LABORATORY CULTURE OF ZEBRA (DREISSENA POLYMORPHA) AND QUAGGA (D. BUGENSIS) MUSSEL LARVAE USING ESTUARINE ALGAE

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1 J. Great Lakes Res. 22(l):46-54 Internat. Assoc. Great Lakes Res., 1996 LABORATORY CULTURE OF ZEBRA (DREISSENA POLYMORPHA) AND QUAGGA (D. BUGENSIS) MUSSEL LARVAE USING ESTUARINE ALGAE David A. Wright, Eileen M. Setzler-Hamilton, John A. Magee, and H. Rodger Harvey The University of Maryland System Center for Environmental and Estuarine Studies Chesapeake Biological Laboratory P. O. Box 38 Solomons, Maryland ABSTRACT Zebra (Dreissena polymorpha) and quagga mussel (D. bugensis) larvae were reared through and beyond metamorphosis in the laboratory on diets of the estuarine algae, Isochrysis galbana T-Iso and Pavlova (= Monochrysis) lutheri. Larvae were successfully spawned and raised in the laboratory for over 1 year with routine survival to settlement. Some adult males reared from an August 1994 spawn reached sexual maturity with active sperm by April Diets of dried Chlorella sp. and Synecococchus sp. were unable to support larvae. Settlement of pediveligers at 22 C occurred at ~ 21 days for zebra mussel and 32 days for quagga mussels. Saturated fatty acids predominated in I. galbana and P. lutheri, whereas polyunsaturated fatty acids (PUFAs) predominated in Chlorella sp. The process to culture dreissenid larvae in the laboratory is very labor-intensive and requires continuous culture of live algae maintained in log growth phase. Larvae are fed every other day at a density of 2 x 10 5 cells ml- 1 for the first week, and then daily densities of 3-5 x 10 5 cells ml -1 thereafter. Water quality is critical. Larvae and juveniles should be stocked at densities of ~ 1 ml -1 and the water must be changed at least 3 x wk -1 in static cultures. Water must be changed at least weekly in tanks holding adult brood stock. Methods of controlling protozoan and rotifer infestation of cultures are also important for successful culture. INDEX WORDS: zebra mussels, quagga mussels, diet, fatty acids, culture, estuarine algae. INTRODUCTION Inadvertent introduction of zebra mussels (Dreissena polymorpha) into the Great Lakes and their subsequent dispersal into the St. Lawrence, Illinois, Mississippi, Ohio, Arkansas, Tennessee, and Hudson rivers and a number of inland lakes in Ontario, Ohio, Indiana, Kentucky, and New York, and the recent identification of a second dreissenid species, the quagga mussel (D. bugensis) (May and Marsden 1992, MacIsaac 1994) has prompted concern over the eventual extent of their colonization in North America (Neary and Leach 1992, Ludyanskiy 1993). At least one species, the zebra mussel has been shown to have major economic and ecological impacts in North America (Nalepa and Schloesser 1993). These mussels are unique to the freshwater molluscan fauna of North America because they possess free swimming larvae which greatly enhance their dispersal through freshwater systems (Griffiths et al. 1991). This contrasts with the freshwater Unionidae which have parasitic larvae, and the freshwater Sphaeriidae in which larvae develop in adults. Particular attention has focussed on the larvae as the most sensitive developmental stage, and the human and environmental factors which might limit its survival, growth and dispersal. Although several laboratories have expended considerable effort on attempts to culture zebra mussels, and the closely related quagga mussel, success has proved surprisingly elusive. Paradoxically, despite the great fecundity of zebra mussels in the natural environment (Walz 1978; Sprung 1990, 1993; Neumann et al. 1993), most researchers have been unable to rear larvae beyond several days after hatching in the laboratory (Nichols 1993). Reports of survival through metamorphosis (usually ~ 3 weeks) are rare and survival to settlement low. Apart from data presented in this paper the most successful attempts to culture zebra mussel larvae have been those of Vanderploeg et al. (1996). Van-derploeg et al. (1996) report maximum survival from D stage to pediveliger stage of 27, 34, and 9% respectively using the marine and freshwater strains of the green alga, Chlorella minutissima, and the cryptophyte, Rhodomonas minuta. Settlement began 2-3 weeks after hatching when larvae reached с 220 µm (Liebig and Vanderploeg 1994). Vander-ploeg et al. (1996) attribute success to settlement to the small size of the algae used (< 6 µm) and their fatty acid profiles (rich in eicosapentaenoic acid, EPA, 20:5(n-3) or docosahexaenoic acid, DHA, 22:6(n-3)). The importance of fatty acid profiles similar to those utilized by marine bivalve species for survival of zebra and quagga mussels (Enright et al. 1986a, b) is

