Differences in strains of Isochrysis of importance to mariculture

AquaCUltuVe ELSEVIER

Aquaculture 123 (1994) 127-135

Differences in strains of kwchryszk of importance to mariculture Gary H. Wikfors”,*, Glenn W. Pattersonb “NOAA,National Marine Fisheries Service, Northeast Fisheries Science Center, 212 Rogers Avenue, Milford, CT 06460-6499, USA bDepartment of Botany, University of Maryland, College Park, MD, USA (Accepted 19 January 1994)

Abstract Algal strains identified as Isochrysis are used widely in commercial mariculture and in rearing marine invertebrates for research purposes. Isochrysis isolates are from several geographic locations, and their relationships to each other are not known. Research reports of Zsochrysisgrowth characteristics, biochemical composition, and nutritional value to invertebrates suggest that a number of these strains differ in terms of their taxonomy and fundamental biology; their practical use as mariculture feeds differs as well. In this note, we discuss origins of some of these strains, describe differences between these from our own and others’ research, and recommend that researchers be cognizant of the cultural history of their Zsochrysisstrains because differences between them will affect their use as mariculture feeds.

1. Introduction Among the most widely-used phytoplankton cultures in mariculture are a number of strains identified as belonging to the genus Isochrysis (Parke, 1949 ). The genus, within the taxonomic class Prymnesiophyceae (Hibberd, 1976)) includes small (ca. 5-7 pm) unicells with two equal flagella, chrysophycean pigmentation, and no cell wall. There have been three described species of Isochrysis: I. galbana (Parke, 1949; Green and Pienaar, 1977)) I. maritima (Billard and Gayral, 1972), and I. Zitoralis (Billard and Gayral, 1972 ), although I. maritima was transferred to Chrysotila lamellosa by Green and Parke ( 1975). The dominant life-history *Corresponding author. 0044~8486/94/$07.00

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stage of I. litoralis is a palmelloid form, therefore this species has received essentially no attention from mariculture research that focuses most often upon suspension culture of microalgae. I. galbana is the species historically first used as a live food in molluscan mariculture (Bruce et al., 1940) and cited most often since. The small size and lack of cell wall render these cells ingestible by small, larval invertebrates and easily digested (Walne, 1964; Ukeles, 1975; Babinchak and Ukeles, 1979). In addition to the described species, strains isolated from tropical and subtropical waters and identified provisionally as Isochrysis are now in wide use, chiefly as live foods for larval bivalves; these strains generally are referred to as T-IS0 (from Tahiti) and C-IS0 (from the Caribbean). Distribution of strains among and between marine research and production facilities has been wide and frequent, and often undocumented. With such distribution, origins and identities of strains may have become lost or confused. Problems arise when culture performance or nutritional value obtained in a particular application differ substantially from expectations based upon published reports or experience of others. When Isochrysis strains of known different identity have been compared directly, differences in such characteristics as temperature tolerance (Ewart and Pruder, 198 1 ), biochemical composition (Brown et al., 1989; Brown, 199 1 ), or nutritional value to invertebrates (Brown et al., 1989; Okauchi, 1990) have been demonstrated. Recent research in our laboratories showed substantial, qualitative differences in the lipid compositions - specifically of sterols and long-chain alkenones - derived from six strains of Isochrysis (Patterson et al., 1994). Of most surprise was the finding that two strains assigned to the same species, I. galbana, had sterol and alkenone compositions that were fundamentally different. In response to. that finding, we describe here origins of the following Isochrysis strains in the Milford Laboratory Microalgal Culture Collection: BL-IS0 (CCMP1323), LB-1292, T-ISO, C-ISO, M-ISO, and UW-330 (CCMP462). We also discuss similarities and differences in biochemical composition of strains and suggest likely implications relative to published literature and the use of Isochrysis strains in mariculture.

