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Copepod diapause eggs: a potential source of nauplii for aquaculture
Aquaculture 201 Ž2001. 107–115 www.elsevier.comrlocateraqua-online
Copepod diapause eggs: a potential source of nauplii for aquaculture Nancy H. Marcus ) , Margaret Murray Department of Oceanography, Florida State UniÕersity, Tallahassee, FL 32306 USA Received 7 September 2000; received in revised form 15 December 2000; accepted 15 December 2000
Abstract The aim of this study was to evaluate the feasibility of producing, storing, and hatching copepod diapause eggs that could be used as a source of nauplii for rearing larval fish. The copepod Centropages hamatus was reared on a mixed diet of four dinoflagellates: Gymnodinium sanguineum, Lingulodinium polyedra, Prorocentrum micans, and Scrippsiella trochoidea, in 19-l carboys mounted on a plankton rotator in a walk-in environmental chamber, set at 158C and a 12L–12D cycle. Eleven experiments were conducted. Following maturation to the adult stage, eggs were produced over a 16–33 day period. Total egg production was assessed in three of the experiments and ranged from 1.2 to 2.4 million eggs. Hatching success was generally ) 80% for eggs stored 4–17 months at 258C. Diapause eggs shipped via overnight express remained viable and were used as a source of nauplii to feed larval fish. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Copepod nauplii; Diapause eggs; Larval fish culture
1. Introduction For years, the aquaculture industry has relied primarily on brine shrimp and rotifers to provide the necessary nutrition for rearing the early life stages of fish. The continued supply of Artemia cysts sufficient to meet the demands of the aquaculture industry is in question however, due to poor yields from the Great Salt Lake, the main source of cysts ŽLavens and Sorgeloos, 2000.. In addition to this potential crisis, brine shrimp and rotifers are not suitable first feeds for all fish larvae. This may be because these food
)
Corresponding author. Tel.: q1-850-644-5498; fax: q1-850-644-2581. E-mail address: [email protected] ŽN.H. Marcus..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 1 4 - 2
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N.H. Marcus, M. Murrayr Aquaculture 201 (2001) 107–115
items are too large Žvan der Meeren, 1991; Pepin and Penney, 1997., or Artemia and rotifers may not elicit a feeding response or they may be difficult to capture since the swimming behavior of prey items makes them more or less susceptible to predation by fish larvae Žvan der Meeren, 1991; Buskey et al., 1993; von Herbing and Gallagher, 2000.. Because of these problems, there has been great interest in the identification of alternative live feeds to increase the variety and survival of fish that can be cultivated. A number of studies have tested the effectiveness of copepods as a food item Žsee Stottrup and Norsker, 1997.. Investigators have generally obtained good growth and survival of fish larvae in the laboratory when wild, net-collected plankton containing an abundance of copepods was tested as food Že.g. van der Meeren, 1991; Holmefjord et al., 1993; Naess et al., 1995.. However, since the abundance and nutritional content of wild plankton can vary temporally and spatially, other studies have explored the feasibility of using pond- or laboratory-cultured-copepods to rear fish larvae Žsee review by Watanabe et al., 1983; Stottrup et al., 1986; Kraul et al., 1992, 1993; Stottrup and Norsker, 1997; Schipp et al., 1999.. These studies have generally shown good growth of the fish larvae using cultured-copepods. Moreover, success has been achieved with some fish species that could not be reared with Artemia andror rotifers, e.g. the golden snapper Lutjanus johnii ŽSchipp et al., 1999.. Despite the growing interest and success obtained using cultured-copepods, they are not used routinely by the aquaculture industry. Copepod cultures are difficult to maintain in ponds or in the laboratory on a continuous basis and the procedures for their large-scale cultivation are still in the developmental stage. This study brings a new perspective to solving the problem of providing a reliable source of copepods for the aquaculture industry. We do not rely on the coincident maintenance of copepod and larval fish cultures, instead we use copepod diapause eggs as a source of nauplii to feed larval fish. One reason brine shrimp and rotifers have been used so successfully in the aquaculture industry is that when these organisms are needed directly, as food or to start food stock cultures, they can be hatched from a plentiful supply of dried resting cysts that were stored for months to years. Copepod diapause eggs Žsee review Marcus, 1996. are analogous to the cysts of Artemia and rotifers. Small quantities Žthousands. of these eggs have been produced in the laboratory to gain insight into the factors that control their production and hatching Že.g. Marcus, 1980, 1982.. Studies of the calanoid copepods Labidocera aestiÕa ŽMarcus, 1982. and Acartia clausi ŽUye, 1985. showed that the production of diapause eggs are determined primarily by photoperiod and temperature. Other investigations showed that the survival of copepod diapause eggs for extended periods Žweeks to months. in the laboratory is affected by temperature and oxygen concentration ŽMarcus, 1980, 1987, 1989.. The aim of this study was to ascertain if the diapause eggs of Centropages hamatus ŽLilljeborg. could be produced in sufficient quantity, stored, and subsequently hatched to provide a source of nauplii to feed larval fish. We chose this species because previous work had clarified the factors that affect the induction, maintenance, and termination of diapause ŽChen and Marcus, 1997; Marcus, unpub.. and wild specimens to initiate cultures were available in the nearshore waters of the northeastern Gulf of Mexico ŽGrice, 1956.. The range of the species is broad, extending northwards from Florida to
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Canada ŽPinhey, 1926., but the planktonic phase is generally limited to cool periods of the year, e.g. in the northeastern Gulf of Mexico, adults are present from November to April ŽGrice, 1956.. Non-diapause eggs are produced from November to April and presumably hatch within a few days in the field, since they typically hatch within 2–3 days in the laboratory at ambient field temperatures ŽChen and Marcus, 1997.. Diapause eggs are produced from January to April ŽChen and Marcus, 1997.. Unlike non-diapause eggs, these eggs do not hatch within a few days, but rather sink to the seabed where they over-summer. While in the seabed, the eggs complete a refractory phase, during which time they do not hatch even if conditions Že.g. temperature, oxygen. are suitable. Eventually, the eggs become competent so that hatching will occur when environmental conditions become appropriate. Marcus and Lutz Ž1998. suggested that the hatching of C. hamatus diapause eggs from the seabed occurs in mid-fall, when the water temperature drops below 208C. If diapause eggs are kept in the laboratory at 258C, they become competent to hatch after approximately 3–4 months of incubation. Hatching occurs within 5–7 days when competent eggs are transferred to 158C.
