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Notice: ©1992 John Wiley & Sons, Inc. This manuscript is an author version with the final publication available at http://www.wiley.com/WileyCDA/ and may be cited as: Lellis, W. A. (1992). A standard reference diet for crustacean nutrition research. VI. Response of postlarval stages of the Caribbean king crab Mithrax spinosissimus and the spiny lobster Panulirus argus. Journal of the World Aquaculture Society, 23(1), 1-7. doi:10.1111/j.1749-7345.1992.tb00744.x

JOURNAL OF THE Vol. 23, No.1 WORLD AQUACULTURE SOCIETY March,1992

A Standard Reference Diet for Crustacean Nutrition Research VI. Response of Postlarval Stages of the Caribbean King Crab Mithrax spinosissimus and the Spiny Lobster Panulirus argus

,I WILLIAM A. LELLIS Division ofApplied Biology, Harbor Branch Oceanographic Institution Inc., 5600 Old Dixie Highway, Fort Pierce, Florida 34946 USA

Abstract Two experiments were condncted to determine if two proposed crustacean reference diets, BML­ 81S or HFX CRD-84, could serve as nutritional standards for early postlarval stages ofthe Caribbean king crab Mithrax spinosissimus or the spiny lobster Panulirus argus. In the first study, after an eight week trial period, survival of postlarval crabs (initial mean weight = 20 mg) was higher (P < 0.05) for crabs fed a control diet of commercial larval fish feed (66.7%) or BML-81S (45.8%) than for crabs fed HFX CRD-84 (16.7%). Final mean weight was also greater (P < 0.05) for crabs consuming the fry feed than for crabs fed BML-81S or HFX CRD-84 (710 vs. 460 and 340 mg, respectively). In the second study, after a ten week trial period, survival of second stage postlarval spiny lobsters (initial mean weight = 0.20 g) was greater (P < 0.05) for animals fed live adult Artemia (93.3%) than for lobsters fed HFX CRD-84 (26.7%) or BML-81S (0%). Lobsters offered the casein-based BML-81S did not consume the feed and died within 25 days. Results suggest that BML-81S may be an acceptable reference diet for Mithrax spinoslssimus, but that neither BML­ 81S nor HFX CRD-84 is adequate for the early postlarval stages of Panulirus argus.

The Caribbean king crab Mithrax spi­ cost-effective diets based upon a complete nosissimus and the spiny lobster Panulirus understanding of the animals' nutritional argus are two tropical marine crustaceans requirements at different life stages. One currently being evaluated for mariculture method to measure progress during diet de­ potential in the Caribbean Basin. The Ca­ velopment is to compare performance of ribbean king crab is a slow-moving omni­ animals fed a test diet with that of animals vore, particularly well suited for grazing on fed a control diet ofdefined chemical com­ fine macroalgae (Bohnsack 1976). The crab position. The purpose of this study was to can complete its short, five-to-seven-day determine if either of two proposed stan­ larval phase without an external food supply dard crustacean reference diets developed (Provenzano and Brownell 1977), and after for H omarus americanus, BML-81 S or HFX molting to first crab will immediately feed CRD-84 (Castell et al. 1989) can be used as on a variety of live and commercially pre­ nutritional standards during diet develop­ pared foods (Brownell et al. 1977; Bolton ment for the early postlarval stages ofMith­ 1987). Spiny lobsters have a longer and more rax spinosissimus or Panulirus argus. complex larval stage, and cultivation through this phase is not yet possible. How­ Materials and Methods • ever, early stage postlarvae have been readi­ ly collected from the wild and subsequently Culture System cultured to maturity using combinations of All experiments were conducted within a various amphipods, gastropods, bivalves 65,000 L closed seawater system main­ and fish as feed (Witham 1973; Serfting and tained by the Division of Applied Biology Ford 1975; Chittleborough 1976). at Harbor Branch Oceanographic Institu­ Future commercial-scale culture ofcrabs tion Inc. (HBOI), Fort Pierce, Aorida, USA. and lobsters will require development of The culture system is housed within 2,700