2 Culture of Zebra and Quagga Mussels reasonable considering the close taxonomic relationship between Dreissena and the brackish water genus Mytilopsis (of which there are two North American species; Abbott 1974). The origin of D. polymorpha and D. bugensis in the brackish water areas of the Black and Caspian seas has led to concern over their potential salinity tolerance in estuarine areas of North America. Some initial studies on salinity tolerance have been reported (Kilgour et al. 1994, Setzler-Hamilton et al. 1995, Wright et al. 1995). Another factor which must be considered is whether existing algal assemblages in tidal freshwater, oligohaline (0.5-5% o ), and mesohaline (5.1-18%c) areas of temperate estuaries would provide suitable food for Dreissena populations. We report here on progress made in culturing D. polymorpha and D. bugensis under laboratory conditions and the success of utilizing marine/estuarine algal species as food sources. MATERIAL AND METHODS BROODSTOCK COLLECTION Adult zebra mussels were collected from Lake Erie near Put-In-Bay, Ohio, and Erie, Pennsylvania, in June 1994 and from Lake Ontario near Buffalo, New York, in July 1994 and May Adult quagga mussels were collected from Lake Ontario near Buffalo and Olcott, New York, in July 1994 and May All experimental animals were transported to Maryland according to required protocols of the Maryland Department of Natural Resources governing their transport. Mussels were held in culture water of 12.0 g CaCl 2 2H 2 O, 10.0 g MgSO 4 7H 2 O, g NaHCO 3 and 110 L of well water, at 17 C with a 14h:10h light/dark cycle. Culture water contained 31 mgl -1 Ca 2+, 10 mg L -1 Mg 2+, 38 mg L -1 SO4 2-, 51 mg L -1 СГ, and 0.4 mg L -1 NO3 -. Additional mussels were maintained at 13 C. Mussel food was provided (per 1,000 mussel individuals of cm length) as 1 g dried Chlorella sp. added to 500 ml of culture water and blended for 10 minutes. SPAWNING Approximately 25 adults were scrubbed of debris under running deionized water and placed for 5 minutes in a 0.03% sodium hypochlorite solution to control protozoan and rotifer populations. Mussels were then rinsed for 2-3 minutes with deionized water and placed into a 10-L polycarbonate spawning chamber filled with 8 L of 22 C culture water. Culture water was aerated and replaced after 30 minutes to remove pseudofeces. A slurry of gonads prepared from 2-3 male mussels in a mortar was added to the aquarium. If spawning did not begin within 30 minutes, water temperature was raised to 24 C by the addition of C culture water. Once spawning began, mussels were placed into 40-L aquaria filled with L of 22 C culture water. When aquaria became cloudy with eggs and/or sperm, spawning mussels were transferred into another 40-L aquarium. This was repeated until spawning ceased (generally 3-4 hours). Each aquarium was filled to 40 L with culture water and strongly aerated. Gametes were examined under a microscope to determine the number of sperm attached to each egg. Because few eggs developed normally when more than 100 sperm were attached per egg, culture water was diluted as necessary to lower sperm concentrations to approximately 100 sperm per egg. CULTURE AND FEEDING Larvae reached the D-hinge stage after hours. Densities of D-hinge larvae in each container were determined by filtering several liters of culture water through a 33 urn Nitex sieve and concentrating to 100 ml. The concentrate was mixed with a plastic stirrer and three 1-mL aliquots were counted using a Sedgwick-Rafter counting chamber. Larval densities were adjusted to ~ 2-3 ml -1. Culture water was changed in each container on the third day and 0.25 ml of a penicillin/streptomycin/ neomycin solution (500 units penicillin, 5 mg streptomycin, and 10 mg neomycin per ml: Sigma #3664) was added to each liter of culture water. Culture water was completely replaced three times weekly and penicillinstreptomycin-neomycin added with each water change in initial experiments. Antibiotics were only used in later experiments when there was evidence of bacterial contamination of culture water. Stock cultures were reared in 40-L aquaria and all experiments were conducted in 4-L plastic beakers. Air stones (one/aquarium) provided aeration in 40-L aquaria; and one Pasteur pipette aerated each 4-L beaker. In initial experiments in 1994, larvae were fed live cultures of algae at a concentration of 1.0 x 10 5 cells ml- 1 every other day until the 15th day after which larvae were fed daily. In later experiments, larvae were fed every other day during the first week and daily thereafter at algal densities of x 10 5 ml -1. Algal densities were gradually increased to x 10 5 cells ml -1 as the larvae grew and settled as juveniles (Table 1). Juveniles 5 months old and older were fed daily at a rate of 8.0 x x 10 6 cells ml -1. Rotifer and ciliate protozoan infestations in larval cultures of older larvae (> 180 µn) were controlled by immersing larvae in a 47

