editor. Freshwater zebra mussel Dreissena potymorpha (Pall.) (Bivalvia, Dreissenidae). Systematics, ecology, practical meaning. (In Russian). Moscow: Nauka Press, pp. 67-108. Sieckel, J. B. 1986. Corbicuta population mortalities: factors influencing population control. Am. Malacol. Bui. Spec. Ed. 2:89-94. Sokolova, N. Y.. E. I. Izvekova, A. A. Lvova & M. I. Sakharova. 1980. Features of benthos forming in small waterbodies using Uchinskoe Reservoir as an example. Tr. Vses. Gidrohiol. O-va. 23:161-170. (In Russian) Spiridonov, Y. 1. 1972. Polarographic investigation of the respiration of Dreissena. In: Topics in physiological and population ecology. 2. (In Russian). Saratov: Saratov University Press, pp. 15-21. Stewart, T. W., J. C. Gafford, J. G. Miner & R. L. Lowe. 1999. Dreissena- shell habitat and antipredator behavior: combined effects on survivor- ship of snails co-occurring with molluscivorous fish. / N. Am. Benlliol. Soc. 18:274-283. Stewart, T. W., J. G. Miner & R. L. Lowe. 1998. Quantifying mechanism for zebra mussel effects on benthic macroinvertebrates: organic matter production and shell-generated habitat. / N. ,A»i. Benthol. Soc. 17:81- 94. Strayer, D. L, 1999. Effects of alien species on freshwater mollusks m North America. / N. Am. Benthol. Soc. 18:74-98. Taylor, M. F. 1980. The physicochemical limnology of Nacogdoches Res- ervoir, Nacogdoches County, Texas. Master of Science Thesis, Stephen F. Austin State University, Nacogdoches, Texas, 179 pp. Zhadin. V. 1. 1946. The traveling shellfish Dreissena. Priroda. 5:29-37. Joimuil of Shellfish Research. Vol. 22, Nii. 1. 493-500, 2003. PATTERNS OF EMERGENCE AND SURVIVAL OF CONCHOPHTHIRVS ACUMINATUS (CILIOPHORA: CONCHOPHTHIRIDAE) FROM DREISSENA POLYMORPHA (BIVALVIA: DREISSENIDAE) ALEXANDP:R v. KARATAYEV,' * SERGEY E. MASTITSKY,- DANIEL P. MOLLOY,' AND LYUBOV E. BURLAKOVA' ^Department of Biology. Stephen F. Austin State University; Nacogdoches. Tews 75962-3003; 'General Ecology Department. Belanisslun State University. 4 Skorynu Ave.. Minsk. 220050 Belarus; and Division of Research & Collections. New York State Museum. Albany. New York 12230 ABSTRACT Thi.s is the first study to quantify the penodic emergence of a Cdihluiplnhinis sp. from its bivalve host. Emergence rates of C. acuininanis from Dreissena polymorpha over the entire 24-day experiment appeared to be directly correlated with infection intensity. The rate of ciliate emergence from individual mussels varied considerably throughout the experiment at both I4'C and 210. It was not uncommon to have a sampling period in which no emergence was observed immediately followed by a period of high emergence, e.g., at I4°C from 0 to 25 ciliates and at 21°C from 0 to 720 ciliates. The total mean number of ciliates that were observed to have emerged from each mussel during the 24-day experiment was significantly higher at 21°C (207 ciliates/mu.ssel) than at 14°C (29 ciliates/mussel). Our experiments suggested that C. acuininanis have a short survival period outside their host. Although we observed a maximum survival period of 144 hr (6 days), most ciliates died within 48 h. KEY WORDS: Conchiipliiliini.y ucuminulus. ciliate, commensal, host, bivalve, Dreissena polxiiiorpha. /.ebra mussel, mantle cavity INTRODUCTION The ciliate Conchophthirus ucwninatus (Claparede & Lach- mann) (Scuticocillatida: Conchophthiridae) is the most common of 34 endosynibionts associated with zebra tnussels {Dreissena poly- morpha (Pallas)) (Molloy et al. 1997). Although tiot known from North America, this ciliate is very common in European zebra mussel populations, including in Bulgaria (Raabe 1934). Denmark (Fenchel 1965), Hungary (Raabe 1950). Macedonia (Raabe 1966). Poland (Dobrzanska 1958). and Switzerland (Claparede & Lach- mann 1858). Its widespread distribution was recently confirined by its presence in all 21 zebra mussel populations surveyed in Belarus (Burlakova et al. 1998, Karatayev et al, 2000a). Among all zebra mussel protozoan symbionts, this ciliate typically has the highest prevalence (i,e,. percentage of mussels with ciliates) and intensity of infection (i.e., number of ciliates per infected mussel) (Molloy et al. 1997, Burlakova et al. 1998. Karatayev et al. 2000a). Conchophthirus acmninatus appears to be very specific to Dreis- sena and has never been reported from any other host. Raabe (1950) never observed it in unionid mussels, even though they were sometimes completely covered by C acwninatus-'miecXeA zebra mussels. Although its feeding on the sperm cells of D. poly- morpha has been documented (Laruelle et al. 1999), C acuminatus is likely a commensal organism which ingests a variety of organic particles present on Dreissena'^ mantle epithelial suifaces (Molloy et al. 1997). C. acwniiuitiis is typically found on the epithelial surfaces of the mantle, gills, visceral mass, and labial palps, and within gill water tubes and suprabranchial cavities (Laruelle et al. 1999). As with other Conchophthirus spp.. C acuminatus appears to have an obligate association with its bivalve host, with the only free-living phase of its life cycle occurring during its transfer to new hosts. The longer these ciliates can live in open water, the greater their success in reaching new hosts, particularly distant zebra mussel populations. An investigation of this free-living phase in the C acuminatus life cycle was the focus of this study. *Corresponding author. E-mail: akaratayev@sfasu.edu In a series of laboratory experiments, we quantified the frequency that these ciliates emerged from zebra mussels and measured their survival rate in open water. The results presented herein are part of an extensive investigation that we. as members of the International Research Consortium on Molluscan Symbionts (Molloy 2003), are conducting to characterize the systematics. biology, ecology, and distribution of Di-eissena\ endosymbionts (Molloy et al. 1996. Molloy et al. 1997, Molloy et al. 2001. Burlakova et al. 1998, Laruelle et al. 1999. Karatayev et al. 2000a. Karatayev et al. 2000b, Karatayev et al. 2002, Laruelle et al. 2002, Fokin et al, 2003). This current study, in particular, will hopefully contribute to a better understanding of the emergence patterns and subsequent free-living phase of C. acuminatus and will thereby provide in- sights into the life cycle of a commensal — a type of symbiont which, relative to parasites and mutualists, has received little re- search attention. MATERIALS AND METHODS Laboratory experiments were conducted during 1998-2002 in the Republic of Belarus using zebra mussels collected at a ca. 1 .5 m depth from the Dnieper-Bug Canal (52°06'N. 26°00'E) and the Svisloch River (53°55'N. 27°32'E). Emergence of C. acuminatus /row D. polymorpha To determine the frequency of emergence of C. acuminatus from zebra mussels, an experiment was conducted in April 1998 in which 48 mussels from the Dnieper-Bug Canal were placed indi- vidually in 20-mL Petri dishes containing a suspension of the alga Sceneilesinus acuminatus (LagerheimI in 10 mL of unchlorinated tap water. For 24 days, half of these dishes were held at 14 (±1 )°C and half at 21 (±1)°C. Mean mussel lengths in the I4°C and 21°C dishes were, respectively, 13.8 mm and 14.3 mm (Tables 1 and 2). Every 2 to 3 days, the water in each dish was transferred to a plankton counting chamber and fresh, unchlorinated tap water and algae were added to each dish. Water in the counting chamber was examined for C. acuminatus using a stereomicroscope (20x), with ciliates counted and discarded. 495 496 Karatayev et al. TABLE 1. Pattern of emergence of C. acuminatus from D. polymorpha at the 14 (±1)°C. \Iussel Number of Ciliates Collected Outside Their Host Infection Mussel Length Total During Intensity No. (mm) Day 3 Days Day 7 Day 10 Day 12 Day 14 Day 17 Day 19 Day 21 Day 24 Experiment on Day 24 1 13,4 6 0 0 1 4 1 1 0 11 7 31 34 2 13.2 1 0 0 1 0 6 1 0 4 6 19 74 3 15.5 1 0 1 2 5 3 0 2 17 24 55 313 4 14.0 3 0 0 1 0 0 1 -) 17 6 30 19 5 14.6 ~i 1 T 3 3 -> -) 0 25 28 68 125 6 13.1 1 0 0 1 0 — 1 I 6 1 12 1 7 13.2 0 0 T 1 ~i 0 2 1 14 5 27 117 8 13.6 0 0 0 0 0 5 1 1 4 4 15 3 9 13.0 0 0 1 0 0 -) 0 ■> 8 24 37 41 10 13.5 1 0 0 3 2 3 ") 4 11 7 33 24 11 14.0 1 0 0 2 1 0 1 0 8 8 21 13 12 14.1 4 6 26 19 0 8 13 4 3 12 95 0 13 14.0 1 0 0 3 0 1 0 1 4 6 16 5 14 14.0 0 0 0 0 -) 0 T T 5 29 40 89 15 14.2 1 0 3 3 1 0 1 3 5 1 19 0 16 13.9 0 0 0 1 0 0 0 1 13 6 21 89 17 14.1 0 0 3 0 0 6 9 5 5 6 34 10 18 13.6 3 0 2 1 1 0 0 0 2 10 19 4 19 13.6 0 1 1 1 0 2 0 0 6 3 14 69 20 13.9 1 3 1 1 T 0 — 1 5 7 21 16 21 13.2 0 2 4 2 I 1 0 4 6 18 38 20 22 13.4 1 0 0 1 0 -) 0 0 4 17 25 77 23 13.6 1 0 1 2 1 0 0 0 0 1 6 11 24 13.6 0 1 0 0 -) 0 1 0 0 5 9 10 Mean 13.8 1.2 0.6 2.0 2.0 1.1 1.8 1.7 1.4 7.6 10.0 29.4 48.5 SE 0.02 0.06 0.06 0.22 0.16 0.06 0.10 0.13 0.06 0.25 0.36 0.83 2.85 To determine infection prevalence and intensity at the begin- ning of the experiment, we dissected 13 14-mm long mussels from the above-mentioned Dnieper-Bug Canal sample. Infection preva- lence and intensity were also calculated at the end of the experi- ment by dissecting the 48 mussels used in the Petri dishes. During dissection, mussel mantle cavities were repeatedly flushed with unchlorinated tap water using a pipette to remove all ciliates from exposed epithelial surfaces. Because C. acuminatus were also present within gill water tubes and suprabranchial cavities, gills were lacerated with forceps and then flushed by pipette. The num- ber of C. acuminatus in all rinse water was determined in a plank- ton counting chamber using a stereomicroscope (20x). Survival of C. acuminatus Outside D. polymorpha Three laboratory experiments were conducted to determine how long C. acuminatus survive outside their host in open water. In all experiments, C. acuminatus were transferred with a pipette into dishes containing water. Dishes were then covered with lids to prevent evaporation and half of them were held at 14 (±1 )°C and the other half at 21 (±1 )°C. Using a stereomicroscope (20x), dishes were inspected until all ciliates had died. Experiment 1 In November 1998, mussels were collected from the Svisloch River and dissected. Ciliates were held in groups of 10 in each of six lO-mL Petri dishes containing 2 mL of unchlorinated tap water and were inspected daily. Experiment 2 In January 2000. 40 C. acuminatus obtained by dissection from zebra mussels collected in Dnieper-Bug Canal were held individu- ally in lO-mL Petri dishes containing 3 mL of unchlorinated tap water. Mortality was scored at 6, 21, 70, and 90 h. Experiment 3 In July 2002. 20 C. acuminatus obtained by dissection from zebra mussels collected from the Svisloch River were held at 14 (±I)°C and 23 (±1)°C in 40 individual 4-mL plastic dishes con- taining 2 mL of filtered ( lOO-jjim mesh net) Svisloch River water. Mortality was scored at 6, 24. 30, 48, and 54 h. During each dish inspection. I mL of water in each dish was replaced with fresh filtered water. Since ciliates may be more sensitive to environmen- tal changes than their hosts (Beers 1959), we followed Beers' suggestion to collect mussels as needed and to use the ciliates at once. Therefore, in experiment 3 we repeated the same exact pro- cedure three times, starting on three consecutive days using ciliates from freshly collected mussels. Data Analysis The Box-Cox procedure (Krebs 1999) indicated that the best transformation to achieve a normal distribution was X' = {X+ \f~. Emergence and Survival C. acuminatus 497 tablp: 2. Dynamics of the emergence of C. acuminatus from D. polymorpha at the 21 (±1)°C \Iu$$el Num ber of CiUates Collected Outside Their Host Infprtinn Mussel Length Total During ■ iiitri. iiuii Intensity No. (mm) Day 3 Day 5 Day 7 Day 10 Day 12 Day 14 Day 17 Day 19 Day 21 Day 24 Kxperiment on Day 24 1 13.1 -) 1) 0 3 (1 0 70 176 15 26 292 0 2 13.1 1 0 17 99 29 9 22 15 16 1 210 0 3 14.0 2 9 2 2 5 3 2 85 171 35 316 7 4 15.5 0 720 186 45 14 5 4 0 12 1 987 63 5 14.0 1 3 6 4 (1 3 31 20 12 5 85 1 6 15.5 9 0 6 120 159 87 156 32 24 47 640 143 7 15.8 1 0 1 -) 0 0 1 5 5 32 47 49 8 15.2 43 9 22 6 1 4 14 7 4 4 114 129 9 14.6 20 -} 0 0 3 9 105 24 37 16 216 114 10 13.0 0 2 0 28 15 9 34 6 9 3 106 168 11 14.3 3 72 3 1 4 11 39 14 24 41 212 732 12 13.5 0 3 10 ") 1 2 0 1 1 5 25 262 13 14.5 1 0 0 0 1 3 66 45 15 7 138 331 14 14.5 1 2 1 T 0 5 8 3 6 11 39 158 15 15.2 9 76 1 1 5 0 6 29 9 6 142 1035 16 13.9 5 0 2 0 4 13 4 4 1 1 34 0 17 15.8 2 55 1 3 6 33 61 150 34 61 406 93 18 14.0 0 7 0 1 2 15 200 29 52 6 312 175 19 14.1 1 0 0 0 0 3 3 3 6 11 27 264 20 13.4 0 1 4 7 11 7 26 8 11 3 78 39 21 13.7 0 5 0 0 3 0 1 6 5 5 25 21 22 13.2 0 3 I 0 1 3 9 39 25 133 215 82 23 13.6 5 1 3 11 10 6 20 72 12 0 140 27 24 15.4 0 8 0 19 24 14 81 10 4 3 163 351 Mean 14.3 4.4 40.8 11.1 14.8 12.5 10.2 40.1 32.6 21.3 19.3 207.0 176.8 SE 0.04 0.39 6.10 1.57 1.30 1.34 0.74 2.16 1.91 1.43 1.23 9.17 10.23 To compare transformed data, we used Welch's approximate t test (or t test if variances were homogeneous) in Statistica software (Windows Release 6.0, StatSoft. Inc.). Effects were considered stati.stically significant at P < 0.05. RESULTS Emergence of(S. acuminatus /rom D. polymorpha The rate of ciliate emergence from individual mussels varied considerably throughout the experiment at both 14°C and 21°C. It was not uncommon to have a sampling period in which no emer- gence was observed, immediately followed by a period of high emergence, e.g., at I4°C from 0 to 25 ciliates (Table I: mussel 5. day 19 vs. day 21) and at 2I°C from 0 to 720 ciliates (Table 2: mussel 4, day 3 vs. day 5). At I4°C. typically s3 ciliates were observed outside a host mussel each sampling day. but this pattern was typically inter- rupted by periods of higher emergence, particularly toward the end of the experiment (Table I ). The mean number of ciliates that were observed outside of the 24 mussels at I4°C ranged from 0.6 to 10.0 ciliates/mussel (Table 1). During the first 19 days of the experi- ment, a mean of 1.5 ciliates was observed outside the 24 mussels at I4°C (Table I ). Dissection data indicated that infection intensity in the 14°C mussels during the experiment remained constant at about 48 ciliates/mussel (day 0 and day 24 intensities of, respec- tively 47.3 and 48.5 ciliates/mussel. Table 1). This indicated that during the first 19 days of the experiment on average ca. 3% (i.e.. 1.5/49.5) of ciliates were outside their hosts on a sampling day. Emergence rates increased toward the end of the I4°C experiment with a mean emergence of 10.0 ciliates/mussel at the termination of the experiment on day 24 (Table I). Because the 24 mussels dissected at the end of the I4°C experiment had a mean infection intensity of 48.5 ciliates/mussel (Table I), this indicated that ca. 17% (i.e., 10.0/58.5) of all ciliates present within the 24 dishes were outside their hosts on day 24. A similar irregular pattern of ciliate emergence was ob.served at 21°C (Table 2). Typically ,°„o° ^'/^/ -^' .^.^ u A AB 1 i Very Poo( Very Bitlei Very Good Very Sweel(<1) Excellenl Very Sweet 0.5 1253.83 14 11.37 <0.001 58.14 14 4.04 <0.001 Block 161.08 2 3.98 <0.05 15.93 2 1.01 >0.1 0.56 2 0.27 >0.5 Errcir 486.26 24 220.63 28 28.81 28 Color Rating (with paint samples) Color Rating (without paint samples) L* (brightness) Treatment 1.58 14 0.77 >0.5 1.13 14 1.^5 >0.1 419.60 14 1.54 >0.l Block 0.41 2 1 .40 >0.1 0.29 2 2.42 >0.1 8.46 2 0.22 >0.5 Error 4.10 28 a* (redness) 1.68 28 b* (yellowness) 543.46 28 Texture Rating Treatment 85.56 14 1.63 >0.1 122.22 14 3.71 <0.005 3.98 14 2.89 <0.01 Block 3.72 2 0.50 >0.5 5.36 2 1.14 >0.1 0..30 2 1.51 >0.1 Error 104.74 28 Firmness Ratmg 65.89 28 Taste Rating 2.76 28 Treatment 1.5.^ 14 0.89 >0.5 13.26 1 3 3.65 <0.005 Block 0.14 2 0.56 >0.5 0.19 2 0.33 >0.5 Error ,^.4.^ 28 7.27 26 510 Pearce et al. TABLE 3. Results of separate 4-way ANOVAs on percent gonad yield, percent gonad water, gonad color rating, L*, a*, b*, gonad texture rating, gonad firmness rating, and gonad taste rating at the end of the 12-wk experiment. Sources of variation are starch type (S), macroalgal meal source (M), (5-carotene concentration (B), block, and error. Source SS DF f-Ratio P-Value SS DF f"-Ratio P-Value SS DF F-Ratio f-Value Yield (%) Water (%) Color Rating (with paint ^ >amples) S 6.17 -) 0.52 >0.5 3.40 2 2.13 >0,1 0.02 1 0.10 >0.5 M 5.18 I 0.88 >0.1 0.34 1 0.42 >0.5 0.32 1 3.77 >0.05 B 44.98 1 7.64 <0.05 0.07 1 0.09 >0.5 0.19 1 2.20 >0,l SxM 8.84 -) 0.75 >0.1 0.38 2 0.24 >0.5 0.01 1 0.01 >0,5 SxB 24.18 T 2.05 >0.1 1.60 1 1.00 >0.1 0.12 1 0.71 >0.5 MxB 0.41 1 0.07 >0.5 0.22 1 0.28 >0.5 0.01 1 0.01 >0,5 SxMxB 1.44 -) 0.12 >0.5 1.32 1 0.83 >0,1 0.05 1 0.30 >0.5 Block 7.72 -> 0.66 >0.5 1 .05 1 0.66 >0.5 0.61 -) 3.58 <0.05 Error 129.53 n 17.53 ->2 1.88 22 Color Rating (without paint samples) L* (brightness) a* (redness) S 0.08 2 0.81 >0.1 8.41 T 0.23 >0.5 1.87 T 0.26 >0,5 M 0.20 1 4.08 >0.05 51.39 1 2.86 >0.l 1.22 1 0.33 >0.5 B 0.23 1 4.70 <0.05 45.70 1 2,54 >0.1 17.78 1 4.87 <0.05 SxM 0.01 1 0.07 >0.5 100.08 1 2,78 >0.05 12.86 T 1.76 >0.1 SxB 0.02 2 0.16 >0.5 27.82 1 0.77 >0.1 6.73 "> 0.92 >0.1 MxB 0.03 1 0.68 >0.1 15.87 1 0.88 >0.1 12.91 1 3.53 >0.05 SxMxB 0.01 1 0.02 >0.5 33.70 2 0.94 >0.1 2.43 1 0.33 >0.5 Block 0.18 1 1.81 >0.1 2.34 2 0.07 >0.5 0.78 2 0.11 >0.5 Error 1.09 T> 396.04 11 80.41 22 b* (yellowness) Texture Rating Firmness Rating S 13.58 -i 3.20 >0.05 0.07 2 0.39 >0.5 0.17 1 0.68 >0.5 M 1.83 1 0.86 >0.1 0.32 1 3.53 >0,05 0.01 1 0.08 >0,5 B 4.93 1 2.32 >0.1 0.07 1 0,78 >0,1 0.28 1 2.21 >0,1 SxM 4(1.25 2 9.49 <0.001 0.31 2 1,69 >0.1 0.14 1 0.53 >0.5 SxB 18.14 2 4.28 <0.05 0.24 2 1,32 >0.1 0.03 1 0.11 >0,5 MxB 24.73 1 11.66 <0.005 0.01 1 0.05 >0.5 0.01 1 0.08 >0,5 SxMxB 1.54 1 0.36 >0.5 0.07 2 0.41 >0.5 0.08 1 0.32 >0,5 Block 1.50 2 0.36 >0.5 0.06 2 0.33 >0.5 0.09 1 0.37 >0.5 Error 46.65 11 2.00 22 2.83 22 Taste Rating S 0.07 2 0.12 >0.5 M 0.54 1 1.84 >0.1 B 0,92 1 3.13 >0.05 SxM 0.59 2 1.00 >0.1 SxB 0.08 1 0.13 >0.5 MxB 0.29 1 1.00 >0.1 SxMxB 0.07 1 0.11 >0.5 Block 0.50 1 0.85 >0.1 Error 6.46 11 out the pigment (Fig, 2A). A 4-way repeated ANOVA examining the effects of time, starch type, macroalgal meal source, and P-carotene concentration on percent gonad yield showed a signifi- cant effect of time, but no other significant main or interaction effects (Table 4). Percent gonad yield increased over time with all pair-wise comparisons among weeks being significantly different except for comparisons between weeks 0 and 4. weeks 0 and 6, and weeks 4 and 6 (Fig. 2B). Gonad Water Mean percent gonad water decreased during the 12-wk experi- ment from 84.2 ± 0.3% at the beginning of the experiment to less than 81.0% in all feeding treatments at the end of 12 wk (Fig. IC). A 2-way ANOVA analyzing the effects of treatment and block on percent gonad water at the end of the experiment showed a sig- nificant effect of treatment (Table 2). All prepared feed treatments (range: 79.5-81.0%). as well as the kelp control (80,9 ± 1.0%) and wild samples taken at the end of the experiment (81.9 ± 0.8%), had significantly lower percent gonad water than the initial sample (84,2 ± 0.3%) (Fig. IC). There were no significant pair-wise dif- ferences in percent gonad water among any of the prepared feeds or kelp control (Fig. IC). A 4-way ANOVA examining the effects of starch type, mac- roalgal meal source, P-carotene concentration, and block on per- cent gonad water at the end of the experiment revealed no signifi- cant main or interaction effects (Table 3). A 4-way repeated ANOVA examining the effects of time, starch type, macroalgal meal source, and p-carotene concentration on percent gonad water showed significant starch type and tiine main effects and a sig- nificant starch type x (B-carotene concentration interaction (Table 4). For feeds with 0 mg kg"' of p-carotene, those containing potato Gonad Enhancement of Strongylocentrotus droebachiensis 511 2 >- ■a to c o O 30 A 2b A -l- 1 B -r 20 15 10 _L - 5 0 200 [ll-Carotene] (mg kg"') 30 25 °^ 20 0) >- CD c o O 15 10 B B A - D E D -t D C 10 12 Time (week) Figure 2. (A) Mean percent gonud yield at week 12 In feeding treatments with and without (i-carotene. Error bars are SE and n = 18. Letters above bars indicate the results of an ANOVA showing significant difference between treatment means. (B) Mean percent gonad yield of all feeding treatments at each sampling interval. Error bars are SE and ;i = 36. Letters above bars indicate the results of a Fisher's LSD multiple comparison post-hoc test showing signiTicant pair-wise differences among weeks. Starch produced gonads with significantly higher percent water than those containing tapioca starch (Fig. 3A). There were no significant differences, however, among starch types for feeds con- taining 200 mg kg"' of P-carotene (Fig. 3A). There were no sig- nificant differences between the two p-carotene concentrations at any of the starch type levels. Percent gonad water decreased over time with all pair-wise comparisons among weeks being signifi- cantly different except for comparisons between weeks 0 and 2. weeks 0 and 4, and weeks 2 and 4 (Fig. 3B). Gonad Color At the end of the experiment, mean color ratings of sea urchin gonads from prepared diet treatments varied between 2.5 ± 0.3 (Tap Kelp 200) and 3.0 ± 0. 1 (Com Rock 0) for ratings done with paint samples and between 2.3 ± 0.2 (Corn Kelp 200) and 2.8 ± 0. 1 (Tap Rock 0) for ratings done without paint samples (Fig. 4A. B). There were no significant differences at the end of the experiment among any of the prepared feeds, kelp control, or wild controls in either color rating data set (Table 2, Fig. 4A, B). Four-way ANOVAs examining the effects of starch type, mac- roalgal meal source, p-carotene concentration, and block on gonad color ratings at the end of the experiment revealed a significant effect of p-carotene concentration for ratings done without paint samples, but not for ratings done with paint samples (Table 3). In both data sets, feeds with P-carotene produced better gonad color than those without the pigment (Fig. 5A). There were no other significant main or interaction effects in either color rating data set (Table 3). A 4-way repeated ANOVA examining the effects of time, starch type, macroalgal meal source, and P-carotene concentration on gonad color ratings done with paint samples showed a signifi- cant effect of time, but no other significant main or interaction effects (Table 4). Gonad color improved over time; sea urchins sampled in weeks 6. 8, 10. 12 had significantly better gonad color than those sampled in weeks 0 and 4 while those measured in weeks 10 and 12 had significantly better gonad color than those measured in weeks 0. 2. and 4 (Fig. 5B). Time also significantly affected gonad color ratings done without paint samples, although the effect of time was dependent on the interaction with macroal- gal meal source (Table 4). For sea urchins fed kelp meal diets, gonad color ratings significantly improved at each subsequent sampling date; week 12 was significantly better than week 6, which was significantly better than week 0 (Fig. 5C). For sea urchins fed rockweed meal diets, gonad color ratings in weeks 6 and 12 were significantly improved from the beginning of the experiment, but there was no significant difference between weeks 6 and 12 (Fig. 5C). There was no significant difference in gonad color ratings between kelp and rockweed meal diets at weeks 0 or 6, but feeds containing kelp meal produced significantly better gonad color ratings than feeds containing rockweed meal by week 12(Fig. 5D). At the end of the experiment, mean values of L* of sea urchin gonads from prepared diet treatments varied between 45.4 ± 2.3 for Com Rock 0 and 55.1 ± 0.4 for Corn Kelp 0 (Fig. 4C). There were no significant differences at the end of the experiment among any of the prepared feeds, kelp control (52.0 ± 1.3), or wild con- trols (0 wk: 49.5 ± 2.8, 12 wk: 44.3 ± 4.0) (Table 2, Fig. 4C). There were no significant main or interaction effects in the 4-way ANOVA conducted on week 12 L* data (Table 3) and only the effect of time was significant in the 4-way repeated ANOVA (Table 4). The mean value of L* was higher in week 12 than in weeks 0 or 6 of the experiment, but only the comparison between weeks 6 and 12 was significantly different (Fig. 6). Mean values of a* (hue or redness) of sea urchin gonads from prepared diet treatments in week 12 varied between 19.2 ± 0.7 for Com Kelp 0 and 23.0 ± 1.5 for Com Kelp 200 (Fig. 4D). There were no significant differences at the end of the experiment among any of the prepared feeds, kelp control (20.3 ± 0.9), or wild con- trols (0 wk: 18.9 ± 1.3, 12 wk: 23.1 ± \A) (Table 2, Fig. 4D). There was a significant effect of P-carotene concentration on a* values at the end of the experiment, but no other significant main or interaction effects (Table 3). Feeds with 200 mg kg"' of P-caro- tene produced significantly higher a* values than feeds with 0 mg kg"' of pigment (Fig. 7A). Only the effect of time was significant in the 4-way repeated ANOVA (Table 4). Values of a* were 512 Pearce et al. significantly higher in week 12 than in weeks 0 or 6 of the ex- periment (Fig. 7B). At the end of the experiment, mean values of b* (chroma or yellowness) of sea urchin gonads from prepared diet treatments varied between 16.2 ± 0.2 for Com Kelp 0 and 21 .5 ± 1 .4 for Corn Rock 0 (Fig. 4E). Mean values of b* for the feeding treatments at the end of the 1 2-wk experimental period and the wild samples collected at the beginning and end of the experiment differed sig- TABLE 4. Results of separate 4-Hay repeated ANOV.\s on percent gonad yield, percent gonad water, gonad color rating, L*, a*, b*, gonad texture rating, gonad firmness rating, and gonad taste rating. Sources of \ariation are starch type (S), macroalgal meal source (M), (i-carotene concentration (B), time (Tl, and error. Source SS DF F-Ratio P-Value SS DF f-Ratio P-Value SS DF F-Ratio P-Value S M B SxM SxB MxB SxMxB Error T TxS TxM TxB TxSxM TxSxB TxMx B TxSxMxB Error S M B SxM SxB MxB S xMxB Error T TxS TxM TxB TxSxM TxS xB TxMxB TxSxMxB Error S M B SxM SxB MxB SxMx B Error T TxS TxM TxB TxSxM TxSxB TxMxB TxSxMxB Error 55.48 23.30 126.68 20.99 40.66 7.78 25.93 799.39 2121.78 55.36 19.80 34.00 34.39 34.50 22.60 60.27 515.00 1 -) 24 6 12 6 6 12 12 6 12 144 Yield (9r) 2 0.83 1 0.70 1 3.80 2 0.32 2 0.61 0.23 0.39 98.88 1.29 0.92 1.59 0.80 0.80 1.05 1.40 >0.1 >0.1 >0.05 >0.5 >0.5 >0.5 >0.5 <0.001 >0.1 >0.1 >0.1 >0.5 >0.5 >0.1 >0.1 Color Rating (without paint samples) 0.10 0.01 0.11 0.01 0.03 0.01 0.04 2.06 1.81 0.09 0.44 0.13 0.01 0.04 0.03 0.06 2.28 9.42 3.20 0.41 19.25 49.47 0.50 1.48 78.89 878.57 53.62 18.44 5.76 36.46 34.72 62.60 0.81 180.88 24 0.59 0.01 1.25 0.02 0.18 0.11 0.23 19.07 0.47 4.58 1.40 0.02 0.22 0.26 0.29 4 48 b* (yellowness) 2 1.43 1 1 2 2 1 ") 24 4 4 2 4 48 0.97 0.12 2.93 7.53 0.15 0.23 116.57 3.56 2.45 0.77 2.42 2.30 8.31 0.05 >0.5 >0.5 >0.1 >0.5 >0.5 >0.5 >0.5 <0.001 >0.5 <0.05 >0.1 >0.5 >0.5 >0.5 >0.5 >0.1 >0.1 >0.5 >0.05 <0.005 >0.5 >0.5 <0.001 <0.05 >0.05 >0.1 >0.05 >0.05 <0.001 >0.5 8.29 0.10 1.72 1.69 8.12 0.09 1.24 24.95 577.15 10.26 2.99 1.24 5.12 10.06 2.53 7.80 80.79 5.11 0.63 13.91 9.69 28.36 21.54 7.22 285.29 145.14 24.40 84.40 31.88 114.50 32.00 10.77 48.34 940.94 0.18 0.14 0.03 0.18 0.13 0.01 0.06 2.24 0.31 0.13 0.19 0.05 0.28 0.43 0.01 0.30 4.00 Water ( ':f ) 2 3.99 1 0.09 1 1.66 2 0.8 1 2 3.91 1 0.09 : 0.60 Color Rating {with paint samples) 24 6 12 6 6 12 12 6 12 144 L* (brightness) 2 0.22 171.46 1.52 0.89 0.37 0.76 1.50 0,75 1.16 1 1 2 24 0.05 1.17 0.41 1.19 1.81 0.30 3.70 0.31 2.15 0.81 1.46 0.41 0.28 0.62 4 48 Texture Rating 2 0.96 24 4 4 2 4 48 1.51 0.29 0.96 0.67 0.03 0.31 1.87 0.39 1.12 0.27 0.85 1.28 0.01 0.90 <0.05 >0.5 >0.1 >0.1 <0.05 >0.5 >0.5 <0.001 >0.1 >0.5 >0.5 >0.5 >0.1 >0.5 >0.1 >0.5 >0.5 >0.1 >0.5 >0.1 >0.1 >0.5 <0.05 >0.5 >0.1 >0.1 >0.1 >0.5 >0.5 >0.5 >0.1 >0.1 >0.5 >0.1 >0.5 >0.5 >0.5 >0.1 >0.5 >0.1 >0.5 >0.1 >0.1 >0.5 >0.1 0.02 0.14 0.35 0.21 0.07 0.06 0.03 3.91 6.25 0.62 0.88 0.22 0.67 0.59 0.23 0.98 15.62 6.43 0.08 10.73 4.98 1.12 6.23 2.05 87.53 129.05 24.17 3.68 9.18 8.27 11.61 7.21 2.40 224.40 0.12 0.01 0.23 0.06 0.03 0.01 0.02 2.86 1.61 0.09 0.01 0.14 0.57 0.04 0.03 0.07 6.82 24 6 12 6 6 12 12 6 12 144 a* "> 1 1 1 24 2 4 2 2 4 4 0.06 0.83 2.16 0.65 0.21 0.35 0.10 9.59 0.48 1.35 0.34 0.52 0.46 0.36 0.75 (redness) 0.88 0.02 2.94 0.68 0.15 1.71 0.28 13.80 1.29 0.39 0.98 0.44 0.62 0.77 0.13 4 48 Firmness Rating 0.51 2 1 1 2 2 1 2 24 4 48 0.11 1.94 0.26 0.14 0.01 0.10 5.66 0.15 0.02 0.50 1.01 0.07 0.10 0.12 >0.5 >0.1 >0.1 >0.5 >0.5 >0.5 >0.5 <0.001 >0.5 >0.1 >0.5 >0.5 >0.5 >0.5 >0.5 >0.1 >0.5 >0.05 >0.5 >0.5 >0.1 >0.5 <0.001 >0.1 >0.5 >0.I >0.5 >0.5 >0.1 >0.5 >0.5 >0.5 >0.1 >0.5 >0.5 >0.5 >0.5 <0.01 >0.5 >0.5 >0.5 >0.1 >0.5 >0.5 >0.5 continued on next page Gonad Enhancement of Strongylocentrotus droebachiensis 513 TABI.F 4. Continufd Source SS DF f-Ratio P-Value Tasie Rating S 0.05 2 0.04 >0.5 M 1.23 1 1.95 >0.1 B 0.01 1 0.01 >0.5 SxM 0.24 T 0.19 >0.5 SxB 0.16 2 0.12 >0.5 MxB 0.07 1 0.11 >0.5 SxM> B 1.10 1 0.87 >0.1 Error 15.14 24 T 0.11 1 0.29 >0.5 TxS 0.07 2 0.09 >0.5 TxM 0.01 1 O.OI >0.5 TxB 2.08 1 5.75 <0.05 T X S X M 2.28 2 3.14 >0.05 T X S X B 0.02 2 0.03 >0.5 TxM> B 1.06 1 2.94 >0.1 T X S X MxB 1.69 T 2.34 >0.1 Error 8.7(1 24 nitlcantly (Table 2). Gonads from wild sea urchins generally had lower b* values than those from sea urchins fed prepared feeds or kelp, although only Com Rock 0. Com Rock 200 (19.5 ± 0.5). and Tap Kelp 200 (20.9 ± 0.4) had significantly higher b* values than wild samples (Fig. 4E). In the 4-way repeated ANOVA, there was one significant main effect (time) and three significant interactions (starch type x p-carotene concentration, time x starch type, and time x macroalgal meal source x (J-carotene concentration) (Table 4). Comparing the two concentrations of (J-carotene at each inter- action level revealed three out of 12 (week 0 data not tested) pair-wise comparisons to be significant; feeds with 0 mg kg"' P-carotene had significantly higher b* values than feeds with 200 mg kg"' P-carotene for Com Kelp and Pot Kelp at week 6 whereas the reverse was true for Tap Kelp at week 12 (Fig. 8). Comparing the three weeks at each interaction level revealed sicnificant dif- ferences for all treatments except Com Kelp 0. Pot Kelp 0. Pot Rock 200. Tap Kelp 0. and Tap Rock 200; weeks 0 and 12 gen- erally had significantly higher b* values than week 6 (Fig. 8). Gonad Texliirc, Firmness, and Taste At the end of the experiment, mean gonad texture ratings of sea urchins fed prepared diets varied between 1 .3 ± 0.1 for Com Kelp 0 and 1 .9 ± 0. 1 for Com Rock 0. Pot Rock 0. and Tap Kelp 0 (Fig. ID). Mean texture ratings for the feeding treatments at the end of the 12-wk experimental period and the wild samples collected at the beginning and end of the experiment differed significantly (Table 2). All prepared feeds, kelp control (1.7 ± 0.2). and wild 0-wk control (1.8 ± 0.2) had significantly lower texture ratings than the wild 12-wk control (2.8 ± 0.3) (Fig. ID). Among the prepared diets. Com Kelp 0 had significantly better gonad texture than Com Rock 0. Com Rock 200 ( 1 .9 ± 0.2), Pot Rock 0, Tap Kelp 0. and Tap Rock 0 ( 1.9 ± 0.2); there were no other signifi- cant pair- wise differences among prepared feeds (Fig. ID). There were no significant main or interaction effects on gonad texture in either the 4-way ANOVA (Table 3) or 4-way repeated ANOVA (Table 4). Mean gonad firmness ratings of sea urchins fed prepared diets varied between 1.9 ± 0.3 for Pot Kelp 200 and 2.5 ± 0.3 for Tap Kelp 0 (Fig. IE). There were no significant differences among any of the prepared feeds, kelp control (1.8 ± 0.2), wild 0-wk control (2.1 ±0.3). or wild 12-wk control ( 1.8 ± 0.1 ) (Table 2. Fig. IE). A 4-way ANOVA examining the effects of starch type, macroalgal meal source, p-carotene concentration, and block on gonad firm- ness ratings at the end of the experiment revealed no significant main or interaction effects (Table 3). Time was the only significant effect in the 4-way repeated ANOVA (Table 4). Gonads were significantly fimier at week 6 than at weeks 0 or 12. but there was no significant difference in gonad firmness between weeks 0 and 12 (Fig. 9). Mean gonad taste ratings of sea urchins fed prepared diets varied between 3.3 ± 0.2 for Pot Rock 0 and Tap Rock 0 and 4.2 ± 0.1 for Pot Kelp 0 (Fig. IF). A 2- way ANOVA revealed sig- JO to c o O yo " H Corn a Pot ■ Tap 8b 80 AB jr. A B rh NS 0 200 [R>-carotene] (mg kg ' B TO TO c o CD 90 85 80 75 B A A A B C D E - 10 12 Time (week) Figure 3. (A) Mean percent gonad water o>tr the 12-wk experiment in feeding treatments with and without (J-carotene with the three difTerent starches. Error bars are SE and n = 42. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing significant pair-wise differences among starch types at each (5-carotene level. "N.S" denotes no significant difference among treatment means. (B) Mean percent gonad water of all feeding treatments at each sampling interval. Error bars are SE and n = .^6. Letters above bars indicate the results of a Fisher's LSD multiple comparison post-hoc test showing significant pair-wise differences among weeks. 514 Pearce et al. AU Hini .•^ ,^ "^ ^ .^ ^ .^ Dark Brown Grey/Black Yellow-Brown Orange-Brown Pale Yellow Pale Orange Bnghl Yellow Bnght Orange E 03 o o o i, A Q ^ O ^ _Q, ^ Q ^ O ^ .^ c?" J^ Dark Brown Grey/Black Yellow-Brown Orange-Brown Pale Yellow Pale Orange Bnght Yellow ;n sfN Bnghf Orange y^^ -r /^ o^ />o -c _5- < ^'-° O^ O^ O^ <5cO Q^ o^a^^-^ o BCD CD 1 CD I ■^ '^ f O^ tt ct° ^ j^ ^ Figure 4. Mean color rating with paint samples (A), mean color rating without paint samples (B), mean CIE lightness (Cl. mean CIE hue ID), and mean CIE chroma (E) for all experimental treatments and wild controls at the end of the experiment. See text for full explanation of gonad quality ratings. Error bars are SE and n = 3. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing significant pair-wise differences among experimental treatments and wild controls within each graph. "NS" denotes no significant differences among treatment means. nificant differences among treatment means at the end of the ex- periment (Table 2). All prepared feeds produced significantly worse tasting gonads than the kelp control (2.3 ± 0.3) while the wild sample collected at the end of (he experiment (2.5 ± 0. 1 1 had significantly better tasting gonads than all prepared feeds except Pot Rock 0 (3.3 ± 0.2) and Tap Rock 0 (3.3 ± 0.2) (Fig. IF). There were no significant pair-wise differences among any of the pre- pared feeds in terms of gonad taste ratings (Fig. IF). A 4-way ANOVA examining the effects of starch type, macroalgal meal source, P-carotene concentration, and block on gonad taste ratings at the end of the experiment revealed no significant main or in- teraction effects (Table 3 ). A four- way repeated ANOVA on gonad taste ratings over time revealed no significant main effects and only one significant interaction — time x p-carotene concentration (Table 4). There was no significant difference between weeks 6 and 12 in gonad taste of sea urchins fed prepared feeds without P-carotene. but gonad taste was significantly better at week 6 than at week 12 for sea urchins given feeds with p-carotene (Fig. IDA). There was no significant difference in gonad taste of sea urchins fed feeds with or without pigment at either week 6 or week 12 (Fig. lOB). DISCUSSION The experiment was begun during the natural spawning season of the green sea urchin. Gonad yields of individuals sampled at the beginning of the experiment were high (15.1 ± 1.0%), but natural populations had completely spawned out by the end of the experi- ment in July (gonad yield = 2.8 ±0.5'7f ). In contrast, experimental sea urchins maintained in the laboratory under ambient tempera- ture and fixed photoperiod and fed prepared diets did not undergo complete spawning. Partial spawning occurred in some individuals early in the experiment as evidenced by a slight drop in gonad yield during the second week of the experiment. This was also observed macroscopically as a number of individuals were leaking gametes early in the experiment. After the second week, however, there was a steady increase in gonad yield at each subsequent sampling period. This increase in gonad yield was not due to increasing water, however, since percent gonad water decreased during the experiment. These results are interesting from a com- mercial culture perspective since it shows that gonads of sea ur- chins fed prepared feeds can be enhanced even during periods of the year when natural populations are spawning. While sea urchins given prepared feeds showed an increase in percent gonad yields over the 12-wk experimental period, individuals fed kelp actually showed a decrease, albeit not significant, over the same time in- terval. Previous experiments have also shown that kelp can be an inferior feed in relation to prepared diets in terms of optimizing gonad yield (Pearce et al. 2002a, Pearce et al. 2002b, Pearce et al. 2002c). While kelp was inferior to prepared feeds in terms of increasing gonad yield, it did produce significantly better tasting gonads than the prepared diets. Gonad Enhancement of Strongylocentrotus droebachiensis 515 "to Qi k_ o o O o Without Samples s Witti Samples NS r^ [+1 0 200 0 200 [n>-carotene] (mg kg ') 2 4 6 8 10 12 Time (week) Dark Brown Grey/Black Yellow-Brown Orange-Brown Pale Yellow Pale Orange Bright Yellow Bright Orange Kelp Rock CO a. E to to '3 o = 2 CD _o O O 1 D 2 Kelp □ Rock N*^ -in ph A B - Dark Brown Grey/Black Yellow-Brown Orange-Brown Pale Yellow Pale Orange Bright Yellow 12 Bright Orange Time (week) Figure 5. (A) Mean color rating («ith and without paint samples) at week 12 in feeding treatments with and without (J-carotene. Error bars arc SE and n = 18. Letters above bars indicate the results of ANOVAs showing significant difi'erence between treatment means. "NS" denotes no significant difference between treatment means. (B) Mean color rating (with paint samples! of all feeding treatments at each sampling interval. Error bars are SE and ii = id. Letters above bars indicate the results of a Fisher's LSD multiple comparison post-hoc test showing significant pair-v\ise differences among weeks. (C) Mean color rating (without paint samples) in feeding treatments with kelp or rockweed meal at the three sampling intervals. Error bars are SE and n = 18. Letters above bars indicate the results of Fisher's LSD multiple comparison post-hoc tests showing significant pair-wise differences among weeks at each level of macroalgal meal source. (D) Mean color rating (without paint samples) at the three sampling intervals for the kelp or rockweed meal treatments. Error bars are SE and ;i = 18. Letters above bars indicate the results of ANOVAs showing significant difference between treatment means. "NS" denotes no significant difference between treatment means. Whereas all prepared feed treatments experienced gonad growth, the rate of increase (range: 0.3-0.8% wk~' ) was somewhat slower than pubhshed rates of increase in previous studies that have used prepared feeds to enhance 5. droebachiensis ( Klinger et al. (1997): 1.4% wk"': Motnikar et al. (1997): 1.2-2.6% wk"'; Havardsson et al. (1999): 0.7-0.8% wk"': Pearce et al. (2002a): 1.2-1.4% wk"'; Pearce etal. (2002b): 0.9-1.3% wk"'; Robinson et al. (2002): 1.6-2.2% wk"'). Differences in rate of gonad increase may be attributed to variations in dietary components, especially protein concentration (de Jong-Westman et al. 1995a) and/or pro- tein source ratio (Pearce et al. 2002b). Time of year may also affect rate of increase in percent gonad yield. In this study, while experi- mental sea urchins maintained in the laboratory did not spawn completely out, gonad yield dropped slightly in the second week of the experiment suggesting partial spawning in some individuals. Partial spawning was also evidenced by the presence of gonads leaking gametes in many individuals early in the experiment. This partial spawning event decreased the overall rate of gonad yield increase. Gonad yield was unaffected by starch type or macroalgal meal source. This result was not surprising given that: ( 1 ) protein ap- 65 60 CO c 55 .^ 50 LU O 45 40 AB -i- 0 6 12 Time (week) Figure 6. Mean CIE lightness (L''') of all feeding treatments at each sampling interval. Error bars are SE and ;; = 36. Letters above bars indicate the results of a Fisher's LSD multiple comparison post-hoc test showing significant pair-wise differences among weeks. 516 Pearce et al. 0 200 [fl-carotene] (mg kg^) 26 24 TO, 22 -carotene] (mg kg ') en c 00 3 B NS ■h D 0 mg kg ' D 200 mg kg ' Very Poor Very Bitter Poor Bitter Satisfactory Bland Good Sweet Very Good Very Sweet (<1) Excellent Very Sweet 6 12 Time (week) Figure II). (A, B) Mean gonad taste rating of feeds with and without pigment (0 and 200 mg kg ' |3-carotene) at weeks 6 and 12. Error bars are SE and n = 18. Letters above bars indicate the results of ANOVAs showing significant pair-wise differences between pigment concentrations. "NS" denotes no significant difference between treatment means. 518 Pearce et al. LITERATURE CITED Andrew. N., Y. Agatsuma. A. Bazhin, E. Creaser, D. Barnes. L. Botstord. A. Bradbury. A. Campbell. S. Einnarsson. P. Gerring, K. Hebert. M. Hunter, S. B. Hur. C. Johnson. M. A. Juinio-Menez, P. Kalvass. R. Miller. C. Moreno. J. Palleiro. D. Rivas. S. Robinson, R. Sleneck. R. Vadas, D. Woodby & Z. Xiaoqi. 2001. Status and management of world sea urchin tlsheries. Oceaiiogi: Mar. Biol. .Aniui. Rev. 40:343- 425. Barker. M. P.. J. A. Keogh. J. M. Lawrence & A. L. Lawrence. 1998. Feeding rate, absorption efficiencies, growth, and enhancement of go- nad production in the New Zealand sea urchin Evechiims chloroticiis Valenciennes (Echinoidea: Echinometridae) fed prepared and natural diets. J. Shellfish Res. 17:158.VLS90. Black. W. A. P. 1950. The seasonal variation in weight and chemical composition of the common British Laminariaceae. J. Mar. Biol. Assoc. UK. 29:45-72. Cook, E. J., M. S. Kelly & J. D. McKenzie. 1998. Somatic and gonadal growth of the sea urchin Psammechinus miliaris (Gmelin) fed artificial salmon feed compared with a macroalgal diet. J. Shellfish Res. 17: 1549-L555. de Jong-Westman. M.. B. E. March & T. H. Carefoot. 1995a. The effect of different nutrient formulations in artificial diets on gonad growth in the sea urchin Slroiigylocenlroliis droebachiensis. Can. J. Zool. 73:1495- 1502. de Jong-Westman, M., P.-Y. Qian, B. E. March & T. H. Carefoot. 1995b. Artificial diets in sea urchin culture: effects of dietary protein level and other additives on egg quality, larval morphometries, and larval sur- vival in the green sea urchin. StrongyloceiUrotus droebachiensis. Can. J. Zool. 73:2080-2090. Fernandez. C. cfc C.-F. Boudouresque. 1998. Evaluating artificial diets for small Paracenlrotus lividus (Echinodermata: Echinoidea). In: R. Mooi & M. Telford, editors. Echinoderms: San Francisco. Rotterdam: Balkema. pp. 651-656. Fernandez. C. & C.-F. Boudouresque. 2000. Nutrition of the sea urchin Paracenlroriis lividus (Echinodermata: Echinoidea) fed different arti- ficial food. Mar. Ecol. Prog. Ser. 204:131-141. Goebel. N. & M. F. Barker. 1998. Artificial diets supplemented with ca- rotenoid pigments as feeds for sea urchins. In: R. Mooi & M. Telford, editors. Echinoderms: San Franci.sco. Rotterdam: Balkema. pp. 667- 672. Griffiths, M. & P. Perrott. 1976. Seasonal changes in the carotenoids of the sea urchin Strongylocentrotus drohachicnsis. Comp. Biochem. Phxsiol. S55:435^M1. Grosjean. P.. C. Spirlet, P. Gosselin. D. Vaitilingon & M. Jangoux. 1998. Land-based, closed-cycle echiniculture of Paracentroms lividus (Lamarck) (Echinoidea: Echinodermata): a long-term experiment at a pilot .scale. J. Shellfish Res. 17:152.3-1531. Havardsson, B.. A. K. Imsland & R. Chnstiansen. 1999. The effect of astaxanthin in feed and environmental temperature on carotenoid con- centration in the gonads of the green sea urchin Strongylocentrotus droebachiensis Miiller. J. World Aquacull. Soc. 30:208-218. Himmelman. J. H. 1984. Urchin feeding and macroalgal distribution in Newfoundland, eastern Canada. Nat. Can. Rev. Ecol. SysL 111:337- 348. Himmelman. J. H. & H. Nedelec. 1990. Urchin foraging and algal survival strategies in intensely grazed communities in eastern Canada. Can. J. Fish. Aqiiat. Set. 47:1011-1026. Keesing, J. K. & K. C. Hall. 1998. Review of harvests and status of worid sea urchin fisheries points to opportunities for aquaculture. J. Shellfish Res. 17:1597-1604. Kelly, M. S.. C. C. Brodie & J. D. McKenzie. 1998. Somatic and gonadal growth of the sea urchin Psammechinus miliaris (Gmelin) maintained in polyculture with the Atlantic salmon. J. Shellfish Res. 17:1557- 1562. Klinger, T. S., J. M. Lawrence & A. L. Lawrence. 1994. Digestive char- acteristics of the sea-urchin Lytechinus variegatus (Lamarck) (Echino- dermata: Echinoidea) fed prepared feeds. / World Aquacull. Soc. 25: 489-496. Klinger. T. S.. J. M. Lawrence & A. L. Lawrence. 1997. Gonad and somatic production of Strongylocentrotus droebachiensis fed manufac- tured feeds. Bull. Aquacult. Assoc. Can. 97:35-37. Larson, B. R., R. L. Vadas & M. Keser. 1980. Feeding and nutritional ecology of the sea urchin Strongylocentrotus droebachiensis in Maine. USA. Mar. Biol. 59:49-62. Lawrence, J. M. 1975. On the relationships between marine plants and sea urchins. Oceanogr. Mar. Biol. Annu. Rev. 13:213-286. Lawrence, J. M. & A. Bazhin. 1998. Life-history strategies and the poten- tial of sea urchins for aquaculture. J. Shellfish Res. 17:1515-1522. Lawrence. J., L. Fenaux, M. C. Corre & A. Lawrence. 1992. The effect of quantity and quality of prepared diets on production in Paracenlrotus lividus (Echinodermata: Echinoidea). In: L. Scalera-Liaci & C. Cani- catti. editors. Echinoderm research 1991. Rotterdam: Balkema. pp. 107-110. Lawrence, J. M., S. Olave, R. Otaiza. A. L. Lawrence & E. Bustos. 1997. Enhancement of gonad production in the sea urchin Lo.xechinus albiis in Chile fed extruded feeds. J. World Aquacult. Soc. 28:91-96. Lawrence. J., M.-B. Regis. P. Delmas, G. Gras & T. Klinger. 1989. The effect of quality of food on feeding and digestion in Paracentrotus lividus (Lamarck) (Echinodermata: Echinoidea). Mar. Behav. Physiol. 15:137-144. Levin. V. S. & V. P. Naidenko. 1987. Artificial diet for laboratory- maintained sea urchin Strongylocentrotus intennedius. Sov. J. Mar. Biol. 13:344-349. MacKay, A. A. 1976. The sea urchin roe industry on New Brunswick's Bay of Fundy coast. Final Report to the New Brunswick Department of Fisheries. Fredericton, New Brunswick. NB 75-1:1-92. Matsuno, T. & M. Tsushima. 2001. Carotenoids in sea urchins. In: J. M. Lawrence, editor. Edible sea urchins: biology and ecology. New York: Elsevier Science, pp. 115-138. McBride, S. C. J. M. Lawrence, A. L. Lawrence & T. J. Mulligan. 1998. The effect of protein concentration in prepared feeds on growth, feed- ing rate, total organic absorption, and gross assimilation efficiency of the sea urchin Strongylocentrotus franciscanus. J. Shellfish Res. 17: 1.56.3-1570. McBride, S. C. J. M. Lawrence, A. L. Lawrence & T. J. Mulligan. 1999. Ingestion, absorption, and gonad production of adult Strongylocentro- tus franciscanus fed different rations of a prepared diet. J. World Aquacult. Soc. 30:364-370. McBride. S. C. W. D. Pinnix. J. M. Lawrence. A. L. Lawrence & T. M. Mulligan. 1997. The effect of temperature on production of gonads by the sea urchin Strongylocentrotus franciscanus fed natural and pre- pared diets. J. World Aquacult. Soc. 28:357-365. McLaughlin. G. & M. S. Kelly. 2001. Effect of artificial diets containing carotenoid-rich microalgae on gonad growth and color in the sea urchin Psammechinus miliaris (Gmelin). J. Shellfish Res. 20:377-382. Motnikar. S.. P. Bryl & J. Boyer. 1997. Conditioning green sea urchins in tanks: the effect of semi-moist diets on gonad quality. Bull. Aquacult. Assoc. Can. 97:21-25. Olave, S., E. Bustos, J. M. Lawrence & P. Carcamo. 2001. The effect of size and diet on gonad production by the Chilean sea urchin Ln.xechinus albus.J. World Aquacull. Soc. 32:210-214. Pantazis, P. A., M. S. Kelly, J. G. Connolly & K. D. Black. 2000. Effect of artificial diets on growth, lipid utilization, and gonad biochemistry in the adult sea urchin Psammechinus miliaris. J. Shellfish Res. 19:995- 1001. Pearce. C. M.. T. L. Daggett & S. M. C. Robinson. 2002a. Effect of binder type and concentration on prepared feed stability and gonad yield and quality of the green sea urchin, Strongylocentrotus droebachiensis. Aquaculture 205:301-323. Pearce, C. M., T. L. Daggett & S. M. C. Robinson. 2002b. Effect of protein source ratio and protein concentration in prepared diets on gonad yield Gonad Enhancement of Strongylocentrotus droebachiensih 519 and quality of the green sea urchin, Strongyloccnlrnliis cirofhcuhieitsis. Aciiuictilmie 214:307-332. Pearce, C. M.. T. L. Daggett & S. M. C. Robinson. 2002c. Optinii/ing prepared feed ration for gonad production of the green sea urchin Strongylocenlrotus ciroebachiensis. J. World Aqiuuidt. Soc. 33:268- 277. Robinson. S. M. C. J. D. Castell & E. J. Kennedy. 2002. Developing suitable color in the gonads of cultured green sea urchins (Strongylo- centrotus droebachiensis). Aquacultiire 206:289-303. Robinson. S. M. C. & L. Colbome. 1997. Enhancing roe of the green sea urchin using an artificial food source. Bull. Aquacult. Assoc. Can. 97:14-20. Sonu, S. C. 1995. The Japanese sea urchin market. NOAA Technical Memorandum. National Marine Fislwries Senice, Soutlnve.'/t Ref>ion 0.30:1-33. Spirlet, C. P. Grosjean & M. Jangoux. 2000. Optimization of gonad growth by manipulation of temperature and photopenod in cultivated sea urchins, Puracenlrotus liridus CLamarck) (Echinodermata). Aqua- culture 185:8.'i-99. Tsushima. M.. T. Kawakami & T. Matsuno. 1993. Metabolism of carot- enoids in sea-urchin Pseudocentrolus depressiLi. Comp. Biochem. Physiol. B 106:737-741. Tsushima, M. & T. Matsuno. 1990. Comparative biochemical studies of carolenoids in sea-urchins - I Comp. Biochem. Physiol. B 96:801-810. Vadas, R. L. 1977. Preferential feeding: an optimization strategy in sea urchins. Ecol. Monogr. 47:337-371. Walker, C. W. & M. P. Lesser. 1998. Manipulation of food and photope- riod promotes out-of-season gametogenesis in the green sea urchin. Strongylocentrotus droebachiensis: implications for aquaculture. Mar. Biol. 132:663-676. Watts. S. A., S. A. Boettger, J. B. McClintock & J. M. Lawrence. 1998. Gonad production in the sea urchin Lytechinus variegatus (Lamarck) fed prepared diets. J. Shellfish Res. 17:1591-1595. Journal «f Shellfish Research. Vol. 22, No. I, 521-.'i25. 20U3. PRODUCTION OF RED SWAMP CRAWFISH {PROCAMBARUS CLARKII) IN EARTHEN PONDS WITHOUT PLANTED FORAGE: ESTABLISHMENT, MAINTENANCE AND HARVEST OF POPULATIONS LOUIS R. D'ABRAMO* AND CORTNEY L. OHS Department of Wildlife and Fisheries. Mississippi State University. Box 9690. Mississippi State. Mississippi ^9762 ABSTRACT Two separate studies were conducted in successive years to evaluate the effect of initial stocking densities and restocking versus natural recruitment during successive years on the production of red swamp crawfish (crayfish). Proeambanis clarkii. in earthen ponds without planted forage. In the first study, permanently flooded ponds (mean depth = 1.05 m) that contained no crawfish populations were stocked with adult populations of red swamp crawfish (assumed male:female ratio of 1:1 ) at initial biomass densities of 168.8, 225; and 281.8 kg/ha. Production was compared with unstocked ponds with recruitment populations arising from animals that remained after harvest of the previous season (initial stocking density of 225 kg/ha). Crawfish populations were fed a commercially available 25'7r crude protein crawfish feed, twice daily and trap harvested. The mean yield and individual harvest weight of natural recruitment ponds ( 1 584 kg/ha) was not significantly different from that of ponds that were initially stocked at the increasing densities (1623, 1755, and 1816 kg/lia, respectively). In the second study, restocking (112 kg/ha) versus natural recruitment and crawfish versus catfish feed were evaluated. Crawfish were harvested by both trap and seine. The mean yield and harvest weight of crawfish harvested from natural recruitment ponds (2374 kg/ha, 19. Ig) was not significantly different from that of crawfish harvested from ponds that were restocked with broodstock (2160 kg/ha, 18.7g). Mean individual weight of harvested crawfish decrea.sed from 28.5 g in October to 18.4 g in July. There were no significant differences relative to the different feeds used. The results suggest that after initial stocking, good management practices should result in sufficient recruitment to obviate restocking while still achieving consistent annual yields. KEY WORDS: crawfish aquaculture in earthen ponds, Proeambanis elarkii. management INTRODUCTION Louisiana provides 90% of the US supply of crawfish (cray- fish), approximately 47,240 mt per year (mean of 1991-1995) from a combination of forage-based culture fisheries and capture fisheries of P. clarkii. the red swamp crawfish (Huner 1997). Tra- ditional culture of red swamp crawfish is based upon a flood and drain management of shallow ponds in association with a planted forage that is the basis for stimulation of the detrital food chain (Huner etal. 1994). The practice of double-cropping crawfish with a forage-feed, such as rice, may not always be possible or preferred in states outside of Louisiana. As commonly practiced in Louisi- ana, double-cropping with a forage limits realization of the true potential of crawfish growth and production because this manage- ment practice actually limits the time when ponds can be flooded during the growing and harvest season. In addition, the presence of forage restricts the mode of harvest to trap (Huner et al. 1994). In addition to Louisiana, commercial culture of the red swamp craw- fish, Proeambanis clarkii is conducted in Mississippi, Texas, Delaware, North Carolina, Arizona, and many other states in the United States (Huner et al. 1994). Two of the major management problems associated with craw- fish culture in systems with planted forage are depletion of suffi- cient amounts of food prior to the end of the growing and harvest season, and chronically low levels of dissolved oxygen. Production ponds are actually shallow flooded fields where rice is planted. In these systems, crawfish consume food derived directly or indi- rectly from the decomposition of the forage during the fall. How- ever, by spring, this food resource is often depleted and a large population of resident crawfish that has yet to be harvested is left without sufficient food resources. As a result, growth during the *Corresponding author. E-mail: Ldabramocacfr.msstate.edu rising spring temperatures cannot be fully realized (Avaull & Brunson 1990). Warm-water temperatures combined with the characteristic decomposition of plant material in this system are conducive to low levels of dissolved oxygen. Levels of dissolved oxygen less than 3 ppm are considered unsuitable for crawfish (Huner et al. 1994). Frequent incidence of low levels of dissolved oxygen induces stress in the crawfish, and feed efficiencies cor- respondingly decrease (Avault & Brunson 1990). Under these ad- verse conditions crawfish may even seek to respire atmospheric oxygen, and even emigrate from the pond. A management approach that eliininates the use of planted forage in crawfish farming is attractive because permanently flooded, deeper ponds permit better control of the quality of water and food within the production system (D'Abramo & Niquette 1991, McClain & Romaire 199,5). Permanently flooded, deeper ponds (D'Abramo & Niquette 1991) will result in water tempera- tures that are cooler and not so susceptible to wide daily fluctua- tion. Transition from an extensive to semi-intensive production system offers resident crawfish populations a potentially more suitable environment, as described by McClaine and Romaire (1995). In an effort to move toward semi-intensive management and more reliable production from year to year in crawfish farming, D'Abramo and Niquette (1991) evaluated the feasibility of seine harvest and feeding of formulated pelleted diets as alternatives to exclusive trap harvest and the use of planted or volunteer forage as a food resource. Studies were designed to determine the best man- agement practices for the production of red swamp crawfish in earthen ponds without planted forage. They observed that two different initial stocking rates of broodstock did not appear to affect mean harvest weight, suggesting a high degree of plasticity and unpredictability associated with production systems based on natural recruitment. 521 522 D"Abramo and Ohs MATERIALS AND METHODS Common Pond Management Practices Levels of dissolved oxygen and temperature for all experimen- tal ponds were recorded daily for the entire harvest season to guide the implementation of different management practices. When the levels of dissolved oxygen were anticipated to decrease below 5 mg/L, pond water was aerated using a 0.5 hp aerator (Air-O-Lator Corporation, Kansas City, MO). Emergency aeration was provided by PTO driven paddlewheels when levels of dissolved oxygen decreased below 3 mg/L. Every third day during May through August. pH was recorded during the late afternoon. If pH values exceeded 9.2, all ponds received an application of cracked com (56.3 kg/ha) to increase the production of CO, and thereby lower pH. Study 1 Before stocking, 1 2 earthen ponds ranging in water surface area from 0.040 to 0.053 ha were treated with a chemical insecticide (Ambush, ICI Agricultural Products) at a concentration of 50 ppb. After 24 h the ponds were drained, filled half way, and again drained to remove any residual insecticide. This procedure effec- tively eliminated any resident crawfish from the designated ex- perimental ponds. Ponds were then filled (mean depth of 1.05 m) and initially stocked with adults (assumed male:female ratio of 1:1) at biomass densities of 168.75. 225, and 281.75 kg/ha into ponds. The latter stocking densities exceeded those recommended for conventional forage based ponds (56-84 kg/ha) in an attempt to determine whether feeding could increase survival of young and total annual harvest. An additional treatment consisted of four ponds that already contained populations compo.sed of animals remaining from the harvest of the previous season (initial first year stocking density of 225 kg/ha) and natural recruitment. Crawfish populations were fed a commercially available 15% crude protein crawfish feed (Arcadiana Choice "25") twice daily. Feeding rates (Table 1 ) were based on an estimated pond biomass and water temperature. Total feed provided per treatment was in proportion to the initial stocking biomass. Crawfish were harvested using pyramid style traps constructed of L91-cm diameter hexagon mesh with 61 -cm extended necks TABLE \. Monthly feeding rates by percent of total and kg/ha/yr. Percent of Total Month Study 1 Study 2 January 0 0 February 0 0 March 15 8 April 15 15 May 25 20 June 20 20 Julv 5 8 August 0 0 September 0 0 October 5 11 November 10 10 December 5 8 Total feed fed (kj ;/ha/year) * ,^700 '■ 2400 = low density; 3200 = mid density; 4000 = high density. and three funnel openings between 3.8 and 4.4 cm. Traps were baited with approximately 150 g of a commercially available bait (Acadiana Choice - Medium), on Monday and Wednesday and harvested on Tuesday, Wednesday. Thursday, and Monday. Craw- fish were harvested a total of 97 days during periods of November 21, 1995 to December 7, 1995 and March 3, 1996 to August 1. 1996. On one day during each week, up to 50 individuals harvested from each pond were randomly selected and individual weight and sex were recorded. Males were classified as sexually active (form I) or sexually inactive (form II) (Hobbs 1974). All ponds were harvested once per month from May to August with a 1.91-cm mesh, knotted nylon seine, 1.5 m in height, that was modified through the attachment of a heavy nylon mud line to the existing lead line. Fifty individual crawfish were randomly selected from each seine harvest of each pond, and individual weight and sex were recorded. Study 2 The relative value of an annual management practice of re- stocking (1 12 kg/ha) versus natural recruitment was investigated. In addition, two feeds, a 25% crude protein formulated crawfish feed (Acadiana Choice 25) and a sinking pelleted catfish feed (2%% crude protein), were evaluated. Eight ponds, previously stocked in the first study, were randomly assigned to each of the two treatments, four ponds per treatment. An additional eight ponds from the previous year were restocked with crawfish (112 kg/ha) at an assumed male:female ratio of 1:1. Four ponds were randomly assigned to each feed thereby providing a 2 x 2 factorial arrangement of treatments. The annual rate of application of the commercially manufactured pelleted feeds was 3,700 kg/ha, pro- portionally distributed over eight months based upon pond water temperature and estimated resident biomass (Table 1). Harvest was by trap and seine. Harvest by trap occurred on 99 days extending over 9 mo, from October 15, 1996 through De- cember 13 and from March 5 to August 1, 1997. All ponds were seine harvested once in May, June, and August. The same traps. trap density, and seine described in study I were used. Trap har- vest occurred on Monday. Tuesday. Thursday, and Friday. After harvest of unbaited traps on Mondays and Thursdays, traps were baited with approximately 150 g of a commercially bait (Acadiana Choice - Jumbo) and harvested on the following Tuesday and Friday, respectively. Compared with study 1, this management protocol used one less day of baiting and modified the two 72 h unbaited soak periods of study 1 to one 48 h and one 72 h. Data were collated to examine the relationship of yield to water tem- perature based upon whether the traps were baited. Once weekly, up to 50 crawfish from each pond representing the different treatments were randomly selected. In addition. 50 individual crawfish were randomly selected from each pond that was seine harvested. In both instances, individual weight, sex. and reproductive form of the males (Hobbs 1974) were recorded. These data were collected to determine the composition of the crawfish population at different times of the year and identify any possible bias in harvest method relative to sex or male reproduc- tive condition. Statistical Methods For study 1 . an analysis of variance ( ANOVA) using SAS (SAS Institute 1988) was used to determine whether significant differ- ences in mean individual weight, total production, and survival Establishment of Red Swamp Crawfish in Earthen Ponds 523 existed among treatments. For study 2. a two-way analysis of variance (ANOVA). using the general linear model procedure (PROC GLM) of SAS (SAS Institute 1988), was used to determine whether significant differences in mean individual harvest weight, total production (kg/hal. and survival (%) existed between treat- ments and whether a significant interaction between factors (feed and stocking procedure) existed. An ANOVA was also conducted to determine whether the proportions of each sex and male devel- opmental stage harvested were significantly different for the treat- ments in each study. All differences were considered significant at P < 0.05. The relationship between trap yields and water tem- perature for traps with and without bait was examined via linear regression and the best fitting lines were then calculated. 1996- 1997 RESULTS Sliidv ] The mean yield of natural recniitmenl ponds ( 1589 kg/ha) was not significantly different from that of ponds that were initially stocked at densities of 168.75. 225. and 281.75 kg/ha ( 162.^, 1755. and 1816 kg/ha. respectively). Mean individual weight of crawfish harvested from natural recruitment ponds (17.3) was also not sig- nificantly different from that of ponds that were initially stocked at densities of 168.75. 225. and 281.75 kg/ha (17.9. 19.1. and 16.9 g. respectively). Yields from seine harvests, individual harvest weights. M/F ratios, and 7r of form I males are presented in Table 2. Seine harvest comprised 13-359^ of the total wet weight harvest per month. The mean percent of form 1 males that were trap harvested increased from 47c in April to 96% in August and declined to 12% by December. When seine harvest was conducted, the proportion of form 1 males was significantly lower than that obtained by trap harvest for the same week (Fig. 1 ). Study 2 The interaction between quality of feed and stocking proce- dures was not significant (P = 0.45). The mean yield of natural recruitment ponds (2374 kg/ha) was not significantly different TABLE 2. Mean yields from individual seine harvests relative to stocking density treatments (Study 1). 1995-1996 May 8 June 5 July 1 July 30 Kg/ha High 40.2 101.9 136.4 71.5 Medium 76.5 108.4 165.6 55.0 Low 58.8 71.9 126.6 73.8 Average wt. (g) High 12.0 13.7 14.8 18.8 Medium 13.0 14.6 15.7 18.3 Low 13.5 15.0 16.4 19.7 M/F ratio High 1.08 0.91 1.04 0.91 Medium 1 .05 0.8 1.02 0.85 Low 1.15 1.46 0.9X 0.94 Form 1 male (%) High 39 42 72 47 Medium 39 37 71 64 Low 42 47 69 51 100 S 40 Week 1997- 1998 100 80 60 40 20 u □ Trap ■ Seine 22 28 Week Figure 1. The relative percentages of form I males harvested by trap and seine during the same week. from that of ponds that were restocked with broodstock (2160 kg/ha). Mean individual weight of crawfish harvested from natural recruitment ponds ( 19. 1 g) was not significantly different from that of restocked ponds (18.7 g) and decreased in all ponds from 28.5 g in October to 18.4 g in July. There were no significant differ- ences in production based upon feeds used. Yields, individual weights. M/F ratios, and % of form I males from seine harvests are presented in Table 3. Seine harvest com- prised 10-40% of the total wet weight harvest per month. The mean percent of form I males in all ponds increased froin 5% in March to 95% in August. The proportion of form II males in seine harvests indicates that they were not harvested in the same pro- portion by trap (Fig. I ). For crawfish harvested by trap, the relationship between mean harvest/trap/day and temperature, with and without the use of for- mulated bait, is presented (Fig. 2). The best fitting lines derived from harvest data obtained from baited and unbaited traps were detemiined. The intersection of the best fitting lines for unbaited and baited traps is 15C. Above this water temperature, the use of formulated bait becomes increasingly more effective than the use of no bail as the divergence of lines indicates. Soak time for baited traps was approximately 24 h. whereas the soak time for un-baited traps was either 48 or 72 h. DISCUSSION Populations of crawfish initially stocked into earthen ponds without planted forage appear to be self-sustaining for successive annual harvests when sufficient food is provided, good water qual- ity is maintained, and the harvest practices outlined in studies 1 524 D"Abramo and Ohs TABLE 3. Mean yields, maleifemale ratios, and percent form I males from individual seine harvests relative to treatments representing different management practices (Study 2). 1996-1997 May 6 June 20 Augl Kg/ha Nat. Rec./crawfish 82 126.2 84 Nat. Rec/catfish 27.8 85.8 54.7 Restockycrawfisli 118.2 144.1 96.2 Restock/catfish 41.2 93.6 105.8 Average wt. (g) Nat. Rec./crawfish 15.6 15.5 17.3 Nat. Rec./catfish 13.5 16.1 14.8 Restock/crawtlsh 15.3 15.1 16 Restock/catfish 14.4 15.3 16.3 M/F ratio Nat. Rec./crawfish l.ll 0.99 Nat. Rec./catfish 0.88 0.69 Restock/crawfish 1.0 0.9 Restock/catfish 0.97 .77 Form I males (%) Nat. Rec./crawfi.sh 48.9 68.4 Nat. Rec./catfish 68.6 66.4 Restock/crawfish 55.7 63.5 Restock/catfish 57.4 66.6 and 2 are conducted. Increases of 33.3% and 66.7% in stocking biomass did not affect total annual production, even with corre- sponding increases in feeding rates. This suggests that a carrying capacity controlled by a variety of abiotic and biotic factors is reached and maintained as crawfish are harvested, i.e. removed from the pond. When ponds are well managed, restocking is un- necessary unless an annual harvest is unexpectedly low. suggesting that insufficient broodstock would be present for satisfactory re- cruitment for the following year. Active harvest by seine revealed that information about popu- lation characteristics collected through passive trap harvest is not accurate. Data obtained from siene harvest show that the propor- tion of form II males in the pond population during May. June, and July is higher than that suggested by data obtained from trap har- vest. Form II males are not caught by trap in the same proportion as they are present in the male population. This observation may be a manifestation of a behavioral hierarchy that exists among differ- ent males due to differential attraction to bait as a source of food. Another behavioral-based explanation is that form II males are more likely to leave the trap after the bait has dissolved as sug- gested by Romaire (1995). At water temperatures below 15°C. use of the commercial for- mulated bait in traps was not effective in this study. Above 15°C. baited traps are more effective, daily harvest exceeding that of unbailed traps by approximately 0.01 kg/trap for every degree Celsius increase. As a result, a difference of 0.06 kg harvested/ trap/day is realized by 20°C and 0.13 kg harvested/trap/day by 30°C. When bait is used, the higher daily yields/trap begin to be consistently realized at a water temperature of s 1 7C and collec- tively contribute to an overall increase in annual yield. The tem- perature-dependent effectiveness of the bait used in this study may be related to its ingredient composition. Effective baits for trap harvest at temperatures <17 C may be achieved through modifi- cation of the ingredient composition. Further research needs to be conducted to evaluate different harvesting efforts, strategies, and formulated baits within specific temperature ranges. The lack of significance of many of the management practices designed to improve production and evaluated in our studies may simply be due to the lack of sufficient control. Production from year to year is based upon a level of recruitment that cannot be totally controlled. However, a management strategy that does not incorporate an annual drain down followed by the planting of forage seems to offer a greater chance for consistent production from year to year. Nonetheless, the draining of conventional ponds for the planting of forage does offer two indirect benefits, control. 0.7 0.6 0.5 H :| 0.4 0.2 0.1 0.0 • Unbaited o Unbaited Predicted o Baited o - Baiteid Predicted • o o 88 ^ " • o o •^-^ o o o *- -^ o 8 o* o o • o 8 - — • to • o • o o • • • • W" .-*-*^^S^ • • •• • o /^ o • o 8* Q ^00 10 15 30 35 20 25 Temperature (C) Figure 2. Linear regression displaying predicted mean yields (kg/trap/day) for harvest at different water temperatures with (;i = 50) and without (H = 51) the use of formulated bait. Establishment of Red Swamp Crawfish in Earthen Ponds 525 if needed, of predaceous fish that unintenlionally become establish in the ponds and the oxidation of anaerobic sediments that may have resuhed. Under the management practices described in this investigation, an annual production of approximately 1700 to 1800 kg/ha can be expected. Higher yields could be realized with the implementation of different harvest strategies. However, these ap- proaches need to be weighed against the labor required. Practices that are designed to increase production may be inherently limited because of density-dependent factors, including cannibalism, for which management has little control. Some increase in production could be realized by methods to reduce the incidence of cannibal- ism. In addition, changes in the distribution of production over an annual cycle may be possible, but the increases may not be realized because only a particular range of production can be realized given the restrictions imposed by the nature of the management system. The quality of food provided can have an effect but once a certain level of quality of feed is attained, similar levels of production will be achieved, because the feeds primarily serve as an indirect source of nutrients. Most of the food provided appears to stimulate secondary productivity within a pond similar to the detrital food chain that is stimulated by the decomposition of planted forage in traditional ponds. The level of secondary productivity is limited and thereby controls the production of crawfish that can be real- ized. Certainly, routine removal by harvest does contribute to higher production. The red swamp crawfish is a species that has many characteristics that make it appealing as an aquaculture spe- cies. However, some management practices are probably not suc- cessful simply because of the restrictions imposed by the nature of the production system. In addition, the innate level of variation among production ponds may preclude identification of significant treatment-related differences that would only be observed with a inordinate number of replicates. Any management practices that do succeed must still remain within the confines of realizing a posi- tive net return. Those management practices that are ultimately identified as being most efficient and cost-effective must be trans- ferred to larger (at least 0.5 ha) ponds to verify applicability. ACKNOWLEDGMENTS The authors thank the staff of the Eastern Unit of the National Warmwater Aquaculture Center, Mississippi State University, par- ticularly Ms. Beth Peterman. Mr. Bubba Groves, and Mr. Angus Irvine, for their assistance in the management of the water quality of the ponds, the feeding of the crawfish, and the harvesting. We also thank Mr. Mack Fondren. Manager of the Eastern Unit, for his cooperation and interest as well as Dr. Patrick Gerard, Department of Agricultural Information Science and Education. Mississippi State University for his guidance in the experimental design and statistical analysis. The research was funded through a special aquaculture grant from the United States Department of Agricul- ture. Mississippi Agricultural and Forestry Experiment Stafion Publication Number J 10230. LITERATURE CITED Avault, J. W. & M. W. Brunson. 1990. Crawfish forage and feeding sys- tems. Rev. Aqualic Sci. .^:1-10. D'Abramo, L. R. & D. J. Niquette. 1991. Seine harvesting and feeding of formulated feeds as new management practices for pond culture of red swamp crawfish. Procambarus clarkii (Girard. 1852). and white river crawfish. / Shellfish Res. 10:169-177. Hobbs. H. H., Jr. 1974. A checklist of the North and Middle Amencan crayfishes (Decapoda: Astacidae and Cambaridae). Smirhsnnian Conli: Zool. 166:1-153. Huner. J. V.. M. Moody & R. Thune. 1994. Cultivation of freshwater crawfishes in North America. In: J. V. Huner. editor. Freshwater craw- fish aquaculture in North America, Europe, and Australia. Families Astacidae. Cambaridae. and Parastacidae. Binghaniton. NY: Haworth. pp. 5-1-V5. Huner. J. V. 1997. The capture and culture fisheries of North American crawfish. World Aquaculture 28:44-50. McClain. W. R. & R. P. Roniaire. 1995. Management considerations for the production of large Procamband crawfish. J. Shellfish Res. 14:553- 560. Romaire. R. P. 1995. Harvesting methods and strategies used in commer- cial Procambarid crawfish aquaculture. / Shellfish Re.s. 14:545-551. SAS Institute. 1988. SAS/STAT Users Guide, release 6.03. SAS Institute, Gary, NC. 1056 pp. Jounud u] Shellfish Rcsuaich. Vol. 22, No. 1, 527-531, 2003. PRODUCTION OF RED SWAMP CRAWFISH (PROCAMBARUS CLARKII) IN EARTHEN PONDS WITHOUT PLANTED FORAGE: EVALUATION OF TRAP AND SEINE HARVEST STRATEGIES LOUIS R. D'ABRAMO, *CORTNEY L. OHS, AND KATHLEEN C. ELGARICO Deparliiicnt of Wildlife ciiul Fisheries. Mississippi State University. Box 9690, Mississippi State, Mississippi 39762 ABSTRACT The et'lect of trap density and trap versus seine harvest on the production of red swamp crawfish were evaluated in earthen ponds without planted forage. Crawfish were harvested from traps at densities of either 81 or 1 2 I/ha. 4x/week. from October through July of the followmg year. In another treatment crawfish were harvested from traps at a density of 12 I/ha when water temperatures were >I9'C and seine harvested when water teinperatures were between 15 and I9°C. Mean annual production ranged from 2173 to 2606 kg/ha, and mean harvest weight ranged from 16.6 to 17.8 g. Total production and catch per unit effon for seine and trap harvests at water temperatures between 15 and 19°C were not significantly different. Mean individual weight of seine harvested crawfish was significantly less (1 1.4 g) than that of trap-harvested crawfish (21.6 and 17.6 g). In a second .study, the effects of different harvesting strategies and two formulated feeds were evaluated. Crawfish were fed either a 32% crude protein, extruded, slow-sinking formulated diet or a 32% crude protein, pelleted sinking diet, and harvested from traps either 3x (81 traps/ha) or 2x ( 121 traps/ha) per week. Trap harvest at 81 traps/ha, 3.x/week and 121 traps/ha, 2x/week produced 2447 and 1884 kg/ha, and a mean individual harvest weight of 18.7 and 19.8 g. respectively. A significantly lower individual weight (16.2 g) was associated with the pelleted sinking feed relative to the extruded, slow-sinking feed. However, mean total production was not significantly different between treatments. Over 90% of the annual yield was harvested from April through October when water temperatures were >19°C. KEY WORDS: crawfish aquaculture in earthen ponds, Procumbanis cUirkii, seine and trap harvest INTRODUCTION The goal of harvest strategies is to enhance efficiency of the labor expended while maximizing production. In traditional for- age-based fanning of the red swamp crawfish (crayfish), labor accounts for 30-70"^ of the total direct operational expenses and is primarily attributed to harvesting. Therefore, efficient harvest based upon conditions of when, how, how often and at what level of effort to harvest, is critical to the economic success of crawfish farming systems. The ainount of labor is affected by trap density, number of harvest days/week, number of trap sets/day (the number of times a trap is prepared, with or without bait, to harvest crawfish daily), and current market price of crawfish (Romaire 1995). The size of harvested crawfish also must be considered because quality of the product relative to market demand should not be compromised in exchange for a reduction in labor. In forage-based ponds for culture of crawfish, when soak time increases, larger but fewer crawfish are harvested from traps (Romaire 1995). There- fore, a harvesting strategy also must consider the number of craw- fish that are harvested at one set. Inverting traps to prevent access when bait is not provided might result in an increase in mean size at harvest and yield because crawfish would be allowed to feed, reproduce, and molt in the pond for longer periods of time during the harvest season. A decrease in catch per unit effort for trap harvest is encoun- tered commonly for consecutive harvest days in production ponds with (McClain et al. 1998) and without (D'Abramo & Ohs 2003) planted forage. Proper management of the harvest schedule could lead to a more consistent yield with a corresponding decrease in labor. Trap density is also a component of an optimal harvest strategy, whereby a reduction in cost per unit effort can be real- ized. Water temperature is also a major factor that infiuences the efficiency of harvest. Temperature-related differences in harvest *Corresponding author: E-mail: Ldabramo@cfr.msstate strategy can also optimize trapping effort. Significant increases in catch per unit effort (CPUE) were achieved with the u.se of com- mercially manufactured baits when water temperature is equal to or exceeds 19 C (D"Abramo & Ohs 2003). A similar relationship between temperature and CPUE using formulated bait was ob- served in production ponds with planted forage (Romaire 1995). Efficient, cost-effective harvest at water temperatures less than 19°C requires a different approach that may include bait, soak time, or method of harvest. A preliminary investigation of the utility of seine harvest of crawfish in production ponds without planted forage was con- ducted by D'Abramo and Niquette (1991). However, consistent yields were not achieved and mean individual weight of harvested crawfish declined because as the number of harvested crawfish accumulated in the seine, the ability of small crawfish to escape through the mesh decreased. D'Abramo and Ohs (2003) used pe- riodic seine harvesfing at pond water temperatures >19°C in an atteinpt to reduce biomass density and density-dependent growth reduction, with the intent to increa.se total annual yield. However, seine harvest may be most effective at water temperatures 19°C, once each in May, June, and July. Pyra- mid traps used in this study were constructed with 1.91 -cm wire mesh (Gulf Coast Wire Products, Kaplan, LA). The traps had three funnel entryways, elongated necks that extended above the water surface, and polyvinyl chloride-retaining rings at the top. Traps were harvested four days per week (Monday, Tuesday, Thursday, and Friday) at temperatures >19°C. Harvests on Tuesday and Fri- day occurred after baiting on the previous days (24 h soak). Har- vests on Thursday and Monday occun'ed with no bait after 48 and 72 h soak times, respectively. Traps were baited with approxi- mately 150 g of a commercially available bait (Gros Rouge, Cargill, Minneapolis, MN). Four ponds were randomly assigned to each treatment stocked previously ( 1 or 2 y). No harvest was conducted when pond water temperatures were below 15°C. Craw- fish were fed a 28% protein extruded, slow-sinking formulated feed for nine months (Table 1 ). Traps were harvested a total of 105 days extending over 1 1 nio. from September 16 through July 31. The seine used for harvest was nylon, 1 .5 m in height, and consisting of 1 .9 cm mesh and was modified through the attachment of a heavy nylon mud line to the existing lead line. Once per week, after harvest, up to 50 crawfish from each pond were randomly selected and individual weight and TABLE \. Monthly feeding rates (percent of total) and total amount of feed fed annually for studies 1 and 2. Percent of Total Month Study 1 Study 2 January 0 7.25 February 0 6.5 March 10 7.0 April 17 11.5 May 15 14.25 June 14 12.5 July 10 7.0 August 0 4.0 September 8 7.75 October 10 8.0 November 10 7.0 December 6 7.25 Total feed fed (kg/ha/y) 4400 5635 sex were recorded. From each seine harvest, fifty individual craw- fish were also randomly selected and individual weight and sex were recorded. Levels of dissolved oxygen and water temperature for each of the experimental ponds were recorded daily for the entire year. If dissolved oxygen was anticipated to decline below 5 mg/L, surface aeration was provided by a 0.5-hp Aquarian aerolators (Air-O- Later Corp.. Kansas City, MO). Additionally, tractor powered paddlewheels were used when the concentration of dissolved oxy- gen was anticipated to decline below 3 mg/L. From May to Au- gust, pH was measured every third day from water samples col- lected from each pond. In June, all ponds were treated with gyp- sum at approximately 182 kg/ha to control the sporadic and rapid increase in pH. The value of different harvesting methods and strategies was compared through calculation of CPUE. The calcu- lations were based upon the assumptions that one worker (laborer) with a boat can harvest 150-300 crawfish traps/h (Romaire 1995), and a crew of three laborers, with the proper equipment, can seine harvest a I ha pond in 1 h. Seine harvest requires the removal of traps. However, no additional investment of labor is necessary if the traps are removed at the same time they are last harvested. The different labor investments required for the different strategies of harvest during water temperatures between 15 and I9°C, were standardized by assuming a 1 ha production pond. CPUE (kg/ha/ laborer/h) was calculated by dividing the total harvest (kg/ha) for the entire period when water temperatures between 1 5 and 1 9^C by the number of harvest days, and then dividing by the number of hours required to complete harvest. Study 2 Twelve earthen ponds were used in the evaluation of the effects of an extruded feed and an increased trap density. There were three treatments, four replicates (ponds) per treatment. Nine ponds had been in continuous production for either 2 or 3 y as part of pre- vious investigations. The remaining three ponds were stocked with a 1:1 ratio of males to females at 1 12.5 kg/ha during July 1998. One of these ponds was randomly assigned to each of the three treatments. The management practices represented by the first treatment were the feeding of a 32% crude protein, pelleted, for- mulated diet, and a trap density of 81 /ha. Traps were harvested 3 days/week, 2 consecutive days, followed by 24 h of soak. Traps were then inverted one day, baited the following day, and then harvested after a 24 h soak time. The second treatment was the same as the first treatment except a 32% crude protein, extruded, slow-sinking, formulated diet was fed. The final treatment con- sisted of the feeding of extruded, slow-sinking formulated diet, a trap density of 121/ha, and 2 consecutive harvest days/week with a 24 h soak time. When traps did not contain bait, they were inverted. Harvest was conducted with the pyramid traps described in study I. Trapping with bait occurred at water temperatures >19°C using a lOO-g piece of formulated bait (Gros Rouge. Cargill, Min- neapolis, MN). When water temperatures were between 15 and I9°C, traps were not baited and remained soaked. Under these conditions, a higher mean harvest weight would be expected be- cause smaller crawfish would have more time to exit out of the trap. Dissolved oxygen concentrations and pH were measured and managed as described in study I . Some management constraints were imposed on trap harvest to maximize return on trapping effort. If the weekly harvest yielded Production of Red Swamp Crawfish in Earthen Ponds 529 <15 kg/ha/treatment, or mean individual liarvest weight of the crawfish was 19C or between 15 and 19 C. The cumulative harvest weights were divided by the number of harvest days, and then divided by the number of laborer hours required for trap harvest. Statistical Analysis A one-way analysis of variance using the general linear model of SAS (Statistical Analysis System, version 8.1, Cary, NC) was used to determine whether differences existed among treatments for mean yields (kg/ha and number/ha), mean individual weights and mean CPUE overall and relative to harvest temperature of the pond water. Significant differences were identified at the P < 0.05 level. RESULTS Sltidy 1 The mean total production (kg/ha), and mean individual harvest weight (g) of crawfish harvested from ponds with a trap density of 81 /ha were not significantly different from those ponds with a trap density of 121/ha (Table 3). Total yield (kg/ha) at water temperatures between 1 5 and 1 9"C was not significantly different among treatments (Table 4). How- ever, the mean individual weight (g) of the crawfish harvested by seine (11.4 g) was significantly less than those harvested from traps at densities of 81/ha (21.6 g) and 121/ha (17.6 g). Seine harvest required a greater amount of labor than trap har- vest and contributed to the lowest CPUE (10.1 kg/ha/laborer hour). TABLE 2. Results of the proximate analysis for extruded, slow -sinking, and pelleted diets fed to crawfish in ponds without planted forage. Component (% Dry Weight) Extruded Slow-Sinking Diet Pelleted Diet Ash 8.4 7.1 Crude protein 37.8 39.1 Crude lipid (acid hydrolysis) 5.4 4.3 Crude fiber 6.7 6.2 Nitrogen-free extract (carbohydrate). by difference 41.7 43.3 TABLE 3. Mean annual production (kg/ha) ± SE and mean individual weight (g) ± SE of harvested crawfish (Study I ). Treatment Total Production (kg/ha) .Mean Individual Weight (g) Kl traps/ha 2606 ± 400 1 2 1 traps/ha. trap < 1 9°C 23 1 8 ± 2 1 1 121 traps/ha. seine < 1 9"C 2I73±239 I7.S + 0.4 16.7 + 0.6 16.6+ I.O CPUE was highest for trap harvest at 121 traps/ha (13.9 kg/ha/ laborer hour) during water temperatures between 15 and 19 C (Table 4). Each of the three seine harvests conducted during the summer months when water temperatures exceeded 19 C in ponds that contained 81 traps/ha yielded between 50 and 100 kg/ha. Greater yield was achieved from a cumulative four day trap harvest than one seine harvest during the same week two of three times. The individual harvest weight of seine harvested crawfish was less than that of trap harvested crawfish collected during the first seine harvest in May. Under an equal number of trap days, the yields for the two different trap densities were similar. Further evaluation of trap density and trapping effort is warranted. Study 2 The mean total production, mean individual weight, and mean number/ha did not differ significantly among treatments (Table 5). A 33% decrease in the number of harvest days for ponds contain- ing 121 traps/ha resulted in a 23% decrease in annual production (kg/ha) and a 27% decrease in total number of crawfish harvested per hectare. CPUE (kg/ha/laborer hourj for the trap harvest con- ducted twice per week was 58% greater than that conducted 3x/ week. At water temperatures between 15 and 19°C mean total pro- duction (kg/ha), mean individual weight, and mean number/ha were not significantly different among treatments (Table 6). To evaluate the feasibility of trap harvest at water temperatures less than I9°C. CPUE was calculated and multiplied by a market price of $2.75 US/kg. Maximum return for one hour of labor to harvest 81 traps/ha three times a week would be 37.8% less than the return realized from harvest of 109 traps/ha twice a week. TABLE 4. Total production (kg/ha), CPUE ( kg/ha/laborer hour), and mean individual weight (g) of crawfish harvested when water temperatures were between 15 and 19 C (trap harvest was conducted for 15 separate days and seine harvest occurred eight different times, study 1 ). Treatment Total Production (kg/ha) CPUE (kg/ha/laborer hour*) Mean Individual Weight (g) 81 traps/ha. trap 1 2 1 traps/ha. trap 121 trap.s/ha. seine 293 284 298 10.5 13.9 10.1 21.6 17.6 11.4 * Assumed labor required: one laborer can harvest 1 50 traps per hour, three laborers can seine harvest a I ha pond in 1 hour. Values are based upon the mean of each treatment. 530 D'Abramo et al. TABLE 5. Mean total annual production (kg/ha) ± SE and mean individual weight (g) ± SE of harvested crawHsh (Study 2). Treatment Mean Total Production (kg/ha) CPUE (kg/ha/laborer hour*) Mean Individual Weight (g) Number/ha 81 traps/ha 3x. ESS 8 1 traps/ha, 3x, PS 121 traps/ha. 2x. ESS 2447 ± 169 2294 ± 309 1884 ±260 13.2 12.4 20.8 18.7 + 0.9 16.2 ±0.5 19.8 ± 1.5 130.764 ±3.781 141.233+ 18.416 95.928 ± 18.878 At trap densities of 81/ha and 121/ha. the total number of harvest days was 100 and 67 days, respectively. Either a e.xtruded slow-sinking (ESS) or a pelleted sinking (PS) diet was fed. * Assumed labor required: one laborer can harvest 150 traps her hour. Values are based upon the mean of each treatment. For trap harvest when water temperature exceeds 19''C. mean total production (kg/ha), mean individual weight, and mean num- ber/ha were not significantly different. However, with a mean increase of 500 kg/ha and 32.000 crawfish/ha. the potential eco- nomic impact is obvious (Table 7). A mean weight increase of 1 g with the decreased trapping effort was not statistically significant. DISCUSSION Large inherent variation of production parameters of ponds within the same treatment does present some problems in the iden- tification of the relative value of different management strategies. This condition is characteristic of the system under investigation, that is, production from one year to the next cannot be directly controlled and is principally determined by recruitment success. Nonetheless, some recommendations can emerge and future areas of investigation can be defined. Results indicate either an extruded, slow-sinking diet or a pel- leted 32-35% crude protein, sinking diet can be fed. and selection should be determined by cost and availability. A sinking catfish diet that is not particularly water stable works as well as a formu- lated diet. These results suggest that stimulation of the detrital food chain may be the best way to serve the nutritional needs of the crawfish as long as a selective harvest schedule is sufficiently intense to remove a satisfactory amount of biomass through time. Further investigation into the use of other alternative feedstuff's is warranted because the cost of feed represents a large proportion of the total operational costs. The results of the two studies suggest that trap density is sufficient at 81/ha and that trap harvest is a better strategy when water temperature is <19°C. Although the catch per unit effort is greater al a density of 121/ha because of less labor for harvest, this apparent benefit must be weighed against the cost of additional traps and the higher production that can be achieved for the entire harvest season when traps are harvested three times/week. The comparatively poor performance of seine versus trap harvest is probably caused by a less-than-efficient de- sign for harvest. A design specific to the harvest of pond raised crustaceans may result in an attractive option. Other potential ap- proaches to enhance yield from seine harvest would be provision of food (bait) just prior to a scheduled harvest, and/or harvest soon after dusk when foraging activity is believed to be highest. An alternative management strategy that needs investigation is a modification in the proportion of trap days per month when trap harvest is conducted at water temperatures >19°C. This procedure would consist of a decrease in trapping effort from March through May. followed by a corresponding increase in effort from June through October. The ultimate goal of this management strategy would be maintenance of equivalent annual production but with the amount of production being proportionately greater when tra- ditional capture and culture fisheries can no longer provide the product. Those management practices that are ultimately identified as being most efficient and cost-effective must be transferred to larger (at least 0.5 ha) ponds to verify applicability. ACKNOWLEDGMENTS The authors thank the staff of the Eastern Unit of the National Warmwater Aquaculture Unit for their assistance in the manage- ment of the water quality of the experimental ponds, the distribu- tion of feed to the ponds, and the harvest of crawfish. We also thank Dr. Patrick Gerard of the Department of Agricultural Infor- mation Science and Education, Mississippi State University for his assistance in the establishment of an experimental design and guid- ance in performance of the appropriate statistical analysis. The research was supported by the U.S. Department of Agriculture through a special grant for aquaculture research. Mississippi Ag- ricultural and Forestry Experiment Station Publication Number J 10249. TABLE 6. Total production (kg/ha), CPUE (kg/ha/laborer hour), and mean individual weight (g) of crawfish harvested when water temperatures were between 15 and 19 C (Study 2). Treatment Total Production (kg/ha) CPUE (kg/ha/man hour*) Mean Individual Weight (g) Number/ha 81 traps/ha, 3x. ESS 81 traps/ha. 3x. PS 121 traps/ha. 2x. ESS 200 126 136 4.5 2.8 6.2 20.7 19.8 20.7 9679 7721 7082 Trap harvest was conducted a total of 24 and 16 days at trap densities of 81/ha and 121/ha, respectively. Either an extruded slow-sinking (ESS) or a pelleted sinking (PS) diet was fed. * Assumed labor required: one laborer can harvest 150 traps per hour. Production of Red Swamp Crawfish in Earthen Ponds 531 TABLE 7. Total annual production (lI9 C iStudv 2). Total Produc tion CPUE Mean Individual Treatment (kg/ha) (kg/ha/man hour*) Weight (g) Number/ha 81 Iraps/ha, 3x. ESS 2246 1(1.0 18.6 121.08."^ 81 trap.s/ha, 3x, PS 2168 15.4 16.0 133,512 121 traps/lia. 2x. ESS 1748 25.5 19.7 88.846 Trap harvest was conducted a total of 76 and 50 days at trap densities of 81/ha and 121/lia. respectively. Either an e.xtruded slow-sinking (ESS) or a pelleted sinking (PS) diet was fed. * Assumed labor required: one laborer can harvest 150 traps per hour. LITERATURE CITED D'Abramo. L. R. & D. J. Niquette. IWl. Seine harvesting and feeding of De Silva. S. S. & T. A. Anderson. 1995. Fish nutrition in aquaculture. formulated feeds as new management practices for pond culture of red London: Chapman & Hall. 340 pp. swamp crawfish, Procambarus cUiikii (Girard, 1852). and white river McClain, W. R., J. J. Sonnier & D. T. Miller. 1998. Effects of vertical crawfish. / Shellfish Res. 10:169-177. substrate on crawfish growth and survival. 90th Annual Research Re- D'Abramo, L. R. & C. L. Ohs. 2002. Production of red swamp crawfish port. Rice Research Station, Louisiana Agricultural Experiment Sta- (Procambarus clarkii) in earthen ponds without planted forage: estab- tion, Louisiana State University Agricultural Center, pp. 481^84. lishment, maintenance, and harvest of populations. J. Shellfish Res. Romaire, R. P. 1995. Harvesting methods and strategies used in commer- 22:340 p. cial Procambarid crawfish aquaculture. J. Shellfish Res. 14:545-553. Journal of Shellfish Research. Vol. 22. No. I. S33-S4{). 2(){)3. DISTRIBUTION, SHELTER FIDELITY, AND MOVEMENTS OF SUBADULT SPINY LOBSTERS iPANULIRUS ARGUS) IN AREAS WITH ARTIFICIAL SHELTERS (CASITAS) ENRIQUE LOZANO-ALVAREZ, PATRICIA BRIONES-FOURZAN, AND MARIA EUGENIA RAMOS-AGUILAR Instituto de Ciencias del Mar y Limiwlogi'a. Uiiidad Acadcniica Puerto Morelos, Universidad Nucionul Auu'moma de Mexico. Ap. Posted 1 152. Caiuiiu. Q. R. 77500 Mexico ABSTRACT In Balu'a de la Ascension, a large bay on the Caribbean coast of Mexico, artificial shelters (casitas) have been used in the fishery for spiny lobsters (Panulinis argiis) for several decades. We selected two bay sites that differed in their ecological characteristics: site I was a protected inner-bay site, rich in benthic vegetation (settlement and postsettlement habitat) and site 2 was a more exposed, outer-bay site, closer to the coral reef tract, with less vegetation and more open hard bottoms. In each site, we explored the size distribution, population density, and patterns of aggregation of lobsters in casitas. as well as the site and shelter fidelity and the short-term movement ranges of individually tagged subadults (mean ± SD carapace length: 68.1 ± 10.9 mm). We expected that, owing to its lush vegetation, site I would have a higher density of lobsters of a smaller mean size than site 2. but that because of the occurrence of casitas in both sites, site and shelter t~idelity and the movement ranges of subadull lobsters would be similar in both sites. As expected, site 1 had significantly more lobsters encompassing a wider size range, but with a smaller mean size, than site 2. Lobsters were also more aggregated beneath casitas in site 1 than in site 2. Subadull lobsters exhibited similar site fidelity and short-term movement ranges in both sites, but a marginally higher shelter fidelity in site 2. However, shelter fidelity in both sites was lower than expected based on studies conducted by other workers in areas with natural shelters only. Although not conclusive, our results suggest that, because casitas might all afford a similar shelter quality to lobsters, lobsters in areas with casitas exhibit lower shelter fidelity and wider movement ranges than lobsters in areas with natural shelters only. KEY WORDS: Pamiliriis argiis. artificial shelters, casitas. site fidelity, shelter fidelity, movements INTRODUCTION The spiny lobster Pamdirus argus (Latreille, 1804), a major fishing resource throughout the Caribbean area, has several onto- genetic shifts in habitat and sociality during its benthic life. After a protracted, oceanic larval phase, the postlarvae of P. tirf^iis settle in shallow, vegetated habitats, where the ensuing algal-phase ju- veniles (6 to 15-20 mm carapace length, CL) remain widely dis- persed, displaying asocial behavior. The postalgal juveniles (15- 20 to approx. 45 mm CL) remain close to the settletnenl habitats but occupy crevice-type shelters and become socially gregarious. The subadults (45-80 mm CL) are more nomadic and may aggre- gate in large shelters but tend to migrate towards nearby coral reef tracts as they approach the adult phase (>80 mm CL). Adults dwell in caves and crevices in coral reefs and rocky bottoms on wide expanses of continental shelf and undergo massive, organized sea- sonal migrations (reviews in Herrnkind 1980, Butler & Hermkind 1997). Shelter availability plays an important role in the survival of spiny lobsters (Smith & Herrnkind 1992. Mintz et al. 1994. Bri- ones-Fourzan & Lozano-Alvarez 2001 ) and much of the individual and social behavior of spiny lobsters revolves around the shelter (Childress & Herrnkind 1996, 2001). Spiny lobsters must balance their need to remain in a shelter to avoid predation with the op- posite need of leaving that shelter to forage (Sih 1992. Vannini & Cannicci 1995) but have the ability to relocate known shelters (Hermkind et al. 1975, Cobb 1981, Nevitt et al. 2000, Lozano- Alvarez et al. 2002). On the other hand, spiny lobsters prefer shelters that allow cohabitation (Spanier & Zimmer-Faust 1988. Eggleston et al. 1990, MacDiarmid 1994), and individuals of P. argus may use conspecifics as cues both to locate and to assess the quality of a shelter (Ratchford 1999, Nevitt et al. 2000, Childress & Henmkind 2001). Vegetated and hard-bottom habitats have a fractal structure. which decreases the amount of shelter for large animals compared with small animals (Morse et al. 1985. Caddy 1986). Paucity of shelter may affect the movements and residence time of spiny lobsters in different ways. In areas poor in shelter, juveniles may exhibit either high rates of nomadism (Hermkind 1980), which increases their risk of predation, or restricted foraging movements, which precludes them from exploiting available food resources (review in Lipcius & Eggleston 2000) and may result in a poor nutritional condition (Briones-Fourzan et al. 2003). Also, shelter scarcity would increase shelter fidelity in spiny lobsters, i.e.. the propensity of lobsters to return to a previously used shelter (Herm- kind et al. 1975. Ratchford 1999). Casitas. or artificial shelters for spiny lobsters, have been em- pirically used for a number of decades in the fishery for P. argus in Bahi'a de la Ascension, a large, shallow bay on the Caribbean coast of Mexico (Briones-Fourzan et al. 2000; Fig. I ). Casitas may increase lobster abundance and biomass in areas with limited natu- ral shelter (Briones-Fourzan & Lozano-Alvarez 2001) by increas- ing protection from predators (Eggleston et al. 1990, Mintz et al. 1994). Casitas used in Bahi'a de la Ascension are scaled to accom- modate mostly subadults and adults (i.e.. lobsters >45 mm CL). but because of their gregarious behavior lobsters that occupy casitas are 10-145 inm CL (Lozano-Alvarez et al. 1991). However, mean size of lobsters is generally larger in "outer-bay" sites (sites be- tween the mouth of the bay and the reef tract, see Fig. 1 ) than in "inner-bay" sites (elsewhere in the bay; Eggleston et al. 1990, Lozano-Alvarez et al. 1991). The area of the bay suitable for using casitas has been divided in parcels (called campos) allotted to the members of the local fishing cooperative. Fishers decide how many casitas and where to deploy them within their campos. Favored substrates are vegetated habitats and hard bottoms. Unvegetated soft bottoms are generally avoided because on these substrates casitas tend to sink or their sheltering space becomes obstructed by sediment (Briones- Fourzan et al. 2000). Therefore, although Camarena-Luhrs et al. (1996) estimated an average of 3.3 casitas ha"' in some bay areas. 533 534 Lozano-Alvarez et al. 19°40'- 19°30'N- Figure 1. Location of the two study sites: an inner-bay site (site 1) and an outer-bay site (site 2) in Bahi'a de la Ascension (Caribbean coast of Mexico). Blacli areas represent the coral reef tract. the distribution of casitas throughout the fishing areas in the bay is highly heterogeneous. Lozano-Alvarez (1993) hypothesized that, in addition to de- creasing predation risk of lobsters, the occurrence of numerous casitas over large expanses could allow spiny lobsters to exploit food resources over more extensive areas, because after their noc- turnal foraging excursions lobsters could retreat into any casita available in their vicinity. Moreover, lobsters foraging close to a casita may be attracted by chemical cues emanating from other lobsters already sheltered in that casita (Ratchford & Eggleston 1998. Nevitt et al. 2000). This hypothesis implies a low shelter fidelity among lobsters occurring in areas with casitas. We explored the lobster density and the pattern of lobster ag- gregation in casitas in two sites in Bahi'a de la Ascension that differed in their environmental characteristics: an inner-bay site (site 1) and an outer-bay site (site 2). Based on previous studies (e.g., Lozano-Alvarez et al. 1991. 1994). we expected 1 ) a smaller mean size and a higher abundance of lobsters in site 1 than in site 2. and 2) larger aggregations of lobsters in casitas in site 1 than in site 2. We also explored the site and shelter fidelity and the short- term movements among casitas of lobsters >45 mm CL (i.e., sub- adults and young adults). Despite the environmental differences between both sites, we hypothesized that, owing to the presence of casitas. ( 1 ) site and shelter fidelity of lobsters would be similar in both sites, and (2) short-term movements of these lobsters would be similar between both sites but greater than those reported for areas with natural shelters only. MATERIALS AND METHODS Study Sites Site 1 was located west of Punta Hualastok, an inner-bay area highly protected from wave surge (Fig. I). The water in this site was very calm and reddish in color as a result of the thick man- grove forests bordering the nearby coasts to the east and south of the site. Depth was 3^ m. The bottom in site I was mostly fine calcareous sand and mud. extensively covered with dense mead- ows of macrophytes that included mixed seagrass {Thalassia testudinum and Syringodium filifonne) and abundant macroalgae, such as Laurencia intricata. Dictyota divaricata. Jania adiuierens, Caulerpa sp.. Halimeda iiicrassota, H. monile. Batophom oersie- dii. and Ripocephalus phoenix. Site 2 was located in an outer-bay zone, leeward of the coral reef tract (Fig. I ) and was more exposed to wave surge than site 1 . Water in site 2 was generally very clear, and depth was 3-3.5 m. The bottom in site 2 was coarse calcareous sand with a few small coral heads and patches of exposed calcareous pavement. The bottom type changed gradually towards the coast, where a few patches of dense vegetation were interspersed with vast expanses of sparse vegetation and open sand. The macrophytes consisted of mixed seagrass with interspersed macroalgae. especially Halimeda spp., Laurencia scoponia, Penicillus diimetosus. Udotea flavelhun. U. conghainata and U. spinidosa. Lobster Sampling In Bahi'a de la Ascension, casitas harbor more lobsters towards the end of the closed season (1 March-30 June), which is reflected in significantly higher catches during the first month (July) than during the rest of the fishing season (August-February) (Lozano- Alvarez et al. 1991 ). Therefore, to avoid bias in our results caused by fishing activities, our study was conducted in June 1990 and May through June 1991. Two divers towed by a boat in a systematic pattern surveyed each of the two sites for casitas. When a casita was found, it was marked with an individually numbered buoy. Casitas were more widely dispersed in site 1 than in site 2. We marked 22 casitas in site 1 and 25 in site 2. The size and shape of the 47 casitas were similar (-1.8 m long x 1.2 m wide x 6-8 cm high) and all were constructed with the same materials (a palm-trunk frame and a ferrocement roof). We delimited the area enclosing the marked casitas in each site with additional buoys, measured the distance between adjacent buoys, and estimated the surface area of each site. This was approximately 25 ha in site 1 and 12 ha in site 2. The delimited areas were surveyed again, but no further casitas were found. Divers censused the lobsters beneath the 22 casitas in site I on six occasions between 15 and 23 June 1990. On each of the first 4 days, all the lobsters sheltering beneath a randomly chosen casita were prodded into the cod-end of a seine net (Lozano-Alvarez et al. 1991. Lipcius et al. 1998). The cod-end was kept underwater at the side of the boat to maintain the lobsters submerged and pro- tected from direct sunlight. Lobsters were then extracted from the net one at a time to determine their sex and to measure CL with calipers (±0.1 mm. between the rostral horns and the posterior margin of the cephalothorax). Subadults (individuals 2:45.0 mm CL) were then tagged and returned to their original casita. Tags consisted of a color-coded flag of adhesive tape held by a rubber band around the carapace between the fourth and fifth pair of pereiopods that allowed for identification of both the individual and the casita from where it was extracted. Over subsequent sur- veys, we recorded the data of resighted lobsters and of the casitas where they sheltered. Lobsters beneath the 25 casitas in site 2 were censused on 15 occasions between May 7 and June 9, 1991. On eight dates be- tween May 7 and 24. all the lobsters from one casita were mea- sured and the subadults tagged. In addition to the color-coded tag. SuBADULT Spiny Lobsters in Areas with Casitas 535 o in CM o CO in CO o un o ■>d- Ln in o CD ID CD O in o 00 in c» o CJ> in Carapace length (mm) I Site 1 (N = 161) HSite2(N = 122) Figure 2. Panulirus argiis. Size distribution of lobsters captured from beneath casitas in an inner-bay site (site 1 ) and an outer-bay site (site 2) in Bahi'a de la Ascension. The arrow indicates the class size from which individuals were tagged. which aicieii in the rapid icientification of the original casita. we also applied to these lobsters individually numbered Australian "spaghetti" tags (Chittleborough 1974), modified for small lobsters (Lozano-Alvarez 1992. Negrete-Soto et al. 2002), on the dorsolat- eral muscle between the cephalothorax and abdomen. With these tags, Lozano-Alvare/ (1992) estimated a tag-related mortality of -5% after three months in individuals of P. argiis over the same size range as in our study. Lobster Density and Patterns of Aggregation in Casitas In each site, lobster population size, losses (death -i- emigration) and immigration were estimated using the Fisher-Ford model (Fisher & Ford 1947), which relies on several tagging and recap- ture dates as well as on multiple recaptures of individuals. When capture-recapture data are scarce, the Fisher-Ford model tends to yield more reliable results than other models based on multiple- recapture data (Bishop & Sheppard 1973, Begon 1979, Lozano et al. 1982, Negrete-Soto et al. 2002). The Fisher-Ford model as- sumes a constant survival rate ((()) but provides a method to test for this assumption (Begon 1979). Because the number and frequency of sampling dates varied between sites, we used only the data from censuses conducted over consecutive days (four dates in site 1 and six dates on site 2) to estimate lobster abundance. This would also increase the probability of a constant survival rate over such short periods. We then obtained the density of lobsters in each site by dividing the number of lobsters estimated by the model over the site area (Begon 1979). We also estimated the density of the por- tion of the lobster population sheltering in casitas in each site by dividing the daily number of lobsters censused beneath casitas over the site area. The propensity of lobsters to aggregate in casitas was analyzed in each site by plotting the number of lobsters in each casita vs. the number of casita surveys over the sampling period (Briones-Fourzan et al. 2000) and fitting the data to a ran- dom distribution. Site and Shelter Fidelity Among Lobsters The percent of tagged lobsters that were resighted at least once in each site was considered as a measure of site fidelity (Butler & Herrnkind 1997). Because consecutive censuses were conducted 1 to 6 days apart, we used two measures of shelter fidelity 1 ) the percent of occasions a tagged lobster returned to the shelter it used the previous day (Ratchford 1999). and 2) the percent of occasions a tagged lobster returned to the shelter it occupied on the previous census date throughout the study periods. We compared site and shelter fidelity of lobsters between sites using contingency table analyses (Zar 1999). Movements of Lobsters Although lobsters may forage following complex, circuitous routes (Jemakoff 1987), we considered as the minimum daily dis- tance moved by a tagged lobster the distance measured on a straight-line between casitas occupied by that lobster on consecu- tive days (Acosta & Butler 1997. Ratchford 1999). We used con- tingency table analyses (Zar 1999) to compare between sites the median daily distance moved by those lobsters that shifted casitas on the first post-tagging day and during the first post-tagging week (Jemakoff et al. 1987). We also measured the angle between ca- sitas occupied by tagged lobsters on consecutive dates with an underwater compass, and analyzed the circular distribution of the angles with a Rayleigh test (Zar 1999) to determine whether lob- sters showed directional or random movements. To assess the movements of lobsters over periods longer than those encom- pas.sed by our study, fishermen were requested to report the cap- ture of tagged lobsters and their location of capture after the open- ing of the fishing season on July the first of each year. RESULTS Size Distribution of Lobsters The number of lobsters extracted from four randomly chosen casitas in site 1 was 161, over a size range of 22.3-99.5 mm CL (mean + SD: 58.7 ± 17.5 mm CL). In site 2, 122 lobsters were extracted from eight casitas. These lobsters were 34.8-96.9 mm CL (mean ± SD: 66.9 ± 10.9 mm CL) (Fig. 2). Mean size of lobsters was significantly different between sites (Student's ?-test with log-transformed data to homogenize variances, t = 5.024. 536 Lozano-Alvarez et al. df = 272, P < 0.0001 ). The difference was the result of the greater occurrence of small, postalgal juveniles (i.e.. juveniles <45 mm CL) in site 1 (Fig. 2). Postalgal juveniles made up 31.7% of lob- sters sampled in site 1 but only 4.5% of lobsters sampled in site 2. When postalgal juveniles were excluded from the comparison, the mean size of subadults was similar in both sites (site 1: 68.15 ± 12.4 mm CL; site 2: 68.25 ± 9.3 mm CL: t = 0.008, df = 215, P > 0.50). Sex ratio was around 1;1 in both sites. Lobster Density and Patterns of Aggregation in C'asitas We tagged and returned to their original casitas 136 subadults and young adults (45.1-97.5 mm CL) in site 1, and 1 17 (45.1-96.9 mm CL) in site 2. Of these, 67 (49.3%) were resighted at least once in site 1 and 71 (60.7%) in site 2. Table I shows the statistics derived from the Fisher-Ford model for each site. As expected, population size, losses and immigrations were higher, but more variable, in site 1 than in site 2. Survival rate ((})) was also higher in site 1 (4) = 0.865) than in site 2 ((}> = 0.745). Based on the estimates of population size, mean ± SD lobster density was esti- mated as 47.7 ± 9.7 lobsters ha"' in site 1 and 25.8 ± 3.8 lobsters ha"' in site 2 (Table 1). These mean densities were significantly different (t = 4.673. df = 6. P = 0.0034). The daily number of lobsters beneath the 22 casitas in site 1 ranged from 295 to 467, yielding a density of 1 1 .8-1 8.7 lobsters in casitas ha"' (mean + SD: 16.6 ± 2.7). In site 2. the daily number of lobsters in the 25 casitas fluctuated between 1 1 1 and 174, yield- ing a density of 9.3- 14.5 lobsters in casitas ha"' (mean ± SD: 13. 1 ± 1.6), significantly different from that of site 1 (t = 3.653. df = 19, P = 0.0017). When considering only those dates included in the Fisher-Ford model, the mean number of lobsters beneath ca- sitas accounted for 34.4% and 52.1%, respectively, of the mean number of lobsters estimated throughout sites 1 and 2. The distribution of lobsters in casitas departed significantly from a random distribution in both sites (site 1: x" = 156.865; P < 0.001: site 2: x" = 40.493; P < 0.001 ). However, lobsters tended to be more aggregated in site 1 than in site 2 (Fig. 3). In site 1 , 52% casitas harbored over 20 lobsters and the maximum number of lobsters sheltering beneath a casita was 60, whereas in site 2 these figures were, respectively, 3% and 40. In both sites, some casitas harbored no lobsters (Fig. 3), but casitas with no lobsters on a given date had lobsters on the following date and vice versa. Site and Shelter Fidelity Among Lobsters Table 2 summarizes the results on site and shelter fidelity of subadult lobsters in both sites. Some lobsters that were not re- sighted on the first few post-tagging days were seen again later, whereas others were never seen again. Site fidelity was higher, but not significantly different, in site 2 than in site 1 . Mean shelter fidelity A (percent of occasions a tagged lobster returned to the same casita it used the day before) did not differ significantly between sites, whereas the difference in mean shelter fidelity B (percent of occasions a tagged lobster returned to the casita it used on the previous census date throughout the study period) was mar- ginally significant. Tlie P values may indicate that the power of the tests was low, but the overall results suggest that lobsters in site 2 exhibited slighdy higher site and shelter fidelity than lobsters in site 1 . Movements of Lobsters On the first post-tagging day, lobsters that shifted casitas moved 58^16 m overnight in site 1 (median distance = 165 m) and 25-290 m in site 2 (median = 108 m). The medians were not significantly different (x-= 2.110: df = \: P = 0.220). During the first post-tagging week the movements remained similar, both within (median of site 1: 133 m; of site 2: 1 10 m) and between sites (x' = 1.100; df = I, P = 0.431). Therefore, lob.sters from both sites exhibited similar movement ranges during the first post- tagging week. Lobsters that used more than two casitas moved 1 55—400 m among casitas over the study periods. The movements of lobsters within site 1 (mean angle ± angular deviation: 154.6° ± TABLE 1. Panulirus argus: statistics of the Fisher-Ford model for spiny lobsters in (a) an inner-bay site (site 1) and (b) an outer-bay site (site 2) in Bahia de la Ascension, Mexico. Sampling Date Spiny Lobsters Population Estimates Captured Tagged Size (N) Losses Emigration Density (Lobsters ha"') Inner-bay (site 1 ) 15 June 16 June 17 June 18 June Mean ± SD Outer-bay (site 2) 07 May 08 May 09 May 10 May 11 May 12 May Mean ± SD 293 410 467 466 175 185 131 146 160 169 37 46 17 0 40 19 9 14 0 0 1196 1435 948 1196 + 246 346 320 355 273 251 320 ± 45 161 194 128 88 82 91 70 64 401 47.8 ■294 57.4 — 37.9 47.7 ±9.7 62 28.8 117 26.7 9 29.6 47 22.8 — 20.9 25.8 ± 3.8 All lobsters were captured from beneath artificial shelters (casitas). Losses are deaths -i- emigration. Density of lobsters was estimated by dividing the population size over the surface area of each site (25 ha in site 1. 12 ha in site 2). SuBADULT Spiny Lobsters in Areas with Casitas 537 (A "55 (0 o c o Q. O N ^ -b ^ b fe A % <^ sO sN ^^ ^^ ^^ ^^ K^ <\ ^% ^ r/> ri> r> T^ nfo X> n% r« 4i 45 Number of lobsters per casita I Site 1 (N = 128) SSite 2 (N = 374) Figure 3. Panulirus argus. Distribution of lobsters beneath casitas in an inner-bay site (site 1) and an outer-bay site (site 2) in Bahia de la Ascension. N is the number of casita surveys conducted throughout the study period in each site. 71.6°) were non-directional (Rayleigh's test: z = 2.162, n = 45, P > 0.10). In contrast, movements of lobsters within site 2 (mean angle ± angular deviation: 82.6° ± 69.1°) were not uniformly dis- tributed around the circle (z = 3.1, « = 42, P < 0.05). These lobsters showed a tendency to move towards the coral reef, which lies at 80° from site 2 (V-test, u = 2.493, n = 42, P < 0.01). Fishermen recaptured 33 lobsters tagged in site 1 during July 1990. 4-8 wk after being tagged. Of these. 17 remained within site 1, but 16 were recaptured 2,000-14,600 m away from this site. In contrast, fishermen recaptured 20 lobsters tagged in site 2 during July 1991 (8-13 wk after being tagged), of which 19 remained within site 2 and only one was caught outside this site (distance not recorded). Lobsters recaptured by fishermen (67.5-84.2 mm CL) had increased 4.3-20.2 mm in 6-13 wk. DISCUSSION As expected, the inner-bay site (site 1 ) had significantly more lobsters encompassing a wider size range, but with a smaller mean size, than the outer-bay site (site 2). Although we sampled site 2 one year later than site 1 , our results are consistent with previous findings. In Bahi'a de la Ascension, larger lobsters occur in many bay areas but are more common in the outer-bay, whereas smaller lobsters commonly occur at higher densities in more protected inner-bay areas, rich in settlement and post-settlement habitats (Eggleston et al. 1990; Lozano-Alvarez et al. 1991, 1994). Similar results have been obtained in shallow areas of northern Quintana Roo (Arce et al. 1997. Sosa-Cordero et al. 1998) and Belize (Acosta 1999). TABLE 2. Panulirus argus: comparisons of site and shelter fidelity of subadult spiny lobsters tagged in an inner-bay site (site 1) and an outer-bay site (site 2) in Bahia de la .Ascension. Mexico. Number of Lobsters Site Tagged Resighted Site Fidelity (%) Shelter Fidelity {%) B Inner-bay (site 1) Outer-bay (site 2) X' value P value 136 117 67 71 49.3 60.7 3.31 0.069 18.4 30.3 3.17 0.074 31.6 47.4 3.94 0.047 All lobsters were captured from beneath artificial shelters (casitas). Site fidelity is the percent of tagged lobsters resighted at least once within the respective site. Two measures of shelter fidelity were considered: (A) the percent of occasions a tagged lobster returned to the same casita it used the day before, and (B) the percent of occasions a tagged lobster returned to the same casita it used on the previous census date. Census dates were 1 to 6 days apart. Degrees of freedom = I in all comparisons. 538 Lozano-Alvarez et al. Density of lobsters beneath casitas was also higher and lobsters were more aggregated in site 1 than in site 2. Lobsters tend to aggregate more beneath large artificial shelters deployed over veg- etated habitats, where juvenile density is higher, than over hard bottoms (Lozano-Alvarez et al. 1994, Mintz et al. 1994, Arce et al. 1997, Sosa-Cordero et al. 1998. Briones-Fourzan et al. 2000. Bri- ones-Fourzan & Lozano-Alvarez 2001 ). This pattern of aggrega- tion may indicate a "guide-effect."' which is a consequence of conspecific attraction related to lobster density (Childress & Herm- kind 2001). Short-term movements (and hence site fidelity) of lobsters could be affected by disturbance caused by capture and tagging (Hermkind 1980). However, initial capture had only short-term effects, and tagging had no additional effect on the movement of individual Jasiis edwardsii (MacDiarmid et al. 1991); capture and handling had no short-term effects on movements of individually tagged P. cygiuis (Jernakoff et al. 1987). and disturbance of lob- sters had no apparent effect on the selection of shelter by other lobsters (Ratchford 1999). Disturbance probably had little effect on our tagged lobsters because there were no significant differ- ences in movement ranges and site fidelity between our study sites, and tag-related mortality was unlikely in either site. Therefore, lobsters that were not resighted may have been predated, moved beyond the boundaries of the sites, or occupied unsurveyed natural shelters throughout the sites. We did not survey the potential natural shelters occurring in each of our sites; this would have been a formidable task given their large surface area. However, it has been shown that benthic vegetation, in addition to providing settlement habitats and feeding areas, may also provide shelter to juvenile P. argiis. In Bahi'a de la Ascension, Lipcius et al. (1998) plotted algal biomass vs. survival of tethered juvenile P. argits (30-75 mm CL) and obtained a hyperbolic habitat-survival function. Their results indicate that even a modest increase of algal biomass. which increases the ar- chitectural complexity of the habitat, significantly enhances the survival of juvenile P. argus. In Belize, greater numbers of juve- nile P. argus moved into and from habitats surrounded by seagrass than those surrounded by rubble, which suggests that vegetated substrates may function as movement corridors for juvenile lob- sters, facilitating their dispersal to areas containing new resources (Acosta 1999). This would explain the greater variations in popu- lation estimates of juveniles in our site 1 compared with site 2. Moreover, Acosta and Butler (1997) found that large juveniles of P. argus have similar survival when sheltering among mangrove prop roots and in coral crevices. Our inner-bay site, in addition to having more benthic vegetation, was close to thick mangrove for- ests; therefore, the higher survival rate estimated for lobsters in site 1 may reflect the additional protection provided by these vegetated substrates. Also, the lesser habitat complexity in site 2. where vegetation was scarce, could underlie the slightly higher shelter fidelity exhibited by lobsters in site 2 compared with site 1 . Herrnkind (1980) devised a conceptual model postulating that lobsters in areas of abundant food and shelter will tend to be residential, whereas lobsters in areas of scarce shelter and disperse food supply will tend to be more nomadic owing to intraspecific competition for shelter. But evidences for a relationship between site and shelter fidelity, lobster size, and shelter abundance remain equivocal (Hermkind et al. 1975, Hermkind 1980, MacDiarmid et al. 1991, Acosta & Butler 1997, Butler & Hermkind 1997, Bri- ones-Fourzan & Lozano-Alvarez 2001). Some studies report that smaller lobsters display stronger shelter fidelity than larger lob- sters, whereas others report that subadults and young adults are more transient and nomadic (which implies a low shelter fidelity) than old adults. However, these evidences have been obtained in areas with natural shelter only. For example, in the case of P. argus. average shelter fidelity A of tagged subadult and young adult individuals was estimated at 38% (range: 15-88%) by Ratch- ford ( 1999), similar to the 42% reported for old adults by Herm- kind et al. (1975). and Acosta and Butler ( 1997) found average den residence times for postalgal P. argus of 2.0 to 4.38 days over five consecutive days (equivalent to a shelter fidelity A of 40-87%). Compared with these values, the average shelter fidelity A of our subadult P. argus (18.4% in site 1; range: 20-60%, and 30.3% in site 2; range: 20-75%) was rather low. The occurrence of casitas could partially explain these results, as proposed by Lozano-Alvarez (1995), because casitas presum- ably reduce competition for shelter by allowing cohabitation of large numbers of individuals. However, based on laboratory ex- periments, Ratchford (1999) suggested that the longer a lobster resides in an area and becomes more familiar with the shelters in that area, the lower its shelter fidelity will appear. This could also explain the overall low shelter fidelity A of our lobsters as well as the marginal difference in shelter fidelity B among lobsters be- tween our sites 1 and 2. The large number of postalgal juveniles cohabiting in casitas with subadults in the inner-bay site 1, rich in settlement and post-settlement habitats, suggests that these sub- adults had probably remained in that area since settlement. But this inner-bay area may cease to be an appropriate habitat once sub- adult lobsters reach a critical size. These subadults would then immigrate to other outer-bay habitats (Cmz et al. 1986, Lozano- Alvarez et al. 1991), thus explaining the distant locations where lobsters tagged in site 1 were recaptured by fishermen a few weeks later. In contrast, individuals beneath casitas in site 2 were mostly subadults, which had probably immigrated recently to this site from other, more vegetated areas. The proximity of the coral reef, the habitat preferred by subadults and adults, could also underlie the more directional movements of subadults towards this habitat in site 2. Some species of spiny lobsters are highly mobile [e.g. PanuU- rus cygnus (Jernakoff 1987. Jemakoff et al. 1987) and P. argus (Hermkind et al. 1975, Ratchford 1999)] and others are more sedentary [e.g. Jasus edwardsii (MacDiarmid et al. 1991) and P. guttatus (Negrete-Soto et al. 2002, Lozano-Alvarez et al. 2002)]. However, even in highly mobile species, the extent of the daily movement range appears to depend on the occurrence of suitable structured shelter. Previous studies estimating the daily move- ments of tagged postalgal juveniles and adults of P. argus have been conducted in areas with natural shelters only. Hermkind et al. ( 1975) used sonic tags to individually track 27 large, adult P. argus (average size approx. 1 10 mm CL) in a coral reef habitat over five consecutive nights. These lobsters typically moved 30-90 m over- night and used three or four dens within 140 m, with a maximum den shift just under 500 m (Hermkind 1980). In shallow coastal areas, postalgal juveniles (average size approx. 37 mm CL) moved 5.4 to 24.5 m overnight when shifting shelters (Acosta & Butler 1997). whereas lobsters 70.6-134.0 mm CL moved 10-185 m ovemight when shifting shelters and up to 270 m among shelters over a period of four weeks (Ratchford 1999). Our subadult P. argus (mean size: 68 mm CL) moved 25—116 m overnight when shifting casitas, and 155^00 m among casitas over the study periods. These movements are greater than those reported by Acosta and Butler (1997) for postalgal juveniles and Ratchford SuBADULT Spiny Lobsters in Areas with Casitas 539 ( 1999) for subadults and adults in areas with natural shelters only, suggesting that the occurrence of casitas does increase the move- ment range of subadult P. argtis. Areas with few natural, appropriate shelters would favor be- havior that allows lobsters to efficiently relocate previously used shelters (Ratchford 1999). Because of their physical properties, casitas allow cohabitation of many individuals over a wide size range and afford — at least in theory — a similar quality of shelter, although the latter may vary somewhat depending on the type of substrate around individual casitas (Meiners-Mandujano 2002). Therefore, areas with numerous casitas would allow lobsters to forage over greater areas by reducing their need to relocate a previously used casita. Moreover, a lobster could be attracted to any nearby casita at the end of its foraging excursion by cues emanating from lobsters already sheltered in that casita (Nevitt et al. 2000, Ratchford & Eggleston 2000, Childress & Herrnkind 2001). This would be reflected in low values of shelter fidelity A and wide short-term movement ranges, as suggested by our results. These results are, however, inconclusive, because to fully test this hypothesis it would have been necessary to compare shelter fidelity and movement ranges of subadult lobsters in areas of the bay with and without casitas. This was unfeasible because an estimated 20,000 casitas occur throughout the lobster habitats in Bahi'a de la Ascension (Briones-Fourzan et al. 2000). However, preliminary results of a controlled field experiment recently con- ducted in the reef lagoon at Puerto Niorelos, Mexico indicate a significant increase in the daily movements of postalgal juvenile P. argus after the introduction of casitas scaled to their size (Meiners- Mandujano 2002, Lozano-Alvarez et al., unpublished data). ACKNOWLEDGMENTS The authors thank F. Negrete-Soto for his invaluable help in the fieldwork. Much appreciated logistic support was provided by the crew of the boat "Fipesco 207." Capt. Daniel Duran, Pedro Men- dez, and Michel Moreno, and the local lobster fisher Manuel Cahuich. DGAPA (Direccion General de Asuntos del Personal Academico, UNAM) provided a scholarship forMERA. This work was partially funded by World Wildlife Fund-U.K. through Aso- ciacion de Amigos de Sian Ka'an, A.C., and Universidad Nacional Autonoma de Mexico (UNAM). LITERATURE CITED Acosta. C. A. 1999. Benthic dispersal of Caribbean spiny lobsters among insular habitats: implications for the conservation of exploited marine species. Conserv. Biol. 13:603-612. Acosta. C. A. & M. J. Butler, IV. 1997. Role of mangrove habitat as a nursery for juvenile spiny lobster. PanuUnis argus. in Belize. Mm: Freshwater Res. 48:721-727. Arce, A. M., W. Aguilar-Davila, E. Sosa-Cordero & J. F. Caddy. 1997. Artificial shelters (casitas) as habitats for juvenile spiny lobsters Pami- linis argus in the Mexican Caribbean. Mar. Ecol. Prog. Ser. 158:217- 224. Begon, M. 1979. Investigating Animal Abundance: Capture-Recapture for Biologists. London: Edward Arnold, 97 pp. Bishop, J. A. & P. M. Sheppard. 1973. An evaluation of two capture- recapture models using the technique of computer simulations. In: M. S. Bartlett & R. W. Hiorns. editors. The mathematical theory of the dynamics of biological populations. London: Academic Press, pp. 235- 254. Briones-Fourzan, P. & E. Lozano-Alvarez. 2001. Effects of artificial shel- ters (casitas) on the abundance and biomass of juvenile spiny lobsters, Panulirus argus, in a habitat-limited tropical reef lagoon. Mar. Ecol. Prog. Ser. 221:221-231. Briones-Fourzan, P., E. Lozano-Alvarez & D. B. Eggleston. 2000. The use of artificial shelters (Casitas) in research and harvesting of Caribbean spiny lobsters in Mexico. In: B. F. Phillips & J. Kittaka. editors. Spiny lobsters: fisheries and culture, 2nd edition. Oxford: Fishing News Books, pp. 420-446. Briones-Fourzan, P., V. Castaneda-Femandez de Lara, E. Lozano-Alvarez & J. Estrada-Olivo. 2003. Feeding ecology of the three juvenile phases of the spiny lobster Panulirus argus in a tropical reef lagoon. Mar. Biol 242:(in press). Butler, M. J. IV & W. F. Herrnkind. 1997. A test of recruitment limitation and the potential for artificial enhancement of spiny lobster (Paiuilinis argus) populations in Rorida. Can. J. Fish, .\quat. Sci. 54:542-463. Caddy, J. F. 1986. Modelling stock-recruitment proces.ses in Crustacea: some practical and theoretical perspectives. Can. J. Fish. Aquat. Sci 43:2330-2344. Camarena-Luhrs. T.. L. Coba-Cetina, A. Aguilar-Perer & W. Aguilar- Davila. 1996. Densidad y abundancia de juveniles de langosta Panu- lirus argus en Bahi'a de la Ascension. Quimana Rod, Mexico. Proc. Gulf Carihh. Fish. Inst. 44:579-593. Childress. M. J. & W. F. Herrnkind. 1996. The ontogeny of social behav- iour among juvenile Caribbean spiny lobsters. Anim. Bchav. 51:675- 687. Childress, M. J. & W. F. Herrnkind. 2001. The guide-effect influence on the gregariousness of juvenile Caribbean spiny lobsters. Anim. Behav. 62:465^72. Chittleborough, R. G. 1 974. Development of a tag for the Western rock lobster. Commonw. Sci. Ind. Res. Org. Div. Fish. Oceanogr. Rep. 56: 1-19. Cobb. J. S. 1981. Behaviour of the Western Australian spiny lobster. Panu- lirus cygnus George, in the field and laboratory. Ausi. J. Mar. Fresh- water Res. 32:399-t09. Cruz, R., R. Brito, E. Diaz & R. Lalana. 1986. Ecologi'a de la langosta (Panulirus argus) al SE de la Isia de la Juventud. II. Patrones de movimiento. Rev. Inv. Mar. 7:19-35. Eggleston. D. B., R. N. Lipcius. D. L. Miller & L. Coba-Cetina. 1990. Shelter scaling regulates survival of Caribbean spiny lobster. Panulirus argus. Mar Ecol. Prog. Ser. 62:70-88. Fisher. R. A. & E. B. Ford. 1947. The spread of a gene in natural conditions in a colony of the moth PamLxia dominula (L.). Heredity 1:143-174. Herrnkind, W. F. 1980. Spiny lobsters: patterns of movements. In J. S. Cobb & B. F. Phillips, editors. The biology and management of lob- sters, vol 1 : physiology and behavior. New York: Academic Press, pp. 349-f07. Herrnkind. W. F.. J. Vanderwalker & L. Barr. 1975. Population dynamics, ecology and behavior of spiny lobsters. Panulirus argus. of St. John. U. S. Virgin Islands. IV: Habitation, patterns of movement and general behavior. Nat. Hist. Mus. Los Angeles County Sci. Bull. 20:31-34. Jemakoff P. 1987. Foraging patterns of juvenile western rock lobster Panulirus cygnus George. J. Exp. Mar. Biol. Ecol. 113:125-144. Jemakoff, P., B. F. Phillips & R. A. Mailer. 1987. A quantitative study of nocturnal foraging distances of the western rock lobster Panulirus cyg- nus George. J. E.xp. Mar. Biol. Ecol. 1 13:9-21. Lipcius. R. N. & D. B. Eggleston. 2000. Introduction: Ecology and fishery biology of spiny lobsters. In: B. F. Phillips & J. Kittaka, editors. Spiny lobsters: fisheries and culture. 2nd edition. Oxford: Fishing News Books, pp. 1^1. Lipcius. R. N., D. B. Eggleston, D. L. Miller & T. C. Luhrs. 1998. The habitat-survival function for Caribbean spiny lobster: an inverted size effect and non-linearity in mixed algal and seagrass habitats. Mar. Freshwater Res. 49:807-816. Lozano-Alvarez. E. 1992. Pesqueria, dinamica poblacional y manejo de la 540 Lozano-Alvarez et al. langosta Pamdinis argiis (Latreille, 1 804) en Bahi'a de la Ascension, Q. R., Mexico. Tesis Doctoral, Univ. Nal. Auton. Mexico. 142 pp. Lozano-Alvarez. E. 1995. Requisites para la introduccion de refugios ar- tificiales en pesquerias de langosta. Rev. Ciihana Im: Pesq. 19:21-26. Lozano-Alvarez. E.. P. Briones-Fourziin & B. F. Phillips. 1991. Fishery characteristics, growth, and movements of the spiny lobster PanuUnis argils in Bahi'a de la Ascension. Mexico. Fish. Bull. 89:79-89. Lozano-Alvarez. E., P. Briones-Fourzan & F. Negrete-Soto. 1994. An evaluation of concrete block structures as shelter for juvenile Caribbean spiny lobsters. Paniilirus argus. Bull Mar. Sci. 55:351-362. Lozano-Alvarez. E.. G. Carrasco-Zanini & P. Briones-Fourzan. 2002. Homing and orientation in the spotted spiny lobster. Pamdirus guttanis (Decapoda: Palinuridae), towards a subtidal coral reef habitat. Crushi- ceana. Lozano. E.. P. Briones. L. Santarelli & A. Gracia. 1982. Densidad pobla- cional de Pamdirus gracilis Streets y P. inflatus (Bouvier) en dos areas cercanas a Zihuatanejo. Gro., Mexico. Ciencia Pesq. 3:61-73. MacDiarmid. A. B. 1994. Cohabitation in the spiny lobster y^iiii edwardsii (Hutton, 1875). Crustaceana 66:341-355. MacDiarmid, A. B., B. Hickley & R. A. Mailer. 1991. Daily movement patterns of the spiny lobster Jasus edwardsii (Hutton) on a shallow reef in northern New Zealand. J. Exp. Mar. Biol. Ecol. 147:185-205. Meiners-Mandujano. C. G. 2002. Importancia de refugios artitlciales tipo casita para juveniles de langosta Panulirus argus (Latreille. 1804): Dinamica de ocupacion y heterogeneidad del sustrato en la laguna arrecifal de Puerto Morelos. Quintana Roo. Tesis de Maestria. Univ. Nal. Auton. Mexico. 59 pp. Mintz, J. D.. R. N. Lipcius. D. B. Eggleston & M. S. Seebo. 1994. Survival of juvenile Caribbean spiny lobster: effects of shelter size, geographic location and conspecific abundance. Mar. Ecol. Prog. Ser. 112:255- 266. Morse. D. R.. J. H. Lawton. M. M. Dodson & M. H. Williams. 1985. Fractal dimension of vegetation and the distribution of arthropod body lengths. Nature 314:731-733. Negrete-Soto. F.. E. Lozano-Alvarez & P. Briones-Fourzan. 2002. Popu- lation dynamics of the spiny lobster Pamdirus gullalus (Latreille) in a coral reef on the Mexican Caribbean. J. Shellfish Res. 21:279-288. Nevitt. G., N. D. Pentcheff, K. J. Lohmann & R. K. Zimmer. 2000. Den selection by the spiny lobster Panulirus argus: testing attraction to conspecifics odors in the field. Mar. Ecol. Prog. Ser 203:225-231. Ratchford. S. G. 1999. The influence of chemical communication on shel- ter selection, shelter sharing, and aggregation among spiny lobsters, Pamdinis argus. PhD Thesis. North Carolina State Univ. Raleigh, NC. 82 pp. Ratchford. S. G. & D. B. Eggleston. 1998. Size- and scale-dependent chemical attraction contributes to an ontogenetic shift in sociality. Anim. Behav. 56:1027-1034. Ratchford. S. G. & D. B. Eggleston. 2000. Temporal shift in the presence of a chemical cue contributes to a diel shift in socilaity, Anim. Behav. 59:793-799. Sih, A. 1992. Prey uncertainty and the balancing of antipredator and feed- ing needs. Am. Nal. 139:1052-1069. Smith. K. N. & W. F. Hermkind. 1992. Predation on eariy juvenile spiny lobsters Panulirus argus (Latreille): influence of size and shelter. / E.\p. Mar Biol. Ecol. 157:3-18. Sosa-Cordero. E.. A. M. Arce. W. Aguilar-Davila & A. Ramirez-Gonzalez. 1998. Artificial shelters for spiny lobsters Panulirus argus (Latreille): an evaluation of occupancy in different benthic habitats. J. E.\p. Mar. Biol. Ecol. 229:1-18. Spanier. E, & R. K. Zimmer-Faust. 1988. Some physical properties of shelter that intluence den preference in spiny lobsters. J. Exp. Mar. Biol. Ecol. 121:137-149. Vannini, M. & S. Cannicci. 1995. Homing behaviour and possible cogni- tive maps in crustacean decapods. J. E.xp. Mar. Biol. Ecol. 193:67-91. Zar. J. H. 1999. Biostatistical analysis, 4th ed. Upper Saddle River, NJ: Prentice Hall. 663 pp + appendices. Jo:iimil of Slu'lljlyh Ri'scairh. Veil. 22. No. 2. .'i41-.S4.S, 200.V TETRAPLOID INDUCTION BY HEAT SHOCKS IN CHINESE SHRIMP, FENNEROPENAEUS CHINENSIS FUHUA LI, JIANHAI XIANG,* XIAOJUN ZHANG. CHANGGONG WU, CHENGSONG ZHANG, LINGHUA ZHOU, AND KUIJIE YU lustiliitc (if Occciiu)loi>y. Cliiiwsc Academy of Sciences. 7 Nanhai Road. Qiiiiidao 26607 f People '.V Republic of China ABSTRACT Tetraploid induction in the Chinese shrimp Fenneropenueus chinensis was studied. Tetraploid larvae were successfully produced through mitosis I inhibition hy heat shock in this species. Proper tiine window for tetraploid induction was optinii/ed. and the highest induction level was inore than 909^ as measured by How cytometry. At spawning temperature of Ih C. the best starting time for heat shocks was 98 to 1 10 min po.stfertili/ation. Tetraploid embryos had less viability compared with diploids. The highest tetraploid level detected at nauplius stage was .IS'/t. Further work is needed to increase the viability of tetraploid larvae. KEY WORDS: tetraploid. heat shocks, flow cytoinetry. Fenncnipfiuwiis chinensis INTRODUCTION Chinese shrimp Feuiicrnpenacits cliiiieusis is one of the most important aquaculture species in China. Because of its meal quality and cold resistance, the Chinese shrimp is one of the best species for shrimp culture. In recent years, the prevalence of vinjs disease has devastated shrimp culture worldwide. Genetic improvetnent is being used to enhance growth rate or disease resistance in culture fish and shellfish. It was reported that triploid shellfish were useful for aquacul- ture because of their sterility, superior growth and improved tneat quality, and increased disease resistance (Allen et al. 1989. Hand et al. 1998. Quo 1999). In fish, triploids were produced to improve growth (Flajshans et al. 1993, Pandian & Koteeswaran 1998), control reproduction, or reduce contamination for transgenic spe- cies (Devlin & Dotialdson 1992, Pandian & Marian 1994). Since triploid induction was rarely 100% effective, the best way to pro- duce triploids is using telraploids to hybridize with diploids (Arai et al. 1993, Guo et al. 1996). Tetraploid production however, is challenging according to the reports to date because of the low viability of tetraploids. Until now, tetraploid production has been successful only in a few species of fish and shellfish (Guo & Allen 1994, Pandian & Koteeswaran 1998, Yang et al. 2000). Theoret- ically, tetraploid induction can be achieved by inhibiting mitosis of fertilized eggs. Through this method, production of tetraploids has been reported in a few fish species (Thorgaard et al. 1981), but there is no report on successful tetraploid production through in- hibiting mitosis I in shellfish. Tetraploid embryos have been pro- duced in the Pacific oyster by heat shock induced mitosis I inhi- bition, but the larvae did not survive beyond tnetamorphosis (Guo et al. 1994). Tetraploids could be produced by inhibiting the first polar body of the eggs from triploids (Guo & Allen 1994, Eudeline et al. 2000, He et al. 2000) or by inhibiting meiosis I of diploid zygotes (Yang et al. 2000, Zhang et al. 2000). Studies on chro- mosome manipulation for cultured shrimp have progressed slowly. Successful triploid production for shrimp was reported in a few species (Xiang et al. 1998, 2001 ; Li et al. 1999, 2002. 2003; Norris et al. 2001). Because of difficulties with artificial fertilization in shrimp, there is to date no way to induce triploids on large scale. The only way to induce triploids in shrimp is to treat fertilized eggs from one shrimp at a time. The need for tetraploids seetBS tnore 'Corresponding author. Fax: -i- 1-532-289-8578; E-mail: jhxiang(9'ms qdio.ac.cn urgent in shrimp than in other species. To our knowledge, there has been only one report about tetraploid induction in shrimp (Xiang et al. 1993). In this study, tetraploid induction was performed and optimal treatment conditions for tetraploid induction were deter- mined in Chinese shrimp. MATERIALS AND METHODS Source of Gravid Shrimp Gravid shrimp were collected from the wild from the Yellow Sea or from an over-wintered population from a hatchery nearby Qingdao. The gravid shrimp were brought into the aquarium of our institute and put into 4 m' tanks. Twenty individuals were put in each tank, where the seawater temperature was set at 12-13°C. At that time, ovaries of the gravid shrimp were at stage IV. The shrimp were kept at I2-I3°C for 3—4 days to acclimate them to the conditions of our laboratory. Then water temperature was raised gradually (0.5°C/day) to the ptoper spawning temperature (16- 18°C). Meanwhile, the normal light cycle for these tanks was reversed according to the method that was developed in our labo- ratory to make shrimp spawn at daytime (Xiang et al. 1993). Gravid shrimp were fed with polychates and fresh clam meat. Collection of Fertilized Eggs Shrimp with good ovary development that would spawn in 1 or 2 days were put into 300-L tanks with controlled temperature and light cycle. According to their behavioral changes, shrimp that would spawn immediately were taken out and put into 20-L con- tainers. Usually the spawning process for gravid shrimp lasts about 10 min. After the spawning, they were placed in larger tanks to be cultured until re-maturation again. Spawned eggs were collected and concentrated for tetraploid treatment. Treatment of Fertilized Eggs Experiments were designed to compare tetraploid induction level under treatments of different starting time, different intensity, and different treatment duration. Heat-shocks were used to inhibit the first mitosis of fertilized eggs. Proper window for starting treatments was determined according to the tetraploid induction efficiency from a 3^ min heat shock of 33-34°C applied at dif- ferent starting times from 90-1 14 min. Tetraploid induction effi- ciency was determined at embryo stage using flow cytometry then induction efficiency under different treatment intensity, including heat-shock temperature and duration time, was compared. For each treatment, about 700-800 fertilized eccs taken out from the con- 541 542 Ll ET AL. 700 »0 300 ISO 400 4fi0 R.I SOO 2n 60 100 160 TOO 260 300 380 400 4B0 FL1 GOO 2n j 4n ^1 300 ■ 260 - 2n f 200 - 1 IBO 100 ' i 4n W L MBbtMWHUw 260 MO ]eO 400 460 FLt BOO 100 ISO 100 tSO 300 ISO 400 4B0 FL1 SOO Figure 1. Flow cytometry analysis of embryos after heat shock treatment starting at different times after fertilization: (a) 90 min, (bl 96 min, (c) 102 min, (d) 106 min, (e) 108 min, and (f) 114 min. centrated eggs were put into 1000 ml beakers containing about 600 mL hot seawater with the desired temperature. The beakers that were used to treat fertilized eggs were put into a water bath with temperature that stabilizes the treatment condition. When the treat- ment was almost finished, the beakers with fertilized eggs were removed from the water bath and most of the hot water in the beakers was removed. Seawater with natural temperature (15- 120 100 80 60 40 20 0 1 8°C) was added to the beakers to change the water temperature to 1 8-20°C. The treated eggs were then incubated at ~20°C. For each treatment, fertilized eggs without any treatment were used as the control group. Usually for each group of experiments, all fertilized eggs for the treatment were from the same gravid shrimp to ex- clude variation in egg quality. After -24 h of incubation, 70-80 embryos were taken out for ploidy detection using flow cytometry. 120 100 MM ■ 2 min ■ 4 min D6 min 32. 5 33 33. 5 90 92 94 96 98 100 102 104 106 108 110 112 starting time (minutes) Figure 2. Tetraploid level detected at the embryo stage when a 3—1 under different beat-shock temperatures and dilTerent duration of min heat shock of 33-34 C was applied at different starting times. treatment. treatment temperature C C) Figure 3. Comparison of tetraploid level detected at embryo stage Tetraploid Induction in Chinese Shrimp 543 300 - coum »0 b 200 • 160 4n lOO ■ 50 0 *t>^ 4G0FLI 600 460rLI 600 Figure 4. Flow cytometry analysis of embryos from telraphiicl Induction treatment under optimized conditions in Cliinese shrimp Fennerope- naeus chinensis. (a) control and (b) tetraploid embryos. and the remaining embryos were kept until they hatched into nau- plii. about 20-30 of which were used for final ploidy analysis. Ploidy Detection Tetraploidy were detected using flow cytometry. For ploidy detection at embryo stage, 70-SO embryos were put together and triturated in 0.2 niL preparation buffer consisting of 2% citrate acid and 0.5% Tween 20 in distilled water. For nauplius stage. 20-30 larvae from each treatment were triturated in preparation buffer. Tissue debris was removed using nylon screen, and 0.7 mL 2 mg/L DAPl solution was added to stain the nuclei. Embryos or larvae from the control groups were treated in the same way and used as diploid controls. Percentages of triploids and tetraploids in the sample were determined by comparing areas of different peaks. Hatching Success For every treatment, percentages of nauplii hatched in the treated and control groups were recorded to determine the rela- tionship between tetraploid levels and hatching levels. Statistical Analysis To compare the effects of different factors such as starting time, treatment intensity, and duration on tetraploid induction. F-test was used to analyze the effect of different factors on the efficiency of tetraploid induction. RESULTS (2-6 min) were tested and compared (Fig. 3). Tetraploid frequency detected at embryo stage rose apparently with extension of treat- ment duration from 2 to 6 min at 32-33. 5°C. There was no sig- nificant difference in tetraploid levels between different treatment temperatures for the same treatment duration in the range of 32- 33.5°C. The data indicated that 32-33.5°C temperature could ef- fectively inhibit the first mitosis of fertilized eggs when treatment duration was 4-6 min. Tetraploid level detected under 34°C was much higher than that at 32-33. 5°C when the treatment lasted for 4 min. There was no difference in tetraploid level between 4 and 6 min treatment at 34°C. It showed that at certain range of treat- ment temperature, proper duration of the treatment was a key factor for tetraploid induction. Evaluation of Effects of Different Factors on Tetraploid Level Starting time, treatment duration and treatment temperature are major factors affecting tetraploid induction. Totally, 5 levels of starting time (79, 81, 85, 91, 96, 100 min), 3 levels of treatment duration (2, 4, 6 min), and 5 levels of treatment temperature (32, 32.5, 33, 33.5, 34°C) were tested. F-test showed that tetraploid level detected at different starting time for the treatment among different treatments and treatment duration (2, 4, 6 min) had sig- nificantly different effects among groups; and that different treat- ment temperature had no significant effects. At low treatment tem- peratures, longer treatment duration increased tetraploid induction Effect of Starting Time on Tetraploid Inducing Rate To determine the optimal window for tetraploid induction, dif- ferent starting times for treatment were tested. Flow cytometry analysis of embryos from treatment starting at different times is shown in Figure 1. Usually two peaks, diploid (2n) and tetraploid (4n or G2 phase of 2n) peaks, were present in each sample. With changes in starting time, the relative 4n area changed greatly. After numerical repeats for each treatment, the optimal starting time for tetraploid induction was determined based on data in Figure 2. The proper starting time for tetraploid induction was 98-1 10 min under a spawning temperature of 16'C. When starting time was at 1 12 min, tetraploid level dropped sharply. The window for tetraploid induction was only about 10 min. Out of this range, the treatment could not effectively inhibit mitosis I. Effect of Different Treatment Duration and Different Temperature on Tetraploid Rate Using the optimized time window for the treatments, different treatment intensities (32-34°C) for different treatiTient duration 0{control) 70-100 30-40 41-50 51-60 tetraploid rate (%) Figure 5. Relationship between tetraploid level and hatching success in Chinese shrimp Fenneropenaeus chinensis. 544 Li et al. efficiency. When the spawning temperature of gravid shrimp was 16''C, the proper starting time for treatment should be 102-1 10 min after fertilization. If the spawning temperature was lower or higher, then the starting time for treatment should be later or earlier. After the induction condition was optimized, tetraploid level detected at embryo stage reached almost 100% (Fig. 4). Relationship Between Tetraploid Rate at Embryo Stages and Hatching Rate Higher treatment temperatures led to more tetraploids. they also led to reduced survival of the treated embryos. There was strong negative correlation between tetraploid induction efficiency and larval survival (Fig. 5). Production of Tetraploid Larvae Although tetraploid levels detected at embryo stages were high, tetraploid embryos experienced problems in hatching. The hatch- ing success rate of tetraploids was low. In our experiments, the highest tetraploidy rate detected at nauplius stage was about 38% (Fig, 6a) while tetraploid levels detected at embryo stage was 55% (Fig, 6b), This result was obtained under spawning temperature of 15.7°C. with a 3-min heat shock at 34°C starting 110 min after fertilization. During the hatching process of tetraploid embryos, some live embryos in membrane were observed, but their mor- phology was abnormal. And when the.se abnormal embryos were selected for detection of ploidy, it was found that most of these embryos were tetraploids (data not shown). DISCUSSION Available data showed that the Chinese shrimp Fennerope- naens chinensis. tetraploids could be produced by inhibiting the first mitosis. Reported methods for producing tetraploids in aquatic animals include inhibiting first mitosis (Thorgaard et al. 1981, Varadi et al. 1999), inhibiting the first meiosis of diploid fertilized eggs (Yang et al. 2000, Zhang et al. 2000), or inhibiting polar body I in eggs from triploids (Guo & Allen. 1994, He et al. 2000). In shellfish, there is no successful production of viable tetraploids by inhibiting mitosis I (Guo et al. 1994), although tetraploid embryos can be produced. In our experiments on shrimp, no tetraploids were produced through inhibiting meiosis. This study showed that tetraploid embryos could be produced at high rate in shrimp. How to make more embryos hatch into nauplii, however, remains a problem that must be solved. The challenge is to improve the treatment conditions so that they lead to high-level production of tetraploids without causing serious damage to treated embryos. It is also possible that tetraploid embryos have limited viability or ability to hatch, and they can be obtained by improving hatching conditions. During tetraploid induction, the exact time of thermal or pressure shock applied to inhibit mitosis I is important. Inhib- iting different processes will lead to different viability according to an analysis of tetraploid induction in fish (Pandian & Koteeswaran 1998). This study showed that heat-shock is effective in inhibiting mitosis I in the Chinese shrimp Fenneropenaeus cliinensis. This shrimp is a temperate species, so it is more sensitive to heat than tropical species. Heat shock has an advantage over chemical treat- ments, in that there is no pollution to the environment. For sub- tropical or tropical species, cold shocks may be more helpful. To our knowledge. Fenneropenaeus chinensis is the only shrimp spe- cies where tetraploid induction has been reported. Until now, there is only one report that tetraploid was produced in this species (Xiang et al. 1993). In earlier reports in this study, tetraploids were induced by cytochalasin B (CB) and ploidy was detected through chromosome counting. In this work, heat shock was used and optimal treatment was identified. The use of flow cytometry as a method for detecting tetraploidy was a key factor in our successful evaluation of heat shock treatment. Compared with chromosome counting, flow cytometry analysis allows rapid and accurate ploidy determination of many experiment groups. The application of flow cytometry techniques has greatly advanced polyploidy research in shrimp (Zhou et al. 1999). Although heat-shocks can effectively inhibit mitosis 1 in shrimp, optimal conditions for the hatching of fertilized eggs need further investigation. Extending treatment duration might increase the tetraploid rate, but reduce hatching success. The proper strat- egy to induce triploids should be to achieve certain high levels of tetraploid and hatching rates. Hatching rates varied from brooder to brooder when the tetraploid rate was the same because of dif- ferent egg quality. There is the common tendency however, that hatching success decreases when tetraploid level increases. In Fen- neropenaeus chinensis. 40-60% tetraploid rate is preferred to ob- tain viable larvae. Although no viable tetraploid post-larvae were obtained, this study showed that high percentages of tetraploids could be produced by heat shock. The optimization of heat shock treatments is an important first step in successful tetraploid induc- tion. Further work is needed to improve the survival of tetraploids so that viable tetraploid shrimp can be eventually obtained. ACKNOWLEDGMENTS The authors thank Dr. Ximing Guo from Rutgers University, USA for his kind instructive comments and revision of this manu- script and Dr. Xiaolin Liu for his help in statistical analysis of data. 2n ^^ifmnt/^t^tu^ti^ t*^ t ^ s 2n 4n iiMi|Uiiiini!iis anguilticmuiauis. Aquacullure 1 17:227-235. Devlin. R. H. & E. M. Donaldson. 1992. Containment of genetically altered fish with emphasis on salmonids. In: C. L. Hew &. G. L. Fletcher, eds. Transgenic Fish. Singapore: World Scientific Publishing, pp. 229-26.5. Eudeline. B., S. K. Allen Jr. & X. Guo. 2000. Optimization of tetraploid induction in Pacific oyster, Crassostrea gigas. using first polar body as a natural indicator. Aquaculture 187:73-84. Flajshans, M., O. Linhart & P. Kvasnicka. 1993. Genetic studies of tench {Tinea tinea L.): induced triploidy and tetraploidy and first perfor- mance data. Ac/iiaeiilmre 1 13:301-312. Guo, X. 1999. Superior growth as a general feature of triploid shellfish: evidence and possible causes. / Shelljish Res. 18:266-267. Guo. X. & S. K. Allen, Jr. 1994. Viable tetraploids in the Pacific oyster. Crassostrea gigas (Thunberg), produced by inhibiting polar body I in eggs from triploids. Mot. Mar. Biol. Biotechnol. 3:42-50. Guo, X., G. A. DeBrosse & S. K. Allen. Jr. 1996. All-triploid Pacific oyster {Crassostrea gigas Thunberg) produced by mating tetraploids and dip- loids. Aqiiaeulttire 142:149-161. Guo. X.. W. K. Hershberger. K. Cooper & K. K. Chew. 1994. Tetraploid induction with mitosis 1 inhibinon and cell fusion in the Pacific oyster {Crassostrea gigas. Thunberg). / Shellfish Res. 13:193-198. Hand. R. E.. J. A. Nell, I. R. Smith & G. B. Maguire. 1998. Studies on triploid oyster in Australia, XI: Survival of diploid and triploid Sydney rock oyster Saeeost?-ea commereialis (Iredale and Roughley) through outbreaks of winter mortality caused by Mikroeytos roiighlevi infesta- tion. J. Shellfi.sh Res. 17:1 129-1 135. He, M, X., Y. G. Lin, Q. Shen. J. X. Hu & W. G. Jiang. 2000. Production of tetraploid pearl oyster {Pinetada martensii Dunker) by inhibiting the first polar body in eggs from triploids. J. Shellfish Res. 19:147-151. Li, F. H., J. H. Xiang. L. H. Zhou. C. G. Wu & X. J. Zhang. 2003. Optimization of triploid induction by heat shock in Chinese shrimp Fenneropenaeus ehinensis. Aquaculture 219:221-231. Li. F. H.. X. J. Zhang. L. H. Zhou. C. S. Zhang. C. G. Wu & J.H. Xiang, 2002. Reproductive performance of triploid shrimp Fenneropenaeus ihincnsis, [Abstracts]. Presented at World Aquaculture 2002, Beijing. People's Republic of China, April 23-27, 2002. Li. Fuhua, L. H. Zhou. J. H. Xiang, X. D. Liu & J. Z. Zhu, 1999. Triploidy induction with heatshocks to Penaeus ehinensis and their effects on gonad development. Chin. J. Oeeanol. Lininol. 17:57-61. Norris, B.. F. Coman & N. Preston. 2001. Effects of triploidy on growth and survival of the Kuruma shrimp Penaeus japonicus. Aquaculture 2001: Book of Abstracts. World Aquaculture Society. 143. Baton Rouge. LA: J. M. Parker Coliseum. Louisiana State University. 478 pp. Pandian. T. J. & R. Koteeswaran. 1998. Ploidy induction and sex control in fish. Hydrohiologia 384:167-243. Pandian. T. J. & L. A. Marian. 1994. Problems and prospect of transgenic fish production. Curr. Sci. 66:635-649. Thorgaard. G. H.. M. E. Jazwin & A. R. Stier. 1981. Polyploidy induced by heat shock in rainbow trout. Trans. Am. Fish. Soc. 1 10:546-550. Varadi, L., L Benko, J. Varga & L. Horvath. 1999. Induction of diploid gynogenesis using interspecific sperm and production of tetraploids in African catfish. Clarias gariepinus Burchell ( 1822). Aquaculture 173: 401-411. Xiang. J.. L. Zhou. F. Li & X. Zhang. 2001. Triploidy induction in pe- naeidae shrimp with special reference to a new chemical inducing reagent 6-DMAP (6-dimethylaminopurine). Aquaculture 2001: Book of Abstracts. World Aquaculture Society, 143. Baton Rouge. LA: J.M. Parker Coliseum Louisiana State University. 705 pp. Xiang. J. H., L. H. Zhou & F. H. Li. 1998. Reproductive and genetic manipulation in the Chinese shrimp. Penaeus ehinensis (OSBECK, 1765). Proceedings of the Fourth International Crustacean Congress. Koninklijke Brill NV. Laiden. The Netherlands, pp. 987-995. Xiang. J. H.. L. H. Zhou, R. Y. Liu. J. Z. Zhu. F. H. Li & X. D. Liu. 1993. Induction of the tetraploids of the Chinese shrimp Penaeus ehinensis. In: C. You & Z. L. Chen. ed. Biotechnology in Agriculture. Kluwer China Science and Technology Press, Beijing, China, pp. 841-846. Yang. H. P.. F. S. Zhang & X. Guo. 2000. Triploid and tetraploid Zhikong Scallop, Chlamys farreri Jones et Preston, produced by inhibiting polar body I. Mar Biotechnol. 2:466-475. Zhang. G.. Z. Wang. Y. Chang, et al. 2000. Tetraploid induction in the Pacific abalone Haliolis discus hannui Ino with 6-DMAP and CB. J. Shellfish Res. 19:540-541. Zhou. L. H.. T Deng. X. J. Zhang. K. J. Yu & J. H. Xiang. 1999. Detection of ploidy in shrimp by flow cytometry. Mar. Sci. 2:42^5. Jtnimal of Slwlljhh Kesearch. Vol. 22. No. 1. 547-553, 2003. SELECTION AND USE OF DIFFERENT DIETS IN A STUDY ON CHINESE SHRIMP, FENNEROPENAEUS CHINENSIS GUOQIANG HUANG, SHUANGLIN DONG,* FANG WANG, AND SHEN MA Murkuhure research laboratory. Fisheries College. Ocean University of China. Qingdao. 266003. People's Republic of China ABSTRACT A 30-day feeding e,\periment was conducted to investigate the dietary selectivity in Chinese shiinip. Fenneropenaeus chinensis. Si.x groups of shrimp with initial body weight of 1.530 ± 0.047 g (mean ± SD. n = 6) were used, in which the first five groups were fed to satiation with single diets of FF, llesh of fish (Sardinella zunasi); SF, tlesh of shrimp (Trachypenaeus curvirostris); CF. foot of clam (Ruditapes vangata); PW, polychaette worm (Neanthes japonica) FD. a commercial formulated diet; and the last group received MD. mi.xed diet. The feeding tfials were conducted simultaneously and shrimp were fed to satiation. The specific growth rate (SGR), food intake (FI), food conversion efficiency (FCE), and apparent dige.stive ratio (ADR) were determined. The results showed thai specific growth rates of dry weight, protein, and energy (SGR^. SGR^. and SGR,.) were highest in the MD fed group, food conversion efficiencies (FCEj, FCEp. and FCR,.) were highest at PW fed group. Food ingestion in terms of dry weight, protein, and energy were significantly higher in CF and MD fed groups than others. The highest ADR was observed in CF fed group. In mixed diet feeding group, percentages of the five ingested diets to the total ingested amount based on dry material, protein, and energy were: FF. 13.07%; SF, 9.60%: CF. 46.45%; PW, 30.88%; and FD. 0%; FF. 15.56%; SF, 11.44%; CF. 45.09%: PW. 27.91%; and FD. 0%; FF. 13.66%; SF, 10.48%; CF, 44.00%; PW, 31,86%; and FD, 0%; respectively. This indicates that Chinese shrimp possess the ability to discriminate different diets. The optimal foraging strategy of Chinese shrimp in this experiment was to gain energy as much as possible to meet energy needs of variable physiologic activities under the premise of maximizing growth. Additionally, the protein sparing effect of dietary E/P ratio and lipid content was also observed in this experiment. KEY WORDS: dietary selectivity. Chinese shrimp. Fenneropenaeus chinensis INTRODUCTION Because of their economic significance to fisheries and impor- tant function in aquatic ecosystems, several studies were con- ducted to investigate feeding habits of shrimp and crab. The most commonly used direct method was to analyze the stomach content or foregut of the animal, from the wild or under culture condition, and evaluate its feeding habits from the composition (Chong & Sasekumar 1981. Phil & Rosenberg 1984, Cockcroft & Mclachlan 1986, Prakash & Agarwal 1989, Nunes et al. 1997, Roy & Singh 1997, Kulkami et al. 1999. Minami 2000, Schwambom & Griales 2000). The activity of different digestive enzymes in the animals was also used to judge their feeding habits (Biesiot & Capuzzo, 1990). In recent years, stable isotope analysis method was also applied in analyzing of the feeding habits of the animals (Newell et al. 1995. Nunes et al. 1997. Schwambom & Griales 2000). Ivlev (1961 ) proposed a selection index to describe the dietary selectiv- ity of fish. Pinn et al. ( 1998) used Strauss" Linear Selection Index to describe in their study the dietary selectivity of two mud-shrimp. Nevertheless, the selectivity of a diet item is affected by such factors as energy content, difficulty of foraging and handling, and so on (Sunaga 1971, Griffiths 1975, Manghagen & Wiederhohn 1982. Mikheev 1984, Buskey et al. 1991, Alam et al. 1996, Meh- ner et al. 1998). The theory of optimal foraging is based on the evolutionary premise that individuals within a population that for- age most efficiently and maximize their net rate of energy intake will possess greater fitness and contribute more genes to future generations (Calow & Townsend 1981 ). It has been found that the dietary selectivity of animals is partly or completely subjected to the law (Kislalioglu & Gibson 1976. Elner & Hughes 1978). The dietary condition of shrimp is variable in wild and exten- sive or semi-intensive cultural waters, and the abundance and com- position of diet vary greatly in different waters and time periods *Corresponding author. E-mail: dongsKs'mail.ouc.edu.cn (Marte 1980, Luna-Marte 1982). In most cases, abundance of its preferred diet is likely to decrease to a low level because of the natural fluctuation or high feeding pressure. Hence, the shrimp cannot select the diet species in accordance with its actual prefer- ence. It is probable that shrimp might ingest the diet species that is not preferred, to release the pressure of starvation or innutrition and satisfy its growth or development. Therefore the methods men- tioned previously, to analyze its feeding habit, are quite difficult to reflect or define its dietary selection or preference. Quantitative study of the preference of some diet items of animals can be really conducted in only controlled conditions when different diet items (include items differing in nutritional composition, origin, size, and so on) are provided. In the wild environment, crustaceans, polychaette worms, and juvenile bivalves are major diet items of Chinese shrimp (Fen- neropenaeus chinensis) (Wang, 1997). In this study Chinese shrimp, widely cultured and distributes in China, were used and five diet items were provided equally in excessive amounts to study the feeding preference of the shrimp and the strategy of its diet selectivity. MATERIALS AND METHODS Rearing Conditions Chinese shrimp were kept in glass aquaria (45 x 30 x 30 cm^. water volume of 35 dm'), and each rearing unit was stocked with four shrimp. The room temperature was controlled by an air con- ditioner, and water temperature was 25 ± 0.5°C. Aeration was provided continuously and 0.50-0.67 of water was exchanged ev- ery other day. Seawater used in the experiment was filtered by composite sand filter. During the experiment, dissolved oxygen of water was maintained above 5.5 mg/L, pH was about 8.0, the water salinity was between 30-33%c, photoperiod of 14 h of light: 10 h of darkness was used. 547 548 Huang et al. Diets Preparation The five diets used in the experiment were: fish flesh (FF) — the flesh of sardine iScinlinella ziinasi) without head, scales, fins, bow- els, and bones: shrimp flesh (SF) — small shrimp (Trachypenaeus cur\nrostris) without head and shell; clam foot (CF) — from {Rii- ditapes varigata): PW, polychaette worm — Neanthes japonica: FD formulated diet — a commercial sold shrimp diet (Sea-Horse Brand. Fujian Mawei Unite Feed Ltd. China) comprised of bean powder, fish powder, shrimp powder, compound vitamins, and compound minerals; MD, mixed diet — equal combination of the five diets. Shrimp were fed diets to satiation. Each diet item was cut into almost the same size as the formulated diet (about 4 mm in length and diameter of 2 mm) before feeding. Biochemical composition of the diets is listed in Table 1. Source and Acclimation of Shrimp The experiment was carried out at the Mariculture Research Laboratory, Ocean University of Qingdao, People's Republic of China. The shrimp used in the experiment were collected from the Tianheng Shrimp Farm, Qingdao. Prior to the experiment, the shrimp were transferred into aquaria and underwent a 6-day accli- matization period during which they were fed with formulated diet (FD) at satiation level twice a day (at about 6:00 and 18:00). Experiment Design After 24 h starvation. ^6 shrimp with an initial wet weight of 1.530 ± 0.047 g (mean ± SD) were selected from acclimated ani- mals and placed in 24 aquaria to form 6 experimental groups fed with different diets of FF, SF, CF, PW. FD, and MD. A complete randomized block design was used to arrange the 24 aquaria of 6 groups. Sample Collection and Analysis Three groups (eight shrimp each) were sampled from the ac- climated shrimp simultaneously while experimental shrimp were selected to determine the initial body composition of the experi- mental shrimp. After the 30-d experiment, the shrimp of all groups were starved for 24 h and then sampled. The shrimp from the individual aquaria were pooled as a sample and there were 24 samples of final shrimp. During the course of the experiment the daily food supply was recorded and uneaten food was collected 3 h after feeding. Feces were collected promptly. Shrimp and food were weighed to the nearest 0.001 g using an electronic balance after carefully blotted with paper towel to remove excess moisture. After the weight was obtained all samples of shrimp, feces, and food were dried in an oven at 70°C to constant weight, homog- enized with a glass mortar, and stored at -20°C. Before chemical compositions were analyzed, the samples were re-dried at 70"C to constant weight. The N content was measured using the Micro-Kjeldahl meth- ods and the crud protein content was calculated by multiplying Kjeldahl N content by 6.25(AOAC, 1984). Crude lipid was deter- mined by the Soxthlet method (AOAC, 1984). ash was determined by combusting dry samples in a muffle furnace at 550°C for 1 2 h (AOAC. 1984). and the gross energy content of dry samples was determined by PARR 1281 calorimeter (PARR Instrument Com- pany. USA). An analysis of each sample was conducted in tripli- cate (three sub-samples for each sample). Calculation of Data Specific growth rate (SGR), food ingestion (FD. apparent di- gestive ratio (ADR), food conversion efficiency (FCE), and Ivlev's index of dietary selectivity (I,) were calculated as follows: SGRw(%/day) = 100(In W-In W„)/T (Ricker 1979) Fl^C^r body weight/day) = lOOC/IT (W, + W„)/2] (Wu et al. 2()()()) ADR (%) = 100(C-F)/C (Smith 1971) FCEw{ •7r) = 100(W,-W„)/C (Matty & Smith 1978) Where W, and W„ were the finial and initial wet weight of the shrimp. T was the duration of growth period in days. F was the dry weight of feces, and C was the dry weight of consumed food. SGR. Fl. ADR. and FCE in terms of dry matter (SGRj, FIj, ADRj, and FCE^). protein (SGRp. FIp, ADRp, and FCEp), and energy content (SGR^, FI^, ADR^. and FCE^) were calculated similariy. I, = (r,-p,)Ar, + p,) (Ivlev 1961) Where r, was the portion of one diet in the total ingested diet, and TABLE 1. Biochemical composition and energy content of experimental diets (Mean ± SE).' Diets Composition FF SF CF PW FD MD" Moisture (%) 77.23 ± 0.38 80.33 ± 1.27 79.35 ±3.15 74.18 ±0.80 7.70 + 0.15 76.57 + 0.10 Protein (%) 83.98+1.12 84.13 ±0.65 68.49 ± 0.59 63.73 + 0.44 42.57 ± 0.50 71.I4±0.18 Lipid (%) 5.18 ±0.01 5.00 ± 0.01 5.96 ±0.01 16.32 ±0.03 9.93 ± 0.02 8.96 ± 0.02 Ash (%) 6.41 ± 0.02 3.2 ±0.01 5.38 ±0.02 6.89 ± 0.02 10.75 + 0.03 5.77 ± 0.02 Energy 22.15 ±0.24 22.95 ± 0.04 19.89 ±0.05 21.66 + 0.09 19.23 ±0.09 21.02 ±0.03 E/P 26.38 ±0.14 27.28 ±0.1 7 29.17 + 0.20 33.99 ±0.17 45.20 ± 0.70 29.54 ±0.11 L/P 0.061 ±0.001 0.059 ±0.001 0.087 ±0.001 0.256 ± 0.002 0.233 ± 0.003 0.126 ±0.001 ' Moisture is percentage content of wet sample, moisture = 100 x (WW - DS)AVW. WW: wet weight. DW: dry weight: Protein. Lipid and Ash are percentage content of dry sample; Unit for energy content is KJ.g"' in dry sample; E/P: energy/protein ratio, unit for E/P is KJ.g''; L/P: Lipid/protem ratio, unit for E/P is g.g"'. •^ Composition of mixed diet was calculated after the experiment basing on the ingested dry weight of the first five diets, it is a weighted value according to the portions of every diet in the total ingested weight in mixed diet fed group. Selective Diets of Fenneropenaeus Chinensis 549 Pj was the portion of one diet in tiie total provided diet r, and p, were calculated in terms of dry matter. Statistical Analysis Statistics were performed using SPSS 10.0 statistical software with possible differences among diet treatment being tested by one-way ANOVA. Tukey's-b multiple range tests was used to test differences between treatment groups. Differences were consid- ered significant at a probability level of 0.05. RESULTS Food Consumption and Feces Table 2 lists the food consumption and feces for the six diet treatments. Shrimp fed with CF and MD consumed significantly more food than the others did. The largest amount of feces in terms of dry matter emerged in MD but it was not significantly larger than FD. However, the largest amount of feces was observed in FD in terms of protein and energy. Growth At the end of the experiment no significant difference existed among CF-, PW-, and MD-fed shrimp in terms of WW. DW, P, and E, and all were significantly higher (df = 5, P < 0.05) than the other three groups (Table 3). FF and SF were the lowest of the six groups in all terms of WW, DW. P. and E. and no significant difference existed between them (Table 3). No signitlcant difference was observed among CF. PW and MD in SGR,,. SGRj. SGR^, and SGR^. respectively, and all were significantly higher than the other three groups. Except for the high SGR„ (2.76 ± 0.06) observed in PW, the highest SGRj (2.99 ± 0.07), SGRp (2.91 ± 0.07), and SGR^. (3.15 ± 0.07) all appeared in MD { Fig. 1 ). FD was significantly higher than SF and FF in SGRj, SGRp, and SGR^. Every parameter of FF and SF was lower than other groups (Fig. I ). Food Conversion Efficiencies Figure 2 illustrates that the FCE in terms of DW, P, and E. PW was significantly higher than other groups in FCEj, FCEp, and FCE,, and it had the highest values of 22.86 ± 1.63, 22.87 ± 1.65, and 21.39 ± 1.49, respectively. FD was significantly higher than other groups except PW in FCEp, and it was not significantly different from CF and MD though it was significantly higher than FF and SM had the lowest FCE (only about 20-25% of PW). MD had significantly lower FCE than PW when four diets were ingested in different portions. Food Ingestion of Six Diet Treatments Fl„, FI(j, FIp, and Fl^ in CF and MD fed groups were 5.58 ± 0.24. 22.89 ± 0.84, 23.62 ± 0.87, 23.59 ± 0.86, and 5.94 ±0.14, 23.28 ± 0.45, 24.97 + 0..50, 24.98 ± 0.48, respectively (Fig. 3). CF and MD were significantly higher than other groups in Fl for all the four terms, and no significant difference between CF and MD was observed (Fig. 3). FI in FD was significantly lower than the others because of its lowest protein content. FI of FF and SF fed groups were the lowest in all measures except protein (Fig. 3). Percent Composition of Ingested Diets and Indexes of Selectivity of Five Provided Diets in MD When five diets were provided simultaneously and in excess, Chinese shrimp ingested four diets of FF, SF, CF, and PW, and no FD was ingested (Fig. 4). Percentages of dry weight and energy consumed by shrimp of FF and SF fed groups were not signifi- cantly different from each other, but the percentage of protein consumed in SF was significantly lower than FF. Among the four ingested diets, CF had significantly higher percentage of dry weight (46.45 ± 1.63). protein (45.09 ± 1.49) and energy (44.00 ± 1.60) than the other three. Percentages of dry weight, protein, and energy of consumed PW were 30.88 ± 2.06, 27.91 ± 1.93, and 31.86 ± 2.08, respectively, and significantly higher than FF and SF. Indexes of selectivity of five provided diets (based on dry mat- ter) in MD treatment were FF, -0.210 ± 0.017: SF, -0.352 ± 0.016; CF, 0.397 ± 0.016; PW, 0.210 ± 0.030; and FD, -I ±0 respec- tively. It indicated that Chinese shrimp performed positive selec- tivity on CF and PW. Negative selectivity on FF and SF was observed, and FD was excluded under experimental conditions. Apparent Digestive Ratio of Diets The highest ADR in terms of dry weight, protein, and energy was in CF (92.97 ± 0.35, 98.05 ± 0.10, and 99.10 ± 0.04, respec- tively). ADR,, ADRp, and ADR, of FD were 77.97 ± 1.92, 86,15 ± 2.06, and 91.10 ± 1.55, respectively, which are significantly lower than other groups (Fig. 5). DISCUSSION Feeding behaviors of shrimp and crab have been studied by methods of analyzing stomach contents or foregut, activity of di- gestive enzymes, and by stable isotope technique (Chong & Sase- TABLE 2. The dry weight (g, DW), energy (KJ, E), and protein (g, P) content of the food consumed and feces for the six diet treatments. Treatments (mean ± SE) FF SF CF PW FD MD Food consumption DW 1.383 + 0.0-18'' 1.378 ±0.163" 3.897 ±0.185" 2.215 ±0.074" 2.020 ±0.1 74" 4.3.M ± o.oes-" P 1.183 ±0.033" 1. 169 ±0.138" 2.681 ±0.127' 1.425 ±0.048" 0.868 ± 0.075" 3.084 ± 0.048'' E 30.352 ± 0.838" 31.610 ±3.729" 77.509 ±3.67f 47.990 ± 1.610" 38.841 ± 3.342" 91.014 ± 1.295" Feces DW O.I 23 ±0.020" 0.234 ±0.018" 0.275 ± 0.023" 0.307 ± 0.030" 0.4-30 ± 0.03 r 0.436 ±0.018' P 0.066 ±0.001' 0.041 ±0.001" 0.052 ±0.001" 0.074 ±0.001'' O.I 14 ±0.003' 0.090 ±0.001'= E 0.473 ± 0.078" 0.742 ± 0.057" 0.704 ± 0.059" 1.350 ±0.133" 3.336 ± 0.243" 2.040 ± 0.084' Values with different letters in the same line were significantly different (df = 5, P < 0.05) from each other. 550 Huang et al. TABLE i. Initial and final shrimp wet weight (g. WW), dry weight (g, DW), protein Ig. P) cuntent and energy (KJ. E) for the six diet treatments. Treatments (mean ±SE) FF SF CF PW FD MD Initial shrimp WW 1.536 ±0.010 1.520 ±0.040 1 .525 ± 0.030 1 .497 ± 0.026 1.519 ±0.021 1.535 ±0.008 DW 0.360 ± 0.002 0.356 ± 0.009 0.357 ± 0.007 0.351 ±0.006 0.356 ± 0.005 0.360 ± 0.002 P 0.243 ± 0.002 0.240 ± 0.006 0.241 ±0.005 0.237 ± 0.004 0.240 ± 0.003 0.243 ±0.001 E 6.802 ± 0.048 6.722 ±0.1 70 6.755 ±0.1 34 6.627 ±0.1 17 6.726 ± 0.090 6.802 ± 0.036 Final shrimp WW 2.057 ±0.108" 2.204 + 0.151- 3.133 ±0.152'' 3.418 ±0.139" 2.371 ±0.136" 3.335 ± 0.096" DW 0.423 ± 0.016" 0.448 ± 0.040" 0.780 ± 0.050' 0.855 ± 0.044'- 0.591 ±0.045" 0.882 ±0.014' P 0.288 ±0.011" 0.292 ± 0.026" 0.517 ±0.033' 0.561 ±0.029' 0.378 ± 0.029" 0.581 ±0.009' E 7.753 ±0.305" 7.777 ±0.687" 15.193 ±0.981' 16.864 ±0.862' 11.229 ±0.862" 17.501 ±0.286' Values with different letters in the same line were significantly different from each other (df = 5. P < 0.05 ). kumar 1981. Phil & Rosenberg 1984. Cockcroft & Mclachlan 1986, Prakash & Agarwal 1989. Biesiot & Capuzzo 1990. Newell et al. 1995, Nunes et al. 1997, Roy & Singh 1997, Minami 2000. Schwamborn & Griales 2000). Because of the variation of food abundance and composition in natural and extensive or semi- extensive cultural water, these results cannot reflect the real food preference of the animals to some dietary organisms. Although Finn et al. (1998) analyzed the selectivity on dietary organisms in two mud crabs by utilizing the Strauss" Linear Index, it was af- fected by such factors as diet density, difficulty of searching, dif- ficulty of handling, and soon (Sunaga 1971, Griffiths 1975. Mang- hagen & Wiederholm 1982, Mikheev 1984, Buskey et al. 1991. Alam et al. 1996, Mehner et al. 1998). The dietary selectivity of Chinese shrimp on five diets with same availability was observed in these studies, and indicates that the Chinese shrimp possess the ability to discriminate different diets. The shrimp selected CF and PW. but ingested almost no FD (Fig. 4). The dietary selectivity of animals are affected by many factors such as dietary energy content, abundance, and difficulty of searching, handling, digesting, and so on. In foraging, animals gain energy by ingesting prey (food) and expand energy in searching and handling prey (food). Composition of diets of some fish ac- corded with the prediction by the theory of optimal foraging in many experimental studies (Kislalioglu & Gibson, 1976; Tytler & Calow, 1984). Feeding behaviors of some crabs on snails and bi- valves also agreed with the Optimal Foraging Theory on the whole (Finer & Hughes 1978. Boynton 1979. Hughes & Finer 1979. Kennedy et al. 1983, Lawton & Hughes 1985. Seed & Hughes 1997). Diets with almost the same encounter probability, size, and difficulty of handling were provided in this study. It was observed that the Chinese shrimp did not exhibit behaviors that maximized the net energy gain in the selection of diets under this experimental condition, and a great deal of CF. which was lower in energy content, was ingested. The correlation coefficient between in- gested percent quantity and per unit energy content of diet was only 0.131. which indicated that no significant correlation existed (/? = 20. P = 0.589). Poor relativity between Ivlev's index of selectivity and per unit energy content of diet was also observed (R = 0.207. /I = 20. P = 0.380). In this experiment, every diet provided to the shrimp was in excess but equally divided. Major factors that affect the dietary selectivity of shrimp are diet nutrition, difficulty of digesting, ufi- lizing rate, attraction to diets, and so on. Shrimp of group MD showed distinct selectivity of different natural diets provided in the experiment. ADR of four natural diets were significantly higher than FD. The highest portion (based on dry weight) of 46.45% was observed in CF (Fig. 4), and the portion of PW (30.88%) was also comparatively higher than those of FF (13.07%) and SF (9.59%). It could be concluded from the results the shrimp preferred diets that met their high growth requirement (such as CF and PW), then they selected diets based on the digestibility (indicated by ADR), namely, they ingested more CF, which had relative higher ADR, Figure 1. Special Growth Rati (SdRl of all diet treatments (Different letters above the bars denote significant differences (P < 0.05) among columns in the same cluster). Where FF = Fish Flesh, SF = Shrimp Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = Formulated Diet, and MD = Mixed Diet: S(;R„, SGR,,, StiRp, and SGR.. are Spe- cial Growth Rates in terms of wet weight, dry weight, protein, and energy of shrimp body respectively; Verical bar = SE (n = 4). Figure 2. Food Conversion Efficiencies (FCE) in diet treatments (Dif- ferent letters above the bars denote significant differences (P < 0.05) among columns in the same cluster). Where FF = Fish Flesh, SF = Shrimp Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = For- mulated Diet, and MD = Mixed Diet: FCE^. FCEp, and FCEe are Food Conversion Efficiencies in terms of dry matter, protein, and energy respectively; Vertical bar = SE (n = 4). Selective Diets of Fenneropenaeus Chinensis 551 IFF @SF ICF IPW ^FD Figure 3. Food Ingestion (FI) of all groups (Different letters above the bars denote significant differences (P < 0.05) among columns in the same cluster I. Where FF = Fish Flesh. SF = Shrimp Flesh. CF = Clam Foot, PW = Polychaette Worm, FI) = Formulated Diet, and MI) = Mixed Diet; FI„, FIj. Fl^, and FI^. are Food Ingestion in terms of wet weight, dry matter, protein, and energy respectively; Vertical bar = SE (n = 4). than PW. The optimal foraging strategy of Chinese shrimp in this study was to gain as much energy as possible to meet the needs of variable physiologic activities, under the premise of ensuring fast growth, not to select diets to gain the highest FCE. In Group MD. the shrimp ingested a large amount of CF. which was easily di- gested (Fig. 5). so that they were able to ingest more diet continu- ously during the period to ma.ximize the dietary energy ingestion. More studies on the effect of feeding attractants of these diets on dietary selectivity are still needed. It was found in a few of studies that the decisive factors af- fecting the utilization, expressed in protein efficiency ratio (PER) and food conversion efficiency (FCE), of diets, were other ingre- dients when dietary protein content was above a reasonable level. These phenomena occurred in fish (Degani & Viola 1987, Viola & Lahav 1991. Erfanullah & Jafri 1995. Company et al. 1999. Morais et al. 2001, Shalaby et al. 2001. Das 1991). and also existed in shrimp and crab (Andrews et al. 1972. Colvin 1976. Sedgwick 1979. Xu & Li 1988). Xu and Li (1988) found, a protein sparing effect of lipid, in a study on the optimal protein, carbohydrate, fibrin, and lipid contents for Chinese shrimp diet that the increase of lipid content significantly promotes PER at all of the three protein contents of 36%, 40%, and 44%. Dietary protein contents of all the diets provided in this study exceeded 40% (Table 1) and could satisfy the demands for protein of Chinese shrimp. The IFF ^SF iCF IPW ■FD(a) Dry Weight Frotein hri.MRv Figure 4. Percent compostion of ingested diets in mixed diet fed group (Different letters above the bars denote significant differences (P < 0.05) among columns in the same cluster). Where FF = Fish Flesh, .SF = Shrimp Flesh, CF = Clam Foot, PW = Polvchaette Worm, FI) = Formulated Diet, and MD = Mixed Diet. Vertical bar = SF (n = 4). Note: FD = 0(a) in all cluster. Figure 5. Apparent Digestive Ratio (.XDR) of every diet group (Dif- ferent letters above the bars denote significant differences (P < 0.05) among columns in the same cluster). Where FF = Fish Flesh, SF = Shrimp Flesh, CF = Clam Foot, PW = Polychaette Worm, FD = For- mulated Diet, and MD = Mixed Diet; ADR^, ADR^, and ADR,, are Apparent Digestive Ratios in terms of dry matter, protein, and energy respectively; Vertical bar = SE (n = 4), energy to protein ratio (E/P) and lipid content, however, varied greatly in different diets. The lipid contents of PW and FD were 16. .32% and 9.93% respectively, and they were higher than other provided diets (Table 1 ). Although the lipid content in shrimp diet should not exceed 10% (Xu & Li 1988, Li 1990), it was found in this study that high lipid content had positive effect on FCE in Chinese shrimp, and the highest FCE was observed in PW, which was the highest in lipid content (16.32%). This result indicates that the shrimp could use more lipid than indicated in other reports, and diet for shrimp should be higher than 10% while protein content was high. E/P of these two diets are 33.99 KJ.g"'and 45.20 KJ.g"' respectively, which are also higher than other diets. It was prob- able that the protein sparing effect of these two parameters sig- nificantly improved the FCEp of PW and FD (reaching 22.87% and 15.46%, respectively). Although shrimp of CF treatment ingested a larger amount diet in terms of DW. P, and E than PW treatment (Fig. 3), their SGR, FCE, and final body weight were not signifi- cantly higher than PW due to the lower lipid content and lower E/P ratio. Munoz and San Feliu (1984) found in an experiment that Japa- nese shrimp, Penaeus japanicits fed on natural diets grew faster than those fed on formulated diet. In this study, the Chinese shrimp fed on FD grew significantly faster than those fed on FM and SM, and slower than those fed on CF and PW. Because the shrimp ingested no FD when it was fed simultaneously with natural diets, it was necessary that natural diets and the formulated diet should not be fed simultaneously in practice to avoid the wasting of for- mulated diet. Because the Chinese shrimp ingested no FD in the MD group, there was no significant difference among FD, FF, SF, and PW groups in FI^, (Fig. 3). Furthermore, FIj of FD was sig- nificantly higher than that of FF, SF, and CF, and the FCE^ of FD was significantly higher than that of other diets except PW (Fig. 2). This result indicates that FD was le.ss contaminating than FF, SF, and CF because of less nitrogen and organic matter loss in the water when these diets were ingested. It was found that fish culture used trash fish as feed, which results in heavier contamination than dry and artificial feeds (Wu, 1995). Considering the heavy con- tamination that can be caused by feeding the shrimp with natural diets in pond culture practice and the diets resource limitation (Wu 1995, Dong et al. 2000), it is reasonable to propose a high quality formulated diet to be used in culture practice. 