© Copyright by the World Aquaculture Society 1992 2 LELLIS square feet ofcovered greenhouse and con­ USA, and their eggs subsequently hatched tains nine 1,800 L circular fiberglass culture at HBOI research facilities in Fort Pierce. tanks. Each culture tank is connected to a Resulting postlarvae were communally separate 750 L primary filtration unit con­ housed and fed a diet of filamentous algae taining a polyester fiber filter, an aragonite and flaked tropical fish food for 30 days, at gravel bed and a 30 watt ultraviolet steril­ which time all remaining crabs were mea­ izer. A small electric pump circulates the sured and divided into groups separated to \ . waterfrom the gravel bed to the culture tank the nearest 1.0 mm carapace length (CL). A at a rate of 15 L/min. Temperature of the total of 144 crabs were selected from the 3 culture water is regulated by means of a mm CL size group (approximate mean wet thermostatically controlled polypropylene weight = 20 mg) and divided into 24 groups heat exchanger located within the gravel bed. of six crabs each. Each group of six crabs Each ofthe nine primary biofilters receives was housed within a 20.5 em diameter ring, a constant exchange of water (2.5 L/min) and all 24 rings were floated on four rafts from a central 40,000 L secondary filtration within the same culture tank. The rings were and reservoir system which contains a series filled with shredded black plastic netting to of fine particulate, carbon and macroalgal provide the crabs with shelter, then ran­ filters. The original system was designed and domly assigned to one of six dietary treat­ constructed by Robert A. Winfree (personal ments (producing four replicates per diet). communication). Water in the system is The six diets tested included two purified supplied from the Indian River lagoon at crustacean diets BML-81Sand HFX CRD­ times when salinity is at or near levels found 84 developed for Homarus americanus in ocean water (35 ppt). (Castell et al. 1989), a semipurified experi­ Within each culture tank, small crusta­ mental crustacean diet HFX EXD-l (Cas­ ceans are individually or communally tell and Kean 1986) and three commercially housed within rings constructed from 10.2 available diets manufactured for feeding em high sections ofpolyvinylchloride (PVC) larval fish (Fry Feed C, Biokyowa Inc., Cape pipe which have a 500 /lm screen attached Girardeau, Missouri, USA), postlarval to the bottom. The amount of space avail­ shrimp (Feed 38-469, Zeigler Bros. Inc., able to each animal (or group) can be ad­ Gardners, Pennsylvania, USA) and guinea justed by using a different diameter PVC pigs (Guinea Pig Pellets, Hartz Mountain pipe to construct the ring. Rings are sup­ Corp., Harrison, New Jersey, USA). The ported in the upper 6 em of the water col­ purified diet BML-81S utilized vitamin-free umn by means ofa pentagonal-shaped float­ casein as the major protein source, whereas ing raft. Four rafts per tank are strapped HFX CRD-84 was based upon a freeze-dried together to form a free-moving circle around concentrate of solvent-extracted deboned a center standpipe, and are rotated beneath crab meal (Castell et al. 1989). The semi­ a stationary overhead sprinkler barby means purified HFX EXD-l diet was similar in oftwo airlifts secured to the inside walls of composition to HFX CRD-84, but con­ the fiberglass tanks. Animals receive clean tained a freeze-dried meal made from de­ recirculated seawater from the sprinkler at shelled cooked crab as the major protein a rate sufficient to exchange the volume of component. Ingredient labels from both the • the rings approximately seven times each larval fish feed and the shrimp diet listed hour. fishmeal as the major dietary component, whereas guinea pig pellets were primarily ( composed of alfalfa, soybean meal and Mithrax Study wheat. Ovigerous female Mithrax spinosissimus Diets were ground, sifted to a 1.0 mm were collected off Sugarloaf Key, Florida, particle size and stored frozen. Crabs were CSRD VI: EFFECTS ON MITHRAX CRABS AND SPINY LOBSTERS 3