2. Material and methods 2.1. Source population C. hamatus were collected from inshore waters Ž- 5 m water depth. near the Florida State University ŽFSU. Marine Laboratory at Turkey Point, FL, USA, with a 153-mm mesh net, transferred to insulated coolers containing seawater, and transported to the FSU main campus for sorting. Several hundred females of C. hamatus were isolated from the samples and transferred with wide mouth pipettes to 2-l beakers containing glass-fiber-filtered ŽGFF; Gelman ArE filters. seawater and a mixture of four cultured dinoflagellates, Gymnodinium sanguineum ŽGSBL. Žformerly G. nelsoni and G. splendens ., Lingulodinium polyedra ŽGP60. Žformerly Gonyaulax polyedra., Prorocentrum micans ŽPRORO., and Scrippsiella trochoidea ŽPERI. Žformerly Peridinium trochoideum.. The beakers were incubated overnight in an environmental chamber at 158C and 12L–12D. The next morning, the contents of the beakers were sieved through a 153- or 243-mm mesh screen to separate the females from the eggs. The females were either discarded or resuspended in clean seawater with food and returned to the incubator for further incubation. The wash water with the eggs was filtered through a 48-mm mesh screen to concentrate the eggs, which were then washed into a 100-ml crystallizing dish containing GFF seawater. The eggs were incubated for 3–7 days at 158C to allow hatching. 2.2. Procedure for the laboratory production of diapause eggs Laboratory cultures of C. hamatus were initiated from either non-diapause eggs or competent diapause eggs. The nauplii that emerged were concentrated by placing the
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dish next to a fiber optics light source that attracts the nauplii. A Pasteur pipette was used to remove and transfer aliquots of the nauplii to another dish containing approximately 100 ml of seawater. Approximately 1 mg Žthe tip of a spatula. of MS-222 ŽSigma, St. Louis, MO, USA. was added to anaesthetize the nauplii so that they could be counted and transferred to a 0.5-l jar containing GFF seawater. Nauplii were not exposed to the anesthetic for more than 30 min. Nauplii were also counted by transferring smaller aliquots directly to a well slide for counting, followed by washing directly into the jar. This avoided the use of anesthetic. 600–2000 nauplii were placed in each jar and transported to the FSU Marine Laboratory in an insulated cooler. At the laboratory, each jar of nauplii was washed into a 19-l polycarbonate carboy containing seawater Žapproximately 15 l. that was filtered through 5-mm mesh bags, and a supply of food. A food mixture of the four dinoflagellates previously mentioned was prepared by adding an appropriate volume of each culture to produce a concentration of 150 cells mly1 speciesy1 carboyy1 for a total concentration of 600 cells mly1 in each carboy. The phytoplankton were grown in 2.8-l Fernbach flasks containing Guillard’s fr2 media in incubators set at 198C and a 20L–4D photoperiod. Cell densities in the flasks were determined with a Neubauer Hauser Hy-Lite Hemacytometer. The carboys were mounted on a plankton rotator in a walk-in environmental chamber set at 158C and 12L–12D photoperiod. After 5–7 days, the contents of each carboy were filtered through a 48-mm mesh sieve to collect the animals. Survivors were transferred to a clean carboy containing 5-mm-filtered seawater and fresh food, and returned to the incubator. When the animals matured, the contents were sieved every 3–4 days as described above, and then re-sieved through a 153 or 243-mm mesh screen to separate the adults from the eggs. For each experiment, two to four carboys were generally maintained for approximately 5–6 weeks and then terminated. Eggs collected from the carboys on a given day were washed into dishes containing GFF seawater, transferred to a 250-ml beaker containing 200 ml of seawater and sonicated ŽBranson Sonifier Cell Disruptor 200. for 60 s at setting a3. This was done to remove debris Žfecal pellets, dead food. that adhered to the eggs. The eggs were then concentrated by sieving through a 48-mm mesh screen, washed into dishes containing GFF seawater and incubated at 158C for 5–7 days. The unhatched eggs were counted Žsee below., distributed into 60-ml hypo-vials ŽPierce Chemical, Rockford, IL, USA. containing GFF seawater, which were then capped with butyl rubber stoppers ŽBellco, Vineland, NJ, USA, Size No. 20. that were secured with electrical tape, and purged of oxygen by bubbling with nitrogen for 30 min. Lutz et al. Ž1992. describe the details of this procedure. The number of eggs placed in the vials ranged from as little as 50 to tens of thousands. The vials were incubated in darkness at 258C so that the eggs would complete their refractory phase and become competent to hatch. Viability was determined by opening a vial, pouring the contents into a crystallizing dish, transferring 100–150 eggs with a fine-tip Pasteur pipette to well compartments in plastic trays, incubating the eggs at 158C for 5–7 days, and counting the hatched and unhatched eggs using a dissecting microscope. The experimental cultures of C. hamatus were initiated either from Ž1. non-diapause eggs produced by females reared in the laboratory or collected in the field, or Ž2. diapause eggs produced by females reared in the laboratory. The procedure for produc-