2. Strains, origins, and affinities Flagellate I, a.k.a., CCMPl323 or BL-IS0 ‘Flagellate I” (Plymouth Laboratory strain designation), isolated in 1938 from a seawater fish pond near Port Erin, Isle of Man, UK by Mary Parke, is the culture from which both the genus and the type species, I. galbana, were described (Parke, 1949). Accordingly, this culture, and those that can be demonstrated to be descended from it, are the only truly authentic cultures of I. galbana. “Plagellate I” was maintained as such at the Plymouth (UK) Laboratory. When Plymouth algal-culture holdings were transferred to the Cambridge Laboratory, the strain designation was changed to “LB 927/l ” (Culture Centre of Algae and Protozoa, 197 1). The CCAP 1971 List of Strains indicates that “LB 927/l” was

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descended from “Plymouth ‘g”‘, but this was amended in a later communication to be the type material (Culture Center of Algae and Protozoa, 1972). “ISO”

At least several times (of which we are aware), algal cultures were provided to researchers in the United States from the Plymouth Laboratory. Correspondence between M. Parke and H.C. Davis at the Milford Laboratory during the early 1950s indicates that some attempted mailings did not survive transit or initial subculture at Milford. Davis ( 195 3 ) acknowledges receipt of “Flagellate I” from Plymouth for use in larval-oyster feeding studies at Milford; however, Davis and Guillard ( 1958) indicate that the Isochrysis “galbana” used in this later study was obtained from L. Provasoli at the Haskins Laboratories in New York. Records of R.R.L. Guillard (pers. commun.) indicate that the strain he obtained from Dr. Provasoli on 17 November 1955, was the same as that used by McLaughlin ( 1958); McLaughlin’s reference states that his I. galbana culture was sent from Plymouth by M. Parke and was rendered bacteria-free at the Haskins Laboratory. Records of I.J. Pintner (pers. commun. ) indicate receipt at the Haskins Laboratory on 15 November 1955, directly from M. Parke, of a strain of Isochrysis galbana designated “Plymouth no. 9”; her notes also confirm that this was the strain used in the work of J.J.A. McLaughlin. Although it is possible that “Plymouth ‘g”’ and “Plymouth 9” designations could have been confused by misreading a label, it seems unlikely that McLaughlin was able to rid the culture of bacteria and provide a subculture to Guillard in 2 days. Guillard’s records show that he was treating a number of cultures, including one labelled “ISO” already at Milford, just prior to his receipt of the Isochrysis culture from Provasoli; it is not clear which of these cultures gave rise to the “ISO” culture deposited in the Milford Collection. It also seems possible that the I. galbana culture deposited in the Milford Laboratory Microalgal Culture Collection by Guillard may have been replaced at a later date with another bacteria-free isolate, although records do not confirm this (Ukeles, pers. commun. ) . The strain designation “ISO” has been used for the culture we have been calling I. galbana since it was deposited at Milford. IS0 and M-IS0 The most troubling discrepancy between strains we analyzed is the fact that CCMP 1323 has sterol and long-chain alkenone compositions consistent with previous analyses at Plymouth of “Flagellate I” (Marlowe et al., 1984; Patterson et al., 1994); records indicate that CCMP1323 is descended from Milford “ISO” (Andersen et al., 199 1). We are confident from our analyses that CCMP 1323 is descended from Plymouth “Flagellate I”, but are uncertain of the true identity of Milford’s “ISO”. We have given Milford’s “ISO” the amended strain designation “M-1SO”; it differs considerably in lipid composition from strains known to be descended from the “type” culture of I. galbana. Specifically, “M-1SO” contains no epibrassicasterol, the principal sterol of “Flagellate I”, and “M-1SO” contains no long-chain alkenones (Patterson et al., 1994). The sterol composition of