552 Huang et al. ACKNOWLEDGMENTS This work was supported by funds from the Chinese National Science Foundation for Talent Youths (Grant no. 39725023). the Project under the Major State Basic Research of China (Grant no. G1999012011) and the National Tenth five-year Scientific and Technological Key Project (Grant no. 2001BA505B-04). LITERATRUE CITED Alam, M. J., K. J. Ang & M. Begum. 1996. Ingestion efficiency of Mac- robrachium rosenbergii (de Man) larvae feeding on Anemia. Moina micrura Kurz and their combination. Aijiuiciilture Research 27:113- 120. Andrews, J. W., L. V. Sick & G. J. Baptist. 1972. The influence of dietary protein and energy levels on growth and survival of penaeid shrimp. Aquaculhire 1:341 -347 . AOAC. (Association of Official Analytical Chemists). 1984. Official meth- ods of analysis: 14th ed. Virginia: Association of Official Analytical Chemists. 1141 pp. Biesiot, P. M. & G. M. Capuzzo. 1990. Digestive protease, lipa.se and amylase activities in stage 1 larvae of the American lobster, Hananis Americamis. Coinp. Biochem. Physiol. 95:47-54. Boynton, J. E. 1979. Optimal prey selection of Lirtoriiia Hitorea by green crabs. Biol. Bull. 157:359-360. Buskey, E. J.. C. Coulter & S. Strom. 1991. Locomotory patterns of mi- crozooplankton: Potential effects on food selectivity of larvae fish. Bull. Mar. Sci. 53:29^3. Chong, V. C. & A. Sasekumar. 1981. Food and feeding habits of white prawn Penaeus merguierisis. Mar. Ecol. Prog. 5:185-191. Calow, P. & C. R. Townsend. 1981. Ecology, energetics, and evolution. In: C. R. Townsend & P. Calow, editors. Physical Ecology: An Evolution- ary Approach to Resource Use. Oxford: Blackwell. pp. 3-19. Cockcroft, A. & A. Mclachlan. 1986. Food and feeding habits of the surf zone penaeid prawn Macropetasma africanus (Balss). Pubblicazioni della Stazione zoological di Napoli I: Berlin. Hamburg: Marine Ecol- ogy. 13 pp. Colvin P. M. 1976. Nutritional studies on penaeid prawns: protein require- ments in compounded diets for juvenile Penaeus indicus. .Aqiiaciillure 7:315-326. Company, R., J. A. Calduch-Giner, J. Perez-Sanchez & S. Kaushik. 1999. Protein sparing effect of dietary lipids in common dentex (Denlex dentex): A comparative study with sea bream (Sparus auraia) and sea bass (Dicentrachus labrax). Aqua. Liv. Res. 12:23-30. Dall, W., D. M. Smith & L. E. Moore. 1991. Biochemical composition of some prey species of Penaeus esculentus Haswell (Penaeidae: Deca- poda). Aquaculhire 96:151-166. Das. K. M. 1991. Studies on digestive enzyme activity of fish Ctenopharyngodon idella. Aquaculture 92:21-30. Degani. G. & S. Viola. 1987. The protein sparing effect of carbohydrates in the diet of eels {Anguitla anguilla). Aquaculture 64:283-291. Dong, S. L., K. H. Pan & U. Brockmann. 2000. Review on effects of mariculture on coastal environment. J. Ocean Univ. Qingdao 30:573- 582. Elner. R. W. & R. N. Hughes. 1978. Energy maximization in the diet of the shore crab, Carcinus maenas. J. Animal Ecol. 47:103-107. Erfanullah & A. K. Jafri. 1995. Protein-sparing effect of dietary carbohy- drate in diets for fingerling Labeo rohita. Aquaculture 136:331-339. Griffiths, D. 1975. Prey availability and food of predators. Ecology 56: 1209-1214. Hughes. R.N. and R. W. Elner, 1979. Tactics of a predator, Carcinus maenas, and morphological responses of the prey, Nucella lapiltus. J. Animal Ecol. 48:65-78. Ivlev, V. W. 1961. Experimental Ecology of Feeding of the Fishes. New Haven: Yale University Press. 302 pp. Kennedy, V. S., C. King & J. A. Blundon. 1983. Blue crab predation of infaunal bivalves: relation to optimal foraging hypotheses. J. Shellfish /?f.f. 3:95. Kislalioglu. M. & R.N. Gibson. 1976. Prey "handling time" and its im- portance in food selection by the 15-sp stickleback, Spinachia spina- cliia (L). J. Exper. Mar. Biol. Ecol. 25:151-158. Kulkami, B. G., V. D. Deshmukh & V. R. Kulkami. 1999. Food and feeding habit of a penaeid prawn Melapenaeopsis stridulans (Alcock 1905). J. Bombay Nat. Hist. Soc. 96:262-267. Lawton, P. & R. N. Hughes. 1985. Foraging behavior of the crab Cancer paguru feeding on the gastropods Nucella lapillus and Littorina lit- torea: Comparisons with optimal foraging theory. Mar. Ecol. Prog. Series 21:\43-\54. Li A. J. 1990. Advances in the nutrition research of Penaeus Chinensis. J. Shaghai Fish. Univ. 7(Suppl.): 16-23. Luna-Marte, C. 1982. Seasonal variation in food and feeding of Penaeus monodon Fabricius (Decapoda, Natantia). Crustaceana 42:250-255. Manghagen, C. & A-M. Wiederholm. 1982. Food selectivity versus prey availability: A study using the marine fish Pomatoschisius microps. Occo/og/a 55:31 1-315. Marte, C. L. 1980. The food and feeding habits of Penaeus monodon Fabricius collected from Makato River, Aklan, Philippines. Crustacea 38:225-236. Matty, A. J. & P. Smith 1978. Evaluation of a yeast, a bacterium and an alga as protein source for rainbow trout. Effect of protein level on growth, gross conversion efficiency and protein conversion efficiency. Aquaculture 14:235-246. Mehner, T., M. Plewa, S. Huelsmann & S. Wonschka. 1998. Gape-size dependent feeding of age-0 perch iPerca fliiviatilis) and age-0 zander iStizostedion luciopercu) on Daphnia galeata. Archiv fuer Hydrobiolo- gie 142:191-207. Mikheev. V. N. 1984. Prey size and food selectivity in young fishes. / Ichihyology 24:66-76. Minami. T. 2000. Predator-prey relationship and trophic levels of the pink shrimp, Pandalus eous. in the Yamato Bank, the Sea of Japan. J. Shellfish Res. 19:553-554. Morals. S.. J. G. Bell. D. A. Robertson, W. J. Roy & P. C. Morris. 2001. Protein/Iipid ratios in extruded diets for Atlantic cod (Gadus morhua L.): effects on growth, feed utilization, muscle composition and liver histology. Aquaculture 203:101-119. Munoz, F. & J. M. San Feliu. 1984. Essay of a commercial diet for Penaeus japonicus as food for the Spanish prawn Penaeus kerulhurus. Inf. Tec. Inst. Invest. Pesq. Bare. 111:12. Newell, R. I. E.. N. Marshall. A. Sasekumar & V. C. Chong. 1995. Relative importance of benthic microalgae, phytoplankton, and mangroves as sources of nutrition for penaeid prawns and other coastal invertebrates from Malaysia. Marine Biology 123:595-606. Nunes. A. J. P., T. C. V. Gesteira & S. Goddard. 1997. Food ingestion and assimilation by the southern brown shrimp Penaeus subtilis under semi-intensive culture in NE Brazil. Aquaculture 149:121-136. Phil, L & R. Rosenberg. 1984. Food selection and consumption of the shrimp Crangon crangon in some shallow marine areas in western Sweden. Marine ecology progress series. Oldendotf 15:159-168. Pinn, E. H., R. J. A. Atkinson & A. Rogerson. 1998. The diet of two mud-shrimps, Calocaris macandreae and Upogebia slellaia (Crusta- cea: Decapoda: Thalassinidea). Ophelia 48:211-223. Prakash, S. & G. P. Agarwal. 1989. A report on food and feeding habits of freshwater prawn, Macrobrachium choprai. Imtum J. Fisheries 36: 221-226. Ricker, W. E. 1979. Growth rates and models. In: W. S., Hoar, D. J. Randall & J. R. BreU. editors. Fish Biology, Volume 8. Academic Press. New York. pp. 677-743. Selective Diets of Fenneropenaeus Chinensis 553 Roy. D. & S. R. Singh. 1997. The food and feeding habits of a freshwater prawn Macrchrachium choprul. Asian Fish. Scl. Metro Manila 10:51- 63. Schwambom. R. & M. M, Griales. 2000. Feeding strategy and daily ration of juvenile pink shrimp {Farfaniepenaeii.s duranim) in a South Florida seagrass bed. Marine Biol. 1. '17: 139-147. Sedgwick R.W. 1979. Influence of dietary protein and energy on growth, food consumption and food conversion efficiency in Penaeus mer- guiensis. AquaeiiUure 16:7-30. Seed, R. & R. N. Hughs. 1997. Chelal characteristics and foraging behavior of the blue crab Callinecles sa/ndiis Rathbun. Esluarine Coast. .Shelf Sci. 44:221-229. Shalaby. S. M., A. Y. El-Dakar & S. I. Ghoneim. 2001. Protein-sparing effect by carbohydrate in diets of rabbitfish Siganus rivulaius ( Book of Abstracts). Aquaculture. Baton Rouge: World Aquaculture Society. 205 pp. Smith, R. R. 1971. A method for measuring dige,stibility and metabolizable energy of feeds. Progr. Fish Ciiltiirist 33:132. Sunaga, T. 1971. Relationship between food consumption and food selec- tivity in fishes: II experimental study on the food selection by juvenile guppy (Poeciliu retieulata). Japan. J. Ecol. 21:67-70. Tylter, P. & P. Calow. 1984. Fish Energetics: New Perspectives, Croom Helm, London and Sydney, pp. 67-98. Viola, S. & E, Lahav. 1 99 1. The protein sparing effect of lysine supple- ments in carp feed and its implication on reduction of water pollution. Fish. Fishbreed. Isr. 24:61-67. Wang, K. X. (editor), 1997. The multiplication and culture of shrimp and crabs. Beijing, China: Agricultural Press of China. 157 pp. Wu. L., S. Dong. F. Wang & X. Tian. 2000. Compensatory growth re- sponse following periods of starvation in Chinese shrimp. Penaeus chinensis. J. Shellfish Res. 19:717-722. Wu. R. S. S. 1995. The environmental impact of marine fish culture: Towards a sustainable future. Mar. Poll. Bull. 31:159-166. Xu X. Z. & A. J. Li, 1988. Studies on the daily requirements and optimum contents of protein, carbohydrate, fiber and fat in the compound diet of Penaeus orientalis. Marine Sciences 6: 1-6. Juuiiial of Shelljisli Rcseiirdi. Vol. 22, No. 2, 555-559. 2003. EFFECT OF SALINITY ON SURVIVAL, GROWTH, AND OXYGEN CONSUMPTION OF THE PINK SHRIMP FARFANTEPENAEVS PAULENSIS (PEREZ-FARFANTE 1967) MONICA Y. TSUZUKI,'* RONALDO O. CAVALLl,' AND ADALTO BIANCHINI- 'Liihoraldrio cle Mariciiltiira. Dcpiirtanwiito Je Oceanogrcifia. Fuuda<;M> Universidude Federal do Rio Grande. Caixa Postal 474, 96201-900. Rio Grande, RS. Brazil: 'Lxiboratorio de Zoofisiologia, Departamento de Ciencias Fisiologicas. Fiiiidd^cio Universidade Federal do Rio Grande, Caixa Postal 474. 96201-900, Rio Grande. RS. Brazil ABSTR.ACT Survival, growth, and o.xygen consumption rates of Farfantepenueus pauleiisis postlarvae (PL) were examined at different salinities. Initially, PL 15 maintained at 30%c salinity were gradually acclimated to 2. 5. 10. 20, and 30%c over 5 days. Afterwards, survival, growth, and oxygen consumption rates of shrimp reared at these salinities were determined over a 42-day experimental period. Lower wet weight and cephalotorax length, and higher mortality rates were observed in shrimp reared at 2%o salinity, especially when compared with those reared at lO'At salinity (P < 0.05). In the range of S'/tr to 30%f salinity, growth was optimized at Iff/n salmity. although this response was not significant. Salinity affected the oxygen consumption rates of F. paiilensis postlarvae. At the beginning of the growth trial, oxygen consumption rate was markedly lower at 27ii salinity than at 10%c or 30%o salinity (P < 0.05). This response was probably associated with a metabolic depression that preceded the shrimp death. Thereafter, oxygen consumption at 2%t salinity showed a nonsignificant increase due to a higher variability of measurements probably associated with a better performance of surviving shrimp, which were tolerant to low salinity levels. At the intermediate salinities (5%c-20'^t). oxygen consumption was higher at 10%f salinity. At the end of the experiment, oxygen consumption reached similar and low levels irrespective of the salinity level. Oxygen consumption rate of shrimp reared at 30^. salinity was constant and close to 5-(iL mg dry weight"' hr"' throughout the experiment. KEY WORDS: shrimp. Faifantepenaeus paiilensis. growth, oxygen consumption, salinity INTRODUCTION FaifaiUepciuiciis paiilensis (Perez-Farfante 1967) is a cold tol- erant shrimp naturally occurring between Mar del Plata. Argentina, and Ilheus, Brazil (D'Incao 1995). It is an important fishery re- source, especially in Southern Brazil, where catches by artisanal fisheries have averaged around 3500 metric tons/yr in the last 40 years. However, unpredictable fluctuations in capture caused by climatic and oceanographic factors (Castello & Moller 1978, D'Incao 1995) usually result in a severe socio-economical prob- lem. Some studies have examined the viability of cultivation and restocking programs with this species (Olivera et al. 1993. Wasielesky et al. 1995, Peixoto et al. 2002). The release and growth of F. paulensis in pen enclosures is routinely carried out at the estuary of the Patos Lagoon, Southern Brazil (Wasielesky 2000). which is characterized by abrupt and wide variations in salinity (Baptista 1984). Salinity is one of the most important environmental factors affecting growth and survival of penaeids as it influences food consumption, conversion efficiency, and metabolic responses (Venkataramiah et al. 1972. Castille & Lawrence 1981. Dalla Via 1986, Staples & Heales 1991. Clark 1992, Brito et al. 2000). The knowledge of the species tolerance limits and optimum salinity levels is necessary to evaluate the viability of F. paulensis culti- vation at variable environmental conditions. Furthermore, it is im- portant to understand the effects of salinity when shrimp is reared in nursery grounds characterized by sudden salinity tluctuations and extreme environmental conditions. Salinity might have an in- direct influence on the survival and growth of postlarvae when they penetrate estuarine areas, and also on the migration of juve- niles back to the ocean. For example. Staples ( 1980) observed that reductions in salinity caused the migration of Fenneropeiiaeiis merguiensis juveniles from nursery grounds to oceanic waters. Salinity tolerance limits and the effects of acclimation to sa- linity on the survival of F. paulensis have already been evaluated (Tsuzuki et al. 2000). However, as the optima! salinity range for growth is narrower than for survival, growth occurs when the metabolic demands for maintenance and feeding activity are sat- isfied. Several studies have analyzed the metabolism and activity in crustacean decapods through oxygen consumption measure- ments (Kutty et al. 1971, Venkataramiah et al. 1974, Venkat- aramiah et al. 1975, Gaudy & Sloane 1981, Du Preez et al. 1992, Villarreal & Rivera 1993). Since the rate of oxygen consumption is modified by changes in the energetic demand for biologic ac- tivities, it is expected that salinity variations would lead to changes in oxygen consumption of shrimp, as demonstrated by Kutty et al. (1971). It is also expected that changes in metabolic rates induced by salinity can affect shrimp growth and production, as pointed out by Dalla Via (1986). In light of discussion earlier, the objective of this study is to investigate the effects of salinity on survival, growth, and oxygen consumption of F. paulensis postlarvae. MATERIAL AND METHODS General Rearing Conditions This study was conducted at the Marine Aquaculture Station "Prof Marcos A. Marchiori" of the Funda^'ao Universidade Fed- eral do Rio Grande (Southern Brazil). Postlarvae (PL) oi Faifante- penaeus paulensis were reared at 22-25°C, 30Sff salinity, and natural photoperiod. In the initial stages of development, PL were fed with newly-hatched Artemia nauplii, and afterwards with Ar- temia nauplii and finely chopped meat of white clam (Mesodesnui maelroides), tlsh (various fresh fish) and squid (llle.x sp). Water of different salinities was obtained by mixing dechlorinated tap water with natural seawater. Salinity was measured with an optical re- fractometer ( I .O'/n precision. Atago Co., Tokyo, Japan). Survival, Growth, and Oxygen Consumption Fifteen-day-old PL were reared in the conditions described ear- lier and were gradually acclimated from 30 to 2. 5, 10, and 20%o 555 556 TSUZUKI ET AL. salinity over a 5-day period, by daily reductions of 6, 5, 4. and 2%c salinity, respectively (Tsuzuki et al. 2000). Postlarvae maintained at 30%o salinity were used as control. After the salinity acclimation period, PL survival and growth in each salinity were examined over 6 wk by stocking 80 PL in a 100-L plastic tank. Shrimp were fed ad lihinim twice a day with a commercial diet containing 45% crude protein (Tetra DoraMarin. Pfizer Co., USA). Every day. pH and temperature were monitored, and organic residuals were si- phoned out from the bottom of the tanks when at least 10% of the water was renewed. Every two weeks, 20% of the animals in each tank were counted and individually weighed to the nearest 0.1 mg (wet weight). Cephalotorax length and dry weight (60°C for 48 h) were measured to the nearest 0.01 mm and 0.1 mg. respectively, at the beginning (» = 48) and at the end of the trial. At the end of the experiment (week 6). all living shrimp were weighed (wet and dry weights) and measured {n = 26-16.^). Cephalotorax length was measured using a stereoscopic microscopy (Nikon. Japan). At the end of the salinity acclimation period, and every 2 wk during the growth trial, oxygen consumption was measured using a Barcroft-Warburg respirometer (Oser 1965). Values were ex- pressed as (xL of O, per mg of dry weight per hr. Statistical Analysis Each treatment was done in triplicate. However, no significant difference was detected between replicates and results were then pooled for further analysis. Differences between replicates and treatments were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey's test. The significance level adopted was 95% (P < 0.05). RESULTS Water temperature throughout the experiment was 24.9 ± 0.1 °C (mean ± SB), while mean values of pH and salinity were 7.5, 7.6, 7.6, 7.8, and 7.9 at 2. 5. 10, 20. and 30%c salinity, respectively. After the five-day acclimation period to different salinities, mean weights (wet and dry weights) and cephalotorax length of 20-day-old PL did not change with the acclimation salinity (P > 0.05). Therefore, all values were pooled and only one mean was calculated. Mean (± SE) wet and dry weight and cephalotorax length was 9.2 ± 0.2 mg. 2.0 ± 0.1 mg, and 2.1 ± 0.0 mm. respec- tively. Survival rates of these PL were higher than 95% and there were also no significant difference between treatments (P < 0.05) (data not shown). However, after two weeks of experiment, sig- nificantly lower survival rates (28.1%) were observed at 2%o sa- linity (results not shown). At this salinity, only 15.8% survival was observed at the end of the growth period (Table 1 ). Figure 1 shows the PL growth as wet weight at different sa- linities throughout the experimental period. From the second to the fourth week of experiment, a higher mean wet weight was ob- served in PL reared at 10%p salinity, especially when compared with those reared at 2%i, 59cc. and 20%^ salinity (P < 0.05). After six weeks of experiment, wet weight of shrimp reared at 2%c salinity was significantly lower than those reared at 10%r salinity (P < 0.05). For salinities between 5%c and SO^ft, PL wet weight was higher at 10%c salinity although this difference was not sta- tistically significant (Fig.l, Table 1). At 2%< salinity, PL dry weight tended to be lower at the end of the growth period, but no significant changes were detected. Cephalotorax length was sig- nificantly smaller in PL reared at 2%c salinity than in those reared at 5%c. or lOVcc salinity (P < 0.05) (Table I ). After the salinity acclimation period, oxygen consumption of PL acclimated to 2%c salinity was lower than that observed in PL acclimated to 10%fi or 30%c salinity. At 2%f' salinity, oxygen con- sumption increased after the second week and reached a maximum value at the fourth week of experiment. Afterwards, a marked drop in oxygen consumption rate occurred. At the intermediate salinities (from 5%c to 20%c), oxygen consumption was higher at 10%c sa- linity until the second week of the experiment although not statis- tically different (P > 0.05). At the end of the growth period, oxy- gen consumption reached similar and low levels (around 4 p-L Oo mg dry weight"' hr~') irrespective of the salinity level tested. Oxygen consumption of shrimp at 30%c salinity was constant and close to 5 |jiL O2 mg dry weight ' hr~' throughout the experiment (Table 2, Fig. 1). DISCUSSION In this study, survival of Faifantepenaeus paiilensis postlarvae (PL) was extremely low ( 15.8% ) after a 6-wk growth period at 2%c salinity. A similar result was verified by Cawthome et al. (1983) when only 34% of Pemieus monodon juveniles survived at that salinity for two weeks. Although Tsuzuki et al. (2000) verified an increase in salinity tolerance of F. paulensis postlarvae with aging (from PL 15 to 30) when PL were directly transferred from 30%o to 27co or 5%o salinity, the low survival rate observed at 2%p salinity in this study indicates that 20-day-old PL were not able to cope with low salinity levels for a long period of time (6 wk). Also, the lower shrimp growth rates observed at 2%c salinity confirms the physiologic disturbance induced by low salinity in F. paidensis PL. Dalla Via (1986) suggested that reductions in shrimp growth at low salinities can be related to a higher energetic expenditure to keep the osmotic equilibrium at these saline conditions. The same author showed that exposure to 10%o salinity for five months re- sulted in reduction (up to 33%) of the ash-free organic content. Therefore, in low salinity environments a significant reduction in shrimp production might be expected. However, this hypothesis can only be considered if one assumes that the food assimilation TABLE 1. Survival, wet and dry weights, and cephalotorax length of Farfantepenaeus paulensis postlarvae reared at different salinities for 6 weeks. Salinity (%r) Survival (%) Wet Weight (mg) Dry Weight (mg) Cephalotorax Length (mm) 5 10 20 30 15.8 ±4.7 (a) 81.3 ±5.2 (b) 88.3 ± 0.6(b) 82.9 ± 9.0(b) 70.0 ± 14.3(b) 102.4: 135.2: 147.2: 140.3 : 140.9: 11.3(u) 5.1 (ab) 4.6 (b) 7.6 (ab) 10.0 (ab) 27.2 ±3.7 (a) 30.8 ± 1.2(a) 33.2 ±1.1 (a) 33.2 ± 1.9(a) 34.2 ± 2.6 (a) 4.8 ±0.4 (a) 5.7 ± 0.2(b) 5.7 ±0.1 (b) 5.1 ±0.2(ab) 5. 1 ± 0.2 (ab) Data are means ± SE (;i = 26-163). Same letters indicate absence of significant difference between salinities (P > 0.05). Growth and Ox-igen Consumption of Farfantepenaeus paulensis 557 160 150 - 140 - 130 120 - 110 100 3 90 - E 80 I 70 S 60 50 40 30 20 10 Time (weeks) Figure 1. Wet weight of Farfantepenaeus paulensis postlarvae reared at different salinities. Data are means ± SE (;i = 26-1631. Different letters indicate sij^niflcant differences between salinities at the same time of cultivation {P < (1.(15). Salinities (%c): O = 2; D = 5; A = 10; V = 20; 0 = 30. rate is not dependent on salinity. Marques & Andreatta (1998) found significant differences in dry matter consumption of F. pau- lensis reared in low salinity levels while Wasielesky et al. (2002) reported that food consumption in this species was not affected by salinity. Therefore, further investigation is needed to clarify this question. It has been demonstrated for several penaeids that higher growth rates usually are observed at salinities ranging from 57(i to 35%c, depending on the species and the ontogenetic phase consid- ered. In Liiopenaeus vannamei PL. higher growth rates occurred at 20^r salinity when compared with those observed al 5'^r and 45"^? salinity (Huang 1983). Bray et al. ( 1994) reported that juveniles of the same species reared at 5%t and \57cc salinity achieved higher increment in wet weight than those reared at 25%c, 35%f. and 49'ycc salinity. Venkataramiah et al. (1974) verified that F. aztecus growth was enhanced at '^.S'/n and 17"y?f salinity. Henning & Le- mos ( 1994) verified that L. schmilli growth was similar at 5'/tf and 30%c salinity, but higher at 10%c salinity. In this study, a better growth rate was observed at 10%f salinity, being significantly dif- ferent from that observed at 2%c salinity throughout the experi- ment. Venkataramiah et al. ( 1975) observed in estuaries where F. aztecus is naturally found, that a higher abundance occurs in sa- linities that are close to the optimum level estimated under labo- ratory conditions. The same fact is observed for F. paulensis dis- tribution in the Patos Lagoon estuary, where a higher abundance is observed in areas with salinities below lO'^f salinity, although shrimp can be found in salinities between 0%6 and 'iVic (DTncao 1991). Before growth occurs, the metabolic demand for maintenance and feeding activity must be satisfied. The knowledge of such demands under different environmental conditions is necessary (Brett 1970). Several studies have used the oxygen consumption measurement to analyze metabolisin and activity in crustacean decapods (Kutty et al. 1971, Venkataramiah et al. 1974, Gaudy & Sloane 1981; Villarreal & Rivera 1993). Since the oxygen con- sumption alters with changes in the energetic demand for biologic activities, it would be expected that salinity variations could lead to changes in oxygen consumption (Kutty et al. 1971). At the beginning of the experiment, the oxygen consumption of 20-day-old PL reared at 27i< salinity was markedly lower com- pared with PL reared at the other salinities tested. This fact prob- ably indicates that PL could be in a metabolic depression stage that would precede death. In fact, a high mortality rate (71.9%) was observed in the first two weeks of the experiment. In Penaeus semisukatus. Clark (1992) also observed a decrease in the respi- ration rate after a salinity reduction from 409ft to \%9ii salinity. He also noticed that shrimp were moribund and died 12 hours after being exposed to the salinity shock. Chen & Fang (1986) consid- ered that the respiratory depression observed in Metapenaeus ensis after a salinity change was caused by a reduction of the w aler flow through the gills to resist the salinity shock, leading to a reduction of the oxygen consumption. In this study, a low oxygen consump- tion was observed throughout the experiment at 2%c salinity, ex- cept after four weeks when a non-significant increase in oxygen consumption was observed. In this case, mortality rates did not significantly change after two weeks of experiment. Therefore, the increase in oxygen consumption observed after four weeks of ex- periment at 2%c salinity could be attributed to a higher variability of the oxygen consumption measurements due to a better perfor- mance of surviving animals, which probably were more resistant to lower salinity levels. Until the second week of the experiment, higher oxygen con- sumption rates were observed in shrimp maintained at \Wcc salin- ity. Concomitantly, higher growth rates expressed as wet weight and cephalotorax length were generally observed for shrimp kept at this salinity. Yagi & Ceccaldi (1984) verified in Palaemon seiratus larvae that the oxygen consumption was maximum in salinities ranging from 25'^f to 30%f. which could be explained by a higher physiologic activity related to larvae food utilization. Moreover, between 25%c and 30%(: salinity the energetic demand for osmoregulation seems to be lower and growth higher. It is important to emphasize that an attempt to correlate energetic ex- penditures for ionic and osmotic regulation with oxygen consump- tion rates is speculative, once the subject is still controversial. Some investigators point out that the energy expended to osmo- regulation can be evaluated by oxygen consumption measurements in aquatic invertebrates (Lofts 1956. Rao 1968). In this case, oxy- gen consumption can be expected to increase for osmoregulators TABLE 2. Oxygen consumption rates (jtL {), mg dry weight' hr ') of Farfantepenaeus paulensis postlarvae reared at different salinities for 6 weeks. Salinity {■7<,) Time of Cultivation (Weeks) -) 3.7 ± 0.6 (a) .'i.3±().y(ah) 5 7.9 ± 2.3 (ab) 8.2 ±1.4 (a) 10 1 3.0 ± 2.8(b) 12.9 ± 3.3 (ab) 2(J 9.1 ±2.6(ab) 8.1 ± 1.3 (ab) 30 6.3 ± 0.4(b) 4.3 ± 0.5(b) 12.3±3..S(a) .^.0±0.8(a) 3.9 ± 0.7(b) 3.6 ±0.5 (a) 5.6±0.9(ab) 4.3 ±0.5 (a) 4.6 ± 0.5 (ab) 4.3 ± 0.5 (a) 4.4±0.y(ab) 4.1 ±0.7 (a) Data are means ± SE (n = 3-6). Same letters indicate absence of signifi- cant difference between salinities (P > 0.05). 558 TSUZUKI ET AL. when the osmotic difference between the hemolymph and the en- vironment increases, resulting in an increase in the metabohc de- mand to keep constant the hemolymphatic concentration. Never- theless, changes in metabolic rates related to salinity are, in most cases, too big to be attributed only to the energetic cost with ionic and osmotic regulation. In this case, it would be difficult to relate oxygen consumption rates exclusively to energetic requirements for osmoregulation (Potts & Parry 1964). Therefore, not only the anisosmotic regulation of extracellular tluids. that seems less likely the general cause of metabolic changes of the organism (Duncan 1966, Kinne 1971 ) should be taken into consideration, but also the isosmotic regulation of the intracellular fluids involving the mo- bilization of organic substances and changes in the energetic needs for ionoregulation (Wheatly 1988). Additionally, the interference of the locomotion activity should be considered (Beamish & Mookherji 1964). The comparatively low and stable oxygen consumption rates of shrimp reared at 30^c salinity, and the low oxygen consumption at the end of the experiment irrespective of salinity levels, indicate more economical respiration rates at salinities where animals are genetically adapted or acclimated for a longer period (Kinne 1971). ACKNOWLEDGMENTS The authors thank Alvaro Montenegro Neto for his technical assistance. This study was supported by the Brazilian CNPq. R. Cavalli and A. Bianchini are research fellows of this agency (Proc. n^ 300131/01-1 and 300536/90-9, respectively). LITERATURE CITED Baptista. J. R. 1984. Flutua^oes diarias e horarias dos elementos; dissolvi- dos. material em suspensao e caracteristicas ffsicas da agua na parte sul da Lagoa dos Patos e praia do Cassino (RS-Brasil). MSc thesis. Rio Grande, Brazil: Universidade do Rio Grande. 100 pp. Beamish, F. W. H. & P. S. Mookherji. 1964. Respiration of fislies with special emphasis on standard oxygen consumption. I. Influence of weight and temperature on respiration of goldfish. Carassitis aiiralus L. Can. J. Zool. 42:161-17.'>. Bray, W. A., A. L. Lawrence & J. R. Leung-Trujillo. 1994. The effect of salinity on growth and survival of Penaeus vawwmei with observations on the interaction of IHHN virus and salinity. Aquacuhure 122:133- 146. Breu, J. R. 1970. Fish-the energy cost of living. In: W. J. McNeil, editor. Marine Aquaculture. Corvallis: Oregon State University Press, pp. 37-52. Brito. R.. M. Chimal & C. Rosas. 2000. Effect of salinity in survival, growth, and osmotic capacity of early juveniles of Fa/fanrepenaeus brasiliensis (Decapoda: Penaeidae). J. Exp. Mar. Biol. Ecoi 2-14:253- 263. Castello. J. P. & O. O. Jr. Moller. 1978. On the relationship between rainfall and shrimp production in the estuary of the Patos Lagoon (Rio Grande do Sul, Brazil). AtUintica 3:67-74. Castille, F. L. & A. L. Lawrence. 1981. The effect of salinity on the osmotic, sodium and chloride concentrations of hemolymph of eury- haline shrimp of the genus Penaeus. Cunip. Biochem. Physiol. 68A: 75-80. Cawthome, D. F., T. Beard, J. Davenport & J. F. Wickins. 1983. Responses of juvenile Penaeus monodon Fabricius to natural and artificial seawa- ter of low salinity. Aquaculture 32:165-174. Chen, I.-M. & L.-S. Fang. 1986. Metabolic and osmotic responses of Metapenaeus ensis (De Haan) subjected to sudden salinity change. In: J. L. Maclean, L. B. Dizon & L. V. Hosillos, editors. The First Asian Fisheries Forum. Manila: Asian Fisheries Society, pp. 629-632. Clark, J. V. 1992. Physiological responses of adult Penaeus semisulcatus (De Haan) to changes in salinity. Comp. Biochem. Physiol. 101 A: 1 17- 119. Dalla Via, G. J. 1986. Salinity responses of the juvenile penaeid shrimp Penaeus japonicus. I. Oxygen consumption and estimations of produc- tivity. Aquaculture 55:297-306. DTncao, F. 1991. Pesca e biologia de Penaeus paulensis na Lagoa dos Patos, RS, Brasil. Atldntica 3:57-66. DTncao, F. 1995. Taxonomia, padroes distribucionais e ecologicos dos Dendrobranchiata (Crustacea: Decapoda) do Brasil e Adantico Ociden- tal. PhD thesis. Brazil: Universidade Federal do Parana. 365 pp. Duncan, A. 1966. The oxygen consumption of Potinnopyrgus jenkinsi (Smith) Prosobranchiata in different temperature and salinities. Verh. Int. Vehr. Limno. 16:1739-1751. Du Preez, H. H., H. Chen & C. Hsieh. 1992. Apparent specific dynamic action of food in the grass shrimp, Penaeus monodon Fabricius. Comp. Bwchem. Physiol. 103A: 173-178. Gaudy, R. & L. Sloane. 1981. Effect of salinity on oxygen consumption in postlarvae of penaeid shrimp Penaeus monodon and P. styliro.ttris without and with acclimation. Mar. Biol. 65:297-306. Henning, O. L. & A. Lemos. 1994. Avalia^ao dos parametros: peso medio final e sobrevivencia de pos-larvas de Penaeus schimitti (Burkenroad, 1963) subtmetidas a diferentes salinidades. Proceedings of VllI Sim- pdsio Brasileiro de Aquicultura. 1 1-14 Outuhro 1994, Piracicaba. Sao Paulo, Brazil, pp. 2-12. Huang, H. J. 1983. Factors affecting the successful culture of Penaeus stylirostris and Penaeus vannamei at an estuarine power plant site: temperature, salinity, inherent growth variability, damselfly nymph predation, population density and distribution, and polyculture. PhD thesis. Texas, EUA: College Station. 221 pp. Kinne, O. 1971. Salinity: animals — invertebrates. In: O. Kinne, editor. Marine Ecology. Vol. 1. Environmental Factors, Part 2. New York: Wiley-lnterscience. pp. 821-995. Kutty, M. N.. G. Murugapoopathy & T. S. Krishnan. 1971. Influence of salinity and temperature on the oxygen consumption in young juveniles of the Indian prawn Penaeus indicus. Mar Biol. 1 1:125-131. Lofts, B. 1956. The effects of salinity changes on the respiratory rate of the prawn Palaemonetes varians (Leach). J. Exp. Biol. 33:730-736. Marques, L. C. & E. R. Andreatta. 1998. Efeito da salinidade sobre o consumo de raf ao, crescimento e sobrevivencia de juvenis do camarao rosa Penaeus paulensis (Perez-Farfante, 1967). Anais do Aquicullura Brasil "98, Recife, PE, Brazil, pp. 804. Olivera. A., E. Beltrame, E. R. Andreatta, A. Silva, S. Winkler da Costa & S. Westphal. 1993. Crescimento do "Camarii rosa" Penaeus paulensis no repovoamento da Lagoa do Ibiraquera, Santa Catarina — Brasil. Pro- ceedings of the 4th Brazilian Symposium on Shrimp Culture, Joao Pessoa, PB, Brazil, pp. 439^51. Oser, B. L. 1965. Hawk's Physiological Chemistry. 14th ed. New York: McGraw-Hill. Peixoto, S., W. Wasielesky, Jr. & L. Louzada, Jr. 2002. Comparative analysis of Faijantepenaeus paulensis and Litopenaeus vannamei cul- ture in extreme southern Brazil. / Appl. Aquacult. (in press). Potts, W. T. W. & G. Parry. 1964. Osmotic and ionic regulation in animals. London: Pergamon Press. Rao, K. P. 1968. Oxygen consumption of rainbow trout (Salmo f;aidneri) in relation to activity and salinity. Can. J. Zool 46:781-786. Staples. D. J. 1980. Ecology of juvenile and adolescent banana prawns, Penaeus meriiuiensis in mangrove estuary and adjacent offshore waters of the Gulf of Carpentaria. II. Emigration, population structure and growth of juvenile. Aust. J. Mar. Freshwat. Res 31:653-665. Staples, D. J. & D. S. Heales. 1991. Temperature and salinity optima for growth and survival of juvenile prawn Penaeus merguiensis. J. Exp. Mar. Biol. Ecoi 154:251-274. Growth and Oxygen Consumption of Farfantepenaeus paulensis 559 Tsuzuki. M. Y., R. O. Cavalli & A. Bianchini. 2000. The effects of tem- perature, age and acclimation to salinity on the survival of Faifaiiw- penaeus paulensis postlarvae. J. World Aquacult. Soc. 31:459-468. Venkataramiah, A.. G. J. Lakhsmi & G. Gunter. 1972. The effects of salinity and feeding levels on the growth rate and food conversion efficiency of the shrimp Pcmwns cizlecus. Proc. World MaricuU. Soc. 3:267-283. Venkataramiah. A.. G. J. Lakshmi & G. Gunter. 1974. Studies on the effects of salinity and temperature on the commercial shrimp, PenueKS aztecus Ives, with special regard to survival limits, growth, oxygen consumption and ionic regulation. Contract Report H-74-2. US. Vicks- burg. Mississippi: Army Engineer Waterways Experimental Station, USA. 134 pp. Venkataramiah. A., G. J. Lakshmi & G. Gunter. 1975. A review of the effects of some environmental and nutritional factors on the brown shrimp Penaeus aztecus Ives in laboratory culture. In: H. Barnes, edi- tor. Proc. lOth European Symposium of Marine Biology. Scotland: Aberdeen University Press, pp. 523-547. Villarreal. H. & J. A. Rivera. 1993. Effect of temperature and salinity on the oxygen consumption of laboratory produced Pemieiis californiensis postlarvae. Comp. Biochcm. Physiol. I06A: 103-107. Wasielesky. W. F. B. 2000. Cultivo de FurjaiUcpenacus paidensis (Deca- poda-Penaeidae) no estiiario da Lagoa dos Patos: Efeitos de parametros ambientais e manejo de cultivo. PhD thesis. Rio Grande. RS. Brazil: Funda(,-ao Universidade Federal do Rio Grande. 199 p. Wasielesky, W., A. Bianchini. C. C. Sanchez & L. H. Poersch. 2002. The effect of teinperature, salinity and nitrogen products on food consump- tion of pink shrimp Farfantepenaeus paulensis. Braz. Arch. Biol. Tech- no!. Wasielesky. W.. R. O. Cavalli. D. Dolci & T. Silva. 1995. Cultivo do cumarao Penaeus paulensis em gaiolas e cercados no estuario da Lagoa dos Patos. RS. Proceedings of the 3rd South-Brazilian Aquaculture Meeting. Ibiruba. RS. Brazil, pp. 14-25. Wheatly. M. G. 1988. Integrated responses to salinity fluctuation. Am. Zool. 2&:65-n. Yagi. H. & H. T. Ceccaldi. 1984. Influence combine des facteurs tempera- ture et salinite sur le metabolisme et la croissance larvaire de la crevette rose Palaemon serralus (Pennant) (Crustacea. Decapoda. Palae- nionidae). Aquaculture. 37:73-85. Journal of Shellfish Rcmirch. Veil. 22, No. 2, 561-56S, 2003. ANATOMICAL DAMAGE TO HUMPBACK SHRIMP, PANDALVS HYPSINOTUS (BRANDT 1851) CAUGHT BY TRAWLING AND TRAPPING P. M. TROFFE, S. ONG, C. D. LEVINGS,* AND T. F. SUTHERLAND Di'partnu'iir of Fisheries and Oceans, Wesr Vancuuver Laboratory 4160 Marine Drive, West Vancouver, V7V-1N6. Canada ABSTRACT We compared the anatomical damage, individual size, total catch, and bycatch when humpback shrimp. Pandaliis hypsiiuniis (Brandt 1851). were harvested using otter trawls, beam trawls, and traps in Simoom Sound. British Columbia. Regional body damage (RBD) and total body damage (TBD) to humpback shrimp were assessed for four major regions of the shrimp body (rostrum, carapace, abdomen, and tailfan). TBD was higher for otter and beam trawling compared with traps, with a significant difference observed between the otter trawling and half-day trapping. After standardizing trawl data by fishing effort (area swept and fishing time). TBD was significantly higher for beam trawl. RBD was significantly different across fishing methods and there were also significant differences among the various body parts. Trawl caught humpback shrimp showed the highest ratio of damaged/total individuals relative to those caught by traps. In general, the carapace and rostrum body regions were more damaged relative to the abdomen and tail fan. The survival of humpback shrimp released after trawling or trapping will depend on the extent of the body region-specific anatomical damage that has occurred and its functional importance. KEY WORDS: damage, otter trawling, beam trawling, trapping, shnmp, fishing gear, bycatch, andaliis hypsinonis INTRODUCTION Studies exploring the use of selective fishing gear are ongoing and past studies have focused on the size and shape of net meshes as well as the use of extruders in trawl nets to separate the target and bycatch species (e.g., DeAheris & Reifsteck 1993, Suuronen et al. 1996. Richard 1999). Most studies comparing fishing gear bycatch have focused on the volume of bycatch and only a few more recent studies have focused on damage to the catch and subsequent survivability of organisms, (e.g., Mensink et al. 2000, Stevens et al. 2000. Bergmann & Moore 2001 ). This study, how- ever, turns a lens to the damage to humpback shrimp. Pandahts hypsinotus, harvested in an inshore ecosystem in Pacific Canada with three different fishing gear types: beam trawl, otter trawl, and traps. Humpback shrimp are caught in directed trap and trawl fisheries in British Colunibiu and are also commonly found as incidental catch in shrimp trawl {Pandahts spp.) and spot prawn {Pandalus platyceros) trap fisheries (Boutillier & Nguyen 1999). No information has been published about fishing gear-related ana- tomic damage caused by these harvesting methods in Pacific Canada. In this study we focused on three objectives: 1 ) the rela- tive total damage to humpback shrimp among fishing methods (total body damage [TBD]); 2) susceptibility to gear-related dam- age among major anatomic regions of shrimp (regional body dam- age |RBD|); and 3) comparison of catches of target and nontarget species among gear types. This study was part of a larger project designed to determine whether trawling or trapping would be a preferable method of harvesting humpback shrimp, as a representative crustacean spe- cies, in an ecosystem-based management system (e.g.. Jamieson & O'Boyle 2001 ). One of the aspects of such a management system would be to avoid "bykill" or unwanted fishing mortality of un- dersized shrimp or nontarget shrimp species by minimizing the practice of discarding bycatch if there were high levels of collat- eral damage during harvest. Previous studies have showed that shrimp trawling can result in damage to benthic habitats (e.g.. ♦Corresponding author. Tel: 604-666-7915: fax: 604-666-3497: E-mail: levingsc@pac.dfo-mpo.gc.ca Hansson et al. 2000). However, data to compare damage by trawl- ing relative to other gear types are not available, and the interaction between, gear type, bycatch. and collateral damage have not been presented to date. MATERIALS AND METHODS Experimental Trawling and Trapping Simoom Sound, an inlet off Fife Sound on the central coast of British Columbia was chosen as the study location (Fig. 1 ). Bottom salinity and temperature ranged between 31.5 to 33.5 psu and 7.5 to 8.7^C, respectively, using a Sea-Bird CTD (model SBE- 911 plus) deployed in October 2001. The surface sediment in Si- moom Sound consists of approximately 90% silt and 21% organic content. Beam trawling, otter trawling, and trapping were used to catch humpback shrimp in Simoom Sound during November 2000 (otter trawl, trap), and February 2001 (beam trawl). Each gear type was deployed in a separate "block" of the seafloor (approximately 700 m by 400 m) characterized by relatively uniform depth and sediment type. The gear used was representative of that used in the commercial fishery and complete details on methods, vessels, and gear dimensions are given eLsewhere (Ong et al. 2002). The shrimp trawl industry in British Columbia has voluntarily adopted a 100% implementation of bycatch reduction devises (BRDs) in their nets since 2000 and all trawl nets used in this study were fixed with rigid type bycatch reduction grids (Department of Fisheries and Oceans 2002). Otter Trawling Six otter trawls were conducted on three transects on Novem- ber 14. 2001 (Fig. 1). with two trawls performed on each of the transects. The water depths ranged between 55 and 60 m. Transects lengths were between 643 and 677 m and each trawl was 10-13 min in duration, not including the time required for net haul-back. The otter trawl net measured 36.8 m long with a head- rope and footrope (without a tickler chain) of 23.8 m and 30.5 m. Codend mesh size was 38 mm. Catches were sorted and counted by species and weighed to the nearest 0.1 kg. Humpback shrimp specimens used for the otter trawl damage assessment (/; = 106) 561 562 Troffe et al. v-\ , British > ^Columbia> Figure 1. Map of Simoom Sound. BC, witli approximate locations of beam trawl, other trawl, and traplines used in this study. were collected from the catch after the codend contents had been placed onto a sorting table. Samples were frozen in labeled freezer bags for later analysis in the laboratory. After collection, care was taken to keep specimens flat to minimize damage because of han- dling. Beam Trawling Beam trawls were completed on three transects, west of the otter trawl lines on February 22, 2001 (Fig. 1), with two trawls conducted on each of the three trawl lines. The trawl duration, length, and depths ranged among 15-17 min, 313 and 660 m, and 46 and 55 m. respectively. The beam trawl net measured 26.6 m in total length, with a headrope and footrope length of 14.0 m and 16.5 m. Codend mesh size was 44 mm. Beam trawl catches were sorted by species, then counted and weighed to the nearest 0.1 kg. Humpback shrimp specimens used in the beam trawl damage as- sessment {n = 132) were collected and frozen using the same techniques described for otter trawling. Trapping Traps were set out twice, east of the otter trawl lines, during November 15-16, 2000, on three transects (62-75 m deep; Fig. 1 ). Two time periods were used. The first set of traps remained sub- merged for approximately 6 h (half-day traps), during the day, and the second set of traps was submerged for 17 h over night (over- night traps). Approximately 40 traps were set on each trap line with spacing of about 1 5 m between each trap. The three transects measured from 558 to 660 m long. Most sets included traps outside the defined block of sea tloor because the groundline used was longer than the predetlned transect length of 500 m. Traps were baited with salmon fish feed pellets, cut-up Pacific herring. Chipea harengus pallasi. and shiner perch. Cymatogaster aggregaui. col- lected on site as bycatch from the trawling experiments. The traps were conical and measured 76.2 x 30.5 x 71.1 cm. with a stretch mesh size averaging 45 mm. Each trap weighed approximately 1.4 kg. On one overnight set. humpback shrimp were only collected from traps that fished the predetermined line. All the other hump- back shrimp were from at least 10 traps within the predetennined line and from a few of the traps extending outside of it. The traps were emptied into a plastic tote and the catches from each trap were then identified to species and counted. Catches of all shrimp species were weighed to the nearest 0. 1 kg. A subsample of the humpback shrimp catch was frozen using the same techniques described for otter and beam trawling. 139 humpback shrimp were collected from the half-day traps and 145 from the overnight traps. Sample Processing Humpback shrimp were thawed in the laboratory for 1-2 h. Weight, length, and sex were recorded for each individual. Lengths were recorded to the nearest millimeter using manual or electronic Vernier calipers. Carapace length was measured from the poste- rior-most part of the orbit to the posterior middorsal margin, and total length, from the tip of the rostrum to the tip of the telson. As several humpback shrimp from the beam trawl were in a transi- tional stage (from male to female phases), their total lengths were calculated based on the relationship between male carapace length and total length. Sexing was accomplished by noting the presence of eggs in the head or abdomen, and by examination of the endo- pods of the second pleopods (Butler 1980). Damage Assessment A table was constructed to delineate the principal body parts in the four major regions (rostrum, carapace, abdomen and tailfan) of the shrimp body, as shown in Butler ( 1980). The body parts chosen for analysis were those that would be required for the survival of a humpback shrimp if it were to be released. Each body part within each of the four major body regions was given a score from 0 to 1.0. with zero being a missing body part, and 1.0 representing a fully intact body part, with intermediate scores representing vary- ing levels of damage, that is. the rostrum is composed of nine body parts and a summed score of 9.0 reflects zero percent damage whereas a score of 6.0 represents. (1 - [6.0/9.0] 1 00). or 33.3% damage. Table 1 describes the codes and damage scores used for each of the body regions and Table 2 depicts the particular body parts and their accompanying functions. Humpback shrimp from each of the fishing methods were assessed for damage using this scheme (Table 1) resulting in data on RBD and TBD. TBD was assessed by summing the damage scores from all body regions and dividing the score by the total number of body parts assessed from each gear transect and expressing the resultant as a percentage. Because of time constraints, some of the trap-caught humpback shrimp were assessed using a low-resolution scheme wherein dam- age to the abdomen was not assessed (Ong et al. 2002). Only data from the high-resolution scheme are presented herein. Statistical Analysis Statistical analysis was performed on the humpback shrimp catch data using Systat'"' v. 10 statistical software. A Bartlett's test for homogeneity of variances was performed on the data set prior to statistical analysis and for parametric analyses, proportional data was arcsine transformed (Zar 1984). A single factor analysis of variance (ANOVA) and Tukey HSD multiple comparison tests were used to test for significant differences in RBD and TBD. In cases where were parametric assumptions were not met. a single factor Kruskal-Wallis ANOVA by ranks was performed. Untrans- p. HYPSiNOTUs Damage by Trawling and Trapping 563 Region Code TABLE 1. List oF humpback slirinip body regions assessed for damage. Body Part Damage Score Rostrum Rl Rostrum 0 = R2 Eye stalk and cornea (R) 0 = R3 Eye stalk and cornea (L) 0 = R4 Antennae 1 (L) 0 = R5 Antennae 1 (R) 0 = R6 Antennae 2 (L) 0 = R7 Antennae 2 (R) 0 = R8 Antennal scale (L) 0 = R9 Antennal scale (R) 0 = Carapace CI Third ma.xilliped (L) 0 = C2 Third maxilliped (R) 0 = C3 Pereiopod I (L) The C4 Pereiopod I (R) 0 = C5 Pereiopod II (L) 0.2 C6 Pereiopod II (R) 0.5 C7 Pereiopod III (L) 0.7 C8 Pereiopod III IR) 0.9 C9 Pereiopod IV (L) 0.9 CIO Pereiopod IV (R) 0.9 CU Pereiopod V (L) 0.9 C12 Pereiopod V (R) 0.9 C13 Carapace itself 0.5 Abdomen Al Somites I-VI 0.5 A2 Pleurons I-V 0.5 A3 Pleopods I(L and R) 0.5 A4 Pleopods II (L and R) 0.5 A5 Pleopods III (L and R) 0.5 A6 Pleopods IV (L and R) 0.5 A7 Pleopods V (L and R) 0.5 Tail Fan Tl Tel son 0 = T2 Uropods (L) 0 = T3 Uropods (R) 0 = completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage. I completely broken off. 0.5 = some damage. I completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage, I completely broken off, 0.5 = some damage. I completely broken off, 0.5 = some damage. I completely broken off, 0.5 = some damage, 1 following scores apply to all pereiopods: broken off below (bob) coxa O.I = bob basis = bob ischium 0.3 = bob merus, 0.4 = bob c; = bob propodus, 0.6 = damaged chela = broken off chela, 0.8 = broken off exopod = broken off epipod, 1 .0 = intact = broken off epipod, 1 .0 = intact = broken off epipod, 1 .0 = intact = broken off epipod, 1 .0 = intact = broken off epipod, 1 .0 = intact = some damage, 1 .0 = intact = some damage, 1 .0 = intact = some damage, 1 .0 = intact = some damage, I.O = intact = some damage, 1 .0 = intact = some damage, 1 .0 = intact = some damage, 1 .0 = intact = some damage, 1.0 = intact completely broken off, 0.5 = some damage. I both broken off. 0.5 = some damage. 1 .0 = i both broken off. 0.5 = some damage, 1 .0 .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .0 = intact .irpus .0 = ntact ntacl Scores for each body region are assessed based on the total score assigned to each body part in the region (e.g.. Rostrum has nine body parts, minmium score = 0, maximum score = 9). formed data were used because arcsine transformation did not change the ranks of the parameters. A parametric ANOVA was al.so used to compare catch weights of humpback and pink shrimp, Pandalus eous (P. borealis), among hairest methods. Four major hypotheses were tested: 1 ) gear-related damage to humpback shrimp was not equal among fishing methods, 2) proportional damage to the four major shrimp body regions differed with fishing methods, 3) total numbers and biomass of humpback shrimp in the catches TABLE 2. List of humpback slirimp body parts according to function. Region Bodv Part Function Rostrum Rostrum Antennae I (antennules) Antennae 2 Antennal scales Carapace 3rd Maxilliped Pereiopod I Pereiopod II Pereiopods III to V Abdomen Abdomen (somites and pleurons) Pleopods Tail Fan Tel son Uropods The head spine that helps deter small predators Detect waterborne smells For touch and to detect approaching predators Provide stability while swimming Holds food while pieces are pulled off with claws; used when sparring uith other shrimps If chelate, it is used to catch small prey Chelate leg with articulated carpus for grooming and retrieving scraps of food Walking legs; pereiopod V may have brushes used for grooming and cleaning eggs With tail fan, the strong muscles are used for fast backward swimming (in escape response) For forward swimming, and to brood eggs Bears the anus; involved in backward swimming Involved in backward swimmins 564 Troffe et al. differed among fishing methods, and 4) individual weight of humpback shrimp was different among fishing methods tested. RESULTS Raw data on damage scores, lengths, and weights for all indi- vidual humpback shrimp together with catch data from each fish- ing gear are presented elsewhere (Ong et al. 2002). Specific analy- ses are summarized below. TBD to Humpback Shrimp Among Fishing Methods TBD tended to be higher in trawls than traps and the otter trawl caught humpback shrimp were significantly more damaged than those from half-day traps (9.9 ± 5.0% vs. 2.0 ±1.1%: P = 0.023). Other comparisons were not statistically significant (P > 0.0: Table 3). A comparison of standardized TBD data between otter and beam trawl methods resulted in a significant difference (5.7 ± 4.1% vs. 23.6 ± 8.6%; P < 0.001). TBD to trap-caught humpback shrimp was standardized by soak time. There was no significant difference (P > 0.05) in total percent damage per hour between traps set out for 6 h in daytime compared with 17 h overnight (Table 3). There were also marked differences in the proportion of individual humpback shrimp that received any damage to the 32 body parts observed. Trawl caught humpback shrimp received the highest ratio of damaged/total individuals (otter 89.9%; beam 78.8%; overnight traps 45.0%; half-day trap 37.4%). RBD to Humpback Shrimp Among Fishing Methods Considering all three fishing methods, the carapace was the most damaged (Table 3) and showed the greatest variability of the four body parts, however, there were differences between gear types, as explained below carapace damage was represented by disfigurement, depression, partial tear-off and detachment from the thorax. There was very weak negative correlation (r^ = 0.049) between the carapace length of humpback shrimp and percent carapace damage across all tlshing methods. There were significant differences (P = 0.047) in carapace damage between fishing methods with otter trawls (16.4% ± 10.0) and beam trawls (10.3% ± 0.4) resulting in higher proportions of carapace damage than humpback shrimp harvested by overnight trap (4.5% ± 1.8) or half-day traps (2.9% ± 1.9). However, there were no pair-wise significant differences (f > 0.05) assessed with Tukey tests (Table 3). Damage to the rostrum differed among fishing gear {P = 0.003; Table 3). Damage to the rostrum of humpback shrimp har- vested by otter trawl was significantly greater (12.0% ± 2.6) com- pared with both overnight traps (5.6% ± 2.4; P = 0.02) and half- day traps (2.6% ± 1.2; f = 0.002). The rank of the proportional damage to the rostrum of humpback shrimp was the same as re- ported for the carapace, with otter trawl (12.0% ± 2.6) incurring the most damage followed by beam trawls (7.2% ± 0.9), overnight traps (5.6% ± 2.4). and half-day traps (2.6% ± 1.2), respectively (Table 3). Regardless of fishing method, the abdomen and tailfan of humpback shrimp were less damaged than the carapace and ros- trum (Table 3). There was a significant difference {P = 0.028) in the damage to the abdomen between gear types, with the beam trawl causing at least five times more damage than any other fishing method, and significantly more than the overnight traps (Tukey test, 0.01 < P < 0.025; Table 3). There were also significant differences in the amount of damage to the tailfan among fishing methods (P = 0.034). Tailfan damage from the otter trawl catch was the highest (3.0% + 1.9), with the beam trawl (1.8% ± 0.9), half-day traps (0.2% ± 0.2). and overnight traps (0.2% ± 0.2) following, respectively (Table 3). As with carapace data, there were no pair- wise significant differences (P > 0.05) when assessed with Tukey tests (Table 3). RBD to Humpback Shrimp by Each Fishing Method Damage to specific humpback shrimp body parts differed within otter trawl activity {P = 0.002; Table 4). The carapace received the highest damage scores and was significant- ly more damaged than the abdomen (Tukey test, P = 0.023) (Table 4). The damage assessments for humpback shrimp caught by beam trawl were similar to those revealed in the otter trawl catch (Table 4). There were significant differences (P < 0.001) in the damage to various shrimp body parts (Table 4). The carapace of the beam trawl caught humpback shrimp had significantly more damage TABLE 3. RBD and TBD data TUKEY Otter Beam Traps Traps ANOVA Gear Type, Body Region Trawl Trawl Overnight Half-Day Gear Type P Values RBD: Carapace 16.4 ±10 10.3 ±0.4 4..5± 1.8 2.9 ±1.9 H = 7.95. P = 0.047* NS RBD: Rostrum 12.0 ±2.6 7.2 ±0.9 5.6 ± 2.4 2.6+1.2 F = 11.14. P = 0.003* O vs HT. 0.002 0 vs OT, 0.02 RBD: Abdomen 0.2 ± 0.2 1.7± 1.7 0.0 ± 0.0 0.3 ± 0.0 H = 9.13, P = 0.028* B vs OT, 0.01 < P < 0.025 RBD: Tailfan 3.00 ±1.9 1.8 ±0.9 0.2 ± 0.2 0.2 ±0.2 H = 8.76. P = 0.034* NS TBD 9.9 ±5.0 7.4 ±0.7 3.4 ± 1.4 2.0± 1.1 F = 5.58, P = 0.023* O vs HT, 0.027 TBD standardized hy total catch (kg) by area swept (km") per hour .'i.7±4.l 23.6 ± 8.6 ND ND F = 21.2, P = 0.001* — TBD standardized by trap gear soak time (hr) ND ND 0.2 ± 0.08 0.3 ± 0.2 NS — Mean percent ± SD; » = 3 for humpback shrimp compared by gear types with ANOVA and Tukey P values; Kruskal-Wallis test applied to nonparameCric comparisons, a = 0.05. * Statistically significant: only statistically significant comparisons given for Tukey tests. O, otter trawl; B, beam trawl; HT, half-day traps, OT, overnight traps. p. HYPSiNOTus Damage by Trawling and Trapping 565 TABLE 4. RBD (mean percent + SD) (h = 3) for humpback shrimp caught by different gear types compared with \NO\ A and Tukey /' values. anova Tl'KEY Gear Type Bod) Region Body Region, P \ alues Otter trawl H = 14.46. P = 0.002* C vs A. 0.02.^ Beam trawl F = 40.08. P< 0.001* C vs R. 0.03 1 C vs A. <0.001 C vs T. <0.001 R vs T. <0.001 R vs A. <0.001 Overnight traps H = 9.24. P = 0.026* R vs A, 0.05 Half-day traps H = 8.69. P = 0.0.34* NS Kruskal-Wallis test applied lo non-parametric comparisons, a = 0.05. * Statistically significant; only statistically significant comparisons are shown for Tukey tests. C. carapace; R. rostrum; A. abdomen; T. tail fan. than the rostrum, abdomen, and tailfan (P = 0.0.31; P < 0.001; P < 0.001. respectively) and the rostrum had significantly more damage than the abdomen and tailfan (P < 0.001. P < 0.001. respectively. Table 4). Trapping caused less damage to humpback shrimp than trawl- ing; however, there were still significant differences among shrimp body parts (Table 4). A Kruskal-Wallis ANOVA with data from the half-day traps suggested there was a significant difference (P = 0.034) in the amount of damage assessed between body parts, but a ranked Tukey HSD test failed to reveal any significant differences {P > 0.05: Table 4). Damage to various humpback shrimp body parts from overnight traps, like the half-day traps. were significantly different (P = 0.026; Table 4). The greatest amount of damage in overnight traps was to the rostrum followed by the carapace tailfan and abdomen. The rostrum and abdomen were significantly different ^P = 0.05) when tested with a ranked Tukey HSD test (Table 4). Assessmenis of Humpback Shrimp Catch and Bycalch On average humpack shrimp catches were significantly higher (P < 0.001; numbers, biomass) on the trap lines relative to the beam and otter trawl transects (Table 5). Highest catches were on the overnight traplines (582, 7.9 kg). The average weight of indi- vidual humpback shrimp was higher for trap-caught animals. (P < 0.0001). Overnight traps collected the largest humpback shrimp (13.7 ± 0.5 g) followed by half-day traps (13.0 ± 0.3 g). otter trawls (8.3 ±0.5 g), and beam trawls (7.1 ± 1.5 g). respectively (Table 5). There were significant differences in the individual weights of humpback shrimp caught by both otter and beam trawl when com- pared with both half-day (P < 0.001) and overnight traps (P < 0.001; Table 5). In addition to humpback shrimp, several other fish and inver- tebrate species were caught. Beam trawl and otter trawl bycatch was dominated by demersal fish, roundfish, and other shrimp spe- cies. Bycatch from the traps included decapod crustaceans, echi- noderms, roundfish and smaller shrimp species (Table 6). The average bycatch of finfish was 4 ± 3 per trapline. 5 1 ± 1 8 per beam trawl, and 376 ± 218 per otter trawl. Pink shrimp were present in the catch of all harvest methods. However, the abundance of this species was significantly higher in the otter trawl catches (P < 0.0001; Table 6). DISCUSSION Effects on Sunival The type and extent of damage to individual shrimp will likely affect survivorship if humpback shrimp are released following their capture, as found for other Crustacea. Stevens (1990) inves- tigated the survival of trawl-caught king crab (Paralithodes camtschaticus) and tanner crdh {Chionocceres bainii and C. opilio) in the Bering Sea. After injuries to both body and legs of the crabs, survival rates in experimental tanks were about 507^ and 75%, respectively, for the two crabs. Lancaster and Frid (2002) docu- mented survival of undersize brown shrimp, Crangon crangon. from an UK beam trawl fishery. They reported low mortality and only the occasional loss of a telson or antennae after the catch was brought aboard and sorted by mechanical riddle. However. Berg- mann and Moore (2001 ) suggested that post-trawling mortality of discarded decapod crustaceans have been underestimated, and showed that damaged decapods had a significantly lower longer- term survival {30% ) than controls (72-83%). Mensink et al. (2000) showed that only 40% of common whelks, Bucciniim iiiniatiiiu. TABLE 5. Mean biomass and counts (±SD) per transect for humpback shrimp catches arranged by gear types (n = 3). Gear Type Otter Trawl Beam Trawl Traps Overnight Traps Half-Day ANOVA Gear Type Mean catch weight (kg) 0.3 + 0.2 3.2+1.6 7.9 ±1.5 6.6 ±1.4 Mean catch count (no.) 41 ±22 458 ± 226 582 ±131 508 ± 99 Mean individual shrimp weight (g) 8.3 + 0.5 7.1 + 1.5 13.7±0.5 13.0±0.3 20.4. />< 0.001* 8.9, P = 0.006* F = 44.0. P< 0.0001^ TUKEY Gear Type, P Values B vsOT. 0.01 O vs HT. 0.002 O vs OT. <0.0001 B vs O, 0.027 O vs HT. 0.015 O vs OT, 0.007 B vs HT, <0.001 B vs OT, <0.001 O vs HT, 0.001 O vs OT. <0.0001 Biomass and counts of various harvests compared separately among gear types with ANOVA and Tukey tests, a = 0.05. * Statistically significant; only statistically significant comparisons are shown for Tukey tests. O. otter trawl; B. beam trawl; OT. overnight traps; HT. half-day traps. 566 Troffe et al. TABLE 6. Mean number of animals harvested from beam trawl, otter trawl, individual trap and trapline catches in Simoom Sound in = 3). Species Beam Trawl Otter Trawl Individual Trap HD Individual Trap ON Trapline HD Trapline ON Common Name Scientific Name Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Mean ± SD Shrimp Humpback Pandalus hypsinotus 441 + 339 41 ±23 13 ±2 15 ±3 508 ± 99.2 582 ± 151 Prawn Puiukdus platyceros — — 0.08 ±0.1 0.4 ± 0.3 3 ±4 17 ± 13 Spiny pink Pciiulalus eous (P. borealis) 42 ± 63 6123+ 1059 0.2 ±0.1 0.2 ±0.1 8.3 + 7 10 ±6 Two-spined crangon Crangon communis 31 ± 10 1 ±0 — — Short-scaled eualid Eiiahis suckleyi 0.2 ± 0.3 — 0.2 ±0.2 7±3 8±8 267 ± 140 Flatfish English sole Pleuroneaes vetulus 0.3 ± 0.3 — — — — — Flathead sole Hippoglossoides elnssodon 26 ± 12 0.7 ± 0.6 — — — — Rock sole Pleuronectes hdineatus 0.5 ± 0.5 — — — — — Arrowtooth flounder Atheresdies stoniias 0.2 ± 0.3 — — — — — Selachii Spiny dogfish Squalus acanthias 0.2 ± 0.3 — — — — — Spotted ratfish Hydrolagus colliei 9±3 — — — — — Longnose skate Raja rhina 0.2 ± 0.3 — — — — — Roundflsh Pacific tomcod Microgadus proxinnis — 0.7 ±0.6 — — — — Pacific herring Cliipea harengus pallasi — 229 ± 29 — — — — Blackbelly eelpout Lxcodopsis pacifica 0.8 ±0.3 — — — — — Lingcod Ophiodon elongarus 0.2 ± 0.3 — — — — — Black cod Anoplopoma fimbriii — — 0.03 + 0.01 0.008 ±0.01 1.0 ±0.0 0.3 ± 0.6 Walleye Pollock Theragra chalcogramma 1.2± 1.6 1.2 ± 1 — — — — Sandlance Ammodytes hexaplerus — 1.5 ±2.6 — — — — Dwarf wrymouth Lyconectes ateutensis — — 0.008 ±0.01 0.05 ± 0.02 0.3 ± 0.6 2.0 ± 1.0 Shiner perch Cymatogaster aggregata 14 ± 13 144.5 ±96 0.05 ± 0.03 0.008 ±0.01 2.3 ± 1.5 0.3 ± 0.6 Showy snailtlsh Liparis pulchellus 0.2 ± 0.3 — — — — — Staghom sculpin Leptocottus annatus — — 0.008 ± 0.01 0.008 + 0.01 0.3 + 0.6 0.3 + 0.6 Prickleback Stichaeidae (Family) — 0.2 ± 0.3 — — — — Capelin Mallolus villosiis 0.2 ±0.3 — — — — — Invertebrates Clams Bivalvia (class) 0.5 ± 0.5 — — — — — Smbby squid Rossia pacifica 0.2 + 0.3 — — — — — Squid Lidigo opalescens — 0.3 ± 0.6 — — — — Flatworm Turbellaria (class) — 0.3 ± 0.6 — — — — Dungeness crab Cancer niagister — — 0.02 ± 0.03 — 0.7 ±1.2 — Decorator crab Majidae (family) — — 0.008 ±0.01 — 0.36 ± 0.6 — Spider crab Majidae (family) — — 0.008 + 0.01 — 0.3 ±0.6 Tanner crab Chionoecetes bairdi — — 0.008 ±0.01 0.008 ±0.01 0.3 ± 0.6 0.3 + 0.6 Sunflower star Pycnopodia helianlhoides — — 0.03 ±0.1 0.05 ± 0 1.3 + 0.6 2.0 + 0.0 Trapline data calculated for 40 traps. ON. overnight traplines sets of approx. 17 h; HD. half-day traplines sets of approx. 6 h; — indicates no catch. harvested by a beam trawl survived after a 6-week experimental period. Over 95% of whelks harvested in baited traps survived, suggesting that trapping was relatively benign compared with trawling. Other models assessing collateral mortalities to benthic megafauna have suggested that 5-39% of annual mortalities in fisheries on the Dutch continental shelf can be attributed to trawl discards, with half of the species observed showing values of greater than 20% annual mortality (Bergmann and van Santbrink 2000). Future studies should calibrate damage assessment with survivorship to determine the level of resolution required to create meaningful damage indices. Possible Causes of Damage It is likely that the relatively severe damage to humpback shrimp from otter trawling was caused by a combination of factors. Otter trawl gear was towed at higher speeds for a relatively long distance (643-677 in), had a larger net opening, a finer cod-end mesh and caught more non-target species than trapping or beam trawling. The otter trawl yielded the smallest catch of humpback shrimp of the three gears, but exhibited the largest bycatch of finfish and pink shrimp, which likely resulted in higher internal net pressures, more scouring by other spiny crustaceans, and ulti- mately more damage to the humpback shrimp catch. However, when total damage to humpback shrimp caught by trawl gear was standardized by total catch weight (kg) by area (km") fished per time (h) TBD to humpback shrimp caught by beam trawl was higher compared with otter trawl caught animals. It is important to standardize damage data in a format that will allow for cross- comparisons to take place between investigations. We have chosen to standardize the data according to catch, fished area, and fishing p. HYPsiNOTUs Damage by Trawling and Trapping 567 duration to incorporate effects imposed by factors such as catch size, duration of fishing activity, and boat speed. BRDs reduction devices were shown to reduce head, carapace, and tail damage to trawl caught penaeid prawns (Salini et al. 2000). These authors also mentioned that prawn damage was higher when large animals were present in the trawls, possibly because of their trashing effect in the codend. In our study, it is possible that the trawls we used might have intlicled more damage to humpback shrimp if BRDs had not been used. Further data are needed to confirm this. Haulup water pressure and bycatch may have also caused dam- age to humpback shrimps in traps. However, as the traps were hauled through the water over a short distance (62-75 m, verti- cally) at a relatively slow rate (about 15 m/minute) and caught fewer bycatch species these factors were lessened relative to trawl- ing. The rostrum and carapace of humpback shrimp from the over- night traps were more damaged than half-day traps possibly be- cause the 17 h soak allowed for more time for the humpback shrimp to collide with each other in efforts to escape. This may have been particularly important at night when shrimp are more active and may have even been attempting to swim off the bottom. Catches were also higher in the overnight sets, leading to more crowding in the traps. However, the differences in damage to the rostrum and carapace for half day and overnight traps were not evident when standardized by soak time. Additional Damage to Humpback Shrimp from Fishing Gear In addition to the fragmenting and crushing injuries we ob- served on the exoskeleton of humpback shrimp, other important but subtle anatomic damage likely occurred during or after capture by both traw I and trap. Examples would be the loss of integumen- tal scales which function as distance receptors (Mauchline et al. 1977) and sensilla (20-500 [jtm in length) found on the antennae, carapace, walking legs, abdomen, telson, and uropods (Heinisch & Wiese 1987). As observed with our methods, the tailfan and ab- dominal region of humpback shrimp were the most resistant to damage (<2% damage), and the tailfan received more damage than the abdomen. Tailfan damage differed among gear types, with the beam trawl resulting in the most damage. The telson, which is borne on the tailfan, was reported to carry two pairs of tuft organs used as chemosensors (Mauchline et al. 1977). In our study, the highest damage scores were assigned to the carapace in all cases except overnight trapping, where the rostrum received the highest damage. The carapace of humpback shrimp also received the high- est cumulative damage scores of any body region surveyed and is likely the most critical part of the shrimp's anatomy for survival as it houses the cardiac, gastric and branchial organs. The visceral or gastric region of lobsters {Homanis americanus) were also iden- tified by Ganz (1980) as particularly vulnerable to damage from otter trawling, especially during the molting phase. Because our work only assessed damage to humpback shrimp when they are not molting, the estimates presented herein are minima. Further work at different seasons would be required to investigate effects on humpback shrimp at different life stages. Humpback Shrimp Catch and Biomass There were considerable differences in the biomass of hump- back shrimp harvested in Simoom Sound among fishing methods. Although they fished over approximately the same distance along the bottom of Simoom Sound (500-700 m) humpback shrimp catches in traps were higher than trawl catches. A similar pattern can be seen in the commercial fishery, with trap vessels usually landing more humpback shrimp compared with trawlers (average 20 metric tonnes (t) vs 10 t per year; Boutillier & Nguyen 1999). Standardization of catches by trawling and trapping vessels may be possible using fuel consumption, but data on specific vessels would be required. The mean individual weights of humpback shrimp caught by trap were significantly higher than those from beam trawl or otter trawl, suggesting that traps were selecting for larger individuals. Wright and Panek (2000) http://www.orst.edu/ Dept/IIFET/2000/papers/wright2.pdf have suggested that there is an inverse relationship between the trap soak-time and the weight of prawns {Pandahis platyceros) harvested. However, we found no significant differences between the individual size of humpback shrimp caught in half-day versus overnight traps. ACKNOWLEDGMENTS Thanks are owed to the Masters of the fishing vessels for their cooperation and assistance during this study. Victor Keong, Beth Piercey, Shane Petersen, and Hugh McLean provided great help in the fieldwork and laboratory analyses. Jim Helfield, Tamara Ro- manuk, Laurie Convey, and Sung-Yun Hong kindly provided com- ments on the manuscript. Dario Stucchi provided the oceano- graphic data for Simoom Sound. Funding for this study was pro- vided by the DFO Environmental Sciences Strategic Research Fund and Science Branch. Pacific Resion. LITERATURE CITED Bergmann. M. & P. G. Moore. 2001. Survival of decapod crustaceans discarded in the Nephrops fishery of the Clyde Sea area. ScotUind. ICES J. Mar. Sci. 58:163-171. Bergmann. M. J. N. & J. W. van Santbrink. 2000. Mortality in megafaunul benthic populations caused by trawl fisheries on the Dutch continental shelf in the North Sea in 1994. ICES J. Mar. Sci. 57:1321-13.31. Boutillier. J. A. & H. Nguyen. 1999. Paiuialus Inpsinoms. Humpback shrimp, A review of the biology and recommended assessment frame- work for a directed fishery. DFO Canadian Stock Assessment Secre- tariat Research Document 99/067. Butler, T. H. 1980. Shrimps of the Pacific coast of Canada. Cun. Bull. Fish. Aquat. Sci. 202:280. DeAlteris. J. T. & D. M. Reifsteck. 1993. Escapement and survival of fish from the codend of a demersal trawl. ICES Mar. Sci. Sym. 196:128- 131. Department of Fisheries and Oceans. 2002. Web site on Shrimp Fishery: Pacific Region, (http://www.pac.dfo-mpo.gc.ca/ops/fm/shellfish/ shrimp/gear.htm) Accessed 2003 Mar 10. Ganz, A. 1980. Otter trawl induced lobster damage evaluation. In: E. A. Yablonskaya, editor. Rhode Island Department of Environmental Man- agement, Completion Report, September 198(1. Wickford. RI: 28 p. Hansson, M.. M. Lindegarth. D. Valentinsson & M. Ulmestrand. 2000. Effects of shrimp-trawling on abundance of benthic macrofauna in Gullmarstjorden. Sweden. .Mar Ecol. Proi;. Ser 198:191-201. Heinisch. P. & K. Wiese. 1987. Sensitivity to movement and vibration of water in the North Sea shrimp, Crangon crangon L. / Crust. Bio. 7:401-413. Jamieson. G. & R. O' Boyle. 2001. Proceedings of the national workshop on objectives and indicators for ecosystem-based management, 27 Feb- ruary-2 March, 2001. Sidney. British Columbia. Canadian Science Advisory Secretariat Proceedings Series 2001/09. 568 Troffe et al. Lancaster. J. & C. L. J. Frid. 2002. The fate of discarded juvenile brown shrimps. Crangon crangon. in the Solway Firth UK fishery. Fish. Res. 58:95-107. Mauchline. J.. Y. Aizawa. T. Ishimaru. S. Nishida & R. Marumo. 1977. Integumental sensilla of pelagic decapod crustaceans. Mar. Biol. 4.^: 149-156. Mensink, B.P., C.V. Fischer. G.C. Cadee. M. Fonds, C.C. Tens Hallers- Tjabbes & J. P. Boon. 2000. Shell damage and mortality in the common whelk, Biicciiium undatum, caused by beam trawl fishery. J. Sea Res. 43:53-64. Ong, S.. C. D. Levmgs. T. F. Sutherland. G. E. Piercey. V. Keong & R. Davis. 2002. Data record on trawling and trappmg effects on humpback shrimp bycatch organisms in Simoom Sound and Northumberland Channel. Can. Dam Rep. Fish. Aqua. Sci. 1084 1 14 p. Richard, G. 1999. An assessment of trawling technology in Canada. Ot- tawa, Ontario, Canada: Fisheries Management. Fisheries and Oceans, 45 p. Salini. J.. D. Brewer, M. Farmer & N. Rawlinson. 2000. Assessment and benefits of damage reduction in prawns due to use of different bycatch reduction devices in the Gulf of Carpentaria. Aust. Fish. Res. 45:1-8. Stevens. B. G. 1990. Survival of king and tanner crabs captured by com- mercial sole trawls. Fish. Bull. 88:731-744. Stevens, B. G.. I. Vining. S. Byersdorfer, & W. Donaldson. 2000. Ghost fishing by Tanner crab iChioiwecetes bairdi) pots off Kodiak. Alaska: Pot density and catch per trap as determined from sidescan sonar and pot recovery data. Fish. Bull. 98:389-399. Suuronen. P.. J. A. Perez-Comas, E. Lehtonen & V. Tschemij. 1996. Size- related mortality of herring (Clupea harengus L.) escaping through a rigid sorting grid and trawl codend meshes. ICES J. Mar. Sci. 53:691- 700. Wright, C. S. & P. Panek. 2000. Effect of soak-time on the trap-selectivity profile and by-kill in prawn-trap fisheries. International Institute of Fisheries Economies and Trade. Oregon State University. July 10-15. 2000. PDF files of proceedings: Accessed 2001 Dec 27. Zar, J. H. 1984. Biostatistical analysis. 2nd ed. Englewoods Cliffs, NJ: Prentice-Hall, Inc.. 7 1 8 p. Journal of Shellfish Research. Vol. 22, No. 2, 569-579, 2003. INTRASPECIFIC AGGREGATION STRUCTURE OF A SHOAL OF A WESTERN MEDITERRANEAN (CATALAN COAST) DEEP-SEA SHRIMP, ARISTEUS ANTENNATUS (RISSO, 1816), DURING THE REPRODUCTIVE PERIOD FRANCESC SARDA,'* JOAN B. COMPANY,' AND ARTURO CASTELLON" ^Institut de Ciencies del Mar (CMIMA-CSIC) and 'Unidad de Teaiologia Marina (CMIMA-CSIC). Passeig Maritim de la Barceloneta 37-49. 0,H003 Barcelona. Spain ABSTRACT The deep-sea rose shrimp, Arisleus amennalus. constitute an important fishery resource in the Western Mediterranean Sea. The spatio-temporal behavioral pattern of A. antennatiis is well-known, with the species forming seasonal aggregations on the middle slope at depths between 400 and 900 m. These aggregations form between late winter and early summer. The object of the present study is to determine the internal structure of shoals of the western Mediterranean (Catalan coa.st) rose shrimp along the slope on the grounds where the species is tlshed (from 400 to 10(X) m) at the tmie of peak density during the reproductive period. Interactions between fishing and research vessel have been used to sample synchromcally and bathymetrically the shoals of the deep-sea shrimp to determine intra and interspecific shoal structures. The results of this study on A. uineiiiiatiis have specifically shown that ( 1 ) The pattern shrimp shoal distribution is such that density rises rapidly in the portion located in the shallower distribution range of this species and then gradually decreases at greater depths; (2) the distribution of this resource straddles both sides of the ecological boundary located at 900 m, though with changes in the sex-ratio and individual size: (3) species coexisting with this shrimp species are concentrated at depths other than the depths of peak shrimp density; (4) commercial trawlers deploy according to the abundance pattern of the resource; and (5) the reproductive portion of the stock is heavily exploited. KEY WORDS: shoals Arisleus aiueimauis. Mediterranean Sea. population structure, aggregation, sex ratio, size frequencies, fisheries. INTRODUCTION The deep-sea rose shrimp, Arisleus anteniuitiis (Risso, 1816) (Crustacea. Decapoda, Dendrobranchiata, Aristeidae), represents an important fishery in the Western Mediterranean Sea (Sarda & Martin 1986, Demestre & Lleonart 1993, Bianchini & Ragonese 1994, Carbonell et al. 1999). This species is a characteristic com- ponent of the demersal muddy bottom community on the middle slope at depths between 400 and 1,200 m (Cartes & Sarda 1993), where Cartes & Sarda ( 1992) and Maynou & Cartes (2000) have defined it as a nektobenthic species of moderate-to-high swimming mobility. However, the distribution of this species is also fished frequently between 400 and 800 m in other Mediterranean areas (Bianchini & Regonese 1994, Carbonell et al. 1999, Papaconstan- tinou & Kapiris 2001, Cau et al. 2002). The distribution of this species is nonetheless considerably broader, reaching at least to depths of 2250 m (Sarda & Cartes 1992, 1993), indicating that the species is eurybathic with a distribution considerably broader than that of other decapod crustacean species. The spatiotemporal behavioral pattern of /I. antennanis is well known, with the species forming seasonal aggregations on the middle slope at depths between 400 and 900 m. These aggrega- tions form between late winter and early summer (Tobar & Sarda 1987, Demestre & Martin 1993, Sarda et al. 1994). Towards the end of summer, the shrimp shoals tend to break up and move inside submarine canyons, with the shrimp being fished at shallower depths (400-700 m) along the margins of the canyons, locations that are less accessible to trawlers (Sarda 1993, Sarda et al. 1994, Sarda et al. 1997). During the period in which this species forms aggregations (late winter to early summer), shoals consist of reproductive adult females. Copulation takes place at the start of the aggregation stage (Relini Orsi, 1980, Sarda & Demestre 1987) with a percentage of *Corresponding author. E-mail; siscu@icm.csic.es males in the population of less than 20% (Sarda & Cartes 1992, Demestre & Martin 1993, Sarda et al. 1994). Tursi et al. (1996) reported that during copulation in late winter, males can be 50% of the population in Ionian Sea. Studies conducted on the catchability of shoals of this species (Sarda & Maynou 1998) have suggested that the shoals take on an elongate shape parallel to the coast. It is exactly at this time when the shrimp stock bears the brunt of fishing effort (Tudela et al. 2003), because shoal formation is at its peak on the part of the slope most readily accessible to trawlers and females attain maximum size, that is, biomass concentration is also at its peak. In addition, marketability of this species is also highest at this time. Studies on schooling in pelagic species (Swartzman et al. 1994. Nonacs et al. 1994, Nottestad et al. 1996) particularly using echo- sounding, and in species in captivity (Pitcher 1983, Pitcher et al. 1985), have been common, but there have been very few such studies on benthic or benthopelagic species. Gordoa & Duarte (1991) considered some Merliiccius species and reported size- dependent schooling behavior. Macpherson & Duarte ( 1991 ) also related size and depth for different fish species and discussed the possible existence of a general size-depth relationship. On the whole, studies on schooling and shoaling behavior have been quite diverse in terms of methodology used, and they have also dealt with a range of different aspects. Furthermore, although shoaling of coastal prawns and migratory displacements relating to their life cycles are well known (Garcia & Le Reste 1987), our literature review has not disclosed any similar studies on shoaling patterns for species dwelling at depths below the margin of the continental shelf Accordingly, the object of the present study was to determine the depth structure of shoals of the Catalan coast rose shrimp, Arisleus antennanis (Risso, 1816), along the slope on the grounds where the species is fished (from 700 to 1000 m) at the time of peak density (aggregation). Bearing in mind, however, that the depth distribution for this species extends across several commu- nity boundaries (down to at least 3000 in in depth; Sarda 2001 ). the 569 570 Sarda et al. role of the noncommercially exploited portion ot the population on the population as a whole has also been discussed. Shoaling struc- ture has been considered in terms of both intraspecific aspects, such as density, size range, and sex ratio, and interspecific aspects, i.e.. density relationships between the rose shrimp and other fish and crustacean species dwelling in the same faunal assemblage, on the basis of depth. Our goal has been to underscore the importance of understanding the intra and interspecific structure of aggrega- tions of marine species as a significant factor in establishing the actual level of vulnerability to exploitation by fisheries. In addi- tion, over and above a simple discussion of the results presented here, this article aspires to be an example of studies of this kind and thus also includes a consideration of ecological and fisheries aspects in the discussion, relating them to the specialized literature. MATERIAL AND METHODS A study was conducted jointly by the RN Garcia del Cid and commercial trawlers on 21 to 23 June 2000 on the "Serola" fishing grounds located off Barcelona (Northwest Mediterranean Sea). where mature females of the deep-sea rose shrimp typically ag- gregate at that time of year (Fig. 1 ). To be able to obtain an instantaneous view of the aggregation structure of a shoal of this deep-water species, operations must be completed in the shortest possible time to avoid variations in re- sponse to sudden environmental changes affecting community structure. The weather was sunny and good over the 48 h in which sampling was performed and remained stable over the course of the survey. The fishing vessels operating in this area are trawlers from the port of Barcelona specialized in the shrimp fishery, with engine power ratings ranging between 800 and 1100 horsepower and lengths between 17 and 21 m. Five fishing trawlers conducted fishing operations during their normal operating hours at depths 41.4- 41.3- 41.2- 41.1 50 m 100 m 200 m 400 m 700 m 900 m ^« ,^v^^^ .nnili.s mordax. as a schooling predator explaining patchy prey. Deep-Sea Res. Part II 41:147-170. Nottestad. L.. M. Aksland, A. Bellestad, A. Femo, A. Johannssen & O. A. Misud. 1996. Schooling dynamics of Norwegian spring spawning her- ring {Cliipea harengus L.) in a coastal spawning area. Sarsia %0:TI1- 284. Pitcher. T. J. 1983. Heuristic definitions of shoaling behaviour. Anim. Behav. 31:611-613. Pitcher. T. J.. A. E. Magurran & J. I. Edwards. 1985. Schoolmg mackerel and herring choose neighbours of similar size. Mar. Biol. 86:319-322. Puig. P., J. B. Company. F. Sarda and A. Palanques. 2001. Responses of deep-water shrimp populations to the presence of intermediate nepheloid layers on continental margins. Deep-Sea Res. II. 48:2195- 2207. Relini Orsi. L. 1980 Aspetti reproducttivi in Aristeus antennatiis (Risso, 1816) (Decapoda Penaeidae) Mem. Biol. Suppl. 10:285—289. Relini. L. & Relini. G. 1979. Pesca e riproduzione del gambero rosso Aristeus antennatiis (Decapoda. Penaeidael nel Mar Ligure. Quail Civ Staz Idrohiol. Milano 7:39-62. Sabatini. A.. M. Mura. M. Murenu & A. Cau. 1999 Daily vertical migration and feeding of Aristeus antennatiis and Aristaeomorpha foliacea in Sardinian Seas. General information and book abstracts of the 7"^ Col- loquium Crustacea Decapoda Mediterranea (6-9 September 1999). University of Lisboa. Sarda. F. 1993. Bio-ecological aspects of the decapod crustacean fisheries in the Western Mediterranean. Aq. Liv. Res. 6:299-305. Sarda. F. 2001. Exploratory survey to collect data of the exploited and virgin stocks of deep-sea shrimp A. antennatiis. of interest to the CFP. Final Report EC DG VIX. Study num. 2000/39. Sarda, F. & J. E. Cartes. 1992. Distribution, abundance and selected bio- logical aspects of Aristeus antennatus in deep-water habitats in NW Mediterranean. B.I. OS. (Macedonia. Greece) 1:59-73. Sarda. F, & J. E. Cartes. 1993. Relationship between size and depth in decapod crustacean populations on the deep slope in the Western Medi- terranean. Deep-Sea Res. 40:2389-2400. Sarda. F. & J. E. Cartes. 1997. Morphological features and ecological aspects of early juvenile specimens of the aristeid shrimp Aristeus antennatus (Risso, 1816). Mar. Freshwater Res. 48:73-77. Sarda. F. & M. Demestre. 1987. Estudio bioecologico de la gamba. Aris- teus antennatus, Risso 1816. en el marCatalan. Invest. Pesq. 51{Suppl. 1):2 13-232. Sarda. F. & P. Martin. 1986. Las pe.squen'as a Catalunya (evolucio en els ullims decennis) in: I'Oceanografia. Recursos Pesqiiers de la Mar Catalana. Quadems d'Ecologia Aplicada, Diputacio de Barcelona. Sen'ei. Medi. Ambient. 9:91-112. Sarda, F. & F. Maynou. 1998. Assessing perceptions: do Catalan fishermen catch more shrimp on Fridays? Fish Res. 36:149-157. Sarda. P., J. E. Cartes & W. Norbis. 1994. Spatio-temporal structure of the deep-water shrimp Aristeus antennatus Risso. 1816 (Decapoda: Aristeidae) population in the Western Mediterranean. Fish. Bull. NOAA 92:599-607. Sarda. F., F. Maynou & L. L. Tallo. 1998. Seasonal and spatial mobility patterns of rose shrimp [Aristeus antennatus Risso, 1816) in the west- ern Mediterranean: results of a long-term study. Mar. Ecol. Prog. Ser. 159:133-141. Stefanescu. C, D. Lloris & J. Rucabado. 1992. Deep-living demersal fishes in the Catalan Sea (western Mediterranean) below a depth of 1000 m. J. Nat. Hist. 26:197-213. Stefanescu, C, D. Lloris & J. Rucabado. 1993. Deep-living fish assem- blages in the Catalan Sea (western Mediterranean) below a depth of 1000 m. Deep-Sea Res. 40:695-707. Stefanescu. C. B. Morales-Nin & E. Massuti. 1994. Fish assemblages on the slope in the Catalan Sea (western Mediterranean): Influence of a submarine canyon. J. Mar. Biol. Assoc. 74:499-512. Swartsman. G.. W. Sluetzle. K. Kulman & M. Powojouwski. 1994. Relat- ing the istribution of pollack schools in the Bering Sea to environmen- tal factors. ICES. J. Mar. Sci. 51:481^92. Tobar. R.. & F. Sarda. 1987. Analisis de las capturas de gamba en los ultimos decenios en Catalufia. Informes Tecnicos del ICM(CSIC) de Barcelona 142:1-20. Tursi. A.. A. Matarrese. G. D'Onghia. M. Panza, P. Maiorano. M. Basanisi. F. Perri. C. A. Marano & F. Casaniassima. 1996 Density, abundance and structure of population of red shnmps. Aristeus antennatus and Aristaemorpha foliacea. in the Ionian Sea (Southern Italy). EC Final Report Contract MED92.0I5 DG XIV: p. 264. Jminuil of Shellfish Reseanh. Vol. 22, No. 2, 58I-5SS. 2UUJ. SEAFOOD DEALERS' SHRIMP-PURCHASING BEHAVIOR AND PREFERENCES WITH IMPLICATIONS FOR UNITED STATES SHRIMP FARMERS FERDINAND F. WIRTH* AND KATHY J. DAVIS Food and Rcsoiiicc Economics Department. Indian River Researcli and Editcali70 cm total length were effective predators, and oysters <70 g wet total weight were preferred, and we used these sizes in subsequent experiments. Salinity did not affect feeding rates. Experiments in 30.000 L raceways indicated that scent did not significantly lower feeding rates. Parametric analyses of factorial experiments on oyster leases at two sites in Barataria Bay. Louisiana, during the fall and spring (periods of the year when fish feeding is most intense), indicated that scent reduced feeding rates by 10% to 20%. but only at one site in one season. Nonparametric analyses corroborated seasonal differences indicated by parametric analyses, but not the scent effect. We therefore conclude that scent froin dead con-specifics is not an effective control strategy under most conditions. Dredge hauls during experiments suggested mortalities to all predators ranging from 63.1% to 92.5% within the first 4 weeks after seeding. The relative mortalities to black drum, southern oyster drills [Snamcmita haeinastoimi) or possibly PerkiiLsiis marinus infections varied among sites, as did temporal patterns of mortality within and among seasons. KEY WORDS: black drum, oysters, deten-ents. olfactory INTRODUCTION Oyster reefs (Crassostreu virginica) are important components of Gulf of Mexico coastal ecosystems, providing shelter for several economically important invertebrates and larval fish, improving water quality, and stabilizing shorelines (Bahr & Lanier 1981, Zimmerman et al. 1989). Oyster production in the northern Gulf of Mexico is greatest in an "optimal" salinity band ranging frotii 5-15 psu in coastal marshes. Survivorship is physiologically constrained at lower salinities, while predation losses are too high to sustain populations at higher salinities (Melancon et al. 1998), except at interlidal sites where oysters have a refuge from predation (Roeg- ner & Mann 1995, O'Beirn et al. 1996. Brown & Stickle 2002). Oysters spawn in the northern Gulf of Mexico as water tem- peratures rise above 25°C in the spring, and then resume spawning as temperatures drop below that level in late summer and fall (Supan 198.3, Banks & Brown 2002). Warmer temperatures in summer months are also associated with increased prevalence of the parasitic protozoan Perkinsus marinus C'Demio'"), which can result in mortality as high as 50% in oyster populations, especially at higher salinities (La Peyre et al. 2003). The Gulf of Mexico oyster industry was developed in the mid- eighteen hundreds and was further spurred by the development of the oyster dredge in the early nineteen hundreds. Oysters are har- vested from public areas (mostly reserved as "seed grounds" where leaseholders can collect small oysters to plant on their leases) and private leases. Seed oysters are planted in the fall, and typically harvested in their second spring when they reach market size. Louisiana currently has approximately 8700 leases covering 419.000 acres under cultivation. Louisiana's oyster production av- erages almost one third of the national production, and the Gulf of Mexico region produces 60% of the national production, worth almost 50 million dollars annually. Coastal zones producing maximal oyster yields are however This research was supported through the Gulf Oyster Industry Program of the National Sea Grant College. changing, as coastal erosion results in saltwater intrusion, shrink- ing the optimal salinity band (Melancon et al. 1998). As salinities increase, predation by black drum (Pogonias cromis) and southern oyster drills {Stramonita haemastoma) increases, as does preva- lence of Dermo. The iinportance of black drum as predators was clearly indicated by a survey of Louisiana oyster leaseholders (Louisiana Department of Wildlife and Fisheries [LDWF] 1999) indicating significant loss in 55% of the leases. Seed oysters may be stressed during transport from seed areas to oyster leases, and they produce scents that attract black drum. Almost 80% of lease- holders who had recently seeded oysters reported significant losses to black drum. Black drum inhabit near-shore and estuarine waters in the Gulf of Mexico (Simmons &. Breuer 1962), mature at the end of their second year, and spawn in coastal passes from February through March. The larvae are transported into estuaries where the juve- niles mature. The largest black drum in the Gulf of Mexico are over 40 years old, and reach 105 cm in length and 29 kg in weight (Sutter et al. 1986, Beckman et al. 1990). Black drum consume over 30 oysters per night (Sutter et al. 1986), with small "seed" oysters planted in leases preferred over natural reefs (Cave 1978, Cave & Cake 1980, Dugas 1986). Oysters are consumed by fish greater than 40 cm in total length, and oyster sizes consumed increase with length. Juvenile black drum feed on a variety of invertebrates (Pearson 1929, Gunter 1945, Darnell 1958). Produc- tion losses may be as high as 1500 sacks per lease in some parishes (LDWF 1999). Most oysters are lost in March, when groups offish return to coastal leases to feed after spawning, or in October, immediately after small seed oysters are bedded. Our long-term goal is to develop deterrents to black drum pre- dation on oyster leases. We first performed preliminary experi- ments to understand basic aspects of the predator-prey interaction, such as the vulnerability of different size classes of oysters, the role that drum body size plays in determining feeding rates and prey-size selection, and how the predator-prey interaction is af- fected by salinity. Based on prior experimental work (Cave 1978), or analyses of diet (Dugas 1986, Luquet 1992), our hypothesis wa.s 589 590 Brown et al. that larger fish would be more effective predators, and small oys- ters most at risk. We also expected reduced feeding rates with lower salinity, because leases in coastal areas experience higher mortality (LDWF 1999). These experiments also determined which sizes of predators and prey and salinities were used in the laboratory olfactory cue experiments discussed in the next para- graph. Second, we compared feeding rates in the laboratory, with or without a black drum carcass; our hypothesis being that alarm substances deter predation, as leaseholders report that carcasses suspended above leases reduce losses (P. Vujnovich Jr., Pers. Comm.). Alarm substances have been shown in other cases to cause avoidance behavior in fish (reviewed in Smith et al. 1994, Mathis et al. 1995, Chivers & Smith 1998). If scent cues reduce feeding rates, scent, or components of scent, could be added above the lease as a deterrent. Third, to determine if olfactory cues were practical deterrents under field conditions, we conducted experiments on commercial leases in Barataria Bay, Louisiana. With the help of a leaseholder, we planted leases with seed oysters with or without drum car- casses. Comparison of predation rates between control and experi- mental plots determined whether scent was an effective deterrent. We placed oysters in trays and recorded their survival at 2 dis- tances from scent sources in plots to determine how effective scents were at a distance, and the role of current in displacing scents. We also made hauls with a dredge to independently esti- mate losses to predation. This design was replicated on two leases, and in both the fall and spring. MATERIAL AND METHODS Preliminary Experiment Predation rates and size preference of black drum were mea- sured in experiments conducted from December 1999 to March 2000 at the LDWF Lyle St. Amant Marine Laboratory on Grand Terre Island, Louisiana. Black drum captured by hook and line or trot lines were held for 5 days at ambient salinities with oyster prey for acclimation, and were then starved for 2 days before experi- ments to standardize hunger levels. Experiments were conducted with a single fish for 5 days in 2000-L tanks with biologic filters. Barataria Bay water was mixed with fresh water to achieve aver- age salinities of 13 (±0.4, standard error of the mean) and 36 (±0.3) psu; water temperature averaged I5°C (±1.0). Oysters were pro- vided in three sizes (10 <50 g total wet mass, 10 between 51-150 g, and 7 >151 g) and were replenished daily. We used two size classes of black drum (30-70 cm (average = 51.4 ± 0.7] and >70 cm [90.6 ± 0.9] total length). Because prey sizes were presented in unison to the fish, they were not independent treatments. We there- fore used a multivariate analysis of variance (Peterson & Reynaud 1989). Effects of predator and prey size, and salinity were evalu- ated in the factorial arrangement of treatments (2 predator x 2 prey sizes X 2 salinities). Laboratory Experiment These experiments were conducted during October to April of 1999 to 2000 (to avoid low dis.solved oxygen concentrations) at the Grand Terre Laboratory in large outdoor raceways (concrete block tanks measuring 10 x 3 x 1 m deep) holding 30,000 L of water. Tanks initially received filtered seawater (ambient salinity aver- aging 27.9 ± 0.7 psu, and temperature averaging 22.4 ± 0.7°C) from Barataria Bay, and water was re-circulated through oyster chip filters during experiments. Two black drums, greater than 70 cm total length, were held in each tank. Fish were measured, tagged, and acclimated in the tank for 1 week before starting experiments. Temperature, dissolved oxygen, and salinity were measured daily. Seventy-five small and 25 medium oysters were added initially. Broken shells or missing animals were counted daily and oysters replenished. For each 5 day long experiment, two tanks were randomly selected as experimental tanks, and two as controls. Experimental tanks had a single black drum carcass suspended in a buriap bag at one end of the tank. After each experiment, fish were removed; the tank was drained and scrubbed, and refilled with filtered water from Barataria Bay. Fish used in experimental treatments were used next in controls (or the reverse) and were either then released or sacrificed and used for the scent deterrents. Numbers of oysters consumed were compared between the two treatments with a one- way analysis of variance. Field Experiment Whereas laboratory experiments aid in understanding predator behavior under controlled conditions, field experiments determine if olfactory cues deter predation under natural conditions and are feasible for industry use. We therefore made arrangements with a leaseholder to conduct experiments on two leases in Barataria Bay with a history of predation problems. The leases were in Lake Grand Ecaille in southeast Barataria Bay (29°35'06"N, 89°33'47"W) with historically high predation levels (Mr. Peter Vujnovich, Jr., pers. comm.), and in Creole Bay in west Barataria Bay (29°35'73"N, 89°34'10"W), a site with intermediate historical levels of black drum predation (Fig. 1 ). The field experiments were replicated twice, once in October 2000 and again in March 2001. At each site, four 60-m diameter, circular plots were seeded by the leaseholder with oysters at densities typical for leases. Each plot was randomly assigned as a control or treatment, and plots were located 100-m apart to minimize infiuence from other treat- ments. Immediately after seeding, two black drum carcasses were enclosed in burlap bags and suspended from a PVC pole in the center of the experimental plots, and plastic "oyster grow out" trays (60 x 50 x 10 cm) each with a minimum of 100 oysters were placed in all four plots to assess predation rates. Three trays were near the center, and one tray was set at the end of three equidistant rays (Fig. 2). Trays were inspected for predation. oysters replen- ished, and the deterrent sources renewed at 1-week intervals, for 4 weeks. The number of oysters gaping (a sign either of oyster drill predation. Brown & Richardson 1987, or possible mortality to Dermo), or missing (presumably either consumed or at least handled and removed from trays by black drum) were recorded at each date. Separate trays (2 per plot) enclosed with 3 cm Vexar and retrieved at the end of the experiment, indicated that natural oyster mortality, or mortality caused by handling, averaged only 2%, and so we have assumed that gaping oysters were mostly the result of predation by oyster drills. Oyster drills were quite common on trays when they were retrieved, averaging from 2.7/tray at the coastal site to 8.5/tray at the estuarine site. Each plot was also sampled with a dredge (0.3 x 0.6 m open- ing) to as.sess oyster densities. Three 30-m long dredge hauls (par- allel to each axis on which trays were .set) were taken in each plot at the start, after 2 weeks, and at the end of the experiment (after 4 weeks), and all oysters were pooled for each plot and date. Data were analyzed in repeated measures factorial analyses of Olfactory Deterrents to Black Drum 591 Figure 1. Map of Barataria Bay, with botli sites where experiments were conducted on commercial oyster leases indicated. variance. Data from trays for each date, or the three dredge hauls, were the repeated variables in the two way design (presence or absence of scent versus two seasons) conducted separately for each site. Prehminary statistical analyses at all sites indicated no sig- nificant effect of distance (e.g., center versus edge of plot. P > 0.08 in all cases) so all trays in a plot were considered replicates. Dependent variables were percent of oysters surviving (n = 12 trays for both plots in each treatment at each site at each date), percent mortality because of black drum, percent of oysters gap- ing, and numbers of oysters retrieved from dredge hauls (n = 2 plots per treatment per site per date). In several cases, data were not nomially distributed, even after log transformation. We there- fore performed a nonparametric factorial test, a 2-way (season x scent treatment) Sheirer-Ray-Hare extension of the Kruskal Wallis test (p. 446. Sokal & Rohlf, 1995). This test is essentially a two- way analysis of variance performed on the ranked data that pro- vides H statistics that test the treatment and interaction effects. Each of the 4 weeks was considered replicates in this analysis. RESULTS Preliminary Experiment Black drum size had a highly significant effect on feeding rate (Table I, Wilk's \ = 0.81, F = 7.9, P = 0.008), with larger fish consuming on average 3.8 oysters and smaller fish only 0.9 oysters per week. Oyster size was also important (Wilks's \ = 0.64, F = 9.4, P = 0.0006). with a Tukey's a posteriori test indicating that the 5.2 small oysters consumed on average was significantly greater than the 1.7 medium oysters consumed. No large oysters were consuined by the fish. In contrast, salinity neither had an effect on feeding rates (Wilk's \ = 0.99, F = 0.43. P = 0.52), nor were any of the interactions between the main treatment effects significant. Based on these experiments, we only used fish larger than 70 cm total length in later experiments, and small or medium sized oysters as prey. Because salinity had no consistent effect, we used ambient salinity water for the laboratory scent experiments. 592 Brown et al. TRAYS WITH SEED OYSTERS LOCATION OF SCENT DETERRENT CONTROL PLOT 1 EXPERIMENTAL PLOT 1 EXPERIMENTAL PLOT 2 CONTROL PLOT 2 Figure 2. Layout of the four circular plots at each of the two sites. Two plots were controls, and two plots had centrally-located black drum carcasses renewed weekly during the 4-week long experiments. Loca- tions of trays containing seed oysters are also indicated. Distances among plots are not to scale. Laboratory Experiment Under laboratory conditions, the presence of the scent of dead con-specifics depressed feeding rates (Fig. 3) but not significantly (F, ,T = 0.9. P = 0.37). Feeding rates were quite variable among replicates, and overwhelmed differences between the two treat- ments. Field Experiment At Creole Bay (Table 2), the repeated measures analysis of variance indicated a strong difference in oyster survival among weeks, and a significant interaction between time and season. The general pattern was for survival rates to increase with time (Fig. 4), although the shape of the curves differed between the fall and spring experiment. Evidently black drum were quickly attracted to the seeded leases, but moved away later (especially in the fall) as seed oysters were depleted on the lease. The significant season TABLE L Average feeding rates |x ± SE, N in parentheses) for 2 size classes of black drum feeding at 2 salinities on 3 size classes of oysters. Fish Salinity Small Medium Large <70cm \n, (5) 1.8 + 0.6 (1 ■if^c (4) 3.5 ± 1.2 0 >70 cm \yi, (6) 10.0 ±4.5 4.0 ±2.6 36%t (3) 5.7 ±4.7 2.7 ±2.7 T3 E V) c o O (0 <0 CO > O Si E 3 Z 40 30 20 10 Control Scent Treatment Figure 3. Number of oysters consumed (x ± SE) in the control and scent treatments in the laboratory experiment. There were 6 replicates in each treatment. main effect still however indicates that mortality rates were much higher overall in the spring than in the fall. Comparison of Tukey's a posteriori tests indicated that survival rates differed among sea- sons for each of the 4 weekly samples. In contrast, the scent treatment was not significant, nor were any of the interactions of scent and other treatments significant. The nonparametric test also indicated a strong difference between seasons (H = 8.5. P< 0.01), but an insignificant treatment (H = 0.3, P > 0.05) and interaction (H = O.I, P>0.Q5) effect. At Lake Grand Ecaille, there were also differences among weeks in oyster survival (Table 2), with a significant interaction between time and season. In the fall, survival rates were high initially, but dropped considerably, probably caused by movement of black drum onto the lease (Fig. 5). The significant season main effect again suggested different mortality rates among seasons, and survival was essentially zero in most of the weeks during the spring, probably caused by high predation rates by black drum present at the site from the initiation of the experiment (Fig. 5). Comparison of Tukey's a posteriori tests indicated significant dif- ferences in survival between seasons, for each of the weeks. There was also a significant treatment effect at this site however, al- though the increased survival in scent treatment plots averaged only 10-20%, and occurred only in the fall, when mortalities were TABLE 2. F values from two-way repeated measures analyses of variance of oyster survival at two sites in Barataria Bay, Louisiana, in two seasons. The repeated measures are four samples through time at each site and season. Source of Variation Creole Bay Lake Grand Ecaille Time Time x Season Time x Scent 3 way interaction Season Scent Scent x Season 80.8** 22 9** 0.4 0.1 117.9** 1.0 0.03 92.4** 94.9** 3.2* 1009.0** 13.7** 5.4* * Significant at P < 0.05 ** Significant at P < 0.01. Olfactory Deterrents to Black Drum 593 100 c ^> '> 3 CO to I. 0) > O Week Figure 4. Percent survival of oysters I x ± SE, pooled over both scent treatments, n = 24) in samples collected during 4 weeks at Creole Bay in l«o seasons. TABLE 3. F values from t\»o-way repeated measures analyses of variance of oysters collected in dredge hauls at tv\o sites in Barataria Bay, Louisiana, in two seasons. The repeated measures (= time) are three dredge hauls through time at each site and season. Source of Variation Creole Bay Time L3.1 Time x Season 2.7 Time x Scent 1.2 3 way interaction L3 Season 7.4 Scent 0.3 Scent X Season 2.9 * Significant < O.OI. Lake Grand Ecaille 32.7** 8.0 1.8 6.6 7.0* 0.1 Lfi much lower than in the spring (Fig. 5). However, a posteriori tests indicated treatment plots differed from controls only for the last week of the experiment in the fall. The nonparanietric test again indicated a significant season effect (H = 15.1, P < 0.01 ). but not a significant treatment (H = 0.4. P > 0.05) nor interaction effect (H = 0.\. P> 0.05). Oysters collected in dredge hauls also declined dramatically through time (Table 3, Fig. 6) corroborating the high mortality rates suggested by the data from the trays deployed in plots. Oyster densities also differed between the two seasons, but there were neither scent treatment effects nor interactions. Oyster survival (estimated by dividing final mean densities by initial densities) varied from 7.5'7f in the fall at Creole Bay to 8.9'7r in the spring. At Lake Grand Ecaille, 36.9% of the oysters survived in the fall, but only 9.7% in the spring. Thus these data also suggest, as did the data from trays, that mortality rates were higher in the spring at the coastal site. Nonparanietric tests were not necessary here as data were normally distributed (SAS, Inc. 1988, procedure Univariate). Mortality caused by black drum (again as estimated by the fraction of shells missing from trays) varied with time and season (Table 4. Fig. 7). At Creole Bay in the fall, mortality due to drum peaked during week 2, and was always at least 50%. In the spring, mortality caused by fish steadily declined. At Lake Grand Ecaille, mortality due to black drum steadily increased through the experi- ment in the fall, and was constant and near 100% in the spring. Conclusions from nonparanietric tests were again similar: no sig- nificant treatment effects occurred at Creole Bay, but a significant seasonal effect (H = 43, P < 0.01 ) occurred at Lake Grand Ecaille. The percentage of oysters gaping in trays also varied with time, and was dependent on season at one of the sites as well (Table 4). At Creole Bay, percentage of oysters gaping was fairly consistent through time in the fall, but increased with time in the spring. At Lake Grand Ecaille. percent of shells gaping was high initially in the fall, but declined through time. Gaping shells were essentially absent in all but the final week in the spring, explaining the strong seasonal effect at this site. The nonparanietric tests re-enforced these conclusions: none of the treatment contrasts were significant {P > 0.05) at Creole Bay, but there was a significant seasonal effect (H = 15.1, P < 0.01) at Lake Grand Ecaille. DISCUSSION Predator Prey liiteraetioii Our data indicate that large black drum are much more signifi- cant predators of oysters than smaller fish, and that smaller oysters are much more at risk. These findings corroborate earlier labora- tory feeding data (Cave 1978), studies of the diet of field caught fish (Pearson 1929, Gunter 1945, Darnell 1958, Dugas 1986), and results of surveys of oyster leaseholders (LDWF 1999). Oyster 100 O) c 80 > > 3 60 (/) (0 40 (1) *^ (0 > O 20 0 12 3 4 Week Figure 5. Percent survival of oysters (x ± SE, n = 12) in samples collected in 2 scent treatments during 4 weeks at Lake Grand Ecaille in two seasons. 3 CO X CO k_ CO >. O 1 60 ♦ CB Fall — ^- CB Spr 120 i S.— '— — — . T — H - GE Spr \ ■,, 'V \ 80 V. V ■•• X » V X * ^ N T \ -Q. X T 40 ?-.. \ " ^ "^--^^^^:^^:^^r:~::3! 1 2 3 Date Figure 6. Number of oysters collected in hauls (x ± SP-, « = 4 plots, pooled over both scent treatments) collected at three dates during field experiments at both sites in two seasons. 594 Brown et al. TABLE 4. F values from two-way repeated measures analyses of variance of oyster percent gaping and mortality caused by black drum at 2 sites in Barataria Bay, Louisiana, in two seasons. The repeated measures (= time) are four samples through time at each site and season. % by Site Source % Gaping Black Drum Creole Bay Time Time x Season 36.7** 7.9** 50.7** 6.3** Time x Scent 0.1 0.1 3 way interaction Season 0.5 3.2 0.8 0.9 Scent 0.1 0 Scent X Season 1.1 1.5 Lake Grand Ecaille Time Time x Season 21.5** 17.6** 50.5** 51.8** Time x Scent 4.1* 3.2* 3 way interaction Season 1.4 48.7** LI 128.3** Scent 0.4 0.1 Scent X Season 0.7 0.4 ' Significant at P < 0.05. ■* Sianificant A P < 0.01. leaseholders reported much higher predation rates on leases seeded with oysters the previous fall in comparison to inactive leases, or active leases that were not seeded the previous fall. Two mecha- nisms could explain this higher loss. First, our experiments suggest these small, individual oysters are easily consumed by fish. In comparison, naturally occurring oyster reefs along the Louisiana coast occur inter-tidally, where predation pressure by fish, stone crabs and oyster drills is reduced because of greater aerial expo- sure, and where oysters grow in aggregations that are hard for fish to feed on (Brown 1997. Brown & Stickle 2002). Second, oysters that are transported on boat decks from state-maintained seed areas on the eastern side of the Mississippi River to commercial leases that are often 40-80 Km away undoubtedly experience stress, and injured oysters produce scents that attract black drum (LDWF 1999). Surprisingly, we found no evidence that reduced salinities al- tered feeding rates. Perhaps salinities below the 13 ppt used in O c o Q. 100 80 60 40 20 0 B^-__ a -_J.r_7_r_m-,^ "^?'<§v . y y^ ^'X^" \X \^ tr JF \ \ i A V \T 1 / ./ ■ CB Fall / \t 1 / "©" CB Spr I -, L- _«... QE Fall J^ - B - GE Spr 2 3 Week Figure 7. Percent mortality (x ± SE. n - 12) caused by black drum at two sites in two seasons in the field experiments on leases. these experiments reduce predation. This salinity occurs at the so-called "conch line" in Lousiana estuaries, north of which oyster drills are not considered serious predators (Bahr c& Lanier 1981, Butler 1985). We also found evidence from the field experiments that losses to oyster drills were higher in the fall particularly at Creole Bay, whereas fish predation rates were higher in the spring at Lake Grand Ecaille. Black drum are apparently replacing nutri- ent reserves lost during spawning in the previous winter, and by voraciously feeding in groups, far outweigh any advantages of oyster growth during the winter months. Our field experiments certainly corroborate that predation can be a considerable mortality source for oysters planted in leases. Losses to drum occurted at high rates in both seasons, but were particulariy high at Lake Grand Ecaille in the spring, where es- sentially all oysters were handled and consumed in most of the weeks. There was also a tendency for mortality to be high initially at Creole Bay. and for the reverse pattern to occur at Lake Grand Ecaille in the fall. These different temporal patterns are probably explained by black drum either being resident on the site at the start of the experiment (for example at Creole Bay in the spring). or being attracted to the site after the leases were seeded (for example at Lake Grand Ecaille in the fall). Other mortality sources for oysters include predation by south- em oyster drills, and mortality to Dermo infections. We hypoth- esize that Dermo infections in these experiments caused minimal mortalities for four reasons: ( 1 ) southern oyster drills were recov- ered in high numbers on trays, especially at the estuarine site in the fall; (2) oysters in covered cages survived well (average of 98%) in all experiments; (3) temperatures were not extreme, as experi- ments were conducted in the spring and fall, not summer months when prevalences are higher in Louisiana oyster reefs (Cook et al. 1998. La Peyre et al. 2003), and (4) Dermo prevalences were low (weighted incidence of 0.2-0.9 on Mackin scale) in seed grounds where oysters were collected to seed our experimental plots (P. Banks, LDWF, Pers. Comm.). Assuming gaping shells were the result of southern oyster drill predation. mortality to these inver- tebrate predators appeared to be greatest in the fall, not spring months, and occurred more frequently at the more estuarine site. Importance of Scent Laboratory experiments did indicate some reduction in feeding in the presence of scent of a dead conspecific. but the effect was overwhelmed by differences in the feeding rates of individual fish in each of the independent experimental replicates. Observations indicated that fish were not fully acclimated to the laboratory settings even after a week. Although raceways were large, fish repeatedly rubbed against walls, and the concrete surface produced scrapes on the fish skin. Cave (1978) kept fish for long periods in aquaria similar to ones in our preliminary experiments, undoubt- edly resulting in better acclimation, and perhaps producing feeding rates that are more comparable to field conditions. Our field experiments did not indicate that scent of dead black drum was a practical feeding detertent. Increases in oyster survival occuned in scent plots at Lake Grand Ecaille. but only during the fall, when mortality rates were overall lower, and survival was increased only by 20% at the most. Black drum feed in groups, and a scent stimulus may have to be maximal to deter these voracious predators, especially when they feed on leases in the spring to Olfactory Deterrents to Black Drum 595 renew energy reserves. We deployed two careasses at the center of a 700-nr circular plot, and it could be argued that deploying larger numbers of carcasses would be more effective. However, this is a small area (selected as the smallest area where an oyster boat could circle and wash off seed oysters onto the sediment). Furthermore, if greater numbers of black drum are fished off leases and de- ployed, the question arises as to whether the improved survival of oysters is caused by the scent stimulus or simply reduced fish abundance and thus reduced predation pressure. ACKNOWLEDGMENTS This research was funded by the Gulf Oyster Industry Prograin of the National Sea Grant College. The authors thank the Louisiana Department of Wildlife and Fisheries Lyle St. Amant Laboratory for providing lodging and a logistical base for the research, also Dr. Frank Truesdale, and Pete Vujnovich Jr. who was instruinental in providing oysters for use in experiments, and in seeding experi- mental leases in Barataria Bay. LITERATURE CITED Bahr. L. M, & \V. P. Lanier. 1981. The ecology of intertidal oyster reefs of the south Atlantic coast: a community profile. US Fish. Wild. Serv. FWS/obs 81/LS. 105. pp. Banks. P. D. & K. M. Brown. 2002. Hydrocarbon effects on fouling as- semblages: the importance of taxal differences and spatial and tidal variation. Mar. Em: Res. 53:31 1--^26. Beckman, D. W., A. L. Stanley. J. H. Render & C. A. Wilson. 1990. Age and growth of black drum in Louisiana waters of the Gulf of Mexico. Trans. Ainer. Fish. Soc. 119:537-544. Brown, K. M. 1997. Size-specific aspects of the foraging ecology of the southern oyster drill. StramouUa haemastoma (Kool 1987). / E.xp. Mar Biol. Ecol. 214:249-262. Brown. K. M. & T. D. Richardson. 1987. Foraging ecology of the southern oyster drill Thais haemastoma (Gray): constraints on prey choice. J. Exp. Mar Biol. Ecol. 114:123-141. Brown. K. M. & W. B. Stickle. 2002. Physical constraints on the foraging ecology of an intertidal snail. Mar. Freshw. Behav. Physiol. 35:157- 166. Butler. P. A. 1985. Synoptic review of the literature on the southern oyster dnll. Thais haemastoma. NOAA Tech. Rep. NMFS No. 35:9. Cave. R. N. 1978. Predator-prey relationships involving the American oyster. Cras.wslrea virginica (Gmelin). and the black drum. Pogonias cromis (Linnaeus), in Mississippi Sound. MSc Thesis. Hammond. LA: Southeastern Louisiana University. 43 pp. Cave. R. N. & E. W. Cake. Jr. 1980. Observations on the predation of oysters by the black drum Pogonias cromis (Linnaeus) (Sciaenidae), Proc. Natl. Shellfish. Assoc. 70:121. Chivers, D. P. & R. J. F. Smith. 1998. Chemical alarm signalling in aquatic predator-prey systems: A review and prospectus. Ecoscience 5:338- 352. Cook. T.. M. Folli. J. Klinck. S. Ford & J. Miller. 1998. The relationship between increasing sea-surface temperature and the northward spread of Perkinsiis marinus (Dermo) disease epidemics in oysters. Est. Coastal Shelf Sci. 46:587-597. Darnell. R. M. 1958. Food habits of fishes and larger invertebrates of Lake Pontchartrain. Louisiana, an estuarine community. PtihI. Inst. Mar. Sci. 5:35.3-416. Dugas. C. N. 1986. Food habits of black drum. Pogonias cromis. in south- east Louisiana with emphasis on their predation of the American oyster, Crassostrea virginica. Contributions of the Marine Research Labora- tory. 1980-1985. LA Dept. Wildl. Fish. Tech. Bull. No. 40. pp. 32-38. Gunter. G. 1945. Studies on the marine fishes of Texas. Piihl. Inst. Mar. Sci. 1:1-90. La Peyre. M. K.. A. D. Nickens. A. K. Volety. G. S. Tolley & J. F. La Peyre. 2003. Environmental significance of freshets in reducing Perk- insiis marinus infection in eastern oysters Crassostrea virginica: po- tential management implications. Mar. Ecol. Prog. Ser. 248:165-176. Louisiana Department of Wildlife and Fisheries. 1999. Black drum preda- tion: results of a survey of Louisiana oysterground lease holders. Pre- liminary Report. 50 pp. Luquet. C. Jr. 1992. Oyster predation by black drum. Unpubl. Report. Louisiana Department of Wildlife and Fisheries, 13 pp. Mathis, A.. D. P. Chivers & R. J. F. Smith. 1995. Chemical alarm signals: Predator deterrents or predator attractants? Amer. Natur. 145:994- 1005. Melancon. E.. Jr.. T. Soniat. V. Cheramie. R. Dugas. J. Barras & M. LaGarde. 1998. Oyster resource zones of the Barataria and Tertebonne estuaries of Louisiana. J. Shellfish Res. 17:1 143-1 148. O'Beim, F. X.. R. L. Walker & P. B. Hefferman. 1996. Enhancement of subtidal eastern oyster, Crassostrea virginica. recruitment using mesh bag enclosures. J. Shellfish Res. 15:313-318. Pearson. J. C. 1929. Natural history and conservation of redfish and other commercial scianidson the Texas coast. Bull. US Bur. Fish. 4:129-214. Peterson. C. H. & P. E. Reynaud. 1989. Analysis of feeding preference experiments. Oecologia 80:82-86. Roegner. C. G. & R. Mann. 1995. Early recruitment and growth of the American oyster, Crassostrea virginica (Bivalvia: Ostreidae) with re- spect to tidal zonation and season. Mar. Ecol. Prog. Ser. 117:91-101. Simmons. E. G. & L. P. Breuer. 1962. A study of redfish. Sciaenops ocellata Linnaeus, and black drum. Pogonias cromis Linnaeus. Publ. Inst. Mar Sci. Univ. Texas 8:184-211. Smith. R. J. F.. A. Mathis. D. P. Chivers & C. Gelowitz. 1994. Alarm signals: a key element in fish predator-prey interactions. / Fish Biol. 45:949-954. SAS. Inc. 1988. SAS User's guide: Statistics. Gary. North Caolina: SAS Institute. Sokal, R. R. & F. J. Rohlf 1995. Biometry, 3rd edition. New York. New York: W. H. Freeman Publishers. Supan. J. 1983. Evaluation of a leased oyster bottom in Mississippi sound. Gulf Res. Rep. 7:261-266. Sutter. F. C. R. S. Waller, and T. D. Mcllwam. 1986. Species profiles: lile histories and environmental requirements of coastal fishes and inver- tebrates (Gulf of Mexico) — Black Drum. Rep. No. Biological-82 (1 1.51). Ocean Springs. MS: Gulf Coast Research Laboratory. Zimmerman. R.. T. Minello. T. Baumer & M. Castiglione. 1989. Oyster reefs as habitat for estuarine macrofauna. .MOAA Tech. Memo NMFS- SEFC 249:\6. Joimial of Shellfish Research. Vol. 22, No. 2, 597. 2003. ABSTRACTS OF TECHNICAL PAPERS Presented at the 56th Annual Meeting NATIONAL SHELLFISHERIES ASSOCIATION (Pacific Coast Section) & PACIFIC COAST SHELLFISH GROWERS ASSOCIATION Newport, Oregon September 27-30. 2002 597 NSA & PCSGA. Newport. Oregon Abstracts, September 27-30. 2002 599 CONTENTS Dan L. Ayres and Ervin J. Schumacker Assessing populations of Pacific razor clams {Silii/iiii panda) along the Pacific coast of Washington State 601 Colleen A. Burge, Yuiclii Eugene Saito and Carolyn S. Friedman Relationships between summer mortality and immune responses in (he Pacific oyster. Crassostrea gigas 601 Melinda D. Chambers, C.S. Friedman, L. Hauser and Glenn R. Vanblaricom Population structure and recovery dynamics of black abalone (Halinlis cnwherodii) at San Nicholas Island. California 601 Aimee E. Christy 2002 monitoring of harmful algae in South Puget Sound and Willapa Bay - species of concern and future considerations 601 Aimee E. Christy and Stuart D. Glasoe Literature review - impacts of urbanization on water quality in shellfish growing areas in Puget Sound, Washington 602 Marion Dumont The history and development of the Puget Sound commercial geoduck industry 602 Ford Evans, Sean Matson, John Brake and Chris Langdon Relative importance of survival and growth rate in determining yields of Pacific oysters. Crassostrea gigas 602 Carl A. Finley, Tliea T. Robbing and Carolyn S. Friedman Life history of an exotic sabellid polychaete Terehrasabella lieteroiincinata: fertilization strategy and influence of temperature on reproduction 602 C.S. Friedman, C.A. Burge, D.P. Cheney, R.A. Elston, A.D. Suhrbier, G.N. Cherr, F.J. Griffin, A. Hamdoun and C.J. Langdon Summer mortality of the Pacific oyster. Crassostrea gigas, along the West Coast of the U.S.: performance of family lines and environmental parameters 603 Carolyn S. Friedman, James D. Moore, Thea T. Rabbins, Beverly A. Braid, Carl A. Finley, Ronald P. Hedrick, Dolores V. Baxa, Karl B. Andree, Eric Rosenblum, Mark R. Vianl, Ronald S. Tjeerdema, Peter L. Haaker, Mia J. Tegner and Luis 1. Vilchis Withering syndrome of abalone in California 603 Graham E. Gillespie, Randy Webb and Todd Johansson .Assessment and management of intertidal clam resources in British Columbia 603 Blaine Griff en, Chris Langdon and Ted DeWitt Feeding rates of the mud shrimp Upogehia piigettensis and implications for estuarine phytoplunkton abundance 604 K. Holsman, P. Sean McDonald. D. Armstrong and J. Ruesink Patterns in intertidal habitat use by subadult Dungeness crab (Cancer magister) 604 Geoff Hosack, David Armstrong, Brett Dumbauld. Brice Semmens and Jennifer Ruesink Seasonal utili-^ation of intertidal habitats by fish in a Washington State ceiastal estuary 604 R. Russ Jones, Carl Schawrz, Bart DeFrietas and Lynn Lee Biomass surveys and active management of intertidal razor clams (Silic/iia patula) at beaches near Massett, Haida Gwaii. Canada 60.5 Matthew J. Krachey and Steven C. Hackett Economics of California's Dungeness crab (Cancer magister) fishery, preliminary results 605 Chris Langdon, Sean Matson, John Brake and Ford Evans The Molluscan Broodstock Program: family-based selection improves yields of Pacific oysters. Crassostrea gigas 605 Heather M. Macrellis, Jennifer L. Ruesink and Brett Dumbauld The role of culture practices in structuring interactions between cultured oysters and native eelgrass 606 Sean E. Matson and Chris Langdon A specific pathogen free culture system for Crassostrea gigas larvae and spat 606 P. Sean McDonald, Gregory C. Jensen and David A. Armstrong Biotic resistance to European green crab. Carcinus maenas, by native analogs in the Northeastern Pacific 606 C. Pearce, T. Daggett, T. Chopin, K. MacKeigan, V. Zitko and S. Robinson Effect of diet on somatic growth of juvenile green sea urchins (Sirongylocentrotiis droebachiensis) 607 Don P. Rothaus, R.E. Sizemore, M.J. Ulrich and Carolyn S. Friedman Trends in pinto abalone (Hatiolis kamtschatkana) abundance at ten sites in the San Juan Islands and management of the species in Washington State 607 600 Abstracts. September 27-30. 2002 NSA & PCSGA, Newport. Oregon Steven S. Rumrill and Victoria K. Poulton Ecological role and potential impacts of molluscan shellfish culture in the estuarine environment of Humboldt Bay. CA 607 B.C. Smith, C.E. Gnie, N.P. Kohn and J.P. Davis The effects of the herbicide Rodeo® on Pacific oyster gametogenesis and tissue accumulation 608 Andrew D. Suhrbier, .Aimee E. Christy, Hector S. Beltran, Daniel P. Cheney, Jonathan P. Davis, Kenneth M. Brooks and Frank J. Smith Mussel growth and food utilization in relation to water quality on a raft system in Puget Sound, Washington 608 Vera L. Trainer, Barbara M. Hickey and Ervin J. Schumacker Results from the Olympic Region Harmful Algal Bloom (ORHAB) Project on the Washington State coast: the value of a collaborative project 608 B. Vadopalas, L.L. LeClair and P. Bentzen Genetic differentiation amongst geoduck clam (Panopea ahnipta) populations revealed by allozyme and microsatellite analyses 609 B. Vadopalas and Don P. Rothaus Trial use of the U.S. Navy Remotely Operated Vehicle (ROV) SORD IV for sampling deep water geoduck clams ( Panopea ahnipta) 609 Donald E. Velasquez, S.F. Burton, D.A. Sterritt and B. McLaughlin Shell condition testing of Dungeness crab in Puget Sound. Washington 609 NSA & PCSGA, Newport, Oregon Abslmcis. September 27-30, 2002 601 ASSESSING POPULATIONS OF PACIFIC RAZOR CLAMS (SIUQUA PATULA) ALONG THE PACIFIC COAST OF WASHINGTON STATE. Dan L. Ayres, Washington Depart- ment of Fish and WildMfe. 48 Devonshire Road. Montesano, WA 98563; and Ervin J. Schumacker, Quinault Department of Natu- ral Resources. PC Box 189. Taholah, WA 98587. Perfect habitat for the Pacific razor clam (Silii/tia panda) is found along the Pacific Ocean beaches in Washington State. To detennine total abundance of razor clams, the newly designed Pumped Area Method recently became the method of choice. This method requires water to be pumped from the surf or a nearby lagoon. This water, as it is directed through a handheld PVC wand, is used to liquefy the sand within an aluminum ring ( '/: square meter in area). The razor clams found float to the surface and are removed, measured and returned. This process is repeated along a randomly selected transect with 6 rings completed every 50 feet. Each transect requires one turn of the tide (5 hours). For each mile of razor clam habitat determined to be on management beach, one transect is completed. The data collected is used to calculate the average number of razor clams per square meter. Using an estimate of the number of square meters of razor clam habitat, the total number of razor clams can be determined. The State of Washing- ton and the Quinault Indian Nation use this jointly determined abundance estimate to co-manage the harvest of razor clams at the Copalis, Mocrocks and Kalaloch manageinent beaches. RELATIONSHIPS BETWEEN SUMMER MORTALITY AND IMMUNE RESPONSES IN THE PACIFIC OYSTER. CRASSOSTREA GIGAS. Colleen A. Burge, Yuichi Eugene Saito and Carolyn S. Friedman, School of Aquatic and Fishery Sciences, University of Washington, Seattle, WA 98195. The Pacific oyster, Crassostrea gigas. has experienced summer mortality events in the Pacific Northwest and Japan since the mid 1950's and in California starting in 1993. Summer mortality events have been linked to multiple stressors associated with planting times and height including extreme dissolved oxygen and tempera- ture fluctuations. Hemocytes are integral in many important physi- ological processes such as nutrient digestion and transport, excre- tion, wound repair and pathogen defense. Cellular defense is the primary immune mechanism of marine invertebrates. Hemocytes found in hemolymph and interstitial spaces function in host de- fense via inflammation, wound repair, encapsulation, and phago- cytosis. Hemocyte performance may contribute to observed dif- ferences in mortality between selected family lines of C. gigas from the Molloscan Broodstock Program (MBP) of Oregon State University. To better understand the differences in oyster perfor- mance, we examined the immune response of oysters from two different MBP families grown at a site with low mortality (Totten Inlet). These families were selected based on mortality rates: high mortality (MBP family 10-116) vs. low mortality (MBP family 10-115). The ability of hemocytes to phagocytose or engulf foreign particles, move towards a chemical stimulus (chemotaxis). and kill Vibrio paraluu'inolylicits was examined. Differences and similari- ties in immune responses between the two groups of oysters will be described. POPULATION STRUCTURE AND RECOVERY DYNAM- ICS OF BLACK ABALONES {HALIOTIS CRACHERODII) AT SAN NICOLAS ISLAND, CALIFORNIA. Melinda D. Chambers, C. S. Friedman, L. Hauser, School of Aquatic and Fisheries Sciences, University of Washington, Seattle, WA 98105; and Glenn R. Vanblaricom, Washington Cooperative Fish and Wildlife Research Unit. School of Aquatic and Fisheries Sciences. University of Washington, Seattle, WA 98105. Populations of black abalone have experienced declines of 85- 99% since the emergence of the disease Withering Syndrome (WS) in 1985. Black abalone populations in the California Channel Islands formerly harbored unprecedented densities. Since 1981 we have collected data that documents the change in abundance on San Nicolas Island. Recent data indicate the first recruitment event since the onset of WS with observations of individuals sized <50 mm. A drift card study conducted in August 2002 on San Nicolas Island, indicated that dispersal was largely localized. Subsequent genetic studies will be conducted throughout 2002 and 2003 to confirm speculation that genetic differentiation corresponds with geographic distance. Tissue samples will be collected from each of the California Channel Islands that support black abalone popula- tions of high density and genetic analysis will be conducted using allozymes and mtDNA. Additionally, further drift card studies will be conducted to improve our understanding of circulation patterns in the Channel Islands and temperature data will be monitored using TidbiT stowaway devices, as elevated sea surface tempera- tures are tightly associated with WS symptoms in black abalone. 2002 MONITORING OF HARMFUL ALGAE IN SOUTH PUGET SOUND AND WILLAPA BAY— SPECIES OF CON- CERN AND FUTURE CONSIDERATIONS. Aimee E. Christy, The Evergreen State College, Olympia, WA 98505. Harmful Algal Blooms (HABs) are natural phenomena and have occurred worldwide for hundreds of years. The impacts of these blooms include both economic and health concerns for shell- fish growers, consumers and the local economies dependent on shellfish resources. Pacific Shellfish Institute (PSI) is a member of the Olympic Region Harmful Algal Bloom (ORHAB) Partnership working to understand HABs and reduce HAB impacts on humans and the environment. In addition to monitoring toxic algae in Willapa Bay, PS! independently monitors plankton communities at several locations in south Puget Sound. Monitoring efforts have detected numerous species of plankton that form HABs and are of special concern to the shellfish industry. Evidence exists that the incidences of problems associated with toxic algae are rising. Pos- 602 Abstracts. September 27-30. 2002 NSA & PCSGA. Newport, Oregon sible explanations for the increased frequency and intensity of blooms include natural dispersal of plankton via currents, climatic changes, nutrient enrichment and the transport of new species in ballast water. Understanding the factors that contribute to HABs. studying life cycles of local species, diligent monitoring and re- sponse efforts, innovative methods for quick and economic toxin detection and the ability to predict HAB occurrences are necessary steps in the protection of human health and shellfish resources. LITERATURE REVIEW— IMPACTS OF URBANIZATION ON WATER QUALITY IN SHELLFISH GROWING AREAS IN PUGET SOUND, WASHINGTON. Aimee E. Christy, The Evergreen State College, Olympia, WA 98505; and Stuart D. Glasoe, Puget Sound Water Quality Action Team, Olympia, WA 98504. In response to population growth, urbanization and worsening bacterial contamination trends throughout the region, the Puget Sound Water Quality Action Team is undertaking a study to better understand the impacts of urbanization on water quality in shell- fish growing areas. A literature review was conducted to assemble available information to determine the cunent understanding of the relationship between urbanization and bacterial contamination in the nearshore environment. Results of the literature search indicate distinct differences in bacteria sources and transport pathways be- tween rural and urban watersheds. The concentration and rapid transport of urban pollutants into receiving waters caused by the conversion of native vegetation to impervious surfaces and drain- age networks is well documented. A number of indicators (imper- vious surface coverage, developed land, population, housing den- sity) are being examined and some appear more significant than others in correlating development and bacterial contamination. Findings encourage protecting natural filtration areas, preserving buffers and native vegetation, disrupting connectivity between im- pervious surfaces and receiving waters, educating the public, and using innovative planning and low-impact development techniques to mimic and preserve natural hydrologic functions. Understanding the relationship between urbanization and water quality will pro- vide the tools necessary to develop in ways that support future growth, natural resources, public health and clean water. THE HISTORY AND DEVELOPMENT OF THE PUGET SOUND COMMERCIAL GEODUCK INDUSTRY. Marion Dumont, Univ. of Washington, Tacoma, Washington. The commercial geoduck industry had its official beginnings in Washington State in 1970, the onset of a decade defined by con- flict, transition and change. The men and women, politicians, har- vesters, leaseholders and government agents were fiercely com- petitive and adventurous, given to quarreling and trouble making. They proved a driving force for an innovative and booming in- dustry. RELATIVE IMPORTANCE OF SURVIVAL AND GROWTH RATE IN DETERMINING YIELDS OF PACIFIC OYSTERS, CRASSOSTREA GIGAS. Ford Evans, Sean Mat- son. John Brake, and Chris Langdon. Coastal Oregon Marine Experiment Station and Dept. Fisheries and Wildlife, Oregon State University, Newport, OR 97365. Data were collected on three cohorts (C-6, C-7, C-9) of unse- lected full-sib Pacific oyster (Crassostrea gigas) families. The roles which individual growth rate and survival play in determin- ing average family yield were investigated. C-6 families were planted subtidally in Yaquina Bay. OR. C-7 families were planted intertidally in Tomales Bay, CA, and subtidally in Yaquina Bay, OR. C-9 families were planted intertidally in Totten Inlet, WA. Once market size, oysters were harvested and yield, average indi- vidual growth rate and survival recorded for each family. Pheno- typic correlation coefficients (rp) between performance characters were estimated within each cohort. Correlation coefficients of per- formance characters between sites were estimated for C-7. Sur- vival was significantly correlated with yield within all cohorts (r = 0.66 to 0.98, p<0.05). Average individual growth rate was significantly correlated with yield in all cases (r^ = 0.65 to 0.93, p<0.05 ) except for C-7 in Yaquina Bay, OR (rp = 0.52. p = 0.20). Correlations between average individual growth rate and survival tended to be higher in cohorts planted intertidally (r^ = 0.70 to 0.71 ) than in cohorts planted subfidally (rp = -0.130 to 0.32). C-7 yield was significantly correlated between Tomales Bay, CA. and Yaquina Bay, OR (rp = 0.88, p<0.01J. Yield stability appeared to be driven primarily by the significant correlation of survival be- tween sites (rp = 0.89. p<0.01). Individual growth was not corre- lated between sites (rp = 0.32, p = 0.51 ). These results indicate the relative importance of survival and growth rate in contributing to yields of Pacific oysters varies between sites and cohorts. The implication of these results on breeding schemes targeting oyster yield will be discussed. LIFE HISTORY OF AN EXOTIC SABELLID POLY- CHAETE, TEREBRASABELLA HETEROUNCINATA: FER- TILIZATION STRATEGY AND INFLUENCE OF TEM- PERATURE ON REPRODUCTION. Carl A. Finley, Thea T. Robbins, California Department of Fish and Game. Bodega Marine Laboratory. P.O. Box 247, Bodega Bay, CA 94923; and Carolyn S. Friedman, School of Aquatic and Fishery Sciences, University of Washington. Box 355020. Seattle. WA 98195. Abalone culture facilities have been devastated by an exotic sabellid, Terehrasabella heterowuinata. following its introduction from South Africa in the late 1980s. Infestations are associated with shell deformities, increased mortality and financial losses. In addition, the potential introduction and establishment of this exotic pest into the natural environment was unknown. The development of an effective management strategy is dependent upon under- standing the life history of this sabellid, including its fertilization NSA & PCSGA, Newport, Oregon Ahstracls. September 27-30, 2002 603 strategy and generation time. In the present study, red abalone, Haliotis rufescens. with single sabellid infestations were isolated in containers al 18°C. This first, parental generation was held in isolation until individuals produced F, larvae, which were subse- quently isolated until indniduals produced a second, F,, genera- tion. In a separate study, uninfesled abalones were exposed to infested abalone at three temperatures typically encountered in California. Transmitted larvae were observed as they developed to specific life stages; initiation of feeding, sexual maturation and production of motile, infestive, larvae. This research demonstrated that isolated individuals are functional hermaphrodites and do pose the threat of producing fully functional offspring and that the gen- eration time of T. heieroiincinata is significantly temperature de- pendent. The aquaculture industry. UC Santa Barbara researchers and Department of Fish and Game (DFG) initiated an aggressive eradication program in 1996, and DFG policy was established in 1997 to prevent further spread of the sabellid. Culling of infested stocks and strict hygiene protocols including freshwater treatment of tanks proved effective in curbing new infestations. Results of recent eradication efforts will be described. SUMMER MORTALITY OF THE PACIFIC OYSTER, CRASSOSTREA GIGAS, ALONG THE WEST COAST OF THE U.S.: PERFORMANCE OF FAMILY LINES AND EN- VIRONMENTAL PARAMETERS. C. S. Friedman, C. A. Burge, University of Washington. Seattle, WA 98195; D.P. Cheney, R. A. Elston, A. D. Suhrbier, Pacific Shellfish Institute. Olympia, WA 98501; G. N. Cherr, F.J. Grimn, A. Hamdoun, Bodega Marine Lab, UC Davis. Bodega Bay, CA 94923: and C. J. Langdon, Hatfield Marine Science Center, OSU, Newport, OR 97365. Mortality of the Pacific oyster, Crassostrea gigas, has occurred in the U.S. west coast and Japan since the mid !950's. Multiple stressors have been implicated as contributing to these mortality events. In an attempt to alleviate the >50'7f annual oyster mortality observed in California and variable losses in Washington state, we examined the interaction between survivorship, growth and stress response of family lines from the Molluscan Broodstock Program (MBP) of Oregon State University, and planting time and height, and selected environmental parameters. To examine differential performance between family lines and planting period three oyster families were each outplanted during Fall 1999, 2000, 2001 and Spring 2000, 2001, 2002 at 2-3 sites in California, 3 sites in Washington (Spring only), and 1 site in Oregon (Spring 2002 only). Fall plants survived significantly more than did oysters planted in the spring (p<0.05) in California. In addition, two fami- lies (one commercial strain and MBP family 10-115) outper- formed MBP family 10-1 16 (pl-t- year classes), which reach densities as high as 4,300 crabs ha ' in subtidal chan- nels during low tides, may migrate during flood tides from subtidal refuges into intertidal habitats to forage. Results of a bioenergetic model for crabs in Willapa Bay, Washington, indicate that inter- tidal foraging may contribute significantly to the energy budget of subadult C. magister and may facilitate the high abundance of crabs observed in large coastal estuaries. We conducted bay wide trapping surveys in intertidal oystershell. eelgrass, and bare mud habitats in order to elucidate patterns of habitat use by subadult crabs, and underwater video was used to observe tidal migrations in these habitats. Significant differences in crab abundance and the magnitude of migrations were observed across habitats, with low- est densities of C. magister occurring in older shell beds concur- rent with high densities of red rock crabs (C productus). Obser- vations of tidal migrations using underwater video suggest that the physical structure of plants may hinder crab movement in eelgrass beds since the number and size (carapace width, CW) of crabs migrating was smallest in this habitat. Although the density of prey species may be less in open mud or sand habitats, the lack of structural hindrance and interspecific competition may render open mud the most valuable intertidal habitat to subadult crabs. The importance of intertidal habitats to subadult crabs has direct im- plications in coastal estuaries of the Northeastern Pacific where anthropogenic and biotie modification of intertidal areas threaten the productivity of intertidal habitats and may adversely impact estuarine populations of C. magister. SEASONAL UTILIZATION OF INTERTIDAL HABITATS BY FISH IN A WASHINGTON STATE COASTAL ESTU- ARY. Geoff Hosack, David Armstrong, School of Aquatic and Fishery Sciences, Box 355020. University of Washington, Seattle. WA 98195; Brett Dumbauld. Washington State Department of Fish and Wildlife. Willapa Bay Field Station, P.O. Box 190, Ocean Park, WA 98640; Brice Semmens and Jennifer Ruesink, Dept. of Zoology, University of Washington, Seattle, WA 98195. Estuaries are regarded as important nursery areas for juvenile marine and anadromous fish. Estuaries also support fisheries and aquaculture and the effects of these activities on fish habitat are NSA & PCSGA, Newport, Oregon Abstracts. September 27-30, 2002 605 becoming increasingly scrutinized under tlie Endaui-ered Species Act and Magnusou-Stevens Act . We are conducting a study to evaluate the importance of the intertidal environment tor juvenile fish within Willapa Bay, Washington with respect to aquaculture. Our objectives are to compare commercially cultivated and uncul- tivated habitats in order to; (1) elucidate potential habitat prefer- ences among juvenile fishes. (2) establish possible mechanisms for habitat preferences, and (3) evaluate the function of intertidal habi- tats for fish foraging, predator avoidance, and mobility behaviors. One-meter high hoop nets were deployed over three habitats (oys- ter culture, eelgrass and unvegetated open mud/sand) to determine habitat preference at three locations in 2001. They were also de- ployed monthly to determine seasonal presence/absence of fish species and at three different tidal elevations in 2002. Preliminary results show that intertidal use by the majority of species exhibits a pronounced increase during late spring and early summer. Few significant differences in habitat use were found, but a prototype two-boat surface trawl was designed and tested in 2002 to further investigate potential differences in utilization of these low inter- tidal habitats and adjacent subtidal channel by juvenile Chinook salmon (Oncorhyncus tshawytscha). Finally, Chinook salmon and shiner perch (Cymatogaster aggregata) have been marked with acoustic tags and held in a large enclosure to observe fine-scale movement over a suite of intertidal habitats. BIOMASS SURVEYS AND ACTIVE MANAGEMENT OF INTERTIDAL RAZOR CLAMS [SILIQUA PATULA) AT BEACHES NEAR MASSETT. HAIDA GWAII, CANADA. R. Russ Jones, Haida Fisheries Program. P.O. Box 98. Skidegate, Haida Gwaii VOX ISO; Carl Schwarz, Department of Mathemat- ics and Statistics. Simon Fraser University, Bumaby, BC V5A 1S6; Bart DeFreitas, Haida Fisheries Program, P.O. Box 87, Mas- sett, Haida Gwaii VOT IMO; Lynn Lee, Marine Toad Enterprises. P.O. Box 74, TIell. Haida Gwaii VOT lYO. Intertidal razor clam populations and biomass were estimated for commercial clam beaches near Massett, Haida Gwaii for the period 1994 to 2000 using a three stage sampling design. Clams were collected by fluidizing the substrate in a 0.5 ni" sampling cylinder. Population was estimated for three size fractions (Shell Length (SL) >4 mm, >20 mm and >90 mm, the latter being the commercial size limit) at three beach sections on 18.8 km of beach accessible to the commercial fishery. Calculations varied consid- erably in some years depending on assumptions about transect length and beach area. There was a record catch of 237 t in the fishery in 2000 that led to concerns by managers about possible overfishing. However surveys indicated that the biomass of clams >90 mm at the start of 2000 was 1876 t (SE 157 t). Biomass was shown to have been at a historic high in 2000 with large numbers of two year old clams in the population, most of which were expected to recruit to the fishery in 2001. The fishery had been passively managed using size limits for many years. However an examination of razor clam gonads showed that only 50% were mature at 87 mm. Beginning in 2001. in addition to the size limit, an annual quota was introduced in the fishery based on the annual bioinass survey and a harvest rate of 12.3% (2/3 of a 1994 estimate of Fmsy)- ECONOMICS OF CALIFORNIA'S DUNGENESS CRAB (CANCER MAGISTER) INDUSTRY. PRELIMINARY RE- SULTS. Matthew J. Kraehey, Department of Fisheries Biology. Humboldt State University, Areata, CA 95521 and Steven C. Hackett, School of Business and Economics, Humboldt State Uni- versity. Areata. CA 95521. The cunent management regime for California's Dungeness crab fishery has led to a derby, with the vast majority of the catch occurring with the first six weeks of the six-month long season. Questions have been raised about the impacts of the derby on industry structure, prices, and product quality. One thrust of our work is to identify baseline economic characteristics of the indus- try under current management. These include value added, product mix, employment, and capital investment in the processing sector, as well as value added by fishermen. Our preliminary findings indicate that, unlike former derby fisheries for tlnfish, the product forms that economically dominate are not suppressed by derby conditions. Moreover the derby fishery promotes large-scale pro- cessing facilities that create important jobs and processing capa- bility for other fish species in economically less robust coastal communities. Ongoing research will focus on fishery participant's opinions on management alternatives, number of traps deployed, and marginal fishing cost. THE MOLLUSCAN BROODSTOCK PROGRAM: FAMILY- BASED SELECTION IMPROVES YIELDS OF PACIFIC OYSTERS, CRASSOSTREA GIGAS. Chris Langdon, Sean Matson, John Brake and Ford Evans, Coastal Oregon Marine Experiment Station and Dept. Fisheries and Wildlife, Oregon State University, Newport. OR 97365. The Molluscan Broodstock Program (MBP) was established in 1995 to improve yields of Pacific oysters on the West coast, U.S., by family-based genetic selection. Parental families (PI) in three cohorts of about 60 families each were selected based on superior live weight and meat yields at harvest. Live weight yields of prog- eny (Fl) from crossing PI selected families were significantly greater than those of non-selected control families in four out of seven trials (ANOVA, p<0.001). resulting in an average gain of 9.5% after one generation of selection. The response to selection was greatest if Fl families were tested at the same site as that used for their parents' selection rather than at a different site. There were weak (p = 0.06; p = 0.04) positive correlations between the yields of families planted at both inter-tidal and sub-tidal sites, indicating strong genotype by environment interaction effects on 606 Ahslnicts. September 27-30. 2002 NSA & PCSGA. Newport. Oregon yield. Nonetheless, it was possible to identify four to six "gener- alist" families that were among the top ten families at both sites. Further evaluation of families across a wider range of environ- ments is needed to determine if the best strategy to improve oyster yields will be to select "generalist" families that perform well along the whole Pacific coast, or whether it will be more effective to develop site-specific lines instead. THE ROLE OF CULTURE PRACTICES IN STRUCTUR- ING INTERACTIONS BETWEEN CULTURED OYSTERS AND NATIVE EELGRASS. Heather M. Macrellis, Jennifer L. Rue.sink, Zoology Department. Box 351800. University of Wash- ington. Seattle. WA 98195; and Brett Dumbauld, Washington State Department of Fish and Wildlife. Willapa Bay Field Station, P.O. Box 190. Ocean Park. WA 98640. The potential for positive interactions between aquaculture spe- cies and native eelgrass {Zostera marina) is the subject of growing interest in the Pacific Northwest. We conducted surveys of cul- tured oysters {Cnissostrea gigas) and Z. marina density to deter- mine the nature of the relationship between these two species, and to determine whether this relationship changes under different cul- ture practices used in Willapa Bay. Washington. Culture practices assessed included ground culture harvested by dredgmg. ground culture harvested by hand, and off-bottom line culture. The role of planting density was also assessed in two separate experiments where small plots were planted with several densities of oyster seed and two year old oysters respectively. Eelgrass production and density were measured throughout the experiments. Results of the survey and experiments will be discussed. A SPECIFIC PATHOGEN FREE CULTURE SYSTEM FOR C. GIGAS LARVAE AND SPAT. Sean E. Matson and Chris- topher Langdon. Hatfield Marine Science Center. Oregon State University. Newport. OR 97365. The Molluscan Broodstock Program (MBP). a selective breed- ing program for the Pacific oyster. Crassosirea gigas. uses a Spe- cific Pathogen Free culture system for all production and mainte- nance of larvae, spat, broodstock and microalgae. This system is necessary to exclude infectious agents of Haplosporidian costale (Seaside Organism. SSO). which has been found in Pacific oysters grown in Yaquina Bay. Oregon, for the safe outplanting of MBP spat in field test sites along the West coast (USA). All seawater entering MBP facilities is filtered through sand, diatomaceous earth, and 20, 5. and l|jLm cartridge filters. Seawater to mass algal cultures and the nursery is also irradiated with UV-light at >30.000 micro- Watts-sec/cni" (MWS) as a back-up precaution. Since the system's inception, no MBP spat have been identified as being contaminated with SSO. or any other infectious agent. A series of laboratory experiments was performed to assess the effects of UV water on larval growth and survival, spat growth and survival, and microalgal culture density. Experiments with oyster larvae indi- cated that both the micro-filtration system and UV water treatment had a significant negative effect on larval growth (p = 0.0001 ). A significant reduction in growth was evident at UV intensities as low as 10.000 MWS (p<0.05). Methods that have significantly improved larval growth, survival, speed to metamorphosis and spat growth within the SPF culture system include substituting a 0.2|jim filter and charcoal for a UV filter when rearing larvae, and the addition of calcium bentonite (2nig/ml/day) or calcium montmo- rillonite (5mg/ml/day) to larvae and spat cultures (p<0.05). BIOTIC RESISTANCE TO EUROPEAN GREEN CRAB, CARCINVS MAENAS, BY NATIVE ANALOGS IN THE NORTHEASTERN PACIFIC. P. Sean McDonald, Gregory C. Jen.sen and David A. Armstrong, School of Aquatic and Fishery Sciences. University of Washington. Seattle. WA. 98195. The notion of "biotic resistance", which holds that characteris- tics of native biota act to prevent establishment and persistence of nonindigenous species, remains a dominant component of invasion biology theory. Yet. studies of nonindigenous marine species have often focused on impacts to the recipient community while ignor- ing effects of the latter on the former. The case of the European green crab. Carcinus maenas. provides one such example; the species has successfully colonized temperate coastal embayments throughout the world and its attendant adverse consequences to nafive biotic communities have been well-documented. However, the distribution and habitat use of C. maenas in the northeastern Pacific is more limited than would be expected based on Atlantic populations, and peak abundances occur only in isolated, back- marsh or high intertidal locations. We conducted a limited survey of crab populations in Bodega Bay Harbor (BBH), California, in 1998. and subsequent intensive sampling was undertaken in 2001 in BBH and other central California estuaries. Results from snorkel surveys and trapping data suggest that C. maenas are largely ab- sent from areas occupied by native ecological analogs (Cancer spp.). Incidence of limb autotomy in C. maenas is significantly higher at BBH sites shared with Cancer spp. than in isolated areas uninhabited by the latter or in Atlantic populations. A series of tethering experiments similarly supports the assertion that preda- tion/aggression by Cancer spp. affects the distribution and habitat utilization of C. maenas. The significance of these interactions to the eventual distribution of C. maenas in the northeastern Pacific is discussed, as well as implications for monitoring and control efforts. NSA & PCSGA, Newport, Oregon Abstracts, September 27-30, 2002 607 EFFECT OF DIET ON SOMATIC GROWTH OF JUVE- NILE GREEN SEA URCHINS (STRONGYLOCENTROTVS DROEBACHIENSIS). C. Pearce, Fisheries and Oceans Canada. Pacific Biological Station. 31 W Hammond Bay Road, Nanaimo, BC V9T 6N7: T. Daggett, Ross Island Salmon Ltd.. P.O. Box 1 304. Grand Manan. NB E5G 4M9; T. Chopin. Centre for Coastal Studies and Aquaculture. Centre for Environmental and Molecular Algal Research, Department of Biology, University of New Bruns- wick Saint John. P.O. Box .5050. Saint John. NB E2L 4L5; K. MacKeigan. V. Zltkos and S. Robinson, Fisheries and Oceans Canada. St. Andrews Biological Station. 53 1 Brandy Cove Road. St. Andrews. NB E5B 2L9. Populations of sea urchins, harvested for their gonads, are in decline worldwide and so research is now focusing on full life- cycle grow out. The objective of this study was to compare the somatic growth rates of juvenile green sea urchins (Strongylocen- trotiis droebachiensis) fed one of seven diets. Sea urchins (test diameter: 4.5 - 10.7 mm) were collected from the wild, held in laboratory tanks supplied with flow-through seawater, and fed ad libitum one of seven diets: (Da prepared diet. (2) Poipliyra pur- purea. (3) Pabnaha pabnata. (4) Enteromorpha linza. (5) a mix- ture of Ulvaria obscura and Ulva lactuca. (6) Lamiiuiria longi- cruris collected from an Atlantic salmon culture site, and (7) Laini- naria longicruris collected from a site uninfluenced by salmon culture. Each diet was randomly assigned to three separate tanks with each tank containing 19 individually housed urchins. Test diameter and whole wet weight measurements from each urchin were initially taken at the start of the experiment and then again once per month for a period of 12 months. Feed type significantly affected growth rate in terms of both diameter and wet weight. Porpbyra purpurea and the prepared diet supported the best growth while Laminaria longicruris collected from a site uninflu- enced by salmon culture was the least effective diet. TRENDS IN PINTO ABALONE (HALIOTIS KAMTSCHAT- KANA) ABUNDANCE AT TEN SITES IN THE SAN JUAN ISLANDS AND MANAGEMENT OF THE SPECIES IN WASHINGTON STATE Don P. Rothaus, R. E. Sizemore, M.J. Ulrich. Washington Department of Fish and Wildlife. Fish Management Program, Central Shellfish Unit, Olympia WA 98501-1091; and Carolyn S. Friedman. University of Washing- ton. School of Aquatic and Fishery Sciences. Seattle WA 98195- 5680. As a result of concerns regarding the stability of pinto abalone (Haliotis kamtschatkana) populations in Washington and the clo- sure of the abalone fishery in neighboring British Columbia, Canada, the Washington Department of Fish and Wildlife (WDFW) established index stations at ten sites in the San Juan Islands. These stations varied in size from 50 m" to 380 m", av- eraging about 220 m~. WDFW divers systematically surveyed each of these stations in 1992, 1994, and 1996. A decrease in total abalone abundance at these ten index stations from 1992 to 1994 (n = 351 to n = 288). along with anecdotal information of popu- lation decline by University of Washington (UW) researchers and WDFW Enforcement personnel, resulted in the closure of the Washington pinto abalone fishery in 1994. Following the closure, a 1996 survey by WDI^W resulted in a combined n = 297. Re- search in other regions indicate that sedentary invertebrates, such as abalone, must be within 1.0-2.0 m of one another (ds0.337ndash;0.15 abalone/nr) for successful fertilization. The average abalone density (d) from one half of the sites surveyed in 1996 contained ds0.15 abalone/m". Based on survey data, and information from abalone fisheries around the world, it is clear that additional stock assessment is needed to analyze the trend in Washington abalone stocks. Addi- tional index sites, early juvenile life history, population genetics, and the potential for enhancement have been propo.sed for study. ECOLOGICAL ROLE AND POTENTIAL IMPACTS OF MOLLUSCAN SHELLFISH CULTURE IN THE ESTUA- RINE ENVIRONMENT OF HUMBOLDT BAY, CA. Steven S. Rumrill and Victoria K. Poulton. Estuarine and Coastal Sci- ences Laboratory. South Slough National Estuarine Research Re- serve. Charieston, OR 97420. The intertidal mudflats of Humboldt Bay. CA. provide habitat for eelgrass {Zostera marina), invertebrates, shellfish, tlnfish, and birds. Humboldt Bay is also the leading producer of Pacific oysters (Crassostrea gigas) in California. We have completed the first year of a 3-year project to identify and quantify the effects of commercial oyster mariculture in tidetlat habitats, eelgrass beds, and invertebrate communities. Experimental oyster long-line spac- ing plots were established for comparison to a ground culture site and 6 reference sites (no oysters). We sampled study plots quar- terly between Aug 2001 -Aug 2002 for presence of eelgrass, oys- ters, and other cover types. We collected infaunal cores, deployed fish traps, and measured water quality, sedimentation, light inten- sity, and oyster growth characteristics. Eelgrass shoot density and percent cover were consistently highest in an eelgrass bed control site, lowest at the 1.5-ft. long-line spacing plot, and most variable at the ground culture site. Eelgrass metrics in the other long-line spacing plots were generally lower but within the range of varia- tion exhibited by the reference sites. Preliminary analysis of in- vertebrate cores has produced a species list of over 70 taxa, many of which are known prey items for estuarine fish. Sedimentation measurements showed no consistent patterns among experimental long-line plots. Oyster growth measurements did not differ sub- stantially between long-line plots; oysters grew 20-35 mm in length and 16-22 mm in width between May and Aug 2002. Light intensity was lower beneath oyster long-lines, but did not differ substantially between the 1 .5 and 5 ft. spacing plots. 608 Abstracts. September 27-30. 2002 NSA & PCSGA. Newport, Oregon THE EFFECTS OF THE HERBICIDE RODEO* ON PA- CIFIC OYSTER GAMETOGENESIS AND TISSUE ACCU- MULATION. B. C. Smith, C. E. Grue, University of Washing- ton, School of Aquatic and Fishery Sciences, Seattle. WA: N. P. Kohn, Battelle Marine Sciences Laboratory. Sequim. WA; and J. P. Davis, Taylor Shellfish Company, Quilcene. WA. In Willapa Bay. WA, Rodeo® (Monsanto Agricultural Co., St. Louis. MO) is being used to control Spartina (Spartina alterni- flora). an invasive cordgrass native to the Atlantic Coast. Spartina alters the tideland habitat by trapping sediment and raising the elevation of the mudflats, thus reducing the available habitat for oyster culture. Rodeo tank mixes include a surfactant to reduce the surface tension of the spray. R-I I is currently the surfactant used in the Bav. R-I I belongs to a class of non-ionic surfactants com- prised of alkylphenol ethoxylates (APEO). Breakdown products of APEOs have been implicated as endocrine disruptors in fish and observed to cause delays in development of oyster veligers. The objectives of our study were to assess whether applications of Rodeo tank mixes 1 ) result in tissue concentrations of glyphosate in oysters that exceed the established tolerance of 3 ppm wet weight edible tissue and 2) impair oyster gametogenesis. To de- termine this. Pacific oysters (Crassostrea gigas) were subjected to five treatments; Rodeo; Rodeo tank mix (with R-11 surfactant); two concentrations of R-1 1; and a control. The oysters were ex- posed for 12 h once a week for 4 wks. Tissue samples were collected for residue analysis of glyphosate, AMPA and APEO and cross-sections of gonadal tissue were collected for histological examination. Initial results indicate that exposure to Rodeo without the surfactant results in concentrations of glyphosate below the established human health criteria. Tissue resides of APEO and an assessment of treatment effects on gametogenesis will be deter- mined this fall. MUSSEL GROWTH AND FOOD UTILIZATION IN RELA- TION TO WATER QUALITY ON A RAFT SYSTEM IN PUGET SOUND, WASHINGTON. Andrew D. Suhrbier, Aimee E. Christy, Hector S. Beltran, Daniel P. Cheney, Pacific Shellfish Institute, Olympia, WA 98501; Jonathan P. Davis, Tay- lor Shellfish Farms, Shelton. WA 98584: Kenneth M. Brooks, Aquatic Environmental Science Lab, Port Townsend, WA 98368; and Frank J. Smith, Northwest Research Associates. Inc., Belle- vue, WA 98007. With an annual production of approximately 3 million pounds live weight on the U.S. west coast, suspended mussel and oyster culture is predicted to increase significantly in coming years. De- scription of the changes associated with the culture of these crops is essential for the siting and evaluation of new culture facilities and in improving yield and production of existing facilities. This research has three general objectives; ( I ) to assess mussel shell growth and meat yield against measured physical, chemical and biological variables; (2) to compare a suite of variables with mea- surements of mussel feeding and biodeposit production; and (3) to collaborate with an on-going nutrient modeling study to estimate potential mussel carrying capacity in an entire farming area. Dur- ing the first year (2001-02) multiple observations were made of water currents, water chemistry, phytoplankton, mussel growth, seston removal and absorption, fouling, and fish utilization at a commercial mussel raft culture site in Totten Inlet, Washington. Certain parameters, such as phytoplankton abundance varied markedly inside and outside the raft units and under differing tidal conditions, although these preliminary data suggest feeding effects on phytoplankton are highly localized and largely contained in the immediate raft system. The second project year (2002-03) will continue the Totten Inlet experiments and add a study site at a commercial mussel farm in Penn Cove. Washington. This research is supported by the Sea Grant Program Office National Marine Aquaculture Initiative grant no. NAI6RG159I. RESULTS FROM THE OLYMPIC REGION HARMFUL ALGAL BLOOM (ORHAB) PROJECT ON THE WASHING- TON STATE COAST. THE VALUE OF A COLLABORA- TIVE PROJECT. Vera L. Trainer, NMFS, Northwest Fisheries Science Center, 2725 Montlake Blvd. E., Seattle WA 981 12; Bar- bara M. Hickey, University of Washington. School of Oceanog- raphy. Seattle WA 98195-7940; Ervin J. Schumacker, Qumault Department of Natural Resources. PO Box 189. Taholah. WA 98587. Harmful Algal Blooms (HABs) became a serious problem to the coast of Washington state in 1991 when blooms of pennate diatoms of the genus Pseudo-nitzschia produced the potent neu- rotoxin, domoic acid. Pacific razor clams, (Siliqua patula). and Dungeness crab, {Cancer magisler). bio-accumulated toxic levels of domoic acid and recreational and commercial fisheries were shut down in many areas. Since 1991 Pseudo-nitzschia blooms have recurred many times along the Washington coast causing suspensions of fisheries with associated economic and cultural losses for coastal residents. Federal funding for HAB monitoring projects since 1991 have been contingent on collaborative efforts that include local stakeholders. The Olympic Region Harmful Al- gal Bloom (ORHAB) project secured federal funding in year 2000 to investigate and monitor HABs along the Olympic peninsula. Participants include state and federal agencies, the University of Washington, non-profit research institutions, commercial shellfish growers, coastal tribes and shellfish managers. The ORHAB proj- ect has made significant findings regarding the physical and chemical processes which create and transport HABs to the Wash- ington coast. An initiation site for Pseudo-nitzschia blooms has been found in the Juan de Fuca eddy region adjacent to Washing- ton state and Vancouver island. Blooms from this area may be transported by storm events to the coast where they are ingested by shellfish. Monitoring and the use of new technologies by ORHAB participants have better protected the public health and paved the NSA & PCSGA, Newport. Oregon Abslracls. September 27-30. 2002 609 way for better understanding of west coast HABs and more etTi- cient means of early detection and monitoring. GENETIC DIFFERENTIATION AMONG GEODUCK CLAM iPANOPEA /lB/ft/Pr.4) POPULATIONS REVEALED BY ALLOZYME AND MICROSATELLITE ANALYSES. B. Vadopalas, Scliool of Aquatic and Fishery Sciences. University of Washington. Seattle. WA 98195; L. L. LeClair, Washington Department of Fish and Wildlife. Olympia. WA 98504; and P. Bentzen, Department of Biology. Dalhousie University. Hali- fax, NS B3H4J1. The genetic population structure of geoduck clams (Panopeci ahnipiii) in inland waters of Washington may affect fishery man- agement and aquacultural practices involving this species. To in- vestigate genetic differentiation in geoduck clams, samples were collected from 16 Washington State sites located in the five Puget Sound subbasins. southern Georgia Strait, and the Strait of Juan de Fuca. A collection from Clarence Strait in SE Alaska was included as an outgroup. Individuals were genotyped at 1 1 allozyme and 7 microsatellite loci. To investigate the level of isolation by distance, we analyzed correlations between pairwise geographic distances and multilocus Fst values. The Freshwater Bay collection in the Strait of Juan de Fuca was differentiated from others at both mi- crosatellite and allozyme loci. For both marker classes, there was no evidence of significant correlation between genetic and geo- graphic distance measures. In contrast to the microsatellite loci, the allozyme loci were in Hardy-Weinburg Equilibrium (HWE). De- viations firom HWE expectations at microsatellite loci were inter- preted as being primarily due to primer site sequence variation rather than population level processes such as inbreeding. These results may be due to stochastic variation in reproductive success and recruitment, and warrant further investigation into temporal genetic differentiation. TRIAL USE OF THE US NAVY REMOTELY OPERATED VEHICLE (ROV) SORD IV FOR SAMPLING DEEP WATER GEODUCK CLAMS (PANOPEA ABRUPTA). B. Vadopalas, School of Aquatic and Fishery Sciences. University of Washington. Seattle WA 98195; and Don P. Rothaus. Wash- ington Department of Fish and Wildlife, Fish Management Pro- gram, Central Shellfish Unh. Olympia WA 98501-1091. The existence of geoduck clams (Panopea abrupta) below the legally fishable depth of 21 m in Puget Sound, Washington has been surmised from video camera drops in one embayment, but the study of population dynamics and genetic relationships has been hampered by the lack of practical deep water sampling methodol- ogy for macrobenthic infauna. We initiated a deepwater geoduck sampling trial using the U.S. Navy ROV SORD IV (Submerged Ordnance Recovery Device). The ROV suction dredge and video system were modified to enhance geoduck excavation and re- trieval. The trial was conducted along a depth gradient seaward of a commercial geoduck bed in central Hood Canal. In four hours of ROV bottom time between 35 and 80 m depth we positively iden- tified and attempted sampling of three geoducks. We obtained two specimens approximately 15 meters apart that share many charac- teristics, including small size, thin valves, and poor viscerosomatic condition. The two animals were of the same age. a result unlikely to arise by chance based on age frequencies in a proximate shallow collection (p<0.01). Genetic analyses indicated that the two clams are most likely full siblings (p<0.001). These findings suggest large variation in year class strength and bias in reproductive suc- cess among spawners. and underscore the need for further inves- tigation into population dynamics and recruitment processes in deep water geoduck. SHELL CONDITION TESTING OF DUNGENESS CRAB IN PUGET SOUND, WASHINGTON. Donald E. Velasquez. S.F. Burton, Washington Department of Fish and Wildlife. 16018 Mill Creek Blvd.. Mill Creek. WA 98012-1296; D. A. Sterritt and B. McLaughlin, Washington Department of Fish and Wildlife. 1000 Point Whitney Road, Brinnon, WA 98320-9899. Since 1997 the State of Washington and the Treaty Tribes have been conducting cooperative shell condition testing of Dungeness crab over a large portion of Puget Sound. The purpose of testing has been to determine when it is best to conduct fisheries during the year and limit the problems associated with handling softshell crab. A number of conclusions have been made regarding the data collected since the program began. The peak molting season for legal male crab differs between subareas within Puget Sound. A pattern where legal-sized crabs finish molting earlier in the year in Central Puget Sound and later in the year for areas adjacent to the Canadian border is apparent. Data also indicate the schedule for the peak softshell period in any given subarea can differ somewhat from year to year. In a few subareas, it is difficult to assign a single softshell period because either the softshell crab are not easily detected or multiple softshell events appear to occur. Possible explanations for the variation of the peak softshell period within Puget Sound will be discussed. Additional observations of syn- chrony and asynchrony in the life cycle of Dungeness crab were made during shell condition sampling and will be covered. THE NATIONAL SHELLFISHERIES ASSOCIATION The National Shellfisheries Association (NSA) is an international organization of scientists, manage- ment officials and members of industry that is deeply concerned and dedicated to the formulation of ideas and promotion of knowledge pertinent to the biology, ecology, production, economics and man- agement of shellfish resources. The Association has a membership of more than 1000 from all parts of the USA. Canada and 18 other nations: the Association strongly encourages graduate students" mem- bership and participation. WHAT DOES IT DO? — Sponsors an annual scientific conference. — Publishes the peer-reviewed Journal of Shellfish Research. — Produces a Quarterly Newsletter. — Interacts with other associations and industry. WHAT CAN IT DO FOR YOU? — You will meet kindred scientists, managers and industry officials at annual meetings. — You will get peer review through presentation of papers at the annual meeting. — If you are young, you will benefit from the experience of your elders. — If you are an elder, you will be rejuvenated by the fresh ideas of youth. — If you are a student, you will make useful contacts for your job search. — If you are a potential employer, you will meet promising young people. — You will receive a scientific journal containing important research articles. — You will receive a Quarterly Newsletter providing information on the Association and its activities, a book review section, information on other societies and their meetings, a job placement section, etc. HOW TO JOIN — Fill out and mail a copy of the application blank below. The dues are 65 US $ per year ($35 for students) and that includes the Journal and the Newsletter! NATIONAL SHELLFISHERIES ASSOCIATION— APPLICATION FOR MEMBERSHIP (NEW MEMBERS ONLY) Name: For the calendar year: Date: Mailing address: Institutional affdiation, if any: Shellfishery interests: Regular or student membership: Student members only — advisor's signature REQUIRED: Make checks (MUST be drawn on a US bank), international postal money orders or VISA for $65 ($35 for students with advisor's signature) payable to the National Shellfisheries Association and send to Nancy Lewis, Bookkeeper, PO Box 350, V.I.M.S. Eastern Shore Lab, Wachapreague. VA 23480, USA. INFORMATION FOR CONTRIBUTORS TO THE JOURNAL OF SHELLFISH RESEARCH Original articles dealing with all aspects of shellfish re- search will be considered for publication. Manuscripts will be judged by the editors or other competent reviewers, or both, on the basis of originality, content, merit, clarity of presentation. and interpretations. Each article should be carefully prepared in the style followed in prior issues of the Journal of Shellfisli Research before submission to the Editor. Papers published or to be published in other journals are not acceptable. Title, Short Title, Key Words, Abstract: The title of the paper should be kept as short as possible. Please include a "short running title'" of not more than 48 characters including spaces, and key words. Each manuscript must be accompanied by a concise, informative abstract, giving the main results of the research reported. The abstract will he published at the beginning of the article. No separate summary should be in- cluded. Text: Manuscripts must be typed double-spaced throughout on one side of the paper, leaving ample margins, with the pages numbered consecutively. Scientific names of species should be underlined or in italics and. when first mentioned in the text, should be followed by the authority. Common and scientific names of organisms should he in accordance with American Fisheries Society Special Publications 16 and 17; Common and Scientific Names of Aquatic Invertebrates from tlie United States and Canada: Molhtsks and CSNAIUSC: Decapod Crus- taceans, or relevant publications for other geographic regions. Abbreviations, Style, Numbers: Authors should follow the style recommended by the sixth edition (1994) of the Council of Biology Editors [CBE] Style Manual, distributed by the American Institute of Biological Sciences. All linear measure- ments, weights, and volumes should be given in metric units. Tables: Tables, numbered in Arabic, should be on separate pages with a concise title at the top. Illustrations: Line drawings should be in black ink or laser print and planned so that important details will be clear after reduction to page size or less. No drawing should be so large that it must be reduced to less than one third of its original size. Photographs and line drawings should be prepared so they can be reduced to a size no greater than 17.3 cm x 22.7 cm. and should be planned either to occupy the full width of 17.3 cm or the width of one column. 8.4 cm. Photographs should be glossy with good contrast and should be prepared so they can be reproduced without reduction. Originals of graphic materials (i.e., line drawings) are preferred and will be returned to the author. Each illustration should have the author's name, short paper title, and figure number on the back. Figure legends should be typed on separate sheets and numbered in Arabic. Digital Figures: Authors may provide digital figures (they are not required); they must be accompanied by hardcopy fig- ures of equal quality, which the printer will use for comparison and backup. If digital figures are supplied, please note the following instructions; • Each piece of art should be saved as its own file. • Files must be one of the following fonnats; TIF, EPS, or JPG. • Each file should be named according to its figure number and format (e.g.. "fig2b.tif'). • Figures must not be embedded in a word-processor or spreadsheet document: the printer cannot use images stored in Word. WordPerfect, Excel, Powerpoint, etc. • Resolution; line shots: 1000 dpi; halftones/grayscales: 300 dpi if no lettering, 500 dpi if figure contains lettering. • Color figures; save the files as CMYK-encoded TIF images (preferred) or CMYK-encoded EPS or JPG images. Color figures have the same resolution requirements a BAV. above. Color illustrations will not be accepted unless the author agrees to cover the cost of associated reproduction and printing. Literature Cited: References should be listed alphabeti- cally at the end of the article. Abbreviations in this section should be those recommended in the American Standard for Periodical Title Abbreviations, available through the American National Standard Institute. 