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ing non-diapause eggs in the laboratory is similar to that described above for producing diapause eggs, except that cultures were maintained at 10L–14D instead of 12L–12D. The procedure for obtaining eggs from field-collected animals was described above. 2.3. Storage of diapause eggs The effect of the duration of storage on diapause eggs was determined by comparing the viability of eggs stored in vials of de-oxygenated seawater at 258C for different periods of time Ž4–22 months.. In some cases, a vial was opened, 100–150 eggs were transferred to determine viability Žas described above. and unused eggs were discarded. In other cases, unused eggs were returned to the vial, the vial was sealed with a stopper, re-bubbled with nitrogen for 30 min, and returned to the darkened incubator for further storage at 258C. The viability of the unused eggs was determined at a later date by re-opening the vial and following the procedure described above. This procedure was repeated as many as six times on a single vial of eggs. The effect of duration of storage on the viability of diapause eggs produced by females reared in the laboratory was also compared to the viability of diapause eggs that were produced by females collected from the field in March and April 1999. The eggs from the field-collected females were harvested in the laboratory over a period of 4 and 3 days, respectively. They were distributed into vials and stored as described above for eggs that were produced by females reared in the laboratory. 2.4. Counting eggs Determining the number of eggs in the carboys was difficult since many thousands were produced on any given day and the eggs’ spines caused them to aggregate in clumps. During the course of this study, we developed a dilution-volumetric protocol to determine egg production. Eggs were placed in a 250-ml beaker and sonicated for 60 s at setting a3. The contents of the beaker were rinsed through a 48-mm sieve to remove debris and concentrate the eggs. The eggs were rinsed into a clean 50-ml polyethylene centrifuge tube and GFF seawater was added until the volume was 30 ml. The tube was shaken gently and 2 ml of egg solution was removed from the 50-ml centrifuge tube and transferred into a 15-ml centrifuge tube. The original 50-ml tube was shaken gently again and an additional 3 ml of the solution was transferred to the 15-ml tube. Five ml of GFF seawater was added to the 15-ml tube to bring the total volume to 10 ml. A 0.5-ml subsample of this solution was removed and placed in the chamber of a Sedgewick-Rafter slide. These eggs were counted using a dissecting microscope. This was repeated two more times and the number of eggs present in the original sample was then calculated as the Žaverage number of eggs in the three 0.5-ml subsamples. = 120. 2.5. Shipment of eggs for feeding trials Vials of diapause eggs were packed in insulated coolers and shipped via overnight FEDEX to the Marine Science Institute, Port Aransas, TX, USA. Nauplii derived from
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the shipped eggs were used successfully in feeding trials with comet, jackknife, and red drum larvae ŽJoan Holt, pers. comm...
3. Results and discussion We conducted 11 experiments from May 1998 to March 2000. Egg production was generally sustained for 16–33 days following a developmental period of 20–26 days. The total number of eggs produced was assessed in three of these experiments using the dilution-volumetric protocol. These experiments were initiated with four carboys, each containing 1200 nauplii. The numbers of eggs produced were 1.2, 1.7, and 2.4 million. The hatching success of eggs was determined for at least one vial of eggs from each of the 11 experiments. Although the number of eggs produced in the different experiments varied Ž21 out of 26 vials tested., hatching success was generally high Ž) 80%. for diapause eggs that were stored for 4–17 months at 258C and opened once ŽFig. 1ŽA... Moreover, the hatching success of diapause eggs remained high despite repeated opening and re-bubbling of a vial ŽFig. 1ŽB... The results indicate that a vial of diapause eggs can be opened and closed at least four times over a period of 15 months with little or no negative effect on hatching success. Some hatching was even noted after 22 months of storage. There was one exception to the generally high hatching success of eggs. In the last experiment, 2.4 million eggs were produced, but the subsequent hatching success of eggs was 0% for six vials that were tested. In two other experiments, a low hatching success was noted for two vials Ž3% and 25%, respectively., but the hatching success of eggs from at least one other vial from each of those experiments was high. Thus, in the context of the whole study, the results for the last experiment are anomalous, but we could not identify an obvious cause for the poor hatch. The hatching success of diapause eggs after 13–15.5 months of storage at 258C was higher for those produced by laboratory-reared females than for those produced by field-collected females ŽFig. 1ŽA... We did not check the short-term Že.g. 4–6 months. survivability of the diapause eggs produced by field-collected females, so it is not known if the eggs were generally of poor quality or whether they were unable to survive extended storage. In the same way that some lipid stores enhance the capacity of adult copepods to survive periods without food ŽSargent and Henderson, 