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“M-1SO” is very similar to that of several Pavlova species (Gladu et al., 199 1). Initially we were concerned that “M-1SO” was a mis-labelled culture of P. lutheri; however, observations with phase-contrast and scanning electron microscopy revealed a short haptonema, a morphological feature typical of Isochrysis, but different in appearance in Pavlova (Green, 1980). In addition, we analyzed a number of sub-strains separated at various times over the past 20 years from the “parent ISO” strain and maintained in parallel culture in a number of different growth media with differing salinities, nutrient contents, etc.; all had similar sterol compositions. If another culture at Milford was mistakenly labelled “ISO”, this occurred prior to 1976 (date of earliest parallel culture). Besides P. Zutheri,we considered the possibility that Dicrateria inornata was mis-labelled, however, sterols of D. inornata clearly are different from those of either “M-1SO” or CCMP1323 (Gladu et al., 1990). Interchange with Prymnesium parvum is unlikely as “M-1SO” has been used in feeding studies with many invertebrates, as well as with tinfish, and no toxic responses were ever obtained. In recognition of demonstrated, qualitative differences between the Milford strain and the “type” strain, we will refer to the renamed Milford strain “M-1SO” as Isochrysis sp. until further taxonomic studies are undertaken to determine if this is, indeed, even in the genus Isochrysis. The Milford “ISO” strain has been the subject of many publications from the Milford Lab over the years, with the species listed consistently as Isochrysis galbana; work conducted before 1955 most likely employed the “Flagellate I” received by H. Davis directly from Plymouth (see above), while that done later, after the bacteria-free culture was obtained by R.R.L. Guillard, must be assumed to have been done with “M-1SO”. A partial listing of studies on the rearing of a number of molluscan species that were conducted with “M-1SO” follows: Loosanoff and Davis, 1963; Rhodes and Landers, 1972; Landers, 1976; Losee, 1978; Goldberg, 1980, 1989. It should be noted that much of the original research on artificial propagation of mollusks (Loosanoff and Davis, 1963) was conducted using algal food cultures that may or may not have been descended from “Flagellate I”. Certainly later work was done with what we are now calling “M-ISO”, including the studies of Ukeles on algal temperature tolerance (Ukeles, 196 1), inorganic carbon utilization (Ukeles and Rose, 1976), and culture for feeding invertebrates (Ukeles, 1973 ). Studies of metal tolerance (Wikfors and Ukeles, 1982; Wikfors et al., 1991a) and biochemical aspects of molluscan nutrition (Wikfors et al., 1992) also report data for “I. galbana” that must be re-evaluated in light of our recent recognition of substantial, qualitative biochemical differences between “M-1SO” and “Flagellate I”. T-IS0 The Tahitian isolate known as “T-1SO” has been referred to in published reports as Isochrysis sp., Isochrysis aff. galbana, and as I. galbana. This strain was isolated from a CNEXO aquaculture pond in Tahiti by K. Haines, who provided a bacteria-free culture to A. Michel in Tahiti; the strain analyzed by Patterson et al. ( 1994) was obtained from Michel by R. Ukeles at Milford in 1977. To our

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knowledge, there is not a published taxonomic (morphological) study of this strain to place it with any confidence in the genus Isochrysis, let alone the species I. galbana. Our lipid analyses (Patterson et al., 1994) demonstrate clear differences in sterol and alkenone composition between CCMP 1323 and “T-ISO”, although the presence in both of long-chain alkenones suggests stronger afftnities between “Flagellate I” and “T-1SO” than between “Flagellate I” and “M-1SO”. Differences between “1. galbana” (source was Guillard, WHO1 Collection) and “TISO” in temperature tolerance (Ewart and Pruder, 198 1 ), fatty-acid, protein, and sugar composition (Brown et al., 1989; Brown, 1991; Grima et al., 1992), and nutritional value to mollusks (Brown et al., 1989; Okauchi, 1990) support our findings of differing lipid composition. Of greatest interest to nutrition of marine animals being reared on diets of these algae is the finding that “T-1SO” lacks the essential fatty acid 20:5n-3, whereas 1. galbana has both essential fatty acids 22:6n-3 and 20:5n-3 (Brown et al., 1989; Grima et al., 1992). It is especially critical for authors to maintain this distinction when publishing research results so that conflicting characteristics are not ascribed to the species I. galbana.

The most recent “ISO” being used as a live food in molluscan and other hatcheries is the Caribbean isolate, “C-1SO”. The strain analyzed by Patterson et al. ( 1994) was obtained in 1987 from M. Davis, Caicos Conch Farm, Providenciales, Turks and Caicos Islands, BWI. R. Glazer had isolated it previously (3 August 1984) from waters near Providenciales. The “C-1SO” strain received at Milford was unialgal but bacterized; it was rendered bacteria-free at Milford and deposited at CCMP. The sterol and long-chain alkenone composition of “C-1SO” is essentially identical to that of “T-1SO”. It seems likely, therefore, that these tropical or sub-tropical isolates may be related more closely to each other than to I. galbana. Once again, no critical morphological or taxonomic studies have been published on “C-ISO”, and hence its identity remains unknown. We refer to it as Isochrysis sp., but even this is without a firm taxonomic basis. LB-1292 In our previous study (Patterson et al., 1994) we analyzed two additional algal strains identified as Isochrysis, the first of which - LB-l 292 from the University of Texas Collection - appears to be similar in sterol and alkenone composition to authentic I. galbana, i.e., “Flagellate I” and CCMP 1323. The LB- 1292 isolate, listed as Isochrysis sp., is ascribed M. Parke, who is thought to have isolated it while at the Plymouth Laboratory; it was deposited in the UTEX Collection as B927-2 (Starr and Zeikus, 1993), and is listed as being identical to “Plymouth 8”, which is consistent with earlier Plymouth Laboratory listings (Guillard, pers. commun. ). The UTEX listing also indicates that LB-1292 is synonymous with “T-1SO”; our sterol and long-chain alkenone analyses do not support this synonymy (Patterson et al., 1994).