1430 Broadway, New York, NY 10018. For appropriate citation format, see examples below; Journal: Watts, R. J.. M. S. Johnson & R. Black. 1990. Effects of re- cruitment on genetic patchiness in the urchin Echinometra mathaei in Western Australia. Mar. Biol. 105;145-151. Book: Claudi. R. & G. L. Mackie. 1994. Practical manual for Zebra Mussel monitoring and control. Boca Raton, FL; CRC Press. 227 pp. Chapter in Edited Book: Davio, S. R., J. F. Hewetson & J. E. Beheler. 1985. Progress toward the development of monoclonal antibodies to saxitoxin; antigen preparation and antibody detection. In: D. M. Ander- son. A. W. White & D. G. Baden, editors. Toxic dinoflagel- lates. Amsterdam: Elsevier, pp. 343-348. Page Charges: Authors or their institutions will be charged $100.00 per printed page. All page charges are subject to change without notice. A handling fee of $50 will be charged for all manuscripts accepted for publication. Proofs: Page proofs are sent to the corresponding author and must be corrected and returned within seven days. Alter- ations other than corrections of printer's errors may be charged to the author(s). Reprints: Reprints of published papers are available at cost to the authors. Information regarding ordering reprints will be available from The Sheridan Press at the time of printing. Cover Photographs: Appropriate photographs may be sub- mitted for consideration for use on the cover of the Journal of Shellfish Research. Black and white photographs and color illustrations will be considered. Corresponding: An original and two copies and electronic copy of each manuscript submitted for publication consider- ation should be sent to the Editor, Dr. Sandra E. Shumway. Department of Marine Sciences, University of Connecticut, 1080 Shennecossett Rd.. Groton. CT 06340. E-mail: sandra. shumway@uconn.edu or sandrashumway@hotinail.com Membership information may be obtained from the Editor or the Treasurer using the form in the Journal. Institutional subscribers should send requests to: Journal of Shellfish Re- search. P.O. Box 465. Hanover, PA 17331. Alexander Y. Karatayev, Sergey E. Mastitsky, Daniel P. Molloy and Lyubov E. Burlakova Patterns of emergence and survival of Conchophthirus acuminatus (Ciliophora: Conchophthiridae) from Dreissena polymorpha (Bivalvia: Dreissenidae) 495 Ronald B. Toll. Robert S. Prezanl and Harold B. Rollins A novel method for locating tagged infaunal bivalves: Submersible pulse technology metal detectors 501 Christopher M. Pearce, Tara L. Daggett and Shawn M. C. Robinson Effects of starch type, macroalgal meal source, and (i-carolene on gonad yield and quality of the green sea urchin Slnmgylocenlmtiis droebachiensis (Miiller) fed prepared diets 505 Louis R. D'Abramo and Cortney L. Ohs Production of red swamp crawfish (Procamharus clurkii) in earthen ponds without planted forage: Establishment, maintenance and harvest of populations 521 Lxjuis R. D 'Abramo, Cortney L. Ohs and Kathleen C. Elgarico Production of red swamp crawfish {Procamharus clarkii) in earthen ponds without planted forage: Evaluation of trap and seine harvest strategies 527 Enrique Lozano-Alvarez, Patricia Briones-Fourzdn and Maria Eugenia Ramos-Aguilar Distribution, shelter fidelity, and movements of subadult spiny lobsters [Pamdirus urgiis) in areas with artificial shelters (Casitas) 533 Fuhua Li, Jianhai Xiang, Xiaojun Zhang, Changgong Wu, Chengsong Zhang, Linghua Zhou and Kuijie Yu Tetraploid induction by heat shocks in Chinese shrimp Feniieropeiiaeus chinensis 541 Guoqiang Huang. Shuanglin Dong, Fang Wang and Shen Ma Selection and use of different diets in a study of Chinese shrimp. Fenneropenaeus chinensis 547 Monica Y. Tsuzuki, Ronald O. Cavalli and Adalto Bianchini Effect of salinity on survival, growth, and oxygen consumption of the pink-shrimp Farfantepenaeus paulensis (Perez-Farfante 1967) 555 P. M. Troffe, S. Ong, C. D. Levings and T. F. Sutherland Anatomical damage to humpback shrimp, Pandalus hypsinotus (Brandt 1851) caught by trawling and trapping 561 Francesc Sardd, Joan B. Company and Arturo Castellan Intraspecific aggregation structure of a shoal of a western Mediterranean (Catalan coast) deep-sea shrimp, Aristeus antennalits (Risso, 1816), during the reproductive period 569 Ferdinand F. Wirth and Kathy J. Davis Seafood dealers" .shrimp-purchasing behavior and preferences with implications for United States shrimp farmers 581 Kenneth M. Brown. Gary W. Peterson. Patrick D. Banks. Brian Lezina, Charles Ramcharan and Michael McDonough Olfactory deterrents to black drum predation on oyster leases 589 Abstracts of technical papers presented at the 56th Annual Meeting of the Pacific Coast Section. National Shellfisheries Association, Newport, Oregon, September 27-30, 2002 597 COVER PHOTO: Sea urchins (Strongylocentroliis droebachiensis, S. frunciscanus. and 5. purpuralus) being used in a gonad enhancement experiment at the Pacific Biological Station (Fisheries and Oceans Canada, Nanaimo, British Columbia, Canada) to test the efficacy of various prepared feeds to produce suitable gonad color, taste, firmness, and texture. Photo: Chris Pearce. The Journal of Shellfish Research is indexed in the following: Science Citation Index®. Sci Search®, Research Alert®, Current Contents*/Agriculture, Biology and Environmental Sciences, Biological Abstracts, Chemical Abstracts, Nutrition Abstracts, Current Advances in Ecological Sciences, Deep Sea Research and Oceanographic Literature Review, Environmental Periodicals Bibliography, Aquatic Sciences and Fisheries Abstracts, and Oceanic Abstracts. JOURNAL OF SHELLFISH RESEARCH Vol. 22, No. 2 September 2003 CONTENTS Fabrice Fernet, Rejean Tremblay and Edwin Bourget Biochemical indicator of sea scallop (Placopecten magellaniciis) quality based on lipid class composition. Part I: Broodstock conditioning and young larvae performance 365 Fabrice Fernet, Rejean Tremblay and Edwin Bourget Biochemical indicator of sea scallop iPliuapecteii magellaniciis) quality based on lipid class composition. Part II: Larval growth, competency and settlement 377 Stephen L. Estabrooks A rapid test for the determination of the spawning status of the bay scallop. Argopecten inadians (Lamarck, 1819) 389 Ruben Avendano-Herrera. Carlos Riquelmes, Fernando Silva. Miguel Avendanod and Rate Irgang Optimization of settlement of larval Argopecten purpuraius usnig natural diatom biofiliiis 393 Enid K. Sichel and Richard C. Karney Adhesives to attach juvenile bay scallops to plastic netting in aquaculture 401 Tao Zhang, Hongsheng Yang, Huayong Que, Guofan Zhang, Shilin Liu, Yichao He and Fusui Zhang Evidence for the involvement of cyclic AMP in the metamorphosis of the bay scallop. Argopecten uiadians (Lamarck ) larvae 403 William J. Dore, Jennifer Farthing and Ian Laing Depuration conditions for great scallops (Pecten niaximus) 409 Oscar Chacon, Maria Teresa Viana, Ana Farias, Carlos Vazquez and Zaul Garcia-Esquivel Circadian metabolic rate and short-term response of juvenile green abalone [Halioiis Jiilgens Philippi) to three anesthetics 415 Sean E. Matson, Jonathan P. Davis and Kenneth K. Chew Laboratory hybridization of the mussels. Mylilus Irossulus and M. galtoprovincialis: Larval growth, survival, and early development 423 Supannee Leethochavalit, E. Suchart Upatham, Kang-Sik Choi, Pichan Sawangwong, Kashane Chalermwat and Maleeya Kruatrachue Ribosomal RNA characterization of non-transcribed spacer and two internal transcribed spacers with 5.8S ribosomal RNA or Perkinsiis sp. found in undulated surf clams (Paphiii uitdiihila) from Thailand 43 1 M. Delgado and A Perez Camacho A study of gonadal development in Ruditapes deciissateu (L.| (Mollusca. Bivalvia), using image analysis techniques: Influence of food ration and energy balance 435 M. Albentosa, M. J. Ferndndez-Reiriz, U. Labarta and A. Perez-Camacho Absorption of biochemical components and feeding behavior with natural and carbohydrate-rich diets in Ruditapes decussatus and Venenipis pidla.sira clams 443 Stephen R. Fegley, Susan E. Ford, John N. Kraeuter and Harold H. Haskin The persistence of New Jersey's oyster seedbeds in the presence of oyster disease and harvest: The role of management 45 1 Jorge Chavez- Villalba, Jean Barret, Christian Mingant, Jean-Claude Cochard and Marcel Le Pennec Influence of timing of broodstock collection on conditioning, oocyte production, and larval rearing of the oyster, Crassdsirea giga\ (Thunberg). at six production sites in France 465 Mi Seon Park, Chang-Keun Rang, Dong-Lim Choi and Bo-Young Jee Appearance and pathogenicity of ovarian parasite Marteitioides clwngmuensis in the farmed Pacific oysters, Crassoslrea gigas. in Korea 475 Gab-Man Park and Ee-Yung Chung Molecular phylogenetics of five Corbicula species determined by partial 28S ribosomal RNA gene sequences 481 Alexander Y. Karatayev, Lyubov E. Burlakova, Thomas Kesterson and Dianna K. Padilla Dominance of the Asiatic clam, Corhiciila Jlumtnea (MUller), in the benthic community of a reservoir 487 CONTENTS CONTINUED ON INSIDE BACK COVER JOURNAL OF SHELLFISH RESEARCH VOLUME 22, NUMBER 3 DECEMBER 2003 The Journal of Shellfish Research (formerly Proceedings of the National Shellfisheries Association) is the official publication of the National Shellfisheries Association Editor Sandra E. Shumway Department of Marine Sciences University of Connecticut Groton, CT 06340 EDITORIAL BOARD Peter Cook (2004) Austral Marine Services Lot 34 Rocky Crossing Road Warrenup Albany. W.A. 6330, Australia Simon Cragg (2004) Institute of Marine Sciences University of Portsmouth Ferry Road Portsmouth P04 9LY United Kingdom Leroy Creswell (2005) University of Florida/Sea Grant 8400 Picos Road, Suite 101 Fort Pierce, Florida 34945-3045 Lou D'Abramo (2004) Mississippi State University Department of Wildlife and Fisheries Box 9690 Mississippi State, Mississippi 39762 Christopher V. Davis (2004) Pemaquid Oyster Company, Inc. P.O. Box 302 1957 Friendship Road Waldoboro. Maine 04572 Ralph Elston (2005) Aqua Technics/Pacific Shellfish Institute 455 West Bell Street Sequim, Washington 98382 Susan E. Ford (2004) Rutgers University Haskin Shellfish Research Laboratory 6959 Miller Avenue Port Norris, New Jersey 08349 Raymond Grizzle (2005) Jackson Estuarine Laboratory Durham, New Hampshire 03824 Karolyn Mueller Hansen (2004) 1524 Bariey Circle Knoxville, Tennessee 37922 Journal of Shellfish Research Volume 22, Number 3 ISSN: 0730-8000 December 2003 www.shellfish.org/pubs/jsr.htm Standish K. Allen, Jr. (2004) Aquaculture Genetics and Breeding Technology Center Virginia Institute of Marine Science College of William and Mary P.O. Box 1346 Gloucester Point, Virginia 23062 Shiriey Baker (2004) University of Florida Department of Fisheries and Aquatic Sciences 7922 NW 71" Street Gainesville, Florida 32653-3071 Bruce Barber (2005) School of Marine Science University of Maine 5735 Hitchner Hall Orono, Maine 04469 Brian Beal (2004) University of Maine 9 O'Brien Avenue Machias, Maine 04654 Neil Bourne (2005) Fisheries and Oceans Pacific Biological Station Nanaimo, British Columbia Canada V9T 6N7 Andrew R. Brand (2005) University of Liverpool Port Erin Marine Laboratory Port Erin. Isle of Man IM9 6JA United Kingdom Eugene Burreson (2005) Virginia Institute of Marine Science P.O. Box 1346 Rt. 1208 Create Road College of William and Mary Gloucester Point, Virginia 23062 Mark Luckenbach (2005) Virginia Institute of Marine Science Eastern Shore Lab P.O. Box 350 Wachapreague, Virginia 23480 Bruce MacDonald (2004) Department of Biology University of New Brunswick Saint John, New Brunswick Canada E2L 4L5 Roger Mann (2004) Virginia Institute of Marine Science Gloucester Point, Virginia 23062 Islay D. Marsden (2004) Department of Zoology Canterbury University Christchurch. New Zealand Jay Parsons (2005) Memorial University Marine Institute Box 4920 St. John's, Newfoundland Canada AlC 5R3 Tom Soniat (2004) Biology Department Nicholls State University Thibodaux, Louisiana 70310 J. Evan Ward (2004) Department of Marine Sciences University of Connecticut 1080 Shennecossett Road Groton, Connecticut 06340-6097 Gary Wikfors (2004) NOAA/NMFS Rogers Avenue Milford, Connecticut 06460 Joiimul oj Shcllftsli Rcscunh. Vol. 22, No. 3. 611-613, 2003. FEB 3 2004 V.'CT:.': Melbourne Romaine Carriker Honored Life Member Melbourne Cuniker, or "Mer" as he is known ti) his many students, colleagues, and friends is a world recognized student of Malacology, and an authority on marine subjects as diverse as functional morphology, biominerah/.ation. larval ecology, and predator- prey interactions. Mel's interest in shellfisheries extends from his intense interest in molluscs, their ecology, biology, and morphology. Scientist, scholar, husband, father, and friend — his career and his life have been punctuated by transition and achievement. Mel's fascinating story began on February 2.Sth, 1915 when he was born in Santa Marta. Colombia. For the first twelve years of his life. Mel li\ed on a coffee plantation (called Vista Nieve) with his American parents. His parents. Myrtle Carmela Carriker de Flye and Melbourne Armstrong Carriker. Jr.. developed and managed the coffee plantation in the Siena Nevada de Santa Marta Mountains. During his early years, Mel lived in an agrarian community among crops of coffee and sugarcane, hnmersed in rugged surroundings, he and his siblings happily lived on the edge of a tropical paradise. When he was ten. Mel began accompanying his father, an accomplished amateur naturalist and ornithologist, on short field trips to collect birds, birds' eggs, and small mammals. Undouhtably, these experiences sparked his interest in the natural world and the seemingly secret lives that animals lead. In 1927, Mel's parents sold the coffee plantation and moved the family to southern New Jersey, taking up residence in Beachwood. His father took a position at the Academy of Natural Sciences of Philadelphia as Associate Curator of Ornithology, and Mel was enrolled in Toms River grade school. After struggling through the depression years with his family, Mel graduated from Toms River High School in 1934. Immediately after graduation, he accompanied his father on an ornithological expedition into Bolivia, South America. This "enviable, exhilarating experience" (p. 273, Carriker 2()()()), reinforced Mel's desire to further his education in the field of zoology, in particular ornithology. In the fall of 1935 Mel entered Rutgers University, New Jersey, majoring in agricultural research and minoring in zoology. During several summers, he worked as the director of aquatic and recreation programs at the Boy Scout Camp, Burton-at- Allaire in southern New Jersey to earn money for college. It was at Rutgers that Mel met Thurlow C. Nelson, his undergraduate adviser and mentor, who offered him an opportunity that shaped his scientific career. Through Nelson's urging. Mel began working on Rutgers' College of Agriculture's houseboat in Barnegat Bay. New Jersey, in the summer of 1938. studying the life history of oyster larvae. In subsequent summers of 1939 to 1941. he continued this pursuit on the "Cynthia." broadening his studies to include the general biology and ecology of oysters. In the fall of 1939. Mel traveled to the University of Wisconsin where he began graduate work with Lowell E. Noland. For his graduate work, he studied the biology of the pond snail, Lynmaeu sU)i>iuilis. a host of the trematode worm that causes swimmer's itch. It was here that Mel began honing his skills as a scientist, studying invertebrate anatomy and physiology, and prepared his first paper on the boring mechanisms of the oyster drill snail. Wisconsin also introduced Mel to one other love, his future wife Scottie McAllister. In 1942. he participated in his first NSA annual meeting, presenting his first scientific paper on oyster-drill boring mechanisms! Mel graduated (in June of 1943) with a doctoral degree in invertebrate zoology and physiological chemistry, and with the rank of ensign in the U.S. Naval Reserve. Immediately after graduating from the University of Wisconsin, he entered the Naval Training School. Harvard University, where he was trained in naval communications. During World War II. he served on a PC 780 ship in the Aleutian and Hawaiian Islands as communications officer. Although naval duty interrupted Mel's career, his love for malacology continued; rumor has it that during his time off Mel would explore the coast around Adak (Aleutian Islands) collecting marine molluscs and their hemolymph to mail to Rutgers 611 612 Ward University for ongoing systematic studies. After the War. he returned to the east coast and accepted a position as instructor in the Department of Zoology at Rutgers in 1946. Mel worked at Rutgers for eight years, being promoted to Assistant Professor before leaving in 1954. During his time as a faculty member at Rutgers, he developed courses (e.g.. estuarine ecology graduate course), taught, and. during the summers, worked with T.C. Nelson and Harold Haskin (see Kraeuter and Ford 1999), investigating the biology of the quahog. In the summers of 1947 to 1949. he returned to the houseboat "Cynthia" in Little Egg Harbor. New Jersey, establishing a research program in shellfish biology that would span his career. From a small laboratory in the stem of the houseboat, he studied quahog ecology and continued researching the shell-boring mechanisms of predatory gastropods. This research was the foundation for several classic published works including. "Critical Review of Biology and Control of Oyster Drills Urosalpinx and Euplewa" (Carriker 1955). and "Interrelation of Functional Morphology, Behavior, and Autecology in Early Stages of the Bivalve Mercenaria mercenarid" (Carriker 1961 ). Mel lived on the boat with his wife Scottie and two children, Eric and Bruce; a happy but nonetheless crowded existence. In 1954. Mel was offered, and accepted, a position as Associate Professor at the University of North Carolina (UNC). Chapel Hill. He taught marine ecology and conducted marine-related research in the Department of Zoology. During the summers of 1953 to 1955 he also conducted research on pond culture of oysters and clams on Gardiner's Island. New York. This work was sponsored by the J. & J.W. Elsworth Oyster Company and the U.S. Fish & Wildlife Service. Mel's work on Gardiner's Island was productive and brought him in contact with shellfish biologist Victor Loosanoff. In 1956. his research on clam larvae was shifted to the UNC Institute of Fisheries Research in Morehead City. Over the next five years Mel interacted with scientists at the Institute and at Duke University Marine Laboratory a few miles away, focusing his research on larval biology and the predatory drilling snails of oysters. In 1961. due to unfriendly politics that can be encountered in academia. Mel left UNC and took a position with the U.S. Bureau of Commercial Fisheries Biological Laboratory. Oxford, Maryland. At the Oxford Laboratory he began working on an emerging disease of oysters known as MSX. and this research consumed all of his time. The move to Oxford, however, was to be short lived. In 1962. Mel was enticed by an offer to head a new systematics and ecology program at the Marine Biological Laboratory in Woods Hole. Massachusetts. The Carriker family moved to Falmouth, Massachusetts, in the fall of 1962. where Mel assumed the position as Director of the Systematics-Ecology Program. The long-term goal of this program was to spearhead research and training in marine systematics and ecology, and enhance the scientific knowledge of organisms in the Cape Cod region. This Program turned out to be "highly successful and functioned productively for ten years" (p. 281. Carriker 2000). One of the most recognized accomplishments of the Program was the publication of a set of keys and check lists of the common invertebrates of, essentially, the waters of southeastern New England. First published in 1964. the "Keys to Marine Invertebrates of the Woods Hole Region" (edited by Ralph I. Smith) provided nonsystematists a useful guide for the identification of many common invertebrates in the region, and were invaluable to students and scientists alike. The first complete revision of these keys in 35 y began in 1999. and the first revised sections can be viewed on the Marine Biological Laboratory's web site. Unfortunately, due to a shortage of funds, the Program was closed in 1972. By then. Mel's reputation as an outstanding marine scientist proceeded him. and he was offered a full professorship at the new College of Marine Studies (CMS). University of Delaware, in Lewes. In the fall of 1972. Mel and his wife Scottie moved to Delaware where he taught, conducted research, and helped shape the CMS graduate program for thirteen years. During this time he studied oyster shell ullrastructure and chemistry as related to shell penetration by oyster borers, taught a course in malacology, and supervised the research efforts of many graduate students (including some from Central and South America). Mel officially retired in February 1985 at the age of 70. receiving the title of Professor Emeritus. After retiring, he served as president of the Delaware-Panama Partners of the Americas: he continues his scholarly contributions through his writings about his family and the science he loves. In 2000. Mel published a book concerning the fascinating history of his family and their coffee plantation titled "Vista Nieve." from which much of this biography has been gleaned. Mel is an accomplished scientist, publishing over 45 abstracts and 160 scientific papers and reports, and coining well-known malacological terms such as the "accessory boring organ" (ABO) of muricids, and the "pediveliger" stage of bivalve molluscs. He has presented technical papers at meetings and chaired scientific session over 255 times. From 1965 to 1977. Mel served as editor for the manuals on the Marine Flora and Fauna series produced by the National Marine Fisheries Service. His dedication to the scientific community is evidenced by the many positions he has held including chairman of the Division of Invertebrate Zoology. American Society of Zoology ( now the Society of Integrative and Comparative Biology): vice-president of the Association of Marine Laboratories of the Caribbean: and president of the Institute of Malacology, the American Malacological Society, and the Atlantic Estuarine Research Society. For almost a 50 y period. Mel has served NSA in various capacities, including: Secretary-Treasurer from 1953 to 1954. Vice President between 1955 to 1957, President from 1957 to 1959, and as a source of trusted advice for many an Executive Committee ever since. As Secretary-Treasurer, he was instrumental in formalizing the regular publication of the Association's meeting notes as the "Proceedings of the National Shellfisheries Association (PNSA)," serving as its first Editor from 1954 to 1957. Mel also served several times on the Publications Committee, including during 1979 to 1980 when the name of the NSA publication was changed from the PNSA to the Journal of Shellfish Research. In 1978, Mel was presented with the Honored Life Member award by NSA. and in 1998 was recognized for his years of dedication and scientific achievement in shellfish research when the first NSA student research award was named in his honor. Presently. Mel serves as Historian of the Association, recently completing an historical account of NSA as it emerged from earlier oyster meetings and groups, titled "Taming of the Oyster" (in press). Throughout his career Mel has been a teacher, researcher, editor, and mentor. He has supervised 35 graduate students ( 17 Ph.D., 18 M.S.) and has served on numerous graduate student committees. Those of us who have had the pleasure of being a student of Mel's know his objective, quiet approach to seemingly unsurmountable problems, and his deft ability to hone a piece of writing — with comments neatly scripted in pencil on just about every page of many a proposal or paper (often to the immediate displeasure of his students) — so Honored Life Member M. R. Carriker 613 that it was clear and concise. Mel is a source of knowledge and encouragement, and continues to mentor, albeit informally, young students, former graduate students, and colleagues at yearly scientific meetings and e\ents. The scientific fields of malacology, shellfish biology, and marine ecology have prospered from his life's work, and all of us who have had the pleasure of interacting with him have benefitted by Mefs wisdom, poise, and grace. J. Evan Ward Groton. Connecticut REFERENCES Carriker. M. R. 2000. Viski Merc. Blue Mantle Press. Rio Hundu. Texas. 313 pp. Kraeuter. J. & S. Ford. 1494. Harold Haley Haskin. Honored Life Member. J. Shellfish Res. 18:337-339. Joiiniul of Shellfish Research. Vol. 22. Nci. ?. (il?-6l7. 2003. Michael Castagna Honored Life Member Michael Castagna. known to almost everyone in NSA as Mike, was horn in Janesvijie. Wisconsin on October 21. 1927. His parents immigrated to this country from Sicily; his father worked in a General Motors factory in Janesville and his mother worked in the home and for a time in a woolen mill. Following graduation from Janesville High School in 1945. Mike joined the Navy, received his initial training in the Great Lakes, and first viewed the ocean when he shipped out for the Pacific. Mike was stationed in Honolulu where he served as a Pharmacist Mate 2nd Class from 1945 until 1949. After leaving active duty in the Navy, he enrolled at Florida State University as an undergraduate where he participated in intercollegiate sports, swimming on the all-Navy swim team. In 1951. with only one semester of study remaining at FSU. Mike was recalled to active duty for the Korean conflict as a Hospital Corpsman 2nd Class. Mike's swimming talents were quickly put to use as he became one of the first Navy divers to use SCUBA, taking part in many of the initial dives that led to the development of the now familiar dive tables. When his tour of duty was over in 1953. he returned to FSU to complete work on his Bachelor of Science degree. While enrolled in school. Mike supported himself by working in the Women's Department of Physical Education. After receiving his B.S. degree in 1953. he was admitted to the graduate program at FSU where he worked on a Master's degree. He completed this degree in 1955 with a study of the distribution and ecology of the hogchoker (Trinecles nniciilatiis) in the Wakulla River under the guidance of Dr. Ralph Yerger. In his first job out of graduate school, many of Mike's talents — swimming, fisheries, biology, and a keen love of the ocean — were used as an Assistant Curator at Marine Studios of Marineland. located just south of St. Augustine. FL. He literally swam with dolphins and was in charge of caring for and treating any of the animals that became ill. At this time Mike and his wife of 48 y. Mary Sperry. got married. Mary worked for many years as a nurse and she and Mike have four children. In 1956. Mike was hired by the Bureau of Commercial Fisheries (BCF) in Boothbay Harbor. ME. to work on the herring investi- gations under Les Scattergood. This job put him back out on the ocean with frequent sampling trips offshore. During the two years Mike spent in Boothbay Harbor, he served in the Naval Reserves and on several occasions was sent to Key West. FL. for Underwater Demolition Training. There, as the oldest member of the team at nearly 30. he was called "Grandpa" by the younger team members, but he went on to graduate with highest honors. In 1958 he left Boothbay Harbor and began work at a small BCF laboratory in Franklin City. VA. This laboratory was placed at the end of a long causeway on a former railroad spur, which extended into Chincoteague Bay. The sheet metal building was built next to the former railroad pier. It was a perfect place for Mike who has both the ability to develop new techniques and a hands-on work ethic. Mike has always had a firm commitment to understanding the fundamental ecology of the area where he was working. This included what was present, where it could be found, general observations on abundance, and life history biology. To that end. he helped to design and fabricate the gear needed to investigate the marine life of the bay. After a short time in Franklin City, he was asked by a fellow Florida State graduate. Bill Hargis. to become the Scientist-in-Charge of the Virginia Institute of Marine Science (VIMS). College of William and Mary laboratory in Wachapreague, VA (At the time it was only known as the "Eastern Shore Laboratory", and it was not until much later that the formal connection to William and Mary was 615 616 Kraeuter established). This new position included moving into a newly constructed, single floor building housing offices, wet and dry laboratories. and two dormitory rooms. This was an inspired choice, because it allowed Mike"s skills, of leadership, mentoring, innovation, and hard work to flourish. Mike began this position in 1962. remained at the Eastern Shore Laboratory moving up the ranks at VIMS until he retired as a Professor and Division Director in 1992. He continues to work at the laboratory as a Professor Emeritus. Mike instituted a prograin to gather basic biological information on the flora and fauna of the local area. He became intimately involved in seeking information froin, and giving information to. the local fishing community and encouraged others to take field trips to the Wachapreague area. In addition to the basic science efforts. Mike coordinated the Eastern Shore components of many oyster trials that were being conducted at VIMS in Gloucester Point. VA. Efforts to control oyster drills with the pesticides Polystream and Sevin. numerous studies on oyster disease and the effectiveness of disease resistant stocks in the higher salinity waters were among the research projects that kept Mike and many others busy with field work. Although his diving skills were not used heavily at Wachapreague. he often helped members of the community find lost gear or clear fouled propellers. The love of diving and natural history were combined when Mike was invited to spend nine days in the Underwater Laboratory Helgoland in the Baltic Sea in 1974. He returned there as a scientific coordinator for a 14 day underwater mission in 1978. In between (1976) he spent five days in a Hydro Laboratory off Freeport, Bahamas. In 1962, Mike hired Paul Chanley and they began a series of investigations into bivalve natural history. This included providing information on spawning times, salinity tolerance, larval development, and other aspects for over 60 species. By the end of Mike's tenure as head of the laboratory. 55 species had been reared to setting and 26 species had been reared through their entire life cycle. Much of this work was done in large garbage cans. Water was exchanged by siphons, but was, from time to time, carried in buckets across the road by hand. When temperatures in the wet laboratory were not high enough to rear larvae, the "culture containers"" were placed on wheeled cans and aligned down the hall between the offices. This Spartan setting was certainly indicative of funding limitations, but it also reflected Mike's frugal, get-the-job-done approach. As a direct result of the efforts to document the various life history parameters of bivalves. Mike developed expertise in hatchery technology and aquaculture. This led to the development of a greenhouse for culturing large quantities of algae via the Wells-Glancy technique and later, in a converted oyster shucking house, to a fairly large nursery for the hatchery output of bay scallops and hard clams. Here again. Mike's ability to design and engineer simple, cost-effective solutions was critical. One of the most enduring images from this hatchery was a heat exchanger crafted from an old whiskey ban-el and salvaged tubing. Mike often said, ""There's no reason to spend $2 on a valve if pinching a hose will work just as well." Already involved with maintaining a large number of oysters in trays, scattered throughout several bays, Mike was well aware of the difficulties with field studies. This reality and the lack of seed caused his early focus on hatchery and nursery work with clams and scallops. The success of this program provided burgeoning numbers of clams and scallops and he began to develop experimental field plantings. Unless they were heavily protected in trays, the early clam plantings were nearly all consumed by crabs, and e\en modest size grow-out experiments required tremendous effort. As an example of Mike's inventiveness, one fall, with a substantial number of clam seed on hand, and the necessity of having to close down the seawater pumps for the winter, Mike happened to glance out the window. Within the past week, the road had been taned and covered with gravel. Most of the gravel had been pushed to the side of the road. Mike decided that, because of the well-documented association of clams with shell beds, gravel inight be a good shell substitute. The gravel was swept from the road, loaded into a scow and placed on an intertidal mud flat of a marsh creek. Clams were planted in this gravel and survival was excellent! Unfortunately, subsequent years' plantings did not survive as well. It took Mike, the Eastern Shore Laboratory staff, input from various waterinen, many clams, a number of years and a lot of trial and error to develop the knowledge of planting size and protective mechanisms to assure consistent results with seed planting. This effort, as with the innovative descriptive work of Chanley and Castagna a decade earlier, established the Eastern Shore Laboratory as a premier place to do research on bivalve shellfish. This reputation was enhanced by the development of a course to teach basic techniques in clam aquaculture, including how to make the gear, to a cadre of individuals. Many of these individuals became leaders in the hard clam aquaculture industry that has spread throughout the east and gulf coasts, now employs hundreds of indi\ iduals and is woilh tens of millions of dollars annually. Mike has authored or co-authored >75 peer reviewed publications, many abstracts, served as editor for two books and was a co-author on a host of reports — including one that has probably been read by more individuals than any work published in the peer-reviewed literature. "'A manual for growing the hard clam Mercenaria mcrccnaria". Field Trips Because of his interest in natural history and his gregarious nature. Mike was always ready to lead a field trip. These were of two types, those for fellow scientists visiting the Eastern Shore Laboratory and those for students. Always the raconteur par excellence Mike had many tales to tell about visits from scientists. One that left a distinct impression was a visit by a distinguished senior scientist from Europe. Mike was impressed by the scientist"s world reputation and wanted to provide a grand tour, which included visiting the habitats on a nearby barrier island. The island had a few cabins that were used primarily on summer weekends, and in the winter for hunting. Mike anchored the boat and indicated they would have to wade ashore. The senior scientist had already figured this out and proceeded to disrobe — completely. Though there were seldom people on the island, passing sport or commercial fishing boats were not uncommon. Mike, thinking that someone might pass by. and wanting to keep the situation as decorous as possible for the laboratory's reputation, handed the individual a towel. The scientist thanked him and proceeded to wrap the towel around his head as a turban and walked ashore. A significant part of the program at the VIMS Eastern Shore Laboratory was the hosting of field trips for students from other Honored Life Member Michael Castagna 617 institutions. This program, which) Mil.1°C) followed by spawning from mid-May to mid-August (>8'C). Although mature eggs were observed in the ovary in July-August, spawning trials suggested a declme in the fecundity of the Bayport population during this period. Two main recruitment events were observed at Mason's Beach (June and August), but only one at Bayport (June). From the data on fecundity and settlement rates, it was estimated that a 100-mm long C. intestinalis (0.6 g dry weight) may produce 12.000 eggs in a season and that recruitment intensity may reach 3.000 individuals m"-. Laboratory predation trials indicated that rock crabs (Cancer irroratus) consumed significantly more C. inlestinalis than did green crabs (Carcinus muenas). A ma.ximum predation rate of 1 1 individuals per day per rock crab (80 mm carapace width) was recorded at peak water temperatures of 18X. In a series of chemical width eradication trials, exposure to 5'"* acetic acid was found to be a more effective strategy for eliminating C. inlestinalis than hydrated lime, saturated brine, or hypochlorite solution. Total mortality was observed following exposure to 5% acetic acid for 15 to 30 s, with no corresponding mortality in the control mussels or oysters. Initial field trials indicated that spraying with acetic acid might prove to be an effective means of eliminating C. inlestinalis under commercial conditions. KEY WORDS: Ciona inlestinalis. tunicates, biofouling, shellfish production, predation INTRODUCTION Ciona intestinalis is a solitary phleobranchiate ascidian, or tu- nicate, which occurs on natural substrates such as rocky bottoms and eelgrass beds, or on artificial structures such as aquaculture gear, marker buoys, dock pilings, and boat hulls (Petersen & Riis- gard 1992, Connell 2000, Ma/ouni et al, 2001), Although native to the northern Atlantic Ocean (Van Name 1945, Plough 1978). this species is now distributed worldwide, most likely as a result of dispersion by shipping activities (Monniot & Monniot 1994, Lam- bert & Lambert 1998). Published accounts indicate that C. intes- tinalis has recently become a serious biofouling problem for many shellfish culture operations including those in Scotland (Karayucel 1997), .South Africa (Hecht & Heasman 1999), and Chili (Uribe & Etchepare 2002). In eastern Canada, the severe impact of C. in- testinalis biofouling was first documented in 1997 at a mussel farm in Lunenburg Bay, Nova Scotia (Cayer et al. 1999). In an unprec- edented recruitment event, this tunicate species heavily colonized the mussel sleeves, causing a substantial reduction in growth and the eventual loss of the crop. Subsequent reports of significant C intestinalis recruitment at several other shellfish growing sites in Nova Scotia suggest that this species has become a widespread biofouling problem. In a similar scenario, the nonindigenous club tunicate Styela ckiva has recently infested several mussel fanns on the eastern coast of Prince Edward Island and is now recognized as a serious threat to the viability of the ttiussel industry (Boothrovd etal. 2002). Information on the basic life-history traits of C intestinalis originates primarily from natural populations in northern European *Corresponding author. E-mail: amalletcs'ns >ympatico.ca waters (Gulliksen 1972, Svane 198.3, Petersen et al. 1995, Petersen et al. 1 997 ). Under these conditions, the life cycle of C. intestinalis is reportedly 12 to 18 mo, with growth and longevity varying in response to temperature and food levels (Millar 1952. Petersen et al. 1995). Growth rates in terms of length are estimated at 1 to 3% day"' or 10 to 20 mm mo ' (Dybern 1965. Petersen et al. 1995). In contrast, reports from Japan indicate that C. intestinalis has a life span of 3 mo in the summer at temperatures of 20 to 26°C, and 6 mo in the winter at 14"C (Yamaguchi 1975). The timing of reproductive activity also varies depending on temperature. In more northerly regions, such as in Sweden, reproductive activity peaks in May and June, whereas in warmer zones, such as Britain, gamete release may occur throughout the year (Dybern 1965, Gul- liksen 1972). Given the various life-history strategies of this spe- cies, it is important to document this basic information for C. intestinalis populations in Atlantic Canada. The primary objective of this study was to develop a strategy to mitigate the impact of C. intestinalis on an oyster culture operation in Lunenburg. Nova Scotia. In contrast to mussel culture, oysters are contained in a cage from which the tunicates can be removed without losing the inventory. Heavy infestations, however, have the potential to depress shellfish growth, and to increase mortality due to competition for food (Lesser et al. 1992) and obstruction of water flow (Uribe & Etchepare 2002). The removal of these tuni- cates from the grow-out structures and oyster inventory is labor intensive, and, in .some cases, disposal of the waste biomass can be costly, A series of field and laboratory experimental trials were undertaken from November 1999 to Noveinber 2000 for the fol- lowing purposes: (1) to document the local distribution of C in- testinalis; (2) to investigate the growth, spawning, and recruitment patterns of this species: and (3) to evaluate possible biological and chemical strategies for eliminating this species from the culture equipment and the oyster inventory. 621 622 Carver et al. MATERIALS AND METHODS Field Ecology Distribution, Growtli, and Condition Several exploratory dives aimed at documenting the local dis- tribution of C. inlestinalis were carried out at the two field sites in Lunenburg Bay, Bayport. and Mason's Beach, in the fall of 1999 and the fall of 2000 (Fig. 1). Two experimental oyster tables with oyster bags containing adult C. intestinalis (year 1999 class) were set up at each of the two grow -out sites (i.e.. Mason's Beach and Bayport) on October 30. 1999 (Fig. 2). Temperature recorders were attached to the tables at each site. The two experimental groups were sampled monthly from November 1999 to May 2000 and then every 3 wk until September 2000. On each occasion, a random sample of 10 indi- viduals was collected from each site to evaluate their condition index. Each individual was measured and dissected to obtain es- timates of wet tunic and wet body weight, and then they were dried overnight at 60°C for 24 h and reweighed. The condition index was calculated as dry body weight divided by total dry weight. In early June 2000. oyster bags with recently recruited indi- viduals were transferred to the experimental tables. Growth in terms of length, whole wet weight, and whole dry weight were estimated for the newly settled year 2000 cohort. Ten individuals from each site were measured, weighed, and then dried. Due to the difficulty in obtaining measurements from individuals in a fully e.xtended position, a relationship was derived between body diam- eter when contracted and body length when alive and fully ex- tended. This was used to estimate the mean body length of the Figure 2. Pliotograph of an oyster table with oyster bags. Tunicates are apparent on the lower side iif the oyster bags. cohort over time. .\ final sample was collected in November 2000 to document the development of the year 2000 class. Reproductive Status Five individuals from each year class at each site were dis- sected and weighed, and the body was fixed in 1% glutaraldehyde and 47f formaldehyde. The samples were then sent to the Diag- nostics Laboratory at the Atlantic Veterinary School (Prince Ed- ward Island) for histological processing. The tissues were embed- 4 ^%^ K.. 0 50 100 y NoC: ~^- 44022 - 44°20' 64°20' 64° 16' Figure I. Map of l,unenburg Bay showing the location of the mussel farm in Upper South Cove, the site of the initial C. intestinalis infestation, and the two experimental sites, Bayport and Mason's Beach. BlOFOULlNG OF CULTURED SHELLFISH BY ClONA 623 ded in paraffin and sectioned (6-fjLm thick), and the sections were stained with hematoxylin and eosin. The histology sections were assessed for reproductive status using a Weibel graticule (two fields per slide). The contents of each field were assigned to five categories: empty of follicle tissue; eggs in early development stage: eggs in late development stage: mature eggs: and regressing eggs. Mature eggs are surrounded by a thick layer called the vi- telline coat, which clearly distinguishes them from immature or developing eggs. These data were used to estimate the proportion of the ovary that contained follicle tissue and the proportion of that area occupied by eggs in various stages of development. The cross- sectional area of the ovary was akso measured using an image analyzer system. Recruitment Four recruitment plates (-200 cm") cut from clean but used oyster bags (4-mm mesh) were attached to the lower side of each oyster table on each sampling occasion. Plates deployed on the previous sampling trip were retrieved and were placed in separate plastic containers filled w ith seaw ater for transfer to the laboratory. The plates were examined with a stereomicroscope to detect the presence of newly recruited juvenile C. imestinalis. The plates were then placed in flowing filtered seawater (50 |j,m) for 2 to 3 wk to allow for the development of very small individuals that may not have been counted initially. The plates were then reassessed. and the maximum of the two counts was retained. The final counts for both sides of each plate were tallied and divided by the avail- able solid area to estimate the intensity of settlement over the previous sampling period. The data were plotted such that any .settlement that was observed at the end of a particular interval was assigned to the midpoint of that interval. Laboratory Trials Larval Development The objective of the first .series of trials (January-May 2000) was to induce natural spawning in the laboratory, to document the various phases of larval development, and to devise a protocol for rearing juveniles. Adult C. imestinalis from the 1999 cohort were collected from the field populations at each sampling event and were transferred to a fiow -through system running at ambient tem- perature with unfiltered water. Spawning trials were undertaken on January 19. February 16. February 28, March 13. March 30, April 18, and May 3. To determine whether the adults possessed com- petent gametes, attempts were made to trigger spontaneous spawn- ing by exposing individuals to a natural-light regimen for 24 h. When this proved unsuccessful, adults were strip-spawned and cross-fertilized to determine whether the eggs were competent. Fertilization trials were conducted at ambient water temperatures (0-6°C). Fecundity A series of five spawning trials were conducted in the quaran- tine unit at the Bedford Institute of Oceanography from May 15 to August 25, 2000, to estimate the fecundity of individuals obtained from the 1999 C. intestinalis cohort at both sites. Several individu- als from the newly recruited 2000 cohort were included in July and August in an attempt to determine the minimum size at which spawning was initiated. The first four trials each lasted from 14 tol8 d (May 15-June 2. June 8-26, July 4-21, and July 25-August 10), but the fifth trial (August 1-^25) was discontinued after 10 days because of technical problems with the water supply system. Five individuals of various sizes from each site were placed in separate 50()-mL Mason jars in a tank of ambient flowing seawa- ter. The water was prefiltered through a 40-(xm mesh to remove any risk of contamination from eggs originating outside the sys- tem. The water level in the main reservoir was adjusted such that the flowing water just cleared the top of each jar: the objective was to allow sufficient flow for gas exchange and particle renewal but not enough to entrain the eggs. Control jars were placed down- stream in the tank to estimate whether eggs were being lost. No eggs were retrieved from the control jars, and observations of fecal deposition suggested that negatively buoyant particles, including eggs, were retained inside their respective jars. The experimental tank was set up approximately 3 m from an east-facing window such that the dawning light each morning would induce normal spawning behavior (Lambert & Brandt 1967). Every second or third day. the individual tunicates were transferred to new jars, and the contents of each old jar were screened through a 60-|xm mesh to retain any eggs ( 150 (xm size) produced over the previous 48 to 72 h. The jar and the screen were well rinsed with filtered seawater to remove any eggs stuck to the surface and were then flushed with hot freshwater to avoid con- tamination between samples. The eggs from each jar were col- lected in a petri dish and were counted using a stereomicroscope. Fecundity was estimated in terms of eggs produced per individual per day over the duration of the trial. At the end of each trial, the surviving individuals were dissected for assessment of dry body weight. Methods of Control Natural Predation A series of predation experiments were set up in flowing sea- water tanks in the quarantine unit at the Bedford Institute of Oceanography, Nova Scotia. Various sizes of C. intestinalis at- tached to weighted pieces of oyster bag were offered to a range of potential predators including starfish (Asterias vulgaris), green crabs (Carcinus maenas), rock crabs {Cancer irroratus). and her- mit crabs (Pagiints acadianus). The first three trials were con- ducted in late January 2000 at water temperatures of 2 to 4°C. The second series of five trials focused on assessing the predation activity of rock crabs versus green crabs at a range of temperatures. Trials were undertaken on February 4 to 14 (2°C), April 13 to May 3 (5°C), July 27 to 31 (15°C), August 8 to 10(18°C), and August 14 to 15 ( I8°C). The crabs ranged in carapace width (CW) from 40 to 100 mm, and the tunicate prey ranged in length from 15 to 125 mm. The duration of the experiments had to be reduced in the later trials to ensure that the supply of prey was not exhausted prior to the end of the trial. Predation rates were calculated in terms of indi\idual tunicates consumed per crab per day. Chemical Treatment A series of physical/chemical eradication trials were under- taken in the laboratory from February to August 2000. The chemi- cals tested included sodium hypochlorite ( 10-60 parts per million), hydrated lime ( 1—4%), saturated brine, freshwater, and acetic acid ( l-57f ). The effectiveness of heated freshwater (40°C and 60°C) for eradicating C. intestinalis was also investigated. Various sizes of tunicates were used in each trial to determine whether younge 624 Carver et al. stages might be eliminated more easily than older stages. Mussels and oysters were also included in the trials to ascertain whether the treatment could potentially be used to remove tunicates from shell surfaces or from gear containing shellfish. RESULTS Local Distribution and Conditions Diving surveys carried out at both sites in the fall of 1999 and the fall of 2000 did not identify any C. inlestinalis attached to natural substrates, including rocks or eelgrass. None were ob- served on local wharf pilings at Baypoit. but there was a substan- tial population attached to the bottom of a floating dock at Mason's Beach. Otherwise, C. intestinalis was only observed attached to oyster tables or suspended culture gear such as mussel sleeves and longlines. Both experimental sites typically have a lower incidence of C. intestinalis than the more sheltered Upper South Cove, the site of the original 1997 infestation, where the conditions tend to be warmer and more productive (Mallet & Carver 1993). Tem- perature profiles for the two e.xperimental sites were virtually iden- tical (Fig. 3). C. intestinalis: 1999 Year Class Growth and Condition Index Estimates of body length and total wet weight per individual for the 1999 year class showed low variation over time or location (mean values: November 1999 69 mm and 5.9 g. respectively; September 2000 76 mm and 6.7 g. respectively). The mean dry weight per individual remained at 0.3 to 0.4 g (range 0.1-0.9 g) for the duration of the study, and the overall relationship between whole dry weight (gl and body length (mm) was estimated as y = 0.0000106X-"* (r- = 0.91). Note that both primary tissues, the outer tunic and the body, are composed of approximately 95% water. Although the smaller individuals did grow from April to September 2000. the mortality of the larger individuals during the summer obscured any population growth trend. During the colder months, the condition index (dry body weight/total dry weight) declined slightly from 44% in November 1999 (6"C) to 40% in late February 2000 (0°C) (Fig. 4). The condition index then increased sharply at both sites to a maximum of 60% at Bayport in late April, and 55% at Mason's Beach in 80 20 Bayport Masons Beach 1 — I — ' Feb I — I — I Apr I — I — I Jun Dec Feb Apr Jun Aug Oct 1999-2000 Figure 4. Condition index (dry body weight/total dry weight) for the year 1999 class of C. intestinalis at Bayport and Mason's Beach. mid-May. At that lime, the ambient water temperature at both sites was in the 6 to 9°C range (Fig. 3). The condition index of C. inlestinalis at the Bayport site declined steadily from late April to early August, stabilizing at 35%). This profile would suggest that spawning started between April 20 and May 13. and continued through June and July. In contrast, the 1999 cohort at Mason's Beach exhibited a slight drop in condition in May but then main- tained a condition index of >50% until mid-July, at which time values declined sharply. If the condition index is related to repro- ductive status, this profile suggests that the major spawning event at Mason's Beach occurred after mid-July or later than at Bayport. Reproductive Status Data on the reproductive status of C. intestinalis were pooled over the two sampling sites. The mean cross-sectional area of the adult ovary increased from 10 mm" in November 1999 to 25 mm" in late Januarv 2()()(). declined slightly in February-March, and then rebounded in April-May to 24 mm". Between May 13 and June 7. the mean size of the ovary fell to approximately 10 mm", where it remained until September. Estimates of the proportion of the ovary occupied by follicle tissue ranged from 55 to 70% from November 1999 to March 2000, increased to 90% in April-May, and then declined to 70% in July (Fig. 5). The follicle area occu- Apr Jun 1999-2000 Figure 3. Temperature profiles for Bayport and Mason's Beach from November 1999 to November 200(1. too 80 60 CD 40 20- D Early Dev Q Late Dev ■ Mature ■ Regressing 4,C 'iLU 6oC 9oC 0.C •^ <$" $? ■•' ^250 eggs ind"' day"' in May-June. However, unlike the Bayport population, the individuals at Mason's Beach continued to produce >100 eggs TABLE 2. Egg pniduction ratus for year 1999 class C. iiilesliiialis (eggs ind day 'l from the two experimental sites overtime. Trial Bayport Mason's Beach Duration (eggs Ind"' d"') (eggs Ind ' d"') May 15-June 2 183 ±80 221 ±98 June 8-June 26 172 ±71 257 ± 70 July 4-July 21 97 ± }4 160 ±40 July 25-August 10 35 ± 15 150 ±.30 Values given as mean ± SE. day"' through July-August. This was consistent with the higher condition index for this population. Larval/Juvenile Development From January 19 to March 30 2000. eggs were obtained by dissection because of failures to trigger spontaneous spawnings. Very few mature eggs were obtained from January through March, and the sperm rapidly lost motility. In the few instances in which mature eggs were obtained, fertilization was generally poor (<10%), and development did not proceed to the larval stage. In the April 18 and May 3 trials, however, larvae were successfully produced both by spontaneous spawning and dissection. As in the earlier trials, the eggs were fertilized at ambient temperature (6- 9°C in April-May) and then were allowed to gradually warm up to 15°C in the dark. The development of C. inteslinalis eggs at 15'C typically took 24 to 36 h. hatching and growth of the tadpole larvae lasted 24 h, followed by settlement and metamorphosis over another 12 h for an approximate total of 3 days to the juvenile stage (see also Berrill 1947). Larvae were successfully settled on plastic petri dishes, where they metamorphosed into juveniles. The dishes were sub- merged in a 10-L tank, and the water was changed every 2 to 3 days. The juveniles proved to be remarkably resilient and survived for weeks with minimal handling/feeding. A series of photos were taken to document the development of C. intestinalis from the egg to the juvenile phase (Fig. 7a, b, c, d. e, and f). It should be noted that the species identity of C. intestinalis was confirmed by the presence of single refringent bodies in the halo of follicle cells that surround the egg (Byrd & Lambert 2000). TABLE 1. Results of first spawning trial (May 15-June 1) indicating the individual variability in daily egg production rate over time. C. intestinalis individuals Here brought in from the t«o Held sites on May 13 and were held in flowing seawaler until Jun 2, Ind May May Mav Mav May Mav Mav Mean Eggs Length Whole Drv No. 15-17 17-19 19-22 22-24 24-26 26-29 29-31 May 31-.lun 2 day"' (mm) Weight (gl Ml 7 292 91 378 615 206 717 37S 317 89 0.41 M2 2 1032 66 120 165 178 72 224 223 65 0.47 M3 0 0 0 0 0 0 54 0 6 56 0.27 M4 0 798 1998 363 0 398 0 44 533 89 o,m M5 0 228 0 6 0 6 5 0 28 79 0.37 Bl 0 0 0 105 74 126 60 102 59 65 0.16 B2 0 3.W IS 1158 453 416 140 1674 491 89 0.53 83 117 344 45 135 281 164 71 135 155 74 0.36 84 42 77 43 0 174 78 80 0 62 61 0.27 85 225 180 III 0 392 0 371 0 148 70 (1.29 Mean 44 329 237 226 215 157 157 256 202 74 0.40 Abbreviations: M = Mason's Beach; 8 = Bayport. 626 Carver et al. D ■B c LLJ BOOH -•- Bayport ■ -o- Masons Beach 600- o ■ • 400- 200- 0- • -1 — 1 1 1 1 ' 1 ' 1 0.2 0.4 0.6 1.0 Whole dry weight (g) Figure 6. Fecundity (eggs ind"' day ') versus wliole dry weight of the 1999 C. iiilesliiialis from Mason's Beach and Bayport from May through August 2000. Data from individuals that died during the trials were not included. C. intestinalis: 2000 Year Class Recruitment Patterns The observed recruitment profiles suggest one settlement event at Bayport and two at Mason's Beach (Fig. 8). Estimates ot settle- ment intensity ranged as high as 47 per 100 cm" of solid collector area, but levels often varied substantially among replicate plates. The timing of the settlement peak at Bayport (May 13-June 29) is consistent with the condition index/spawning profile for the year 1999 class (Fig. 4). In the case of Mason's Beach, the timing of the second recruitment peak (Aug 3-24) closely followed the decline in condition observed at that site (Fig. 4). The absence of a decline in condition in May-June may indicate that the first recruitment event at Mason's Beach was related to an influx of larvae from other areas such as Bayport or Upper South Cove. However, the fecundity trials confirmed that Mason's Beach adults were capable of producing eggs from mid-May onward. Recruitment plates de- ployed from August 24 to September 19 exhibited some new settlement at Mason's Beach, but no juveniles were observed on the plates deployed from September 19 to November 29. 2000. Growth Rate The growth rate of the first year 2000 cohort in terms of body length was relatively steady from mid-July through to mid- September and then decreased, possibly due to declining water temperature or the onset of maturity (Fig. 9). Continued growth in terms of whole dry weight through October-November was appar- ently related to an increase in body weight as opposed to length (Fig. 10). Whereas profiles of mean body length were similar for the two sites, estimates of whole dry weight were consistently higher at Mason's Beach than at Bayport. At Mason's Beach in November 2000, the mean body length was 96 mm. the whole wet weight was 1 1 g, and the whole dry weight was 0.7 g. These values were consistently higher than those for the year 1999 class the previous November. Individuals from the second year 2000 cohort at Mason's Beach had a body length of 36 mm. a mean wet weight of 0.5 g, and a mean dry weight of 0.05 g on November 29, 2000. The overall relationship between whole dry weight (g) and body length (mm) for the year 2000 class was estimated as y = 0.0000080 lx--'(r- = 0.93). Reproductive Status Profiles of the percentage of follicle area as well as the pro- portion of the follicle area occupied by mature eggs increased rapidly between July 19 and August 3. 2000 (Fig. 1 1 ). At that time, the tlrst year 2000 cohort had a mean length of 47 mm and a mean whole dry weight of approximately 0.1 g. Although dry weight continued to increase through the fall, gonad area increased only slightly to approximately 10 mm", and follicle area remained at 70%. Over the same period, the proportion of mature eggs declined from 40 to 20% in late November. Final values for all three re- productive indices were slightly higher than those for the 1999 year class recorded 1 y previously. Fecundity A few individuals from the year 2000 class did produce eggs in the July-August fecundity assessment trials. Estimates were typi- cally 20 mm in she!! 628 Carver et al. 50 40 e " 30 o o 20 10 0-t Bayport — Masons Beach T 1 1 1 T" Apr May Jun Sep Oct 2000 Figure 8. Projected recruitment profiles for the two year 2000 cohorts of C. intestinalis at Bayport and Mason's Beach. length (SL) were typically uiiiilYected b> the acetic acid spray/dip. but control mussels <10 mm SL died in one comparative trial. Other chemical methods were consistently less effective at eradicating tunicates. Exposure to hydrated lime for 8 min was 70% effective, whereas saturated brine was only 20% effective over the same exposure time. Solutions of sodium hypochlorite at concentrations up to 60 parts per million for as long as 20 min had no impact on tunicate survival. Exposure to freshwater for 1 min resulted in only 10% mortality. Longer exposure times may be more effective, but under field conditions this may not be practical. A 1-min exposure to 40°C freshwater was 100% effective at eradi- cating C. intestinalis. but the European oyster (40 mm SL) and one of the two mussels (50 mm SL) also died. The second phase of the eradication trials was to test the ef- fectiveness of acetic acid treatment on C. intestinalis attached to oyster grow-out bags in the field. It should be noted that these trials were preliminary and were only assessed at a qualitative level. To administer the treatment, the acetic acid solution was placed in a garden-spraying unit. Goggles, gloves, and appropriate clothing were used, and care was taken to ensure that the bag being sprayed was located downwind. Although there was a slight smell, the fumes were rapidly dispersed in the open air. The treatment pro- tocol followed was similar to that developed in the laboratory trials: 5% acetic acid spray for .^0 s followed by air exposure for - Bayport — Mason's Beach 0 8 - r 0 6 - 0 ^"\^ 5 04 - 0.2 - ^^ - ' 1 1 1 1 1 1 1 1 I 1 I -r- 1 1 Jun Jul Aug Sep 2000 Oct Nov Dec Figure 9. Increase in body length (mm) over lime for the year 2000 cohort of C. intestinalis at Bayport and Mason's Beach. Figure 10. Increase in whole dry weight (g) over time for the year 2000 cohort of C. intestinalis at Bayport and Mason's Beach. .^0 s. Note that the C. intestinalis individuals had ample time to fully contract before being exposed to the treatment. The preliminary trials were earned out on August 24 and Sep- tember 19. 2000. The oyster bags were covered with a heavy settlement of 70 to 80-mm-long year 2000 individuals. In each case, individuals on one third of the bay were covered to act as controls. It should be noted that the health of these control indi- viduals was apparently not affected by either the nearby applica- tion of the acetic acid or the subsequent mortality of their imme- diate neighbors. The effectiveness of the treatment varied from 60 to 100%, depending largely on the density of the settlement (qualitative assessment). In the second field trial (September 19). 10 European oysters (40 mm SL) were placed in the bags while the acetic acid treatment was administered. On average, 80% of the oysters were alive when the bag was examined on November 29. Although these small-scale experiments were in no way conclusive, the re- sults were sufficiently promising to warrant further trials. DISCUSSION Natural Distribution In northern Europe, substantial natural populations of C. intes- tinalis are found in eelgrass beds or attached to rocky substrates (Dybem 1965, Gulliksen 1973, Petersen & Riisgard 1992). Diving surveys in 1999-2000 aimed at documenting the local distribution of C. intestinalis at the two study sites in Lunenburg found no individuals attached to natural substrates. Two independent sur- veys of the Bayport area carried out in August 2000 and August 2001 also failed to locate any C. intestinalis on eelgrass or rocky bottom areas (Barry MacDonald, Department of Fisheries and Oceans, pers. comm.). It would appear that the distribution of this species is generally restricted to man-made structures, such as floating docks and aquaculture gear. The sudden proliferation of the C. intestinalis population in Lunenburg is a classic example of the potential impact of man- made structures on the settlement and survival of sessile inverte- brate species (Connell & Glasby 1999). Various Australian studies comparing the species assemblages found on pier pilings and pon- toons versus adjacent natural substrates have suggested that the introduction of artificial structures may effectively increase local species abundance and diversity (Butler & Connolly 1996, Glasby BlOFOULING OF CULTURED SHELLFISH BY ClONA 629 Figure II. Roproductive status of the first year 2000 eiihort of C. inlesiinalis: proportion of the ovary that contained follicle tissue with early development, late development, or mature eggs. 1999. Connell & Glasby 1999. Connell :(l()0). In particular, soli- tary ascidians such as C. intestinalis were typically more abundant at marinas than at reference locations (Glasby 1997). Ascidians in general have been recognized as the dominant biofouling organism on oysters grown in rope culture in L'Etang de Thau (France) (Mazouni et al. 2001). The conspicuous absence of C. intestinalis from natural sub- strates suggests that manmade stmctures may function as a refuge from predation. Field studies in Denmark and Norway have re- ported that variations in spatial abundance of this species are linked to predation by sea stars {.Aslerias niljcns). plaice (Pleu- ronectes platessa). and cod (Gctdus morhua) (Gulliksen 1972. Gul- liksen & Skjaeveland 1973). Natural predators include jellyfish, sea stars, rock crabs, hermit crabs, dog whelks, and surface- feeding fish (Gulliksen 1972. Yamagiichi 197.'i. Svane 198.3. Ole- ,sen et al.l994). Recently settled juvenile stages may also be sus- ceptible to dislodgment by surface grazers such as gastropods and sea urchins (Svane 1983). Predation trials conducted in the present study demonstrated that the rock crab. C. irroratus. can rapidly excise the body tissues of C. intestinalis from the heavy tunic and may consume as many as 1 1 ind day"' during the summer months. M-\ 10- — 6 c g I 4 CD Green Crab Rock Crab Temperature {°C) Figure 12. Predation rates of two crab species (ind crab"' day" intestinalis (35-80 mm long! for a range of temperatures. 20 ) on C. Field observations also suggested that predators, in particular crabs, were actively reducing the abundance of C. intestinalis on the upper surface of the oyster bags but were apparently unable to access individuals attached to the underside of the bags (Fig. 2). Other surface-feeding predators such as sea stars may also play a role in controlling the distribution of small individuals, but. apart from the crab activity, there was no indication of any significant predation pressure on the larger lunicates. Life History Traits Field observations suggested that most of the individuals from the 1 999 year class died prior to November 2000. This pattern of mortality, apparently as a result of natural senescence, was con- sistent with life span estimates of 12 to 18 mo for C. iniestiuatis in Scandinavian waters (Petersen et al. 199.'i). Similar to Lunenburg, reports from Sweden indicate that C. intestinalis. which settles in the summer, spawns the following spring and dies during the win- ter ( Dybern 1 965 ). Reports from Japan suggest the life span of this species is apparently determined by cumulative environmental temperature (Nomaguchi 1974). Thus, individuals that settle early in the summer, such as those in the first year 2000 cohort at Lunenburg, may die al a younger age than those that settle in late summer. Estimates of growth rate in terms of body length for the year 20(J0 cohort were approximately 20 mm mo"' from July through September, which is similar to estimates from Swedish (Petersen et ai. 1995) and Chilean waters al 12 to 21 mm mo"' (Uribe & Etchepare 2002). Observations on maximum size in terms of body length (100-140 mm) were higher than the 60 mm reported for Japanese waters (Yamaguchi 1975). Perhaps this species can attain a larger body size under colder conditions. The results of the histological assessment and the spawning trials indicated that individuals that settled in May-June were ca- pable of initiating egg production and spawning by August of the same year. This was consistent with observations from Sweden where two breeding generations of C. intestinalis have been found to co-occur in populations living close to the surface (Dybern 1965). D 15-35 mm ■ 35-80 mm ■ 80-125 mm 50 70 90 Crab carapace width (mm) Figure 13. Predation rates of various sizes of rock crabs (ind crab" day"') on a range of sizes of C intestinalis. 630 Carver et al. Yamaguchi ( 1975) also reported that C. intestiualis reached sexual maturity within 2 mo of settlement in winter, and within 1 mo at higher summer temperatures. This variability confirms that repro- ductive capability is size-dependent rather than age-dependent (Pe- tersen et al. 1995). Gulliksen (1972) concluded that the lowest temperature for the production of cionid larvae in Norwegian populations was in the range of 6 to 8°C. or comparable to their deep water winter tem- peratures. This was generally consistent with observations from the present study, which indicated possible gonad regression in January-February at <3°C, gametogenesis in March-April-May at 4 to 8°C. and production of competent gametes from mid-May onward when ambient temperatures exceeded 8''C. The juvenile settlement data indicated that C. intestiualis popu- lations in adjacent inlets may differ in their spawning and recruit- ment patterns. In Bayport. the recruitment peak was observed in May-June, whereas at Mason's Beach recruitment peaks were re- corded in May-June and again in early August. The liming of the first peak was consistent with the histological data, indicating the presence of mature eggs in both populations in early May. and the spawning trials, suggesting that these two populations were ca- pable of releasing eggs. However, unlike the Bayport population. the condition index for the Mason's Beach population remained relatively high until mid-July, suggesting that they did not spawn in May-June. It should be noted that the use of the condition index (body dry weight/total dry weight) as an index of spawning may be misleading. Petersen et al. (1995) found that this index reflected the level of growth but did not link it to spawning activity. It is possible that the relatively high condition index values for the Mason's Beach population in June-July were related to higher food levels at that site rather than to a delay in the onset of spawning activity. It remains unclear whether the first recruitment event at Mason's Beach originated from larvae produced by the local popu- lation or from other spawning populations such as those in Bayport and Upper South Cove. Unlike many shellfish species that spawn over an interval of weeks, C. intestiualis can apparently spawn continuously over a 3-mo period (mid-May through mid-August). Information on re- productive status combined with estimates of fecundity illustrated the duration and intensity of the spawning events at the two sites. On the basis of these data it was estimated that one adult tunicate (100 mm long, 0.6 g dry weight) can produce on average 150 eggs day"' for 60 days for a total of 12.000 eggs per year. This was consistent with the estimate of Petersen and Svane (1995). who suggested a conservative figure of 10.000 eggs ind^' over a sea- son. In contrast, Yamaguchi (1975) reported that adult C. intesti- nalis in Japanese populations released 2000 to 3000 eggs per night, every other night, and that the total fecundity of a single specimen was estimated conservatively at 100.000 eggs. At this stage, it is impossible to estimate egg to juvenile survival rates, but observa- tions of dense aggregations of tunicates on any floating surface, up to 3000 ind m~-, suggest that the population has considerable potential to expand. Management Strategies There are few published reports on strategies for controlling the proliferation of tunicates on shellfish culture gear. In general, growers who use nets recommend husbandry procedures such as changing the gear more often, using power sprayers, or treating bags with antifouling compounds. Other suggestions include ex- posing tunicates to air. fresh water, lime, or saturated brine dips (90 parts per thousand) followed by air (Shearer & Mackenzie 1997). Among mussel growers who use sleeving material rather than nets, there are few cost-effective solutions. Suggestions from anecdotal reports include air-drying lines overnight. //; situ pres- sure washing with a bleach solution, and stabbing individual tu- nicates. Although feasible in the case of small farms or low-level infestations, these options are logistically impossible in the case of large-scale operations. An alternative strategy is to develop a site-specific manage- ment plan for minimizing the level of settlement; for example, growers in South Africa re-sleeve their mussels immediately fol- lowing the recruitment of C. intestiualis (Hecht & Heasman 1999). Based on the recommendations of the present study, the Nova Scotian company involved in oyster culture adjusted its work schedule to mitigate the impact of C. inlestinalis on its operation. In particular, the major annual cleaning/changing of the shellfish and the culture gear was postponed from May to September when the heaviest settlement had passed; this strategy has since proved to be a reasonably cost-effective option for the company. Because C. intestinalis tends to occur in highly aggregated distributional patterns (Havenhand & Svane 1991. Svane & Havenhand 1993, Petersen & Svane 1995), the annual eradication of the broodstock population from the culture gear may reduce future recruitiuent levels. Encouraging natural predation is always a preferred strategy for pest management In aquaculture (e.g.. Enright et al. 1983) but may only have a limited application in this instance. At an estimated recruitment level of 25 ind 100 cm"~. 3000 tunicates may settle on one oyster bag; even at a consumption rate of 1 1 tunicates day"' at peak water temperatures, it would take one crab 273 days to clean one bag. Moreover, tunicates that have settled directly on the shell- fish inventory are not accessible. Field observations suggest, how- ever, that natural predation, possibly by rock crabs, may play an important role in reducing the abundance of tunicates during the winter. Another potential control method that has yet to be inves- tigated is based on the hypothesis that recently recruited C. intes- tinalis may be vulnerable to dislodgement by surface grazers such as periwinkles (Littorina littorea). Enright et al. (1983) reported that the addition of periwinkles to lantern nets containing Euro- pean oysters resulted in a significant reduction in biofouling levels. Chemical treatment protocols including lime and brine immer- sion have been developed for the purpose of eliminating the foul- ing tunicate Molgida sp. from oyster spat collector units (MacNair & Smith 1998). Laboratory trials undertaken in the present study indicated that acetic acid was considerably more effective than more traditional methods for eliminating C intestinalis. However, it should be noted that the use of acetic acid dips or sprays under field conditions should be carefully evaluated to ensure personal safety as well as containment and/or neutralization of the chemical so as to minimize any environmental impact. This study has shown that the ascidian C. intestinalis is well adapted to conditions on the Atlantic coast of Nova Scotia, being capable of developing mature eggs and spawning at temperatures upward of 8°C. It can apparently tolerate a wide range of envi- ronmental conditions and has the potential to rapidly establish substantial populations on floating structures. The presence of sus- pended or off-bottom shellfish culture operations that offer refuge from natural predators may inadvertently promote its survival. Given the tendency of C. intestinalis to attach to the hulls of ships. BlOFOULING OF CULTURED SHELLFISH BY ClONA 631 local maritime traffic will likely facilitate its dispersal to other sites in Atlantic Canada over the next few years ACKNOWLEDGMENTS We would like to thank Dr. Ellen Kenchington [Department of Fisheries and Oceans (DFO)] for providing access to laboratory facilities for sample processing, and Dr. Ken Freeman (DFO) for his advice on the design of the laboratory trials. Dr. Dan Jackson (DFO) contributed one of the photographs and demonstrated the use of the image analyzer system. Dr. Peter Strain (DFO) was very helpful in as.sessing the potential environmental impact of certain chemicals. Staff from Lunenburg Shellfish Inc. provided field sup- port in sampling the experimental tables and conducting the pre- liminary eradication trials. Special thanks are extended to Dr. Donald Douglas. Industrial Technical Advisor, for his contribution to the experimental design of this project, and to the National Research Council Industrial Research Assistance Program for par- tial funding. LITERATURE CITED Berrill, N. J. 1947, The de\elopment and growth of Ciona. J. Mar. Biol. Ass. U. K. 26:616-625. Boothroyd. P. A.. N. G. MacNair. T. Landry. A. Locke. & T. J. Davidson. 2002. Dealing with an aquatic invader: the clubbed tunicate {Sr\ela clava) in Prince Edward Island waters. Bull. Aquacul. Assoc. Can. 102:98-99. Butler, A. J. & R. M. Connolly. 1996. Development and long term dy- namics of a fouling assemblage of sessile marine invertebrates. B/o- fouling 9:\Sl-209. Byrd. J. & C. Lambert. 2000. Mechanism of the blocl^ to hybridization and selfing between the sympatric ascidians Ciona iniesiinalis and Ciona savignyi. Mot. Reprod. Dev. 55:109-116. Cayer. D.. M. MacNeil & A. G. Bagnall. 1999. Tunicate fouling in Nova Scotia aquaculture: a new development. / Shellfish Res. (abstract! 18:327. Connell. S. D. 2000. Floating pontoons create novel habitats for subtidal epibiota. J. Exp. Mar. Biol. Ecol. 247:183-194. Connell. S. D. & T. M. Glasby. 1999. Do urban structures influence local abundance and diversity of subtidal epibiota? A case study from Syd- ney Harbour. Australia. Mar. Environ. Res. 47:373-387. Dybem. B. I. 1965. The life cycle of Ciona intestinalis (L.)/ typica in relation to the environmental temperature. Oikos 16:109-131. Enright, C. D. Krailo, L. Staples. M. Smith. C. Vaughan. D. Ward, P. Gaul, & E. Borghese. 1983. Biological control of fouling in oyster aquaculture. / Shellfish Res. 3:41^14. Glasby, T. M. 1997. Analysing data from post-impact studies using asym- metrical analyses of variance: a case study of epibiota on marinas. Atisi. J. Ecol. 22:448-459. Glasby. T. M. 1999. Differences between subtidal epibiota on pier pilings and rocky reefs at marinas in Sydney. Australia. Est. Coast. Shelf Sci. 48:281-290. Gulliksen. B. 1972. Spawning, larval seulement, growth, biomass, and distribution of Ciona intestinalis L. (Tunicata) in Borgenfjorden, North-Trondelag. Norway. Sarsia 51:83-96. Gulliksen. B. 1973. The vertical distribution and habitat of the ascidians in Bjorgenfjorden. North-Trondelag. Norway. Sarsia 52:21-28. Gulliksen. B. & S. H. Skaeveland. 1973. The sea-star Airfnui rubens L.. as predator on the ascidian Ciona intestinalis (L.) In Borgenfjorden. North-Trondelag, Norway. Sarsia 52:15-20. Havenhand. J. N. & I. Svane. 1991. Roles of hydrodynamics and larval behaviour in determining spatial aggregation in the tunicate Ciona intestinalis. Mar. Ecol. Prog. Ser. 68:271-276. Hecht. T. & K. Heasman. 1999. The culture uf Myiilus galloprovincialis in South .Africa and the carrying capacity of mussel farming in Saldanha Bay. World Aquaadmre 30:50-55. Karayucel. S. 1997. Mussel culture in Scotland. World Aquaculture 28:4- 10. Lambert. C. C. & L. Brandt. 1967. The effect of light on the spawning of Cuma intestinalis. Biol. Bull. 132:222-228. Lambert. C. C. & G. Lambert. 1998. Non-indigenous ascidians in southern California harbors and mannas. Mar. Biol. 130:675-688. Lesser. M. P.. S. E. Shumway. T. Cucci. & J. Smith. 1992. Impact of fouling organisms on mussel rope culture: interspecific competition for food among suspension-feeding invertebrates. / Exp. Mar. Biol. Ecol. 165:91-102. MacNair. N. & M. Smith. 1998. An investigation into the effects of lime and brine immersion treatments on Molgula sp. (sea grape) fouling on oyster collectors on Prince Edward Island. P.E.I. Tech. Rep. 219:13 pp. Mallet. A. L. & C. E. Carver. 1993. Temporal production patterns in various size groups of the blue mussel in eastern Canada: spatial, temporal, stock and age variation. Mar. Ecol. Prog. Ser. 67:35-41. Mazouni. N.. J.-C. Gaertner. & J.-M. Deslou-Paoli. 2001. Composition of biofouling communities on suspended oyster cultures: an in situ study of their interactions with the water column. Mar. Ecol. Prog. Ser. 214:93-102. Millar. R. H. 1952. The annual growth and reproductive cycle in four ascidians. J. Mar. Biol. Assoc. U.K. 31:41-61. Monniot. C. & F. Monniot. 1994. Additions to the inventory of eastern tropical Atlantic ascidians: arrival of cosmopolitan species. Bull. Mar. Sci. 54:71-93. Nomaguchi. T. A. 1974. Seasonal variations in life span of the ascidian Ciona intestinalis. E.xp. Geront. 9:231-234. Olesen, N. J.. K. Frandsen. & H. U. Riisgard. 1994. Population dynamics, growth and energetics of jellyfish {Aurelia aurita) in a shallow tjord. Mar. Ecol. Prog. Ser. 105:9-18. Petersen. J. K. & H. U. Riisgard. 1992. Filtration capacity of the ascidian Ciona intestinalis and its grazing impact in a shallow fjord. Mar. Ecol. Prog. Ser. 88:9-17. Petersen. J. K. & I. Svane. 1995. Larval dispersal in the ascidian Ciona intestinalis (L.): evidence for a closed population. J. E.xp. Mar. Biol. Ecol. 186:89-102. Petersen. J. K., O. Schou. & P. Thor. 1995. Growth and energetics in the ascidian Ciona intestinalis. Mar. Ecol. Prog. Ser. 120:175-184. Petersen. J. K.. O. Schou. & P. Thor. 1997. In situ growth of the ascidian Ciona intestinalis (L.) and the blue mussel Mytilus edulis in an eelgrass meadow. J. Exp. Mar. Biol. Ecol. 218:1-11. Plough, H. H. 1978. Sea squirts of the Adantic continental shelf from Maine to Texas. Baltimore. MD: Johns Hopkins University Press. 1 18 PP- Shearer. L. W. & C. L. MacKenzie. 1997. Effects of salt solutions of different strengths on oyster enemies. Fish. Aquaculture 15:97-103. Svane. I. 1983. Ascidian reproductive patterns related to long-term popu- lation dynamics. Sarsia 68:249-255. Svane. I. & J. N. Havenhand. 1993. Spawning and dispersal in Ciona intestinalis. Mar. Ecol. 14:53-66. Uribe. E. & 1. Etchepare. 2(.)02. Effects of biofouling by Cioiui intestinalis on suspended culture of Argopecten purpuralus in Bahia Inglesa. Chile. Bull. .Aquacul. Assoc. Can. 102:93-95. Van Name, W. G. 1945. The North and South American ascidians. Bull. .Am. Mus. Nat. Hist. 84:1^76. Yamaguchi. M. 1975. Growth and reproductive cycles of the marine foul- ing ascidians Ciona intestinalis. Stxela plicata. Botrylloides violaceus. and Leptoclinum mitsiikurii at Aburatsubo-Moroiso. Mar. Biol. 29: 253-259. Jiiiiniiil of Shclljhli Rcscunh. Vol. 22. No. 3. 633-63!J. 2UU3. FOULING ORGANISMS OF THE BLUE MUSSEL MYTILUS EDULIS: THEIR EFFECT ON NUTRIENT UPTAKE AND RELEASE A. R. LEBLANC,' T. LANDRY," AND G. MIRON '* 'Departciiwn! dc Biolofiie. Univcrsite dc Mancton. Moinion. NB. Canada. El A 3E9: 'DepartineiU of Fisheries and Oceans Canada, Science Biancli Gnlf Fislieries Centre. Monctoii. NB. Canada EIC 9B6 ABSTRACT The effects of fouling organisms are a cause for concern among mussel growers. On Prince Edward Island. Canada, mosi of the foulers are sedentary filter feeders, and are. therefore, potential competitors with cultured mussels for available resources. This could translate into a reduction in meat yield in mussels. Laboratory experiments were carried out in July. September, and December 2001 to determine the relative iinpact of fouling organisms on the uptake and release of nutrients. The uptake of chlorophyll 0, and the production of ammonium, phosphate, nitrate, nitrite, and organic matter were investigated. There were some significant differences in chlorophyll a uptake between mussel/fouler units and mussels alone. The mean (±SE) chlorophyll a uptake by mussel/fouler units was 5.05 ± 1.48. 8.53 ± 0.94. and 12.87 ± 1.03 L/h. respectively, for July. September, and December. The mean consumption by mussels only was 5.37 ± 1.19. 8.72 ±0.83. and 9.64 ±0.97 L/h for each of the experiments. Foulers increased ammonia production before water temperatures dropped in the fall (end of Septemher-early October). Mussel/fouler units released mean amounts of ammonia of 58.42 ± 10.01 and 667.54 ± 252.69 L/h. respectively, in July and September, while mussels alone did not produce ammonia in July, and in September they released 103.10 ± 13.25 L/h. There was a significant production of phosphate by mussels in July (6.67 ± 2.96 L/h) and in December (46. 1 1 ± 3.02 L/h). and by the mus.sel/fouler units in December (27.95 ± 1 .8 /h). In the presence of foulers. the nitrite production was 16.01 ± 6.53 L/h. In its absence, however, nitrite consumption was 17.09 ± 5.63 L/h. Mussel/fouler units consumed nitrate (4.25 ± 1.47 L/h), however, there was no significant difference when foulers were absent (0.86 ± 1.44 L/h). There was a significant consumption of organic matter by foulers in summer only (6.22 ± 1.38 L/h). Foulers have the potential to prolong phytoplankton blooms by increasing the production of inorganic nutrients, especially ammonia. This study shows that the effect of foulers on mussels may not be as great as previously thought, and it may not be profitable to invest time and money in trying to reduce them. A£) WORDS: Tracadie Bay Myiilus eciiilis. blue mussel, epifauna. fouling organisms, chlorophyll a. ammonia, phosphate, nitrate, nitrite, feces. INTRODUCTION Culture of the blue mussel. Mytiliis edidis Linneaus 1758. on Prince Edward Island (PEI). Canada, began in the 1970s and grew into a $25 million per year industry by 2002. Thi.s expansion can be attributed to an increase in the number of mussel grow-out sites accompanied by the rapid development of husbandry practices. Presently, however, no new grow-out sites that can support mussel culture are available on PEL and it seems that any further devel- opment of a sustainable industry relies on the optimization of productivity at the farm level (Thomas Landry, pers. comm.). Mussel socks are fouled by different species of marine inver- tebrates. Some of these foulers are filter feeders that compete with mussels for resources and therefore represent an additional strain on a system. In this context, the fouling of culture units by various epifaunal species is an issue that warrants further attenti(m. It is well-documented for molluscs such as scallops and oysters grown in nets or cages that the settlement of fouling organisins can restrict water flow to a point where both food availability and growth are negatively affected (Claereboudt et al. 1994. Lodeiros & Himmelman 1996. Taylor et al. 1997). PEI mussels, however, are cultivated on longline systems, which consist of subsurface buoyed backlines permanently anchored at each end. Mussels are suspended in socks along the backlines. Little is known about the effects of the fouling community on the growth of mussels on longline systems. In theory, since the fouling organisms tend to settle directly on mussel shells, they may obstruct the opening o'i the valves, thereby interfering with feeding (Lesser et al. 1992. Lodeiros & Himmelman 1996). Moreover, because most foulers *Corresponding author. E-mail: mirongCs'umonclon.ca are sedentary filter feeders (Arakawa 1990. Lesser et al. 1992, Lodeiros & Himmelmann 1996. Taylor et al. 1997. Mazouni et al. 1998a. Mazouni et al. 1998b. Cayer et al. 1999. MacNair & Smith 1999. Uribe & Etchepare 1999), it is possible that they contribute to the depletion of the phytoplankton biomass at culture sites. Despite these possibilities, evidence suggests that foulers do not significantly limit the yield of mussels cultured in suspension on backlines (Beristain & Malouf 1988, Lesser et al. 1992). More- over, it has been suggested that the fouling coinmunity may in fact enhance seasonal phytoplankton blooms by altering nutrient fluxes (Mazouni et al. 1998a. Mazouni et al. 1998b) in a favorable way. Such an effect is plausible, given that the metabolic wastes re- leased by fouling organisms may introduce nutrients into the water column that would otherwise not be available to the phytoplankton community (Kasparet al. 1985. Dame et al. 1991. Prins & Smaal 1994. Smaal & Zurburg 1997. Mazouni et al. 1998a. Mazouni et al. 1998b, Landry 2002). The goal of this study was to investigate the relative uptake of food and the release of nutrients by foulers commonly found on cultured mussels in Tracadie Bay, PEI. Our experimental approach was based on measuring food (i.e., seston and chlorophyll a) intake and nutrient (i.e., ammonia, phosphate, and nitrate) relea.se in two study groups (mussels and mussel/fouler units) during the ice-free period. Results are compared with previous work on mussels, and an attempt is made to relate laboratory findings to applied methods used in mussel culture. MATERIAL AND METHODS Experimental Design Mussel socks were collected from Tracadie Bay. PEI. Canada in July. September, and November 2001. During each trip, four 633 634 LeBlanc et al. socks were collected and transported to the Ellerslie Hatchery, PEL At the hatchery, a small quantity [mean quantity (±SE) 30 ± 9 to 902 ± 82 mg ash-free dry weight] of foulers and 30 mussels of approximately the same size were carefully removed by hand from each of the four socks. Foulers and mussels were placed in individual mesh (window screening) bags (four mussel/fouler units) and were maintained alive using running water from the Bideford Estuary. Water temperatures were 23°C in July, 16°C in September, and 3°C in December, and salinity was 28 parts per thousand for all experiments. After a short acclimation period (<1 wk), the experimental animals were transferred to 12-L flow- through containers. Four containers with no animals were used as controls. All containers were connected to a single supply tub that was continuously fed sand-filtered seawater. Water tlow was set at about 300 niL/min. Immediately after the introduction of animals into the contain- ers, 1-L water samples were taken from the supply tub (input) and also from the output spout of all containers. Thereafter, additional I -L samples were taken every hour during a 7-h period. At the end of this time, feces were collected from the bottom of the contain- ers. Foulers were separated from mussels and were frozen for subsequent determination of weights (i.e.. ash-free dry weight AFDW). Mussels (the same individuals as in the mussel/fouler units), on the other hand, were subjected to a short reacclimatiza- tion period (' was estimated in this study, using information of H^ and A'. Data used in all cases are size-at-age. Ocean Country Latitude H^ (mm) A' or-' I Age Range Source Atlantic Uruguay 30=50' 75.98 0.39 3.35 270 1-9 Atlantic Uruguay 36=40' 81.15 0.31 3.31 96 1-7 Atlantic Araentine 39=24' 74.70 0.42 3.37 197 1-9 Atlantic Araentine 39°47' 68.69 0.50 3.37 75 1-8 Atlantic Argentine 41°50' 74.18 0.38 3.32 83 1-10 Atlantic Argentine 41°50' 69.93 0.37 3.25 87 1-8 Atlantic Argentine 42°30' 59.76 0.49 3.25 152 1-7 Atlantic Argentine 43°53' 66.32 0.50 3.34 79 1-8 Atlantic Argentine 44°00' 75.59 0.54 3,49 124 1-8 Atlantic Argentine 46°47' 65.67 0.63 3.43 65 1-7 Atlantic Argentine 49°50' 62.65 0.40 3.19 89 1-8 Atlantic Argentine 52=00' 54.66 0.58 3.24 91 1-8 Atlantic Argentine 54=30' 54.90 0.39 3.07 90 1-7 Pacific Chile 5}'00' 66.(10 0.14 2.78 95 1-11 This study This study Valero (2002: in Ciocco et al.. 2003) Waloszek & Waloszek (1986) Valero (2002: in Ciocco et al. 2003) Waloszek & Waloszek (1986) Waloszek & Waloszek (1986) Waloszek & Waloszek (1986) Waloszek & Waloszek (1986) Waloszek & Waloszek (1986) Waloszek & Waloszek ( 1986) Waloszek & Waloszek (1986) Waloszek & Waloszek (1986) L'rban ct Tesch (1996) study). II from Argentina an(j one from Chile (Table 1). The growth inde.x (b' = 2\ogj„{L^^J log/,;A' (Pauly & Munro 1984) was calculated and used to assess growth performance. The relation- ships between growth parameters and latitude (centesimal units) were tnodeled by linear and nonlinear fitting procedures, and the model with the best goodness of fit selected. RESULTS Scallops at latitude 36°40'S grew significantly faster than at latitude 35°50°S (Fig. 1). The non-linear fitting of the VBGF explained 93% (36°40'S) and 84% (35°50'S) of the variance, and all parameters were signitlcant. except /,, at latitude 35 = 50'S (Table 2). Results of likelihood ratio tests showed that the overall VBGF significantly differed between latitudes (x" = 45.234. df = 3. P < 0.0001 ). Testing of the remaining null hypotheses showed that the H^ and K parameters did not differ significantly (x" test, df = \.P> 0.05). Conversely, a significant difference between l,, values was strongly indicated, either in isolation (x" test, df = 1, P < 0.01) or in combination v\ith the other two VBGF parameters (Table 3). Marked differences in mean-length-at age at earlier ages, notably age 1 (ANOVA test; p < 0.01; Cochran test for 35°50S 36°40'S Figure waters 4 5 6 7 8 9 10 AGE (years) I, Growth models for the scallop Z. palagonica in Uruguayan Details are provided in Table 2. homoscedasticity C = 0.68; P = 0.49) could explain the observed differences between curves (see Fig. I ). The large-scale analysis showed that both H, and (}>' were inversely correlated with latitude (/• = -0.80; P < 0.0006 for H.^ and r = -0.63: P < 0.015 for (j)'). but K was not (/■ = 0.03: P = 0.91) (Fig. 2). Values of H,- were in the range 55 to 81 mm, with the lowest value at 54°30'S and the highest at 36'40'S (this study; Table 1 ). DISCUSSION Scallops grew significantly faster at the southern limit of Uru- guayan waters when compared to the northern border. Gutierrez & Defeo (2003) also showed that muscle weight increased linearly towards the southern end of Uruguayan waters, whereas the har- vestable stock (ie, individuals >55 mm H). mean individual height and maximum height increased asymptotically in the same direc- tion. Defeo & Brazeiro ( 1994) found hardly any scallops north of the range considered here (between 35=50'S and 35 'OO'S) and. where few specimens were caught, individual height also tended to be low. This is in agreement with the higher estimate of H-^ found at 36°40'S (81.15 mm H) when compared with that at 35°50'S (75.98 mm H). The occurrence of lower abundances and sizes at the nonhem distribution end has been ascribed to habitat TABLE 2. Z. palagonica. Results of the >on Bertalanffy growth models fitted by nonlinear least squares for scallops of Uruguayan waters. Significant \alues {P < (I.OI) are highlighted in bold and italics. Latitude 35°50'S Parameters Estimate (SEl Latitude 36°40'S Estimate (SE) F H,_ (mm) A-(yr-') 'o (y) R- 75.98(1.60) 0.39(0.03) -0.01 (0.11) 0.84 0.0000 0.0000 0.9336 0.0000 81.15(2.81) 0.31 (0.04) -0.65(0.19) 0.93 0.0000 0.0000 0.001 1 0.0000 Scallop Growth in the Southwestern Atlantic 645 TABLE 3. LikelihiKid ratio tests comparing von Bcrtalanff) parameter estimates for the two scallop beds in Uruguayan waters. Results of the RSS (residual sum of squaresl. the \" test and associate statistics are shown on the basis of the seven null hvpotheses tested (columns 3 to V). assuming thai the listed parameter or a c -> -> P 0.0000 0.1980 0.2007 0.0069 0.32.^4 0.0166 0.0007 unsuitability and scarcity of food (Gutierrez & Defeo 200.3). In spite of tlie above, the likelihood ratio test indicated that major between-latitude differences in the VBGF could be ascribed to parameter /„. which jointly with H.^ strongly determines the form of the VBGF (Haddon 2001 ). Differences between curves could be attributed to \ariations in length-at-age at ages 1 to 5. notably age 1, where individual sizes-at-age at 36°40'S were greater than at 35°50'S (see Fig. 1). Growth parameters of Z. patcifionica showed clear latitudinal patterns: asymptotic height H , and the overall growth performance 4>' both significantly decreased from north to south in the SAO. The results of our study are consistent with the pattern found at a large-scale, and indirectly reaffirm the annual nature of growth ring formation. These results pro\ide additional evidence for the large-scale patterns found for Argentinean waters, where a signifi- cant decrease in H^_ was estimated as latitude increases (Valero 2002. Ciocco et al. 2003). Between-latitude differences in growth rate have been attributed to variations in environmental param- eters, such as temperature and food availability (Ciocco et al. 2003 and references therein). Valero (2002) presented new evi- dence on the effects of factors acting at small spatial scales (eg. intrabed scale), seasonal cycles and year-to-year variation in growth in this species, which were mainly related to variations in temperature and oceanographic regimes. These factors seem to be critical in explaining growth variations in other scallop populations (see eg MacDonald & Thompson 1985, Schick et al. 1988 and papers in Shumway 1991). Latitudinal differences in growth rate could also be explained by density-dependence operating at the scale of scallop beds, a mechanism already proposed by Orensanz et al. (2003) to explain intra and inter-cohort variations in popu- lation dynamics. Ciocco et al. (2003) showed that for high-density patches occurring in Argentine waters, individuals at high concen- trations are affected by density-dependence e\en in small areas (Lasta & Bremec 1998). The low growth performance in high- density beds in southern waters of the SAO (as denoted by ') provides additional support to the density-dependence hypothesis, which was also suggested by Gutierrez & Defeo (2003) for Uru- guayan scallop grounds. Finally, differences between Atlantic and Pacific estimates could be attributed to different environmental scenarios and the fact that the species occurs in relatixely shallow waters in the Pacific. 42 46 LATITUDE SOUTH 50 54 58 42 46 LATITUDE SOUTH Figure 2. '/.. palagonica. Regression lines (±95'7f CI) between latitude (centesimal units! and (al asymptotic height and (hi the growth per- formance inde.v cj)'. (•) Lruguay; ( . ) Argentina and (■) Chile. 646 Defeo and Gutierrez Managemenl Implications The significant latitudinal gradient in growth rate detailed here could have implications for fishery management, as spatial varia- tion in population dynamics and life history traits are used to provide area-based estimates of potential yield (Caddy 1975) and to implement spatially explicit management measures (eg, rotation of fishing areas and reproductive refugia: Orensanz & Jamieson 1998. Catilla & Defeo 2001). This should call for a spatially dis- crete analysis of population dynamics and other life history traits, the sun'ounding environment, and the fishery. In this setting, map- ping of density and related population processes is worthwhile as a way of forecasting the spatial features of the stock, and to assess the economic potential of the fishery (Caddy 1989a. b). ACKNOWLEDGMENTS This paper was written during the M.Sc. thesis of Nicolas Gutie- rrez. We are especially grateful to Dr. Raiil Palacios for his advice on the application of the likelihood ratio test. Nestor Ciocco and Juan Valero kindly provided us scallop growth estimates for Ar- gentinean waters. Two referees gave us useful suggestions that improved the paper. Financial support from DINARA and PEDECIBA Uruguay is gratefully acknowledged. LITERATURE CITED Bertalanfty. L. von. 1938. A quantitative theory of organic growth. Hum. Biol. 10:181-213. Caddy. J. F. 1975. Spatial model for an e.xploited shellfish population, and its application to the Georges Bank scallop fishery. J. Fish. Res. Bd. Can. .32:1305-1328. Caddy. J. F. 1989a. A perspective on the population dynamics and assess- ment of scallop fisheries, with special reference to the sea scallop. Placopeclen mai^ellanicus. Gmelin. In: J. F. Caddy, editor. Marine in- vertebrate fisheries: their assessment and management. New York: Wiley, pp. 559-590. Caddy. J. F. 1989b. Recent developments in research and management for wild stocks of bivalves and gastropods. In: J. F. Caddy, editor. Marine invertebrate fisheries: their assessment and management. New York: Wiley, pp. 665-700. Caslilla. J. C. & O. Defeo. 2t)01. Latin-.American henthic shellfisheries: emphasis on co-management and experimental practices. Rev. Fisli Biol. Fisheries 11:1-30. Cerrato. R. M. 1990. Interpretable statistical tests for growth comparisons using parameters in the von Bertalanffy equation. Can. J. Fish. .Aqiial. Sci. 47:1416-1426. Ciocco. N. F.. M. L. Lasta. M. Narvarte. C Bremec. E. Bogazzi. J. Valero & J. M. Orensanz (Lobo). 2003. Argentina. In: S. E. Shumway. editor. Scallops: biology, ecology and aquaculture, 2nd Edition. Amsterdam: Elsevier: in press. Defeo. O. & A. Brazeiro. 1994. Disiribucion. estructura poblacional y relaciones biometricas de la vieira Zygochlamys patagonica en aguas uruguayas. Com. Soc. Malac. Urug. 66-67 (VII):362-367. Gutierrez. N. & O. Defeo. 2003. Development of a new scallop Zy- gochlamys patagonica fishery in Uruguay: latitudinal and bathymetric patterns in biomass and population structure. Fish. Res. 62:21-36. Haddon. M. 2001. Modelling and quantitative methods in fisheries. Boca Raton: Chapman and Hall/CRC 406 pp. Kimura. D. K. 1980. Likelihood methods tor the von Bertalanffy growth curve. Fish. Bull. 77:765-775. Lasta. M. & C. Bremec. 1998. Zygoclilaniys patagonica in the Argentine sea: a new scallop fishery. J. Shellfish Res. 17:103-1 1 1. Lasta. M.. J. Valero, T. Brey. & C. Bremec. 2001. ZygochUinivs patagonica beds on the Argentinian shelf Part 11: Population dynamics of Z. pa- tagonica. Arch. Fish. Mar. Res. 49:125-137. Orensanz. J. (Lobo) & J. Jamieson. 1998. The assessment and management of spatially structured stocks. Can. Spec. Publ. Fish. Aqiiat. Sci. 125: 441-159. Orensanz. J. M.. A. M. Parma. T. Turk & J. Valero. 2003. Dynamics, assessment and management of exploited natural populations. In: S. E. Shumway. editor. Scallops: Biology. Ecology and Aquaculture. 2nd Edition. Amsterdam: Elsevier: in press. Palacios, R. 1994. Individual growth and dynamics of living and extinct soft shell clam (Mya arenaria) populations in Grays Harbor. Washing- ton, and ecological interactions with Dungeness crab (Cancer niagis- ler). PhD. Thesis. University of Washington, xi 210 pp. Pauly. D. & J. L. Munro. 1984. Once more on the comparison of growth in fish and invertebrates. Fishhyle 2(1):21. Schick. D. F.. S. E. Shumway & M. A. Hunter. 1988. A comparison of growth rates between shallow water and deep water populations of scallops. Placopecten magellaniciis (Gmelin. 1 97 1 1 in the Gulf of Maine. Amer. Malac. Bull. 6:1-8. Shumway, S. E., editor. 1991. Scallops: biology, ecology and aquaculture. Develop. Aquae. Fish. Sci. 21. Amsterdam: Elsevier. 1096 pp. Valero. J. L.. 2002. Temporal and spatial growth variation and natural mortality estimation with an integrated dynamic model for the patago- nian scallop {Zygochlamys patagonica). MSc. Thesis, University of Washington. 154 pp. Waloszek. D. 1991. Chlamys patagonica (King & Broderip. 1832). a long 'neglected' species from the shelf off the Patagonian Coast. In: S. E. Shumway & P. A. Sandifer. editors. An international compendium of scallop biology and culture. Selected papers from the VII International Pectinid Workshop, National Shellfisheries Association. The World Aquaculture Society. Parker Coliseum. Louisiana State Univ.. Baton Rouge. USA. pp. 256-263. Waloszek. D. & G. Waloszek. 1986. Ergebnisse der Forschungsreisen des FFS "Walther Heiwig" nach Siidamerika. L.XV. Vorkommen. Re- produktion. Wachstum und mogliche Nutzbarkeit von Chlamys pata- gonica (King & Broderip, 1832) (Bivalvia, Pectinidae) auf dem Schelf von Araentinien. Arch. Fish. Wiss. 37:69-99. J,,iinuil „f Shellfish Research. Vol. 22. No. 3. 647-654. 200.^. INTERMEDIATE CULTURE OF KING SCALLOP {PECTEN MAXIAWS) IN SUSPENSION IN CAGES: EFFECT OF STOCKING DENSITY AND DEPTH G. ROMAN.* A. LOURO, AND J. P. DE LA ROCHE lustinito Espcwol dc Ovcaiioiiiafia. Ministcrio clc Ciciicia y Tecnologia. Ccntro Ocecmogrcifico de A Coniiui. P. O. Box 1.^0. 15080. A Coniila. Spain ABSTRACT Scullop spal settled on collectors were grown ui suspended cages in O Grove. (Ria de Arousa. Galicia. nonhwest .Spain) and in Fuengirola. (Malaga, Andaluci'a. southern Spain). Mean (±SDl spat heights of 20.4 ± 3.7 mm (Fuengirola. September 1998) and 26.6 ± 5.8 mm (O Grove. November 1998). were stocked al densities ranging between 25 and 2()0/cage"' (=200-1600 spat nV\. and at depths of 6 and 10 m in O Grove, and between 13 and 25 m in Fuengirola. Even low stocking densities were found to affect scallop growth, therefore juveniles (>35 mm) were used to set up new cultures at lower stocking densities (12 and 24 juveniles/cage"' ) at the end of winter (Fuengirola) and at the beginning of spring (O Grove). The most rapid growth took place at Fuengirola, where the mean height reached on May 19. 1999. was 63.9 ± 4.1 mm compared with a mean height of 51.2 + 4.5 mm for the O Grove spat on May 27. 1999. KEY WORDS: Andalucia. density, depth. Galicia, intermediate culture. Peeten iiniMmus. suspension culture INTRODUCTION Attempts are currently being made in several European coun- tries to cultivate Peeten maxinnis on a commercial scale (Fleury et al. 1997), using both hatcheiy-produced spat (Norway and France) and spat captured by natural settlement on collectors (Ireland and Scotland). Pectinids (pectinoid form sensit Waller 1992) cultivated in suspended cages grow slowly after a certain size (Slater 1995), possibly because of the differences in the conditions in the cages and in the natural habitat (recessed in sediment), although waves have been observed to have a negative effect on Eitvola ziczac and Nodipecten nodosus (Freites et al. 1999). Because of these diffi- culties, the present trend in Europe is for intermediate culture, usually in suspension, followed by the seeding of juveniles of different sizes, depending on the conditions in each area, on the sea floor. However, in certain areas the environmental conditions or the techniques result in the successful use of suspended culture to grow scallops to commercial size (Roman & Fernandez 1991. Gallagher 1999, Cano et al. 2000). The desired final size of the Juveniles maintained in interme- diate culture will obviously depend on the method of on growing. In the case of seabed culture, the size required depends on the environmental conditions (i.e.. sediment, current, and predators) and varies from region to region: 30 mm in France (Fleury et al. 1995); and 50 mm in Norway, Ireland, and Scotland (Fleury et al. 1997). The size required for ear-hanging culture is more stan- dardized. Generally, scallops are not ear-hung until they have reached at least 55 mm shell height (Ventilla 1982, Dadswell & Parsons 1991, O'Connor et al. 1999). Gallagher (1999) considers the minimum size for ear-hanging culture to be 50 mm, whereas Cano et al. (2000) used juveniles of between 51.3 and 64.3 mm height. Roman and Fernandez (1991 ) describe ear-hanging culture in Galicia, where scallops of heights of between 60 and 70 mm are used. In order for the scallops to reach the size required for the on-growing stage, they must undergo a period of intermediate culture in mesh trays or cages, which are usually suspended in the water, although cages are also placed on the seabed (Dao et al. *Corresponding author. Fax: +34-981-229077: E-mail: guillermo.roman@ co.ieo.es 1996). Subsequently, scallops are seeded on the seabed (Fleury et al. 1997), are suspended using the ear-hanging technique (Paul 1988, Roman & Fernandez 1991. Gallagher 1999). or are held within lantern nets, cages, or other artifacts (Cano et al. 20(J0). There are many reports in the literature on the effects on growth and survival of the use of mesh enclosures (O'Connor et al. 1999) and of the stocking density and depth at which pectinid spat are cultured (Cote et al. 1993. Duggan et al. 1973. Leighton 1979, Parsons & Dadswell 1992, Rhodes & Widman 1984. Wallace & Reisnes 1984, 1985, Roman et al. 1999, Cano et al. 2000, Freites et al. 1995). Most of these studies refer to aequipectinoid pectinids (form sensu Waller. 1991 ). the natural habitat of which is similar to the conditions of suspended culture. Scallops are commercially produced in two areas of Spain: Galicia (in northwest Spain): and in the province of Malaga (An- daluci'a, in southern Spain). Cultivation on the seabed is not pos- sible for legal and social reasons, and only suspension culture is feasible. The aims of the present study were to determine, in each area, the optimum conditions (in terms of depth and stocking den- sity) required for the intermediate culture of spat and juveniles in suspended cages to obtain scallops of a suitable size for ear- hanging culture (=60 mm height), and to compare the growth and survival of spat cultured in the different areas. In addition, the possibility of cultivating scallops to commercial size in suspended cages in Galicia was evaluated, which is an aspect that has been studied previously in Malaga (Cano et al. 2000). MATERIALS AND METHODS Study Area The study was carried out at two sites, in O Grove, Ria de Arousa. in the Atlantic Ocean (Galicia, in northwest Spain), and in Fuengirola. in the Alboran Sea (western Mediterranean, Malaga, Andalucia, in southern Spain) (Fig. 1 ). Environmental Conditions The temperature and levels of chlorophyll (/ were measured, using a conductivity-temperature-depth (CTD) recorder, every week in O Grove and every fortnight in Fuengirola. Salinity also was recorded in O Grove. 647 648 Roman et al. O Grove (Galicia) , V 4»0' -i_. Atlantic f > Jy Ocean > J / — 40° 0' / ) 1 1 Spain / 40° 0' - /< Mediterranean Z' Sea 1 1 Fuengirola (Malaga) lOOKms Fijiure 1. Locations of the experimental intermediate culture of P. maxiiniis. Animals Scallops of up to 30 mm are considered to be spat, and between 30 and 60 mm they are considered to be juvenile. In both areas, the spat was obtained by natural settlement on collectors. Sampling The cages were raised periodically so that the height of the scallops (measured to the nearest millimeter using calipers) and the number of dead could be recorded, and they then were resus- pended. Suspended Culture The spat were cultured in circular rigid plastic cages (40 cm diameter. 10 cm height. 10 mm mesh size). In O Gro\e. the cages were hung from a raft, whereas in Fuengirola they were anchored, following the scheme outlined by Cano et al. (2000). Spat growth was greatly affected by stocking density, therefore new experi- ments were started in the spring in both areas using juveniles at lower stocking densities of 12 and 24 scallops/cage"'. The experi- ments were carried out in duplicate (Table 1). Statistical Methods Mean sizes (height) were compared by factorial analysis of variance (ANOVA) using stocking density and depth as factors. Normality was previously checked using the Kolmogorov- Smimov test, and the homogeneity of variance was checked by Bartlett's test. The differences in size were compared a posteriori using a Newman-Keuls test (a = 0.05). except when there was interaction between factors. Comparisons between pairs of samples were made using a Student's t test. Arcsin transformation was used to compare percentage survival. TABLE 1. Description of experimental intermediate culture of P. maximus. Date Area Start End Initial Size (mm)" (Mean ± sd) Density Culture n" Scallops Cage' Initial Coverage ( Vc l'' Depth Culture (m) Intermediate culture of spat Fuengirola 9/24/98-2/24/99 O Grove 1 1 / 1 7/98-4/6/99 Intermediate culture of juvenile Fuengirola 2/24/99-5/19/99 O Grove 4/6/99-.'S/.^0/99 204 ± 3.7 25/50/100/200 6.5/13.0/26.0/52.0% 13/18/23/26 26.6 ±5.8 25/50/100 11.1/22.1/44.2% 6/10 52.1 ±5.0 12/24 20.4/40.7% 13/18/23/26 39.9 ±3.2 (6 m) 12/24 11.9/23.9% (6m) 6/10 414 ±3.9 (10 m) 12.9/25.7% (10m) ' Presented as mean ± SD. ' Experimental design 2x2 factor. Intermediate Culture of Pt:cT[-:N maximus 649 RESULTS Envinnititfiiltil C 'omlitions O Grove There was a slight temperature inversion with depth in winter. ho\\e\er. during the rest of the year the temperature was sHghtly higher in the first 6 ni. In general, the temperatures were very similar throughout the year, at both depths, ranging between 1 1°C and 18°C, with only occasional differences of >1°C (Fig. 2). Sa- linity ranged between M7co and .'^5.5^i throughout most of the study period, except in May 2000. when minimum values of 32.0%f and 33.1%c were registered at depths of 6 and 10 m. re- spectively. There was a trend toward slightly higher levels of chlorophyll a in the surface layers of water from the end of autumn until the beginning of spring, but from May onward increasing levels were found at depth. There were large variations in the le\els. with minimum \alues of approximately 1 p-g L~'. and of 2 and 3 |a.g L at 6 and 10 m. respectively maximum value: (Fig. 3 1. Fuengirola High temperatures ( 17-18. 5°C) were registered at the begin- ning of September, followed by a maximum of 2 1 "C at the end of September. From the end of October until May. the temperature varied between 14"C and 16"C (Fig. 2). The levels of chlorophyll a observed were rarely < 1 (xg L" ' . with peaks of between 2 and ? fj-g L"' in September. October, and February, and particularly between April and May. As there was strong vertical mixing dur- ing the period of the study, there was very little variation with depth, with only a slight trend toward lower temperatures and chlorophyll a levels at lower depths (Fig. 3). Growth O Grove Spat culture. On March 2. 1949. the mean heights ranged between 35.6 and 39.8 mm. depending on the culture conditions. There were significant differences in the heights achieved at the different stocking densities (25 scallops/cage"' > 50/cage"' > 100/ cage"'), but there were no significant differences associated with depth (Fig. 4). One month later, on April 6. 1999. only very small increases in size were registered, with mean heights ranging be- tween 36.4 and 41.4 mm. depending on the culture conditions. Again, growth was affected by stocking density but not by depth. Scallops cultivated at a stocking density of 25/cage"' were sig- nificantly larger than those cultivated at higher stocking densities, whereas there was no difference in the size of scallops cultured at the two higher stocking densities (50 and 100/cage"'l. The mean coverage was 25.6% at 25/cage~'. 45.0% at 50/cage"'. and 86.0% coverage at lOO/cage"'. The spat growth experiment finished on this date, and a new experiment, using juveniles at lower stocking densities, was started. Juvenile culture. This experiment was begun on April 6. 1999, using scallops previously grown at stocking densities of 25/cage~' (the mean heights reached by scallops culti\ated at depths of 6 and 10 m were 39.9 ± 3.2 and 41.4 ± 3.9 mm. respectively). The new stocking densities were 12 and 24/cage~', at the same depths as before (i.e., 6 and 10 ni). The initial coverage was 11.9% and 23.9% and 12.9% and 25.7%, respectively, at 12 and 24 spat/ cace"' at 6 and 10 m. 22 20 s 16 - 14 12 - 10 -e — O Grove ( 1 Dm depth) -^ — O Grove ( 6m depth) -A- - - Fuengirola (13m depth) 24-07 12-09 01-11 21-12 09-02 31-03 20-05 09-07 28-08 17-10 06-12 25-01 15-03 04-05 23-06 12-08 1998 1999 2000 Figure 2. Interannual variation of temperature in U Grove and Fuengirola. 650 Roman et al. et) 4 a. 1>3 £ ■2 2 -3K — O Grove ( 6m depth) -© — O Grove ( 10m depth) ■A- • - Fuengirola (13m depth) 0 24-07 12-09 01-11 21-12 09-02 31-03 20-05 09-07 28-08 17-10 06-12 25-01 15-03 04-05 23-06 12-08 1998 1999 2000 Figure 3. Interannual variation of Chlorophyll a in O Grove and Fuengirola. 45 40 ? E - 35 en 6 m- 25/q lOm-25/q 30 25 6 m- 50/q 10m- 50/q 6m-100/q lOm-lOO/q 11/11/98 11/12/98 10/01/99 09/02/99 11/03/99 10/04/99 Figure 4. Growth of P. maxiimis spat on intermediate culture in O Grove. -c^ 6 m- 24/q -^-lOm-24/q 6 m- 12/q 10 m- 12/q 31/03/99 19/06/99 07/09/99 26/11/99 14/02/00 04/05/00 Figure 5. Growth of P. maximus juvenile on intermediate culture in O Grove. Intermediate Culture of Pecten maximus 651 Depth affected the growth of the juveniles hetween May 27 and September 28. 1999 (the jii\eniles maintained at a depth of 10 m reached a larger size than that cultivated at 6 m; Fig. 5). On February 1 7. 2000. there were no effects associated with depth, but on May .■'0, 2000. a depth effect was once again observed (ANOVA: Table 2). The effect of stocking densit> on grov\th rate was obsersed from September 28. 1999. until the end of the experiment on May 30, 2000. with larger scallops being obtained at the louer stocking density (Fig. 5). Very little growth was registered between Febru- ary 17 and May 30. 2000. On February 17. 2000. the mean height reached ranged between 66.1 and 73.9 mm, depending on the culture conditions, whereas on May 30. 2000, it ranged between 67.1 and 76.4 mm. In May. 9.2% of the scallops cultured under the most favorable conditions (i.e., 12 scallops/cage^' at 10 m depth) had reached commercial size. (ANOVA; Table 2). T.4BI,E 2. Effect of depth (6 and 10 m) and stocking density (25, 50, and 100 per cage"') on the growth of king scallop in O Grove. Ria de .\rousa. Fuengirola Spat culture. Scallops were sampled on November 23. 1998. Jantiary 27. 1999, and February 24. 1999. Faster growth rates always were observed at lower stocking densities. The effect of depth was not clear, although growth rates were generally higher at shallower depths (Fig. 6). A posteriori analysis of data was not carried out because there was interaction between factors each month. At the end of the experiment, on February 24. 1999, the mean heights of the spat at each stocking density (pooled for the different depths) were 36.7 ± 5.9 {200/cage"'), 42.1 ± 6.6 (100/ cage-'). 48.2 ± 5.9 (50/cage-'). and 53.0 ± 6.2 mm (25/cage-'). Juvenile culture. A new experiment was started in February, using stocking densities of 12 and 24 juveniles/cage"'. The initial mean size was 52.1 ± 5.0 mm. Monthly sampling was carried out on March 23, 1999, April 21. 1999. and finally on May 19, 1999, when the scallops had reached a suitable size for ear-hanging culture (overall mean height 63.9 mm I and the experiment was finished (Fig. 7). Significant differences in size were observed from March onward that were related to stocking density. Growth was not affected by depth (ANOVA; Table 3). Survival Source of F P Variation Df Ratio Value Newman-Keuls Test March 2. 1999 Depth 0.04 0.852 D6 ni = D lOm Stocking density I 42.34 0.003* SD 25 cage"' > 50 cage"' > lOO/cage"' Density x depth 2 3.97 0.080 Apnl 6. 1999 Depth 1.80 0.229 D 6 m = D 10 m Stocking density ; 14..S7 0.005* SD 25cage"' > 50 cage"' = 100 cage"' Density x depth I 1.75 0.252 May 27. 1999-' Depth 8.09 0.047* D 6 ni< D 10 m Stocking density 0.43 0.547 SD 12 cage-' = SD 24 cage"' Density x depth 0.28 0.624 July 20. 1999 Depth 12.48 0.024* D 6 m < D 10 m Stocking density 2.44 0.193 SD 12 cage"' = SD 24 cage-' Density x depth 0. 1 1 0.757 September 28. 1999 Depth 1 5.77 0.074 D 6 m = D 10 m Stocking density 1 9.67 0.036* SD 12 cage"' > SD 24 cage"' Density x depth 1 0.00 0.996 February 17. 2000 Depth 5.26 0.084 D 6 m = D 10 m Stocking density 25.69 0.007* SD 12 cage-' > SD 24 cage"' Density x depth 0.05 0.831 May 30, 2000 Depth 20.41 0.011* D 6 m < D 10 m Slocking density 129.58 0.000* SD 12 cage"' > SD 24 cage"' Density x depth 0.63 0.472 D. depth; SD. stocking density; DF, degrees of freedom. "After April 6 new stocking densities were used (12 and 24 per cage"') * Indicates a significant result. P < 0.05. O Grove Spat culture. The survival rates between November 1998 and April 1999 were 100% at stocking densities of 25 and 50 spat/ cage-' at both depths, and 90% at a stocking density of 100 spat/ cage"' at both depths. Juvenile culture. The survival rate ranged between 73.5% and 87.0% (Table 4). The multifactorial ANOVA revealed interaction between factors, and a posteriori analysis was not carried out. Fuengirola Spat culture. The survival rate ranged between 91% and 100%'. Juvenile culture. In May. the survival rate ranged between 83.3% and 95.8% (Table 5). There were no significant differences in mortality associated with density or depth. DISCUSSION Effect of Depth In Fuengirola. the environmental conditions showed very little difference at the different depths tested. Possibly because of this. -e— 25 -a— 50 -6—100 -o— 200 12/09/98 01/11/98 21/12/98 09/02/99 31/03/99 Figure 6. (irowlh of/", inaxiimis spat at four densities, depth pooled, on intermediate culture in Fuengirola. 652 Roman et al. -0—12 -B— 25 70 1 65 60 55 50 45 10/05/99 09/06/99 09/02/99 11/03/99 10/04/99 Figure 7. Growth of P. maximus ju\enile at two densities, depth pooled, on intermediate culture in Fuengirola. there was very little differenL-e in growth at different depths ot either spat or juveniles. In O Grove, during the period of spat culture no differences were observed in temperature, levels of chlorophyll a. or growth at the different depths. However, during the period of juvenile culture in spring and summer of 1999. growth was faster at a depth of 10 m than at 6 m. possibly because of higher levels of chlorophyll a TABLE 3. Effect of depth (13, 18. 23, and 26 ni) and stocking density (25, 50, 100, and 200 per cage"') on the growth of king scallop in Fuengirola, Malaga. Source of Variation Df F Ratio P Value Newman- Keuls Test November 23. 1998 Stocking density Depth Density x depth January 27. 1999 Stocking density Depth Density x depth February 24. 1999 Stocking density Depth Density x depth March 23. 1999» Stocking density Depth Density x depth April 21. 1999 Stocking density Depth Density x depth May 19. 1999 Stocking density Depth Density x depth 3 3 9 3 3 9 3 3 9 1 3 3 I 3 3 ] 3 3 64.06 10.11 7.47 790.28 331.28 24.12 1163.68 199.7? 8.11 6.37 1.35 0.23 5.43 1.05 1.49 43.46 1.13 3.41 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* 0.036* 0.326 0.876 0.048* 0.422 0.289 0.000* ()..W4 0.074 SD 12 > SD 24 SD 12 > SD 24 SD 12 > SD 24 D. depth; SD. stocking density. " After February 24, new stocking densities were used (12 and 24 juveniles per cage"'). * Indicates a significant result. P < 0.05. TABLE 4. Survival rales of juveniles grown at different stocking densities and depths in O Grove, Ria de Arousa. 6 m 10 m 12 juveniles 24 Juveniles 12 juveniles 24 juveniles Date cage"' cage"' cage"' cage"' 5/27/1999 99.8 100 100 100 7/20/1999 96.9-' 100" 100" 100" 9/28/1999 92.7" 95.8" 100" 91.8" 2/17/2000 83.3 92.7 93.5 83.7 5/20/2000 78. l-''^ 84.4"^ 87,0" 73.5-' Presented as ';;-. Values with a common superscript letter do not differ significanlly in each month. at that time, as the temperature varied very little at the different depths. Similar results have been reported by Roman et al. ( 1999) ior Aequipectcu openulahs. The results confirm those of Lodeiros and Himmelman (1994). who observed that in temperate and northern regions food availability is more important than tempera- ture for the growth of pectinids. The results, however, contrast with those of Laing and Utting (2001 ). who suggest that tempera- ture is the most important factor for the growth of scallop seed in the sea. However, they worked with a wider temperature range (5-23°C) than that recorded in the present study (14-2rC in Ftiengirola: 13-I8°C in O Grove). From autumn until the following spring, the rate of growth of the scallops maintained at 6 m was slightly higher than that of scallops cultivated at 10 m. and there were no longer any signifi- cant differences. The higher growth rate in the scallops maintained at 6 111 may have been partly due to the sharp decrease in levels ol chlorophyll a at 10 m between September and October, and partly due to compensatory growth. In May 2000. the growth rate of the scallops maintained at 10 m was again higher than that of the scallops maintained at 6 m, although the reasons for this were not clear. The slight decreases in salinity that were registered may have been sufficient to produce differences in the giowth rates. Fouling of the cages and of the scallops may be an important factor affecting the growth rate, although no specific study has been carried out to test this. In O Grove, minimum temperatures (I2-13°C) and levels ot chlorophyll a (1-1.5 [jlL"') were registered during the period of spat culture (November 17. 1998. to April 6. 1999). However. TABLE 5. Surv ival rate of spat and juveniles grown at different stocking densities, depths, and dates in Fuengirola, Malaga. Stocking Density (Spat/Cage) 26 m 23 m 18 ni 13 m Spat. Febniary 1999 25 .SO UK) 200 Juveniles. May 1999 24 12 100 100 96.0 ± 2.0 94.0 ± 2.0 100 94.0 ±2.0 100 100 100 100 9S.0 ± 2.0 92.0 + 2.0 92.0 ± 4.0 100 96.0 ± 2.0 9 1 .0 ± 4.0 93.8 ±6.3 92.8+1.0 89.6 ±2.1 88..? ± 5.2 93.8 ±2.1 95.8 ±0.0 83.3 ±16.7 95.8 ±4.2 Presented as mean (±SD) %. Intermediati; Culture of Pixtkn maximus 653 growth rates of between 0.131 and 0.089 mm d~' were recorded between November and March, when scallops were not handled, and of only between 0.047 and 0.024 mm d~' between March and April. The decrease in the growth rate may have been caused by stress according to Laing et al. ( 1999). who observed an increasing level of stress in scallops disturbed monthly during a period when there was little food available for growth. In the previous winter months, undisturbed scallops grew faster, even with lower or simi- lar food availability and temperature levels. Effect of Slocking Density and Areal Coverage When spat reached a height of appro.ximately 35 mm. lower growth rates were recorded in both areas at stocking densities of 50/cage"' and higher (corresponding to at least 38% areal cover- age). In O Grove, there were no significant differences in the growth of juveniles (those of initial size range of 39.9—11.4 mm on April 6, 19991, until 6 mo after the .start of the experiment (September 28. 1999. height reached .57-65 mm), despite the different stocking densities used ( 12-24 juveniles/cage"' ). However, in Fuengirola, where growth was faster, significant differences in growth rates at different stocking densities were observed in the first month after the start of the cultivation period. Different growth rates were apparent when areal coverage reached 23% and 47%. respectively, for 12 and 24 scallops/cage"' (height, approximately 55 mm). In O Grove, juveniles scallops maintained at 12/cage"' from September onward showed a significantly higher growth rate than those maintained at 24/cage^'. These densities corresponded to mean pooled areal coverages of 29.7% and 54.6%. respectively. Growth almost ceased between February and May 2000 at both stocking densities, when areal coverage was 39.7% at 12 scallops/ cage''. For the range of sizes used in this study. P. maxiiiuis showed low growth rates at areal coverages of between 30% and 40%. This is in accordance with the guidelines for growing scallops in net culture (i.e., that the area of the floor space occupied by scallops should not exceed 33%: Paul et al. 1981). When growing aequipectenoid pectinids (A. operciilaris. Placopecten mageUani- ciis. and Argopecten irradians). higher coverage rates can be used, but the same is not true for pectinoid Pectinids. Perhaps because they are not byssus-attached. there is a higher incidence of biting, and they are more affected by sea swell. Comparison Between Areas In both regions, there was sustained growth of spat during the winter. From November 1998 until May 1999. growth in the cul- tures held under the most favorable conditions (i.e.. those held at a stocking density of 25/cage"' as spat, and at 12/cage"' as juve- niles) was higher in Fuengirola (final mean size 64.7 ± 4.5 mm) than in O Grove (final mean size 52.6 + 4.1 mm; (P < 0.001 by / test). The faster growth rates in Fuengirola may have been due to the higher availability of food (measured as chlorophyll a). Fur- thermore, the temperature in Fuengirola was higher, as during most of the experimental period it ranged between 14°C and 16°C. compared with between 12^0 and 13"C in O Grove. In Fuengirola. during the first 2 mo of cultivation (September 24 to November 23. 1999) the spat increased in height from 20.4 to 34.9 mm (mean values), at the same time as the maximum temperature was recorded {2rC). However, in natural populations in Galicia. we have observed the formation of false growth rings between September and October (Roman, unpub. results). These rings are associated with an arrest in growth that coincides with the maximum temperatures that occur during the year (~18.5-20°C). It is therefore possible that the scallops in Malaga are genetically adapted to the higher temperatures. In O Grove, the rate of growth between February and April was very slow; by this date scallops reach the size when juveniles maintained in suspension stop growing, as has been described in other areas. According to Slater (1995). the cultivation of P. iiia.xi- iiiiis in baskets or cages is easily carried out until the spat reach a size of 45 mm. but thereafter growth is retarded. Although Cano et al. (2000) obtained scallops of commercial size (100 mm length) after 18 mo of cage culture in Malaga, only a small proportion of the spat culture in O Grove reached commercial size when main- tained in cages. In O Grove, the scallops detached from collectors in November and cultivated in cages had grown sufficiently (>60 mm) by the following September to be ear-hung; taking into ac- count that in the latter months of cage culture (until May 2000) growth was very slow, it may be advisable to begin ear-hanging culture in September. The culture of P. nni.xiiiuis in suspension is complicated and is influenced by many factors, not all of which have been thoroughly studied or are well understood. In addition to the factors usually considered, such as food availability, temperature, stocking den- sity, and depth, other factors such as handling frequency and foul- ing of both shells and cages, and the interactions among these also should be taken into account. This species lives recessed in the sediment and under natural conditions is not usually heavily fouled. However, when grown in suspension the animals are heav- ily fouled, apparently more than other epifaunal pectinids. such as A. operciilaris and Chlamys viiria. The effect of fouling should be studied because, as well as the negative effects (i.e., competition and reduction of water flow), there may be positive effects, such as the prevention or reduction of mobility within the cages, thereby reducing biting and malformations. ACKNOWLEDGMENTS This study was financed by Fondo Europeo para el Desarrollo Regional (FEDER) grant IFD I997-020I-C03-01 in Galicia. by the Junta de Andalucia in Fuengirola and by the Institute Espeiiol de Oceanografia (lEO) in both areas. The CTD data for O Grove were provided by the Centro de Control da Calidade do Medio Marino. We also thank Recursos Mariuos Grovenses (REMAGRO) for the loan of facilities, and for the help provided for Carmen Presas. Carmen Vazquez. Juan Fernandez. Teresa Garcia, Lourdes Fernandez, and the fishermen from Los Boliches. LITERATURE CITED Cano, J.. M. J. Campos & G. Roman. 2000. Growth and mortality of the king scallop grown in suspended culture in Malaga. Southern Spain. Aqiiacullurc IiiU'nuilioncil 8:207-225. Cote. J.. J. H. Himmelman. M. Claereboudt & J. C. Bonardelli. 1993. Influence of density and depth on the growth of Juveniles sea scallops (PUicopeaen mugellanicu.s) in suspended culture. Can. .1. Fish. Aqmitic. Sci. 50:1857-1869. Dadswell. M. J. & G. J. Parsons. 1941. Potential for aquaculture of the sea scallop. Placopecten maj>ellaniciis (Gmelin. 1791) in the Canadian Maritimes using naturally produced spat. In: S. E. Shumway & P..A. 654 Roman et al. Sandifer. editors. World Aquaculture Workshops. No. 1 . Baton Rouge. LA: World Aquaculture Society, pp. 300-307. Dao. J. C, J. Barret, N. Devauchelle. P. G. Fleury cS: and R. Robert. 1996. Rearing of scallops (Peclen maximus) in France, from hatchery to intermediate culture, results of a 10 year programme ( 1983-1993). In: G. Gajardo & P. Coutteau. editors. Improvement of the coinmercial production of marine aquaculture species: Proceedings of a workshop on Fish and Mollusc Larviculture. Santiago de Chile: Impresora Creces. pp. 1 21-134. Duggan, W. P. 1973. Growth and survival of the bay scallop, Argopecten irradians. at various locations in the water column and at various densities. Proc. Nat. Shellfish Ass. 63:68-71. Fleury. P.-G., C. Halary & J. C. Dao. 1995. The intermediate culture of Pecten mci.xinnis in Brittany (France). In: P. Lubet. J. Barret & J. C. Dao. editors. 8th International Pectinid Workshop. Cherbourg. France. May 22-29, 1991. IFREMER. ACTES de COLLOQUES-n = 17-1995, pp. 95-100. Fleury. P.-G.. J. C. Dao. J. P. Mikolajunas, D. Minchin, M. Norman & 0. Strand. 1997. Concerted action on scallop seabed cultivation in Europe (I993-I996): Final report (1997) AIR 2-CT 93-1647. Freites. L.. B. Vera. A. Velez & C. Lodeiros. 1995. Efecto de la densidad sobre el crecimiento y la supervivencia de juveniles de Euvola (Pecten) ziczac (L.) bajo condiciones de culti\'o suspendido. Cit: Mar. 21 :361-372. Freites, L.. J. Cote. J. H. Himmelman & J. Lodeiros. 1999. Effects of wave action on the growth and survival of the scallops Euvola ziczac and L\ropecten nodosus in suspended culture. / E.xp. Mar. Biol. Ecol. 239:47-59. Gallagher, J. 1999. Survival and growth of ear-hung scallop (Pecten ma.xi- mits) in Mulroy Bay. Book of abstracts, 12th International Pectinid Workshop, May 5-11. 1999. Bergen. Norway, pp. 139-140. Laing. I.. & P. F. Millican. & N. H Earl. 1999. Effect of sampling fre- quency on growth and sunival of juvenile scallops (Pecten ma-\imus) In: Book of abstracts. 12th International Pectinid Workshop. May 5-11. 1999, Bergen, Norway, pp. 47-48. Laing. I., & S. D. Utting. 2001. Cultivating king scallop. In: Proceedings of 2001 scallop Odyssey, 13th International Pectinid Workshop. Co- quimbo, Chile, April 18-24, 2001, pp. 48-49 Leighton. D. L. 1979. A growth profile for the rock scallop Hinnites nniltirugosiis held at several depths off La Jolla. California. Mar. Biol. 51:229-232. Lodeiros. C, & J. H. Himmelman. 1994. Relations among environmental conditions and growth in the tropical scallop Euvola (Pecten) ziczac (L.) in suspended culture in the Golfo de Cariaco. Venezuela. Aqua- culture 119:345-358. O'Connor, S. J.. M. P. Heasman & W. A. O'Connor. 1999. Evaluation of alternative suspended culture methods for the commercial scallop. Pecten fumatus Reeve. Aquaculture 171:237-250. Parsons. G. J., & M. J. Dadswell. 1992. Effect of stocking density on growth, production and survival of the giant scallop, Placopecten ina- gellanicus. held in intermediate suspension culture in Passamaquoddy Bay, New Brunswick. Aquaculture 103:291-309. Paul, J. D. 1988. Cultivation of the Scallop Pecten maxitmis using the techniques of ear hanging. Technical report No. 326. SFIA. Marine Fanning Unit (ARDTOE). Paul, J. D., A. R. Brand & J. N. Hoogesteger. 1981. Experimental culti- vation of the scallops Chlamys opercularis (L) and Pecten luaximiis (L.) using naturally produced spat. Aquaculture 24:31—44. Rhodes. E. W., & J. C. Widman. 1984. Density-dependent growth of the bay scallop Argopecten irradians irradians. in suspension culture. Int. Counc. Explor. Sea 18:1-8. Roman, G. & I. Fernandez. 1991. Ear hanging culture of scallop (Pecten ma.ximus (Linnaeaus 1758)) in Galicia. In: S. E. Shumway and P. A. Sandifer. editors. An international compendium of scallops biology and culture: Worid Aquaculture Workshops. No. 1. Baton Rouge. LA: World Aquaculture Society, pp. 322-330. Roman, G., M. J. Campos, C. P. Acosta & J. Cano. 1999. Growth of the queen scallop (Aequipecten opercularis) in suspended culture: intlu- ence of density and depth. Aquaculture 178:43-62. Slater, J. 1995. An initial ear hanging trial with Pecten nuixinius in Mulroy Bay. Book of Abstracts. 10th International Pectinid Workshop. April 27-May 2, 1995. Cork. Ireland, pp. 146-147. Ventilla, R. F. 1982. The scallop industry in Japan. Adv. Mar. Biol. 20: 309-382. Wallace. J. C. & T. G. Rein.snes. 1985. The significance of various envi- ronmental parameters for growth of the Iceland Scallop (Chlamys is- landica). in hanging culture. Aquaculture 44:229-242. Waller, T. R. 1991. Evolutionary relationships among commercial scallops (Mollusca: Bivalvia: Pectinidae). In: S. E. Shumway, editor. Scallops: Biology, ecology and aquaculture: Developments in Aquaculture and Fisheries Science, vol. 21. Amsterdam: Elsevier, pp. 1-73. Jcniriial uj Skclljhh Research. Vol. 22. No. 3. 655-660. 2003. INTRASPECIFIC GENETIC VARIATION IN MITOCHONDRIAL 16S RIBOSOMAL GENE OF ZHIKONG SCALLOP CHLAMYS FARRERI XIAOYU KONG,' ZINIU YU,' "* YAJUN LIU.' AND LINLIN CHEN' College of Fisheries. Oeeaii University of China. Qingdiio 266003. Peoples Repuhlie of China: 'Haskin Shellfish Research Laboratory. Institute of Marine and Coastal Sciences. Rnigers University. Port Norris. New Jersey 08349 ABSTHACT A 592 base-pair fragment of the mitochondrial 16S ribosomal gene in 47 Zhikong scallop {Clilamys faireri) specimens was sequenced to examine its intraspecific genetic variation and geographic structure. These samples were collected from six populations [four from China, and one each from South Korea (SK) and Japan] across its range. Thirty-one nucleotide positions were found variable, and twenty-three haplotypes were detected in all samples, which showed that more I6S rDNA variation existed in C. farreii when compared with several other oyster species. Analysis at the intrapopulation level showed that the SK sample had the richest sequence diversity. However, an analysis of haplotype frequency distribution and analysis of molecular variance indicated that little geographic structure was present among all samples, and an absolute majority (99.659^) of the genetic variation was distributed within populations, suggesting that the populations in this study may belong to a single panmictic unit. A relatively smaller distribution range and various currents may account f(.>r sufficient gene flow amS Genetic Variation of 16S rDNA in Zhikong Scallop 657 TABLE 2. The sequence indices of intrapopulatlun level of 16S rRNA gene in Zhikong scallop C.farreri. DL YT RC QD SK JP No. polymorphic sites 3 8 7 7 14 6 No. haplotypes 4 7 6 6 8 6 Haplotype diversity 0.750 1.00(1 0.893 0.893 1. 000 0.929 Nucleotide diversity 0.0017.5 0.0()4IS 0.00296 0.00326 0.00.591 0.00314 Average No. nucleotide differences 1.0357 2.4762 1.7500 1.9286 3.5000 1. 857 1 cial catch practice in Gunsan. SK. and in Kana/awa. JP. respec- tively (Fig. I ). Total DNA was extracted from adductor muscle tissue using a standard phenol/chlorofomi method (Sambrook et al. 1989). 16S fragments of the 16S rDNA were amplified using a pair of uni- versal primers: 16sar-L/16sbr-H: 5'-CGCCTGTTTATCAAAAA- CAT-375'-CCGGTCTGAACTCAGATCACGT-3' (Palumbi 1991). Amplification of the products was performed using a PTC- 100 thermal cycler (MJ Research. USA). The 100-(xL amplification reaction contained the following: 2.0 mM MgCU; 200 (xM each dNTP; 0.2 (jlM each primer: 2.3 (jlL of template DNA: 2.5 units of Taq polymerase (Sangon. China) with supplied buffer. For all amplifications, a hot-start polymerase chain reaction (PCR) was initiated by the addition of polymerase and primers following an initial 2-min denaturization at 80°C. The PCR cycling profile was as follows: 35 cycles at 94°C for 45 sec. 50°C for 1 min. and at 72°C for 1 min: with a final extension at 72°C for 7 min. C» X 165 33 3^0 JO / M*- 82. fas ,f-o 260 176 160 — -ifr3 Gc- i' l' ' 3J7 3^ I 69 UO ^ J--- Figure 2. \ median network diagram elucidating the relationship of the 23 haplotypes of the 16S rRNA gene in the /Jiikong scallop ('. farreri. Haplotype codes are dellned in Table 1. Sequencing PCR products were purified using UNIQ-5 Column PCR Prod- uct Purification Kit (Sangon, China), were ligated into pMD18-T vector by following the instruction of the Takara DNA Ligation Kit. version 2 (Takara, Japan), and were used to transform com- petent JM109 Escherichia coli cells using standard protocols. Re- combinant colonies were identified by blue-white screening. In- serts of the correct size were detected via restriction enzyme di- gestion by EcoRl and HindUX. Vector DNA containing the desired insert was further purified using the Pharmacia EasyPrep Kit (Sweden). Sequencing was performed for both strands of every sample on an ABI PRISM 377XL DNA Sequencer using ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction Kit with AmpHTaq DNA Polymerase, FS (Perkin Elmer). DaKi Analysis Initially. 16S sequences from individual specimens were aligned with CLUSTAL X (Thompson et al. 1997) and then were assigned a haplotype on the basis of discrete combinations of nucleotide sites. Population-specific haplotypic diversity (Nei & Tajima 1981) and nucleotide diversity (Nei 1987) were quantified, respectively. A haplotype median network diagram describing the relationships of observed haplotypes was built using Network 3.1.1.1 (Riihl 1999. Bandelt et al. 1999). All populations were nested into three groups (China. SK. and JP), respectively, and then analysis of molecular variance was conducted to determine the genetic differentiation of the populations with ARLEQUIN (Schneider et al. 1997). Haplotype frequency distributions also were analyzed by exact test (Raymond & Rousset 1995) with the same software. Genetic differentiation at different hierarchical lev- els was assessed by statistics (Weir & Cockerham 1984). A pairwise matrix of interpopulation nucleotide divergences (Nei 1987) among all populations was calculated, and it was used to construct an unweighted pair group method with arithmetic mean (UPGMA) phenogram employing the NEIGHBOR program, and the tree was drawn using the drawgr.-v.m program in the PHYLIP package (version 3.56C: Felsenstein 1989). RESULTS Sequences of the 592-base pair 16S rRNA gene of all 47 speci- mens were obtained, and 3 1 nucleotide positions were found vari- able. Twenty-three haplotypes were detected among all samples, and their frequencies are shown in Table 1 . Haplotype A and B were the most common ones and were observed in all populations. Their frequencies were 29.8% and 17.0%, respectively. Haplotype C was shared by three populations (DL, YT, and RC), haplotype M was present only in the SK and JP populations, and haplotype N was observed in both the SK and RC piipulations. All others were 658 Kong et al. TABLE 3. Analysis of molecular variance of 16S rRNA gene haplotypcs in Zhikong scallop C.farreri. Source of Variation Df % Total * Statistics P Value Among groups Among samples within groups Within samples 3 41 2.00 0.00 (-1.67) 99.67 Jf^ Figure i. Cyst of I'erkiiisus sp. trophozoites encapsulated by a hemocytc within the jjill of a Manila clam. Bar, l(( |un\. H&E. qiiciitiy III the mantle and labial palps. Parasites were ot'len asso- ciated with tissue hemocytDsis (Fig. I) and occurred as single or multiple trophozoites (Fig. 2). In severe infections, the parasites were more abundantly distributed in the tissues, including the vas- cular sinuses around the digestive diverticula. Broad areas of the subepithelial connective tissues were composed of solid masses of parasite cysts in the most severe infections. In many cases, the parasites were contained within a thin-walled cyst formed by one to several host cells (Fig. 3). Such encapsulations contained up to 10 protozoan cells and associated heniocytosis. The parasites were often characterized by the presence of an eccentric vacuole (Fig. I and ?i}. characteristic of Perkinsiis sp. trophozoites. Confirmation of Perkinsus sp. by ISH The genus-Perkinsiis SSUrRNA gene probe PerkspVOODIG demonstrated strong hybridization to Perkinsus sp. cells in all of the tissue sections, except those of P. qiigwadi infecting P. yes- soeiisis (Table I and Fig. 4A-I). No hybridization to parasite cells of other genera was observed. ISH of parasite cells in tissue sec- tions of infected Korean Manila clams with this genus-Perkinsiis probe confirmed the genus level affiliation of the parasites in our sample of juvenile Korean Manila clams (Fig. 5). DISCUSSION We report the confirmation by ISH assays and histology of Perkinsus sp. infections in Manila clam seed proposed for the introduction into Mexican waters and the subsequent transport to growout sites on the Pacific coast of the United States. This is the first confirmation by a molecular diagnostic probe of Perkinsus sp. infection of Korean Manila clams As a result of these findings, the plan for importation of these clams was rejected by the shellfish producer, and no Korean seed clams were imported to the west coasts of Mexico or the United States. However, the ready avail- -'■--vT";'''- Uiy/'-ti^::. ■•.*V; ■?!» v.v 2 ».. 4G* ..v» Figure 4. Tissues sections of host tissues reacted with the yenus-ZVrAmvHv probe Perksp7IIO b> ISH. Positi^ely stained l'crkinsu\ sp, parasites are shown by arrows. (Al P. marinus in C. rirgiuica intestine (bar, 11) pni). (15) Perkinsus sp. in ('. paeijicus (bar. It) pnil. (Cl /'. atlanlieus in R. deeussalus (bar, 10 pm). (D) P. olseni in //. laevigata gill and mantle (bar, 25 pm). (F) Perkinsus sp. in M. halthiea (bar 25 pml. (F) Perkinsus sp, in Japanese V. pliilippiuarum (bar, II) pml. (G) P. ehesapeaki in M. tireuarin (bar It) pm), (Hi A", mediterraneus n. sp. in (). edulis (bar, 10 pm). (Il /'. qugwadi in P. yessoensis (no hybridization observedl (bar. 25 pml. 664 Elston et al. Figure 5. Tissue section ol Korean Muuilu ilani rtaitud witli gtnus- Perkiiistis probe Perksp7()0DlG by ISH. Positively stained Perkiiisus sp. parasites arc shown by arrows. Bar, Id pni. ability ot such infected seed clams from Korean or Japanese pro- ducers requires vigilance to ensure that no such importations take place into areas thai are free of the pathogen, such as the west coasts of North and Central America. Reports of lethal Perkiihsiis sp. infections in European and eastern Asian Manila clams from latitudes as far north as that of northern Oregon, confirm the high likelihood that such infections, if introduced, could persist and be transmitted, with damaging results to both wild and cultured clam stocks along the Pacific coasts of North and Central America. This study demonstrated that infection prevalence in seed clams ranging from 16 to 32 mm SL can be nearly ]Q09r and that high parasite intensities cause significant histologic damage to the organs of infected clams, particularly the gills. Choi and Park (1997) studied five species of Korean clams for infections by Perkinsus sp. using Ray's fluid thioglycollate me- dium (Ray 1966) and found infected Manila clams along the south coast of Korea. While no infection occuned in clams of <15 mm SL. nearly 100% infection prevalence occurred in clams of >20 mm SL. Park et al. ( 1999) reported mass mortality of Manila clams along the west and south coasts of Korea over a period of several years, which was associated with Perkinsus sp. infections. They reported 100% infection prevalence in 142 clams from Komsoe Bay on the west coast of Korea with moderately severe mean parasite intensities of 2.87 based on the infection intensity scale of Choi et al. ( 1989). A negative con-elation was found between the intensity of Perkinsus sp. infections and the clam condition index, while clam size was positively correlated with infection intensity. Maeno et al. ( 1999) reported Perkinsus sp. parasites in Manila clams from an inner bay of the western part of Japan in April 1998. using genus-P(^/A/;i,v//.v-specific antibodies. These authors con- cluded that the parasites were Perkinsus sp. based on a positive reaction with both single and clustered trophozoites. Hamaguchi et al. (1998) have reported the first detection of Perkinsus sp. in Japanese Manila clams. Anecdotal information that we received from the Korean supplier of the seed clams and their Japanese customers indicated that the Manila clam seed had been trans- ported from the Korean source to Japan for at least 20 y with no unusual mortalities or loss of growth reported. This anecdotal re- port and the multiple reports of the Perkinsus sp. parasite occuiring about 1997 or 1998 in Japan and Korea suggest that it could have been a new introduction to the Korean clams, as well as the Japa- nese clams, at about this time. Manila clams and other bivalve species from Europe reportedly have been infected with Perkinsus sp., as follows: P. atlantieus from the Mediterranean coast of Spain (region of the Ebro Delta, Tarragona, Spain) infected R. plulippinarum (Sagrista et al. 1996); Manila clams from the Lagoon of Venice in northeast Italy in- fected with a Perkinsus sp. (DaRos et al. 1998): and P. atlantieus infected the carpet shell clam (R. decussatus) from European lo- cations (Ordas et al. 2000). Villalba et al. (2000) reported a sig- nificant conelation between the SL of/?, deeussalus and P. atlan- tieus infection intensity. No clams of <20 mm SL were infected, and the highest seasonal parasite intensities occurred in spring and late summer to early autumn. The relationship of Perkinsus sp. in European waters to the Perkinsus sp. found in Korea and Japan is unknown at this time. Nonetheless, this and other studies cited in this report indicate the presence of this damaging parasite in Korean and Japanese Manila clams, confirmed first in this study by histology and then defini- tively by the Perkinsus sp. -specific probe presented for the first time in this article. This knowledge can be used to prevent the iniintentional introduction of this parasite to west coast of North and Central America. We urge that the science presented in this article be applied by shellfish growers, and by natural resource and conservation managers to prevent such a damaging introduction. ACKNOWLEDGMENTS N. A. Stokes, K. L. Hudson, K. Apakupakul, and R. M. Ham- ilton provided expert technical assistance in the performance of ISH assays. Perkinsus sp. -infected mollusc histologic samples were generously provided by C. Azevedo. S. M. Bower, E. M. Burreson, C. L. Goggin, F. G. Kern, and Y. Maeno. Parasitic dinoflagellate-infected crustacean tissue samples were provided by J. D. Shields and G. D. Stentiford. This work was supported in part by National Oceanic and Atmospheric Administration (NOAA) Sea Grant funding of project NA86RG0037 to CFD. This work is also a result of research sponsored in part by NOAA Office of Sea Grant, U.S. Department of Commerce, under grant No. NA96RG0025 to the Virginia Graduate Marine Science Consor- tium and the Virginia Sea Grant College Program, and under grant No. NA016RG2207 to the Maryland Graduate Marine Science Consortium and the Maryland Sea Grant College Program. The U.S. Government is authorized to produce and distribute reprints for governmental puiposes. notwithstanding any copyright nota- tion that may appear hereon. VIMS contribution #2575. LITERATURE CITED American Fisheries Society. 1998. Common and scientific names of aquatic invertebrates from the United States and Canada: Molliisks. 2nd ed. American Fisheries Society Special Publication 26. Bethes- da, MD. Azevedo, C. 1989. Fine slnicture of Perkinsit.'. tiilciniieus n. sp. (Apicum- plexa, Perkinsea) parasite of the clam Riuliiape.s cleciissatiis from Por- tugal. J. Purasitol. 75:627-635. Blackbourne. J.. S. M. Bower & G. R. Meyer. 1998. Perkinsus qui<\\adi sp. nov. (incertae sedis). a pathogenic protozoan parasite of Japanese scal- lops. Patinopecten yessoensis. cultured in British Colombia. Canada. Can. J. Zool. 76:942-953. Bower. S. M. & G. R. Meyer. 2002. Morphology and ultrastructure of a prolistan pathogen in the haemolymph of shrimp {Puinlulus spp.i in the northeastern Pacific Ocean. Cun. J. Zool. 80:1055-1068. P!:kki.\sus sp. in Manila Clam Juveniles 665 Casas. S. M.. A. Grau. K. S. Reece. K. Apakupakul. C. Azevedo & A. Villalba. 2003. Perkiiisiis meililernmeiis n. sp. a prntistan parasite of the European flat oyster Osirea ediili.s (L.) from the coast of Balearic Islands, Mediterranean Sea. Dis. Aqiiat. Org. in press. Choi. K. S. & K. I. Park. 1997. Report on the occurrence of Perkinsiis spin the Manila clams, Riiditapcs philippinanmi in Korea, t Korean) J. AqiiMulnire 10:227-237. Choi. K.-S.. E. A. Wilson, D. H. Lewis, E. N. Powell & ,S. M. Ray. 1989. The energetic cost of Perkinsiis miiriniis parasitism in oysters: quanti- fication of the thioglycellate method. / Shellfish Res. 8:125-131. Coss, C. A., J. A. F. Robledo & G. R. Vasta. 2001. Description of Perk- insiis undrewsi n. sp. isolated from the Baltic clam [Mueomu Inilthiea) by characterization of the ribosomal RNA locus, and development of a species-specific PCR-based diagnostic assay. J. Eiikanol. Microbiol. 48:52-61. Couch. J. A. 1967. Concurrent haplosporidian infections of the oyster. Cnissoslrea virginiea (Gmelin). J. Parasiiol. 53:248-253. DaRos, L., M. G. Marin, N. Nesto & S. E. Ford. 1998. Preliminary results on a field study on some stress-related parameters in Tapes philippi- narum naturally infected by the protozoan Perkinsiis sp. Mar. Eiiv. Res. 46:249-252. Dungan, C. F.. R. M. Hamilton. K. L. Hudson. C. B. McCollough & K. S. Reece. 2002. Two epizootic diseases in Chesapeake Bay commercial clams. Mya arenaria and Tagelus pleheius. Dis. Aqiiat. Org. 50:67-78. Field. R. H. & P. L, Appleton. 1995. A Hemarodinium-like dinofiagellate infection of the Norway lobster Nephrops noiTegiciis: observations on pathology and progression of infection. Dis. Aqiiat Org. 22:1 15-128. Goggm, C. L., K. B. Sewell & R. J. G. Lester. 1989. Cross-infection experiments with Australian Perkinsiis species. Dis. Aqiiat. Org. 7:55- 59. Hamaguchi, M., N. Suzuki. H. Usuki & H. Ishioka, 1998. Perkinsiis pro- tozoan infection in short-necked clam Tapes 1.= Rmlilapes) phiUppi- miriim in Japan. Fish Pathol. 33:473^80. Haskin, H. H., L. A. Stauber & J. G. Mackin. 1966. Minehinia nelsoni n. sp. (Haplosporida. Haplosporidiidae): causative agent of the Delaware Bay oyster epizootic. Science 153:1414-1416. Liang, Y.-B., X.-C. Zhang. L.-J. Wang. B. Yang. Y. Zhang & C.-L. Cai. 2001. Prevalence of Perkinsiis sp. in the Manila clam Riiditapes phil- ippinaniin along northern coast of Yellow Sea in China. Oceanol. el Liiniwl. Sinica 32:502-511. Mackin. J. G.. M. Owen & A. Collier. 1950. Preliminary note on the occurrence of a new protistan parasite, Dennocystidiiim inarimim n. sp. in Crassostrea virginiea (Gmelin). Science 1 1 1:328-329. Maeno, Y., T. Yoshinaga & K. Nakajima. 1999. Occurrence of Perkinsiis species (Protozoa. Apicomplexa) from Manila clam Tapes pltilippi- iiariini in Japan. Fish Pathol. 34:127-131. Murrell. A.. S. N. Kleeman. S. C. Barker & R. J. G. Lester. 2002. Syn- onymy of Perkinsiis olseni Lester & Davis, 1981 and Perkinsiis atlan- ticiis Azevedo, 1989 and an update on the phylogenetic position of the genus. Perkinsiis. Bull Eur. Assoc. Fish Pathol. 22:258-265. Navas. J. 1.. M. C. Castillo. P. Vera & M. Ruiz-Rico, 1992. Principal parasites observed in clams. Riiditapes decussates (L.), Riiditapes phil- ippinanmi (Adams et Reeve), Venerupis pullastra (Montagu), and Venerupis aureus (Gmelin ), from the Huelv a coast ( S.W. Spain ). .Aqua- culture 107:193-199. Ordas. M.. A. Ordas. C. Beloso & A. Figueras. 2000. Immune parameters in carpet shell clams infected with Perkinsus atlanticus. Fish Shellfrth Immunol. 10:597-609. Pacific Coast Shellfish Growers Association. 20(.)3. West Coast shellfish production. Olympia. WA: Pacific Coast Shellfish Growers Associa- tion. Available at: www.pcsga.org. Park, K. L, K. S. Choi & J. W. Choi. 1999. Epizootiology ot Perkinsus sp. found in the Manila clam. Riiditapes philippinuruiii in Komsoe Bay, Korea. / Korean Fish. Soc. 32:303-309. Pacific Shellfish Institute. 2001. Manila Clam Mortality and Health Evalu- ation. Final report to Saltonstall-Kennedy Program, National Marine Fisheries Service, U.S. Department of Commerce on Grant No. Grant Number: NA96FD0I94. Olympia. WA: Pacific Shellfish Institute. Ray, S. M. 1966. A review of the culture method of detecting Dennocys- tidiiim mariniim. with suggested modifications and precautions. Proe. Natl. Shellfish A.ssn. 54:55-69. Sagnsta, E., M. Durfort & C. Azevedo. 1996. Ultrastructural data on the life cycle of the parasite, Perkinsus atlanticus (Apicomplexa). on the clam, Riiditapes philippinaruiii. in the Mediterranean. Sci. Mar. Bare. 60:283-288. Shaw, B. L. & H. I. Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster, Crassostrea virginiea (Gmelin). Can. ./. Zool. 35:325-347. Shields. J. D. 1994. The parasitic dinonagellates of marine crustaceans. Ann. Rev. Fish. Dis. 4:241-271. Stokes, N. A. & E. M. Burreson. 1995. A sensitive and specific DNA probe for the oyster pathogen Haplosporidiiim nelsoni. J. Eiikaryot. Micro- biol. 42:350-357. Stokes, N. A. & E. M. Burreson. 2001, Differential diagnosis of mixed Haplosporidiuni costale and Haplosporidiiim nelsoni infections in the eastern oyster, Crassostrea virginiea. using DNA probes. J. Shellfish Res. 20:207-213. Villalba, A., S. M. Casas, M. J. Carballal & C. Lopez. 2000. Effects of perkinsiosis on the clam Riiditapes decussatus industry of Galicia (NW Spain). J. Shellfish Res. 19:649. JcKriwI oj Shellfish Resenrch. Vol. 22, No. 3, 667-674. 2003. TOLERANCE AND RESPONSE OF MANILA CLAMS, VENERUPIS PHIUPPINARVM (A. ADAMS and REEVE, 1850) TO LOW SALINITY RALPH A. ELSTON.'* DANIEL P. CHENEY." BRIAN F. MACDONALD,' AND ANDREW D. SUHRBIER- ^Pacific Slu'lljisli liiMiiiite. PO Box 687. Cailsborg. Washiniito)i 98324: 'Pacific Shellfish Institute. 120 State Ave. N.E., No. 142. Olympia. Wa.shington 98501-0600; ^Washington Department of Fish and Wildlife. 600 Capitol Wa\ North. Ohmpiu. Washington 98504-3200 ABSTR.ACT Til delerniine under what conditions winter mortalities of the Manila clam (Veiierupis philippiiianim. A. Adams and Reeve. 1850) might be the result of excessive exposure to low salinities, a series of experiments was conducted. Clams were exposed to various concentrations of salinity to determine their physiologic lower limit of tolerance to salinity concentration, the duration they could withstand lethal or marginal low salinities through the mechanism of shell closure, and diagnostic structural changes in tissues indicative of low salinity exposure. Salinities of ^ 10 parts per thousand (ppt) were not tolerated in long-term exposures of 13 groups of clams. This lethal low salinity was also confirmed by the exposure of clams with a resection of a portion of shell. A salinity of 12.5 ppt was considered marginal, and various proportions of the different populations were able to tolerate this salinity, while no significant mortality occurred at ^15 ppt. Clams could withstand lethal low salinities of 5 ppt and 10 ppt for between 6 and 8 days, but all populations exposed to lethal low salinities for 14 days and then placed at high ambient salinity (-31 ppt) showed a high cumulative mortality. Clams may not die until .several days after exposure to lethal low salinity followed by placement in a recovery tank at their normally tolerated high salinity. We found no significant difference in the responses of several groups of clams to the marginal salinity of 12.5 ppt when exposed at temperatures of 6°C, 12°C, and 18°C. Histologic examination showed that the following sequential changes occurred in the digestive gland in clams exposed to 10 and 12.5 ppt for between 2 and 14 days: loss of granulation of the digestive tubular absorptive cells: swelling of these cells and occlusion of the tubular lumina: and finally the shedding of necrotic tubular epithelium into the digestive gland tubular lumina. KEY WORDS: Venerupis {Tapes) iiluHi^pmanim. low salinity tolerance, Manila clam INTRODUCTION Over 3000 tons of Manila clams (Venenipis philippinaniin. A. Adams and Reeve. 1850), valued at over $22 million (US dollars), were produced on the west coast of the United States in 2000 (Pacific Coast Shellfish Growers Association 2003). Most produc- tion occurs in Washington, but clams are also produced in Cali- fornia. Oregon, and British Columbia. Canada. An unfilled domes- tic and overseas demand is driving attempts to increase the pro- duction of this clam. In addition, a significant Manila clam seed production industry has developed, with production facilities in Washington. Oregon. California, and Hawaii. Native littleneck clams iProtothaca stainiiiea. Conrad 1837) are also produced in Washington and Alaska, but production is limited due to a short shelf life, a lower price for the producer, and the preference of consumers for the Manila clam. One constraint to the growth of the Manila clam industry on the west coast of the United States is the occurrence of sporadic mor- tality and poor growth due to unknown causes. With some excep- tions, mortalities are usually reported between November and March. Freezing damage may be a factor in Manila clam mortali- ties during the winter (Bower 1992). No highly pathogenic infec- tious diseases of Manila clams are known to occur on the west coast of North America (Elston et al. 2003). Clams may be reared in locations near freshwater streams or rivers with occasional high outflows in winter. We therefore sus- pected that at least some of the reported winter mortality events could be the result of exposure to salinities below the physiologic tolerance of the clam or from exposures to low salinity of duration longer than that for which clams can maintain shell closure. The clams burrow into the substrate, and clam deaths may only be observed at some time after the mortalitv event. A sur\'ev of the *Corresponding author. E-mail: aquatech (sHilypen.com literature revealed limited information on the low-salinity toler- ance of juvenile and adult Manila clams (Kim et al. 2001. Kurata 2000. Numaguchi 1998). Therefore, we conducted the studies re- ported here (1) to determine the lowest salinity at which Manila clams from several populations could survive over an extended time period, (2) to determine the duration of exposure that adult and juvenile clams can survive when exposed to lethal and mar- ginal low salinities. (3) to determine the relationship of water temperature to clam survival at a marginal low salinity, and (4) to determine histologic changes that could be used to diagnose the exposure of clams to low salinity. Taxonomic references to the Manila clam (also commonly re- ferred to as the Japanese littleneck clam) in the scientific literature are particularly confusing. We have designated it Venenipis phil- ippiiuirum in accordance with the Committee on Scientific and Vernacular Names of Molluscs of the Council of Systematic Ma- lacologists. American Malacological Union (American Fisheries Society 1998). The common name of Manila clam is also found in the literature, and. apparently in reference to the same species, the clam is associated with scientific designations of Tapes philippi- naniin. Riiditapes philippiiuinn. Tapes semidecussatus. and, less recently, as Tapes japoniea. MATERIALS AND METHODS Apparatus for Low Salinity Exposure Assessment We conducted initial salinity exposure experiments in static aerated aquaria over a 3-day period. In these experiments, the clams were not fed. However, the majority of experimental evalu- ations of low-salinity effects were made in two flowing seawater systems that we designed and built for this purpose, and that were operated at a commercial shellfish hatchery facility in Quilcene, Washington, where the ambient salinity ranged from 29 parts per thousand (ppt) to 32 ppt. These systems provided several tlovv- 667 668 Elston et al. through tanks capable of holding large numbers of test animals at constant levels of reduced salinities (up to four treatments simul- taneously) for extended periods of lime. This system was later modified to allow for multiple temperature treatments across a single salinity. In the initial configuration of this system, sand-filtered seawa- ter and unchlorinated fresh water were pumped into separate head- tanks (-200 L) the levels of which were kept constant by stand- pipes and float valves. A coiled length of vinyl tubing was used as a heat exchanger for the fresh water line to help equalize the temperature of the two water sources. Each of these two tanks fed a manifold fitted with four outlets restricted by variously sized orifices that flowed into mixing tanks. Each mixing tank (-2()L) flowed in turn into a treatment tank (~40L) where the test animals were held. Altering the sizes of each orifice feeding into the mix- ing tanks thereby controlled the salinity of the water within each treatment tank. A continuous flow of mixed algal food species provided by the commercial hatchery production system was in- troduced into the saline headtank at a rate sufficient to allow ex- cess food in all treatments. Airstones were used in each mixing tank to ensure the adequate mixing of the two water sources and to maintain dissolved oxygen saturation prior to the water being al- lowed to enter the treatment tanks. Salinity loggers and periodic manual checks were used to track treatment salinity and tempera- ture levels. Overall, the actual salinities varied no more that ±1.0 ppt from target salinities based on logger checks and spot manual checks, with the exception of two instances where actual salinity was 2.2 ppt higher than the target or 1 .2 ppt lower than the target. Flow apertures were checked, and any salinity deviations ap- proaching or greater than I ppt from target were corrected at least twice per week during experiments. Method of Testing Clams Initially, we tried to maintain shell opening by inserting wooden wedges between the \alves. but we abandoned this method because the clams usually rejected the wedges, although the method has been used successfully in other species such as Mytilus edulis (Shumway 1977). Alternatively in the initial experi- ment, we cut a wedge-shaped opening in the shell of clams (Fig. I ) to force exposure of tissues to the exposure salinities. While this method appeared to have some utility for determining physiologic tolerance to low salinity levels, it was time-consuming and success required extensive operator practice to avoid damage to soft tis- sues. Therefore, we abandoned this method in favor of long-term exposures (4 wk) to evaluate physiologic adaptation or lack thereof to various salinity concentrations. Clams were obtained from locations in Washington. California, and Hawaii, and were placed in trays in the flowing seawater tanks without sand. Water temperatures were maintained at a constant level within experiments but varied between experiments from 10.0 to I4.0°C, except for the trial in which we tested the effect of temperature on tolerance to a marginal low salinity concentration. Clams were removed from the experiments and considered dead when their shells gaped and they were unresponsive to prob- ing. Alternatively, clams that were counted as alive when returned to recovery tanks at ambient salinity had active shell adduction and extension of the siphons. Two experiments were conducted to observe the histologic ef- fects of low-salinity exposures on the gills and digestive gland of clams, two organs that in preliminary experiments appeared sen- sitive to low-salinity exposure. Adult clams [40-50 mm shell length (SL)] were used in both experiments, which lasted 9 days and 14 days, respectively, with samples collected at the initiation of the study and at 2, 4, 7, 9, and 14 days of exposure to 10 ppt and 12.5 ppt, along with control clams at ambient (-30 ppt) salinity. We compared the 4-wk mortality rate for the groups containing two replicates (Table I) using the probability density function for a binomial distribution (Samuels & Witmer 1999). Analysis of variance was not used as it did not meet the requirements for normality and sample size. The probability density function for the binomial distribution is ,/'(.v) /)'(l -/J)\.v = 0,l,2. Fiyiiru 1. Manila ilani with portion ol shill resected to isolate the physiologic response to low salinities. where n is the number of trials and /) is the probability of "suc- cess."" Applied to the data in Table I. there are only two outcomes for each clam, dead or alive, with dead clams corresponding to the success of a trial. For example, to test whether there is any sig- nificant mortality difference between two locations (e.g., Stoney Point-Willapa and Little Skookum Creek in the 10 ppt treatment), the probability of success for the Stoney Point-Willapa site is estimated as p,, = 13/30 = 0.4333. Our null hypothesis (/y„) is p = /)||. And the alternative hypothesis (//,) is p > p^. The P value for observing .v > 2 1 is P(X a 2 1 ) = 0.0002, where X is a random variable following a binomial distribution with 30 trials and the probability of success for each trial is 0.4333. Since the P value for testing Hf, vs //, is so small, we reject the null hypothesis (at least at a 5% level of significance). This means that there exists a significant difference in the mortality rate at the two different locations. Where applicable, results also were compared using t- tests and determination of 95% confidence inter\ als for sequential time points in serially sampled experiments. RESULTS Measurement of Physiological Tolerance to Low Salinity hy I'arliat Shell Removal Figure 2 shows the results when clams with a shell wedge removed were exposed to six salinity levels for 3 days in a .static aquarium at 10.5°C, followed by a 19-day recovery period in flow- ing seawater. There was no mortality in either of the control groups (shell cut or intact clams). Although the salinity treatment at 20 ppt Manila Clam Low Salinity Tollrance 669 TABLE 1. Cumulative mortality of Manila clams held in desi)>nated salinity concentrations for 4 \vk^ Replicates H Salinity Concentrations Testedt (%) Clam Source? 25ppt 20 ppt 17.5 ppt 15 ppt 12.5 ppt 10 ppt Oakland Bav 30 3 (1 0 97 Chelsea ground 30 0 0 7 67 Chelsea yearling 30 0 0 0 67 California nur.serv seed clams 30 20.0 13.3 66.7 100 Survivors of low-salinity event 30 6.7 1 0.0 16.7 90.0 Hawaii nursery seed clams 50 0 6 s: 100 Little Skookum Creek 2 15 0 3 ± 4.7 27 ± 9.4 73 ± 4.7 Little Skookum Slough 2 15 0 0 7±0 80 ± 6.7 Chelsea Seafarms Creek 2 15 0 0 27 ± 9.4 80 ±12.7 Chelsea Seafarms Beach-N 2 15 3% ±4.7 0 30 ± 4.7 93±4.1 Stoney Point-WiUapa 2 15 7% ±0 7 + 0 13 ±5.0 43 ± 2.0 Oakland Bay 2 15 0 0 80 ±18.9 97 ± 4.7 Thorndyke Bay 2 15 0 17±4.7 7 ±9.4 97 ± 4.7 * The results arc Ihe cuniulatn e mortality rate afier 4 u k exposure at the indicated salinity. Further mortality was observed in many groups within 7 days after the 4-wk exposure, when the clams were placed in an ambient (30 ppt) salinity tank. Replicated treatment results are expressed as the a\'erage ± SD. t Target salinity concentrations are shown. + All clam sources are from Washington except as noted and. except as noted, arc adult clams with 40 to 50 mm SL. resulted in a cumulative mortality rate of 20Vf and the salinity treatment at 1.5 ppt resulted in a 10% loss, these losses were attributed to nontreatment effects. It was clear from this experi- ment that 5 ppt and 10 ppt were lethal low salinities from which a 3-day exposure resulted in lOOVr mortality within 17 days postex- posure. Most of the clanis in these two groups died between .3 and 6 days after removal from the static salinity treatment tanks. Physiological Tolerance to Low Salinity Measure by Exposures of 4 Week Duration Table 1 shows that there was relatively little mortality in any group tested at 15 ppt or higher, in comparison with control group mortalities, and no significant differences were found between the tested groups at these higher salinities. At 12.5 ppt salinity, the binomial distribution test showed a significant difference at the 5% level between Stoney Point-Willapa and Little Skookum Creek, and between Chelsea SeaFaniis Beach-N and Oakland Bay. In terms of significant difference at the 5% level, the mortality of clams at 1 2.5 ppt can be split into three groups that are different 100% 80% 60% 40% _ 20% 0% » —♦—5 5 ppt _,_10 4ppt _4_15 9ppt _,_20 5ppt ^i(-25 1 ppt -^ Shell cut • 30 1 ppt -Shell intact -30,1 ppt ^t 12 14 17 18 19 Days post exposure (+) in recovery tank after 3 day exposure to indicated salinity Figure 2. Experiment 1 results showing the respon.se of Thorndyke Bay adult clams with shell wedge removed to six salinity concentra- tions in a 3-day static tank exposure in = 10 clams per group; 41 ± 1.9 mm mean SL; test temperature 10 to H C). from each other: ( 1 ) Thorndyke Bay. Little Skookum Slough, and Stoney Point Willapa: (2) Little Skookum Creek. Chelsea Sea- Farms Creek, and Chelsea SeaFamisBeach-N; and (3) Oakland Bay. At 10 ppt salinity, there is a significant difference (5% level) in salinity between Stoney Point-Willapa and Little Skookum Creek. Stoney Point-Willapa is significantly different from the rest of the group. At the 57c level of significance for the 10 ppt expo- sures, the seven locations can be split into three groups, which are significantly different from one another: ( 1 ) Stoney Point-Willapa; (2) Little SkookuiTi Creek, Little Skookum Slough, and Chelsea SeaFarms Creek; and (3) Chelsea SeaFamis Beach-N. Thorndyke Bay, and Oakland Bay, Overall. Table 1 shows that, of the 13 groups of clams, in all but one group very few clams could survive a 4-wk exposure to 10 ppt salinity. However, the ability to survive at a salinity of 12.5 ppt varied greatly between groups of clams. Unlike Oakland Bay clams, adult clams from Thorndyke Bay (Fig. 3) were tolerant to salinity of 12.5 ppt. The highest mortality rate at 15 ppt was 17% and apparently was due either to factors other than salinity or to within-group variation since the mortality rate of the same group of clams (Thorndyke Bay) at 12.5 ppt was only 7%. The clams were removed from the .salinity exposures after 4 wk to ambient salinity (-28 ppt) for I v\'k. during which additional mortality occurred at 10 ppt and 12.5 ppt, bringing the mortality rate in all 10 ppt groups to nearly 100%, possibly as a result of partial ac- climation to the lower salinity and the inability to readapt quickly to the higher salinity. Only the 4-wk mortality rates are shown in Table 1. The 4-wk exposure of the Thorndyke Bay clams demon- strated that mortality reached nearly 100% after 3 wk of exposure at a salinity of 10 ppt. Figure 3 also shows that differences in mortality rate in clams from the Thorndyke Bay population at 12.5, 15.0. and 17.5 ppt were not statistically significant (P < 0.05), We sampled two paired groups of clams (Chelsea Seafarms and Little Skookum) from locations near intermittent high-flow streams and paired locations distant from the freshwater sources. Location near the creek outflows did not correspond to lower mortality at 4 wk. and. in fact, the Little Skookum Creek clanis had 670 Elston et al. ro 60% 5 3 40% TO -I F :^o% D o 0% -♦-lOppt _B_12 5ppt r^- _^15ppt / ^i_17.5 ppt / y .^^ ,, — ^ i^4^.^ 1 00% 0 12 3 4 5 Four Weeks of Exposure and One Week in Ambient Salinity Tank (30 ppt) Figure 3. Cumulative mortality of Thorndyke Bay adult clams held at four salinity levels for 4 \>k and a fifth week at ambient salinity (-30 ppt). Each treatment was replicated twice with 15 clams in each rep- licate group (41 ± l.** mm SL; vertical bars are 95% confidence in- tervals; test temperature 11-13'C). TO ■e o > E O 1 3 5 7 9 11 13 15 Days Post Exposure Figure 5. Thorndyke Bay adult clams exposed to the lethal low salinity of 10 ppt for various durations show a graded mortality response that is directly correlated with the duration of exposure and exhibit the ability to survive longer than clams similarly exposed to the lethal low salinity of 5 ppt (Fig. 5| (h = 15 clams per treatment and control at ambient salinity: 41.7 ± 2.9 mm SL; test temperature 10-11 C). a significantly higher mortality rate than did the Little Skookum Slough clams. Overall, the results establish that 12.5 ppt is a marginal salinity for most populations of clams, with variable numbers of individu- als able to survive this salinity concentration, and some popula- tions, such as the Oakland Bay clams, contained very few clams able to survive at 1 2.3 ppt. Duration of Lethal and Marginal Salinities That Can Be Survived Groups of 15 clams each |41.7 ± 2.9 mm average (±SD) SL] from Thorndyke Bay were exposed to lethal low salinities in flow- ing seawater of 5 ppt and 10 ppt for intervals ranging from 2 to 14 days and returns to ambient salinity (-28 ppt). The postexposure mortality results (Figs. 4 and 5) showed that at 5 ppt a mortality response occurred in all groups exposed for >8 days. The response was graded, but there was nearly a 50% greater mortality rate at 12 days of exposure in comparison to 1 0 days of exposure. No mor- tality was seen in clams exposed for 2. 4. or 6 days to 5 ppt. At 10 ppt a mortality response occurred in all groups exposed for SIO days, and the response was graded from 10 to 14 days of exposure. No mortality was seen in clams exposed for 2. 4. 6. or 8 days at 10 100% ppt. The results demonstrate that a population containing a high proportion of marginal ( 1 2.5 ppt) salinity-tolerant clams (Table 1 ) could withstand 5 ppt exposure for 6 days without losses, and exposure to 10 ppt for 8 days without losses. We conducted two additional experiments to evaluate the du- ration of tolerance to lethal and marginal salinities. We compared the response of two groups of adult clams to the lethal low salinity concentration of 10 ppt followed by placement in an ambient sa- linity (-28 ppt) tank. This included one population that showed a low proportion of individuals with tolerance to marginal low sa- linity (Oakland Bay clams) and another group of clams believed to contain individuals with a high degree of tolerance to low salinity (clams from Totten Inlet. Washington). Clams were exposed for durations of betw een 1 and 1 4 days, and were observed over a total of 4 wk, including the time in the low-salinity exposure tank and the ambient salinity (-28 ppt) recovery tank (Figs. 6 and 7). These results indicated that no mortality occurred in Totten Inlet clams held for 7 days at 10 ppt but that an intermediate mortality rate of 40% occurred in Oakland Bay clams held for 7 days at 10 ppt. Nearly 100% mortality occurred in both groups of clams held at 10 ppt for 14 days. An experiment was conducted using juvenile clams to examine their duration of tolerance to lethal (10 ppt) and marginal (12.5 ppt) low salinities, followed by a return to the ambient salinity. The results showed that when exposed to salinity of 10 ppt (Fig. 8) 80% 60% E o 40% 20% 1 3 5 Figure 4. Thorndyke Bay adult clams exposed to the lethal low salinity of 5 ppt for various durations show a graded mortality response that is directly correlated with the duration of exposure (« = 15 clams per treatment and control at ambient salinity: 41.7 ± 2.9 mm ,SL; test temperature 10-11 C). week 2 weeks 3 weeks 4 weeks Weeks including exposure and recovery periods Figure 6. Totten Inlet adult clams exposed to salinity of 10 ppt for intervals ranging from 1 to 14 days followed by a recovery period in ambient ( 30 ppt) salinity (;; = 2(1 clams per group: 46.1 ± 5.3 mm SL: test temperature 10-11 C). Manila Clam Low Salinity Tolerance 671 start 1 week 2 weeiss 3 weeks 4 weeks Weeks including exposure and recovery periods Figure 7. Oakland Bay adult clams txposed to salinity <>f 10 ppt for intervals raufiiu); tnim 1 to 14 davs lolloHed In a recovery period in anihient ( M) ppt) salinity (» = 20 clams per jjroup: 43.6 ± 3.'> mm SL; test temperature 10-11 C(. there was no significant difference in cumulative mortality be- tween exposures for 1. 2. 4, and 7 days, which all had cumulative mortality rates below 20%, but that a 14-day exposure resulted in about a 90% cumulative mortality rate within 2 wk after placement in the recovery tank. At 12.5 ppt, the mortality responses were similar, but there was higher variability among replicate groups, and the mortality rate was not statistically significantly different between any of the treatment groups. The final mortality rates for all treatments at 12.5 ppt were very similar to those resulting from similar exposures of clams to salinities of 10 ppt. The high vari- ability was not due to the smaller size clam replicate (5 mm SL compared with 1."^ mm and 14 mm SL) being more sensitive to 12.5 ppt salinity, because one of the larger size groups showed very high mortality compared with the other two groups. Effect of Temperature on Tolerance to Marginal Salinity We examined the effect of temperatures from 6 to 18 "C on the survival of seed clams exposed to the marginal salinity of 1 2.5 ppt (Fig. 9) There were no statistically significant differences in cu- mulative mortality rate over the 4-wk exposure period. Mortality was highest at 4 wk in the 6°C treatment, but this was due to a high cumulative mortality rate in one of three replicate groups. The maximum cumulative mortality rate was 20% over the 4-wk ex- posure period. Evaluation of Histological Changes at Lethal and Marginal Salinities Although the histological observations were variable between individuals, clear trends emerged that can be useful in the pre- sumptive diagnosis of low salinity exposure. The following se- quential changes occurred in the digestive glands of clams exposed 100% 1 -*- 1 Day J . f 80% -B-2Day / •' L 60% -A— 4 I3ay / 40% 20% -M-'Day / -»— 14Day / 1 0%^ start 1 week 2 weeks 3 weeks 4 weeks Time Including Exposure and Recovery Figure S. Replicate groups of 20 juvenile clams, each exposed for various durations to lethal salinity concentration of 10 ppt and a re- covery period at ambient salinity i~M) pptl. Three seed replicate groups measured 5 ± 1.5 mm, 1.' ± 1.,^ mm, and 14 ± 1.4 mm SI. each (test temperature 10-11 C). 40% r; 30% 20% 10% -♦_Ambient(~28ppt) _B_ 18 C -*-12°C _«_6°C 1 [ i 1 a — ""^ ^ F— ' L ^"T^ — i 0%_ 0 12 3 4 Weeks of Exposure at 12 5 ppt Figure 9. Evaluation of the effect of three temperatures on juvenile clam survival at the marginal salinity of 12.5 ppt. Three clam groups: group 1, two replicates of 22 clams each (5 ± 1.5 mm SL); group 2, three replicates of 25 clams each ( 13 ± 1.3 mm SL); and group 3. three replicates of 25 clams each (14 ± 1.4 mm SL). to 10 ppt and 12.5 ppt: loss of granules in the absorptive cells (presumably tVom lack of feeding) (Figs. 10 and 1 1 ); swelling of the absorptive cells of the digestive gland so that the luminal spaces of the terminal digestive tubules became occluded (Fig. 12); and sloughing of absorptive cells of the digestive tubules into the cell luinina where they appeared as necrotic cells and cellular debris (Fig. 13). Loss of granules in the digestive tubular absorp- tive cells occurred within 2 days of exposure to both 10 ppt and 12.5 ppt salinity. In the shorter experiment, the digestive tubule absorptive cell luminu remained patent and normal-appearing through 7 days of exposure to both 10 ppt and 12.5 ppt, but by 9 days of exposure the luminal spaces were occluded in all clams exposed to 10 ppt salinity, and in about one half of the clams exposed to 12.5 ppt salinity. At 9 days, mild evidence of digestive absorptive cell sloughing was noted in a few clams exposed to 10 ppt only. In the 14-day experiment, using two different gioups of :yS^'^'°^^ . i I ■a... /Vk** -^^^ vGt^ - . ' .i'^ ^ . _ - -t Figure 10. Histological section of normal Manila clam digestive gland showing granules in absorptive cells and open digestive tubular lumina (arrows). Bar, 2(1 pni, H&K. 672 Elston et al. • './=)(;«• w ' -J-** ' " • o '■J ' ^ ^'- ^.^J ' .<:• ^^, Figure 11. Histological section of normal Manila clam digestive gland without granules in absorptive cells but with patent digestive tubular lumina (arrows). Bar. 2(1 pni. H&E. '^"' r- jp «-»' 'A ® .o 0 «^ • -•■' ©c '#5 «' J ^> ^.'N / » •?.f Figure 13. Histological section of digestive gland from Manila clam exposed to 12.5 ppt salinity for 14 days showing shed necrotic absorp- tive cells in the digestive tubular lumina (arrows). Bar, 20 |jm, H&E. clams, about half of the clams exhibited swelling and luminal occlusion of the digestive tubule absoiptive cells after 4 days of exposure to both 10 and 12.5 ppt salinity. A similar proportion showed these changes at 7 days as well as mild cell sloughing at the lower salinity concentration. By 14 days of exposure, clams from one group had uniformly sloughed and necrotic cells in the tubular lumina. while in the other group about one half of indi- viduals had occluded swollen luniina and one half had shed ne- it; ;<3' cv -*ff .rx. ... ^r, 1 1^ ^' '' .*/ .' 4 ' •'•'-r:^ 3 ^ 7 ■■ (^^A J •^-' r- 1 •. iT .. - ;. > ! ^ fe-'. •i-^ i- '^.