3 Wright et al. 0.03% sodium hypochlo-rite solution for 2-3 minutes and rinsed for about 1 minute under running deionized water. An alternate method of controlling rotifer and ciliate infestations in juvenile mussel cultures was by immersing the mussels in = 29% o salinity (Instant Ocean ) for 5 minutes and then rinsing well in deionized water. Frequent water changes, daily if necessary, also greatly reduced the incidence of protozoan and rotifer infestations. The estuarine algal species, Isochrysis galbana T-Iso, CCMP 1324, Isochrysis galbana C-Iso, CCMP 1323, and Pavlova (= Monochrysis) lutheri, were cultured at salinities of 8-9% o obtained by diluting Patuxent River water (ambient salinities = 13% o ) with laboratory well water. In December and January, two freshwater algal species, Scenedesmus sp. and Neochloris oleatundans, were cultured, respectively. Algae were grown in f/2 medium (Guillard 1975) at 20 C, 14:10 L:D cycle, in 20- L glass containers with aeration supplied through glass tubing. Light was provided by four, 48-inch 40- watt fluorescent bulbs and algae was harvested during exponential-phase growth. Both freshwater species as well as dried Chlorella were used to supplement the feeding of juvenile zebra and quagga mussels. Larvae were fed only I. galbana T-Iso. Experiments were undertaken with 3-d-old zebra and quagga mussel larvae to determine survival and settlement on different algal diets. Four algae were used; Isochrysis galbana T-Iso and I. galbana C-Iso, Pavlova lutheri, and dried Chlorella sp. as a control. Feeding experiments were conducted in triplicate in 4-L plastic beakers stocked with larvae at a density of one larva ml -1. Experimental procedures are described elsewhere (Wright et al. 1995). FATTY ACID ANALYSIS Samples of each algal food were collected onto precombusted GF/F filters for analysis of total fatty acids. Lipids were extracted three times from algae using dichloromethane:methanol (1:1) with probe sonication as described by Harvey et al. (1987). After mild alkaline hydrolysis with 0.2N methanolic KOH the extracts were acidified and polar lipids were partitioned into hexane:diethyl ether (9:1). Fatty acids were converted to methyl esters using BF 3 in methanol with gentle heating (30 min at 50 C). Fatty acids were quantified by capillary gas chro-matography (GC) using a Hewlett Packard 5890A instrument with flame ionization detection. A (5%-phenyl)-methyl silicone fused capillary column (DB-5, 30 m, 0.32 mm I.D., 0.25 µn film thickness) was employed with hydrogen as the carrier gas. The temperature program was 50 C to 120 C at a rate of 10 C min -1 followed by 4 C min -1 to 300 C. A dedicated data system (Chemstation-Hewlett Packard) was used to quantify the fatty acids by comparison of peak areas with an internal standard, nonade-canoic acid. Structural identification was performed by comparison of the mass spectrum of the different GC peaks with a standard MS library (ref. library). Mass spectra were acquired with a capillary GC interfaced with a Hewlett Packard 5970B mass-selective detector operating at 70eV with an acquisition over the amu range. The temperature program was the same as the GC with the exception helium was the carrier gas. Structural confirmation of fatty acids used a series of authentic standards and mass spectral interpretation. RESULTS 48 FEEDING AND CULTURE Zebra and quagga mussel larvae were reared through and beyond metamorphosis (settlement), beginning in August 1994, on individual diets of Isochrysis galbana C-Iso or Pavlova lutheri. It was not possible to rear zebra mussel larvae through metamorphosis using dried Chlorella, Synechococ-cus sp., or an algal paste of I. galbana C-Iso and I. galbana T-Iso or P. lutheri. Diets of settled juveniles were supplemented with the freshwater algae Scenedesmus sp. and Neochloris oleatundaras in some experiments. Zebra mussel larvae in the controlled feeding experiment reached metamorphosis and settlement on diets of both I. galbana T-Iso and P. lutheri. No larvae fed dried Chlorella sp. survived