1977 Tropical Pacific

pre 1964 Temperate Atlantic

Norris Mussel Point, Pacific Grove, CA 1966 Pacific Parke Port Erin, Isle of Man, UK 1938 Plymouth “I” is “type” culture

Glazer CCF, Providenciales, BWI 1985 Tropical Atlantic

Haines COP, Tahiti

UK

Isochrysis sp. Jacobson, 1990 1. Andersen CCMP462 2. Waaland, J. uw-330’

Isochrysis sp. Guillard, 1956 1. Provasoli Haskins 2. McLaughlin Haskins 3. Parke Plymouth “I”

Isochrysis sp. Davis, 1988 1. Davis CCF C-ISO’

lsochrysis sp. Michel, 1977 1. Michel COP T-ISO” 2. Haines UTEX T-IS0 3. Martin COP seawater samp.

bochrysis sp. Starr, 1990 1. Starr UTEX LB- 1292’ 2. SAG8 LB 927-2 3. Plymouth “8”

Isochrysis galbana Jacobson, 1990 1. Andersen CCMPl 323a 2. Guillard WHO1 ISOb 3. Guillard Milford ISO” 4. Provasoli Haskin@ 5. McLaughlin Haskins 6. Parke Plymouth “‘I”= Parke Port Erin, Isle of Man, UK 1938 Plymouth “I” is “type” culture

uw-330

M-IS0

C-IS0

T-IS0

LB- 1292

Microalgal Culture Collection

BLISO

on Isochrysis strains in the Milford Laboratory

“CCMP, Center for the Culture of Marine Phytoplankton, Bigelow Lab. for Marine Science, W. Boothbay Harbor, ME, USA. ‘WHOI, Woods Hole Oceanogr. Inst., Woods Hole, MA, USA (collection discontinued). “Milford, NOAA, NMFS, Milford, CT, USA. dHaskins Laboratories, New York, NY, USA (collection discontinued). “Plymouth Laboratory, UK (collection discontinued). ‘UTEX, Department of Botany, Univ. Texas at Austin, Austin, TX, USA. %AG, Sammlung van Algenkulturen der Universitat Gottingen, Germany. aCOP, Centre Oceanologique du Pacific, Papeet, Taravae, Tahiti. ‘CCF, Caicos Conch Farm, Providenciales, Turks and Caicos Islands, BWI. ‘UW, University of Washington, Seattle, WA, USA.

Date Notes

Isolated by Location

Taxon Deposited by Intermediate locations

Table 1 Background information

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uw-330

The second strain, UW-330 (CCMP462), is a very interesting alga from a sterolcomposition standpoint for two reasons: ( 1) it contains cholesterol as the principal sterol, which is unusual among the algae (Patterson, 1992)) and (2) there is a very large amount of cholesterol on a per-cell basis. This strain was isolated in 1966 from Pacific Grove, California waters by Norris ( Andersen et al., 199 1) . UW-330 grows very poorly in standard algal growth media, relative to the other strains, but its composition suggests great promise as a feed for mollusks that may have difficulty metabolizing dietary sterols that differ in structure appreciably from cholesterol (Wikfors et al., 1991b). Use of UW-330 in mariculture awaits studies to better define its physical and chemical requirements for population growth at commercial production levels. 3. Summary Records of Isochrysis “geneologies” discussed above are summarized in Table 1. Results of biochemical studies are at odds with records showing identical histories of presumed Isochrysis gulbuna strains in the Milford Microalgal Culture Collection and at the Center for the Culture of Marine Phytoplankton. The purpose of this note is, thus, three-fold: first, to notify the mar&culture community of our recent findings concerning the Milford strain of “ISO”, now renamed “MISO”, so that the considerable body of literature employing this strain can be better evaluated in relation to work done with authentic Isochrysis gdbana, i.e., “Flagellate I” and its descendants. Second, we wish to stress fundamental differences in composition between the “T-1SO” and “C-1SO” strains and I. galbana and to urge authors to not refer to these strains as I. galbana until taxonomic studies are undertaken. Finally, we suggest that taxonomic studies of the “lsochlysis” strains used widely in mariculture are long overdue and offer an open plea to algal taxonomists to provide valid descriptions of these strains so that confusion about them does not continue to interfere with their use in mariculture. Acknowledgements