^^ . : « o ■ ^0, Q> "©. Figure 12. Histologk;il Mition of digestive gland from Manila clam exposed to 10 ppt salinity for 4 days showing swelling of the digestive tubular cells and occluded lumina (arrows). Bar, 20 (im, H&K. erotic cells into tubular lumina at 10 ppt salinity. At salinity of 12.5 ppt and 14 days of exposure in both groups, about one half of the clams had swollen occluded tubular lumina. and about one half had sloughed necrotic cells in the tubular lumina. The gills showed sloughing of epithelium in exposed clams, but. due to the random planes of section typical in histological preparations of the gill and the fact that at least mild epithelial loss was observed in apparently normal clams, the gills were a less reliable measure of low salinity exposure and were therefore not systematically evaluated. DISCUSSION Other limited studies of juvenile or adult Manila clams have shown similar low salinity tolerance. Kurata (2000) found that when tested at a temperature of I °C. salinities below 15 ppt limited the survival of Manila clams. Manila clam larvae have been found to have an optimal salinity range of 20 to 30 ppt in hatchery studies (Robinson & Breese 1984). Numaguchi (1998) reported that D- hinge Manila clam larvae could survive for 72 h at 12 ppt but that swimming was abnormal. Larvae did not survive at 8 ppt, but at salinities of S|5.5 ppt survival and swimming behavior did not differ from those of control larvae held at higher salinities. On the other extreme of salinity, Shpigel and Fridman (1990) found that Manila clams (referred to in the article as Tapes semidecussatiis) grew well in a salinity of 41 ppt. Lethal and Marginal Salinity Concentrations These experiments clearly showed that salinity of slO ppt is a lethal concentration for Manila clanis. at least for all of the popu- lations of clams used in this study. Although we placed clams in a recovery tank after the fourth week, we have reported only the mortality that occurred after 4 wk at constant salinity. The fact that additional mortality occurred in the fifth week may indicate at least a partial adaptation to the lower salinity followed by an inability to adapt quickly to the higher salinity experience in the fifth week. Manila Clam Low Salinity Tolerance 673 This, in fact, represents likely environmental conditions that may compound the mortality effect of long-term low-salinity exposure. In fact, our other experiments showed that clams exposed to 10 ppt for only 2 wk (Figs. 6 and 7) or less (Figs. 4 and 5) and then removed to ambient high salinity (-28 ppt) succumbed at a high rate. Therefore, the survival of clams held in low salinities (10 to 12.5 ppt for extended periods (e.g., 4 wk) may depend on the rate at which they are reacclimated to higher salinities. A salinity concentration of 12.3 ppt was shown to be a marginal concentration in which the survival of clams over a 4-wk period followed by 1 wk in a recovery tank was highly variable between populations and even within replicated groups. The average per- centage mortality rate at 12.5 ppt ranged from 7 to H2'/r. Standard deviations were typically very high in replicated groups, indicating the high variance within given populations for survival at 1 2.5 ppt. The striking difference between two populations is demonstrated by the Thorndyke Bay clams (tolerant to 12.5 ppt) and the Oakland Bay clams (intolerant to 12.5 ppt). We were not able to statistically link high survival at 12.5 ppt to specific locations wheie the clams seemed likely to have adapted to low salinity due to freshwater inflows near the clam beds. However, the Thorndyke Bay clams, which had the greatest survival at prolonged exposure to 1 2.5 ppt. are located near streams that may occasionally subject them to low-salinity conditions. The results seem to indicate that most clam populations contain some individuals with the ability to with- stand 1 2.5 ppt for extended time periods. Clams from many of the locations tested are the result of planting hatchery-produced juve- nile clams and represent possibly mixed as well as undocumented heritage, which may, in part, explain the variation in the propor- tions of individuals that can survive at 12.5 ppt in various popu- lations of Manila clams. However, if one had the objective of selecting clams with resistance to low-salinity concentrations, it would seem advisable to use a population such as the Thorndyke Bay clams as a founder population, since it appears to be enriched with individuals capable of withstanding a marginal salinity of 12.5 ppt. Kim et al. (2001 ) reported that Manila clams recovered a typi- cal endogenous circatidal rhythm of oxygen consumption when placed in reduced salinity as low as 15 ppt but not at salinities below 10 ppt. These authors concluded that Manila clams cannot maintain normal metabolic activity below 15 ppt. They also re- ported that all clams exposed to 5 ppt were dead within 7 days. The authors apparently did not evaluate the metabolic activity of clams at 12.5 ppt. The results of our study suggest that some clams may be able to respire normally at 12.5 ppt, based on their long-term survival at this salinity concentration. Mechanism of Response lo Low-Saliiiily Concentration in Manila Clams In regard to low-salinity effects on Manila clams, our working hypothesis was that resistance to low salinity consists of two fea- tures: a physiological capacity of the tissues lo tolerate a particular low salinity; and a survival response, consisting of the time for which the clam can maintain a closed shell condition, thus exclud- ing lethal low salinities, as has been shown to occur in other bivalve species. For example. Shumway and Youngson (1979) showed that shell closure oi Modiolus modiolus (Linnaeus 1758) occurred at 60% seawater. Burrell (1977) hypothesized that the greater resistance to low salinity in Merceiniria inercenaria (Lin- naeus 1758) in comparison to Eastern oysters (Crassustrea vii- ginica. Gemlin 1791) was due to the ability of clams to maintain shell closure for a longer period of time. Clearly, the survival response is complex, and depends both on aspects of the clam"s metabolism (e.g., capacity for anaerobic metabolism) and possibly on environmental factors that remain undelined, although, surpris- ingly, temperature did not appear to affect the response, at least within the parameters of the experiment conducted in this study. Our results suggest that while Manila clams can successfully resist lethal low salinities for a period of time, they are probably constantly testing salinity either by active subtle valve opening or seepage of low-.salinity seawater into the mantle cavity. The fact that we observed swelling of digestive gland tubular absorptive cells at 4 days of exposure to both 10 and 12.5 ppt salinity, com- binations of exposure time and salinity that we also showed to be clearly survivable. indicates that the clams do not totally exclude lethal and marginal low salinities during exposure by shell closure, although it is clear that they limit the exposure of their tissues to the low salinities by shell closure. Effect of Temperature on Tolerance to the Marginal Salinity of 12.5 ppt We were not able to demonstrate any significant effect of tem- perature on the tolerance of three groups of clams to the marginal salinity of 12.5 ppt, even though the populations tested included those that showed moderate to high mortality rates when exposed to 12.5 ppt in earlier experiments. However, Cain ( 1973) reported that survival was reduced in larval Rimgia cuneata at high tem- perature-low salinity combinations. Laing and Child (1996) showed that 6°C, the lowest temperature that we tested, was com- patible with the growth of Manila clams, while Mann (1979) showed that growth and spawning occurred at 18^C, the highest temperature that we tested. Structural Response of Tissues to Exposure to Lethal and Marginal Low Salinities These experiments provided data that can be used for the di- agnosis or forensic evaluation of clams that are suspected of ex- posure to lethal or marginal low salinities. Individual variation in response is probably a result of the extent to which individuals open and test the ambient salinity or, conversely, their ability to remain tightly closed when they sen.se lethal or marginal salinities. In either case, the results showed that relatively short-term expo- sure (i.e., between 4 and 14 days) to salinity of 10 or 12.5 ppt resulted in the swelling of the absorptive cells of the digestive tubules, presumably from the absorption of hypoosmotic .seawater, followed by the sloughing or loss of these cells into the digestive tubular lumina. Mortality Due to Lethal Low Salinity Exposure May Occur Over Several Weeks While our experiments on the duration of tolerance to 10 ppt, a lethal low salinity, showed that only about 7 days of exposure was required for a significant (approaching 100% in many cases) mortality response, all of the experiments in which clams were exposed and then placed in a recovery lank at ambient salinity tended to show a sharp increase in obvious shell gaping (our cri- teria of mortality) after placement in the recovery tank. The long- term (4-wk) exposures, for example, showed that while some of the clams were in the exposure tanks at 10 ppt they maintained shell closure and appeared normal for up to 4 wk followed by a 674 Elston et al. sharp rise in the mortality response during the fifth week when the clams were placed in an ambient salinity recovery lank. The reason for this is not known, but it may be due to the fact that the low salinity stimulates a strong shell closure response that disappears when the stimulus is removed (i.e.. the clams are placed in the recovery tank). However, structural damage to tissues, as demon- strated in this study, as well as stressful metabolic alterations (e.g.. depletion of free amino acids) are significant and. in fact, are irreversible much earlier, although they are not manifested in the obvious death of the clam at gaping, until it is returned to an environment where the stimulus for protective shell closure is removed. Whatever the basic underlying mechanism, the results from this study show that the obvious mortality response to lethal low-salinity exposure may be delayed, depending on the salinity regimen and perhaps other factors. Therefore, as a practical appli- cation of our results, it would be incorrect to assess a clam popu- lation immediately after, for example, a 7-day exposure to salinity of s 10 ppt and assume that clams showing tight shell closure were unaffected. It would be more accurate to assess the clam popula- tion several weeks later and. best of all. to additionally obtain tissue samples for histological analysis during and at intervals after the exposure to the low-salinity regimen. Reasons for Control Mortality Losses Control mortalities were generally <20'7f and often near zero. However, a control mortality rate even approaching 20% is a vex- ing issue and is one that will require further investigation to elu- cidate the causes. Clearly, the clams were held in a somewhat artificial environment in that a sedimentary substrate was not pro- vided due to the necessity to evaluate their condition frequently and could have contributed to the losses. We determined from an extensive histological examination of the clam populations used in this experiment and from other studies that there were no signifi- cant known infectious diseases of Manila clams present. ACKNOWLEDGMENTS This work was supported in whole by a grant from the Salton- stall-Kennedy Program, the National Marine Fisheries Service, the U.S. Department of Commerce entitled "Manila Clam Mortality and Health Evaluation" (grant number NA96FD0194). The assis- tance of Mr. Kevin Ford in the administration of the grant is gratefully acknowledged. The provision of space, water, and bi- valve food supply to conduct these experiments by Taylor Re- sources Company at their shellfish hatchery in Quilcene. Wash- ington, made the study possible, and the cooperation of Mr. Paul Taylor. Dr. Jonathan Davis, and Mr. Ed Jones in this endeavor is appreciated. The assistance of Dr. Dane Wu with statistical analy- sis is acknowledged. The efforts of Ms. Heidi Elston and Ms. Kendra Kinnan in the maintenance of the clam tanks and the enumeration of experimental clams is gratefully acknowledged. LITERATURE CITED American Fisheries Society. 1998. Common and scientific names of aquatic invertebrates from the United States and Canada: mollusks. 2nd ed. Special Publication 26.Bethesda. MD: American Fisheries Society. Bower. S. M. 1992. Winter mortalities and histopathology in Japanese littlenecks {Tapes philippiiuinim (A. Adams and Reeve. 1850) in Brit- ish Columbia due to freezing temperatures. J. Shellfhli Res. 1 1:255- 26.1 Burrell, V. G. 1977. Mortalities of oysters and hard clams associated with heavy runoff in the Santee River System. South Carolina in the spring of 1975. Proc. Natl. Shellfish. As.wc. 67:35-13. Cain. T. D. 1973. The combined effects of temperature and salinity on embryos and larvae of the clam Rangia cuneala. Mar. Biol. 21:1-6. Elston. R. A., C. Dungan. T. Meyers. & K. Reece. 2003. Perkinsus sp. infection risk for manila clams. Venerupis philippinarum (A. Adams and Reeve. 1850) on the Pacific Coast of North and Central America. / Shellfish Res. 22:661-665. Kim. W. S.. T. H. Huh. S. H. Huh, & T. W. Lee. 2001 . Effects of salinity on endogenous rhythm of the Manila clam, Ruditapes philippinarum (Bivalvia: Veneridae). Mar. Biol. 138:157-162. Kurata, M. 2000. Tolerance of the Japanese littleneck clam Riulilapes phillipinarum to low salinity and dissolved oxygen at low temperatures, (in Japanese). Sci. Rep. Hokkaido Fish. Exp. Stn. 58:17-2L Laing. L & A. R. Child. 1996. Comparative tolerance of small juvenile palourdes {Tapes descnssatus L.) and Manila clams {Tapes philippi- narum Adams and Reeve) to low temperature. ./. £v/). Mar. Biol. Ecol. 195:267-285. Mann, R. 1979. The effect of temperature on growth, physiology, and gametogenesis in the Manila clam Tapes philippinarum (Adams & Reeve, 1850). / £v/7. Mar. Biol. Ecol. 38:121-133. Nuniaguchi. K. 1998. Preliminary experiments on the influence of water temperature, salinity and air exposure on the mortality of Manila clam larvae. Ac/i/acif/;. Int. 6:77-81. Pacific Coast Shellfish Growers Association. 2003. West coast shellfish production. Available at: www.pcsga.org. Olympia, WA: Pacific Coast Shellfish Growers Association. Robinson, A. M. & W. P. Breese. 1984. Gonadal developmeni and hatch- ery rearing techniques for the Manila clam Tapes philippinarum (Ad- ams and Reeve). / Shellfish Res. 4:161-163. Samuels, M. L. & J. A. Witmer. 1999. Statistics for the life sciences. Indianapolis, IN: Prentice Hall. 638 pp. Shpigel, M, & R. Fridman. 1990. Propagation of the Manila clam {Tapes semidecussatus) in the effluent of fish aquaculture ponds in Eilat, Is- rael. Aquacidlure 90:1 13-122. Shumway. S. E. 1977. Effect of salinity fiuciuation on the osmotic pressure and Na*, Ca"*. and Mg"^* ion concentrations in the heniolymph of bivalve mollusks. Mar. Biol. 41:153-177. Shumway, S. E. & A. Youngson. 1979. The effects of fluctuating salinity on the physiology of Modiolus demissus (Dillwyn). / Exp. Mar. Biol. Ecol. 40:167-181. .Itniniiil ,>f Shellfish Research. Vol. 22, No. 3. 675-6S0. 21)U3. ON TWO NEW MACROSCOPIC INDEXES TO EVALUATE THE REPRODUCTIVE CYCLE OF ENSIS MACHA (MOLINA, 1782) OLGA L. ARACENA.* IRENE M. LEPEZ, JAVIER SANCHEZ, ANGELICA M. CARMONA, LUCILA MEDINA, AND ALEJANDRA SAAVEDRA. Deparkiiiicnti) dc Oceanoiinifia. L'nlvcrsickul clc Conccpcldn. Casilla 160-C. Concepciihi. Chile ABSTRACT We describe the reproducllve cycle of razor clam Ensis maeha. during 1996 and 1997. in the Golfo de Arauco. Chile (37°l4'S-73'2y'W) ba.sed in the variation of the monthly averages of common and new macroscopic and microscopic indexes and scales. The common macroscopic indexes are weight ratio of soft tissues to valve weight or Somatic Valve Index and. weight ratio of the soft tissues to total weight or Somatic Tissue Index. The new Macroscopic Index and scale are the quantification of the width of the posterior foot or Morphometric Index and the quantification of digestive gland cover with ovary tissue plus the degree of ovary development or mature morphometric scale. The microscopic indexes consist of the quantification of ripe gamete over the bulk of the gonadic tissues, previously treated in formalin and without stain or gametic index and the same quantification over histologic preparations or Gametic Histologic Index. The somatic valve index and somatic tissue index results are not adequate to describe the reproductive cvcle of this specie; however, the Morphometric Index and Mature Morphometric Scale are very useful. These last two methods, in addition to the Gametic Index and Gametic Histologic Index, show that the razor clam reproductive cycle, over these 2 years, is characterized by a resting period from March through July, and a progressive development of the gametes between August and October. The spawn starts in November and is widespread until February. The index dropped abruptly during November 1 997, showing a spawn rate more intensive than the previous year, which may be related to anomalous temperatures for the region. The reproductive E. macha cycle described here, is similar to the Eiisis minor cycle in the Manfredonia Gulf, in Italy and the Eitsis siliqua of Vilamoura on the southern coast of Portugal, but it is different to that observed for others authors in the Region X during 1 994, and in the Golfo de Arauco and other locations of southern Chile during 1997. KEY WORDS: gametic and gonadic indexes, reproductive cycle, razor clam. Ensis INTRODUCTION The razor clam Ensis macha (Molinu. 1782) is a bivalve shell- fish distributed from Caldera to Magallanes in the Chilean coast and up to San Mati'as Gulf in the Argentine coast. It is found in the shallow sandy bottom living buried deeply in the sand favored by its shape and a large foot. Razor clam fishery became an important resource, exploited mainly for exportation and includes activities of artisanal fisher- man, mediators and canning enterprises that commercialize the product mainly to Spain and Japan. The fishery started in south Chile in 1988. with a catch reaching 1.741 tones from Region X only. After that it reached a maximum landing of 8.617 tones in 1991 with contributions from the X and VIII Regions, but this year the landings in Region X diminished while the landings from Region VIII were increasing until 1993. Later on. the total land- ings diminished to 6.1 15 tones in 1999 when 88.3% came from the Region VIII, 10,1% from the Region X and the rest from the VII, XII, and IV Regions which has been slowly incorporated but at very small rate (Semapesca 2000). This reduction in the landings led to additional fishery management and the support of the re- search projects to cover the basic biology and fishery aspects of this resource looking towards future aquaculture. In this article, the background of the reproduction of this spe- cies in the Golfo de Arauco, Region VIII, Chile, during 1996 and 1997 is given. It has been characterized through macroscopic and microscopic methods in common use, plus two new methods not described previously, here proposed as and easy application, vali- dated with the histology of the female ovary. (Lepez et al, 1997a, 1997b, Aracenaet al. 1998a), MATERIALS AND METHODS Samples of about 60 adults of £. macha (S15 cm valve length) were taken monthly during 1996 and 1997 for the study of scale and macroscopic indexes. For microscopic indexes, monthly samples of 20 adults were taken during 1 996, and monthly samples of 30 adults during 1997. All samples taken during 1996 were selected in the landing zone of Tubul, Golfo de Arauco (37°I4'W- 73 '29'W) and during 1997 were collected on board of artisanal boats in the same Golfo, E. macha is gonochoric. The males have white-grey gonads with a homogeneous texture, while females show ovaries of a white-cream color and granular texture, especially when they are close to spawning. The sex was always corroborated with micro- scopic observation of ovary tissue smears. In the mature razor clams, as in many bivalves, the ovaries extend dorsally over the digestive gland and the anterior adductor muscle, showing a simple way to determine the sex and the de- velopment stage. The ovaries invade the ventral zone of the vis- ceral complex, the posterior part of the foot and form a cord in the inner channel of the eatable foot. To evaluate the mature stage, we applied the following mac- roscopic indexes: (i) Somatic Valve Index lS\T) SVI-- DVV'S*IOO DWV vv here DWS is the dry weight of the soft body parts and DWV is the dry weiahl of the valve. (/(') Somatic Tissue Index iSTI) STI = - DVV.S*10() *Corresponding author. E-mail: oaracena@udec.cl & ilepez@udec.cl where DWS is the dry weight of the soft body parts and DWT is the total dry weiuht. 675 676 Aracena et al. (Hi) A new Macroscopic Maturity Scale (MMS) Estimate the covering of the ovary over and around the diges- tive gland, on a scale of 1 to 4: 1. Ovary covers 1/4 of the digestive gland. 2. Ovary covers 1/2 of the digestive gland. 3. Ovary covers 3/4 of the digestive gland. 4. Ovary covers totally the digestive gland. Points were also assigned to the progressive development of the gonadic tissue. 1. No development; no observable gonadic tissue or very scarce and transparent. 2. hitermediate developmental stage; average bulk and granu- late aspect. 3. Very developed; shows maximum bulk and granulate as- pect. Mixing these two scales, we obtain the mature stages from Table I and the monthly average as follows: 1=1 Anterior adductor muscle Left gill 2)mM5, MMS = where MMS, is the value of the scale assigned to the individual /. and II is the total number of individuals counted every month. In addition, the respective variance can be obtained from: ^(MMS, - MMS)- VARiMMS)-- iiin- 1) (iv) A new Morpliometric Index (MI) The MI is obtained from the measureitient of the width of the posterior area of the foot, under the visceral complex, showing the degree of invasion of the gonad tissue. To this purpose, we made a cut in the posterior area of the foot, as shown in Figure 1. to measure its width. The monthly Ml is the average of the width of each individual (MI/) on the total number of individual mea- sured (/i) .Ml. Ml- and its respective variance: VAR (MI) : ^(M/, - A//)" /)("- I) TABLE 1. Maturity stages of the razor clam: Macroscopic Maturity Scale (MMS). Covering Developed Foot Cut to measure foot width Figure I. Ventral view of the soft body of £. madia showing the cut in the posterior part of the foot where the width measure is made to obtain the Morphometric Index. Other tw(i microscopic indexes were; )■; Gametic Index (Gl) The Gl corresponds to the proportion of ripe gametes in rela- tion to other kinds of cells in the bulk of the unstained ovary tissue smeared on a slide. The proportion of mature cells for each individual (Gl,) was obtained by applying 10 times a microscope integration plate ot 10(_) points to the mass and quantifying; Maturity Stage (MMS) Gl, a, iii where, 01, is the proportion of ripe gametes for individual /. a, is the number of ripe oocytes of the total elements in the individual /. and III is the total number of quantified elements. With this, we obtain the monthly average of Gl as follows: Gl- and its respective variance; VAR(GI)- I.O,. 2(G/,-G/)' /!(/!- II (>■/) Gametic Histologic Index (GHI) The GHI is the same prior proportion but on histologic prepa- rations stained with Gallegos embedding in Hystosec. previously embedding in chloroform and mounted in Entellan (Con 1960, modified by Delpi'n, personal communication). The proportion of matures gametes for each individual (GHI,) was obtained applying four times a integratiiin plate of 100 points on the histologic cuts and quantifying; GHI, in where GHI,. is the proportion of ripe gametes of each individual, a, is the number of ripe oocytes of the total elements in the indi- vidual / and m is the total number of quantified elements. With this obtain the monthly average of GHI. following: ^GHI, CHI-- and the variance is: REPRODLicTivb Indexes for Ensis macha 677 (CHI-GHir VAR[GHI] = n{n- 1) SVl ami STI. were used for the 1996 series on male and temale razor clams plus GI only on female clams. MMS and MI were used in the two series of samples, only on female. The GHl was used onl\ ill 1997. for female clams. All these inde.\es and scales were applied to individuals larger than 15 cm \'al\e length, because Reyes et al. (1995) had previ- ously defined the mean size of first sexual maturity al 14 cm salve length for razor clam of the Region X. To detect significant differences between the indexes and scales and between males and females for 1996. a Kruskall-Wallis test was carried out (P > 0.05 ). Same statistical test was carried out for the 1997 informatiiin and also multiple comparisons test (Least Significant Difference) to validate the macroscopic scale MMS with the GHI index (Aracena el al. 1998b). To explain the tendencies in the reproductive behavior of razor clam described in this article, complementary oceanography infor- mation available (temperature, salinity, density) for the Region VIII was used (Salamanca. 1997). RESULTS From a total of 1.502 individuals analyzed during the years 1996 and 1997. the observed ratio of females to males was always slightly smaller with 40.3% and 41.3%, respectively (Table 2). although this difference was obviously never significant. The Kruskall-Wallis test to detect differences between the in- dexes and macroscopic scales was applied, among males and fe- males for 1996. but it was not significant (P < 0.05). It was there- fore possible to compare results of two years, even though in 1997 only females were considered. Both SVI and STI showed similar tendencies during the year 1996 (Fig. 2c and d). being different to the other indexes and scales applied in that same period. Maximum levels were observed from March to May and low values from June through December. The SVI oscillated between 34.34 and 50.16 with variance between 9.63 and 79.08. The STI o,scillated between 25.00 and 33.31. with variance somewhat smaller, between 3.13 and 16.2S. The GI is an index applied directly on gonadic tissues and it is considered together with the GHI, that are the best to describe the evolution of the gametes. From February to .lune the first index stayed very low (Fig. 2e). with values between 0.02 and 0.04. rising later on to a maximum of 0.21 in October and then down again apparently through January or February of the next year. The variances were always lower than 0.01 . except during December of 1995. The MMS and the Ml followed similar lendencies in 1996 (Fig. 2a and b). The MMS fluctuated between 3.36 in March and 8.86 in October, with variances between 0.35 and 4.62. The MI have TABLE 2. Sexual proportions of the razor tlam: Golfo de Arauco. Female CXf ) Male (%) Total (N) 1996 1947 40.3 41..^ .S.S.7 S34 (i6S values from 3.98 mm (March) and 6.43 mm (September), with variances between 0.39 and 1 .48. During 1997 (Fig. 3), the GHI showed a very similar tendency to the GI of the previous year, but the descent after the maximum of October (0.253) it was very abrupt, falling to 0.017 in Decem- ber, with equally smaller variances of 0.01. The same might be said of the MMS and Ml of that year. To validate the MMS. the average value of the GHI in 1997. was calculated for each value of the scale, with its variance and confidence intervals (Table 3) and then, the multiple comparisons test (Table 4), indicated that stages 2, 3. and 4 of the MMS have a very similar GHI and thus could only be considered one. The stage 5 and 6 are very similar to each other but stage 7 is different. In this way and for practical effects the MMS could be simplified to the following three states: 1. 0 and '2 of the digestive gland is covered by the ovary; volume and granulation are minimal or intermediate. 2. 1/2 of the digestive gland is covered by the ovary; voliuiie intermediate and granulation are at their maximum. 3. The digestive gland is completely covered by the ovary; volume and granulation are at tlieir maximum. According to these data, the reproductive cycle of the razor clam in the Golfo de Arauco. is characterized by a resting time between March and June of every year (autumn and early winter), a gradual increase of the maturity of the ovary starting in this last month and reaching a maximum in October (spring), followed by a single spawn period that begins in November and may finish in December or be prolonged until February of the next year. DISCUSSION AND CONCLUSION Considering that the index GI and GHI. are the only ones that represent the changes that happen at the level of the gametic tissue, we may conclude that the index SVI and STI are not good to define the reproductive cycle of this specie because through its evaluation the gonadic tissue was not separated from the body tissue. In experiments carried out by Sastry (1968). with Aequipeaen irra- dians Lamarck, by Bayne (1975) with several species of bivalve and for Lowe et al. (1982) with Mytilus ediilis L. among many other authors, they showed that a nutritious transfer takes place from the digestive gland into the ovary, or since nutritional tissue from mantle toward the gametic tissue lowering the change of volume and weight in the ovary when the total body weight is considered. However, the index MI and the simplified scale MMS are good descriptors of the reproductive cycle of razor clam because they are obtained from the observation and measurement of the ovary, they are faster, easier and of low cost. Regarding the MMS. even though the ovary cover on the digestive gland is a simple measure to carry out and to be standardized, the gonadal volume and granu- lation, are very subjective parameters. For this reason, between these two indexes we recommend the MI index as a macro.scopic index because is easy to obtain and quantify. The GI is a low cost method as well, although not so simple or quick, but since it is a direct quantification of the gonadic tissue it is more advisable than the macroscopic index. As observed in Figs. 2 and 3. the reproductive cycle of E. macha during 1997 follows a similar tendency to the one observed during 1996. but with a more marked fall between October and December indicating a shorter, and more intense and synchronous spawning than in the previous year. This may be associated with salinity and temperature anomalies that affected the coastal areas 678 Aracena et al. II - 10 - 9 - 8 - 7 - 4 - 3 - 2 - a) Die Jan Feb Mar AprMay Jun Jul Aug SepOct Nov months Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov months 60 55 50 45 -; >40 00 35 - 30 25 20 c) d) Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov months 20 Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov months 0.4 0.3 e) 0.2 0.1 - 0.0 -O.I Die Jan Feb Mar AprMay Jun Jul Aug Sep Oct Nov months Figure 2. Monthly averages and variances of scale and maturity index of razor clam, Golfo de Arauco, 1995-1996. a, MMS = Macroscopic Maturity Scale: b, Ml = Morphometric Index; c, SVI = SomaticA'alve Index; d. STI = Somatic/Total Weight Index; e, Gl = Gametic Index in the bulk of gonadal tissue without stain. of the Region VIII dtiring 1997 (Salamanca. 1997). Table 5 shows that the spring-summer period 1997 was warmer and with less saline water (on the average) than in a '"normal" year as the one that was detected in 1981 for the same area (Llancamil 1982). The winter conditions are similar among the two studies. During the years 1996 and 1997 the razor clam of Golfo de Arauco in the Region VIII, registered a cycle of annual maturity with only one spawning period between November-December cor- responding to the late spring early summer of the southern hemi- spheie and this is very similar to the razor clams of the northern hemisphere. Thus for Ensis minor (Chenu) of the Manfredonia Gulf in Italy Casavola et al. (1994) describe a cycle of annual maturity with a longer resting period between May and October. The gametogenic activity starts in December and finish in March. and spawning between these last months and April, spring of the north hemisphere. Caspar & Monteiro ( 1998) point out that Ensis siliqua (L.) from the south coast of Portugal has an annual gametic cycle with an extended inactive period from June to October, the gametogenesis activity starting in December with a maturity peak ill March. Spawning starts in this last month and shows a maxi- Reproductive Indexes for Ensis macha 679 TABIK 4. Multipk' comparison tt-sls (LSD) lor seM'ii gonadit matiirily stages (MMS) of £. macha compared with the Gametic Index (GHIl during 1997. Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Die Figure 3. Maturity index and scale of ra/.or clam. Golfo de Arauco. 1997. a, MMS = Macroscopic -Maturity Scale; b, Ml = Morfometric Index: c. GHI = Gametic Histologic Index. mum in April, which may be extended through May. They also add that the males and females have a synchronous gonadal develop- ment similar to E. iiuicha. In the Gormanstow bed of E. siliqiui in Ireland. Fahy ( 1999) found that gonadal cycle are fairly similar to the same species off the Portuguese coast, hut Ireland clams spawns later in the year. Reyes et al. (1995) found thai the largest evacuations of razor clam gametes take place at the end of September. November. February, and March in the Region X of Chile, which is in late spring and throughout summer. This difference may be related to the oceanographic conditions in the area because the fiords in the Region X are very different to the Golfo de Arauco. Urban (1996) describe an annual reproductive cycle with a short spawning season in summer for E. macha. from Chile at 36' S, very similar to our finding. However, Avellanal et al. (2002). in a study of the reproductive cycle of £. macha in the south of Chile. used a very different methodology consisting of the assigning of six stages of gonadic organization to histologic preparations to determine the reproductive cycle for this species at Tubul (Golfo de Arauco) between November of 1996 and 1997. They found that 20% of the females presented ovaries partially spawned in Febru- ary of 1997 and l()09f presented a partial spawning in March and April. Between June and July, there was a quick recovery of the ovaries and 40% of the samples presented partial spawning in August, a percentage that increased to 100% in November and December of the same year. This apparent difference between the reproductive cycle de- TABLE 3. Statistics of GHI for each state of Macroscopic Maturity Scale Maturity Stage (MMS) 2 3 4 5 6 7 T 0.40 0.37 0.00 0.00 0.00 3 0,40 0.9.5 0,011 0.00 0.00 4 0..^7 0.95 0.00 0.00 0.00 5 0.00 0.00 0.00 0.12 0.00 6 0.00 0.00 0.00 0.12 0.01 7 0.00 0,00 0.00 0.00 0.01 scribed by ,\vellanal et al. (2002) and this work, both on the same population and period, probably would be due to different methods for evaluating the state of development of the gametogenesis. In both cases, a massive spawn between November and December of 1997 is described. Other results by Avellanal et al. (2002) indicate that the spawn- ing of Ensis macha in Corral (39°50'S-73''28'W) was similar to thai one described by the same authors for Tubul. But. the cycle of this species in Ancud (4r50'S; 73°47'W). was different because the partial spawning started in January of 1997 and reached 100% in April. June, and July. Later on a recovery of the ovary was observed to reach 100% of mature individuals in December of that year. These authors also found in Tubul and Corral a positive conelation between the percentage of mature females and the chlo- rophy 11 a and a negati\e correlation among the percentage spawn- ing and the chlorophyll a. In Ancud, the percentage of mature females was highest when the temperatures were increasing and the spawning reached a maximum when the temperatures were low. According to Avellanal et al. (2002). this relations points to the important influence that temperature and the quantity of food can have in the energy balance, transfer of nutrients and other processes occurring during the gametogenesis. Finally, we conclude that the quantification method of the stage of maturity of the ovary of the razor clam, not described previ- ously, which closely reflects the gametic cycle of Ensis macha are MI and MMS. being easier, fast and of low cost. These two meth- ods have also been used to define the reproductive cycle of Tage- lus domlieii (Lamarck. 1818) (Lepez el al. 1997b) whose ovaries are also diffuse in the visceral complex and may be adapted for other similar species. TABLE 5. Comparative chart of oceanography parameters in Coliumo Bay (average conditions). (MMS) of razor clam: Golfo de .\rauc(i .lanuary -De Lcmber 1997. PlaceAVater Mass Temperature C Salinity xlO"^ GHI Averaged GHI Variance Confidence Interval Maturity Scale (MMS) Subantartic Water (SAW) Subsurface Equatorial Water (SSEW) >n.o 9^13 <34.3 >34.4 -) 0.063 0,0004 0.015 Coliumo Bay (Llancamil 1982) 3 0.07S 0.0010 0.008 Winter 12.07 32.76 4 0.078 0.0015 0.007 Spring 11.22 34.20 5 0.143 0.0075 0.019 Coliumo Bay (Salamanca 1997) 6 0,207 0.0031 0.016 Fall-Winter 12.57 33.44 7 0,262 0.0010 0.025 Spring-Summer 14.88 33.69 680 Aracena et al. ACKNOWLEDGMENTS We thank H. Moscoso for his help in fieldwork, M. Canales for help in manuscript, and Dr. J. Stuardo for the review and sugges- tions of an earlier version of this article. Financial suppon: Fondo de Investigacion Pesquera FIP 95-20A and Fondo de Fomento al Desarrollo CientiTico y Tecnologico. FONDEF D96/1095. LITERATURE CITED Aracena, O. L., A. Carmona & L. Medina. 19ySa. La navaja en la VIII Region. Documento N°ldel Proyecto FONDEF D96/1095: Desarrollo del cultivo de la navaja iEnsis macha) en la VIII Region. Universidad de Concepcion-Instituto de Fomento Pesquero. 14 pp. Aracena, O. L., A. Carmona, L. Medina & I. Lepez. 1998b. Ciclo repro- ductive de Ensis macha (Molina, 1782) en base a indices gonadicos macroscopicos y su validacion histologica. Resiimenes XVIII Congreso de Ciencias del Mar, Iquique: 141 pp. Avellanal, M. H.. E. Jaramillo, E. Clasing, P. Quijon & H Contreras. 2002. Reproductive cycle of the bivalves Ensis macha (Molina, 1782) (So- lenidae), Tagehis dombeii (Lamarck, 1818) (Solecurtidae) and Midinia eiiiilis (King. 1831 ) (Mactridae) in southern Chile. The Veliger 44:33- 44. Bayne, B. 1975. Reproduction in bivalves molluscs under environmental stress. In: J. Vemberg, editor. Physiological ecology of estuanne or- ganisms. Columbia: University of South Carolina Press, pp. 259-277. Casavola, N., E. Rizzi. G. Marazo & C. Saracino. 1994. Ciclo nproductivo e biometria di Ensis minor (Chenu) (Bivahia: Solenidael nel golfo di Manfredonia. Oehatia 11:439-449. Con, H. J. 1960. Staining Procedures. 2nd ed. Baltimore: Williams & Wilkins, pp. 67-68. Fahy, E. 1999. A new fishery for razor clams [Ensis silii/iia) on Ihe east coast of Ireland. / Shellfish Res. 18:715. Caspar, M. B. & C. C. Monteiro. 1998. Reproductive cycles of the razor clam Ensis siliqua and the clam Venus striatula off Vilamoura. South- em Portugal. J. Mar. Biol. Ass. U. K. 78:1247-1258. Lepez, I, M., O. L. Aracena, A. Carmona. A. Espinoza, L. Fuentes, J. Sanchez & A, Cerda. 1997a. Caracterizacion bioecondmica de las pesqueri'as de huepo (Ensis macha) y navajuela (rage/iis dombeii) en la VIII Region. Informe Final Proyecto F.I. P. 95-20A. Convenio U. de Concepcidn- FEREPA Bio Bio: 87 pp. Lepez, I. M., J. Sanchez, O. L. Aracena, M. A. Carmona & A. Saavedra. 1997b. Condicion reproductiva y sexual de los sUvks de navaja y navajuela en Tubal y Lirquen, VIII Region. XVII Congreso de Ciencias del Mar, Santiago. Resumen, 199 pp. Llancamil. L. A. 1982. Variacion estacional inviemo-pnmavera de temper- atura, salinidad y de oxi'geno disuelto en la Bahia Coliumo (36"32'S: 72°57'W). Tesis para optar al titulo de Biologo Marino. Universidad de Concepcidn. Fotocopiado, pp. 1-94. Lowe, D. M., M. N. Moore & B. L. Bayne. 1982, Aspects of the gamete- genesis in the marine mussel Mytihis edulis L. J. Mar. Biol. Assoc. U.K. 62:133-145. Reyes, A., N. Barahona. A. Carmona, C. Rojas. E. Arias. V. Pezo, V. Asencie & E. Lozada. 1995. Diagnostico de las Principales Pesqueri'as Nacionales Bentiinicas. Ill, IV y X Region. 1994. Informe Tecnice CORFO-IFOP. 96 pp. + Anexos. Salamanca, M. A. 1997. Serie de liempo quincenal de las condiciones oceanograficas en Bahia Coliumo. VIII Region, Chile. Informe interne, U. de Concepcidn, Mimeografiado: 14 pp -i- Tablas y Anexos. Sastry. A. N. 1968. The relationships among food, temperature, and gonad development of the bay xaWops Aequipecten irradians Lamarck. Phys- iol. Zool. 41:44-53, SERNAPESCA. 2000. Anuario estadistico de pesca. Chile: Servicio Na- cional de Pesca, Ministerio de Economia, Fomento y Reconstruccidn. 291 pp. Urban. H.J. 1996. Population dynamic of the bivalves Venus annqua. Tagelus dombeii. and Ensis macha from Chile at 36 degree S. / Shell- fish Res. 15:719-727. Journal ot Slwllflsli Rfsciiirli. Vol. 22. No. ,^. 6XI-fiSS. 2003. POPULATION GENETICS OF TWO BIVALVE SPECIES (PROTOTHACA STAMINEA AND MACOMA BALTHICA) IN PUGET SOUND, WASHINGTON MICAELA SCHNITZLER PARKER.'* PETER A. JUMARS.' AND LARRY L. LECLAIR' University of Wusliington School of Oceanogiapliy. Cumpiis Box 357940. Seattle. Washington 9S 195-7940; 'Darling Marine Center, University of Maine. 193 Clark's Cove Road. Walpolc. Maine 04573: and ^Washington Department of Fish and Wildlife. 600 Capitol Wax North. Olxmpia. Washington 98501-1091. U.S.A. ABSTRACT Allo/yme polymorphlsm.s from individuals ol Prototkuca slaminea and Mmoma balrliica were examined electropho- retically and scored at five loci. Both species were sampled at three sites located in different hydrologically defined basins of Puget Sound, Washington. Highly significant differences in allele frequencies among the three P. siumineci populations were found at all five loci. Significant differences in allele frequencies were detected consistently at only one locus among the M. hcilthica populations. Genetic distances between the three P. smminea populations, determined using both Cavalli-Sforza and Edwards ( 1967) chord distance and Nei's (1972) genetic distance measures, revealed the South Sound population as the genetic outlier. This pattern is consistent with the hydrology of the Puget Sound basins and the mixing that occurs at the sills between basins. Two to four of the allozyme loci demonstrated heterozygote deficiencies in P. .stamiiieci. depending on population. Only one locus exhibited a heterozygote deficiency in each of the three M Iniltliicu populations. Potential contributing factors to the heterozygote deficiencies include a temporal Wahlund effect, selection, and null alleles. When data were corrected for the presence of a putative null allele, conclusions about population differentiation did not change. KEY WORDS: population, genetics, allozymes, bivalves, Pioloiluua stumiiwa. Puget Sound, Macoina hallliica INTRODUCTION Early genetic studies of marine populations found little evi- dence for genetic differentiation over large geographic distances. It was generally believed that open aquatic environments permit ex- tensive dispersal of planktonic larvae, i-esulting in little genetic heterogeneity over wide spatial scales (e.g., Buroker et al. 1979, Crisp 1978. Gooch et al. 1972). This notion was soon challenged, however, by several studies presenting compelling evidence for population structure even along open coastlines (Scheltema 1975, Burton 1983). Increasingly, studies now find that any number of factors can contribute to population differentiation in apparently open systems. Populations may be defined not only by their re- productive mode (Hellberg 1996), but by hydrological forcing (Reeb & Avise 1990), chemical gradients (Koehn et al. 1976. Ma et al. 2000), or changes in source populations (Kordos & Burton 1993), Population subdivision is evident even among the bivalves, whose long-lived planktonic larvae might otherwise be equated with high dispersal potential (e.g., Mariani et al. 2002). Other examples of genetic differentiation in marine populations over both small and large spatial scales are reviewed in Shaklee and Bentzen (1998). Collectively, these studies demonstrate that re- productive and dispersal strategies are not the only determinants of genetic differentiation among marine populations. In this study, we examined the potential for hydrological forc- ing to promote differentiation of broadcast spawners with plank- totrophic larvae in a small estuarine system, Puget Sound, Wash- ington, is a fjord-like estuary composed of five contiguous basins with constrictions and sills that strongly influence the tidally- driven currents. The basins fall into two categories: well-mixed with rapidly circulating water masses (Admiralty Inlet, Main ba- sin, and Southern basin), or stratified with slow-moving water masses (Hood Canal and Whidbey basin; Fig. 1 ). Ebbesmeyer et al. ( 1988) proposed that as much as 50% of the water in each basin ■■"Corresponding author. E-mail: micaela@u. washington.edu is recirculated back into the basin of origin because of intense mixing at the sills. This recirculation includes the upper layer of the water column (10-30 in deep) where planktonic larvae of marine invertebrates are commonly found, possibly leading to par- tial restriction of larvae to their basin of origin. Such a barrier to dispersal could create genetically differentiated subpopulations among basins. Fevi' population genetic studies of marine invertebrates have been conducted in Puget Sound despite the presence of many managed commercial and recreational fisheries. Grant and Utter (1988) examined allele frequencies from two polymorphic loci in the intertidal gastropod NiicclUi [Thais ] lamellosu at several sites within Puget Sound, adjacent waters and along the open coasts of Oregon and Washington. They found evidence for population sub- division at various geographic scales, however the differences were attributed primarily to the nonplanktonic life history of this species. A more limited study in Puget Sound involving a species with a planktonic larval stage, the bivalve Saxidomus giganteus. found a geographic dine in populations in one of the two allozyme loci examined (Johnson & Utter 1973). Unfortunately, this study did not investigate any differences that might be attributed to sepa- ration by the hydrologically defined basins. Our objective in this study was to test whether the bivalves Protothaca staminca (Conrad) and Macoiiui balihica (L.) exhibit evidence of genetic differentiation in Puget Sound consistent with its unique hydrology. Both species broadcast spawn between April and September with planktotrophic larvae that feed for weeks prior to settlement. Given the long planktonic larval phase of the two species and the small length scale of Puget Sound (on the order of 130 km), one might expect genetic homogeneity in the absence of any physical baniers to dispersal. Differentiation of the popula- tions inight suggest that recirculation of water masses at the sills contributes to partial isolation of populations in Puget Sound. To determine whether the hydrology of Puget Sound is the principle mechanism for any observed differentiation we chose two species that share similar reproductive and dispersal strategies yet have 681 682 Parker et al. '^^^VvSkagit Bay -, Wnidbey Basin Admiralty Inlei f'l ) -''^ "■ ^ r' Edmonds / \J. \ ^s^-j"^ ,■ Ma/r. Basin Priest PtTo""*e } V Figure 1. Sampling sites for Prnlnlhaca slamiiiea (Edmonds. Potlatch, Priest Pt.) and Maconui hallhica (Skagit. Potlatch. Tolmiel in Puget Sound, Washington. disparate adult characteristics. Prointlnicti staininea occurs from the Aleutian Islands of Alaska to Baja California; is a suspension feeder preferring coarse sand to gravel substrate; attains a maxi- mum valve length of around 7 cm; and is preyed upon primarily by starfish, moonsnails, and octopuses. Macoma balthica. conversely, is circumboreally distributed; may switch between surface-deposit and suspension feeding; prefers muddy substrate; may inhabit brackish waters; in Puget Sound, rarely exceeds 2 cm in length; and is preyed upon primarily by flounder, crabs and sea birds. By examining two species with similar reproductive and dispersal strategies but with different adult characteristics, we hoped to as- sess the influence hydrology may have on population distributions of different species in this estuary. We examined allozyme polymorphisms at five presumptive gene loci in each of the two species of intertidal bivalve clanis: Protothaca staininea and Macoma balthica. Genotype and allele frequencies from each species were then compared among three of the hydrologically defined basins of Puget Sound, Washington. METHODS Field Sampling Protothaca staininea were collected at Potlatch (Hood Canal Basin; /; = 94), Priest Point (Southern Basin; /; = 114). and Edmonds (Main Basin; ;; = 114) between March 1 and Sept. 7. 1998. Macoma balthica were collected from Potlatch (n = 113). Tolmie Park at Big Slough (Southern Basin; n = 1 16), and Skagit Bay (Whidbey Basin; n = 132) between March 2, 1998 and June 29, 1999 (Fig. 1). All samples were obtained during low low tide along 100- to 500-m transects running parallel to the shore. Care was taken to sample individuals from the full extent of their range in the intertidal zone as well as across size classes. The length of the right valve of P. staininea specimens sampled ranged from 9 mm to 57 mm and for M. hallhica from 4.3 min to 17 mm. M. balthica specimens included the white, pale pink, and dark pink color morphologies. The clams were transferred live in ambient seawater to the laboratory. Immediately upon arrival, foot muscle, ctenidium, digestive gland, mantle, and adductor muscle were dis- sected from each P. staininea. The tissues from each clam were then combined in a single test tube. Because of the small size of the Macoma clams, they were stored whole (minus shell) in indi\ idual test tubes. All samples were stored at -80''C for subsequent elec- trophoretic analysis. Electrophoresis Following the methods of LeClair and Phelps (1994), tissue samples were homogenized in TC-1 gel buffer (Shaw & Prasad 1970) and centrifuged at 1.000 g for 5 min. Supernatants were absorbed with filter-paper wicks (Schleicher & Schuell no. 470) and used for starch gel electrophoresis. Details of the electropho- retic method are described in Aebersold et al. (1987) and Harris & Hopkinson ( 1976). Gels were run in a refrigerator at 8°C. Enzyme and gene nomenclature follow the guidelines of Shaklee et al. ( 1990). Both species were assayed for allozyme polymorphisms on four different buffer systems: CAME 6.8 (LeClair & Phelps 1994, modified from Clayton & Tretiak 1972); LiOH-RW (Ridgeway et al. 1970). TRIS-GLY (Holmes & Masters 1970); and TC-4 (buffer "a" of Schaal & Anderson 1974). The following enzyme/buffer combinations were tested: aspartate aminotransferase (AAT), isocitrate dehydrogenase (IDHP), malate dehydrogenase (MDH). malic enzyme (MEP). phosphogluconate dehydrogenase (PGDH). and phosphoglycerate kinase (PGK) on CAME 6.8; esterase-D (ESTD). formaldehyde dehydrogenase (FDHG). nucleoside- triphosphate pyrophosphatase (NTP). octopine dehydrogenase (OPDH). and strombine dehydrogenase (STDH) on LiOH-RW; alanine aminotransferase (ALAT). arginine kinase (ARGK). ESTD. glucose-6-phosphate isomerase (GPI). lactate dehydroge- nase (LDH). mannose-6-phosphate dehydrogenase (MPI). cytosol nonspecific dipeptidase (PEPA). tripeptide aminopeptidase (PEPB). peptidase-S (PEPS), phosphoglucomutase (PGM). STDH. and triose-phosphate isomerase (TPI) on TRIS-GLY; adenosine deaminase (ADA), aconitate hydratase (AH), glyceraldehyde-3- phosphate dehydrogenase (GAPDH). PEPA. and proline dipepti- dase (PEPD) on TC-4. Of the 25 enzymes assayed, activity of six (AAT. ESTD. GPI. IDHP. PGDH, PGM) were well resolved and indicated encoding by polymorphic loci (more than one allelic form detected). These enzymes were subsequently screened in all clams except AAT. which was screened only in P. staininea. and IDHP, which was screened only in M. balthica. Allelic variants are designated by their electrophoretic mobility relative to the most frequent variant encountered during the initial screening. Variants preceded by a minus sign indicate cathodal migration. Data .Analysis The population genetics software GENEPOP version 1.2 (Ray- mond & Rousset 1995a) was used to run analyses of population differentiation and heterozygote deficiency or excess relative to Hardy-Weinberg equilibrium. For testing population differentia- tion, both "genie" and "genotypic" tests were run. The genie test is used to determine whether allelic distributions are identical across populations. Contingency tables for each locus were tested using the R X C Fisher test to arrive at an unbiased estimate of the P value (Raymond & Rousset 1995b). The genotypic test is used to determine whether genotypic distributions are identical across populations. Although less powerful, the genotypic test is more appropriate when alleles within individuals are not independent, which may occur when there is nonrandom mating (Goudet et al. PopuLATKJN Genetics of Bivalves 683 19%). For this test, an unbiased estimate of the P value is achieved by using the G-based test (Goudet et al. 1996) on contingency tables for each locus. Tests for both heterozygote deficiency and excess are concerned with the same H,,, random union of gametes. For both tests, the unbiased P value was estimated using the score test (U test: Rousset & Raymond 1995). Because of the presence of rare alleles, defined as having frequencies <0.005 (Hartl & Clark 1997). the exact tests used by GENEPOP are more appro- priate than the comnmnly used x" test because the results will not be biased b> rare alleles (Guo & Thompson 1992). Expected het- erozygosities (//g), fixation indices (F,s) and the extent of popu- lation divergence (F^-^) were also calculated for each locus in each population using GENEPOP. The f-statistics used by GENEPOP follow Weir & Cockerham (1984). GENEPOP was also used to test for genotypic linkage disequilibria. The program BIOSYS-1 (Swofford & Selander 1981) was used to determine Cavalli-Sforza and Edwards (1967) chord distances and Nei's (1972) genetic distances. Finally, when individuals without a banding pattern are observed, yet are not conclusively null homozygotes. the fre- quency of a putative null allele can be estimated using (H^, - Ho)KH^ + H(,). where Wj. and //<, refer to the expected and ob- served heterozygosities, respectively (Brooktleld 1996). Using this algorithm allele frequencies for the populations of both species were corrected for the presence of a null allele. RESULTS Stains for GPI and PG.M were most successful on the Tris-Gly buffer .system: PGDH. AAT. and IDHP on CAME 6.8: ESTD on LiOH-RW. In each species, two private alleles (alleles only de- tected in one population) were found: GPI*- 1 7 (P. staminea. Pot- latch). AAT*-1500 {P. staminea. Edmonds). IDHP*I50 (M. bal- thica. Skagit). PGDH* 1 14 (M. hallhica. Skagit). Four rare alleles occurred in P. skimiiwa populations and eight in M. balthica popu- lations (Table 1 ). Expected heterozygosities (//p) and fixation indices {F^^) var- ied widely in both species depending on the locus (Table 1 ). No- tably. f,s values for the P. staminea population at Edmonds were consistently higher than values for the population at Potlatch or. with most loci, at Priest Point suggesting strong heterozygote de- ficiencies in this population. The tests for Hardy-Weinberg equi- librium revealed significant heterozygote deficiencies (P < O.O.S) in up to four of the five loci in the P. staminea populations (Table 2). Only at the ESTD* locus was a significant heterozygote deficiency detected in the M. balthica populations (P < 0.001; Table 2). In neither species was a heterozygote excess detected. For both species, locus pairs were also tested for genotypic linkage disequilibrium within each population. A significant link- age disequilibrium suggests the genotypes at different loci are not independent. Linked loci may be an indication of inbreeding. After applying a sequential Bonferroni correction (Ury. 1976). only one population (P. staminea. Edmonds) had loci with significant link- age disequilibria. The two locus pairs demonstrating a significant disequilibrium were: GPI* and AAT* (P < 0.001 ) and AAT* and £5rD-2*(P< 0.005). With both the genie and genotypic tests, we found strong evi- dence for population differentiation among all three P. staminea populations at all loci (P < 0.001; Table 3). Both chord and Nei's distances indicated that the populations from Edmonds and Pot- latch are more closely related than either is to the Priest Point population (Table 4). When distances were determined locus by locus, four of five loci were in agreement with this pattern. Fg^ \ alues for the P. staminea populations ranged from 0.07 {PGM*) to 0.13 {ESTD*). In the M. balthica populations, both the genie and genotypic tests demonstrated differentiation at one of the five loci {PGDH* Table 3). The genie test revealed an additional differentiation at the ESTD* locus (Table 3). Ff^^ values for M. balthica ranged from -0.002 {PGM*) to 0.009 {ESTD*). Because of the lack of differ- entiation among M. balthica populations at most loci, distance measures were not significant (data not shown). To determine whether heterozygote deficiencies had any effect on the population differentiation tests, the allele frequencies were recalculated to account for the potential presence of a null allele. An indication of null alleles is a null homozygote demonstrating no banding pattern. In the P. staminea samples, absence of enzy- matic activity occurred with only one individual from Priest Pt. when stained for GPI and two individuals from Edmonds when stained for ESTD and PGM. In the M. balthica samples, absence of enzymatic activity occurred in three individuals from Skagit Bay (all using the stain for IDHP. one additionally did not stain for ESTD) and four individuals from Potlatch (all using the stain for ESTD. one additionally did not stain for PGDH). Because this absence of activity could also have been caused by tissue degra- dation, staining inconsistencies, or tissue samples that are too small (for M. balthica). we could not conclusively assign these individuals as null homozygotes. It is possible to estimate the frequency of a putative null allele based on the heterozygote de- ficiency in a population. Following Brookfield (1996). allele fre- quencies were corrected in each population to account for the presence of a null allele and the genie and genotypic tests re-run. The level of population differentiation observed did not decline for either species. On the contrary, both the chord and Nei's genetic distances increased slightly with the addition of the null allele (between I and 30% increase, data not shown). DISCUSSION Evidence for Distinct Populatiinis of P. staminea But Mot M. balthica Both Piotothaca staminea and Macoma balthica are free- spawning bivalves, with feeding larvae that spend about 3—4 wk in the plankton. These larvae are the dispersal propagules. largely at the mercy of local horizontal currents. Given the similar reproduc- tive and dispersal strategies of P. staminea and M. balthica. one might expect consistency in the level of population differentiation of these species when exposed to the same estuarine currents. The population structure of these two species, however, is very differ- ent in the complex estuarine system of Puget Sound. Washington. Populations of P. .staminea were found to be highly differentiated at all loci surveyed, whereas the M. balthica populations were significantly different at only one locus using both the genie and genotypic tests. While it is possible that allozymes are not variable enough to detect differences between the populations of M. bal- thica. it is likely that .species-specific selective pressures also play a role in structuring these populations. Piotothaca staminea and Macoma balthica occupy very differ- ent ecological niches. It is possible that these two species experi- ence different selective pressures in Puget Sound from the physical environment or from local predators, including humans (van der Veer et al. 1998. Ejdung & Elnigren 199S. Chew & Ma 1987). P. 684 Parker et al. TABLE L Allele frequencies at loci for Protothaca slaminea and Macoma balthica individuals from three locations in Puget Sound, \VA Locus, allele Prololhaca slaminea IjOcus. Macimia balthica Potlatch Edmonds Priest Pt. allele Potlach Skagit Tolmie CPl -17 0.011 0.000 0.000 -22 0.004* 0.004* 0.000 14 0.074 0.039 0.138 8 0.009 0.019 0.022 36 0.420 0.237 0.170 38 0.434 0.481 0.457 58 0.35 1 0.202 0.589 66 0.128 0.092 0.116 11 0.112 0.167 0.085 100 0.376 0.385 0.353 100 0.032 0.285 0.013 130 0.049 0.019 0.052 127 0.000 0.070 0.004* (N) (113) (130) (116) (N) (94) (114) (112) «E 0.654 0.614 0.652 H^ 0.685 0.791 0.600 f.s -0.014 -0.027 -0.097 F,s 0.177 0.358 -0.012 PGM 66 0.101 0.108 0.090 38 0.018 0.008 0.030 85 0.261 0.171 0.232 62 0.159 0.129 0.156 100 0.314 0.230 0.602 86 0.053 0.057 0.065 ii: 0.245 0.387 0.076 100 0.611 0.621 0.593 132 0.080 0.104 0.000 124 0.124 0.159 0.113 (N) (94) (111) (105) 154 0.035 0.027 0.043 «E 0.761 0.750 0.574 (N) (113) (132) (115) f.s 0.148 0.267 0.238 «E 0.585 0.570 0.609 fis 0.107 0.097 0.000 AAT -1500 0.000 0.004* 0.000 70 0.004* 0.000 0.004* -700 0.293 0.180 0.009 82 0.062 0.036 0,039 -100 0.670 0.798 0.947 94 0.013 0.008 0,000 500 0.021 0.004* 0.044 100 0.903 0.933 0,953 900 0,016 0.013 0.000 124 0,018 0.020 0.004^^' (N) (94) (114) (114) 150 0.000 0.004* 0.000 Hb 0.467 0.332 0.101 (N) (113) (126) (116) f.s 0.112 0.207 -0.043 He 0.182 0.129 0.091 Fis 0.124 0.017 0.152 PGDN -1100 0.048 0.024 0.253 62 0.004* 0.004* 0.000 -600 0.425 0.524 0.552 74 0.022 0.068 0.030 -100 0.495 0.423 0.155 82 0.157 0.209 0.129 300 0.011 0.014 0.041 90 0.511 0..^92 0.500 1000 0.022 0.014 0.000 100 0.305 0.316 0.341 (N) (93) (104) (97) 114 0.000 0.011 0.000 He 0.575 0.548 0.599 (N) (111) (131) (116) /^■s 0.178 0.299 0.333 He 0.625 0.700 0.619 F,s 0.063 0.052 0.025 ESTD-2 75 0.000 0.045 0.004* 90 0.081 0.116 0,078 84 0.202 0.134 0.425 100 0.720 0.633 0.759 92 0.069 0.290 0.294 106 0.199 0.251 0.164 100 0.723 0.513 0.276 (N) (105) (129) (116) 111 0.005 0.018 0.000 He 0.443 0.526 0.393 (N) (94) (112) (114) f.s 0.448 0.485 0.519 «E 0.433 0.635 0.659 f,s 0.166 0.270 0.002 N = the number of clam.s scored in each collection. Frequencies in bold indicate priva(e alleles. Asterisks (*) indicate rare alleles (frequencies <0.005). Hf = e.xpected heterozygosities; f,; = fixation index for individuals wi[hin each population. sliiiiiiiwa has a larger adult si/,e and often occupies niueh more sandy substrates than M. haltluca. .Sanche/.-Salazar et al. (1987a, 1987b) demonstrated the inllueiiee both tidal elevation and shore crabs can have on the population structure of the bivalve. Ceras- loilvniui I'l/iilc. The lecreational harvest of P. stamiiwa in Puget Sound may also contribute to selective pressures in this species. In addition, harvesting of P. staininea may reduce its effective popu- lation size (yV(.). contributing to differentiation of populations Population Genetics oi- Biv.xlves 685 TABI.K 2. Probability values for the te.st of heterozjgote dcricienc) relative to Hardy-Wcinberg expectation.s at each locus for each population Protolhaca slamiiiea Locus Macoma balthica Locus Potlach Edmonds Priest Pt. Potlach Skagit Tolmie GPI 0.096 <0.001* 0.347 GPI 0.333 0.835 0.942 PGM 0.084 <0.00l* 0.035* PGM 0.100 0.403 0.173 PGDN <0.001* 0.057 <0.001* PGDN 0.123 0.097 0.474 AAT 0.019* 0.030* 1 .000 IDHP 0.087 0.447 0. 1 49 ESTD-: 0.008* <0.00l* 0.383 ESTD <().001* <0.001* <0.001* Asterisks (*) indlcule signiricuiil hetero/ygole deliciencies (P < 0.05). through genetic drift. M. halthicci is too small to attract recreational or commercial interest and may therefore also have a much larger A'^,. Additionally, neither selective pressures nor genetic drift may be strong enough to dri\'e population differentiation of M. haltliica if there are sufficient migrants to homogenize the populations (Hartl & Clark 1997). Exchange of individuals between populations may be facili- tated by larval behavior. The planktonic larvae of many estuarinc invertebrates do not behave as strictly passive particles, instead exhibiting selective transport in horizontal currents mediated by vertical migration (Morgan 1995). Although the most extensive research has focused on crustaceans (e.g.. Sandifer 1975. Cronin 1982. Forward et al. 1995). a few studies have confirmed selective larval transport among bivalves (Wood & Hargis 1971. Manuel et al. 1997). It is possible that P. suiininea and M. balthica larvae exhibit divergent swimming behaviors that could affect their trans- port out of their respective estuarine basins of origin in Puget Sound. Unfortunately, there have not been any studies investigat- ing vertical migration behavior of P. staminea larvae. Work by Roegner (2000) suggests that the larvae of M. balthica are pas- sively distributed. However, there is evidence for selective post- metamorphic drifting of M. balthica juveniles (Beukenia & de Vlas 1989). Byssal threads attached to these post-larvae provide drag and lift allowing transport on horizontal flow. A recent study of Macoma spp. post-larval distributions in the York River estuary of the Chesapeake Bay strongly suggests that this life-history stage exerts a behavioral control over position in the water column (Gar- rison & Morgan 1999). Because byssal thread-drifting has not been demonstrated in P. staminea. one possibility is that M. balthica populations in Puget Sound are less differentiated due to selecti\e thread-drifting of the post-metamorphic juveniles. TABLE 3. Probability values for the genie and genotypic tests for population differentiation of three Prolothaat ■ilominca and three Macoma balthica populations Protolhaca staminea Macoma balthica Genie Genotypic Genie Genotypic Locus test test Locus test test GPI <0.001* <0.001* GPI 0.467 0.524 PCM <0.001* <0.001* PGM 0.589 0.681 AAT <0.()01* <0.001* IDHP 0.273 0.320 PGDN <0.001* <0.001* PGDH 0.011* 0.007* ESTD-2 <0.()01* <0.()01* ESTD 0.043* 0.140 P. staminea Populations May Be Constrained by Puget Sound Hydrology Because we found substantial differentiation among Pro- tolhaca staminea populations, we hypothesize that gene flow be- tween these populations may indeed be restricted. The chord dis- tances as well as Nei's genetic distances suggest that the popula- tions of P. staminea in Hood Canal and the Main Basin are more similar to each other than either is to the South Sound population (Table 4). Hydrology of the Puget Sound estuary supports the hypothesis of South Sound isolation. Cokelet et al. (1991 ) deter- mined that as much as 52% of the water entering Admiralty Inlet from Puget Sound is recycled back through mixing at the sill (Fig. 1). This retluxing coupled with their proximity suggests a large potential for exchange between Hood Canal and the Main Basin. Cokelet et al. ( 1991 ) also estimated that the longest residence times in Puget Sound are for waters originating in the southernmost reaches of the Sound. Populations from the South Sound and Main Basin might therefore be restricted in their ability to exchange indi\'iduals. In fact, there are two minor sills and one major sill (at Tacoma Narrows) between the Priest Point population in South Sound and the Edmonds population in the Main Basin. Recently, the slow flushing times of South Sound have been implicated in the die-off of a number of benthic species, perhaps due to pollutant retention (Ebbesmeyer et al.. 1998). It remains to be seen whether the refluxing of South Sound waters is directly preventing dis- persal of P. staminea larvae. There is. however, a correlation be- tween the observed genetic pattern and the expected circulation pattern of Puget Sound. Deviations from Hardy-Weinberg Equilibrium Heterozygote deficiencies are commonly found with a variety of molecular methods, especially in marine bivalve populations (Raymond et al. 1997. Gaffney 1994. Zouros & Foltz 1984. Singh TABLE 4. (Jenetic distance measures for the three Protolhaca staminea populations (Edni = Edmonds; Pot = Potlatch: PPt = Priest Pt.) Protolhaca staminea. all loci Edm-Pot Edm-PPt Pot-PPt CSE NEI 0.1997 0.0723 0.3141 0.1947 0.2858 0.2085 Asterisks (*) indicate significant differences [P < 0.05). CSE = Cavalli-Sforza and Edwards (19671 chord distance; NEI = Nei's distance from Nei (1972). 686 Parker et al. & Green 1984). Often, heterozygote deficiencies are indicative of reproductive isolation resulting in inbreeding. Additional causes ascribed to heterozygote deficiencies are wide ranging but may include aneuploidy. molecular imprinting, genotype-dependent spawning, selection, population mixing, null alleles, scoring bias, and tissue degradation. Aneuploidy. molecular imprinting, and ge- notype-dependent spawning have not been reported for either of these species and there is little evidence to support these phenom- ena in bivalves. Heterozygote deficiencies resulting from spatial population mixing do not seem likely either, given the large sam- pling area (100- to 500-m transects), high abundances, and long pelagic phases of these two species in Puget Sound. However, it is possible that we encountered temporal popula- tion mixing since we likely sampled over several generations by sampling over a wide range of sizes. It has been hypothesized that the chance reproductive success of free-spawners may lead to large variances in the genetic composition of each successive generation due to random drift (Hedgecock 1994). The result is a small num- ber of individuals contributing disproportionately to the next gen- eration. Sampling across these generations may lead to temporal population mixing, also know as a temporal Wahlund effect. To maintain differences between year-classes, selection and/or assor- tative mating may also be occurring (HartI & Clark 1997). Similar to Ruzzante et al. (1996). we investigated the effect of pooled age-classes on Hardy-Weinberg equilibrium by dividing the P. staminea individuals into large- and small-size classes and re- testing for heterozygote deficiencies. For all populations, the num- ber of loci with heterozygote deficiencies decreased in both size classes except one (small class, Potlatch) compared with popula- tions that had both size classes pooled (data not shown). This suggests that pooling the size classes may have contributed to the observed heterozygote deficiencies. Selection may also act to reduce the number of heterozygotes in a population. An ongoing debate in bivalve genetics is the apparent paradox between observations of hybrid vigor and heterozygote deficiencies. Individuals in both the laboratory and natural field populations demonstrate strong correlations between heterozygos- ity and fitness-related traits, e.g.. size, growth rate, and reproduc- tive capacity (Hedgecock et al. 1996, Zouros 1987). Yet field populations of many bivalve species are heterozygote deficient. One possible explanation is genotype-dependent larval mortality (Singh & Green 1984, Zouros & Foltz. 1984). Investigating the timing of the heterozygote deficit, Fairbrother and Beaumont (1993) found heterozygote deficiencies in a cohort of newly settled mussel (Mytilus edulis) spat, concluding that the loss of heterozy- gotes must have occurred during the larval stage or early settle- ment. Singh (1982) suggested that selection might act against the more heterozygous, faster-growing larvae because of their in- creased food requirements during the critical period of larval de- velopment. If plankton abundances are not high during this period. these larvae face a greater mortality. This phenomenon has yet to be investigated in either P. staminea or M. balthica. Finally, the presence of null alleles may also contribute to the observed deficiencies. It is possible that either true null alleles or artifacts, such as insufficient tissue or staining inconsistencies, caused the deficiency in the one locus (ESTD) across all Macoma halthica populations. However, heterozygote deficiencies occuiTed in most loci and in all populations of Protolhaca staminea. sug- gesting null alleles are not sufficient to explain the observed de- ficiencies in this species. For these populations, selection and in- breeding due to partial reproductive isolation could explain the deficiencies we observed. In addition, it is possible that we en- countered a temporal Wahlund effect in the P. staminea popula- tions. Importantly, when all other allele frequencies were con'ected for the presence of a null allele and the analytical tests re-run. the population differentiation conclusions did not change. CONCLUSIONS Many factors may contribute to population differentiation of marine invertebrates in Puget Sound. To prevent genetic homoge- neity over such a small geographic scale, however, selective forces must be strong, gene flow must be restricted, and/or temporal variance of the populations must be extreme. Environmental fluc- tuations can be dramatic in the estuarine ecosystem. Extremes of salinity and temperature can be found over small spatial scales. In such a heterogeneous environment, selection may take the form of both physical and biological constraints. They may act in concert to vary pressures on adult clams or the recruiting larvae. Popula- tions may vary from generation to generation simply due to pulsed recruitment or sweepstakes sampling from the previous generation. Factors that might limit gene flow between populations in this estuary include large-scale retlux via mixing at sills, larval behav- ior, or small-scale circulation patterns such as nearshore eddies. We have demonstrated a correlation between the population dif- ferentiation of P. staminea and the circulation pattern of Puget Sound warranting further study of the effects of Puget Sound hy- drology on larval dispersal. The hydrology of Puget Sound, how- ever, does not ensure differentiation in every species. In stark contrast to P. staminea. we have shown that M. luiltliica popula- tions reveal little differentiation among the same basins. The amount of differentiation between sites is highly species depen- dent, and therefore population dynamics should not be generalized based on reproductive characters alone. ACKNOWLEDGMENTS The authors thank Paul Bentzen for his insights and expertise with the population genetic analyses and Fred Utter and Tatiana Rynearson for their helpful comments in reviewing the manuscript. They also thank Cherril Bowman and Norm Switzler for assisting with the preliminary phase of the lab analysis. This work was funded by National Science Foundation Grant OCE 9617701. LITER.\TllRE CITED Aebersold. P. B.. G. A. Winaii.^. D. J. Teel. G. B. Milner & F. M. Utter. 1987. Manual for starch gel electrophoresis: a method for the detection of genetic variation. NOAA Tech. Rep. NMFS 61:19. Beukema. J. J. & J. de Vlas. 1 989. Tidal-current transport of ihread-drifting postlarval juveniles of the bivalve Macoma Inilrliica from the Wadden Sea to the North Sea. Mar. Ecol. Prog. Ser 52:19.V200. Brookfield. J. F. Y. 1996. A simple new method for estimating null allele frequency from heterozygote deficiency. Mol. Ecol. 5:453-455. Biiroker, N. E.. W. K. Hershberger & K. K. Chew. 1979. Population ge- netics of the family Ostreidae. I. Intraspecific studies of Crassosirea gigas and Succostrea comrnercialis. Mar. Biol. 54:157-169. Burton. R. S. 1983. Protein polymorphisms and genetic differentiation of marine invertebrate populations. Mar. Biol. Lett. 4:193-206. Cavalli-Sforza. L. L. & A. W. F. Edwards. 1967. Phylogenetic analysis: Models and estimation procedures. Evolution 32:550-570. Chew, K. K.. and A. P. Ma. 1987. Species profiles: life histories and en- Population Genetics of Bivalves 687 vironmental requirements of coastal fishes and invertehrates (Paelt'ie Northwest) — eomnion littleneck elam. U.S. Fish Wildlife .Service Biol Rep 82 (11.78). Clayton, J. W. & D. N. Tretiak. 1972. Amine-citrate buffers lor pH control in starch gel electrophoresis. J. Fish. Res. Board Can. 29:1 169-1 172. Cokelet. E. D.. R.J. Stewart & C. C. Ebbesmeyer. 1991. Concentrations and ages of conservative pollutants in Puget Sound. Proceedings; Puget Sound Research ■91. Seattle. WA. pp. 99-108. Crisp. D. J. 1978. Genetic consequences of different reproductne strategies in marine invertebrates. In: B. Battaglia and J. .A. Beiu-dmore. editors. Marine organisms: genetics, ecology, and evolution. New York: Ple- num Press, pp. 237-273. Cronin, T. W. 1982. Estuarine retention of lar\ae of the crab RliiihniiHi- nopeus Ininisii. Est. Coast. Shelf. Sci. 15:207-220. Ebbesmeyer, C. C. R. A. Hannan & R. J. Stewart. 1998. Po.ssible impacts of tidal retluxing on southern Puget Sound benthos. Proceedings: Puget Sound Research ■98, Seattle. WA. vol. 1, pp. 267-275. Ebbesmeyer, C. C. J. Q. Word & C. A. Barnes. 1988. Puget Sound: A fjord system homogenized with water recycled over sills by tidal mix- ing. In: B. Kjerfve. editor. Hydrodynamics of estuaries 2: Estuarine case studies. Boca Raton. FL: CRC Press. Ejdung, G. & R. Elmgren. 1998. Predation on newly settled bivalves by deposit-feeding amphipods: a Baltic Sea case study. Mar. Ecoi Proi;. Ser. 168:87-94. Fairbrother. J. E. & A. R. Beaumont. 1993. Heterozygote deficiencies in a cohort of newly settled Mytilus edulis spat. J. Mar. Biol. Assoc. U. K. 73:647-653. Forward, R. B.. Jr., R. A. Tankersley, M. C. de Vries & D. Rittschof 1993. Sensory physiology and behavior of blue crab (Callincctes sapidtis) postlarvae during horizontal transpon. Mar. Fnwli. Bcliuv. PInsioi 26:233-248. Gaffney. P. M. 1994. Heterosis and heterozygote deficiencies in marine bivalves: more light? In: A. Beaumont, editor. Genetics and evolution of aquatic organisms. London: Chapman and Hall, pp, 146-153. Garrison, L. P. & J. A. Morgan. 1999. Abundance and vertical distribution of drifting, post-larval Macoma spp. (Bivalvia: Tellinidae) in the York River, Virginia, USA. Mar. Ecol. Prog. Ser. 182:175-185. Gooch, J, L.. B. S. Smith & D. Knupp. 1972. Regional survey of gene frequencies in the mud snail Nassarius ohsoletiis. Biol. Bidl. 142:36- 48. Goudet. J.. M. Raymond, T. De Meeus & F. Rousset. 1996. Testing dif- ferentiation in diploid populations. Genetics 144:1933-1940. Grant. W. S. and F. M. Utter. 1988. Genetic heterogeneity on different geographic scales in Niicella lamellosa (Prosobranchia, Thaididae). Mahicologia 28:275-287. Guo, S. W. & E. A. Thompson. 1992. Performing the exact test of Hardy- Weinberg proportion for multiple alleles. Biometrics 48:361-372. Harris. H. & D. A. Hopkinson. 1976. Handbook of enzyme electrophoresis in human genetics. New York: Elsevier. Hartl, D. L. & A. G. Clark. 1997. Principles of population genetics. Sun- derland. MA: Sinauer Associates. Inc. Hedgecock. D. 1994. Does variance in reproductive success limit effective population sizes of marine organisms'? In: A. Beaumont, editor. Ge- netics and evolution of aquatic organisms. London: Chapman and Hall, pp. 122-134. Hedgecock. D., D. J. McGoldrick. D. T. Manahan. J. Vavra. N. Appclmans & B. L. Bayne. 1996. Quantitative and molecular genetic analyses of heterosis in bivalve molluscs. J. E.xp. Mar. Biol. Ecol. 203:49-59. Hellberg, M. E. 1996. Dependence of gene flow on geographic distance in two solitary corals with different larval dispersal capabilities. Evoliitioii 50:1167-1175. Holmes, R. S. & C.J. Masters. 1970. Epigenetic interconversions of the multiple forms of mouse liver catalase. FEBS Lett. 1 1 :45^8. Johnson. A. G. & F. M. Utter. 1973. Electrophoretic variation of adductor muscle protein and tetrazolium oxidase in the smooth Washington clam, Saxidomus giganteus (Deshayes 1839). Anini. Blood Grps. hio- chem. Genetics. 4:147-152. Koehn, R. K.. R. Milkman & J. B. Mitton. 1976. Population genetics of marine pelecypods. 4. Selection, migration and genetic differentiation in the blue mussel Mytilus edulis. Evolution 30:2-32. Kordos. L. M. & R. S, Burton. 1993. Genetic differentiation of Texas Gulf Coast populations ol the blue crab Callincctes sapidiis. Mar. Biol. 117:227-233. LeClair, L. L. & S. R. Phelps. 1994. Genetic characteristics and relation- ships of five razor clam {Siliijua panda Dixon) populations along the Pacific coast of North America. J. .Shellfish Res. 13:207-216. Ma. X. L.. Cowles. D. L. and R. L. Carter. 2000. Effect of pollution on genetic diversity in the bay mussel Mytilus gulloprovincialis and the aconi barnacle Balanus glandula. Mar. Environ. Re.i. 50:559-563. Manuel, J. L.. C. M. Pearce & R. K. O'Dor. 1997. Vertical migration for horizontal transport while avoiding predators: IL Evidence for the tidal/ diel model from two populations of scallop (Placopecten magellaniciis) veligers. 7. Plankton Res. 19:1949-1973. Mariani. S.. V. Ketmaier c& E. de Matthaeis. 2002. Genetic structuring and gene tlow in Ceraslodernia glaucum (Bivalvia: Cardiidae): evidence from allozyme variation at different geographic scales. Man Biol. 140: 687-697. Morgan. S. G. 1995. Life and death in the plankton: larval mortality and adaptation. In: L. McEdward. editor. Ecology of marine invertebrate larvae. Boca Raton: CRC Press, pp. 279-322. Nei. M. 1972. Genetic distance between populations. Am. Nat. 106:283- 292. Raymond. M. & F. Rousset. 1995a. GENEPOP (version 1.2): population genetics software for exact tests and ecumenicism. J. Heredity 86:248- 249. Raymond, M. & F. Rousset. 1995b. An exact test for population differen- tiation. Evolution 49:1280-1283. Raymond. M.. R. L. Vaanto, F. Thomas. F. Rousset, T. de Meeus & F. Renaud. 1997. Heterozygote deficiency in the mussel Mytilus edulis species complex revisited. Mar. Ecol. Prog. Ser. 156:225-237. Reeb. C. A. & J. C. Avise. 1990. A genetic discontinuity in a continuously distributed species: Mitochondrial DNA in the American oyster. Cras- sostrea virginica. Genetics 124:397^06. Ridgeway, G. J., S. W. Sherburne & R. D. Lewis. 1970. Polymorphisms in the esterases of Atlantic herring. Trans. Am. Fish. Sac. 99:147-151. Roegner. G. C. 2000. Transport of molluscan larvae through a shallow estuary. J. Plankton Res. 22:1779-1800. Rousset, F. & M. Raymond. 1995. Testing heterozygote excess and defi- ciency. Genetics 140:1413-1419. Ruzzante, D. E.. C. T. Taggart & D. Cook. 1996. Spatial and temporal variation in the genetic composition of a larval cod {Gadus inorhua) aggregation: cohort contribution and genetic stability. Can. J. Fish. Aqiiat. Sci. 53:2695-2705. Sanchez-Salazar, M. E.. C. L. Griffiths & R, Seed. 1987a. The effect of size and temperature on the predation of cockles Cerastoderinu edide (L.) by the shore crab Curciiiiis maenas (L.). J. Exp. Mar. Biol. Ecol. 111:181-193. Sanchez-Salazar, M. E.. C. = L. Griffiths & R. Seed. I987h. The interac- tive roles of predation and tidal elevation in structuring populations of the edible cockle. Cerastoderma ediile. Estiiar. Coast. Shelf. Sci. 25: 243-260. Sandifer, P, A. 1975. The role of pelagic larvae in recruitment to popula- tions of adult decapod crustaceans on the York River estuary and adjacent lower Chesapeake Bay. Virginia. Est. Coast. Mar. Sci. 3:269- 279. Schaal, B. A. & W. W. Anderson. 1974. An outline of techniques for starch gel electrophoresis of enzymes from the American oyster Crassostrea virginica Gmelin. Tech Rep Georgia Mar Sci Center. 74-3. 1 8 pp. Scheltema, R. S. 1975. Relationship of larval dispersal, gene-flow and natural selection to geographic variation of benthic invertebrates in estuaries and along coastal regions. In: L. E. Cronin. ed. Estuarine research. New York: Academic Press. Inc.. pp. 372-391. Shaklee. J. B. & P. Bentzen. 1998. Genetic identification of stocks of marine fish and shellfish. Bull. Mar. Sci. 62:389-621. 688 Parker et al. Shaklee. J. B., F. W. Allendorf. D. C. Morizot & G. S. Whitt. 1990. Gene nomenclature for protein-coding loci in fish. Tmns. Am. Fish. Soc. 119:2-15. Shaw. C. R. & R. Prasad. 1970. Starch gel electrophoresis of enzymes - A compilation of recipes. Biochem. Genet. 4:297-320. Singh, S. M. 1982. Enzyme heterozygosity associated with growth at dif- ferent developmental stages in oysters. Can. J. Genet. Cytol. 24:451- 458. Singh. S. M. & R. H. Green. 1984. Excess of allozyme homozygosity in marine molluscs and its possible biological significance. Malacologiu 25:569-581. Swofford, D. L. & R. B. Selander. 1981. BIOSYS-1: a FORTRAN pro- gram for the comprehensive analysis of electrophoretic data in popu- lation genetics and systematics. J. Heredity 72:281-283. Ury. H. K. 1976. A comparison of four procedures for multiple compari- sons among means (pairwise contrasts) for arbitrary sample sizes. Technometrics 18:89-97. Van der Veer, H. W., R. J. Feller. A. Weber & J. I. Witte. 1998. Importance of predation by crustaceans upon bivalve spat in the intertidal zone of the Dutch Wadden Sea as revealed by immunological assays of gut contents. J. Exp. Men: Biol. Ecol. 231:139-157. Weir. B. S. & C. C. Cockerham. 1984. Estimating F-statistics for the analy- sis of population structure. Evolution 38:1358-1370. Wood, L. & W. J. Hargis, Jr. 1971. Transport of bivalve larvae in a tidal estuary. Fourth European Marine Biology Symposium, Bangor, Wales. Zouros, E. 1987. On the relation between heterozygosity and heterosis: an evaluation of the evidence from marine moUusks. In: C. L. Markert. editor. Isozymes: Current topics in biological and medical research. New York: Alan R. Liss, Inc.. pp. 255-270. Zouros, E. & D. W. Foltz. 1984. Possible explanations of heterozygote deficiency in bivalve molluscs. Malacologia 25:583-591. Jmirmil oj Shcttfish Hcscanh. Vol. 22. No. 3. bii9~Mb. 2()()3. SHELL REPAIR OF MECHANICALLY INDUCED FRACTURES IN MERCENARIA MERCENARIA UNDER EXPERIMENTALLY SUBOPTIMUM CONDITIONS RICHARD R. ALEXANDER' AND ROBERT M. BARON' Department of Geological and Marine Sciences. Rider University. Lawrenceville. New Jersey: and -/nsitute of Marine and Coastal Studies. NOVA Southeastern University. Fort Lauderdale. Flmida ABSTRACT Sixty hand-tonged, harvested specimens of Mencnariii nierceiuirici from wild stocl< in Raritan Bay. New Jersey, measuring 34 to 43 mm in dorsal venlrul length, were apportioned among buckets of sediments submerged in predator-e.xcluded flow -through tanks. Experimental sediments simulate substrata found native to hard clams and included: ( 1 ) well-sorted sand. 1 2) pure mud. (3l. an admixture of equal volume of shell-free sand and Jiiud. (4) an admixture of 759c sand and 25'7f .shell hash, and (5) an admixture of ISVc mud and 25'y'r shell hash. Hand-excavated clams reburrowed monthly for one year. Progressively dysoxic interstitial pore water beneath the sediment interface mediated burrowing conditions. Shells of live specimens in progressively blackened sands became chalky in appearance with ornamentation completely abraded and/or etched away. Upon sacrifice, 30 (50%) specimens revealed fractures in the valve interior that radiated from the ventral (24), posterior (four), and anterior (two) margins, whereas only five of 36 (\49c) specimens in the unburrowed "control" group showed anthropogenically (harvesting and machine-sorting) induced microfractures at the ventral margin. Mean annual dorsal-ventral shell accretion was negligible under these experimentallv suboptimal conditions. Distribution of fractured specimens among the five experimental substrata is statistically random, although, paradoxically, more clams that reburrowed in mud than sand-shell hash had internally repaired valves. Severity of fractures is evidenced by stuccoed cracks that encroached within a cm of the dorsal hinge and others that bifurcated and deflected though the adductor inuscle scars. Converged fractures in one rebunowed specimen removed a large triangular wedge of shell that proved lethal. Nevertheless, repaired fractures did not fail under the strain of repeated re-burrowing. KEY WORDS: Mcrct'imria menenoria. burrowing, fracture, repair, abrasion INTRODUCTION Lethal and sublethal shell fractures in Mercenaria mercenaria have been primarily atttibuted to durophagous predators. The toll these molluscivores inflict on this commercially valuable species has been reviewed by Krauter (2001 ). Dredging activity also may sublethally fracture shells of commercially valuable clams as ob- served in commercially harvested Glyeymeris s^lycymeris (Ramsey et al. 2000). Ensi.s silitjua (Caspar et al. 1994), Solen sp. (Bergman & Hup 1992). and Arctica islandica (Wilbaard & Klein 1994). Scar frequencies have been used to attempt reconstruction of the history of past shellfishing pressure. Another possible non- predatory cause of shell fracture in bivalves is burrowing, although such shell-fracturing mechanical processes have been infrequently investigated experimentally. Checa (1993) illustrated specimens of the thin-shelled deep bivalve.? Lutraria liitraria. Panopea gylcy- meris. and Soleciirtus strigalatits with scars of repaired cracks induced by reburrowing by individuals that were prone to exca- vation by winter storm waves. However, repair of burrowing-induced fractures and its fre- quency has not been documented in a shallow-burrowing, thicker- shelled, and coiTimercially harvested clam, such as Mercenaria mercenaria. The extent to which such mechanically induced frac- tures can be repaired is unreported. Appreciable abrasion of the ventral margin of M. mercenaria has been documented in trans- plant experiments (Pannella & MacClintock 1968, Rhoads & Panella 1970. Kennish 1978). Repeated burrowing may chip the commissure margin of some young adults of M. mercenaria. thereby providing a site for initiation of dorsally propagated frac- tures. Conceivably, sediment texture and cohesiveness could in- fluence both sediment loading against the valves (Checa 1993) and/or the likelihood that shell shards become occluded between the valves during the repeatedly opening and adduction of the valves. Reburrowing may provide the additional stress on valves marginally chipped by the commercial excavation, handling, and sorting processes. Raked specimens, jostling against each other in transport and sorted by conveyor-belt into bags of commercially graded sizes may bear very slightly chipped margins that could become the initiation sites of fractures if the clams are afforded an opportunity to reburrow. Conceivably, sediment texture may be causally related to frequencies of (1) anthropogenically induced microfractures that are propagated through the valve during rebur- rowing, and/or (2) burrowing-induced microfractures that are fur- ther expressed during repeated penetration of the sediment. Sedi- ment texture may also influence interstitial water chemistry medi- ated by sediment porosity and permeability. Substrates of different mean grain sizes and degree of sorting have different porosity and permeability properties. Suboptimum interstitial conditions be- neath the sediment surface where the clam bun'ows also may in- fluence both shell fracture propagation and the ability of the mantle to repair cracks. Accordingly, this investigation experimentally focuses on the repair of nonpredatory shell fractures in young adults of M. mer- cenaria that repeatedly burrow into various textured sediments. The testable, refutable null hypotheses are { 1 ) that microfractures possibly initiated by anthropogenic excavation and handling are repaired prior to or during reburrowing activity. (2) that the bur- rowing process also initiates microfractures that are repairable, (3) repaired fractures withstand the strain induced by reburrowing, (4) that no significant difference in the frequency of fractures results from reburrowing in different textured sediments, and (5) that no significant change in valve thickness and external ornamentation resulted from re-burrowing in different textured sediments. METHODS AND MATERIALS Within Raritan Bay. New Jersey-New York, commercial shell- fish beds, some situated in depths above effective storm wave base, include sediments characterized as mud, shell, gravel, sand, and sand-mud that host \arying densities of M mercenaria. To test 689 690 Alexander and Baron the effect that sediment texture has on shell abrasion, chipping, and fracture-initiation or propagation in M. mercenaria. fivel2-L buckets of sediment were submerged in each of two 690-L flow through tanks at the NOAA Laboratory at Sandy Hook. New Jer- sey, which pumps in water from Raritan Bay. Each bucket was filled with a substratum to a 14 cm depth, resulting in the sediment surface recessed about 4 cm form the top of the bucket. Enclosed substrate included one of five types of sediments and shell hash native to Raritan Bay to simulate the various substrata naturally occupied by M. mercenaria. The five sediment categories included ( I ) sieved, intertidal sand void of any gravel size grains and shell fragments. (2) pure mud. (3). an admixture of shell-free intertidal sand (50% by volume) and mud (507f by volume). (4) an admix- ture of 75% by volume of beach sand and 25% by volume of shell hash, and (5) an admixture of 75% by volume of mud and 25% by volume shell hash. Shell hash included shards of razor clams {Eiisis direcliis). blue mussels [Myliiiis cjiilis). surfclams iSpisiila solidissima), and hard clams (M. mercenaria) created by mortar and pestol. The longest dimension of any shell shard did not ex- ceed 4 mm. Admixtures of sediment types were thoroughly mixed with a trowel to homogenize the substrates. Two replicates of each substratum were created, one for each flow through tank. Sixty hand-raked, machine-sorted, specimens of /W. mercenaria obtained from a depuration plant operating in Raritan Bay were measured dorsal-ventrally ( = shell length), and perpendicular to the hinge line at the point of maximum curvature or maximum cross-sectional height ( = shell height) to the nearest 0. 1 mm by means of electronic vernier calipers. All specimens ranged from 34 to 43 mm in dorsal-ventral length. Initial scrutiny of the specimens revealed no hairline fractures expressed on the valve exteriors. A separate batch of 36 machine-sorted specimens from the depura- tion plant, measuring 34^1 mm in DV length, were held in an aquarium without sediment for four weeks and then sacrificed to determine if commercial har\esting and handling could have ini- tiated any interior fractures in the shells prior to reburrowing. Among the 60 experimental clams, six specimens were assigned to each of the 10 buckets of substrata and placed reclining on one valve in a clockwork arrangement (12. 3. 6. and 9 o'clock with two specimens at the center) on the sediment in May 1998. Acclima- tion to the conditions in the tanks occurred during the ensuing summer months. Monitoring of changes in the shell dimensions and external surface appearance commenced in October 1998 and lasted through October 1999. The flow of water discharged into each tank was maintained at nearly 20 cm/s. Discharge occurred from eight 3-mm diameter perforations along the length of 30-mm diameter pipe that jetted water into the tank. These perforations are too narrow to allow metamorphosed clam predators to enter the tanks. Nevertheless. tanks were checked monthly for incidental invasions. None were found. Water exited the tanks from two vertical oriented, overflow drains at each end of the 70-cm deep tank. Twice a month the dissolved oxygen, salinity, temperature, and pH were recorded for each tank by means of a portable hydrolab. A Marsh McBimey current meter checked the flow velocity jetting from perforations in the tube in the tank twice a month. The tanks were not dosed with any algal extract to enhance clam growth during the experi- ments. The clams were excavated by hand from their buckets monthly, and their shell length, and height recorded after any adhering sedi- ment was washed off from the valve exteriors. This procedure was followed monthly from October 1998 until October 1999 when the clams were sacrificed. No data were collected in May 1999. Dead specimens were cleaned and examined for abrasion, fracture, and repair. No specimen showed infestation with the boring sponge Cliona sp. Fractures and repairs among M. mercenaria at the end of ex- perimental interval were described and categorized as to ( I ) frac- ture expression (crack visible on interior or exterior of valve, or both). (2) valves affected by fracture (right, left, or both). (3) number of fractures per valve. (4) length of fractures. (5) fracture initiation site at, or very near the valve margin (ventral, posterior, anterior). (6) fracture propagation inward from the valve margin (diagonal, curved, right angled deflections, merging and/or bifur- cating), and (7) state of fracture repair (internally stuccoed cracks or unrepaired). A Goodness of fit test determines (1) if sublethal fractures occur randomly among specimens in different textured sediments. (2) if fractures occur randomly around the shell margin (posteriorly, ventrally. or anteriorly), and (3) if fractures propagate in a restricted pathway. A t test determined ( I ) if fractured and iinfractured specimens differ according to valve thickness at the ventral margin and (2) if repair condition (stuccoed vs. unrepaired) differs according to fracture length. Additionally, at the conclusion of the 12-month monitoring period, the valve suiface of each surviving clam was examined under magnification and the degree of shell abrasion and/or surfi- cial etching categorized according to the relief of the concentric lamellae as ( 1 1 abrasion-negligible. (2) abrasion/etching — slight; wear restricted to ventral area. (3| abrasion/etching — moderate; shiny, bare patches over central and ventral valve area, and (4) abrasion/etching — extensive; obliteration of concentric lamellae over most of valve surface area. It should be noted that abrasion and etching must be distinguished from ontogenetic changes in shell micro-ornament over the valve surface. A swath of the central valve of M. mercenaria inherently lacks micro-ornamentation in adulthood, although the entire valve surface of many juveniles to young adults possess fine concentric ribbing. RESULTS AND ANALYSIS Among the 36 "control" specimens held in an aquarium and sacrificed after 4 weeks, tlve showed microfractures radiating dor- sally from the ventral margin that were most probably induced anthropogenically during raking, transport, and/or machine- sorting. None showed any signs of repair. The pH in the experimental tanks holding the sixty specimens fluctuated from 7.0 to 8.0 during the 16 mo interval (Fig. I). The dissolved oxygen ranged from 3.4 to 6.7 mg/L o\er the same time frame (Fig. 1). Temperature changed seasonally, peaking in the summer at 25°C. and dropping to a low of 8°C in the winter months (Fig. 1 ). Salinity fluctuated (sub) parallel with temperature, ranging from a high of 28 ppt in November 98 to a low of 22 ppt in March 99 (Fig. I). Current velocity from the pipe perforations ranged between 16 cm/s and 24 cm/s over the 16-month interval (Fig. I) Of the sixty experimental specimens. 30 were fractured sub- lethally (Fig. 2A-G) and one lethally (Fig. 2H). Of those fractured sublethally. the crack was visible on the valve interior exclusively in 20 specimens. For 10 specimens, the fracture was evident on both the valve interior and, faintly, on the exterior (Fig. 2A; Table I ). In 12 specimens, the fracture occurred in both opposing valves (Table 1; Fig. 2A). Eleven specimens had a crack in the right valve only and seven had a fracture in the left valve only (Table I). Shhll Repair in Reburrowlu Hard Clams 691 — A — Temperature (deg. C) Current Velocity (cm/sec) Salinity (ppt) DO (ppm) 30 29 28 27 26 25 24 23 WI 22 at 3 21 n 20 > 19 18 — z 17 .2 16 'u 14 _u 13 o 12 '£ 11 < 10 9 8 7 6 5 4 3 - - -ffl- - - pH Q. P' t), PO Q p-o' q p' \ ffiffl^^'^^^^'ffi'^BS^ffl *-a-A 'A-6-' I I I I I I I I I I I 1 I I r Month and Year Figure I. Monthly fluctiiutions in miinitiiri-d ahlolic variables in flo» tlirouHh tanks with \l. iiurciiiaria at NOAA laboratory. Sandy Hook, New Jersey. Fracture length varied from 4 mm to 38 mm. Very long, stuccoed fractures propagated to within 5 mm of the dorsal margin of the clam (Fig. 2B). One specimen had three fractures in one valve and five specimens had two fractures in one valve. Short cracks (.'i-lO mm) that extended dorsally from or near the ventral margin to the pallial line, but not beyond, were unrepaired ( = unstuccoed; Table 1). However, cracks longer than l.'imm. extending dorsally beyond the pallial line, were repaired by the mantle (Table 1 ). Fractures were initiated at or very near the ventral margin (Fig. 2A and B) in 24 specimens, at the posterior margin (Fig. 2C) in four specimens, and the anterior margin in two specimens. Good- ness of fit test revealed that this distribution is nonrandom (Table 1 ). Thirteen fractures radiated inward on a diagonal from the ven- tral valve margin, slightly oblique to the dorsal-ventral axis (Fig. 2D; Table I ). Six fractures curved and three had sharp right angle deflections cutting through the edge of the adductor muscle scars in two instances (Fig. 2E-F; Table 1 ). Two fractures bifurcate near the center of the valve (Fig. 2G). Two cracks converge inward from the ventral margin. One convergence of cracks resulted in a lethal fracture (Fig. 2H). Thirty-six specimens (62'?^) have chipped ventral margins. Nevertheless, only 13 of the 30 fractures radiated dorsally from a chipped point on a valve margin (Fig. 3A and B). Resecretion of a small, v-shaped wedge to fill in the chipped ventral margin accoinpanied mortaring of the fracture in one specimen (Fig. 3C). Seventeen fractures became fainter between the pallial line and the ventral margin, and cannot be traced to the very edge of the shell. Although the highest frequency of sublethal shell breakage oc- curred in clams burrowing into pure mud (9 of 12), the distribution of fractures is statistically randoin (Fig. 4). Ventral margin thick- ness had no bearing on which valves fractured (Table 1 ). Fractures were just as likely to be confined to one valve as to be mirrored in both valves (Table 1). Furthermore, valve fractures occurred mostly in mud-burrowing specimens (Fig. 4). and appears to be to be independent of the degree of external valve abrasion, which is most severe in sand-shell burrowing specimens (Fig. 5). Two thirds of the specimens that bunowed in sand had the concentric lamellae obliterated on all areas of the valves (Fig. 3D), whereas 18 specimens of the 24 that burrowed into mud and mud-shell had only slight ventral abrasion (Fig. 5). Accretion along the ventral margin was suppressed under these experimental conditions. Mean annual increa.se in dorsal-ventral shell length varied from only 0.45 mm in clams kept in sand to 1.3 mm for clams kept in mud (Fig. 6). Clams reared in sand and shell-sand showed an annual decrease (<0.05 mm) in cross- sectional shell height whereas clams reared in mud and shell-mud showed an annual increase of -0.3 mm in cross-sectional shell height (Fig. 7). DISCUSSION These experiments indicate that monthly reburrowing by young adults increases the risk of either self-induced shell-breakage or the propagation of fractures induced anthropogenically. The fact that 30% of the experimental clams had fractures when sacrificed but only 14% of the control group had fractures indicates that the burrowing process is responsible for initiation and propagation for many, if not the majority of fractures. These experiments do not. however, indicate a threshold of reburrowing frequencies at which fracturing is likely to be initiated or expressed. The rate of repair of the fractures also cannot be precisely established, although repair of fractures induced by burrowing possibly occuiTed between inonthly rebunowing episodes. In a separate study, seed of M. merceiiaria 15-25 mm in dorsal-ventral length were able to resecrete 2- to 3-mm long notches beveled by a high-speed Dremel in the anterior, posterior, and ventral valve margins within 2 weeks while living caged on an intertidal flat in North Carolina (Fig. 8; Alexander c& Dietl. in preparation). Serra- tions or contiguous scallops in the valve posterior (Fig. 3E) may be lethally inflicted on young adult, intertidal M. merceiiaria by wad- ing birds (Krauter 2001). Conditions on the North Carolina mud- flat subjected to tidal flushing facilitated rapid repair with preclu- sion of predators. The shell repair processes may have been re- tarded under suboptimum laboratory conditions at Sandy Hook, New Jersey. Furthermore, internal fractures inay be stuccoed (Fig. 2) at a different rate than notches of the valve margin are filled in by resecreted shell (Fig. 8). Nevertheless, the repair of the internal fractures within one month, i.e.. between reburrowing sequences, is a realistic estimate given the much shiirter time it takes to repair notches around the valve inargin. Regardless of the timing of initiation of the fractures, or their 692 Alexander and Baron Figure 2. Expression of internal, stuccoed, sublethal fractures, and lethal breakage in valves of M. mercenaria that repeated!) reburrowed in sediment. Width of bar = one cm. A, External and internal expression of sublethal fractures in opposing valves. B, Stuccoed fracture radiating from posterior-ventral margin to within 5 mm of dorsal hinge. C, Stuccoed fracture radiating from posterior margin. D, Stuccoed Hnear fracture from ventral margin. E, Stuccoed ventral fractures that merge and dellect through edge of adductor muscle scar. F. Stuccoed fracture that makes right angle deflection through adductor muscle scar. G, Bifurcating, stuccoed fracture radiating fnmi ventral margin. H, Lethal fractures that merged, resulting in removal of large triangular piece of valve. propagation, during the months of reburrovving. these experiments complement the experimental results of Checa (1993) who dem- onstrated that reburrowing only onee fractured the valves of the deep-burrowing Solecurtus strii^akinis. However, fractures in the shallow-buiTowing M. mercenaria were not necessarily invariably induced by sediment-loading against the hardclam valve exteriors as advocated by Checa (1993) for S. sirigalatus. First, the experi- mental hard clam specimens never burrowed deeper than 10 cm (maximum sediment depth 14 cm) in contrast to the deep burrow- ing (>40 mm beneath the sediment surface), thin shelled clams studied by Checa (1993). Second, three times as many fractures are visible on the in the interior of the valve rather than the exterior of M. mercenaria. Yet all of Checa's (1993) illustrated examples show external expression of the fractures. Eschewing those speci- mens fractured by anthropogenic handling, these observations are congruent with the argument that closure of the valves on sediment grains or shell shards introduced between the valves fractured the ventral margin and valve interior of many if not most of the speci- mens. The high percentage of cracks (677f ) that did not propagate from the valve interior to be expressed on the valve exterior indi- cates that fracture propagation was halted at the annual growth increment discontinuities in the shell microstructure of M. merce- naria. The valve microstructure consists of overlapping layers of crossed lamellar aragonite (Boggild 1930) bounded by organic films (Pannella & Maclintock 1968. Rhoads & Pannella 1970. Kennish 1980). Although all but six of the fractures were initiated near the ventral margin, the fact that 1 7 of the 30 cracks did not radiate from a chipped point on the valve margins, but instead disappear within 1 to 2 mm of ventral margin, suggests that chip- ping of the margin is not invariably the progenitor of fractures. The faint expression of the fractures in the area between the pallial line and the ventral margin coincides with the thicker part of the shell relative to shell thickness dorsal to the pallial line. Fractures may have originated dorsal to the pallial line, dissipating before crack- ing the entire thicker area between the pallial line and the ventral margin. Although contrasting sediment textures did not statistically sig- nificantly differentiate the frequency of fractures among this sample of M. mercenaria (Fig. 4). the greater frequency of sub- lethal fractures among clams that burrowed in mud (nine) vs. sand (three) and shell-mud (six) is counterintuitive. If adduction of the valves upon clasts introduced between the valves during burrow- ing caused the fractures, the probability of encountering shell Shell Repair ln Reburrowed Hard Clams 693 TABLK 1. Distribuliun uiid iiiurphulogy of fractures induced b) burrowing of 60 specimens of Merceitaria merceiiaria. Mean ventral valve thickness of fractured vs. unfractured shells Mean length for stuccoed vs. unplastered shell fractures Location of fracture initiation on valve marsin Expression of Valve interior Valve exterior Both sides of fracture only = 20 only = 0 valve = 10 Fractures = Unfractured = 1.5 mm 1.5 mm Stuccoed = Unplastered = 20 mm 12 mm Posterior Anterior Ventral margin = = 4 margin = ■> margin = ■)^ Fracture-affected Right = 1 1 valves Left Both 12 Propagation of Dorsal-xentrally Dorsal-ventrally Rt. angle fracture straiizht = 5 curved = 7 deflection = 3* Merging and branching = Diagonal to dorsal-ventral axis = 13 r test; P = 0.71; Accept Ho (means are equal) ; test; P < 0.001; Reject Ho (means are unequal) Goodness of Fit; x" = 29.6 with 2 df; Reject Ho at P = 0.01 (nonrandom distribution) Goodness of Fit; x" = 20.0 with 2 df; Reject Ho at /> = 0.01 (nonrandom distribution) Goodness of Fit; x" = 1- ^''h 2 df; Accept Ho (distribution random) Goodness of Fit; x' = 10.22 with 4 df; Reject Ho at P = 0.05 (nonrandom distribution) * Two stuccoed fractures cut across muscle scar area. shards during burrowing would be highest in the sediment admix- tures with ly/c by volume shell hush. Furthermore. buiTowing in sand increased external shell abrasion, including the ventral mar- gin. (Fig. 5). but any ensuing chipping of the ventral margin did not increase the frequency of fractures propagated dorsally. As previously noted, only 13 of the 30 fractures can be traced from a chipped point on the posterior-ventral margin. One possible ex- planation is that the initial commercial excavation and handling of the specimens induced the fractures, and more specimens with microfractures were fortuitously placed on the muddy versus the sandy susbstrata. Given the probability of the low percentage (14%) of specimens with fractures induced before the bun'owing experiment commenced, based on extrapolation from the control group, it is unlikely that a preponderance of the lew clams frac- tured before commencement of the experiments were experimen- tally placed on mud. It should be noted that the interstitial water in the sand and sand-shell hash had become blackened during the experiments with accumulated fecal tnatter in the sediment interstices a few cm beneath the sediment surface before the conclusion of the experi- ments. This accumulation of organic matter occurred despite hand- tilling of these sediments each month during excavation of the specimens. Valve surfaces became slightly chalky in appearance, but if the valve skeletal microstructure was altered and mechani- Figure ,1. \ alves of A/, merceiiaria with chipped margins, resecreled val>e wedges, and degree of abrasion following miinlhjy reburrowing into sediment. \\ idth of bar = one cm. .\, Specimen with chipped ventral margin from which crack radiates dorsallv. B, \ enlrallv chipped margin with faint expression of dorsally radiating fracture. Note also abrasion of concentric micni-ornament limited to ventral margin of specimen. C, Specimen with secreted wedge at ventral margin filling In small triangular piece of shell removed bv cracks. Internally, fractures are stuccoed. D, Complete obliteration of micro-ornament on valve exterior of specimen that monthly reburrowed into sand. K, I'redator-lnduced. contiguous divots at posterior shell margin 694 Alexander and Baron Reburrowed Mercenaria mercenaria [3 Repaired Fracture; mean valve thickness = 1.5 mm H Lethal Fracture; mean valve thickness = 1.5 mm ■ Unfractured; mean valve thickness = 1.5 mm Experimental Sediment Substratum Figure 4. Frequency of fractures among specimens of M. mercenaria that reburrowed monthly in various sediment textures. Distribution Is random according to Goodness of Fit test (x" = 4.52 with 4 df). cally weakened by the change in interstitial water chemistry, it didn't facilitate the initiation of more fractures than specimens that rebuiTOvved in muds (Fig. 4). Clams that repeatedly reburrowed in mud did not show the same degree of loss of surface ornament (Fig. 5). Reburrowing in abrasive sand, accompanied by etching of the shell exterior by the interstitial water did significantly re- tard the expected annual increase in cross-sectional shell height relative to that shown by clams burrowed in mud and mud-shell hash (Fig. 7). A valve thickness threshold may exist at which shell fracture due to burrowing does not occur (Table 1 ). but it could not be unequivocally established by this investigation. All of the speci- mens in this investigation that cracked had a valve margin thick- ness along the dorsal-ventral axis of less than 2.0 mm just ventral to the pallial line. The four specimens with a ventral margin valve thickness greater than 2.0 mm did not bear fractures. This inves- tigation deliberately used similar size young adults (mean DV length 37 mm; std dev. 4 mm) to minimize ontogenetic (age) effects on experimental results. Expanded experiments should use a wide range of hard clam sizes to determine if a size threshold for burrow ing-induced fracture exists. The question can be raised as to whether young adult (30—40 min in dorsal-ventral length) hard clams show such reburrowing- induced fractures naturally in their native substrata, or if the fre- quency of repair in the experimental clams is merely an artifact of shell fatigue under suboptimum conditions in sediments in holding tanks where they reburrowed monthly. A specimen of A/, merce- naria collected from the field shows very siinilar internal fractures to Figure 3A, but this is only one individual out of .'iOO specimens re-examined from a collection analyzed for repair scars from Tuckerton NJ (Alexander & Dietl, 2001 ). Se\eral specimens have fractures similar to those in Figure 2, but they lack the stuccoed thread-like ridge o\er the crack. Without the stuccoed repair ridge, it cannot be determined if the crack occuued during the life of the clam or during its post-mortem, transportational history. Greg Dietl (personal communication) forwarded a photograph of a farm- raised hard clam from North Carolina that has an internal fracture and repair in both valves similar to Figure 2B. These anecdotal occurrences of internal, stuccoed fractures from field collections belie the high frequency of fracture and repair found in the ex- periments. The disparity suggests that the anthropogenic handling of the specimens and/or the strain induced by monthly reburrowing contributed to internal fracturing of the shell in the experiments. Regardless, these artificial experimental stresses did not pre- clude repair of the fractures by the mantle tissue. Given that many repairs were probably followed by re-burrowing episodes, the stuc- coed repair process is sufficiently strong to enable the overwhelm- ing majority of clams to repeatedly stress the valves during rebur- rowing without a repaired fracture failing lethally. Whatever the percentage of fractures induced anthropogenically before the re- burrowing experiments commenced, which based on the control group could be approximately \4'7c. or 7 specimens, the repairs withstood the repeated strain in the shell due to reburrowing as many as 1 2 times. Thirty specimens had fractures in the valves, but only one specimen fatally cracked its valves during monthly re- burrowing over a 12-month period (Fig. 2H). The dysoxic pore water beneath the sediment surface, and diminished supply of plankton flowing through the holding tanks may have contributed to the severely retarded accretionary growth (Fig. 6), but these DEGREE OF VALVE ABRASION 03 negligible Q slight - ventral [D moderate - ventral & central ■ extensive - all surface area 12 n Experimental Sediment Substratum Figure 5. Frequency of various degrees of valve abrasion for speci- mens that reburro\>ed monthly into various sediment textures. Shi I L Rfpair in Rkburrowed Hard Clams 695 Mercenaria mercenaria One Way ANOVA p < 0.079 1.4 1.2 ti t« . 4 c .2 ■a ■o c ■o 3 s c n (0 c V) E V) 1 3 T3 V E 3 E Experimental Sediment Substratum Figure 6. Mean increase (delta mm) in dorsal-ventral length (mm I of specimens of M. mercenaria that reburrowed monthly in various tex- tured sediments. **Mean increase is signillcantlv greater tha// value for sand (P = O.Oll and sand-shell (/' = ().02l according to Fisher's PSLD test. Mean ventral margin thicknesses are not signit'icantly dif- ferent among specimens reburrovving into different sediments based on analysis of variance (ANOVA; P = 0.067). Sample size = 60. suboptimum conditions did not prevent the mantle from stuccoing the fractures. This investigation on hardshell clams also shows that Checa's (1993) investigation on burrow ing-induced fractures and repair is not necessarily a phenomenon restricted to thin-valved, deep- burrowing clams, although the frequency of reburrowing necessary to fiacture the valves may be an order of magnitude higher for thick shelled clams and less likely to occur naturally in their native habitats. This investigation should prompt bivalve functional inor- phologists interested in shell biomechanics to search for internally stuccoed fractures in field surveys of shells of a variety of ven- erids, not just M. niercciniria. Just as external shell repairs in commercially valuable clams may be an indicator of shellfishing pressure (Bergman & Hup 1992, Caspar et al. 1994, Witbaard & Klein 1994, Ramsey et al. 2000), frequency of stuccoed micro- fractures expressed on the interior of valves may indicate the his- tory of both naturally and anthropogenically caused excavations and reburrowing episodes experienced by a clam population. Re- pair frequencies also may reflect the physiochemical conditions beneath the sediment surface in which commercially valuable clams reburrowed. Fracture repair may have impact on accretion- ary growth rates of hardclams yet to reach harvestable sizes. CONCLUSIONS Reburrowing into the substrata by M. mercenaria may either induce sublethal shell fractures, or further propagate fractures in- duced by anthropogenic excavation and handling processes. Tex- ture of the sediment (sand, mud. sandy mud, shelly sand, shelly mud) may not necessarily differentiate frequencies of burrowing- Mercenaria mercenaria One way ANOVA P< 0.0018 E E, £ '5 I Q) w < c' (0 0) S .35 .3 .25 .2 .15 .1 .05 0 05 T3 C (0 w TJ 3 E ■a c (S u I ■a 3 E ■o c 10 in •a 3 E Experimental Sediment Substratum Figure 7. Mean net change (delta mm) in shell height in cross- sectional, lateral profile for M. mercenaria that reburrowed monthly in different textured sediments. **Mea« value significantly greater vs. sand and sand-shell, (P < 0.01), as well as mud-sand iP = 0.025) ac- cording to Fisher's PSLD test. ##MeaH values significantly greater vs. sand, shell-sand, and mud-sand (P = or < 0.01) according to Fisher's PSLD test. Sample sizes = 60. propagated fractures. Nevertheless, adduction of valves on sedi- ment grains and shell shards can induce the strain that initiates or propagates fractures; sediment-loading against the valve exterior is not the likely culprit of fracture propagation in the shallow- bunowing Mercenaria mercenaria. Shell surface micro-ornament may be completely abraded and/or corroded away by repeated reburrowing in organic-rich sands with dysoxic pore water condi- tions beneath the sediment surface. Nevertheless, such abraded and etched shells are no more susceptible to fractures than shells of clams that repeatedly reburrowed in muds. Fractures are most often initiated at or very near the ventral margin, rather than shell posterior or anterior margin. Fractures that do not extend dorsal to the pallial line are not likely to be repaired. Fractures may extend beneath the adductor muscle and be B \ Figure 8. Resecreted shell in notched anterior (A), ventral (B), and posterior (C) margins of seed of .1/. mercenaria after 2 weeks while kept caged on a muddy sand tidal fiat near Masonboro Inlet, North Carolina. Notches created by a Dremel in early October 2001. Width of line is 2 cm. 696 Alexander and Baron repaired. Fractures are mostly likely repaired (stuccoed) between monthly burrowing episodes, given rates of shell regeneration in marginally notched specimens. These repaired fractures withstand the strain induced by repeated burrowing as evidenced by the fact that only one of 30 fractures failed lethally. ACKNOWLEDGMENTS We thank Barb Boyd and Bruce Boyd of the Marine Academy of Science and Technology of Mommouth County. New Jersey. for dredging of the sediment samples, collection of shells for cre- ation of shell hash, periodic monitoring of the abiotic conditions in the flow through tank, as well as providing access to the NOAA labora- tory at Sandy Hook. Jonathan Radcliffe and Daniela Zima. students of the MAST, assisted in the measurements of the clams and monitoinng of the abiotic conditions in the flow- through tanks. We appreciate the ciitical suggestions of Greg Dietl. which greatly improved the manu- script. Finally, thanks to the New Jersey Baymens Association for donating the hardshell clams used in these experiments. LITERATURE CITED Ale.xander. R. R. & G. P. Dietl. 2001. Shell repair frei.|uencies in Neu Jersey bivalves: A Recent baseline for tests of escalation with Tertiary mid-Atlantic congeners. Pcilnios 16:354-371. Bergman. M. J. N. & M. Hup. 1992. Direct effect of beanitrawling on macrofauna in a sandy sediment in the southern North Sea. ICES. 7. Mm: Sci. 49:5-11. Boggild, O. B. 1930. The shell structure of mollusks. Del Kaiigelifit' Danske Viedenskabemes Selskab Skrifter 9 raekke bind II. part 2. Math. Afd 9 Raekke 223- 326. Checa, A. 1993. Non-predatory shell damage in Recent deep-endobenthic bivalves from Spain. Pohieoiieogv PciUu'tHlinitilol. PnUieoccol. 100: 309-331. Caspar. M. B.. C. A. Richardson & C. C. Monteiro. 1994. The effects of dredging on shell fonnation in the razor clam Eiisis siliqiia from Bar- rinha. southern Portugal. J. Mar. Biological Assoc. U. K. 74:927-938. Kennish, M. 1 978. Effects of thermal discharge on mortality of Mercenaria mercenaria in Barnegat Bay. New Jersey. Environ. Geol. 2:223-254. Kennish. M. 1980. Shell microgrowth analysis. Mercenaria mercenaria as a type e.xample for research in population dynamics. In: D. C. Rhoads & R. Lutz. editors. Skeletal growth of aquatic organisms. Topics In geobiology. New York: Plenum, pp. 255-294 Kraeuter. J. N. 2001. Predators and predation. In: J. N. Kraeuler & M. Castagna. editors. Biology of the hard clam. Developments in aqua- culture and fisheries science. Amsterdam: Elsevier Science, pp. 441- 589 Pannella. G. & C. MacClintock. 1968. Biological and environmental rhythms reflected in molluscan shell growth. In.- D. B. Macurda. editor. Paleobiological aspects of growth and development: a symposium. Pn- leomol. Soc. Mem 2. J. Paleonlol. 42(Suppl):64-80. Ramsey. K.. M. J. Kaiser. C. A. Richardson. L. O. Veale & A. R. Brand. 2000. Can shell scars on dog cockles iCIycymeris glycymeris L.) be used as an indicator of fishing disturbance? J. Sea Res. 43:167-176. Rhoads. D. C. & G. Panella. 1970. The use of molluscan shell growth patterns in ecology and paleoecology. Lethaia 3:143-161. Witbaard, R. & R. Klein. 1994. Long-term trends on the effects of the southern North Sea beamtrawl fishery on the bivalve mollusk Arctica islandica L. (Mollusca. bivalvia). ICES. J. Mar. Sci. 51:99-105. JiniriHil ol Shell full Rcseciivli. Vol. 22, No. 3. 697-703. 2UU3. IDENTIFICATION AND INCORPORATION OF GROWTH AND SURVIVAL BOTTLENECKS IN ECONOMIC MODELS OF NORTHERN QUAHOG (HARD CLAM), MERCENARIA MERCENARIA MARICULTURE JONATHAN H. GRABOWSKI,'* SEAN P. P0VVP:RS,- ' AND MARK HOOPER^ 'University of North Carolina at Chapel Hill. Insiitiiie of Marine Sciences. Morehead City. North Carolina 2S557: 'Department of Marine Sciences. University of South Alabama, Mobile. Alabama 36688: 'Dauphin Island Sea Lab. Dauphin Island, Alabama 36528; ^Hooper Family Seafoods. Smyrna, North Carolina 28579 ABSTRACT Research ihal identifies potential bottlenecks in survival and growth penalties during the different phases of clam grow-out is necessary to iiia.\imize the profitability of clam aquaculture and reduce pressure on already threatened wild stocks along the Atlantic coast of the Eastern United States. In this study, initial planting density (489. 729. and 972 clams m"-) did not affect survival (64.8-77.5%) during the first year of clam grow-out. Clams planted at the lowest density outgrew (greater final shell length. SL, and individual clam volume) those planted at higher densities: therefore, clam growth was density dependent during the first year of grow-out. In the second experiment, size of clams (26.2, 32.5. 37.7. and 42.(.) mm SLi planted after year one did not affect survivorship (92.3-96.6%) or growth (36.2, 41.7. 45.1, and 49.2 mm SL, respectively). Evaluations of the economic feasibility of clam culture demonstrated that clams planted at intermediate densities would result in the greatest return on the initial investment. To increa.se the robustness of our economic feasibility analysis to interannual variations in clam survivorship and growth during the initial year of grow-out. we pert'ormed an identical analysis with data from an eariier study. Taken together, these studies bracket a realistic range of survivorship and growth during the initial year of clam grow-out: low survivorship and growth (this study) and high survivorship and growth (earlier study). Based on this range, the estimated expected profitability ranged from $4893 to $7717 per 100.000 seed clams. In contra.st to aquaculture of other bivalve species (e.g., oysters), our analysis demonstrates that the profitability of clam aquaculture is fairiy robust to substantial variations in market prices primarily as a result of the development of methods over the last decade that enable relatively high survivorship with moderate growth penalties. KEY' WORDS: Mfrcunariu nwicenaria. northern qiiahog. h;ud cUim. aquaculture. survivorship, growth, density dependence, eco- nomic feasibility INTRODUCTION Bivalve aquaculture holds great promise in contributing to the goal of sustainable and dependable production of seafood. Whereas aquaculture of some marine species, primarily fish and shrimp, is associated with a host of negative environmental effects (e.g.. increased biological oxygen demand as a result of fecal production (Silver! & Sowles 1996. Paez-Osuna et al. 1998. Tovar et al. 2000), habitat loss associated with construction of shoreline aquaculture facilities (Hopkins et al. 1995, Paez-Osuna 2001 ), and introduction and propagation of pathogens (HaiA-ell el al. 1999)), negative environmental effects of bottom or near-bottom culture of bivalves are relatively minor (Kaiser et al. 1998. Nay lor et al. 2000). In fact, aquaculture of bivalves may contribute positively to the local environment. Removal of phytoplankton as a result of filter feeding may improve water clarity in coastal areas, thus promoting the growth of sea grasses, which serve as essential habitat for fish and crabs. Because many coastal estuaries have experienced increased eutrophication in recent decades (Paerl et al. 1998), bivalve aquaculture could assist wild populations of filter feeders remove excess nutrient loading. Despite a relatively reliable market for northern quahog (hard clam). Mercenaria mercenaria and the minimal environmental ef- fects of bivalve aquaculture, hard clam aquaculture in many areas, including North Carolina, has yet to reach its potential (Diaby 1997). Two of the primary obstacles hindering establishment and * Corresponding author. Present address: University of Maine at Orono. Darting Marine Center. 193 Clarks Cove Road. Walpole. Maine 04573. E-mail: jgrabow@maine.edu expansion of economically viable hard clam aquaculture are ( 1 ) restrictive regulations by states and (2) low and/or unpredictable yields of clams on leases. The latter obstacle largely results from heavy predation of seed clams (Carriker 1959. Castagna & Kraeu- ter 1981. Peterson et al. 1995. Kraeuter et al. 1998), lower grovvth rates of clams associated with many practices adopted to exclude predators (Sumnierson et al. 1995. Grabowski et al, 2000). mor- tality induced by clam diseases, variation in the quality of lease sites for clam growth, and the frequency of natural perturbations (e.g.. hurricanes and floods). Further hindering the development of successful aquaculture initiatives is the relative paucity of eco- nomic feasibility models that couple relevant biological informa- tion with econoiTiic assessments. Specifically, bioeconomic mod- els that identify and incorporate major survival and/or growth bottlenecks during the entire grow-out phase while allowing for fluctuations in market price are of critical importance in develop- ing an industry that is competitive to wild harvest. Profitable clam culture requires planting clams at densities far above those found under natural conditions. If not mitigated, such aggregations of potential prey items can greatly increase predator efficiencies, resulting in severely reduced clam survival (Carriker 1959, Eldridge et al. 1976). Methods to reduce clam mortality rates have involved identifying threshold seed sizes for planting and appropriate times to plant seed clams in the field (Menzel et al. 1976. Whetstone and Eversole 1978. Manzi et al. 1986. Peterson et al. 1995. Marelli & Arnold 1996. Grabowski et al. 2000). Further reductions in predation have been achieved by planting clams in gravel, nylon-mesh bags, or cages and possibly by using biological controls (Castagna & Kraeuter 1977. Eldridge et al. 1979, Walker 1984, Bisker & Castagna 1989, Summerson et al. 1995, Kraeuter et al. 1998, Fernandez et al. 1999). Because most of these protec- 697 698 Grabowski et al. tive measures typically reduce clam growth (Grabowski et al. 2000). effective grow-out requires balancing increased sur\ivor- ship with subsequent growth penalties. In a previous study, we quantified clam growth and survivorship in bottom beds versus tented bags and determined that tented bags increased survivorship but reduced growth rates of clams (Grabowski et al. 2000). We also determined that expected additional revenue from increasing survivorship should more than compensate for potential lost rev- enue as a consequence of slower growth rates during the first year in tented bags. Eldridge et al. (19791 noted that survival rates were greater for clams planted at higher initial densities. Eldridge et al. ( 1979) also found that clams planted at higher densities can take up to an extra 12 mo to achieve legal size in South Carolina, which could jeopardize the economic feasibility of clam aquaculture. Yet it is uncertain whether increasing planting density during the first year will affect survivorship or growth (i.e.. if these processes are density dependent) enough to counterbalance associated reduc- tions in costs of clam grow-out. Aquaculture research has traditionally focused on the early stages of clam grow-out (Peterson et al. 1995). .Although clam mortality in the wild and in culture operations is typically greatest during postlarval and early juvenile life history stages, survivor- ship and growth rates of larger clams may be size or density dependent (Eldridge et al. 1979). Further empirical tests are nec- essary to determine whether the size of larger clams will affect growth rates when planted at intermediate densities. Culture stud- ies often assume mortality is extremely low after the initial stages and use estimated mortality rates for the final stages when evalu- ating the protltability of differing types of clam grow-out. Quan- tifying survivorship and growth during the later stages of clam grow-out is necessary to evaluate whether these assumptions are valid and to enhance the reliability of economic models that proj- ect the profitability of clam culture. Even if mortality is relatively minor after the initial hatchery phase, small differences in survi- vorship and growth at later stages may be critical in determining profitability under marginal market conditions. Consequently, ef- fective crop management requires identifying culture techniques during each phase of grow-out that increase revenues relative to costs. In this study, we examined potential growth penalties and/or survival bottlenecks within the first two years of clam grow-out. Included in this effort were experiments designed to quantify the relationships between seed clam planting density and growth w hen using methods that offer substantial predator protection. In par- ticular, we tested whether clam planting density during the first year of grow-out in nylon bags, a widely used predator exclusion technique in bivalve aquaculture. influences clam survivorship, individual growth, and total yield. Further, we examined whether differences in growth after one year of grow-out are propagated throughout the second year or if compensatory growth reduces size variation in older clams (Peterson 1979). Finally, results from both of these experiments were incorporated into a cost-benefit analysis designed to examine the profitability of manipulating planting den- sities within a range of empirically derived survivorship and growth conditions under varying market conditions. MATERIALS AND METHODS Experimental Grow-Out In August 2000, seed clams (4—6 mm) were obtained from Atlantic Farms, Inc.. Charleston, South Carolina, and placed into a nursery system on the premises of Hooper Family Seafood. Smyrna, North Carolina. In October 2002. seed clams were sieved on a 10 mm screen to obtain clams of mean 13.7 mm shell length (SL), with SL being the maximum measurement along the anteri- or-posterior axis. Seed clams were planted at three densities (700, 1 050, and 1 400 clams per bag ) in three sets of 1 0 nylon bags, mesh size 9.4 mm (stretch) and measuring 1.2 x 1.2 m. Each of the three sets of nylon bags corresponded to one of the three densities of clams. We planted seed clams in nylon-mesh bags because this method resulted in greater survivorship and was more viable eco- nomicallv than bottom beds (Grabowski et al. 2000). A random sample of 50 clams w as measured for SL from four of the 1 0 nylon bags of each initial density at the inception of the experiment. A one-factor ANOVA confirmed that the initial SL of the three den- sity classes was not significantly different (F,„ = 1.2: P = 0.35). Nylon bags were interdispersed randomly on North Carolina shell- fish lease 570 D in Midden's Creek, Smyrna, North Carolina. Each nylon bag was sealed with a cable tie, staked down on each comer, and raised in the center with a 30-cm-long PVC stake that pro- jected 20 cm above the substrate surface. In January of 200 1 . the center stake in each nylon bag was removed. In October of 2000, one-year-old clams grown out under simi- lar methods and at the same lease site as described in the previous paragraph were collected. Clams were graded by shell thickness using slotted graders (1.5. 1.9, 2.2, and 2.5 cm bar spacing) into four distinct size classes (small, mean SL = 26.2 mm; medium, mean SL = 32.5 mm; large, mean SL = 37.7 mm; and extra large, mean SL = 42.0 mm). We then planted six sets of 500 clams of each size class in 1.2 x 1.2 m bottom beds (24 total beds) and covered the beds with 7 mm polypropylene mesh. Random samples of 50 clams were measured for SL from three of the six bottom beds for each size class. A one-factor ANOV.A confirmed that the initial SL of the four size classes were significantly dif- ferent (F3S = 129.2; P < 0.0001). The clams were planted in shallow water (2.5 cm shell thick- ness) after one additional year of grow-out. For those clams that did not attain legal size after two years, we estimated the time to legal size by ( 1) determining which size category they grew into after the second year of growth and (2) projecting future growth by determining the proportion of two-year-old clams in each of the size classes that would grow to legal size after one or more addi- tional years of grow-out. Using this series of calculations, we were able to project the time duration of clam grow-out and the timeline of harvests for clams planted at each density during the first year of grow-out (75% legal after 48 months). Clams in North Carolina typically grow to legal size in two to four years depending on several physical and biological factors associated with grow-out location. Clams that achieved legal size in each projected year of grow-out were multiplied by a price of IS? per clam, the average market price in North Carolina for clams at or just above the legal size over 1998-2001. and discounted at an annual rate of 39r. The costs (i.e.. labor, disposable supplies, equipment, bottom- water lease, electricity, and seed clams) of planting 100.000 seeds were estimated from records of Mark Hooper's clam culture op- erations over the past half-decade. Based on informal surveys of other clam culturists in North Carolina, we are confident that Hooper's operations are representative of hard clam culture in the region. Costs of equipment such as nylon bags and bottom-bed materials were factored in under two scenarios; ( 1 ) actual, all costs incurred and (2) annualized, equipment costs projected over a five-year lifespan (i.e.. equipment would be used for future crops). To evaluate the robustness of hard clam aquaculture to fluctuation in market price, we calculated the break-even clam price at which revenues still could meet or exceed expected costs given the pro- jected streamline of clam harvests. The break-even price (P^) was calculated as follows: P.= Costs ^H*\/i\ +d)' (1) We next compared results frotii the first year of grow-out to those from our previous study (Grabowski et al. 2000) to deter- mine how variability in growth and survival in the first year of grow-out influences the profitability of clam aquaculture. In 1999. seed clams were planted at a density of 700 clams per bag using a similar range of seed sizes (Grabowski et al. 2000); therefore, we compared economic estimates derived from survivorship and growth parameters of the first year of grow-out in 1999 to tho.se in 2000 (this study). For each year, actual and annualized costs were subtracted from revenues, which were calculated using a price of 18e and a discount rate of 3%. Finally, we compared the profit- ability and break-even price of 1999 versus 2000 operations. RESULTS Experimental Grow-Out Initial planting density did not affect percent survivorship among the three density treatments (F,,, = 1.6. P = 0.23). and survivorship ranged from 71. 291- (low density) to 77.5% (medium density) and 64.8% (high density). Initial planting density did af- fect SL after one year of grow-out (Fig. I; one-factor ANOVA F2.27 = 8.7. P = 0.001). Shell length in low-density bags was significantly greater than SL for both mediuin- and high-density clam bags {P < 0.05 for both comparisons), but medium- and high-density treatments did not differ (P = 0.59). Initial planting density also influenced individual clam volume (one-factor ANOVA F-, ,7 = 6.9. P = 0.004). which was also significantly greater in low-density bags than in either medium- or high-density clam bags (P < 0.05 for both comparisons). Individual clam vol- ume for medium- and high-density clam bags did not significantly differ (P = 0.26). Finally, initial planting density did not affect the total clam volume per sample (one-factor ANOVA F, n-, = 1.5, P = 0.25). which ranged from 2034 mL/replicate (low density) to 2500 mL/replicate (medium density) and 2559 mL/replicate (high density). In the second experiment, we tested w hether clam planting size after one year of growth affects survivorship and growth during the second year of clam grow-out. Clam planting size marginally af- fected survivorship after the second year of grow-out (Fig. 2; one-factor ANOVA F, ,(, = 2.8, P = 0.06). Post hoc comparisons indicated that the small size class had significantly lower survival 28.0 - I 27.0 - £ 26.0 - ot S 25.0 .J =5 24.0 23.0 22.0 21.0 I where H, is the number of clams harvested in year i. d is the discount rate (3%), and costs are as mentioned previously. Low Medium High Initial Planting Densit>- Figure I. Final clam shell lengtii after 1 y of grow-out in nylon bags: low density, 26.6 mm; medium density, 24.3 mm; high density, 23.2 mm. Frror bars are +1 SE in = 10 for each planting density). Letters above bars signify post hoc results (different letters denote significant differences al P < 0.05, Fisher's PLSD). 700 Grabowski et al. 50 Small (26.2 mm) Medium (32.5 mm) Large (37.7 mm) Extra Large (42.0 mm) Initial Clam Size Figure 2. Clam survivorship after the second year of clam grow-out: small, 92.3%; medium, 96.5%; large, 96.6%; extra large, 95.9%. Er- ror bars are -fl SE (;i = 6 for each clam size). Letters above bars signify post hoc results (different letters denote significant differences at P < 0.05, Fisher's PLSD). than the other three size classes (P < 0.05 for all three compari- sons), and that the three larger sizes did not differ from each other {P > 0.05 for all three comparisons). Clam planting size signifi- cantly affected clam SL after the second year of grow-out (Fig. 3; one-factor ANOVA F, ,o = 160.1. P < 0.0001). The ranking of final SL was consistent with differences in initial clam planting size (P < 0.0001 for all comparisons). The results of both volume measurements were consistent with the results from the analysis of final clam size (SL). Clam planting size also influenced individual volume per surviving clam (one-factor ANOVA F, ,0 = 160.6, P < 0.0001 ) and total volume of surviving clams per replicate (one- factor ANOVA F, - 139.3, P< 0.0001). Economic Analyses Cost-benefit analysis determined that planting clams at an in- termediate density of 1050 clams per bag during the first year of grow-out resulted in the greatest projected return on the invest- ment. Clams planted at the intermediate density were 25.1% and 33.2% more profitable than clanis planted at the high ( 1400 clams per bag) and low (700 clams per bag) densities, respectively (Table 1 ). Annualizing equipment expenses over a more realistic time period of five years increased overall profits by an average of 18.9% in 2000. After annualizing equipment costs, cost-benefit analysis again determined that profits were greatest from clams planted at the intermediate density (Table I ). Under this scenario, profits from clams raised at low densities were slightly greater than profits of clams at high densities (Table I ). The break-even price ranged from I l.Otf (low) to 9.2«! (medium) on the actual expenses and 9.2v^ (low) to 7.9v; (medium) when expenses were annualized (Table I ), which was substantially lower than the price ( 18^^) used to calculate projected revenues. Projected profits after the initial year of grow-out in 1999 (high survivorship and growth) were 72.3% higher for actual expenses and 57.7% higher for annualized expenses than profits based on data from 2000 (poor survivorship and growth). a 1 Small (26.2 mm) (37.7 mm) Extra Large (42.0 mm) Initial Clam Size Figure 3. Final clam size after the second year of clam grow-out: small, .16.2 mm; medium, 41.7 mm; large, 49.2 mm; extra large, 45.1 mm. P>ror bars are -i-l SE in = 6 for each clam size). Letters above bars signify post hoc results (different letters denote significant differ- ences at P < 0.05, Fisher's PLSD). DISCUSSION Empirical assessments of clam aquaculture have attempted to identify the magnitude and scope of survival bottlenecks and growth penalties associated with differing culture methods and techniques. Unfortunately, methods that increase survivorship of- ten are associated with subsequent growth penalties (Grabowski et al. 2000). Assessment of the economic consequences of survival bottlenecks and growth penalties associated with each culture method is necessary to maximize the profitability of hard clam culture ventures and to determine the price levels where revenues exceed costs. In this study, we determined the degree to which survivorship and growth were affected by ( I ) seed planting density during the first year of grow-out and (2) size of 1-y old clams in the second year of grow-out. Both of these experiments wei'e in- corporated into cost-benefit analysis of hard clam culture to iden- tify if any reductions in survivorship or growth penalties associ- ated with planting density during the first year of grow-out would impact profitability. Although survivorship did not vary statistically with planting density, planting clams at the higher density ( 1400 clams per bag) reduced survivorship by 12.7% and 6.4%. in comparison to me- dium- (1050) and low-density (700) bags, respectively. Similar results (i.e., clam survivorship is unaffected by planting density during the first year of grow-out if clams are protected from pre- dation) have been shown by other studies (Summerson et al. 1995, Fernandez et al. 1999). Eldridge et al. (1979) found in South Carolina that seed clams (13.0 mm SL) planted at low densities experienced higher mortality rates, which they attributed to pre- dation rather than competitive exclusion. These studies suggest that if survival bottlenecks in clam culture exist as a consequence of clam density, they occur at smaller sizes and during the hatchery or nursery phases of grow-out. Furthermore, choosing an appro- priate method of grow-out that protects clams against local preda- tors might be more important to patterns of survivorship than initial planting density (Summerson et al. 1995). It is important to note that although these relatively small differences in survivor- ship rarely meet a formal statistical threshold for detecting differ- Economic Viability of Hard Clam Culture 701 TABLE 1. Kcunumic cvuluuliun of (A) pluntin;; clams at difterent dcnsitits and (H) tariatiun in the first year of grow-out ( IW) vs. 2000 results). (A) Planting Clams at Different Densities in the First Year of Grow-out (700, 1050, and 1400 per bag) Actual Annaulized" Planting! J Density:" Low ($1 Medium ($) High 1$) Low ($) Medium ($) High ($) Cosloflabor (SlO/h) 3,453 3.145 2.561 3,453 3.145 2.561 Supplies 243 195 171 243 195 171 Equipment 1.236 983 849 247 197 170 Electricity \5i) 150 150 150 150 150 Lease price 10 10 in 10 10 10 Clam seed 1,000 1.000 1.000 1,000 1,000 1.000 Total costs 6.042 5.483 4.741 5,103 4.697 4.062 Projected revenues 9,995 10.683 8,897 9,995 10.683 8.897 Net present value (NPV) 3.904 5.200 4.156 4,893 5,987 4,835 Break-even price 0.110 0.092 0.096 0.092 0.079 0.082 (B) Comparison of Projected Range of Earnings from 1999 and 2000 Data (irovvth and .Survivorship Comparison Grow-Out Year 1999 2000 Initial planting size (SL) Survivorship after 1 y Mean size (SL) after I y 13.9 mm 90. 1 '7r 32.4 mm 13.9 mm 71.2% 26.6 mm Actual 1999 ($1 2000 ($) Total costs Projected revenue'' Net present value (NPV) Break-even price 6.599 6,092 13.326 9,995 6.728 3.904 0.089 O.IIO Annualized" 1999 ($) 2000 ($1 5,610 5,103 13,326 9,995 7,717 4,893 0.076 0.092 " Annualized costs accounts for equipment expenditures that were spread over 5 y. "Clams were planted at 700 per bag in 1999; therefore, 1999 results are compared to the low density irealnient in 200(1. " Revenues were estimated at a price of $0. 18/clam and 3% annual discount rate. ences, the economic impact of these declines still affect profitabil- ity of clam culture, particularly in years of poor growth and sur- vivorship. Increasing clam density did negatively impact clam growth (both in terms of SL and volume): clams planted at the low (700 clams per bag or 489 clams m~") density grew larger than clattis planted at either of the higher ( 1 050 and 1400 clams per bag or 729 and 972 clams m"") densities. Total clam yield at the end of the first year of grow-out did not differ among the three treatments because clams at the low density grew larger than clams at either of the higher densities, thus providing further evidence that clam growth was density dependent. Eldridge et al. (1979) showed in South Carolina that seed clams planted in oyster trays protected with 9.0 mm mesh cloth at a density of 290 clams m"" were significantly larger than similar-size clams planted at 869 or 1 1.59 clams m""". Fernandez et al. ( 1999) planted larger (21.1 mm) seed clams in 10.5 mm nylon mesh bags (1.2 x 1.2 m) at Oak Hill. Florida, at densities of 520, 694, and 866 clams m"" and found no difference in SL among the density treatments after nine months of growth. However, they did find a greater proportion of legal-size clams in the low-density treatment than in the medium and high densities. Differences in findings between Fernandez et al. ( 1999) and our study could be explained by their use of a narrower density range, larger initial seed size, or protective-mesh size. More plau- sibly, differences between the quality of grow-out conditions (e.g.. physical/chemical variables, phytoplankton supply, abundance of fouling organisms) between areas used by Fernandez et al. ( 1999) and our study explain the dichotomy in findings. In areas that experience marginal growth conditions, clam cultures would ex- perience more pronounced density-dependent growth penalties (Powers & Peterson 2000). Our findings demonstrate that methods which effectively re- duce clam predation not only reduce clam growth but also result in more pronounced growth penalties at higher densities where food limitation is presumed to be more inten.se. If risk of clam kiss to theft or from hurricane damage is great, the interest rates and thus the rate of inflation are high, or \olatility in clam prices is con- siderable, growth penalties associated with planting clams at high densities may decrease overall profitability and add substantially more risk to grow-out success. Results from the second experiment suggest that survival is very high and largely independent of size during the second year of clam grow-out. Eldridge et al. (1979) also reported high survi- vorship of clams after the initial six months of grow-out. Thus, clam mortality is predominately an issue for clam growers during the nursery phase and the first year of grow-out. Differences in individual clam size and total clam yield in volume after two years of grow-out were in direct proportion to differences after one year of growth. Therefore, growth penalties resulting from culture tech- niques in year I are propagated unmodified through the additional 702 Grabowski et al. years of grow-out. The results of our study as well as that of Eldridge et al. (1979) demonstrate that investigation of the tem- poral sequence of growth penalties and survivorship can produce more precise crop management and further the profitability of aquaculture endeavors. Economic analyses suggest that planting clams at intermediate (729 clams/m~") densities should increase the profitability of clam culture operations. Therefore, the reductions in operating costs associated with planting at higher densities are initially greater than the lost revenues from growth penalties and lower clam sur- vivorship. However, increasing clam densities even further (972 clams/m"-) during the first year of growth eventually results in reduced profit margins as a consequence of survivorship and growth penalties. If market demand for clams does not meet recent increases in clam production from several southern coastal states within the eastern United States, clam prices could continue to fall and threaten the viability of aquaculture operations. Planting clams at intermediate densities should reduce the threat of clam prices dropping beneath the break-even price, which should be of great concern to potential clam growers. Given the fairly modest value of our calculated break-even price (7.6 to 9.2c per clam with annualized costs), our analysis demonstrates that profitability of clam aquaculture could be robust to substantial variations in market prices. The relatively low cost per clam produced results primarily from the development of methods over the last decade that enables relatively high surxivor- ship with moderate growth penalties. Further identification of and reductions in the magnitude of growth penalties and survival bottlenecks in clam aquaculture may lead to additional increases in the profitability of these operations. These enhancements coupled with realistic economic models for clam aquaculture operations should continue to stimulate expansion of bivalve mariculture. In turn, further expansions may indirectly reduce harvest pressure and thus provide greater opportunity for natural recovery of wild stocks. ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of B. Wood- ward. M. Dolan. D. Kimbro. A. Baukus, K. Sullivan, and R. Wa- gaman in the field. The manuscript benefited from comments pro- vided by S. E. Shumway and anonymous reviewers. Support for this research was provided by the North Carolina Fisheries Re- source Grant Program administered by the North Carolina Sea Grant College Program and by the state of North Carolina. LITERATURE CITED Bisker. R. & M. Castagna. 1989. Biological control of crab predation on hard clams Mercenaria nwrcenurki (Linnaeus 1758) by the toadfish Opsanus Ian (Linnaeus) in tray cultures. J. Shellfish Res. 8:33-36. Carriker. M. R. 1959. The role of physical and biological factors in the culture of Crassostreti and Mcncniirici in a salt-water pond. Ea>l. Monogr. 29:219-266. Castagna, M. & J. N. Kraeuter. 1977. Mcireitaria culture using stone ag- gregate for predator protection. Proc. Nut. Shellfish Assoc. 67:1-6. Castagna. M. & J. N. Kraeuter. 1981. Manual for growing the hard clam Mercenaria. VIMS special report in applied marine science and ocean engineering no. 249. 110 pp. Day, R. W. & G. P. Quinn. 1989. Comparisons of treatment^ after an analysis of variance in ecology. Ecol. Monogr. 59:433-463. Diaby. S. 1997. Economic analysis of Nonh Carolina's coastal fishing industry: A preliminary assessment tor Carteret County. North Carolina Department of Environment. Health and Natural Resources, Division of Marine Fisheries, Report #319, Morehead City, NC. Eldridge, P. J., A. G. Eversole & J. M. Whetstone. 1979. Comparative survival and growth rates of hard clams Mercenaria mercenaria. planted in trays subtidally and intertidally at varying densities in a South Carolina estuary. Proc. Nar. Shellfish Assoc. 69:30-39. Eldridge, P. J., W. Waltz, R. C. Gracy & H. H. Hunt. 1976. Growth and mortality rates of hatchery seed clams, Mercenaria mercenaria. in protected trays in waters of South Carolina. Proc. Nar. Shellfish Assoc. 66:13-20. Fernandez, E. M., J. Lin & J. Scarpa. 1999. Culture of Mercenaria mer- cenaria (Linnaeus): Effects of density, predator exclusion device, and bag inversion. J. Shellfish Res. 18:77-83. Grabowski, J. H.. S. P. Powers & M. Hooper. 2000. Balancing tradeoffs between predator protection and associated growth penalties in aqua- culture of northern quahogs, Mercenaria mercenaria {Linnaeus, 1758): A comparison of two common grow-out methods. J. Shellfish Res. 19:957-962. Harvell, C. D.. K. Kim. J. M. Burkholder. R. R. Colwell. P. R. Epstein. D. J. Grimes, E. E. Hofmann. E. K. Lipp, A. D. M. E. Osterhaus. R. M. Overstreet, J. W. Porter. G. W. Smith & G. R. Vasta. 1999. Emerging marine diseases-climate Iniks and anthropogenic factors. Science 285: 1505-1510. Hopkins. J. S.. M. R. Devoe & A. F. Holland. 1995. Environmental im- pacts of shrimp farming with special reference to the situation in the continental United States. Estuaries 18:25-42. Kaiser. M. J.. 1. Laing, S. D. Utting & G. M. Burnell. 1998. Environmental impacts of bivalve mariculture. J. Shellfish Res. 17:59-66. Kraeuter. J. N.. S. Fegley. G. E. Flimlin. Jr. & G. Mathis. 1998. The use of mesh bags for rearing northern quahog (hard clam). Mercenaria mer- cenaria. seed. J. Shellfish Rf.v. 17:205-209. Manzi, J. J., N. H. Hadley & M. B. Maddox. 1986. Seed clam. Mercenaria mercenaria. culture in an experimental-scale uptlow nursery system. .Ac/nacnlliire 54:301-311. Marelli, D. C. & W. S. Arnold. 1996. Growth and mortality of transplanted juvenile hard clams, Mercenaria mercenaria. in the Northern Indian River Lagoon, Florida. J. Shellfish Res. 15:709-713. Menzel, R. W.. E. W. Cake, M. L. Haines, R, E. Martin & L. A. Olson. 1976. Clam mariculture in nonhwest Florida: A field study of preda- tion. Proc. Nat. Shellfish As.soc. 65:59-62. Naylor. R. L., R. J. Goldburg, J. H. Primavera. N. Kautsky. M. C. M. Bevendge, J. Clay. C. Folke, J. Lubchenco. H. Mooney & M. Troell. 2000. Effect of aquaculture on world fish supplies. Nature 405:1017-1024. Paerl. H. W.. J. L. Pinckney, J. M. Fear & B. L. Peierls. 1998. Ecosystem responses to internal and watershed organic matter loading: Conse- quences for hypoxia in the eutrophying Neuse River Estuary, North Carolina. Mar. Ecol. Prog. Ser 166:17-25. Paez-Osuna. F. 2001. The environmental impact of shrimp aquaculture: Causes, effects, and mitigating alternatives. Environ. Manage. 28:131- 140. Paez-Osuna. F.. S. R. GueiTero-Galvan & A. C. Ruiz-Fernandez. 1998. The environmental impact of shrimp aquaculture and the coastal pollution in Mexico. Mar. Pollution Bull. 36:65-75. Peterson. C. H. 1979. Predation, competitive exclusion, and di\ersity in the soft-sediment benthic communities of estuaries and lagoons. In R. J. Livingston, editor. Ecological Processes in Coa.stal and Marine Sys- tems. New York: Plenum Press, pp. 233-264. Peterson, C. H., H. C. Sunimerson & J. Huber. 1995. Replenishment of hard clam stocks using hatchery seed: Combined importance of bottom type, seed size, planting season, and density. J. Shellfish Res. 14:293-300. Powers. S. P. & C. H. Peterson. 2000. Conditional density dependence: The flow trigger to expression of density-dependent emigration in hay scal- lops. Linniology anil Oceanography. 45:727-732. Economic Viabilit'i' of Hard Clam Culture 703 Silvert, W. & J. W. Sowles. 19%. Modelling environmental impacts ol marine fint'ish aquaculture. J. App. Icthyol. 12:73-81. Summerson, H. C. C. H. Peterson & M. Hooper. 1995. Aquacultural pro- duction of northern quahogs, Mercenuriu merceiiaria (Linnaeus, 1758): High water temperature.s in the nursery and growth penalties of predator control by gravel. / Shellfish Res. 14:25-31. Tovar, A., C. Moreno. M. P. Manuel-Vez & M. Garcia-Vurgas. 200(1, Environmental impacts of intensive aquaculture in marine waters. Wa- ter Res. 34:334-342. Underwood, A. J. 1981. Techniques in analysis of variance in experimental marine biology and ecology. OceiinoKi'. Mar. Biol. Ann. Rev. 19:51,^- 605. Walker, R. L. 1984. Effects of density and satiipling time on the growth of the hard clam. Mercenaria mercenaria. planted in predator-free cages in coastal Georgia. Nauliliis 98:1 14-1 19. Whetstone. J. M. & A. G. Eversole. 1978. Predation on hard clams Mer- cenaria mercenaria by mud crabs Pannpeus licrhstii. Pnx . Nat. Shell- fish Assoc. 68:42-f8. Joiinuil of SlwUthh Research. Vol. 22. No. 3, 703-709. 20(13. A STUDY OF THE NOAH'S ARK SHELL {ARCA NOAE LINNAEUS 1758) IN MALI STON BAY, ADRIATIC SEA MELITA PEHARDA.' JAKSA BOLOTIN,' NEDO VRGOC,' NENAD JASPRICA," ANA BRATOS/ AND BOSKO SKARAMUCA" Uwititutc of Occaiioi^rciphy and Fisheries. S. I. Mcstrovica 63. 21 000 Split. Croatia: -Institute of Oceanoiiraphv and Fisheries. D. Jiide 12. 20000 Dubrovnik. Croatia: ^Collesiitin Rafiiisiiuon. Cira Carica 4. 20000 Dubrovnik. Croatia ABSTRACT A Mud\ of the Noah's Arks (Aicu none) was conducted in Mah .Slon Bay. between Novemher 20111 and Novenitier 2002. Noah's Arks were collected monthly for the analysis of the condition index (CI I. and every 2 mo for biometric measurements. CI was related to seawater temperature, salinity, and chlorophyl a levels, which were measured every 2 wk. Throughout the study, 39% of the ,4. mnw were >50 mm in size. Based on length frequency distribution, a modified Von Bertalanffy growth equation was constructed: L, = 79.19 [1-e"" '■"'''"']. Using the modal sizes estimated from the length frequency distributions, the estimated population growth rates of the shell were greater than the individual growth rates estimated from shell sections. Low values for the CI were recorded m December and January, and also in the period from .luly to October. The highest condition values were recorded from April until June. KEY WORDS: Bi\alvia. Ann noae. biometrics, condition index INTRODUCTION In recent years, an increase in the collection and aqiiactillure of bivalves from the family Arcidae has occurred (Food and Agri- culture Organization 2002a. Food and Agriculture Organization 2002b). In 1991. a total of 69.700 metric tons (MT) of bivalves from the Arcidae fainily were collected from natural populations around the world, while in 2000 94,518 MT were landed in Cuba. "Venezuela, Korea, Mexico, Japan. Indonesia, Fiji, and the Philip- pines (Food and Agriculture Organization 2002a). In addition, >33O,00O MT of Scapluirca l>roiigluonii and Anadara i-ramdosa was cultured in 2000. mostly in China, Malaysia, Thailand, and Korea (Food and Agriculture Organization 2002b). Arcidae spe- cies are also fast becoming important fished species in some new regions, such as along the eastern coast of the United States (McGraw et al. 2001, Power & Walker 2002). The Noah's Ark shell {Area noae Linnaeus 1758) is a coin- mercially important bivalve that is distributed in the eastern At- lantic Ocean, the Mediterranean Sea. the Black Sea, and the West Indies (Nordsieck 1969), It lives attached with a solid byssus on rocks or shells, and is widely distributed and locally common in the Adriatic Sea (Hrs-Brenko & Legac 1996). The species is com- mercially exploited and, until the end of the Second World War, constituted an important component of the diet of local populations (Hrs-Brenko 1979, Zavodnik 1997). In the late 1940s, due to a catastrophic mortality caused by an unknown agent, the A. noae fishery in the Adriatic Sea collapsed (Hrs-Brenko 1980). Although the fishery has never returned to the annual catch rate of >600 MT of the 1940s (Hrs-Brenko 1980), it is still one of four major com- mercially exploited bivalves in the eastern Adriatic (Benovic 1997). Due to an increasing number of tourists and a subsequent in- crease in demand for seafood products, the A. noae fishery re- ported in this article has recently intensified in the Croatian part of the Adriatic. In a lecent study of A. noae shell sections (Peharda et al. 2002). it was found to be a slow-growing bivalve. .4. noae can live for >I6 y. a feature that makes it potentially susceptible to overfishing. However, the research conducted by Peharda et al. *Corresponding author. E-mail; melita@izor.hr (2002) investigated only the growth of the shell, but did not in- vesdgate the population structure of this species. The research undertaken in this article had the objective of gaining a better understanding of seasonal changes in the A. noae population struc- ture and condition index (CI), data that are crucial for monitoring the sustainability of the A. noae fishery in the Adriatic. MATERIALS AND METHODS Mali Ston Bay is an extended bay located in the southeastern Adriatic Sea (Fig. 1 ). It is characterized by strong marine currents, underwater freshwater springs, and abundant and constant sedi- mentation that influences the formation of soft-mud sediments (Simunovic 1981 ). The concentration of nutrients is high due to the high freshwater input (Vukadin 1981. Caric et al. 1992). Analysis of phytoplankton abundance and zooplankton community structure indicate that the bay is a naturally moderately eutrophic ecosystem (Vilicic 1989. Lucie & Krsinic 1998). The sampling station for the study was located in part of Mali Ston Bay called Bistrina. The study was based at Bistrina marine station between No- vember 2001 and November 2002. Noah's Arks were collected from the seabed by scuba divers at depths of between 2 and 4 m. Sampling was conducted once a month for the analysis of CI (;; = 30) and every 2 mo for biometric measurements. The following parameters were measured for each specimen: length (L). height (H). and width (Wd) in millimeters: and dry flesh weight and wet weight of shell in grams. Flesh was dried at 60°C to constant weight, and the following CI was calculated according to the method of Davenport and Chen ( 1987): C.I. = Dry flesh weight/Shell weight x 100 Temperature and salinity were measured at a depth of 2 m twice a month with a WTW (Ft. Myers, FL) multiline hydrographic probe. Seawater samples for chlorophyll a (Chi a) analysis were collected twice a month at the same depth using Niskin (General Oceanics, Miami, FL) water bottles. Samples of 0.5 dnv^ were filtered using Whatman (Kent, U.K.) GF/F glass-fiber filters and were subse- quently stored at -20°C. The Chi a level was determined fluoro- metrically using a Turner TD-70() Laboratory Fluorometer (Sunnyvale. CA), and was calibrated with pure Chi a (Sigma Chemical. St. Louis. MO) after homogenization and 90'7f acetone 705 706 Peharda et al. Figure I. Location of Mali Ston Bay and Bistrina. extraction (24 li at room temperature) of filters, following the method of Strickland and Parsons ( 1972). Spearman's correlation analysis and regression were applied to describe the biometric characteristics of the shell and body tissue. and to determine the degree of association with the CI and envi- ronmental conditions. A nonparametric Kruskal-Wallis test was used to examine monthly changes in CI. Length frequency data were analyzed using the FiSAT statistical package (Food and Ag- riculture Organization-The International Center for Living Aquatic Resources Management (ICLARM). Rome. Italy). Data were smoothed using the running average of three classes, and the Pow- ell-Welherall method (Wetherall 1986) was applied to estimate asymptotic length {LJ. The method of Bhattacharya (1967) was used to separate a composite distribution into separate cohorts, while the method of the sum of squared errors was used to deter- mine the curvature parameter (k) of the modified von Bertalanffy growth equation L, = L^ [ 1 -e (SpaiTC & Venema 1992). RESULTS The seawater temperatures ranged from 7.2'C (January 2002) to 25.8°C (June 2002). Seawater temperatures >20°C were re- corded between June and mid-September (Fig. 2). The lowest sa- linity values were recorded during sampling in July |26.9 practical salinity units (psu)| and October 2002 (28.8 psu). The highest D. 3 c Q. 1 E 40 35 30 25 20 15 10 5 0 V ■0.22 .♦ -.^ + 0-20 0.18 0 16 r 0 14 0.12 0.10 - 0-08 0.06 0.04 0.02 0.00 N D 2001 J F M A M J J A S 0 2002 Chi a N D ♦ Salinity ■ Temperature Fiuure 2. Seasonal variation in the salinity (psu), temperature ("O. and Chi a levels (mg m"') in Mali Ston Bay. salinity value recorded in this study was .^7.1 psu (March 2002). Chi (( values ranged from 0 mg m"' (December 2001 ) to 0.094 mg nr' (April 2002). The minimum shell length recorded during the 1-y study was 6 mm. while the maximum was 80 mm [mean (±SD) length 4.'i.04 ± 13.68). Only 1% {n = 14) of measured individuals were longer than 70 mm. 6% in = 96) were longer than 60 mm. and 39% {II = 589) were longer than 50 mm. Shell height values ranged from 3 to 44 mm (mean 23. 1 7 ± 6.59 mm), and shell width ranged from 3 to 51 mm (24.81 ± 7.24 mm). Length frequency histo- grams, according to sampling months, are shown in Figure 3. The polynomial type of length distribution is visible in all the presented graphs, indicating the presence of several age classes. Using the length frequency distributions, up to eight cohorts were separated according to the method of Bhattacharya (1967) (Table 1). The asymptotic length (L^j of A. noae was estimated at 79.91 mm. while the calculated curvature parameter (k) was 0.342 y"' (r" = 0.992). According to the von Bertalanffy growth equa- tion obtained. .4. iiocic reaches a length of 60 mm in its 5th year of life, while it takes over 10 y to grow to its asymptotic length (Fig. 4). The relationship between length and height could be described using the following equation: H = 4.33 -I- 0.418 L (/( = 1531; r- = 0.75; P < 0.001 ). while the equation Wd = 3.32 + 0.477 L {n = 1531; r- = 0.82; P < 0.001) described the relationship between shell L and Wd. The calculated values of r"^ indicate the degree of variation in the shape of the shells. The relationship between shell weight and length could be described using the following equation W = 0.01* L' "" (;; = 390; r" = 0.79: P < 0.001 ). A. noae has negative allometric growth, meaning it grows proportionally more in length than in H. Wd. total weight, or flesh weight with increase in age (Table 2.) Seasonal differences in body CI are shown in Figure 5. Low mean ratios of dry flesh weight and shell weight (<9) were re- corded in December and January, and also in the period from July to October. The highest ratio values (-11) were recorded from April until June. A sharp decrease in CI was noted between June and July. Observed monthly changes were statistically significant (Kruskal Wallis H = 126.95; P < 0.001 ). There were no statisti- cally significant correlation between CI and temperature (r = -0.369; P = 0.468) and Chi (( (r = 0.036: P = 0.477). while a negative correlation was found between CI and salinity (r = 0.137; P = 0.007). DISCUSSION The Bay of Mali Ston is the largest bivalve aquaculture area in the eastern Adriatic Sea. with a long tradition of collecting marine organisms and their aquaculture over several centuries, and. ac- cording to some authors, even from the time of the Roman Empire (Basioli 1968). Although A. noae is one of the main bivalve spe- cies traditionally collected in this area, there are no data on its biometry, population structure, or seasonal changes in CI at this location. The current study confirms previous observations that A. noae is variable in shape (Valli & Paro\el. 1981. Poppe & Goto 2000. Peharda et al. 2002). Negative allometric growth noted for A. noae in the Gulf of Trieste (Valli & Parovel 1981) also was confirmed in this study. According to the literature. A. none can grow up to 90 mm, hut usually it grows up to 70 mm in size (Parenzan 1974. Hrs-Brenko 1980. Poppe & Goto 2000, Peharda et al. 2002). In this study, the A Stud\' of Arca noae in the Adriatic Sea 707 (a) November (d) May N=269 X=42.0+10.4 Length (mm (b) January _ 6 N=85 c X=48.3±13.0 .il.lJ _ 6 -S s >. 4 o 10 20 30 40 50 60 70 80 10 20 30 40 50 60 70 80 Length (mm) (c) March N=200 X=40.0±15.0 _ 6 >, 4 0) '^ == 2 J? 1 ^ 0 0 10 20 30 40 50 60 70 80 Length (mm) (e) July N=200 X=44.7±16,2 10 20 30 40 50 60 70 80 Length (mm) (f) September >. 4 N=188 X=40.6±13.0 10 20 30 40 50 60 70 80 Length (mm) o- 2 N=219 X=45.3±12.6 0 10 20 30 40 50 Length (mm) 60 70 80 Figure 3. Length-frequency histograms for A. iioae collected in (al November 2001, (b) January 2002. (cl March 20(12. (d) May 2002, (el July 2002. and (f) September 2002. largest indi\idual had a length of 80 iiini. but \ery few animals longer than 70 mm were recorded. Length frequency distributions recorded in 1977 and 1978 on the west coast of the Istria peninsula (Hrs-Brenko 1980) are similar to that recorded in this study. The relatively high percentage of .4. noae longer than 50 mm indicates good survival, a stable population, and a potentially good spawn- ing stock (Hrs-Brenko 1980). The parameters of the \on Bertalanffy growth equation ob- tained in this study show that it takes >10 y for A. noae to grow to its asymptotic length. Mistri et al. (1988) found similar results for a related species. Scapluiira inaequivalvis, which was introduced by ballast water into the northern Adriatic. S. iiiaec/iiivtilvis grows slowly and needs >10 y to reach its maximum theoretical length (Mistri et al. 1988). A slow growth rate was also shown for the ark TABLE L Cohorts split using Battacharya's method, according to sampling months. Cohort November January March May July September 1st 10.89 (4.474) 16.09 (3.323) 20.43 (2.829) 10.00(3.101) 13.19 (3.3S5) 16.34(2.447) 2nd 21.37 (3.122) 25.95 (4.085) 28.28(3.711) 18.67 (2.717) 22.87 (2.997) 24.96 (2.980) .^rd 31.78 (3.585) 33.45 (2.085) 38.68 (3.640) 28.50 (3.926) 30.25 (3.694) 34.63 (4.276) 4ili 41.31 (4.243) 42.93 (4.052) 47.06 (2.959) 39.68(5.951) 39.70 (4.639) 44.50 (5.219) .^ih 50.33 (3.358) 53.18 (2.812) 53.47 (2.898) 49.38 (3.510) 48.95 (2.104) 52.95 (5.6,39) fith 57.54 (2.686) 58.27 (2.190) 62.63(3.921) 56.79 (4.219) 58.71 (5.702) 64.36 (2.579) 7tli 64.65 (3.934) 63.38 (3.157) 64.81 (3.256) 74.94 (3.44S) Sth 71.75(1.779) Values given as mean length (SD). 708 Peharda et al. 90 80 70 "E 60 ^50 O) 40 -I 30 20 10 0 TABLE 2. Parameters of log,,, regressions of biometric parameters. 012345678 Relative age (years) Figure 4. (Jrowth eur>e for A. none fitted using the von Bertalanffy growth equation L, = 79.19 [i_e" '^"-'"']. shell Noctia poiulcrosa, which was shown to attain a market size when it is 8+ years old and could li\'c tor 15 y (McGraw et al. 2001). The presented resuUs in this study indicate that .4. iioae might be a somewhat faster growing species than previously found using shell sections (Peharda et al. 2002). According to this study, the asymptotic length of A. none is larger (80 mm) than the asymptotic length that was obtained from the analysis of shell sections (6.5 mm). Similarly, the calculated curvature parameter (k) was also larger (0.34), than the k value (0.17) in Pehai'da et al. (2002). which indicates a faster growth rate. However, it is important to keep in mind that the Bhattacharya method applied in this study is useful for splitting a composite distribution into separate normal distributions in cases in which there are several age groups (co- horts) and is less reliable for longer living species (Gayanilo & Pauly 1997). On the other hand, the study of shell sections is usually limited to a smaller number of specimens (Richardson 2001), and it is possible that the earlier study by Peharda et al. Dependent Independent \ ariable \ariable a b r- 11 H L -0.04 0.85 ±0.010* (I.S30 \5}\ Wd L -0.09 0.90 ± 0.009* 0.864 I5,M Total weiaht L -2.04 1 .92 ± 0.050* 0.794 390 Flesh weight L -2.61 1.94 ±(1.(165* 0.695 390 * Values given as mean ± SE (departure from isometry at P < 0.01 ). (2002) was somewhat infiuenced by sample size. Fuilher research should compare these two methods and examine the possibility of determining growth parameters using mark-recapture techniques. The results obtained for the CI show that there are seasonal changes in body weight. The ma.Kimum values were recorded dur- ing the period from April until .lune. The minimum values of CIs were recorded in December and January, at the end of the sumtner, and at the beginning of the fall. The first minimum value is prob- ably related to temperature stress and a period of reduced feeding, as it is a period when we also recorded the lowest seawater tem- peratures. The later minimal value might be attributed to a period following spawning. Reduction in body CI in September and Oc- tober was attributed to a summer spawning in related species. S. hroiiiihroitii (Park et al. 2001 ) and .S". imictiuivalvis (Mistri et al. 1988). respectively. According to Graeffe (190.3) and Vatova (1928. 1949). the spawning of A. none in the Adriatic Sea occurs in May and June. In the Gulf of Trieste, this species has a prolonged spawning season, with peaks occurring in March and September (Valli & Parovel 1981). Our data on seasonal changes in body weight in- dicate that the spawning activity of A. noae in Mali Ston Bay might be the most intense in June. This is further supported by the ob- servation of small individuals in the November samples. D 13 0 Q t B Q 5 R B Q ^ B R Nov Dec Jan Feb l^ar Apr IVIay Jun Jul Aug Sep Oct Nov 2002 Figure 5. Monthly variations in CI of .1. iwac. CI = ratio between dry llesh weight and shell weight, with values given as percentages. A Study of Arca noah in thf, Adriatic Sea 709 According to our results, salinity is the only environmental factor that correlates with CI throughout the year. This result is similar to the findings of Park et al. (2001 ), who did not record a coiTclation between the CI of S. hnnighloiiii and temperature and Chi (( level, and have recorded a positive correlation between CI and salinity. Although during our study period Chi a concentra- tions were about 10 times lower than the lowest values recorded by Jasprica and Caric (1997), previously established patterns of sea- sonal variation of phytoplankton in the semi-closed bays along the eastern Adriatic Sea and our values are in agreement with the presented data (Vilicic & Stojanoski 1987). Increased temperature values, in addition to available nutrients (Caric pers. comm.), fa- \i)red phytoplankton development in April, when an increase in A. iKhw CI was registered. According to the thermohaline conditions and Chi a levels analyzed, Mali Ston Bay may be considered to be an ecologically stable location suitable for the growth of ,4. iiocw. ACKNOWLEDGMENT The authors express their gratitude to the Ministry of Science and Technology of the Republic of Croatia for funding this project, and to Zeljko Bace and Vladimir Onofri for providing technical support. Special thanks to C. A. Richardson for valuable assistance with data analysis and preparation of the manuscript. LITERATURE CITED on the eastern shores of the Adriatic Basioli. J. 1968. Shell-breedin^ Pomorski zbornik 6:179-218. Benovic. A. 1997. The history, present condition, and future of the mol- luscan fisheries of Croatia. In: C. L. MacKenzie, Jr.. V. G. Burrell, Jr.. A. Rosenfield & W. L. Hobart, editors. The history, present condition, and future of the niolluscan fisheries of North and Central America and Europe, vol. 3. Europe NOAA Technical Report NMFS 129. Wasli- ington. DC: US Department of Commerce, pp. 217-226. Bhattacharya, C. G. 1967. A simple method of resolution of a distrihulion into Gaussian components. Biometrics 23:1 15-13.'i. Caric, M., N. Jasprica & D. Vilicic. 1992. Nutrient and chlorophyll <; concentrations in Gruz and Mali Ston Bays (Southern Adriatic). Rapp. Comm. Int. Men Meclit. 33:367. Davenport, J. & X. G. Chen. 1987. A comparison of methods for the assessment of condition in the mussel {Myiilus ediilis L.). J. Mull. Studies 53:293-297. Food and Agriculture Organization. 2002a. Fishery statistics: capture pro- duction, vol. 90. 2000, Rome: Food and Agriculture Organization. 617 pp. Food and Agriculture OrganizatK)ii. 2002b. Fishery statistics: aquaculture pro- duction, vol. 90. 2000. Rome: Food and Agriculture Organization. 178 pp. Gayanilo. F. C. Jr. & D. Pauly. 1997. FAO-ICLARM stock assessment tools (FiSAT): Reference manual. FAR Computerized Information Series (Fisheries). No. 8. Rome: Food and Agriculture Organization. 262 pp. Graeffe, E. 1903. Uebersichte der Seethiere des Golfes von Triest. VI Mollusca. Arh. Zoot. Inst. Wien Zool. Stti. Trieste 14:89-136. Hrs-Brenko, M. 1979. Shellfish as food: the natural populations of edible shellfish in the Adriatic Sea. Prehmmbeno-tehnoloska reviju 17:125-130. Hrs-Brenko, M. 1980. Preliminary survey of populations of the bivalve Noah's Ark {Area none. Linne) in the northern Adriatic Sea. Aqiuuiil- mre 2l:357-.363. Hrs-Brenko. M. & M. Legac. 1996. A review of bivalve species in the eastern Adriatic sea: II. Pteromorphia [Arcidae and Noetidae). Nal. Croat. 5:221-247. Jasprica. N. & M. Caric. 1997. A comparison of phytoplankton biomass estimators and their environmental correlates in the Mali Ston Bay (Southern Adriatic). Mar. Ecol. Puhbl. Sin. Zool. Napoli 18:35-50. Lucie, D. & F. Krsini(:. 1998. Annual variability of mesozooplankton as- semblages in Mali Ston Bay (Southern Adriatic). Period. Biol. 100: 43-52. McGraw. K. A., M. Castagna & L. L. Conquest. 2001. A study of the arkshell clams, Noetia ponderosa (Say 1 822) and Anadara ovalis ( Bru- guiere 1789). in the oceanside lagoons and tidal creeks of Virginia. ./. Shellfish Res. 20:185-195. Mistri, M.. R. Rossi & V. U. Ceccherelli. 1988. Growth and production of the ark shell Scaphaira inaeijiiivalvis (Bruguiere) in a lagoon of the Po river delta. Mar. Ecol. Puhbl. Stn. Zool. Napoli 9:35-f9. Nordsieck, F. 1969. Die europaischen Meeresmuscheln (Bisalvia). Stutt- gart: Fischer Verlag. 256 pp. Parenzan, P. 1974. Carta d'identita delle conchiglie del Mediterraneo: Bi- valvi: Parte prime. Taranto, Italy: Ed. Bios Taras. 277 pp. Park. M. S.. C -K. Kang & P.-L. Lee. 2001. Reproductive cycle and bio- chemical composition of the ark shell Scapharca broughtonii (Schrenck) in a Miuthern coastal bay of Korea. J. Shellfish Res. 20: 177-184. Peluirda. M.. C. A. Richardson. V. Onofn. A. Bratos & M. Crncevic. 2002. Age and growth of the bivalve Arca iioae L, in the Croatian Adriatic Sea. J. Moll. Stud. 68:307-310. Poppe. G. T. & Y. Goto. 2000. European seashells. vol. II Scaphopoda. Bivalvia, Cephalopoda. Hackenheini: ConchBooks. 221 pp. Power. A. J. & R. L. Walker. 2002. Growth and gametogenic cycle of the blood ark. Anadara ovalis (Bruguiere. 1789) in coastal Georgia. J. Shellfish Res. 21:157-162. Richardson, C. A. 2001. Molluscs as archives of environmental change. Oceunogi: Mar. Biol. Ann. Rev. 39:103-164. Sparre, P. & S. C. Venema. 1992. Introduction to tropical fish stock as- sessment: Part 1. Manual. FAO Fish Technical Paper (revision 1). vol. 306. Rome: Food and Agriculture Organization. 376 pp. Strickland. J. D. H. & T. R. Parsons. 1972. A practical handbook of seawater analysis. Bull. Fish. Res. Bd. Can. 167:1-31 1. .Simunovic. A. 1981. Biolosko-ekoloska istrazivanja jestivih skoljkaSa Malostonskog zaljeva. In: J. Roglic & M. Mestrov, editors. Zbornik radova savjetovanja "Malostonski zaljev prirodna podloga i drustveno valoriziranje." Dubrovnik: JAZU, Znanstveni savjet za zastitu prirode. pp. 252-267. (in Croatian). Valli, G. & C. Parovel. 1981. Aspects de la reproduction et de la biometrie chez Arca noae L. (Mollusca. Bivalvia). Rapp. Comm. Int. Mer Medii. 27:135-136. Vatova, A. 1928. Compendio della Flora e Fauna del Mare Adriatico presso Rovigno. Mem. R. Com. Talass. Ital. 143:1-614. Vatova. A. 1949. La fauna bentonica delFAlto e Medio Adriatico, Nova Thalassia 1:1-110. Vilicic. D. 1989. Phytoplankton population density and volume as indica- tors of eutrophication in the eastern part of the Adriatic Sea. Hydro- hiologia 174:117-132. Vilicic, D. & L. Stojanoski. 1987. Phytoplankton response to concentration of nutrients in the central and southern Adriatic Sea. Acta Adriat. 28:7.3-84. Vukadin. I. 1981. Hidiogralska svojstva Malostonskog zaljeva i susjednog mora u periodu 1980-1981 godine. In: J. Roglic & M. Mestrov, editors. Zbornik radova savjetovanja "Malostonski zaljev prirodna podloga i dru.stveno valoriziranje". Dubrovnik: JAZU, Znanstveni savjet za zas- titu prirode, pp. 52-65. (in Croatianl. Wetherall. J. A. 1986. A new method for estimating growth and mortality parameters from length-frequency data. ICLARM. Fishbyte 4:12-14. Zavodnik. D. 1997. Non-conventional seafood sources at the eastern Adri- atic sea markets. In: B. Finka. editor. Thousand years from first men- tion of fisheries in Croats. Zagreb: Croatian Academy of Arts and Sciences, pp. 637-656. (in Croatian). Jiiiiiiuil iif Shell fish RiSi-anh. Vol. 22. No. 3, 711-714. 2()(U. PRESENCE OF GIANT POLYMORPHIC CELLS IN CRASSOSTREA GIGAS CULTURED IN BAHIA FALSA, BAJA CALIFORNIA NW MEXICO JORGE CACERES-MARTiNEZ* AND REBECA VASQUEZ-YEOMANS Laboratoiio cle Biologi'a v Patologia tie Organisnio.s Aciidticos del Departumento de Aciiiculliiia. Cciitro de Investigcicii'm Cienlifica v de Ediicacldn Superior de Emcmidu. Apdo. Posnd 2732. 22800. Ensenada Baja Ccdifornia. Mexico ABSTRACT The culture of the Japanese oyster Crassoslrea aigas is a successful commercial activity In Northwest Mexico. Since 1997. hi!;h mortality outbreaks have occurred in the area without apparent reasons. In thi.s study we found gill erosions during clinical observations, hemocyle infiltration into the tis.sues at the histopathological level, and in some cases we detected the presence of giant polymorphic cells. In general, conditions mentioned above, including the presence of Trichodiiia sp. and especially the presence of giant polymorphic cells matches with the signs of the gill disease caused by an icosahedral DNA virus (Gill Necrosis Virus infection) first recorded in the Portuguese oyster Crassoslrea angidala and in the Japanese oyster C. ninas in Europe. However, the Transmission Electron Microscopy (TEM) analysis of damaged tissues did not reveal the presence of viral particles. KEY WORDS: Crassosrrea i^lsas. giant polymorphic cells, mortality, gill necrosis \irus infection (GNV). trichodines. INTRODUCTION The Japanese oyster Crassostreci gigas is one of the most widely cultured mollusks in the world. This species has been in- troduced from its original area in Japan, to countries such as Aus- tralia. France. Holland, Spain, Portugal, Thailand, to the Pacific coast of the United Sates, and United Kingdom (Bardach et al., 1982: Edwards, 1997). In 1973, C. gigas was introduced in several coastal lagoons in the states of Sonora, Baja California Sur, and Baja California, in Northwest Me.xico. including Bahi'a Falsa. B.C. The oyster seed was obtained from The Laboratory of the Lumi Indians in Marietta, Washington U.S.A. (Islas Olivares. 1975). In Bahi'a Falsa, this species was cultured in floating rafts. At present, the culture is produced in racks, bags, and occasionally some stocks are maintained directly on the bottom. Currently, the annual production in the region is around 1,622 metric tons with a value of 2,4 million dollars. Around 1.800 workers are involved in this activity and Bahi'a Falsa contributes \6Vr of this figure (Anuario Estadistico de Pesca. 2001). The industry depends on the impor- tation of oyster spat from Oregon. Washington, and California, USA. Since 1997, several high mortality outbreaks of C. gigas. in- cluding seed, juveniles, and adults, have occurred in Sonora and Baja California Sur. In April 1998, mortality outbreaks began to be recorded in Bahi'a Falsa, B.C. Unusual inortality has remained in the region. Several causes have been attributed to these mortalities: 1 ) unusual high temperatures and conditions produced by El Nino in 1997 and 1998. 