1986., the biochemical composition of diapause eggs could affect their ability to survive long-term storage. Food quality has been shown to affect the viability of non-diapause copepod eggs Že.g. Ianora et al., 1996. and undoubtedly affects their biochemical composition as it does copepodite stages ŽGraeve et al., 1994.. Thus, the difference in hatching success may have been due to differences in biochemical composition resulting from differences in the diet of the laboratory-reared and field-collected females. Other results from the present study also suggest that diet influences the viability of diapause eggs. For the females that were collected from the field in March, the hatching success of eggs collected on days 1, 2, 3, and 4 in the laboratory was 19%, 13%, 9%, and 54%, respectively. Similarly, for the females that were obtained in April, the hatching success of eggs collected on days 1, 2, and 3 in the laboratory was 20%, 48%, and 65%, respectively. Although we have no data on the diet the animals experienced in the field,
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Fig. 1. ŽA. Percentage hatch of eggs pre-incubated in vials for different periods of time at 258C. 100–150 eggs were removed from a vial and incubated at 158C to assess viability. Hatching was determined after 5–7 days. These data represent the hatching success of eggs the first time a vial was opened. Like symbols represent vials of eggs from the same experiment but collected and stored on different days. Filled symbols represent experiments started from diapause eggs. Empty symbols represent experiments started from non-diapause eggs. Shaded symbols represent diapause eggs obtained from females collected in the wild in March and April 1999. Results of the last experiment in which six vials were tested but no eggs hatched are not shown. ŽB. Percentage hatch of eggs pre-incubated in vials for different periods of time at 258C. 100–150 eggs were removed from a vial and incubated at 158C to assess viability. Hatching was determined after 5–7 days. These data represent the hatching success of eggs from vials that were opened, closed, and re-bubbled with nitrogen one or more times. Like symbols represent the hatching success of eggs from a specific vial. Filled and shaded symbols represent experiments started with diapause eggs. Empty symbols represent experiments started from non-diapause eggs.
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it probably differed from the food offered to the females during the egg production period in the laboratory. Thus, this switch in diet may have altered the biochemical composition of the eggs and gradually improved their capacity to survive extended storage. Support for this explanation comes from Tester and Turner Ž1990. who showed that the time it takes for ingested food to be converted into egg material varies among copepod species. In their study, the time ranged from 9.5 to 91 h.
4. Conclusion Copepod nauplii are the natural food of most marine fish larvae. Studies conducted to date on the effectiveness of copepods as a food for cultivated larval fish have relied on specimens collected in the wild, from enclosed ponds or reared in the laboratory. The approach reported in this study relies on laboratory culture, but cultivation of the fish larvae is no longer dependent on the simultaneous maintenance of copepod cultures. It is evident that copepod diapause eggs can be produced in the laboratory, stored, and hatched when nauplii are needed to feed larval fish. The opportunity now exists to test the nauplii as an effective food item for rearing species of fish and invertebrates that have been difficult to cultivate. If the nauplii prove to be effective for rearing species that have been problematic, then the next challenge will be to develop a large-scale system to produce greater quantities of diapause eggs. In addition, attention should be given to increasing the variety of copepods that can be reared from diapause eggs, since C. hamatus may not be suitable for tropical fish and invertebrate species that must be reared at temperatures warmer than 258C.
Acknowledgements This work was accomplished with the laboratory and technical assistance of Garry Glover, Jennifer Kelly, and Nicadia Gilles. J. Holt provided comments on the manuscript. Florida Sea Grant College Program Grant No. RrLR-A-22 to N. Marcus and J. Holt supported the research.
References Buskey, E.J., Coulter, C., Strom, S., 1993. Locomotory patterns of microzooplankton: potential effects on food selectivity of larval fish. Bull. Mar. Sci. 53, 29–43. Chen, F., Marcus, N.H., 1997. Subitaneous, diapause, and delayed-hatching eggs of planktonic copepods from the northern Gulf of Mexico: morphology and hatching success. Mar. Biol. 127, 587–598. Graeve, M., Kattner, G., Hagen, W., 1994. Diet-induced changes in the fatty acid composition of Arctic herbivorous copepods: experimental evidence of trophic markers. J. Exp. Mar. Biol. Ecol. 182, 97–110. Grice, G., 1956. A qualitative and quantitative seasonal study of the Copepoda of Alligator Harbor. Florida State Univ. Stud. No. 22, Papers from the Oceanogr. Inst. No. 2, 37–76. Holmefjord, I., Gulbrandsen, J., Lein, I., Refstie, T., Leger, P., Harboe, T., Huse, I., Sorgeloos, P., Bolla, S., Olsen, Y., Reitan, K.I., Vadstein, O., Oie, G., 1993. An intensive approach to Atlantic halibut fry production. J. World Aquacult. Soc. 24, 275–284.