We wish to express our appreciation to R. Ukeles, I.J. Pintner and R.R.L. Guillard, who shared freely information from their personal records of algal strains and for reviewing a draft of this report. We also thank E.N. Wikfors, R. Robohm, and A. Calabrese for their comments. References Andersen, R.A., Jacobson, D.M. and Sexton, J.P., 1991. Provasoli-Guiilard Center for the Culture of Marine Phytoplankton - Catalog of Strains. Provasoli-Guillard Center for the Culture of Marine Phytoplankton, West Boothbay Harbor, ME, USA, 98 pp.

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Babinchak, J. and Ukeles, R., 1979. Epifluorescence microscopy, a technique for the study of feeding in Crassostrea virginica veliger larvae. Mar. Biol., 5 1: 69-76. Billard, C. and Gayral, P., 1972. Two new species of Zsochrysiswith remarks on the genus Ruttnera. Br. Phycol. J., 7: 289-297. Brown, M.R., 199 1. The amino-acid and sugar composition of 16 species of algae used in mariculture. J. Exp. Mar. Biol. Ecol., 145: 79-99. Brown, M.R., Jeffrey, SW. and Garland, C.D., 1989. Nutritional aspects of microalgae used in aquaculture; a literature review. CSIRO Marine Laboratories Report 205, CSIRO, Canberra, A.C.T., 44 PPBruce, J.R., Knight, M. and Parke, M.W., 1940. The rearing of oyster larvae on an algal diet. J. Mar. Biol. Assoc., U.K., 24: 337-374. Culture Center of Algae and Protozoa, 1971. List of Strains. Culture Center of Algae and Protozoa, Cambridge, UK, 73 pp. Culture Center of Algae and Protozoa, 1972. Amendments to the 1971 list of strains. Culture Center of Algae and Protozoa, Cambridge, UK 4 pp. Davis, H.C., 1953. On food and feeding oflarvae of the American oyster, C. virginica. Biol. Bull., 104: 334-350. Davis, H.C. and Guillard, R.R.L., 1958. Relative value of ten genera of micro-organisms as foods for oyster and clam larvae. Fish. Bull. U.S. Fish Wildl. Serv. Vol. 58, Fish. Bull. 136. U.S. Government Printing Office, Washington, DC, pp. 293-304. Ewart, J.W. and Pruder, G.D., 198 1. Comparative growth of Zsochrysisgalbana Parke and Zsochrysis aff. galbana, clone T-IS0 at four temperatures and three light intensities, J. World Maricult. Sot., 12: 333-339. Gladu, P.K., Patterson, G.W., Wikfors, G.H., Chitwood, D.J. and Lusby, W.R., 1990. The occurrence of brassicasterol and epibrassicasterol in the chromophycota. Comp. Biochem. Physiol., 97B: 49 l494. Gladu, P.K., Patterson, G.W., Wikfors, G.H. and Lusby, W.R., 1991. Free and combined sterols of Pavlovagyrans. Lipids, 26: 656-659. Goldberg, R., 1980. Biological and technological studies on the aquaculture of yearling surf clams. Part I. Aquaculture production. Proc. Natl. Shellfish. Assoc., 70: 55-60. Goldberg, R., 1989. Biology and culture of the surfclam. In: J.J. Manzi and M. Castagna (Editors), Clam Mariculture in North America. Elsevier, Amsterdam, pp. 263-276. Green, J.C., 1980. The line structure of Pavlovapinguis Green and a preliminary survey of the order Pavlovales (Prymnesiophyceae). Br. Phycol. J., 15: 151-191. Green, J.C. and Parke, M., 1975. New observations upon members of the genus Chrysotila Anand, with remarks upon their relationships within the Haptophyceae. J. Mar. Biol. Assoc. U.K., 55: 109-121. Green, J.C. and Pienaar, R.N., 1977. The taxonomy of the Order Isochrysidales (Prymnesiophyceae) with special reference to the genera Zsochrysis Parke, Dicrateria Parke and ZrnantoniaReynolds. J. Mar. Biol. Assoc. UK, 57: 7-17. Grima, E.M., Perez, J.A.S., Sanchez, J.L.G., Camacho, E.G. and Alonso, D.L., 1992. EPA from Zsochrysisgalbana. Growth conditions and productivity. Process Biochem., 27: 299-305. Hibberd, D.J., 1976. The ultrastructure and taxonomy of the Chrysophyceae and Prymnesiophyceae (Haptophyceae): a survey with some new observations on the ultrastructure of the Chrysophyceae. Bot. J. Linn. Sot., 72: 55-80. Landers, W.S., 1976. Reproduction and early development of the ocean quahog, Arctica islandica, in the laboratory. Nautilus, 90: 88-92. Loosanoff, V.L. and Davis, H.C., 1963. Rearing of bivalve mollusks. Adv. Mar. Biol., 1: 1-136. Losee, E., 1978. Influence of heredity on larval and spat growth in Crassostrea virginica. Proc. World Mar&It. Sot., 9: 101-107. Marlowe, LT., Green, J.C., Neal, A.C., Brassell, SC., Eglinton, G. and Course, P.A., 1984. Long chain (n-C3+&) alkenones in the Prymnesiophyceae. Distribution of alkenones and other lipids and their taxonomic significance. Br. Phycol. J., 19: 203-216.