2) the presence of toxins in the environment produced by microalgae or other organisms, 3) pollution, 4) the quality and quantity of food (phytoplankton), and 5) pathogens, or synergic conditions by the joint action of two or more of the above factors (Caceres-Martinez 2000, Hoyos 2000). This work presents the results of a clinical and histopathological survey of C. gigas cultured in Bahia Falsa. B. C. and the possible relation of the observations and parasites with mortality outbreaks present in the bay. MATERIALS AND METHODS The study was conducted in Bahi'a Falsa. Baja California, Mexico from July 1997 to June 1998 (monthly samplings). Two localities. Agromarinos. in the middle area of the bay. and Alfon- sos, in the inner area of the bay, were sampled (Fig. 1 1. Rack and bag cultures are used in Agromarinos, whereas bottom culture is used in Alfonsos. In each locality and culture condition, 30 com- mercial size oysters were collected (mean total shell length, 12.45 cm ± 5.5 in the Agromarinos" racks, mean total shell length 10.46 cm ± 5.5 in the Agromarinos" bags and Alfonsos"). In all three culture conditions, oysters are exposed to air during low tide. Live oysters were transported to the laboratory and all fouling organ- isms were removed with a brush and a stream of seawater. Oysters were placed in a Petri dish, opened, and the intervalvar water and oyster flesh were examined for the presence of parasites under a dissecting microscope. The soft body of the oysters were removed from the shell and fixed whole in Davidson"s fixative (Shaw & Battle 1957) for at least 24 h An anterior transverse section in- cluding the digestive gland, mantle, and gills was taken. Tissue samples were embedded in paraffin wax and were sectioned and stained with hematoxylin and eosin (Shaw & Battle 1957). Tissue analysis and measurements were made with a micrometer placed in an optical microscope. Prevalence of parasites and lesions were considered as the number of infested or wounded oysters/number of oysters examined xlOO. Intensity was considered as the number of lesions or parasites per damaged or infected oysters in the Present address of both authors: Instituto de Sanidad Acui'cola. Calle 9na y Gastelum No. 468 Local 14. Zona Centro. C.P. 22800. En.senada, Baja California, Mexico. *Corresponding author. E-mail jcaceres@cicese.mx V ..' 1 1 \- Baja California \ ^^. Falsa \ AlfoniLH ■( ^-\ A^^-T^ \\ j^ 30^5 > / ^^ 1 \^ Sonora ^ / /^ \mexico / / I \ / Baja CaliTornia Sur ^-j ^v^. c? Figure 1. Map showing the region, where Crassoslrea gigas culture is conducted in North Western Mexico, and the Bahia Falsa, where the study was performed. Black dots indicate the sampling sites. 711 712 Caceres-Martinez and Vasquez-Yeomans B 50 p r 40 e V 3 30- 1 e n 20. c e 10- Gill inflamation 1-30 1 r^ r Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun 50- Trichodina sp. 40_ Salinity ppt 30- 20- 1-40 • 30 • 20 Jul Aug Sep Oct Nov Dec Jan Feb Mar Apr May Jun 1997 1998 30- 40-1 Figure 2. A, The combined mean values and standard error of prevalence and intensity of gill intlammaticm of C. gigas from all three cultures conditions during the study period. The dark triangle indicates when the oyster culturist noted the mortality outbreak. B, Mean values and standard error of prevalence and intensity of Trichodina sp. in C. gigas from the three cultures conditions during the study. Temperature and salinity values are shown as a line on the top of A and B, respectively, and their scale \alues are placed in the right side of the graphics. il :aw tf» % ,. * ♦Pn * ♦ ,fe* ♦ ' ^ . "* Figure i. A. Strong gill inllammation in C. gigas with destruction of gill tllaments. tissue rupture with massi\e inllltration of heniocytes (h) and the presence of some giant polymorphic hypertrophic cells (gc), scale bar = KM) (jm. B, Detail of a gill lesion showing the polymorphic hypertrophic cells (gc) with basophilic inclusion (bi), picnotic nucleus (pnl and heniocytes around the lesion (h). Haematoxylin and eosin, scale bar = 2U fim. Giant Polymorphic Chlls in Crassostrea g/gas 713 sample. For the estimation of the gill lesions the following scale was used: (1) light gill intlammation, low infiltration of hemocytes and the gill structure unaltered; (2) medium gill intlammation. infiltration of hemocytes within the gill filaments and interlamellar septum; (3) Strong gill inflammation. massi\e infiltration of hemocytes. swelling of gill filaments and necrosis of the gill tissue. After this study period, two additional samplings were earned out. one after a mortality episode occurred in the Bahia Falsa, in June 20(10. During this sampling 30 surviving oysters were col- lected, opened in the field, and the condition of the gills was observed. Photographs of the gills were taken with a camera placed on a stereomicroscope and the degree of gill damage was deter- minate. All gills were fixed in Davidson's fluid and processed for histopathological analysis as mentioned above. The last sampling was carried out in Nov ember 2000. Ten oysters from an area of the bay where mortalities were common were reviewed and those showing symptoms of gill erosion were separated and a small pieces from the eroded area of the gill was removed and fixed in 3% glutaraldehyde in 0.1 M sodium cacodylate buffer. pH 7.8. for 4 h at 4"C. Fixed tissues were washed for 1 2 h at 4'"C in the same buffer and cut into 1 mm' pieces. These pieces were then postfixed in buffered Wr OsOj for 4 h at the same temperature, dehydrated through a series of ethanols, and embedded m Epon. Sections (90 nni thickness) were cut and stained with 5% uranyl acetate for 30 min and lead citrate for 2 min and observed with a Transmission Electron Microscope (TEM) operated at 75 kV at the Instituto de Investigaciones Marinas. Vigo. Spain. RESULTS Histologic slides showed from light to strong gill intlammation. There were no differences among the prevalence and intensity of gill intlammation of oysters collected from different culture con- ditions (Kruskal-Wallis test. H = 2.42. P = 0.29 and H = 0.97. P = 0.61, respectively). The combined mean values of prevalence and intensity of gill inflammation from the three culture conditions are shown in Figure 2. There was a positive correlation between the prevalence and intensity of gill intlammation (r = 0.72. P = 0.007) and there was a general increase in the prevalence and intensity of gill inflammation from the beginning to the end of the study period (Fig. 2). In two cases. April in the Alfonsos" and June in the Agromarinos" rack, we detected cellular lesions reminiscent of the gill necrosis virus infection (GNV) caused by an icosahedral cytoplasmic deoxyribovirus (Comps. 1988): occun'ence of giant polymorphic cells (ranging from 25 to 30 |xm in diameter) con- taining basophilic granules and some oval hypertrophied nucleus and hemocytes accumulation in the lesion (Fig. 3). Also. Tri- chodina sp. were detected in the mantle cavity and gills of C. gigcis. Their prevalence and intensity was similar in oysters taken from the three different culture conditions (Kruskal-Wallis test. H = 0.9. P = 0.63 and H = 0.77. P = 0.67. respectively). The combined mean values of prevalence and intensity of Trichodimt sp. from the three culture conditions are shown in Fig. 2. There was a positive correlation between the prevalence and intensity of Triclwdina sp. (r = 0.77, P = 0.003) and there was a general increase in the prevalence and intensity of Trichodina sp. from the beginning to the end of the study period (Fig. 2). Temperature and salinity remained between the tolerance limits of this oyster spe- cies (Pauley et al. 1988; Fig. 2). There was no statistical correlation between gill lesions and Trichodina sp. presence; however, there was a trend of an increase in gill lesions and Tricliodina sp. pres- ence from the beginning to the end of the study period (Fig. 2). Figure 4 shows a varying degree of gill erosion in surviving oysters sampled in June 2000, from normal appearance (0 de- grees), to very eroded appearance (4 degrees). Histologic analysis revealed the presence of picnotic nuclei in cells at the distal edge of the gill filaments where erosion occurred but no Trichodine presence. Curiously, no evidence of necrotic areas was observed in the eroded areas of gill filaments; however, deformations of the distal edge of the gill filaments were evident (Fig. 4). The preva- lence of the lesions was 1009f and its intensity value was medium. These lesions showed inflammation of tissue and cicatrisation The TEM study did not reveal the presence of giant polymorphic cells and viral particles. DISCUSSION According to Comps (1988), virus infection has been associ- ated with major diseases of oysters of the genus Crassostrea. These infections include the GNV affecting the Portuguese oyster and. to a lesser degree, the Japanese oyster cultured in Europe. Figure 4. DiUcrinl (k};riis of «!!! inisioii in surviving C. gigas. A, Normal appearance (n). light eroded appearance (lei. B, Medium eroded appearance (me). C Highly eroded appearance ihe). 714 Caceres-Martinez and Vasquez-Yeomans This author remarked that the gill necrosis virus causes, mainly in the Portuguese oyster, an evolutive ulceration of the gills, includ- ing cellular hypertrophy and severe hemocyte infiltration. Mortali- ties have been observed in the most serious cases. In this study, we found clear histologic evidence of giant polymorphic cells, which have been associated with GNV infection; however, we did not detect characteristic damage of GNV on the gills because we failed to look for it. It is important to mention two points: 1 ) this study was concluded three months after an unusual mortality in the bay was recorded, and 2) no moribund oysters were sampled. How- ever, in the second sampling (June 2000) when we went specifi- cally looking for gross signs of the GNV infection; we detected the erosion of the gill filaments, which is the characteristic gross sign of the GNV infection. In spite of the failure to reveal viral particles by TEM possibly because of the sample process, the status of the gill tissues of surviving oysters (cicatrisation), and the difficulties in finding those particles in fixed tissues, evidence of giant poly- moiphic cells in C. g/go.v cultured in the bay and region showed an obligated line of research and the possibility that a virus may be involved in oyster mortality outbreaks. It is known that herpes-like viruses in several oyster species have been associated with high mortality rates (Le Deuff & Renault 1999). However, the presence of Trichodina sp. was observed in a similar condition when the unusual mortality of C. angiilata occurred in France. On that oc- casion, it was thought that Trichodina sp. could be the cause of oyster gill lesions (Besse 1968), Afterwards, it was found that Trichodina sp. might have been a secondary invader of oysters that were suffering from virus-caused gill necrosis (Bower et al. 1994). The trend of the increase in prevalence and intensity of Triclwdina sp., from the beginning to the end of the study period, is consistent with the increase of gill lesion observed and a secondary invasion of the protistan. Oyster culturists from Bahia Falsa first reported a mortality outbreak in April 1998. and gill inflammation increased from November 1 997 to the end of the study period. It is possible that the presence of giant polymorphic cells or the gill inflamma- tion condition could have been present in the oysters of the bay earlier; however, an unknown factor may favor the increase of gill lesions at the end of the study period. Temperature and salinity remained between the tolerance limits of the oyster species and the culture technique seems to be independent of gill lesions, and the parasite prevalence and intensity observed. Further studies using molecular tools are being conducted to confirm the presence of virus in oysters from the region and its possible relationship with mortality outbreaks. ACKNOWLEDGMENTS The authors thank M.C. Jose Angel Olivas Valdez and Oc. Sergio Curiel Ramirez for sample processing; Dr. Antonio Figueras from Instituto de Investigaciones Marinas de Vigo. Spain, for TEM analysis; and Consejo Nacional de Ciencia y Tecnologia from Mexico for financial support throughout the project number 39.V^P-B. LITERATURE CITED SAGARPA. Gobierno de Mexico. Anuano Estadistico de Pesca. 2001. Poder Ejecutivo Federal. Bardach, J. E.. J. H. Ryther & W. O. Mclarney. 1982. Aquaculture. John Wiley & Sons. Inc., 741 pp. Besse, P. 1968. Resultats des quelques observations sur une affection bran- chiale des huitres [Crassosuea an;iidaut Lrnk). Bull. Acad. Veterinmri' de France 41:87-91. Bower, S., S. E. McGladdery &. I. M. Price. 1994. Synopsis of infectious diseases and parasites of commercially exploited shellfish, Ann. Rev. Fish Dis. 4:1-199. Caceres-Martinez. J. 2000. Resultados de los analisis patoliigicos efectua- dos a ostiones del Pacifico relacionados con mortalidades masivas. Foro Regional Sobre la Problematica del Cultivo de Moluscos Bivalvos en el Noroeste de Mexico. Veiliuno de enero del 2000. Hemiosillo. Son. Mexico. Comps, M. 1988. Epizootic diseases of oysters associated with viral in- fections. In: W. S. Fisher, editor. Disease processes in marine bivalve molluscs. Bethesda, MD: American Fisheries Society, Special publi- cation 18, pp. 23-37. Edwards, E. 1997. Molluscan fisheries in Britam. In: C. L. MacKenzie. Jr.. V. G. Burrell. Jr.. A. Rosenfield. W. L. Hobart. editors.. The history. present condition, and future of the molluscan fisheries of North and Central America and Europe. Volume 3, Europe. U.S. Depl. Commer.. NOAA Tech. Rep. 129. 240 p. Hoyos. C. F. J. 2000. Antecedentes de las mortalidades masivas de ostion Japones y otros bivalvos en 1997-1999. Foro Regional Sobre la Prob- lematica del Cultivo de Moluscos Bivalvos en el Noroeste de Mexico. Veiliuno de enero del 2000, Hermosillo, Son. Mexico. Islas Olivares. R. 1975. El o.stion japones Cra.ssoslrea gigas en Baja Cali- fornia. Cienc. Mar. 15:21-38. Le Deuff. R. M. & T. Renault. 1999. Purification and partial genome characterization of a herpes-like virus infecting the Japanese oyster, Crassostrea gigas. J. Gen. Vir. 80:1317-1322. Pauley. G. B.. B. Van Der Raay & D. Troutt. 1988. Pacific oyster. Bio- logical Report 82 (11.85). Species profiles: life histories and environ- menlal requirements of coastal fishes and invertebrates (Pacific North- west). Washington. DC: Fish and Wildlife service. U.S. Department of the Interior. 28 p. Shaw. B. L. & I. H. Battle. 1957. The gross microscopic anatomy of the digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J. Zoul. 35:325-346. Joiirmil ofShrlllhli Risciinh. Vol. 22, No. 3, 715-720, 2003. IN VIVO AND IN VITRO APPROACHES TO THE ANALYSIS OF GLYCOGEN METABOLISM IN THE PACIFIC OYSTER, CRASSOSTREA GIGAS CLOTHILDE HEUDE BERTHELIN.'* BRUNO FIEVETr GAEL LECLERC,' PIERRE GERMAIN,- KRISTELL KELLNER,' AND MICHEL MATHIEU' 'Laboraloire de Biologic et Biotechnologies Marines. UMR IFREMER "Phxsiologie et Ecophysiologie des Mollusques marins. " Univcrsite de Caen Basse-Noniiandie. 14 032 Caen cedex. France: -Laboraloire d'Etndes Radioecologiqnes de la Fa(,adc Atlantiqne. Institnt de Radioprolection et Si'irete Nucleaire, BPIO. Rue Max Pol Foncliet. 501 M) Chcrhowg-Octeville. France ABSTRACT Seasonal variations of glycogen and prolein metabolism m the Pacific oyster Cnissastmi f;iiiii\ were investigated in vivo using a radiolabeled glucose injection technique and were compared with in vitro experiments on vesicular cells. Protein metabolism appeared stable during a gametogenetic cycle, whereas glycogen metabolism in low was found to be clearly dependent on the sexual cycle, with decreasing incorporation during gonadal tubule development. The in vivo results correlated well with data from in viti-o experiments on vesicular cells, which correspond to the animal's glycogen storage compartment, KEY WORDS: Pacific oyster. Crassa\lrca fiiK'ts. gametogenesis. glycogen, storage tissue. //; vivo, bioassay INTRODUCTION 111 the Pacific oyster. Crassostrea gigas. as in mosl bivalves, glycogen is one of the major energetic fuels for gametogenesis (Bayne et al, 1982. Gabbott & Whittle 1983, Ruiz et al. 1992; Mathieu & Lubet 1993), On the west coast of Europe, gametoge- nesis in C. gigas follows an annual cycle: gonial mitosis occurs in autumn and early winter in the gonadal tubules: the gonad devel- ops in winter and spring: and in summer, the ripe gonad is ready for sequential spawning in July or August, depending on the rear- ing site. The biochemical composition of the whole animal and isolated organs was previously studied, and glycogen levels were determined (Walne & Mann 1975, Robert et al. 1993, Berthelin et al, 2000b), Glycogen storage and mobilization activities were tightly correlated to the reproductive cycle. Histologic studies showed a seasonal inverse relationship between the increase of the gonadal tubules and the regression of the storage tissue in the gonadal area. Moreover, glycogen was stored during autumn and early winter while gonadal tubules regressed, and was subse- quently mobilized during active gametogenesis (Berthelin el al. 2000a. 2000b). In the oyster, the biochemical mechanisms of glycogen storage and mobili/ation in relation to reproductive activity remain poorly documented in comparison with other models like the marine mus- sel. Mytihis ediilis (Houtteville 1974. Pipe 1987. Lenoir 1989). Recently, an in vitro bioassay was developed for C. gigas to mea- sure glucose incorporation into glycogen in vesicular cells, the glycogen storage compartment (Berthelin et al. 2(.)0()a). The //; vitro approach provided some valuable information on the cellular mechanisms of glycogen metabolism in vesicular cells, but it may not reflect the true metabolism of reserve cells in vivo. First, cel- lular dissociation could damage receptor protein structure leading to glucose uptake modifications, coinpared with physiologic con- ditions in the whole animal. Second, the incubation conditions may not reflect the seasonal variations in the natural environment. For these reasons, the glycogen metabolism of cupped oysters was investigated using an in vivo approach based on injections of '■*C-labeled clucose into the adductor muscle. After defining the *Corresponding author. E-mail: heude(3>ibfa.unicaen,fr optimal experimental conditions, glucose incorporation was stud- ied in the whole animal in relation to the annual sexual cycle. Finally, in vivo results were matched with in vitro data obtained from isolated vesicular cells. MATERIAL AND METHODS Animals Pacific oysters (C gigas. 3 years old) were obtained from a commercial oyster farm in Saint-Vaast-La-Hougue. Normandy. France. The animals were kept in aerated seawater at 13°C throughout each experiment. The animals were starved for 24 h prior to all in vivo experiments. In vivo Bioassay Conditions of Injection Two days before injection, both valves of the oyster were notched beside the adductor muscle, paying attention not to dam- age the muscle, A preliminary experiment was conducted to evalu- ate the diffusion of the injected solution into the animal tissues: 200 ^^.L of neutral red in sterile seawater was injected into the adductor muscle. The oysters, kept in 0.5 L tanks, were dissected 45 min or 3.5 h after injection. Seawater coloration in each tank was controlled, and neutral red diffusion was observed in each animal. [U-'^C] Glucose Injection For labeling experiments, the injected solution included 50 (xL of |LI- '""Cl glucose and 1 50 |jlL of D-glucose ( 1 3 niM). resulting in a final glucose concentration increase of approximately 1 mM in the hemolymph. according to the standard of Livingstone and Claike ( 1983). For each condition, six animals were injected and were kept in aerated seawater at I3°C. Control animals were ana- lyzed just after injection to estimate nonspecific radioactivity. Af- ter incubation, soft parts were separated from shells that had been individually collected in 50-mL tubes and blended (ultra-turrax, Labosi. France). Animal tissues were stored at -20'C before sample treatment. For each animal, the blended tissue sample vol- ume was adjusted to 40 niL with sodium hydroxide (0,006 N) and thoroughly homogenized. Different [U-'''C1 glucose doses were tested (0,5. I, 5. and 10 (iCi). Kinetic measurements of incorporation in protein and gly- 715 716 Berthelin et al. cogen also were performed (after 0. 7.5. 16, 24, 48, and 72 h of incubation). Seasonal variations of incorporation were studied. Total Radioactivity Determination Fi\e hundred microliters of potassium hydro.xide (0.3 N) was mixed with 500 jjlL of a blended tissue sample, and 250 p.L of this mixture was diluted in 4 mL of scintillation fluid (Optiphase. Hisafe II 2*51 Wallac. France, EG and G division instruments) were analyzed for '"'C activity (Packard scintillation counter. France). [U-"C] Glycogen Content Five hundred microliters of a blended tissue sample was mixed with 500 p.L of 10% trichloroacetic acid, and precipitated proteins were discarded by centrifugation (8000 t;, 10 min, 4'C). Seven hundred microliters of the supernatant was transferred to a 5-mL tube containing 10 mg of unlabeled oyster glycogen (Sigma- Aldrich. France) as a carrier and 4 mL of absolute ethanol. After overnight precipitation at 4°C. glycogen was collected by centrifu- gation (2500 i;. 10 min, 4°C), and the pellets were washed three times with absolute ethanol containing D-glucose (0.1 M). Glyco- gen pellets then were dried and resuspended in 500 p.L ol potas- sium hydroxide (0.3 N). Radioactivity was determined in 250 |xL of glycogen suspension diluted in 4 mL of scintillation ftuid. [U-^CI Protein Content Protein content also was determined in each tissue sample as follows: 500 |j.L of oyster extract was mixed with I mL of potas- sium hydroxide (0.3 N) and 3 mL of 10% trichloroacetic acid. After overnight precipitation at 4°C, protein pellets were collected by centrifugation (3000 g, 10 min. 4°C), were washed three times in trichloroacetic acid, and were resuspended in potassium hydrox- ide (1 niL, 0.3 N). Radioactivity was determined in 250 |jlL of protein suspension diluted in 4 iiiL ol scintillation fluid. In Vitro Approach Preparation of Vesicular Cell Suspension Oysters were maintained on ice during the dissection, were opened by sectioning the adductor muscle, and were rinsed thor- oughly with sterile seawater. The labial palps were dissected, rinsed three times in sterile sea water, and decontaminated for one night in 50 niL of Leibovitz culture medium (Leibovitz LI 5; NaCI 340 mM, KCl 50 mM. Hepes 20 mM (pH 7.4). 1100 mOsm, filtered on a Millipore 0.22-|j.m filter] supplemented with antibi- otics (streptomycin 100 mg L~'. penicillin 60 mg L~'. gentamycin 50 mg L~', and nystatin 8.2 mg L"'). Vesicular cell isolation was performed as previously described by Berthelin et al. (2000a). Dissociated cells were diluted in Lei- bovitz. culture medium to obtain 3 x 10"' cells mL"' and were distributed into 24-well culture plates. Significant survival was evaluated with the MTT [3-(5.5- dimethyl-thiazol-2-yl)-2.5- diphenyl tetrazolium bromide] reduction assay (Mosmann 1983, Coulon 1993). [U-'"'C] Glucose Incorporation into Glycogen by Vesicular Cells Glucose incorporation measurement was derived from Berthe- lin et al. (2000a). In each well, 500 \xL of vesicular cell suspension (3 X 10'' cells mL"') was mixed with 50 |jiL of (U-'-'Cj glucose (0.5 (xCi. specific activity: 150-260 mCi mmoP') and 50 |jiL of unlabeled D-glucose (1.5 mM). After incubation (7 h, I5"C), the well contents were transferred into 1.5-mL microtubes and centri- fuged (8000 ^. 10 min. 4"C), resulting in cell disruption. Three hundred microliters of supernatant was transferred into a 5-mL tube containing unlabeled oyster glycogen (10 mg) and then was mixed with 4 mL of cold absolute ethanol. Glycogen was precipi- tated o\ eniight and rinsed three times with a solution of D-glucose in absolute ethanol (0.1 IVI). Glycogen pellets were dried and di- luted in 500 p,L of distilled water. An analysis of radioactivity was performed on 200 |j.L of glycogen suspension diluted in 4 mL of scintillation fluid. Blanks without cells or without radioactive labeled glucose were tested, and control samples were prepared by stopping the incubation immediately after [U-'"'C] glucose addition. Data Analysis Results were expressed as the mean ± SD. Each value is the mean of six replicates. A nonparametric test (Kruskall-Wallis test) followed by a multiple comparison test (Newnian-Keuls test) also was performed to determine the significant differences between samples (Scherrer 1984). RESULTS Injection Into the Adductor Muscle A preliminary experiment using neutral red as a visual tracer led to an estimate of its diffusion in seawater. After injection, no seawater coloration was observed. Moreover, after 45 min, the digestive cardiac sinus, the adductor muscle, and the gills were stained red, whereas the palps and mantle appeared colorless. Three hours after injection, the gills were still stained, and the palps and mantle also appeared red. However, the digestive cardiac sinus was faded. Reco>ered Radioactivity It represented 35 to 85*5^ of injected radioactivity with large individual variations due to injection efficiency. Because of these variations, [U-'''C] glucose incorporation into protein and glyco- gen were expressed as the percentage of recovered radioactivity for each animal. [U-'''C] Glucose Incorporation Four doses of [U-'"*C] glucose were tested: 0.5 \x.Ci (0.0185 MBq) and 1.0 p.Ci (0.037 MBq) values were chosen by reference to previous in vitro experiments (Berthelin et al. 2000a); and 5 p.Ci (0.185 MBq) and 10 fjiCi (0.37 MBq) were tested considering the potential dilution of radioactive material in the whole animal. After 24 h of incubation, irrespective of the amount of injected labeled glucose, 8 to 10% of recovered radioactive carbon was found in the protein fraction, and about 2% was incorporated into glycogen (Fig. I). Ten microcuries (0.37 MBq) of radioactivity was used routinely in all subsequent experiments to keep sensitivity as high as possible, since low levels of labeled glycogen were expected at certain periods of the year. The kinetics of radioactive carbon incorporation into proteins is presented in Fig. 2a. The incorporation rate was maximal during the first 16 h of incubation. After 48 and 72 h, I 1% and 12.2%, respectively, of radioactive carbon was incorporated into proteins. For the same animals, glucose incorporation into glycogen in- creased linearly during the first 24 h of incubation (Fig. 2b) and reached a maximal value of 1.6% of recovered radioactive carbon Analysis of Glycogen Metabolism in Crassostrea gigas 717 JL. O.SmCi ■ Protein D Glycogen i i A. Figure I. Fraction of recovered radioactivity ineasured in protein and glycogen depending on the quantity of radioactivity used (^C'il. Results are expressed as tfie percentage of recovered radioactivity in oyster ±SD. after 4S h (this iiicuhatinn time was chosen for further experi- ments). This maximal value \ aried in the range of 1 .6 to 29c for the eight experiments performed. Seasonal Variations of /;/ l7i'o Carbon Incorporation into Proteins The radioactive carbon fraction incorporated into proteins was measured over the annual cycle with an incubation time of 48 h. The radiolabeled protein fraction was found to represent 7 to 1 I'/r of recovered radioactivity according to month (Fig. 3). With re- spect to protein metabolism, two statistically different groups were observed: one from January 1 to October 1; and the second, with reduced metabolism, from November 1 to March 2 [P < 0.05 (Kniskall-Wallis and Newman-Keuls tests)]. Ill Vivo and In Vitro Glucose Incorporation into Glycogen In vivo results (Fig. 4) showed that glycogen represented be- tween 0.6% and 1.9% of recovered glucose incorporation in the oyster, depending on the season (injected amount ()..17 MBq; in- >-< o > o o O •5 to S? a 10 20 30 40 hours 50 60 0 3n b overed tivity 2- 1 - ■ ■ r 1- o .-^^^^^^ > 0< , *^^ 10 20 30 40 50 60 70 hours Figure 2. Kinetics of '^C incorporation, (a) Incorporation of '''C' into proteins, (bl Incorporation of '''C into glycogen. Results are expressed as the percentage of recovered radioactivity ±SD. 718 Berthelin et al. 14 n 12 10 "? £ a. 0 I 1 1 A JL B B I B M JFMAMJJASONDJ__ Figure 3. Seasonal variation of carbon incorporation in protein fraction. Results are expressed as the percentage of recovered radioactivity ±SD. Groups A and B are statistically different. cubatioii time 41S h). In the first year, labeling was maximal in February ( 1.9%). decreasing progressively to 0.6% in July, before increasing during the autumn and returning to the maximal value the following March (with a lower value in February of the second year) (P < 0.03). The stages of development of the gonadal and storage tissues in the gonadal area (Heude Berthelin et al. 2001) are overlaid on Fig. 4. In vitro measurements were performed on vesicular cells from February 1 to October 1 (Fig. 5). These values show that ;;; vitrn incorporation was maximal in February (1.5 nmol per 1.5 x 10'' cells), decreasing to an undetectable level in July and August, and finally rising again during the following autumn (P < 0.05). DISCUSSION The investigation of different metabolic pathway.s in bivalves has been based mainly on //( vitro techniques, due to the anatomic characteristics of these animals. These in vitro approaches have o r. T3 on o T3 o » >. o 3,0 - 2,5 2,0 1 ,5 - I ,0 0,5 0,0 I I I I I M M O N M ST developed regression regressed development developed regression GT gonial mitoses development regression rest gonial mitoses development spawn Figure 4. (top) Seasonal variation of carbon incorporation in glycogen fraction. Results are expressed as the percentage of recovered radioac- tivity ±SD. (bottom) Gametogenesis and storage tissue development in oysters (Saint Vaast la Hougue, France) |redra\vn from histologic data published In Heude Berthelin et al. (20111 )|. Analysis of Glycogen Metabolism in Crassostrea gigas 719 3.0 n f= 2,5 - F M A M J J A S O Figure 5. Changes of [l'-'''C| glucose incorpr>rati()n into gl>cogen iiieasurtd both in vitro and in vivo. The results are expressed in nanomoles of glucose incorporated per 1.5 x 10'' cells for in ritru experiments, and as a percentage of recovered radioactivity for in viro experiments (mean ± SD; n = 61. revealed specific information about metabolism at the scale of isolated cells or specialized tissue samples maintained in strictly controlled conditions. However, tissue or cell preparation steps associated with the chosen artificial conditions may disturb the metabolic activity of the sample relative to its true state in the whole animal. The present study aimed to compare seasonal variations of glucose incorporation into glycogen measured by in vitro bioassay in C. gigas (Bertheiin et al. 2000b) with in vivo levels of incor- poration. This incorporation was measured after the injection of '""C glucose into the adductor muscle using an experimental pro- cedure originally used for the artificial infection of oysters with pathogens (Hervio et al. 1992). Protein metabolism was first analyzed by measuring the incor- poration of radioactive carbon into proteins. Radiolabeled proteins represented 7 to I l^f of recovered radioactivity. Whereas experi- mental conditions were significantly different (i.e.. tracer concen- tration, incubation time, and injection procedure), these data may be compared with the results reported by de Zwaan et al. ( 1975) for the mussel Mxtilus edulis in aerobic conditions: these authors found that proteins accounted for ll^r of radioactivity. In the oyster, the annual pattern of '"'C incorporation into proteins ap- peared rather stable throughout the complete gametogenetic cycle (from January 1 to October 1 ). Following the gametogenetic cycle (from November I ) incorporation was also stable, but slightly lower (1%). By comparison with this relative stability, glycogen metabo- lism shows some significant variations: the ''^C incorporation into glycogen ranged from 0.6 to 1.9'7f of the radioactivity. Glycogen storage decreased during gonadal development from February to July and increased after the spawning event when the oysters were in the sexual resting stage. These results were then compared with in vitro data obtained during a previous gametogenetic cycle (Ber- theiin et al. 2000b). Because of possible interannual bias, addi- tional in vitro measurements also were performed within the same year as the /;; i'/i(; experiments. Whatever the approach. '""C glucose incorporation into glyco- gen presents the same annual pattern. Indeed, the observed varia- tions correlated with seasonal changes in glycogen content re- ported in C. gigas (Mann 1979. Robert et al. 199.^. Almeida et al. 1997) and confirm that glycogen storage occurs during eariy ga- metogenesis to support the energetic cost of the reproductive effort (Ruiz et al. 1992. Mathieu & Lubet 1993. BertheUn et al. 2000b. Heude Bertheiin et al. 2001). The mobilization of glycogen also was observed in vivo in early spring and autumn. This matching between in vivo and in vitro data is essential to verify the previous (/; vitro approach to the study of seasonal variations in glycogen metabolism: the \n vitro bioassay should be considered as an ad- justed technical approach to investigate the cellular mechanisms involved in the regulation of these changes of metabolism. Moreover, the correlation between the pattern of glycogen me- tabolism in the whole animal (i.e.. the in vivo approach) and the gonadal vesicular cells suggests that in the oyster glycogen me- tabolism occurs mainly in the specialized storage tissue located in the gonadal area (Bertheiin et al. 2000a), and that this metabolism is the main source of energy for reproductive effort. Further ex- periments should now be carried out to improve different technical aspects of the /;/ vivo procedure. With the current in vivo proce- dure, glucose is supplied directly into the hemolymph sinus with- out taking into account digestive assimilation or possible short- term storage in the digestive gland (Bertheiin et al. 2000b). Im- provement may result from the use of labeled microalgae or coated beads. In addition, organ dissection should be considered to quan- tify the respective role of each coinpartinent involved in glucose metabolism. ACKNOWLEDGMENTS The authors would like to thank C. Costil for essential ad\ ice on all the statistical aspects of this study, and I. Probert for his expert linguistic guidance. LITERATURE CITED Almeida. M. J., J. Machado & J. Coimbra. 1997. Growth and biochemichal Bayne. B. L.. A. Bubel, P. A. Gahbon, D. R. Livingstone. D. M. Lowe & composition of Crassostrea gigas (Thunberg) at three fishtarm earthen ponds. J. Shellfisli Res. 16:455-462. M. N. Moore. 1982. Glycogen utilisation and ganietogenesis in Mytilus edulis (L.). Mar. Biol. ten. .^:89-I05. 720 Berthelin et al. Berthelin, C K. Kellner & M. Mathieii. 2000a. Histological cliaracteriza- tion and glucose incorporation into glycogen of the Pacific oyster Cra.s- sostreu gigas storage cells. Mm: Biowdtnol. 2:136-145. Berthelin. C, K. Kellner & M. Mathieu. 2000b. Storage metabolism m the Pacific oyster (Crassoslrea gigas) in relation to summer mortalities and reproductive cycle (West Coast of France). Comp. Binchcm. Physinl. 125B:.359-.369. Coulon, I. 1993. Mise au point d'un systeme controle de culture de cellules de coeur d'huitre Cnissostrea gigas: Application au test de la toxicite des produits chimiques en milieu aquatique. These de doctorat es Sci- ences. Paris-Grignon, France: Institut National Agronomique. De Zwaan. A.. A. De Bont & J. Kluytmans. 1975. Metabolic adaptation on the aerobic-anaerobic transition in the sea mussel MmHiis ciliitis (L.l. Proceedings of the 9th European Marine Biology Symposium, pp. 121- 138. Gabbott. P. A. & M. A. Whittle. 1985. Glycogen synthetase in the sea mussel Myiiliis ediilis L: II. Seasonal changes in glycogen content and glycogen synthetase activity in the mantle tissue. Comp. Biochcni. Physiol. 836:197-207. Hervio. D. 1 992. Contribution a Tetude de Boiuiiniu oslreae [Ascctospora I. protozoaire parasite de I'huitre Osriea edulis (Bivalvia). et a I'analyse des interactions hote-parasite. These de doctorat. Clermond-Ferrand. France: Universite de Clermond-Ferrand. Heude Berthelin. C. J. Laisney. J. Espinosa, O. Martin. G. Hernandez, M. Mathieu & K. Kellner. 2001. Storage and reproductive strategy in Crassostreu gigas from two different growing areas (Normandy and the Atlantic coast. France). Invert. Reprod. Dev. 40:79-86. Houtteville, P. 1974. Contribution a I'etude cytologique et experimentale du cycle annuel du tissu de reserve du manteau de Myrilas cdidis (L.). These de Doctorat. Caen. France: Universite de Caen. Lenoir, F. 1989. Mise au point de techniques de dissociation, de purifica- tion et de culture cellulaires chez la moule Myriliis edidis (L.). Appli- cation a I'etude des regulations du metabolisme du glucose et du gly- cogene dans les cellules a glycogene ( = cellules vesiculeuses). These de Doctorat. Caen. France: Universite de Caen. Livingstone, D. R. & K. R. Clarke. 1983. Seasonal changes in hexokinase from the mantle tissue of the common mussel Myrilus edulis (L.). Comp. Biochem. Physiol. 746:691-702. Mann. R. 1979. Some biochemical and physiological aspect of growth and gametogenesis in Ciassosrrea gigas and Osirea edulis at sustained elevated temperatures, J. Mar. Biol. Ass. U. K. 59:95-100. Mathieu. M. & P. Lubet. 1993. Storage tissue metabolism and reproduction in inarine bivalves: A brief review. Invert. Reprod. Dev. 23:123-129. Mosmann. T. 1983. Rapid colorimetric assay for cellular growth and sur- vival: application to proliferation and toxicity assays. J. Immunol. Methods 65:55-63. Pipe, R. K. 1987. Ultrastructural and cytochemical study on interactions between nutrient storage cells and gametogenesis in the mussel Mytilus edulis. Mar. Biol. 96:519-528. Robert. R.. G. Trut. M. Borel, M. & D. Maurer 1993. Growth, fatness and gross biochemical composition of the Japanese oyster Crassostrea gi- gas in stanway cylinders m the bay of Arcachon. France. Aijuaculture 110:249-261. Ruiz. C. D. Martinez. G. Mosquera, M. Abad & J. L. Sanchez. 1992. Seasonal variations in condition, reproductive activity and biochemical composition of the flat oyster. Osirea edulis. from San Cibran (Galicia. Spain). Mar. Biol. 112:67-74. Scherrer. 6. 1984. 6iostatistiques. Quebec. Canada: Gaetan Morin. 850 pp. Walne. P. R. & R. Mann. 1975. Growth and biochemical composition in Ostrea edulis and Crassostrea gigas. In: H. 6arnes. editor. Proceedings of the 9th European Marine Biology Symposium. Aberdeen. Scotland. UK: Aberdeen University Press, pp. 587-607. Journal of Shcllfisl, Rcsccuih. Vol. 22. No. 3. 721-731, 2003. EFFECTS OF TEMPERATURE AND FEEDING REGIMES ON GAMETOGENESIS AND LARVAL PRODUCTION IN THE OYSTER CRASSOSTREA GIGAS JORGE CHAVP:Z-VILLALBA,"* JEAN-CLAUDE COCHARD/ MARCEL LE FENNEC," JEAN BARRET,- MARTHA ENRIQUEZ-DIAZ,' AND CARLOS CACERPIS-MARTINEZ'^ ^ Unite Mixte de Recherche (U.M.R.) Centre National de Reclierclw Scientifiqne (C.N.R.S.) 6539: Institiit Universitaire Europeen de la Mer. 292H0: Ploiizane. France: 'Laboratoirc de Physiologie des Inrertehres. Institute Fram^ais de Recherche poin- L' exploitation de la Mer (IFREMER) Centre de Brest. BP 70. 29280 Ploiizane. France: ^Centre de Investii^aciones Bioldgicas del Noroeste (CIBNORj. GiiaYinas Unit. AP 349. Giiuynuis. .Sonora H5465. Mexico: ^Universidad Aittononui de Baja California Sitr. AP I9-B, La Paz. Baja California Snr (B.C.S.) 23080, Mexico ABSTRACT The effect of feeding regimes and temperature on the beginning of gametogenesis in the Pacific oyster Crassoslrea ,i;/,i;c/.v (Thunherg) was examined under laboratory conditions. Oysters from two different culture sites in France. Baie des Veys (Department Manche) and La Tremhlade (Department Charente-Maritime). were collected in January 2000 and exposed to four treatments, nivolving a period of maintenance at lO'C with or without feeding followed by a conditioning period at I9°C with or with feeding. Routine conditioning procedures at 19°C (direct conditioning), with or without food, were performed at the same time and were used as controls. Oocyte size was used to describe the evolution of gametogenesis in all treatments. Contrasting responses were noticed between samples from Baie des Veys (B V-oysters) and La Tremblade (LT-oysters). BV-oysters containing more tissue reserves than specimens from the other location used carbohydrates to support gametogenesis. while LT-oysters used proteins to fuel oocyte development. During the initial period at 10°C, fed BV-oysters began gametogenesis and produced mature oocytes, while unfed BV-oysters began gametogenesis, but at a slower rate. Fed LT-oysters began gametogenesis at 10°C. whereas unfed LT-oysters remained unchanged (early gametogenesis stage) during the cold phase and only initiated gametogenesis when the temperature was increased. Oysters conditioned without food produced significantly less oocytes than specimens conditioned with food, but no differences in larval yield (D-larvae) were detected amongst the different conditions and sampling locations. Only LT-oysters kept without food throughout the experiment did not produce oocytes at the end of the conditioning period. These experiments demonstrate that oocyte production in C. )>igas is dependent upon food supply and temperature, but that oocyte quality under controlled conditions appears to be related to stored reserves in natural settings. KEY WORDS: conditioning. Crassostivci gigiis. food, gametogenesis, temperature INTRODUCTION Acciinnilation of reserves in the Pacific oyster Crassoslrea gi- gas (Thtinhcrg 179.3) take.s place in autumn ancJ winter. an(J the first signs of the beginning of gametogenesis are observed in Janu- ary when temperature is still descencjing (Chavez-Villalba et al, 2002a). The influence of food supply and tetnperature on the re- production cycle of bivalves has been noted by many authors (DinamanI 1987, Ruiz et al. 1992). However, there are few data on the influence of environmental factors on gametogenesis in C. gigas. The studies on this topic for the scallop Aeqiiipecten irra- dians concenlriciis (Sastry 1979) and eastern oyster Crassoslrea virginica (Thompson et al. 1996) suggest that in the early phase of gametogenesis, bivalves require an adequate food supply, as well as a suitable temperature to stimulate gonad growth. These authors proposed that, under inadequate food conditions, tissue reserves are used for maintenance metabolism rather than gametogenesis. and that food supply appears to be less critical after certain mini- mum reserves have accumulated in the gonad. Gonad maturation then occurs at a rate that is dependent on temperature. Gametoge- nesis in oysters is directly coiTelated with water temperature (Mu- ranaka & Lannan 1984). However. Sastry (1968) found that low temperature can be inhibitory in well-fed scallops held at I5^C that already had started early gametogenesis. but oocytes did not enter into normal growth until exposed to higher temperatures (20°C). *CorTespondmg author. E-mail; jechavezCs'cibnor.mx Thus, normal reproductive development requires a minimum tem- perature and an adequate food supply Temperate bivalves exhibit a marked seasonal cycle in the syn- thesis, accumulation, and use of biochetiiical energy reserves. In general, reserves are stored during periods of high food availability (late summer and fall), at which time the major energy require- ments for somatic and germinal growth have already been satis- fied. Stored reserves are used to initiate gametogenesis and to maintain metabolism during periods of low food availability (Thompson et al. 1996). Berthelin et al. (2000) found that glycogen accumulation in the gonad of C. gigas occurs in fall and winter, and this compound serves as a substrate to support gametogenesis. In this way. oysters can partially uncouple temporal food avail- ability with gamete production, allowing gametogenesis to start when food supply is at a miiiiminii (winter). The accumulation and use of stored reserves in bivalves depend on the state of gonad development, the influence of en\ ironinental parameters on metabolic activities, and the nutritional value of food supplied during conditioning. In French hatcheries, the be- ginning of broodstock conditioning of C. gigas starts in December to obtain viable gametes and larvae by the end of January (Chavez- Villalba et al. 2002b). Conditioning procedures consist of feeding abundantly the oysters for 7 wk at a warm temperature ( 19°C). The use of this technique allows animals to be conditioned from De- cember until April, producing an increase of viable gametes and larvae with time (Chavez-Villalba 2001). Additionally, it was found that there were sotne groups of oysters that can produce 721 722 Chavez-Villalba et al. viable oocytes without being fed during the conditioning proce- dures, and that larval hatching rates from unfed oysters were not significantly different, compared with fed animals. However, the nutritional stress produced by partial or complete deprivation of food can substantially alter the biochemical composition of bi- valves. Whyte et al. ( 1990) demonstrated that oysters exhibit dif- ferent biochemical composition if deprived of food. Different experiments in our laboratory (Chavez-Villalba 2001. Chavez-Villalba et al. 2002a. Chavez-Villalba et al. 2002b. Chavez-Villaba et al. 2003) have shown that gametogenesis in C. gigas seems to be an integrated response to different environmen- tal factors, in which temperature and food supply play significant roles. In this study, the effects of food and temperature in the period at the beginning of gametogenesis. as well as the biochemi- cal changes in soft tissues produced by the effect of these param- eters, were investigated. To achieve these objectives, we studied two oyster populations: one from Baie des Veys (BV) where oys- ters have low spawn rates as a result of low summer temperatures; and the other from La Tremblade (LT) where en\ironmental con- ditions allow full spawning in summer. MATERIAL AND METHODS Experimental Conditions At the end of January 2000. two 600-oyster samples were taken from two different culture sites in France where they had been raised in plastic mesh bags on iron tables. These animals were initially collected from the Bassin d'Arcachon (44''41.8'N. I°8.3'W). In the Bassin. the water temperature varies from 7.5°C in January to 22°C in August, and salinity records go from 26 to 33i7(:c during the year. Juveniles then were grown at Le Morbihan (47°35.5'N, 3°I.3'W) until they were 18 mo old and subsequently were dispatched to one of two culture sites, where they were raised for about I y (Fig. I). In Le Morbihan. water temperature fluctu- ates from 3 to 5°C in the winter to 20 to 22^C in the summer, and salinity values remain stable throughout the year (34. 8-35. 4*^*^?). One of the culture sites is on the Baie de la Seine in the BV in the Departments of Manche and Calvados (49°2I.5'N. r6.9'W). In this zone, the average temperature in January is about 6°C. about I7°C in August, and during spring and summer temperature in- creases on the surface. Salinity records in the bay are always under 34.5'/rc. showing a decreasing pattern toward the coast (30^f ). The other site is located on the Atlantic coast in the estuary of the Seudre River at LT (45°3I.6N. I°I.7W) in the Department of Charente-Maritime (Fig. I). In this area, the temperature varies from 7°C in winter to 22°C during the summer, and salinity records are closer to I5%f (www.ifrenier.fr). Oyster samples were transported from the culture sites to the Brest-IFREMER center where the samples from the two sites were divided into several groups. Fifty animals from each site were exposed to standard conditioning (I9°C with ample food). Two more 50-oyster groups from each site were exposed to standard conditioning (I9°C) but without food. These groups were condi- tioned (direct conditioning) from February 8 to March 30, 2000, and were used as controls. Four groups of 2.'iO-oysters each (two from BV and two from LT) were placed in maintenance tanks at IO''C. One group from each site was fed continuously with the same diet as that used for conditioning, and one group from each site was maintained with- out food. These maintenance treatments were sustained for 60 days (8 February-9 April. 2000). At the end of this period, each group was divided into two subgroups that were conditioned under two different treatments, with and without food (Fig. 2). For the conditioning experiments, seawater temperature in the tanks was increased IC per day until it reached I9°C (heating period), and the photoperiod was adjusted to 16 h of daylight and 8 h of night (spring conditions). Oysters were fed a diet coiumonly used in experimental hatcheries for conditioning: a mixture of two micro-algae species ( 10" cells of each species per day per animal) from monospecific cultures of hiichrysis aff. galbana Green (Clone T-Iso: Tahiti Isnchnsis) and Clnwroceros calcitnuis Ta- kano. Sampling English Channel^ _,_^ r^ "' r Baie de^^ys ^1 ^ ■ — ^ Manche O ^- -48° *"^ Baden (Intermediate culture)V^^ FRANCE Atlantic \^^ Ocean '^ < La Tremblade) i Charente- -Maritime Bassin d Arcachon k — 44° (Spat collection)! 0° 1 Figure 1. Location of C. gigas sites. During the direct conditioning, the groups were sampled (20 oysters per site) two times: the first sample was obtained before the heating period ( 10°C), and the second sample was taken after 6 wk at I9°C. For the four treatments, oyster samples (20 from each group) were taken at the beginning, in the iniddle. and at the end of the period at IO°C. The last samples were obtained at the end of the four conditioning procedures (Fig. 2). Froiri each sample (20 oysters). 10 specimens were used for histologic examination, and the other 10 were used for biochemical analyses. Semi-Quantitative Histology Procedures in this part of the study generally followed the methods of Chavez-Villalba et al. (2002a, 2002b). Oysters used for histology were opened, and a section of approximately 1 cm^ of visceral mass was taken from above the pericardial area and fixed in Bouin's solution for at least 48 h. Samples were dehydrated with a series of ethanol treatments of increasing concentration, cleared in toluene, and embedded in paraffin following a standard proce- dure. Sections 3 p-m wide were cut. mounted on glass slides, and colored with Groat's hematoxylin and eosin Y solution (Martoja and Martoja-Pierson 1967). The histology slides were examined under a microscope connected to a video camera to determine Gametogenesis and Larval Production in Chassostrea cncAS 20 -, Conditioning at 19 °C 723 16 12 8 4 0 - f • o o o -s. a -a I c G o With food ^« ^^ > w WHh food ^^ ^ ^ ,r< o ? 0) # •Without food ^* ^ ^ft y^ T° ^k Winter conditions (10 °C) ....4 ^ Q. Witfifood ^^ ^t" E ^^ L w (U Wittiout food ^* ^ .,< ^ Wittiout food ^B .^t* ^^ Oyster groups ^ Samplin g 1 1— 1 1 1 1 0 20 40 1 1 1 60 80 1 1 1 100 1 120 Days Figure 2. Experimental conditions of the four treatments (black circles = samples taken for histologic and hiochemical studies). B = BV specimens; T = I,T specimens. Starting date is Kebruarj 8, 2(((M). oocyte size and frequency, and gametogenic activity. Recorded images were processed by digital image analysis. Oocytes were measured and histology classified following the description by Lango-Reynoso et al. (2000). These operations were conducted on 1 00 randomly chosen oocytes per oyster, and measurements followed a standard bias reduction procedure for selecting measurement fields. Transects of gonad preparations were oriented to maximize coverage of the larger vertical or hori- zontal oocyte field axis. All oocytes with a well-defined germinal vesicle in a field were measured, and every oocyte measured was assigned to a reproductive stage based on the diameter and histo- locic characteristics of the sonad (Table 1 1. In the case of male oysters, the evolution of the spermatogenesis was described ac- cording to the histologic characteristics of the gonad. Three de- velopmental stages were recognized (Table 1). Biochemical Analyses We used 10 oysters per sample for biochemical analyses. Specimens were dissected, and soft tissues were divided into two sections: gonad-digestive gland portion (called "gonad"); and the remaining tissue (called "meat"). All samples were ground by adding 1 niL of distilled water per gram of tissue at 5°C in an ice bath. A 400-|a.L aliquot was used for lipid detennination using the TABLE 1. Reproductive stages in the female and male oyster C gigas: Cytologic characteristics corresponding to each stage arc included. .Stages Stage Interval (Mm) Histologic Description Females Early gametogenesis Growing Mature Degenerating Males Early gametogenesis Growing Mature ,^.0± 12.0 Follicles are elongated and often isolated in the abundant connective tissue, with walls consisting of primary oocytes of homogeneous size. I2.1-.^0.0 Start of oocyte growth. A large range of oocyte size at all gametogenic stages can be observed, including some free oocytes. Interfollicular connective tissue disappears. 30. 1— H.O Follicles of relatively homogeneous size completely filled with mature oocytes with distinct nucleus. 41.1-60.0 Follicles containing degenerating oocytes, often elongated in shape, sometimes broken. Obvious redevelopment indicated by increased number of primary oocytes. Abundant connective tissue containing elongated follicles with walls consisting of germinal epithelium with some spermatogonia and spermatocytes Connective tissue is reduced, follicles become larger, and normal sequences of spermatogenesis are observable with spermatocytes 1 and 11. spermatides. and some spermatozoids organized in the lumen ConnecUve tissue almost disappeared. Follicles tilled with packages of spermatozoids oriented with tails toward the follicle lumen Reproductive stages in female specimens are based on an oocyte diameter (p.m) interval (Lango-Reynoso et al. 2000). 724 Chavez-Villalba et al. Bligh and Dyer (1959) method. Carbohydrates were analyzed in a 300-|xL sample by the method of Dubois et al. ( 1956). and proteins were analyzed in a 300-(j.L aliquot by the method of Lowry et al. (1951). Tissue dry weights were calculated from the macerate of each sample; 2 niL were emptied into preweighed aluminum con- tainers and dried in an oven at 80°C for 48 h. Finally, aluminum containers were reweighed after cooling in a desiccator. Given that no significant differences were observed between total dry weights of oysters (from BV and LT locations) at the beginning and by the end of the experiments, the dry weight per- centages of each biochemical compound were multiplied by the total dry weight of each tissue sample to express the results in milligram equivalence of each biochemical compound per total dry weight of the tissue (gonad and meat). Oocyte PrDcluclioii and Larval Yield Estimation (I)-Lanae) Oocyte production and larval yield estimations followed the recommendations of Chavez-Villalba et al. (2()()2a). Oysters from each group were taken from the experimental tanks at the end of conditioning procedures; 20 animals per group were opened, and their sex was determined by observing a fresh smear sample from the gonad under a microscope. After this procedure, females and males were separated, and gametes from both sexes were recov- ered using the scarification technique described by Allen and Bushek (1992). The gonads of all oysters were scarified by a light incision of the gonadal tegument. Oocytes were collected in bea- kers by rinsing the gonad with filtered seawater. The oocytes were passed through a 60-|xm sieve to eliminate undesirable material. Mature oocytes were retained in a 20-|jim sieve. These were rinsed several times and placed in 2- or 5-L beakers. To determine oocyte production, three 50-|xL samples per group were examined and counted under a profile projector. Males underwent the same pro- cedure, but spermatozoa suspensions were examined under a mi- croscope for motility. Batches of spermatozoa of low motility were discarded. A minimum of three batches was mixed together and 10 to 20 niL were used for fertilization. Oocytes were fertilized in 5-L beakers and checked for normal progress about 0.5 to 1 h later (Robert and Gerard 1999). After fertilization, an equal number of embryos from all oysters of each group were pooled together and placed, one group per tank, in 150-L tanks at a 33 embryos mL"' concentration. After 48 h. the tanks were emptied, and the larvae were recovered by siev- ing. Three 50-(j.L larvae samples from each tank were taken for larval yield estimation (number of D-larvae after 48 h of culture/ initial number of embryos). Data Analysis The oocyte proportion corresponding to each reproductive stage was calculated according to Lango-Reynoso et al. (2000). and the arcsine was transformed (Snedecor and Cochran 1972) for each oyster. The logarithms of oocyte production data were cal- culated. The transformed proportions and logarithms were com- pared using the Kruskal-Wallis test. A two-way analysis of vari- ance (ANOVA) test was used to examine the effect of origin and feeding regimen on early, growing, and mature oocyte categories for the direct conditioning. A three-way ANOVA test was run to analyze the following: ( 1 ) the effect of time (between days 30 and 60). origin, and feeding regimen on the different oocyte categories during the period at 10°C; (2) the effect of time (beginning and end of conditioning), origin, and the four tested conditions on the oo- TABLE 2. Mean proportion" of different oocyte stages: early gametogenesis, growing, and mature in C. gigas under direct conditioning «ith and without food, and four treatments during a second phase of conditioning. Day (I End of Conditioning (Day 56) Oyster Sample M M Characteristics Direct conditioning BV LT 69 hi 31 M 6 .^0 7 \\k at I9°C 51 41 4h 72 19 38 Conditioning WF Conditioning WOF Conditioning WF Conditioning WOF Period at IOC Day 0 Day 3(1 Day 6() Oyster Sample M M M Knd of Conditioning (Day 110) E G M Characteristics Four treatments BV LT 69 63 37 58 42 0 33 66 1 -) 84 16 0 (lU 40 t) 0 11 VU 10 u 38 62 0 5 96 4 0 87 Li 0 10 57 It 26 12 76 28 27 34 43 76 73 88 13 67 72 56 0 Treatment Treatment Treatment Treatment Treatment Treatment Treatment Treatment 1 (WFAVF) 2 (WFAVOF) 3 (WOFAVF) 4 (WOFAVOF) 1 (WFAVF) 2 (WF/WOF) 3 (WOFAVF) 4 (WOFAVOF) Presented as %. E, early gametogenesis; G, growing; M. mature; WF. with food; WO. without food. " Mean of values found in a 10-oyster sample. Gametogenesis and Larval Production in Crassosthha gigas 725 cytes (early, growing, and mature) during the conditioning pliase; (3) the effect of origin, time, and feeding regimen on the lipid, protein, and carbohydrate content in the gonad and ineat of oysters maintained at lO'C for 60 days; and (4) the effect of origin, time, and four tested conditions on the lipid, protein, and carbohydrate content in the gonad and meat of oysters during the conditioning procedures. Statistics were analyzed at a significance level a = 0.03. RESULTS Oogenesis Direct Conditioning The proportions of the different oocyte categories tound at the beginning and the end of direct conditionings are presented in Table 2. Mature oocytes were observed in all BV and LT groups at the end of conditioning, but statistical results (Table 3) showed a significant effect of feeding regimen and place of origin. There was a higher proportion of mature oocytes in specimens condi- tioned with food, and BV oysters produced a higher proportion of mature oocytes than LT specimens. Four Tested Conditions The proportions of the oocyte categories found during the cold phase and at the end of conditioning in both BV and LT oysters for TABLE 3. Results of two-Hav and three-way ANOVA tests for early, groHing, and mature oocyte categories, and for biochemical content in the gonad and meat of oysters C. gigas, respectively (direct conditioning). Source of Variation Df MS F P Oogenesis Early gametogenesis stage Factor A (feeding regime) 1 1840.1 8.53 0.0079 Factor B (origin) I 1144.1 5.54 0.0280 Interaction (A x B) I 111 0.05 0.8225 Growing stage Factor A (feeding regime) 1 475.2 345 0.0768 Factor B (origin) 1 68.5 0.5 0.4882 Interaction (A x B) 360.2 2.61 0.1202 Mature stage Factor A (feeding regime) 1 6201, fs .34.8 ().()()()() Factor B (origin) 1615.2 9.05 0.0065 Interaction (A x B) I 78.9 0.44 0.5128 Biochemistry Meat Factor A (origin) 1 .03E6 ,54.2 0.0000 Factor B (feeding regime) 936.7 0.05 0.8246 Factor C (time) 433395 22.7 0.0000 Interaction (A x B) 814.4 0.04 0.8.366 Interaction (A x C) 28714 1.5 0.2217 Interaction (B x C) 1179.6 0.06 0.8040 Gonad Factor A (origin) 2.25E6 126 0.0000 Factor B (feeding regime) 324793 18.2 0.0000 Factor C (time) 349092 19.5 0.0000 Interaction (A x B) 7675.7 0.43 0.5132 Interaction (A x C) 1445.8 0.08 0.7765 Interaction (B x C) 339690 18.9 0.0000 DF. degrees of freedom; MS, mean .square: F, ratio; and P, probability. all conditions are summarized in Table 2. Statistical analyses (Table 4) for the period at lO'C showed a significant effect of feeding regimen, place of origin, and time. In the treatment with food, there was a higher proportion of growing oocytes than in the treatment without food. The production of growing oocytes was significantly higher in BV oysters, and there was a significant increase of growing oocytes over time. For the conditioning pe- riod, we found a significant effect of treatment (the proportion of mature oocytes was significantly lower in treatment 4) and time (mature oocytes increased significantly with time). Nevertheless, there was not a significant effect of place of origin for tiiature oocytes (Table 4). Spermatogenesis The number of male oysters found in this study did not allow observation of any pattern of change in spermatogenesis. The number of males detected in the four treatments is presented in Table 5. Biochemistry Direct conditioning. Place of origin and time had a significant effect on the biochemical content in both the gonad and meat of oysters (Table 3). Specimens from BV had a higher content of biochemical compounds in the gonad and meat coirtpared with oysters from LT, and the biochemical content increased signifi- cantly over time in the conditioning procedures. Feeding regitnen also had a significant effect but only on the biochemical content in the gonad. There was a higher content of proteins, lipids, and carbohydrates in the gonad of oysters conditioned with food (Table 6). Four tested conditions. Three-way ANOVA during the phase at 10°C (Table 4) showed a significant effect of feeding regimen, place of origin, and time on the biochemical content of the gonad, and a significant effect of place of origin and time on the bio- chemical content in the meat. A significantly higher biochemical content was found in the gonads of oysters maintained with food. No significant differences were noted relating biochemical content in the meat of animals kept with or without food. Significant biochemical differences in meat and gonad tissue favored the BV oysters. The biochemical content increased significantly over time. Statistical analyses (Table 4) during the conditioning procedures showed a significant effect of treatment, place of origin, and time on the biochemical content of oyster gonads. For meat, a signifi- cant effect of place of origin favored the BV specimens. Concern- ing conditioning effects on the biochemical content of gonads, the highest content was found in treatment I (BV-1 and LT-1) com- pared with the other treatments, and there were no significant differences between treatments 2 and 3. The lowest concentration was observed in treatment 4. Finally, the biochemical content was significantly higher in oysters from BV. and compound concen- trations increased significantly over time (Figs. 3 and 4). Oocyte Production and Larval Yield Direct conditioning. LT oysters conditioned without food did not produce any oocytes at the end of the direct-conditioning phase. In the other three treatments, BV oysters produced more oocytes than LT specimens. Concerning the yield of larvae (D- larvae), similar values were obtained (85% and 78%) for fed oys- ters coming from both sources and a yield of larvae (62%) in BV .specimens conditioned without food. There were no significant 726 Chavez-Villalba et al. TABLE 4. Results of three-way ANOVA tests for early, growing, and mature oocyte categories, and for biochemical content in the gonad and meat of oysters C. gigas in the period at WC and conditioning. TABLE 4. continued Source of Variation Df MS Oogenesis Period at 10°C Early gametogenesis stage Factor A (origin) Factor B (feeding regime) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Growing stage Factor A (origin) Factor B (feeding regime) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Mature stage Factor A (origin) Factor B (feeding regime) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Conditioning Early gametogenesis stage Factor A (treatment) Factor B (origin) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Growing stage Factor A (treatment) Factor B (origin) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Mature stage Factor A (treatment) Factor B (origin) Factor C (time) Interaction (A x Bl Interaction (A x C) Interaction (B x C) Biochemistry Period at 10°C Meat Factor A (origin) Factor B (feeding regime) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Gonad Factor A (origin) Factor B (feeding regime) Factor C (lime) 1 1016.3 4.36 0.0491 1 1S08.2 7.76 0.0111 1 2378.y 10.2 0.0043 1 21.03 0.09 0.7668 1 7.38 0.03 0.8604 1 131.58 0.56 0.4607 1 993.1 4.33 0.0499 1 1 768.2 7.71 0.0113 1 2343.1 10.2 0.0043 1 25.6 (J. 11 0.7417 1 9.51 0.04 0.8406 1 120.9 0.53 0.4758 1 2.57 0.45 0.5079 1 4.3 0.76 0.3938 1 2.57 0.45 0.5079 1 4.3 0.76 0.3938 1 2.57 0.45 0.5079 1 4.3 0.76 0.3938 3 2193.3 16.9 0.0000 1 2152.8 16.6 0.0000 1 16.M0 126 0.0000 3 353.2 2.73 0.0523 3 552.8 4.27 0.0087 2 39.2 0.3 0.5842 3 546.5 2.97 0.0395 1 354 1.92 0.1711 1 561.4 3.05 0.0862 3 340.6 1.85 0.1486 3 2121.5 11.5 0.0000 1 560.7 3.04 0.0864 3 2396.8 26.7 0.0000 1 701.3 7.81 ().()()71 1 29084 324 0.0000 3 55.9 0.02 0.6030 3 2337.6 26 1 0.0000 1 614.6 6.85 0.0114 1 2.73E6 100 0.0000 1 21011 0.77 0.3816 1 224169 8.22 0.0048 1 8410.4 0.31 0.5796 1 5842.8 0.21 0.6442 I 2307.9 0.08 0.7716 1 1.9E6 229 0.0000 1 149781 18 0.0000 1 1270S6 15.3 0.0008 c tnlinucd Source of Variation Df MS Interaction (A x B) Interaction (A x C) Interaction (B x C) Conditioning Meat Factor A (treatment) Factor B (origin) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) Gonad Factor A (treatment) Factor B (origin) Factor C (time) Interaction (A x B) Interaction (A x C) Interaction (B x C) 1 39084 4.7 0.0318 1 20100 2.42 0.1222 1 96709 11.6 0.0008 3 32563 1.02 0.3829 1 5.02E6 158 0.0000 1 147545 4.63 0.0321 3 651.59 2.14 0.0950 3 10153 0.32 0.8119 1 236027 7.41 0.0068 3 492786 17.9 0.0000 I 5.5 1E6 201 0.0000 1 308652 11.3 0.0009 1 32763 1.2 0.3113 3 307844 11.2 0.0000 1 324.5 0.01 0.9134 Df, degrees of freedom; MS, mean square; F, ratio; and P. probability. differences in oocyte production among the three treatments in this part of the experiment (Fig. 5). Four tested conditions. The highest oocyte production oc- curred in oysters fed during the experiment, in particular in speci- mens under conditions BV-I and BV-3. Specimens raised under condition LT-4 (without food) did not produce any oocytes by the end of the e.\periment. The highest yield of larvae was detected in BV and LT oysters that were not fed during the cold phase and in oysters raised with food during conditioning. The lowest yield of larvae was observed in specimens from BV maintained under treatment 1, even though the mean oocyte production in these oysters was 42.5 million. The statistical analysis showed that treat- ments involving feeding produced significantly more oocytes than did treatments involving oysters kept without food, with the high- est values favoring BV oysters. There are no significant differ- ences in oocyte production among unfed oysters (Fig. 5). DISCUSSION Oocytes in early gametogenesis and growing stages in BV oys- ter samples were observed at the beginning of February in our laboratory in previous experiments (1999). These oocyte catego- ries were detected later in LT oysters (Chavez-Villalba et al. 2002), which is in agreement with the results of Lango-Reynoso (1999), who found oocytes in the growing stage in oysters from Marennes-Oleron (near LT) by the end of February 1998. In this study, oocytes in early gametogenesis and growing stages in both oyster samples were detected at the beginning of the direct con- ditioning (February 2000), showing that the oysters in LT began gametogenesis earlier in the year. Even though similar proportions of early gametogenesis and growing oocytes were measured at the beginning of conditioning in both samples, by the end of condi- tioning lower proportions of mature oocytes were found in LT oysters in both treatments, indicating differences in the environ- mental patterns regulating the beginning of gametogenesis be- tween these samples. With decreasing latitude, the temperature rec^uired for the ini- Gametogenesis and Larval Production in Ch-^ssostrea gigas 727 TABI.K 5. Number of male oysters (C. gigas) and their respective developmental stage found during the four treatments. 10 C 0 Davs 30 Davs 60 Days BV LT B\ LT BV LT End C 1 10 Days BV LT Treatment 1 Early gametogenesis Growing Mature Treatment 2 Early gametogenesis Growing Mature — Treatment 3 Early gametogenesis Growing Mature Treatment 4 Early gametogenesis Growing Mature End C. end ot conditioning. tiation of the ganietogenic cycle increases, and. as a result, repro- ductive cycles occur later in the year (Barber and Blake 1983). This study showed different responses between northern and southern oysters to food and temperature during the period con- sidered, such as the beginning of gametogenesis in C. gigas. Oys- ters from BV (in the north of France), within a high productivity ecosystem (Goulletquer et ul. 1996). acclimated to colder water than southern populations, developed mature oocytes after 60 days TABLE 6. Protein, carbohvdrate. and lipid content (ing/e(|ui\alent tissue") in the gonad and meat of C. gigas samples from t"o culture sites at the beginning and by the end of the direct conditioning experiment conducted under two types of conditions: with and without food. Conditioning Oyster Sample Tissue Compound Beginning End Conditioning Characteristics BV Gonad Meat LT Gonad Meat Carbohydrates Proteins Lipids Carbohydrates Proteins Lipids Carbohydrates Proteins Lipids Carbohydrates Proteins Lipids 283 ± 27 264 ± 1 1 113 ±7 195 ± 2:1 253 ± 17 45 ±2 5+1.5 47+12 8±2 15 ±3.5 93 ± 1 1 3 1 + 4.5 211 + 16 With lood 256 ± 37 Without food 732 ± 58 With food 307 + 28 Without food 278 ± 30 With lood 119 ±23 Without food 144 ±28 With food 206 ± 32 Without food 557 ± 34 With food 502 ±31 Without food 128 ±16 With food 106 ±20 Without food 70 ±9 With food 16 ±5 Without lood 341 ±.% With food 63 ±9 Without food 73±1I With food 66 ±37 Without food 36 ±6 With food I8±7 Without food 235 ± 30 With food 262 ± 47 Without food 40 ±5 With food 53 ± 20 Without food " Presented as mean ± SE in a sample size of 10 oysters. 728 Chavez-Villalba et al. 0) cn E 0) *-» c o u 15 o E O o £0 ••♦■ Carbohydrates Meat Proteins Treatment 1 4- Lipids Gonad Treatment 2 — « Treatment 3 Treatment 4 120 30 60 90 120 Time (days) Figure i. Protein, carbohydrate, and lipid content in the meat and gonad of C gigas from the BV site during the four treatments (10°C for the period of O-fit) days, and conditioning at 19 C for the period of 6(>-110 days). at 10°C when maintained with food. When l (U 60 (D 40 c- 03 _1 20 0 50 fe 40 X c o o 30 3 ■n o o. 20 (II >^ (> o O 10 -II- T T T T r^ rn L "^ 1 //- w wo w wo BV LT Direct conditioning BV LT Treatments Figure 5. Oocyte production and larval yield (D-larvae) of C f^igas conditioned in February to March 2000 (direct conditioning at 19°C: W ■■ with food; WO = s>ithout food). age composition during autumn and winter, but with the highest amounts in the gonad from April to June. Even though BV oysters were exposed to conditions that affected physiologic activities, they have an important quantity of reserves, using carbohydrates as the principal source to support gametogenesis under all conditions. In contrast. LT oysters used proteins to fuel gamete development. Probably, southern oysters used proteins because they have a net loss of glycogen during the winter, when it may be catabolized to meet maintenance requirements during poor food conditions (Deslous-Paoli & Heral 1988). Whyte et al. (1990) found that protein can contribute more than carbohydrates for the metabolic processes of oysters maintained in unfavorable food conditions. Moreover, Barber and Blake (1983) found that the source of re- productive energy for Argopccten imuUans over its latitudinal range could be affected by food supply and metabolic rates. These authors suggest that, with decreasing latitude, the bay scallop has a greater metabolic rate as well as a smaller food supply, with less energy available for reproduction. This concept may be true for this study if northern oysters are in more favorable food conditions than southern populations, and that metabolic rates in this species are influenced by temperature (Bougrier et al. 1995) BV and LT oysters conditioned with food produced more oo- cytes by the end of the conditioning period than those conditioned without food. Similar results in C. gi^os were obtained by Rob- inson (1992). These results indicate that the oocyte quantity pro- duced under controlled conditions is dependent on the food offered during conditioning, since the oysters fed during the phase at lO^C. but conditioned without food, have produced significantly fewer oocytes than animals conditioned with food, but maintained with- out food during the cold phase. Oocyte production was signifi- cantly lower in the oysters kept with food during the direct- conditioning procedure than in oysters maintained in treatments 1 and 3 (BV-1, BV-3 and LT-1, LT-3, respectively). If it is consid- ered that oysters in treatments 1 and 3 were maintained for 60 days at the same temperature as that at the beginning of the experi- ments (10°C), then the difference in terms of oocyte production may indicate that animals during the cold phase continue their oogonie multiplication with or without the influence of food. It would be interesting to study changes at the cellular level and try to quantify oogonie multiplication under similar experimental con- ditions. Le Pennec et al. ( 1990) found a significant relationship between the lipid index of oocytes in Pecieii nicLxiiniis and the parameters involved in the endotrophic phase of larval rearing. They empha- size that D-larvae and the anomaly rates of prodisoconch I are strongly related to the mean lipid index; the greater the lipid con- tent in the oocytes, the greater the quality of larval rearing required in the first 2 days of culture. We observed that lipids accumulated in the gonads of oysters conditioned with food, and these animals produced more oocytes than oysters maintained without food, but the larval yield of the two groups was similar. Muranaka and Lannan (1984) observed higher fecundity rates in oysters condi- tioned with food when compared with oysters conditioned without food. Nevertheless, the results of this study did not show signifi- cant differences in larval yields between samples conditioned with or without food. On the contrary, the lowest larval yield was found in the oysters kept in condition 1 . These observations suggest that lipid reserves in unfed oysters are maintained even in conditions of the absence of food, and, although there are few oocytes in these animals, these gametes will yield good quality D-larvae. In previ- ous experitnents, Chavez- Villalba et al. (2003) observed that unfed oysters not only yielded good-quality D-larvae but that larvae pre- sented similar growth and survival patterns as larvae from fed animals throughout 19 days of trials. Thus, oocyte quality seems to be related not only to food quality during conditioning, but also to reserves accumulated in nature prior to experiments. Gametogenesis and Larval Production in Chassostrea gigas 731 In this study, oysters having the same place of origin show flexible reproductive patterns that are responses to \arying envi- ronmental factors, most notably food availability. Northern oys- ters, having a larger reserve stock than southern oysters, initiate gamete development in conditions of low temperature, which con- firms that the beginning of gametogenesis is not dependent on thermal conditions. The amount of gametogenic material is also dependent on food supply, but oocyte quality seems to depend, to a large extent, on accumulated reserves. The differences found in this study are that the stored reserves in BV oysters are used to initiate gametogenesis and to maintain metabolism under low food a\ ailability. while LT oysters operate closer to their energetic limit at the production site, and require supplementary energy from spring planktonic blooms to continue gametogenesis and to pro- duce \ lable oocytes and larvae. ACKNOWLEDGMENTS We thank Consejo Nacional de Ciencia y Tecnologi'a (Mexico) for a scholarship to Jorge Chavez-Villalba for doctoral studies at the Universite de Brelagne Occidentale. France. Experimental work was supported by IFREMER/Contrat Universitaire Univer- site de Bretagne Occidentale (UBO) project No. 98/2.'i21426. We are grateful to Christian Mingant for \ery helpful technical assis- tance during the experiments. The editing staff at Centro de In- vestigaciones Biologicas del Noroeste (CIBNOR) reviewed and improved the English text. LITERATURE CITED Allen. S. K. & D. Bushek. 1992. Large scale production of triploid oysters Crassostrea vlrginica (Gnielin) using "stripped" gametes. Aqticicitltiire 103:241-251. Barber, J. B. & N. J. Blake. 1983. Growth and reproduction of the hay scallop. Argopecten iriadians (Lamarck) at its southern distributional limit. / Exp. Mar. Biol. Ecol. 66:247-256. Barber. J. B.. S. E. Ford & R. N. Wargo. 1991. Genetic variation jii the timing of gonadal maturation and spawning of the Eastern oyster. Cnis- sostrect virginica (Gmelin). Biol. Bull. 181:216-221. Berthelin. C, K. Kellner & M. Mathieu. 2000. Storage metabolism in the Pacific oyster { Cra.Mo.sY/prt gigas) in relation to summer mortalities and reproductive cycle (West coast of France). Coinp. Biochcin. Pliy.siol. 125:359-369. Bligh, E. G. & W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Bioch. Physiol. 37:911-917. Bougrier. S.. P. Geairon. J. M. Deslous-Paoli. C. Bacher Ik. G. Jonquieres. 1995. Allometric relationships and effects of temperature on clearance and oxygen consumption rates of Crassostrea gigas (Thunberg ). .Aqua- culture 134:143-154. Chavez-Villalba, J.. 2001. Conditionnement experimental de I'huitie Cras- sostrea gigas. Doctoral Thesis. Brest. France: Universite de Bretagne Occidentale. 186 pp. Chavez-Villalba. J.. J. Barret. C. Mingant. J. C. Cochard & M. Le Pennec. 2002a. Autumn conditioning of the oyster Cras.sostrea gigas: .\ new approach. .Aqiiaculture 210:171-186. Chavez-Villalba. J.. J. Pommier. J. Andriamiseza. S. Pouvreau. J. Barret. J. C. Cochard & M. Le Pennec. 2002b. Broodstock conditioning of the oyster Crassostrea gigas: origin and temperature effect. Aquaculiure 214:115-130. Chavez-Villalba. J.. J. Barret. C. Mingant. J. C. Cochard & M. Le Pennec. 2003. Influence of timing of broodstock collection on conditioning, oocyte production, and larval rearing of the oyster Crassostrea gigas (Thunberg) at six production sites in France. / Shellfish Res. Deslous-Paoli. J. M. & M. Heral. 1988. Biochemical composition and energy \alue of Crassostrea gigas (Thunberg) cultured in the bay of Marennes-Oleron. Aquat. Living Resour. 1 :239-249. Dinamani. P. 1987. Gametogenic patterns in populations of Pacific oyster. Crassostrea gigas. in Northland. N. Z. .Aquaculiure 64:65-76. Dubois. M.. K. Gilles. J. Hamilton. P. Rebers & F. Smith. 1956. Colori- metric method for determinations of sugars and related substances. Anal. Chem. 28:350-356. Goulletquer. P.. J. P. Joly. A. Gerard. E. Le Gagneur. J. Moriceau. J. M. Peignon. S. Heurtebise & P. Phelipot. 1996. Performance of triploid Pacific oysters Crassostrea gigas (Thunberg) reared in high carrying capacity ecosystem: Survival, growth and proximate biochemical com- position. Haliotis 25:1-12. Lango-Reynoso. F.. 1999. Determination de la sexualite chez I'huitre Cras- sostrea gigas (Thunberg. 1793). Doctoral Thesis. Brest. France: Uni- versite de Bretagne Occidentale. 139 pp. Lango-Reynoso. F.. J. Chavez-Villalba. J. C. Cochard & M. Le Pennec. 2(J00. Oocyte size, a means to evaluate the gametogenic development of the Pacific oyster. Crassostrea gigas (Thunberg). Aquaculture 190: 18.3-199. Le Pennec. M.. F. Gueguen. J. C. Cochard. Y. M. Paulet & G. Dorange. 1990. Relations entre le contenu lipldique des ovocytes de Pecten ma.\imus (Mollusque. Bivalve) et les performances des larves en el- evage. Haliotis 10:101-113. Lowry. O.. N. Rosenhrough, A. Farr & R. Randal. 1951. Protein measure- ments with the folin phenol reagent. J. Biol. Chem. 193:265-275. Luhet. P. 1976. Ecophysiologie de la reproduction chez les mollusques lainellibranches. Haliotis 7:49-55. MacDonald. B. A. & R. J. Thompson. 1988. Intraspecif'ic variation in growth and reproduction in latitudinally differentiated populations of the giant scallop Placopecten magellanicus (Gmelin). Biol. Bull. 175: 361-371. Martoja. R. & M. Martoja-Pearson. 1967. Initiation aux techniques d'histologie animale. Paris: Mason et Cie. Muranaka. M. S. & J. E. Lannan. 1984. Broodstock management of Cras- sostrea gigas: Environmental influences on broodstock conditioning. /I c/HflCH/nire 39:217-228. Pastoureaud. A.. M. Herat. P. Prou. D. Razet & P. Russu. 1996. Particle selection in the oyster Crassostrea gigas (Thunberg) studied by pig- ment HPLC analysis under natural food conditions. Oceanol. Acta 19(l):79-88. Robert. R. cS: A. Gerard. 1999. Bivalve hatchery technology: The current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maxinms in France. Aquat. Living Resour. 12:121-130. Robinson. A. 1992. Dietary supplements for reproductive conditioning of Crassostrea gigas kumamoto (Thunberg). I. Effects on gonadal de\el- opment. quality of ova and lar\ae through metamorphosis. / Shellfish Res. Il(2):437^t4l. Ruiz. C. M. Abad. F. Sedano. L. O. Garcia-Martin & J. L. Sanchez-Lopez. 1992. Influence of seasonal environmental changes on the gamete pro- duction and biochemical composition of Crassostrea gigas (Thunberg) in suspended culture in El Grove. Galicia. Spain. J. E.xp. Mar. Biol. Ecol. 155:249-262. Sastry, A. N. 1968. The relationships among food, temperature, and gonad development of the bay scallop .Aequipecten irradians Lamark. Phys- iol. Zool. 4 1 :44-53. Sastry. A. N. 1979. Pelecypoda (excluding Ostreidae). In: J. C. Giese & J. S. Pearse, editors. Reproduction of marine invertebrates, vol. 5. New York: Academic Press. Thompson. R. J.. R. I. E. Newell. V. S. Kennedy & R. Mann. 1996. Reproductive processes and early development. In: V. S. Kennedy. R. 1. E. Newell. & A. F. Eble. editors. The eastern oyster Crassostrea virginica. College Park. MD: Maryland Sea Grant Book. pp. 335-370. Whyte, J. N. C, J. R. Englar & B. L. Carswell. 1990. Biochemical com- position and energy reserves in Crassostrea gigas exposed to different levels of nutrition. Aquaculture 90:157-172. J.mriHil of Shclljhl' Research. Vol. 22. No. 3. 733-73&. 2003. TWO SPECIES OF OYSTER LARVAE SHOW DIFFERENT DEPTH DISTRIBUTIONS IN A SHALLOW, WELL-MIXED ESTUARY PATRICK BAKER* Viri^iiila Institute oj Murine Science. College of William and Mary. Gloucester Point. Virginia. 23062 ABSTRACT The vertical distribution of late stage, or pediveliger. larvae of several bivalve mollusks was examined in a west Florida estuary. The study site was an artificial canal, and the water was shallow (1.5 m) and well mixed, with only modest cuncnts. Pediveligers of three bivalve taxa were collected: the eastern oyster Crassostrea virginica: the crested oyster OsHea ec/iu'stris: and unidentified shipworms (Teredinidae). Despite the shallow and well-mixed water column, larvae exhibited vertical zonation. with most larvae of all three species collected from lower in the water column. The larvae of C viri^inicu and shipworms showed no significant effect of time of day. but larvae of O. eqiiestris reversed their distribution pattern at night, with most larvae being near the surface. Pediveliger larvae were not behaving as neutrally buoyant particles but appeared to regulate their depth even in this well-mixed and shallow water column. Given that the larvae of the two oyster species were probably coinpelent to settle, their vertical distribution patterns do not fit what has been reported about their adult depth distribution. KEY WORDS: Crassostrea viri;iiuca. estuary, larvae. Oslrea cc/iwslris. pediveliger. plankton. Teredinidae INTRODUCTION A variety of studies over the years have attempted to address the issue of whether larval distribution in estuaries is controlled mainly by hydrologic forces, or whether there is a significant larval behavioral component that also affects distribution. For some crus- tacean larvae, the case seems to be fairly well made that behavior plays a large part in planktonic distribution, usually (but not al- ways) for late-stage larvae or post-larvae (.Shanks 1986. 1995. Benfield & Aldrich 1992. Gherardi 1995). Bivalve mollusks also have been the focus of studies on larval distribution in estuaries, but there is no consensus in the literature on whether bi\alve veligers are distributed as neutrally buoyant particles or whether behavior significantly affects their distribu- tion. Like crustacean larvae, bivalve larvae clearly exhibit oriented swimming, at least in the laboratory (Feeny 1984, Hidu & Haskin 1978). .Some field studies have appeared to show nonrandom bi- valve larval distribution, relative to hydrodynamic processes (Tremblay & Sinclair 1990, Shanks et al. 2002. Baker & Mann 200.^). Compared with crustacean postlarvae. however, bivalve pediveligers are small and slow swimming, and Banse (1986) questioned whether the weak swimming rates observed for these larvae are sufficient to produce distribution patterns. The distribu- don of bivalve larvae in estuaries may be attributed to hydrody- namic processes alone in some cases, if larvae are treated as neu- trally buoyant particles (Wood & Hargis 1971, Mann 1988). This author examined the above question (i.e.. does bivalve larval distribution in an estuary have a behavioral component?) under the most restrictive conditions possible for an estuarine sys- tem. The estuarine system in question was simple in shape (an artificial inlet), very shallow, and well mixed throughout the study, although it was a low-energy system. Only late-.stage bivalve lar- vae were included in the study. If bivalve larvae behave as neu- trally buoyant particles, their distribution should be fairly even throughout the water column (allowing for boundary-layer ef- fects), and the species should have similar distributions. 'Present address: Department of Fishenes and Aquatic Sciences. Institute of Food and Agricultural Sciences. University of Florida. P.O. Box 110600. Gainesville. FL 32611. E-mail: pbaker@mail.ifas.un.edu MATERIALS AND METHODS Research was conducted at the Harbor Branch Oceanographic Institute, near Foil Pierce. FL. in May 1993. The study site was about halfway along a I -km artificial canal that opened into the Indian River Lagoon. The sides of the canal were concrete and steel seawalls, heavily fouled by eastern oysters. Crassostrea rir- ginica. and the mean water depth at the wall were about 1 m. gradually increasing toward the center of the canal. The observed currents were mostly tidal, with velocities near the seawalls of 1 to .^ cm s"'. and the tidal range was up to 0.5 m. Plankton was sampled with two modified 12-V bilge pumps, each rated at 1 800 L h~ ' . Power came from a standard 1 1 0- V outlet with a transformer to regulate voltage. Pumps were suspended about 2 m out from the canal wall, where the mean water depth was about 1.5 m. One pump was maintained at a depth of about 20 cm above the bottom, which was determined by preliminary samples to be the maximum depth achievable without entraining significant quantities of sediment. The other pump was adjusted for each sampling episode to a depth of about 20 cm below the surface. Mann (1986) and Mohlenberg (1987) found no avoidance of a plankton pump intake by bivalve mollusk larvae, which swim slowly compared with many /ooplankton. Water from each pump was delivered by a garden hose to a separate sieve on the banks of the canal. Each sieve consisted of a 400-|ji,m coarse filter and a 150-|xm final filter on which the sample was retained. Plankton was sampled twice daily, at mid-morning (full daylight) and mid-evening (after nightfall), for about 2 h at a time. The volume sampled at each depth was calculated from the time, to the nearest minute, multiplied by the mean pumping rate. The pumping rate was estimated before and after each sample, for each pump, by the time required to fill a 20-L container. (If sam- pling episodes included high or low water, the pumping rate mea- surements also were taken then and factored into volume calcula- tions.) Samples were taken into the laboratory, and bivalve larvae were counted and identified to the lowest possible taxonomic level. The identification of oyster pediveligers (C virgiiiica and Os- trea eqiiestris) was verified by collecting newly settled juveniles on shell-strings (Haven & Fritz 1985) that had been immersed at the study site for <24 h. marking individuals, and letting them grow in the canal for .several weeks. By the end of this time. O. 73.3 734 Baker ecjuestris shells had developed the diagnostic dorsal-marginal den- tition, or chomata (Galtsoff & Merrill 1962). Only samples that had six or more pediveligers of a given taxa from the two pumps combined were used in the analysis. Data for each pump were converted to proportions of total larvae of a given species collected in a sampling episode and were arcsine-square root-transformed prior to statistical analysis (Zar 1996). Analysis of variance tests were used to test null hypotheses of equal pro- portions of larvae collected by either pump (top vs. bottom) at either time of day (morning vs. evening), with no interaction (Zar 1996). RESULTS Two species of oyster larvae were collected in plankton samples on the majority of days sampled: the eastern oyster. C. virgiiiica: and the crested oyster, O. equvstris. Pediveligers. or late-stage larvae, of these species could be distinguished on the basis of shape (O. equestris pediveligers were nearly identical to those of C. virguiica in size but were more rounded, with a broader, less pronounced umbo). Living pediveliger larvae were clearly distinguishable on the basis of color. C virgiiiica pedive- ligers at this site were tan to brown and opaque, while O. equestris pediveligers were transparent except for their visceral masses, which were green to brown. The only other common bivalve lar- vae were shipworms (Teredinidae) of unknown species, which were treated in this study as if they were a single taxon. Uniden- tified pediveligers of other bivalve taxa were occasionally col- lected. The abundance of all three species was highly variable, but fairly low. C. virginica and O. equestris reached peak densities of just over 1 2 per m \ but teredinids peaked at less than half of that. All three taxa showed peak densities near the beginning of the study. Density data for all three taxa from the lower intake are shown in Fig. 1. The plankton pumps at the two sample depths did not collect equal densities of larvae, for any species. About 85% of C. vir- giiiica pediveligers and 759f of teredinid pediveligers were col- lected from the bottom pump, and time of day had no significant effect. During the day. the distribution patterns for O. equestris pediveliger larvae appeared to be similar to the above taxa. but at night 61% of O. equestris pediveligers were collected by the near- surface pump. Thus, for O. equestris. abundance differed signifi- cantly for neither time of day nor depth, but the interaction of depth and time of day was significant at a = 0.05. The proportions for each species collected for each time and daylight treatment are presented in Table 1. and the results of the analysis of variance are presented in Table 2. DISCUSSION The above study ro.se serendipitously from an attempt to locate an estuarine environment in which oyster pediveliger larvae (C virginica) were randomly distributed throughout the water column, for a separate study (Baker 1993). Clearly, nonrandom distribution complicates the effort to quantify the larval supply. Yet. even in this highly simplified estuarine environment, in <2 m of water, all three bivalve taxa exhibited strong vertical distribution patterns. The vertical distribution patterns from this study were similar to those observed for C. virginica and teredinid larvae in a more complex estuarine environment in Virginia (Baker 1993). The ma- jor difference noted from that prior study was the effect of time of day on the distribution of O. equestris larvae; no effects of time of 14 12 10 8 6 4 2 Crassostrea virginica * • Sti • •> ♦« 100 200 300 400 500 600 700 (0 c O 0. 16 14 12 10 8 6 4 2 « Ostrea equestris ♦ ♦ ♦ « ♦ ♦ # ♦ • X * ♦ ♦ ♦ • ♦ ♦ ♦ ♦ -•- _^ ♦ ♦^» ♦ 100 200 300 400 500 600 700 Teredinidae 0 . — • #4. * •>»... • •♦♦ ♦ 100 200 300 400 500 600 700 Elapsed Time (h) Figure 1. .\bundance (density per cubic meter) of three taxa of bivalve pediveligers at the Harbor Branch Oceanographic Institute canal dur- ing May 1993. from the near-bottom plankton pump. day were reported for any species in the Virginia study. The sparseness of pediveliger larvae also was noted by Carriker ( 1 95 1 ), who collected only 56 pediveligers from > 14.500 C. virginica larvae across six samples. TABLE 1. Mean proportional ( % ) abundances of three taxa of bivalve pediveligers at two times (morning vs. evening) and two depths (top vs. bottom! in the Harbor Branch Oceanographic Institute canal during May 1993. Taxon Depth Morning Evening All Times C. vir^intLii Top 14.6 (18.8) 16.8 (30.3) 15.5 (24.5) Biittom 85.4 (18.8) 83.2 (30.3) 84.5 (24.5) (H = 19) (" = 13) in = 32) O. equestris Top 18.6(29.4) 61.0(41.9) 33.2 (38.6) Bottom 814(29.4) 39.0(41.9) 66.8 (38.6) (" = 19) Ui = 10) (« = 29) Unidentitled teredinids Top 23.2 (39.1) 26.8 (334) 24.2 (38,3) Bottom 76.8 (.39.1) 73.2 (33.4) 75.8 (38.3) (» = 20) (n = 8) (n = 28) SDs are given in parentheses. Bivalve Larval Depth Distribution in an Estuary 735 TABLE 2. Summary of analyses of variance for the effects of time of day (morning vs. evening) and depth (lop \s. bottomi on proportional abundance of three ta\a of bivalve pediveligers in the Harbor Branch Oceanographic Institute canal during May I'n^. Source DF Seq SS Adj SS Adj MS F Value P Value Analysis of variance Time of day for C. vlrfiinica 1 7.779 7.779 7.779 1.89 0.174 Depth 1 46.685 36.225 36.225 8.81 0.004 Time x depth EiTor 1 58 7.967 238.417 7.967 238.417 7.967 4.111 1.94 0.169 Total 61 300.848 Analysis of variance Time of day Depth for O, ecjitestris 1 1 10.701 41.905 10.701 15.269 10.701 15.269 1.39 1.98 0.244 0.165 Time x depth E[Tor 1 54 52.386 416.069 52.386 416.069 52.386 7.705 6.80 0.012 Total 57 521.062 Analysis of variance Time of day Dep(h for teredinids 1 1 0.0161 16.6334 0.0161 10.9201 0.0161 10.9201 0.02 11.26 0.898 0.001 Time X depth Error 1 52 0.7875 50.4180 0.7875 50.4180 0.7875 9.9696 0.81 0.372 Total 55 67.8550 Seq SS = sequential sum 1 >r squares; Adj SS = adjusted sum of squares; Adj MS = adjusted mean square. Several authors have reported the vertical stratification of bi- valve larvae in estuaries (Nelson 1927. Perkins 1932. Wood & Hargis 1971. Sekiguchi et al. 1991 ), although they did not attempt to demonstrate that this was due to larval behavior. Vertical strati- fication or the migration of bivalve larvae also has been observed in the absence of estuarine stratification (Tremblay & Sinclair 1990. Raby et al. 1994), but those studies were in systems signifi- canlK deeper than l.S in. Dekshenieks et al. { 1996) modeled C. virt^inica larval distribu- tion in the water column of a well-mixed estuary, and predicted, as observed here, that the majority of late-stage larvae would be within a meter of the benthos. As larvae grow, they sink faster (due to an increased shell/cilia ratio), and the swim-sink behavioral pattern observed for this species by Hidu and Haskins (1978) would result in a net sinking rate for older larvae, according to the model (Dekshenieks et al. 1996). The above model, however, does not include bottom avoidance; larvae must either increase swim- ming rates in response to the benthos or spend a certain amount of time resting on the benthos. The latter behavior (except for benthic explorations by competent-to-settle larvae; Prytherch 1934. Cran- field 1973) has not been reported, and increased contact with the benthos also exposes the larva to a new guild of predators (Breese & Phibbs 1972. Steinberg & Kennedy 1979. Cowden et al. 1984. Osman et al. 1989. Andre et al. 1993). It is likely, therefore, that size-related sinking/swimming ratios provide only a partial expla- nation for pediveliger distribution in C. virginica. O. equestris pediveligers. which in this study were about the same size as C. virginica pediveligers. were not constrained to the lower reaches of the water column by the weight of their shell, at least not during the night. If pediveliger larvae were no more than negatively buoyant particles, they could not remain in the water column in a low- energy environment. If they were neutrally buoyant particles, they would be distributed evenly in a well-mixed water column. None of the species observed in this study were evenly distributed, and one species (O. equestris) differed from the others, altering its depth distribution on a diurnal cycle. Thus, while neutral buoyant (nodels may be sufficient to describe broad distribution patterns (Wood & Hargis 1971. Mann 1988). ciliated larvae are clearly not inert particles, and species-specific larval behavior must be in- voked to describe at least some scales of distribution. ACKNOWLEDGMENTS Funding for this study was provided by the Commonwealth of Virginia through the Virginia Institute of Marine Science Bivalve Ecology program. The Smithsonian Marine Station (then at Link- port) and the Harbor Branch Oceanographic Institute (HBOI) gra- ciously provided us with the use of their facilities for this study. Technical assistance was pro\ ided by Sherry Reed and other mem- bers of the Smithsonian Marine Station staff. Gratitude is also expressed to the alligators in the HBOI canal for restraining their territorial and predatory tendencies when I had to enter the water at night to service equipment. Andre, C, P. R. Jonsson & M. Lindegarth. 1993. Predation on settling bivalve larvae by benthic suspension feeders: The role of hydrodynam- ics and larval behaviour. Mar. Ecol. Pmi'. Scr 97:183-192. Baker. P. 1993. Quantification of settlement and recruitment processes in bivalve mollusks. Ph.D. Thesis. Williamsburg. VA: College of Vv'illiam and Mary. 381 pp. LITERATURE CITED Baker. P. & R. Mann. 2003. Late stage bivahe larvae in a well-mixed estuary are not inert particles. Estuaries 26:837-845. Banse. K. 1986. Vertical distribution and hori/onlal transport of planktonic larvae of echinoderms and benthic polychaetes in an open coastal sea. Bull. Mar Sci. 39:162-175. Benfield, M. C. & D. V. Aldrich. 1992. Attraction of postlar\al Penaeus 736 Baker aztecits Ives and P. seriferus (L.) (Crustacea: Decapoda: Penaeidae) to estuarine water in a laminar-How choice chamber. / £v/). Mar. Biol. Ecol. 156:39-52. Breese. W, P. & F. D. Phibbs. 1972. Ingestion of bivalve molluscan larvae by the polychaete annelid Polydora ligni. Veliger 14:274-275. Carriker, M. R. 1951. Ecological observations on the distribtition of oyster larvae in New Jersey estuaries. Ecol Monogr. 21:19-38. Cowden, C, C. M. Young & F.-S. Chia. 1984. Differential predation on marine invertebrate larvae by two benthic predators. Mar. Ecol. Prog. Ser. 14:145-149. Cranfield. H. J. 1973. Observations on the behaviour of the pediveliger of Ostrea ediili.s during attachment and cementing. Mar. Biol. 22:203- 209. Deshieneks, M. E.. E. E. Hofmann, J. M. Klinck & E. N. Powell. 1996. Modelling the vertical distribution of oyster larvae in response to en- vironmental conditions. Mar. Ecol. Prog. Ser. 136:97-110. Feeny, C. F. 1984. Effects of salinity on the vertical distribution of the larvae of Crassostrea virginicu (Gmelin) and Ostrea equestris (Say). J. Shellfish Res. 4:88-89. Galtsoff. P. S. & A. S. Merrill. 1962. Notes on shell morphology, growth, and distribution of Ostrea equestris Say. Bull. Mar. Sci. Gulf Carihh. 12:234-244. Gherardi. F. 1995. Hermit crab larval behavior: Depth regulation in Dis- corsopagurus schmitti. J. Exp. Mar. Biol Ecol. 192:107-123. Haven, D. & L. W. Fritz. 1985. Setting of the American oyster Crassostrea virginica in the James River, Virginia. US.^: Temporal and spatial distribution. Mar. Biol 86:271-282. Hidu, H. & H. H. Haskin. 1978. Swimming speeds of oyster larvae Cras- sostrea virginica in different salinities and temperatures. Estuaries 1: 252-255. Mann, R. 1986. Sampling of bivalve larvae. In: G. S. Jamieson & N. Bourne, editors. North Pacific workshop on stock assessment and man- agement of invertebrates. Canadian Special Publications in Fisheries and Aquatic Sciences. 92, pp. 107-116. Mann, R. 1988. Distribution of bivalve larvae at a frontal system in the James River, Virginia. Mar. Ecol Prog. Ser. 50:29— +4. M0hlenberg. F. 1987. A submersible net-pump for quantitative zooplank- ton sampling; comparison with conventional net sampling. Ophelia 27:101-110. Nelson, T. C. 1927. Report of the Department of Biology of the New Jersey State Agricultural Expenment Station, for the year ending June 30, 1926. New Brunswick, NJ: State of New Jersey, pp. 103-1 13. Osman, R. W., R. B. Whitlatch & R. N. Zajac. 1989. Effects of resident species on recruitment into a community: larval settlement versus post- settlement mortality in the oyster Crassostrea virginica. Mar. Ecol Prog. Ser. 54:61-73. Perkins. E. B. 1931. A study of oyster problems in Bamegat Bay: Report of the Department of Biology of the New Jersey State Agricultural E.xperiment Station, for the year ending June 30, 1930. New Bruns- wick. NJ: State of New Jersey, pp. 25-47. Prytherch, H, F. 1934. The role of copper in the setting, metamorphosis, and distribution of the American oyster, Ostrea virginica. Ecol. Monogr. 4:47-107. Raby. D.. Y. Lagadeuc. J. J. Dodson & M. Mingelbier. 1994. Relationship between feeding and vertical distribution of bivalve larvae in stratified and mi.xed waters. Mar. Ecol. Prog. Ser. 103:275-284. Shanks, A. L. 1986. Vertical migration and cross-shelf dispersal of larval Cancer spp. and Randalia oniata (Crustacea: Brachyura) off the coast of southern California. Mar. Biol. 92:189-199. Shanks. A. L. 1995. Oriented swimming by megalopae of several eastern North Pacific crab species and its potential role in their onshore mi- gration. J. Exp. Mar. Biol. Ecol 186:1-16. Shanks, A. L., J. Largier, L. Bnnk, J. Brubaker & R. Hooff 2002. Obser- vations on the distribution of meroplankton during a downwelling event and associated intrusion of the Chesapeake Bay estuarine plume. J. Plankton Res. 24:391-416. Steinberg. P. D. & V. S. Kennedy. 1979. Predation upon Crassostrea virginica (Gmelin) larvae by two invertebrate species common to Chesapeake Bay oyster bars. Veliger 22:78-84. Tremblay, M. J. & M. Sinclair. 1990. Diel vertical migration of sea scallop larvae Placopecten magellanicus in a shallow embayment. Mar. Ecol Prog. Ser. 67:19-25. Wood, L. & W. J. Hargis. Jr. 1971. Transport of bivalve larvae in a tidal estuary. In: Crisp, editor. Fourth European Biology Symposium. New York: Cambridge University Press, pp. 29—44. Zar. J. H. 1996. Biostatistical analysis. 3rd ed. Upper Saddle River, NJ: Prentice-Hall. 918 pp. Joiinml of Shellfish Research. Vol. 22. No. 3. 737-746. 2(XI3. DIOXIN/FURAN AND POLYCHLORINATED BIPHENYL CONCENTRATIONS IN EASTERN OYSTER {CRASSOSTREA VIRGINICA, GMELIN) TISSUES AND THE EFFECTS ON EGG FERTILIZATION AND DEVELOPMENT M. L. VVINTERMYER* AND K. R. COOPER Rutgers, The Stare University of New Jersey. Joint Groclinite Proi;nini In Toxicology. Pi.scataway. New Jersey ABSTItACT A 10-mo field study was conducted to evaluate the bioaccumulatioii of dioxins/furans and polychlorinated biphenyls (PCBs) in transplanted adult eastern oysters {Crassoslrea virginica. Gemliii) to Newark Bay and the Raritan Complex. New Jersey. Adult oysters (mean size 86.4 ± 14.2 mm) were deployed from September 2000 until June 2001. Oysters transplanted to Newark Bay, Anhur Kill, and Sandy Hook. NJ. accumulated 3.2/2.1. 1.3/1.7. and 0.15/2.3 parts per trillion (pptr) of 2.3.7.8-Tetrachlorodibenzo- /)-dioxin (TCDD)/2.3.7.8- Tetrachlorodibenzo-p-furan. respectively. In addition, oysters transplanted to Newark Bay. Arthur Kill, and Sandy Hook. NJ. had bioaccumulation levels of 68.6. 64.5. and 35.3 parts per billion total PCBs. respectively. The number of fertilized eggs (±SD) from strip spawned transplanted oysters from Newark Bay. Arthur Kill, and Sandy Hook. NJ. was 107 (±6.00). 54 (±36.1 1), and 1 13 (±13.61 ). respectively, and the number of unfertilized eggs was 164 (±25.6). 178 (±15.9). and 97 (±39.9). respectively. The number of veliger larvae that resulted from fertilized eggs ((7 = 100) was 3 (±1.7). 4 (±2.31), and 82 (±12.2). respectively, for Newark Bay. Arthur Kill, and Sandy Hook. NJ. Survival data from a laboratory study using an acute static 48-h hi vivo and ex vivo exposure regiment to 2.3.7.8-TCDD showed that exposure to 2 pptr dioxin caused adverse effects on egg fertilization and development. Exposure to dioxin-like compounds at the low parts per trillion ranges can result in altered gonadal development and altered embryonic development. AT;)' WORDS: Crassoslrea virginica. dioxins/furans. egg fertilization, polychlorinated biphenyls. transplant study INTRODUCTION Since the early 1970s there has been concern about the impacts of 2,3,7,8-Tetrachlorodibenzo-/)-dioxin (TCDD) and related com- pounds because of their potential hazard to humans and animals. TCDD is a byproduct of anthropogenic processes such as paper and chemical manufacturing, incineration, the manufacturing of pesticides and herbicides, the production of iron and steel, and enzymatic reactions in sewage sludge (Rappe 1992. Alonso et al. 1996, Poland et al. 1982). The most important source of TCDD for humans is food, especially diary products, meat, and fish (Pohja- virtaet al. 1994. EPA 2000). Concern about TCDD stimulated numerous studies to assess its behavior in the environment and its effects on living organisms. Studies conducted in contaminated areas have shown a positive correlation between dioxin levels in animals and their soil contact (Pohjavirta et al. 1994). Studies in aquatic model ecosysteins also have shown that TCDD and other organochlorine pollutants bio- accumulate in organisms in concentrations approximately equal to those in the sediment (Isensee et al. 1975. Chen et al. 2002). The effects of TCDD on feeding, growth, and development are most pronounced in young, growing organisms compared with adults (ASTM 1994. Davis & Herber 1969. Calabrese et al. 1973. Capuzzo. 1989, Capuzzo 1996). Because of the lipophilicity of these compounds, they are associated with lipid stores and high lipid-containing tissues (Cooper 1989. EPA 2000). Prior to spawn- ing, bivalves have a high lipid and glycogen content in gonadal tissue. Therefore, the spawning status of the bivalve would affect the amount of dioxin present over the spawning season in a similar fashion to that observed in fish (Capuzzo 1989. Vashchenko et al. 1993. Bayne et al. 1972, Bayne et al. 1978). Oysters release their gametes into the water column: therefore, planktonic lar\ae will have limited exposure to TCDD via water *Corresponding author. E-mail: margyw@eden.rutgers.edu due to the low water solubility of dioxin (EPA 2000.). Newly settled bivalve spat and adult bivalve molluscs may be exposed to TCDD through their sediment contact and feeding on resuspended materials, while the developing eggs would receive the inajority of exposure from the adult female (Cooper 1989). Bivalve embryos begin to accumulate TCDD at the two-cell embryonic stage (ASTM 1994). This may explain the sensitivity of young, growing organisms to low-level concentrations of dioxins. There has been limited work on the bioaccumulation of dioxin in the eggs of aquatic organisms. Isensee and Jones (1975) re- ported no effect of 2,3,7,8-TCDD exposures on snail egg survival, but there was a reduction in the number of viable eggs. There have been several studies on both resident and migratorv species of fish and crustaceans in New Jersey. Aquatic organisms in the tidal Passaic River were found to contain elevated levels of TCDD in the edible tissue, ranging from 38 parts per trillion (pptr) in the American eel (Angiiilla rostrata) to 476 pptr in the blue crab {Callinectes scipiihis) hepatopancreas (Tucker and Prince 1993). Cooper et al. (1993) found that (he TCDD levels in the .Arthur Kill organisms accumulated within higher trophic levels. For example, the soft-shell clam {Mya arenaria) contained 6.9 pptr TCDD, and the killifish (Fimdiilus heteroclitiis) contained 100 pptr TCDD, total body burden. Changes in the gonadal tissue of bivalves after exposure to a wide variety of pollutants such as oil, heavy metals, and lipophilic organic compounds have been reported (Vashchenko et al. 1993, Capuzzo 1996, Moore et al. 1980. Gardner et al. 1991. Lowe & Pipe 1985. 1986. 1987: Capuzzo & Leavitt 1988: Lowe. 1988; Moore 1988. Widdows & Johnson 1988). For instance, oocyte mass resorption observed in the sea urchin as well as other inver- tebrates at prespawning is considered to be a reaction to pollution (Vashchenko ct al. 1993, Lowe & Pipe 1985. 1986. 1987. Capuzzo 1996). The abnormal development of oocytes, and altered egg shape and size have been correlated with polluted sites (Winter- myer 1998, Lowe & Pipe 1985). The accumulation of pollutants in 737 738 WiNTERMYER AND CoOPER bivalves can cause stress. Capuzzo (1996) reported that pollution- induced sites can lower biochemical reserve, and contribute to poor egg quality and fertilization rates in bivalves. Bayne et al. (1972. 1978) similarity reported that under stressful conditions the mussel (Mytiliis ediilis) produced fewer and smaller eggs, and that larvae that developed from the gametes of stressed adults had a lower growth rate. In a study comparing egg size and larval sur- vival of the hard-shell clam (Mercenario mercenaria) and the bay scallop (Argopecten irradians). Kraeuter et al. ( 1982) reported that for both species, smaller eggs (20-25 jjim) had a significantly less than expected survival rate, while larger eggs (35—14 |j.m) had a significantly greater than expected survival rate. Intermediate size eggs (25-35 \i.m) showed no difference between the expected and observed survival rates. The objectives of this study were to transplant adult oysters into sites contaminated with different levels of dioxin and dioxin-like compounds to measure the effects on egg development and fertil- ization, and to evaluate the potential for restoring oyster popula- tions into the New Jersey bay area. METHOD AND MATERIALS Deployment Adult eastern oysters (n = 180) were purchased from Prince Edward Island, Canada, and were transplanted in September 2000 at three study sites (n = 60 per site): Newark Bay, NJ; Arthur Kill, NJ; and Sandy Hook Bay, NJ (reference site). The oysters v\ere determined to be disease free by histologic examination prior to deployment. Oyster bags (/i = 2) were suspended in the water column in Sandy Hook Bay located north of the bridge connecting the Highlands entrance to Sandy Hook State Park. For the Arthur Kill site, oyster bags (/; = 2) were suspended in the water column from General Anline Works building dock (longitude 74"12.312W. latitude 40°36.647N) in Elizabeth, NJ. For the Newark Bay site, oyster bags (/) = 2) were suspended in the water column from an abandoned dock on Shooter's Island (longitude 74°09.7S8W, latitude 40 38.482N) in Newark, NJ (Fig. 1). Each oyster was filed, numbered (1-60), and weighed (in grams), and the dimensions were measured [i.e., length, width, and height (in millimeters)] prior to being placed into marked, mesh polyethylene bags (0.5 x 0.5 inch mesh). Each site was equipped with two bags containing 30 oysters each suspended into the water column 1.8 to 2.4 m (6-8 feet) below the water surface. The depth was selected to avoid low-tide exposure and icing during the win- ter. Oyster bags were collected in June 2001. terminating the 10- mo field study. Oysters were wet weighed immediately upon col- lection, and were prepared for tissue chemical analysis, histologic evaluation, and fertilization assays. Chemical Analysis Samples of shucked oysters (50 g, /( = 7) from each site were sent to Triangle Laboratories (Research Triangle Park, NC) for dioxin, furan. and polychlorinated biphenyl (PCB) tissue analysis. Samples were analyzed by high-resolution chromatography and high-resolution mass spectrometry [method 1613B (9/97) and modified method 680 (11/85), Triangle Laboratories], Tissues were sent in labeled amber-colored jars and were frozen during shipment. Histologic Evaluation Oysters from each site (/; = 15) were selected randomly for histologic evaluation. Shucked oyster samples were preserved in a 10% phosphate formalin buffer for several days followed by 70% ethanol. Transverse cuts were made with a scalpel through the mid-visceral region of the oyster to obtain a segment approxi- mately 5 mm thick. Segments were embedded in paraffin after processing (i.e., dehydration and clearance through an alcohohxy- lene series). Sections (6 |j.m) were cut and stained with Harris' hematoxylin and eosin. Histologic grading was based on a scale New Jersey Atlantic Ocean Sandy Hook Site Figure 1. Locations of New Jersey field study sites in the Newark/Raritan Bay Complex. Dioxin/Furan and Polychlorinated BiPHENYL Concentrations in Eastern Oyster Tissues 739 from mild (T) to severe (TTT) for lesions. iiinaniin;Uion-likc re- sponses, and infectious diseases. Gonad condition was graded according to Kennedy (1977): Stage 0 = resting stage Stage 1 = early development Stage II = later development Stage III = sexual maturity Ilia = maturity Illb = spawning IIIc = redevelopment Hid = recently spent Tissues evaluated were gills, mantle, adductor muscle, kidney/ heart, digestive gland, and gonadal condition. Fertilization Assay: Strip Spawning Field Study A total of six ripe oysters from each site were strip spawned (male = 3, female = 3). Eggs and sperm were extracted from the gonadal region using a scalpel and lightly lacerating the gonad (Allen et al. 1989). Collected eggs were sieved on a 25-|ji.m screen and were washed with seawater collected from the respective site. Eggs were viewed under a microscope for maturation before being fertilized with the collected oyster sperm (sperm was diluted to 50 mL). Once sperm (1 niL) was added to the egg suspension (200 eggs per mL), the eggs were set aside for 1 h before being assayed to allow for fertilization. The total number of fertilized and unfer- tilized eggs, in three 1-mL replicate samples, was ascertained be- fore eggs were dispensed into petri dishes. To each 10-mL glass petri dish (/; = 3 per site). 10 mL of the site-collected water and fertilized eggs (« = 100) from each site were dispensed into the appropriate petri dish. Fertilized eggs were allowed to develop for 48 h at room temperature without aeration or food. After 48 h, the larvae were sieved on a 53-p.m screen, and the number of larvae that had developed to the straight hinge stage was counted. (i.e., control. 2.0 pptr. and 20.0 pptr groups) were placed into separate recirculating seawater systems 24 h after the injections. All oysters were reinjected on day 14 of the study according to the procedure described above. This procedure was performed to maintain dioxin concentrations in the oysters over 28 days (Win- termyer 1998). Treatment groups were strip spawned on day 28 according to the procedure described above (field study). Eggs (10 eggs per mL) from each treatment group were fertilized with sperm (1 mL; sperm was diluted to 100 mL) collected from the corresponding treatment group. Ex vivo. The 48-h static ex vivo assay consisted of control eggs (9 eggs per mL) fertilized with control sperm (1 mL: sperm was diluted to 100 mL). Glass exposure beakers (150 mL) (n = 3) consisted of 0.1 mL of nominal 2.0 pptr TCDD. and 0.1 mL of nominal 20.0 pptr TCDD and 0.0 pptr TCDD. respectively. To each treatment beaker, a 10-mL egg suspension and a 2-mL sperm suspension were added, and allowed to set for 2 h for fertilization. Both 48-h //) vivo and ex vivo assays were conducted in 20-mL glass petri dishes. Fertilized eggs (10 mL) from each treatment group was pipetted into individual petri dishes (/; = 20 per group) and were incubated at 22°C for 48 h. After 48 h, each petri dish in both the in vivo and e.x vivo assays was examined for the number of fertilized and unfertilized eggs, as well as for the number of living and dead larvae and their development stages. Radiolabeled Compounds -'[HI 2,3,7,8-TCDD (34.7 Ci/mM, 98% pure by high- performance liquid chromatography, with carbons 1 and 6 radio- labeled) was purchased from Chemsyn Science Laboratories (Le- nexa. KA). Oysters were exposed to 0.996 pg/g (2 pptr) or 27.7 pg/g (20.0 pptr) of -'[Hj-TCDD via adductor muscle injection. All ""[Hj-TCDD values were based on equivalents. RESULTS Laboratory Study In vivo. Adult eastern oysters (Crassostrea virginica) were purchased from Haskin Shellfish Research Laboratory (Rutgers University, Piscataway, NJ). Oysters (/( = 32) were exposed to two treatments of tritium-labeled 2.3.7..S-TCDD via adductor muscle injections. The study was conducted for 28 days to allow the circulation and distribution of dioxin throughout the oyster. This time period was selected based on results obtained from a distribution study using 2,3,7,8-TCDD (Wintermyer 1998). Oys- ters (/! = 48) were weighed (mean weight 50 g), numbered and notched, and their dimensions were measured (i.e., height, length, and width). Oysters were notched on the left side of the valves for access to the adductor muscle. Control oysters (n = 16) were injected (via adductor muscle) with 100 p.L (0.1 mL) of 20 parts per thousand filtered seawater. The nominal 2.0 pptr treatment group {n = 16) was injected with 100 |xL (0.1 mL) of 0.996 pg/g ^[H]-TCDD. The nominal 20.0 pptr treatment group (;; = 16) was injected with 100 \xL (0.1 niL) of 27.7 pg/g '[HJ-TCDD. '|H]- TCDD equivalents were based on radioactivity in 0. 1-mL injection volumes in a 50-g oyster (pg/g) in = 3). All oysters were placed on absorbent paper for 1 h before being put into 76-L aquarium tanks for 24 h. This procedure was performed to allow the dis- charging and recirculation of dioxin by the oysters. Oysters were not fed 24 h before or 24 h after the injections. Treatment groups Deployment and Retrieval In this study, a total of six bags containing eastern oysters was transplanted to the Newark Bay and the Raritan Bay Complex from September 2000 until June 2001. Oysters transplanted to Newark Bay for 10 mo had the second highest increase in total weight gain (-1-6 g). Oysters transplanted to Arthur Kill had a decrease in total weight gain (-10.9 g), and oysters transplanted to Sandy Hook Bay had the highest increase in weight gain (-t-10.3 g). There was not a significant difference in shell growth among the Newark Bay. Arthur Kill, or Sandy Hook transplanted oysters over the 10-mo field study (Table 1). Tissue Analysis Oyster tissues were analyzed for dioxin. furan, and PCB ana- lytes. Newark Bay oysters had the highest tissue levels of 2,3,7,8- TCDD (3.2 pptr), total TCDD (16.5 pptr). total TCDF (93.8 pptr). and total PCBs [1.7 parts per billion (ppb)]. Arthur Kill trans- planted oysters had the second highest tissue levels of 2,3,7,8- TCDD (1,3 pptr), total TCDD ( 13.3 pptr), total TCDF (56.7 pptr), and total PCB (64.5 ppb). Sandy Hook oysters had the lowest levels of 2.3.7.8-TCDD (0.15 pptr), total dioxin (2.5 pptr). total furan (47.6 pptr). and total PCBs (35.3 ppb) (Tables 2 and 3). 740 WiNTERMYER AND COOPER TABLE 1. Deployment and retrie^al data from C. virginica transplanted to Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ, field sites.' Temp. Salinity Sites Date No. of Oysters'" (X) (ppt) Weight (g) H (mm) 1, (mm) \\ (mm) Deployment Newark Bay 9/12/00 60 18.5 20 57.5 ± 15.3 81.4 ± 13.6 45.8 ±5.0 19.8 ±2.8 Anhur Kill 9/12/00 60 19.5 20 66.8 ± 19.9 88.7 ± 14.0 46.9 + 7.4 20.5 + 2.9 Sandy Hook 9/12/00 60 18 23 68. 1 ± 25 89.7 ± 15.0 46.6 ± 5.0 20.8 ±4.0 Retrieval Newark Bay 6/1/01 47/13'' (2 bags recovered) 14.3 16 63.5+18.0 81.7 ± 13.2 45.4 ±4.8 19.5 ±2.6 Arthur Kill 6/1/01 45/15'^ (2 bags recovered) 17.3 16 55.9 ± 13 88.2+ 13.8 46.2 ±7.7 20.8 ± 3.0 Sandy Hook 6/1/01 25/5' (1 bag recovered) 14.6 20 78.4 ±26 89.4 ± 14.7 46.1 ±5.5 20.5+4.2 Presented as mean + Sd, unless otherwise indicated. " H, height; L. length; W, width; ppt. pans per thousand. '' Number of oysters per site; two bags per site. " Number of live oysters/number of dead oysters. Histologic Evaluation Oysters transplanted to Newark Bay showed moderate signs of epithelial-severe hyperplasia, while oysters transplanted to Arthur Kill showed signs of severe epithelial-severe hyperplasia with some cells (>4) showing mitotic division, and connective tissue displaying areas of focal fibrosis. Oysters transplanted to Sandy Hook showed signs of slight epithelial-severe hyperplasia. Only the transplanted oy.sters to Arthur Kill were observed to have a haplospoiidiuin nelsoni (MSX) infection in the digestive gland and mantle tissues (Table 4). All transplanted oysters showed slight- to-moderate gill hyperplasia ("clubbing"). Oysters transplanted to Newark Bay and Arthur Kill showed an alteration in gill cilia shape, size, and orientation. The cilia had a thickened appearance and an alteration in cilia length resulting in a distinct whip-like appearance (approximately six times the length of normal gill cilia). Gross Body Evaluation Oysters transplanted to Newark Bay had semi-developed go- nadal tissue. The gonadal area had a slightly cream-colored ap- pearance, and the oysters appeared to be of moderate health and were plump. The shell interior had a white, iridescent color and had no obvious scarring or discoloration. Oysters transplanted to TABLE 2. Oyster tissue analysis for dioxins/furans at Newark Bay, N,I, Arthur Kill, NJ. and Sandy Hook Bay, NJ, during a 10-mo water suspension field study. Analytes Newark Bay Concentration (pptr)" Arthur Kill Concentration (pptr)'' Sandy Hook Concentration (pptr)' 2,3,7.8-TCDD 1,2,3.7.8-PeCDD 1,2.3.4.7.8-HxCDD 1,2.3,6,7,8-HxCDD 1.2,3.7,8.9-HxCDD 1.2,3.4,6.7,8-HpCDD 1.2,3.4,6.7,8.9-OCDD 2.3,7,8-TCDF 1.2.3.7.8-PeCDF 2,3,4,7,8-PeCDF 1,2,3.4,7,8-HxCDF 1,2,3.6.7.8-HxCDF 2.3,4.6.7.8-HxCDF 1.2,3.7.8.9-HxCDF 1.2,3.4,6.7,8-HpCDF 1, 2,3.4,7,8,9- HpCDF 1,2,3.4.6.7.8.9-OCDF Total TEFs'

4) showing mitotic division and connective tissue displaying areas of focal fibrosis. Oysters transplanted to Sandy Hook showed signs of slight epithelial-severe hyperplasia. The epithelial-severe hyperplasia could be interpreted as preneoplastic in nature, however, further research is needed to verify that these lesions can progress to a neoplastic condition. Only the Arthur Kill oysters were observed to have a moderate-to-severe MSX infec- tion in the digestive gland and mantle tissues (Table 4). Sandy TABLE 5. Summary of the strip-spawning assay from Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ, l(l-mo field study (September, 2000-June, 2001 ). Newark Bay, NJ Arthur Kill, NJ Sandy Hook. NJ Weight of oysters at time of deployment (g) (9/00) Weight of oysters at termination of study (g) (6/01) % lipid (6/01) Egg size fertilized vs. unfertili/ed (|x at 40x) (n = 5) Total number of fertilized eggs' Total number of unfertilized eggs" Number of veligar larvae after 48 h" 57.5 ± 15.3 (n = 60) 63.5+ lS.4(n = 45) 0.3 64 (xm fertilized 56 (Jim unfertilized 107 ±6.00 164 + 25.6 3± 1.7 66.8 ± 19.9 (n = 60) 55.9+ 13.1 (n = 47) 0.2 64 |j.m fertilized 48 fjim unfertilized 54 ± 30. 1 1 178+ 15.9 4 ± 2.3 1 68.1 ±24.4(11 = 60) 78.4 ±25.6 I n 25) 0.6 64 (xm fertilized 56 (im unfertilized 113± 13.61 97 ± 39.9 82 ± 12.2 Presented as mean + SD, unless otherwise indicated. "Numbers repre,sent the average of 1-mL replicate samples (n = 3). '' Number of veligar larvae resulting from approximately 100 fertilized eggs (n 3 replicates). Dio.xin/Furan and Polychlorinated Biphenyl Concentrations in Eastern Oyster Tissues 743 ■ fertilized eggs 0 unfertilized eggs = veliger larvae Transplant sites Figure 2. The percentage of fertilized eggs, unfertilized eggs, and veliger lar\ae resulting from the strip-spawning assay using transplanted ii>sttrs from Newark Bay, NJ, Arthur Kill, NJ, and Sandy Hook Bay, NJ ( lO-mo field study, Septemher 2000-June 2001 1. Numher of fertilized and unfertilized eggs are averages of 1-niL replicates (three per site). Numbers of veliger larvae are those resulting frcmi 100 fertilized eggs after 4X h (three sites). #* (light), fertilized egg groups that are significantly different (/' < 0.05: ANON A); #* (dark), unfertilized egg groups that are significantly different [P < 0.05; ANOVA); a, veliger larvae groups that are significantly different (P < 0.05; ANOVA); NB, Newark Bay; AK, Arthur Kill; SH, Sandy Hook. Hook oysters had fully developed gonads and were in a prespawn- ing state. Newark Bay and Arthur Kill oysters were slightly mod- erately underdeveloped due to a lack of gonadal development com- pared with Sandy Hook oysters at the time of collection (Table 4). All transplanted oysters showed slight-to-moderate gill hypeijila- sia (clubbing). Oysters transplanted to Newark Bay and Arthur Kill showed an alteration in gill cilia shape, size, and orientation. The cilia had a thickened appearance and an alteration in cilia length resulting in a distinct whip-like appearance (approximately six times the length of normal gill cilia). This alteration in gill cilia could be a result of chronic exposure over time. The lesions ob- served in the transplanted oysters would be consistent with those resulting from chronic exposure to chemicals. The lesions are not pathoneumonic but are consistent with a wide variety of chemical and physical irritants. Oysters transplanted to the Newark Bay site had the second highest increase in weight gain (-F 6 g). percentage of lipids (0.3%), egg fertilization (39.5%). and larval development (0.03%). Oysters transplanted to the Arthur Kill site had a decrease in weight over the 10-mo study (-10.9 g). the lowest percentage of lipid content (0.2%), the lowest percentage of egg fertilization (23.3%). and a decrease in larval development (0.04%). Oysters transplanted to the Sandy Hook site had the greatest increase in weight gain (-1-10.3 g). the highest percentage of lipids (0.6%). the highest percentage of egg fertilization (53.770). and the highest percentage of larval development (84%) (Table 5, Fig. 2). Weight gain and the per- centage of lipid content of the oyster contribute greatly to egg development and production, egg fertilization success, and larval development (Capuzzo 1996, Capuzzo & Leavitt 1988, Lowe 1988, Moore 1988). Oysters transplanted to Sandy Hook had the highest level of fitness followed by oysters transplanted to Newark Bay and Arthur Kill, based on lesion grading, intlammatory-like responses, infectious disease states, weight gain/loss, and the de- gree of gonadal development. Results from the strip-spawning assay using oysters trans- planted to Newark Bay. Arthur Kill, and Sandy Hook. NJ. showed TABLE 6. Summary of an acute static 48-h in vivo and ex vivo strip-spawning bioassay for C. virginica exposed to 2 and 20 pptr 2,3,7,8-TCDD. Initial (Egg) .After 48 h (Veliger Larvae) Number of Fertilized Eggs Number of Unfertilized Eggs Number Dead after 48 h Stage of Development Number Alive after 48 h Stage of Development" Control in vivn 19h ± 143 2 ± U.63 39 ± 1.45 Trochophore. egg. and D-stage 2 pptr in vivo 1 .^2 ± 3. 1 2 1 66 ± 3.80 3 1 X ± 3.45 Egg 20 pptr »! i/\fi'' 6 ±0.801 660 ±16,2 663+17.94 Egg Control t-A vivo 1 94 ± 2. 1 7 4 + 0.84 48 + 2. 1 0 Trochophore. egg, and D-stage 2 pptr ex vivo 13 + 0.489 420 ± 10.8 423 ± 12.0 Egg and D-stage 20 pptr o- VIVO 16 ±0.410 803 ±27.3 810 ±27.6 Egg and D-stage 1 5y + 1 .66 0 3 + 0.52 150 + 2.36 D-stage NA D-stage D-stage 10 ±0.513 Trochophore and D-stage 9 + 0.510 D-stage Presented as mean + SD. unless otherwise indicated. N.A. not applicable. In vivo represents eggs expo.sed to TCDD during gametogenesis and ex vivo represent eggs exposed to TCDD in petri dishes during fertilization (n 20 for each group). Table taken from Wintermyer ( 1998). " Stage of fertilized egg (Loosanoff and Davis 1963). Viable control eggs were fertilized with 20 pptr spemi. 744 WlNTERMYER AND COOPER •a a> N s c 3 «o" O) O) 0) ■o o _N t .(1* c o 0) Q. 120 100 80 g» "35 > ■o c re w O) a> 60 40 20 in VIVO ^ a' ^ ex VIVO I /\^\.''' ■ fertilized eggs s unfertilized eggs Qveliger lar\«e Figure 3. The percentage of fertilized eggs, unfertilized eggs, and veliger larvae resulting from an acute static 4S-h /'/; vivo and f.v vivo strip-spawning assay using C. virgiiika exposed to 2 and 20 pptr 2,3,7,8-TCDD. "in vivo, eggs exposed to TCDD during gametogenesis; *ex iii'o, eggs exposed to TCDD in petri dishes during fertilization in = 20 for each group). Table from Winterniyer (1998). that the majority of eggs collected from female oysters at the Newark Bay and Arthur Kill sites were not viable (Fig. 2). This study shows that 60.59r and 76.7%. respectively, of eggs collected from Newark Bay and Arthur Kill transplanted oysters were not fertilized, and of the eggs that were fertilized (39.5% and 23.3%, respectively) only 0.03% and 0.04%. respectively, of the eggs developed to the straight-hinge stage. Most fertilized eggs did not develop beyond the zygote stage. The strip-spawning assay for oysters transplanted to Sandy Hook showed that .'i3.7% of the eggs were fertilized, and of those eggs 84% developed to the straight- hinge stage (Fig. 2). This study was perfonned to evaluate the potential for restoring oysters in to the bay area. Based on the field study and strip-spawning assay, transplanting oysters into the Newark bay and Arthur Kill sites at this time would not result in successful recruitment of the bay area. However, the Sandy Hook site would be an ideal area for oyster restoration. In the laboratory studies, the 2.0 pptr and 20.0 pptr treatment concentrations of 2,3,7, 8-TCDD used in the 48-h acute in vivo and ex vivo studies were based on tissue concentrations that were re- ported from the soft-shelled clam (Mya arenaria) living in New- ark. NJ. (11-20 pptr TCDD) and Tuckerton. NJ. (0.1-0.6 pptr) (Brown et al. 1993) and on sediment samples from Newark Bay (20 pptr). Arthur Kill (10 pptr), and Tuckerton (0.5 to 1.0 pptr) (Brown et al. 1993). In this study using C. virgiiiica. there was an observable decrease in the number of fertilized eggs within the 2 and 20 pptr TCDD treatment groups. In Fig. 3. controls for the in vivo and e.x vivo assays had high rates of egg fertilization and larvae development to the straight-hinge stage (80.3%). The 2.0 pptr ill vivo assay had a 47.8% egg fertilization rate, but a 100% mortality rate at the zygote development stage. In the 20.0 pptr //; vivo assay to which viable control eggs were fertilized with 20.0 pptr sperm, there was very little fertilization (0.901%), which re- sulted in a high egg mortality rate (99.6%). The 20-pptr treatment group did not have any female oysters remaining due to toxicant- induced stress and mortality by the end of the 28-day period. The 2.0 pptr e.x vivo and 20.0 pptr e.x vivo assays also had low fertil- ization, which resulted in high egg mortality (Fig. 3). In both the 48-h acute in vivo and e.x vivo studies, there was a large decrease in the number of fertilized eggs respective to treatment group compared with the controls. Within treatment groups (nominal 2.0 pptr TCDD and 20.0 pptr TCDD), there were 52 to 99% unfertil- ized eggs. Eggs that were fertilized had a 98 to 100% mortality rate and did not develop beyond the zygote stage. In contrast, the control eggs had an 80% survival rate to the straight-hinge stage (Table 6, Fig. 3). This laboratory study is important in understand- ing the effects of 2,3, 7, 8-TCDD independent of other lipophilic compounds on oyster gametogenesis and egg fertilization. We can- not state that the field study results were solely due to 2,3,7,8- TCDD, but laboratory studies demonstrate that TCDD can result in a significant decrease in gametogenesis and egg viability. CONCLUSION In conclusion, this study was designed to investigate two points of interest: (1) the dioxin/furan and PCB concentrations in the eastern oyster during gametogenesis and the effects on egg fertil- ization and development; and (2) to evaluate the potential for restoring oysters back into the New Jersey bay area. Oysters trans- planted to Sandy Hook, NJ, had the greatest weight gain, percent- age of lipid content, percentage of egg fertilization, and percentage of larval development to the straight-hinge stage, followed by oysters transplanted to Newark Bay and Arthur Kill. NJ. The labo- ratory in vivo and e.x vivo strip-spawning assays showed that ex- posure to compounds such as dioxin can accumulate in animal tissues and can interfere with normal metabolic processes that affect gonadal development and egg fertilization. While we cannot separate the effects of different gonadal development on strip- spawning fertilization and larval development, the laboratory stud- ies support the effect of 2,3, 7, 8-TCDD on gonadal development at levels observed in the field. This study demonstrated that dioxins. furans. and PCBs are still bioavailable in the Newark Bay estuary. The levels approach con- centrations that in the laboratory result in impacts on gonadal development and egg viability. This study clearly demonstrates that 2. 3. 7. 8-TCDD effects gonadal development and egg viability in the eastern oyster in a similar fashion to fish species. ACKNOWLEDGMENTS The authors would like to thank Michael Stringer and the NY/ NJ Baykeeper Program for helping with the deployment and re- trieval of the oyster bags for the field study reported in this article. Dioxin/Furan and Polychlorinated Biphenyl Concentrations in Eastern Oyster Tissues 743 Allen, S.. S. Downing & K. Chew. 1989. Hatchery manual for producing triploid oysters. Seattle, WA: Wa,shington Sea Grant Program. Univer- sity of Washington Press, pp. 1-20. Alonso, K. R. 1996. In-vivo exposure to 2.3,7,8-TCDD results m delayed reproductive maturation in the domestic chicken. Oriiamtlmhii^en Com- pounds 29:195-199. American Society for Testing and Material (ASTM). 1994. Standard guide for static acute toxicity tests starting with embryos of four species of saltwater bivalves molluscs. Am. Soc. Tesiiiii; Mater. £724:22.^-240. Belton. T. J.. R. Hazen. B. E. Ruppel. K. Lockwood, R. Mueller, E. Steven- son & .1. J. Post. 1985. A study of dioxin (2.3,7,8-TCDD) contamina- tion in select finfish, crustaceans, and sediments of New Jersey water- ways: final repon. Trenton. NJ: Office of Science and Research, New Jersey Department of Environmental Protection. Bayne, B. L., D. L. Holland, M. N. Moore. D. M. Lowe & J. Widdows. 1 978. Further studies on the effects of stressing in the adult on the eggs of Mytiliis ediilis. J. Mar. Biol. Assoc. U. K. 58:825-841. Bayne, B. L. 1972. Some effects of stress in the adult on the larval devel- opment of Mytiliis ediilis. Nature 237:459^63. Brown, R.. K. Cooper. A. Cristini. C. Rappes & P. Bergqvisl. 1993. Poly- chlorinated dibenzo-p-dioxins and dibenzofurans in Mya arenaria in the Newark/Raritan Bay estuary. Eviron. To.x. Chem. 13:523-528. Calabrese. A.. R. Collier, D. Nelson & J. Maclnnes. 1973. The toxicity of heavy metals to embryos of the American oyster Crussostreu virginicu. Marine Biol. 18:162-166. Cappuzzo, J. M., J. Farrington, P. Rantamaki, C. Clifford, B. Lancaster, D. Leavitt & X. Jia. 1989. The relationship between lipid composition and seasonal differences in the distribution of PCBs in Mvtilus edulis L. Marine Environ. Res. 14:201-228. Capuzzo McDowell. J. 1996. The bioaccumulation and biological effects of lipophilic organic contaminants. In: V, S, Kennedy, Roger Newell & Albert Eble, editors. The eastern oyster Crassostrea virginica. College Park, MD: Maryland Sea Grant College Publication, University of Maryland System. Capuzzo, J. M. & D. F. Leavitt. 1988. Lipid class composition of the digestive gland of Mytilus edulis and Carcinus maenas in response to contaminant gradients. Mar. Ecol. Prog. Ser. 46:139-145. Chen, W., L. Zhang, L. Xu, X. Wang. L, Hong & H, Hong. 2002. Residue levels of HCHs, DDTs and PCBs in shellfish from coastal areas of ea.st Xiamen Island and Minjiang Estuary, China. Marine Pollution Bulletin 45:385-390. Cooper. K. 1989. Effects of polychlorinated dibenzo-p-dioxins and poly- chlorinated dibenzofurans on aquatic organism. Ai/iiatic Sciences 1: 227-242. Cooper, K., J. Schell. T. Umbreit & M. Gallo. 1993. Fish embryo toxicity associated with exposure to soils and sediments contaminated with varying concentrations of dioxins and furans. Marine Environ. Res. 35:177-181. Davis. H. C. & H. Hidu. 1969. Effects of pesticides on embryonic devel- opment of clams and oysters and on growth of the larvae. Fisheiy Bull. Fish Wildl. Sen: U. S 67:383-t04. Environmental Protection Agency (EPA). 2000. Exposure and Human Health Reassessment of 2,3.7. 8-Tetrachlorodibenzo-p-Dioxin (TCDD) and Related Compounds, unpublished manuscript. Isensee, A. R. & G. E. Jones. 1975. Distribution of 2.3.7.8-tetrachloro- dibenzo-p-dioxin (TCDD) in an aquatic model ecosystem. Environ. Sci. Tclmol. 9:668-672. Kennedy, V. S. 1977, Reproduction in Mytilus edulis aoteanus and Aulu- comya maorians (Mollusca: Bivalvia) from Taylors Mistake, New Zealand. N. Z. J. Mar. Freshwater Res. 1 1:255-267. Kennedy, V. S. & H. I. Battle. 1964. Cyclic changes in the gonad of the American oyster, Crassostrea virginica (Gmelin). Can. ./. Zool. 42: 305-321. LITERATURE CITED Kraeuter, J. N„ M. Castagna & R. van Dessel. 1982. Egg size and larval survival of Merenaria mercenaria (L.) and Argopecten irradians (Lamarck). / £.vp. Mar. Biol. Ecol. 56:3-8. Loosanoff, V. & H, Davis. 1963. Rearing of bivalve moUusks. In: F. S. Russell, editor. Advances in marine biology, vol. 1 . London and New York: Academic Press, pp. 1-133. Moore. M. N.. D. R. Livingstone. P. Donkin. B. L. Bayne. J. Widdows & D. M. Lowe. 1980. Mixed function oxygenase and xenobiotics detoxi- cation/toxication systems in bivalve molluscs. Holgol. Wiss. Meere- sunters 33:278-291. Moore. M. N. 1988. Cytochemical responses of the lysosomal system and NADPH-ferrihemoprotein reductase in moUuscan digestive cells to en- vironmental and experimental exposure to xenobiotics. Mar. Ecol. Prog. Ser. 46:81-89. Gardner. G. R.. P. P. Yevich. J. C. Harshbarger & A. R. Malcolm. 1991. Carcinogenicity of Black Rock Harbor sediment to the eastern oyster and trophic transfer of Black Rock Harbor carcinogens from the blue mussel to the winter flounder. Environ. Health Perspectives 90: 53-66. Lowe. D. M. 1988. Alterations in cellular structure of Mytilus edulis re- sulting from exposure to environmental contaminants under field and experimental conditions. Mar. Ecol. Prog. Ser. 46:91-100. Lowe. D. M. & R. K. Pipe. 1985, Cellular responses in the mussel Mytilus edulis following exposure to diesel oil emulsions: Reproductive and nutrient storage cells. Marine Environ. Res. 17:234-237. Lowe, D. M. & R. K. Pipe. 1986. Hydrocarbon exposure in mussels: A quantitative study on the responses in the reproductive and nutrient storage cell systems. Aquatic To.xicol. 8:251-265. Lowe. D. M. & R. K. Pipe. 1987. Mortality and quantitative aspects of storage cell utilization in mussels. Mytilus edulis. following exposure to diesel oil hydrocarbons. Marine Environ. Res. 22:243-251. New Jersey Department of Environmental Protection, Division of Science Research and Technology. 1999. Dioxin levels in New Jersey, pp. 1-3. Available at: www.state.nj.us/dep/dsr/ New Jersey Department of Environmental Protection (NJDEP). 1996. New Jersey coastline shapefile. Trenton, NJ: Office of Information Re- sources Management (OIRMi, Bureau of Geographic Information and Analysis (BGIA). Available at: www.sate.nj.us/dep/gis/digidownload/ zips/statewide/coast.zip New Jersey Department of Environmental Protection and Energy, Division of Science and Research. 1990. Polychlorinated biphenyls (PCBs), Chlordane, and DDTs in selected fish and shellfish from New Jersey waters, 1986-1987. In: P. Hauge. P. Morton, M. Boriek, & G. Casey, editors. Results from New Jersey's Toxics in Biota monitoring pro- gram, pp. 1-65 New Jersey Department of Environmental Protection and Energy. Division of Science and Research. Polychlorinated biphenyls (PCBs). chlordane. and DDTs in selected fish and shellfish from New Jersey waters. 1988- 1991. In: P. Hauge. editor. Results from New Jersey's Toxics in Biota monitoring program, pp. 1-95. Pohjanvirta. R. & J. Tuomisto. 1994. Short-term toxicity of 2.3.7. 8-TCDD in laboratory animals: Effects, mechanisms, and animal models, Phar- macol. Rev. 46:483-549. Poland. A. & J. C. Knutson. 1982. 2.3.7.8-TCDD and related halogenated aromatic hydrocarbons: Examination of the mechanism of toxicity. Ann. Rev. Pharmacol. To.xicol. 22:517-554. Rappe. C. 1992. Sources of exposure, environmental levels and exposure assessment of PCDDs and PCDFs. Dioxin '92. Boston. MA. E.xtended .\hstracts 9:5-8. Triangle Laboratories Incorporated. 2001. Method I6I3B (9/97) for di- oxin/furan analytes and modified method 680 ( 1 1/85) for PCB analytes. Research Triangle Park, NC: Triangle Laboratories Inc. 746 WiNTERMYER AND COOPER Tucker. B. & R. Prince. (1993). Draft: Issues paper-dioxin and related compounds: New York-New Jersey Harbor Estuap,' Program. HEP Toxics Work Group, New York. Van den Berg, M., J. De Jongh. H. Poiger & J. Olson. 1994. The toxico- kinetics and metabolism of polychlorinated dibenzo-p- dioxins and dibenzofurans and their relevance for toxicity. Critical Rev. Toxicdl. 24:1-73. Vashchenko, M & P. Zhadan. 1993. Ecological assessment of marine en- vironment using two sea urchin tests: Disturbance of reproduction and sediment embryotoxicity. Sci. Total Environ. (Suppl) 1235-1245. Widdows, J. & D. Johnson. 1988. Physiological energetics of Mytiliis I'dulis: Scope for growth. Mar. Ecol. Prog. Ser. 46:1 13-121. Wintennyer, M. 1998. Tissue distribution of 2.3,7.8-Tetrachlorobenzo-p- dioxin (TDCC) in the adult eastern oyster (Crassostrea virginica) and its effect on egg and larval survival. MS thesis dissertation. Camden, NJ: Rutgers University. Journal of Shellfish Research. Vol. 22, No. 3, 747-752. 2003. AN IMPROVEMENT TO THE DETERMINATION OF MEAT CONDITION INDEX FOR THE EASTERN OYSTER CRASSOSTREA VIRGINICA (GMELIN 1791) GEORGE R. ABBE* AND BRIAN W. ALBRIGHT Academy of Natural Sciences Exliiarine Research Center, 10545 Mackall Road, St. Leonard, Maryland 2()6S5 ABSTRACT The meat condition index (MCI) of a bivalve is a numerical representation of the quality of its soft tissue. Based on the percentage of the internal shell volume occupied by a bivalve's soft body tissue, the enumeration of a quantitative index is possible. Early methods sought to measure the shell cavity volumetrically; however, this technique is both slow and difficult to perform accurately. In 1982. Lawrence and Scott developed a method to determine the MCI for oysters gravimetrically, in which shell cavity capacity was determined by the difference between whole oyster weight and empty shell weight after drying for 24 h This technique was not only as accurate as the volumetric method, but it was faster and easier. However, since any water contained within the shells themselves (not between them) was included in the initial whole oyster weight, it seemed logical that this should be included in the weight of empty shells as well. Drying shells for 24 h could inake the calculated shell cavity appear larger, resulting in reduced meat condition. To determine the significance of weighing shells after 0 h versus 24 h of drying, the MCls were determined by shell cavity casts and were compared with MCIs determined by the two gravimetric methods. Weighing shells immediately after processing (0 h) was determined to more accurately estimate cavity volume whenever shells lost >3% of their weight due to drying. Of 1749 oysters examined from the Patuxent River. Maryland, over 3 y, 74% lost >3'7f of their shell weight. Several other sites in the Chesapeake Bay were also exainined. yielding similar results. Weighing shells at 0 h not only increased accuracy for most of the oysters examined, but also saved time, as shells did not need to be held for an additional 24 h. Differences in shell morphology and fouling community structure may intluence shell porosity, favoring one technique over the other. KEY WORDS: oyster. Crassosrrea virgiiiicti. meal condition. Chesapeake Bay INTRODUCTION The meat condition index (MCI) of a bivalve i.s a iiunierical representation of the quality (i.e., nutritive status or "fatness") of its soft tissue. Quantitative methods of determining the meat con- dition of bivalves have been conducted by various researchers as far back as the early 1900s. Crosby and Gale ( 1990) presented a brief history of the development of condition indices, which men- tioned the works of Moore (1908), Milroy (1909), Grave (1912). Higgins (1938), Haven (1962), Walne (1970), Lawrence and Scott (1982). and Hawkins et al. (1987). Mo.st of these indices were calculated using a formula that relates the weight of the soft tissue to the shell cavity volume. The Hopkins formula (Higgins 1938) used dry tissue weight (g) x lOO/intemal cavity volume (cm'). In many of the earlier methods, shell cavity volume was determined by displacemetit. until Lawrence and Scott ( 1982) showed that the weight of the whole oyster in air (g). less the weight of the etnpty valves in air (g). gave a very close approximation of cavity volutne (cm ). Their fomiula for determining MCI became: MCL dry soft tissue wt (g) X 100 internal shell cavity capacity (g) Although some authors prefer to use dry soft tissue weight x 1000 (Hawkins et al. 1987, Crosby & Gale 1990). this formula simply results in a condition inde.x value an order of magnitude larger than that of Lawrence and Scott ( 1 982). Internal shell cavity volume (cm') and shell cavity capacity (g) are the same when the cavity contents are assumed to have a density of 1 g cm"'. This index represents meat quality or nutritive status while not necessarily reflecting the health of the indiv idual. A fat oyster or one with a high MCI generally has a rich creatiiy color due to stored glycogen reserves and shows little of the internal organs beneath the mantle (Fig. I A). An oyster of lesser qualilv will show *Corresponding author. E-mail: abbeCs'acnatsci.org some internal structure because the mantle is thinner and more transparent (Fig. IB). A very poor quality oyster will be watery and almost entirely clear. Condition indices of oysters (Crassostrea viri^inicii) in the Chesapeake Bay normally display u cyclical pattern, with the high- est levels occurring in late fall and winter, and the lowest levels occurring in late summer after spawning is completed, but low conditions may occur at any time of year, possibly indicating a disease problem or unfavorable environmental conditions (Haven 1962. Abbe & Sanders 1988). In fact. Scott and Middaugh (1978), Scott and Vernberg (1979), and Lawrence and Scott (1982) all suggested the use of an MCI to monitor the effects of waterborne pollutants. Low salinity may have the opposite effect on condition since gametogenesis may be depressed or halted at salinities be- tween 5 and 7.5%c (Butler 1949. Loosanoff 1953). Oysters that do not ripen their gonads, and thus fail to spawn, may attain higher condition indices than would otherwise be observed if gametes are resorbed or if the energy intended for gamete production is tun- neled into glycogen production. A visual assessment of oyster meats, at least in some primitive form, has probably been con- ducted almost as long as humans have consumed oysters. As an index based on opacity due to glycogen content, however, visual condition indices are overly subjective and somewhat impractical. Lawrence and Scott (1982) determined shell cavity capacity by weighing the whole oyster after air drying for 4.*^ to 60 min. and then subtracting the weight of the valves after air drying for an additional 24 to 30 h After using this method for more than 15 y to detertnine the MCIs of the eastern oyster C. virginiea (Gmelin. 1791 ). we questioned its accuracy when shells were dried for 24 h. We suspected that the elapsed titne betv\een when the oyster is shucked and when the valves are weighed might have a major effect on the calculated value of shell cavity volume. When the whole oyster is weighed, there is a certain amount of water within the shells (not between them) in the spaces created by shell-boring animals. Since this weight is included in the initial weight of the 747 748 Abbe and Albright Figure 1. A fat oyster (A) with high MCI and one of lower meat condition (B). Note that the mantle along the left edge of B is trans- parent allowing shell structure to be observed beneath it. whole oyster, it should also be included in the weight of the empty valves because it does not represent any part of the internal shell cavity. Drying shells for 24 h could make the calculated shell cavity volume appear larger, resulting in a reduced meat condition. We expected that valves might continue to lose weight as they dried over time for up to several days, although most of the weight loss would probably occur during the first 24 h. We suspected, therefore, thai it might be necessary to weigh the empty valves immediately after the oyster is opened and the soft tissue removed to obtain a more accurate determination of shell cavity capacity or volume. The amount of water weight within the valves also depends on the size (age) of the oyster as well as the number and size of the organisms living in (not on) the shells, which may include boring sponge (Cliona sp.) and mud worms (Polydora sp.) Young oysters have smaller shells to be inhabited by boring organisms and less time for their shells to be colonized. Older and larger oysters have more shell surface area to hold boring organisms and more time for their shells to be colonized. Regardless of size, however, if shells were weighed immediately after the meat was removed, the water weight in the shells should have a minimal effect on the calculated cavity volume, and thus on the condition index. We conducted several experiments (one that examined 1749 oysters over 36 mo) to investigate and quantify the effect of water-weight loss and drying time on oyster MCI. MATERIALS AND METHODS Drying Time Thirty oysters were collected from the Holland Point oyster bar in the Patuxent River (Fig. 2 1 near Benedict. Maryland, in March 1997. They were cleaned of external fouling organisms and scrubbed with a nylon brush in the field. They were held in water until they were returned to the laboratory, where they were held in a raceway of running filtered river water at ambient salinity (10-14 parts per thousand). They were kept in water until processed, since it is critical that they release no cavity fluid when weights are used to determine cavity volume. Oysters were removed from water, rinsed, blotted dry, numbered, measured for right valve length (height), and weighed before they could gape and lose fluid. Once weights were determined, the loss of some cavity fluid by animals that gaped had no effect on subsequent measurements. Oysters were then shucked into preweighed beakers and dried to constant Patuxent River # Monthly Sampling ■ Annual Sampling 10 km v^ Figure 2. Locations of 12 natural oyster bars in the Patuxent River where oysters were collected during 1997-2(100: Teague Point (TP); Holland Point (HP): Macks Hallow (MH); Broad Neck (BN); Jacks Marsh (JM); Gatton (GAT); Peterson (PT); Hellen (HEL); Hawks Nest (HN); Town Creek (TO: Southeast Middleground (SM): and Little Cove Point (LCP). weight at 60 to 70^C (5-7 days). Dry meat weight was measured to the nearest 0.001 g. After shucking, the internal surfaces of the valves of each oyster were wiped dry. and the valves were weighed immediately to the nearest 0.001 g. They were weighed again after 6. 24. 48. and 72 h Weight Loss To determine whether valves weighed immediately after oys- ters were shucked (0 h) or after drying for 24 h resulted in the closest estimate to true cavity volume, it was necessary to first determine the true volume as accurately as possible. Several meth- ods were tried, all of which were discussed by Crosby and Gale (1990), but most gave highly variable results and were time- consuming. The best technique that we found used a liquid casting medium of known density that was poured into each valve until slightly overfilled. As the liquid began to harden, the two valves were realigned and pressed tightly together so that excess casting material was squeezed out. Valves were banded to keep them tightly together until the cast was solid, which took only a few minutes. When the cast was hard, it was removed from the shells, the flashing was trimmed from the edges, and the volume of the cast was then determined volumetrically by displacement or gravi- metrically by weighing and dividing by the density of the casting medium. MCIs were determined for 169 oysters using 0 and 24 h valve weights and cavity volumes using casts. From March 1997 to February 2000, monthly sampling of oys- ters from four beds (Holland Point, Gatton, Hellen, and Southeast Middleground) and annual sampling from eight additional beds (Teague Point. Macks Hollow. Broad Neck, .lacks Marsh. Peter- son, Hawks Nest, Town Creek, and Little Cove Point; in the Patux- Improvement in Determining Oyster Condition Index 749 ent River (Fig. 2) was conducted witfi MCIs determined for 1 749 oysters using shell weights at both 0 h and after drying for 24 h. Following the analysis of Patuxent River oysters, oysters were examined from two sites in the upper Chesapeake Bay (Eastern Bay and near Shady Side) and from two tributaries of the Potomac River (Wicomico and St. Mary's Rivers) (Fig. 3) to determine whether oysters from these areas were similar in weight loss to those from the Patuxent River. Approximately 50 oysters were collected and analyzed from each of these four areas. RESULTS Drying Time Oysters used to determine drying time ranged from 72 to iS9 mm shell length (SL), with a mean (±SD) SL of 80.4 ± 4.1 mm. Whole weights were 89.6 to 163.0 g, with a mean weight of 128.2 ± 23.2 g. The mean shell weight at 0 h was 104.4 g. and 101.5 g after drying for 6 h (a loss of 2.8'7r). Shells continued to lose weight out to 72 h when they averaged 100.0 g (Fig. 4A), although the rate of decrease declined over time. As shell weight decreased o\er 72 h. calculated cavity volume increased from 23.8 to 28.2 cm', resulting in a decrease in mean condition from 9.5 at 0 h to 8.1 after 72 h (Fig. 4B). However, since the mean condition had already decreased to 8.2 after just 24 h, the decrease over the next 48 h was minimal. Weight Loss When the percentage shell weight losses were averaged by 2-mni SL increments, the means ransied from 2.87r for oysters of Figure 3, Other sites in Maryland from which oysters were collected during 20nO including Kastern Bay (EB), near Shady Side (SS), the Wicomicd Ri\er (W K), and the St, Mary's River (SMR). I- I LU X w < LU X LU Q z z o Q z o o I- < LU < LU 1 lU A 105 n 100 - 1 11 1 \ -| 95 - 9.5 - r-| B 9.0 - 8.5 r-| 8.0 -1 0 6 72 24 48 HOURS Figure 4. Mean shell weight over time as shells dried (A) and resulting loss in condition index (B). Drying was nearly complete after 24 h. hut continued to 72 h, 62 mm SL to 5.09? for those of 110 mm SL, and they exhibited a highly significant relationship (P < 0.001 ) between SL and weight loss (Fig. 5). Figure 5 also shows that the three smallest groups were well below the regression line. The individual percentage weight loss for all 1749 oysters, however, ranged from as little as 1.2% to as much as 13.0%, with distribution skewed to the right, although most were in the 2 to 8% range (Fig. 6). Shells were weighed at 0 and 24 h, and calculated cavity vol- O X in if) UJ o cr UJ Q. Y = 0899 + R-' = 0 760 p<0001 0.039 X 60 70 80 90 100 110 SHELL LENGTH (mm) Figure 5, Linear regression of SL (hy 2-mni increments) and shell weight loss after drying for 24 h. 750 Abbe and Albright 2 3 4 5 6 7 8 9 10 11 12 PERCENT SHELL WEIGHT LOSS Figure 6. Percent weight loss frequency for sliells dried for 24 h. More tlian 74% of tlie 1749 oysters examined lost at least 3^} of their initial shell weight (darker bars). umes were compared, with "true" volumes determined by casts for 169 oysters (Fig. 7). Shells weighed at 0 h underestimated true volume by about 5.8% and remained nearly unchanged as shell weight loss increased from 1 to 12% (Fig. 7A). Shells weighed at 24 h. however, estimated a cavity volume that was fairly close to true volume when shell weight loss was 1 to 2%, but as weight loss increased to 12%. cavity volume was overestimated by as much as 20% (Fig. 7B). The point at which the underestimation of cavity volume by 0-h shell weight equals the overestimate of cavity vol- ume by the 24-h weight was at a shell weight loss of slightly <3%. Thus. 3% allowed a fairly conservative breakeven point, below which the 24-h weight gave better estimates of cavity volume and above which the 0-h weight gave better estimates. Since cavity volume is the denominator in the MCI equation. o > LU o o > 10 -10 t 30 > < a: O a: o a: 20 10 Shells Weighed at 0 Mr Y = -6 310* 0 257X R' = 0,054 Shells Weighed after 24 Hr B Y = -0 451 ♦2 423X R' = 0,591 p < 0.001 23456789 10 11 PERCENT SHELL WEIGHT LOSS 12 Figure 7. Linear regressions of the percentage error in cavity volume and the percentage shell weight loss for shells weighed at II h (A) and after 24 h (B). overestimated cavity volume results in underestimated meat con- dition by an equal ainount. Although 0-h weights overestimated meat condition by about 5.8% (range -2 to 15%), they remained relatively constant over the range of weight loss from 1 to 12%. However, the 24-h shell weights underestimated meat condition by about 7.5% (range 10 to -20%). and the estitnates became worse as the pei'centage of shell weight loss increased. Meat condition determined gravimetrically was correlated with meat condition determined by casts for 169 oysters after 0 h (Fig. 8A) and after 24 h drying (Fig. 8B). and both gave significant correlations. The 24-h shell weights had a highly significant r" of 0.952 {P < 0.001). but the 0-h shell weights were slightly better with an r' of 0.979 (P< 0.001). Because the oysters used in this analysis were all native to the Paluxent River, the possibility existed that oysters from elsewhere in the Chesapeake Bay might show different weight loss properties after drying for 24 h and might reduce the validity of using 0-h shell cavity volumes. In order for this technique to be valid else- where, the average shell weight loss after drying for 24 h should exceed 3%. For those areas examined. 24-h weight losses were >3% in all cases. Eastern Bay oysters lost 4.6%, Shady Side oys- ters lost 5.3%, the Wicomico River oysters lost 6.1%, and the St. Mary's River oysters lost 8.3%. All of these were greater than the 4.3% for Patuxent oysters, indicating that this technique would be valid in at least those areas, but probably in many other areas of the state, and perhaps other areas of the east coast, as well. DISCUSSION The value of gravimetric meat condition ineasurements has been demonstrated by Lawrence and Scott ( 19S2) and Crosby and Gale (1990). Dry weight measured at 24 h provides an excellent estimate of cavity volume with comparisons between cavity ca- pacity (in mL) by water displacement and cavity volume (in cm'') 16 SHELLS WEIGHED AT 0 HR 14 , -j^v ' i 12 . A ^J^ _l < 10 .sir Q^ 8 ol^ H • Jtf^ LU ^ ^ -> b ^ >^ Y = 0,203 + 1,032 X y^ R- = 0,979 y p< 0.001 o ^ - ^ Q 1 1 1 1,1.1 ^ 14 SHELLS WEIGHED AFTER 24 HR ^ ^ 19 ' ^' ' ^ 10 . B 1' 'tS^ ' lU VJ* ■ ' ' e 8 - y^ ' o ^yfT ' ^ 6 - •^' 4 ^"^ Y = 0.122 + 0.910 X y^ R' = 0.952 2 : f^ ^ p< 0,001 1 . > . 1 , 1 , 1 . 1 2 4 6 8 10 12 MCI DETERMINED BY CASTS 14 Figure 8. Linear regressions of MCI determined gravimetrically and by cast. For shells weighed at 0 h (A) the points fit the line slightly better than for shells weighed at 24 h (B). Improvement in Determining Oyster Condition Index 751 Figure 9. An oyster with a smooth shell typical of one that will lose i9c of its shell weight after drying for 24 h. Insets show enlarged detail. yielding correlation coefficients of 0.93 to 0.98 for oysters from three sites in South Carolina (Lawrence & Scott 1982). Our 24-h dry weights also yielded a correlation coefficient of 0.98, but 0-h dry weight coefficients reached 0.99 (Fig. 8A and B). Either method appears to yield a good estimate of meat condition, but since the water in the shell pores was weighed initially, there is no reason to exclude it in the second weight. The arbitrary removal of a variable amount of weight (water) artificially increases the vol- ume (capacity) of the shell cavity, which in turn artificially and unnecessarily decreases the meat condition. In addition to slightly increased accuracy, the use of 0-h weights means that all the shell weighing is completed within minutes. There is no need to allow shells to dry overnight and to return the next day to weigh them again. While this technique appears to be an improved method for estimating the meat condition of C. virginica in much of the Mary- land Chesapeake Bay. in terms of time and accuracy, it has not been tested elsewhere. The amount of water in the shell depends on porosity, which can be a function of oyster size, rate of growth, boring organisms, and shell structure. Small oysters generally lost less weight after 24 h. as a percentage of 0-h weight, than larger oysters because their shells had less time for shell-boring organ- isms to inhabit them. Oysters with smooth shells (Fig. 9) will often lose ^3% because there are few places for water to enter the shell. Oysters with rough shells (Fig. 10) will generally lose >?>% and sometimes a great deal more (up to IjVr). If the shells lose an average of just <3% of their weight from shell water, then a 24-h shell weight is the best estimator of cavity volume and thus of MCI. However, if shells lose >y/( . then the 0-h weight proves to be the best estimator. It should be relatively easy to determine whether oysters from any particular area lose (on average) >3% or <3% of their shell weight upon drying for 24 h, and thus determine which method is more accurate for that site. Investigations of condition index have been conducted with Cnissostrea gigas on the west coast (Schumacker et al. 1998, Brett Dumbauld, Wash- ington Department of Fish and Wildlife, pers. comm.l. and since the shell structure and porosity of C. gigas may differ from that of C. rilginica. we await the results of these investigations. ACKNOWLEDGMENTS Funding for this project was provided by the Academy of Natu- ral Sciences. An early draft was reviewed by B. Dumbauld. and the authors thank him for his helpful suggestions. We also appreciate the critical comments of an anonymous reviewer. The senior au- thor would like to acknowledge the efforts of junior author Brian Albright who was instrumental in the design and management of much of this project from the beginning. Brian passed away fol- lowing surgery in October 2001. at the age of 36, before we com- pleted this manuscript. LITERATURE CITED Abbe. G. R. & J. G. Sanders. 1988. Rapid decline in oyster condition in Ihe Patuxent River. Maryland. J. Shellfish Res. Ti.iT-.Sg. Butler, P. A. 1949. Gametogenesis in the oyster under conditions ot de- pressed salinity. Biol. Bull. 96:26.V269. Crosby. M. P. & L. D. Gale. 1990. A review and evaluation of bivalve condition index methodologies with a suggested standard method. J. Shellfish Res. 9:233-237. Grave. C. 1912. A manual of oyster culture in Maryland. Board of Shellfish Commis-sioners of Maryland. 4th Report, pp. 279-348. Haven. D. 1962. Seasonal cycle of condition index of oysters in the York and Rappahannock Rivers. Proc. Nat. Shellfish. A.-isoc. 51:42-66. Hawkins. C. M.. T. W. Rowell &. P. Woo. 1987. The importance of cleans- ing in the calculation of condition index in the soft-shell clam. M\a arenaria (L.). / Shellfish Res. 6:29-36. 752 Abbe and Albright Higgins. E. 1938. Progress in biological inquiries, 1937. Bulletin of the U. S. Bureau of Fisheries Administration Report No. 3(1. Washington. DC: U.S. Government Printing Office, pp. 1-70. Lawrence, D. R. & G. L Scott. 1982. The determination and use of con- dition index of oysters. Estuaries 5:23-21 . Loosanoff. V. L. 1953. Behavior of oysters in water of low salinities. Proc. Nat. Shellfish. Assn. 1952:135-131. Milroy. J. A. 1909. Seasonal variations in the quantity of glycogen present in samples of oysters. Sci. Invest. Land. Sen 2. 17(6). Moore. H. F. 1908. Volumetric studies of the food and feeding of oysters. Bull. U. S. Bur. Fish. 28:1297-1308. Schumacker. E. J.. B. R. Dumhauld & B. E. Kauffman. 1998. Investiga- tions using oy.ster condition index to monitor the aquatic environment of Willapa Bay Washington. J. Shellfish Res. 17:338-339. Scott. G. L & D. P. Middaugh. 1978. Seasonal chronic toxicity of chlori- nation to the American oyster. Crassastreu virgiuica. In: Water chlo- nnation. vol. 2: Environmental impact and health effects. Ann Arbor. Ml: Ann Arbor Science Publishers, Inc., pp. 31 1-328. Scott. G. I. & W. B. Vemberg. 1979. Co-occurring chlorine produced oxi- dants in seawater and their effect on the growth, survival and physiol- ogy of the American oyster. Crassostrea virginica (Gmelin): evidence for synergistic effects with seasonal temperature stress. In: W. B. Vem- berg. F. Thurberg. A. Calabreese & F. J. Vemberg. editors. Marine pollution: functional responses. New York: Acadeinic Press, pp. 4L1- 435. Walne. P. R. 1970. The seasonal variation of meat and glycogen content of seven populations of oysters Osirea edulis L. and a review of the literature. Miu. Agric. Fish. Food. Fish. Invest. Ser. 2. 26(3). Journal of Shctlfi.sh Rcseanh. Vol. 22. No. 7,. 15i-lbl. 2003. EFFECTS OF OYSTER REEFS ON WATER QUALITY IN A TIDAL CREEK ESTUARY KIMBERLY A. CHESSMAN, MARTIN H. POSEY,* MICHAEL A. MALLIN, LYNN A. LEONARD, AND TROY D. ALPHIN University of North Carolina at Wilmiiii^ton Center for Marine Science. 5600 Marvin K. Moss Lane. Wilniini^ton. Norlli Carolina 2,^409 ABSTRACT The importance of oyster filtering in moderating aspects of water quality has received increased attention over the past several years. This sttidy examined the intluence of intertidal oyster reefs on chlorophyll a. fecal coliform bacteria, and total suspended solid concentrations under field conditions in a tidal creek estuary. Oyster reefs of varying live oyster density were sampled during summer 2002, winter 200.1. and spring 201B. Water samples were taken upstream and downstream of each reef as well as over a mud Oat control area on an ebb tide and analyzed for concentrations of these water column constituents. Summer data showed consistent, significant decreases in chlorophyll a concentrations as water moved over the reefs, usually by 10-25%. Fecal coliform counts were frequently lower downstream, by up to 45%. but were much more variable and not statistically different in most cases. Data taken in winter, when temperatures and oyster feeding rates were lower, showed less consistency in upstream versus downstream patterns. In spring, chlorophyll « decreases were less frequent than in summer, but significant fecal coliform decreases were more frequent. Total suspended solid concentrations were not changed by the presence of oyster reefs during any season. Data from this study indicate that feeding by oysters and changes in water How caused by the presence of reefs may both play a role in reducing chloi-ophyll a and bacterial concentrations in the water column. KEY WORDS: Cnissoxtrcii virginicu. fecal coliform bacteria, chlorophyll a. tidal creek INTRODUCTION Increasing coastal papulations and watershed development have led to concerns over water quality for both shellfishing and human contact waters. Among the water quality concerns in coastal areas are water-borne pathogens, eutrophication, increased turbidity, and sediment loads. Nutrients, sediments, and pathogens enter natural water bodies through runoff and can have both human health and ecosystem-level impacts. Microbial pathogens, particularly those from human and animal feces, can pose concerns for human health (Grimes 1991). Fecal coliform bacteria, indicators of pathogens associated with human and animal wastes, have been shown to be positively conelated with impervious surface cover in a watershed as well as with nitrate and orthophosphate concentrations (Mallin et al. 2000) and turbidity (Pommepuy et al. 1992, Mallin et al. 2000), and inversely correlated with salinity (Goyal et al. 1977, Mallin et al. 1999, Mallin et al. 2000). Suspended solids and turbidity can contribute to survival and even growth of fecal coliform bacteria by providing protection from light, an organic substrate, and a mechanism for transport downstream (Gerba & McLeod 1976, Pommepuy et al. 1992. .Sayler et al. 1975). Rainfall events have also been coiTelated with increases in fecal coliform concentrations (Goyal et al. 1977. Struck 1988. Howell et al. 1995) due to runoff inputs. Increasing sedimentation and turbidity are concerns not only for their role in the survival of fecal coliforms, but also because of their effects on water column irradiance. Suspended solids and turbidity can prevent light from penetrating the water column and thus can negatively impact the growth of primary producers such as rooted aquatic macrophytes. benthic microalgae. and phy- toplankton (Cordone & Kelley 1961). Benthic cominunity struc- ture, including the occurrence of shellfish beds, can be affected through burial by sediments and interference with filter feeding (Loosanoff & Tommers 1948, Posey 1990, Shumway 1996). Eutrophication. caused mainly by nutrient loading, can also 'Corresponding author. E-mail: poseymCs'uncw.edu ha\e detrimental effects on ecosystems (Nixon 1995. Brickeret al. 1999). Direct effects of eutrt)phication include initial increases in chlorophyll and primary production, changes in phytoplankton and macroalgal communities, and loss of seagrass (Burkholder 2001, Cloern 2001). Indirect effects include changes in water transpar- ency, nutrient cycling, benthic communities, and food web struc- ture (Cloern 2001. Posey et al. 2002). The.se effects are moderated by system attributes, with some areas being more sensitive to nutrient loading than others (Cloern 2001. Posey et al. 2002). In response to the potential deterioration of water quality as- sociated with watershed development, natural measures are being examined as possible remediation techniques. Several recent stud- ies have concentrated on the role of bivalves in regulating sus- pended particulate loads in estuarine systems. Models based on laboratory studies of bivalve filtration rates predict that bivalves, when sufficiently abundant in shallow waters, can control phy- toplankton biomass (Cloern 1982. Officer et al. 1982, Gerritsen et al. 1994). These models, however, are often based on high esti- mates of feeding rates from laboratory trials and fail to take into account variability in bivalve feeding rates under field conditions or bivtilves' release of nutrients, which could actually stimulate phytoplankton growth. Oyster feeding rates can be affected by temperature, salinity, suspended solid concentrations, and other factors (Shumway 1996). While filter feeding is hypothesized to remove substantial amounts of particulate matter, removal may also be caused by physical effects of oyster reefs on water flow (Dame 1987). The presence of reefs can cause eddies and turbu- lence, which lead to the settling of fine particles. Field studies regarding removal of particulate matter by oyster reefs are somewhat limited. Dame et al. (1984, 1985. 1989) and Dame & Dankers (1988) found significant decreases in total or- ganic carbon, particulate organic carbon, total suspended solids, nitrite-i-nitrate. and chlorophyll a. Ammonium concentrations in- creased downstream of oyster reefs, suggesting a role for oyster reefs in nutrient cycling (Dame et al. 1984. 1985. 1989: Dame & Dankers 1988; Nelson et al. 2003). In one sitidy. tidal creeks with oysters did not show significantly lower chlorophyll a levels than 753 754 Cressman et al. creeks without oysters (Dame & Libes 1993); however, another study found significantly lower chlorophyll a (especially pho- totrophic flagellates I in creeks with oysters (Wetz et al. 2002). The eastern oyster. Crassostrea virginica (Gmelin). is a filter feeder that is widely believed to reduce the amount of particulate matter in the water column. Field evidence to support this idea is limited, however, and no field tests of fecal coliform reductions over oyster reefs have been published. The research described here assessed the impacts of intertidal oyster reefs on suspended solids, chlorophyll a. and fecal coliform bacteria in a human-impacted tidal creek and also examined whether live oyster density over natural ranges influenced rates of seston removal. MATERIALS AND METHODS Study Site Six natural, intertidal oyster reefs were examined in Hewletts Creek, southeastern North Carolina. Hewletts Creek is an anthro- pogenically impacted tidal creek with a watershed that is approxi- mately TC/f developed, with 18% impervious surface coverage (Mallin et al. 2000). The reefs used in this study were bar reefs approximately 10 m wide and were selected to provide a gradient of ambient live oyster density from "low'" (79 live oysters m~") to "high"" (167 live oysters m~"-. Table 1) based on live densities available in the study area. Because the amount of shell hash covering oyster reefs may contribute to physical effects on water flow, reefs with different amounts of shell cover were used. Two of the reefs had low dead shell cover (approximately 60-80% of the reef consisted of live oysters, and the rest of the substrate was exposed sediment); the others were completely covered by live and dead shell. All reefs were located near a channel in the creek to ensure sufficient flow and were at least 5 m distant from other reefs. Reefs were not located immediately adjacent to marsh, thus reducing potential effects of sedimentation associated with marshes. A mud flat area immediately upstream of the selected reefs was used as a no-oyster control. The mud flat area lacked any shell cover, was more than 20 m distant from oyster reefs, and was dominated by sediment of similar grain size (fine sands) as that adjacent to the studied oyster reefs. The vertical height and vertical complexity of each reef were measured, as they may impact physi- cal effects such as flow velocity (Lenihan 1999, Posey & Alphin. unpubl. Table 1 ). Reef height was measured while water covered the crest of the reef by recording the depth of water over the crest and subtracting this from the depth of water covering the edges of the reef. Vertical complexity was calculated by allowing a 1 m long chain to conform to the vertical contours of the reef and measuring the actual horizontal distance covered by the chain. Com- plexity was quantified as the ratio of straight distance after conform- ing to the contours divided by 1 m. Values for complexity range from 0 to 1. with smaller values indicative of higher complexity. Because flow speed can affect bivalve growth and filtration (Lenihan et al. 19961 as well as sediment deposition, it was im- portant to characterize the flow regimen of each reef in this study. Flow measurements were taken with a Marsh McBirney, Inc., (Frederick. MD) Flo-mate Model 2000 handheld current meter once in the summer and during sample collection in winter and spring. Further, because oyster reefs may cause settling of fine particles, it was desirable to determine whether sediment compo- sition was different upstream versus downstream of the reefs in this study. Sediment samples were taken at approximate upstream and downstream water column sampling locations during a low tide in June 2003, and grain size fractions were determined using a Beckman LS Coulter Counter (Miami. FL). Sampling Fecal coliform and chlorophyll a concentrations in tidal creeks have been shown to be highest at approximately mid-to-low tide (Mallin et al. 1999). Additionally, significant decreases in chloro- phyll a concentrations downstream of a created oyster reef near the study area were observed 3 h after high tide (Nelson et al. 2003). To increase the likelihood of detecting effects, water samples were taken as close as possible to mid-ebb tide (generally about 2 h after high tide). Samples were taken from a canoe to avoid disturbing sediment. All sampling was conducted on ebb tides with a pre- dicted range of 0.9-1.1 m after a high tide of approximately 1 m. Water depth was less than 35 cm on the upstream and downstream sides of the reef at the time of sampling and only a few cm of water were present over the crest, thereby maximizing the amount of water that came into contact with the oysters. Samples were taken at two locations upstream and two loca- tions downstream of each reef. The two upstream samples were approximately 1 m apart from each other, as were the downstream samples. Upstream samples were taken at mid-depth in the water column. Because dye studies conducted prior to sampling showed that water from mid-depth flowed up over the crest of the reef and stayed near the surface, downstream samples were taken just under the surface of the water. Downstream samples were taken before upstream samples to avoid the collection of sediments that had been stiired up by prior sampling. For the same reason, the first reef sampled in a day was downstream of the second reef. Sampling of the six reefs, as well as a mud-bottom control area. TABLE 1. Physical characteristics of oyster reefs used in the study. Live oyster densities (m"") were measured in Summer 2002 and Spring 2003. Also indicated is ''i shell cover, which is indicative of the amount of dead sliell covering the reef. Width is the distance water traveled over the reef between upstream and downstream sampling locations; height is the vertical difference between the crest and base of the reef. Sunmier Density Spring Density % Shell Length Width Height Vertical Reef (per m -) (per m~') Cover (m) (m) (m) Complexity 1 79 132 100 14.5 13.5 0.29 0.68 2 113 129 100 10.0 15.0 0.15 0.64 3 114 150 60 13.0 8.0 0.40 0,68 4 116 163 80 13.0 9.5 0.50 0.75 5 129 176 100 13.0 S.O 0.30 0.70 fi 167 IS3 100 17.7 5.5 0.65 0.73 Oyster Reef Effects on Water Quality 755 was accomplished over a period of three days during each sam- pHng period, with two reefs sampled per day. Sampling was con- ducted twice per season during summer 2002 (once in July and once in August) and spring 2003 (twice in May. approximately two weeks apart). Due to low concentrations of water column constitu- ents as well as weather limitations, only one sampling set was conducted in winter 2003 (February). Sampling within 24 h of ruin was avoided due to potential effects of storm water runoff on water column constituents. In winter, however, there were such low con- centrations of the water column constituents of interest that it was necessary to sample after a rain event, in addition to the scheduled sampling period, to have sufficiently high chlorophyll a and fecal coliform concentrations to allow detection of potential effects. The two reefs with the highest live-oyster density and the mud tlat control area were all sampled the day after a rainfall of approxi- mately 3 cm in February 2003. Chlorophyll ci samples were taken in triplicate into 125-mL opaque plastic bottles. A fourth bottle was used to ensure collec- tion of enough water for total suspended solids (TSS) analysis. Fecal coliform samples were collected using autocluved 50()-mL glass bottles. All samples were kept on ice until they were filtered. Water remaining after filtration of fecal coliforms and chlorophyll a was combined and stored at 4 C until it could be used in analysis of TSS. Originally, this project was intended to focus on changes in turbidity rather than TSS. However, initial attempts to measure turbidity met with methodologic difficulties, and TSS analysis was added to the study in the second summer sampling period. Sample Processing Fecal coliform and chlorophyll a samples were filtered upon return to the laboratory and within 6 h of collection. Fecal coliform bacteria concentrations were determined according to the mem- brane filter procedure, using niFC medium (Sparks, MD) (APHA 1995). Chlorophyll a samples were filtered through Gelman (Clifton, NJ) A/E glass fiber filters with 1.0 jxm pore size. The filters were wrapped individually in aluminum foil and frozen in a sealed container with desiccant. Concentrations were determined tluorometrically (Welschmeyer 1994) within three weeks. TSS were analyzed gravimetrically (APHA 1995) using 500 mL of water from each sampling location. TSS were filtered through predried Gelman A/E 47 mm diameter glass fiber filters with 1.0 p.m pore size. Statistical Analysis The parameters of chlorophyll ci and fecal coliform concentra- tions were tested for normality and nonheterogeneity of variances. Variances upstream and downstream of reefs were nonheteroge- neous for both parameters. However, neither showed a normal distribution, even after standard transformations, leading to the use of nonparametric tests. Kruskal-Wallis tests were used (Sokal & Rohlf 1445) to test upstream versus downstream concentrations of the sampled variables and to determine whether they were signifi- cantly different across each individual reef for each sampling pe- riod. In all other analyses, which involved concentration changes of variables and not the non-normally distributed concentrations themselves, parametric methods were used. Multiple regression was used to determine whether the concentration changes of the studied variables were related to live oyster density, mean up- stream fiow speed, tidal range, and the time elapsed between high tide and actual sampling. An ANOVA was used to test for differ- ences between the high-shell and low-shell reefs of the same live oyster density. A /-test was used to test for overall reef effects within a season (i.e., did the reefs show consistently decreased concentrations downstream?). All analyses used SAS (SAS Insti- tute. Inc. 1989). RESULTS SniniNvr Mean chlorophyll a concentrations ranged from 2.3-10.6 p-g L"' over the reefs and mud tlat during the summer sampling pe- riods. Mean fecal coliform concentrations ranged from 1 .3-54.8 colony forming units (CFU) 100 mL"'. Total suspended solid concentrations ranged from 10-27 mg L"'. Temperature was ap- proxiinately 25-27°C and salinity ranged from 30-36 ppt at the study site during these sampling periods. Chlorophyll ci was significuntly lower downstream of reefs than upstream in summer for 9 of 1 2 comparisons (two comparisons for each of the six reefs: Table 2). This overall reef effect was sig- nificant for all reefs combined {P = 0.002), for high-shell-cover reefs (P = 0.023) and for low-shell-cover reefs (P = 0.053). Each reef demonstrated a significant decrease in chlorophyll a at least one of the two times it was sampled over the summer. There was no significant difference in percent removal of chlorophyll a be- tween the high-shell-cover and low-shell-cover reefs of the same live oyster density (P = 0.52). The control was sampled only once during suinmer, and at that lime chlorophyll a was significantly lower downstream than upstream (P = 0.010). Changes in chlo- rophyll a concentrations were not significantly related to live oys- ter density (Fig. lA) or tidal range. Fecal coliform concentrations were often lower dow nstream of reefs than upstream (8 out of 12 comparisons), although only two differences were statistically significant and there was not a sig- nificant overall reef effect (P = 0.22). Fecal coliform concentra- tions were higher downstream on the mud fiat than upstream, but this difference was not significant. Changes in fecal coliform con- centrations were not significantly related to live oyster density (Fig. IB) or tidal range. There was no significant difference in percent fecal coliform removal between the high-shell and low- shell reefs of the same live oyster density iP = 0.86). Because of difficulties encountered when measuring turbidity. TSS concentrations were added to sampling during the second summer sampling period. There were three instances of lower (24-38%) TSS concentrations downstream of reefs, two instances of higher (25^3%) concentrations downstream, and one instance with very little change. The mud tlat showed no change in TSS concentration. Due to a lack of replication (only two samples upstream and two downstream), no statistical test could be run on the differences across each reef or the mud tlat. There was no significant overall reef effect on TSS concentrations {P = 0.44). Changes in TSS concentrations were not significantly related to live oyster density (Fig. IC) or tidal range, and percent change was not significantly different between the high-shell and low-shell reefs of the same live oyster density {P = 0.80). Winter Mean chlorophyll a concentrations ranged from 0.3-1 .5 p.g L"' over the reefs and control during the winter sampling period. Mean fecal coliform concentrations ranged from 0.2-8.0 CFU 100 mL ' over the reefs and 22.5-36.7 CFU 100 mL ' over the nonreef mud flat area (control). Temperature was approximately 4 C and salin- 756 Cressman et al. TABLE 2. Results of Kruskal-Wallis ttsts on upstream vs. downstream concentrations of chlorophyll a (chl) and fecal coliform hacteria (fc) concentrations. Significant differences are in bold. All sinnificant changes were reductions (lower downstream) evcept for one, designated with a. Each reef was sampled twice in summer 2002 and spring 2003 and once in winter 2003. The mudHat was sampled only once in summer, and reef 6 was sampled tw ice in winter. Reef TABLE 2. (Continued!. K-W Season Parameter df Chi-square /•-Value Summer 1 chl 10 5.SI0 0.016 Summer 2 chl 10 0.SI9 0.366 Winter chl 5 .V667 0.056 Spring 1 chl 10 0.85(1 0.357 Spring 2 chl 10 9.000 0.003 Summer 1 fc 9 3.427 0.064 Summer 2 fc 10 0.315 0.575 Winter fc 10 4.046 0.044 Spring 1 fc 8 0.099 0.753 Spring 2 fc 10 0.660 0.417 Summer 1 chl 9 0. 