through metamorphosis. High mortality caused by rotifer predation compromised results of this feeding experiment and the experiment was discarded. At the D-hinge stage, ~ 96h after fertilization, zebra and quagga mussel larvae averaged 85 ± 8.1 urn and 80 ± 7.2 µm, respectively (n = 10). Zebra mussel larvae grew ~ 9 µm d -1 from D-hinge stage (4 d) until settlement. Growth rates (increases in shell length) of 45-d-old juveniles from the "23 Day ZM" experiment averaged 10 µm d -1 (Table 1). Shell lengths of these juveniles increased on average, 54 µm d -1 from Day 45 to Day 75, and 52 µm d -1 from Day 75 to Day 169. The most successful culture of zebra mussels occurred when fed Isochrysis galbana T-Iso during 16 of the first 20 days. Thereafter, I. galbana T-Iso was supplemented with either I. galbana C-Iso or Pavlova lutheri. This culture exceeded 50% settlement after 28 days, based on the number of 3-d-old D-hinge larvae. The most successful quagga mussel culture in 1994 was also fed almost exclusively I. galbana T-Iso and had more than 60% survival for 20 days, although this fell to 25% by the time of settlement. However, in July 1995 cultures of quagga mussels (fed exclusively I. galbana T-Iso) exceeded 85% survival (and > 220 µm; 29 days after fertilization). In the feeding experiment begun in December 1994, mean survival of quagga larvae fed P.

4 Culture of Zebra and Quagga Mussels lutheri, I. galbana C-Iso, and I. galbana T-Iso exceeded 15% after 20 days (Table 2), although few larvae survived in the Chlorella sp. treatment. Larvae fed I. galbana T-Iso had the greatest survival; mean survival after 36 days was 44 ± 20.3% (± 2SE; n = 3). Due to intrasample variability, percent survival was not significantly different (P < 0.05) from that seen at 20 days (29 ± 10.4%; n = 3). Seventy percent of surviving quagga larvae had settled by 36 days (27% of the initial number at the beginning of the experiment). Nine percent (± 6.0%; n = 3) of the larvae fed I. galbana C-Iso were still alive on Day 36, although none had settled. Maximum shell lengths for the I. galbana T-Iso fed larvae and the I. galbana C-Iso fed larvae were 215 ± 6.4 µm (± 2SE) and 158 ± 19.5 µm respectively, a highly significant difference (p < 0.01) in larval size between the two groups. There were no survivors after 36 days among larvae fed exclusively P. lutheri. TABLE 1. Feeding growth and settlement of young zebra (ZM) and quagga (QM) musssels in the laboratory. Larvae were fed daily except for the first week when larvae were fed every other day. 1Experiment designation refers to age of larvae at initiation of salinity tolerance experiments in our laboratory. Data presented in Table 1 are for control mussels reared in fresh culture water. See Wright et al. (1995) for results of salinity tolerance experiments. Zebra mussel larvae grew more quickly and settled in less time than quagga mussels. Routinely, zebra mussel larvae began settlement by about day 21 (slower than the time reported by Vanderploeg et al. 1996) and settlement was > 99% complete by Day 30. Quagga mussel larvae under identical conditions began settlement on or about Day 32. Settlement in this species was > 95% complete by Day

5 FATTY ACIDS IN ALGAE Wright et al. Table 3 shows the relative distribution of fatty acids of Pavlova lutheri, Isochrysis galbana T-Iso, Isochrysis galbana C-Iso (cultured in the laboratory), and Chlorella sp. (obtained as powder from an independent supplier). Fatty acid class distribution (Table 4) shows a preponderance of saturated fatty acids in P. lutheri, I. galbana T-Iso, and I. galbana C-Iso. In cultured assemblages, saturated and monosaturated (chiefly 18:1) fatty acids were present in equal proportion. In powdered Chlorella sp., polyunsaturated fatty acids (PUFAs) accounted for 67% of the total fatty acids, with linoleic acid (18:2(n-6)) and linolenic acid (18:3(n-3)) dominant. Saturated fatty acids consisted primarily of 20:0, 18:0, and 14:0. The latter was present only in trace amounts in Chlorella sp. Isochrysis galbana T-Iso was the sole source of 18:5 and 21:0. Only P. lutheri was the sole source of 20:4, 20:5, 22.0, and TABLE 2. Survival and settlement of quagga mussel (D. bugensis) larvae fed on single species algal cultures. TABLE 3. Relative distribution (%) of fatty acids in four cultures of algae. *tentative assignment Footnote: Fatty acids are described by the length of the carbon chain: no. of double bonds (position of double bands in fatty acids). 50