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Ianora, A., Poulet, S.A., Miralto, A., Grottoli, R., 1996. The diatom Thalassiosira rotula affects reproductive success in the copepod Acartia clausi. Mar. Biol. 125, 279–286. Kraul, S., Nelson, A., Brittain, K., Ako, H., Ogasawara, A., 1992. Evaluation of live feeds for larval and postlarval mahimahi Coryphaena hippurus. J. World Aquacult. Soc. 23, 299–306. Kraul, S., Brittain, K., Cantrell, R., Nagao, T., Ako, H., Ogasawara, A., Kitagawa, H., 1993. Nutritional factors affecting stress resistance in the larval mahimahi Coryphaena hippurus. J. World Aquacult. Soc. 24, 186–193. Lavens, P., Sorgeloos, P., 2000. The history, present status and prospects of the availability of Artemia cysts for aquaculture. Aquaculture 181, 397–403. Lutz, R., Marcus, N.H., Chanton, J., 1992. The effects of low oxygen concentrations on the hatching and viability of marine calanoid copepod eggs. Mar. Biol. 114, 241–248. Marcus, N.H., 1980. Photoperiodic control of diapause in the marine calanoid copepod, Labidocera aestiÕa. Biol. Bull. 159, 311–318. Marcus, N., 1982. Photoperiodic and temperature regulation of diapause in Labidocera aestiÕa ŽCopepoda: Calanoida.. Biol. Bull. 162, 45–52. Marcus, N.H., 1987. Differences in the duration of egg diapause of Labidocera aestiÕa ŽCopepoda: Calanoida. from the Woods Hole, Massachussetts region. Biol. Bull. 173, 169–177. Marcus, N.H., 1989. Abundance in sediments and hatching requirements of eggs of Centropages hamatus ŽCopepoda: Calanoida. from the Alligator Harbor region, Florida. Biol. Bull. 176, 142–146. Marcus, N.H., 1996. Ecological and evolutionary significance of resting eggs in marine copepods: past, present, and future studies. Hydrobiologia 320, 141–152. Marcus, N.H., Lutz, R., 1998. Longevity of subitaneous and diapause eggs of Centropages hamatus ŽCopepoda: Calanoida. from the northern Gulf of Mexico. Mar. Biol. 131, 249–257. Naess, T., Germain-Henry, M., Naas, K.E., 1995. First feeding of Atlantic halibut Ž Hippoglossus hippoglossus . using different combinations of Artemia and wild zooplankton. Aquaculture 130, 235–250. Pepin, P., Penney, R.W., 1997. Patterns of prey size and taxonomic composition in larval fish: are there general size dependent models? J. Fish Biol. 51, 84–100 ŽSupplement A.. Pinhey, K., 1926. Entomostraca of the Belle Isle Strait expedition, 1923, with notes on other planktonic species. Part 1. Contrib. Can. Biol. N.S. 3, 1–55. Sargent, J.R., Henderson, R.J., 1986. Lipids. In: Corner, E.D.S., O’Hara, S.C.M. ŽEds.., The Biological Chemistry of Marine Copepods. Clarendon Press, Oxford, pp. 58–108. Schipp, G.R., Bosmans, J.M.P., Marshall, A.J., 1999. A method for hatchery culture of tropical calanoid copepods, Acartia spp. Aquaculture 174, 81–88. Stottrup, J.G., Norsker, N.H., 1997. Production and use of copepods in marine fish larviculture. Aquaculture 155, 231–247. Stottrup, J.G., Richardson, K., Kirkegaard, E., Pihl, N.J., 1986. The cultivation of Acartia tonsa Dana for use as a live food source for marine fish larvae. Aquaculture 52, 87–96. Tester, P.A., Turner, J.T., 1990. How long does it take copepods to make eggs? J. Exp. Mar. Biol. Ecol. 141, 169–182. Uye, S., 1985. Resting egg production as a life history strategy of marine planktonic copepods. Bull. Mar. Sci. 37, 440–449. van der Meeren, T., 1991. Selective feeding and prediction of food consumption in turbot larvae Ž Scopthalmus maximus L.. reared on the rotifer Brachionus plicatilis and natural zooplankton. Aquaculture 93, 35–55. von Herbing, H., Gallagher, S., 2000. Foraging behavior in early Atlantic cod larvae Ž Gadhus morhua. feeding on a protozoan Ž Balanion sp.. and a copepod nauplius Ž Pseudodiaptomus sp... Mar. Biol. 136, 591–602. Watanabe, T., Kitajima, C., Fujita, S., 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture 34, 115–143.
Copepod diapause eggs: a potential source of nauplii for aquaculture Nancy H. Marcus ) , Margaret Murray Department of Oceanography, Florida State UniÕersity, Tallahassee, FL 32306 USA Received 7 September 2000; received in revised form 15 December 2000; accepted 15 December 2000
Abstract The aim of this study was to evaluate the feasibility of producing, storing, and hatching copepod diapause eggs that could be used as a source of nauplii for rearing larval fish. The copepod Centropages hamatus was reared on a mixed diet of four dinoflagellates: Gymnodinium sanguineum, Lingulodinium polyedra, Prorocentrum micans, and Scrippsiella trochoidea, in 19-l carboys mounted on a plankton rotator in a walk-in environmental chamber, set at 158C and a 12L–12D cycle. Eleven experiments were conducted. Following maturation to the adult stage, eggs were produced over a 16–33 day period. Total egg production was assessed in three of the experiments and ranged from 1.2 to 2.4 million eggs. Hatching success was generally ) 80% for eggs stored 4–17 months at 258C. Diapause eggs shipped via overnight express remained viable and were used as a source of nauplii to feed larval fish. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Copepod nauplii; Diapause eggs; Larval fish culture
1. Introduction For years, the aquaculture industry has relied primarily on brine shrimp and rotifers to provide the necessary nutrition for rearing the early life stages of fish. The continued supply of Artemia cysts sufficient to meet the demands of the aquaculture industry is in question however, due to poor yields from the Great Salt Lake, the main source of cysts ŽLavens and Sorgeloos, 2000.. In addition to this potential crisis, brine shrimp and rotifers are not suitable first feeds for all fish larvae. This may be because these food
)
Corresponding author. Tel.: q1-850-644-5498; fax: q1-850-644-2581. E-mail address: [email protected] ŽN.H. Marcus..