G.H. Wikfors, G. W. Patterson / Aquaculture 123 (1994) 12 7-l 35

135

McLaughlin, J.J.A., 1958. Euryhaline chrysomonads: nutrition and toxigenesis in Prymnesium parvum, with notes on Zsochrysisgalbana and Monochrysis lutheri. J. Protozool., 5: 75-81. Okauchi, M., 1990. Food value of Zsochrysisaff. galbana for the growth of pearl oyster spat. Nippon Suisan Gakkaishi, 56: 1343. Parke, M., 1949. Studies on marine flagellates. J. Mar. Biol. Assoc. U.K., 28: 255-288. Patterson, G.W., 1992. Sterols of algae. In: G.W. Patterson and W.D. Nes (Editors), Physiology and Biochemistry of Sterols. American Oil Chemists Society, Champaign, IL, pp. 118- 157. Patterson, G.W., Tsitsa-Tsardis, E., Wikfors, G.H., Gladu, P.K., Chitwood, D.J. and Harrison, D., 1994. Sterols and alkenones of Zsochrysis.Phytochemistry, 35: 1233-1236. Rhodes, E.W. and Landers, W.S., 1972. Growth of oyster larvae, Crassostrea virginica, of various sizes in different concentrations of the chrysophyte, Zsochrysisgalbana. Proc. Natl. Shellfish. Assoc., 63: 53-59. Starr, R.C. and Zeikus, J.A., 1993. UTEX - the culture collection of algae at the University of Texas at Austin 1993 list of cultures. J. Phycol., 29 (Suppl.): l-106. Ukeles, R., 196 1. The effect of temperature on the growth and survival of several marine algal species. Biol. Bull., 120: 255-264. Ukeles, R., 1973. Continuous culture - a method for the production of unicellular foods. In: J.R. Stein (Editor), Handbook of Phycological Methods, Culture Methods and Growth Measurements. Cambridge University Press, London, pp. 233-254. Ukeles, R., 1975. Views on bivalve larvae nutrition. In: K.S. Price (Editor), Proc. First Int. Conf. on Aquaculture Nutrition. University of Delaware, Lewes, DE, pp. 127-162. Ukeles, R. and Rose, W.E., 1976. Observations of organic carbon utilization by photosynthetic marine algae. Mar. Biol., 37: 1 l-28. Walne, P.R., 1964. The culture of marine bivalve larvae. In: M. Wilbur and C.M. Yonge (Editors), Physiology of Mollusca, I. Academic Press, New York, pp. 197-2 10. Wiklors, G.H. and Ukeles, R., 1982. Growth and adaptation of estuarine unicellular algae in media with excess copper, cadmium or zinc and effects of metal-contaminated algal food on Crassostrea virginica larvae. Mar. Ecol. Prog. Ser., 7: 191-206. Wikfors, G.H., Neeman, A. and Jackson, P.J., 1991a. Cadmium-binding polypeptides in microalgal strains with laboratory-induced cadmium tolerance. Mar. Ecol. Prog. Ser., 79: 163-170. Wikfors, G.H., Gladu, P.K. and Patterson, G.W., 199 lb. In search of the ideal algal diet for oysters: recent progress, with emphasis on sterols. J. Shellfish Res., 10: 292 (abstr.). Wikfors, G.H., Ferris, G.E. and Smith, B.C., 1992. The relationship between gross biochemical composition of cultured algal foods and growth of the hard clam, Mercenaria mercenaria (L. ). Aquaculture. 108: 135-154.