6 Culture of Zebra and Quagga Mussels TABLE 4.Distribution of classes of fatty acids in four cultures of algae Isochrysis galbana Fatty Acid Classes Pavlova lutheri (C-Iso) (T-Iso) Chlorella sp. Saturated Monounsaturated Polyunsaturated Bacterial Total A small amount of the odd carbon length fatty acid 15:0 diagnostic of bacteria (e.g., Kaneda 1991) was found in all algae, although 17:0 was only detected in Chlorella sp. The higher bacterial value found in Chlorella sp. may be due to its storage over 1 year compared with cultured algae. DISCUSSION Success or failure of algae as a food source depends upon a variety of factors including size and fatty acid profile of the algae. In particular, fatty acids of the (n-3) series contain essential dietary factors for marine animals as only phytoplankton are capable of their de novo synthesis (Fraser et al. 1989). Dietary studies with oyster larvae have suggested that (n-3) polyunsaturated fatty acid content is of particular importance in characterizing a nutritious diet (Chu and Dupy 1980; Enright et al. 1986a, b). Reports on lipid metabolism of freshwater zooplankton are much less common although some information is available and the importance of (n-3) as well as (n-6) is again stressed (Conklin and Provasoli 1977). Although a low incidence of settlement in zebra mussel larvae fed Pavlova lutheri was recorded in October 1994, the growth and survival of Dreissena polymorpha was consistently better when fed Isochrysis galbana T-Iso. Similar observations were made with D. bugensis. It is not immediately apparent from its fatty acid profile why Isochrysis galbana T-Iso represents the most successful diet. For example, P. lutheri contained a similar amount of docosahexaenoic acid (22:6) as Isochrysis galbana T- Iso and was the only source of eicosapentaenoic acid (20:5) in cultures. The relatively low percentage of 22:6 and lack of 20:5 in Isochrysis galbana T-Iso cultures is contradictory to the work of Lang-don and Waldock (1981) and Enright et al. (1986a, b) who stressed the importance of these PUFAs as constituents of algal diets for juvenile oysters. Enright et al. (1986a) found that Chaetoceros gracilis fed as a monoculture to juvenile oysters resulted in a growth rate 1.8 times higher than oysters fed Isochrysis galbana T-Iso. They noted that C. gracilis had a high 22:6 content (2.5%). Enright et al. (1986a) obtained best oyster growth rate using a mixed algal diet of I. galbana T-Iso, C. gracilis, C. simplex, P. lutheri, and Phaeodactylum tricornutum in equal cell numbers. Using this diet, growth rates were 1.9 times higher than using I. galbana T-Iso alone. From the data reported here it seems unlikely that the success of culturing D. polymorpha can be explained solely by the level of n-3 PUFAs in the diet. However, we did not analyze for carbohydrate and proteins in the current work. Webb and Chu (1983) found that the nutritional status of algae correlated better with total protein than with total lipid or carbohydrate content. The relative success of I. galbana T-Iso as a food source cannot be explained simply on the basis of size considerations. All of the algal species used here were selected for their small size and, therefore, acceptability to larval Dreissena. In fact the upper end of the I. galbana T-Iso size range (3-6 µm) included the largest of the cultured cells. We have insufficient data to be certain of differences in dietary preference between D. polymorpha and D. bugensis. However, we believe distinct differences in the conditioning cycle between the two species exists. In mixed stocks of the two dreissenids collected from northeastern Lake Erie the last week of June 1994 and held under identical conditions, D. polymorpha lost spawning condition faster than D. bugensis. These stocks were held at 13 C and fed almost exclusively powdered Chlorella sp. supplied in similar density, often in mixed species containers. We were unable to spawn D. polymorpha from the beginning of November 1994 and this stock was replenished in Spring However, D. bugensis were ripe with 100% spawning success through April 1995 and D. bugensis and D. polymorpha broodstock collected in May 1995 remained ripe with 100% spawning success as of this writing. Stocking densities used in experiments (± 1 larva ml -1 ), and in stock cultures (± 2-3 larvae ml -1 ), were based on previous experience rearing 51