fed to excess twice daily (0900 hand 1700 A total of75 individually housed lobsters h) seven days per week. Uneaten food and (initial mean weight 0.20 ± 0.04 g, 6.4 ± other waste material was removed by si­ 0.4 mm CL) were floated on four rafts with­ phoning each ring prior to the 0900 h feed­ in the same culture tank, and each lobster ing. Water temperature was recorded twice was randomly allotted to one offive dietary daily and maintained at 27.0 ± 0.5 C. Sa­ treatments (producing 15 replicates per diet). linity was measured weekly using a refrac­ Randomization was accomplished by draw­ tometer (Aquafauna Biomarine Inc., Haw­ ing an identification numberassigned to each thorne, California, USA) and averaged 34.5 lobster from one bucket and matching it ± 0.5 ppt. Dissolved oxygen levels within with a diet identification number drawn the rings remained at near saturation levels from a second bucket. Diets included live due to surface agitation ofwater falling from adult Artemia, BML-8lS, HFX CRD-84, the overhead sprinkler bar (oxygen mea­ HFX EXD-l and the same postlarval sured periodically with a YSI Model 58 ox­ shrimp feed used in the crab study. For­ ygen meter, Yellow Springs Instrument Co., mulated feeds were ground, sifted to a par­ Yellow Springs, Ohio, USA). The pH ofthe ticle size of 2 mm and stored frozen. Arte­ water in the system was determined bi­ mia were cultured on a diet of yeast and weekly with an Orion digital pH meter Spirulina. Lobsters were fed to excess twice Model 211 (Orion Research Inc., Boston, daily (0900 hand 1700 h) seven days per Massachusetts, USA) and averaged 8.1 ± week and uneaten food and feces were re­ 0.1 during the trial. Total ammonia was de­ moved by siphon prior to the 0900 h feed­ termined using the Hach Nessler procedure ing. Shed exuvia were removed and mea­ (Hach Chemical Co., Ames, Iowa, USA) and sured to obtain the carapace length during never exceeded 0.1 mg/L. A 13: 11 light: the previous postlarval stage, then returned dark regime was maintained by overhead to the ring for the lobster to consume. All fluorescent lights. After eight weeks oftrial, lobsters were weighed within five to seven all surviving crabs were counted and days post ecdysis, after surface water was weighed after removal of surface water by first removed by lightly blotting the animal lightly blotting the animals on paper towels. on a paper towel. Statistical Analyses Lobster Study Growth data from both experiments were Spiny lobster pueruli (first stage postlar­ subjected to analysis of variance (Snedecor vae) were collected from floating artificial and Cochran 1980) using 4 replicates per habitats (Little and Milano 1980) at several diet in the crab study and 15 replicates per locations around the island ofAntigua, Brit­ diet in the lobster study. Ifsignificant treat­ ish West Indies, and subsequently trans­ ment effects were indicated by ANOVA, ported to HBOI, Fort Pierce, Florida, USA, Fisher's LSD was used to establish a rank while in their second postlarval stage (Lewis order (Milliken and Johnson 1984). Surviv­ et al. 1952). Thereafter, each lobster was al data were arcsine transformed prior to individually housed within a 10.5 em di­ analysis for the crab study (Snedecor and ameter PVC ring (l0.5 em deep, 500 Jim Cochran 1980), whereas differences in sur­ screen attached to bottom) provided with a vival were determined by Chi-square for the small mesh tube for shelter. All lobsters were lobster study (Cochran and Cox 1957). Nor­ fed an excess of frozen adult Artemia for malized biomass increase (NBI) was cal­ five days prior to initiation of the experi­ culated by dividing the difference between ment. Otherwise, culture systems and water initial total biomass and final total biomass quality parameters were similar to that de­ by the initial number of animals in each scribed for the Mithrax study. experimental group (Conklin et al. 1975). 4 LELLIS