1 38 0.711 Summer 2 chl 10 8.768 0.003 Winter chl 10 1.000 0.317 Spring 1 chl III 1.169 0.280 Spring 2 chl 10 4.373 0.037 Summer 1 fc 10 6.322 0.012 Summer 2 fc 10 1.664 0.197 Winter fc 9 0.222 0.637 Spring 1 fc 10 0.523 0.470 Spring 2 fc 10 0.241 0.624 Summer 1 chl 10 8.366 0.004 Summer 2 chl 7 5.492 0.019 Winter chl 10 4.083 0.043 Spring 1 chl III 8.366 0.004 Spring 2 chl 9 5.307 0.021 Summer 1 fc 10 0.007 0.934 Summer 2 fc 8 0.702 0.402 Winter fc 10 2.898 0.089 Spring 1 fc 10 6.657 0.010 Spring 2 fc 10 3.718 0.054 Summer 1 chl 10 8.426 0.004 Summer 2 chl 10 4.790 0.029 Winter chl 10 5,978 0.015 Spring 1 chl 10 4.333 0.037 Spring 2 chl 10 4.889 0.027* Summer 1 fc 10 0.058 0.810 Summer 2 fc 9 0.533 0.465 Winter fc 10 0.946 0.331 Spring 1 fc 10 0.410 0.522 Spring 2 fc 10 0.235 0.628 ity ranged IVotii 17-35 ppt ;tl the stridy site during this sampling period. Turbidity was very low, ranging frotn 1.5-5.0 NTU. and TSS concentrations ranged from 1.8-7.5 mg L"'. Because concentrations of the studied water column constitu- ents were so low, the two highest live-oyster-density reefs (both with high dead shell cover) and the mud flat were also sampled after approximately 3 cm of rain, when the creek water level was higher than normal. After this rain event, mean chlorophyll ci con- centrations ranged from 1.8-2.6 |jLg L"' and mean fecal coliform concentrations were approxitnately 146-516 CFU 100 tiiL"'. Tem- perature was 4°C and salinity ranged from 15-29 ppt among sites Reef Season Parameter df Chi-square K-W P-Value Mudnm Summer I Summer 2 Winter Spring 1 Spring 2 Summer 1 Summer 2 Winter Spring 1 Spring 2 Summer 1 Suminer 2 Winter 1 Winter 2 Spring 1 Spring 2 Summer 1 SuiTimer 2 Winter 1 Winter 2 Spring 1 Spring 2 Summer Winter Spring 1 Spring 2 Summer 2 Winter Spring 1 Spring 2 chl chl fc fc fc fc fc chl chl chl chl chl chl fc fc fc fc fc fc chl chl chl chl fc fc fc fc 10 8.396 10 8.640 10 0.000 10 1.331 10 5.843 10 2.857 10 0.103 10 0.244 8 2.455 10 0.026 10 2.929 10 5.810 10 3.008 10 1.637 10 0.232 10 0.164 9 1 .656 9 7.569 10 5.507 10 0.007 10 6.564 10 1.713 10 6.610 10 3.209 10 0.058 10 3.274 9 0.307 10 6.587 10 5.043 10 0.000 0.004 0.003 1 .000 0.249 0.016 0.091 0.749 0.622 0.1 17 0.871 0.087 0.016 0.083 0.201 0.630 0.686 0.198 0.006 0.019 0.933 0.010 0,191 0.010 0.073 0.810 0.070 0.580 0.010 0.025 1 .000 on the same day. Water flow speed was higher than normal after the rain event. This was due partly to a larger tidal range than was normally sampled ( 1.5 tn versus a usual range of 0.9-1.1 ni) as well as flow effects from storm water runoff. Turbidity was com- parable to spring and suinmer turbidity, ranging from 7.8-12.5 NTU. TSS concentrations were 9.0-15.4 mg L"'. During the regular winter sampling period, there were 2 sig- nificant decreases {P < 0.05) in chlorophyll ci concentrations over the reefs (Table 2). These differences were observed over low- shell-cover reefs, but there was not a significant difference be- tween these reefs and the high-shell-cover-reef of the same density (P = 0.564). A /-test did not show a significant overall reef effect on this variable for all reefs combined (P = 0.691), for high-shell- cover reefs (P = 0,582), or for low-shell-cover reefs (P = 0,323). 0\er the mud flat, there was no significant change in chlorophyll II. Changes in chlorophyll a in winter were not significantly telated to live oyster density (Fig. 2A), mean flow speed upstream of the reefs, or change in flow speed. After the rain event, both reefs and the nuid flat showed slight, nonsignificant increases in chlorophyll a. In the regular winter sampling period, fecal coliforms were lower downstream than upstream five times (out of seven coin- parisons; the highest-density reef was sampled twice in winter), but this overall reef effect was not significant for all reefs com- bined (P = 0.26), for high-shell-cover reefs (P = 0,22), or for low-shell-cover reefs (P = 0,86). Two of the fecal coliform de- creases were significant, and these occurred over the highest- Oyster Reef Effects on Water Quality 737 c o o o c 03 o O 0) O) c CO c o o o 0) en c 10 1 0 60 -10 -20 -30 °~ -40 o I 20 c -20 -40 -60 60 40 20 60 -20 -40 -60 60 Chlorophyll a ♦ 80 100 120 140 160 180 ♦ ♦ ♦ Live Oyster Density (m'-) Fecal Conforms 80 100 ►120 140 160 180 Live Oyster Density (m"') TSS 80 ♦ 100 120 140 160 180 ♦ Live Oyster Density (m"-) Figure 1. Water column constituents as related to live ovster density, summer 2(HI2: Percent changes in chlorophyll a, fecal coliforms. and TSS. Negative numbers represent a lower concentration downstream of the reef than upstream. density reef (/> = 0.019) and the lowest-density reet'(P = 0.044; Table 2). Fecal coliform concentrations significantly decreased over the mud flat {P = 0.010) during this sampling period. Changes in fecal coliform concentrations were not correlated with live oyster density (Fig. 2B). upstream flow speeds, or changes in flow. There was no significant difference between percent change in fecal coliform concentrations between the high-shell and low- shell reefs of the same live oyster density (P = 0.67). After the rain event, fecal coliform concentrations were el- evated above nonrain conditions. Due to crowding of the petri dishes on which the bacteria were grown, the counts were not considered reliable enough for statistical analysis. However, it was apparent that fecal coliform concentrations were highest over the mud flat (approximately 400 CFU 100 mL"'). lower over the highest-density reef, which was slightly downstream of and adja- cent to the mud flat (approximately 360 CFU 100 mL"'), and lowest over the most downstream reef (approximately ISO CFU 100 niL"'). During the regular winter samplmg period, TSS concentrations were higher (25-36*^) downstream of reefs as compared with upstream on three occasions. TSS concentrations were moderately lower {\0%) once, and twice were only slightly (<5'^»-) lower downstream. Given the low TSS concentrations during this sam- pling period, however, an increase of <1 mg L"' could translate to a 30% change. There was no significant overall reef effect on concentration changes {P = 0.252). Upstream to downstream changes in TSS concentrations were not significantly related to live oyster density (Fig. 2C), flow speed of water upstream of the reefs, or changes in flow .speed during the winter sampling period. There was no significant difference in TSS change between high- shell and low-shell reefs of the same live oyster density {P = 0.744). TSS concentrations were 0J9r higher downstream than 758 Cressman et al. g CO Chlorophyll a c 70 1 o ro 50 - c o o o 30 - O c o c o O 0 c o O) c -50 to ^ O S5 -100 i;!0 130 ♦ 140 150 ♦ 160 170 180 190 Live Oyster Density (m'^ TSS 40 30 c 20 o o 10 c o n D) C -10 U s« -20 120 f30 140 150 160 170 180 190 Live Oyster Density (m"^) Figure 2. Water column constituents as related to live oyster densit>, winter 20IL^: Percent changes in chlorophyll a, fecal coliforms, and TSS. Negative numbers represent a lower concentration downstream of the reef than upstream. upstream over the liighest-density reef after the rain event, but were 30% higher over the second-highest-density reef. On the mud flat. TSS concentrations were approximately \\% lower down- stream. Spring Mean chlorophyll a concentrations ranged from 1.3-7. 1 |j.g L"' over the reefs and 2.0-12.2 |xg L~' over the mud flat during the spring sampling period. Mean fecal coliform concentrations ranged from 8-330 CFU 100 niL"' over the reefs and mud flat. Fecal coliform counts were higher during the first spring sampling period than the second due to a long rainy period preceding sam- pling. Samples were not taken within 24 h of rain, but the earlier rain did affect the water column. Temperature was approximately 24°C, and salinity ranged from 19-2.'i ppt during the flrst spring sampling and 30-34 ppt during the second spring sampling period. Turbidity ranged from 5.8-9.8 NTU over both spring sampling periods. In spring, there were six significant decreases and one signifi- cant increase in chlorophyll (/ concentrations across the reefs (Table 2). There was not an overall reef effect on chlorophyll fl concentrations for all reefs combined {P = 0.18), for high-shell- cover reefs (P = 0.19), or for low-shell-cover reefs (P = 0.28). Chlorophyll ii changes also were not significantly related to live oyster density (Fig. 3A). flow speed upstream of the reefs, change in flow speed, or how long after the high tide samples were taken. There was no significant difference in percent removal of chloro- phyll (( between high- and low-shell-cover reefs of similar live oyster density (P = 0.45). Ten of 12 comparisons showed fecal coliform concentrations that were lower downstream than upstream in spring. Three of these decreases were significant (Table 2). as was the overall reef effect (P = 0.009). The mud flat showed a significant {P = 0.025) downstream decrease in fecal coliforms during one of the two spring sampling periods. Changes in fecal coliform concentrations were not significantly related to live oyster density (Fig. 3B). flow o O O 10 0 -10 -20 - -30 -40 -50 120 Oyster Reef Effects on Water Quality A Chlorophyll a 759 130> ♦ 140 150 ♦ 160 170 ♦ 180* 190 Live Oyster Density (m"-) Fecal Conforms 10 c o 2 0 c OJ o c o -10 O c (n -20 C31 c CO -30 O oS -40 o O g O) c CO 120 130 ♦ ♦ 140 150 ♦ 160^ 170 180 190 ♦ ♦ Live Oyster Density (nr^) TSS 15 10 5 0 -5 -10 -15 J ♦ ♦ ♦ 120 130 140 150 160' 170 A 180 190 Live Oyster Density (m"-') Figure 3. Water column constituents as related to live oyster density, spring 2003: Percent changes in chloropli>ll a. fecal coliforms, and TSS. Negative numbers represent a lower concentration downstream of the reef than upstream. speed upstream of reefs, or changes in flow. A /-test did show significantly decreased fecal coliform concentrations downstream of oyster reefs in spring for all reefs combined (P = 0.009). High-shell-cover reefs did not show this overall effect (P = 0.10); the pattern was driven by the low-shell-cover reefs (P = 0.012). However, high- and low-shell-cover reefs of similar live oyster density did not show significantly different patterns of fecal coliform removal in spring (P = 0.16). TSS did not exhibit a significant pattern with respect to the variables examined in spring. Out of 12 comparisons, downstream TSS concentrations were higher seven times, lower three times, and unchanged twice. There was not a significant overall reef effect on TSS concentration changes (P = 0.29). TSS concentra- tions were higher downstream once over the mud flat and re- mained unchanged during the other spring sampling period. The observed changes in TSS concentrations were not significantly related to live oyster density (Fig. 3C). water flow speed upstream of the reefs, or changes in flow. Percent removal of TSS was not significantly different between high- and low-shell-cover reefs of the same live oyster density (P = ().5A). Overall During the warm seasons of summer and spring, chlorophyll a was significantly lower downstream of reefs than upstream a total of I .^ times (out of 24 observations). Only once was it significantly higher. In summer, chlorophyll a concentrations were significantly lower downstream of oyster reefs than upstream (P = 0.002) overall. In spring, however, there was no significant overall reef effect. Fecal coliforms were reduced the majority of the time dur- ing the warm seasons ( 18 of 24 comparisons), but only 4 of these decreases were statisticallv sisznificant. In summer, this overall reef 760 Cressman et al. effect was not statistically significant, but it was significant in spring {P = 0.009). Water flow varied somewhat from reef to reef. The lowest observed flow over the parts of the reef from which samples were taken was 6 cm s' . Flow velocity reached 22 cm s" ' over the other reefs. The three-dimensional cuirent study showed increases in flow speed over the crest of three of the reefs and decreases over the crests of the other three reefs. However, differences in flow speeds between reefs were not significantly related to changes in the water column constituents. Vertical complexity did not differ among the reefs (Table 1 ). Over five of the six reefs, downstream sediments contained a greater amount of coarse sediment than upstream (by S-H'/f; Table }•). The mud flat did not exhibit the same distribution of sediment texture. DISCUSSION The presence of oyster reefs caused significant reductions in chlorophyll a and fecal coliform bacteria concentrations in this study. Effects on chlorophyll were greatest in summer, whereas effects on fecal coliforms were strongest in spring when bacterial counts were highest. The decreases in chlorophyll concentrations were consistent with previous studies showing that bivalve beds can have significant effects on the overlying water column (Dame et al. 1984. 1985. 1989: Asmus & Asmus 1991 ). and there has not been any previous investigation regarding effects of oyster reefs on fecal coliform concentrations. In this study, oyster reefs did not have any clear, consistent effects on TSS concentrations. Haven and Morales-Alamo ( 1970) found that a doubling of the number of oysters in an experimental tank led to an approximate doubling of removal rates of particulate matter. Changes in sus- pended particulate concentrations, then, should be significantly related to live oyster density if oyster feeding is the sole or over- riding factor in particulate removal. In this study, such a relation- ship was not observed. One possible explanation for this observa- tion is a threshold effect, some critical density of live oysters at which a measurable effect can be detected. Alternatively, the re- lationship between changes in seston and live oyster densities may be detected only over a greater density range and spatial scale. The oyster reefs used in this study provided only a small range of live oyster densities, especially after a large spatfall in summer 2002 (Posey & Alphin. unpubl.). Thus, the examined range of live oys- ter densities may have been too narrow for a density relationship to be detected. Because the changes in concentrations of the stud- ied water column constituents were not significantly related to flow speeds or changes in flow speed across the reefs, it is TABLE 3. Sediment eomposition. as '7t fine sediment (detlned as less than 63.41 fini diameter), upstream and downstream (el)b tide) of the oyster reefs. Reef % Fine Upstream '7c Fine Downstream 1 2 3 4 5 6 Mudflat 40 38 72 85 41 64 40 32 21 70 78 30 51 41 unlikely that the observed changes were due solely to flow speed. Live oyster lengths near the study site averaged 65 mm (Har- well. Posey & Alphin, unpubl.). Using the methods of Dame ( 1972). the mean dry weight for these oysters was calculated to be 1.33 g. NewelFs (1988) estimate of oyster clearance rates of 5 L h~' g"' were used to calculate the potential volume of water that could be cleared by each oyster reef in this study. In summer, flow velocities upstream of the oyster reefs ranged from 6-21 cm s"', and the oysters on the reefs could potentially clear only 5-15% of the water moving over them. Many of the observed chlorophyll a differences in summer were greater than the potential filtration capacity of the oysters on the reefs based on these estimates (up to 30% removal), suggesting that either oyster feeding rates are higher than NewelTs (1988) estimate or that factors other than oyster feeding (i.e.. other filter feeders or physical effects) are important in particulate removal. Additional calculations of approximate clearance rates, assum- ing 100% efficiency of particle removal, were made using the observed summer decreases in chlorophyll a concentrations. These rates ranged from 3-18 L h~' g"' across the reefs. The mean was 10 L h~' g~', which is consistent with Jordan's ( 1987) laboratory estimate. Oysters do not remove all particles from water with 100% efficiency, however, so this estimate may be conservative. Other filter feeders, such as mussels, were not abundant on these oyster reefs and therefore cannot account for the larger than expected effects. Even though flow velocities did not decrease downstream of the reefs, particle trapping within the reef crest may have occuned in flow shadow /ones between oyster clumps. This explanation is consistent with chlorophyll a and fecal coliform data in that the reefs that consistently showed significant decreases in chlorophyll it and fecal coliform concentrations were the reefs with low shell cover (i.e., low areas floored by mud). These were also the reefs with the lowest flow velocities (approximately 8 cm s"' ). Dame et al. (1985) and Dame (1987) found that most material uptake over an oyster reef in North Inlet occurred when flow was less than 15 cm s"' and attributed this to a combination of biofil- tration and sedimentation. Lower flow speeds could contribute to removal of particles by increasing the time water is in contact with the oysters and thus increasing their ability to filter particulates; it could also be that particles settled out of the water at these lower speeds. Preferential ingestion of chlorophyll (microalgae) by oys- ters (Ward et al. 2000, Wetz et al. 2002) may interact with low flow velocities to produce the strongest effects on this parameter, consistent with the significant reduction in chlorophyll concentra- tions over these reefs but low influence on TSS concentrations. Oyster reefs ha\e been shown to play a role in nutrient cycling in tidal creeks by releasing NH/ (Dame et al. 1984, 1985, 1989; Dame & Dankers 1988; Nelson et al. 2003). As such, it could be argued that chlorophyll a concentrations should actually be higher downstream of reefs than upstream. Ammonium released by bi- valves can be taken up by phytoplankton and lead to increased phytoplankton biomass. Asmus and Asmus (1991) made this ar- gument for sy.stems impacted by a mussel bed, though their field study showed significant decreases in phytoplankton biomass across the bed. Increased phytoplankton production due to nutrient release is also a possibility for oyster reefs. However, there is a lag time of a few hours before the ammonium shows up as primary production in the water column, and any increased production may be appearing further downstream of the reefs than the location of sample collection for this study. In terms of the parameters exam- ined in this study, the only change that would be immediate Oysti;r Rhhf Effects on Water Quality 761 enough lo detect as water Hows over the oyster reefs is particle remo\al. hccal colit'orni concentrations v\ere often lower downstream of reefs than upstream, but the differences were rarely significant. The overall reef effect of decreased fecal coliform concentrations was significant in spring but not suinmer, the opposite of the effect for chlorophyll a. Fecal coliform counts are e.\tremely variable, necessitating large changes before a significant effect can be de- tected. Because fecal coliform concentrations were higher in spring than in summer and winter, differences were slightly more detectable. C. virgiitka filters unattached bacteria with an efficiency of only 5% {Langdon & Newell 1990). However, fecal coliforms have been associated with turbidity and suspended sediments in the water column (Sayler et al. 1975. Pommepuy et al. 1992. Mallin et al. 2000) and inay be removed with suspended particulate matter through either filtration or settling. In this study, fecal coliform counts did not have consistent relationships with either turbidity or TSS. Changes in fecal coliform concentrations were not significantly related to live oyster density, fiow speeds, or changes in flow speed across the reefs. None of these factors is readily apparent as the most influential one. and changes in fecal coliform concentrations are likely due to a combination of factors. Changes in TSS concentrations did not exhibit any significant patterns relative to the variables examined in this study. Due to a lack of replication, statistical tests could not be used to determine whether changes across a single reef were significant. However, tests could be run to detect overall reef effects within a season, and none of these were significant for TSS. Changes in TSS were not consistently positive or negative in any season. Water temperature in winter was 4°C, lower than the minimum temperature (5°C) at which oysters typically feed (Galtsoff 1928. in Shumway 1996). Chlorophyll a and fecal coliforms were con- sistently decreased in the warm seasons of summer and spring, but neither showed a consistent effect in winter. Feeding effects are suggested by a lack of consistent change in water column con- stituents during winter, even when concentrations were high enough to detect a difference (after the rain event). The fact that the presence of oyster reefs frequently led to significant decreases in chlorophyll ci and fecal coliform concen- trations, but rarely reduced total suspended solids, leads to specu- lation that selective feeding by oysters occurred. In laboratory experiments, oysters have been shown to feed selectively on high quality food particles (Loosanoff 1949, Newell & Jordan 1983). In South Carolina tidal creeks. Wetz et al. (2002) found preferential feeding on phototrophic fiagellates. but not heterotrophic flagel- lates, bacterioplankton, or cyanobacteria. Although the current study was not designed to investigate selectivity, these results do suggest that it occurs to a degree in these systems. Flow conditions may also have contributed to changes in water column constituents; particles may have settled over the crest of the reefs (also suggested bv Dame 1987). Differences in bottom sediment composition, however, may be due to larger-scale flow patterns. Sediments were finer on the sides of the reefs that were upstream during ebb tide (downstream during flood tide). Faster flow during ebb tide than flood tide would lead to greater depo- sition of fine particles during flood tide than ebb (as suggested by Dame 1987), which could explain the observed differences in sedi- ment texture. In Bradley Creek, a tidal creek in southeastern North Carolina, current velocities were 14—55% higher on flood than ebb tides (Angelidaki 1997). Howexer. high velocities lasted longer on the ebb tide than flood tide (Angelidaki 1997). possibly causing more sediment to settle on the flood tides. This study did not examine effects of oyster reefs during flood tides because chloro- phyll (/ and fecal coliform concentrations are highest during ebb tides (Mallin et al. 1999). reflecting upland drainage influences. While there was never a significant difference for changes of chlorophyll ii. fecal coliform. or TSS concentrations between high- shell-cover and low-shell-cover reefs, the reefs themselves showed different patterns of effect. The reefs with low shell cover were also the reefs with lowest flow velocities and showed consistent removal of fecal coliforms in spring, whereas the other reefs did not. Vertical complexity was approximately equal between all reefs, and complexity may be a more important component in flow effects than the presence of shell itself. Multiple factors could be responsible for the observed effects on chlorophyll a. fecal coliform, and TSS concentrations. Both filtration by oysters and flow patterns over oyster reefs could contribute to particle removal in tidal creek ecosystems. CONCLUSIONS Significant changes in concentrations of chlorophyll a and fecal coliform bacteria were detected during warm seasons, even when effects on TSS concentrations were not observed. None of the examined variables were significantly related to live oyster den- sity, flow speed, or change in flow speed across reefs, suggesting possible threshold effects. Oyster reefs do have detectable effects on chlorophyll a and fecal coliform concentrations under field conditions, though effects vary temporally. The degree of removal suggests physical mechanisms for removal in addition to filtration effects. ACKNOWLEDGMENTS This work was supported by North Carolina Sea Grant (R/MER 46 to M. Posey andT. Alphin and R/MG 0213 toT. Alphin and M. Posey), the New Hanover County Tidal Creeks Program and the new center for marine science. The authors thank the Benthic Ecology Lab (B. Allen. M. Anderson. R. Barbour, B. Boutin. H. Harwell, T. Molesky, B. Noller, M. Owens, and J. Vinson) and the Aquatic Ecology Lab (H. CoVan, V. Johnson, T. MacPherson, M. Mclver, D. Parsons, and D. Wells) at the UNCW Center for Ma- rine Science for assistance in the field and laboratory. Additional thanks go to A. Croft and J. O'Reilly. LITERATURE CITED Angelidaki. K. 1997. Sediment and nutrient transport in a soulheaslern North Carolina tidal creek. Masters Thesis. University of North Caro- lina at Wilmington. 96 pp. APHA. 1995. Standard methods for the cxaminalion of water and waste- water. 19th ed. Washington. DC: Amenean Puhhc Health Associa- tion. 1041 pp. Asnius. R. M. & H. Asnius. 1991. Mussel beds: hmiling or promoting phytoplankton' i. E\p. Man Biol. Ecol. 148:215-232. Bricker. S. B.. C. G. Clement. D. E. Pirhalla. S. P. Orlando & D. R. G. Farrow. 1 999. National estuarine eutrophication wssessment. Effects of nutrient enrichment in the nation's estuaries. Silver Spring. MD: Na- tional Oceanic and Atmospheric Administration. 71 pp. 762 Cressman et al. Burkliolder. J. M. 2001. Beyond algal blooms, oxygen deficits and fish kills: chronic, long-term impacts ot nutrient pollution on aquatic eco- systems. In: L. Bendell-Young & P. Gallaugher. editors. Waters in peril. Norwell. MA: Kluwer Academic Press, pp. 103-125. Cloem. J. E. 1982. Does the benthos control phytoplankton biomass in South San Francisco Bay? Mar. Ecol. Prog. Ser. 9:191-202. Cloem, J. E. 2001. Our evolving conceptual model of the coastal eutro- phication problem. Mar. Eta!. Prcig. Ser. 210:223-253. Cordone. A. J. & D. W. Kelley. 1961. The influences of inorganic sediment on the aquatic life of streams. Cnl. Fish & Game 47:189-228. Dame. R. F. 1972. Comparison of various allometric relationships in in- tertidal and subtidal American oy.sters. Fish. Bull. 70:1 121-1 126. Dame. R. F. 1987. The net flux of inorganic matter by an intertidal oyster reef Com. Shelf Res. 7:1421-1424. Daine. R. F. & N. Dankers. 1988. Uptake and release of materials by a Wadden Sea mussel bed. / E.xp. Mar Biol. Ecol. 118:207-216. Dame, R. & S. Libes. 1993. Oyster reefs and nutrient retention in tidal creeks. / E.\p. Mar. Biol. Ecol. 171:251-258. Dame. R. F., J. D. Spurrier & T. G. Wolaver. 1989. Carbon, nitrogen and phosphorus processing by an oyster reef Mar. Ecol. Prog. Ser. 54: 249-256. Dame, R. F., T. G. Wolaver & S. M. Libes. 1985. The summer uptake and release of nitrogen by an intertidal oyster reef Neiherlaiuls J. Sea Res. 19:265-268. Dame, R. F.. R. G. Zingmark & E. Haskin. 1984. Oyster reefs as processors of estuarine materials. / Exp. Mar. Biol. Ecol. 83:239-247. Galtsoff P. S. 1928. The effect of temperature on the mechanical activity of the gills of the oyster iOstrea virgiiiica Gmelin). / Gen. Phys. 11:415-431. Gerba, C. P. & J. S. McLeod. 1976. Effect of sediments on the survival of Escherichia coli in marine waters. Appl. Environ. Microbiol. 32:114— 120. Gerritsen, J., A, F, Holland & D. E. Irvine. 1994. Suspension-feeding bi- valves and the fate of primary production: An estuarine model applied to Chesapeake Bay. Estiuuies 17:403—116. Goyal. S. M., C. P. Gerba & J. L. Melnik. 1977. Occurrence and distribu- tion of bacterial indicators and pathogens in canal communities along the Texas coast. Appl. Environ. Microbiol. 34:139-149. Grimes, D. J. 1991. Ecology of estuarine bacteria capable of causing hu- man disease: A review. Estuaries 14:345-360. Haven. D. S. & R. Morales-Alamo. 1970. Filtration of particles from sus- pension by the American oyster Crassoslrea virginica. Biol. Bull. 139: 248-264. Howell. J. M.. M. S. Coyne & P. Cornelius. 1995. Fecal bacteria in agri- cultural waters of the Bluegrass region of Kentucky. J. Environ. Qual. 24:411-419. Jordan. S. J. 1987. Sedimentation and remineralization associated with biodeposition by the American oyster Crassoslrea virginica (Gmelin). Doctoral dissertation. University of Maryland. College Park. 200 pp. Langdon. C. J. & R. I. E. Newell. 1990. Utilization of detritus and bacteria as food sources by two bivalve suspension feeders, the oyster Cras- soslrea virginica and the mussel Geukensia ilemissa. Mar. Ecol. Prog. Ser 58:299-310. Lenihan. H. S. 1999. Physical-biological coupling on oyster reefs: how habitat structure influences individual performance. Ecol. Monogr. 69: 251-275. Lenihan. H. S.. C. H. Peterson & J. M. Allen. 1496. Does flow speed also have a direct effect on growth of active suspension-feeders: An experi- mental test on oysters. Limnol. Oceanogr. 41:1359-1366. Loosanoff V. L. 1949. On the food selectivity of oysters. Science 1 10:122. Loosanoff V. L. & F. D. Tommers. 1948. Effect of suspended silt and other substances on rate of feeding of oysters. Science 107:69-70. Mallin, M. A., E. C. Esham. K. E. Williams & J. E. Nearhoof 1999. Tidal stage variability of fecal coliform and chlorophyll a concentrations in coastal creeks. Mar Poll. Bull. 38:414-422. Mallin. M. A., K. E. Williams. E. C. Esham & R. P. Lowe. 2000. Effect of human development on bacteriological water quahty in coastal water- sheds. Ecol. Appl. 10:1047-1056. Nelson. K. A., L. A. Leonard. M. H. Posey. T. D. Alphin & M. A. Mallin. 2003. Transplanted oyster (Crassoslrea virginica) beds as self- sustaining mechanisms for water quality iniprovenient in small tidal creeks. J. Exp. Mar. Biol. Ecol. Newell. R. L E. 1988. Ecological changes in Chesapeake Bay: Are they the result of overharvesting the American oyster Crassoslrea virginica'! in: M. P. Lynch and E. C. Krome. editors. Understanding the estuary: Ad- vances in Chesapeake Bay research. Publication 129. Solomons. MD: Chesapeake Research Consortium, pp. 536-546. Newell. R. I. E. & S.J. Jordan. 1983. Preferential ingestion of organic material by the American oyster. Crassoslrea virginica. Mar. Ecol. Prog. Ser 13:47-53. Nixon. S. W. 1995. Coastal marine eutrophication: A definition, social causes, and future concerns. Ophelia 41:199-219. Officer. C. B.. T. J. Smayda & R. Mann. 1982. Benthic filter feeding: A natural eutrophication control. Mar. Ecol. Prog. Ser. 9:203-210. Pommepuy. M.. J. F. Guillaud. E. Dupray. A. Derrien. F. LeGuyader & M. Cormier. 1992. Enteric bacterial survival factors. Walcr Sci. Technol. 25:9.VI03. Posey. M. H. 1990. Functional approaches to soft-substrate communities: How useful are they? Rev. Aquai. Sci. 213:343-356. Posey. M. H.. T. D. Alphin. L. B. Cahoon. D. G. Lindquist. M. A. Mallin & M. B. Nevers. 2002. Top-down vs. bottom-up limitation in benthic communities: direct and indirect effects. Estuaries 25:999-1014. SAS Institute. Inc. 1989. SAS/STAT user's guide. Version 6. 4th ed. Gary. NC: SAS Institute. Inc. 943 pp. Sayler. G. S.. J. D. Nelson. Jr.. A. Ju,stice & R. R. Colwell. 1975. Distn- bution and significance of fecal indicator organisms in the upper Chesapeake Bay. Appl. Microbiol. 30:625-638. Shumway. S. E. 1996. Natural environmental factors. In: V. S. Kennedy & R.I.E. Newell, editors. The eastern oyster Crassoslrea virginica. Col- lege Park. MD: Maryland Sea Grant, pp. 467-513. Sokal. R. R. & F. J. Rohlf 1995. Biometry: The principles and practice of statistics in biological research. New York: W. H. Freeman and Com- pany. 887 pp. Struck. P. H. 1988. The relationship between sediment and fecal coliform levels in a Puget Sound estuary. J. Environ. Health 5550:403—407. Ward. J. E.. J. S. Levinton. S. E. Shumway & T. L. Cucci. 2000. Influence of diet quality on pre-ingestive feeding strategies of bivalves: connect- ing pallial cavity function to ecosystem processes. J. Shell. Res. 19:606. Welschmeyer. N. A. 1994. Fluorometric analysis of chlorophyll a in the presence of chlorophyll /' and phaeopigments. Limnol. Oceanogr. 39: 1985-1993. Wetz. M. S.. A. J. Lewitus. E. T. Koepfler & K. C. Hayes. 2002. Impact of the eastern oyster Crassoslrea virginica on microbial community struc- ture in a salt marsh estuary. Ai/ual. Microh. Ecol. 28:87-97. Joimuil oj Slu'llfisli Research. Vol. 22. No. 3, Ity-lUh. 2003. EXPRESSION OF HSP 70 IN EXPERIMENTALLY METAL-EXPOSED EUROPEAN FLAT OYSTERS OSTREA EDULIS ISABELLE BOUTET.' ARNAUD TANGUY," MICHEL AUFFRET,' NEDZAD MUJDZIC.' AND DARIO MORAGA'* 'Lahonitoire des Sciences cle I'Emirowwiuent Marin (LEMAR), UMR-CNRS 6539. Insiimt Univer.sitaire Eiiropeen de la Men Universire de Bretagne Occidentale. Place Nicolas Copemic. 292iS(). PUnizane. France: and 'Ha.-ikin Shellfish Research Lahoraloiy. 6959 Miller Avenue. Port Norris. New Jersey 0SJ49 .\BSTR.\CT The heat shock protein 70 family is eomposed of both environmentally inducible (Hsp) and constitutively expressed (Hsc) members. The expression of Hsp70 was investigated in the European tlat oyster Ostrca cdulis exposed to different metal concentrations. By using a polyclonal antibody developed in our laboratory for a recombmant HspVO of the oyster Crassostrea gigas. the soluble HspVO level in O. editlis was found metal dose dependent. An exposure to copper did not induce Hsp70 synthesis in either gills or digestive gland. A decrease of Hsp70 was observed in gill from cadmium-exposed animals, whereas digestive gland tissue showed an increase. KEY WORDS: heat shock protein 70. 0\lrfci eihitis. ELISA. expression, quantification, metal accumulation. INTRODUCTION The cellular stress response is involved in protecting organisms from damage caused by e.xposure to a great variety of stressors, including temperature, heavy metals, and other xenobiotics. The stress response entails the rapid synthesis of heat shock proteins (HSPs) to protect the proteins against denaturation (Lindqiiist & Craig 1988. Sanders 1993). HSPs were first described in Droso- phila husckii (Ritossa 1962) and the genes encoding the Droso- phila Hsp were among the first eucaryotic gene to be cloned (Craig et al. 1979). The major and the most highly conserved and studied of the HSPs in all organisms is the 70-kDa protein family (HSP70) because ot its implication in protein chaperoning (Gething & Sam- brook 1992) and acquired tolerance processes (Lindquist & Craig 1988, Clegg et al. 1998). The genes encoding Hsp70 are highly conserved in evolution and contain both heat-inducible (Hsp) and constitutive genes (Hsc). both of which encode stress proteins under nomial conditions (Hightower 1993, Wood et al. 1998). The types of studies conducted on stress proteins in aquatic organisms are highly variable (Sanders 1993, Gourdon et al. 1998). The synthesis of Hsp70 and induction of thermo-tolerance has been demonstrated in the Pacific oyster, Crassostrea giiius iSham- seldin et al. 1997, Clegg et al. 1998, Gourdon et al. 2000) and in the mussels Mytiliis ediilis and Mytilus galioproviiicialis ( Sanders 1988. Snyder et al. 2001 ). Piano et al. (2002) showed a rapid and significant synthesis of the inducible Hsp69 in thermal stressed flat oyster Ostrea edulis, but no significant variations in the constitu- tive isoforms level (Hsp72 and Hsp77). Recently, we characterized two HSPVO genes and quantified soluble HSPVO by enzyme-linked immunosorbent assay (ELISA) in C. gigas exposed to metals in the laboratory (Boutet et al. 2003). In this previous study, we showed that soluble HSP70 level decreased in tissues of experimentally metals-exposed oysters. In the present work, the expression of HspVO and Hsc70 pro- teins in different organs of the European flat oyster, Osirca edulis. exposed to a concentration gradient of metals under experimental conditions was quantified by ELISA, using a polyclonal antibody ♦Corresponding author. E-mail: Dario.MoragaCsHiniv-brest.fr for a recombinant HscV2 of C. gigas developed in the laboratory (Boutet et al. 2003). MATERIALS AND METHODS Oyster Collection and Maintenance Adult European tlat oysters. O. edulis (3 years old; 7-8 cm), were purchased from an oyster farm of Mont Saint-Michel Bay (France) and maintained for one week in aerated 0.22-|j.m filtered seawater before experimentation. All the experiments were con- ducted in a temperature-controlled rooin (15°C) at a salinity of 349fc. Groups of 25 oysters were exposed to two metals, one es- sential (Cu-*) and the other toxic (Cd"""). Each metal was applied from a stock solution ( 100 mM) at each of two final concentrations (0.4 \xM and 4 p.M) and also in a mixture (0.2 (jiM each) for 15 days. The metal doses were chosen according to those found in most contaminated French estuaries. A group of 25 oysters was maintained in seawater. without metals, as a control. Seawater was renewed every day and oysters were fed with microalgae (Isoch- n-sis galbaiui) every two days. The metals were reapplied to the appropriate concentrations after every water change. Protein Extraction from Oyster Tissues On days 0. 1.2. 3, 5, V, and 15 of the experiment, gills and digestive gland from exposed and control oysters (/! = 3 for all samples) was harvested after oyster killing and homogenized in protein extraction buffer (150 mM NaCl, 10 mM NaH^POj, I mM phenylmethanesulfonyl fluoride, pH V.2) according to the protocol described by Tedengren et al. (1999). Samples were then centri- fuged at 12.000 g for 10 min at 4''C and supernatant fractions containing soluble proteins were collected in fresh tubes. Total soluble proteins were quantified using the D^ Protein Assay kit (Bio-Rad) with dilutions of Bovine Serum Albumin (Sigma) as the standai'd. Optical density was measured at 620 nm using a micro- plate reader. Metal .Analysis Pools of soft body excised from three oysters per sample day were mineralized with suprapure nitric acid. Concentrations of 763 764 BOUTET ET AL. cadmium and copper were measured in each tissue sample using tlie potentiometric stripping metliod (Riso et al. 1997. Boutet et al. 2002). 66.2 kDa Weslern Biol Analysis The cross-reactivity of the anti-CgHsc72 IgG antibody de\el- oped in our laboratory (Boutet et al. 2003) was tested by Western blot as follows. Samples of O. edidis (control and cadmium- exposed) proteins were electrophoresed on 12% SDS- polyacrylamide gel and electrotransferred to PVDF-membrane (Bio-Rad). The membrane was blocked for Ih with blocking buffer (0.1 M Tris. 5% nonfat dry milk) and then incubated with Tris buffer containing anti-CgHsc72 antibody (1/125 diluted) for 1 h with gentle agitation at room temperature. The membrane was washed twice for 10 min with washing buffer (0.1 M Tris. 0.02% Tween 20) and incubated with Tris buffer containing 1/1.000 di- luted polyclonal anti-rabbit IgG horseradish peroxidase- conjugated (Sigma) for 1 h with gentle agitation at room tempera- ture. Again the membrane was washed twice with washing buffer, the reactive band was visualized by staining with 2.4 mM ot 3-amino-9-ethyl-carbazole (Sigma) dissolved in 50 mM acetate buffer (0.2 M acetic acid. 0.2 M sodium acetate, pH 5) containing 5% of MW-dimethyl Formamide (Sigma) and 12%f of H,Oo. ELISA Microtiter plates were coated with 20 |xg per well of total proteins extracted from the digestive gland and gills of control and experimentally exposed oysters. HSP70 concentrations were quan- tified by ELISA developed in C. gigas using rabbit anti-CgHsc72 IgG and recombinant CgHsc72 as a standard (Boutet et al. 200.^). Statistical Analysis The variations in metal and Hsp le\el during the experiment were analyzed by analysis of covariance (a = 0.05) using CSS Statistica (Statsoft). RESULTS Metal Quantification in Oyster Tissues Copper and cadmium concentrations in tissues of oysters ex- perimentally exposed to Cu"* and Cd"* showed a significant time- dependent increase (compared with controls) during the 15 days of the experiment. Copper concentrations in the tissues of oysters exposed to 4 \iM or 0.4 |xM of Cu"* increased from 0.17 to 0.73.10"^ M/g wet weight tissue (M/gwwt) and 0.17 to 0.37. 10^^" M/gwwt. respectively. Dosing with 4 (jlM or 0.4 (xM of Cd"* resulted in an increase of Cd concentration in the gills from less than 0.01 to 0.31. lO"*" M/gwwt and 0.01 to 0.075.10"" M/gwwt. The concentration of metals in tissues of oy.sters exposed to a mixture of the two metals increased from O.OI to 0.025.10 ' M/gwwt for Cd. while copper concentration did not vary. Cross-Reactivity of Anti-CgHsc72 Antibody With O. eduih Proteins The Western blot revealed a high cross-reactivity of our anti- CgHsc72 antibody with O. cdidis HSP70 (Fig. 1). Two bands appeared on the membrane at a molecular weight of 68 and 70 kDa, confirming the specificity of the antibody with Heat Shock Protein 70 of this oyster species. M 1 2 Figure 1. Western Ulot (((digestive gland protein sample from control (lane 1) and cadmium-exposed oyster (lane 2) electrophoresed and probed with anti-Cghsc72 antibody. Marker (M) is SDS-PAGE Stan- dard broad range (Bio-Rad Laboratories, Hercules, CA), Quantification of Heat Shock Proteins 70 by ELISA Application of the ELISA to protein samples extracted from gill and digestive glands of control oysters showed significant differences between these tissues in basal level of Hsp70. Quan- tities of 46.5 ± 2.6 and 59.3 ± 3.4 mg Hsp/g"' protein, correspond- ing to approximately 4.7 and 5.9% of total proteins, were measured respectively in the gills and the digestive gland of control oysters. Hsp levels decreased significantly (compared with the control, a = 0.05) in the gill of oysters exposed to a mixture of the two metal and ^^^x.M of Cd (Fig. 2. A and B). A decrease (not signifi- cant) of Hsp70 levels in the gill of oysters exposed to copper was also observed. In contrast, a significant increase of Hsp concen- tration occurred in the digestive gland of animals exposed to 0.4 |jiM of Cd (Fig. 2E). No differences were observed in gills of individuals exposed to 0.4 p.M of Cd (Fig. 2B) or to Cu (Fig. 2C) and in digestive gland of oysters exposed to a mixture of metals (Fig. 2D), to 4|j.M Cd (Fig. 2E) or to Cu (Fig. 2F). A stronger dosage-effect of cadmium was observed as either a decrease or increase of Hsp levels in the two organs. No dosage-effect of copper could be demonstrated in either organ. DISCUSSION In this study, we quantified soluble HSP70 by ELISA in ex- perimentally metal-exposed O. edulis. The cross-reactivity of the purified rabbit anti-CgHsc72 IgG demonstrated here with OeHspJO supported the suitability of using these reagents to quan- tify HSP70. An increase in intensity of the 70 kDa bands was also observed in digestive gland of a cadmium-exposed oyster, in agreement with measurement of Hsp70 by ELISA. Now. our re- sults showed that HSP70 level is different in gill and digestive gland (4.7 vs. 5.9% of total protein). We previously reported a concentration of about 6% in the oyster C. giga.s (Boutet et al. 2003). and Feige and Polla (1994) observed a general HSP level of about 5% under normal conditions (without stress) in other organ- isms. In comparison to these basal levels, the quantification of soluble HSP70 in experimentally exposed O. edulis showed a metal-dosage response. A decrease of soluble HSP70 was ob- served in gills of oysters exposed to the highest concentration of cadmium or to a mixture of the two metals, in spite of a significant increase of metal concentration in the tissues. In contrast, an ex- posure to the lowest cadmium concentration induced an increase of HSP7() in digestive gland. Furthermore copper did not modify HSP70 levels in oyster tissues. We previously showed that metal exposure induced a significant decrease of HSP70 in tissues of C. gigas with the same treatments (Boutet et al. 2(X)3). Veldhuizen- Tsoerkan et al. (1991) found no variation in HSP70 in M. edulis caged in seawater with various concentrations of cadmium, like the response in copper-exposed O. edulis. In contrast. Lewis et al. (2001 ) showed an inhibitory effect of metals, particularly copper. Hsp70 Expression in Metal-Exposed Ostrea edulis 765 a. ai a. V) I 5 10 exposure duration (days) D 100 - - -a ■ - control — «^ 0.2mMCu +0.2 \iM Cd 5 10 exposure duration (days) B 5 10 exposure duration (days) control 4pMCd 0.4 pM Cd 5 10 exposure duration (days) S Q. a. M X 5 10 exposure duration (days) -^■- control — • — 4 pM Cu -a-- 0 4pMCu S 10 exposure duration (days) Figure 2. QiiantifKation of HSP70 (mean ± SKl by KLIS,\ in the nills l.\. B, and t) and in the digestive gland (D. E, and F) of O. edulis exposed to copper and cadmium l.\ and Dl, cadmium (B and K), and copper (C and F). in the seaweed Eiiternmorplici liilestiiuilis. These aulhors observed that high levels of copper appeared to damage protein synthesis. therefore impairing the HSP70 response. In our experiment, a de- cline of HSP70 was observed in cadmium- and a cadmium-copper mixture exposed oysters. A similar HSP70 synthesis inhibition was observed in earthworms. Liimhriats terrcstris. exposed to a variety of metals (lead, cadmium, and copper; Nadeau et al. 2001 ). When exposure approaches lethal levels, such as 4 p,M in our experi- ments, the average degradation rate of HSP70 will exceed its syn- thesis rate because of cytopathologic damage, such as ruptured 766 BOUTET ET AL. membranes, in many cells (Triebskorn & Kohler 1996. Quig 199S). The fact that the gills are the first barrier to metals could explain why this organ was more affected by the toxic effect of metals and showed a higher decrease in HSP70 concentration. At sub-lethal levels, our work showed an increase of HSP70 in re- sponse to exposure, in agreement with results described by Snyder et al. (2001). These authors showed a significant increase of HSP70 in cadmium-contaminated mussels. M. editlis. and limpets. Collisella peltci. and in heat-shocked and oil-exposed mussel. M. galloprovincialis. and abalone. Hidiotis riifescens. The ELISA developed in a previous study in C. gigas allowed us to specifically and rapidly quantify HSP70 proteins in tissues from marine mollusks. This immunologic method has the advan- tage of quantifying the protein of interest, unlike the commonly used Western blot analysis (Clegg et al. 1998. Nadeau et al. 2001 ), which gives only a semi-quantitative estimation of HSP70 amounts. Furthermore, this study showed that O. edulis displayed a differential response to the level of metal contamination. Ac- cording to the present study and a previous work on metallothio- nein in this species (Tanguy et al. 2003), the oyster O. edulis do not seem to be an appropriate indicator for studying environmental contamination. ACKNOWLEDGMENTS This research program was supported by the Region Bretagne and the CE program FAIR DISENV CT98-4129: "Environmental factors and shellfish diseases." We are grateful to Jean-Michel Escoubas who purchased the Crassostrea gigas hsc72 cDNA clone. Thanks are also due to Dr. Ricardo Riso for metal analysis in oyster tissues, to Brenda J. Landau for useful English correc- tions, to Dr. Louis Quiniou for his help with use of the CSS Statistica. and to Monique Briand for editing the figures. LITERATURE CITED Boutet. I.. A. Tanguy. M. Auffret. R. D. Riso & D. Moraga. 2002. Immu- nochemical quantification of metallothioneins in marine mollusks: characterization of a metal exposure bioindicator. Environ. Toxicol. Clu-m. 21:1009-1014. Boutet. I., A. Tanguy. S. Rousseau. M. Auffret & D. Moraga. 2003. Mo- lecular identification and expression of heat shock cognate 70 (hsc70l and heat shock protein 70 (hsp70) genes in the Pacific oyster Crasso- strea gigas. Cell Stress Chap. 8:76-85. Clegg. J. S.. G. N. Cher. E. Ritlcin & C. S. Friedman. 1998. Induced thermotolerance and the heat shock protein-70 family in the Pacific oyster Crassostrea gigas. Mol. Mar. Biol. Biotech. 7:21-30. Craig, E. A., B. J. McCarthy & S. C. Wadsworth. 1979. Sequence orga- nization of two recombinant plasmids containing genes for the major heat shock-induced protein of D. mekmogaster. Cell. 16:575-588. Feige, U. & B. S. Polla. 1994. Hsp70 - a multi-gene, multi-function family with potential clinical applications. Experientia. 50:979-986. Gething. M. J. & J. Sambrook. 1992. Protein folding in the cell. Nature. .\^5:33-i5. Gourdon. I.. M. C. Guenn & J. Torreilles. 1998. Cellular and molecular mechanisms of the stress response from marine bivalvia. C R, Soc. Biol. 192:749-774. Gourdon, I., L. Gricourt. K, Kellner. P. Roch & J. M. Escoubas. 2000. Characterization of a cDNA encoding a 72 kDa heat shock cognate protein (Hsc72) from the Pacific oyster, Crassostrea gigas. DNA Seq. 11:265-270. Hightower. L. E. 199.^. A brief perspective on the heat-shock response and stress proteins. Mar. Environ. Res. 35:79-83. Lewis. S.. M. E. Donkin & M. H. Depledge. 2001. Hsp70 expression in Enteromorpha intestinalis (Chlorophyta) exposed to environmental stressors. A^Ho;. Toxicol. 51:277-291. Lindquist, S. & E. A. Craig. 1988. The heat-shock proteins. Ann. Rev. Genet. 22:631-677. Nadeau. D.. S. Corneau. I. Plante. G. Morrow & R. M. Tanguay. 2001. Evaluation for Hsp70 as a biomarker of effect of pollutants on the earthworm Luinbriciis terrestris. Cell Stress Chap. 6:153-163. Piano. A., C. Asirelli. F. Caselli & E. Fabbri. 2002. Hsp70 expression m thermally stressed Oslrea edulis. a commercially important oyster in Europe. Cell Stress Chap. 7:250-257. Quig. D. 1998. Cystein metabolism and metal toxicity. .Altern. Med. Rev. 3:262-270. Riso. R. D.. P. Le Corre & C. J. Chaumery. 1997. Rapid and simultaneous analysis of trace metals {Cu, Pb and Cd) in seawater by potentiometric stripping analysis. Anal. Chim. Acta. 351:83-89. Ritossa. F. 1962. A new puffing pattern induced by heat shock and DNP in Drosophila. Experientia. 18:571-573. Sanders. B. M. 1988. The role of the stress proteins response in physi- ological adaptation of marine molluscs. Mar. Environ. Res. 24:207- 210. Sanders. B. M. 1993. Stress proteins m aquatic organisms: an environmen- tal perspective. Crit. Rev. Toxicol. 23:49-75. Shamseldin. A. A.. J. S. Clegg, C. S. Friedman. G. N. Chenr & M. C. Pillai. 1997. Induced thermotolerance in the Pacific oyster. Crassostrea gigas. J. Shell. Res. l6:487-f9l. Snyder. M. J.. E. Girvetz & E. P. Mulder. 2001. Induction of marine mollusc stress proteins by chemical or physical stress. .Arch. Environ. Contain. Toxicol. 41:22-29. Tanguy. A.. I. Boutet. R. D. Riso. P. Boudry. M. Auffret & D. Moraga. 2003. Study of two metallothionein genes in the European fiat oyster Ostrea edulis as a potential ecological tool for environmental monitor- ing. Mar. Ecol. Prog. Ser. 257:87-97. Tedengren, M., B. Olsson, O. Reimcr. D. C. Brown & B. P. Bradley. 1999. Heat pre-treatment increases cadmium resistance and HSP 70 levels in Baltic Sea mussels. Aquat. Toxicol. 48:1-12. Triebskorn. R. & H. R. Kohler. 1996. The impact of heavy metals on the grey garden slug Deroceras reticnlatinn (Miiller): metal storage, cel- lular effects and semi-quantitative evaluation of metal toxicity. Envi- ron. Polliil. 93:327-343. Veldhuizen-Tsoerkan, M. B.. D. A. Holwerda, A. M. T. de Bont. A. C. Smaal & D. I. Zandee. 1991. A field study on stress indices in the sea mussel, Mytilus edulis: application of the stress approach in biomoni- toring. Arch. Environ. Contain. Toxicol. 21:497-504. Wood. L. A.. I. R. Brown & J. H. Youson. 1998. Characterisation of the heat shock response in the gills of sea lampreys and a brook lamprey at different intervals of their life cycles. Coinp. Biochem. Physiol. 120A: 509-518. Jonnuil ,ij Shellfish Research. VoL 22. No. 3. Ibl-llX. 2003. IS BEAUTY IN THE EYE OF THE BEHOLDER? DEVELOPMENT OE A SIMPLE METHOD TO DESCRIBE DESIRABLE SHELL SHAPE FOR THE PACIFIC OYSTER INDUSTRY JOHN BRAKE. FORD EVANS, AND CHRIS LANGDON* Coastal Oregon Marine Experiment Station and Department of Fislieries and Wildlife, Hatfield Marine Scienee Center. Oregon State University. Newport. Oregon 97365 ABSTRACT Shell samples of Pacific oysters (Crfi.v.vo.vfcco ,t;',i;'") were evaluated from three different U.S. West coast farms. Industry experts described each shell sampled as being either desirable (good) or undesirable (bad). There were slight differences in the categorization of good and bad oysters among farms, but common trends were evident. The ratio of greatest shell depth to greatest shell length (D/L) was found to be more effective in categorizing good and bad shell shapes compared with other descriptors. Good oysters had a mean D/L of 0.316. whereas the bad oysters had a significantly lower mean of 0.219 iP < 0.001). Using the threshold value of D/L > 0.25 for good oysters. 85.6% of all sampled oysters were correctly assigned to good and bad categories, as defined by industry participants. The use of D/L and greatest shell width to greatest shell length (W/L) may be beneficial in distinguishing shell .shape quality and allow for the rapid assessment of many sampled oysters. These findings have implications in the development of industry standards for shell shape; furthermore, such standards would be useful in designing oyster breeding programs to improve shell shape. KHY WORDS: shell, shape, oyster. Crassosirea gigas. standards, industry INTRODUCTION Product quality is becoming more important as production of Pacific oysters (Crassostrea gigas) increases and competition for lucrative markets rise. Shell morphology often provides consumers w ith their first impression of product quality. Many shellfish in- dustries recognize shape as a valuable marketing tool. For the Atlantic Canadian oyster industry. Section 65 of the Canadian Food Inspection Agency's Fish Inspection Regulations outlines four different shape classes (differentiated by length to width ra- tios) by which Eastern oysters. Crassostrea virginica. are to be sold. In certain regions of France, growers have to sign a contrac- tual agreement with the Shellfish Professional Organization in which they agree to not sell oysters (Crassosirea gigas) of a cer- tain shape (determined by a previously reported forinula; Galtsoff 1964). In exchange, these growers are able to market oysters using that region's trademark (Goulletquer, personal communication). Such industry quality control has provided successful and favor- able product label identification within the inarketplace. In con- trast, classification of desirable and undesirable shell shapes has not been objectively defined by the U.S. West coast oyster indus- try. The development of industry standards by which shape can be objectively defined would be of use to the West coast oyster in- dustry in assessing the effects of different culture practices and genetic stocks on shell shape. In addition, shell shape may become increasingly important in product label identification and industry quality assurance. The objective of this research was to use simple linear measurements to characterize the shape of Pacific oyster [Crassosirea gi.qas) shells, classified as being either desirable (good) or undesirable (bad) in appearance by industry experts. MATERIALS AND METHODS Experimental oysters (commercially farmed oysters) were sampled from three (A. B. and C) commercial oyster farms and divided into two groups (approximately 50 per group) of either good or bad shell shape by each farmer. Oysters provided by farms A and C were grown intertidally in mesh oyster bags, while *Corresponding author. E-mail: chris.langdon@oregonstate.edu samples provided by farm B were grown subtidally in lantern nets. All shell measurements were performed at the Hatfield Marine Science Center in Newport. OR. Sample oysters were shucked to obtain the left valve, or "halfshell." Greatest shell length, greatest shell width, and greatest shell depth were measured for all oysters. All size measurements were made using Vernier calipers to the nearest 0.1 mm. Analysis of variance was used to determine whether good- and bad-shaped oysters differed for any of the three linear measure- ments (greatest shell length, width, and depth). Analyses were performed using data from within each farm site. Normality was tested using the Kolmogorov-Smirnov method (SPSS Inc.. 2000. Chicago. IL). These analyses were repeated for data pooled across all farms. Absolute measures, such as greatest shell length, width, or depth, are less useful in categorizing oysters by shape because of variation in oyster size at harvest. To eliminate the confounding effects of size on shape it is usual to apply proportional measures (Reist 1985). As a result, two descriptors were generated to char- acterize shell shape: ratios of depth to length (D/L) and width to length (W/L). Normality of D/L and W/L values were determined using the Kolmogorov-Smirnov method (SPSS Inc.. 2000, Chi- cago, IL). The ability of each variable to discriininate good from bad oysters was determined using data collected froin each farm site separately and with data pooled across all farm sites. A com- parison of the total percentage of correct assignments of sampled oysters (into good or bad groups) was then used to determine which of the descriptors (D/L, W/L, and three previously described formulae: Wada (1986); Galtsoff (1964): Imai and Sakai (1961) resulted in the most accurate discrimination between good and bad shaped oysters. RESULTS All variables were normally distributed after log transformation (Kolmogorov-Smirnov. P > 0.05), except for shell depth measured in the sample from farm B (P = 0.014). However, due to robust- ness of ANOVA and the large sample size (ii = 99), this departure from normality may be ignored for the purpose of analysis (Ram- sey & Schaffer 2002). Good and bad shaped oysters differed in length, width, and depth at all farm sites (P < 0.05) except at farm 767 768 Brake et al. C. where the two groups of oysters only differed in length (Table 1 ). When significant differences occurred, good oysters tended to be both deeper and wider than bad oysters. Good oysters were significantly shorter in length (P < 0.051 than bad oysters at farms B and C: however, good oysters were significantly longer than bad oysters at farm A. Table 2 lists the means and standard deviations of D/L and W/L ratios for each individual farm, and for the pooled good and bad oyster samples. Based on the ratio D/L. good oysters were significantly deeper than bad oysters at all farms {P < 0.05; Table 2). Good oysters were 1 .35, 1 .88, and 1 .36 times as deep, per unit length, as bad oysters at farms A. B, and C. respectively. Good oysters were 1.06, 1.10, and 1.22 times as wide, per unit length, as bad oysters at farms A, B, and C, respectively (P < 0.05). Although there were differences in mean D/L and W/L ratios among farms, the trends (larger values for good oysters than bad oysters) within sites were similar. To obtain a robust sample that would best represent different farms across industry, the data from all farms were pooled to evaluate the average differences between good and bad shell groups across all sites. Good oysters had a mean W/L of 0.689 and D/L of 0.316, whereas bad oysters had significantly lower means of 0.597 and 0.219 respectively (P < 0.001 ). Table 3 lists reported shape descriptors for assignment of oys- ters into good and bad groups. Previously used thresholds for separating good and bad shapes, as well as the values maximizing the percent coixect assignment in the current study are given. Cor- rect assignment was maximized for D/L at 0.25, with 92.6% of all good oysters falling above the value of 0.25 (depth was at least 0.25 of shell length), while 78.8% of all bad oysters fell below this value. Using the Atlantic Canadian shell shape guidelines, shells with a value of length / width <1.75 would be termed as either ■■fancy" or ""choice", the top two of four possible categories ot oyster shells. Table 3 shows the percentage of oysters correctly assigned to their proper good or bad groups by using D/L > 0.25, W/L, and the Atlantic Canadian threshold of good >1.75 (as well as the value for which percent correct assignment was maximized, 1 . 1 2). In addition, a formula (Galtsoff 1964) used as a standard for the Irish and French industry (using a threshold value for good shell shape of >3, and the value of maximum correct assignment of 3.5) was compared along with a previously described formula for shell convexity (Wada 1986). Percent correct assignment for con- vexity was maximized at a value of 0.3 1 5 when this descriptor was applied to the current data. The index of shell depth described by Imai and Sakai (1961) maximized the percentage of oysters cor- rectly assigned to both good and bad groups at a value of 3 1 .6. TABLE 1. Shell length, width, and depth measurements of good- and bad shell-shaped oysters from three commercial U.S. West coast oyster farms. TABLE 2. Observed ratios of depth/length (D/L) and width/length (W/L) of good- and bad shaped-oyster shells from three commercial U.S. West coast oyster farms. Length ( Good mm) Bad Depth ( Good mm) Bad Width (mm) Farm Good Bad A Mean 101. LS* 95.99 30.41* 21.64 71.50* 61.56 SD 7.10 8.35 3.39 3.89 5.78 7.09 B Mean 77.62* 90.86 27.15* 17.29 52.82* 50.61 SD 5.00 13.32 3.96 3.41 4.71 6.49 C Mean 90.66* 117.12 27.07 26.57 60.37 61.33 SD 7.L^4 23.58 4.33 6.52 8.58 6.35 Sample D/L W/I. Standard Standard Farm Type Mean Deyiation Mean Deviation A Good 0..W2 0.037 0.711 0.084 Bad 0.226 0.040 0.646 0.092 B Good 0.351 0.052 0.683 0.072 Bad 0.194 0.046 0.571 0.125 C Good (1..^0I 0.055 0.669 0.097 Bad 0.235 0.079 0.547 0.136 Pooled Good 0.316 0.053 0.689 0.087 Bad 0.219 0.057 0.597 0.123 * Means were significandy different (P < 0.05: ANOVAi. All mean good-shaped samples within a site were statistically larger than bad-shaped samples (P < 0.05, ANOVA). A threshold value of D/L > 0.25 (for good oysters) was the most effective at correctly assigning oysters to their respective categories, with 85.6% of all oysters being correctly assigned (Table 4). This threshold was more effective at correctly assigning good oysters to good groups (90.9% correct) than bad oysters to bad groups (77.8% correct). The index of shell depth (Imai & Sakai 1961) was also effective at correctly assigning good oysters (84.2%) and bad oysters (82.0%). correctly assigning 84.4% of the total oysters sampled. The ratio of W/L, using the threshold value of >0.63 (for good oysters) correctly assigned 70.6% of all oysters. When the ratios of D/L and W/L were applied simultaneously, only 30.0% of all oysters were correctly assigned by both ratios (Table 4, Fig. I ). The Atlantic Canadian guideline was less effec- tive in discriminating good from bad oysters using the threshold value for good oysters of >1.75 (65.7%), resulting in an overall maximum correct assignment of 70.6%. The measure of convexity (Wada. 1986) was effective at correctly assigning good oysters (86.1%). but not bad oysters (35.9%). correctly assigning 61.8% of the total oysters sampled. Using the previously applied threshold and the value of percent maximum assignment, the Galtsott for- mula was effective at correctly assigning bad oysters (96.4% and 98.8% respectively), but ineffective at assigning good oysters (0% and 0%). and only assigned 49.2% and 50.4% of all oysters cor- rectly. DISCUSSION Members of the U.S. West coast oyster industry have subjec- tively identified shell depth and width, relative to length, as the two most important factors in determining the quality ot an oyster halfshell. Most oyster growers identified a long and skinny shape (typically called "'rabbit ears") as being undesirable, with a deep and wide halfshell being more desirable. These distinctions, when relative shell size is considered, are described by the ratios of D/L and W/L. Previous work has shown the abUity to categorize bivalve shape regardless of absolute size. Day et al. (2000) used stepwise discriminant analysis and principal component analysis to show that relative size of the umbo cavity was the most useful character for identification of sympatric Saccostrea species. The authors reported success in identifying species using the non-lethal mea- Method to Describe Desirable Shell Shape 769 TABLE 3. Descriptors used in the current study to compare percent correct assignment of oysters into farm-specified good and bad shell-shape groups. Previously \ alue Maximizing Employed Percent Correct Descriptor Expression Species Threshold Assignment Reference D/L Depth/length Crassoslrea gigus na 0.25 Current study W/L Width/length Crassoslrea gigas NA 0.63 Current study Atlantic Leneth/width Crassoslrea 1.75' 1.58 Section 65 of CHA's Canadian viri^inica Fish Inspection Regulations Galtsoff (Length depth i/width Crassoslrea Crassoslrea virginica 3 3.5 Galtsoff 1 964. Heath & Wilson 1999 Convexity Width/( length width depth) Pinclada fucala marlensii NA 0.315 Wada 1986 Index of (Depth/mean of width and Crassoslrea NA .M,6 Iniai & Sakai 1961 Shell Depth length) X 100 gigas ■■ Separates top two of four possible shape categories; details in results section. sures of total oyster depth, right valve length, and right valve width. In addition. Wilding et al. (1998) reported that hinge length was the only one of four investigated shell measures that provided a clear distinction among groups of the scallop, Pecten maximus. The various formulae (Tables 3 and 4) used to separate good froin bad oysters confirm the importance of depth in determining optimal oyster shape. The two relationships that were inost effec- tive at correctly assigning good and bad oysters (D/L and an index of shell depth; Imai & Sakai 1961 ) had only depth in the numera- tor. These descriptors were more effective than the ratio of W/L. a previously described shell convexity assessment method (Wada 1986). the formula used as a guideline in the Irish and French industry (Galtsoff 1964). and the Atlantic Canadian guideline (Table 3). Culture environment has an important effect on shell shape (Carriker 1996, Boulding & Hay 1993, Seed 1968). It is commonly believed by growers that oysters subjected to movement by fre- quent disturbance tend to grow deeper halfshells. The outer edge of the shell is repeatedly broken off and subsequently grown back with the net result being that the oyster grows more quickly in terms of depth than length. This process is commonly referred to as "pruning." Few attempts have been made to investigate genetic effects on shell shape or to improve bivalve shell shape using selective breed- ing in aquaculture. Wada ( 1986) reported on first, second and third generation responses to selection for shell width and shell convex- ity for the Japanese pearl oyster, Pictada fucata martensii. Selec- tion for shell convexity and shell width was effective and Wada obtained a realized heritability of 0.467 for shell convexity after two generations of selection, pro\ iding evidence that shell shape in the Japanese pearl oyster may be improved by selective breeding. The current study suggests that width may not be as important as depth in determining quality. It is conceivable, however, that if a breeding program were to select oysters without consideration of width, the result could be a less desirable deep and narrow oyster. As both depth and width have been anecdotally described as being important (by industry) to the quality of a halfshell, it may be prudent to consider both relationships to determine the true quality TABLE 4. Percent correct assignment of good and bad oysters based on D/L, W/L, and Atlantic Canadian Measure, the Galtsoff measure, an index of shell depth, and a measure of shell convexity using values for maximum percent correct assignment, unless stated otherwise. Percent Correct .Assignment Sample Type Depth/I.ength (D/L) Index of Shell Depth Width/I.ength (W/I.) D/L + W/L° .Atlantic Canadian'' Atlantic Canadian Convexity Galtsoff* Galtsoff Good oysters Bad oysters Good -f bad oysters 90.9 77.8 85.6 84.2 84.5 84.4 74.5 64.7 70.6 56.4 3.1 30.0 89.1 42.0 65.7 74.5 64.7 70.6 86.1 35.9 61.8 0 96.4 49.2 0 98.8 50.4 A description of the measures and the maximum percent correct assignment values for each are given in Table 3. ' Maximum percent correctly assigned by both D/L and W/L. ''Threshold value described in the literature; not the value maximizing percent correct assignment given in Table 3, 770 Brake et al. 0.6 0.5 0.4 0.3 0.2 0.1 0 ■ Good ° Bad ■ mg ■■*- o I* "■a* ^ ' ■ ■ D W OP D D D C*, g' i' _ a 4 o D D D O 0.2 0.4 0.6 0.8 1.2 L/W Figure 1. Ratios of depth/length (D/L) and width/length (\V/L) of good- and bad-shaped oyster shells from three commercial West coast U.S. oyster farms. Lines represent threshold values of VV/L > 0.63 and D/I. > 0.25 maximizing percent correct assignment. of an oyster halfshell. The use of the D/L and W/L thresholds simul- taneously was ineffective in correctly assigning oysters to good or bad groups. The index of shell depth (lamai & Sakai 1961) might be useful in this regard as it incorporates shell width, and should there- fore exclude any abnormally deep and narrow oyster shells. A method to evaluate shell shape quality that is both simple and reliable could be of great value to the U.S. West coast oyster industry. Growers could objectively compare practices to find which culture methods tend to influence shell shape in a positive way. Oysters grown in different areas commonly have different shape characteristics. Using an objective method, site differences could also be as.sessed, with a grower being able to determine whether particular sites produce oysters with a better shape. An- other possible long-term benefit of having an objective comparison could be the establishment of industry shape standards. This would allow producers and consumers to use a common scale of shell shape measurement. The Atlantic Canadian oyster industry has realized the benefits of such a set of standards. For example. "fancy" oysters are defined in section 65 of the Canadian Food Inspection Agency's Fish Inspection Regulations as having a length not exceeding one and one-half times its greatest width, and as not being abnormally flat, thin-lipped, or malformed. Consum- ers can therefore go to several different famis or retailers and pur- chase "fancy" oysters, knowing that they share a common shape. An important consideration in a method to characterize shell shape is the practicality of the methodology. Heath and Wilson (1999) used computer assisted image analysis to assess shell shape and size in Crassostrea gigas. Although they demonstrated that this method could be used to separate oysters into categories ac- cording to general shape specifications, the required equipment might be cost prohibitive and this method does not allow for as- sessment of oysters in the field. The ratio of D/L and the index of shell depth used in the current study would, therefore, be more practical to separate good from bad shells compared with using image analysis. In summary, industrial-scale assessment and selective breeding programs both require methods to efficiently determine the value of an oyster in terms of shell shape. The D/L ratio and the index of shell depth show promise in this regard. The D/L threshold of <0.25 separates most of the good and bad oyster halfshells, while requiring only two simple linear measurements. The index ot shell depth (lamai & Sakai 1961) was nearly as effective and has the advantage of incorporating width, which might eliminate any ab- normally deep and narrow oysters. The current data suggests that depth is likely the most important measure to evaluate oyster shell shape quality. Consideration of W/L might also be prudent in shell shape assessments to avoid narrow deep oysters. The current study only investigated differences between good and bad shell samples from three farms; therefore, future work should include samples from more industry participants. ACKNOWLEDGMENTS The authors thank Ebru Onal. David Stick. Drew Mosher, Sean Matson, Salina Gaskill, and Dave Jacobson, for technical assis- Method to Describe Desirable Shell Shape 771 tance during the project. Thanks also to Olivier Drean for help in measuring many sample shells. Special thanks are given to the generous support afforded by the donor farms. Taylor Shellfish Farms Inc., Westcott Bay Sea Farms Inc., and Oregon Oyster Farm Inc. This project was funded by a special L'SDA-CSREES grant to the Molluscan Broodstock Program, Oregon State University. LITERATURE CITED Boulding, E. G. & T. K. Hay. 199.^ Quanlitalive genetics of shell form of an intertidal snail: constraints on short-term response to selection. £10- Uuum 47:576-592. Carriker. M. R. 1996. The shell and ligament. In: V. S. Kennedy. R. E. I. Newell, and A. F. Eble, editors. The eastern oyster, Crassostrca vii- ginica. pp. 75-168. College Park. MD: Maryland Sea Grant College Publication, pp. 75-168. Day, A. J.. A. J. S. Hawkins & P. Visootiviseth. 2000. The use of al- lozymes and shell moqihology to distinguish among sympatric species of the rock oyster Saccostrea in Thailand. Aquaculmre 187:51-72. Galtsoff. P. S. 1964. The American oyster, Crassoslrea virginica (Gmelinl. U.S. Fish. Wildl. Sen: Fish. Bull. 64:1^80. Heath, P. L. & J. H. Wilson. 1999. Assessment of Pacific oyster. Cms- sotrea gigas (Thunberg), size and quality using a computer-based shape analysis technique. Aquae. Res. 30:299-303. Imai. T. & S. Sakai. 1961. Study of Japanese oyster, Crassoslrea gigas. TohokuJ. Agric. Res. 12:W1-\12. Ramsey, F. L. & D. W. Schaffer. 2002. The statistical sleuth: A course in methods of data analysis. Pacific Grove. CA: Duxbury, 742 pp. Reist. J. D. 1985. An empirical evaluation of several univariate methods that adjust for size variation in morphometric data. Can. J. Zool. 63: 1429-1439. Seed, R. 1968. Factors influencing shell shape in the mussel Mxtihis eilulis. J. Mar. Biol. Ass. U. K. 48:561-584. Wada, K. T 1986. Genetic selection for shell traits in the Japanese pearl oyster, Pinctada fucada martensii. Aquaculture 57:171-176. Wilding, C. S., J. W. Latchford & A. R. Beaumont. 1998, An investigation of possible stock structure in Pecten maximus (L.) using multivariate morphometries, allozyme electrophoresis and mitochondrial DNA polymerase chain reaction-restriction fragment length poymorphism. J. Slwllfish Res. 17:131-139. JoKinal oj Shellfish Research, Vol. 22, No. 3, 773-77.'i. 2003. SHOULD SLOW GROWING PEARL OYSTER {PINCTADA MARGARITIFERA) SPAT ("RUNTS") BE DISCARDED? JOSIAH H. PIT* AND PAUL C. SOUTHGATE Pearl Oyster Researeh Group School of Murine Biology and Aquacultiire. James Cook University. Townsville. Qiieenshiud 4HI I . Australia ABSTRACT In this laboratory, hatchery-produced Piiictiula luariinntiferd ju\endes are routinely graded at 3.5 mo of age. when .spat of <5 mm ("runts") are generally discarded. This anicle reports on an experiment to assess the relative growth rates of three size classes (<5, 5-10, and >10 mm) of hatchery-produced blacklip pearl oyster (P. iimrftaritifeni) spat from the same cohort. The three size classes were classified as runts, normal growers, and fast growers, and had mean (±SE; n = 30) dorso-venlral shell heights (DVHs) of 4.5 ± 0. 1 . 8.6 ± 0.3, and 1 2.8 ± 0.2 mm, respectively, at the start of the 4-mo experiment. The mean DVH at completion of the study for each initial size cla.ss (<5, 5-10, and >10 mm) was 24,6 ± 0.4, 32.3 ± 0.4, and 35.6 ± 0.4 mm, respectively. All differed significantly from each other {P < 0.001 ). The mean incremental increases in DVH for each size class (<5. 5-10, and >I0 mm) over the 4-mo period was greatest in oysters from the 5-10-mm size cla.ss (mean DVH 23.3 ± 0.4 mm) and lowest in oysters from the <5-mm size class (mean DVH 20.0 ± 0.5 mm). Incremental increases in DVH were significantly different between oysters from the <5-iTim size class and those from the larger size classes. The mean (±SE) percentage increase in DVH was greatest in oysters from the <5-mm size class (448 ± 17%) and lowest in oysters from the >10-mm size class (178 ± 7%). A number of oysters in the <5-nim size class grew very rapidly during the experiment and reached the same DVH as oysters in the larger size classes. This study shows that, given appropriate conditions, runts are capable of similar growth rates as larger spat. It may therefore be inappropriate to discard pearl oysters, which are classed as runts (<5 mm) at grading (3.5 mo). Furthermore, it is suggested that grading be delayed until 5 to 6 mo when a greater proportion of oysters are likely to be in the larger size classes. A'£>' WORDS: pearl oyster. Pimlada inuri;aritifcra. spat, runts, growth INTRODUCTION The growth of cultured bivalve molluscs is highly variable during hatchery and nursery culture, and variation in growth can occur among individuals of the same age reared under identical conditions (Newkirk 1981), Small differences in the size of spat can become large differences in juvenile size (Mason et al. 1998), and the greater the time required by slow growers to reach com- mercial size increases costs and reduces profitability (Askew 1978), Pearl oysters need to reach a minimum shell size before being used for pearl production. This size is generally reached at appro.ximately 2 y of age. As such, maximizing growth rate and minimizing growth variation are important factors in pearl oyster cultivation, A large variation in growth rate is evident for pearl oysters reared under identical conditions. For example, 43-day-old black- lip pearl oyster (Pinctada margarilifera) spat have been reported to range in size from 1 to 5 mm in dorso-ventral shell height (DVH) (Pit & Southgate 2000). and from <2 to 23 mm DVH at 3.5 mo of age (Southgate & Beer 1997), To minitnize the size varia- tion in pearl oyster spat. Rose (1990) recommended continual grading to separate fast growers frotn slow growers. Slow-growing pearl oyster spat are often discarded. In this laboratory, hatchery- produced P. margarilifera are routinely graded at 3.5 mo of age, when spat <5 mm ("runts'") are generally discarded. "Runting" may result from unfavorable culture conditions, and, if this is the case, runts may be capable of good growth rates if provided with appropriate culture conditions. Given the high cost of hatchery production and the high value of pearl oyster spat, it is in the interest of pearl oyster fanners to maximize the number of spat from a given cohort that are eventually used for pearl production. The aim of this study was to determine whether slow-growing P. margaritifera spat remained as runts or whether they are capable *Corresponding author. E-mail: Josiah.Pit@jcu.edu.au of similar growth rates as normal spat when provided with appro- priate conditions. MATERIALS AND METHODS This study was conducted at the Orpheus Island Research Sta- tion of James Cook University, north Queensland, Australia ( 180°35' 146°29'E), and larvae and spat were cultured according to the methods described by Southgate and Beer ( 1997) and Pit and Southgate (2()()()). At 43 days of age, when spat had a mean (±SE) DVH of 2.8 ± 0. 1 ttim (range 1-5 mm), they were transferred from the hatchery to the ocean where they were held in suspended mesh trays at a depth of 6 ni (Southgate & Beer 1997). Spat were graded at 3.5 mo of age into three different size classes. <5, 5 to 10, and >I0 mm, which, for the purpose of this study, were classified as runts, normal growers, and fast growers, respectively. The mean DVH in = 30) of P. margaritifera in the <5-. 5-to-lO- and >10-mm size classes were 4.5 ± 0.1. 8.6 ± 0.3. and 12,8 ± 0.2 mm. respectively, and these differed significantly from each other (F,;,? = 285.42; P < 0.001). Thirty P. marga- ritifera spat from each size class were individually fixed to the bottoms of each of three replicate plastic mesh trays (60 x 35 x 10 cm) using a waterproof cyanoacrylate adhesive (Loctite 454 gel. Loctite Australia, Caringbah, New South Wales. Australia). This minimizes oyster aggregation (Friedman 1999. Pit 1998). which can significantly affect growth (Friedman & Southgate 1999). To minimi/.e the disturbance to spat and to maximize growth rates, oysters were not measured during the 4-n)o study; however, trays were cleaned in situ every month to remove external fouling or- ganisms (Pit & Southgate in press). Cleaning involved the manual scrubbing of the outside surfaces of the trays. Trays were not cleaned internally, but were moved gently up and down in the water column to remove any silt and mud that had accumulated inside the trays. Oysters from each tray were measured for DVH at the end of the study. Data were analyzed using a one-way analysis of variance to 773 774 Pit and Southgate determine whether P. margarilifeni from different size classes differed in size (DVH) at the completion of the study. Assumptions of homogeneity and normality were met (Zar 1984). The rates of growth among the three size classes were also assessed using nonparametric analyses to determine whether differences existed. Significant differences were identified using the Tukey"s test and the Dunnett's T.^ for the parametric and nonparametric tests, re- spectively (Zar I4S4). RESULTS On completion of the study, the mean (±SE) DVH for each initial size classes (<5, 5-10, and >I0 mm) were 24.6 ± 0.4, .^2..^ ± 0.4. and 35.6 ± 0.4 mm, respectively. All differed significantly from each other (F, ^-, = 167.67; P < 0.001 ) (Fig. 1 ). The mean incremental growth in DVH (n = 30) for each size class «5. 5-10, and >10 mm) over the 4-mo period was greatest in oysters from the 5-IO-nim size class (23.3 ± 0.4 mm) and was lowest in oysters from the <5-mm size class (20.0 ± 0.5 mm). Incremental shell growth was significantly greater in the two larger size classes (F2 87 = •5-99; P < 0.001) (Fig. 2). Weekly growth rates averaged 1.25, 1.42, and 1.48 mm, respectively, for the <5-. 5-I0-, and >10-mni size classes. However, the mean percentage increases in DVH for each size class (<5. 5-10, and >I0 mm) over the 4-mo period was greatest in oysters from the <5-mm size class (448 ± 17%) and lowest in oysters from the >IO-mm size class (178 ± 7%), while oysters in the 5-IO-mm size class increased by 308 ± 12%. A number of oysters in the <5-mm size class grew very rapidly and achieved DVH measurements within the ranges of those shown by oysters in the two larger size classes. DISCUSSION P. nuiriiariufera used in this study were hatchery-reared ani- mals of the same age that were cultured under identical conditions. However, when oysters were transferred from the hatchery to the nursery at 6 wk of age, their DVH ranged from 1 to 5 mm (mean 2.8 ± 0.1 mm). It is unclear whether such size variation resulted from environmental factors, genetic factors, or a combination of both. Factors that have previously been suggested to cause such size variation in bivalves include fluctuations in water quality and food quality (environmental), as well as egg and larval quality (genetic) (Gallager & Mann 1986, Rose 1990. Mason et al. 1998, Devakie & Ah 2000, Nicolas & Robert 2001 ). Size variation was also evident during early nursery culture prior to grading when oysters ranged in size from 2 to 23 mm. Again, it is unclear whether size variation at grading reflected a continuation of llic size variability observed in the hatchery, or 4U - a F ^U- E " S^'^^ ■ '^c — 20 ^ ^ ^"^ , - ' X , -^ > ^- ^^ a 10 - 0 - --■•' -1 ^ <5 mm 5-10 mm — - — >10 mm 3.5 75 Age (months) Figure 1. Changes in mean (±SK: n = M)) DVH of P. margarilifera juveniles In different size classes l<5, 5-10, and >l(l mm) culliired for 4 mo at Orpheus Island. Means with the same superscript are not significantly different (P > 0.05). E 24 1 E. 23 - X > 22 - Q 21 - c « 20 - O) c 19 - re r 1 0 0 <5 mm >10 mm 5-10 mm Oyster size class Figure 2. Mean (±SF) change in DVH of P. iiiari;ahlifera juveniles in different size classes (<5, 5-10, and >1() mm) cultured for 4 months at Orpheus Island. Means with the same superscript arc not significantly different [P > 0.05). whether subsequent environmental factors were also involved. The negative impacts of poor growing conditions on pearl oyster growth rates during nursery culture are well documented. For ex- ample, pearl oysters aggregate to form clumps in culture units (Southgate & Beer 1997, Friedman & Southgate 1999). This re- sults in a greater size range of individuals within a cohort and a higher proportion of smaller oysters when compared with oysters grown in conditions that prevent clumping (Friedman & Southgate 1999. Southgate & Beer 2000). The smaller oysters in the former group are thought to be those that are bound into clumps of oysters, and, as a result, have impaired access to good water flow and food availability (Friedman & Southgate 1999). The growth rates of P. niariiaririfeni spat recorded in this study were clearly influenced by initial size class, suggesting that genetic factors were more influential on initial spat size than were envi- ronmental factors. In a similar study with Pacific oysters. Collet et al. (1999) demonstrated a positive relationship between larval and postmetumorphic growth, indicating a genetic rather than environ- mental basis for slower growth in postmetamoi-phic bivalves. In contrast. Mason et al. (1998) reported that growth variation in Sydney rock oyster spat was not affected by initial size class and suggested that initial differences in size resulted from "temporary environmental stunting" rather than from genetic factors. Similar findings have been reported for edible oysters (Newkirk 1981, Newkirk & Haley 1982) Hatchery production of pearl oysters is expensive, and it is clearly in the interest of pearl oyster farmers to maximize the number of spat from a given cohort that can be used for pearl production. However, the use of smaller spat, which take a longer time to reach a size suitable for pearl production, becomes an economic issue. Pearl farmers must consider the benefits of maxi- mizing the number of usable oysters from a cohort of spat, against the increased time required for slower growers to reach pearl pro- duction size. Prior research at the culture site used in this study reported growth rates for P. maiiiaritifera during nursery culture ranging from 3.66 mm mo'' (in trays) to 4.86 mm mo"' (in pocket nets) (Southgate & Beer 2000). Assuming similar subsequent growth rates for the three size classes of oysters used in this study, it is possible to estimate the time required for each size class to reach a pearl production size of 110 mm DVH. On this basis, oysters in the 5-10- and >10-mm size classes would reach I 10 mm at 19 to 24 mo and 19 to 23 mo of age, respectively. However, oysters in the <5-mm size class would require 2 1 to 27 mo to reach this size (110 mm DVH). The costs involved in culluring oysters from the smaller size class for this additional time, however, may outweigh the costs of increasing oyster numbers by additional Slow Growinc; Pbarl Oyster Spats 775 hatchery production or the purchase ot'juveniles. hi a similar study with Crassostrea virginica. O'Beirn and Luckenbach (2000) noted that the use of runts for the oyster industry would be feasible, given good growing conditions, but that it may not warrant the invest- ment of extra time and resources. When provided with good growing conditions, oysters in the <5-mm size class grew at a significantly slower rate than those in larger size classes. Nevertheless, certain individuals from the <5- mm size class did attain sizes within the overall si/e ranges of oysters in the larger size classes. This suggests that some runts may not always remain runts and indicates that such individuals are likely to have been affected by environmental stunting. Clearly, at first grading (3.5 mo of age), it is not possible to identify those P. margaritifera individuals in the <5-mm size class that are capable of growth rates allowing them to catch up to larger individuals within a cohort. Culling runt oysters at this stage would result in the loss of oysters that could subsequently be used for pearl pro- duction. A second grading at approximately 5 to 6 mo of age. however, would allow such individuals to be identified. This would maximize the number of oysters used for pearl production from a given cohort of juveniles. A similar outcome might also be achieved through more appropriate spat collector design. .Spat are generally transferred from the hatchery to the field on spat collec- tors and remain on them until grading (.Southgate & Beer 19^7). Spat collectors that provide more uniform environmental condi- tions are likely to result in a more tiniform size range of spat at grading. Hatchery production of P. imirgaiilijcni in many developing Pacific nations is often constrained by limited resources (South- gate & Beer 1997) and cannot be conducted on a routine basis. In these cases, it is preferable to use as many oysters as possible from each cohort of hatchery-produced spat. The results of this study indicate that modifications to the current protocols may allow in- creases in the number of P. margunlifera from a given cohort that can be used for pearl production. ACKNOWLEDGMENTS This study was conducted as part of project FIS 97.^1. "Pearl Oyster Resource Development in the Pacific Islands," which was funded by the Australian Centre for International Agricultural Re- search. The authors thank the staff at the Orpheus Island Research Station of James Cook University for technical assistance during the study. LITERATURE CITED Askew. C. G. 1978. A generalised gniwlh and nuirtalily niddcl lor assess- ing the economics bivalve culture. Aqmuultiirc 14:9 1-104. Collet. B.. P. Boudry, A. Thebault. S. Heurtebise. B. Morand. & A. Gerard. 1999. Relationship between pre- and post-metamorphic growth in the Pacific oyster Crasso.slrea ,?/,?a.s (Thunberg). Aciiuiculliirc 17,'i:21.'i- 226. Devakie. M. N. &. A. B. Ali. 2U00. Salinity-temperature and natnlional effects on the setting rate of larvae of the tropical oyster, CiiissDstreu iredalei (Faustino). Acjiiaciihure 184:105-1 14. Friedman. K. J. 1999. Pearl culture using wild-caught spat of hlacklip oysters (Piiichula mcirgariiifera) in Solomon Islands. PhD Thesis, School of Marine Biology & Aquaculture. James Cook University. Townsville, Australia. Friedman. K. J. & P. C. Southgate. 1999. Growout of blacklip peari oys- ters. Piucliuhi ntargarinfera collected as wild spat in the Solomon Islands. / Shellfish Res. 18:159-167. Gallager. S. M. & R. Mann. 1986. Growth and survival ot larvae of A/cr- cenuiia inerceiuiria (L. ) and Crassosrreu viii;iiiiai (Gmelin) relative to broodstock conditioning and lipid content of eggs. Aiiiiaiiilturc 56: 10.5-121. Mason, C. J., D. D. Reid, & J. A. Nell. 1998. Growth characlerislics of Sydney rock oysters, Saccostrea commercialis in relation to si/e and temperature. J. Exp. Men: Biol. Ecol. 27:155-168. Newkirk, G. F. 1981. On the unpredictability of bivalve growth rates: is a slow growing juvenile oyster a runt for life. In: C. Claus. N. De Pauw. & E. Jaspers, editors. Nursery culture of bivalve molluscs. Proceedings of the International Workshop on Nursery Culturing of Bivalve Mol- luscs, February 24-26, Ghent, Belgium, pp. 211-218. Newkirk, G. F. & L. E. Haley. 1982. Phenotypic analysis of the European oyster Oslrca echilis L: relationship between larval period and postset- ting growth rate. J. Exp. Mar. Biol. Ecol. 59:177-184. Nicolas. L. & R. Robert. 200 1 . The effect of food supply on metamorphosis and post-larval development in hatchery reared Peclen iiiumiiiiis. .Aqua- culture 192:347- .159. O'Beirn. F. X. & M. W. Luckenbach. 2000. A study investigating the potential of an alternate seed source for Virginia aquaculturists. / Shellfish Res. 19:653-654. Pit. J. H. 1998. Factors affecting growth and survival of the blacklip pearl oyster (Pinctada margaritifera. Linneaus) during early nursery culture. Honours thesis. School of Marine Biology & Aquaculture. James Cook Univershy, Townsville, Australia. Pit. J. H. & P. C. Southgate. 2000. When should pearl oyster, Pincuula margaritifera (L.). spat be transferred from the hatchery to the ocean? Aquaculture Res. 3\:71i-nH. Pit. J. H. & P. C. Southgate. In press. Fouling and predation: how do they affect growth and survival of the blacklip pearl oyster. Pinctuda mar- garitifera, during nursery culture? Ac/uaculture Int. 1 1:545-555. Rose, R. A. 1990. A manual for the artificial propagation of the silverlip or goldlip pearl oyster, Pinctada maxima, (Jameson) from Western Aus- tralia. Fisheries Department Western Australian Marine Research Laboratories. North Beach. Western Australia. 41 pp. Southgate. P. C. & A. C. Beer. 1997. Hatchery and eariy nursery culture of the blacklip pearl oyster (Pinctada margaritifera L.). J. Shellfish Res. 16:561-567. Southgate. P. C. & A. C. Beer. 2000. Growth of blacklip pearl oyster {Pinctada margaritifera L.) juveniles using different nursery culture techniques. Aqiuicullure 187:97-104. Zar. J. H. 1984. Biostatistical analysis. 2nd ed. Upper Saddle River. NJ: Prentice Hall. 718 pp. Journal ,<) Shellfish Research. Vol. 22. N(i. 3. 111-11^). 2003. CORROSION CASTING OF THE DIGESTIVE DIVERTICULA OF THE PEARL OYSTER, PINCTADA FUCATA MARTENSII (MOLLUSCA: BIVALVIA) TAKESHI HANDA* AND KEN-ICHI YAMAMOTO Department of Applied Aqiiabiology. National Fisheries University. 2-7-1 Nagata-honinachi. Shiinonoseki. )'aiihii;uchi 759-6595. .lapan ABSTRACT We examined corrosion casting as a means of studying the digestive organ in the pearl oyster Pinctada fiieaui nuinensii and other molluscs. The cast was made with resin that mixed hardener (Mercox MA) and prepolymerization methyl methacrylate (MercoxCL-2R). In pearl oysters, the resin was injected through the polyethylene tubing within 3 min, after the animal sufficiently relaxed in 0.4 niM MgCl, solution. It was left at least I h in the .seawater and hardened. Then, it was treated with 20% NaOH for I day at room temperature. As a result, it was po.ssible to cast from the mouth to the anus, including the ducts and tubules of the digestive diverticula. Using the same method, the castings of digestive organ in other molluscs, Scapharca broughtonii. Crassoslrea gigcis. Meretrix hisoria (Bivalvia), and Haliotis discus (Gastropoda), were completed as well as the pearl oyster. KEY WORDS: corrosion cast, digestive organ, digestive diverticula, duct, tubule INTRODUCTION Molluscs absorb food and nutrients, secrete digestive enzymes, and store the nutrients in the digestive diverticula that develops at the circumference of the stomach. The digestive diverticula is connected with the stomach by ducts (Owen I95fia. 1995b, Pur- chon 1957. 1958. I960). The structure of the tip of the digestive diverticula is shown as a terminal vesicle (Owen 1955a, 1995b. Nakajima 1956). Yonge (1926) demonstrated the structure of the stomach and the main ducts of the digestive diverticula with a cast made in gelatine in the Pacific oyster. Cras.wstrfa gigas. but the secondary ducts and tubules were not cast. Corrosion casting is well suited to study the three-dimensional structure of the cardiovascular-respiratory system; however, there is little information provided on the structure of the branch and connection in the secondary ducts and tubules with casting. Infor- mation on the structure of digestive diverticula will be useful for the research of taxonomy and the function of digestive diverticula. This study examined corrosion casting as a means of charac- terizing the whole digestive organ, especially digestive diverticula, with prepolymerization methacrylate in some molluscs with spe- cial emphasis on the pearl oyster, Pinctada fucaia martensii. MATERIALS AND METHODS Pearl oysters were obtained from a farm in Tsushima, Na- gasaki prefecture. After cleaning the shell valves, they were reared for 5-10 days in running seawater filtered to remove particles >0.5 [j.m. The experiments were conducted in 60 pearl oysters (mean shell length; 64.4 ± 5.8 mm (SD), shell height; 72.8 ± 4.3 tnm, shell width: 24.6 ± 1.6 mm, and total wet weight; 40.3 ± 6.3 g). The resin used was red prepolymerization methyl methacrylate (Mercox CL-R. Oken Shoji) and hardener (Mercox MA, Oken *Corresponding author. E-mail: handat@nsh-u.ac.jp Shoji). When both reagents were mixed at 5-20%, the resin started to gradually solidify after about 5 min. Therefore, they were mixed just before injection to the digestive organ. After the pearl oyster was relaxed enough in 0.4 mM MgCl, solution (Nainba et al. 1995), the left shell valve was removed and the mantle was dis- sected to expose the labial palp. Polyethylene tubing ( 1 inm in outer diameter, 20 cm length, Hibiki No. 3), which inflated the tip spherically in order to prevent the counterflow of the resin, was inserted about 5 mm from the mouth to the esophagus. Then, 4 niL of resin were injected within 3 min w ith a plastic syringe of 5 mL capacity. The tubing was sealed with the flame to .stop the resin overflowing, and the injected pearl oyster was returned to the seawater. After it was left at least for 1 h and the resin hardened, the pearl oyster was immersed in 20% NaOH solution for I day at room temperature, and then washed with tap water. The completed corrosion castings were preserved in the 0.1% sodium azide. We also examined the injection of the resin from the anus, the casting to the pearl oyster which was preserved in formalin, and the addition of methyl methacrylate (Nisshin EM) in order to lower the viscosity of the resin. We also cast other mollusks; ark shell, Scapharca broughtonii. Pacific oyster C. gigas. clam. Meretrix hisoria (Bivalvia). and abalone. Haliotis discus (Gastropoda) using this method. RESULTS AND DISCUSSION The cast was easily made from the mouth to the anus (Fig. 1 ), Within the digestive diverticula, various features of the casting were observed, such as the tubule that surrounded the stomach (Fig. I A), the main duct (Fig. IB), the ducts of digestive diver- ticula without tubules (Fig. IC). and the ducts and tubtiles (Fig. ID, E). The cast was also showed the stomach and the orifice of the ducts of the digestive diverticula that were illustrated by Yonge (1926). The ducts and the tubules, which Owen (1955a, 1955b) and Nakajima (1956) showed, were also observed (Fig, IE). 777 778 Handa and Yamamoto Figure 1. The corrosion cast of the digestive organ in the pearl oyster, Pinctada fiicala martensii. A, the whole of digestive organ; B, the main duct; C, the stomach and the main duct without the tubule; D, the main duct and the tubule; E, the secondary duct and the tubule. The a and b represent the right and left aspects, respectively. Ksophagus I O), digestive diverticula (D), stomach (Si, Intestine (I), anus (AN I, main duct (MD), secondary duct (SD), and tubule (T). Bars in A, B, and C = I mm, bar in D = l«(l (jm, and bar in E = 1 \im. Injecting the resin wliicli contained the hardener at 20%. it always filled the ducts and tubules, terminal spaces (Fig. lA). In low concentration at 5*^. the resin reached the ducts (Fig. IB and C). After casted, the observation was not often easy because there was hardly contrast on the castings. Then, they were immersed in 20%^ NaOH solution at 60°C. As a result, many contrasts (e.g., red to pink) emerged (Fig. IB). When the methyl methacrylate was added to the resin to lower the viscosity, the castings come apart to pieces in 20% NaOH solution for the proteolysis and could not be completely made. Previous studies have demonstrated the opening and closing of the tubule in the digestive diverticula synchronized with tidal pe- riods (Morton 19.S6. Morton 1970. Owen 1972). circadian rhythms (McQuiston 1969, Morton & McQuiston 1974, Robinson & Lang- ton 1980), and food intake (Morton 1969, McQuiston 1969, Mor- ton 1979. Robinson & Langton 1980). In this study, the castings seems to be not inlluenced the conditions of the tubule, because they were made regardless of them. The food particles are transported to the tubules by the ciliary movement in the stomach (Owen 1955a, 1955b, Purchon 1957, 1958, 1960), and ciliary and/or muscular movement of the diges- tive diverticula (Owen 1955a, 1995b). Castings were similar whether resin was injected from the anus or the mouth. It was not possible to make casts with animals preserved in formalin. Thus the resin is probably transported to the tubules by not only the injected pressure but also similar functions of sending the particles from the stomach to tubules. We also examined the corrosion casting of the digestive organs in 4 molluscs. As the results, it was possible to cast and observe the ducts and tubules in ark shell 5. broughtonii (Fig. 2A). Pacific oyster C. gigas (Fig. 2B), clam M. htsoria (Fig. 2C), and abalone H. discus (Fig. 2D). Therefore, these methods are applicable to cast the digestive organ in molluscs. The detailed structure of digestive diverticula is suggested by the histological method, but it is very difficult to indicate the distribution of ducts and tubules, or to grasp a sense of the three- dimensional aspects of the structures. This casting method accu- rately shows the whole structure of digestive diverticula, for ex- ample, the positional relation to the stomach, and the features of branch and connection of ducts and tubules. Information on the digestive diverticula map will be very important for the criteria for classification, and also useful to investigate the function of diges- tion and absorption through the digestive canal, especially in the digestive diverticula and stomach. Corrosion Casting of thf Digestive Divertici'La 119 Figure 2. The casting preparation of the digesti\e organ of four molluscs. A, ark shell Scapharca hroiighloiiii: B. pacific oyster Crassostrea gigas: C, clam Meretrix liisoria (Bivalvial; D. abalone Haliolis discus (Gastropoda). The a and b represent the right and left aspects, respectively. The c and d represent the dorsal and ventral views. rcspecti>ely. Anus lANl. stomach (S). digestive diverticula (DD). Oesophagus (O). intestine (1). The cannula (Cnl is polyethylene tubing which was used for the injection of resin. Bars = 1 mm. McQuiston, R. W. 1969. Cyclic activity in the digestive diverticula of Lusaea rubra (Montagu) (Bivalvia: Eulamellibranchia). Proc. Malm: Sac. Land. 38:483-192. Morton. B. 1969. Studies on the biology of Drcissena pah inorplia Pall. 11. Correlation of the rhythms of adductor activity, feeding, digestion and excretion. Proc. Malac. Soc. Land. 38:401-414. Morton, B. 1970. A note on the cytological structure and function of the digestive diverticula of Mocoma ballhica cortelaled with the rhythm of the tide. Malac. Rev. 3:115-119. Morton. B. 1979. The biology, ecology and functional aspects of the organ feeding and digestion of the S.E. Asian mangrove bivalve. Enigmonia aenigmatica (Mollusca: Anomiacea). J. Zaol. Ltvid. 179:437—466. Morton, B. & R. W. McQuiston. 1974. The daily rhythm of activity in Teredo navalis linnaeus correlated with the functioning of the digesti\ e system. Forma et functio 7:59-80. Morton. J. E. 1956. The tidal rhythm and action of the digestive system of the lamellibranch Lasaea rubra. J. Mar. Biol. Ass. U. K. 35:503-586. Nakajima. M. 1956. On the structure and function of the mid-gut gland of mollusca with a general consideration of the feeding habitats and sys- tematic relation. Jpn. J. Zool. 1 1 :469-566. Naiiiba, K., M. Kobayashi.. S. Aida., K. Uematsu.. M. Yoshida.. Y. Kondo LITERATURE CITED & Y. Miyata. 1995. Persistent relaxation of the adductor muscle of oyster Crassostrea gigas induced by magnesium ion. Fisheries Sci. 61:241-244. Owen. G. 1955a. Observations on the stomach and digestive diverticula of the lamellibranchia I. The Anisomyaria and Eulamellibranchia. Quart. J. Micr. Sci. 97:517-537. Owen. G. 1955b. Observations on the stomach and digestive diverticula of the lamellibranchia 11. The Nuculidae. Quart. J. Micr. Sci. 97:541-567. Owen, G. 1972. Lysosomes. peroxisomes and bivalves. Sci. Prog. O.xf. 60:299-318. Purchon. R. D. 1957. The stomach in the lllibranchia and pseudolamelli- branchia. Proc. Zool. Soc. Land. 129:27-60. Purchon. R. D. 1958. The stomach in the Eulamellibranchia; Stomach type IV. Proc. Zool. Soc. Umd 131:487-525. Purchon. R. D. 1960. The stomach in the Eulamellibranchia: Stomach type IV and V. Proc. Zool. Soc. Land. 135:431-489. Robinson. W. E. & R. W. Langton. 1980. Digestive in a subtidal popula- tion of Mercenaria iiiercenaria (Bivalvia). Mar. Biol. 58:173-179. Yonge. C. M. 1926. Structure and physiology of the organs of feeding and digestion in 0.s7/-<'« <'(/»//,v. J. Mar. Biol. Ass. U. K. 14:295-386. .loiiriuil nfSJii-Ufish RcKcavch. Vol. 22, No. 3. 7S1-7S7. 2(K).V MITOCHONDRIAL DNA REVEALS GENETIC DIFFERENTIATION BETWEEN AUSTRALIAN AND INDONESIAN PEARL OYSTER PINCTADA MAXIMA (JAMESON 1901) POPULATIONS JOHN A. H. BENZIE,"* CAROLYN SMITH,' AND KETUT SUGAMA' ^Australian Institute of Marine Science. PMB No 3. Tonnsville, Queensland 4810. Australia; 'Centre for Marine and Coastal Studies. The University of New South Wales. Sydney. NSW 2052. Australia: and ^Gondol Research Institute for Aquaculture. PO Bo.x 140. Singaraja. Bali. Indonesia ABSTRACT A total of 234 individual silver-lipped pearl oyster (Pinclada maxima) from six populations in Australia and two populations in Indonesia were analyzed for genetic variation within a 680-base pair region of the mitochondrial DNA COI gene using restriction fragment length polymorphism analysis. The Indonesian populations were markedly different from all Australian popula- tions examined, and the differences were greater than that expected on the basis of their geographical separation. In contrast with this broader regional pattern of genetic differentiation, the Australian populations sampled were not significantly differentiated from one another, and a high degree of connectivity was observed among Western Australian pearl oyster populations. In addition, these genetic data show that Western Australian P. maxima populations have a closer tie to those from the northern Australian coast than with populations in Indonesia. This regional pattern of genetic separation is evident despite the proximity of Indonesia to the eastern Indian Ocean locations sampled and the potential for dispersal afforded by the southward currents of the Indonesian throughflow. KEY WORDS: aquaculture, biogeography. fisheries population genetics. INTRODUCTION gement, Indo-Pacific. mitochondrial DNA. pearl oyster. Pmctada maxima. The silver-lipped pearl oyster. PiiiclmUi maxiina (Jameson 1901) is found in Southeast Asia and northern Australia and pro- vides the basis for the strong south sea pearling industry (Shirui 1994). Although there is increasing use of hatchery stock, the industry in Western Australia is still dependent upon the collection of wild shell and upon the effective management of wild stocks. Early work by Johnson and JoU (1993) showed marked differences in allozyme frequencies in pearl oyster populations collected from northern and Western Australia (WA), suggesting that these needed to be managed separately. Despite significant genetic dif- ferences detected between two northern populations of P. maxima. which are separated by as little as 320 km. Johnson and Joll ( 1993) found no differentiation between the two WA populations that were sampled some several hundred kilometers apart. On the basis of this limited sampling of just two populations, the pearl oyster stocks within this important pearl producing region were consid- ered essentially panmictic. Significant genetic differences have also been detected between populations of several other species of pearl oyster over a range of spatial scales. Pinclada fiicala (Gould 1850). Pinclada albimi (Lamarck 1819), and Pinclada macidala (Gould 1830) were shown to be differentiated between sites less than 100 km apart (Wada 1982), Small genetic differences have also been observed between both widespread (Durand ct Blanc 1986. 1989) and geo- graphically closer populations (Benzie & Bailment 1994) of the black lip pearl oyster Pinclada margaritifera (Linnaeus 1758). Finally a genetic study of Pinclada radiata (Leach 1814) revealed significant differentiation between sites less than 33 km apart (Beaumont & Khamdan 1991 ). This collection of studies suggests the potential for population substructure within the P. maxima pearl oyster stocks, which extend for thousands of kilometers along the WA coast. The maternally inherited mitochondrial DNA (mtDNA) ana- *Corresponding author. E-mail: j.benzie@unsw.edu, au lyzed in the present study has a smaller effective population size than the allozyme genetic markers used by Johnson and Joll in their 1993 study and. as such, is inore sensitive to the effects of genetic drift and consequently often affords greater sensitivity for detecting genetic differences in population studies. Given the im- portance of the WA pearl oyster stocks to the Australian pearling industry, a sensitive genetic study of WA population substructure with more extensive spatial coverage than that of Johnson and Joll (1993) was undertaken. In addition, the analysis of collections from both Indonesia and northern Australia, two potential long- distance sources of recruits, allowed larger scale connectivity to be assessed. Populations of a number of marine species from northwest Australia have closer genetic affinities with Pacific rather than Indian Ocean populations despite being situated geographically in the Indian Ocean (Benzie 1999), These results are consistent with a connection via the strong cutxents of the Indonesian throughflow which move south from Indonesia towards Australia. Given that P. maxima is a broadcast spawner with a larval life of 2 to 3 wk (Shirai 1994). this species is potentially capable of dispersal over long distances. For this reason the present study examines the extent to which WA populations may derive recruits from both Indonesia and northern Australia. The present article reports the genetic structure of P. maxima stocks using mtDNA to determine local population structure within WA and the extent of connectivity to both Indonesian and northern Australian populations. MATERIALS AND METHODS Sample Collection Between 27 and 30 adult P. maxima were analyzed from each of six populations in Australia and two populations in Indonesia. Samples of adductor muscle were collected from P. maxima oys- ters aboard pearling industry vessels between February 1998 and November 1999, Samples were obtained in Northern Australia to the west of Darwin and in Western Australia from the Lacepede Islands. 80 Mile Beach (shallow water). 80 Mile Beach (deep 781 782 Benzie et al. water). Port Hedland, and Exmouth Gulf (Fig, 1). The 80 Mile Shallow collections were made inshore at less than a 10 m depth from the Northern end of 80 Mile beach. The 80 Mile Deep col- lections were made at a similar latitude but from a more offshore site at -30 m depth. The two Indonesian populations, Madura and Sumbawa Island, were collected in November 1999. Live animals were delivered by road to Gondol Fisheries Station and held in flowing sea water tanks before dissection. Adductor muscle samples were immediately snap frozen in liquid nitrogen after collection. DNA Extraction and Polymerase Chain Reaction (PCR) DNA was e.xtracted from using a CTAB extraction procedure modified from Adamkewicz and Harasewych (1996) in which small cubes of frozen muscle (-0.5 cm^) were ground in pre- warmed (60°C) CTAB extraction buffer (29^ CTAB. 27c polyvi- nylpyrrolidone. 100 niM Tris-HCl pH 8.0. 1.4 M sodium chloride, 20 niM EDTA) to which proteinase K was added to a final con- centration of 0.5 mg/niL. After overnight incubation at 60°C, samples were heated to 90°C for 20 min before addition of RNase A (0.1 mg/mL) and a 1-h incubation at 37°C. DNA was then extracted and precipitated using standard phenolxhloroform: isoamyl alcohol methods as per Sambrook et al. (1989). Echinoderm universal primers for the Cytochrome Oxidase I (COD gene (Col, fwd: 5' ATA ATG ATA GGA GGR TTT GG 3' and Col. Rev: 5' GCT CGT GTR CTA CRT CCA T 3' (Williams 1997) were used to amplify a 680-base pair segment of that gene. PCR reactions were conducted with 2 ng/p.L DNA in a IX PCR buffer containing 1.5 mM MgCK. 0.03 units/jiL Taq DNA poly- merase (Qiagen. Australia). 200 |jiM dNTPs, and 0.5 |jlM each primer. Thermocycler conditions were 94°C for I min (one cycle), followed by 94°C for 1 min. 45°C for 1 min. 72°C for 1 min, 30 s (30 cycles), with a final 4°C hold. Fifty-microliter PCRs were performed in a Perkin-Elmer 9700 thermocycler. Restriction Fragment Length Polymorphism (RFLP) Analysis Of the 39 restriction enzymes tested, only fi\e (D/))j1I. Eco0\90 I. Fokl. HcicUl. and NlaW) produced polymorphic fragment pat- terns, and these were used to survey RFLP variation within the amplified region of the COI gene. Overnight digest reactions con- tained 5 [X.L of PCR product and -0.03 units/ |jiL restriction enzyme (New England Biolabs. Beverley, MA) in a 15-|jlL reaction with IX buffer as per the enzyme manufacturer's instructions. Digest fragments were separated on 3% agarose gels (2% GibcoBRL agarose- 1 000, F/r Progen DNA grade agarose) at 4-5 volts/cm for up to 5 h with repeated photography of ethidium bromide-stained gels throughout the running period. Fragment sizes were estimated by regression against standard size markers and for each restriction enzyme the unique fragment patterns were given an alphabetical assignation (Table 1 ). The position of each restriction enzyme site producing the unique fragment patterns was identified by DNA sequencing of several individuals and a composite profile of the <:>Qr^=^''CZ:x^''^ s^ Lacepedes =^7v , ^tj-- WESTERN \> AUSTRALIA 500km Port Hedland Haplotype 1 K'-'J Haplotype 2 ^H Shared Private to Indonesia Private to Australia Figure 1. Pie diagrams illustrating the frequencies of the major haplotype or haplotype groups differentiating the eight P. maxima populations. Pearl Oystkr COI Genetic Structure 783 TABLE 1. Mitochondrial DNA restriction fragment sizes observed anions 234 f'iiiclada maxima from Australia and Indonesia Enzvme Haplotvpe Fragment Sizes (bp) DpnW EciM\m\ lok\ HiiA\ MalV 454.226 263.226.14? 226,158.145.105.45 408.226.45 680 590,93 603,77 680 353.250.77 448.155.77 299.29 1 ,5 1 .42 590.5 1 .42 632.51 388.249.43 388.155.94.43 43 1 .249 543,94.43 presence/absence of each site was constructed tor each animal tor all restriction enzymes (Table 2). Statistical Analyses The DA program in REAP (McElroy et al. 1992) was used to estimate haplotvpe diversity (h) and nucleotide diversity (tt) within populations and nucleotide divergence (d^-, ) among popu- lations (Nei & Tajima 1981 ). Spatial structuring of the populations was investigated using programs in ARLEQUIN (Schneider et al. 2000). AMOVA (Excoffier et al. 1992) was used to calculate sT (analogous to F^i )• N^.m. and to perform hierarchical analysis of (bsT- The MXCOMP program in NTSYS (Rohlf 1997) was used to calculate the Mantel test (Mantel 1967) to measure the degree of association between the matrix of pairwise ^-^ comparisons and the geographic distance between populations. Significance levels for simultaneous multiple tests were adjusted following Rice (1989). Further analysis, such as mismatch distributions, tests of neutrality, and timing of population expansion were not conducted because the small number of sites covered by the RFLP data would result in large errors and low statistical power. A character state matrix showing the presence or absence of presumptive restriction sites created using programs in REAP (McElroy et al. 1992) was used to construct unrooted, phylogenies using the maximum likelihood method in the RESTML program in PHYLIP, which assumes a Jukes-Cantor model of evolution (Felsenstein 1993). and the parsimony method implemented in PAUP (Beta Version 4.0b2; Swofford 19901. RESTML was set to find the best tree with global rearrangement of subtrees and input order of the haplotypes jumbled three times. In PAUP. restriction sites were treated as relaxed Dollo characters with gains weighted twice as heavily as losses (McMillan & Bermingham 1996). One thousand optimal trees were found using a heuristic search with the tree bisection and reconnection branch swapping algorithm and the 50% majority rule consensus was applied to obtain a single con- sensus tree. RESULTS The survey identified 16 composite haplotypes among the 234 samples (Table 2. Fig. 1). Two haplotypes (12.59^ accounted for 91% of the individuals assayed (213 individuals). Nine haplotypes (56.3%) were unique, accounting for 3.9%- of the animals. The other five haplotypes (31.3%) were each represented by only two or three animals. At the population level, nine haplotypes were private (i.e.. occuired in only one population) while at the regional level, eight haplotypes were private to Australia (62% of all hap- lotypes found in Australia) and three private to Indonesia (38%). Genetic Diversity Within I'opulalians On average, the Indonesian populations had higher levels of genetic diversity than the Australian ones, with Darwin having the lowest level of all (Table 3). The pattern was seen most clearly in the data for haplotype diversity (h). which was two times greater in the Indonesian populations (mean h = 0.520) than in the Aus- tralian populations (mean h = 0.246). Nucleotide diversity (tt) was also two times greater in the Indonesian populations (mean -n = 0.0097) than in the Australian populations (mean tt = 0.0046). Genetic Differentiation Among Populations The most common haplotype ( 1 ) was more frequent in Austra- lian populations, where it comprised 79-93% of the individuals assayed compared with 17-27% in the Indonesian populations. The next most common haplotype (2) was more frequent in Indo- TABLE 2. Composite mtDNA haplotypes observed among 234 Pinctada maxima from Australia and Indonesia Composite Haplotvpe Number DpnW £foOI09l Fokl Haem iV/olV abed c Igh ,Jk Inin 1 1011 0 001 111 Oil "1 1011 1 001 Ml 111 3 1011 1 000 111 111 4 1011 0 001 101 on 5 1011 0 Oil 111 oil 6 1111 1 001 111 111 7 1011 0 001 1 1 1 111 8 1011 0 000 111 oil 9 0011 0 001 111 oil 10 0001 0 001 111 oil 11 1011 1 101 111 111 12 1011 0 (101 111 010 13 1011 1 001 101 111 14 1011 1 001 1 1 1 101 15 1011 i 001 111 (III 16 1011 0 001 100 OKI 1110 1 112 031 211 Presence (1) or absence (Ol of restriction sites was inferred from banding paltems obtained from single digestions of extracted total DNA with each of the five restriction enzymes (Table 1 ). Bold numbers at the base of the table indicate the number of times a cutting site was lost in the maximum likelihood phylogeny. The 16 haplotypes represented 14 putative cutting sites, two of which were present in all animals surveyed. In the maximum likelihood phylogeny one site (j) was lost three times and two (h. 1) were lost two times. The remainder were lost once (nine sites). 784 Benzie et al. TABLE 3. Measures of genetic diversity within pupubtions: number of haplotypes (h,,), the ratio of (h,,) to the number of individuals sampled («,): [(H|,/«i)]. haplotype diversity I//), and nucleotide diversity (71) within each of eight populations of the pearl oyster Pinctada maxima Population II "h njn, h (±)SE) 7T Madura 29 3 0.17 0.458 (±0.102) 0.(.)()S6 Sumbawa 30 5 0.17 0.582 (±0.079) 0.0107 Darwin 30 2 0.07 0.129 (±0.(.)79) 0.0026 Lacepedes 30 4 0.13 0.251 (±0.102) ().()()5I 80 Mile Deep 29 4 0.14 0.200 (± 0.098) 0.0028 80 Mile Shallow 29 6 0.21 0.374 (±0.1 13) 0.0078 Port Hedland 27 3 0.11 0.271 (±0.105) 0.0048 Exmouth 30 5 0.17 0.253 (±0.104) 0.0045 Average 4.25 (±0.49) 0.15 (±0.02) 0.315 (±0.056) 0.0059 (±0.0011) All populations 234 16 0.07 nesian populations where it comprised 60-72% of individuals compared with 3-11% in Australian populations. These data, and the fact that three haplotypes were private to Indonesia and eight were private to Australia, suggest considerable regional differen- tiation among populations (Fig. 1 ). There were highly significant pairwise cts^ values between the two Indonesian populations and all the Australian populations, the mean 4>sr heing 0.562 (Table 4). There was no significant differentiation among populations within Australia (with the exception of Darwin and some Western Aus- tralian sites) or among populations within Indonesia. The signifi- cant differentiation of Darwin and some Western Australian popu- lations (mean 4)^^ = 0.038) was an order of magnitude less than that for the Indonesian-Australian comparisons. A hierarchical AMOVA analysis, partitioning variation within populations, be- tween populations within regions (Indonesia and Australia), and between regions, confirmed that all of the genetic variation oc- curred within populations (47%). and between regions (33%: P < 0.05). When pairwise ^-j- against distance in km for the total data set was significant (r = 0.69. P < 0.001). However, when the data were decomposed into comparisons either between or within regions, there was no significant relationship between ^y and geographical separation among Australian popu- lations (;■ = 0.56. P = 0. 156). There was not enough data to allow a test within Indonesia. The pattern of connectivity among popu- lations (using the effective number of migrants per generation (N^,,„) as the measure of exchange) emphasizes the strong connec- tion within regions and the limited exchange between regions (Fig. 3). Haplotype Phylogeiiy The 50% consensus tree based on parsimony analysis showed little structure and no deep relationship between haplotype group- ings related to their geographical distribution (Fig. 4). The maxi- mum likelihood network (not illustrated) was dominated by two star-like nodes each centered on one of the two most common haplotypes (haplotypes I. 2). Both stars included haplotypes found in either Australian and Indonesia or both regions, and most hap- lotypes differed by only one restriction site change from the dom- inant haplotypes. DISCUSSION RFLP analysis of a portion of the mitochondrial COI gene has provided strong evidence for high levels of dispersal among WA populations of P. nuixinia confirming the findings of Johnson and Joll (1993) based on nuclear markers (allozymes). The analysis also showed clearly that the WA populations weie more closely connected to northern Australian populations than to Indonesian ones. There has been movement of pearl oysters by the cultured pearl industry, largely from some WA wild sites to farms in the Northern Territory, but it is highly unlikely that the pattern ob- TABI.E 4. Pairwise F.,, among eight populations of the pearl oyster Pinctada maxima Madura Sumbawa Darwin Lacepedes 80 Mile Deep 80 Mile Shallow Port Hedland Sumbawa -().()23"' — Darwin 0.684*** 0.617*** — Lacepedes 0.569*** 0.497*** 0.046"- — 80 Mile Deep 0.657*** 0.585*** 0.000"- -0.000"- — 80 Mile Shallow 0.521*** 0.456*** 0.050*** -0.008"- 0.010"- — Port Hedland 0.560*** 0.487*** 0,057* -0.028"- 0.005"- -0.024"- Exmouth 0.592*** 0.521*** 0.038"- -0.022"- -0.013"- -0.012"- * P < 0.05; *** P < 0.001 : "' not significant. -0.024"- Pearl Oyster COI Genetic Structure 785 0.9 0.7 - 0.5 0.3 ^ 0.1 -0.1 0 500 2500 1000 1500 2000 Dista nee (km ) Figure 2. F^, graphed as a function of tlie geographical separation of the population pairs. Comparisons with Indonesian populations have been given a different symbol (triangles). served in this study is related to those stock movements. Using allozymes. Johnson and Joll (1993) noted clear genetic differences between the WA populations and those from Oxley Island in the Northern Territory despite large numbers of WA animals having been introduced to a farm within HO km of Oxley Island. The present study sampled wild populations in the Darwin region that were geographically more distant from the nearest farm supporting the suggestion that the connectivity observed between the Darwin and WA populations is unlikely to be a reflection of stock transfer. The le\el of di\ergence of P. nuixiina populations using the mitochondrial COI gene RFLP data (assuming a 2% divergence per million years) suggests present day mixing between all Aus- tralian populations but isolation of Indonesian and Australian populations during the Pleistocene at least 100.000 years ago. Cau- tion needs to be applied to these interpretations because of the limited data available in the RFLP analysis and the large errors inherent in these types of estimates in any case (Edwards & Beerli 2000, Kishino et al. 2001, Zhivotovsky 2001). Nevertheless, these data present a consistent picture of divergence between Indonesian and Australian populations well before the last low sea level stand around 12,000 years ago. Therefore, despite the apparent possibil- ity for migration of marine invertebrate larvae from Indonesia on strong southerly flowing currents the limited gene exchange ob- served between Indonesian and WA populations of P. maxinui is consistent with the strong westward deflection of the Indonesian 4'5^ ,'^ _^^-r^-C-'' Nem (mtPNA) <1 1-15 15-100 >100 Figure 3. .Map illustrating the le\els of gene flow between P. maxima populations estimated from mtDNA. throughflow just south of the Indonesian arc which makes it un- likely that this cuiTent would reach the coastal regions of Australia. In population genetic analysis of the giant tiger prawn Penaeus moiiodon (Fabricius 1798) also using RFLP analysis of mtDNA, Benzie et al. (2002) showed a closer relationship between the WA population of P. monodon and those from northern and eastern Australia, and a clear distinction from Indonesian and Philippines populations. The Philippines sample was to some extent interme- diate between Indonesian and Australian samples, suggesting links to Southeast Asia primarily via eastern Southeast Asian and east- ern Australian populations and also linkages to WA via northern Australia. There were no samples of P. maxima available from the Philippines or elsewhere in Southeast Asia or eastern Australia for the present study but the pattern observed in P. maxima over the range surveyed is consistent with that for P. moiwd(m. A strong genetic divide between the Pacific and Indian Ocean populations found in several species of marine invertebrates may involve considerable shifts in gene frequency as well as deep di- visions in haplotype phylogeny (Benzie 1999, Barber et al. 2000). In contrast, genetic distances between the Pacific and western Australian populations of marine invertebrate species may be an order of magnitude less (Benzie 1999) and in mtDNA markers may involve differences in the frequency of relatively closely re- lated haplotypes (Williams & Benzie 1997, 1998, Benzie et al. 2002). The fact that Indonesian and northernAVA populations off. maxima did not show deep divergence of COI haplotypes associ- ated with geographical region is consistent with these studies. The fact that genetic diversity is higher in southeast Asian populations of P. maxima than in Australian populations is also consistent with a general trend of decreasing genetic diversity outwards from southeast Asia to more geographically distant sites (Benzie et al. 2002). Marked genetic differences between WA and Indonesian pearl oyster stocks contrasts with considerable gene exchange over thou- sands of kilometers among WesteiTi Australian populations. The mtDNA variation in P. maxima populations between Indonesia and Australia suggests a strong influence of biogeographical events at the regional scale. Future assessments of larger scale patterns of dispersal of this species should include samples from elsewhere in the Indian Ocean and from additional locations in Southeast Asia and from eastern Australia. ACKNOWLEDGMENTS This work was supported by grant 97/.344 from the Fishing Research and De\elopment Corporation (FRDC) in Australia. We thank the Arrow Pearling Company, Broome Pearls, Maxima Pearling Company, Morgan and Co. Pty Ltd, Norwest Pearling, Paspaley Pearling Co.. Pearl Coast Di\ers Pty Ltd.. The Gun Char- ter Fishing, and the Pearl Producer's Association for their assis- tance with the project. Thanks also to Serena Sanders, Rick Scoones, Mick Buckley, Helen O'Donogahue, and officers from Western Australian Fisheries and Northern Territory Fisheries for their assistance. We also thank the staff of the Gondol Fisheries Research Centre of the Indonesian Government, particularly Dr Haryanti and Sari Budi Moria, and E. Bailment and S. Uthicke from AIMS, for their collaboration in sampling pearl oysters from Indonesia. We thank Megan Johnson, Lesa Peplow. Christine Clegg. and Melissa Merrit for technical assistance and Lee Ann Rollins for assistance with statistical analysis of the results. In part this work made use of the bioinformatics facilities of the Austra- lian National Genomic Information Service (ANGIS). 786 Benzie et al. M S D L 8D 8S PH E All Rank (29) (30) (30) (30) (29) (29) (27) (30) (234) 3.5 □ 17.2 26.7 93.3 86.7 89.7 79.3 85.2 86.7 70.5 1 3.5 3.7 • 8 □ 3.5 3.5 1.3 3 3.3 0.4 5 0.4 5 .2 □ 72.4 60.0 6.7 3.5 6.9 II. I 3.3 20.5 □ 3.3 3.3 0.8 4 6 □ 6.7 3.5 0.4 5 0.4 5 3.3 0.4 5 n Haplotype shared by Australia and Indonesia ■ Haplotype private to Indonesia Figure 4. Percentage frequencies of tlie 16 composite mtDNA haplotypes observed among P. maxima individuals, and the 50''f majority rule con-sensus tree (from KMM) maximum parsimony networks) indicating the relationships among the haplotypes. Numbers in parentheses imme- diately below the location codes indicate the number of individuals assayed. The column of numbers immediately to the right of the tips of the branches of the tree is the composite haplotypes listed in Table 2. The columns on the far right give the percentage of each haplotype in the total population, and their rank abundance, respectively. Locations are as follows: M. Madura; S, Sumbawa; D. Darwin; L, Lacepedes; 8D, 8(( Mile Beach deep: 8S. 80 Mile Beach shallow; PH. Port Hedland; E, Exniouth Gulf. REFERENCES Ad^imkewic/. S. L. & M. G. Harasewyeli. 1996. Systematics and biogeog- raphy of the genus Doiui.x (Bivalvia: Donacidael in Eastern North America. Am. Malucol. Bull. 13:97-103. Barber. P. H.. S. R. Palunibi. M. V. Erdmann & M. Kasim Moosa. 200(1. A marine Wallace's line? Nature 496:692-693. Beaumont, A. R. & S. A. A. Khamdan. 1991. Electrophorelic and moipho- Pearl Oysti-;r COl Genetic Structure 787 metric (.haracter-. in population differentiation of the pearl oyster Pincuida nuluila (Leach), from around Bahrain. / Miilhi.\ctin Suul. 57:433^41. Benzie, J. A. H. 1949. Genetic structure of coral reef organisms — ghosts of dispersal past. Am. Zooi 39:I3I-I4.'i. Benzie. J. A. H. & E. Bailment. 1994. Genetic differences among hlack- hpped pearl oysleri Piiictacla marguririfenn populations in the Western Pacific. Aqiiaci(Uiiie \21:\A5-\Sb. Benzie, J. A. H., E. Bailment, A. T. Forbes, N. T. Demetriades, K. Sugama. Haryanti & S. Moria. 2002. Mitochondrial DNA variation in Indo- Pacific populations of the giant tiger prawn. Pomcus nunuijim. MdI. Ecol. 11:2253-2569. Durand. P. & F. Blanc. 1986. Divergence genetique che/ un hivahe marin tropical: Pinchula marai>rinfent. In: Biologic des Populations. Lyon: Coll. Natl.. CNRS. Lyon. Universile Claude Bernard. Lyons, pp. 323- 333. Durand, P. & F. Blanc. 1989. Diversite genetique che/ un bivalve marin tropical: Pinchula iiuiragriiifera (Linne. 1758). Bull. Soc. Zaol. Fr. 113:293-304. Edwards, S. V. & P. Beerli. 2(100. Perspective: Gene divergence, popula- tion divergence and the variance in coalescence time in phylogeo- graphic studies. Evolidion 54:1839-1854. Excoffier. L., P. Smouse & J. Quattro. 1992. Analysis of molecular vari- ance inferred from metric distances among DNA haplotypes: applica- tion to human muochondrial DNA restriction data. Generics 1.11:479- 491. Felsenstein. J. 1993. (Phylogeny inference package) version 3.5c. Distrib- uted by the author. Seattle: Department of Genetics, University of Washington. Johnson, M. S. & L. M. Joll. 1993. Genetic Subdivision of the pearl oyster Pinctmla maxima (Jameson, 1901) (Mollusca: Pteriidae) in Northern Australia. Ai<\l. J. Mar. Freshwaler Res. 44:519-526. Kishino. H., J. L. Thome & W. J. Bruno. 2001. Performance of a diver- gence time estiination method under a probabilistic model of rate evo- lution. Mol. Biol. Evol. 18:352-361. Mantel, N. 1967. The detection of di.sease clustering and a generalised regression approach. Cancer Res. 27:209-220. McElroy, D.. P. Moran, E. Berminghani. & I. Komtleld. 1992. REAP: an integrated environment for the manipulation and phylogenetic analysis of restriction data. J. Hered. 83:157-158. McMillan, W. O. & E. Berminghain. 1996. The phylogeographic pattern of mitochondrial DNA variation in Dall's porpoise Phocacnaides dulli. Mol. Ecol. 5:47-61. Nei. M. & F. Tajima. 1981. DNA polymorphism detectable by restriction endonucleases. Genetics 97:145-163. Rice. W. R. 1989. Analysing tables of statistical tests. Evolution 43:223- 225. Rohll. F. J. 1997. NTSYSpc. version 2.02. Exeter Software, Applied Bio- statistics Inc., Setauket, New York. Sambrook, J., E. F. Fritsch & T. Manlatus. 1989. Molecular cloning: a laboratory manual. 2nd ed. New York: Cold Spring Harbor Laboratory Press. 1659 pp. Schneider, S., J.-M. Kueffer. D. Roessli. & L. Excoffier. 2000. ARLE- QUIN Manual ver 2.0. A software for population genetic analysis. Geneva. Switzerland: Genetics and Biometry laboratory. University of Geneva, Shirai. S. 1994. Pearls and pearl oysters of the world. Okinanwa. Japan: Marine Planning Company. Swofford, D. L. 1990. PAUP: Phylogenetic analysis using parsimony, ver- sion 3.0. Champaign. IL: Illinois Natural History Survey. Wada, K. T. 1982. Inter- and intraspecific electrophoretic variation in three species of the pearl oysters from the Nansei Islands of Japan. Bull. Natl. Res. Inst. Ac/iiaciilture 3:1-10. Williams, S. T. & J. A. H. Benzie. 1997, Indo-West Pacific patterns of genetic differentiation in the high-dispersal starfish Liiickia laevitiata. Mol. Ecol. 6:559-573. Williams, S. T. & J, A, H. Ben/ie. 1998. Evidence of a biogeographic break between populations of a high dispersal starfish: congruent re- gions within the Indo-West Pacific defined by colour morphs, mtDNA and allozyme data. Evolution 52:87-99. Zhivotovsky. L. A. 2001. Estimating divergence time with the use of nii- crosatellite genetic distances: impacts of population growth and gene flow. Mol. Biol. Evol. 18:700-709. Jcmnuil of Shellfish Research, Vol. 22, No. 3. 789-794, 2U03. SHALLOW-WATER DISTRIBUTION AND POPULATION CHARACTERISTICS OF STROMBUS GIGAS AND S. COSTATUS (GASTROPODA: STROMBIDAE) IN BOCAS DEL TORO, PANAMA ALEXANDER TEWFIK ' AND HECTOR M. GUZMAN "* ^ Dcpariiiu'iit i>f Bioloi^x. McGIII University. 1205 Ave. Dr. Penfield. Montreal, Canada. H3A IBl: -Smithsonian Tropical Research Institute, Unit (ms. APO AA 34002. USA .ABSTRACT Extensive visual surveys for the economically and ecologically significant queen conch iSlroiiilnis gigas) and milk conch (Stromlms cosralus) were conducted within the Bocas del Toro archipelago. Overall population densities are among the lowest recorded in the region (S. gigas 1.43 conch ha"': S. costatus 1.27 conch ha"'), and are likely the result of overexploitation by both commercial and subsistence fishing. The very low adult densities (S. gigas 0,30 conch ha"') and the lack of reproductive behaviors observed are a serious concern when one considers the "Allee effect" and the resultant negative per capita population growth rates reported elsewhere in the literature. This information has provided some of the rationale for establishing the recently announced 5-y ban on conch exploitation on the Caribbean coast of Panama. KEY WORDS: queen conch, stock assessment, overfishing, Panama, Allee effect, Siromlms INTRODUCTION StroiiihKs f^igds Linnaeus, 1758, and Strombus costatus Gme- lin, 1791. are two herbivorous gastropods of the family Stromhidae that inhabit shallow seagrass meadows (SGs). sand beds, and algal flats throughout the Caribbean. Queen conchs have long been val- ued for their meat and shell, and were first harvested in the Ca- ribbean by the Lucayans and Arawaks during pre-Columbian tunes (Brownell & Stevely 1981, Berg & Olsen 1989). Local commer- cial and subsistence use of both conch species has continued to this day and on occa.sion still provide a primary source of protein in some fishing communities. During the last 30 y, the overall harvest of queen conch has increased substantially, driven largely by international export as well as growing resident populations and increasing tourism in the Caribbean region (Berg & Olsen 1989. Tewfik 1997). Conch is commercially exploited in at least 22 countries throughout the region, and is often consumed only as a luxury food item due to its relative rarity and high market value (Mulliken 1996. Theile 2001 ). The shell products of several strombids are also sought after and are well recognized in the tourist industry of many Caribbean nations. Present landings of conch meat in the region are now in excess of 13.000 metric tons (Food and Agriculture Organization of the United Nations 2000). However, it should be noted that Food and Agriculture Organization landings are for all "Strombid conchs" and may therefore include several species. Significant landings of other strombids. including S. costatus. are likely to be occurring in places such as Mexico (Gil 1994. Theile 2001). The fear of the disappearance of commercial Queen conch fisheries has prompted 5. gigas to be included under appendi.x 2 of the Con- vention for the International Trade of Endangered Species (CITES) in 1992. Most recently. CITES has initiated a "significant trade review" for the species (Theile 2001 ). The San Bias and Bocas del Toro archipelagos are the main areas of conch fishing in Panama (Marians 1997). Limited data are available for the total number of conch landings in Panama, such landings being considered incidental to the spiny lobster harvest, with the latest figure being 1 16 metric tons in 199S (Martans 1997. Autoridad Maritima de Panama 1999). No specific regulations exist for the harvest of either 5. gigas or S. coslalus in Panama. *Corresponding author. E-mail address: gu/.manh@naos.si.edu however, the use of scuba gear is prohibited for the harvest of any marine resource (Martans 1997). Aside from the role that conchs serve in both local and regional economies, their populations pro- vide critical links between primary producers and higher-level consumers within near-shore marine communities throughout their range (Stoner & Waite 1991, Stoner et al. 1995). The following article will describe the abundance, population structure, morphology, and spatial distribution of S. gigas and 5. costatus, which have been heavily exploited over the last few decades in the Bocas del Toro archipelago. The consequences of this exploitation on future recruitment will also be discussed. Fi- nally, some brief comments will be made regarding the potential interaction that may exist between the two strombids defined here, with special attention to the spatial partitioning of these species over shallow, near-shore seagrass-sand-algal complexes that are typical of many areas of the Caribbean. MATERIALS AND METHODS The study was conducted over a 47,158-ha area of shallow water (<10 m) habitats in the Bocas del Toro archipelago between February and September 2000, A comprehensive description of the sea bottom topography, climate, geology, and reef distribution of the archipelago are available in several other publications (Rod- riguez et al, 1993. Greb et al. 1996, Guzman & Guevara 1998). The entire shallow (<10 m) coastal zone, inespective of habitat type, was divided into 240 2 x 2-km grid squares, of which 120 grids or sites were randomly selected and surveyed. Within each site, three replicate belt transects (100 x 6 m) were surveyed by two divers (width 3 x 3 m each) at each of two different depth strata (0.5-5 and 5-10 m). In total, each site had 1800 m" per depth strata or 3600 m" in total area surveyed. All strombids located within a transect were counted and mea- sured for total shell (siphonal) length (SL), maximum shell width, and lip thickness (at mid-lateral region approximately 40 mm from the edge) to the nearest millimeter using a caliper. Adult status was assigned to all conchs with a lip thickness >4 mm (Appeldoorn 1988). The depth and major substrate/habitat type where the strom- bids were located was also noted. The substrate/habitat types were classified according to a predefined typology that included only the most common habitats: algal plain (AP); SG; sand plain (SP); and coral rubble (CR) (Table 1 ). All data sets were analyzed using 789 790 Tewfik and Guzman TABLE 1. Substrate/habitat categories used in characterizing all sites surveyed within the Bocas del Toro archipelago, Panama. Habitat Code Description Algal plain AP Seagrass meadow SG Sand plain SP Coral rubble CR Fine mud. coarse sand, rubble, shell bottom dominated by benthic algal cover {Pi'iiicilliis spp.. Caulcr/ui spp.. Dasyclaihis spp.. Halimedii spp.. LUlorea spp.. Puilina spp.. Luureiiciu spp.) Coarse sand bottom dominated by Turtle {Thalassia tesmdinum.) and Manatee {Syriiigocliiiiu filifiinne.) grass. Coarse sand bottom with sparse or no benthic algae or seagrass cover. Rubble, shell fragment bottom with sparse cover of macro and encrusting algae. parametric statistics in .SYSTAT. version 10.2 {Systat Software Inc.. Richtiiond. CA). The density distribution of the two species was mapped using Geographical Information System. A digital classification for the area of study was based on a combination of digital images frotn three sources: topographic maps at a scale of 1 :50,0()0; color aerial photographs at a scale of 1:25.000; and LANSAT TM-.'i satellite images (Guzman & Guevara 2002). Density data were integrated using the programs MIP (Micro Images Inc.. Lincoln. NE), ver- sion ."^.1 (Map and Image Processing System), and ArcView, ver- sion .^.0. RESULTS Shallow marine environments ( E E, 25- S 20- ■§ 15- i- 9- 10- —1 0 5- sz CO n- B • 50 100 150 200 250 Shell Length (mm) 300 350 Figure 2. SL versus shell width (A) of S. gigas (R' = O.'J.V') and .V. costatus {R- = (I.KVJl, and SL >ersus shell width (B) of ,S'. gigas (R- = 0.202) and S. costatus (R- = 0.533) in Bocas del Toro, Panama. Distribution and Ahiindance of Strombus in Panama 791 Figure 3. Density distribution (individuals/ha) of S. gigas (A) and S. costaliis (B) over the shallow water (<10 m) in Bocas del Toro, Panama. The MPA is denoted by the polygon. encompasMng 1.7% (805 ha) of the total area. The lowest densities ( 1-10 conch ha~') were found in 19 scattered areas: two inside the marine protected area (MPA), Cayos Zapatillas (474 ha with 6 conch ha~' ), and near the southwest side of the park (480 ha with 3 conch ha~') (Fig. 3a). The highest densities for S. costalus (41- 50 conch ha"' ) were located northwest of Bastimentos Island in an area of 125 ha (0.3%) (Fig. 3b). The lowest densities for this species (1-20 conch ha"') were observed in 11 relatively small areas (7.5%), one of which occurred inside the MPA (761 ha with 14 conch ha"') (Fig. 3b). The distribution of strombids by the two depth strata vastly favored the shallower of the two (0-5.0 m) with 84% and 98%, respectively, of S. gigas and S. costatus being found in these areas. The most favored habitat/substrate type for both species was SGs (>70%i), with a relatively even distribution of the remaining indi- viduals among AP, CR, and SP areas (Fig. 4). When examining site occupation among the two species, S. gigas appears to have a broader distribution than S. costatus (24 vs. 14 sites), and the number of co-occupied sites was limited to just 4.2%', or 5 sites of the total 120 sites surveyed (Fig. 3). AP CR SG Habitat / Substrate Type Figure 4. Habitats occupied by S. gigas and S. coslaliis in Bocas del Toro. Panama. See Table 1 for habitat descriptions. DISCUSSION The long-term, heavy exploitation of strombid populations within the shallow water habitats of the Bocas del Toro archi- pelago have likely contributed to the overall densities of S. gigas (1.43 conch ha"'), which are among the lowest reported in the region (Table 2). Considerably less information is available for S. costatus in the literature, however, the densities observed here (1.27 conch ha"') are considered low when compared with that of Bermuda (2.6 conch ha"') (Berg et al. 1992) and the .Southwest Dominican Republic (50-200 conch ha"') (Tewfik, unpubl. data). It is suspected that the densities of S. costatus began to decline only after the populations of the larger and more valuable fisheries species, S. gigas. were already at low levels. Although this study has no information available on conch densities of <10 m, it did intensively survey habitats that are known to be important for conch as nursery and breeding areas throughout the region (Randall 1964, Sloner & Ray 1996. Tewfik et al. 1998. Stoner 2003). We suspect that areas down to >20 m may also have low densities, given the considerable capabilities of artisanal free divers that have been observed in Panama and other areas of the Caribbean (Martans 1997, Bene & Tewfik 2001 ). this despite the refuge that deeper waters might provide for adults. The low densities of conch and the lack of reproductive activity ob- served during this study become quite serious when one considers the "Allee effect," as described by Stoner and Ray-Culp (2000). Negative rates of per capita population growth were shown to occur below critical population levels. Specifically, mating (pair- ing and copulating) never occurred when adult densities fell below 56 conch ha"', and spawning never occurred with densities below 48 conch ha"'. Again, no such reproductive activities were ob- served during the entire 8 mo (February-September) of this study, which covered the intense spring and summer reproductive period for conch (Randall 1964, Buckland 1989. Stoner et al. 1992, Tew- fik et al. 1998). This has serious implications for the future levels of local recruitment and rebuilding of depicted populations, even with the establishment of MPAs and strict enforcement of fisheries regulations. The spatial distribution of the two-strombid species was con- centrated in the shallow (<5 m) SGs and is slightly surprising, given that these areas are the most accessible to local fishers. Another interesting element of the spatial distribution is that there was relatively little overlap (5 sites) out of the 33 sites occupied by either species (Fig. 3). This begs the question of whether there may 792 Tewfik and Guzman TABLE 2. Comparison of mean densities of S. gigas in tiie Caribbean determined by visual surveys. Location Conch ha Reference Antigua and Barbuda Bahamas Little Bahamas Bank Great Bahamas Bank Bermuda Belize Dominican RepubMc Florida Keys Haiti Honduras Cayos Cohinos Jamaica Pedro Bank (1994) Pedro Bank (1997) Morant Bank (1996) Mexico Panama Puerto Rico US Virein Islands Juveniles Adults (lip >4 mm) 1983/83 Unprotected Bank (1983/1983) Protected Bank (1991/1994) Protected Shelf ( 1991/1994) 1988 1989 Sub-legal (<15 cm) Legal (>15 cm) Juvenile (del Este 1996) Adults (del Este 1996) Juvenile (del Este 1997) Adults (del Este 1997) Juvenile (del Este) Adults (del Este) Juvenile (Jaragua) Adults (Jaragua) 1987-1988 1990 Juveniles (Gonave Island) Adults (Gonave Island) Rochelios Bank Western end Juveniles Adults Juveniles (Artisanal Zone) Adults (Artisanal Zone) Juveniles (10-20 m) Adults (10-20 m) Juveniles (20-30 m) Adults (20-30 m) Juveniles (Artisanal Zone) Adults (Artisanal Zone) Juveniles (10-20 m) Aduhs (10-20 m) Juveniles (0-10 m) Adults (O-IO m) Juveniles (10-20 m) Adults (10-20 m) Juveniles (20-30 ni) Adults (20-30 m) Cozumel (1989) Cozuniel (1995. after closure) Bocas del Toro (0-10 m) Southwest (1985/1986) West (1995) East (1996) St. Croix (1981) St. Thomas/St. John (1981) St. Thomas/St. John (1990) 13.5 3.7 28.5 20.8 53.6 96.0 0.5 2.9 14.4 14.9 283.0 4.5 22.5 1.6 14.4 0.6 53.0 0.6 2.4 1.5 10.0 0.0 15.0 160.0 7.3 7.3 15.0 73.6 51.2 152.3 73.7 202.9 221.0 93.0 466.0 48.0 482.1 10.9 59.9 101.1 31.8 214.5 89.0 830.0 1.4 8.1 4.2 7.2 7.6 9.7 12.3 Tewtlk et al. (2001) Tewfik et al. (2001) Smith & Neirop (1984) Smith & Neirop (1984) Stoner & Ray (1996) Stoner & Ray (1996) Berg et al. (1992) Berg et al. (1993) Appeldoom & Roike (1996) Appeldoorn & RoIke (1996) Delgadoet al. (1998) Delgado et al. (1998) Delgado et al. (1998) Delgadoet al. (1998) Torres & SuUivan-Sealy (2000) Torres & Sullivan-Sealy (2000) Posada etal. (1999) Posada et al. (1999) Berg & Glazer (1995) Berg & Glazer (1995) Haitian Fisheries Division (pers. com.) Haitian Fisheries Division (pers. com.) Haitian Fisheries Division (pers. com.) Haitian Fisheries Division (pers. com.) Tewfik etal. (1998) Tewfik etal. (1998) Tewfik (1996) Tewfik (1996) Tewfik (1996) Tewfik (1996) Tewfik (1996) Tewfik (1996) Tewfik & Appeldoom (1998) Tewfik & Appeldoom (1998) Tewfik & Appeldoom (1998: Tewfik & Appeldoom (1998) Stephens (1997) Stephens (1997) Stephens (1997) Stephens (1997) Stephens (1997) Stephens (1997) Martinez Vasquez (1995) Martinez Vasquez (1995) This study Torres Rosado (1987) Mateo et al. (1998) Mateo etal. (1998) Wood & Olsen (1983) Friedlander et al. (1994) Friedlander et al. (1994) be a true partitioning of suitable habitats and resources between the two congeneric herbivores due to some form of competitive inter- action. Berg et al. (1992) explained the differences in population distribution between the two species as being due to differences in habitat preference, and to the processes of larval dispersion, reten- tion, and recruitment (see Stoner 2003). However, a true competi- tive interaction (exploitative or interference) may also be possible, as has been investigated for other groups of trophically similar benthic plants (Williams 1987) and animals (Williams 1981, Teg- ner& Levin 1982, Keller 1983). Distributiiiii and Abundance of StronihK.s in Panama 793 In summary, this study concentrated on the population charac- teristics of two common strombids o\er their critical shallow water nursery and breeding habitats. Both species appear to be severely overexploited within the archipelago. The present low densities, combined with the suspected Allee effect, ultimately resulting in decreased recruitment levels, could severely restrict recovery. In- formation from this study will be combined with other surveys of macrophyte (see Stoner 2003). algal, and other invertebrate distri- butions to begin to understand the overall benthic community dy- namics within the archipelago and elsewhere. Finally, it is hoped that this baseline information mav also be useful in assessing the success of the nationw ide 5-y ban on conch harvest that is under consideration by the Panamanian government. ACKNOWLEDGMENTS This research was partially funded by the Fundacicin Natura, the Fundacion Proteccion del Mar (PROMAR). and the Smithsonian Tropical Research Institute. A. Domingo. C. Guevara, L. Partridge, and W. Pomaire provided invaluable assistance in the field. C. Mufioz developed the map in Geographical Information System. The authors thank the Government of Panama for provid- ing all necessary permits to work in the country. LITERATURE CITED Alcolado. P. M. 1976. Growth, morphological variations in the shell, and biological data of the conch ('Cobo') Stiombus gigas L. (Mollusca, Mesogastropoda). Serie Oceanologica No. 34. Havana. Cuba: Aca- demia de Ciencias de Cuba. Instituto de Oceanologica. 36 pp. Autoridad Maritima de Panama. 1999. Compendio estadistico 1998: direc- cion de planificacion y desarrollo del sector maritimo. Panama City. Panama: Autoridad Maritima de Panama. 159 pp. Appeldoom. R. S. 1988. Age determination, growth, mortality, and age of first reproduction in adult queen conch. Stmnihus gigtis. off Puerto Rico. Fish. Res. 6:363-378. Appeldoom. R. S. & W. Rolke. 1996. Stock abundance and potential yield of the queen conch resource in Belize. Report to the CARICOM Fish- enes Research Assessment and Management Program (CFRAMP) and Behze Fisheries Department. Belize. 30 pp. Bene. C. & A. Tewfik. 2001. Fishing effort allocation and fishermen's decision-making process in a multi-species small-.scale fishery: Analy- sis of the conch and lobster fishery in the Turks and Caicos Islands. Hum. Ecol. 29:157-186. Berg. C. J.. Jr. & D. A. Olsen. 1989. Conser\ation and management of queen conch {Strombus gigas) fisheries in the Caribbean. In: J. F. Caddy, editor. Marine invertebrate fisheries: their assessment and man- agement. New York: John Wiley and Sons, pp. 429^142. Berg. C. J., F. Couper, K. Nisbet. & J. Ward. 1992. Stock assessment of Queen conch, Strombus gigas, and Harbour conch, 5. costatus. in Ber- muda. Proc. GulfCarib. Fish. Inst. 41:433^38. Berg, C.J., Jr., J. Ward. B. Luckhurst. K. Nisbet. & F. Couper. 1993. Observation of breeding aggregations of the queen conch. Strombus gigas. in Bermuda. Proc. Gulf Carib. Fish. Inst. 42:161-171. Berg, C.J. & R. A. Glazer. 1995. Stock assessment of a large marine gastropod [Strombus gigas) using randomized and stratified towed- diver censusing. ICES Mar. Sci. Symp 199:247-258. Brownell, W. N. & J. M. Stevely. 1981. The biology, fisheries, and man- agement of the queen conch. Strombus gigas. Mar. Fish. Rev. 43: 1 - 1 2. Buckland, B. J. 1989. Reproduction and growth of the queen conch. Strom- bus gigas. off St. Christopher and Nevis in the Eastern Caribbean. M.Sc. Thesis. University of Guelph. Guelph. ON. Canada. 52 pp. Delgado. G. A.. M. Chiappone. F. X. Geraldes. E. Pugibet. K. M. Sullivan. R. E. Torres. & M. Vega. 1998. Abundance and size frequency of queen conch in relation to benthic community structure in Parque Na- cional del Este. Dominican Republic. Proc. Gulf Carib. Fish. Inst. 50:1-31. Food and Agriculture Organization of the United Nations. 2000. State of the world fisheries and aquaculture. Rome, haly: Food and Agriculture Organization of the United Nations. Available at: http:/Avww. fao.org/ DOCREP/003/,X8002E/X8002E00.htm Friedlander. A.. R. S. Appeldoom, & J. Beets. 1994. Spatial and temporal variations in stock abundance of queen conch. Strombus gigas. in the U.S. Virgin Islands. In: R.S. Appeldoom & B. Rodriguez, editors. Strombus gigas: queen conch biology, fisheries and mariculture. Ca- racas. Venezuela: Fundacion CientiTica Los Roques. pp. 51-60. Gil. L. A. R. 1994. Analisis de la evolucion de la pesqueria del caracol en dos estados de la peninsula de Yucatan. Mexico y en una cooperativa de Pescadores. In: R. S. Appeldoom & B. Rodriguez, editors. Strombus gigas: queen conch biology, fisheries and mariculture. Caracas. Ven- ezuela: Fundacion Cientifica Los Roques. pp. 1 13-124. Greb. L., B. Saric. H. Seyfried, T. Broszonn. S. Branch. G. Gugau. C. Wiltschko. & R. Leinfelder. 1996. Okologie ind sedimentologie eines rezenten rampensystem an der Karibikkiiste von Panama. Profil. Band 10. Stuttgart. Germany: Universitat Stuttgart. 168 pp. Guzman, H. M. & C. A. Guevara. 1998. Arrecifes coralinos de Bocas del Toro, Panama: I. Distribucion. estmctura y estado de conservacion de los arrecifes continentales de la Laguna de Chiriqui y la Bahi'a .Mm- irante. Rev. Biol. Trop. 46:601-622. Guzman. H. M. & C. A. Guevara. 2002. Annual reproductive cycle, spatial distribution, abundance, and size structure of Oreaster reticulatus (Echinodermata: Asteroidea) in Bocas del Toro. Panama. Mar. Biol. 141:1077-1084. Keller, B. D. 1983. Coexistence of sea urchins in seagrass meadows: An experimental analysis of competition and predation. Ecology 64:1581- 1598. Marians, R. 1997. Explotacion de la cambombia, Strombus gigas en Panama. In: J. M. Posada & G. Garci'a-Moliner. editors. International Queen conch conference. San Juan, Puerto Rico: Caribbean Fisheries Management Council, pp. 112-113. Martinez Vasquez. D. 1995. tQue pasa con las poblaciones de caracol en Cozumel? Informe CRIP. Puerto Morelos 2(2). Mateo. I.. R. Appeldoom. & W. Rolke. 1998. Spatial variations in .stock abundance of queen conch. Strombus gigas (Gastropoda: Strombidae), in the west and east coast of Puerto Rico. Proc. GulfCarib. Fish. Inst. 50:32-48. Mulliken, T. A. 1996. Status of the queen conch fishery in the Caribbean. TRAFFIC Bull. 16:17-28. Posada. J.. I. Mateo. & M. Nemeth. 1999. Occurrence, abundance and length frequency distribution of Queen conch. Strombus gigas (Gas- tropoda) in shallow waters of Jaragua National Park. Dominican Re- public. Carib. J. Sci. 35:70-82. Randall. J. E. 1964. Contributions to the biology of the queen conch Strom- bus gigas. Bull Mar. Sci. 14:246-295. Rodriguez, E.. R. Almanza. & R. Alvarado. 1993 Situacion biofisica y ambiental de la Provincia de Bocas del Toro. In: S. Heckadon-Moreno, editor. Agenda Ecologica y Social para Bocas del Toro. Panama City, Panama; Impresora Continental, S.A. Panama, pp. 55-72. Smith. G. B. & M. van Neirop. 1984. Distribution, abundance, and poten- tial yield of shallow-water fishery resources of the Little and Great Bahama Banks: UNDP/FAO Fisheries Division Project BH.V82/002. Rome. Italy: Food and Agriculture Organization of the United Nations. 78 pp. Stephens. I. 1997. An assessment of the queen conch [Strombus gigas) population around the Morant Cays. Jamaica. London UK: Queen Mary and Westfield College/Kingston. Jamaica: Fisheries Division. Stoner. A. W. 2003. What constitutes essential nursery habitat for a marine 794 Tewfik and Guzman species? A case study of habitat form and function for queen conch. Mar. Ecol. Prog. Ser. 257:275-289. Stoner, A. W. & J. M. Waite. 1991. Trophic biology of Srroinbiis gigas in nursery habitats: diets and food sources in seagrass meadows. J. Moll. Stud. 57:451-460. Stoner. A. W., V. J. Sandt. & I. F. Boidron-Metairon. 1992. Seasonality of reproductive activity and abundance of veligers in queen conch, Strom- bus gigas. Fish. Bull. 90:161-170. Stoner. A. W.. M. Ray & J. M. Waite. 1995. Effects of a large herbivorous gastropod on macrofauna communities in tropical seagrass meadows. Mar. Ecol. Prog. Ser. 121:125-137. Stoner. A. W. & M. Ray. 1996. Queen conch iStrombiis gigas) in fished and unfished locations of the Bahamas: positive effects of a marine fishery reserve on adults, juveniles, and larval production. U.S. Fisli. Bull. 94:551-565. Stoner. A. W. & M. Ray-Culp. 2000. Evidence for AUee effects in an overharvested marine gastropod: density-dependent matmg and egg production. Mar. Ecol. Prog. Ser 202:297-302. Tegner. M. J. & L. A. Levin. 1982. Do sea urchins and abalones compete in California? In: A. A. Balkema, editor. International Echinoderms Conference. Tampa Bay. Florida. Rotterdam. The Netherlands, pp. 265-271. Tewfik. A. 1996. An assessment of the biological characteristics, abun- dance, and potential yield of the queen conch [Stroiiibus gigas L.) fishery on the Pedro Bank off Jamaica. M.Sc. Thesis. Wolfville. NS. Canada: Acadia University. 139 pp. Tewfik. A. 1997. Life history, ecology, fisheries, stock status, and man- agement measures of the queen conch. Strombus gigas. CARICOM FisheriesRes.DOC. 19:84-117. Tewfik. A. & R. S. Appeldoorn. 1997. Queen conch (Strombus gigas) abundance survey and potential yield estimates for Pedro Bank. Ja- maica: technical report. Kingston. Jamaica: Fisheries Division, 31 pp. Tewfik. A.. H. M. Guzman. & G. Jacome. 1998. Assessment of the queen conch. Siromhus gigas. (Gastropoda: Strombidae) population in Cayos Cochinos. Honduras. Rev. Biol. Trap. 46:137-150. Tewfik. A.. S. Archibald. P. James. & L Horsford. 2001. Antigua and Barbuda Queen Conch Abundance Survey (1999). CARICOM Fishery Report. Doc. 7. 30 pp. Theile. S. 2001. Queen conch fisheries and their management in the Ca- ribbean. Technical report to the CITES Secretariat. Cambridge. UK: TRAFFIC Europe. 95 pp. Torres. R. E. & K. M. Sullivan-Sealy. 2000. Abundance, size frequency and spatial distribution of queen conch (S. gigas) in southeastern Do- minican Republic: a four-year population study in Parque Nacional del Este. Proc. Gulf Carib. Fish. Inst. 53:. Torres Rosado. Z. A. 1987. Distribution of two mesogastropods. the queen conch. Strombus gigas Linnaeus, and the milk conch. Strombus costa- tus Gmelin. in La Parguera. Lajas. Puerto Rico. M. S. Thesis. May- agiiez. Puerto Rico: University of Puerto Rico. 37 pp. Williams. A. H. 1981. An analysis of competitive interactions in a patchy environment. Ecology 62:1 107-1 120. Williams. S. 1987. Competition between the seagrasses Thalassia testudi- iium and Syringodium filiforme in a Caribbean lagoon. Mar. Ecol. Prog. Ser. 35:91-98. Wood. R. & D. A. Olsen. 1983. Application of biological knowledge to the management of the Virgin Islands conch fishery. Proc. Gulf Carib. Fish. Inst. 35:112-121. Joimml of Shclljhh Research. Vol. 22. N,.. 3. 79S-S()0. 2003, WHEN IS THE ABALONE HALIOTIS DISCUS HANNAI INO 1953 FIRST ABLE TO USE BROWN MACROALGAE? HIDEKI TAKAMI.' ' DAISUKE MURAOKA,' TOMOHIKO KAWAMURAr AND YOH YAMASHITA' ' Tohoku National Fisheries Research Instiliiie. Fisheries Research Agency, Shinhama, Shiogama. Miyagi 9S5-00U1. Japan: -Ocean Research Institute. The University of Tokyo. Minawiclai. Nakano. Tokyo 164-8639. Japan: and ^ Kyoto University Graduate School of Agriculture. Fisheries Research Station. Nagahaina. Maizuru. Kyoto 624-0R31. Japan ABSTRACT The dietary value of microscopic algal stages (gametophyte and juvenile sporophyte) of a brown alga Laminaria jiiprmica. Areschoug 1851 and of the benthic diatoms Cyliiulniilwcii closlehiim (Ehrenberg) Reimann and Lewin 1964. andAclimmtlws langipes Agardh 1824 were examined for different developmental stages of Huliotis discus lumniii Ino 1953 (0.4-2.9 mm shell length (SL)| to determine the size at which abalone begin to use macroalgae efficiently. Most individual abalone showed active feeding behavior, but there was considerable variation in growth of abalone between different algae and developmental stages of abalone. The growth rates of smaller post-larvae (0.4-1.2 mm SL) fed gametophytes and juvenile sporophytes of L. japonka. or A. longipes were significantly lower than those fed C closteriwn. In contrast, juvenile sporophytes of L. juponica and A. longipes produced significantly faster growth in larger postlarval abalone (>1.8 mm SL) than gametophytes of L. japcmica or C. closterium. Postlarvae in all developmental stages fed C. closterium actively grazed and efficiently ingested diatom cells. However, the relative dietary value of C. closterium decreased as abalone grew, probably because feeding efficiency on this diatom decreased because of its low cell volume and thin film-like colonies. Smaller post-larvae (0.4-1.2 mm SL) grazed repeatedly on the same .surface of gametophytes. juvenile sporophytes of L. juponica. or on A. longipes without detaching these algae, whereas larger post-larvae (>1.8 mm SL) detached and ingested large amounts of whole cells of these algae. Postlarval abalone 01.8 mm SL) began to use L. japonica gametophytes and juvenile sporophytes at approximately the same size at which morphologic changes occurred in their radulae, which enabled the ingestion of macroalgae. KEY WORDS: phyte benthic dialoni. brown alga, dietary value, gametophyte. growth. Haliotis discus liannin. postlarval abalone. sporo- INTRODIICTION Survival and growth rates in early life stages of abalone Hali- otis discus banned Ino 1953 are considerably affected by food type and the ability of individuals to use available food (Kawainura & Takami 199.^. Kavvamura et al. 1995, Seki 1997. Takami et al. 1997a. 1997b. Takami et al, 2000. Sasaki & Shepherd 2001, Takami 2002). Understanding the abalone' s early life feeding hab- its considered to be important in improving the rearing techniques in abalone hatcheries and also in understanding the factors con- trolling natural recruitment (Kawaniura et al. 1998a. Sasaki & Shepherd 2001, Takami 2002). As young of//, discus liannai grow, the main food sources shift frotn benthic diatoms to macroalgae (Kawamura et al. 1998a, Takami 2002). For postlarval abalone. benthic diatoms are the principal foods. The dietary value of diatoms for postlarvae is significantly different between diatom species or strains and is controlled largely by the ingestibility and digestibility of diatoms. Limited diatoms produce high digestion efficiencies and thus rela- tively rapid postlarval growth (Kawamura & Takami 1995. Kawa- mura et al. 1995, 1998a, 1998b, Roberts et al. 1999a). Attachment strength of diatoms is one of the factors that affects diatom digest- ibility for postlarval abalone (Kawamura et al. 1995. 1998a. 1998b. Roberts et al. 1999a). Very tightly attached diatoms, such as Cocconeis spp. and Achnanlhes spp.. require considerable force to be detached from substrata and are usually ruptured if dislodged. In contrast, many diatoms with low adhesive strength are ingested without cell rupture, and the majority of ingested cells pass *Corresponding author. E-mail: htakaiiii@affrc.go.jp through the gut alive and unbroken. There are some exceptional diatom species, such as Cylindnitheca closterium (Ehrenberg) Rei- mann and Lewin 1964. which has low attachment strength but is subject to high digestion efficiencies and supports rapid growth of postlarvae. probably because of its weak silica frustule. which is easily broken (Kawamura et al. 1995. 1998a. 1998bl. Cocconeis spp.. which have a high attachment strength and a relatively high dietary value for postlarval H. discus hannai larger than -0.8 mm shell length (SL; Kawamura et al. 1995. Takami et al. 1997a), are often dominant in the habitat of postlarval abalone in the natural environment (Kawamura et al. 1992. Takami 2002) and are used for rearing postlarvae in abalone hatcheries (loriya & Suzuki 1987, Suzuki et al. 1987). Benthic diatoms, such as Cocconeis spp., are probably one of the important diets for postlarval abalone in their natural habitat. In contrast, it has been suggested that juvenile abalone of more than 10 mm SL do not graze Cocconeis species if more favorable foods are available (loriya & Suzuki 1987. Suzuki et al. 1987). This is because Cocconeis spp. are not efficient food sources for these larger juveniles because their low-volume cells and prostrate growth form provides little energy (Takami et al. 1996). Large juveniles (>10 mm SL) and adult H. discus hannai prefer to feed on brown macroalgae especially Laminaria spp. (Sakai 1962. Kikuchi et al. 1967. Uki 1981 ) and show rapid growth rates when fed these algal species (Kikuchi et al. 1967. Uki 1981. Uki et al. 1986). Evidence from natural habitats suggests that the diet of abalone becomes dominated by macroalgae as juveniles grow (Tomita & Tazawa 1971. Shepherd & Cannon 1988). However, it is not clear at what size H. discus hannai begin to use macroalgae. Moreover, most of the food value experiments of brown macroal- gae for abalone have been conducted with mature algae whose 795 796 Takami et al. tolerance to herbivory may be different from juvenile algae (Van Alstyne et al. 1999, 2001). From the standpoint of physical as- pects, small abalone may be able to ingest juvenile macroalgae more easily than mature macroalgae. In this study, we compared the dietary value of microscopic algal stages (gametophyte and juvenile sporophyte) of Lciminaria japonica Areschoug 1851 and benthic diatoms for different devel- opmental stages of W. discus hannai to determine the size at which abalone begin to use macroalgae efficiently. MATERIALS AND METHODS Reproductive fronds of L. japonica were collected from the subtidal zone. Hokkaido Japan in October 2000. To obtain zoospores, fragments (2-3 cm~) of reproductive fronds were rinsed with sterilized seawater and placed separately in glass culture ves- sels containing sterilized seawater. To obtain zoospores, fertile fragments (2-3 cm~) of the desired algae were rinsed with steril- ized seawater and placed separately in 200-mL glass beakers con- taining sterilized seawater. Newly liberated zoospores were pipet- ted to 50-niL polystyrene or 200-mL glass beakers containing PESI medium (Tatewaki 1966). Beakers were kept in a growth chamber at I5°C and 43-1 13 nE/m'/s on a 12:12 LD cycle, and zoospores were allowed to settle on to the surface of the beaker. The settlement density was 25-30 zoospores/mm~. Any diatom contaminants were not observed in the beakers. After 5-8 days of incubation, morphologic differences were observed between fe- male and male plantules. Two types of microscopic algal stages of L. japonica (haploid gametophytes and diploid sporophytes) were used for the experiments. Gametophytes were kept in a growth chamber at 25°C to inhibit maturation, whereas the sporophytes were kept at 15°C to promote maturation (Yabu 1964). Juvenile sporophytes were allowed to grow until the size of thalli reached 0.5-1 mm in length. Gametophytes grew prostrate across the sur- face of the vessel, whereas juvenile sporophytes grew erect and formed three-dimensional colonies. Benthic diatoms Cylindrotlieca closteriuni and Achnanlhes lon- gipes Agardh 1 824 were also used as food items for abalone. These benthic diatoms were isolated from an abalone nursery tank at Tohoku National Fisheries Research Institute, Miyagi Japan, and were grown following the methods of Kawaniura et al. ( 1995). Larval abalone were hatched m May and October 2000 at the Yamagata Sea Farming Association (Yamagata. Japan) and reared using the method of Uki and Kikuchi (1984). Four days after fertilization at 20'C, the veliger larvae were transported to Tohoku National Fisheries Research Institute within 4 h. Competent larvae were transferred to 200-mL glass beakers with 150 mL of au- tocaved filtered (0.45 (jim: Millipore HA) natural seawater (FSW) containing 150 (xg/mL each of penicillin G sodium and strepto- mycin sulphate BP. These larvae were induced to metamorphose by the addition of I jjlM 7-amino butyric acid (Takami et al. 2000). Four days after metamorphosis induction, an adequate number of C. closteriuin cells were added as a food supply. The rearing beakers were incubated in light at 31-53 jiE/m'/sec on a 12:12 LD cycle. These abalone were maintained as a source of experimental animals, by adding supplementary C. closteriuin cells and replac- ing the water every 3—4 days with new FSW without antibiotics. All chemicals were obtained from Wako Pure Chemical Industries (Osaka, Japan). Si.\ experiments were conducted using different size classes of abalone. Detailed information on the experiments is presented in Table I . Before each experiment, abalone were dislodged with a fine needle from the stock beakers and placed into a 50-mL poly- styrene dish with 25 mL of FSW containing 6 mg/L of GeO, without food for a period of 2 days in the dark. GeO, effectively inhibits the proliferation of diatoms attached to abalone and does not affect the survival and growth of animals (Takami et al., 1997b). Most C. closteriuni cells ingested by abalone were di- gested, so any contamination by live diatom cells from abalone feces was negligible (Kawamura & Takami 1995, Kawaniura et al. 1995, Roberts et al. 1999a). Active postlarval abalone were placed into 50-mL polystyrene (Exp. I-IV) or 200-mL glass beakers (Exp. V, VI) in which each algal diet was available (Table I ). Beakers were submerged in a 35-L tank. Beakers containing experimental animals and algal di- TABLE 1. Details of experimental treatments. Initial Shell Length Rearing Duration of Exp. of .\balone Temperature Experiment Number Number of No. Algal Species Algal Type ((jm, mean ± SE) ( C) (days! of Rearing .Abalone per Beaker I Liiimiiiina japonica Gametophyte 458 ± 6.4 20 ±1 7 3 5 Cylimiroiheca closteriiim Benthic diatom 447 ± 6.9 20 ±1 7 3 5 U Achmmlhes loiigipes Benthic diatom 674 ± 5.4 20 ± 1 7 5 10 Cylimiroiheca closteriiim Benthic diatom 657 ± 5.5 20 ± I 7 5 10-12 III Laminaria japonica Gametophyte 898 ±21 20 ±1 8 3 5 Unninaria japonica Juvenile sporophyte 860 ± 17 20 ±1 8 3 5 Cylindrotheca closteriuin Benthic diatom 854 ± 23 20 ±1 8 3 5 IV Laminaria japonica Gametophyte 1092+14.7 20 ±1 7 3 5 Laminaria japonica Juvenile sporophyte 1152 ±28.0 20 ± 1 7 3 5 Cylindrotheca closteriuin Benthic diatom n21±21.2 20 ± 1 7 3 5 V Unninaria japonica Gametophyte 2008 ±51.6 17+ 1 10 3 5 Laminaria japonica Juvenile sporophyte 2106 + 81.4 17± 1 10 3 5 Cylindrotheca closteriuin Benthic diatom 1874 ±78.7 17± 1 10 3 5 VI Laminaria japonica Juvenile sporophyte 2894 ±153 17± 1 10 3 2 Achnanthes longipes Benthic diatom 2809+ 137 17± 1 10 3 2 Cylindrotheca closteriuin Benthic diatom 2711 ±90 17+ 1 10 3 -) When Does an Abalone Begin to Use Macroalgae? 797 ets were covered with a 200 (Exp. I-IV)- or a 600 (Exp. V, VlVixni nylon mesh to allow water exchange. Incoming filtered (1 |xm) natural seawater was maintained at a flow rate of approximately 2.4 L/min into the tanks. The rearing temperatures were set at a temperature that abalone of specific developmental stages encoun- ter in the Miyagi coast. Because the spawning season of H. discus hamuli in the area is from late summer to mid autumn, postlarvae encounter decreasing temperature as they age. SL of live individu- als in each experiment was measured to the nearest 10 [ji,m using a monitor and video camera system with an image analyzer, con- nected to an inverted microscope (Exp. I-Vl) or a dissecting mi- croscope (Exp. V. VI) at the beginning and at the end of the experiment. The feeding behavior of abalone was observed at in- tervals of 1-3 days using an inverted microscope. The differences between survival and growth rates of treat- ments were tested using Student's r test (Exp. I. II) or Tukey- Kramer multiple comparison test (Exp. III. VI). Sur\ i\al data were arcsine-transformed before analysis to normalize the data. RESULTS In all experiments, considerable variation was found in the growth rates of abalone between both algal types and developmen- tal stages of abalone (Fig. 1 ). even though most individuals were observed actively feeding. The growth rates of postlarval abalone of 0.4— 1.2 mm SL that were fed gametophytes and juvenile sporo- phytes of L. japonica were significantly lower (14-17 (j.m/day) Exp.l (0.4-0.£ T ' mm) n T =3 ■ 1 Exp.ll (0.4-0.7 mm) n=5 b • n=5 . 1 LiG Cc Exp III (0 80-9 mm) "=3 b ■ — 1 — m LjG IjS Cc 120- Exp, VI (2-8-2,9 r=3 mm) ion • T" i n=3 80 ■ 60 ■ 1 r4 40 • 20 n- — 1— ■ — 1 — Ij S Al Cc Figure 1. Growth of six developmental stages of postlarval Haliolis discus hannai Ipm per day) fed ganietophjles of iMiniitaria japonica (Lj (;), juvenile sporophytes of L. japonica (Lj Si, the benthic diatom Achnanllies longipes (,\ll, or the benthic diatom Cylindrolhcca cluste- riuni (C'cl. Numbers in parentheses indicate the range of Initial shell length of abalone used for each experiment. Each bar represents mean ± SE with the number of replicates. Letters on the top of each column indicate the results of Student's / test (Exp, I, II) or Tukey Kramer multiple comparison (Exp, III-VIl tests; columns with different letters represent means that are statistically different (/' < 0,(15). than those of postlarvae fed the benthic diatom C. closterium (21- 44 jim/day; Fig. 1 ; Exp. I. III. IV. P < 0.05). Differences in growth rates between postlarvae fed C. closterium and gametophytes or juvenile sporophytes were larger for postlarvae of 0.8-1 .2 mm SL (Fig. 1: Exp. Ill, IV) than of 0.4-0.5 mm SL (Fig. 1: Exp. I). Growth rates were not significantly different between postlarvae fed gametophytes and juvenile sporophytes in Exp. I. 111. and IV {P > 0.05). For larger postlarvae (> 1.8 mm SL), juvenile sporo- phytes produced significantly faster mean growth (81-95 jxm/day) than gametophytes (41 (xm/day, Exp. V, P < 0.05) or C. closterium (58 |xm/day. Exp. VI. P < 0.05). Postlarvae of 0.6-0.7 mm SL (Fig. 1; Exp. II) fed A. longipes showed significantly lower growth rates (9 (xm/day) than those fed C. closterium (33 p.m/day. P < 0.05). In contrast, the mean growth rate of post-larvae of 2.8-2.9 mm SL (Fig. 1: Exp. VI) fed A. loiii;ipes was significantly higher ( 100 ixm/day) than that of post- larvae fed C. closterium (58 (a.m/day. P < 0.05). The postlarvae that were fed C. closterium actively grazed and efficiently ingested diatom cells in all the experiments. Smaller postlarvae <1.2 mm SL (Exp. I-IV) grazed repeatedly on the same area of gametophytes. juvenile sporophytes. or A. longipes uithout detaching these algae. We could not directly observe the ingestion of algal diets by larger postlarvae (>1.8 mm SL; Exp. V, VI) ingested algal diet or not because most of the abalone stopped feeding when we tried to observe them under the microscope. However, we concluded that larger postlarvae ingested large amounts of gametophytes and sporophytes of L. japonica because these algae were almost completely cleared from the substratum and many feces remained. Ruptured cells of A. longipes were observed in the fecal pellets of postlarvae in Exp. VI but not in Exp II. Significantly lower survival rates were detected when smaller postlarvae were fed L japonica gametophyte (Exp. I) and A. lon- gipes (Exp. II: Fig. 2. P < 0.05) rather than C. closterium. In Exp. III-VI, the survival rates of individuals were generally high (80- 100%) except for Exp. IV (Fig. 2) when many contaminant pro- tozoans were observed in all rearing beakers. DISCUSSION The results of this study show that dietary values of gameto- phytes and juvenile sporophytes of a brown alga, L japonica, vary depending on the developmental stage of abalone. Most smaller postlarvae (<1.2 mm SL) could not etTiciently detach either ga- metophytes and sporophytes when feeding. In contrast, larger post- larvae (> 1 .8 mm SL) detached and ingested these brown algae and showed comparable or faster growth rates than those fed a benthic diatom. C. closterium (Fig. 1 ). In the experiments using smaller postlarvae < 1.2 mm SL (Exp. 1-IV). abalone fed C. closterium showed the highest growth rates. This diatom species has a weak silica frustule and low attachment strength; therefore, abalone can ingest and break the diatom cells resulting in high ingestion and digestion etTiciencies of abalone. The differences in growth rates between postlarvae fed L. japonica and C closterium were more marked on animals of 0.8-1.2 mm SL (Exp. III. IV) than those of 0.4-0.5 mm SL (Exp. I). These results correspond to the changes in abalone feeding (Kawamura et al. 1998a). The energy source of postlarvae is graduall) transferred from yolk supply to particulate food after metamorphosis at a size of -0.4—0.5 mm SL. Young postlarvae can grow using mucus materials secreted from diatoms (Kawamura & Takami 1995) and 798 Takami et al. 100 ' Exp. (0.4.0, T ° S mm) T ' 3 3 50 • 0 • 1 LjG Exp.lll (0.8-0.9 mm) n.3 n=3 L o f *■ [ ^H ■ 0 1 1 P — ""i 1 1 1 loo- se ■ (1 ■ Exp. IV (1.0-1.2 — 1 1 — 1 — mm) =3 Tn=3 o II LjG lis Cc L|G US Cc 100 ■ Exp.V(1.8-2 2mm) n=3 100 • Exp VI (2 8-2.9 mm) n=3 1 o 0 ° ■" w* 50 H 50 - 0 • — 1 — 0 - — 1 — £^ — 1 — LiG US LjS Figure 2. Survival rates of six developmental stages of postlarval Hali- Otis discus hannai fed ganietophytes of Laminaria japonica (Lj G), juvenile sporophytes o{ L. japonica (Lj S). the benthic diatom Achnan- thcs longipes (Al), or the benthic diatom Cylindrolheca closterium (Cc). Numbers in parentheses indicate the range of initial shell length of abalone used for each experiment. Each bar represents mean ± SE with the number of replicates. Letters on the top of each column indicate the results of Student's / test (Exp. \. II) or Tukey Kramer multiple comparison (Exp. III-VI) tests; columns with different letters represent means that are statistically different (/• < 0.05). macroalgae, such as crustose coralline algae (CCA; Daume et al. 1997, Kitting & Morse 1997, Takami et al. 1997b) supplemented by residual yolk supply fTakami et al. 2000. Roberts et al. 2001 ) and possibly absorption of dissolved organic matter (Shilling et al. 1996). At around 0.6-0.8 mm SL, postlarvae become responsive to the digestibility of diatom diets and grow more rapidly on effi- ciently digested diatoms (Kawamura et al. 1995. Takami et al. 1997a, Kawamura et al. 1998b, Roberts et al. 1999a). Dietary benefits are size dependent in postlarval abalone (Roberts et al. 1999a). Postlarvae of 0.6-1-2 mm SL who were fed ganietophytes, juvenile sporophytes, and A. longipes could not get adequate en- ergy sources for rapid growth because of the difficulty in ingestion of these algae and possibly insufficient amount of secreted mucus (Kawamura & Takami 1995, Kawamura et al. 1998a). The growth of postlarval H. discus hannai of 1.3 mm SL fed thinly sliced fronds of brown alga ihuhiria pinnatifula was com- parable to that of abalone fed diatoms (Sakai 1976). H. discus discus of 3— i mm SL fed softened fronds of U. pinnatifula (Fujii & Yotsui 1989) and germlings of macroalgae (Maesako et al. 1984) also showed good growth rate (72-1 10 (im/day). Takami et al. (1998) reported that postlarval H. discus hannai of 1 mm SL had a suite of enzymes useful for digesting brown algal polysac- charides and these enzyme activities increased rapidly from ap- proximately 2 mm SL, suggesting they could use macroalgae if they could ingest the algal fronds. The ingestion efficiency of postlarvae on algal diets is determined by the radula morphology (Roberts et al. 1999a, 1999b, Kawamura et al. 2001). The major ontogenetic changes in the radula structure occur around 1-2 mm SL for postlarval H. discus hannai (Kawamura et al. 2001). For example, the adult complement of five pairs of lateral teeth was completed by 1 .9 mm SL. A rapid increase in the clearance angle of the radula (Padilla 1985) was observed in postlarval H. discus hannai between 1-2 mm SL (Kawamura et al. 2001). Postlarvae >1 mm SL develop radula teeth w ith positive clearance angles that are more suitable for cutting rather than just sliding across the substratum. Larger post-larvae have well-developed outer lateral teeth (L3-L5 teeth), which appear to be used to cut the elastic macroalgae and three-dimensional growth forms of benthic diatom such as A. Umgipcs. In H. discus liannai larger than 1 .5 mm SL, the L3-L5 teeth become longer and more pointed (Kawamura et al. 2001). The results of this study show that postlarval H. discus liannai begin using ganietophytes and juvenile sporophytes almost at the same size at which major morphologic changes in radula occur. The relative dietary value of C. closterium decreased as post- larvae grew (Fig. 1). Three-dimensional A. longipes colonies and juvenile L. japonica sporophytes provide a much higher biomass per unit area than low volume, two-dimensional C. closterium films, once post-larvae are able to detach and ingest them. The significant difference in growth rate between post-larvae fed ju- venile sporophytes and ganietophytes (Fig. 1; Exp. V) might be caused by differences in the algal growth forms, because gameto- phytes also show prostrate growth form. In Exp. I and IL the survival rates of postlarvae fed ganieto- phytes of L japonica (66.7%) and A. longipes (65.6%) were sig- nificantly lower than those fed C. closterium (92.7-93.3%; Fig. 2). This low survival was not considered to be caused directly by starvation because abalone at these stages could survive more than 15 days of food deprivation (Takami & Kawamura. unpubl.). There is a possibility that the diffusive boundary layer (DBL), where diffusion dominates molecular transport, severely affects survival of post-larval abalone because oxygen concentrations in the dark may be reduced whereas algal secondary metabolites increase affecting water quality (Searcy-Bemal 1996, Roberts et al. 2000). The DBL "water quality" probably depends upon culture condition, and the algal strains and species used. Algal species with three-dimensional growth forms have a thicker DBL than the diatoms that form flat film-like colonies (Roberts et al., 2000); therefore, the DBL produced by A. longipes may have had reduced "water quality" and affected survival of the smaller postlarvae. Another possibility is that the combination of nutritional stress and water quality stress causes mortality more quickly than seen from starvation in clean containers. Larval H. discus hannai settle preferentially on CCA in the natural environment, and grow on CCA for several months (Saito 1981, Sasaki & Shepherd 1995, 2001, Takami 2002). Food sources from CCA include the alga's surface polysaccharides and epithe- lial cell contents, which can keep postlarvae alive but are not adequate to support rapid growth (Garland et al. 1985. Daume et al. 1997. Kitting & Morse 1997, Takami et al. 1997b). CCA rely on grazing by herbivores to prevent their surfaces from being covered with competitively superior algae (Paine 1980. Steneck 1982). Grazing-resistant algae with strongly adhesive prostrate forms such as benthic diatoms Cocconeis spp. tend to dominate under high grazing pressures by relatively large gastropods (Kesler 1981, loriya & Suzuki 1987, Suzuki et al. 1987, Steinman et al. 1989, Kawamura et al. 1992). Because grazing gastropods occur at high densities on CCA (Ayling 1981, Choat & Schiel 1982, Kawa- When Does an Abalone Begin to Use Macroalgae? 799 mura et al. 1992. Takami. 2002). Cocconeis spp. are ot'leii domi- nant and appear to be the main food sources for early life stages of abalone on CCA (Kawamura 1994. Takami 2002). However. Coc- coneis films probably become energetically inadequate as juvenile grow, and juvenile abalone come to rely on three-dimensional algal populations for food (Takami et al. 1996. Kawamura el al. 1998a). The germling or ju\enile stage of macroalgae is generally sus- ceptible to grazing by herbivores (Lubchenco 197S. Robles & Cubit 1981. Lubchenco 1983. Dayton 1983. Dean et al.. 1989. Asano et al. 1990. Paine 1992. Martinez & Santelices 1998, Van Alstyne et al. 1999. 2001). Therefore, newly recruited juvenile algae may find it difficult to grow on CCA surfaces. However, northern Japanese Laminarian species have prodigious reproduc- tive output, consequently they have considerable potential tor dense recruitment if grazing pressure is low (Yendo 1911. 