0044-8486r01r$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 0 1 . 0 0 5 1 4 - 2
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items are too large Žvan der Meeren, 1991; Pepin and Penney, 1997., or Artemia and rotifers may not elicit a feeding response or they may be difficult to capture since the swimming behavior of prey items makes them more or less susceptible to predation by fish larvae Žvan der Meeren, 1991; Buskey et al., 1993; von Herbing and Gallagher, 2000.. Because of these problems, there has been great interest in the identification of alternative live feeds to increase the variety and survival of fish that can be cultivated. A number of studies have tested the effectiveness of copepods as a food item Žsee Stottrup and Norsker, 1997.. Investigators have generally obtained good growth and survival of fish larvae in the laboratory when wild, net-collected plankton containing an abundance of copepods was tested as food Že.g. van der Meeren, 1991; Holmefjord et al., 1993; Naess et al., 1995.. However, since the abundance and nutritional content of wild plankton can vary temporally and spatially, other studies have explored the feasibility of using pond- or laboratory-cultured-copepods to rear fish larvae Žsee review by Watanabe et al., 1983; Stottrup et al., 1986; Kraul et al., 1992, 1993; Stottrup and Norsker, 1997; Schipp et al., 1999.. These studies have generally shown good growth of the fish larvae using cultured-copepods. Moreover, success has been achieved with some fish species that could not be reared with Artemia andror rotifers, e.g. the golden snapper Lutjanus johnii ŽSchipp et al., 1999.. Despite the growing interest and success obtained using cultured-copepods, they are not used routinely by the aquaculture industry. Copepod cultures are difficult to maintain in ponds or in the laboratory on a continuous basis and the procedures for their large-scale cultivation are still in the developmental stage. This study brings a new perspective to solving the problem of providing a reliable source of copepods for the aquaculture industry. We do not rely on the coincident maintenance of copepod and larval fish cultures, instead we use copepod diapause eggs as a source of nauplii to feed larval fish. One reason brine shrimp and rotifers have been used so successfully in the aquaculture industry is that when these organisms are needed directly, as food or to start food stock cultures, they can be hatched from a plentiful supply of dried resting cysts that were stored for months to years. Copepod diapause eggs Žsee review Marcus, 1996. are analogous to the cysts of Artemia and rotifers. Small quantities Žthousands. of these eggs have been produced in the laboratory to gain insight into the factors that control their production and hatching Že.g. Marcus, 1980, 1982.. Studies of the calanoid copepods Labidocera aestiÕa ŽMarcus, 1982. and Acartia clausi ŽUye, 1985. showed that the production of diapause eggs are determined primarily by photoperiod and temperature. Other investigations showed that the survival of copepod diapause eggs for extended periods Žweeks to months. in the laboratory is affected by temperature and oxygen concentration ŽMarcus, 1980, 1987, 1989.. The aim of this study was to ascertain if the diapause eggs of Centropages hamatus ŽLilljeborg. could be produced in sufficient quantity, stored, and subsequently hatched to provide a source of nauplii to feed larval fish. We chose this species because previous work had clarified the factors that affect the induction, maintenance, and termination of diapause ŽChen and Marcus, 1997; Marcus, unpub.. and wild specimens to initiate cultures were available in the nearshore waters of the northeastern Gulf of Mexico ŽGrice, 1956.. The range of the species is broad, extending northwards from Florida to
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Canada ŽPinhey, 1926., but the planktonic phase is generally limited to cool periods of the year, e.g. in the northeastern Gulf of Mexico, adults are present from November to April ŽGrice, 1956.. Non-diapause eggs are produced from November to April and presumably hatch within a few days in the field, since they typically hatch within 2–3 days in the laboratory at ambient field temperatures ŽChen and Marcus, 1997.. Diapause eggs are produced from January to April ŽChen and Marcus, 1997.. Unlike non-diapause eggs, these eggs do not hatch within a few days, but rather sink to the seabed where they over-summer. While in the seabed, the eggs complete a refractory phase, during which time they do not hatch even if conditions Že.g. temperature, oxygen. are suitable. Eventually, the eggs become competent so that hatching will occur when environmental conditions become appropriate. Marcus and Lutz Ž1998. suggested that the hatching of C. hamatus diapause eggs from the seabed occurs in mid-fall, when the water temperature drops below 208C. If diapause eggs are kept in the laboratory at 258C, they become competent to hatch after approximately 3–4 months of incubation. Hatching occurs within 5–7 days when competent eggs are transferred to 158C.