Results and Discussion ofseveral other live algae. Furthermore, ad­ ditions of frozen Artemia or one of several Mithrax Study flaked feeds to the basic diet ofdiatoms did Eight-week survival of postlarval Mith­ not significantly enhance animal survival or rax spinosissimus fed the casein-based ref­ growth. A subsequent study conducted at erence diet BML-81S was comparable to HBOI (unpublished data) indicated that that of crabs fed either the semipurified Biokyowa Fry Feed C (fry feed) produced crabmeal diet HFX EXD-l or the com­ the highest (P < 0.05) rate of growth and mercial fry feed, but less (P < 0.05) than biomass increase among I I feeds tested on that ofcrabs consuming guinea pig feed (Ta­ postlarval crabs, including a control group ble 1). Survival of crabs fed the purified fed live diatoms. Therefore, Fry Feed C was crab-protein based research diet HFX CRD­ used as the control diet in the present study. 84 was quite poor (16.7%). Final mean Unfortunately, however, Fry Feed C is a weight was equal among crabs fed BML­ commercial formulation of complex feed 81 S, HFX CRD-84, HFX EXD-I and ingredients, which renders the diet unsuit­ shrimp feed, but greater (P < 0.05) for crabs able for use in defining nutritional require­ consuming the commercial fry feed. The ments. least amount of individual weight gain oc­ The generally positive response of crabs curred among crabs fed the guinea pig pel­ to the BML-81S casein formula indicates lets. Normalized biomass increase, which that this diet may be useful as a standard expresses the combined effect ofdiet on both for comparison or as a starting basal feed survival and weight gain, was highest (P < for nutritional experiments with postlarval 0.05) for crabs fed the fry feed, equal among Mithrax crabs. Future use ofHFX CRD-84 animals fed BML-81S, HFX EXD-l, shrimp is not recommended, however, because feed and guinea pig pellets, but lower (P < overall survival and growth ofcrabs fed this 0.05) for crabs consuming HFX CRD-84. diet was poor. The reason for the better re­ In an initial study designed to determine sponse ofcrabs to the HFX EXD-l diet than the dietary needs of postlarval Caribbean to the more purified HFX CRD-84 diet is king crabs, Bolton (1987) found that crabs unknown, considering that the ingredient fed live benthic diatoms had a higher rate composition and proximate analysis ofboth of growth and survival than crabs fed one diets are similar. Perhaps the process ofex­ tracting lipids from the crab protein con­ TABLE 1. Comparison of eight-week survival and tained in HFX CRD-84 removed a nutrient growth ofearly juvenile Mithrax spinosissimus fed essential for M. spinosissimus, or reduced different diets.a the palatability ofthe feed. It is also possible Mean that the lipid extraction process left a chem­ Survival weight NBIb ical residue in the purified crab protein which Diet (%) (mg) (mg) negatively affected crab performance. This B BML-SIS 45.S BC 460 162B contention, however, is not supported by D BC c HFX CRD-S4 16.7 340 3S the work of Bordner (1989), who reported HFX EXD-I 54.2 ABC 480 B 221 B Fry Feed 66.7 AB 7l0A 446 A high survival among the crab Cancer ma­ Shrimp Feed 41.7 c 350 BC I 34BC gister when fed HFX CRD-84. Guinea Pig Feed 70.8 A 260c 166B The fact that crabs attained a moderate SE 10.8 100 51 level of performance when offered ground ) a Means not sharing a common superscript are sig­ guinea pig feed as their sole source of nu­ \ nificantly different (P < 0.05). trition presents some positive connotations b Normalized Biomass Increase = for commercial Mithrax production. Except final total biomass - initial total biomass for a small quantity of animal fat, guinea initial number ofanimals pig feeds are manufactured entirely with CSRDVI: EFFECTS ON MITHRAX CRABS AND SPINY LOBSTERS 5