1919). The season of se.xual reproductive in L. jciponica in Miyagi is from late autumn to mid winter when grazers' activities are relatively low due to the low water temperature. By this tiine. most of the 0-y-old abalone are more than 2 mm SL (Sasaki & Shepherd 1995, 2001. Takami 2002). a size at which they can efficiently ingest ju\ enile sporophytes of L. japonica. Juvenile L. japonica may be an important food source for these abalone at this early life stage. ACKNOWLEDGMENTS We thank Hiroyuki Kawakami of Yamagata Sea Farming As- sociation for providing the hir\al abalone. The critical readings of this manuscript by Christopher Clarke and Rodney Roberts are gratefully acknowledged. This study was supported in part by a grant-in-aid (Development of seed production and releasing tech- niques for stock enhancement of marine resources considering the conservation of ecosystem) from the Ministry of Agriculture. For- estry and Fisheries. Japan. LITERATURE CITED Asano. M.. S. Kikuchi & T. Kawamura. 1990. Effect of small hervivorous sea-snails on survival rates of the young laminariales plants (in Japa- nese with English abstract). Bull. Tohoku Nail. Fish. Res. lusl. 52:65- 71. Ayling. A. M. 19X1. The role of biological disturbance in temperate suh- tidal encrusting communities. Ecology 63:830-847. Choat. J. H. & D. R. Schiel. 1982. Patterns of distribution and abundance of kirge brown algae and invertebrate herbivores in subtidal regions of norlhcrn New Zealand. J. Exp. Mar. Biol. Ecol. 60:129-162. Daume. S.. S. Brand & W. J. Woelkerling. 1997. Effects of post-larval abalone {Hciliolis rubra) grazing on the epiphytic diatom assemblage of coralline red algae. Moll. Res. 18:119-130. Dayton. P. K. 1985. Ecology of kelp communities. Aniui. Rev. Ecol. Sysr. 16:215-245. Dean. T. A.. K. Thics & S. L. Lagos. 1989. Sur\ival of juvenile giant kelp: the effects of demographic factors, competitors, and grazers. Ecology 70:48.3-495. Fujii. A. & T. Yotsui. 1989. Rearing young abalone. 3 mm shell length. h\ feeding Viva perlusa and salted Undaria piwmiifida (in Japanese with English abstract). Bull. Nagasaki Pref. Inst. Fish. 15:13-15. Garland, C. D.. S. L. Cooke, J. F. Grant & T. A. McMeekin. 1985. Inges- tion of the bacteria on and the cuticle of crustose (non-articulated) coralline algae by post-larval and juvenile abalone (Halioris nilwr Leach) from Tasmanian waters. J. Exp. Mar. Biol. Ecol. 91:137-149. loriya. T. & H. Suzuki. 1987. Changes of diatom community on plastic plates used for rearing of abalone Nordotis discus (in Japanese with English abstract). Suisanztishoku 35:91-98. Kawamura. T. 1994. Studies on the variation mechanisms of marine henthic diatom communities (in Japanese). PhD Thesis. University of Tokyo, 221 pp.. Tokyo, Japan. Kawamura. T. & H. Takami. 1995. Analysis of feeding and growth rate of newly metamorphosed abalone Haliotis discus hannai fed on four spe- cies of benthic diatom. Fisheries Sci. 61:357-358. Kawamura. T.. R. D. Roberts & H. Takami. 1998a. A review of the feeding and growth of postlarval abalone. J. Shellfish Res. 17:615-625. Kawamura. T.. R. D. Roberts & C. M. Nicholson. 1998b. Factors affecting the food value of diatom strains for post-lar\al abalone Haliotis iris. Aquaculture 160:81-88. Kawamura. T.. T. Saido. H. Takami & Y. Yamashita. 1995, Dietary value of benthic diatoms for the growth of post-larval abalone Haliotis discus hannai. J. E.xp. Mar. Biol. Ecol. 194:189-199. Kawamura, T. H. Yamada. M. Asano & K. Taniguchi. 1992. Benthic diatom colonizations on plastic plates in the sublittoral zone off Oshika Peninsula. Japan (in Japanese with English abstract). Bidl. Tohoku Natl. Fish. Res. Inst. 54:97-102. Kawamura. T.. H. Takami. R. D. Roberts & Y. Yamashita. 2001. Radula de\elopment in abalone. Haliotis discus hannai. from larva to adult in relation to feeding transitions. Fisheries Sci. 67:596-605. Keslar. D. H. 1981 . Periphyton grazing by Aminicola liinosa: an enclosure- exclosure experiment. / Freshwat. Ecol. 1:51-59. Kikuchi. S.. Y. Sakurai. M. Sasaki & T. Ito. 1967. Food values of certain marine algae for the growth of the young abalone Haliotis discus han- nai (in Japanese with English abstract). Bull. Tohoku Reg. Fish. Res. Lab. 27:93-100. Kitting. C. L. & D. E. Morse. 1997. Feeding effects of postlarval red abalone. Haliotis riifescens (Mollusca: Gastropoda) on encrusting cor- alline algae. Moll. Res. 18:183-196. Lubchenco. J. 1978. Plant species diversity in a manne intertidal commu- nity: importance of herbivore food preference and algal competitive abilities. /Im. Nat. 112:23-39. Lubchenco. J. 1983. Littorina and Fucus: effects of herbivores, substrate heterogeneity, and plant escapes during succession. Ecology 64:1 1 16- 1123. Maesako. N.. S. Nakamura & T. Yotsui. 1984. Food effect of brown and green algae of eariy developmental stage and blue green algae for the growth of the juvenile abalone. Haliotis discus Reeve (in Japanese with English abstract). Bull. Nagasaki Pref. Inst. Fish. 10:53-56. Martinez. B. & B. Santelices. 1998. Selective mortality on haploid and diploid microscopic stages of Lessonia nigrcscens Bory (Phaeophyta. Laminariales). J. E.xp Mar Biol. Ecol. 229:219-239. Padilla. D. K. 1985. The structural resistance of algae to herbivores: a biomechanical approach. Mar Biol. 90:103-109. Paine. R. T. 1992. Food-web analysis through field measurement of inter- action strength. Nature 355:73-75. Roberts. R. D.. T. Kawamura & C. M. Nicholson. 1999a. Growth and survival of postlarval abalone (Haliotis iris) in relation to development and diatom diet. J. Shellfish Res. 18:243-250. Roberts. R. D.. T. Kawamura & H. Takami. 1999b. Morphological changes in the radula of abalone (Haliotis iris) during post-larval development. / Shellfish fics. 18:637-644. Roberts. R. D.. T. Kawamura & H. Takami. 2000. Diatoms for abalone culture: a workshop for abalone farmers. Cawthron Report 547. 28 pp. Roberts. R. D.. C. Lapworth & R. Barker. 2001 . Effect of starvation on the growth and survival of post-larval abalone (Haliotis iris). Aquaculture 200:323-338. Robles, C. D. & J. D. Cubit. 1981. Influence of biotic factors in an upper intertidal community: effects of grazing diptera larvae on algae. Ecol- ogy 62:\5i6-\541. Saito. K. 1981. The appearance and growth of 0-year-old Ezo abalone. Bull. Jap Soc Sci. Fish. 47:1393-1400. 800 Takami et al. Sakai, H. 1976. Culture of juvenile abalone after forming the first respi- ratory pore using macro-algae as a food sources (in Japanese). Siiisdii- zoshoki, 23:145-148. Sakai. S. 1962. Ecological studies on the ahalone Halioiis discus lumitai Ino-I. Experimental studies on the food habit (in Japanese with Enghsh abstract). Bull. Jpn. Soc. Sci. Fish. 28:766-779. Sasaki. R. & S. A. Shepherd. 1995. Larval dispersal and recruitment of Halioris cliscu.s lumiial and Tegula spp. on Miyagi coasts. Japan. Mar. Freshwcuer Res. 46:519-529. Sasaki. R. & S. A. Shepherd. 2001. Ecology and post-settlement survival of the Ezo abalone. Haliotis discus hamuli, on Miyagi coasts. Japan. ./. Shellfish Res. 20:619-626. Searcy-Bernal. R. 1996. Boundary layers and abalone postlarval culture: Preliminary sludies. Aquaculliire 140:129-137. Seki, T. 1997. Biological studies on the seed production of the northern Japanese abalone. Hciliotis discus lunuiai Ino (in Japanese and English abstract). Bull. Tohoku Nail. Fish. Res. Inst. 59:1-71. Shepherd. S. A. & J. Cannon. 1988. Studies on southern Australian balonc (genus Halioiis) X. Food and feeding of juveniles. J. Malacol. Soc. Aust. 9:21-26. Shilling. F. M.. O. Hoegh-Guldberg & D, T. Manahan. 1996. Sources of energy for increased metabolic demand during metamorphosis of the abalone Haliotis rufescens (Mollusca). Biol. Bull. 191:402—412. Steinman, A. D.. C. D. Mclntire. S. V. Gregory & G. A. Lamberti. 1989. Effects of irradiance and grazing on lotic algal assemblages. / Phycol. 25:478^85. Steneck. R. S. 1982. A limpet-coralline alga association: adaptations and defenses between a selective herbivore and its prey. Ecolof>y 63:507- 522. Suzuki. H.. T. loriya. T. Seki & Y. Aruga. 1987. Changes of algal com- munity on the plastic plates used for rearing the abalone Haliotis discus liaiuhii. Nippon Suisan Gakkaishi 53:2163-2167. Takami, H. 2002. A study on the feeding, growth and survival in early life stages of an abalone Haliotis discus hannai (in Japanese). PhD Thesis. University of Tokyo. 220 pp.. Tokyo. Japan. Takami. H.. T. Kawamura & Y. Yamashita. 1996. Dietary value of benlhic diatom for the growth of juvenile abalone Haliotis discus hannai (in Japanese with English abstract). Suisanzoshoku 44:21 1-216. Takami. H.. T. Kawamura & Y. Yamashita. 1997a. Survival and arowth rates of post-larval abalone Haliotis discus hannai fed conspecific trail mucus and/or benthic diatom Cocconeis scutellimi var. pana. Aqua- culture 152:129-138. Takami. H.. T. Kawamura & Y. Yamashita. 1997b. Contribution of dia- toms as food sources for post-larval abalone Haliotis discus baniuii un a crustose coralline alga. Moll. Res. 18:143-151. Takami, H.. T. Kawamura & Y. Yamashita. 1998. Development of polysaccharide degradation activity in postlarval abalone Haliotis iZ/.v- cus hannai. J. Shellfish. Res. 1 7:723-727. Takami. H.. T. Kawamura & Y. Yainashita. 2000. Starvation tolerance of newly metamorphosed abalone Halioiis discus hannai. Fisheries Sci. 66:1180-1182. Tatewaki. M. 1966. Formation of a crustaceous sporophyte with unilocular sporangia in Scylosiphon lomentaria. Phycologia 6:62-66. Tomita. K. & N. Tazawa. 1971. On the stomach contents of young abalone Haliotis discus hannai Ino. in Rebun Island. Hokkaido (in Japanese with English abstract). Sci. Rep. Hokkaido Fish. Exp. Sin. 13:31-38. Uki. N. 1 98 1 . Food value of marine algae of order Laminariales for growth of the abalone. Halioiis discus hannai (in Japanese with English ab- stract). Bull. Tohoku Reg. Fish. Res. Lab. 42:19-29. Uki. N. & S. Kikuchi. 1984. Regulation of maturation and spawning of an abalone, Haliotis (Gastropoda), by external environmental factors. Aquacullure 39:247-261. Uki. N.. M. Sugiura & T. Watanabe. 1986. Dietary value of seaweeds occuiTing on the Pacific coast of Tohoku for growth of the abalone Halioiis discus hannai. Bull. Jpn. Soc. Sci. Fish. 52:257-266. Van Alstyne. K. L.. J. M. Ehlig & S. L. Whitman. 1999. Feeding prefer- ences for juvenile and adult algae depend on algal stage and herbivore species. Mar Ecol. Prog. Ser 180:179-185. Van Alstyne. K. L.. S. L. Whitman & J. M. Ehlig. 2001. Difference in herbivore preferences, phlorotannln production, and nutritional quality between juvenile and adult tissues from marine brown algae. Mar. Biol. 139:201-210. Yabu, H. 1964. Early development of several species of Laminariales In Hokkaido. Mem. Fac. Fish. Hokkaido Univ 12:1-72. Yendo. K. 1911. The development of Costaria. Undaria and Laininaria. Ann. Bol. 25:691-715. Yendo, K. 1919. A monograph of genus Alaria. J. Coll. Sci. Tokyo Imp. Univ. 43:1-145. Joiimul of Shellfish Research. Vol. 22. No. 3, 8UI. 2003. PROCEEDINGS OF WORKSHOP ON REBUILDING TECHNIQUES FOR ABALONE IN BRITISH COLUMBIA Nanaimo, B.C. Canada January 14-16. 2003 Guest Editor Alan Campbell Department of Fisheries and Oceans Pacific Biological Station Nanaimo. British Columbia V9T 6N7 CANADA 801 Journal nf Slwllfish Rcscanh. Vol. 22. No. 3. S()3. 2003. PREFACE The lolknving 6 papers and 1 1 abstracts published in this issue of the Journal of Shellfish Research are part of 17 presentations delivered at an international workshop on rebuilding techniques for abalone in British Columbia (BC) held at Nanaimo. BC. Canada. January 14 to 16. 2003 (Campbell & Heimstra 200.^). The decline of northern (pinto) abalone {Hulinlis kanitscliatkaua) stocks since the late 1970s has prompted fishery closure since 1990. listing this species as "threatened" by the Committee on the Status of Endangered Wildlife in Canada in 1999. two interna- tional workshops (Campbell 2(X)0. Campbell & Heimstra 2003). and development of a national recovery strategy for H. kamtwhcil- kana in BC (Toole et al. 2002). Over exploitation and declines in wild abalone populations base occurred m mam parts of the world and the methods for suc- cessfully rebuilding wild stocks are still in the developmental stage. The workshop discussed community stewardship projects, aquacul- ture. out planting and restocking, wild stock manipulation, and moni- toring tools and evaluation performance indicators as methods for abalone rebuilding. The 6 papers published in this volume represent some of the presentations at the workshop. The papers underwent the stringent refereeing and review process required by this jour- nal. I thank the authors and the many referees for their efforts and co-operation for reviewing and revising the manuscripts. ALAN CAMPBELL Editor LITERATURE CITED Campbell. A., editor. 2000. Workshop on rebuilding abalone stocks in British Columbia. Can. Spec. Publ. Fish. Aquat. Sci. 130. 158 pp. Campbell. A. & L. Hiemstra.. editors. 2003. Proceedings of the workshop on rebuilding techniques for abalone in British Columbia. Can. Tech. Rep. Fish. Aquat. Sci. (In press) Toole. J.. B. Adkins. E. Bomhold. J. Boutillier. G. Caine, A. Campbell, A. Castledine. L. Convey. C. Cote. P. Coulson, T. Down. K. Francis. H. Gill. R. Harbo. H. Holmes. B. Jubinville. D. Lawseth, B. G. Lucas, A. Morgan, G. Parker & J. Rogers. 2002. National Recovery Strategy for the Northem Aba- lone {Haliotis kamtschatkana) in British Columbia Fisheries and Oceans Canada, (http://www-conim.pac.dfo-mpo.gc.ca/pages/consultations/ fisheriesmgmt/abalone/AhaloneRecovStrategy_e.html. 22 pp. 803 Joiinnil ofSluilfish Hcscunh. Vol. 22. No. 3, iS()5-SI0. 2U0.i. UPDATE ON EMERGING ABALONE DISEASES AND TECHNIQUES FOR HEALTH ASSESSMENT SUSAN M. BOWER Departiuciii of Fislieries and Oceans. Pacific Biologic Station. Nanaimo. British Cohiiuhia. \VR 5K6 ABSTRACT This article presents a review of new diseases and additional information on known pathogens of abalone that were encountered in the last few years as a result of increasing efforts towards the culture of abalone around the world and concurrent investigations into abalone health. A novel haplosporidian was associated with high mortalities (82. ,^-90%) of cultured juvenile paua t.Hiiliotis iris) in New Zealand. Disease outbreaks among cultured abalone in Tasmania. Australia were associated with two species of Viljrio {V. Imn-eyi and V. splciulidiis I) and a Flciv(ilnicterium-\ike bacterium with stress factors precipitating the diseases in most cases. The agent of withering syndrome responsible for mass mortalities of black abalone (W. cracherodii) in southern California was identified as the Rickettsiales-Iike prokaryote "Camiuiatus Xenohaliotis califomiensis". The exotic sabellid polychaete that seriously impacted abalone culture in California was named Terehrasalyelta heterouncinuta and experimentally found to reproduce at low temperatures but with a significantly temperature-dependant generation time (a developmental cycle of 298 days at ll.2°C in comparison to 165 days at 15.6°C). To assess the health of cultured abalone. histologic examinations are essential. For histology, tissue samples (less than 1 cm thick) should be fixed in Davidson's solution or 10"^ formalin in seawater such that there is at least 10 parts fixative to 1 part tissue. Histopalhology will not only indicate the presence of infectious agents but can be useful for monitoring the suitability of the diet and aquaculture environment. These assessments will benefit abalone aquaculture and provide assurance that only healthy animals are used in stock rehabilitation programs. KEY WORDS: disease, parasites, abalone. Huliotis INTRODUCTION The decline tif wild stocks of abalone around the world and the increasing demand for this product in the market place has in- creased efforts in the culture of various abalone species and inter- est in rehabilitating wild stocks. Concurrent with this increased attention to abalone, awareness of the various infectious diseases of abalone has arisen. Prior to 2000, six severe diseases associated with mortality in various species of abalone had been reported in the literature (Table I Bower 2000). Since thai lime, knowledge of .some of these diseases has increased and other infectious diseases have been detected. Various investigations have revealed that infectious diseases can have a significant negative impact on the aquaculture of aba- lone (Elston & Loekwood 1983. Oakes & Fields 1996, Bower 1987a, Li et al. 1998. Lizarraga-Partida 1998, Nishimori et al. 1998. Ruck & Cook 1998, Kuris & Culver 1999. Caceres Martinez et al. 2000. Moore et al. 2000b. Diggles el al. 2002). Because infectious diseases can be equally disastrous if inadvertently in- troduced into new locations by stock rehabilitation efforts that involve the translocation of abalone, it is important to be aware of the available information on diseases to circumvent complications (Sinderman 1988). The current low abundance of wild stocks of northern abalone [Haliotis kamtschatkaiun in British Columbia has stimulated the culture of this species and the development of plans for rehabilitation. In conjunction with this effort, abalone will be examined for infectious diseases. Thus, it is prudent to have information on diseases that have affected abalone around the world and on procedures used to assess abalone health at hand. This paper presents information on abalone diseases that was pub- lished since the review by Bower (2000) and describes techniques that can be implemented to examine abalone for infectious disease. Although directed towards concerns for northern abalone. the in- Phone: E-mail: BowerSCa'dfo-mpo.gc.ca formation provided herein is directly applicable to all abalone species regardless of locatiini. UPDATE ON ABALONE DISEASES .Since 2000. new significant diseases of abalone have been encountered during efforts to culture abalone in various parts of the world and new information has been published on previously known diseases. In New Zealand, a novel haplosporidian was associated with high mortalities (82.5-90'7f) of cultured juvenile paua {Haliotis iris) at one farm in the eastern Bay of Plenty (Diggles et al. 2002). Runts were more severely affected but. all infected abalone showed weak adherence to the substrate, had a shriveled foot with pale blister-like lesions on the foot and mantle, and failed to right themselves when turned over. In lightly infected abalone. uni- to multinucleate plasmodia (up to 1.3.5 (xm in length) occurred in the connective tissue surrounding the gut, amongst glial cells adjacent to the nerves of the mantle and foot and within the gill lamellae. In heavy infections, numerous plasmodia were present in the hemolyinph, gills, heart, kidneys, mantle, foot, epipodium, and connective tissue of the digestive gland. Spore formation was not observed but sporocyst-like bodies were found amongst plasmodia in the right kidney of an adult paua collected from the wild (Hine et al. 2002. Diggles et al. 2002). Research into this new pathogen is on going in New Zealand. Various bacteria have also been isolated from cultured abalone experiencing disease and mortalities. In Tasmania. Australia, dis- ease outbreaks among cultured abalone {Haliotis rubra. H. laevi- gata and their hybrids) were associated with two species of Vibrio (V. haireyi & V. splendidiis I) and /•7I8°C) seem to be required for the de- velopment of withering syndrome in abalone exposed to "Caiuli- datiis .Xenohaliotis californiensis" (Moore et al. 2000a. 2000b). This pathogen was transmitted between abalone by injection and bath exposure to post-esophagus homogenates prepai'ed from in- fected abalone and by cohabitation with abalone exhibiting with- ering syndrome (Moore et al. 2001b. Friedman et al. 2002). Ex- amination of hemocyte activity indicated that the hemocytes of infected H. cracherodii were more chemotactic but were less able to engulf and destroy foreign particles, which may contribute to the mortality associated with withering syndrome (Friedman et al. 2000b). Intramuscular injection and oral administration of oxytet- racycline was effective in reducing the losses of infected abalone Disease update and screening of abalone 807 (Friedman et al. 2003). However, other antimicrobials (chloram- plienicol. clarithromycin, and sarafloxicin) had no measurable af- fect on the disease (Friedman et al. 2000a). In addition to the new information on severe diseases of aba- lone, observations on other parasites were also published since the previous review by Bower (2000V A renal coccidia was reported from H. midae in South Africa (Mouton 2000) and Murgolisii'lla haliotis was detected in H. nifesceiis from Baja California, Mexico (Caceres Martinez & Tinoco-Orta 2001). Two species of spionid polychaetes (mudworms). Boccardia bwxi and Polydora hopluni. were associated with severe blistering in the shell and 50% or greater mortality among cultured abalone at several sea-based fa- cilities in southern Tasmania. Australia (Lleonail et al. 200.^). Three species of shell boring clams (Lilhophaga arishihi. Lirlm- phaga pliinnda. and PcuitcUa caiimdi) were found boring in tlic shell of H. fiilgens and two of these species (L. uiistata and L. phiiiiuhi) were also observed infesting the shells of H. cornigata from the vicinity of Isla de Cedros on the west coast of Baja California, Mexico (Alvarez-Tinajero et al, 2001). Nollens et al. (2002) reported that endoscopy applied to anesthetized H. iris was more accurate than radiography and ultrasonography for the de- tection of the shell lesions caused by the invasion of a fungus (described by Grindley et al. 1998), Although endoscopy was in- vasive, apparently no discernible effects on survival of the aba- lone, attributable to the procedure, were observed 8 months after screening (Nollens et al. 2002). ABALONE DISEASE CONCERNS IN BRITISH COLUMBIA Although some and possibly all of the diseases of abalone detected in other parts of the world have the potential of occurring in British Columbia, to date, only one infectious disease of concern has been detected. The protistan Lidnriiuhidoides ludiotidis was involved in the demise of an attempt to culture northern abalone. H. kamtschatkana. in British Columbia in the early 198()s (Bower 1987a. 1987b). This parasite is only known to be lethal for abalone smaller than 5 mm in shell length. Because of the relatively large size of this parasite (about 10 jxm in diameter) and the translucent nature of the tissues of small abalone {<3 mm shell length), in- fected abalone can easily be detected by examination w ith a com- pound light microscope. Detection of L. ludiotidis in small abalone begins when the culture containers are being cleaned. Moribund abalone that are either weakly attached to the substrate or have fallen to the bottom and are not attempting to right themselves should be sampled. The foot and head of infected abalone will have lost tissue integrity and appear swollen (look puffy). Squash the abalone between a glass slide and coverslip. and examine squashed tissues under a com- pound microscope. In comparison to normal/health abalone (Fig. la), the tissues of infected abalone will be filled with stationary spherical protists (-10 |xni in diameter, see Fig, lb) many of which may be undergoing binary fission (semispherical specimens with a clear central dividing line, see Fig Ic). If infection with L. haliotis is suspected, samples should be submitted to a competent authority for confirmation. Prior to submission, abalone should be preserved for histologic examination as described later. Also, immediate steps should be taken to mitigate potential spread of the disease. The spread of L ludiotidis within an abalone culture facility or between facilities can be mitigated by applying good husbandry techniques. Essentially, abalone and equipment should not be transferred between tanks, water exchange between tanks should be avoided and personnel must be careful to not facilitate cross contamination. Although L. ludiotidis is resistant to many disin- fectants, it can be destroyed by a 20 min exposure to 23 ing sodium hypochlorite (chlorine) per liter of sea water (Bower 1989). Treat- ments applied to infected abalone in the past (Bower 1989) have proven problematic. If this parasite should again appear in an / E 4 ? 4-\ - b' Figure 1, Unstained wet mount squashes of Juvenile abalone, Haliotis kamlsihalkaiui. about 2 mm in shell length, (a) Head region of a normal uninfected specimen showing the eye (El and shell (Si. Scale bar = KtO jim, (bl .Same magnilkation of a specimen heavily infected with numerous iMbyrinlhyUndes haliotidis (P) liberated from and embedded in tissues of the head region— the eye is swollen due to loss of tissue integrity caused by the parasite. Scale bar = 1(10 |im. (c) Magnification of infected foot muscle showing numerous L. haliotidis some of which are in the process of dividing by binary fission (I)). Scale bar = 25 jim. 808 Bower abalone culture facility, research will be required to identify effi- cacious methods of control. ASSESSMENT OF ABALONE HEALTH As for all other mollusks. infectious diseases of ahalone are becoming more evident with increased efforts towards the culture of various species of abalone around the world. Disease can often be circumvented in the culture environment by implementing knowledge gained from research on the cause of the disease. Also, when cultured abalone are to be used in rehabilitation efforts, specimens placed into the natural environment must be in good health for the endeavor to have a chance of success. Because few specific diagnostic tools are available for detecting diseases of abalone and assessing abalone health, standard procedures of his- tologic examination must be used. Although the microscopic ex- amination and interpretation of histologic preparations of abalone tissues requires extensive specific knowledge and experience, the preparation of the tissues for histology can be performed with minimal training and equipment. Following is a brief description of the procedures and materials required to prepare abalone for histologic examination. Abalone for histologic examination should first be examined fresh, all abnormalities noted, and shell length measured. For his- tology, appropriate tissue samples must be chemically preserved (Table 2). Regardless of the preservative used, it is critical that tissue samples are less than 1 cm thick, that there is at least 10 parts preservative to 1 part tissue and that the tissues are placed in the preservative as soon as possible after collection. After 24 to 48 h in the preservative, tissues should be transfen'ed to 70% ethanol for storage until further processing or shipping to a pathologist for examination. Abalone of small size and some organs in larger abalone should not be dissected because of damage caused to the tissue in the dissection process. As a general guideline, abalone less than 5 mm in shell length should be preserved intact. Abalone from about 5 mm to 3 cm in shell length should be removed from the shell prior to being preserved whole (ie. with no further dissection). Abalone greater than 3 cm in shell length must be removed from the shell and tissues dissected (Fig. 2). The region of the digestive gland, stomach, crop, and heart kidney complex (Fig. 2b.c) should not be dissected prior to fixation because of damage to these delicate tissues caused by the dissection process. The part of the abalone consisting mainly of the foot muscle can be discarded unless le- TABLE 2. Formulation of two preservatives used to fix abalone tissues for histological examination. Tissues must be fixed as soon as possible after the abalone is removed from the water. Prior to fixation. record all lesions observed on each specimen and confirm that there is at least 10 parts preservative to every 1 part of abalone tissue. -•~%'*isf>. Davidson's Solution (Shaw & Battle 19571 10'7r Formalin in Sea Water 400 niL glycerin, and 800 niL formaldehyde 1 200 mL 95?^ ethanol 1 200 mL sea water Just prior to use add I part glacial acetic acid to every 9 parts of the above mixiuie. 1 part fomialdehyde 9 parts sea water i ■9:0gr % iK \"^<^ epipodial ' tentacles -digestive gland stomat C Figure 1. Dorsal views of an abalone. (a) Orientation of features on outer surface of shell, (b) Body parts .A (head) and B (viscera) should be preserved for histologic examination and part C (foot I can be dis- carded. The lines indicate where the tissues should be cut to avoid unnecessary disruption to the delicate visceral organs, (c) Major or- gans underlying the shell, (dl Major organs underlying the respirato- ry, excretory, and reproductive organs. Images b, c and d were modi- fied from Bullough (1958). sions are detected or withering syndrome is suspected. Lesions or other tissue abnormalities should be described and location noted because they are usually not evident after the tissue has been chemically preserved. If the lesion is on the foot (part of the abalone usually not preserved for histologic examinations), a rep- resentative sample, which is no greater than a I cm cube, should be added to the preservative. If withering syndrome is suspected, a 3-5 mm cross section of the foot muscle should be preserved. Preserved tissues can be stored for prolonged periods (years). However, histologic examinations should be conduced as soon as possible to expedite the use of resulting interpretations. For histo- logic examination, the tissues must be processed and stained for microscopic examination as described and illustrated by Howard and Smith (1983). In brief, the fluids in the tissues must be re- moved and replaced with paraffin wax. The wax embedded tissues are then cut into about 6-p.m thin slices (sections) and the sections mounted on a glass slide and stained. The cells in the resulting stained histologic sections are then examined microscopically for abnoniialities and obser\ations correlated with the notes taken prior to sample preservation. Histologic examinations will usually reveal the presence of synibionts and parasites. Knowledge and experience on the iden- tification of these organisms is used to determine which can cause diseases of concern. Once an infectious agent has been identified, steps to prevent further spread within a culture facility or to the natural environment can be implemented. In addition to detecting pathogens, histologic examinations can also be useful in assessing Disease update and screening of abalone 809 the suitability of the culture environment. The morphology of tis- sue cells can indicate the suitability of the diet or an increased intensity of synibionts (either bacteria or protists) can signify un- suitable parameters in the habitat. Until other more specific and sensitive assays are available to detect disease agents and assess abalone health, histologic examination will ser\e as a valuable tool for optimizing abalone culture conditions and avoiding unneces- sary losses during rehabilitation efforts. LITERATURE CITED del C. Alvarez-Tinajero, M.. J. Caceres-Marti'nez & J. G. Gon/ale/- A\iles. 2001. Shell boring clams in the blue abalone Haliotis fidgens and the yellow abalone Haliotis cornigahi troin Baja California. Mexico. J. Shellfish Res. 20:889-893. del C. Alvarez-Tinajero, M.. J. Ceceres-Martinez & J. G. Gonzales- Aviles. 2002. Histopathological evaluation of the yellow abalone Hcili- otis corrugala and the blue abalone Htiliolis fliigeiis from Baja Cali- fornia. Mexico. J. Shellfish Res. 2l:82.s-8.^0. Andree. K. B., C. S. Friedman. J. D. Moore & R. P. Hedrick. 2000. A pol\ merase chain reaction assay for the detection of genomic DNA of a Rickeusiales-like prokaryote associated with withering syndrome in California abalone. J. Shellfish Res. 19:213-218. Antonio. D. B., K. B. Andree. J. D. Moore. C. S. Friedman & R. P. Hedrick. 2000. Detection of Rickettsiales-like prokaryotes by in situ hybridization in black abalone. Haliotis cracherodii. with withering syndrome. J. Im-ertehr. Pathol. 75:180-182. Bower. S. M. 1987a. Lahyiiitthiiloicles haliotiilis n.sp. (Protozoa: Laby- rinthomorpha), a pathogenic parasite of small juvenile abalone in a British Columbia mariculture facility. Can. J. Zool. 65:1996-2007. Bower. S. M. 1987b. Pathogenicity and host specificity of Lahyrintlui- loides haliotidis (Protozoa: Labyrinthomoipha). a parasite of juvenile abalone. Can. J. Zool. 65:2008-2012. Bower. S. M. 1989. Disinfectants and therapeutic agents for controlling Lahyrinthuloides haliotidis (Protozoa: Lab\'rlnthomorplKi). an abalone pathogen. Aqiiacultiire 78:207-215. Bower. S. M. 2000. Infectious diseases of abalone (Haliotis spp.) and risks associated vMth transplantation. Can. Spec. Piihl. Fish. Aijiiat. Sei. 130: 111-122. Bullough, W. S. 1958. Practical invertebrate anatomy. London: MacMillan and Co. Ltd.. 483 pp. Caceres-Martinez, J. & G. D. Tinoco-Orta. 2001. Symbionts of cultured red abalone. Haliotis nifescens from Baja California, Mexico. J. Sliell- fi.'ih Res. 20:875-881. Cacere.s-Marti'nez, J.. C. Alvarez Tinajero. Y. Guerrero Renteria & J. G. Gonzalez Aviles. 2000. Rickettsiales-like prokaryotes in cultured and natural populations of the red abalone Haliotis nifescens. blue abalone. Haliotis fiilgens. and the yellow abalone Haliotis corrugala from Baja California. Mexico. Abstract. J. Shellfish Res. 19:503. Day. R., C. Culver. A. Kuris. A. Belcher & D. Morse. 2000. The parasite Terehrasabella heteroncinuta (Polychaeta) manipulates shell synthesis in Haliotis nifescens. Abstract. / Shellfish Res. 19:507. Diggles. B. K.. J. Nichol. P. M. Hine. S. Wakefield. N. Cochennec- Laureau. R. D. Robens & C. .S. Friedman. 2002. Pathology of cultured paua Haliotis iris infected with a no\el haplosporidian parasite, with .some observations on the course of disease. Dis. .\qiiat. Org. 50:219- 231. Dixon. M. G.. T. Hecht & C. R. Brandt. 1991. Identification and treatment of a Clostridium and Vibrio infection in South African abalone. Hali- otis midae L. J. Fish Dis. 14:693-695. Elston, R. & G. S. Lockwood. 1983. Pathogens of vibriosis in cultured juvenile red abalone. Haliotis nifescens Swainson. J. Fish Dis. 6: 1 II- 128. Finley, C. A. & C. S. Friedman. 2000. Examination of the geographic distribution of a Rickettsia-VAe prokaryote in red abalone. Haliotis nifescens in northern California. Abstract. J. Shellfish Res. 1 9:5 1 2-5 1 3. Finley. C. A.. T. J. Mulligan & C. S. Friedman. 2001. Life history of an exotic sabellid polychaete. Terehrasabella heteroiincimita: fertilization strategy and influence of temperature on reproduction. / Shellfish Res. 20:88.^-888. Fitzhugh. K. & Q. W. Rouse. 1999. A remarkable new genus and species of fan worm (Polychaeta: Sabellidae: Sabellinae) associated with some marine gastropods. Inveriebr. Biol. 118:357-390. Friedman. C. S.. K. B. Andree, K. A. Beauchamp, J. D. Moore. T. T. Rohbins. J. D. Shields & R. P. Hedrick. 2000a. "Candidatus Xenohali- otis californiensis". a newly described pathogen of abalone. Haliotis spp.. along the West Coast of North America. //;/. J. Syst. Evol. Mi- crobiol. 50:847-855. Friedman. C. S.. T. Rohbins. J. L. Jacobsen & J, D. Shields. 2000b. The cellular immune response of black abalone. Haliotis cracherodii Leach, with and without v\ithering syndrome. .Abstract. J. Shellfish Res. 19: 514. Friedman, C. S.. W. Biggs. J. D. Shields & R. P. Hedrick. 2002. Trans- mission of withering syndrome in black abalone. Haliotis cracherodii Leach. J. Shellfish Res. 21:817-824. Friedman. C. S.. G. Trevelyan. T. T. Rohbins. E. P. Mulder & R. Fields. 2003. Development of an oral administration of ovytetracycline to control losses due to withering syndrome in cultured red abalone. Hali- otis nifescens. Aqiiacultiire. 224:1-23. Grindley. R. M.. J. A. Keogh & C. S. Friedman. 1998. Shell lesions in New Zealand Haliotis spp. (Mollusca. Gastropodal. ./. Shellfish Res. 17:805- 811. Handlinger. J.. D. Taylor & J. Carson. 2001. Flarohacteriiim-hke infection of abalone. Book of abstracts. European Association of Fish Patholo- gists. Tenth International Conference '"Diseases of fish and shellfish." Trinity College Dublin. Ireland. September 9-14. 2001. p. P209. Handlinger. J.. J. Carson. L. Donachie. L. Gabor & D. Taylor. 2002. Bacterial infection in Tasmanian farmed abalone: causes, pathology, farm factors, and control options. Handbook and abstracts, fifth sym- posium on diseases in Asian aquaculture. Queensland. Australia. No- vember 24-28. 2002. p. 139. Hayward. C, R. Lester, S. Barker. H. McCallum. A. Murrell & S. Klee- man. 2002. Transmission of Perkiiisus olseiii among w ild blacklip aba- lone in South Australia. Handbook and Abstracts. Fifth Symposium on Diseases in Asian .Aquaculture. Queensland. .Australia. November 24— 28. 2002. p. 139. Hine. P. M.. S. Wakefield. B. K. Diggles. V. L. Webb & E. W. Maas. 2002. Ultiastructure of a haplosporidian containing Rickettsiae, associated with mortalities among cultured paua Haliotis iris. Dis. Aquat. Org. 49:207-219. Howard. D. W. & C. S. Smith. 1983. Histological techniques for marine bivalve mollusks. NOAA Tech. Mem. NMFS-F/NEC 25. 97 pp. Kuris. A. M. & C. S. Culver. 1999. An introduced sabellid polychaete pe.st infesting cultured abalones and its potential spread to other California gastropods, hnertehr. Biol. ll8:39l-;03. Lester. R. J. G.. S. N. Kleeman, S. C. Barker & H. I. McCallum. 2001. Epidemiology of Perkinsiis olseni, pathogen of abalone. Book of Ab- stracts. European Association of Fish Pathologists. Tenth International Conference Diseases of Fish and Shellfish. Trinity College Dublin. Ireland. September 9-14. 2001. p. O-006. Lleonart. M.. J. Handlinger & M. Powell. 2003. Spionid mudworm Infes- tation of farmed abalone {Haliotis spp.). Aquaculture 221:85-96. Li. T.. M. Ding. J. Zhang. J. Xiang & R. Liu. 1998. Studies on the pu.stule disease of abalone {Haliotis discus hannai Ino) on the Dalian coast. J. Shellfish Res. 17:707-711. Li/arraga-Partida. M. L.. C. Anguiano-Beltran. R. Searcy-Bernal & E. Vazquez-Moreno. 1998. Bacterial water quality in abalone farms of Baja California. J. Shellfish Res. 17:689-692. Loubser. N. C. & N. Dormehl. 2000. The use of ultrasound in the treatment no Bower of sabellid infestations in South African abalone. Abstract. J. Shellfish Res. 19:524. Moore, J. D.. T. T. Robbins & C. S. Friedman. 2000a. The role of a Rickellsia-like prokaryole in withering syndroine in California red aba- lone, Haliotis nifescens. Abstract. / Shellfish Res. 19:525-526. Moore, J. D., T. T. Robbins & C. S. Friedman. 2000b. Withering syndrome in farmed red abalone Haliotis nifescens: thermal induction and asso- ciation with a gastrointestinal Rickettsiales-like prokaryote. J. Aqiiiit. Anim. HIrh. 12:26-34. Moore. J. D., G. N. Cherr & C. S. Friedman. 2001a. Detection of -Can- didants Xenohaliotis califomiensis' (Ricketlsiales-like prokaryote) in- clusions in tissue squashes of abalone {Halidlis spp.) gastrointestinal epitheUum using a nucleic acid fluorochrome. Dis. .Aqiiat. Org. 46: 147-152. Moore, J, D., T. T. Robbins, R. P. Hedrick & C. S. Friedman. 2001b. Transmission of the Rickettsiales-like prokaryote 'Candidatiis Xeno- haliotis califomiensis" and its role in withering syndrome of California abalone, Haliotis spp. / Shellfish Res. 20:867-874. Mouton, A. 2000. Health management and disease surveillance in abalone, Haliotis midae. in South Africa. Abstract. / Shellfish Res. 19:526. Nicolas, J, L., O. Basuyaux, J. Mazurie & A. Thebault. 2002. Vibrio carchariae, a pathogen of the abalone Haliotis tuherculata. Dis. Aqiial. Org 50:35-43. Nishimori, E., O. Hasegawa, T. Numata & H. Wakabayashi. 1998, Vibrio carchariae causes mass mortalities in Japanese abalone, Sulcuhts di- versicolor supratexia. Fish Pathol. 33:495-502. Nollens, H. H., J. C. Schofield, J. A. Keogh & P. K. Probert. 2002. Evalu- ation of radiography, ultrasonography and endoscopy for detection of shell lesions in live abalone Haliotis iris (MoUusca: Gastropoda). Dis. Aquat. Org. 50:145-152. Oakes, F. R. & R. C. Fields. 1996. Infestation of Haliotis nifescens .shells by a sabellid polychaete. Aquaciihure 140:139-143. Ruck, K. R. & P. A. Cook, 1998. Sabellid infestations in the shells of South .African molluscs: implications for abalone mariculture. J. Shellfish Res. 17:693-699, Shaw, B. L. & H. I. Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster Crassosirea virginica (Gmelin). Can. J. Zool. 35:325-347. Sindemiann, C. J. 1988. Disease problems created by introduced species. In: C. J. Sindermann & D. V. Lightner, editors. Disease diagnosis and control in North American marine aquaculture. Amsterdam: Elsevier, pp. 394-398. Tai-wu, L., J. Xiang & R. Liu. 2000. Studies on phage control of pustule disease in abalone Haliotis discus hannai. Abstract. J. Shellfish Res. 19:535. Jimnml of Slwllfi.sh Rcicunh. Vol. 22, No. 3. Sll-SIH. 2003. FECUNDITY AND SEASONAL REPRODUCTION OF NORTHERN ABALONE, HALIOTIS KAMTSCHATKANA, IN BARKLEY SOUND, CANADA A. CAMPBELL, J. LESSARD, AND G. S. JAMIESON Fisheries and Oceans Canada, Science Branch, Pacific Bialoi^ical Slatimi. Nanainio. B.C. V9T6N7, Canada ABSTRACT Fecundity, si/e at maturity and seasonal reproduction of noilliem or "pinto" abalone, Halious kamischatkana. from exposed "surf"' areas and more sheltered, productive abalone habitat were investigated in Barkley Sound. Examination of histologic sections of gonads indicated that si^e at maturity occurred at a smaller size for the stunted 'surf ' abalone than for abalone from more sheltered areas. Gonad index and stages showed that gonads were mainly ripe and that most abalone spawned during April to July. Although there were smaller abalone with ripe eggs from the "surf area than those from the sheltered area, abalone females of comparable si/e from both areas had similar egg numbers. However, there were larger females with considerably higher fecundity from the sheltered areas than from the "surf' areas. Implications of transplanting "surf"' abalone to productive habitats to increase growth and fecundity rates are discussed in the context of population rebuilding attempts for H. kamtschaikana. which is listed by the Committee on the Status of Endangered Wildlife in Canada as a "threatened" species in Canada. KEY WORDS: abalone, Huliotis kainisciuitkaihi. fecundity, reproduction, size at maturity INTRODUCTION The nofthern or "pinto" abalone. Huliotis I 0.05) in mean egg density between different locations on the ovaries of five northern abalone. Other studies (eg. Giorgi & DeMartini 1977. Wells & Keesing 1989) found eggs homogeneously distributed throughout the ova- ries in other abalone species. Fecundity, or total number of eggs per female, was estimated as the product of mean egg density and total ovary weight. The relation between fecundity (F) and shell length (L) was expressed with the natural log transformed linear regression equa- tion: loe,. F = log,. A -I- B loa,, L (2) where the coefficients A and B were estimated using the least squares method. Analysis of covariance (ANCOVA) was used to test for the homogeneity of slopes and elevation coefficients of the log transformed data regressions (Zar 1984) of fecundity of north- em abalone collected from different areas. All ANCOVA com- parisons for fecundity and SL, in similar size groups, between areas Willis Island. Island 39, and south east QCI (Campbell et al. 1992) indicated there were no differences [P < 0.05) between slopes or elevations, so size at fecundity data were combined into one equation. The relation between fecundity (F) and total abalone drained wet weight (W) was expressed with the linear equation NORTHHRN ABALONB REPRODUCTION 813 F = A + B W. where the coefficients A and B were estimated using the least squares method. Potential total egg production per m" (E) was estimated as: E = 2f,P,S,(N,/N) X X o n — ' — I — ' — I — ' — \ — ' — T" 0 20 40 60 80 SHELL LENGTH (MM) 100 Figure 2. Size specific mean gonad index (percent) of H. kamtschat- kana from Willis Island (()) and Island 39 (Xl during June 1991. 814 Campbell et al. than those from Willis Island (.'50 mm SL) (Fig. 3. Table 2). Ex- amination of histologic sections indicated that the smallest mature abaione were 42.6 and 49.4 mm SL and the largest immature individuals were 50.8 and 57.0 mm SL at Island 39 and Willis Island, respectively, during June 1991 to 1992. Maturity occurred for most abaione at sizes greater than 65 mm SL. Although the color of whole gonads could be differentiated to determine sex, histologic examination indicated that not all gonads were mature in the 32 to 57 mm SL range. Fecundity For females with ripe gonads, both mean oocyte diameter (231 |jLm ± 2 SE, (1 = 6) and mean density or number of eggs per g (178.386 ± 3,360 SE, /; = 33) did not significantly differ (two way 7 9 14 10 5 10 14 7 08 1 LLI - a: Z) 0.8- 1- < ^ 0.6- z o - 1- 0.4- o a. O a: 0.2- a. - 0.0- 0 — 1 — ' — I — ' \ ' I ' I 20 40 60 80 100 SHELL LENGTH (MM) 1.0n LU § 0.8 0.6 S 0 O a. O a: Q. 4- 0.2- 0.0 9 18 10 2 ~i — ' — \ — ' — I — ' — r~ 20 40 60 80 SHELL LENGTH (MM) 100 Figure 3. Proportion mature at mid point of each 5-mni shell length class of//. kanUschatkaim (sexes combined I from (.\l Willis Island and (B) Island .W. Numhers next to s>mbols are sample sizes. See Tahle 2 and text for information on predicted equations for size at maturitj curves TABLE 2. Equation coefficients for size at maturity equation from //. kamtschatkana sampled in Barkley Sound. See text for equation details and Fig. 3 for data. Values in brackets are approximate 95% confidence intervals. R" = coefficient of determination. Equation Coefficients Site A B R- Willis Island Island -^9 :?. SO? (±6.475) 12.S63(±4.973) 0.438 (±0.129) 0.204 (±0.1 ID 0.987 0.943 ANOVA. P > 0.05) between abaione of different sizes or between those from Willis Island and Island 39. ANCOVA comparisons indicated there was no difference (P > 0.05) in slopes or intercepts in the linear relation of log^, transformed gonad weight (g) and SL between Willis Island and Island 39. Therefore, data from both locations were combined into one equation: log^. (gonad weight, g) = -13.4807 + 3.48 log,. (SL) (R- = 0.82, P<0.01. n = 33) estimated by the least squares method. Given that ( 1 ) egg densities were independent of abaione size and eggs were distributed ho- mogeneousl) throughout the ovai^. and (2) there was a relation- ship between the preserved ovary wet weight and SL. the fecundity of H. kamtschatkana could be related to SL or total abaione weight (Table 3). Although there was considerable variation in fecundity between indi\'iduals of similar size, the increase in fecundity in relation to increases in SL and weight of H. kamtschatkana was highly correlated (Fig. 4, .see Table 3). The smallest female (40 mm SL) from Island 39 had 90,594 eggs, the largest female (125 mm SL) from Willis Island had 3.0 million eggs, and the largest female (144 mm SL) from southeast QCI had 11.3 million eggs (Campbell et al. 1992). Potential Population Egg Production The cumulated proportion of sizes was higher for small size classes of H. kamtschatkana from Island 39 than from Hankin Island (Fig. 5). This was reflected in a higher cumulated potential egg production from the 50 to 80 mm SL class from Island 39 than from Hankin Island (Fig. 6). TABLE 3. Equation coefficients for the fecundity (F, number of eggs per female) and shell length (I. in mm) log transformed equation log.F = log,..\ Blog^L for //. kamtschatkana. and fecundit> and total weight (\\ in g) linear equation F = A BW. Areas: I = Willis Island and Island .'9 combined (this study), 2 = Willis Island and Island 39 (this study) combined with south east Queen Charlotte Islands (Campbell et al. 1992). Independent \ ariable -Areas Equation Coefficients B R- n Shell length Total weight -1.5203 3.5137 -2.2152 3.6789 108.463 14588.5 -411.590 25179.5 0.843 33 0.915 .56 0.803 33 0.910 .56 R- = coefficient of detennination, n = sample size. Northern Abalone Reproduction 815 UJ q: LU Q. CO o CD LU Ll_ O (/) 10- - +-f 10- - 5- / - w o - ^^% ^^ 0- 1 , , 1 r-rf^^^^ ' ' 1 III. 0 50 100 SHELL LENGTH (MM) 150 Figure 4. Kccundit> at shell leiislh of feniule //. kumlsclialkaiia from Willis Island (Ol and Island 39 (X) durinK .June 1991 (this study), and from Queen Charlotte Islands l + l during June I99(t (after Campbell et al. 1992). See Table 3 for regression equation coefficients. Despite meun ahaloiie density estimates being higher at Island 39 (0.750/nr) than at Hankin Island (0.4()4/m-) dunng June to July 2002, the estimated total potential egg production per unit area (millions of eggs per m~) was higher at Hankin Island (0.26) than at Island .^9 (0.20) (Fig. 6) due to higher fecundity of large abalone (2100 mm SL) present at Hankin Island. In contrast, if we as- sumed a similar hypothetical abalone density of l.O/iir for both areas, estimated total potential egg production (millions of eggs I.U-| z o 1- oa- q: o - CL O OR- ir Q. _ Q 111 04- H 5 - -) ^ 0.2- 3 O " 0.0- 0 50 100 SHELL LENGTH (MM) 150 Figure 5. Cumulated proportion size frequency by 5-mm .SL classes of H. kamtsvhalkanu from Hankin Island )()) and Island .W (X) during June 2002. Lines fitted to data by a spline smoother with cubic equa- tions (SYSTAT 2000). HI Q^ 1- Lli 0 3^ ^ LU DC < 3 O CO 0.2- a: LU Q. - CO CD CD 0 1- LU Ll_ o . CO z O 00- _j 50 100 SHELL LENGTH (MM) 150 Figure 6. Cumulated total potential egg production (millions per nr) by 5-mm SL classes of H. kamlschalkaiia from Hankin Island (()) and Island 39 (X) based on mean density estimate of 0.404 and 0.750 aba- lone/m", respectively, during June to July 2002. See text for method to calculate potential total egg production. Lines fitted to data by a spline smoother with cubic equations (SYSTAT 2000). per m") would be niore than twice as much for Hankin Island {0.65) than for Island .^9 (0.27). DISCUSSION Shell lengths of H. kmntschatkana sampled from Island 39 were all less than 100 mm, typical of abalone from a "surf area with less than optimal habitat conditions (Sloan & Breen 1988, Emniett & Jamieson 1989). In contrast, there was a larger range of sizes of abalone from the moderately exposed areas of Willis and Hankin Islands. The largest abalone encountered in this study was a 12."^ iT)m SL female from Willis Island. Low numbers of large abalone (5100 mm SL) found in this study and in other recent surveys (eg. Lucas et al. 2002) are in sharp contrast to the many (51%) large H. kaintschatkana (maximum 146 mm SL, /; = 1305) sampled by Quayle (1971 ) in Barkley Sound during 1963 to 1964. Seasonal reproduction of H. kunuschatkana occurred mainly from April to June at Willis Island and Island 39 in our study, similar to that found by Quayle ( 1971 ) in other islands in Barkley Sound. Warmer conditions in January to June 1992 than in 1991 may have caused abalone to spawn earlier in 1992 than in 1991. Our study also confirms that there may be a few abalone that are ripe throughout the year that may be able to spawn (see review by Sloan & Breen 1988). Intra- and inter-specific variability in ga- metogenesis and annual spawning periods is cornmon for many abalone species and spawning may partly depend on local condi- tions (eg, temperature, storms, food quality, and abundance) (New- iT)an 1967. Webber & Giese 1969. Shepherd & Laws 1974. Paul et al. 1977. Hayashi 1980, Sloan & Breen 1988, Wells & Keesing 1989, Stekoll & Shirley 1993. Hooker & Creese 1995, Sasaki & Shepherd 1995, Wilson & Schiel 1995). 816 Campbell et al. This study recorded the lowest 50% size at maturity (44 mm SL from Island 39) to date for wild H. kaintschatkana. The 50% maturity at 50 mm SL at Willis Island was similar to that found by Quay le { 1 97 1 ) for abalone generally in Barkley Sound. Maturity of wild H. kamtschatkana further north of Barkley Sound (eg, QCI and Alaska) was found to vary between 50 to 64 mm SL (Larson & Blankenbeckler 1980 referenced in Sloan & Breen 1988. Paul & Paul 1981, Campbell et al. 1992). We confirm Quayle's (1971) observation that although sexes could be differentiated by color of gonads at small sizes (e.g., 32 mm SL) the start of sexual maturity was observed at larger sizes (smallest mature individual we ob- served was 42.6 mm SL). Size at sexual maturity for an abalone species can vary between locations (Shepherd & Laws 1974) de- pending on various factors such as food quality and availability and different temperature regimes (Kikuchi & Uki 1974a. Kikuchi & Uki 1974c, Kikuchi & Uki 1975, Paul et al. 1977, Paul & Paul 198 1 ). Fecundity estimates were similar for H. kamtschatkana of equivalent shell lengths from Willis Island, Island 39 and southeast QCI, and the number of eggs increased exponentially with in- creases in shell size. Fecundity estimates of//, kaml.schalkana (this study. Campbell et al. 1992) are within the range reported for other abalone species. The largest number of eggs reported for a H. kamtschatkana was 11.56 million eggs for a 139-mm SL female (Campbell et al. 19921. High fecundity has been reported for other abalone species — 25.4 million eggs for a 175-mm SL //. midae (Newman 1967), 12.6 million eggs for a 190.5-mm SL H. rufe- scens (Giorgi & De Martini 1977). Methods to estimate fecundity vary from estimating the total number of eggs in ovaries by weight (Newman 1967, Poore 1973. Giorgi & DeMartini 1977, Hayashi 1980, Wells & Keesing 1989, this study) and by volume (Sains- bury 1982, Prince et al. 1987, McShane et al. 1988) to counting the number of eggs spawned (Kikuchi & Uki 1974a. Kikuchi & Uki 1974b, Kikuchi & Uki 1974c. Kikuchi & Uki 1975, Hayashi 1980, Olson 1980. Caldwell 1981, Tutschulte & Council 1981, Ault 1985, Clavier 1992). Although fecundity was estimated as the total eggs present in an ovary prior to spawning in this study, probably not all eggs may be spawned within a spawning event (Poore 1973, Giorgi & DeMartini 1977. Ault 1985). Caldwell ( 1981 ) found that H. kamtschatkana females at 101 and 135 mm SL spawned an estimated 0.03 and 2.3 million eggs, respectively, in 1.5 to 2.5 h in the laboratory, which were lower than the mean total eggs (2.6 and 7.5 million eggs) estimated in ovaries of individuals of the same size from our study. The relationship between fecundity and SL for various abalone species has been described either as curvilinear (Newman 1967, Poore 1973, Giorgi & DeMartini 1977, Hayashi 1980. Wells & Keesing 1989. Wilson & Schiel 1995, Litaay & De Silva 2001. this study) or as linear (Poore 1973. Sainsbury 1982. Prince et. al. 1987, McShane et al. 1988). The relationship between fecundity and weight of the whole abalone has been considered as curvilinear (Ault 1985. Litaay & De Silva 2001) or as linear (New- man 1967. Teener etal. 1989, Shepherd et al. 1991, Shepherd et al. 1995). Northern abalone from exposed "surt" areas were capable of reproducing and, at the equivalent size, potentially have similar fecundity per unit area as abalone from more sheltered areas. Size at maturity and fecundity, size composition and density are im- portant in determining the total potential contribution of an aba- lone population in an area. Given similar densities, larger abalone in moderately sheltered areas have a potentially higher reproduc- tive contribution than smaller, slower growing abalone in high ■'surf' exposed areas. The importance of having sufficient numbers of large abalone for reproductive output has been emphasized in the fishery context (Breen 1986, Tegner et al. 1989, Shepherd & Baker 1998) and for evaluating marine protected areas as conser- vation tools for abalone (Edgar & Barrett 1999. Wallace 1999, Rogers-Bennett et al. 2002). Fertilization and recruitment success of various abalone species may also be density dependent (Clavier 1992. McShane 1995a. McShane 1995b. Babcock & Keesing 1999). Implications of transplanting abalone from "poor" to "good" habitats to increase survival, growth, and reproductive potential, as a rebuilding technique for H. kamtschatkana in BC. are compli- cated. Emniett and Jamieson (1988) concluded that transplanting large sizes of "surf' northern abalone from exposed sites to more productive areas was biologically and economically feasible if survival of the transplanted abalone was reasonably high. They did not evaluate methods to potentially enhance reproductive output or recruitment in the transplant areas. Tegner (1992. 1993. 2000) reported on a transplant of reproductively mature green abalone, H. fiilgens. in California with subsequent strong evidence of suc- cessful local recruitment until the brood stock were poached. The long-term success of a brood stock transplant is dependent on adult survival and density for fertilization success, and local hydrody- namics for larval settlement (Babcock & Keesing 1999). Future attempts to rehabilitate H. kamtschatkana in BC by transplanting "surf' abalone will require pilot experiments to test transplant methods for their feasibility to determine measurable success in increased population survival, growth, reproduction, and recruit- ment. The choice of recipient "good" sites would include criteria such as identifying locations with complex substrates, moderate to low exposure, and availability of suitable algal food for optimal abalone growth and survival. Abundance and distribution of H. kamtschatkana in exposed and moderately sheltered areas are not well known and need to be estimated prior to large scale rebuilding efforts in local areas of coastal BC. ACKNOWLEDGMENTS The authors thank J. Bagshaw. D. Brouwer. W. Carolsfeld. B. Clapp. D. Cooper. G. Dovey, S. Gazetas. W. Harling. S. Head, P. Ladynian, J. Lash, L. Lee, B. Lunn, P. Menning, F. Merilees. A. Phillips, J. Rogers, J. Whang, T. White, for technical assistance. Parks Canada personnel for logistical support. W. Hajas for sta- tistical advice, and F. Wells and G. Gillespie for providing helpful comments to improve earlier drafts of this paper. Partial funding was provided by the Species at Risk Interdepartmental Recovery Fund. LITERATURE CITED Ault. J. S. mS.'i. Some quantitative aspects of reproduction and growth of the red abalone. Haliotis nifescens Swainson. J. World Maricui Soc. 16:398-425. Babcock. R. & J. Keesing. 1999. Fertili/alion biology of the abalone Hali- otis Uievif>ata: laboratory and field studies. Can. J. Fish. Aqiuit. Sci. 56:1668-1678. Breen. P. A. 1986. Management of the British Columbia fishery for north- ern abalone iHaliolis kamlschcitkwm). Can. Spec. Puhl. Fish. Aquat. Sci. 92:300-315. Breen. P. A. & B. E. Adkins. 1980. Spawning in a British Columbia population of nortlieni abalone. Haliotis kaini.scharkana. The Veliiier 23:177-179. Northern Abalone Reproduction 817 Caldwell. M. E. 1981. Spawning, early development and hybridization of Huliotis kamtsclwlkaiui Jonas. MSc. thesis. Uni\ersity Washington. vi+55 pp. Campbell. A. 2000. Review of nonhern abalone. Haliotis kumisclhilkana. stock status in British Columbia. Ciin. Spec. Piihl. Fish. Ac/iiat. Sci. 130:41-50. Campbell, A.. 1. Manley & W. Carolsfeld. 1992. Size at maturity and fecundity of the abalone, Haliotis kamtschatkana, in northern British Columbia. Can Maims. Rep. Fish. Aquat. Sci. 2169:47-65. Clavier, J. 1992. Fecundity and optimal sperm density for fertilization in the ornier {Haliotis tuherculata L.). In: S. A. Shepherd. M. J. Tegner& S. A. Guzman del Proo. editors. Abalone of the world: biology, fish- eries and culture. Oxford. U. K: Biackwell Scientific Publications Ltd. pp. 86-92. Cripps. K. & A. Campbell. 1998. Survey of abalone populations at Dallain Point and Higgins Pass, central coast of British Columbia. 1995-96. Can. Manns. Rep. Fish. Aquat. Sci. 2445 31 pp. Donovan, D. A. & T. H. Carefoot. 1997. Locomotion in the abalone. Haliotis kamtschatkana: pedal moiphology and cost of transport. J. E.xp. Biol. 200:1145-1153. Donovan, D. A. & T. H. Carefoot. 1998. Effect of activity on energy allocation in the northern abalone. Haliotis kamtschatkana Jonas. J. Shellfish Res. 17:729-736. Edgar, G. J. & N. S. Barrett. 1999. Effects of the declaration of marine reserves on Tasmanian reef fishes, invertebrates and plants. J. E.xp. Mar. Biol. Ecol. 242:107-144. Emmett. B.. & G. S. Jamieson. 1988. An experimental transplant of north- ern abalone. Haliotis kamtschatkana. in Barkley Sound. British Co- lumbia. Fish. Bull. (US) 87:95-104. Giorgi. A. E. & J. D. DeManini. 1977. A study of the reproducti\e biology of the red abalone, Haliotis mfescens Swainson. near Mendocino. Cali- fornia. Calif. Fish Game 63:80-94. Hayashi. I, 1980. The reproductive biology of the Omer, Haliotis tuher- culata. J. Mar. Biol. Ass. U. K. 60:415^30. Hooker. S. H. & R. G. Creese. 1995. Reproduction of paua. Haliotis iris Gmelin. 1791 (Mollusca: Gastropoda), in north-eastern New Zealand. Mar. Freshwater Res. 46:617-622. Jamieson, G. S. 2001. Review of status of nonhern abalone. Haliotis ka- mtschatkana, in Canada. Can Field Nat. 115:555-563. Kikuchi. S. & N. Uki. 1974a. Technical study on artificial spawning of abalone. genus Haliotis I. Relation between water temperature and advancing sexual maturity of Haliotis discus hannai Ino. Bull. Tohokii Reg. Fi.sh. Res. Lah. 33:69-78. Kikuchi. S. & N. Uki. 1974b. Technical study on artificial spawning of abalone, genus Haliotis 111. Reasonable sperm density for fertilization. Bull. Tohoku Reg. Fish. Res. Lab. 34:67-71. Kikuchi. S. & N. Uki. 1974c. Technical study on artificial spawning of abalone, genus Haliotis V. Relation between water temperature and advancing sexual maturity of Haliotis discus Reeve. Bull. Tohoku Reg. fwft. Res. Lab. 34:77-85. Kikuchi. S. & N. Uki. 1975. Technical study on artificial spawning of abalone. genus Haliotis VI. On sexual maturity of Haliotis gigantea Gmelin under artificial conditions. Bull. Tohoku Reg. Fish. Res. Lab. 35:85-90. Lessard, J.. A. Campbell & W. Hajas. 2002. Survey protocol for the re- moval of allowable numbers of northern abalone. Haliotis kamtschat- kana, for use as broodstock in aquaculture in British Columbia. Cana- dian Science Advisory Secretariat Res. Doc. 2002/126. 41 pp. (http:// www.dfo-mpo.gc.ea/csas/). Litaay. M. & S. S. De Silva. 2001. Reproductive performance indices based on physical characteristics of female blacklip abalone Haliotis rubra L. / Shellfish Res. 20:673-677. Lucas. B. G.. A. Campbell. D. Brouwer. S. Servant & N. Webb. 2002. Survey of northern abalone. Haliotis kamtschatkana, populations in southea.st Barkley Sound, British Columbia. July 2000. Revised. Can Manus. Rep. Fish. Aquat. Sci. 2623. 1 1 pp. McShane. P. E. 1995a. Recruitment variation in abalone: its importance to fisheries management. Mar. Freshwater Res. 46:555-570. McShane. P. E. 1995b. Estimating the abundance of abalone: the impor- tance of patch size. Mar. Freshwater Res. 46:657-662. McShane. P. E.. K. P. Black & M. G. Smith. 1988. Recruitment processes in Haliotis rubra (Mollusca: Gastropoda) and regional hydrodynamics in southeastern Australia imply localized dispersal of larvae. J. Exp. Mar. Biol. Ecol. 124:175-203. Mottet. M. G. 1978. A review of the fishery biology of abalones. Wash. State Dep. Fish. Tech. Rep. 37:iv -i- SI pp. Nash. W. 1992. Determination of appropriate size limits by egg-per-recruit analysis for the blacklip abalone {Haliotis rubra) in Tasmania. In: S. A. Shepherd. M. J. Tegner. and S. S. Guzman del Proo. editors. .Abalone of the worid: biology, fisheries and culture. O.xford. U. K.: Biackwell Scientific Publications, pp. 318-338. Newman. G. G. 1967. Reproduction of the South Africa abalone Haliotis midae. S. Africa Div. Sea. Fish. Invest. Rep. 64:24pp. Olson. S. 1980. Shellfish enhancement project. Part II: Invertebrate aqua- culture. NOAA. NMFS. Interim report for Project 1-144-R. Washing- ton State Rep. Fish. 32 pp. Paul. A. J. & J. M. Paul. 1981. Temperature and growth of maturing Haliotis kamtschatkana Jonas. The Veliger 23:321-324. Paul. A. J., J. M. Paul, D. W, Hood & R, A. Neve. 1977. Observations on food preferences, daily ration requirements and growth of Haliotis kamtschatkana Jonas in captivity. The Veliger 19:303-309. Poore, G. C. B. 1973. Ecology of New Zealand abalones, Haliotis species (Mollusca: Gastropoda) 4. Reproduction. N. Z. J. Mar. Freshwater Res 7:67-84. Prince. J. D.. T. L. Sellers. W. B. Ford & S. R. Talbot. 1987. E.xperimental evidence for limited dispersal of haliotid larvae (genus Haliotis; Mol- lusca: Gastropoda). ,/. E.xp. Mar. Biol. Ecol. 106:243-263. Quayle, D. B. 1971. Growth, morphometry and breeding in the British Columbia abalone, (Haliotis kamtschatkana Jonas). Fish. Res. Board Can. Tech. Rep. 279. 84 pp. Rogers-Bennett. L.. P. L. Haaker. K. A. Karpov & D. J. Kushner. 2002. Using spatially explicit data to evaluate marine protected areas for abalone in southern California. Conservation Biol 16:1308-1317. Sainsbury. K. J. 1982. Population dynamics and fishery management of the paua, Haliotis iris. I. Population structure, growth, reproduction, and mortality. N. Z. J. Mar. Freshwater Res. 16:147-161. Sasaki. R. & S. A. Shepherd. 1995. Larval dispersal and recruitment of Haliotis discus hannai and Tegula spp. on Miyagi Coasts. Japan. Mar. Freshwater Res. 46:519-529. Shaw. B. L. & H. 1. Battle. 1957. The gross and microscopic anatomy of the digestive tract of the oyster Crassostrea virginica (Gmelin). Can. J. Zool. 35:325-347. Shepherd. S. A. 1986. Movement of the southern .Australian abalone Hali- otis laevigata in relation to crevice abundance. Ausl. J. Ecol. 1 1:295- 302. Shepherd. S. A. 1988. Studies of southern Australian abalone {Haliotis}. VIll. Growth of juvenile H. laevigata. .Aust. J. Mar. Freshwater Res. 39:177-183. Shepherd, S. A. & }. L. Baker. 1998. Biological reference points in an abalone {Haliotis laevigata) fishery. Can. Spec. Pubi Fish. .Aquat. Sci. 125:235-245. Shepherd, S. A. & H. M. Laws. 1974. Studies on southern Australia aba- lone (genus Haliotis) II. Reproduction of five species. Ausl. J. Mar. Freshwater Res. 25:49-62. Shepherd, S, A,. S, A, Guzman del Proo, J. Turrubatiates. J. Belmar. J. L. Baker & P. R. Sluczanowski. 1991. Growth, size at sexual maturity, and egg-per-recruit analysis of the abalone Haliotis fulgens in Baja California. The Veliger 34:324-330. Shepherd. S. A.. M. J. Tegner & S. A. Guzman del Proo. editors. 1992. Abalone of the world: biology, fisheries and culture. Oxford. U. K: Biackwell Sci. Publications. Shepherd. S. A.. J. L. Baker & D. W. Johnson. 1995. Yield-per-recruit and 818 Campbell et al. ega-per-recruit analyses of the Omani abalone. Hciliotis marine. Mar. Fre.shwater Res. 46:663-668. Sloan, N. A. & P. A. Breen. 1988. Northern abalone. Halwtis kaiimchai- kana. in British Columbia: fisheries and synopsis of life history infor- mation. Can Spec. Piibl. Fish. Aqiiat. Sci. 10.3. 46 pp. StekoU. M. S. & T. C. Shirley. 1993. In siiii spawning behavior of an Alaskan population of pinto abalone. Haliolis kaiinschatkaiia Jonas, 1845. The Veliger 36:95-97. SYSTAT. 2000. SPSS Inc.. Chicago, IL. USA. Tegner, M. J. 1992. Brood stock transplants as an approach to abalone stock enhancement. In: S. A. Shepherd. M. J. Tegner & S. S. Guzman del Priio, editors. Abalone of the world: biology, fisheries and culture. Oxford. UK: Blackwell Science Publications, pp. 461—173. Tegner, M, J. 1993. Southern California abalones: can stocks be rebuilt using marine harvest refugia? Can. J. Fish. Aqiiat. Sci. 50:2010-2018. Tegner. M. J. 2000. Abalone (Haliotis spp.) enhancement in California: what we've learned and where we go from here. Can. Spec. Piibl. Fisli. Aquat. Sci. 130:61-71. Tegner, M. J., P. A. Breen & C. E. Lennen. 1989. Population biology of red abalone. Haliotis rufescens. in southern California and management of the red and pink. H. cornigata. abalone fisheries. Fisli. Bull. 87: 3 1 -3-339. Tutschulte, T. & J. H. Connell. 1981 . Reproductive biology of three species of abalone [Haliotis) in southern California. The Veliger 23:195-206. Webber, H. H. & A. C. Giese. 1969. Reproductive cycle and gametogen- esis in the black abalone Haliotis cracherodii (Gastropoda: Prosbran- chiata). Marine Biology 4:152-159. Wallace. S. S. 1999. Evaluating the effects of three forms of marine reserve on northern abalone populations in British Columbia. Canada. Conser- vation Biol. 13:882-887. Wells, F. E. & J. K. Keesing. 1989. Reproduction and feeding in the abalone Haliotis roei Gray. Aust. J. Mar. Freshwater Res. 40: 1 87-197. Wells. P. E. & P. Mulvay. 1995. Good and bad fishing areas for Haliotis laevigata: a comparison of population parameters. Mar. Freshwater Res. 46:591-598. Wilson. N. H. F. & D. R. Schiel. 1995. Reproduction in two species of abalone (Haliotis iris and H. aiistralis) in southern New Zealand. Mar. Freshwater Res. 46:629-637. Worthington. D. G. & N. L. Andrew. 1998. Small-scale variation in de- mography and its implications for an alternative size limit in the fishery for blacklip abalone (Haliotis rubra) in New South Wales. Australia. Can. Spec. Piibl. Fish. Aquat. Sci. 125:341-348. Young, J. S. & J. D. DeMartini. 1970. The reproductive cycle, gonadal histology, and gametogenesis of the red abalone. Haliotis rufescens (Swainson). Calif. Fish and Game 56:298-309. Zar. J. H. 1984. Biostatistical analysis. New Jersey: Prentice Hall Inc. 718 PP- Joiirihil of Slicllfiih Research. Vol. 22, No. 3. 819-823. 2(103. ESTIMATING JUVENILE NORTHERN AB ALONE {HALIOTIS KAMTSCHATKANA) ABUNDANCE USING ARTIFICIAL HABITATS BART DEFREITAS HaUla Fisheries Program. P.O. Box 87. Ma.sset, B.C. VOT I MO Canada ABSTRACT This, .study asscsse;. the use of anificial concrete block habitats that provide standardized sample areas for measuring the abundance of northern abalone (Haliotis kamtschalkana Jonas) in comparison to 10 randomly selected l-m" quadrant samples where all movable rocks were examined for cryptic abalone. A total of 278 abalone were measured within artificial structures and juvenile abalone (£50 mm shell length. SL) were the most abundant size class. Juvenile abalone used artificial structures at greater mean densities (abalone/nr) than nearby natural habitat (1,27 ± 0,25 SE versus 0,07 ± 0,09 SE) and emergent abalone (>50 mm SL) used artiUcial habitats at similar densities as they did in nearby natural habitats (0,38 ± 0,09 SE versus 0,44 ± 0,10 SE). Juvenile abalone abundance was significantly different between sites but not within sites, suggesting artitlcial structures showed promise in their ability to detect area specific differences in recniitment and to easily measure juvenile abalone abundance, KEY WORDS: ju\enile, abalone. cryptic, artificial habitat, recruitment. Hulioln kaiiilschalkaiui INTRODUCTION Northern abalone [Hidiotis kannsclnilkiinii) fisheries in British Columbia (BC) remain closed to commercial, recreational, and First Nations groups since 199(3 due to conservation concerns (Campbell 2000), Dive surveys conducted by Fisheries & Oceans Canada (DFO) at inde.x sites in BC estimated that northern abalone abundance had declined by more than 75% during 1978 to 1984 and continue to remain low (Breen & Adkins 1979 1981. Winther et al, 1995. Campbell et al, 2000). In April 1999. the Committee on the Status of Endangered Wildlife in Canada (COSEWIC) listed northern abalone as "threatened", meaning Hkely to become en- dangered if limiting factors are not reversed. The most significant factors inhibiting northern abalone recov- ery are illegal harvests and poor recruitment (Campbell 2000), Recruitment, defined as the number of juvenile abalone growing and surviving to the adult population each year, may be insuffi- cient as a result of critically low adult densities (Shepherd & Brown 1993, Shepherd & Partington 1995) that reduce reproduc- tive success due to low fertilization of gametes (Alice et al, 1949), Other processes that may reduce abalone recruitment include variation in timing and intensity of gamete production, larval pre- dation, and post-larval mortality (McShane 1992. 1995), Recruit- ment processes for northern abalone are not well understood (Breen 1986. Sloan & Breen 1988), Increasing the abundance of existing wild northern abalone populations in BC is the long-term goal of the northern abalone national recovery strategy (Toole et al. 2002). One component of the strategy is to conduct abalone research and rebuilding experi- ments that inay lead to increased breeding success, recruitment, and population densities. To evaluate the success of various re- building experiments, it will be necessary to measure changes in abalone recruitment by quantifying the abundance of juveniles. Artificial collectors have been successful at measuring the in- tensity of abalone larval settlement (Keesing et al, 1995. Nash et al, 1995) but require high maintenance, a considerable titne in- vestment to sort samples and appropriate larval identification ex- pertise. Other larval settlement survey techniques such as under- water magnification (Shepherd & Turner 1985). anesthesia (Prince & Ford 1985). and suction (McShane & Smith 1988) also require great diving and sample sorting efforts. In California. Davis ( 1995) used artificial concrete block habitats that provided standardized sample areas to monitor juvenile abalone recruitment. Coiuparing results from previous juvenile abalone surveys that required the destruction of natural habitat {Tegner et al, 1989). Davis (1995) was able to provide surrogate juvenile abalone habitat and produce an index of abalone recruitment. This article describes the design and testing of artificial con- crete block habitats over a 12-month period at 6 sites in Haida Gwaii (Queen Charlotte Islands), BC, The objectives were to de- termine if concrete block habitats provided surrogate habitat for juvenile northern abalone and if so. the ability of artificial habitats to quantify juvenile ubalone abundance in different locations. To determine if juvenile abalone abundance within artificial habitats was representative of nearby natural habitats, invasive surveys of natural abalone habitats during the same time period were com- pared, MATERIALS AND METHODS Twenty-four artificial concrete block habitats were tested at 6 sites located at Lyell, Faraday and Murchison Islands (Fig, 1), These sites are within the Haida Gwaii Juan Perez Sound abalone stewardship area, where annual ecological assessments, abalone population surveys, and mark-recapture monitoring were con- ducted during 1998 to 2003 (Jones et al, 2003), The general area currently supports average densities of 0.35 emergent abalone/m" and 0,17 emergent youth (<70 mm shell length. SL) abalone/m" (Campbell et al, 2000), The artificial habitat design used is a modification of that de- scribed by Davis (1995), Each habitat provides about 3,5 m~ of surface area and consists of 24 concrete mini-blocks haphazardly oriented within a modified commercial crab trap (Fig, 2), Standard 20 cm X 20 cm X 40 cm concrete blocks were cut into quarters longitudinally to produce four individual mini-blocks. Discarded commercial crab traps measuring approximately 1 m in diameter and 0,3 m in height were altered by removing the central "fishing" component, leaving a structurally effective frame of corrosive re- sistant metal enclosed with stainless steel mesh. Diamond-shaped openings within the wire mesh frames were approximately 66 mm X 91 mm and tested with empty shells to confirm their permeabil- ity to abalone measuring less than 66 mm SL, Each structure also possessed a prefabricated entry or exit hole measuring 102 mm in diameter that was permeable to all abalone sizes and a hinged lid that allowed access to load, remove, and examine concrete mini- blocks during artificial habitat deployment and sampling. In July 2001. 24 habitats were deployed by belaying each intact 819 820 DeFreitas LEGEND • Artificial Habitat Sites ■ Natural Habitat Sites •■ Hecate Strait •■ ^ • ^1 Lyell I. Ramsay I. Figure 1. Artificial and Columbia. natural abalone habitat study sites in northern Juan Perez Sound abalone stewardship area. Haida Gvvaii, British unit from the dive support vessel to the ocean floor. Divers repo- sitioned each structure with an industrial airlift bag. Within a site, 4 habitats were oriented parallel to shore in depths of 4 to 9 m and from 7 to 30 m apart. The habitats were randomly located within areas dominated by small boulders and cobble encrusted with red coralline algae. No anchoring mechanisnts were used to secure the units in place, because each unit weighed approximately 120 kg and possessed a stable base. Divers visually inspected artificial concrete habitats for struc- tural integrity in February 2002 and thoroughly surveyed each unit in situ during May and July 2002. A pair of divers sampled arti- ficial habitats by removing and examining each concrete mini- brick for abalone. All abalone found were measured for maximum SL to the nearest millimeter and empty abalone shells were also measured and removed. After all bricks were examined, they were haphazardly repositioned within the metal frame. No special effort ^i TABLE 1. Total number of abalone found in 4 artificial habitats at each of 6 sites during surveys in May and July 2002. Kigure 2. Artificial habitat design. May July Site Alive Dead Alive Dead 1 17 1 24 1 "> 44 1 38 4 3 5 2 3 2 4 12 0 10 1 5 37 3 31 0 6 37 3 20 1 Totiil 152 10 126 y Estimating Juvenile Hauot/s kamtschatkana Abundance 821 to 60- 50- 40 30- 20- 0 ll Mean = 42.6 mm SE = O.S mm n =278 ll..l .. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Shell Length (mm) Figure 3. Size-frequency distribution of :ib;ilone measured within artitlcial habitats during May and July 2002. was made to remove or monitor abaloiie adhering to bricks as the bricks were replaced back into the wire mesh containers. To estimate the abundance of juvenile abalone occupying natu- ral habitats, sampling was conducted within 10 iir of area at 4 artificial habitat sites and 4 additional random sites (see Fig. 1 ). At randomly selected locations throughout the available abalone habi- tat at each study site, divers invasively searched 10 l-m"^ quadrats for all hidden and exposed abalone. This method in\i)lved looking on the undersides of all movable rocks but did not include any destruction of natural habitat as care was taken to return any dis- turbed rocks to their original position. Diver efficiency in search- ing natural habitats was not measured. RESULTS All 24 artificial habitats contained abalone (/; = 152. mea/( = 6.3 ± 0.95 SE abalone/container) during the first survey in May 2002. ten months after installation. During the second survey in July 2002, all but 2 artificial habitats contained abalone (n = 126, mean = 5.3 ± 0.87 SE abalone/container). There was no signifi- cant difference in mean abalone/container for either total abalone abundance (/-test, t = -0.84, d.f. = 46, P > 0.406) or total juve- nile abalone (s50 mm SL) abundance {/-test, t = -0.47. d.f. = 34, P > 0.643) between the two sample periods. A total of 278 abalone and 19 empty shells were counted and measured within artificial habitats during the study (Table I ). Ju- venile abalone (S50 mm SL) accounted for 75.4% (n = 224) of all those measured, while only 3.6% (n = 15) were more than 70 mm SL and considered to be mature. The smallest and largest abalone found in artitlcial strtictures were 15 mm and 100 mm SL. The average abalone size was 42.6 mm SL (Fig. 3) and 56.4 mm SL for all empty shells. On average, each artificial habitat required 12.5 min for a pair of divers to completely survey. The mean density of juvenile, mature, and all-si/ed abalone within artificial habitats was 1.27, 0.06, and 1.65 abalone/m". re- spectively (Table 2). Juvenile abalone densities in artificial habi- tats were significantly different between sites (one-way ANOVA, F = 8.409. d.L = 5,35. P < 0.001 ). but not within sites, suggest- ing differential recruitment to these locations. A total of 82 abalone were counted and measured within natu- ral habitat samples. Juvenile abalone accounted for 13.4% {ii = I I ) of all those measured, while 64.6% (n = 53) were mature. The smallest and largest abalone found in natural habitats were 14 mm and 124 mm SL. The average abalone was 79.3 mm SL (Fig. 4) and 75.2 mm SL for empty shells. Mean abalone densities during the May and July surveys were similar (ANOVA, F = 0.819, d.f. = 1,15. P = 0.38) and there was no difference in total abalone densities between artificial habitat sites (sites 1-6) and additional random sites (sites 7-10). The mean density of juvenile, mature and all-sized abalone measured with natural habitats were 0.07. 0.33, and 0.51 abalone/m", respectively, (Table 3). Natural habitat samples were located at a mean depth of 3.08 ± 0.08 m datum (min = -0.4 m, max = 4.8 m). Juvenile abalone densities measured within artificial habitats were compared with natural habitat samples. At sites 1 to 4. where TABLE 2. Mean number and densities (#/m") of abalone in 4 artificial habitats at each of 6 sites surveyed in Mav and ,lulv 2002. No. of Abalone Abalone Density (#/nr) Site <50 mm SL >70 mm SL All Sizes 1 2().-S 1.00 0.00 1.46 1 41.0 2.14 0.14 2.93 3 4.0 0.29 0.00 0.29 4 II.O 0..39 0.18 0.79 5 .14.0 2.25 0.00 2.43 6 28.5 1.64 0.04 2.04 Mean 23.2 1.27 0.06 1.65 SE 4.1 0.25 0.03 0.29 Standard errors shown are for site groups (/i = 12). 822 DeFreitas 12 -I 10 - 8 - u 1 ' t to Mean = 79.3 mm SE = 2.9 mm n =82 4 - 2 - 0 - II .. ■ 1 il III. 0 10 20 30 40 50 60 70 80 90 100 110 120 130 Shell Length (mm) Figure 4. Size-frequency distribution of abalone measured wittiin natural habitat samples during May and Jul> 2002. both artificial and natural habitat samples were conducted within an area greater than 10.000 m", juvenile abalone densities mea- sured within artificial habitats were significantly greater than those within natural habitat samples (r-test. t = 3.049. d.f. = 14. P = 0.009). When all locations were included in the comparison, ju- venile abalone densities measured within artificial habitats re- mained significantly greater than those within natural habitat samples (Mest. t = 5.597, d.f. = 26. P < 0.001). There was no significant difference in juvenile abalone densities measured in natural habitats at sites 1 to 4 when compared with sites 7 to 10. indicating that the presence of artificial structures at sites 1 to 4 did not influence juvenile abalone abundance in surrounding natural habitats. DISCUSSION The artificial habitat design tested in this study provided sur- rogate habitat for both juvenile and mature northern abalone. Within 10 months of installation, native abalone had discovered and occupied each of the 24 artificial habitats. The similar number TABLE 3. Mean number and density (#/ni-) of abalone in 10 natural habitat samples at each of 8 sites surveyed in May and July 2002. Site No. of Abalone Abalone Density (#/m-) <50 mm SL >70 mm SL All Sizes 1 8.0 2 1.0 3 6.5 4 3.0 7 3.5 8 6.0 9 6.0 10 7.0 Mean 5.1 SE 2.99 0.15 0.05 0.05 0.00 0.05 0.05 0.20 ().()() I). 07 0.09 0.45 0.45 0.10 0.25 0.55 (1.40 0.35 0.10 0.33 0.07 0.80 0.65 0.30 0.35 0.60 0.60 0.70 0.10 0.51 0.10 Standard errors shown are for site groups (/i 16). of abaUme occupying artificial habitats in July suggested the con- crete blocks continued to provide preferred shelter throughout the summer months when surrounding food abundance is high and good quality alternative natural habitats were available. The spe- cific length of time required for artificial materials to condition and attract abalone was difficult to determine due to the limited num- ber of sample periods. The concrete materials appeared to be suit- able for northern abalone within 7 months based on observations of abalone occupying most artificial habitats during structural in- spections in February 2002. The conditioning time of this material was consistent with Davis (1995) who found "juvenile native H. nifescens and H. comigata inhabited artificial habitats within 4 months of deploymenf" in California. As indicated in Figure 3, juvenile abalone were the most abun- dant size class occupying artificial habitats. Both the small mesh size and high substrate complexity may have contributed to the size selectivity by limiting access and suitable shelter for abalone greater than 70 mm SL. Juvenile abalone densities measured within artificial habitats were significantly different between sites but similar within sites. This apparent ability of artificial structures to quantify juvenile abalone abundance within standardized sample areas at different locations may provide the feedback re- quired to gauge the success of future stock restoration experiments. Benefits of the modular artificial habitat design tested here in- cluded the low cost of construction, ease of deployment, durability within high energy subtidal environments, and most importantly, their ease of being dismantled and reconstructed by divers in situ. without the destruction of natural habitat. In this study, juvenile abalone recruitment measured within artificial habitats was not representative of recruitment measured within nearby natural habitat samples. At sites 1 to 4, juvenile abalone abundance measured within artificial habitats was signifi- cantly greater than natural habitat samples, ranging in magnitude from 4.3 times greater at site 3 to 42.9 times greater at site 2. Although each natural habitat sample was randomly located within good quality juvenile abalone habitat and the mean juvenile aba- lone density found within natural habitats was similar to Campbell et al. (2000), natural habitats provided little consistency with sub- Estimating Juvknile Haliotis kamtschatkana Abundance 823 strate composition and hence, the lower abundance of sheltered habitat. Specific factors that made the artificial structures attractive to abalone were not investigated experimentally but were likely due to the consistent and availability of good quality sheltered habitat provided by the concrete blocks. Based on observations, additional factors that may have influenced the abundance of aba- lone in artificial habitats included easily accessed algal food grow- ing on concrete bricks and a mesh frame that may have excluded large predators such as Sunflower seastars (PYCiiopciiia hcliiiii- thoides). The measured abundance of juvenile abalone within artificial habitats may haxe been at their annual spring peak, as surveys were only conducted during May and July, a similar time of year that Davis (1995) measured a peak in abalone recruitment. To calibrate artificial habitats into better juvenile abalone abundance instruments, it will be necessary increase the number of surveys and monitor fluctuations in abalone abundance throughout the year. Only by comparing the changes in abalone abundance from winter to summer can the magnitude of localized recruitment events be determined. The use of artificial habitats as a standardized sampling instru- ment to estimate the abundance of cryptic juvenile abalone was supported by this research. The haphazardly oriented concrete blocks provided preferred habitat for juvenile abalone and the metal frame covered with wire mesh provides structural integrity and allowed each sampling unit to be quickly deployed or reposi- tioned. A pair of divers could easily sample units in situ, with no destruction to either natural habitat or abalone adhering to concrete bricks. For proposed abalone rebuilding experiments, artificial habitats of this design can be used as an initial release site for cultured juveniles, as an affordable method of determining base- line juvenile abundance along coastlines of interest, and as a means to quantify changes in juvenile recruitment that may be due to experimental stock enhancement. ACKNOWLEDGMENTS The author thanks the many divers who participated during field activities. Alan Campbell. Russ Jones, and Ron Ydenberg who provided helpful comments during the experimental design and analysis stages. This work was financially supported by the Haida Tribal Society. Fisheries and Oceans Canada's Subvention Grants Research Program, Environment Canada's Habitat Stew- ardship Program for Species at Risk, and the Centre for Wildlife Ecology at Simon Eraser University. LITERATURE CITED Allee. W. C, A. E. Emerson, O. Park. T. Park & K. P. Schmidt. 1949. Principles of animal ecology. Philadelphia: Saunders. 837 pp. Breen. P. A. 1986. Management of the British Columbia fishery for nonh- em abalone {Haliotis kainrsdiatkaiui). Can. Sfiec. Pulil. Fisli. Aqtial. Sci. 92:300-312. Breen, P. A. & B. E. Adkins. 1979. A survey of abalone populations on the east coast of the Queen Charlotte Islands. August 1978. Fi.\li. Mar. Sen: Manuscr. Rep. 1490:12.'^. Breen, P. A. & B. E. Adkins. 1981. Abalone surveys and tagging conducted during 1979. Can. Manuscr. Rep. Fisli. Aijual. Sci. 1623. 88 pp. Campbell. A. 2000. Review of northern abalone. Haliotis icamischulkana stock status in British Columbia. Can. Spec. Piilil. Fisli. Aquat. Sci. 130:41-50. Campbell, A.. D. Brouwer. J. Rogers & D. Miller. 2000. Abalone resurvey in southeast Queen Charlotte Islands. 1998. Can. Manuscr. Rep. Fish. Aquat. Sci. 2528. 30 pp. Davis, G. E. 1995. Recruitment of juvenile abalone {Haliotis spp.) mea- sured in artificial habitats. Mar. Freslnw Res. 46:549-554. Jones. R.. B. DeFreitas. N. A. Sloan. L. Lee. K. Von Boetticher & G. Martin. 2003. Abalone stewardship in Haida Gwaii: forging a long- term commitment. Can. Tech. Rep. Fish. .Aquat. Sci. 2482:5-19. Keesing, J. K.. R. Grove-Jones & P. Tagg. 1995. Measuring settlement intensity of abalone: results of a pilot study. Mar. Freslnw Res. 46: 539-543. McShane, P. E. 1992. Early life history of abalone: a review, pp. 120-138. In: S. A. Shepherd, M. J. Tegner & S. A. Guzman del Proo. editors. Abalone of the wodd: biology, fisheries and culture. Oxford. UK: Blackwell Scientific Publications Lid, McShane. P. E. 1995. Recruitment variation in ahalone: its importance to fisheries management. Mar. Fresim: Res. 46:555-570. McShane, P. E. & M. G. Smith. 1988. Measuring recruitment of Haliotis rubra Leach (Gastropoda:HaIiotidae): comparison of a novel method v\'ith two other methods. Aust. J. Mar. Freshwater Res. 39:331-336. Nash. W. J., J. C. Sanderson, J. Bridley. S. Dickson & B. Hislop. 1995. Post-larval recruitment of blacklip abalone (Haliotis rubra) on artificial collectors in southern Tasmania. Mar. Freshwater Res. 46:531-538. Prince, J. D. & W. B. Ford. 1985. Use of anaesthetic to standardize effi- ciency in sampling abalone populations (genus Haliotis: Mollusca:Gas- Iropoda). Aust. J. Mar. Freshwater Res. 36:701-706. Shepherd. S. A. & J. A. Turner. 1985. Studies on southern Australian abalone (genus Haliotis). VI. Habitat preferences and abundance and predators of juveniles. J. E.\p. Mar. Biol. Ecol. 93:28.5-298. Shepherd. S. A & L. D. Brown. 1993. What is an abalone stock: implica- tions for the role of refugia in conservation. Can. J. Fish. Aquat. Sci. 50:2001-2009. Shepherd, S. A & D. Partington. 1995. Studies on southern Australian abalone (Genus Haliotis). XVI. Recruitment, habitat and stock rela- tions. Mar Freshw. Res. 46:669-680. Sloan, N. A. & P. A. Breen. 1988. Northern abalone. Haliotis l' RhPRouucTiuN IN Hatchery-Raised White Abalonh 827 Female Male TABLE I. Percentage of abalone spawned and gamete production using ultra>iolet and hydrogen peroxide. GI 2 2+ 3 2 2+ 3 Ultraviolet + Temp. Shock Number Tested 9 11 5 11 5 Percent .Spawned 11 46 70 63 IIKI SO Number of CJametes X 10' 1.3 0.2-10.2 1.3 76-3.210 16.3-1,686 924-7.018 Hydrogen Peroxide Number Tested 10 11 4 9 11 20 Percent Spawned 70 36 50 67 64 95 Number of Gametes X 10' 0.07-1.1 0.-39-1.3 0.33-0.90 no significant difference existed in the SL of the males and females (P = 0.43). Also, Pearson's x" tests revealed that there was no significant difference in the number of males in the population compared with the number of females (P = 0.09). Spawning The smallest female that spawned measured 25.6 mm SL, whereas the smallest male was 23.1 mm SL. There was no differ- ence among different size classes of individuals with regard to the occuiTence of spawning (Pearson's x" test, P = 0.42). Greater than 95% of the eggs and sperm examined appeared normal, with no obvious defects. Significantly more males than females spawned in both treatments (Pearson's x" test. P < 0.001 under UV treatment, P = 0.009 under H,Oo treatment) (See Table I ). How- ever, there was no significant difference in the number of males that spawned in either UV or H-,0, (P = 0.07), nor was there a difference between the number of females that spawned in either of these treatments (Pearson's x" test, P = 0.08). During the course of this study, males began releasing sperm early in the hydrogen peroxide treatment. The seawater solution was immedi- ately decanted to minimize exposure to the hydrogen peroxide. In doing so, some sperm were lost and thus it was impossible to obtain accurate gamete counts. For males in the UV treatment, no correlation existed between size and number of sperm released (Pearson correlation coeffi- cient, r = 0.306). The largest amount of sperm (5.6 and 7 x lO'') were released by abalone that measured 27.8 and 31.2 mm SL. respectively, while an intermediately sized male released only 7.6 X lO' cametes. An average number was 1 .5 x lO'' gametes released 0 10 20 30 Shell Length (mm) Figure 1. Shell length — whole wet live weight relation for white aba- lone. Kquation for power curve: Weight = (1.00(11 * Shell Length-''"'''^ (R- = 0.9957, » = 317). per male abalone. Similarly, for females no correlation existed between shell length and the number of eggs released, which ranged between 65 and 10,237 eggs per individual (Pearson cor- relation coefficient, r = 0.079). Because of large variation within treatments, a Mann-Whitney t/-test indicated that there was no difference in the amount of eggs released between treatments (P = 0.09), although the average number of eggs released in the UV and H-,0, treatments was 2,880 and 673, respectively. DISCUSSION Growth of white abalone for the first year in the hatchery averaged 30.0 to 43.4 |j.ni/day, yielding an average 16 mm SL. (minimum 7.0 mm SL to maximum 30.3 mm SL). This was some- what less than hatchery growth rates expected for red and green abalone but was similar to that of pink abalone (McCormick, pers. obse.). No significant difference was observed in the frequency of males and females in the population of I -year-old abalone exam- ined (Pearson's x" test, P = 0.502). This trend is considered common in organisms of many phyla, including invertebrates, where males and females reach maturation at approximately the same size and age. Analysis of 600 legal adult white abalone from the Channel Islands yielded a sex ration of 1 : 1 over all size classes (Tutschulte 1976, Tutschulte & Connell 1981). The present work shows the onset of sexual maturation is sig- nificantly earlier than previously suggested, and hatchery-raised abalone, as young as 1 y in age (23-25 mm SL), are capable of spawning. Tutschulte and Connell (1981) proposed that the mini- mum age required for white abalone to reproduce is 4 y. However, the estimate was based on a sample of only three individuals smaller than 130 mm SL (Tutschulte 1976). The red abalone is a close relative to white abalone (Yang et al. 2000). Studies of sexual maturation of red abalone in northern California, (Giorgi & DeMartini 1977) have shown that wild red females matured at a minimum of 39.5 mm SL, whereas males were at least 84 mm SL before reaching sexual maturity. They also found that the onset of sexual maturation precedes that indicated by visual observation of the gonad. When examined histologically, animals as small as 25-50 mm SL were found to contain spermatozoa or oocytes. A similar phenomenon was found in the ormer. (//. tiibenulara) where spermatozoa were present in animals as small as 28 mm SL but were not observable until the animals reached a size of 40 mm SL (Pena 1986). The smallest red abalone spawned by Auit (1985) were 65 mm SL for males and I 10 mm SL for females. 828 McCORMICK .\ND BROGAN Hatcher\-raised white abalone mature at sizes smaller than their wild coiniterparts. Hatcher>' conditions, most notably the con- tinuous presence of abundant food sources, no doubt promote the early sexual maturation of white and some other species of aba- lone. Ault (1985) demonstrated this effect with red abalone and after conditioning them in the laboratory for 90 days. Conditioned animals spawned at minimum sizes of 55 mm and 60 mm SL for males and females, respectively. These sizes were 85% and 55% the length of the smallest wild spawners (see earlier). Ault (1985) also found that the improved diet of laboratory conditioned aba- lone increased fecundity. Laboratory conditioned red abalone less than 100 mm SL were as fecund as wild animals up to 140 mm SL. Over the course of the last 2 decades, we have noted that crops of hatchery-raised red. green, and pink abalone start to mature at about 50 mm SL. This was less than wild animals but still twice the size of spawning white abalone in the present study (T. McCor- mick, pers. obse.). Tutschulte (1976) found that large (88 to 159 mm SL) wild white abalone were more fecund than either pink or green abalone. We now know that white abalone in the hatchery also mature at a smaller size. Mature gonads were observed in laboratory populations in animals as young as 10 months in age. Although the gametes were not tested for viability (due to permit constraints) no obvious defects were observed and we expected that the eggs and sperm would be as viable as those of older animals. The implied higher fecundity of hatchery-raised white abalone may have an impact on enhancement efforts for this species. When hatchery-raised white abalone are used for enhancement efforts the number of animals that ultimately make a reproductive contribu- tion depends upon the natural annual mortality rate (M) and the time required to reach sexual maturity. Even in natural communi- ties, M is high for newly recruited abalone and may vary from 1-10 between populations (Schiel 1992. Shepherd & Breen 1992). For hatchery-raised abalone M is often higher than that of wild populations (Rogers-Bennett & Pearse 1998). Survival and con- sequent reproductive contribution may be improved by increasing the size of abalone at release. To date, many enhancement efforts utilizing hatchery-raised abalone have focused on abalone averag- ing 20-30 mm SL, with larger animals having greater survival (see summary in McCormick et al. 1994). Production of larger animals requires longer growout times and increased cultivation expense. Earlier sexual maturity and higher fecundity rates of hatchery- raised abalone may be another way of increasing reproductive contribution. We have shown that hatchery-reared white abalone mature at a much younger age and smaller size than anticipated. Providing white abalone with abundant food sources in the hatch- ery could make their initial reproductive contribution equivalent to that of animals much larger, as Ault (1985) observed. Observations in our laboratory indicated that, unlike their wild counterparts that have highly synchronized maturation and spawn- ing cycles, small hatchery-raised adults were apparently capable of spawning for much of the year. After first maturing in the late winter and early spring, the present crop of young adults remained gravid for over a year. Large wild adult abalone synchronize gonad maturation, culminating in a short spawning period in late winter (Tutschulte 1976. Tutschulte & Connell 1981 ). As with some other abalone species (Tutschulte & Connell 1981, Newman 1967, Poore 1974, and Shepherd & Laws 1974), food availability may regulate periodicity of the reproductive cycle. CONCLUSIONS This work defines the lower limit of sexual maturation in cul- tured white abalone. Field studies suggested that wild white aba- lone matured at 4 to 6 years in age at a size 80 mm SL or greater (Tuschulte 1976). At 25 mm SL. white abalone in our hatchery were sexually mature at a much smaller size than other species reared in North American hatcheries. Ault (19851 noted the same phenomena for red abalone in which wild abalone were approxi- mately twice as large when sexually mature than hatchery condi- tioned animals. If the same relationship holds true for white aba- lone, we expect that wild white abalone could begin spawning at 50 mm SL. Tutschulte (1976) noted that white abalone have greater variation in reproductive success than do pink or green abalone and would benefit from an early age at sexual maturity and long life span. This would give them more opportunities for suc- cessful spawning. The present data seems to support this argument. Studies quantifying survivorship of a range (25-100 mm SL) of young adult white abalone after outplanting into the marine habitat are necessary. Field research will be needed to document survival and changes in gonad maturation as young hatchery-raised adult white abalone acclimate to seasonal cycles of temperature and food abundance in their natural environment. Recruitment events and seasonal changes in gonad bulk will provide additional infor- mation on long-term impact of food resources on the abalone. ACKNOWLEDGMENTS The authors thank Peter Haaker. Ian Taniguchi and other per- sonnel from the California Department of Fish and Game for col- lection of adult white abalone for CIMRI. Carl Demetropouolos cultivated red algae used as one of the feeds for the abalone. We also thank Carolyn Friedman and Alan Campbell for valuable suggestions and criticism of the manuscript. This work was sup- ported, in part, by grants from Reliant Resources, Inc. and the Ventura County Fish and Game Commission. The opinions pre- sented in this article are those of the authors and not the funding agencies. LITERATURE CITED Anon. 2001. Endangered and threatened species: endangered status for white abalone. Federal Register 66. National Oceanographic and At- mospheric Administration. Ault. J. 1985. Some quantitative aspects of reproduction and growth of the red abalone. Haliotis nifescens Swainson. / World Mariculliire Sac. 16:398^25. von Bertalanffy. L. I960. Principles and theory of growth. In: W. W. Wowinski. editor. Fundamental aspects of normal and malignant growth. Amsterdam: Elsevier, pp 137-259. Boolootian, R. A.. A. Famianfarmaian & A. C. Giese. 1962. On the re- productive cycle and breeding habits of two western species of Hali- otis. Bio. Bull. 122:183-193. Cox. K. W. 1960. Review of the abalone m Caldomia. Calif. Fish & Game 46:381. Davis. G. E., P. L. Haaker & D. V. Richards. 1998. The perilous condition of white abalone, Haliotis sorenseni. Bansch. 1940. / Shellfish Res. 17:871-875. Davis, G. E., P. L. Haaker & D. V. Richards. 1996. Status and trends of Earl^' RhPRODUCTiON IN Hatcher>-Rai.sed White Abalone 829 white abalone at the California Channel Islands. Tnin.s. Am. Fish. Soc. 125:42-48. Evans. F. & C. J. Langdon. 2000. Co-culture of dulse Palmariu mollis and red abalone Haliotis nifescens under limited tlow conditions, .\quacul- ture 185:137-158. Giorgi, A. E. & J. D. DeMartini. 1977. A study of the reproductive biology of the red abalone, Haliotis rufescens Swainson, near Mendocino. Cali- fornia. Calif. Fish and Game 63:80-94. Haaker. P. L. 1994. Assessment of abalone resources at the Channel Is- lands. In: W. L. Halvorson & G. J. Maender, editors. The Fourth California Islands Symposium. Santa Barbara: Museum of Natural His- tory, pp. 83-95. Hobday, .A. & M. Tegner. 2000. Status review of white abalone [Haliotis sorenseiu) throughout ils range in California and Mexico. NOAA Tech- nical Memorandum NMFS. NOAA-TM-NMFS- SWR-035. 90 pp. Hobday, A., M. Tegner & P. Haaker. 2000. Over-exploitation of a broad- cast spawning marine invertebrate: Decline of the white abalone. Re- views in fish biology & fisheries. The Netherlands. Kluwer Academic Publishers. 22 pp. Ino, T. 1952. Biological studies on the propagation of Japanese abalone (genus Haliotiis). Bull. Tokai Reg. Fish. Res. Lab. 5:1. Inoue, M. 1976. Abalone. In Japan Fish Coop. Assoc. .Aqiiavulture Hand- book 1:19-61. Kikuchi, S. & N. Uki. 1974. Technical study on artificial spawning of abalone. genus Haliotis. II. Effect of irradiated sea water with ultra- violet rays on inducing to spawn. Bull. Tohokii Reg. Fish. Res. Lab. 33:79. Levin, M. 1991. Land-based polyculture of marine macroalgae and pacific salmon. MSc thesis. Oregon State University, Corvallis. OR. 62 pp. McCormick. T. B. 2000. Abalone {Haliotis spp.l aquaculture: present sta- tus and a stock enhancement tool. Can. Spec. Publ. Fish. Aquat. Sci. 130:55-60. McCormick. T. B., K. Herbinson. T. S. Mill & J. Altick. 1994. A review of abalone seeding, possible significance and a new seeding device. Bull. Mar. Sci. 55:680-693. Morse, D. E., H. Duncan, N. Hooker & A. Morse. 1977. Hydrogen per- oxide induces spawning in mollusks, with activation of prostaglandin endoperoxide synthetase. Science 196:298. Morse, D. E., N. Hooker & A. Morse. 1978. Chemical control of repro- duction in bivalve and gastropod mollusks. III. An inexpensive tech- nique for mariculture of many species. Proc. World Maricult. Soc 9:543. Newman, G. G. 1967. Reproduction of the South African abalone, Haliotis midae. Div. Sea Fish. S. Afr. Invest. Rep. No. 67. Pena. J. B. 1986. Preliminary study on the induction of artificial spawning in Haliotis coccinea canariensis Nordsieck (1975). Aquacidtiire 52: 35-42. Poore, G. C. B. 1974. Ecology of New Zealand abalones, Haliotis. spp. (Mollusca: Gastropoda) 4. Reproduction. NZJ. Mar. Freshwater Res. 7:67-84. Rogers-Bennett, L. & J. S. Pearse. 1998. Experimental seeding of hatchery- reared ju\enile red abalone in northern California. J. Shellfish Res. 17(3):877-880. Saito. K. 1979. Studies on the propagation of ezo abalone, Haliotis discus haniiai Ino - I. Analysis of the relationship between transplantation and catch in Funka Bay coast. Bull. Jpn. Soc. Sci. Fish 46:695-704. Schiel, D. R. 1992. The enhancement of paua (Haliotis iris Martyn) popu- lations in New Zealand. In: S. A. Shepherd, M. J. Tegner & S.A. Guzman del Proo. editors. Abalone of the w orld: biology, fisheries and culture. S.A. Fishing News Books. Oxford. U. K: Blackvvell Science Ltd. pp. 370-383. Seki. T. & H. Kanno. 1980. An advanced biological engineenng system for abalone seed production. In: International symposium on coastal pa- cific marine life. October 15-16. 1979. Bellingham. Washington: Western Washington State College. Shepherd, S. A. & P. A. Breen. 1992. Mortality in abalone: its estimation, variability and causes. In: S. A. Shepherd, M. J. Tegner & S. A. Guzman del Proo. editors. Abalone of the world: biology, fisheries and culture. S.A. Fishing News Books. Oxford, U. K: Blackwell Science Ltd. pp. 276-304. Shepherd, S. A. & H. M. Laws. 1974. Studies on the southern Australian abalone (genus Haliotis) II. Reproduction of five species. Aust. J. Mar. and Freshwater Res. 25:49-62. Tegner, M. J.. L. V. Basch & P. K. Dayton, 1996. Near-extinction of an exploited marine invertebrate. Trends. Ecol. Evol. 1 1(7):278-280. Tong, L. J. & G. A. Moss. 1992. The New Zealand culture system for abalone.. In: S.A. Shepherd, M. J. Tegner & S. A. Guzman del Proo, editors. Abalone of the world: biology, fisheries and culture. Fishing News Books. Oxford. U. K: Blackwell Science Ltd. pp. 370-383. Tong. L. J.. G. A. Moss & J. Illingworth. 1987. Enhancement of natural populations of the abalone Haliotis iris, using cultured larvae. .Aqua- culture dl-.dl-ll. Tutschulte. T. 1976, The comparative ecology of three sympatnc abalones. PhD. Dissertation, San Diego: University of California. Tutschulte, T. & J. H. Connell. 1981. Reproductive biology of three species of abalone (Haliotis) in southern California. The Veliger 23:195-206. Uki, N. & S. Kikuchi. 1982. Influence of food levels on maturation and spawning of the abalone. VII. Comparative examinations of rearing apparatus for conditioning of adult abalone. Bull. Tohoku. Reg. Fish Res. Lab. 45:45-53. Yang, Z., W. J. Swanson & V. D. Vacquier. 2000. Maximum-likelihood analysis of molecular adaptation in abalone sperm lysine reveals vari- able selective pressures among lineages and sites. J. Molec. Biol. Evol. 17:1446-1455. Jiniriuil of ShcUfish Rcscanh. Vol. 22, No. 3. 831-838. 2003. DISTRIBUTION AND ABUNDANCE OF HAUOTIS KAMTSCHATKANA IN RELATION TO HABITAT, COMPETITORS AND PREDATORS IN THE BROKEN GROUP ISLANDS, PACIFIC RIM NATIONAL PARK RESERVE OF CANADA T. TOMASCIK' AND H. HOLMES^ ^ Parks Canada. Western Canada Serx'ice Centre. 300-300 West Georgia Street, Vancouver. BC. Canada V6B 684: -Parks Canada. Pacific Rim National Park Reserve. Box 280 Uchielet. BC. Canada VOR 3A0 ABSTR.ACT Ba.seline mfomiation on the distrihutioii and abundance of Hiiliolis himlsclmlkwm wa.s obtained throughout the Broken Group Islands (BGI) in shallow- (2-5 m) and deep-water (6-9 m) habitats. The study demonstrates that abundance of northern (pinto) abalone varied spatially throughout the area and with depth. The shallow habitats in the study area supported significantly higher densities (0.18 abalone/m" ± 0.02 5£) of northern abalone when compared with deep habitats (0.10 abalone/m" ± 0.02 Sf). Maximum and minimum sizes of northern abalone measured in BGI were 132 and 4 mm shell length (SL). respectively. There were significant differences in abalone SL among the 5 island groups and the 2 depth /ones. Juvenile abalones were more abundant in the deep habitat than in the shallow habitat. A significant correlation was detected between abalone densities and the relative index of exposure. There was a positive correlation between abalone size and the abundance of benthic macroalgae and an inverse relationship between abalone size and the abundance of red sea urchins {StnmgylocentiDtus fiimci.scaniis). A positive correlation between abalone and red sea urchin densities was observed. Seven percent of juvenile abalone (<45 mm SL) was found under the red sea urchins" spine canopy. Distribution and abundance of selected invertebrate species associated with northern abalone including its known predators (ie. sea stars, crabs, octopuses) were assessed. The abundance of northern abalone was inversely correlated with predator abundance and density of benthic macroalgae. Detailed surveys of associated organisms and substrate types suggest that the distnbulion and abundance of northern abalone is a complex function of community interactions and substrate habitat characteristics. KEY WORDS: Northern (Pinto) abalone. Halititis kaiiil.'.clhilkami. red sea urchins, competitors, predators, habitat, distribution INTRODUCTION Large, mobile invertebrates, such as abalone and sea urchins, are an important component on subtidal rocky reefs in the coastal waters of British Columbia. The northern (or pinto) abalone iHali- Otis kamtscliatkana Jonas. 1845) is found distributed from Alaska (Paul & Paul 1981) to California (Sloan & Breen 1988) along the west coast of North America. Historically. H. kamtschatkana was widely distributed in British Columbia with preference to semi- exposed to exposed coastal habitats where they graze mainly on attached or drift macroalgae and diatoms. Abalone are slow grow- ing and long-lived gastropods, characterized by patchy distribu- tion, sporadic recruitment, density dependent reproduction and short larval period (Hobday et al. 2001 ). They are dioecious broad- cast spawners with peak reproductive activity during the summer (Breen & Adkins 1980). During spawning events abalone aggre- gate in shallow subtidal areas to maximize fertilization success, which depends on their aggregation density (Babcock & Keesing 1999). It is now recognized that northern abalone is particularly vulnerable to overexploitation because of this life history strategy. The coastal First Nations of British Columbia have a long history of harvesting northern abalone for a wide range of uses, ranging from subsistence harvests to use in native art and cultural activities (Stewart 1977). The first record of modern commercial abalone fishery in British Columbia dates to the early 1900s (Sloan & Breen 1988). Prior to the invention of SCUBA, the abalone fishery targeted mainly the intertidal populations; thus subtidal areas were in effect natural refugia. The use of SCUBA to harvest abalone started in the 1950s, but was generally restricted to few operators. Abalone commercial landings in British Columbia peaked in 1977 to 1978 (428-433 tons, respectively) and then continued to decline. Northern abalone was targeted by recre- ational and commercial dive fisheries until 1990, when the fishery was closed due to major stock declines (Campbell et al. 2000). The purpose of the 1990 commercial fishery closure in British Colum- bia was to allow the abalone populations to rebuild. However. numerous stock assessment surveys by Department of Fisheries and Oceans Canada (DFO) during the 1990s have shown no evi- dence of recovery (Campbell 2000). As a result. H. kamt.schatkana was designated as threatened in 1999 by the Committee on the Status of Endangered Wildlife in Canada (COSEWIC). Recovery strategy and action plans are now in place to assist in rebuilding the northern abalone population to sustainable levels. This study is part of that strategy. The present study was conducted in the Broken Group Islands (BGI). which are part of the Pacific Rim National Park Reserve of Canada (PRNPR). A recent DFO survey of abalone populations in southeast Barkley Sound, adjacent to BGI, provided no evidence of recovery of abalone populations since the province-wide closure in 1990 (Lucas et al. 2002). The mean reported density of 0.1 abalone/m~ is significantly lower than the mean density of 0.52 abalone/m" reported by Emmett and Janiieson (1988) from the same area prior to the 1990 closure. The objective of the present study is to provide baseline information for the managers of PRNPR on the distribution and abundance of northern abalone throughout the BGI at two depth zones (shallow: 2-5 m below chart datum: and deep: 6-9 m below chart datum). The study was designed to explore the association of abalone with other compo- nents of their subtidal habitats, by providing key information on the distribution and abundance of organisms associated with the species, including its major known predators. The study forms the baseline against which to compare future response of abalone populations to sea otter {EnlnJni lutris Linnaeus, 1758). recolo- nization of BGI, and to the expected increase in climate variability associated with climate change. MATERIALS AND METHODS Description of Study Sites The BGI Archipelago is located on the Pacific coast of Van- couver Island in Barkley Sound, roughly between latitudes 831 832 TOMASCIK AND HOLMES 48°57.683'N and 48°50.233'N, and longitudes 125°12.700'W and 125°24.700'W (Fig. 1). Based on geographic and oceanographic features, the BGI were sub-divided into 5 island groups and strati- tied in two depths. The 5 island groups were: Group I. Hand: Group 2, Doddi Group 3. Clark; Group 4, Wouwer: and Group 5. Gibraltar (see Fig. 1). The tidal range within the BGI is approxi- mately 3.8 m. Based on past studies by DFO. each of these island- groups was stratified into shallow (2-3 m below chart datum) and deep (6-9 ni below chart datum) zones reflecting the distribution of northern abalone populations (eg, McShane & Naylor 1995, Sloan & Breen 1988. Campbell et al. 1998. Campbell et al. 2000, Lucas et al. 2002). SAMPLING PROTOCOL A 200 X 200 m geo-referenced grid was laid over each of the 5 island groups using ArcGIS 8.x software. All 200 x 200 m blocks that intersected a shoreline or offshore reefs within each island group were sequentially numbered. The number of blocks that intersected a shoreline or an offshore reef ranged from 58 in Group 3 to 295 in Group 5. Random selection without replacement was used to select 4 sampling locations (ie, blocks) in Group 1. 5 locations in Group 2. 4 locations in Group 3. 4 locations in Group 4. and 5 locations in Group 5. for a total of 22 sampling locations (see Fig. I ). A relative index of exposure was computed for each site following procedures described by Ekebom et al. (2002). At each location, 2 sites approximately 30 m apart were sampled. Sampling was conducted by 2 dive teams. Sampling at each site was conducted by randotnly placing 1 nr quadrats along 25 m virtual transects that were laid randomly parallel to the depth contour. Two transects were sampled within each depth zone at each site. The position of transects within each depth zone was determined by randomly selecting two specific Figure \. Map of the Broken Croup Islands within the Pacific RIni National Park Reserve located on the west coast of Vancouver Island. British Columbia, Canada. Dark lines represent (he rough boundaries of the 5 groups in which randomly chosen study locations were set up. Black dots and associated numbers indicate the number and position of each study location in the survey. The 5 geographic groups were: Group I. Hand; Group 2, Dodd; Group 3, Clark; Group 4, Wouwer; and Group 5, Gibraltar. Distribution and Abundance of H. kamtschatkana 833 depths within each zone (shallow zone: 2. 3. 4, and 5 m; deep zone: 6, 7, 8, and 9 m). Ten random 1 m" quadrats were sampled along each 25 m virtual transect. The positions of the 10 random quadrats along each transect were determined by randomly selecting 10 numbers between 1 and 25. The random quadrat selection was conducted prior to the survey and marked on underwater recording sheets that were specific for each sampling transect. The starting point of each transect was selected haphazardly by swimming along the depth contour and dropping the quadrat after about a 1 to 2 min swim. Once the starting point was determined the divers proceeded to flip the 1 m~ quadrat along the virtual transect until they reached the first predetermined randomly selected position. Quadrat sampling included divers carefully lifting up (but not re- moving) all large macrophytes from the quadrat area to facilitate the systematic search for both emergent and cryptic specimens. Rocks were not removed or turned over in this survey. Once sam- pling of the quadrat was completed divers proceeded to flip the quadrat to the next randomly selected position along the virtual transect. This procedure was repeated until all 10 quadrats were sampled, or divers were forced to surface due to safety consider- ations. Abalone and red sea urchin (Su-ongylocen1rotus franciscanus Agassiz. 18631 counts, including maximum shell length (SL) and test diameter (TD) measurements in mm. were recorded in all 10 quadrats m each transect. The green sea urchin. Slrongyhicentiotus droebachiensis (O.F. Miiller, 1776) and the purple sea urchin. Strongylocentrotiis inirpitnitiis (Stimpson. 1857) were also re- corded. The number and size of juvenile abalone found under the red sea urchin spine canopy were also recorded in each quadrat. Predator densities, including dungeness crab (Cancer magistcr Dana, 1852), red rock crab {Cancer productiis Randall, 1839), octopus (Enlcroctopiis clofleiiu Wiilker. 1910), and sunflower sea star (Pycnopodia heliantlwides (Brandt, 1835)), were recorded in all quadrats along each transect. For octopuses, either individuals or inhabited dens were counted. Sea otters were not observed in the study area. Densities of benthic macroalgae were estimated in 5 randomly selected 1-m" quadrats (from the original 10 quadrats) along each transect. Because of time constraint, ease of taxonomic identifica- tion and reporting efficiency, the following macroalgae were in- cluded: (1) Macrocystis inlegrifolia Bory, 1826; (2) Nereocyslis luetkeana (Mertensi Postels et Ruprecht. 1840; (3) Fnciis spp.; (4) Eisenia arborea Areschoug, 1876; (5) Hedopliylluni sessile (C. Agardh) Setchell, 1901; (6) Aganiiu cliitluarmii Dumortier, 1822; (7) Pteiygophora californica (Ruprecht, 1852); (8) other browns; and (9) green algae. In each quadrat, the macroalgae were identi- fied and counted. Algal holdfasts were counted for all species except M. inlegrifolia for which the number of stipes was used. The following substrate cover types were defined in the present study: (1) encrusting coralline algae, (2) articulated coralline algae. (3) brown algae, (4) green algae, (5) bryozoans, (6) sponges. (7) other invertebrates, and (8) sand. The percentage cover of each substrate type was quantified in 3 randomly selected 1 ni~ quadrats (from the original 10 quadrats) in each transect using a point- intersect method. This method involved the use of a quadrat, which was permanently marked along one side with 20 points (5 cm apart), and a I m PVC bar that was permanently marked with 5 random points. Sampling involved placing the 1 m PVC bar across the quadrat from 3 randomly chosen points along the side of the quadrat and recording the substrate type that was found under each of the 5 points on the PVC bar. The three random points along the side of the quadrat were chosen earlier and were marked on re- cording sheets. Each quadrat was sampled with 15 points (45 points per transect ). STATISTICAL ANALYSES All data analyses were conducted using the NCSS statistical package (Hint/e 2001). Tests of normality and homogeneity of variance were performed on all data sets using normal probability plots and modified Levene equal-variance test, respectively. Square root (SQRT) and ARCSINE(SQRT(X)) transformations were performed as appropriate (Zar 1996) and the assumptions were tested again on the transformed data sets to verify the success of the transformations. A nonparametric Kruskal-Wallis one-way ANOVA on ranks was used to compare abalone and red sea urchin densities, as well as red sea urchin test diameters, among the island groups and depth zones, since no transformation was able to normalize the data. This non-parametric procedure tests the null hypothesis that all medians are equal and is an accepted substitute for one-way ANOVA. Where significant (P < 0.05) among group differences were indi- cated, we used the Kruskal-Wallis multiple-comparison Z-value test to find specific among group differences. The Z-values are appropriate for testing whether the medians of any two groups are significantly different. One-way ANOVA (fixed model) was used to compare abalone shell lengths (untransformed) among the 5 island groups and be- tween the two depth zones. To identify specific among group difference we used the Tukey-Kramer multiple-comparison test, which examines all pairs of group means. The Kolmogorov- Smirnov goodness of fit test was used to compare abalone and red sea urchin size frequency distributions between the shallow and deep zones. The nonparametric Spearman Rank Correlation was used to assess the relationship between the relative exposure index and abalone densities, since transformations failed to normalize the data. The test produces a correlation coefficient (/\), which may range from -1 to 1, and it has no units (Zar 1996). The parametric Pearson Product-moment Correlation analysis was used to identify significant relationships between the abundance of northern aba- lone (SQRT transformed) and the various substrate cover types (ARCS1NE(SQRT(X) transformed). This procedure was also used to examine relationships among abalone. red sea urchin, predator, and macroalgae densities (SQRT transformed). The test produces a simple correlation coefficient (/), which is unitless and ranges from -1 to 1. Simple linear regression analyses were used to assess the relationships between abalone size (untransformed), red sea urchin densities (SQRT transformed), and benthic macroalgae den- sities (untransformed). One location (group 1. location 2l was excluded from these regression analyses because no abalone were found at this location. RESULTS Dislribution and Abundance of Abalone Northern abalone were present at all island groups surveyed in this study, although in varying densities (Table 1 ). The result of the Kruskal-Wallis one-way ANOVA on ranks indicated significant differences (f < 0.05) in abalone densities among the 5 island groups. The Kruskal-Wallis multiple-comparison Z-value test re- vealed that abalone densities in Group 3 were significantly higher (P < 0.05) than at all other groups. There were no significant 834 TOMASCIK AND HOLMES TABLE 1. Northern abalone, red sea urchin, predator and macroalgae densities (mean ± SE) at the 5 island groups and 2 depth zones in the study. The numbers in parentheses are sample size - n (ie. number of quadrats), ii for red sea urchins and predators is same as for abalone. Island Group Abalone (Nuniber/ni") Red Sea Urchin (Number/m") Predators (Number/m") Macroalgae, (Number/m") Group 1 Shallow Deep Group 2 Shallow Deep Group 3 Shallow Deep Group 4 Shallow Deep Group 5 Shallow Deep Total Shallow Deep 0.143 H 0.163 d 0.117 d 0.131 d 0.16.';d 0.044 d 0.266 d 0.288 d 0.240 d 0.104 d 0. 1 54 d 0.045 d 0.085 d 0.135 d 0.025 : 0.147 d 0.180: 0.100: 0.03 (280) 0.05(160) 0.04(120) 0.03 (320) 0.03 (230) 0.03 (90) 0.04 (320) 0.06(170) 0.05(150) 0.02 (240) 0.03(130) 0.02(110) 0.02 (353) 0.03(193) 0.01 (160) 0.01 (1513) 0.02 (883) 0.02 (630) 0.596 : 0.700 : 0.458 : 0.456 : 0.370 : 0.678 : 2.538 : 3.059 : 1.947: 1.242: 0.915: 1.627: 0.244: 0.264 : 0.219: 0.997 : 1.005: 0.987 : 0.06 0.12 0.08 0.07 0.07 0.17 0.13 0.19 0.16 0.12 0.14 0.20 0.06 0.10 0.06 0.05 0.07 0.07 0.200 d 0.225 d 0.167: 0.228 : 0.261 : 0.144: 0.166: 0.141 : 0.193: 0.208 : 0.208 : 0.209 : 0.241 : 0.306 : 0.163: 0.210: 0.233 : 0.176: 0.03 0.04 0.04 0.03 0.03 0.04 0.03 0.03 0.04 0.04 0.05 0.05 0.03 0.04 0.04 0.01 0.02 0.02 0.943 : 0.725 : 1 .233 : 4.176: 5.042 : 1.867: 0: 0: 0: 2.565 : 3.072 : 1.927: 4.486 : 4.367 : 4.627 : 2.534 : 2.883: 2.038 : 0.17(140) 0.21 (80) 0.29(60) 0.58(165) 0.76(120) 0.51 (45) 0(160) 0(85) 0(75) 0.43(124) 0.62(69) 0.58(55) 0.42(181) 0.55(98) 0.66(83) : 0.19 (770) : 0.27 (452) ; 0.24 (318) differences in abalone abundance among the other 4 groups {P > 0.05). Mean abalone densities in the shallow zone (0.18 ± 0.02 SE) were almost twice as high than in the deep zone (0.10 ± 0.02 SE). The Kruskal-Wallis Z-value test revealed significant differences (P < 0.05) in northern abalone densities between the shallow and deep zones. The mean SL of H. kamtschatkana measured in this study was 59.4 mm (± 2.0 SE\ n = 222). The results of one-way ANOVA revealed significant differences in the mean SL of northern aba- lone among the 5 island groups. The mean SL of abalone in Group 3 was significantly smaller {P < 0.05) than those of Groups 2. 4. and 5 (Table 2 and Table 3). Largest abalone were found in Group 2 followed by Group 4. No differences (P > 0.05) in abalone size were found between groups 1 & 3. 1 & 4, 2 & 5, and 4 & 5. The area with the highest densities of abalone (i.e.. Group 3) was also the area with the smallest abalone. In general, the mean SL of abalone in the shallow depth zone (0 = 64.7 mm ± 2.4 SE) was significantly larger than in the deep-water habitat (0 = 46.3 mm ± 3.2 SE) (one-way ANOVA; F = 18.1; P < 0.001). The results of the Kolmogorov-Smimov test indicated that differences in aba- lone size frequency distributions between the shallow and deep zones were statistically significant (P < 0.001) (Fig. 2). Distribution and Abundance of Red Sea Urchins The red sea urchin iS. franciscaiuis) was the most abundant echinoid in the study area. The abundance of both green (S. droe- bacliiensis) and puiple (5. piirpiirains) sea urchins was so low (i.e.. 17 and 5 individuals, respectively) that they were left out of the analysis. Red sea urchins were found in all island groups (Table 1 ). Significantly higher mean red sea urchin densities (urchins/m") were found in Group 3 than anywhere else in the study area (Table 4). No significant differences in red uichin densities were observed between groups I & 2 and groups 2 & 5. For all areas combined, red sea urchin mean densities were not different between the shal- low-water zone (1.01 ±0.07 SE, n = 833 ) and the deep-water zone (0.99 ± 0.07 SE. II = 630) (Kiiiskal-Wallis Z-test; ; = 1.936; P >0.05). The results of the Kruskal-Wallis Z-value test revealed signifi- cant differences {P < 0.05) in red sea urchin TD among the 5 island groups (see Table 2; Table 5). The mean TD of red sea urchins in Group 3 was significantly smaller (80.4 mm ± 1.0 SE) when com- pared with other groups, with the exception of Group 5. Red sea urchins in Group 4 had largest mean TD (91.3 mm ±1.1 SE). No TABLE 2. Summary statistics (mean ± SE) for maximum shell length (mm) of northern abalone and maximum test diameter (mm) of red sea urchin at the 5 island groups and 2 depth zones in the study. Sample size (ie, number of individuals measured) in parentheses. Island Abalone Red Sea I'rchin Group Shell Length (mm) Test Diameter (mm) Group 1 51.2 ±4.3 (38) 87.5 ± 4.0 (li56) Shallow 57.5 ±6. 1(24) 82.0 ±5.5 (82) Deep 40.4 ±3.6 (14) 95.8 ±5.3 (54) Group 2 83.0 ±4.7 (42) 88.9 ±2.7 (143) Shallow 84.7 ±4.6 (38) 92.4 ± 3.4 (84) Deep 66.3 ± 23.5 (4) 83.9 ±4.3 (59) Group 3 46.1 ±2.5 (87) 80.4 ± 1.0(811) Shallow 49.9 ± 3.2 (50) 82.8± 1.2(517) Deep 41.0 ±3.8 (37) 76.0 ±1.5 (294) Group 4 62.7 ±6.1 (25) 91.3 ±1.1 (301) Shallow 61.3 ±7.0 (20) 91. 8± 1.6(179) Deep 68.4 ± 13.4 (5) 90.4 ± 1.4(122) Group 5 72.4 ± 4.4 (30) 83.6 ±6.0 (75) Shallow 73.0 ±4.9 (26) 96.0 ±8.5 (35) Deep 68.5 ±10.0 (4) 72.8 ±8.3 (40) Total 59.4 ±2.0 (222) 84.2 ±0.8 (1466) Shallow 64.7 ±2.4 (158) 84.3 ± 1.1 (845) Deep 46.3 ±3.2 (64) 84. 1 ±1.2(621) DiSTRlBLTION AND ABL'NDANCE OF H. KAMTSCHATKANA 835 TABLE 3. Results of one-way ANOVA and the Tuke\-Kranier multipk'-comparison test to discern statistically si^nltlcant differences in the shell length Imnu of northern abalone amon;; the 5 island groups in the study, (iroup designation as in Figure I. Source Term Sum of DF Squares Mean Square F-Ratio A: Group S(A) Total (Adjusted) Total 4 217 221 46536.75 I4SS23.5 195360.2 11634.19 6S5.S224 16.96 NS NS NS NS I'rob Level (1.01 )0* * Term significant a( alpha = 0.05 Tukey-Kramcr Multiple Comparison Test Group 12 3 4 5 Represents significant difference at least at P < 0.05 level. NS indicates no significant difference between groups. This report provides multiple com- parison tests for all pairwise differences between the means. relative index of exposure was not correlated with macroaigae densities and other substrate cover types. Encrusting coralline al- gae were a dominant substrate cover type in all groups, ranging from 50% to 867f (Table 6). In Groups 1. 3, and 5 encrusting coralline algae occupied more than H)% of the available substrate. The highest percent cover by encrusting coralline algae was mea- sured m Group 3, where they covered 85.9% of the substrate. The percent cover of encrusting coralline algae in Group 3 was sig- nificantly higher that in any other group (Kruskal-Wallis multiple comparison Z- value test; P < 0.05). Articulated coralline algae represented relatively low percentage of substrate throughout the study area, ranging between 2% to 6%. 0 20 40 60 80 100 120 140 significant differences in the TD of red urchins were found be- tween groups 1 ct 2. 1 & 4. 1 c% 5. 2 & 4. and 3 & 5. There were no differences in red sea urchin TD between the two depth /ones (Kruskal-Wallis Z-test: ; = 0.443; P > 0.05). The si/.e frequency distribution of red sea urchins in BGI for both depth zones were combined (Fig. 3), since the Kolmogorov-Smirnov goodness of fit test indicated no differences (Dinn = 0.06, P > 0.05) in size frequency distribution between the two depth zones. Habitat Relationships The relative index of exposure was positively correlated with abalone densities (r, = 0.61, P < 0.003: n = 22). red sea urchin densities (r, = 0.54. P < 0.01; ;; = 22) and with encrusting coralline algae (i\ = 0.44. P < 0.05; n = 22), but was inversely related to predator abundance (r, = -0.45; P < 0.05; /; = 22). The TABLE 4. Kruskal-Wallis multiple comparisons Z-value test to discern statistically significant differences of red sea urchin {S. franciscanus). densities (# individuals/nr) among 5 island groups. Numbers represent '/.■values for the Bonferroni Test (Hintze, 20(11). Bold numbers indicate significant differences among groups at /* < 0.1(5. Group designation as in Figure 1. Group 1 2 3 4 5 1 — 2 2.29 — 3 13.80 16.65 — 4 7.78 6.09 9.33 5 3.9(1 1.62 18.67 7.71 Bonferroni Test: Medians significantly {P < 0.05) different if Z-valiw 2.81 LU m 0 20 40 60 80 100 120 140 30i 25 2& 15 ia 5 JlllhJ ■ 0 20 40 60 80 100 120 140 SHELL LENGTH (MM) Figure 2. Size frequency distributions of northern abalone {H. ka- mlschatkana) from BtJI measured during the study. (.\) .\ll abalone measured during the study in BGI at both shallow and deep zones, (B) abalone measured in shallow zones, (C) abalone measured in deep zones. The size frequency distributions at the shallow (B) and deep (C) zones were significantly different (Kolmogorov-Smirnov goodness of fit test Dmii = 0.32: P < 0.001 ). The vertical axes represent number of abalone per size class. 836 TOMASCIK AND HOLMES TABLE 5. Kruskal-VVallis multiple comparisons Z-ralue test to discern statistically significant differences in the test diameter (mml of red sea urchin iS. fraiuiscaniis) among 5 island groups. Numbers represent Z-raliies for the Bonferroni Test iHintze, 2001). Bold numbers indicate significant differences among groups at P < 0.05. Group designation as in Figure 1. Group 1 2 3 4 5 1 — 2 0.61 — 3 3.83 4.73 — 4 0.32 0.40 5.75 5 2.68 3.22 0.25 3.24 — Bonferroni Test: Medians significantly different if Z-iuluf > 2.8070 Community Relationships The results of simple linear correlation analysis revealed a sig- nificant positive relationship between abalone and red sea urchin densities (r = 0.48. P < 0.05; n = 22). A significant inverse relationship was found between abalone and predator densities ir = -0.61; P < 0.01; /? = 20). as well as between abalone and benthic macroalgae densities (r = -0.43; P < 0.05; n = 22). A significant negative correlation was also found between red sea urchin and benthic macroalgae densities (/■ = -0.69; P < 0.001; n = 22). While encrusting coralline algae showed a strong positive correlation with red sea urchin densities (/■ = 0.75; P < 0.001; /; = 22), they showed no correlation with abalone densities {P > 0.05). The results of simple linear regression analysis levealed a signifi- cant inverse relationship between abalone size and red sea urchin densities (r = 0.33, P < 0.001; n = 21). Furthermore, simple linear regression found a significant positive relationship between abalone size and abundance of benthic macroalgae (r = 0.54, P < 0.001; n = 21). DISCUSSION The results of this study concur with recent surveys by DFO (Lucas et al. 2002). The estimated mean density of abalone in this study (0.15/m") is similar to the mean abalone density (O.lO/m^) a: LU OQ 30(>i 250^ 20a 150 ioa 5a Jlj I I.. 0 30 60 90 120 150 180 TEST DIAMETER (MM) Figure 3. Size frequency distribution of red sea urchins iS. fruncisca- ;i»v) from B(;i measured during the study. Test diameter frei|uencies from shallow and deep zones were combined. The vertical axis repre- sents number of red sea urchins per size class. reported by Lucas et al. (2002) from an adjacent area only a few kilometers away. These values are in sharp contrast to mean den- sity values reported from the north coast of British Columbia be- tween 1978 and 1984 (0.65 to 2.86 abalone/nr, .Sloan & Breen 1988). Surveys conducted in the Queen Charlotte Islands in 1978 reported densities of up to 28 abalone/m" (Breen & Adkins 1979). The size range of abalone in Barkley Sound changed from 51 to 146 mm SL in 1964 (Quayle 1971) to 38 to 119 mm SL in 2000 (Lucas et al. 2002). The size range recorded in this study was 4 to 132 mm SL, with a mean of 59.4 mm SL. Roughly 10% of the abalone population measured in this study was more than 100 mm SL, while 58% of the sampled abalone population was more than 50 mm SL (Fig. 2A). Although northeni abalone reach sexual maturity between 50 to 55 mm SL (Sloan & Breen 1988). juvenile abalone represented 42% of the sampled population. This sug- gested that abalone recruitment was occurring, albeit at relatively low numbers. The low densities of abalone. as well as low abundance of large size indi\iduals recorded in this study may be related to several Island Group TABLE 6. Summary statistics for percentage cover of eight (8) substrate cover types measured during the study. EC AC BA 75.7 4.7 0.4 (±1.9) (±1.0) 53.3 4.9 (±2.2) (±0.9) (±3.8) (±0.6) 85.9 0 (±1.3) (±0.7) (±0) 50.; 5.2 10.0 (±2.8) 70.2 (±1.9) (±1.2) (±0.9) (±1.6) 2.8 (±0.5) Substrate Types GA BR SP 0.6 11.0 0.2 tO) (±0) (±0.4) (±1.4) (±0.1) 0.7 22.5 0.1 (±0.3) :1.9) (±0.1) 0.1 6.3 0 (±0.1) (±0.1) (±0.9) (±0) 0.1 4.2 9.0 (±0.1) :1.1) (±1.5) (±0) 0 1.8 12.0 0.3 (±0) (±0.4) (±1.4) 5.8 (±1.1) 15.6 (±1.8) 3.7 (±0.7) 13.7 (±1.9) (±0.9) 252 279 288 219 309 First number is the mean % cover; second number ui brackets is ± standard error (.SE). Acronyms; EC. encrusting coralline algae; AC, articulated coralline algae; BA. brown macroalgae; GA, green macroalgae; BR. bryozoans; S. sand: SP. sponges (Porifera); O. other invertebrates. /;. sample size (# of quadrats). Island Group designation as shown in Figure 1. Distribution and Abundance of H. kamtschath\na 837 factors, such as human exploitation (i.e.. poaching), competition, predation, starvation, disease, ditferenlial mortality, or environ- mental factors. Ocean-climate variability may also play a role in keeping abalone populations at their current low levels. Tegner et al. (2001 ) demonstrated a strong link between declines in landing of red abalone (Hiiliotis nifesccns Swainson. 1822) in southern California and increased variability in sea surface temperatures (SSTs) associated with El Nino events that affect kelp abundance. However, the inverse relationship between abalone and predator densities found in this study suggests that predation may be an important factor contributing to present day structure of abalone populations. The low abundance of large abalone (>nO mm SL) suggests that large abalone may be more susceptible to predation than small individuals (<70 mm SL). Predators may be preferen- tially selecting larger individuals, or as abalone reach larger size they may loose the ability to either outrun or hide from the preda- tors. In contrast, Watson (2000). citing Sloan & Breen (1988), suggested that only sea otters and human exploitation seem to have a significant impact on the abundance and size of abalone popu- lations. There was a significant positive conelation between abalone and red sea urchin densities. This is in contrast to studies con- ducted in California, where consistent negative correlations be- tween H. nifescens and red sea urchins were found, suggesting spatial inter-specitlc exclusion between these two species (Karpov et al. 2001). The red sea urchin is viewed as perhaps abalone's most important competitor for space and food, since both species are grazers competing for the same food resource and space. The positive relationship therefore suggests that competition for food and space may not be direct, and that abalone may be in some way benefiting from their association with the red sea urchin. The positive relationship between abalone and red sea urchins in this study may be partly a function of lower population densities than those reported previously. For example, Watson (1993) reported that mean red sea urchin densities in Barkley Sound between 1988 to 1989 were about 6.9 urchins/m", which is about 7 fold higher than at present. Current abalone densities in the BGI are about 4 times lower that pre-closure (Emmett & Jamieson 1988). At these low densities direct competition between abalone and red sea ur- chins may not be apparent. However, we also found a significant negative correlation between abalone size and red sea urchin abun- dance. This negative relationship suggests that the urchins may be exerting .some degree of influence on abalone populations through their effect on either encrusting coralline algae or benthic mac- roalgae. Densities of both species exhibited a significant negative correlation with macroalgae abundance. Although abalone size also showed a strong positive relationship with benthic algal abun- dance, food availability may have played a key role in this inter- relationship. The significant positive relationship between red urchins and encrusting coralline algae and the negative relationship with benthic macrophytes suggests that the presence of sea urchins may in some way benefit abalone through their maintenance of a habitat that is preferred by Juvenile abalone. Sloan & Breen (1988) sug- gested that abalone settlement occurs on encrusting corallines in deeper water and that juveniles and adults migrate upwards as they grow. Sasaki & Shepherd (2001) showed that ezo abalone (Hali- olis discus himinii Ino, 19.'i2) settled on encrusting coralline algae and moved into shallow Eisenia forest as they aged. Several other studies have also suggested that abalone prefer to settle on sub- strates dominated by encrusting coralline algae (Shepherd & Turner 198.'>, McShane 1995). However, the primary food source for abalone, essential for rapid growth of post-larval abalone, may be the associated diatoms rather than the encrusting coralline algae themselves (Takami et al. 1997). Encrusting corallines were found to occupy about 71% of the substrate in the deep zone (6-9 m), which was statistically higher than the 66% cover in the shallow zone (2-5 m). Vance (1979) suggested that echinoids play a key role in struc- turing algal turf communities by removing encrusting inverte- brates, thus promoting the growth of encrusting coralline algae. This study found a strong negative correlation between encrusting coralline algae and "other inveilebrates". By maintaining encrust- ing coralline algae free of other invertebrates, the red sea urchin may indirectly influence abalone settlement rates and perhaps post settlement survivorship. However, the present study supports ear- lier studies in British Columbia that found no significant associa- tion of juvenile abalone with red sea urchin spine canopy (Sloan & Breen 1988), even though there was a positive correlation in the densities of these two species. In contrast, several recent studies around the world have shown that juveniles of some Hiiliotis spe- cies have a strong association with the sea urchin spine canopy (eg. Day & Branch 2002). We found only six juvenile abalones (<45 mm SL) under the red sea urchin spine canopy, which represents only 7% of all juvenile abalones recorded in this study. Rogers- Bennett & Pearse (2001) reported that one third of juvenile aba- lone inside a marine protected area was found under the spine canopy of sea urchins. ACKNOWLEDGMENTS The authors thank Rick Holmes, Pete Clarkson, Bob Hansen, Sebastian Marcoux, and Angus Simpson (Pacific Rim National Park Reserve of Canada, PRNPR), as well as, Doug Brouwer and James Pegg (Pacific Biologic Station [PBS]. Nanaimo. DEO) for conducting abalone surveys and field support. Joanne Lessard and Ian Muifitt (PBS) for cotiiputing the index of exposure, and Greg MacMillan and Steve Lobay (Western Canada Service Centre. Parks Canada) for their technical support. The authors thank Alan Campbell (PBS) and Larry Harbidge (Chief of Resource Conser- vation PRNPR) for their continuous support in this interdepart- mental research project. This manuscript was greatly improved by comments from three reviewers and A. Campbell. This project was funded by the Species at Risk Interdepartmental Recovery Fund Prottrani. LITERATURE CITED Babcock, R. & J. Keesing. 1999. Fertilization biology of the abalone Hali- oris liu'vigata: laboratory and field studies. Can. ./. Fisli. Aquai. Sci. 56:1668-1678. Breen. P. A. & B. E. Adkins. 1979. A survey of abalone populations on the east coast of the Queen Charlotte Islands. August 1978. Fisli. Mar. Serv. MS Rep. 1490:125. Breen, P. A. & B. E. Adkins. 1980. Spawning in a British Columbia population of northern ahalone. Haliotis kaimschatkana. Veliger 2.3: 177-179. Campbell, A. 2000. Review of northern abalone. Halimis kannscluiikana. stock status in British Columbia. Can. Spfc. Piihl. Fish. Ai/iiai. Sii. 130:41-50. 838 TOMASCIK AND HOLMES Campbell. A.. B. Lucas & G. Parker. 2000. Discussion on an experimental approach tor northern abalone stock rebuilding in British Columbia, Can. Stock Assessment Secretariat Res. Doc. 2000. 047. Campbell. A.. I. Winther, B. Adkins. D. Brouwer & D. Miller. 1998. Survey ot the northern abalone IHaliotis kumrsclialkaiia) in the central coast of British Columbia. May 1997. Can. Stock Assessment Secre- tariat Res. Doc. 98/99. Day, E. & G. M. Branch. 2002. Effects of sea urchins ( Parechiniis ungii- losiis) on recruits and juveniles of abalone (Halious iiiidae). Ecologiccil Monograph 72:133-149. Ekebom. J.. P. Laihonen & T. Suominen. 2002. Measuring fetch and es- timating wave exposure in coastal areas. The Changing Coast. 6th International Conference EUROCOAST/EUCC. Porto - Portugal, pp. 155-159. Emmett. B. & G. S. Janiieson. 1988. An experimental transplant of north- ern abalone. HalioU.s kamtschatkana. in Barkley Soimd, British Co- lumbia. Fish. Bull. (US) 87:95-104. Hintze. J. 2001. NCSS and PASS. Number Cruncher Statistical Systems. Kaysville. Utah. Hobday, A. J.. M. J. Tegner & P. L, Haaker. 2001. Over-exploitation of a broadcast spawning marine invertebrate: decline of the white abalone. Review of Fish Biology unci Fisheries 10:493-514. Karpov, K. A., M. J. Tegner, L. Rogers-Bennett, P. E. Kalvass & I. K. Taniguchi. 2001. Interactions among red abalones and sea urchins in fished and reserve sites of northern California: implications of coinpe- tition to management. J. Shellfish Res, 20:743-753. Lucas, B. G., A. Campbell, D. Brouwer. S. Servant & N. Webb. 2002. Survey of northern abalone. Haliotis hnntschatkanci. populations in southeast Barkley Sound, British Coluinbia, July 2000. Can. Manuscr. Rep. Fish. Aqiial. Sci. 2623: 1 1 . McShane, P. E. 1995. Recruitment variation in abalone [Haliotis spp.): its importance to management. Mar. Freshwater Res. 46:555-570. McShane, P. E. & J. R. Naylor. 1995. Depth can affect post-settlement survival of Haliotis iris (Mollusca: Gastropoda). J. E.xp. Mar. Biol. Ecol. 187:1-12. Paul. A. J. & J. M. Paul. 1981. Temperature and growth of maturing Haliotis kamtschatkana Jonas. Veliger 23:321-324. Quayle, D. B. 1971. Growth, morphometry and breeding in the British Columbia abalone (Haliotis kamtschatkana Jonas). Fish. Res. Board. Can. Tech. Rep. 279:84. Rogers-Bennett, L. & J. S. Pearse. 2001. Indirect benefits of manne pro- tected areas for juvenile abalone. Conservation Biology 15:642-647. Sasaki, R. & S. A. Shepherd. 2001. Ecology and post-settlement survival of the ezo abalone, Haliotis discus hunnai. on Miyagi coasts, Japan. J. Shellfi.sh Res. 20:619-626. Shepherd, S. A. & J. A. Turner. 1985. Studies on southern Australian abalone (genus Haliotis). VI. Habitat preference and abundance and predators of juveniles. J. E.xp. Mar. Biol. Ecol. 93:285-298. Sloan, N. A. & P, A. Breen. 1988. Northern abalone, Haliotis kannschat- kana. in British Columbia: fisheries and synopsis of life history infor- mation. Can. Spec. Publ. Fish. Aquat. Sci. 103. 46 pp. Stewart, H. 1977. Indian fishing. Early methods on the northwest coast. Douglas Maclntire, Vancouver, Toronto. Seattle: University of Wash- ington Press. 181 pp. Takami, H.. T. Kawamura & Y. Yamashita. 1997. Contribuium of diatoms as food source for post-larval abalone Haliotis discus hannai on a crustose coralline alga. Molluscan Research 18:143-151. Tegner. M. J.. P. L. Haaker. K. L. Riser & L. I. Vilchis. 2001. Climate variability, kelp forests, and the southern California red abalone fish- ery. / Shelithh Res. 20:755-763. Vance, R. R. 1979. Effects of grazing by sea urchin. Cenlrostephanus coronatus, on prey community composition. Ecology 60:537-546. Watson. J. C. 1993. The effects of sea otter iEnhydra lutris) foraging on shallow rocky communities off northwestern Vancouver Island. British Columbia. PhD. dissertation, Santa Cruz: University of California. 169 pp. Watson. J. C. 2000. The effects of sea otters [Enhydra lutris) on abalone (Haliotis spp.) species. Can. Spec. Publ. Fish. Aquat. Sci. 130:123-132. Zar. J. H. 1996. Biostatistical Analysis 3rd ed. Upper Saddle River New Jersey: Prentice Hall. 662 pp. Jiiiinial Hi Slit'llfish Rc.sccinh. Vol. 22. No. .\ 839-847. 2003. IMPLICATIONS OF HIGH LEVELS OF GENETIC DIVERSITY AND WEAK POPULATION STRUCTURE FOR THE REBUILDING OF NORTHERN ABALONE IN BRITISH COLUMBIA, CANADA RUTH E. WITHLER, ALAN CAMPBELL. SHAORONG LI, DOUG BROUWER, K. JANINE SUPERNAULT, AND KRISTINA M. MILLER Fisheries and Oceans Canada. Science Brancli. Pacific Biolo^iic Station. 3190 Hannnand Rax Road. Nanaimo. BC. Canada V9T 6N7 ABSTRACT In the past 25 year.s. the abundance of northern ubalone (Haliotis kaimschatkana) has dechned by 80% in British Columbia (BC). leading to concern over a possible loss of genetic diversity and fragmentation of breeding aggregates in the species. Abalone from 31 sites in BC and one site in southeastern Alaska were surveyed for variation at eight polymorphic inicrosatellite loci. The high level of Hf. characterizing all samples resulted in a large estimated effective population size for northern abalone l>3.5().00()). consistent with high estimates for the historical average number of migrants entering abalone aggregations each generation (-20-125). Hierarchical analysis of gene diversity revealed that 99.6% of genetic variation was contained within abalone samples and only 0.4% partitioned among samples. Approximately half of the variation was accounted lor by differences between abalone of the Queen Charlotte Islands. Alaska and those from central, southern British Columbia while the other half was caused by differences among samples within the two regions. Little allele frequency variation was observed among size classes or between repeat samples from sites sampled in more than 1 year. The results indicated that, historically, northern abalone aggregations did not represent isolated breeding units and any disruption of gene flow that may have been caused by recent low abundance levels cannot yet be detected. These results are discussed with respect to rebuilding efforts to be undertaken for northern abalone within BC. KEY WORDS: Halioiis kuintscluukaiHi. genetic variation, microsatellite. gene tlow. inbreeding, population structure INTRODUCTION Exploited species of abalone throughout the world have suf- fered severe declines in abundance (Davis et al. 1992. Prince & Shepherd 1992) and some are close to extinction (Davis et al. 1998). The northern or pinto abalone {Halicitis kainischatkuna). which inhabits shallow coastal waters of the northeastern Pacific Ocean from southern California to Alaska, declined in abundance by 75% to 80% in British Colutiibia (BC) between 1978 and 1990 (Campbell 2000). Abalone abundance did not increase with imple- mentation of a complete harvest closure in 1990 and northern abalone was listed as a "threatened" species (i.e.. one likely to become in imminent danger of extinction or extirpation if limiting factors are not reversed) by the Comtnittee on the .Status of En- dangered Wildlife in Canada in 1999 (COSEWIC 2(.)(J0). Two factors identified as major threats to northern abalone recovery were low recruitment levels and continued (illegal) harvest. In this study, we undertake a genetic assessment of population structure in northern abalone as an element of a comprehensive recovery plan for the species in BC. Northern abalone are distributed in patches on exposed and semi-exposed rocky coastal areas in BC. Species at low abundance partitioned into isolated small populations are at risk for extirpa- tion and extinction from stochastic demographic, environmental, and genetic factors. The biology of northern abalone makes it vulnerable to all three types of processes. Spawning is usually restricted to summer months (i.e., May to August), and the pelagic larval stage is of short duration, varying frotn 4 to 8 days with local factors such as teinperature ( 14'-'C to 1()°C) (Sloan & Breen 1988). The current low abundance and low densities of mature abalone (Campbell 2000) may rellect not only the historical commercial harvest but also adverse environmental conditions likely to have hindered successful recruitment over the period 1975 to 198.3 (Breen 1986). In turn, low abalone abundance hinders successful spawning because external fertilization requires high-density ag- gregations of tiiature individuals (Babcock & Keesing 1999). Fi- nally, reduced spawning success may lead to disruption of the larval-mediated gene flow among spawning aggregates that typi- cally offsets a loss of diversity within local populations. Expected results would include increased inbreeding within, and genetic drift among, local populations. The level and distance of larval dispersal mediate both detno- graphic and genetic processes in sedentary marine organisms. Lar- val dispersal levels for abalone species are generally not known but are apparently sufficiently low to ensure that demographic processes occur on a local scale (i.e.. recruitment is primarily local, ranging from a few meters or kilometers) and sufficiently high enough to prevent strong genetic differentiation over large geo- graphic ranges (Brown 1991, Hamm & Burton 2000, Huang et al. 2000). Nevertheless, the genetic studies have provided evidence for different scales of population structure in abalone species, even those that are sympatric. This indicates factors such as habitat utilization, spawning season, and larval duration may also intlu- eiice abalone population structure. For blacklip abalone. H. rubra, sampled along the southern coastline of Australia, genetic data indicated there was "isolation by distance", but even the most geographically distant (>1000 ktii) populations were genetically similar. The F^^ value for this spe- cies estimated frotn allozymes was 0.022 (Brown 1991 ) and from microsatellltes was 0.077 (Huang el al. 2000). Greater microspatial genetic heterogeneity was observed in Australian greenlip aba- lone, H. laevigata, a species with a more patchy distribution than H. rulnii. but the estimated F^x value (0.014) was not greater than that of the blacklip abalone (Brown & Murray 1992, Shepherd ct Brown 1993). Microspatial variability contrasting with genetic ho- mogeneity was also evident in the sympatric Roe's abalone. H. roci. for which an F^x value of 0.009 was estiinated from samples collected over almost .3000 km of coastline (Hancock 2000). Differences in population structure have also been observed in two sympatric abalone species of California. Red abalone, H. rufe- seens, from northern and southern California were little differen- 839 840 WlTHLER ET AL tiated at allozyme loci, in mitochondrial DNA sequence, or at a single microsatellite locus, with the F<;t value of 0.012 estimated from allozyme data not significantly different from zero (Gaffney et al. 1996. Kirby et al. 1998, Burton & Tegner 2000). In contrast, significant genetic differentiation among samples of black abalone. H. cracherodii. in central California (F<. 0.039) was attributed to a restricted spawning season that limits larval dispersal (Hanim & Burton 2000). These results indicated that immigration from distant sources was unlikely to be sufficiently great to accelerate recovery in the depleted black abalone populations of southern California, estimated to have declined in abundance by as much as 97% (Altstatt et al. 1996). Low levels of intraspecific variation may make the partitioning of genetic diversity within abalone species most amenable to ex- amination with highly polymorphic, rapidly evolving microsatel- lite loci (Huang et al. 2000. Withler 2000). In the present study, we survey variation at eight polymorphic microsatellite loci in north- ern abalone collected from 3 1 sites in BC and one site in southeast Alaska. The objectives of this study are to determine levels of genetic variation within and amona aggregations of northern aba- lone in BC, and to estimate effective population sizes and inbreed- ing levels for the species. We examine the genetic data for evi- dence of recent bottlenecks in population abundance that might have reduced genetic variation within, or increased variation among, extant abalone aggregations and incorporate the genetic data into recommendations for conservation efforts likely to ben- efit the northern abalone of BC. MATERIALS AND METHODS Epipodial tissue samples from adult abalone were collected from 31 sites within BC and one site in southeast Alaska between 1998 and 2002 (Table 1, Fig. 1). Abalone were collected in 2 different years at six sites. SCUBA dive teams searched for emer- gent or exposed (visible on rocks) individuals because most are easily found, whereas immature abalone tend to be cryptic (Camp- bell 1996). Samples from abalone within 10 to 200 m were used to represent each collection area. The small epidodial tissue sample removal from each abalone was considered non-destructive, caus- ing no mortality to the abalone (A. Campbell unpublished data on TABLE L Locations, years and sample sizes of Haliotis kamtschatkana collections for microsatellite DNA analysis. Site Latitude Longitude Year West coast Vancouver Island Elbow Island Vargas Island Dempster Island Hankin Island Turret Island Austin Island Deer Group Islands Bamfield Inlet Georgia Strait Denman Island Queen Charlotte Strait Alert Bay BC central coast Cranstown Point Nalau Passage Simonds Group Iroquois Island Stryker Island Nowish Islands Higgins Passage Lotbiniere Bay Hankin Point Freeman Passage Kitasu Bay Mosquito Island Rennison Island Kingkown Inlet Queen Charlotte Islands Louscoone Inlet Montserrat Bay Skincuttle Inlet Faraday Island Virago Sound Bruin Bay Carpenter Bay Alaska Sitka Sound 48 -S4.060 49 09.429 48 54.000 48 ?.S.000 48 54.000 48 51.370 48 53.000 48 49.000 49 28.883 50 35.000 51 22.500 51 47.000 51 57.800 52 02.895 52 05.990 52 3 1 .000 52 28.500 53 01.372 53 42.400 53 49.300 52 32.500 51 50.195 52 51.308 53 29.685 52 07.692 52 06.227 52 20.780 52 .36.770 54 04.000 54 10.017 52 13..M1 57 03.100 125 16.556 125 57.729 125 16.000 125 22.000 125 20.000 125 19.100 1 25 08.000 125 08.000 124 41.209 126 55.000 127 46.500 1 28 06.500 128 16.700 128 19.445 123 23.207 128 26.000 128 45.500 129 31.770 130 24.610 130 31.600 128 49.300 1 28 09.900 129 20.540 130 27.559 131 14.127 130 59.170 131 14.260 131 27.800 1 32 3 1 .000 132 58.752 131 03.260 135 20.500 2000 2000 2002 2002 2002 2002 2002 2001 1999. 2000 2000 1999 1999 1999.2001 1999 1999.2001 1999 1999 2000, 2001 2000, 2001 2000, 2001 2001 2001 2001 2001 1999 1998 1998 1998 1998 1999 2002 1999 45 70 170 170 180 ISO 3(1 90 45, 85 40 110 115 80,70 no 90, 20 112 90 28. 90 55. 25 75. 85 35 110 95 85 130 70 73 72 70 90 90 95 Genetic Diversity in Northern Abalone 841 Figure 1. Map showing locations of Haliotis kamtschatkana sample collections made in British Columbia and southeast Alaska between 1998 and 2002. a laboratory experiment). Samples were stored in 95% ethanol prior to DNA extraction using DNeasy kits (Qiagen. Valencia. CA). Variation at eight microsatellite loci isolated from northern abalone (Hka\2, Hka2%, Hka40, Hka4?,, Hka4S, Hka56, Hka65. Hka&5) was surveyed using the primers and protocols outlined by Miller et al. (2001 ). The microsatellite loci consisted of di-. tri- and tetra-nucleotide repeat sequences (Table 2). Alleles at each locus were generally differentiated by the number of basepairs (bp) of the predominant repeat unit, but alleles differentiated by a single base pair were observed and scored without binning at two imper- fect dinucleotide loci (WAy(48 and Hkii65). Allele frequencies for all samples surveyed in this study are available at . Analysis of the allelic and genotypic frequency data was car- ried out using the Genetic Data Analysis (GDAl program of Lewis & Zaykin (2000). GENEPOP version 3. Id (Raymond & Rousset 1995) and FSTAT version 2.9,3.2 (Goudet 2001). Genotypic frequencies at each locus in each sample were tested for conform- ance to Hardy Weinberg equilibrium (HWE) distributions in GENEPOP. Weir & CockerhanVs (1984) F^t values were com- puted over all samples and on a pairwise basis between samples using FSTAT. The significance of the multilocus F^,- value over all samples was determined by jackknifing over loci. FSTAT was used to measure the "allelic richness" (allelic diversity standard- ized to a sample size of 15) for each sample and to perform Mantel's (1967) regression of the pairwise F^.^ values on geo- graphic distance to test for "isolation by distance" among abalone samples. Geographic distances were measured as the shortest di- rect distances between sites. Pairwise F^-y values were clustered with the neighbor-joining algorithm to provide a dendrogram of the genetic relationships among abalone samples. The pairwise average number of migrants (N»;) between samples was estimated by the private alleles method of Barton and Slatkin (1986) using GENEPOP and with the expres- sion F^T = l/(4N»i -t- I), a relationship based on the assumption of island model of population structure (Whitlock & McCauley 1999). The effective population size (N^) for northern abalone was calculated from expected heterozygosity (H^) values for the eight microsatellite loci using the relationship N^, = (l/[l-Hg]^-l)/8ti„ 842 WiTHLER ET AL TABLE 2. Microsatellite loci examined in samples of Haliotis kamtschatkana from 32 locations In British Columbia and Alaska. The total numher (A) and size range (in base pairs) of alleles, the expected (H^ ) and observed (H„) heterozygosity values, the Fs, value and the inbreeding coefficient (f,,) calculated over all samples for each locus are shown. The effective population size (N^) estimated from H^ is also shown. Size Range Locus Repeat A (bpl Hp H„ FsT fi. N^IOOO //fa; 12 di S2 171-377 0.92 0.89 0.000 0.03 19(1 Hka2S di 37 183-271 0.94 0.57 0.001 0.40 350 HkaAO di 37 112-210 0.91 0.85 0.001 0.07 150 Hka43 tetra 24 163-263 0.88 0.87 0.005* 0.01 90 HkaAS di 68 93-250 0.97 0.71 0.002* 0.27 1390 Hka56 di 35 93-164 0.92 0.86 0.001* 0.07 190 Mr/65 di 58 115-250 0.95 0.87 0.001 0.09 500 HkaS5 tri 49 122-390 0.89 0.49 0.000 0.45 100 Mean - 49 - 0.92 (1.76 0.002* 0.17 370 ' P < 0.05. where yi is the mutation rate for the microsatellite loci (Lehmann et al. 1998). Little is known of the mutation rate of microsatellite loci in invertebrate organisms except Drosophila. in which the observed rate (-lO"*"! is much lower than in mammals (-lO'^'l. N^. for northern abalone was estimated in this study using the conser- vative assumption that p. = 10""*. with recognition that N^, values are 100 times greater if the true value is 10""^. Hierarchical analyses of allele frequency variation were carried out with nested ANOVA (random effects model) as described by Weir ( 1996) using GDA. The significance of differences in allele frequencies between pairs of samples collected in different years at each of six sites was examined. Similarly, the significance of allele frequency differences attributable to two geographic regions iden- tified in the dendrogram based on genetic distances (the Queen Charlotte Island [QCI| and southeastern Alaskan sites versus re- maining sites from coastal BC) was tested in a hierarchical model with sample sites nested within regions. Heterogeneity among size classes within samples was investi- gated in abalone from 16 sites (within year samples). Abalone from each site were divided among 4 size classes based on shell length; up to 50 mm. immature; 51 to 69 mm, transition of imma- ture to mature; 70 to 99 mm. mature; and more than 99 mm. fishery; defined by size at maturity estimates by Campbell et al. (1992). For each site, abalone from between 2 and 4 of the size classes were obtained. Each of the size groups contained a range of ages whose growth rates could have been influenced by local environmental conditions; less than or equal to 2 to less than or equal to 4 y (<50 mm SL). between 2 and 7 y (51-69 mm SL). between 3 and 14 y (70-99 mm SL) and more than 6 or more than 14 y (>99 mm SL) estimated from (Fig. 8 in Sloan & Breen 1988). The maximum age of H. kamtschatkana is not known, but individuals reach ages of 30 y and older (Breen 1980). Thus, the potential number of cohorts contained within each size class in- creases with size class. Allele frequencies in the two or three size classes containing the most abalone at each site were analyzed by ANOVA to examine the possibility that small numbers of adults contribute to recruitment in individual cohorts of northern abalone. leading to low genetic variability within cohorts and significant variation among cohorts within abalone aggregations. The allelic richness and inbreeding coefficient was estimated using FSTAT for each size group containing at least 20 abalone from each site. RESULTS Genetic Variation Within Populations All microsatellite loci examined were highly polymorphic, ex- hibiting high numbers of alleles and high values of both observed (Hq) and expected (H^.) heterozygosities (Table 2). Genotypes at all eight loci showed an excess of homozygotes in comparison to those expected under HWE, but the level of heterozygote defi- ciency varied greatly among loci (Table 2). Estimates of f,„ (the level of population subdivision and inbreeding if the excess of homozygotes was due entirely to assortative mating) ranged from 0.01 at HkaAi to 0.45 at Hka^5. Differences in allele frequencies between pairs of samples col- lected in 2 different years from each of six sites were not signifi- canKFs 7,,,, = 1.75. P> 0.10 1. The Fj.;- ^'^lues between the sample pairs ranged from 0 to 0.003. with an average value of 0.001. In each case, samples from the same site were combined for further analysis. All 32 samples of northern abalone displayed high levels of allelic diversity (mean numbers of alleles observed over all loci) and the standardized number of alleles, termed allelic richness, averaged 14.4 over all samples and did not differ among samples (f>0.10) (Table 3). Each locus was characterized by between two and seven common alleles, with frequencies of common alleles rarely exceeding 0.25 in a sample. None of the loci possessed a single allele that was present at the highest frequency in all samples. Although allelic diversity was high, private alleles (those observed in a single sample) were rare. Of the 390 alleles observed over all eight loci, only 30 were private and each was present at a frequency of less than 0.025 in the single sample in which it was observed. Average Hq by sample ranged from 0.73 to 0.79 (mean of 0.76). but in all cases was less than the Hg. which was essen- tially 0.92 for all samples (Table 3). Thus, the estimated f,„ value varied much less among samples (from 0.14-0.21) than among loci. The great range of f,,. values among loci and the consistency of the f|,. values for a given locus among samples indicate that population structure was not the sole explanation for the large observed heterozygote deficits at HkalS. Hka4^, and HkaiiS. Using the mammalian microsatellite mutation rate (lO"'^) and H[. values estimated for the abalone microsatellite loci of this study, we obtained locus-specific estimates of effective population Genetic Diversity in Northern Abalone 843 TABLE 3. (lenetic >ariation within samples of Haliotis kamlschalkana sampled from locations in British Columbia and southeast Alaska. The average number of alleles (A,,), standardized allelic richness (Aj,), and expected (H^ ) and observed (H,,) levels of heterozygosity are shown for each sample. The inbreeding coefficient calculated over all loci (f,^ — all loci) and over the five loci at which there was no evidence of non-amplifying alleles (f,., — 5 loci) are also shown. fu (All fi, (5 Site N Ap Ar Hr H„ loci) loci) West coast Vancouver Island Elbow Island 45 22.6 14..^ 0.92 0.78 0.15 0.08 Vargas Island 70 25.9 14,1 0.91 0.74 0.19 0.09 Dempster Island 170 32.4 14. .S 0.92 0.76 0.17 0.06 Hankin Island 170 32.0 14.3 0.92 0.76 0.18 0.07 Turret Island 180 31.9 14.4 0.92 0.73 0.21 0.08 Austin Island 180 31.5 143 0.92 0.74 0.19 0.06 Deer Group Islands 30 19.0 14.4 0.93 0.74 0.20 0.06 Bamfield 90 27.1 14.6 0.92 0.77 0.17 0.02 Georgia Strait Denman Island 130 29.9 14.4 0.92 0.77 0.17 0.03 Queen Charlotte Strait Alert Bay 40 21.5 14.3 0.92 0.78 0.16 0.06 BC central coast Cranstown Point 110 29.6 14.9 0.93 0.79 0.15 0.04 Nalau Passage 115 30.9 14.6 0.92 0.78 0.16 0.06 Simonds Group 150 31.9 14.5 0.92 0.74 0.20 0.08 Iroquois Island 110 31.6 14.8 0.92 0.78 0.16 0.04 Stryker Island 110 28.1 14.2 0.92 0.77 0.16 0.04 Nov\ish Islands 112 28.9 14.3 0.92 0.73 0.20 0.08 Higgins Passage 90 27.1 14.2 0.92 0.76 0.18 0.05 Lotbiniere Bay 118 29.9 14.3 0.92 0.76 0.17 0.07 Hankin Point 80 27.5 14.2 0.92 0.77 0.16 0.05 Freeman Passage 160 32.8 14.7 0.92 0.79 0.14 0.04 Kitasu Bay 35 20.0 14.2 0.92 0.76 0.17 0.01 Mosquito Island 110 30.5 14.6 0.92 0.77 0.17 0.06 Rennison Island 95 28.1 14.3 0.92 0.78 0.16 0.06 Kingkown Inlet 85 26.3 13.9 0.92 0.74 0.19 0.05 Queen Charlotte Islands Louscoone Inlet 130 32.4 14.8 0.92 0.77 0.17 0.05 Montserrat Bay 70 28.0 14.7 0.92 0.76 0.17 0.04 Skincuttle Inlet 73 27.6 14.3 0.92 0.75 0.18 0.02 Faraday Island 72 26.4 14.2 0.92 0.74 0.19 0.04 Virago Sound 70 26.4 14.2 0.92 0.79 0.14 0.06 Bruin Bay 90 29.6 14.6 0.92 0.78 0.14 0.02 Carpenter Bay 90 29.9 l?.l 0.93 0.76 0.18 0.06 Alaska Sitka Sound 95 28.5 145 0.92 0.76 0.17 0.05 Total/Mean 3345 2S.3 14.4 0.42 0.76 0.17 0.05 size (N^.1 ranging from 90.000 to 1 .390.000 and a mean value of 370.000 (Table 2). Use of the possibly more realistic mutation rate of 10"'' provides estimates 100 times larger. Genetic Variation Among Size Groups Within Samples Allele frequencies did not differ significantly among size classes within each site (Foy ,200 = 1-39. P > 0.05) and size accounted for none of the variation observed within and among the samples of abalone subdivided into size classes. The mean allelic richness of the individual size samples (14.3 alleles) was the same as that of the total samples indicating that there was not reduced di\ersity within cohorts. Fewer ages (year classes) contributed to the smaller (immature and transition) than the larger (mature and fishery) abalone size classes. However, neither allelic richness nor the inbreeding coefficient varied among size classes (both P > 0.05). providing little evidence that individual cohorts were the products of small numbers of or highly related abalone parents. The lack of allele frequency variation among size groups also indicated that the number of abalone participating in individual spawning events was not extremely low. Genetic Variation Among Samples The Fsy value calculated over the eight loci among all samples was low but significantly greater than zero (0.002; SE 0.000). E.xamined on a single locus basis. Fjy values ranged from 0.000 to 0.003. and were significantly greater than 0 for three of the eight loci examined (P < 0.05) (Table 2). There was no strong geographic clustering of samples apparent in the dendrogram (Fig. 844 WiTHLER ET AL 2). The seven QCI and single Alaskan samples clustered together, but the central coast, Georgia Strait and west coast Vancouver Island samples did not cluster geographically. The hierarchical analyses of gene diversity indicated that 99.6% of the observed genetic variation occurred within samples and only 0.4% was at- tributable to differentiation among samples. Of the differentiation among samples, approximately half (0.2% ) was due to differences between the two regions (QCI/Alaska vs. coastal BC) and the other half to differences among samples within regions. The effect of region was not highly significant (F, 3.46, 0.05 0.05) The regression of all pairwise ¥^y values on geographic dis- tance was significant (P < 0.05), but geographic distance ac- counted for very little of the observed variation in F^t values (r" = 0.11) (Fig. 3A). The distinctiveness of the QCI and Alaskan samples and their relatively great geographic distance from many of the remaining samples accounted for the relationship between geographic and genetic differentiation. With the QCI and Alaskan samples removed from the data set, there was no relationship (P = 0.67) and distance accounted for less than 1% of the observed variation in F^-^ values (Fig. 3B). For this set of coastal BC popu- lations, pairwise Fs-p values did not exceed 0.005 (note the change in the F^^ scale between Figs. 3 A and 3B ), and F^,- values of 0 were t^ Bruin Bay Sitka Sound Faraday Island Louscoone Inlet I Virago Sound Montserrat Bay J Skincuttle Inlet Carpenter Bay J Freeman Passage Deer Group Islands Lotbinlere Bay I Elbow Island Alert Bay I Denman Island Austin Island Rennison Island Nalau Passage KItasu Bay Turret Island HIgglns Passage Iroquois Island Vargas Island _r" Cranstown Point \ I Nowjsh Islands KIngkown Inlet Dempster Island Hankin Point Mosquito Island Bamfield Inlet Hankin Island Stryker Island Simonds Group 0.001 Figure 2. Neighbor-jolninR dendrogram of relationships among Hali- otis kamtschatkana samples based on pairwise F^, values. Samples from the Queen Charlotte Islands and southeast Alaska cluster inde- pendently from samples from coastal British Columbia locations. 0.01 1 0 008 - 0.006 • 0004 . i':t 0002 £<&^^ 0 €^S^ 200 400 600 800 1000 1200 1400 DISTANCE (km) B 0005 0.004 800 DISTANCE (km) Figure 3. Regression of pairwise values of genetic ditferentiation (F^p) on geographic distance for (Al all Haliolis kamtscltalkana samples and (B) coastal British Columbia samples only (excluding samples from the Queen Charlotte Islands and southeast Alaska). Note the difference in y-axis scale between A and B. observed between pairs of samples over the entire range of geo- graphic separation from 1 to 700 km. Using the entire data set, the regression of Fj^- on geographic distance intersected the average F^-j value between repeat samples from the same geographic location at the y-axis intercept, a value of 0 km in geographic distance. This would suggest a neighbor- hood size of less than 1 km, the smallest distance by which samples in this study were separated. However, using the data set for coastal BC sites only, the regression of F^-r on distance was essentially a straight line (slope = 1.2 x lO"'), indicating an average pairwise Fj^ of 0.0008 over the entire 700 km range. This line coincided with the F^-^ value of 0.001 obtained between re- peated samples from the same site and suggested that the entire coastal range of BC sampled in this study, exclusive of the QCI, constituted a single genetic neighborhood. The average number of migrants per generation into the aba- lone aggregations represented by each sample was estimated by the private alleles method as 18.7, a number consistent with the observed lack of genetic differentiation among samples. This value changed little when only QCI/Alaskan samples (22.5) or only non- QCI samples (22.6) were considered. Calculating the average number of migrants using the standard expectation for the rela- tionship between F<;-p and Niii provided an estimated 125 migrants entering abalone aggregations each generation. DISCUSSION Northern abalone throughout BC and southeast Alaska were characterized by very high levels of microsatellite DNA variation J Genetic Diversity in Northern Abalone 845 within and very low levels of differentiation among spawning aggregates. Less than 1% of genetic variation was attributable to differences among samples and little geographic structure was ob- served. The lack of strong differentiation among sample locations. between repeated samples from single locations and among aba- lone of different size classes within samples suggested that gene How among abalone breeding aggregations throughout BC has been extensive. If the recent low abundance of abalone has dis- rupted historical patterns of gene flow, it is not yet evident among abalone of the age groups encompassed in this study. During the last Cordilleran glaciation of North America, which ended approximately 12.000 y ago, the QCI and Alaskan coastal regions may have provided refugial habitat for terrestrial and ma- rine organisms (Warner et al. 1982). Thus, northern abalone throughout much of coastal BC and those of the QCI (and perhaps northern BC and southeast Alaska) may be descendants of differ- ent refugial populations. Two distinctive clades in mitochondrial DNA sequences of the littorinid snail Littorina subronindata throughout BC and Washington have been attributed to dispersal from separate glacial refugia (Kyle & Boulding 1998, Kyle & Boulding 2000). The small differences in microsatellite allele fre- quencies between coastal and QCI samples of northern abalone may reflect either historical isolation in separate refugia or more recent restrictions of gene flow between coastal and QCI habitats. Even if extant abalone are descendants of different refugial popu- lations, the high level of intraspecific variability and low level of Hitersample differentiation indicated that refugial population sizes were large and limited genetic divergence occurred during isola- tion, or that gene flow has occurred since the glacial period. All of the microsatellite loci examined in this study exhibited an excess of homozygosity such as that observed in surveys of other mollusks, including abalone species (Brown 1991, Hara & Kikuchi 1992, Beaumont et al. 1993, Huang et al. 2000, Perez- Losado et al. 2002). For abalone, the deficiencies generally have been attributed to inbreeding. In northern abalone. more variation was observed among loci than among samples in the level of heterozygote deficiency, indicating that locus-specific factors such as non-amplifying alleles were also involved. Thus, some level of inbreeding may have occurred in northern abalone. as in other abalone species, the level of which is best estimated by those loci showing the least evidence of non-amplifying alleles (i.e. those loci with genotypic frequencies closest to HWE). The average inbreeding coefficient over all samples for the five loci closest to HWE was O.O.S. This may represent the typical level of inbreeding in northern abalone populations. High levels of local larval recruitment or asynchronous spawn- ing on a small geographic scale may have contributed to inbreed- ing in H. kamtschalkana. as suggested for blacklip abalone (Huang et al. 2000). However, the analysis of allelic differentiation among size classes of northern abalone within samples provided no evi- dence of the increased genetic differentiation among cohorts and the reduced genetic diversity within cohorts expected under "sweepstakes-style" recruitment success (Hedgeeock 1994). Ac- cording lo this model, spatial and temporal variability in recruit- ment success may lead to detectable genetic drift among cohorts and to "chaotic genetic patchiness", in which samples in very close proximity are as genetically differentiated as ones very far apart (Larson & Julian 19991. Although proximal samples of northern abalone in coastal waters were as different as distal ones, all samples were highly polymorphic and little differentiated. Samples representing individual si/e and restricted age groups were as al- lelically "rich" as samples containing all size classes from a single location. Moreover, the average F^x value between size classes within sites (0.001 ) was the same as that between repeat samples from the same site and that between coastal sites. Thus there was no evidence that the successful spawners at any given time were sufficiently small in number or closely related to result in accel- erated genetic drift. Instead, the coastal abalone populations in this study could be considered to form a single genetic neighborhood, with genotype distributions showing no departure from those ex- pected under panmictic mating. The abalone of the QCI and south- eastern Alaska may constitute a second, only slightly differentiated neighborhood. The high abundance of rare alleles in all northern abalone samples (>809'f of alleles were present at frequencies <0. 1) sug- gested that populations have existed at long-term stable sizes (ie. not suffered recent bottlenecks) (Luikart et al. 1998). This obser- vation and the high estimates of effective population size indicated that the small local aggregations of mature abalone observed in census studies (Wallace 1999, Campbell 2000) did not represent genetically isolated breeding units. "Cryptic" abalone. not recently included in census counts, possibly also contributed to reproduc- tion in northern abalone. However, it is evident that local northern abalone aggregations have been connected by gene flow as the result of larval dispersal. The strong genetic homogeneity of northern abalone, a seasonal spawner, contrasts with results obtained for the black abalone. in which higher levels of genetic differentiation were attributed at least in part to the limited spawning season and strong seasonal differences in oceanographic patterns in the coastal waters of Cali- fornia (Hamm & Burton 2000). The lack of genetic structure in northern abalone is more similar to the low level of genetic dif- ferentiation observed in the red abalone of California, which spawns throughout the year (Burton & Tegner 2000) and in three sympatric abalone species inhabiting the waters of southern Aus- tralia. The Australian blacklip, greenlip. and Roe's abalone all show low levels of genetic differentiation over spatial scales as large or larger than those encompassed in the present study (Brown 1991, Brown & Mun-ay 1992, Hancock 2000). Small-scale genetic heterogeneity coupled with large-scale ho- mogeneity in Roe's abalone was attributed to predominantly local recruitment, with the high gene flow resulting more from large effective population sizes than from large migration rates (Han- cock 2000). Hancock also suggested that rare cases of successful long-distance dispersal might play a role in maintaining the ob- served large-scale genetic homogeneity. Little small-scale hetero- geneity was observed among samples of northern abalone. The lower Fsy values observed over short distances in northern abalone suggest that the N^ of this species is larger, or that larval dispersal is greater, than that observed for Roe's abalone. Given the high densities observed for Roe's abalone (Hancock 2000). it seems unlikely that the N^. for northern abalone exceeded that for Roe's abalone even before the recent decreases in abundance. The large estimated numbers of successful migrants among the samples in this study support the idea that dispersal may contribute more to the low observed levels of differentiation in northern abalone than in many other species. Whether successful larval dispersal in northern abalone occurs on a regular basis or is predominantly the result of rare, but highly effective, long distance dispersal events is not known. Marine species with extended longevity possess a "storage ca- pacity" for genetic variation in the face of fluctuating environments 846 WlTHLER ET AL in the large cohort of adults produced from each strong recruitment (Warner & Chesson 1985, Ellner & Hairston 1994. Ellner 1996, Gaggiotti & Vetter 1999). Each large cohort effectively "stores" many genotypes within the reproductive population over many spawning periods that are capable of contributing to both popula- tion size and genetic diversity when favorable spawning and re- cruitment conditions return. However, extended periods of low reproductive or recruitment success may be masked in genetic surveys heavily influenced by the genetic variability being stored in. but not transmitted from, the older age groups. The analysis of genetic variation in different size classes of abalone at several sites in this study provided no indication that younger abalone were less diverse than older ones, but sampling of the younger ages did not include newly recruited "cryptic" individuals. In the black abalone of southern California, recruitment failure was observed after aba- lone abundance dropped by approximately 509c (Richards & Davis 1993). Because of the longevity of northern abalone individuals, it is essential that recruitment be measured to determine current lev- els of reproductive success. Long-term genetic monitoring of newly recruited abalone would reveal the loss of genetic diversity and population fragmentation that might follow a disruption of gene tlow at lov\' abundances, but only some years after the fact. Options for rebuilding abalone abundance in BC include main- taining fishery prohibitions, aggregation of reproductive adult aba- lone in the wild to increase density and improve reproductive success, and out-planting of hatchery-raised juvenile abalone to the wild to enhance recruitment. The possibility of disrupting natural population structure in northern abalone by aggregating adults or out-planting juveniles over geographic areas larger than the small aggregations monitored for stock assessment purposes appears un- likely given the low level of microsatellite differentiation observed in this study. However, studies on other marine molluscan organ- isms have provided indications that both adaptive genetic and non- genetic inducible phenolypic changes may be typical responses to different environments (Kim et al. 2003. Trussell & Smith 2000). Rearing abalone in "common-garden" conditions to assess differ- ences in fitness-related traits may be required to determine at what geographic scale, if any. adaptive differences occur, but it seems likely that transplanting northern abalone will be limited more by disease transfer than by genetic concerns. Two other concerns associated with the out-planting of hatch- ery produced organisms are the random loss of genetic diversity due to a limited number of spawners and, if the broodstock is maintained in the hatchery over generations, the de\ elopment of a strain that is not well adapted to survival and reproduction in the wild. Hatchery strains that are intended for reseeding into natural populations should be carefully monitored to ensure that high lev- els of genetic variation are maintained, and should be open popu- lations that incorporate naturally produced individuals on a regular basis. Genetic monitoring may also contribute to evaluation of the success of enhancement efforts (Burton & Tegner 2000). This study has indicated that, in a genetic sense, northern abalone in BC are poised for recovery under favorable environmental circum- stances. Whether or not active intervention in abalone reproduction is undertaken, prudent management activities would include the identification, protection, and monitoring of spawning aggregates (and recruits) on a regional basis to examine both demographic and genetic parameters for signs of population recovery or decline. ACKNOWLEDGMENTS The authors thank B. Lucas. S. Carignan. B. DeFrietas, J. Dis- brow, R. Gurr. J. Harding. M. McNab, T. Norgard, D. Miller, and D. Woodby for help with sample collections. Drs. Ellen Kench- ington and Nicholas Elliott provided helpful suggestions for im- provement of the paper. LITERATURE CITED Altstatt. J. M.. R. F. Ambrose. J. M. Engle. P. L. Haaker. K. D. Lafferty & P. T Rainiondi. 