2. Material and methods 2.1. Source population C. hamatus were collected from inshore waters Ž- 5 m water depth. near the Florida State University ŽFSU. Marine Laboratory at Turkey Point, FL, USA, with a 153-mm mesh net, transferred to insulated coolers containing seawater, and transported to the FSU main campus for sorting. Several hundred females of C. hamatus were isolated from the samples and transferred with wide mouth pipettes to 2-l beakers containing glass-fiber-filtered ŽGFF; Gelman ArE filters. seawater and a mixture of four cultured dinoflagellates, Gymnodinium sanguineum ŽGSBL. Žformerly G. nelsoni and G. splendens ., Lingulodinium polyedra ŽGP60. Žformerly Gonyaulax polyedra., Prorocentrum micans ŽPRORO., and Scrippsiella trochoidea ŽPERI. Žformerly Peridinium trochoideum.. The beakers were incubated overnight in an environmental chamber at 158C and 12L–12D. The next morning, the contents of the beakers were sieved through a 153- or 243-mm mesh screen to separate the females from the eggs. The females were either discarded or resuspended in clean seawater with food and returned to the incubator for further incubation. The wash water with the eggs was filtered through a 48-mm mesh screen to concentrate the eggs, which were then washed into a 100-ml crystallizing dish containing GFF seawater. The eggs were incubated for 3–7 days at 158C to allow hatching. 2.2. Procedure for the laboratory production of diapause eggs Laboratory cultures of C. hamatus were initiated from either non-diapause eggs or competent diapause eggs. The nauplii that emerged were concentrated by placing the
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dish next to a fiber optics light source that attracts the nauplii. A Pasteur pipette was used to remove and transfer aliquots of the nauplii to another dish containing approximately 100 ml of seawater. Approximately 1 mg Žthe tip of a spatula. of MS-222 ŽSigma, St. Louis, MO, USA. was added to anaesthetize the nauplii so that they could be counted and transferred to a 0.5-l jar containing GFF seawater. Nauplii were not exposed to the anesthetic for more than 30 min. Nauplii were also counted by transferring smaller aliquots directly to a well slide for counting, followed by washing directly into the jar. This avoided the use of anesthetic. 600–2000 nauplii were placed in each jar and transported to the FSU Marine Laboratory in an insulated cooler. At the laboratory, each jar of nauplii was washed into a 19-l polycarbonate carboy containing seawater Žapproximately 15 l. that was filtered through 5-mm mesh bags, and a supply of food. A food mixture of the four dinoflagellates previously mentioned was prepared by adding an appropriate volume of each culture to produce a concentration of 150 cells mly1 speciesy1 carboyy1 for a total concentration of 600 cells mly1 in each carboy. The phytoplankton were grown in 2.8-l Fernbach flasks containing Guillard’s fr2 media in incubators set at 198C and a 20L–4D photoperiod. Cell densities in the flasks were determined with a Neubauer Hauser Hy-Lite Hemacytometer. The carboys were mounted on a plankton rotator in a walk-in environmental chamber set at 158C and 12L–12D photoperiod. After 5–7 days, the contents of each carboy were filtered through a 48-mm mesh sieve to collect the animals. Survivors were transferred to a clean carboy containing 5-mm-filtered seawater and fresh food, and returned to the incubator. When the animals matured, the contents were sieved every 3–4 days as described above, and then re-sieved through a 153 or 243-mm mesh screen to separate the adults from the eggs. For each experiment, two to four carboys were generally maintained for approximately 5–6 weeks and then terminated. Eggs collected from the carboys on a given day were washed into dishes containing GFF seawater, transferred to a 250-ml beaker containing 200 ml of seawater and sonicated ŽBranson Sonifier Cell Disruptor 200. for 60 s at setting a3. This was done to remove debris Žfecal pellets, dead food. that adhered to the eggs. The eggs were then concentrated by sieving through a 48-mm mesh screen, washed into dishes containing GFF seawater and incubated at 158C for 5–7 days. The unhatched eggs were counted Žsee below., distributed into 60-ml hypo-vials ŽPierce Chemical, Rockford, IL, USA. containing GFF seawater, which were then capped with butyl rubber stoppers ŽBellco, Vineland, NJ, USA, Size No. 20. that were secured with electrical tape, and purged of oxygen by bubbling with nitrogen for 30 min. Lutz et al. Ž1992. describe the details of this procedure. The number of eggs placed in the vials ranged from as little as 50 to tens of thousands. The vials were incubated in darkness at 258C so that the eggs would complete their refractory phase and become competent to hatch. Viability was determined by opening a vial, pouring the contents into a crystallizing dish, transferring 100–150 eggs with a fine-tip Pasteur pipette to well compartments in plastic trays, incubating the eggs at 158C for 5–7 days, and counting the hatched and unhatched eggs using a dissecting microscope. The experimental cultures of C. hamatus were initiated either from Ž1. non-diapause eggs produced by females reared in the laboratory or collected in the field, or Ž2. diapause eggs produced by females reared in the laboratory. The procedure for produc-
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ing non-diapause eggs in the laboratory is similar to that described above for producing diapause eggs, except that cultures were maintained at 10L–14D instead of 12L–12D. The procedure for obtaining eggs from field-collected animals was described above. 2.3. Storage of diapause eggs The effect of the duration of storage on diapause eggs was determined by comparing the viability of eggs stored in vials of de-oxygenated seawater at 258C for different periods of time Ž4–22 months.. In some cases, a vial was opened, 100–150 eggs were transferred to determine viability Žas described above. and unused eggs were discarded. In other cases, unused eggs were returned to the vial, the vial was sealed with a stopper, re-bubbled with nitrogen for 30 min, and returned to the darkened incubator for further storage at 258C. The viability of the unused eggs was determined at a later date by re-opening the vial and following the procedure described above. This procedure was repeated as many as six times on a single vial of eggs. The effect of duration of storage on the viability of diapause eggs produced by females reared in the laboratory was also compared to the viability of diapause eggs that were produced by females collected from the field in March and April 1999. The eggs from the field-collected females were harvested in the laboratory over a period of 4 and 3 days, respectively. They were distributed into vials and stored as described above for eggs that were produced by females reared in the laboratory. 2.4. Counting eggs Determining the number of eggs in the carboys was difficult since many thousands were produced on any given day and the eggs’ spines caused them to aggregate in clumps. During the course of this study, we developed a dilution-volumetric protocol to determine egg production. Eggs were placed in a 250-ml beaker and sonicated for 60 s at setting a3. The contents of the beaker were rinsed through a 48-mm sieve to remove debris and concentrate the eggs. The eggs were rinsed into a clean 50-ml polyethylene centrifuge tube and GFF seawater was added until the volume was 30 ml. The tube was shaken gently and 2 ml of egg solution was removed from the 50-ml centrifuge tube and transferred into a 15-ml centrifuge tube. The original 50-ml tube was shaken gently again and an additional 3 ml of the solution was transferred to the 15-ml tube. Five ml of GFF seawater was added to the 15-ml tube to bring the total volume to 10 ml. A 0.5-ml subsample of this solution was removed and placed in the chamber of a Sedgewick-Rafter slide. These eggs were counted using a dissecting microscope. This was repeated two more times and the number of eggs present in the original sample was then calculated as the Žaverage number of eggs in the three 0.5-ml subsamples. = 120. 2.5. Shipment of eggs for feeding trials Vials of diapause eggs were packed in insulated coolers and shipped via overnight FEDEX to the Marine Science Institute, Port Aransas, TX, USA. Nauplii derived from
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the shipped eggs were used successfully in feeding trials with comet, jackknife, and red drum larvae ŽJoan Holt, pers. comm...