feedstuffs derived from traditional agricul­ to accept the diets. After day 30, mortalities tural crops. These feeds are generally higher among lobsters consuming shrimp feed re­ in carbohydrate and fiber, lower in protein sulted from the inability of the lobsters to and lipid, more easily obtained and more completely remove themselves from the old economical than diets formulated with ma­ exoskeleton during ecdysis. This phenom­ rine proteins or algae. enon, known as molt-death syndrome, has been described for the lobster Homarus Lobster Study americanus (Bowser and Rosemark 1981) Response of postlarval spiny lobsters to and is presumed to be related to the nutri­ formulated feeds was uniformly poor in tional composition of the diet. The exact comparison to that of lobsters consuming cause(s) of this problem, however, remains live adult Artemia (Table 2). Overall sur­ unidentified. vival for lobsters fed pelleted diets was only Final mean weight and length of survi­ 22%, which included 100% mortality among vors were greater (P < 0.05) for lobsters fed animals offered the casein-based BML-8IS Anemia. This resulted from the combined reference diet. The majority ofdeaths among effect oflonger intermolt periods and small­ pellet-fed lobsters occurred without appar­ er incremental size gains per molt for lob­ ent cause between 10 and 30 days after ini­ sters fed the formulated diets. There were tiation offeeding (Fig. 1). Formulated feeds no differences in final size, intermolt period were observed to be poorly accepted by the or weight and length gains per molt among lobsters, suggesting that starvation was the lobsters fed the pelleted diets. major cause of death among pellet-fed an­ An explanation for the poor acceptance imals in this study. No lobster was ever seen of the formulated feeds during the first 30 eating the BML-81S diet, even though they days is lacking. Spiny lobsters have been often "attacked" the feed as it entered the shown to be particularly responsive to a water. Presumed starvation-related mortal­ number of low molecular weight dietary ities decreased dramatically after day 30 of compounds which seem to function as the experiment, as surviving lobsters began chemosensory feeding stimulants (Ache et

TABLE 2. Effect ojdiet on ten-week survival, growth. postmolt size increase and intermolt period ojpostlarval Panulirus argus."

Diet Brine BML HFX HFX Shrimp shrimp SIS CRD-S4 EXD-I feed SE Survival (%) 93.9 A O.OC 26.7 8 40.08 20.0 8C Weight Initial (g) 0.20 O.IS 0.19 0.20 0.22 0.02 Final (g) 0.99A 0.33 8 0.37 8 0.33 8 0.20 Carapace length Initial (mm) 6.3 6.4 6.3 6.4 6.5 0.2 Final (mm) 9.6A 7.18 7.58 7.38 0.6 Total no. molts 49 12 16 II Molts/surviving lobster 3.4A 2.0 8 2.38 2.38 0.4 ) Increase/molt Weight (%) 57.4 A 25.0 8 20.6 8 27.2 8 21.1 8 6.5 Length (%) 12.SA 6.1 8 6.4 8 7.78 5.58 2.3 Intermolt (d) ISA 328 298 348 2.5 a Means not sharing a common superscript are significantly different (P < 0.05). 6 LELLIS