1996. Recent declines of black abalone Halioiis crack- emdii on the mainland coast of central California. Mar. Ecol. Prog. Ser. 142:18.'i-192. Babcock. R. & J. Keesing. 1999. Fertilization biology of the abalone Mali- oris laevigata: laboratory and field stadies. Can. J. Fish. Aquat. Sci. 56:1668-1678. Barton, N. H. & M. Slatkin. 1986. A quasi-equilibrium theory of the distribution of rare alleles in a subdivided population. Heredity 56:409- 415. Beaumont. A. R., C. Morgan. S. Huelvan. A. Lucas & A. D. Ansell. 199,'!. Genetics of indigenous and transplanted populations of Pecten ma.xi- mi(s: No evidence for the existence of separate stocks. J. Exp. Mar. Biol. Ecol. 169:77-88. Breen, P. A. 1980. Measuring fishing intensity and annual production m the abalone fishery of British Columbia. Can. Tech. Rep. Aquat. Fish. Sci. 947. 49 pp. Breen. P. A. 1986. Management of the British Columbia fishery for north- ern abalone {Haliotis kamtscliatk. Mar. Biol. Ecol. 254:235-274. Hancock. B. 2000. Genetic subdivision of Roe's abalone. Haliotis roei Grey (Mollusca: Gastropoda), in south-western Australia. Mar. Fresh- water Res. 51:679-687. Hara. M. & S. Kikuchi. 1992. Genetic variability and population structure in the abalone. Halioris ili.scKs hannai. Bull. Tohoku reg. Fisli. Res. U,h. 54:107-114. Hedgecock. D. 1994. Does variance in reproductive success limit effective population sizes of marine organisms? In: A. R. Beaumont, editor. Genetics and evolution of aquatic organisms. London: Chapman and Hall. pp. 122-134. Huang. B. X., R. Peakall & P. J. Hanna. 200(1. Analysis of genetic structure of blacklip abalone ^Halioris rubra) populations using RAPDs. mini- satellite and microsatellite markers. Mar. Biol. 136:207-216. Kim, S. J., M. Rodriguez-Lanetty. J. H. Sub & J. I. Song. 2003. Emergent effects of heavy metal pollution at a population level: Litinrina hrcv- icula a study case. Mar. Pollul. Bull. 46:74-80. Kirby. V. L.. R. Villa & D. A. Powers. 1998. Identification of microsat- ellites in the California Red Abalone, Haliotis rufescfiis. J. .Shellfish Res. 17:801-804. Kyle. C. J. & E. G. Boulding. 1998. Molecular genetic evidence for parallel evolution in a marine gastropod. Littorina subrotundata. Proc. R. Sac. Loiul. (Ser. B) 265:303-308. Kyle, C. J. & E. G. Boulding. 2000. Comparative population genetic struc- ture of marine gastropods (Littorina spp.) with and without pelagic larval dispersal. Mar. Biol. 137:835-845. Larson. R. J. & R. M. Julian. 1999. Spatial and temporal genetic patchiness in marine populations and their implications for fisheries management. CalCOFI Rep. 40:94-99. Lehmann, T.. W. A. Hawley. H. Grebert & F. H. Collins. 1998. The effective population size of Anopheles gumbiae in Kenya: implications for population structure. Mol. Biol. Evol. 15:264-276. Lewis, P. O. & D. Zaykin. 2000. Genetic data analysis: computer program for the analysis of allelic data. Version 1.0 (dl5). Free program dis- tributed by the authors over the internet from the GDA Home Page at hltp://alleyn. eeb.uconn.edu/gda/ Luikart, G.. F. W. Allendorf .l.-M. Cornuet & W. B. Sherwin. 1998. Distortion of allele frequency distributions provides a test for recent population bottlenecks. J. Heredity 89:238-247. Mantel. N. 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27:209-220. Miller. K. M., K. Laberee, K, H, Kaukinen. S. Li & R. E. Withler. 2001. Development of inicrosatellite loci in northern abalone (Haliotis kiuiitseliatkanu). Mol. Ecol. Notes 1:15-17. Perez-Losada. M., A. Guerra. G. R. Carvalho. A. Sanjuan & P. W. Shaw. 2002. Extensive population subdivision of the cuttlefish Sepia ojfici- fialis (Mollusca: Cephalopoda) around the Iberian Peninsula indicated by microsatellite DNA variation. Hered. 89:417-424. Prince, J. D. & Shepherd. 1992. The Australian abalone fisheries and their management. In: S. A. Shepherd, M. J. Tegner & S. A. Guzman del Proo. editors. Abalone of the worid: biology, fisheries and culture. Oxford. UK: Blackwell Scientific Publications Ltd. pp. 407^26. Raymond, M. & F. Rousset. 1995. GENEPOP (Version 1.2): Population genetics software for exact tests and ecumenism. Heredity 86:248-249. Richards. D. V. & G. E. Davis. 1993. Early warnings of modern population collapse in black abalone, Haliotis eracherodii. at the California Chan- nel Islands. / Shellfish Res. 12:189-194. Shepherd, S. A. & L. D. Brown. 1993. What is an abalone stock: impli- cations for the role of refugia in conservation. Can. J. Fish. Ac/ual. Sci. 50:2001-2009. Sloan. N. A. & P. A. Breen. 1988. Northern abalone, Haliotis kamtschat- kana. in British Columbia: fisheries and synopsis of life history infor- mation. Can. Spec. Publ. Fish. Aquat. Sci. 103. 46 pp. Trussell. G. C. & L. D. Smith. 2000. Induced defenses in response to an invading crab predator: an explanation of historical and geographic phenotypic change. Proc. Natl. Acad. Sci. USA. 97:2123-2127. Wallace, S. S. 1999. Evaluating the effects of three forms of marine reserve on northem abalone populations in British Columbia. Canada. Conser- vation Biol. 13:882-887. Warner, B. G., R. W. Mathewes & J. J. Clague. 1982. Ice-free conditions on the Queen Charlotte Islands. British Columbia, at the height of the Late Wisconsin glaciation. Science 218:675-677. Warner. R. R. & P. L. Chesson. 1985. Coexistence mediated by recruitment fluctuations: a field guide to the storage effect. Am. Nat. 125:769-787. Weir. B. S. 1996. Genetic Data Analysis II. Sinauer Associates. Inc. Sun- derland. Massachusetts. Weir. B. S. & C. C. Cockerham. 1984. Estimating F-statistics for the analysis of population structure. Evolution 38:1358-1370. Whitlock. M. C. & D. E. McCauley. 1999. Indirect measures of gene flow and migration: FSTit I/(4Nm-t-l). Heredity 82:117-125. Withler. R. E. 2000. Genetic tools for identification and conservation of exploited abalone (Haliotis spp.) species. Can. Spec. Publ. J. Fish. Aquat. Sci. 130:101-110. Workshop on Rebuilding Techniques tor Abulone in British Columbia Abstracts 849 STATUS OF STEWARDSHIP PROJECTS ABALONE STEWARDSHIP IN HAIDA GWAII: FORGING A LONG-TERM COMMITMENT. Russ Jones and Bart I)e- Freitas, Haida Fisheries Program, PO Box 9S, Skidegate, Haida Gwaii. BC Canada VOX ISO; Norm Sloan. Gwaii Haanas National Park Reserve / Haida Heritage Site. PO Box 37. Queen Charlotte City, Haida Gwaii. BC Canada VOX ISO; Lynn Lee. World Wild- life Fund. PO Box 74. Xlcll. Haida Gwaii. BC Canada VOX lYO; Kimiku von Boetticher. Haida Gwaii Marine Resource Group Association. P.O. Box 6S0. Massett. Haida Gwaii, BC Canada VOX IMO: and Greg Martin. Laskeek Bay Conservation Society. PO Box 867; Queen Charlotte City. Haida Gwaii. BC Canada VOX ISO. Local stewardship is a possible solution to the vexing problem of rebuilding over fished northern abalone (Halioti.s kaintschat- kana) stocks. Northern abalone fisheries in British Columbia were closed coastwide in 1990 but stocks have failed to rebuild and the species became federally listed as "threatened" in 1999. We de- scribe 3 years of community-based stewardship effort in Haida Gwaii to rebuild abalone and prospects for recovery over the long- term. Steps taken include forging a community partnership through regular meetings of a core group and development of a Community Action Plan. Xhe Action Plan's goal is to rebuild abalone popula- tions sufficiently to support both Haida traditional and recreational food fisheries. Specific initiatives include public education, cur- ricula development, establishment of two large abalone steward- ship areas and a research area, creation of an Abalone Watch (coastal surveillance) program, and research diving to test rebuild- ing approaches and monitor recovery. The community response has been positive, but it is too soon to confirm whether there have been changes in human attitude and increases in abalone popula- tions. Xhe challenge is to maintain community interest and com- mitment over the long-term to allow results to be manifested. Much will depend on setting achievable stock rebuilding reference points both for the stewardship areas and Haida Gwaii. As well, the prospect of the return of the sea otter (also listed as a "threat- ened" species) that is a keystone species in the kelp forest ecosys- tem and a predator of northern abalone could result in reduced abalone abundance despite stewardship efforts. THE KITASOO ABALONE STEWARDSHIP PROJECT: SMALL PROJECT, BIG HOPES. Joel Harding, Kitasoo Fish- eries Program. 95.'i Comox Rd.. Nanaimo, BC V9R 3J7 Canada. Xhe Kitasoo Fisheries Program (KFP), operating out of the remote coastal First Nations community at Klemtu, has been work- ing since 199.5 to gain an understanding of northern abalone (Hcili- oti.s kamischatkaiia) population demographics within the Kila.soo/ Xaixais traditional teiTitory. Xhe traditional territory is large with a ont small village of 3.50 people. Xhe objective of the KFP is to develop capacity within the Kitasoo/Xaixais Nation, to foster ac- tive community participation in the conservation and management of the fisheries resources in the region. Xhe program covers manv species, including salmon, herring, manila clams, urchin, prawn, sea cucumber Porphyra. and abalone. Ov er exploitation and deple- tion of northern abalone stocks has brought this species to the tbrefroiit of the program. A significant portion of the Kitasoo/ .Xaixais traditional territory has been surveyed for remnant abalone populations and information shared with Fisheries and Oceans Canada has helped to document the post-closure distribution of abalone. Inventory surveys were initiated in 1995 at sites through- out the area. A study site was established in south Nowish Inlet during 1999. Since then, data on abalone growth from tagging, habitat requirements, predator, and competitor relationships to abalone abundance, have been recorded. In 2001, the KFP and the Habitat Stewardship Program initi- ated the Kitasoo Abalone Stewardship Project, with the purpose of expanding the scope and capacity of abalone rebuilding efforts while joining with local stewardship initiatives, such as education and monitoring campaigns. Xhe main objective of this program is to rehabilitate local abalone populations to self-sustaining levels within the Kitasoo/Xaixais traditional territory. Xhe level of com- munity support and participation will determine the success of the program. Community workshops and follow-up meetings, since 2002. have raised awareness and encouraged local participation in project initiatives. Ongoing outreach efforts include project up- dates on the community radio channel, distribution of material to visitors and tourists, and youth education activities. Xhe project has promoted participation by supporting those able to combine local food fishing activities with voluntary monitoring. Xhe KFP used local knowledge and past survey infiirmation to establish two new stewardship areas to provide sites for evaluation of wild stock manipulation as a means to increase abalone densities and repro- ductive success. In addition, artificial cement habitats (condos) are being evaluated as an index tool to monitor juvenile recruitment and abundance. Xo date, juvenile abalone have been found in the condos. but whether the abalone density data from the condos are representative of wild resident juvenile densities or are useful as an index tool to monitor changes in juvenile abundance over time is still unclear. Xhe negative effect of illegal harvesting on recovery efforts is likely substantial. Increased local monitoring and decentralization of enforcement power, from Fisheries and Oceans Canada to com- munity-based programs, would benefit the abalone resource, stew- ardship programs, enforcement agencies, and communities in- volved. Xhe KFP is a strong proponent of information exchange on this project and is eager to develop working partnerships with other abalone stewardship groups. Xhe project is taking an eco- system-based approach to abalone recovery where all work under- taken is within the constraints of the natural environment. 850 Abstracts Workshop on Rebuilding Techniques for Abalone in British Columbia ABSOLUTELY ABALONE: HABITAT STEWARDSHIP PROGRAM FOR THE PINTO ABALONE ON THE WEST COAST OF VANCOUVER ISLAND. Anne Stewart. Banitield Huu-ay-aht Community Abalone Project. Bamfield Marine Sci- ences Center. Bamfield. British Columbia. Canada, VOR IBO Pinto or northern abalone {Haliotis kamtschatkana) were har- vested traditionally at low tide, for millennia on the west coast of Canada. After intense diving harvests and an inability of manage- ment strategies to control harvests, the pinto abalone was desig- nated as a threatened species in 1999. The Bamfield Huu ay aht Community Abalone Project (BHCAP) was fomied in response to a request for proposals, to work on abalone recovery on the west coast of Vancouver Island. The two key elements of the project strategy are to engage the community in abalone recovery and to operate a successful abalone hatchery for out-planting abalone. The objective of the project is the promotion of Pinto abalone recovery through conservation, education, and community engage- ment. Collaborations have been established with Fisheries and Oceans Canada. Canadian uni\ersities, the Nuu chah nulth Tribal Council and the Pacific Rim National Park Reserve. Members of BHCAP are the Huu ay aht First Nations. Bam- field Community School Association and the Bamfield Marine Sciences Center. The Huu ay aht First Nations have a goal of restoring abalone to the point where they can harvest for food and ceremonial use. The Community School Association is invoh ed in building capacity. The Bamfield Marine Sciences Center, a non- profit society with five western Canadian universities as members, has a mandate for research and education in marine sciences. They provide a base of operation for both the abalone education and research programs and the co-ordination of the dive program for abalone surveys, collections, and out-planting. Public education is a major component of the program and 3.500 students per year, including students from school, college and universities, adult programs, and the Community School learn about abalone conservation biology. Raising the profile of the stewardship project at public events also inspires concern for aba- lone habitat and the kelp forest ecosystem for thousands of people. The Ocean Link website (www.oceanlink@island.net) provides a wealth of information on abalone and this project and had over 8 million visits during 2001. To reduce abalone poaching, the Huu ay aht First Nations crews patrol traditiimal territories and Coast Watch members keep a look out for poaching. Fishers, boaters, crews, lodge operators, and dive operators are also part of the Abalone Coast Watch. Future plans include out-planting projects in conjunction with Fisheries and Oceans Canada, sourcing funding options and con- tinuation of education, outreach and community engagement to strengthen community involvement. This last aspect is especially important to reduce illegal harvesting. This project is a fine ex- ample of First Nations and non-First Nations groups working to- gether. With hard work and co-operation, the future of a healthy and sustainable abalone community and ecosystem is possible. STATUS OF ENFORCEMENT HAVE WE GOT PROBLEMS. Bryan Jubinville. Conservation Protection Branch, Fisheries and Oceans Canada. Labieux St.. Nanaimo, BC V9R 5¥.b Canada Do we have problems? Yes we do — on 2 fronts: (1 ) the con- tinued illegal harvesting of northern abalone [Haliotis kaiulschat- kiiini) and (2) the reduction of reports on illegal activity. Essen- tially the same core group of fisheries officers has been enforcing the abalone fishery closure and has advocated protection of north- ern abalone in British Columbia (BC) since the closure started in 1990. We have received considerable support from other jurisdic- tions both within and outside the province, from stakeholders, the public, and federal Science and Fish Management branches. In the early 1990s, the conservation and protection of abalone started slowly, with the development of public and infor)nant contacts, the creation of an awareness campaign using multi-lingual posters, media contacts, court cases, impact statements, convictions, imagi- native sentencing efforts by lawyers and judges, and video clips. Pi'otectio)! has piogressed which includes the ability to identify the species DNA footprints, the development of abalone stewardship groups, and the fostering of contacts with the public. Public sup- port in enforcement is critical. When the public observes, records and reports poaching activity to enforcement officers, all reports are examined and the infor)iiation provided assists in developing a file and an investigation. Although some of the infoiniation re- ceived may prove to be of little value, some can be of significant value resulting in a conviction. Six years ago. fisheries officers would receive numerous calls each year, which they would investigate. A file would be created and, if possible, poachers prosecuted with success. I do not know of a file that we have failed on when we have had the accused in possession of northern abalone. However, recently important in- formation being provided has diminished. Is it because the amount and the quality of effort by the fishery officers in the field have increased resulting in an exceptional job of enforcing the closure in BC? Aie the illegal harvesters now more reluctant to poach aba- lone because of the increased detenence created'.' I would like to think that these are the answers, but I am realistic enough to know there is a bigger picture in terms of the global problem of poaching abalone. Recently, South Africa reported that the annual seizure of illegal abalone had exceeded the legal harvest. I have to conclude that our northern abalone is under similar pressure. So where are the general public reports of illegal activity? I believe that we need another method of getting the message out to the public to increase the information being sent to us. The Haida Gwaii stewardship emphasizes that "e\ery tip counts'" and the guardians will be docu- menting and sending reports. This should foster credibility for Fisheries and Oceans Canada and the guardians in the communi- ties. Perhaps in addition to the telephone, word of mouth, and other workshop on Rebuilding Techniques for Abalone in Britisli Columbia Abstracts 851 means of communicalnit;. such as an Internet tip hue. would be helpful. Do we have a problem? Yes, we do and one aspect oi the problem is the recent reduced information provided on illegal aba- lone harvesting actisity. We need the assistance of the public and from communities throughout coastal BC in observing and report- ing poaching incidents. AQUACULTURE RECENT PROGRESS IN HATCHERY PRODUCTION OF PINTO ABALONE. HALIOTIS KAMTSCHATKASA, IN BRITISH COLUMBIA, CANADA. Christopher M. Pearce. Fisheries and Oceans Canada, Pacific Biologic Station. 3 1 90 Ham- mond Bay Road. Nanaimo. BC V9T 6N7. Canada; Pelle Agerup. Malcolm Island Shellfish Co-operative. 430 First Street. Box 229. Sointula, BC VON 3E0. Canada; Abayomi Alabi, Probiotic Solu- tions. 7143 Blackjack Drive. Lantzville. BC VOR 2H0. Canada; Davvn Renfrew, Bamfield Huu-ay-aht Community Abalone Proj- ect. Bamfield Marine Sciences Center. Bamfield. BC VOR 1 BO. Canada; John Rosser. Malcolm Island Shellfish Co-operative. 430 First Street. Box 229. Sointula. BC VON 3E0. Canada; Guy Whyte, Bamfield Huu-ay-aht Community Abalone Project. Bam- field Marine Sciences Center. Bamfield, BC VOR IBO. Canada; and Fu Yuan. Island Scallops Ltd.. 5552 West Island Highway. Qualicum Beach. BC V9K 2C8. Canada. In July 1999. Fisheries and Oceans Canada issued a Request for Proposals for 18-mo pilot projects that would develop land-based hatchery rearing techniques for the pinto abalone. Hciliaiis ka- mtschatkana. A percentage of the cultured juveniles produced were to be utilized for wild stock rebuilding. Six projects were initially appro\'ed and fi\e proceeded with the collection of wild broodstock for the purpose of developing hatchery techniques. Of these projects, three were successful at rearing substantial numbers of juveniles (ie. Bamfield Huu-ay-aht Community Abalone Proj- ect, Island Scallops Ltd., and Malcolm Island Shellfish Coopera- tive). Their techniques for broodstock conditioning, spawning, lar- val rearing, larval settlement, and early juvenile grow out are sum- marized in this review paper. Adult broodstock were conditioned with wild kelp (Lximinaria saccluiriiia. Macrocystis integrifolia. Nerencystis hietkeana) and spawned using hydrogen peroxide, temperature shock, and/or UV-treated seawater. Larvae were reared in tlow-through or static systems at 1I°C to I5°C at a density of 1 to 9 larvae ml"' and settled on wavy or flat plastic sheets covered with natural biofilms of various ages. Early juve- niles fed on benthic diatoms and were later converted to kelp and/or prepared diets. Grow out time to commercial size is pre- dicted to be 4 to 6 years. To date, these three projects have pro- duced approximately 170.000 juvenile abalone of various sizes. FIELD RESEARCH NIGHT AND DAY SURVEYS OF A NORTHERN ABA- LONE, HALIOTIS KAMrSCHAThA.\A. POPl'LATION IN EAGLE BAY, BRITISH COLUMBIA. James P. Mortimor, Caitlin R. Henderson, Bamfield Marine Sciences Center, Bamfield B.C. VOR IBO Canada; and Glen R. D. Elliott. Bamfield Huu ay aht Community Abalone Project. Bamfield B.C. VOR 1 BO Canada. This study, initiated by the Bamfield Huu ay aht Community Abalone Project, attempted to establish characteristics of behavior and site selection, for possible out-planting of northern abalone {Haliotis kiiinlsclmikami). Diurnal and nocturnal surveys were un- dertaken to determine population estimates of emergent juvenile and adult abalone at one small area in Barkley Sound. While using conventional methodology the survey conducted was intensive and small scale in nature, contrasting with previous studies that estab- lished abalone population estimates over broader areas. No clear community association was identified, however, behavioral and physical constraints were established. Recommendations for in- creasing out-planting effectiveness include out-planting juvenile abalone at night, between 4 and 6 m below chart datum, on struc- turally complex substrates. TRENDS IN PINTO ABALONE {HALIOTIS KAMTSCHAT- KAN A) ABUNDANCE IN THE SAN JUAN ISLANDS AND MANAGEMENT OF ABALONE IN WASHINGTON STATE. D. P. Rothaus. Washington Department of Fish and Wildlife, Marine Resources. 16018 Mill Creek Blvd. Mill Creek. WA 98012-1296 USA; and C. S. Friedman. School of Aquatic and Fishery Sciences. University of Washington, Box 355020 Se- attle, WA 98195 USA, Northern abalone are contagiously distributed in shallow, rocky, exposed, and kelp covered habitats from Sitka Alaska to Monterey California. In Washington State, abalone are found in the San Juan Islands. Strait of Juan de Fuca, and northern coastal waters. They are a slow growing species, reproductively mature at 25 to 50 mm shell length (SL) depending on location. In 1984. the sport harvest was estimated at 38.200 abalone annually and by 1991 this had increased to 40.934. Before 1992. regulations al- lowed a sport fishery for abalone of 90 mm SL or greater with a harvest limit of 5 abalone per day. and an abalone iron (for re- moval of abalone from rocks) was required. From 1992 to 1 994, the allowable harvest was 3 abalone per day. minimum size 102 mm SL. and an abalone iron and calipers were required. A total closure was instituted in August of 1994. Stocks have declined in both British Columbia and Washington State. leading to the listing of northern abalone as a "Threatened Species" in Canada and as a "Species of Concent" in the USA. Common concerns and potential 852 Abstracts Workshop on Rebuilding Techniques for Abalone in British Columbia trans-boundary issues suggest co-operative restoration efforts be- tween BC and Washington State may be valuable. Surveys conducted throughout the San Juan Islands, 1979 to 1982. with timed 15-min dives, have provided baseline informa- tion on abalone abundance and size for this area. Twenty-three of these sites were again surveyed between 1990 and 1991. Abalone density at 1 site increased, at 4 sites stayed the same, at 9 sites decreased, and at 9 sites no abalone were found. The overall den- sity decrease was appro.ximately 50%. Locating the original 23 sites was problematic and may have been a factor in the dramatic decrease observed. Even with the potential problems with this comparison, the magnitude of the apparent decline, combined with the anecdotal information from sport divers and University of Washington researchers, raised serious concerns about the health of abalone stocks in Washington. Further surveys were required to adequately evaluate the apparent trend in abalone abundance. As with other areas of the world, illegal harvesting is considered to have a major impact on the abalone stocks in Washington State. In 1992, 10 permanent abalone index dive stations were estab- lished around the San Juan Islands. The sites ranged in size from 50 m" to .^80 ni", averaging 220 ni" with depths between 0 to .^0 ft MLLW. Abundance and size of emergent abalone were deter- mined over the whole site (census) with dives of 180 to 340 min bottom time. A declining trend in total abundance for all sites was observed 1992 to 1994 (/i = 351 to n = 288), with no statistical difference. Following the fishery closure in August of 1994. the 1996 survey results were n = 297. Additionally, no significant difference in the mean shell length over time was observed. The average density of half the sites surveyed in 1996 was less than 0.15 abalone/nr. Research indicates that sedentary invertebrates, such as abalone. must be within 1 .0 to 2.0 m of one another (0.33-0.15 abalone/m') for successful fertilization. Therefore, low population levels can lead to inability for gametes to cross-fertilize resulting in recruitment failure. In Washington State, data shows that half of the index stations have abalone densities below the level for successful recruitment. Recovery efforts include a captive broodstock project initiated in 2002 for the development of hatchery techniques using 80 aba- lone collected from Lopez Island. Sixteen percent mortality in the broodstock has occurred over the subsequent 5 months of captiv- ity. Anecdotal information and quantitative survey data suggest a decline in abalone populations in Washington State. Data from index stations show a gradual decrease in abundance at 6 of 10 sites but no overall significant change in abundance from 1992 to 1996. Some of the current index stations report abalone densities below the minimum density levels that are needed for successful recruitment. Additional stock assessment studies will include the re- evaluation of the 10 index sites in February 2003, more frequent (yearly) assessment of abalone abundance at the 10 index sites, development of a better survey method si) that population esti- mates can be obtained, creation of additional index sites in the Strait of Juan de Fuca. and initiation of juvenile abundance sur- veys. Genetics studies, in collaboration with Canadian scientists, will include analysis of relatedness between sites and between individuals within a site. The captive broodstock project will con- tinue to culture abalone using techniques to maximize genetic diversity and to compare behavior of hatchery reared animals in normal versus "natural" tanks. Should surveys show continued instability in abalone popula- tions, management plans would be developed for abalone stock restoration, potentially including out-planting of hatchery raised juveniles and aggregation of adults. Public information meetings and scientific workshops will be held in co-operation with the Puget Sound Restoration Fund to raise public awareness. REHABILITATION METHODS OVERVIEW OF ABALONE STOCK ENHANCEMENT IN NEW ZEALAND AND LESSONS FROM LABORATORY STUDIES OF ABALONE LARVAL SETTLEMENT AND POST-LARVAL FEEDING. R. Roberts, Cawthron Institute. Prisate Bag 2, Nelson. New Zealand. E-mail:nxlneyCs'cawthron.org.nz; and N. Andrew. NIWA, PC Box 14-901, Kilbirnie. Wellington, New Zealand, E-mail:andrew@niwa.cri.nz In New Zealand, abalone catch from commercial, recreational, traditional, and illegal harvest is approximately 1700 t per year. Fishing effort is controlled by catch limits, minimum size ( 1 25 mm shell length. SL). and method restrictions. Since 1999. the com- mercial fishery has been reduced in several main fishing areas through quota cuts and voluntary reduction in catch entitlement. Inxesligations have begun that may provide alternatives to further quota cuts including temporary closures, larger minimum harvest size, and release of hatchery reared juveniles or larvae. Catch reporting has been modified to provide data at high spatial reso- lution, as population dynamics in abalone can vary over small spatial scales and recruitment may be localized. A project under- way is to determine the reason(s) for the large number of stunted, sub-legal size animals. (inea is a small (<70 mm SL) cryptic species that is not landed in the coiinnercial harvest. The most substantial study of abalone reseeding in New Workshop on Rebuilding Techniques tor Abalone in British Columbia Abstracts 853 Zealand produced very promising results at some of 8 sites, with the best site showing 54'* sur\i\'al of lO.OOO of 7 to 12 mm SL seed, 2 years after release. Apparent survival was higher at 2 y than at 1 y, illustrating the difficulty in obtaining accurate survival estimates for cryptic life-stages. In New Zealand reseeding studies, a large proportion of juve- nile mortality often occurred soon after release. Burial by sand was a major cause of mortality in three of four studies. Lower sur\ i\ al and naive behavior from hatchery seed compared with wild juve- niles was observed in each of .^ studies. Predation was considered in only I studs and found to be minor. Naive beha\ ior of hatchery abalone may be reduced if the hatchery encouraged appropriate abalone beha\ior, (eg. by providing shelters to maintain cryptic behavior) using strong light cycles to encourage feeding at night, and exposing abalone to predators periodically to maintain defense responses. Two small trials of larval release have been carried out in New Zealand. In the first trial, 300,000 larvae were released in a 50-m2 gully. Minimum survival was 0.4'v'f after 3 months and the calcu- lated cost of each surviving animal was USS0.I4. indicating this method could be economically viable. In a second trial, mesh- tented seatloor areas of I m" were seeded with 20,000 larvae. Only 10% of larvae settled and minimum survival after 5 months was 0.06% resulting in the cost of each surviving abalone of US SO. 80, indicating this method would be uneconomic. Laboratory studies on larval settlement and post-larval feeding have provided insights into larval reseeding and natural recruit- ment. Abalone larvae are capable of attaching and crawling prior to metamorphosis. Haliotis iris will attach from 4 days of age and metamorphose at 7 to 8 days at 17 °C. Abalone can delay meta- morphosis for 2 to 3 weeks at 17°C to 20°C. In cold water, both the pre-competent period, and the ability to delay metamorphosis would be extended. Hence, potential larval dispersal may be wider than previously assumed for abalone. Larvae become increasingly responsive to metamorphosis cues as they age, so older larvae are more likely to metamorphose close to the point of release. Crustose coralline algae are the most ef- fective settlement-inducer for most abalone species, but larvae often resume swimming after landing on corallines, particularly less preferred species. Resumption of swimming could lead to transport out of the study area with consequences for survival estimates. Abalone of less than 5 mm SL consume the biofilm on coralline algae. Abalone less than 0.8 mm SL will scoop up loose diatoms, bacteria, and coralline secretions, competing with many generalist grazers. Abalone of 0.8 to 5.0 mm SL develop radula teeth spe- cialized for gouging, increasing their grazing capability and reduc- ing competition. In animals more than 5 mm SL, the radula is further specialized and the diet expands to coralline crusts, macro- algae, sea grasses, and drift particles, further reducing competition. The modest carrying capacity of corallines for young abalone should be taken into account when deciding release densities in larval reseeding. Visible signs of star\ation have been described from laboratory studies and reported in post-larvae from natural habitat. Areas with good recruitment are not necessarily recruitment saturated. However, reseeding may not be successful if there is strongly density-dependant mortality at some stage of life. Little is known about the prevalence or intensity of density-dependant mor- tality in abalone — whether it can be strong enough to negate re- seeding returns, or how it may vary spatially, temporally, or be- tween species. Sites that previously had good recruitment and a strong fishery but cuiTcntly suffer from recruitment failure should be ideal for reseeding. Though the results of New Zealand seeding studies are encouraging, more research is needed, especially to determine the factors conlrollina survival after release. A REVIEW OF ABALONE ENHANCEMENT AND REHA- BILITATION IN SOUTH AFRICA. Peter Cook, Zoology De- partment, University of Cape Town, South Africa. Current ad- dress: Center of Excellence in Natural Resources Management, Albany WA 6330, Australia. The South African coastline is approximately 3,000 km long with very few bays, inlets or sheltered areas. The exposed coastline limits opportunities for mariculture. However, the commercial har- vest of abalone is threatened by a high level of illegal poaching and this situation has provided support for a successful farming indus- try for Haliotis midae. Abalone farming has expanded rapidly in South Africa and, by 2004, annual production is expected to ex- ceed 600 tonnes per year. Most farms have hatcheries and this leads to excess production of juveniles that could be used for enhancement or ranching. Wild populations, consisting of six spe- cies of abalone, occur on the southwest, south and southeast coasts of South Africa. Although the west coast is a highly productive area with extensive kelp beds and high wave action, abalone occur naturally only in the southernmost sections. Experiments to determine the feasibility of abalone enhance- ment and ranching in South Africa were carried out at Port Nol- loth, on the northwest coast. This site was chosen due to the presence of abalone fossils, the presence of high densities of ur- chins— both indicating appropriate environmental conditions for abalone — and the availability of security from a diamond mine in the vicinity. The site was over 300 km from any natural abalone population, assuring that any animals in the area were froin the ranching experiments. Anticipated problems with the sea ranching included release mechanisms that could cause mortalities, predation after release, monitoring success (% survival), and assessing economic viability. To reduce the mortality caused by handing during the transporta- tion and release of abalone, special release mechanisms were de- vised. These devises consisted of PVC pipes halved lengthwise and glued to a Perspex sheet. Both ends of the PVC covered with mesh after the abalone entered the devices naturally. The devices 854 Abstracts Workshop on Rebuilding Tecliniques for Abalone in British Columbia were transferred intact without handling the individual animals. The devices were attached to concrete blocks at the experimental site in the late afternoon. Twenty-four hours later, the mesh was removed and the abalone were allowed to exit at will. This pro- cedure also provided protection from predators for the first 24 h while the abalone became acclimatized to the environment. At four experimental sites, 500 or 800 abalone of approxi- mately 14 mm shell length were released per site. Growth and survival were monitored over 24 months and there was signifi- cantly slower growth through the summer compared with the win- ter months suggesting that the timing of the release is important to both survival and growth. Survival is reported in Table 1. The maximum mortality occurred during the first twenty-four hours after release and many remained in the transport devices for extended periods. Even at good sites there was little dispersal from the release area. Abalone appeared to seek out urchins for protec- tion and were often found under the urchin spines. However, the presence of urchins did not appear to ensure a high survival rate since at one site no surviving abalone were found in March 1W7. Factors affecting survival are complex, variable, and inter-related, displaying a variable hierarchy of importance. For ranching to be economically \ iable a survival rate of 5'7f tol0% for out-planted juveniles is required. Preliminary results suggested that, in certain circumstances, survival rates in excess of 30% could be obtained with seeded animals with particularly good survival being obtained when precautions are taken to reduce han- dling stress and at sites where sea urchins were present. Later work at similar sites, however, produced contrasting results and it was concluded that an extremely coinplex interplay between many dif- ferent factors affected survival. Of these, the presence of sea urchnis was only important at certain sites, whilst, at other sites, the present of optimum-si/ed boulders seemed to replace that requirement. Overall, the size of seeded animals was the most important factor that influences sur- vival, larger seed having better survival rates. Following the dem- onstration that, under certain circumstances, ranching could be economically feasible, genetic implications of this operation were investigated. Using mt-DNA markers, it was shown that, not only could animals from dilferent geographic regions be shown to be genetically differentiated but, in addition, distinct genetic differ- TABLE 1. Survi>al of released abalone. Simple April September March Survival to 6 Sites Released 1996 1996 1997 Months Site A .soo 97 40 0 27.4% SiteB 500 117 70 19 39.2% SiteC 800 - 124 99 27.8% SiteD 800 - 145 60 25.6% Average 307r ences between naturally occuiTing and hatchery reared animals was also apparent. REBUILDING CALIFORNIA ABALONE POPULATIONS. Haaker, P. L., I. Taniguchi. California Department of Fish and Game. 4665 Lampson Avenue. Suite C. Los Alamitos. CA. 90720 USA; J. Butler. NOAA Fisheries. Southwest Fisheries Science Center (NWFS). La Jolla. CA. USA; and N. Wright. California Department of Fish and Game. 4665 Lampson Avenue. Suite C. Los Alamitos, CA. 90720 USA. Since 1997. all seven California abalone species have been closed to commercial and recreational fishing south of San Fran- cisco Bay. California abalone occur throughout the coastal marine environment from the intertidal zone to deep offshore reefs, pre- senting a challenge for the development of an abalone recovery plan. A successful plan needs to address the various characteristics of abalone and include an approach that can be applied as broadly as possible using available resources and a method for evaluation of progress. Assessment of remaining abalone stocks is of immediate im- portance. Assessment goals have been established to address and prevent extinction of the abalone. to restore resource sustainability. and to rebuild resources to fishery sustainability levels. Assess- ment must include the identification of remnant populations, es- tablishment of locations for further evaluation, and determination of locations for enhancement. The range of abalone is often specific, extensive, and with patterns of distribution. For each species, information on landings from fishing effort is accumulated into catch block areas which are used to determine the most extensive and the best habitat for abalone and used to determine research index sites. Population density is an essential element of the assessment; however, when numbers are low standard density surveys are uninformative. Free- form searches can yield more individuals than transect constrained surveys and data on individuals, such as size, can be collected. Shell length (SL) is a population indicator, where the occur- rence of a broad size range, even at low numbers, is evidence of reproduction and growth. Size surveys are conducted using 3 size categories. 0 to 100 mm SL. 100 mm SL to minimum legal size (MLS), and MLS to maximum size. Since the 0 to 100 mm SL category includes the cryptic population, which is difficult and destructive to assess, it is only used for occasional determination of settlement. When a broad range of sizes is present, quantitative surveys are used to determine emergent abalone densities. A mini- mum viable population target size is an emergent population of 2,000 abalone per hectare, at all index locations. When an average of 6,600 abalone per hectare at three out of four of the index locations is reached, a fishery could be considered. Survey criteria and modifications should be made according to species characteristics. For example, surveying intertidal popula- tions with a GPS can provide the position of each abalone. White abalone, currently surveyed using free form dives, could be sur- Workshop on Rebuilding Techniques for Abalone in British Columbia Abslraets 855 veyed using an ROV or submarine to pro\ ide geographic position. Multi-beam sonar sea tloor maps are being generated by Depart- ment of Fish and Game and. together with data from a ROV. could be used to construct benthic habitat maps. Abalone populations are at extremely low levels throughout southern California. Some remaining populations are so dispersed that successful natural reproduction is unlikely and enhancement may be the only remaining method of intervention. Enhancement techniques include aggregation of abalone, translocation of indi- viduals from remote source populations, and out-planting of cul- tured juveniles and competent larvae. At the present, aggregation is seen as the only viable method of enhancement. In abalone recovery, there are several challenges that must be addressed as part of the recovery process. Some challenges must be considered before the recovery process can proceed. The pres- ence of sea otters precludes a fishery of abalone and most other marine invertebrates. In the abalone recovery plan, any areas either currently or previously occupied by sea otters are excluded from assessment. Disease has severely affected abalone stocks. The black aba- lone was virtually extirpated from southern California by wither- ing syndrome. Warmer seawater temperatures enhance withering syndrome, which is a concern for translocation projects. A para- sitic sabellid worm, which causes shell growth disruption and de- formity, was introduced into California aquaculture facilities and currently a prohibition exists on the out-planting of abalone from non-certified facilities. Genetic questions need to be addressed, prior to translocation of abalone from different bio-geographic zones and using cultured animals for enhancement of natural populations. Poaching of abalone is a serious problem in California. Recov- ery site criteria should include a low likelihood of illegal activity. Part of recovery is establishing large, dense populations and groups of individuals to facilitate reproduction. It is precisely those conditions that are good for poaching. Optimal locations would include remote islands, and mainland locations within limited ac- cess reserves. Marine Protected Areas. MPAs. offer one of the best opportu- nities for abalone restoration activities. Recently. California estab- lished a number of MPAs at the Channel Islands National Marine Sanctuary and most include ongoing abalone study sites and ap- propriate abalone habitat. Each abalone species has specific environmental requirements, which must be addressed to optimize successful recovery. South- ern California is at the northern end of the range of pink, green, and white abalone, and the southern end of red abalone range. Red abalone growth and reproduction is depressed during warm water periods, but they can survive until temperatures decline. Pink and green abalone prefer warmer water which may allow their popu- lations to extend farther northward with increasing sea water tem- peratures. Environmental effects complicate other factors. If sea- water temperatures increase farther north, withering syndrome may become infectious in northern populations. Our major challenge in rebuilding abalone stocks is to return at least part of the abalone population to a natural situation, where bio-diversity and natural selection can be effective. Jounuil of Shellfish Research. Vol. 22. No. 3. 857-863. 2003. SIZE AT MATURITY OF FEMALE AMERICAN LOBSTERS FROM AN ESTUARINE AND COASTAL POPULATION SUSAN A. LITTLE* AND WINSOR H. WATSON, III Zooloiiy Departineut & Center for Marine Bioloi^y. University of New Hunipshire. Durham. New Hampshire 03824 ABSTRACT The size at which female lobsters reach sexual maturity was determined for two populations that inhabit waters along the coast of New Hampshire. One group was captured in the Great Bay estuary, where water temperatures in the summer typically average between 1 7°C and 20°C. The other group of lobsters resided in coastal waters, near the Isles of Shoals, where the water temperature was much colder during the summer ( 1 1-15"C). Maturity was assessed using criteria that included the following: ovarian classification; abdominal width/carapace length (CL) ratio; and the size frequency distribution of berried females. All the techniques yielded similar results and consistently demonstrated that female lobsters in the estuary matured at a smaller size than those in colder coastal waters. The smallest mature females from Great Bay were 72 mm in CL. with iWr reaching se.xual maturity by 83 mm CL and all beconung mature by 89 mm CL. The smallest mature female from the Isles of Shoals area was 77 mm CL, with 50% mature by 86 mm CL and all mature by 93 mm CL. The difference in the proportion of mature lobsters in the estuarine versus coastal populations was much greater in the smaller size classes than in the larger size classes, suggesting a mi.xing of the two populations, most likely due to females from Great Bay migrating into coastal waters. KEY WORDS: cslu.irv. Hoiiniiiis itmericiiniis. lobster, sexual maturitv INTRODUCTION The American lobster. Hoinunts anicncanus (Milne-Edwards) is the most commercially valuable species harvested in the north- west Atlantic Ocean (NMFS 2002). Although lobsters are most abundant in coastal waters, estuarine populations are common and have been investigated from Canada to Massachusetts (Thomas iy6S. Thomas & White 1969. Munro & Theriiaull 19S-3. Reynolds & Casterlin 1985. Jury et al. 1995: Howell et al. 1999; Watson et al. 1999). One population that has received considerable attention is located in the Great Bay estuary in New Hampshire. Howell et al. (1999) have demonstrated that, like the lobsters in the Iles-de- l-Madeleine in Canada (Munro & Therriault 19S.3). the sex ratio is skewed toward males throughout the estuary, with the greatest proportion of male lobsters found in the portions of the estuary furthest from the coast. It has been proposed that the skewed sex ratio in the estuary is the result of the differential seasonal migra- tion of mature female lobsters out of the estuary (Watson et al. 1999). To ensure that there are enough mature females in a given lobster population, a minimum legal size has been established. This allows a given proportion of the females to reach sexual maturity and reproduce at least once befoi-e they are landed. The size at which 50% of the females from an area are mature (50% maturity) is often used as a reference point because most models indicate that when the minimum size is set at this value sufficient recruits will be produced to sustain the fishery. Currently, the minimum size limit in the inshore waters of New Hampshire is 83 mm carapace length (CL). There is a wide range of sizes over which female lobsters reach maturity. The smallest size at 50% maturity. 70 to 74 mm CL. is found in western Long Island Sound (Briggs & Mushacke 1979), and the largest size. 110 to 120 mm CL. is found in the Bay of Fundy (Templeman 1936. Groom 1977. Campbell 1983). It has been suggested that a number of different factors infiuence the size *Corresponding author. E-mail: slittle (sunh.edu at which female lobsters mature, including nutrient availability (Lawton & Lavalli 1995). fishing pressure (Polovina 1989. Landers et al. 2001 ). and temperature (Templeman 1936. Temple- man 1944. Aiken & Waddy 1980. 1986. Estrella & McKiernan 1989. Fogarty 1995). Increases in all. or any, of these factors results in a decrease in the size at which females reach sexual maturity. Temperature is thought to be the most influential of these factors because it is known to directly affect the growth rates of lobsters, with development occurring more quickly with increased temperature (Aiken & Waddy 1976). The rate of ovarian development is primarily controlled by summer water temperature, with little development occurring throughout the winter months (Templeman 1936). Thus, in areas with warmer water in the summer, lobsters reach sexual maturity at smaller sizes. Estuaries, such as the Great Bay estuary in New Hampshire, are characterized by large daily and seasonal fluctuations in tempera- ture and salinity. In the Great Bay estuary, the water temperature in the summer is approximately IO°C higher than in New Hamp- shire coastal waters (Short 1992). Given the apparent influence of water temperature on the rate of inaturation of female lobsters, we hypothesized that female lobsters in the Great Bay estuary would reach sexual maturity at a smaller size than those in coastal waters, such as near the Isles of Shoals, which are located 1 1 km away from where the Great Bay estuary empties into the Gulf of Maine (Fig. 1). To test our hypothesis, we determined the size at maturity for 92 lobsters collected in the Great Bay estuary with 106 lobsters collected near the Isles of Shoals. A comparison of the results yielded by analyzing (1) the size distribution of berried females, (2) the size of female abdomens relative to their length, and (3) the stage of eggs removed from the ovaries yielded the same pattern. Female lobsters from the estuarine site matured at a smaller size than those from the coastal site, probably due to the influence of warmer summer water temperatures on their growth and develop- ment. 857 858 Little and Watson 43 ID- 70° «■ Figure 1. The two study sites are marl\ed witli an X [Great Bay Es- tuary and Isleof Slioals (II l8°C were summed for each location by adding together the num- ber of degrees that exceeded S' C for each day of the year and summing them for the entire year. Maturity Assessments Dissections Lobsters were collected from two areas (Fig. i ) by commercial fishermen and by University of New Hampshire personnel using standard traps. The first site consisted of the upper region of the Great Bay estuary (i.e.. Great Bay. Little Bay. and the upper Pis- cataqua River), and the second site included waters near the Isles of Shoals. Lobsters were collected in 1991. 1992. 1994. and 2002. The lobsters from each site were divided into l-mm size classes rang- ing from 66 to 110 mm CL. A total of 92 lobsters were dissected from Great Bay. and a total of 106 from Isles of Shoals. Female, nonovigerous. lobsters were examined, using multiple criteria, to determine whether they were sexually mature. For each animal, the CL and the width of the second abdominal segment were measured in millimeters, and the molt stage was recorded by examining the carapace and pleopods. One pair of pleopods then was removed for examination under a dissecting microscope to determine the cement gland stage (Aiken & Waddy 1982) and whether lobsters were in a premolt condition (Aiken 1973). A small circular incision then was made just behind the eye socket to access the anterior end of one of the ovaries. Several eggs were removed, and their size range and color were recorded. An egg stage was assigned to each lobster based on criteria established by Aiken and Waddy (1980). Whether a female was sexually mature, or not. was determined using a combination of criteria, with ovarian stage as the primary tool. Any females with resorbed oocytes were considered to be mature, as these are an indication of prior spawning. Of the fe- males without resorbed oocytes, those with ovaries that were at stage 4 and higher were also considered to be mature. The size range for stage 4 ovaries is different in the spring (stage 4b) than in the fall (stage 4a) due to the timing of development, and this was taken into account. Those females with ovaries at stage 2 and below were considered to be immature. To determine the maturity of those with stage 3 ovaries, we considered cement gland stage as well as egg stage. If a female lobster with stage 3 ovaries had cement glands that were at stage 3 or greater, then the lobster was considered to be mature. To determine the size at which SC/c of the females from each area were mature, a nonlinear regression of percent mature for each l-mm CL size class was carried out using the statistical program, SYSTAT. The following equation was used: p = (1/(1 -I- exp(-bO*(L-hl ))) where p is the proportion mature, bO is the curve shape parameter, L is the carapace length, and bl is the size at 50% maturity (es- timated as a starting point for calculations by the user). The pro- gram estimated values of bO, based on the data set. until it found the best-fit curve. This resulted in sigmoid curve from which bl could be calculated with a 95% confidence interval. A statistical comparison of the regression lines that resulted from each popu- lation of lobsters was made to determine whether they were sig- nificantly different from each other. Sea Sampling Data Sea-sampling data were obtained from LIniversity of New Hampshire research traps, and during trips on commercial lobster boats in 1990 to 1993 and 2002 at each location. The data collected included CL. width of the second abdominal segment, sex. and whether females were ovigerous. A total of 8199 lobsters were examined during these sea-sampling trips. Abdominal Width A ratio of abdomen width to CL (ABD/CL ratio) was calcu- lated for each female, and these were averaged for each l-mm CL Size at Maturity of Femalh American Lobsters 859 size class. A plot then was made of CL versus this ratio for each size class. A nonlinear polynomial regression of these data was created for each site using SYSTAT. The following equation was used: ABD/CL = a + bx + cx'^2 + d\'^3. where x = CL. SYSTAT then estimated the values of a. b. c. and d to most closely fit the curve to the data. To determine the inflection point of the curve, which represents the point at which the rate of change in the ABD/CL ratio is greatest, and therefore approximates the size at which SO'/c of the feinales have reached maturity, the second de- rivative of the original equation, y = 2cx + 6dx. was calculated. That equation was then set to equal zero and was solved for x. yielding the equation x = -2c/6d. Then, the c and d values from SYSTAT were used to solve for x (the CL at 50% maturity) (Landers et al. 2001 ). The size at 50% maturity that was estimated by this method was compared with that obtained by dissection for the estuarine and coastal lobster populations to determine whether the abdominal width estimates fell within the 95% confidence intervals of the dissection estimates. months (June-August; Great Bay 995; Isles of Shoals 404). The difference in degree-days between the two sites for these 3 mo accounted for 75% of the difference in degree-days for the entire year. During this period, the mean water temperature averaged 12.5°C at Isles of Shoals and 19=C in Great Bay. Maturity Assessments Dissections Nonlinear regressions of CL versus percent mature, as deter- mined by dissections, were used to calculate the size at 50% ma- turity for each site (Fig. 3a). The size at 50% maturity for females obtained from waters near the Isles of Shoals was 85.9 mm CL (95% confidence interval 85.3-86.5; n = 106). Fifty percent of females from Great Bay were mature at 83 mm CL (95% confi- dence interval 80.6-85.4 mm; ;; = 92). A comparison of the two regressions showed that they were significantly different from each other (P < 0.001 ). The smallest mature female captured near Berried Female Size Frequency Distributions From the sea-sampling data, a size frequency distribution of berried females, as well as a plot of the overall size frequency distribution of the population was made for each area. The plots of overall size frequency were divided into the proportions that were male and female in each size class so that the proportion that was female at a given size class could be compared with the proportion of females that were berried at that same size class. For each plot the average size, the SEM. size range, and sex ratio were calcu- lated for comparison. The size distributions for the overall popu- lation and for only berried females were compared between sites using a x" test of independence. RESULTS A Comparison oj tlsluarine Versus Coastal Water Degree-Days There was a large difference between the number of annual degree-days (>8°C) in the Great Bay estuary (1532) compared to those in the waters near the Isles of Shoals (738) (Fig. 2). The greatest difference in temperature occun'ed during the summer 25 n A. .-^ ?0 u 01 73 lb m Cl 10 E ,100 mm CL, while 20% of berried females from waters near the Isles of Shoals were >100 mm CL. Nevertheless, despite these differences, the distribution of sizes of berried females was not sianificant between the two sites (P = 0.067). A 10 o Si E Great Bay Berried Females 8 6 4 ^ 2 0 II I n=98 avg. slze=84.8 ± 0.6 size range=72-107 OLnoinoLOOLnomomom i^r^coooairooo^^csicNcoco B 14 12 O 8 •5 6 J3 •* 12 Carapace length (mm) Isles of Shoals Berried Females n=152 avg. size=91.7 ±1.0 size range=77-138 llllilllllllllllllll o U-) n in o in o in CO O) O) CT) o o '- Cv] Csl CO CO Carapace length (mm) Figure 4. Size frequency histoHranis of berried females from ( A ) Great Bay and (B) Isles of Shoals (/' = 0.1)67 x" test of independence). The size range of the overall lobster population at the Isles of Shoals site was 48 to 144 mm with a mean size of 8 1 ± 0. 1 mm CL in = 3337; Fig. 5b). while the size range of the population from the Great Bay site was 38 to 1 13 mm CL, with an average size of 78 ± 0.1 mm CL (;; = 4862; Fig. 5a). The size frequency distri- bution of all lobsters was significantly different between the two sites (P < 0.05). The Great Bay population includes more small lobsters <65 mm CL (6%) than the Isles of Shoals population (3%), and the Isles of Shoals site has more legal lobsters >83 mm CL (277(1) than the Great Bay estuary (18%), particularly those >I00 mm CL (2% at Isles of Shoals, <1% at Great Bay). The most striking difference between these sites is the sex ratio, as reported by Howell and Watson (1999). The overall proportion of females at the Isles of Shoals site (64 ^c) was much larger than that in the Great Bay estuary population (35%), and this was increasingly true at larger sizes. The percentage of females in the Great Bay estuary fluctuated between 30% and 40% but dropped to <30% at sizes >82 mm CL, and no females >96 mm CL were captured in the Great Bay estuary. In contrast, the proportion of females near the Isles of Shoals increased with size class, so that 80% of the lobsters >96 mm CL were female. DISCUSSION All three methods used to assess the size at maturity of female American lobsters (i.e., egg stage, ABD/CL ratios, and benied female size frequency distributions) indicate that female lobsters from the Isles of Shoals mature at a larger size (50% = 85.9 mm CL) than those from the Great Bay estuary (50% = 83 mm CL). even though the two populations are <14 km apart. One of the major differences between these two locations is water tempera- ture. The Great Bay estuary (1532 annual degree-days) is signifi- cantly warmer than the Isles of Shoals study site (738 degree- days), with the greatest difference in temperature (74% of the total difference in degree-days) occurring in the summer months. We conclude that this increased temperature accelerates the rate of development of females in the Great Bay estuary, thereby causing them to reach sexual maturity at a smaller size. This finding once again supports the theory first put forth by Templeman (1936) that summer water temperatures determine size at maturity. The small difference in size at maturity reported is similar to a larger scale pattern observed along the entire range of the American lobster. For example, 50%' of female lobsters from Long Island Sound reach maturity at 70 to 74 mm CL (Briggs & Mushacke 1979), while those from the Bay of Fundy do not reach maturity until 1 10 to 120 mm CL (Templeman 1936, Groom 1977, Campbell 1983). While the size at 50% maturity for female lobsters from Great Bay is significantly different (P < 0.001 ) than that of females from Isles of Shoals, it is clear from the maturity ogives (Fig. 3) that the greatest difference in the two populations exists in the smaller size classes. This may be due to the mixing of mature females from Great Bay with those from the coast, as mature females migrate out of the estuary. As reported by Howell et al. ( 1999), the proportion of females in Great Bay (35%) is much smaller than that near the Isles of Shoals (64%), and this difference is most pronounced in the larger size classes. In fact, the proportion of females in Great Bay begins to decline above the 82-mm CL size class (Fig. 4), which is approximately the size at which lobsters are reaching maturity. As proposed by Watson et al. (1999) and Howell et al. (1999), it would be advantageous for females to move out of the estuary for optimal egg development and survival of larvae. While Size at Maturity of Female American Lobsters 861 500 w 400 - B o 300 o 200 100 35 500 (/I 400 - 1 00 mm CL at Isles of Shoals vs. 1 % > 1 00 mm CL in Great Bay) and more small berried females in Great Bay (SOVr <85 mm CL in Great Bay vs. 10% >85 mm CL near the Isles of Shoals). Therefore, it is likely that the size frequency distributions of ber- ried females in both study sites were not significantly different due to the low sample size of berried females in the Great Bay estuary {J! = 98). This assumption is supported, in part, by the fact that the size frequency distributions of the overall populations (;; = 4862 for the estuary) at the two sites were significantly different (P < 0.05). As with the berried female size frequency distributions, the bulk of this difference can be accounted for by the lack of large lobsters in the Great Bay estuary (100 mm). As dis- cussed earlier, these data support the hypothesis that as lobsters reach se.xual maturity they migrate out of the estuary into deeper water (Watson et al. 1999, Howell et al. 1999). While mature females probably undergo this migration shortly after reaching sexual maturity, giving rise to the skewed sex ratios observed in the estuary in size classes >80 mm CL and the low number of large berried females, male lobsters eventually move into coastal waters as well, as indicated by the scarcity of any lobsters >I00 mm CL in the Great Bay estuary. Our results indicate that while there is a small difference in the size at which females from the two sites reach maturity, that dif- ference is small, suggesting that these are not two distinct popu- lations. There appears to be mixing between the two areas, par- ticularly among the sexually mature lobsters. Thus, despite the small differences in size at maturity, it is probably not necessary to implement different management measures for each area. The size at which half of the females mature from both sites approximates the minimum size limit, and thus it appears to be appropriate to maintain adequate egg production and recruitment to satisfy the FIO requirement. ACKNOWLEDGMENTS We are deeply indebted to Dr. Michael Lesser for providing us with water temperature data for the Isles of Shoals; Jaimie Wolf for helping access the National Estuarine Research Reserve Sys- tem (NERRS) water temperature database for the Great Bay estu- ary: Chris Becker, for her help with some of the maturity dissec- tions and; Dr. Chris Neefus for his assistance with constructing the ogives and clarifying other statistical analyses. We would like to offer special thanks to both Al Vetrovs and Dr. Hunt Howell for their help collecting so much of the data, and Ed Heaphy for allowing us to collect sea sampling data aboard his vessel the Lady Martha. Finally, as with so many of our projects, we would like to thank all the students who helped collect data over the course of this project. This work was made possible as a result of grants from National Oceanic and Atmospheric Administration (Sea Grant) and the Northeast Consortium to W. H. W. It is contribution num- ber 408 in the Center for Marine Biology/Jackson Estuarine Labo- ratory series. LITERATURE CITED Aiken. D. E. 1973. Proecdysis, setal development, and molt prediction in the American lobster [Homanis americcvuis). J. Fish. Res. Board Can. 30:1337-1344. Aiken, D. E. & S. L. Waddy. 1976. Controlling growth and reproduction in the American lobster. Proc. World Maricidl. Soc. 7:415-430. Aiken, D. E. & S. L. Waddy. 1980. Reproductive biology. In: J. S. Cobb & B. F. Phillips, editors. The biology and management of lobsters, vol. I. New York: Academic Press, pp. 215-276. Aiken. D. E. & S. L. Waddy. 1982. Cement gland development, ovary maturation, and reproductive cycles in the american lobster Homarus americaiuts. J. Crustacean Biol. 2:315-327. Aiken. D. E. & S. L. Waddy. 1986. Environmental mduence on recruit- ment of the American lobster. Homarus americanus: a perspective. Can. J. Fislh Aqua!. Sci. 43:2258-2270. Briggs, P. & F. Mushacke. 1979. The American lobster in western Long Island sound. N. Y. Fish Game J. 26:59-86. Briggs. P. T. & F. M. Mushacke. 1980. The American lobster and the pot fishery in the inshore waters off the south shore of Long Island. New York. N. y. Fi.^h Game J. 27:156-178. Campbell. A. 1983. Growth of tagged lobsters {Homarus americanus) off Port Maitland. Nova Scotia 1948-1980. Can. Tech. Rep. Fish. And Aqtuii. Sci. 1232:1-10. Campbell, A., D. E. Graham, H. J. MacNichol & A. M. Williamson. 1984. Movements of tagged lobsters released on the continental shelf from Georges Bank to Baccaro Bank 1971-73. Can. Tech. Rep. Fisli. And Aqual. Sci. 1288:1-16. Campbell. A. & A. Stasko. 1986. Movements of lobsters {Homarus ameri- canus) tagged in the Bay of Fundy, Canada. Marine Biol. 92:393—404. Cooper. R. & J. U/mann. 1971. Migrations and growth of deep-sea lob- sters, Homarus americanus. Science 171:288-290. Cooper, R. & J. Uzmann. 1980. Ecology of juvenile and adult Homarus. In: J. S. Cobb & B. F. Phillips, editors. The biology and management of lobsters, vol. 2. New York: Academic Press, pp. 97-142. Ennis, G. P. 1980. Size-maturity relationships and related observations in Newfoundland populations of the lobster (Homarus americanus). Can. J. Fish. Aquat. Sci. 37:945-956. Estrella. B. T. & D. J. McKieman. 1989. Catch-per-unit-eftort and bio- logical parameters from the Massachusetts coastal lobster {Homarus americanus) resource: Description and trends. Washington. DC: U.S. Department of Commerce. National Oceanic and Atmospheric Admin- istration Technical Report NMFS SSRF 81. Fogarty, M. J. 1995. Populations, fisheries and management. In: J. Factor, editor. Biology of the lobster, Homarus americanus. New York: Aca- demic Press, pp. 1 1 1-137. Fogarty, M. J.. D. V. D. Borden & H. J. Russell. 1480. Movements of tagged american lobster. Homarus americanus. off Rhode Island. Fish- eiy Bull. 78:771-780. Groom. W. 1977. Lobster project, 1977, Grand Manan Island: Fish'N'Ships, notice to Bay of Fundy Fishermen. New Brunswick De- partment of Fishing Information, Fredericton. New Brunswick, pp. 1-10. Howell. W. H.. W. H. Watson. Ill & S. H. Jury. 1999. Skewed sex ratio in an estuarine lobster {Homarus americanus) population. J. Shellfish Res. 18:193-201. Jury. S. H., W. H. Howell & W. H. Watson, HI. 1995. Lobster movements in response to a hurricane. Mar. Col. Prog. Ser. 119:305-310. Krouse. J. 1973. Maturity, sex ratio, and size composition of the natural population of american lobster, Homarus americanus. along the Maine coast. Fisheiy Bull. 71:165-172. Landers. D. F., Jr., M. Ke.ser & S. B. Saila. 2001 . Changes m female lobster {Homarus americanus) size at maturity and implications for the lobster resource in Long Island Sound, Connecticut. Marine Freshwater Res. 52:1283-1290. Law ton. P. cS: K. L. Lavalli. 1995. Postlarval. juvenile, adolescent and adult ecology. In: J. R. Factor, editor. Biology of the lobster. Homarus americanus. New York: Academic Press, pp. 47-88. Size at Maturity of Femalk American Lobsters 863 Munro. J. & J. C. Therriault. 1983. Migrations saisonnieres du homarii {Homurus cimericuiuis) entre la cote et les lagunes cjes Iles-de-la- Madelelne. Can. J. Fish. .Aqucil. Sci. 40:905-918. National Marine Fisheries Service (NMFS). 2002. Available at: littp:// www.st.nnifs.gov/plsAvebpK/MF_ANNUAL_LANDlNGS.RESLILTS Polovina. J. J. 1989. Density dependence in spiny lobster, Fainiliriis mar- ginatus. in the Northwestern Hawaiian Islands. Can. J. Fi.sli. Aqiiat. Scl. 46:660-665. Reynolds. W. W. & M. E. Casterlin. 19S5. Vagile niacrotauna and the hydrographic environment of the Saco River Estuary and adjacent v\ a- ters of the Gulf of Maine. Hyclrobiologia 128:207-215. Short. F. T.. editor. 1992. The ecology of the Great Bay Esluary. New Hampshire and Maine: an estuarine profile and bibliography. Wash- ington. DC: Coastal Ocean Program Publication. National Oceanic and Atmospheric Administration. 222 pp. Skud. B. & H. Perkins. 1969. Size composition, sex ratio, and size at maturity of offshore northern lobsters. Washington, DC: U.S. Fish and Wildlife Service Special Scientific Report — Fisheries No. 598. Templeman, W. 1936. Local differences in the life history of the lobster {Honuiru.'i americaniis) on the coast of the Maritime Provinces of Canada. J. Biol. B,i. Can. 2:41-88. Templeman. W. 1944. Abdominal width and sexual maturity of female lobsters on the Canadian Atlantic coast. J. Fisli. Re\. Bd. Can. 6:281- 290. Thomas, M. L. H. |9(iS. Overwintering of american lobster. Hoinanis americaniis. in burrows in Bideford River, Prince Edward Island. / F/.s7i. Res. Board. Can. 25:2725-2727. Thomas. M. L. H. & G. N. White. 1969. Mass mortality of estuarine fauna at Bideford P.E.I, associated with abnormally low salinities. / Fish. Res. Board Can. 26:701-704. U/niann. J. R.. R. Cooper & K. Pecci. 1977. Migration and dispersion of tagged American lobsters. Honuirus americaniis. on the southern New England continental shelf Washington. DC: National Oceanic and At- mospheric Administration Technical Report NMFS SSRF-705. Watson. W. H. III. A. Vetrovs & W. H. Howell. 1999. Lobster movements in an estuary. Marine Biol. 134:65-75. Journal of Shellfish Research. Vol. 22, No. 3. S65-87I. 200,^. GREEN CRAB (CARCINUS MAENAS LINNAEUS) CONSUMPTION RATES ON AND PREY PREFERENCES AMONG FOUR BIVALVE PREY SPECIES KELLY C. FALACIOS'* AND STEVEN P. FERRARO" t College of Oceanic and Atmospheric Sciences. Oregon Stale University. 104 Ocean Admin Bidg.. Corvallis. Oregon 97331: and 'U.S. Enviroiuuental Protection Agency. 2111 S.E. Marine Science Drive. Newport. Oregon 97365-5260 ABSTRACT Laboratory experiments were conducted to determine green crab. Carciinis maenas. consumption rates on and prey preferences among lour bivalve species: Olympia oysters (Ostrea concbaphila Carpenter), Japanese littleneck clams {Veiienipis philippinanim A. Adams and Reeve), bent-nosed macoma clams (Macoina iiasiiia Conrad), and California softshell clams {Crypiomya californicu Conrad) of different sizes. The bivalve size classes tested ranged in length from 10-14 mm to 33-37 mm. Consumption rate and prey preference experiments were conducted by allowing one starved (48 h) green crab (55-75 mm carapace width) to feed ad libitum on bivalve prey for 16 h. All tests were conducted in 38-L aquaria containing sand substrate 13 cm deep. A total of either 60 or 30 individuals of each prey species were offered without replacement in each test. Mean green crab consumption rates varied depending upon the prey species and size class. For bivalve prey of similar size, Olympia oysters were consumed at a higher rate than bent-nosed macoma clams and Japanese littleneck clams, while Olympia oysters and California softshell clams were consumed at about the same rate. Green crabs preferred Olympia oysters to both bent-nosed macoma clams and Japanese littleneck clams by ratios ranging from 2:1 to 28:1. depending upon the prey size. Small California softshell clams were preferred to small bent-nosed macoma clams by a ratio of 8:1. The mean total biomass of Olympia oysters and bent-nosed macoma clams eaten was 2.31 g • d~'. Our results show that green crabs are capable of consuming large quantities of all four bivalve species tested, and that on bare sand substrate Olympia oysters are at greater risk of green crab predation than bent-nosed macoma clams and Japanese littleneck clams, and California softshell clams are at greater risk than bent-nosed macoma clams. KEY WORDS: Carciiuis inaeiia.'i: consumption rates: Ciyptonna californiea: Macoma na.siiki: Ostrea coiichaphila; prey preference; Venerupis plulippinanim. INTRODUCTION The green crab, Curciniis maenas. a species native to Europe, has recently invaded Pacific Northwest (PNWl estuaries (Dum- bauld & Kauffman 1998, Hunt et al. 1998, Yumada 2001 ). Green crabs prey heavily upon bivalves (Ropes 1968. Davies et al. 1980, Parache 1980, Dare & Edwards l98l,Elner 1981, Dare et al. 1983, Grosholz & Ruiz 1995. Mascaro & Seed 2000). Ropes (1968) and EIner (1981) attributed the decline in the softshell clam {Mya areuaria Linnaeus) fishery along the northeastern coast of the United States to invasive green crabs. Studies by Grosholz and Ruiz (1995. 1996) suggest and Jamieson et al. (1998) have pre- dicted that invasive green crabs could impact bivalve populations in PNW estuaries. The objectives of this study were to estimate green crab con- sumption rates on four bivalve prey species inhabiting PNW es- tuaries and to determine green crab prey preferences among these prey species under controlled laboratory conditions. Consumption rate experiments were conducted on one to three size classes of the four bivalve species to determine the effect of prey species and prey size on consumption rates. Prey preference experiments were conducted with two or three bivalve species of similar size. The bivalve species tested were the Olympia oyster {Ostrea con- chaphila). the Japanese littleneck clam [Venerupis plulippinanim). the bent-nosed macoma clam (Macoma nasiita). and the California softshell clam iCrypiomya californiea). Olympia oysters are native to and were once widely distributed throughout PNW estuaries but now. probably primarily because of overharvesting (Baker 1995, Robinson 1997. Cook et al. 2000). only remnant natural and *Current address: Elkhorn Slough Foundation. P.O. Box 267, Moss ing. CA 95039. tCorresponding author. Land- culture populations remain. Bent-nosed macoma clams and Cali- fornia softshell clams are common native PNW bivalves. The Japanese littleneck clam is a nonindigenous species that has been naturalized and is cultured in PNW estuaries for its commercial value. MATERIALS AND METHODS Green crabs used in our experiments were collected from Yaquina Bay. OR (44 °37 'N, 124 °02 'W) with crab traps de- ployed subtidally and baited with salmon scraps. Prior to their use in an experiment, the crabs were fed a standardized diet of squid while being held submerged in individual containers in now- through water tables in the U.S. Environmental Protection Agency Laboratory at the Hatfield Marine Science Center. Newport. OR. The flow-through water system supplies fresh filtered or unfiltered seawater from Yaquina Bay. Only intermolt crabs were used as experimental subjects to avoid possible behavioral differences as- sociated with molting. The size range of green crabs in our ex- periments (55- to 75-mm carapace width. CW) reflected the size range of crabs collected in the field. Olympia oysters and Japanese littleneck clams used in our ex- periments were obtained from the Olympia Oyster Company. Shel- ton. WA. Bent-nosed macoma clams and California softshell clams used in our experiments were collected from Yaquina Bay. Experimental bivalves were measured and divided into size classes (Table I ). Shell length was measured as the distance from the hinge (umbo) to the furthest edge of the shell. Bivalves were held in the laboratory in water tables supplied with unfiltered. flow- through seawater prior to their use in our experiments. The bi- valves appeared healthy and did not lose weight or die while being held. 865 866 Palacios and Ferraro TABLE 1. Bivalve species and prey size classes used in the consumption rate" and prey preference'' experiments Size (Class) Oljmpia Oyster 19-23 mm' California Softshell Clam Bent-Nosed Macoma Clam Japanese Littleneck Clam Small (1) Medium (II) Large (111) 1(;H4 mm"' 26-30 mm"" 33-37 mm 12-15 mm-"" 18-21 mm"" ,ih 14-18 mm" 22-26 mm '" Consumption Rate and Prey Preference Experimental Protocol and Data Analysis Both consumption rate and prey preference laboratory experi- ments were performed in 38-L (50 cm x 25 cm x 30 cm) glass aquaria placed in flow-through water tables. Sand, that had been air-dried for at least five days and sieved through a 1 .0-mm mesh screen, was placed in each aquarium providing 13 cm of substrate depth. Each aquarium was continuously supplied with fresh, fil- tered Hatfield Marine Science Center seawater with out flow near the top through a mesh cover. At the short (25 cm) end of each aquarium a clear plastic partition was installed about 10 cm into the aquaria to separate the green grab predator from the bivalve prey during a 24-h pre-e.xperimental acclimation period. This setup created staging and feeding areas in each aquarium. Semiopaque visual barriers were placed on the vertical sides of each aquarium to minimize external influences on predator and prey behavior. Seawater temperature (range, I2-16°C) and salinity (range, 32-33 ppt) were monitored during every experiment. The light regimen was fixed using a timer and matched the natural daylight regimen (I4L:I0D). The consumption rate and prey preference tests were each of 16-h duration, beginning with 10 h of darkness followed by 6 h of light. Prior to each experiment, each experimental crab was starved for a total of 48 h: 24 h in its holding container plus 24 h in the staging area. The bivalve prey were measured and placed in the feeding area of the aquaria and allowed to acclimate to the test conditions at least 18 h before the beginning of each test. The same basic protocol was used in both the consumption rate and prey preference experiments. One experimental crab was used in each test, and each crab was used only once. After 24 h in the staging area, partitions between the staging and feeding area were removed and crabs were allowed to feed acl libitum on one bivalve prey species (consumption experiments) or two or three bivalve prey species (preference experiments) without replacement for 16 h. At the end of each test all bivalves were removed from each aquarium and whole, live bivalves were counted and remeasured. The number of individuals of each species eaten was determined as the number originally available minus the number of whole, live individuals remaining at the end of each test. In the consumption rate tests, the feeding area originally contained 60 bivalves (prey) of the same species and the same size class. In the prey preference tests, the feeding area originally contained either 60 or 30 bi\ alves of similar size of each of two or three species. The total number of tests performed was constrained by bivalve prey availability. Due to laboratory space limitations or prey availability, a maximum of twelve tests could be run at the same time. Tests were randomly assigned among aquaria, and each experiment was completed within one month. Seven replicated (;i = -1-8) consumption rate experiments and five replicated (;; = 3-8) pairwise and one rep- licated {n = 4) three-way prey preference experiments were con- ducted (Table 1). Differences in mean consumption rates (number bivalves eaten in 16 h) between two prey species or size classes were tested by /-tests after confirming the parametric assumptions of normality and homogeneity of variances (Sokal & Rohlf 1995). Differences in mean consumption rates among three prey species or three size classes were tested by analysis of variance and Tukey's test. or. when the data failed to meet the parametric assumptions, by an approximate test of the equality of means using the Games and Howell method (Sokal & Rohlf 1995). Prey preference was in- ferred using single classification G-tests with Williams" correction (Sokal & Rohlf 1995) by determining if the observed proportion of prey species eaten differed from the expected ratio (1:1 and 1:1:1 for two and three prey species, respectively) if there was no pref- erence. Bivalve Biomass Estimates Meat weight-length relationship models for Olympia oysters and bent-nosed macoma clams were developed by regressing the logarithms of the biomass (g. flesh dry wt) of 50 individual Olym- pia oysters (18-38 mm shell length) and 30 individual bent-nosed macoma clams (12-22 mm shell length) on shell length (mm). We did not have a sufficient number of indi\ iduals of different shell lengths to generate biomass-length relationships for Japanese littleneck clams and California softshell clams. The flesh of each bivalve was removed from the shell, placed in a pre-weighed dry- ing tin. and dried in an oven for 48 h at 70°C. Upon removal from the oven, the tins were kept in a dessicator. allowed to cool, and re-weighed. Flesh dry weight was determined by subtracting the weight of the drying tin from the total weight (dried flesh -i- drying tin). Biomass-length regression models were used to convert the known length of individual Olympia oysters and bent-nosed ma- coma clams eaten in our consumption rate and prey preference experiments to biomass. The individual biomass estimates were summed to estimate the total bivalve biomass of each species consumed in each test. ANOVA was used to test for differences among the mean total bivalve biomass eaten in our Olympia oyster and bent-nosed macoma clam consumption rate and prey prefer- ence experiments. RESULTS Consumption Rate Experiments The number of bivalve prey eaten in our consumption rate tests ranged from zero large Japanese littleneck clams to fifty-four small California softshell clams. Mean (SE) green crab consumption rates and results of analysis of variance comparing mean consump- tion rates across prey species within a size class and across dif- ferent size classes within prey species are presented in Table 2. The rank order of green crab mean consumption rates for bivalve Green Crab Feeding on Four Bivalves 867 TABLE 2. Prey species," prey size class,'' sample size (;;). and mean (SK) number of bivalves eaten by one 48-h starved green crab in 16 h and ANOVA results in tests of five hypotheses of no sifinillcanl ditTirences betHeen/among mean consumption rates for different prey species of similar size [H|, ( la-lc)l and for different size classes of the same prey species [H,, (2a and 2b)| Prey Species Size Class OO I 00 II 00 ni BN I BN n cs I JL in No. Consumed Mean (SE) Consumption Rate (per day) H„ (la) H„ H„ H„ H„ (lb) do (2a) (2bl A A A A B 8 41.5(5,24) 62.3 A 17.4 (L74) 26.1 10.3 (0.82) 15.5 17.7(2.61) 26.6 B 7.4(1.56) 11. 1 43.4 (2.99) 65.1 A 1.8(0.49) 2.7 B A B Different letters (A. B) in the columns nidicate statistically significant different iP < 0.05) means. °00 = Olympia oyster; BN = bent-nosed macoma clam: CS = California softshell clam; JL = Japanese littleneck clam. "See Table 1. species by size class (I, II, III; see Table 1 ) was Olympia oyster (I) = California softshell clam (I) > bent-nosed macoma clam (I), Olympia oyster (II) > bent-nosed macoma clam (II). and Olympia oyster (III) > Japanese littleneck clam (III). The rank order of green crab mean consumption rates for different size classes of the same bivalve species were Olympia oyster (1) = Olympia oy.ster (11) > Olympia oyster (III), and bent-nosed macoma clam (I) > bent-nosed macoma clam (II). Prey Preference Experiments When two bivalve prey species were present, green crabs ate, on average. 16x more small Olympia oysters than small bent- nosed macoma clams. 2x more small Olympia oysters than small Japanese littleneck clams. 8x more small California softshell clams than small bent-nosed macoma clams. 3x more medium Olympia oysters than medium bent-nosed macoma clams, and 28x more large Olympia oysters than large Japanese littleneck clams (Table 3). When three bivalve prey species were present, green crabs ate, on average, small California softshell clams, small Olympia oysters, and small bent-nosed macoma clams in a 6:4:1 ratio (Table 3). The proportions of the prey species eaten were all significantly different from 1:1 or 1:1:1 (Table 3). indicating strong green crab prey preferences among the bivalve species tested. Bivalve Biomass Estimates Regressions of the logarithm of Olympia oyster and bent-nosed macoma clam flesh dry weight on their shell lengths (mm) were: log (Olympia oyster dry wt. g) = -2.40 + 0.048 Olympia oyster shell length, r = 0.79. P < 0.001. and log (bent-nosed macoma clam dry wt, g) = -2.26 -I- 0.077 bent-nosed macoma clam shell length, ;- = 0.95. P < 0.001. Using the regression equations above, we converted the shell lengths of the Olympia oysters and bent-nosed macoma clams eaten in our consumption rate and prey preference tests to indi- vidual oyster or clam biomass. Individual biomass estimates of consumed prey were then summed to estimate the total biomass of Olympia oysters and bent-nosed macoma clams eaten in each test. There were no significant differences (ANOVA. P > 0.05) among the mean total biomass of bivalves eaten in our Olympia oyster and bent-nosed macoma clam consumption rate and prey preference experiments (Table 4). The grand mean total biomass of Olympia oysters and bent-nosed macoma clams eaten in these experiments was 1.54 (±0.10) g • 16 h"'. which extrapolates to 2.31 g ■ d"'. DISCUSSION Consumption Rates This is the first published report of green crab consumption rates on Olympia oysters, bent-nosed macoma clams, and Califor- nia softshell clams. Parache ( 1980) previously reported green crab consumption rates on Japanese littleneck clams. For a given bi- valve prey size, green crab (55-75 mm CW) consumption rates were highest for California softshell clams and Olympia oysters, intennediate for bent-nosed macoma clams, and lowest for Japa- nese littleneck clams (Table 2). smaller individuals of each prey species were consumed at a faster rate than larger individuals (Table 2), and the mean total biomass of Olympia oysters and bent-nosed macoma clams consumed was 2.31 g • d~' (Table 4). Crab consumption rates on bivalves can vary depending on the crab (species, size, hunger level, and health), the bivalve (species, TABLE 3. Prey preference ratios of green crabs for t«o or three bivalve prey species" of comparable size and results of G-tests comparing the observed versus the expected ratios if there was no prey preference Observed Size Preference Expected Ratio If G Class" Ratio n No Preference Statistic P 16 00:1 BN 3 1:1 94.5 <0.001 2 OO: 1 JL 5" 1:1 29.7 <0,001 8CS:1 BN 6 1:1 118 <0.001 II 3 OO: 1 BN 4 1:1 34.9 <().001 111 28 00:1 JL 8 1:1 82.4 <0,001 6CS:4 00:1 BN 4 1:1:1 93.2 <().()01 All tests were replicated (/i) and 16-h duration. " OO = Olympia oyster; BN = bent-nosed macoma clam; CS = Cali- fornia softshell clam; JL = Japanese littleneck clam. " See Table 1 . ■^^ Thirty individuals of each bivalve prey species were originally available in each replicate test. In all other experiments. 60 individuals of each bivalve prey species were originally available in each replicate test. 868 Palacios and Ferraro TABLE 4. Mean total bivalve biomass (g) consumed b> one 48-h starved green crab in 16 h in consumption rate and prev preference experiments with Olympia oysters (OO) and bent-nose macoma clams (BNl Biomass Prey Size Consumed (gl Species Class'' Exp eriment"" /( Mean (SE) OO I C 4 1.70(0.183) OO n C 8 1.54(0.350) OO m C 8 2.01 (0.235) BN I C 8 1.10(0.116) BN II C 8 1.35(0.759) 00 + BN 1 P 3 1.55(0.124) 00 + BN II P 4 1.63(1.067) "See Table 1. •"C = Consumption; P = Prefere nee. size, density, siiell strength, and morphology), and the experimen- tal conditions (water temperature, duration, with or without prey refuge, with or without prey replacement, etc) under which the rates are measured (Jubb et al. 1983, Arnold 1984, Sanchez- Salazar et al. 1987. Juanes 1992. Ebersole & Kennedy 1995, Mas- caro & Seed 2000. Yamada 2001). Our experiments were con- ducted in aquaria with sand substrate to approximate prime oyster and clam culture habitat in the field. Further research is needed to estimate green crab consumption rates and prey preferences on bivalves in other PNW estuarine habitats (e.g.. salt marsh, eelgrass, burrowing shrimp). We attempted to minimize potential confound- ing variables in our experiments, and to obtain near maximum estimates of average green crab consumption rates under environ- mental conditions as similar as possible to those in the field. We only experimentally varied the bivalve prey species and size (Table 1 ). The predator crab species, size (55-75 mm CW), num- ber (one), and initial hunger level (48 h starved) were constant, and environmental conditions (water temperature, salinity, photope- riod, etc.) were held constant at levels matching local field condi- tions. The bivalve prey were placed on sand substrate ( 1 3 cm deep) and given time ( 18 h) to acclimate and orient themselves naturally on and in the sediment. Thus relative differences in predator for- aging times for the different prey species are subsumed in our consumption rates. Sixty bivalve prey were available at the begin- ning of each consumption rate test, and. on average, twenty whole, live bivalve prey remained at the end. Mean bivalve prey densities, therefore, decreased but remained high (446-131 m"") throughout the tests, thus minimizing the effect of decreasing prey density on crab consumption rates. Bivalves eaten during the experiments were not replaced as newly introduced bivalves would tend to be more vulnerable to predation than the original bivalves that had time to acclimate and bury. Since starved green crabs consume prey more rapidly in the first three feeding hours (Jubb et al. 1983). our tests were run for 16 h to better refiect longer term, average rates. Our experimental light regimen ( 10D:6L) approximates the green crab's natural foraging cycle (Klein Breteler 1976, Elner 1981). In Table 5 we summarize the experimental conditions and results of this and other green crab consumption rate studies. In Parache"s (1980) laboratory experiments, green crabs (50- 69 mm CW) consumed 0.2-0.7 Japanese littleneck clams (23.5 mm) • d"', whereas in our experiments green crabs (55-75 mm CW) consumed Japanese littleneck clams (22-26 mm) at an aver- age rate of 2.1 clams ■ d"'. We used one crab in each test as compared with Parache's three, and our prey densities were higher (Table 5). Aggressive competition is high among green crabs, especially in the presence of food (Kaiser et al. 1990; Sneddon et al. 1997). and green crab consumption rates decrease with decreas- ing prey density (Walne & Dean 1972). Our estimates of green crab consumption rates on Japanese littleneck clams, therefore, better reflect rates when green crab densities are low and clam densities are high, whereas Parache's ( 1980) estimates may reflect rates when green crab densities are higher and clam densities are somewhat lower. Green crabs consumed 19-37 mm Olympia oysters in our ex- periments al an average rate of 15-62 d"' (Tables 2 and 5). This consumption rate is much higher than the £ 2.75 d"' reported by Dare et al. ( 1983) for 19-37 mm Pacific oysters (Cnissostrea gigas Thunberg) and the 1.1 d"' reported by Mascaro and Seed (2000) for 5—40 mm edible oysters iOstrea edulis Linnaeus) (Table 5). Differences in the experimental conditions (Table 5) preclude di- rect comparisons of these results. Nevertheless, such large differ- ences in consumption rates suggests that green crabs can eat Olym- pia oysters at a faster rate than other oysters, perhaps due to dif- ferences in shell strength or morphology (Mascaro & Seed 2000). Green crabs consumed bent-nosed macoma clams and Japanese littleneck clams at a slower rate than similar sized Olympia oysters and California softshell clams (Table 2). Bent-nosed macoma clams and Japanese littleneck clams in our experiments buried into the sediment, some along the sides of the aquaria where they were observed at the maximum sediment depth of 13 cm. California softshell clams buried just below the surface, and Olympia oysters remained on the surface. Slower green crab consumption of deeper burying bivalve species supports the premise that burying provides greater refuge from predation. Blue crab (Callinecles sapidiis Rathbun) consumption rates were also less on deeper-burying bi- valves (Blundon & Kennedy 1982, Ebersole & Kennedy 1995). Green crab consumption rates on bent-nosed macoma clams were less than those on similar size Olympia oysters on a numeri- cal basis (Table 2), but not significantly different on a total bio- mass basis (Table 4). These results suggest that crab consumption rales, measured as number of prey eaten per hour, may have been largely a function of the crab's hunger level. Initial hunger levels of our experimental crabs were the same (48-h starved). But as crabs ate prey, their hunger levels must have decreased, and, logi- cally, the rate of decrease would be more closely related to bio- mass of prey than number of prey consumed. Our length-biomass regressions (see Results. Bivalve Biomass Estimates) show that bent-nosed macoma clams have a greater flesh biomass than Olvmpia oysters of the same length. The hunger level of a starved green crab feeding on bent-nosed macoma clams, therefore, would decrease at a faster rate than if the same crab fed on the same number of similar size Olympia oysters. The total biomass of Olympia oysters and bent-nosed macoma clams eaten in our ex- periments (Table 4) exceeded the approx. 0.8 g of dry blue mussel (Mytiliis edulis Linnaeus) flesh required to satiate green crabs (70- 75 cm CW) (Jubb et al. 1983). It, therefore, appears that the crabs in our consumption rate experiments ate to satiation, but that more individual Olympia oysters than bent-nosed macoma clams of the same size had to be eaten to reach satiation and to maintain ap- proximately the same hunger level thereafter. Green crab consumption rates on larger Olympia oysters were less than those on smaller Olympia oysters, and green crab con- sumption rates on larger bent-nosed macoma clams were less than Green Crab Feeding on Four Bivalves 869 TABLE 5. (ireen crab consumption rate studies on bivalve prey \\ith rales standardized tu mean number consumed in 24 h Crab Size Prey Prey Size Consumption Tank Size Number Prey Prey Time Sediment Hepth Citation (mm) Species" (mm) Rale (24 h ') (cm) Offered Replaced Id) ( cm ) Palacios & Ferraro 55-75 Oc 19-23 62.3 50 X 25 60 No 0.7 13 (This study) 55-75 Oc 26-30 26.1 50x25 60 N(i 0.7 13 55-75 Oc 33-37 15.5 50x25 60 No 0.7 13 55-75 Mn 12-15 26.6 50x25 60 No 0.7 13 55-75 Mn 18-21 11.1 50x25 60 No 0.7 13 55-75 Cc 10-14 65.1 50x25 60 No 0.7 13 55-75 Vp 22-26 2.7 50 X 25 60 No 0.7 13 Walne & Dean (1972) 60-69 Mm 14-20 3.22 27 X 18 15 Yes 7 0 60-69 Me 24-32 4.83 27 X 18 15 Yes 7 0 Elner & Hughes (1978) 60-65 Me 5-35 13 43x23 90 Yes 11 0 Parache (1980)'' 50-59 Vp 8 1.71 50 X 50 50 No 8 8-10 50-59 Vp 14 2.78 50x50 50 No 8 8-10 50-59 Vp 23.5 0.17 50x50 30 No 8 8-10 60-69 Vp 8 2.88 50x50 50 No 8 8-10 60-69 Vp 14 5.88 50x50 50 No 8 8-10 60-69 Vp 23.5 0.71 50x50 30 No 8 8-10 Dareetal. (1983) 65 Cg 19-23 2.75 28x18 2-10 Yes 4-10 0 65 Cg 26-30 1.75 28x18 2-10 Yes 4-10 0 65 Cg 34-37 1.00 28x18 2-10 Yes 4-10 0 Jensen & Jensen (1985) 6 Ce 2-6 7.00 7x7 30 No 1 3 Sanchez-Salazar 65-70 Ce 13 3 @ 9°C 50x30 40 Yes 5-10 5 et al. (1987) 9 @ 15°C Mascaro and Seed 55-70 Me 5-+0 12.0 30 X 20 35 Yes -10 0 (2000) 55-70 Oe 5-40 1.1 30x20 35 Yes -10 0 55-70 Cg 5-40 2.1 30 x 20 35 Yes -10 0 55-70 Ce 5-40 10.1 30 X 20 35 Yes -10 0 ^ Oc = Ostrea conchaphila: Mn = Macoma nasuta: Cc =Ciyptomya califonica; Vp =Venerupis (previously, Ruditapes) phUippinarum): Mm = Mercenaria mercenaria (Linnaeus); Me = Mytilus ediilis: Cg = Crassosirea gigas: Ce = Cerastodenna ediile: Oe = Ostra eduHs. ^ Parache's experiments had three crab predators per tank. All other reported experiments had one crab per tank. those on smaller bent-nosed macoma clams (Table 2). There was no significant difference, however, in the mean total biomass of larger and smaller prey consumed (Table 4). Dare et al. (1983). Jubb et al. (1983), and Sanchez-Salazar et al. (1987) also found prey species specific, inverse relationships between green crab consumption rates and bivalve prey size within the range of con- sumable prey sizes. Such relationships probably hold generally because more time, on average, is needed to handle and eat larger bivalve prey (Jubb et al. 1983; Sanchez-Salazar et al. 1987). while crabs need to eat fewer individuals to reach satiation when con- suming larger prey. Green Crab Prey Preferences Green crabs exhibited prey species preferences based on the proportional number of similar size individuals eaten in our ex- periments. In tests with two prey species, Olympia oysters were preferred to bent-nosed macoma clams and Japanese littleneck clams of similar size, and small California softshell clams were preferred to small bent-nosed macoma clams (Table 3). In a three- way test, small California softshell clams, Olympia oysters, and bent-nosed macoma clams were preferred in a ratio of 6:4; 1 (Table 3). These results indicate that, on bare sand substrate, Olympia oysters are more susceptible to green crab predation than bent- nosed macoma clams and Japanese littleneck clams, and California softshell clams are more susceptible to green crab predation than bent-nosed macoma clams. Factors that influence crab prey preferences include the prey encounter rate, the time and energy the crabs expend to handle and eat the prey, and the nutrient and energetic value of the prey (Elner & Hughes 1978; Ebersole & Kennedy 1994). Our study was not designed to determine the relative importance of these factors. However, because green crabs are tactile and chemosensory hunt- ers (Cohen et al. 1995), and their prey preference ratios (Table 3) were almost always consistent with the bivalve prey burial depths observed in our experiments (Olympia oysters < California soft- shell clams < bent-nosed macoma clams = Japanese littleneck clams), prey encounter rates were probably an important factor. A summary of the results and experimental conditions under which our study and other studies on green crab prey preferences on bivalves is presented in Table 6. Jensen and Jensen (1985) found that juvenile green crabs preferred small cockles (Cerasto- denna ediile Linnaeus) to small Baltic macoma clams (Macoma balthica Linnaeus), and they concluded that juvenile green crabs could be responsible for the decline of small cockles and changes in benthic macrofaunal diversity in the Wadden Sea. Cohen et al. (1995) found that green crabs preferred brackish-water corbula clams (Potainocorbula amurensis Schrenck) to Japanese littleneck clams and mussels (Mytilus spp.) of similar size. Cohen et al. (1995) speculated that green crab predation might lead to a de- crease in brackish-water corbula clams and an increase in benthic diversity in San Francisco Bay. Grosholz and Ruiz ( 1995) showed that green crabs preferred larger individuals of two Niitricola (pre- viously, Transennella) clam species, and they predicted that green 870 Palacios and Ferraro TABLE 6. Green crab prey preference studies with bivalve prey and results presented as ratios Citation Crab Size (mm) Prey Species" Prey Size (mm I Preference Ratio Tank Size (cm or L) Number Prey Offered" Time (days I Sediment Depth ( cm ) Palacios & Ferraro 55-75 Oc:Mn 12-23 16:1 50 X 25 (This study) 55-75 Oc:Mn 18-30 3:1 50 X 25 55-75 Oc:Vp 14-23 2:1 50 X 25 55-75 Oc:Vp 22-37 28:1 50 X 25 55-75 Cc:Mn 10-15 8:1 50 X 25 55-75 Cc:Oc:Mn 10-23 6:4:1 50x25 Jensen & Jensen (1985) 11 Ce:Mb 2-6 7:1 7x7 Cohen et al. (1995) 55-60 Pa:Ms 10-20 1:1 25 X 25 55-60 Pa:Vp 10-20 3:1 25 X 25 55-60 Ms:Vp 1(3-20 16:1 25x25 55-60 Pa:Vp 10-20 8:1 25 X 25 55-60 Pa:Ms 10-20 5:1 25x25 Grosholz & Ruiz (1995) 44-61 Nc«l&>3): <1 & >3 52(Nc&Nt>3): 40L Nt«l cS:>3) 1 (Nc&Nt <1) 120 120 60 120 120 180 30 30 30 30 30 30 40 0.7 0.7 0.7 0.7 0.7 0.7 1 0.08 0.08 0,08 0.08 0.08 2 13 13 13 13 13 13 3 0 0 0 6 6 0 "00 = Osrrea concluiphilir. Mn = Macomu nasuur. Vp = Venerupis (previously, Riiditapes) philippinarum: Cc = Cnproiiixa culifitnica: Ce = Cerasroderma editle: Mb = Macoiiw halrhica: Pa = Potamocorbula amuiensis: M^ = A/\7(/h,v spp.; Nc = Niitrlcola {pre\iouf,\y TranseuelUi) ccinfiisa (S. Gray). Nt = Niitricola (previously. Tmnsenella) kinrilla (Gould). " Numbers are totals for all hivaUe prey species, and all studies were conducted without prey replacement. crabs will impact Niiuicohi clain populations and benthic commu- nities in West Coast embayments. The impact of green crabs on bivalve populations in PNW estuaries will depend upon niany factors, including green crab abundance, distribution, predators, competitors, and recruitment success. Currently, poor recruitment appears to be the main factor limiting green crab abundances in PNW estuaries (Yamada 2001 ), Intra- and interspecific predation and competition for food and/or shelter could also limit their abundance and spatial distribution (McDonald et al, 1998. Moksnes et al, 1998, Yamada 2001. Jensen et al. 2002). If green crab populations increase, however, their potential direct impact is high, as they are capable of consuming large quantities of ecologically and economicall\ important PNW bivalve species (Tables 2 and 4), In bare sand habitat, at or near surface dwelling bivalve species (e,g,, Olympia oysters. California softshell clams) are probably at greater risk of green crab predation than deeper dwelling species (e.g.. bent-nosed macoma clams. Japanese littleneck clams; Table 3), Heavy green crab predation on bivalves could also have substantial indirect effects on benthic niacrofaunal community structure and composition (Griisholz et al. 2000). ACKNOWLEDGMENTS This research was conducted under a U.S. EPA National Net- work for Environmental Management Studies Fellowship (No, U-9 1 529 1 -01-0) to KCP, The authors thank C, Hunt, D. Berube. and Z. Bassett for field and lab assistance, the Olympia Oyster Company for donating Japanese littleneck clams, and S, Yamada for reviewing an earlier draft of the manuscript. The U.S. EPA, Office of Research and Development funded this research, which has been subjected to agency review and approved for publication. Mention of trade names or commercial products does not consti- tute endorsement or recommendation for use. LITERATURE CITED Arnold, W. S. 1984. The effects of prey size, predator size, and sediment composition on the rate of predation of the blue crab Callinecres sapi- dus Rathbun. on the hard clam. Merccnarin mercenaria (Linne). J. Exp. Mar. Biol. Ecol. 80:207-219. Baker. P. 1995. Review of ecology and fishery of the Olympia oyster, Ostrea lurida. with annotated bibliography. ,/. Shellfish Res. 14:501- 518. Blundon. J. A. & V. S. Kennedy. 1982. Refuges for intaunal bivahes from blue crab, Callinectes supidiis ( Rathbun ), predation in Chesapeake Bay. J. E.xp. Mar. Biol. Ecol. 5:67-82. Cohen, A. N,. J. T, Carlton & M. C. Fountain. 1995. Introduction, dispersal and potential impacts of the green crab Carcinus maenas in San Fran- cisco Bay, California. Mar. Biol. 122:225-237. Cook, A, E,. J, A, Shaffer. B. R. Dumbauld & B. E. Kauffman. 2000. A plan for rebuilding stocks of Olympia oysters (Ostreola conchaphila. Carpenter 1857) in Washington state. / Shellfish Res. 19:409^12. Dare, P. J. & D. B. Edwards. 1981, Underwater television observations on the intertidal movements of shore crabs. Carcinus maenas. across a miidtlat. J. Mar. Biol. Assoc. U. K. 61:107-116. Dare, P. J., G. P. Davies & D. B. Edwards. 1983. Predation on juvenile Pacific oysters (Crassostrea gigas) and mussels (Mytilus ednlis L.) by shore crabs {Carcinus maenas/L. Thunberg). Fisheries Research Tech- nical Report. Number 73. Ministry of Agriculture. Fisheries and Food Directorate of Fisheries Research. Lowestoft, Great Britain. Davies, G, P., P. J, Dare & D. B. Edwards. 1980. Fenced enclosures for the protection of seed mussels [Mytilus edulis L.) from predation by shore crabs (Carcinus maenas L.). Fisheries Research Technical Report. Number 56. Ministry of Agriculture, Fisheries and Food Directorate of Fisheries Research, Lowestoft, Great Britian. Dumbauld, B. R. & B. E. Kauffman. 1998. The nascent invasion of green crab (Carcinus maenas) in Washington State coastal estuaries. / Shell- fish Res. 17:1267. Ebersole. E, L. & V. S. Kennedy. 1994. Size selection of Atlantic rangia clams. Rangia cuneata. by blue crabs. Callinectes sapidus. Estuaries 17:668-673. Green Crab Feeding on Four Bivalves 871 Ebersole, E. L. & V. S. Kennedy. 1995. Prey preferences of blue crabs CalHnectes sapidiis feeding on three bivalve species. Mar. Ecol. Prog. Ser. 118:167-177. EIner. R. W. 1981. Diet of the green crab. Carcinu.s maenas (L.) from Port Hehert. Southwestern Nova Scotia. / Shellfish Res. 1:89-94. Elner. R. W. & R. N. Hughes. 1978. Energy maximization in the diet of the shore crab, Carcinus maenas. J. Aiiim. Ecol. 47:103-1 16. Grosholz. E. D. & G. M. Ruiz. 1995. Spread and potential impact of the recently introduced European green crab. Carcinus maenas. in central California. Mar. Biol. 122:239-247. Grosholz. E. D. & G. M. Ruiz. 1996. Predicting the impact of introduced marine species: lessons from the multiple invasions of the European green crab. Biol. Consen: 78:59-66. Grosholz, E. D., G. M. Ruiz. C. A. Dean. K. A. Shirley, J. L. Maron & P. G. Connors. 2000. The impacts of a nonindigenous marine predator in a California bay. Ecology 81:1206-1224. Hunt, C, S. B. Yamada & N. Richmond. 1998. The arrival of the European green crab. Carcinus maenas. in Oregon estuaries. J. Shellfish Res. 17:1265. Jamieson, G. S., E. D. Grosholz. D. A. Armstrong & R. W. Elner. 1998. Potential ecological implications from the introduction of the European green crab. Carcinus maenas (Linneaus), to British Columbia. Canada. and Washington. USA. J. Nail. Hist. 32:1587-1598. Jensen, G. C. P. S. McDonald & D. A. .Armstrong. 2002. East meets west: competitive interactions between green crab Carcinus maenas. and native and introduced shore crab Hemigrapsus spp. Mar. Ecol. Prog. Ser. 225:251-262. Jensen, K. T. & J. N. Jensen. 1985. The importance of some epibenthic predators on the density of juvenile benthic macrofauna in the Danish Wadden Sea. / E.xp. Mar Biol. Ecol. 89:157-174. Juanes, P. 1992. Why do decapod crustaceans prefer small-sized niolluscan prey? Mar Ecol. Prog. Ser 87:239-249. Jubb, C. A.. R. N. Hughes & T. ap Rheinallt. 1983. Behavioural mecha- nisms of size-selection by crabs, Carcinus maenas (L.) feeding on mussels, Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 66:81-87. Kaiser, M. J., R.N. Hughes & D. G. Reid. 1990. Chelal morphometry, prey-size selection and aggressive competition in green and red forms of Carcinus maenas (L.). J. E.xp. Mar. Biol. Ecol. 140:121-134. Klein Breteler, W. C. M. 1976. Migration of the shore crab, Carcinus maenas. in the Dutch Wadden Sea. Nelh. J. Sea Res. 10:338-353. Mascaro. M. & R. Seed. 2000. Foraging behavior of Carcinus maenas (L.): comparisons of size-selective predation on four species of bivalve prey. J. Shellfish Res. 19:283-291. McDonald, P. S., G. C. Jensen & D. A. Armstrong. 1 998. Green crabs and native predators: possible limitations on the West Coast invasion. J. Shellfi.sh Res. 17:1283. Moksnes. P.-O., L. Pihl & J. van Montfrans. 1998. Predation on posllarvae and ju\ eniles of the shore crab Carcinus maenas: importance of shelter. size and cannibalism. Mar. Ecol. Prog. Ser. 166:211-225. Parache, A. 1980. Les relations "proie — predateur" entre le crabe vert Carcinus maenas et la palourde Rudilapes philippinarum. Bull. Mens. Spec. Sci. Tech. Nov. 1980:299-308. Robinson, A. M. 1997. Molluscan fisheries in Oregon: Past, present, and future. In: C. L. MacKenzie Jr., V. G. Burrell Jr., A. Rosenfield & W. L. Hobart, editors. The history, present condition, and future of the molluscan fisheries of North and Central America and Europe. Volume 2. Pacific Coast and supplemental topics. NOAA/NMFS Technical Report 128, pp75-87. Ropes, J. W. 1968. Feeding habits of the green crub. Carcinus maenas. Fish. Bull. 67:183-203. Sanchez-Salazar, M. E., C. L. Griffiths & R. Seed. 1987. The effect of size and temperature on the predation of cockles Cerastoderma edule (L.) by the shore crab Carcinus maenas (L.). J. Exp. Mar. Biol. Ecol. 111:181-193. Sneddon. L. U., F. A. Huntingford & A. C. Taylor. 1997. The mfluence of resource value on the agonistic behavior of the shore crab. Carcinus maenas (L.l. Mar. Freshwater Behav. Physiol. 30:225-237. Sokal, R. R. cS: R. J. Rohlf. 1995. Biometry: the principles and practice of statistics in biological research. 3rd ed. W.H. Freeman and Company, New York, p. 887. Walne, P. R. & G. J. Dean. 1972. Experiments on predation by the shore crab, Carcinus maenas L., on Mytilus and Mercenaria. J. Cons. Int. E.xplor Mer. 34:190-199. Yamada, S. B. 2001. Global invader: The European green crab. ORESU- B-01-001. Sea Grant, Oregon: Oregon State University, 123 pp. Joiinial ol Slirllfixh Ri:\carch. Vol. 22. No. 3. 873-880. 2003. HISTOPATHOLOGY AND PREVALENCE OF THE PARASITIC DINOFLAGELLATE, HEMATODINWM SP, IN CRABS {CALLINECTES SAPIDUS, CALLINECTES SIMILIS, NEOPANOPE SAYl, LIBINIA EMARGINATA, MENIPPE MERCENARIA) FROM A GEORGIA ESTUARY MICHAEL SHEPPARD," ANNA WALKER.- MARC E. FRISCHER,' AND RICHARD F. LEE'* 'Skuhnvay Institute of Occauoiiwphy. 10 Ocean Science Circle. Savannah. Georgia 31411: -Department of Pathology. Mercer University School of Medicine. Macon. GA 31207: ^Murine Science Program. Savannah State University. Savannah. Georgia 31404 ABSTRACT This study reports on seasonal vanations in the prevalence and intensity of HemiUodin'mm sp. infection in the blue crab {Callinectes sapidus). spider crab {Lilvnia eimirgimilu). a xanthid crab (Neopanope sayi). stone crab {Mcnippe mercenaria). and lesser blue crab (Callinectes i»n(7/.!) collected from Wassaw Sound on the Georgia (USA) coast. During the fall of each year there has been a peak in the prevalence of Hematodinium in L emarginata and N. sayi. while in C. sapidus there have been infection peaks in both fall and spring. There was a much lower frequency of infection in M. mercenaria and C. sinnlis. Based on comparisons of I8S rRNA gene sequences of Hemaiodiniiim sp.. it appears that the Hematodinium sp. found in spider and stone crabs are the same or very closely related to the Hematodinium isolated earlier from the blue crab. Morphologically, most parasites were in the mononuclear trophont form, although occasional binucleated and multinucleated forms were observed. The highest numbers of Hematodinium sp. were found in the gills where parasites were present extracellularly within vascular spaces. The parasite infiltrated cardiac and skeletal muscle in an interstitial pattern, but did not invade individual myofibers. Our findings suggest that Hemalndinium sp. is impacting the blue crab population in Wassaw Sound and is responsible for the disappearance of C. sapidus in the summer months, allowing other opportunistic crab species to invade the niche vacated by C. sapidus. KEY WORDS: prevalence, disease. Hcnuiunlummh crab, intensity, estuary. Georgia INTRODUCTION Henuiliitliniiiiii pcrezi is a parasitic dinotlagellate that was first reported in 1931 in two crab species, the green shore crab, Carci- nus maenas, and the harbor crab. Liocarcinus depurator. along the French coast (Chatton & Poisson. 1931 ). Infection with this para- site has since been shown to produce a spectrtim of disease ranging from asymptomatic carriage to death. The parasite proliferates in crustacean hemolymph, consuming hemocyanin, along with other hemolymph proteins and possibly hemocytes (Love et al. 1993, Field & Applcton 1995. Field et al. 1992). Hemolymph taken from heavily infected animals subsequently does not clot. The parasite also infiltrates other tissues, including cardiac and skeletal muscle (Hudson & Shields 1994, Shields & Squyars 2000), Morbidity appears to depend on the burden of organisms. Heavily infected crabs become lethargic, possibly due to hypoxemia and compro- mise of cardiac and skeletal muscle. If not preyed upon, they often succumb to the overwhelming infection. Since the work of Chatton and Poisson (1931) on diseased crabs in France, there have been reports of crustaceans infected with Hematodinium sp. in Australia (Australian blue crab, Piirlii- niis pelagiciis: sand crab, Portimus pelagicits: mud crab, Scylla serrata; coral crab. Trapezia aerolata [Hudson & Lester 1994, Hudson & Shields 1994, Shields 1992, Hudson et al. 1993]). Alaska (Tanner crab. Chionoecetes IniinI (Meyers et al. 1987. 1994]), Scotland (Norway lobster, Ncphraps nonegiciis (Field et al. 1992]) eastern Canada (snow crab, Chionoecetes opilio [Taylor & Khan 1995]) and the eastern United States (blue crab, Calli- nectes sapidus: rock crab, Cancer irroratus: Jonah crab. Cancer borealis: lady crab, Ovalipes ocellatus: amphipods, Leptocheinis pinguis, Ampelisca vadorum [Johnson 1986, MacLean & Rudell *Corresponding author: Richard F. Lee, Skidaway Institute of Oceanog- raphy. 10 Ocean Science Circle. Savannah. GA 31411. E-mail: dickt* skio.peachnet.edu 1978. Messick 1994, Newman & Johnson 1975 j). The life cycle of Hematodinium sp. in blue crabs is complex and involves several different stages, including dinospores, prespores, trophonts, and Plasmodia (Messick 1994, Shields 1994). While Hematodinium sp. has been found in blue crabs, C. sapidus. collected on both the Atlantic and Gulf coasts of the United States (Messick 1994. Messick & Shields 2000. Messick et al. 1999, Newman & Johnson 1975, Shields & Squyars 2000), there have been few reports of this parasite in other crab species from the south Atlantic coast of the United States. The present study repoils on seasonal variations in the prevalence and intensity of Hematodinium sp. infection among the blue crab {Callinectes sapidus], spider crab (Libinia emarginata), xanthid crab (Neopan- ope sayi), stone crab (Menippe mercenaria). and lesser blue crab {Callinectes similis) collected from a coastal Georgia estuary (Wassaw Sound. Fig. I). Histologic examination of tissues from diseased blue, spider and stone crabs was peiformed to study the pattern of the infection and immune response of the different hosts. The parasites from each of the three crab species were morpho- logically very similar. The genetic similarity of the parasites in the three crab species was confirmed by sequencing the I8S rRNA gene. MATERIALS AND METHODS Collection. Preparation. Fixing, and Staining of Hemolymph Crabs were collected in the spring and fall from the Wassaw Sound estuary by trawling or with traps baited with menhaden. Crabs were bled at the hemal sinus with a 1-ml syringe. Hemolymph samples were applied to poly-L-Iysine-coated micro- scope slides as described by Messick (1995). fixed in Bouin's fluid, and stained with Mayer's hematoxylin and eosin (Luna 1968). Fixed and stained slides were examined at xlOOO with a Nikon Eclipse 6400 microscope equipped with a Nikon xlOO I.3NA oil objective. Hematodinium sp. was identified based on 873 874 Sheppard et al. 31 » 31 s^ 31 x^< 31-5 -81.3 -HI 2 -Hll -«ll) -80.9 Figure 1. Study site, Wassaw Sound in coastal Georgia. moijihologic similarities to blue crab HemaUHlinium sp. on slides authenticated by G. Messick (NOAA. Oxford. MD). Prevalence, expressed as a percentage, using the definition for this term given by Margolis et al. (1982), was the number of crabs infected with Hfiiuitodiniiim sp. divided by the number of crabs examined times one hundred. Infection intensity was the percentage of Hemato- diwn sp. cells counted ainong a total of 300 cells from the hemolymph from an individual crab. Average intensity for a sam- pling period was the sum of the intensities of infected crabs di- vided by the number of infected crabs. Fixing and Staining of Tissues Representative portions of tissues were dissected for histologic examination from 10 infected blue crabs, 3 spider crabs, and 1 stone crab. Tissues were fixed in zinc formalin, processed for routine light microscopy and embedded in paraffin. Five- micrometer sections were cut. mounted on glass slides, stained with hematoxylin and eosin, coverslipped and examined by one of us (ANW). Stages of Hematodinium SP. Identification of the different forms of Hcinaliuliniiim sp. was based on our own observations and the observations of others, including Appleton and Vickerman (1998). Hudson and Shields (1984), and Shields and Squyars (2000). The trophont or vegetative form oi Hcinatodiniuin sp. is 8 to 12 p.m in diameter. It has a fairly high nuclear cytoplasmic ratio with the nucleus 7 to 9 |j.m in diameter. Nuclear chromatin varies from appearing rather homogenously dispersed throughout the nucleus to being condensed into structures that resemble chromosomes at metaphase. Trophonts generally possess a single nucleus, but oc- casional, otherwise typical forms appeared to have two nuclei. The Plasmodium is larger than the trophont form ranging in size from 20 to 50 p.m in its longest dimension. Plasmodia are characteristically multinucleated. An elongated, slipper-shape form is referred to as a vermiform Plasmodium; the nuclei in this form are usually arranged in a single file along the long axis of the parasite. There are also more rounded forms that resemble tro- phonts, but have much greater cytoplasmic volumes and are mul- tinucleated. Dinospores are notably smaller than trophont forms, 3 to 6 |i.m in diameter, and are uninucleate. Molecular Identification and Detection of Hematodinium in Crab Hemolymph The specific diagnosis of Hematodinium sp. in crabs was rou- tinely made using a recently developed Polymerase Chain Reac- tion (PCR) assay (Gruebl et al. 2002). Hemolymph (O.-'i-LO riiL) was collected as described above using a sterile chilled syringe and transferred to sterile 1.5-ml microfuge tubes. Anticoagulant was not required if the hemolymph was kept cool. Total DNA was extracted and purified from hemolymph samples as previously described by Gruebl et al. (2002) using the DNeasy^^^' Tissue Kit (Qiagen) and the Heitiatodiniimi-specific primers Hemat-F-1487 (5'-cct ggc teg ata gag ttg) and Hemat-R-I654 (5'-ggc tgc cgt ccg aat tat tea c) to detect Hematodinium. These primers specifically amplify a 195 bp fragment of the 18S rRNA gene from Hemato- dinium. PCR was performed using GenAMP 97(X) or 2400 PCR thermal cycler systems (Perkin Elmer). Amplified gene fragments were visualized and sized by agarose gel electrophoresis in 1.2% gels stained with GelStari® nucleic acid stain (Cambrex). The pres- ence of the correct sized amplicon was routinely taken as evidence of Hematodinium infection. To confirm the identity of the parasites detected in each crab species, representative 195 bp PCR amplicons were sequenced. In addition, nearly the complete 18S rRNA gene sequence ( 1682 bp) from the parasite detected in the spider crab was sequenced and compared with the known Hematodinium 1 8S rDNA fragment that was amplified from DNA purified from a highly infected spider crab (95-98% intensity) using the previously described primers Univ-F-15 (5'-ctg cca gta gtc ata tgc) and Hemat-R-i6.S4 (5'-ggc tgc cgt ccg aat tat tea c) (Gruebl et al, 2002). Sequencing was facilitated by cloning the amplified 18S rRNA gene fragments into the PCR 2.1-TOPO cloning vector using a TOPO^"^' Cloning Kit, Hematodinium Infection in Georgia Crabs 875 Version J (Invitrogen) following the manufacturer's instructions. The plasmid was isolated and purified from E. coli using the High Pure Plasmid Isolation Kit iBoehringer Mannheim) following the manufacturer's instructions. Plasmid concentrations were esti- nialed by fluorometry after staining with PicoGreen® (Molecular Probes) using a TD-700 tluorometer (Turner Designs). Sequencing was accomplished by automated sequencing using the sequencing primers described in Griiebl et al. (2002) with a Beckman CEQ 2000XL DNA Analysis System. Sequencing reactions were facili- tated by using a CEQ DTCS dye terminator cycle sequencing quick start kit, following the protocols recommended by the manu- facturer (Beckman Coulter). Sequence analysis was accomplished using the Beckman CEQ 2000XL Sequence Analysis software, version 4.3.9. RESULTS I'rivak'iicc and Intensity of Hematodinium sp. in Crabs from Wttssaw Sound The prevalence and intensity of Heinatocliiiiiim sp. infection were determined in five crab species collected in Wassaw Sound during different seasons over several years (Table I. Figs. 2. 3). Prevalence at a time period, expressed as a percentage, is defined as the number of crabs infected with Hematodinium sp. divided by the number of crabs examined times 100. Intensity in a crab was the percentage of Hematadinium sp. cells in the hemolymph. Av- erage intensity for a sampling period was the sum of the intensities of infected crabs divided by the number of infected crabs. The TABLE L Prevalence and intensity of Hematodinium sp. in Callineetes similis, Neopanope suyi, and Menippe mereenaria. Average Collection Number Prevalence Intensity" Species Data of Crabs (%) (%) C. similis May. 200U 15 0 — Aug.. 2000 12 0 — Oct., 2000 17 0 — June. 2001 12 0 — Oct., 2001 14 7 11 June, 2002 18 0 — N. sa\i March. 2000 5 0 — Aug. 2000 4 0 — Sept.. 2000 8 6.^ 32 Oct.. 2000 5 40 22 March. 2001 3 0 — Oct.. 2001 7 43 12 March. 2002 4 0 — Oct.. 2002 6 33 26 M. mereenaria March, 2000 4 0 — Aug., 2000 5 0 — Oct.. 2000 10 0 — May, 2001 4 0 — June, 2001 8 13 29 Oct., 2001 7 0 — June, 2002 16 0 — " Average intensity for sampling period was the sum of the intensities of infected crabs divided hy the number of infected crabs. Infection intensity was the percentage of Henniiodium sp. cells counted among a total of 300 cells from the hemolymph from an individual crab. 1999 I 2000 I 2001 I Figure 2. Monthly crab catches and the prevalence and average in- tensity of Hematodinium infection in blue crabs. Callineetes sapidus. collected during l'W9-20(t2 in Wassaw Sound, .\sterisks indicate that infected crabs were not detected in that sampling period. average intensities of Callineetes similis. Neopanope sayi. and Menipppe mereenaria are reported in Table 1 . for Callineetes sapi- dus in Figure 2 and for Lihinia etnarginata in Figure 3. Among the crab species collected, highest prevalences were found in C. sapi- dus. L. emarginata and A', sayi. In C. .sapidns. infection peaks occurred in late spring and fall of each year; moreover, there was an almost complete disappearance of crabs during the summer (Fig. 2). Crabs collected in the winter months of 1999 to 2001 were not infected, but the disease was found in crabs collected during the unusually warm winter of 2001 to 2002. During peak infection periods, prevalence reached 40% with average intensity as high as 80%. Heavily infected L. emarginata were collected each fall for 3 years, but only during one spring (spring 2002) were infected crabs found (Fig. 3). L emarginata normally enter Wassaw Sound in the fall and are common throughout the winter and early spring, and then retreat into cooler, deeper waters in the late spring and sum- Prevalance and intensity of hematodinium infection in spider crabs collected during 2000 - 2002 in Wassaw Sound. a ■»"■ I •g Pwvjbncc(%> Figure 3. Prey alence and ay erage intensity of Hematodinium infection in spider crabs. Lihinia emarginata. collected during 21)110-21102 in Wassayy Sound, .\sterisks indicate that infected crabs were not de- tected in that sampling period. 876 Sheppard et al. men Infected N. sayi were only found in the fall even though this species is a year round resident of Wassaw Sound (Table 1 ). In contrast to the high prevalences and intensities of Hemato- diniwn sp. found in C. sapidus. L. emarginaui. and N. sayi. only one infected Menippe mercenaha and one infected CalUnectes similis were found during the study (Table 1 ). The trophont form was the only form observed in the hemolymph from infected L emarginata and M. mercenaria. While the trophont was the most common form in C. sapidus. the plasmodia form was regularly seen in C. sapidus during peak infection periods. Dinospores were observed in three infected C. sapidus and one infected TV. sayi. Molecular Identification o/Heniatodiniuni Representative 195 bp 18S rRNA gene fragments amplified from both L. emarginata and M. iiwi-cenaria had a 1 00% sequence similarity to comparable gene fragment of the Heinatodinium sp. found in C. sapidus. Based on these comparisons, the parasite identified in these species was confirmed to be Heinatodinium sp. and is likely the same species that occurs in the blue crab. To confirm to the species level the identity of the Heinatodinium sp. found in the spider crab, a larger 18S rRNA gene fragment ( 1682 bp) was amplified, cloned, and sequenced. This resulting sequence exhibited a 99.6'7f base pair similarity to the previously sequenced Heinatodinium sp. (Genbank Accession #AF286023) isolated from the blue crab. By convention, sequence similarities in the 18S rRNA gene greater than 98% are indicative of the same species (Hillis & Dixon 1991). Therefore it can be concluded from these observations that the same species of Heinatodinium occurs in M. mercenaria. L. emarginata. and C. sapidus. Pathologic Findings in Infected Crabs Libinia emarginata The three crabs examined varied in their burden of organisms from light to heavy. In the lightly infected crab, the gills contained occasional trophont forms intermixed with equal numbers of granulocytes. There were occasional mononuclear and multinucle- Figure 4, Gill from a heavily infected spider crab, Libinia emarginata. The vascular spaces of the gills contain many trophont forms of the parasite and a few host hcmocytes, (Hematoxylin and eosin: original magnification: xKMHI). ated trophont forms on the abluminal side of the hepatopancreas. In the heart there were rare mononuclear trophonts and multinu- cleated forms. The skeletal muscle was largely spared. In the mod- erately infected crab, the gill tissues demonstrated mononuclear trophonts in the larger vascular spaces at the base of the gills. There were scattered granular and agranular hemocytes present, but these were considerably outnumbered by parasites. In the heav- ily infected spider crab, there were numerous mononuclear and multinucleated trophont forms dispersed along the vascular spaces of the gills: few hemocytes were present (Fig. 4). Some skeletal muscle fibers appeared fragmented; there were also interstitial clusters and infiltrates of parasites and foci of myofiber necrosis (Fig. 5A). The hepatopancreas was heavily infected with the tro- phont forms on the abluminal side of the tubules and in vascular spaces (Fig. 5B). There were no parasites within the hepatopan- creatic cells or within the tubular lumina. Menippe mercenaria The crab examined was heavily infected. Most of the parasites were in the mononuclear trophont form, although occasional bi- .4^ V .* 25(1 ^0 y> ■»V ••fS ••* »v ,»:-^*> i»l> • ' ■it 25(1 Figure 5. Heavily infected spider crab, Libinia emarginata. i\) Inter- stitial infiltrates of the parasite in the skeletal muscle. The heniocytic response is minimal. Some muscle fibers lack nuclei: there are foci of apparent destruction in association with the parasites (arrow). (B) Hepatopancreatic vascular spaces are filled with parasites, but no in- filtration of the glandular epithelium is seen. (Hematoxylin and eosin: original magnifications: x40(>). Hematodin/um Infection in Georgia Crabs 877 nucleated and multinucleated forms were observed. The highest concentration of Heiitcitodiiuitm sp. was in the gills where the parasites were dispersed along the vascular spaces (Fig, 6A). Crab hemocytes. primarily granulocytes, were present in these vascular spaces although they were far outnumbered by the parasite. He- matodiniiinm sp. were concentrated on the abluminal sides of the hepatopancreas and in its vascular spaces. Both granular and agranular hemocytes were present in the heart; some had infiltrated the cardiac muscle along with Hematodiniuin sp. (Fig. 6B). There was, in addition, a single focal plaque-like aggregate of parasites and granulocytes on the surface of cardiac muscle. Skeletal muscle contained only a few Hematodiniuin sp. in connective tissue ex- ternal to muscle fibers. Gonadal tissue appeared to be free of the parasite. Callinecles sapidus In lightly infected crabs (less than 2% of hemolymph cells were parasites) there was a strong cellular response to Hematodinium sp., as evidenced by scattered aggregates of granulocytes, which formed encapsulating nodules in gill, hepatopancreas. and cardiac muscle (Fig. 7A,B). The nodules in the hepatopancreas were found l\ 10(1 ll. m - - % 0 d %-**3^ ■^ L 0 ^ < Figure 6. I.Al Gill troiii an Infected stone crab. Meiiippe mercenariu. Trophont forms of the parasite and host hemocytes are present « ithin the vascular space. (Bl Cardiac tissue from the same crab. Interstitial inflltrate of trophonts and host hemocytes. (Hematoxylin and eosin; original magnifications: xlOOO). Figure 7. Lightly infected blue crab, Callinecles sapidus. (A) The vas- cular spaces of the gills contain abundant granular and agranular hemocytes and occasional hemocylic nodules. A few trophont forms are present ( arrow). (Bl A cluster of hemocylic nodules on the ablu- minal side of the hepatopancreas. (Hematoxylin and eosin; original magnifications: A. x400; B, xlOOO) on the abluminal side and there was no invasion by parasites of the hepatopancreatic glandular epithelium or tubular lumina. Only mononuclear trophont forms were observed. In heavily infected crabs, several parasites but few hemocytes were found in vascular spaces and within tissues (Figs. 8, 9). Plasmodia (Fig. 8A). mononuclear and binuclear trophonts were noted in both the hemolymph and cardiac muscle. Dense infiltrates of parasites were noted on the abluminal side of the hepatopan- creas (Fig. 8B). but even with heavy infection there was no evi- dence of hepatopancreatic epithelial or tubular luminal invasion by (he parasite. In the hepatopancreatic region of one moribund ani- mal, there were large numbers of a smaller parasite form that possessed a polymorphic nucleus; these may have been dinospores (Fig. 9A). In addition to high concentrations of parasites in bivascular spaces, parasites infiltrated cardiac and skeletal muscle. Focal muscle necrosis was present (Fig. 9B); hemocyte nodules were rare or absent. Parasites were present in the tissues adjacent to the gonads, but not within gonadal tissues. 878 Sheppard et al. # V. 9^ "■'.,•.*. «^ 10i W 9 9 "» ^ I" ... ^ ,'■? -<*■■ ^., '%' *5# ^ .. M ^♦% *■' ^ ., 10 u Figure 8. Heavily infected blue crab, ( iillinccles sapidus. (Al The >as- cular spaces of the gills of this crab contain many Plasmodia; few hemocytes are present. (B) Hepatopancreatic tissue from another heavily infected animal. This vascular space is filled with parasites, mainly in the trophont form. (Hematoxylin and eosin; original mag- nifications: A, x400; B, xlOOO) DISCUSSION Prior to 1999, Wassaw Sound on the Georgia coast supported a robust, year-round commercial blue crab fishery. Since the stud- ies began in 1999. there have been high Hciiuiiocliniiiiii sp. preva- lences in Ccillliu'iles sapidus. Lihiiiia I'nhiri^iinilu. and Neopanope sayi. In addition to a peak each fall, a peak in HematocUnium sp. prevalence in C. sapidus also occurred during the spring months. Associated with the increased prevalence of Hematodiniuin sp. in the spring was the disappearance of C. sapidus from Wassaw Sound during the summers of 3 successive years (Fig. 2). These observations suggest that high mortality secondary to Hemato- diniuin infection in the spring led to the near absence of C. sapidus in the summer. During the summer months, blue crabs were abundant in low salinity areas near freshwater rivers in coastal Georgia (Lee. unpubl.). We hypothesize that female blue crabs found each fall for the past 4 years in Wassaw Sound were return- ing through Wassaw Sound from low salinity areas to spawn in the ocean. Other seasonal studies on prevalence o\' Hcnuitodiniiini sp. have Figure 9. Heavily infected blue crab, Callinecles sapidus. (A) Hemocytic nodules were rare in most of the heavily infected animals. This one is on the abluminal side of the hepatopancreas. Also present are thousands of small forms of the parasite, possibly dinospores. (Bl Infiltrates of the parasite in cardiac muscle. Both trophont and Plas- modia forms are presenl. Focal coagulative muscle necrosis (arrows) has occurred. (Hematoxylin and eosin; original magnit'ications: x400) been conducted on crabs in different coastal areas. A seasonal study of Hematodinium sp. infection in C. sapidus collected from coastal bays of Maryland showed a peak of infection each fall. Prevalences reached 80'7f at this time, while the disease was almost undetectable from March thru May (Messick & Shields 2000). Seasonal studies of Henialodiniuni sp. were conducted in the Nor- way lobster (Nephwps norvegicus) off Scotland (Field et al. 1998) and the tanner crab {Cluonoectes hairdi) off Alaska (Eaton et al. 1991, Love et al. 1993). The peaks oi Hematodinium sp. infection in both species occurred in the late spring and summer, with de- clines in infection noted during the fall and winter. These studies, along with our own findings, indicate that the seasonality of He- matodinium infection can vary among different crustacean species in the same area and among species from different areas. We have shown that Hematodinium sp. can be transmitted when an uninfected crab feeds on an infected crab (Lee et al. unpubl.). Both C. sapidus and L. emarginata are aggressively cannibalistic. We noted a much lower frequency of infection in Menippe mercenaria and Callinecles similis. We speculate that the indolent feeding behavior of M. mercenaria and C. similis Hematodinium Infection in Georgia Crabs 879 account for their low Heinutodiniuin sp. prevalence during periods when there is both high prevalence and intensity of HcnuiUhliiiiimi sp. among other crab species. Other explanations for the varying prevalence of Hematodinium sp. in different crab species include the possibility that Hematodinium sp is more virulent for certain species, possesses tropism for particular crab species, or that the immune systems of C. similis and M. mercenaria are more effec- tive in limiting Hematodinium sp. infection. Another important factor may be crab densities, since we find that Hematodinium epidemics occur in areas where there are high densities of either C. sapidus or L. emarginata (Sheppard. Lee, and Fischer, unpubl.). Some marine diseases are well correlated with host densities (Richardson et al. 1998), but in other diseases there is no relation- ship (Powell et al. 1999). Only two Hematodinium spp.. H. perezi (Chatton & Poisson 1931 ), and H. australis (Hudson & Shields 1994), have been char- acterized. While the parasite in C. sapidus has been referred to as Hematodinium perezi (Messick 1994. Shields & Squyars 2000), Messick and Shields (2000) suggest that the parasite in C. sapidus be referred to as Hematodinium sp. until more comparisons have been made with the type species. Based on the sequence of frag- ments of the 18S rRNA gene, it appears that the Hematodinium sp. found in L. emarginata and M. nwrcenaria are the same or very closely related to Hematodinium sp. isolated from C. sapidus (Gruebl et al. 2002). It thus appears likely that the infection can be readily transmitted among various crab species in our study area. Histopathologic studies of Hematodinium sp. infections include ('. sapidus from coastal bays of Maryland (Messick 1994), Por- tunus pelagicus from the eastern seaboard of Australia (Hudson & Shields 1994) and Cliionoeeetes luiirdi from southeast Alaska (Meyers et al. 1987). The histologic changes described in infected gill and muscle tissues of the animals in tho.se studies are similar to those seen in the tissues of the infected crabs in our studies. Hematodinium sp. was present extracellularly within the vascular spaces of gills. The parasite produced interstitial infiltrates in car- diac and skeletal muscle but did not invade individual myofibers (Figs. 4-5. Hudson & Shields 1994, Meyers et al. 1987). Focal muscle necrosis was apparent in some of our infected crabs. Myers et al. (1987) noted pathologic changes in muscle cells of heavily infected Tanner crabs, including loss of cross striations and cyto- plasmic eosinophilia. Parasitic infiltrates and muscle necrosis would likely compromise the structure and function of these or- gans and thereby contribute, along with the hemocyanin depletion, to the lethargic behavior exhibited by heavily infected animals. The presence of encapsulating nodules in lightly infected C. sapidus and their absence in non-infected crabs is of interest since the response of crustaceans to large foreign bodies is encapsulation by circulating hemocytes (Galloway & Depledge 2001. Holmblad & Soderhiill 1999). In heavily infected animals, the hemocyte population appeared depleted, suggesting that large numbers of parasites can overwhelm the host's ability to contain the infection. Whether such animals are immunocompromised by pre-existing conditions or the parasites gain a proliferative advantage due to environmental circumstances awaits further study. In addition, we have found that bacteria often colonize the hemolymph of heavily parasitized animals (Sheppard, unpublished data). Such secondary invaders may hasten the demise of these impaired hosts, since they cannot mount an adequate hemocyte response. Our results suggest that Hematodinium sp. is impacting the blue crab populations in Wassaw Sound and is largely responsible for the disappearance of C. sapidus during the summer months. As the population of C. sapidus in Wassaw Sound has decreased there have been increases in the populations of other crab species, such as C. similis. Ovalipes ocellalus, Petrolisthes armatus. and Are- naeus cribrarius (Sheppard, unpubl.). ACKNOWLEDGMENT These studies were supported by the NOAA National Sea Grant College Marine Environmental Biotechnology Program (Grant NA06RG0029). LITERATURE CITED Appleton. P. L. & K. Vickerman. 1998. In vitro cultivation and develop- mental cycle in culture of a parasitic dinotlagellate [Hciiuuodiniiiin sp.) associated with mortality of the Norway lobster [Neptirops nonexieus) in British waters. Parasitology 116:115-130. Chatton, E. & R. Poisson. 1931. Sur I'existence, dans le sang des crabes, de peridiniens parasites: Hematodinium perezi n.g.. n.sp. (Syndinidae. CR Seances Soc. Biol. Paris 105:553-557. l:aton. W. D.. D. C. Love. C. Botelho, T. R. Meyers, K. Imamura & T. Koeneman. 1991. Preliminary results on the seasonality and life cycle of the parasitic dinoflagellate causing bitter crab disease in Alaskan tanner crabs iCIiionoecetes iHiirdi). J. Invert. Patliol. 57:426-434. l-ield. R. H.. C. J. Chapman, A. C. Taylor. D. M. Neil & K. Vickerman. 1992. Infection of the Norway lobster Nepliropos nonegicus by a Hematodinium-Vike species of dinoflagellate on the west coast of Scot- land. Dis. Aqiiat. Org. 13:1-15. Field. R. H., J. M. Hills, R. J. A. Atkinson. S. Magill & A. M. Shanks. 1998. Distribution and seasonal prevalence of Hematodinium sp. in- fection of the Norway lobster {Nepl}rops noiregiciis) around the west coast of Scotland. ICES J. Mar Sci. 55:846-858. Galloway, T. S. & M. H. Depledge. 2001. Inimunoloxicity in invenebrates: measurement and ecotoxicology relevance. Ecoloxicol 10:5-23. Gruebl. T. M.E. Frischer, M. Sheppard. M. Neumann, A.N. Maurer & R.F. Lee. 2002. Development of an 1 8S rRNA gene-target PCR-based di- agnostic for the blue crab parasite Hematodinium sp. Dis. .\quat. Org. 45:61-70. Hillis. D. M. & M. T. Dixon. 1991. Ribosomal DNA: Molecular evolution and phylogenetic inference. Quart. Rev. Biol. 66:411-453. Holmblad. T. & K. Sciderhall. 1999. Cell adhesion molecules and antioxi- dative enzymes in a crustacean, possible role in immunity. .Acjuucullure 172:111-123. Hudson, D. A. & R. J. G. Lester. 1994. A parasitological survey of the mud crab Scylla serruta (Forskal) from southern Moreton Bay. Queensland, Australia. Aquuciilture 120:183-199. Hudson. D. & J. D. Shields. 1994. Hematodinium australis n. sp.. a para- sitic dinotlagellate of the sand crab Porumus pelagicus from Moreton Bay. Australia. Dis. Aquat. Oci;. 19: 109-1 19. Hudson. D., N. Hudson & J. D. Shields. 1993. Infection of Trapezia spp. (Decapoda: Xanthidae) by Hematodinium sp. (Duboscquodinida: Syn- dinidae): a new family record of infection. J. Fish Dis. 16:273-276. Johnson, P. T. 1986. Parasites of benthic amphipods: dinoflagellates (Du- boscquodinida: Syndinidae). Fisli. Bull. 84:605-614. Love, D. C. S. D. Rice. D. A. Moles & W. D. Eaton. 1993. Seasonal prevalence and intensity of Bitter Crah dinotlagellate infection and host mortality in Alaskan Tanner crabs Cliionoecetes hairdi from Auke Bay, Alaska. USA. Dis. .Aqual. Org. 15:1-7. Luna, L. G. I96S. Manual of Histologic Staining Methods of the Armed Forces Institute of Pathology. 3rd ed. New York: McGraw-Hill. MacLean, S. A. & C. L. Ruddell. 1978. Three new crustacean hosts for the parasitic dinoflagellate Hematodinium perezi (Dinotlagellata: Syn- dinidae). / Parasitol. 6:158-160. 880 Sheppard et al. Margolis. L.. G. W. Esch. J. C. Holmes. A. M. Kris & G. A. Schad. 1982. The use of ecological terms in parasitology (report of an ad hoc com- mittee of the American Society of Parasitologists). J. Parasiiol. 68; 131-133. Messick. G.A. 1994. Hemalodiniwn perezi infections in adult and juvenile blue crabs Calliuecles sapidus from coastal bays of Maryland and Virginia. USA. Dis. Aqual. Org. 19:77-82. Messick. G. A. 1995. Laboratory techniques to detect parasites and dis- eases of blue crab. Ciillincctes sapidus. In: J. S. Stolen. T. C. Fletcher. A. Smith. J. T. Zelikoff. S. L. Kaatari, R.S. Anderson. K. Soderhall & B. A. Weeks, editors. Techniques in Fish ImmunologY-4. Fair Haven: SOS Publications, pp. 187-198. Messick, G. A. & J. D. Shields. 2000. The epizootiology of the parasitic dinoflagellate Hematodiniu sp. in the American blue crah CalHnectes sapidus. Dis. Acpiat. Org. 43:139-152. Messick. G. A.. S. J. Jordan & W. F. Van Heukelem. 1999. Salinity and temperature effects of Hematodinium sp. in the blue crab CalHnectes sapidus. J. Shellfisli Re.s. 18:657-662. Meyers. T. R.. T. M. Koeneman. C. Botelho & S. Short. 1987. Bitter crab disease: a fatal dinoflagellate infection and marketing problem for Alaskan tanner crabs Chionoecetes bairdi. Dis. Aquat. Org. 3: 193-216. Meyers, T. R., D. V. Lightner & R. M. Redman. 1994. A dinoflagellate- like parasite in Alaskan spot shrimp Pandalus planceros and pink shrimp P. borealis. Dis. Acjiial. Org. 18:71-76. Newman. M. W. & C. .A. Johnson. 1975. A disease of blue crabs (CalH- nectes sapidus) caused by a parasitic dinotlagellate. Hcmuiadiniuni sp. J. Para.titol. 63:554-557. Powell. E. N.. J. M. Klinck. S. E. Ford. E. E. Hofmann & S. J. Jordan. 1999. Modeling the MSX parasite in eastern oyster (Crossostrea vir- ginica) populations. III. Regional application and the problem of trans- mission. J. Shellfish Re.s. 18:517-537. Richardson. L. L.. R. B. Aronson. W. M. Golberg, G. W. Smith. K. B. Kitchie. J. C. Halas. J. S. Feinfold & S. M. Miller. 1998. Florida's mystery coral-killer identified. Nature 392:557-558. Shields. J. D. 1992. The parasites and symbionts of the blue sand crab. Portunus pelagicus. from Moreton Bay, Australia. / Crust. Biol. 12: 94-100. Shields, J. D. 1994. The parasitic dinotlagellates of marine crustaceans. Aim. Rev. Fish Dis. 4:241-271. Shields, J. D. & C. M. Squyars. 2000. Mortality and hematology of blue crabs, CalHnectes sapidus. experimentally infected with the parasitic dinoflagellate Hematodinium perezi. Fish. Bull. 98:139-152. Taylor. D. M. & R. A. Khan. 1995. Observations of the occurrence of Hematodinium sp. (Dinoflagellata: Syndinidae), the causative agent of bitter crab disease in Newfoundland snow crab {Chionoecetes opilio). J. Invert. Pathol. 65:283-288. Jourmil of Shflljhh Kcsi'unh. Vol. 2:, No. 3, 88l-88fi, 2003. THE ROLE OF MACROALGAL BEDS AS NURSERY HABITAT FOR JUVENILE BLUE CRABS, CALLINECTES SAPIDUS CHARLES E. EPIFANIO,* ANA I. DITTEL. RAYMOND A. RODRIGUEZ, AND TIMOTHY E. TARGETT Graduate College of Marine SmJIes. Uiilversin of Delaware. 700 Pilottowu Road. Lewes. Delaware 19958 ABSTRACT We itnestigaled the role of macroalgal beds as juvenile habitat for the blue crab CalUnectes supiihis. A 2-year study was conducted in Rehoboth Bay. a lagoonal estuary in the Middle Atlantic Bight along the east coast of North America. Sea grass meadows do not occur in Rehoboth Bay. and submersed aquatic vegetation consists entirely of macroalgae. Quantitative samples were collected from both vegetated and open (unvegetated) habitat with a throw trap. Results indicate that inacroalgal beds provide important habitat for juvenile blue crabs, beginning at settlement and continuing until the crabs reach a carapace width of about 30 mm. Average abundance of juveniles in macroalgal beds was 7 times greater than in adjacent open habitat, and maximum abundance in the beds reached weekly mea;i values >90 crabs m"^ during periods of high recruitment in early autumn. Mean size of individual crabs was 15 inm carapace width when sampling began in May. These crabs had settled the previous autumn and had over-wintered in the bay. Mean size continued to increase through early summer, and the crabs had reached a inean carapace width >30 mm by August. These 30-mm crabs disappeared frotii the beds in inid-August and were replaced by newly metamorphosed juveniles 25°C), and was minimum (<15°C) in early November 1999. Water was typically well oxy- genated, and levels rarely fell below 5 mg L '. Supersaturated values as high as 15 mg L"' were occasionally measured in mac- roalgal beds on calm, sunny days. Abundance of juvenile blue crabs was significantly greater in inacroalgal beds than in adjacent open bottom (/-test, tij = 3.1, P < 0.001). When calculated for all stations in 1998, mean abun- dance in niacroalgae was 7.3 m"" (±10.1 ), while open areas had an abundance of 1.0 m"" (± 1.9). The large standard deviations re- sulted from seasonal differences wherein the abundance of crabs in macroalgal beds increased markedly in September (Fig. 2). Results from 1999 showed strong seasonal variation in both numbers and size of juveniles in macroalgal beds. Mean abun- dance from May through early .August was always <10 crabs in"", but increased to levels >20 crabs m"" by mid-August and reached values >90 m'" in September and October (Fig. 3). The mean carapace width of juveniles was somewhat less than 15 mm when sampling began in May and approached 30 mm by late July (Fig. 4). The large juveniles disappeared from the beds in mid-August and were replaced much smaller crabs (<10 mm). These small individuals dominated the population throughout the remainder of the study period and were still abundant when sampling ended in early November. Macroalgal beds were well developed during both years of the investigation, with a median standing crop of niacroalgae of ap- proximately 150 g m"". This is within the range of values typical for niacroalgae at high nutrient levels (e.g., Schneider & Searles 1977, De Busk et al. I9S6). Of the 99 vegetated stations sampled over the two years of the study. 40 had a greater proportion of green algae and 59 a greater proportion of red. Among these, 1 8 stations were pure stands of red species, while only 3 stations were pure stands of green forms. Analysis of all stations pooled over the 2 y of the investigation showed a significant positive correlation between abundance of crabs and total standing crop of macroalgae (Table 1). However, there was no correlation between abundance of crabs and the dry- weight ratio of green to red algae at the respective stations. Sepa- rate analysis of early-season data and late-season data gave results that were similar to those for the entire data set. As expected, the gut contents of crabs collected from all three habitats showed a wide variety of prey items (Table 2). These included a number of crustacean groups, bivalve and gastropod mollusks, polychaetes, vascular and macroalgal plant material, and considerable amounts of highly digested tissue that we were un- able to assign to any particular taxonomic group. Regardless of this July August September Figure 2. Abundance of juvenile blue crabs Callinectes sapidus in mac- roai^al beds in Reiiobotli Bay, Delaware. Solid bars are weekly mean abundance in 1998. Error bars = one standard deviation. 884 Epifanio et al. 200 120 - Figure 3. Abundance of juvenile blue crabs Calliiucles sapidus in niac- roalgal beds in Rehoboth Bay, Delaware. Solid bars are weekly mean abundance in 1999. Error bars = one standard deviation. taxonomic diversity, crustaceans were the dominant stomach com- ponent in crabs from each of the three sampUng sites. However, the taxonomic groups comprising this crustacean component var- ied greatly among crabs from the three respective habitats. For example, crab body parts accounted for nearly 30% (by volume) of the stomach contents of juvenile C. siipidus collected from marsh habitat adjacent to Delaware Bay. but never exceeded 13% in either of the other two habitats. In contrast, a miscellaneous group that we called "other crustaceans" composed almost 50% of the stomach contents of crabs collected from tide flat habitat in Dela- ware Bay. This group consisted of harpacticoid copepods, palae- monid and crangronid shrimp, and crustacean body parts that could not be assigned to any particular taxon. Crabs collected from mac- roalgal beds in Rehoboth Bay differed most notably from the other two habitats in the very low proportion of crab body parts in their gut contents and in the high proportion of amphipods in their ID n o May June July Aug Sept Oct Figure 4. Size of juvenile blue crabs {Callinectes sapidus) in seaweed beds in Rehoboth Bay. Solid bars are weekly mean carapace width in 1999. Error bars = one standard deviation. stomachs (>30%). This was remarkable because amphipods were entirely absent from the identifiable gut contents of crabs from the other two sampling sites. DISCUSSION Results of our investigation indicate that macroalgal beds pro\'ide important habitat for juvenile blue crabs, beginning at settlement and continuing until the crabs reach a c;irapace width >30 mm. Average abundance of juveniles in macroalgal beds was approximately 7 times greater than on adjacent open bottom, and maximum abundance in the beds reached weekly mea;; values >90 crabs nr^ during periods of high recruitment in early autumn. Mean size of indi\ idual crabs was about 15 mm in carapace width when sampling began in May. Because settlement of blue crabs in this region occurs almost exclu- sively in late summer and autumn (Jones & Epifanio 1995). the crabs collected in May apparently had settled during the previous autumn and had over-wintered in Rehoboth Bay. Mean size continued to increa.se through early summer, and the crabs had reached a mean carapace width >30 mm by mid-summer. The 30-mm crabs disap- peared from the beds in mid-August and were replaced by newly metamorphosed juveniles <10 mm in carapace width. These small crabs had probably settled in the beds as megalopae and had under- gone metamorphosis soon thereafter (Orth & van Montfrans 1987, Jones & Epifanio 1995). Very small crabs were common in the beds throughout September and were still abundant when sampling was completed at the end of October. Mean size of the crabs did not increase during this period, probably a result of overlapping cohorts of new recruits. However, there was considerable variation in abun- dance among stations (note the high standard deviations in Fig. 4). which undoubtedly reflects the patchy nature of settlement in the bay. This was probably a result of the patchy distribution of megalopae in the water column (Natunewicz & Epifanio 2001), rather than some difference in the attractiveness among beds (Brumbaugh & McCon- naugha 1995). Earlier work in the Little Egg Harbor-Great Bay system along the coast of New Jersey (100 km noilh of Rehoboth Bay) also addressed the importance of macroalgal beds as juvenile habitat (Wilson et al. 1990). This system is similar to our study site, but has ample sea grass meadow in addition to macroalgal beds (Sog- ard & Able 1991). As in our investigation, early-season abundance at the New Jersey site was on the order of 5-10 crabs m"" in vegetated habitat and considerably lower on open bottom. More- over, the general pattern of seasonal abundance of different size classes was similar to that in Rehoboth Bay. However, late-season sampling in New Jersey did not find the extremely high abundance of newly settled crabs seen at our study site, perhaps reflecting a greater distance from the very large spawning stock of blue crabs in Delaware Bay (Garvine et al. 1997). This difference aside, it appears that macroalgal beds generally pro\ ide nursery habitat for blue crabs that is comparable to that of sea grass meadows. For example, there was little difference in mean abundance of juveniles in macroalgal and sea grass habitats in the Little Egg Harbor-Great Bay system; in fact, the abundance of crabs was slightly higher in macroalgal habitat (Sogard & Able 1991). Likewise, mean abundance in macroalgal beds at our study site in Rehoboth Bay was sitnilar to that in sea grass meadows in Chesapeake Bay, and general patterns in seasonal occuixence were nearly identical (e.g., Orth & van Montfrans 1987). The present investigation has provided a much more detailed de- scription of the utilization of macroalgal habitat than was previously Macroalgal Bkds as Juvenile Habitat eor C. sah/dus 885 T.ABLE 1. Correlations betHutn abundunce of ju\eiiilt bluu crabs {C'alliiiecles sapidiis) and two proptrtits (algal standing crop and Iht ratio of green to red macroalgae) of macroalgal beds in Rehoboth Bay, Delaware, USA. Full Season Earl) Season Late Season r /' n r P n r P n [nx.4 [QxR 0.379 -0.103 <0.001 0.3 1 3 99 99 0.443 -0.034 0.002 0.824 45 45 0.575 -0.184 <0.001 0.183 54 54 Data were analyzed separately for full season, early season, and late season (see text). Correlalon statistics: /■ = Pearson product-moment correlation coefficient. P = probability of rejecting a correct null hypothesis, n = number of coordinate observations. Variables: [C] = crab abundance. A = algal standing crop. R = ratio of green to red algae (dry weight). avuilable. Fur example, our results inipl\ iIkiI iiiacmalgai beds are important settlement sites blue crab megalopae in autumn and further de[ini[istrate the consequent role of the beds as nurseries for the ear- liest juvenile stages. In addition, our analysis shows that juvenile blue crabs use beds of red and green macroalgae with equal propensity and that abundance of crabs in a bed increases in propoition to the stand- ing crop of macroalgae. Moreover, these relationships are eqtially valid during early season when the population is dominated by over- wintered crabs or later in the season when beds are poptilated entirely by newly settled juveniles. Supplemental to their provision of complex benthic structure, macroalgal beds may also be important as a source of primary production that supports growth of juvenile blue crabs. Results of our analysis indicate that crustacean body parts dominated the gut contents of crabs collected from all three nursery habitats consid- ered in this investigation. However, the taxonomic groups repre- sented within this dietary category varied considerably among habitats. Of primary relevance is the fact that amphipods were the dominant component in the stomach contents of crabs collected from macroalgal beds, but were entirely absent from the identifi- able gut contents of crabs from marsh or tide flat environments. Available evidence in the present investigation is restricted en- TABLE 2. Mean percentage by volume of prey items in gut contents of juvenile blue crabs iCallinccles 'iapidiis) collected from three different nursery habitats. Gut Contents Macroalgae Marsh Tide Flat Amphipods 31.3 0,0 0,0 Crab body parts 3.3 27.4 12.9 Other crustaceans 13.0 19.0 46.1 Bivalves mollusks 2.8 3.5 0.0 Gastropod mollusks 9.7 1.5 15.7 Polychaeles 1.1 6.1 4.2 Foraminiferans 0.8 0.0 0.0 Macroalgal material 4.3 3.7 1.5 Vascular plant material 0.8 8.3 1.0 Highly digested material 32.3 30.2 16.8 Sand grains 0.4 0.3 1.8 Macroalgal beds were located in Rehoboth Bay. Delaware, USA. Marsh and tide tlal habitats were located in nearby Delaware Bay. Explanation of selected gut-content categories: Crab Body Parts = items identified to the infraorder Brachyura; Other Crustaceans = items identified as harpacti- coid copepods, palaemonid and crangonid shrimp, or simply to the sub- phylum Crustacea; Highly Digested Material = organic material uniden- tifiable to a taxonomic group. tirely to gut-content analysis, which has an inherent bias in favor of material that is refractory to digestion. Nevertheless, the high proportion of amphipods in the guts of crabs from macroalgal beds is in striking opposition to the complete lack of this taxon in the gut contents of juveniles from the other two environments and strongly suggests a major difference in diet between crabs from macroalgal habitat and either of the other habitats. Because of the very high abundance of amphipods in macroal- gal beds in Rehoboth Bay (Timmons & Price 1996). it is reason- able to conclude that the amphipods found in crab stomachs ana- lyzed in our study originated in the beds themselves. Moreover, the common taxa of amphipods found within these macroalgal beds (various species in the families Gammaridae, Amphitoidae, and Bateidae) graze directly on macroalgae, which constitute the main portion of their diets (Watling & Maurer 1972, Macko et al. 1983, Parker et al. 1993, Lotze & Worm 2000, Kamermans et al. 2002). Thus, it is likely that macroalgal production is an important com- ponent of the food web supporting juvenile blue crabs in estuarine systems like Rehoboth Bay. This is in contradiction to results of earlier work with juvenile shrimp in mangrove nurseries, which has shown a link with pri- mary production originating in benthic diatoms, rather than with production emanating from the inangroves (Stoner & Zimmerman 1988, Newell el al. 1995. Dittel et al. 1997). Likewise, previous work with juvenile blue crabs in salt marsh environments has demonstrated at least partial dependence on benthic diatom pro- duction and only indirect linkage to production by emergent marsh plants (Dittel et al. 2000). Investigations of sea grass systems have come to varying conclusions concerning the role of indigenous primary production in supporting growth of juvenile crabs (e.g.. Fry & Parker 1979, Hughes & Sherr 1983), but a recent review finds little evidence that sea grass productit)n per se is a major contributor (France 1996). When considered as a whole, the results of our investigation provide credible evidence that macroalgae beds constitute critical nursery habitat for juvenile blue crabs in areas where seagrass beds are lacking. Moreover, the value of this habitat may include a direct trophic linkage between primary production originating in the macroalgae; this has not been demonstrated in other plant- based nursery habitats used by juvenile blue crabs. ACKNOWLEDGMENTS The research was supported by funds from the Di\ ision of .Soil & Water Conservation and the Division of Fish and Wildlife. Delaware Department of Natural Resources and Environmental Control, from the Wallop-Breaux Program of USF^'. and from the Marsh Ecology Research Program (MERP) (no. G98-04A). 886 Epifanio et al. LITERATURE CITED Brumbaugh. R. D. & J. R. McConaugha. \995. Time to metamorphosis of blue crab CaUinectes sapidiis megalopae: effects of benthic macroal- gae. Mar. Ecol. Prog. Ser. 129:113-118. DeBusk, T. A.. M. Blakeslee & J. H. Ryther. 1986. Studies on the outdoor cultivation of Ulvci hicliicu L. Bat. Mar. 29:381-386. Dennison. W. C, R. J. Orth. K. A. Moore. J. C. Stevenson. V. Carter. S. KoUar. P. W. Bergstrom & R. A. Batiuk. 1993. Assessing water quality with submersed aquatic vegetation. Habitat requirements as barometers of Chesapeake Bay health. Bioscience 43:86-94. Dittel, A. I. 1993. Cambios en habitos de CaUinectes arcuatus (Crustacea: Decapoda) en el Golfo de Nicoya. Costa Rica. Rev. Biol. Trap. 41: 639-646. Dittel. A. I., C. E. Epifanio & C. Natunewicz. 1996. Predation on mud crab megalopae. Panopeus lierhstii H. Milne Edwards: effect of habitat complexity, predator species, and postlarval densities. J. Exp. Mar. Biol. Ecol. 198:191-202. Dittel, A., C. E. Epifanio. L. A. Cifuentes & D. L. Kirchman. 1997. Carbon and nitrogen sources for shrimp postlarvae fed natural diets from a tropical mangrove system. Estiiar. Coast. Shelf Sci. 45:629-637. Dittel. A. I., C. E. Epifanio. S. M. Schwalm. M. S. Fantle & M. L. Fogel. 2000. Carbon and nitrogen sources for juvenile blue crabs. CaUinectes sapidiis. in coastal wetlands. Mar. Ecol. Pros'. Ser. 194:103-112. Duarte. C. M. 1995. Submerged aquatic vegetation in relation to different nutrient regimes. Ophelia 41:87-1 12. Duarte. C. M. & C. L. Chiscano. 1999. Seagrass biomass and production: a reassessment. Aquat. Bot. 65:159-174. France. R. L. 1996. Stable isotopic survey of the role of macrophytes in the carbon flow of aquatic foodwebs. Vegetatio 124:67-72. Fry. B. & P. Parker. 1979. Animal diet in Texas seagrass meadows: C-13 evidence for the importance of benthic plants. Estuur. Coast. Mar. Sci. 8:499-509. Garvine. R. W.. C. E. Epifanio, C. C. Epifanio & K.-C. Wong. 1997. Transport and recruitment of blue crab larvae: a model with advection and mortality. Estiiar. Coast. Mar Sci. 45:99-111. Giesen, W. B. J. T., N. M. van Katwijk & C. den Hanog. 1990. Eelgrass condition and turbidity in the Dutch Wadden Sea. .Aquat. Bot. 37:1 \- 85. Heyman. W. D. & B. Kjerfve. 1999. Hydrologica! and oceanographic con- siderations for integrated coastal zone management in southern Belize. Environ. Manag. 24:229-245. Mines, A. H.. R. N. Lipcius & A. M. Haddon. 1987. Population-dynamics and habitat partitioning by size. sex. and molt stage of blue crabs CaUinectes sapidiis in a subestuary of Central Chesapeake Bay. Mar. Ecol. Prog. Ser. 36:55-64. Hughes. E. H. & E. B. Sherr. 1983. Subtidal food webs in a Georgia estuary: I3C analysis. J. Exp. Mar. Biol. Ecol. 67:227-242. Jones. M. B. & C. E. Epifanio. 1995. Settlement of brachyruan megalopae in Delaware Bay: a time series analysis. Mar. Ecol. Prog. Ser. 125: 67-76. Kamermans, P.. E.-J. Malta. J. M. Verschuure. L. Schrijvers. L. F. Lentz & A. T. A. Lien. 2002. Effect of grazing by isopods and amphipods on growth of Ulva spp. (Chlorophyta). Aquat. Ecol. 36:425-433. Kushlan, J. A. 1981. Sampling characteristics of enclosure throw traps. Trans. Am. Fish. Soc. 110:557-562. Lavery, P. S.. R.J. Lukatelich & A.J. McComb, 1991. Changes in the biomass and species composition of macroalgae in a eutrophic estuary. Estiiar Coast. Mar. Sci. 33:1-22. Lotze, H. K. & B. Worm. 2000. Variable and compleitientary effects of herbivores on different life stages of bloom-forming macroalgae. Mar. Ecol. Prog. 5er. 200: 1 67- 1 75 . Macko, S. A., M. L. F. Estep & W. Y. Lee. 1983. Stable hydrogen isotope analysis of foodwebs on laboratory and field populations of marine aniphipods. J. Exp. Mar. Biol. Ecol. 72:243-249. Moore, K. A., D. J. Wilcox & R. J. Orth, 2000. Analysis of the abundance of submersed aquatic vegetation communities in the Chesapeake Bay. Evm<(nfs 23:115-127. Natunewicz, C. C. & C. E. Epifanio. 2001. Transport of crab larval patches in the coastal ocean. Mar. Ecol. Prog. Ser. 222:143-154, Newell. R. I.. N. Marshall & A. Sasekumar. 1995. Relative importance of benthic microalgae, phytoplankton, and mangroves as sources of nu- trition for penaeid prawns and other coastal invertebrates from Malay- sia. Mar. Biol. 123:595-606. Orth. R.J. & K. A. Moore. 1983. Chesapeake Bay: An unprecedented decline in submerged aquatic vegetation. Science 222:51-53. Orth. R. J. & J. van Montfrans. 1987. Utilization of sea grass meadow and tidal marsh creek by blue crabs CaUinectes sapidus. \. Seasonal and annual variations in abundance with emphasis on post-settlement ju- veniles. Mar. Ecol. Prog. Ser. 41:283-294. Pardieck, R. A.. R, J, Orth. R. J. Diaz & R. N. Lipcius. 1999. Ontogenetic changes in habitat use by postlarvae and young juveniles of the blue crab. Mar. Ecol. Prog. Ser. 186:227-238. Parker. T.. C. Johnson & A. R. O. Chapman. 1993. Grammarid amphipods and littorinid snails have significant but different effects on algal suc- cession in littoral fringe tidepools. Ophelia 38:69-88. Pihl. L. & R. Rosenberg. 1982. Production, abundance, and biomass of mobile epibenthic marine fauna in shallow waters, western Sweden. J. E.xp. Mar. Biol. Ecol. 57:273-301. Price, K. S. 1998. A framework for a Delaware inland bays environmental classification. Environ. Monit. Assess. 51:285-298. Schneider. C. W. & S. B. Searies. 1977. Standing crop of benthic macroal- gaes on the Carolina continental shelf In: Proceedings of the Interna- tional Macroalgae Symposium Vol. 9. Princeton. NJ: Science Press, pp. 29.V.301. Schneider. C. W. & R. B. Searies. 1979. Standing crop of benthic seaweeds on the Carolina continental shelf. In: Proceedings of the International Seaweed Symposium Vol 9. pp. 293-301. Shepherd. S. A.. A. J. McComb. D. A. Buthuis. V. Neverauskas. D. A. Steffensen & R. West. 1989. Decline of sea grasses. In: A. W. D. Larkum, A. J. McComb & S. A. Shepherd, editors. Biology of sea grasses. Amsterdam: Elsevier, pp. 346-349. Sogard. S. M. 1992. Variability in growth rates of juvenile fishes in dif- ferent estuarine habitats. Mar. Ecol. Prog. Ser. 85:35-53. Sogard, S. M. & K. W. Able. 1991. A comparison of eelgrass. sea lettuce macroalgae. and marsh creeks as habitats for epibenthic fishes and decapods. Estiiar Coast. Mar. Sci. 33:501-519. Stoner. A. W. & R. J. Zimmerman. 1988. Food pathways associated with penaeid shrimps in a mangrove-fringed estuary. Fi.\h. Bull. 86:543- 55 1 . Szedlmayer. S. T. & K. W. Able. 1996. Patterns of seasonal availability and habitat use by fishes and decapod crustaceans in a southern New Jersey estuary. Estuaries 19:697-709. Targett, T. E., C. E. Epifanio & R. A. Rodriguez. 1999. The importance of sea lettuce and other marine macroalgae to fishes and macroinverte- brates of Delaware's inland bays. Report to DNREC. Dover. DE: Di- vision of Soil and Water Conservation and Division of Fish and Wild- life. 89 Kings Highway. 32 pp. Tinimons. M. & K. S. Price. 1996. The macroalgae and associated fauna of Rehoboth and Indian River Bays, Delaware. Bot. Mar. 39:231-238. Valiela. I.. J. McClelland. J. Hauxwell. P. J. Behr. D. Hersh & K. Foreman. 1997. Macroalgal blooms in shallow estuaries: controls and ecophysi- ological and ecosystem consequences. Lininol. Oceanogr. 42:1105- 1118. Watling. L. & D. Maurer. 1972. Marine shallow water amphipods of the Delaware Bay area. USA. Cru.stac. Suppl. 3:251-266. Wikson. K. A.. K. W. Able & K. L. Heck Jr. 1990. Habitat use by ju\ enile blue crabs: A comparison among habitats in southern New Jersey. Bull. Mar. Sci. 46:105-114. Journal of Slwllji.^h Research. Vol. 22, No. 3, 8S7-S92, 2003. ISOLATION AND MOLFXULAR CHARACTERIZATION OF VITELLIN FROM THE MATURE OVARIES OF THE PRAWN LITOPENAEUS VANNAMEI CELIA VAZQUKZ-BOUCARD,'* HUMBERTO MEJIA-RUIZ,' FERNANDO ZAMUDIO," VANIA SERRANO-PINTO,' AND HECTOR NOLASCO-SORIA' 'CIBNOR-Centro de Investigaciones Biologicas del Noroeste. S.C. P.O. Box 128. La Paz 23000, BCS. Me.xico; and ~lns1ituto de Biotecnohgia-UNAM. Aveuida Universidad #2001. Col. Chamilpa C.P. 62210. Ciieniavaca Morelos. Me.xico ABSTRACT Vjtellins from ovaries in shrimp Litopenaeus vannamei were examined by polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis, crossed- Immunoelectrophoresis, chromatography (Sepliarose CL 2B and hydrox- ylapatite columns), and high-performance liquid chromatography. Using these methods, two forms of vitellin (Vtl and Vt2) were observed in ovaries (oocyte 1 10 |j.ml. The vitellins identified appear to be lipoglycoproteins. Similar vitellin polypeptide composition was observed in the two forms of vitellin. with molecular weights of approximately 60. 90, 95, 100, 140, and 160 kDa. Policlonal antibodies against the two forms of purified protein were prepared, and their specificity was demonstrated by radial immunoprecipi- tation and Western blotting analysis. The PI and P2 peptides from N-terminal 100 kDa and 60 kDa polypeptides were highly similar to regions of proline 20 and glycine 63.5 residues of crustacean vitellogenins. KEY WORDS: ovary, shrimp, vitellin. lipovilellin. vitellogenesis. L vannamei INTRODUCTION Vitellin is the major yolk protein accumulated in developing oocytes of a female crustacean. Yolk protein is the source of nutrition for development of embryos and larvae. The vitellin from ovaries and vitellogenin from the hemolymph have been charac- terized for several species of penaeids [Penaeiis japonicus. Vazquez Boucard et al. 1986; P. monodon. Quinitio et al. 1990; P. semisidcatiis. Browdy et al. 1990; Tom et al. 1992 and Lubzens et al. 1997; P. monodon, Chen & Chen 1993; Chang et al. 1993 and Chang et al. 1994; P. chinen.sis. Chang & Jena 1995; Chang et al. 1996). In vertebrates and several invertebrates, vitellogenin trans- ported into the blood or hemolymph is considered the precursor of vitellin. In the Crustacea, it is still uncertain whether vitellogenin is the precursor of vitellin, even though intraovarian synthesis has been demonstrated by Yano and Chin/ei (1987), Rankin el al. (1989), Fainzilber et al. (1992), and Khayat et al. (1994). Recent molecular studies showed that specific vitellogenin niRNA was expressed in both the ovary follicle cells and the hepalopancreas parenchymal cells of penaeid shrimp P. japouicas (Tsutsui et al. 2000) and Macrohrachiiim rosenbergii (Soroka et al. 2000). Vi- tellin has been found in subepidermal adipose tissue of penaeids P. japonicus (Vazquez Boucard 1985), P. longiro.stris (Tom et al. 1987a), and P. semi.\itkatus (Fainzilber et al. 1992), but their role in active synthesis of these compounds is not confirmed. Fainzil- bert et al. (1992) confirmed the double synthesis of vitellin. in the hepalopancreas and the ovary, but in different proportions depend- ing on ovarian maturity. Khayat et al. (1994) suggested that ovarian vitellin and hepalopancreas vitellogenin are the products of one gene. Litopenaeus vannamei is an important commercial species in Mexico and other countries. The failure of ovarian maturation is an obstacle for reproduction control. Accordingly, purification and characterization of vitellin from mature ovaries of L. vannamei were the objectives of this study. Using this information, we will be able to undertake molecular studies in vitellogenin gene expression by several tissues of *Corresponding author. E-mail: cboucardCacibnor.mx Litopenaeus vannantei. The complete primary structure of vitello- genin has been elucidated for several crustaceans. The vitellogenin amino acid sequences of Marsupenaeus japonicus (Tsutsui et al. 2000), Metapenaeus ensis (Tsang et al. 2003), Penaeus semisul- catus (GenBank accession number AY05I3I8). Chera.x cjuadri- carinatus (Abdu et al. 2002), and Macrobrachiimx rosenbergii (Yang et al. 2002) share several conserved regions. These are irtore than 2,500 residues long, and vitellins are derived from each vitellogenin. MATERULS AND METHODS Preparation of Ovarian Homogenale Mature (110 |j.m oocyte) and immature female prawns (35 |jim oocyte) were obtained from Acuacultura Mar, La Paz, B.C.S., Mexico, Ovaries were rinsed and homogenized in glassware at 4°C with 0.05 M Tris. 0.5 M NaCl, and 5 niM EDTA (pH 7.0). Pro- lease inhibitor cocktail (Sigma P-2714) was added (0.005%) to the extraction buffer, just before use. The homogenate was centrifuged at 10.000 g for 15 min at 4"C (Beckman ultracentrifuge, Pasadena, CA). The supernatant was frozen at -70°C until analysis. Electrophoresis For identification of vitellins in the \itellogenic female, the ovary homogenates were separated by native PAGE on 6% poly- acrylamide gel in TRlS-glycine buffer (pH 8.8). The vitellin frac- tion was characterized by sodium dodecyl sulfate polyacrylainide gel electrophoresis (SDS-PAGE; 7,5% polyacrylamide gel). A so- lution of 0.5 M TRIS-HCI (pH 6.8). 1% SDS. 1% 2-mercaptoeth- anol, I09f glycerol, and 0.05% bromophenol blue was used as dissociation buffer. Molecular masses of native proteins and dis- sociated subunit polypeptides were determined by comparison of the relative mobility of molecules to those of molecular mass markers. The molecular masses of polypeptides were determined by native PAGE (precast gel gradient polyacrylamide 4-20% Bio- Rad) with a kit containing midrange protein molecular mass stan- dards: p-thyroglobulin (669 kDa), ferritin (440 kDa). catalase (232 kDa). lactate dehydrogenase (140 kDa). and albumin (67 kDa; Pharmacia Fine Chemical. Uppsala. Sweden). The molecular 887 BOUCARD ET AL. masses of standard proteins on SDS-PAGE were myosin (200 kDa). p-galactosidase (1 16 kDa). phosphorylase (97 kDa). serum albumin (66 kDa). and ovalbumin (45 kDa; Bio-Rad. Richmond. CA). The gel was stained with Coomassie brilliant blue R-2.5() and Silver Stain Plus kit (Bio-Rad) for proteins. Sudan black B for lipids, and periodic acid-Schiff s reagent for carbohydrates. Preparation of Aniisera Rabbits were immunized with 6'7r page purified L. vannamei specific ovarian yolk polypeptides. Small gel portions were cut vertically from both extremes and stained with Coomassie brilliant blue R-250 to reveal the migration distance of proteins. These portions were placed next to the rest of the gel without stain at the same level, and the gel vitellin band was cut horizontally. The two proteins (50 |jLg) separated from the polyacrylamide gel were ho- mogenized with NaCl (0.9%), emulsified with complete Freund's adjuvant, and injected at multiple sites on the backs of rabbits. Boosters of 120 |xg of antigen emulsified with incomplete Fre- und's adjuvant were injected at intervals of two weeks. Purification of Vitellin Litopenaeus vannamei vitellin was purified according to Chang et al. (1996. 1993). The ovarian extracts were gel filtered in a Sepharose CL-2B column (Pharmacia Fine Chemicals. Uppsala. Sweden: 100 cm x 1.8 cm i.d.) equilibrated in 0.01 M TRIS buffer with 2 mM phenylmethylsulphonyl tfuoride (pH 7.0). and eluted in the same buffer at flow rate 18 niL/h. Effluent was collected in 2.4-mL fractions, and the absorbance of each fraction was mea- sured at 280 nm. Each concentrated peak (PM 10 membrane. Ami- con. Danvers. MA) was analyzed by immunodiffusion precipita- tion and PAGE {5% gel). The vitellin peak was applied to a hy- droxylapatite column (Bio-Rad. Richmond. CA. #732-0085) using a 0.01 M potassium phosphate buffer (PPB), pH 7.0, with 2 mM phenylmethylsulphonyl fluoride with stepwise gradients of 0.01 M. 0.10 M. 0.20 M. and 0.35 M. The flow rate was 18 mL/h and the fraction size was 2.4 mL. Immunoprecipitation and PAGE of concentrated peaks (PM 10 membrane. Amicon, Danvers, MA) were also analyzed. The concentrated vitellin peak was further separated by high-performance liquid chromatography (HPLC. Beckman Spherisorb) equilibrated with 0.2 M sodium sulfate in 0.1 M sodium phosphate pH 6.5. The tlow rate was 1 mL/min. Immunologic Procedures Immunodiffusion precipitation proceeded according to Outch- terlony (1948). Agar gel (1%, 2 mm thick) was prepared on a glass slide. Vitellin antisera and samples were put in separate wells 0.9 cm apart and put in a humid chamber (4"C) for 48 h. After washing (3 x 6 h) in 0.9% NaCl, the gel was stained with Coomassie blue R250. For crossed Immunoelectrophoresis analysis of vitellins in the vitellogenic female, the homogenates of ovaries were separated by agarose gel (1%) in 0.02 M veronal buffer (pH 8.6). The gel portion enclosing the antigen was cut and placed on a glass slide (6.5 x 10 X 0.1 cm). The slide was then covered with 6.5 ml of \% agarose in veronal buffer and 1% anti-vitellin antibodies of L. vannamei. After 18 h of migration at 2 volts/cm. the slide was washed with 9% NaCl and colored with Coomassie blue. The immunoreactivity of the subunits of vitellin with vitellin antisera was examined by Western blotting. After purification of vitellin in a hydroxylapatite column, effluent containing the fourth peak was analyzed by SDS-PAGE (7.5% polyacrylamide gel in TRIS-glycine buffer. pH 7.2, 1% SDS). Proteins in the polyacryl- amide gel were transferred to polyvinylidene diflouride (PVDF. Immobilon transfer membranes. Bio-Rad. Richmond. CA) with a mini transblot electrophoretic transfer cell (Bio-Rad #170-3930) using 25 mM TRIS, 192 mM glycine, 20% methanol buffer. Ni- trocellulose paper was immersed in the following solutions: 5% blotting grade blocker (Bio-Rad # 170-6404) in TBS buffer (0.15 M NaCl. 10 niM. TRIS). antisera against vitellin (1/3000). and goat anti-rabbit IgG-alkaline phosphatase conjugate (1/3000). Color was developed using diaminobenzidine in TBS buffer. N-Tenninal Amino Acid Sequence After purification, the vitellin was analyzed by SDS-PAGE: 7.5% polyacrylamide gel in Tris-glycine buffer. 1.5 M. thioglyco- lateO.l M(pH7.2). 10% SDS. A solution of Tris-Hcl. 312.5 mM; Na2 EDTA. 10 mM (pH 6.9): 15% SDS: and 0.5 M sucrose was used as dis.sociation buffer at 37°C for 10 min. The proteins in the polyacrylamide gel were transferred to Sequi Blot PVDF (polyvi- nylidene diflouride) membrane (Bio-Rad. Richmond. CA) with a mini transblot electrophoretic transfer cell (Bio-Rad #170-3930) using a 25 niM TRIS. 192 M glycine. 20% methanol buffer. The membrane was stained for 5 min with PVDF Coomassie blue R-250. and destained for 10-15 min with PVDF destain solution. The 100 and 60 kDa bands were cut and N-terminal sequenced in a protein/peptide sequencer at the Molecular Medicine Laboratory Biotechnology Institute UNAM, Mexico (DF). RESULTS The native gel electrophoresis patterns of ovarian extracts (pre- cast gel gradient polyacrylamide 4-20%) of mature and non vitel- logenic L. vannamei females showed a specific protein from ma- ture females with an apparent molecular mass of 500 kDa. The protein contained carbohydrates and lipids, based on staining by Sudan black B (Fig. 1) and periodic acid Schiff s reagent, respec- tively. However, when the sample was centrifuged before electro- phoresis, and the gel was stained with Sudan black B, we observed two female-specific lipoproteins of nearly the same molecular size (Fig. 1 ). The crossed immunoelectrophoretic pattern of the ovarian extract of mature L. vannamei females with antiserum against ovarian extract of the same species is shown in Fig. 2. There were two precipitation lines in the ovarian extracts of vitellogenic shrimp (Vtl and Vt2). Two proteins peaks in ovarian homogenate of mature females were obtained from gel filtration chromatography in a Sepharose CL-2B column (Fig. 3). The concentrated second peak showed a single band considered vitellin (500 kDa) and another more nega- tively charged non-vitellogenic band. The second peak from gel filtration chromatography had a specific immunodiffusion precipi- tation line that reacted with antisera against vitellin of L. van- namei. but the first peak did not (results not shown). The concen- trated second peak was separated into four peak fractions, by hy- droxylapatite column chromatography. Electrophoretic analysis revealed two proteins with very approximate migration coefficient (Fig. 4). The third (Vtl) and fourth (Vt2) peaks separated by hydroxylapatite column chromatography, the mature female hemolymph. and vitellogenic ovarian extracts were recognized by polyclonal anti-Vt antibodies raised against L. vannamei (Fig. 5). To confirm these results, the third and fourth peaks were com- bined, concentrated, and separated by reverse-phase chromatogra- VlTELLINS FROM OVARIES IN SHRIMP L VANNAMEI 889 Vt kDa 669 440 232 140 67 12 3 4 Figure 1. Native-PAGE of Litopenaeus mnnamei o\arits. Precast gel gradient 4-20'7f. (B) Lane 1: silver stain ovarian extract mature females: Lane 2: silver stain ovarian extract nonvitellogenic females; Lane 3: molecular marker: Lane 4: Sudan black B stained ovarian extract mature females after centrifugation. phy (HPLC). revealing two peaks with retention times of 14.53 and 19.55 niin (Fig. 6). Characterization of Purified Vitellin The third (Vtl ) and fourth (Vt2l peaks from the hydroxylapa- tite column, analyzed by SDS-PAGE, both showed six polypeptide subunits. The molecular weights of the subunits were estimated at 60. 90. 95. 100. 140. and 160 kDa. To confirm the subunits cor- responding to each vitellin. Western-blotting was conducted with the anti-Vt antibodies raised against L. vaniiamei. thus confirming that they were molecules composed of six similar polypeptide subunits (Fig. 7). Protein Sequencing We sequenced the N-terminal ends of the 100 kDa and 60 kDa subunits, and two amino acid residue sequences. PI (GQVSLA- Vt1 Vt2 Figure 2. Crossed immunoelectrophoretic pattern of ovarian extract L. vannamei using vitellin specific polyclonal antibodies. (\ tl and V't2 vitellins) Fraction number Figure 3. Elution profiles of ovarian homogenates from a Sepharose CL-2B gel filtration column equilibrated and eluted with 0.01 M Tris buffer. Flow rate: 18 niL/h. Fraction size: 2.4 ml. P.4GE (5% gel). Blue Coomassie stain. Peak 1: High molecular weight proteins. Peak 2: Vitellin (Vt) and contaminate proteins. PEFALGXTVE) and P2 (APXGADVPSKG) respectively, were obtained. PI and P2 were aligned to each vitellogenin reported (Fig. 8), and similarity specific to two regions was observed. The conserved residues ""P and ^"S aligned with P2. and the conserved residues """^G and "'"E aligned with PI. This suggests that PI and P2 are derived from a vitellogenin, as in M. japonicus (Tsutsui et al. 2000) and Metapenaeus ensis (Tsang et al. 2003). DISCUSSION One specific protein, with an approximate molecular weight of 500 kDa, was identified by electrophoresis (PAGE 6% and gradi- ent gel 4-20% ) in the ovaries of Litopenaeus vannamei females in vitellogenesis. The characteristics of this fraction (lipo-glyco- protein) were similar to those of penaeid vitellins. but did not exist 100 50 l»M t4-t6 Fraction number Figure 4. The second peak fraction from Sepharose column was frac- tioned in a hydroxylapatite column equilibrated with 0.01 M PPB buffer. Step-wise gradients of 0.01 M, O.IOM, 0.20.M. and 0.35\I PPB buffer. Flow rate: 18 mL/h. Fraction size: 2.4 ml. Peak 1 and 2: con- taminate proteins. Peak 3 (Vtl) and 4 (Vt2l. 890 BOUCARD ET AL. A Figure 5. Inimunoprecipitation of mature female ovary (11 mature female lieinolymph (2, 6| hemolvmph male {^) third peak hvdroxyl- apatite column (4) fourth peak hydroxylapatite column (5) reacted to specific antiserum against vitellin (7) from L. vanmiiiiei. in the ovaries of immature females. Only one form of vitellin has been detected in Panipeiiaeiis langimstris (Tom et al. 1987b). P. monodon (Quinitio et al. 1990, Chang et al. 1993), P. semisidcatus (Tom et al. 1992, Lubsenz et al. 1997), Metapenaeiis ensis (Qiu et al. 1997). P. Japonicus (Vazquez Boucard 1986, Kawazoe et al. 2000), and P. vaiiinimei (Tom et al. 1992, Garcia Orozco et al. 2002). However, when we centiifuged the mature female ovary sample before loading the electrophoretic gel (gradient 4/30%), and stained it with Sudan black B, we confirmed two lipoproteins. Absorbance 0.0400 - 0.0300 - Vt2 \ vtl / ^vti \Vt2 0.0200 - i 0.0100- 0.0000 _J u 1 10.00 1 20.00 1 30.00 Minute Figure 6. Analytical HPLC of combined third Vtl and fourth Vt2 peak obtained from a hydroxylapatite column. Flow rate, absorbance at 280 nm. B kDa 200 '*«-'=?^!»tl"- :,,i;:^«.j..i :;*.;,' ftjf. -116 ■a, .,, . 97 -66 >iV;v*,ii':vi;;K; ,-^n'iiVf;,-'V':>''SJ;i 1 2 Figure 7. \. Western blotting analysis of vitellins purified by hydrox- ylapatite column (third and fourth peakl; B. SD.S-PAGE (7.5 "7, ) of (I) third and fourth peak of hydroxylapatite column. (2| molecular marker Similarly, the crossed immunoelectrophoresis showed two lines of precipitation with L. vaniuwiei anti-vitellin antibodies. After fur- ther separation by hydroxylapatite column and HPLC, two main vitellin peaks were seen also, which might correspond to the vi- tellins detected by native gradient gel electrophoresis and crossed- immunoclectrophoresis. Denatured SDS gel electrophoresis of the native vitellin isolated (Vtl and Vt2) showed six subunits with VTG_ VTG^ vtg' vtg] vtg' Pi' MARJA: PENSM; 'CHRQU : ^METEN : ^MACRO : LPEVA : LPEVA: 20 JL ;£NGADLPRCSrE. ;f NISADLPRCSrE. I P FGOTTPVC S IE . ;PElEDEAPRCSrE. ^JHPSGTNLCSKE. ;PXGADVP. .SKG. 635 .AFAF(a