7 Wright et al. oysters, Crassostrea virginica, and other marine bivalve larvae. Densities utilized are less than 10 eggs of D. polymorpha ml -1 used by Sprung (1989) and those recommended by Bayne (1965) for Mytilus edulis larvae (3-10 larvae ml -1 ). Jespersen and Olsen (1982), however, reared larvae of M. edulis at densities between 0.1 and 0.2 larvae ml -1. Egg densities at time of fertilization may exceed optimal holding densities for larvae, although we did not exceed 10 eggs ml- 1 in this study. Presumably, abnormal egg development at sperm:egg ratios higher than 100 results from polyspermy. Although no detailed investigation of polyspermy in Dreissena was made in the current study, it appears that the threshold for polyspermy in dreissenids is lower than, for example, the eastern oyster Crassostrea virginica where a sperm:egg ratio of 1,000:1 was exceeded before polyspermy occurred (Alliegro and Wright 1983). Based on the appearance of D. polymorpha after 3 days, Sprung (1987) noted only small changes in percentages of abnormal larvae over a broad range of egg:sperm ratios. Densities of algae used to feed larvae and juveniles (2 x 10 5 cells ml -1 every other day for the first week, increasing to 3-5 x 10 5 cells ml -1 ) are higher than those used by other investigators. Vanderploeg et al. (1996) fed larvae algae at a concentration of 1-2 mm 3 L -1 (l mm 3 L -1 = 21,000 Isochrysis galbana cells ml -1 ). Sprung (1984) found increased growth rates of Mytilus edulis larvae fed I. galbana up to densities of = 10 4 cells ml" 1, followed by a decrease or stabilization in growth rates at denser algal cultures. Sprung suggested this decline in growth rates might be due to hampering of the feeding apparatus of larvae by excessively high numbers of algal cells and the sensitivity of larvae to algal metabolites. Bayne (1965) found optimum growth of M. edulis larvae fed I. galbana at concentrations of 10 5 cells ml -1, whereas Jespersen and Olsen (1982) used a mixed culture of Isochrysis and Pavlova (= Monochrysis) and recommended food concentrations of 4-5 x 10 4 cells ml -1. Growth rates of dreissenid larvae were ~ 9 µm d -1 until settlement and µm d -1 for juveniles. These rates are less than average growth rates of mm d -1 and mm d -1 for cohorts of zebra mussels larvae in fall 1988 and spring 1989 in Lake St. Clair, respectively (Mackie 1993). Successful aquaculture of Dreissenid larvae can be a relatively straightforward procedure and one which can be carried on throughout the year in a laboratory. Rearing of mussels is very labor-intensive however, and does require continuous culture of live algae maintained in log growth phase. We fed larvae every other day at a density of 2 x 10 5 cells ml -1 for the first week, and then daily at algal densities of 3-5 x 10 5 cells ml -1. Algal densities can be gradually increased to 8 x x 10 6 cells ml -1 for juvenile mussels several months old. The practical limiting factor to mussel culture is likely to be the labor required to produce sufficient algal stocks to feed juveniles to the maximum amount mussels can ingest. Therefore, to keep adult brood stock in spawning condition for prolonged periods, live algal cultures may need to be supplemented with dried Chlorella sp. In addition, water quality is critical. We recommend stocking larvae at densities of =1 ml -1, and changing water at least 3x wk -1 in static cultures of larvae and juveniles, and at least weekly in tanks holding adult brood stock. ACKNOWLEDGMENTS This research was funded by the National Sea Grant Exotic Species Research Program with additional funds from Maryland Sea Grant. We thank J.T. Hageman, Ohio State University, F.T. Stone Laboratory, L. Chalker-Scott, Buffalo State University, and C. Murry, Pennsylvania Fish and Boat Commission for assistance in collecting zebra and quagga mussels, and R. Klauda, Maryland Department of Natural Resources for help in obtaining necessary permits to hold and rear mussels in the laboratory. This research was conducted with the assistance of R. Andrewson, S. Cassidy, and J. Kraly. We thank M. Ederington for the fatty acid analyses. Publication No of the Center for Environmental and Estuarine Studies, The University of Maryland System. 52 REFERENCES Abbott, R.T American Seashells. The Marine Mollusca of the Atlantic and Pacific Coasts of North America. New York, New York: Van Nostrand Rein-hold Co. Alliegro, M.C., and Wright, D.A Polyspermy inhibition in the oyster, Crassostrea virginica. J. Exp. Zool. 227: Bayne, B.L Growth and the delay of metamorphosis of the larvae of Mytilus edulis (L). Ophelia 2:1-47. Chu, F.-L.E., and Dupy, J.L The fatty acid composition of three unicellular algal species used as food sources for larvae of the American oyster (Crassostrea virginica). Lipids 15:

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