100 \------~\..------'" ARTEMIA 90 ~ 80 "'l \ "..... 70 ~ \~ 60 -l ~ 50 ~"'...- ..... 5 L -<> HFX EXO-1 0::: 40 ::::) ------~, (/) 30 HFX CRO-84 20 \.------.. SHRIMP FEED 10 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 DAYS ON EXPERIMENT FiGURE I. Effect ofdiet on ten- week survival ofsecond stage postlarval spiny lobsters Panulirus argus. al. 1976; Johnson and Ache 1978). These ster pueruli and to John Castell for supply­ compounds may not have been present or ing experimental diets BML-81S, HFX adequately released from the feeds used in CRD-84 and HFX EXD-1. This is HBOI the present study. Future research into the Contribution No. 778. role of chemical attractants in the feeding behavior ofPanulirus argus may eventually Literature Cited lead to the formulation of more acceptable Ache, B., Z. M. Fuzessery and W. E. S. Carr. 1976. reference diets. However, attraction to a po­ Antennular chemosensitivity in the spiny lobster, tential food source and consumption ofthat Panulirus argus: comparative tests ofhigh and low item are not necessarily coupled events. Be­ molecular weight stimulants. Biological Bulletin cause BML-81S was least accepted as a food lSI :273-282. Bohnsack, J. A. 1976. The spider crab Mithrax spi­ source among the diets tested, it may be nosissimus: an investigation includingcommercial useful as a basal formulation during chemo­ aspects. Florida Scientist 39:259-266. sensory attractant experiments with Panu­ Bolton, C. A. 1987. The effects of different diets on lirus argus. It is also possible that wild caught the survival and development of larval and post­ spiny lobster postlarvae are not very flexible larval Mithrax spinosissimus Lamark. Masters thesis. Florida Institute of Technology, Mel­ in selection of food items, and may require bourne, Rorida, USA. an adaptive weaning period during conver­ Bordner, C. E. 1989. A standard reference diet for sion from live organisms to dry feed. crustacean nutrition research. V. Growth and sur­ Results of this study suggest that neither vival ofjuvenile Dungeness crabs Cancer magis­ BML-81S nor HFX CRD-84 are adequate ter. Journal ofthe World Aquaculture Society 20: reference diets for early postlarval stages of 118-121. Bowser, P. R. and R. Rosemark. 1981. Mortalities of Panulirus argus. However, a reduced rate cultured lobsters, Homarus, associated with a molt ofmortality near the end ofthe feeding trial death syndrome. Aquaculture 23: 11-18. may indicate that one ofthese formulations Brownell, W. N., A. J. Provenzano, Jr. and M. Mar­ may be consumed and serve as a satisfactory tinez. 1977. Culture of the West Indian spider crab (Mithrax spinosissimusi at Los Roques, Ven­ source of nutrition during a later stage of ezuela. Proceedings of the World Mariculture So­ development. ciety 8:157-168. Castell, J. D. and J. C. Kean, editors. 1986. The Acknowledgments crustacean nutrition newsletter, volume 3(1). Hal­ This research was supported by a Harbor ifax Fisheries Research Laboratory, Halifax, Nova Scotia, Canada. Branch Oceanographic Institution Postdoc­ Castell, J. D., J. C. Kean, L. D'Abramo and D. E. toral Fellowship. Appreciation is extended Conklin. 1989. A standard reference diet for to Scott Bannerot for collection ofspiny lob- crustacean nutrition. I. Evaluation of two for- CSRD VI: EFFECTS ON MITHRAX CRABS AND SPINY LOBSTERS 7

mulations. Journal of the World Aquaculture So­ sters, Panulirus argus to the Florida Keys. Florida ciety 20:93-99. Marine Research Publication No. 37, St. Peters­ Chittleborough, R. G. 1976. Growth ofjuvenile Pan­ burg, Florida, USA. ulirus longipes cygnus George on coastal reefs Milliken, G. A. and D. K Johnson. 1984. Analysis compared with those reared under optimal envi­ of messy data. Volume I: Designed experiments. ronmental conditions. Australian Journal of Ma­ Van Nostrand Reinhold Company, New York, rine and Freshwater Research 27:279-295. New York, USA. Cochran, W. G. and G. M. Cox. 1957. Experimental Provenzano, A. J., Jr. and W. N. Brownell. 1977. designs, 2nd edition. John Wiley and Sons, Inc., Larval and early post-larval stages of the West New York, New York, USA. Indian spider crab, Mithrax spinosissimus (La­ Conklin, D. K, K. Devers and R. A. Shleser. 1975. mark) (Decapoda: Majidae). Proceedings of the Initial development of artificial diets for the lob­ Biological Society of Washington 90:735-752. ster, Homarus americanus. Proceedings of the Serfting, S. A. and R. F. Ford. 1975. Laboratory cul­ World Mariculture Society 6:237-248. ture ofjuvenile stages ofthe California spiny lob­ Johnson, B. R. and B. W. Ache. 1978. Antennular ster Panulirus interruptus (Randall) at elevated chemosensitivity in the spiny lobster, Panulirus temperatures. Aquaculture 6:377-387. argus: amino acids as feeding stimuli. Marine Be­ Snedecor, G. W. and W. G. Cochran. 1980. Statistical havior and Physiology 5:145-157. methods. Iowa State University Press, Ames, Iowa, Lewis, J. B., H. B. Moore and W. Babis. 1952. The USA. post-larval stages of the spiny lobster Panulirus Witham, R. 1973. Preliminary thermal studies on argus. Bulletin of Marine Science ofthe Gulf and young Panulirus argus. Florida Scientist 36: 154­ Caribbean 2:324-337. 158. Little, E. J., Jr. and G. R. Milano. 1980. Techniques to monitor recruitment of postlarval spiny lob-