3. Results and discussion We conducted 11 experiments from May 1998 to March 2000. Egg production was generally sustained for 16–33 days following a developmental period of 20–26 days. The total number of eggs produced was assessed in three of these experiments using the dilution-volumetric protocol. These experiments were initiated with four carboys, each containing 1200 nauplii. The numbers of eggs produced were 1.2, 1.7, and 2.4 million. The hatching success of eggs was determined for at least one vial of eggs from each of the 11 experiments. Although the number of eggs produced in the different experiments varied Ž21 out of 26 vials tested., hatching success was generally high Ž) 80%. for diapause eggs that were stored for 4–17 months at 258C and opened once ŽFig. 1ŽA... Moreover, the hatching success of diapause eggs remained high despite repeated opening and re-bubbling of a vial ŽFig. 1ŽB... The results indicate that a vial of diapause eggs can be opened and closed at least four times over a period of 15 months with little or no negative effect on hatching success. Some hatching was even noted after 22 months of storage. There was one exception to the generally high hatching success of eggs. In the last experiment, 2.4 million eggs were produced, but the subsequent hatching success of eggs was 0% for six vials that were tested. In two other experiments, a low hatching success was noted for two vials Ž3% and 25%, respectively., but the hatching success of eggs from at least one other vial from each of those experiments was high. Thus, in the context of the whole study, the results for the last experiment are anomalous, but we could not identify an obvious cause for the poor hatch. The hatching success of diapause eggs after 13–15.5 months of storage at 258C was higher for those produced by laboratory-reared females than for those produced by field-collected females ŽFig. 1ŽA... We did not check the short-term Že.g. 4–6 months. survivability of the diapause eggs produced by field-collected females, so it is not known if the eggs were generally of poor quality or whether they were unable to survive extended storage. In the same way that some lipid stores enhance the capacity of adult copepods to survive periods without food ŽSargent and Henderson, 1986., the biochemical composition of diapause eggs could affect their ability to survive long-term storage. Food quality has been shown to affect the viability of non-diapause copepod eggs Že.g. Ianora et al., 1996. and undoubtedly affects their biochemical composition as it does copepodite stages ŽGraeve et al., 1994.. Thus, the difference in hatching success may have been due to differences in biochemical composition resulting from differences in the diet of the laboratory-reared and field-collected females. Other results from the present study also suggest that diet influences the viability of diapause eggs. For the females that were collected from the field in March, the hatching success of eggs collected on days 1, 2, 3, and 4 in the laboratory was 19%, 13%, 9%, and 54%, respectively. Similarly, for the females that were obtained in April, the hatching success of eggs collected on days 1, 2, and 3 in the laboratory was 20%, 48%, and 65%, respectively. Although we have no data on the diet the animals experienced in the field,
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Fig. 1. ŽA. Percentage hatch of eggs pre-incubated in vials for different periods of time at 258C. 100–150 eggs were removed from a vial and incubated at 158C to assess viability. Hatching was determined after 5–7 days. These data represent the hatching success of eggs the first time a vial was opened. Like symbols represent vials of eggs from the same experiment but collected and stored on different days. Filled symbols represent experiments started from diapause eggs. Empty symbols represent experiments started from non-diapause eggs. Shaded symbols represent diapause eggs obtained from females collected in the wild in March and April 1999. Results of the last experiment in which six vials were tested but no eggs hatched are not shown. ŽB. Percentage hatch of eggs pre-incubated in vials for different periods of time at 258C. 100–150 eggs were removed from a vial and incubated at 158C to assess viability. Hatching was determined after 5–7 days. These data represent the hatching success of eggs from vials that were opened, closed, and re-bubbled with nitrogen one or more times. Like symbols represent the hatching success of eggs from a specific vial. Filled and shaded symbols represent experiments started with diapause eggs. Empty symbols represent experiments started from non-diapause eggs.
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it probably differed from the food offered to the females during the egg production period in the laboratory. Thus, this switch in diet may have altered the biochemical composition of the eggs and gradually improved their capacity to survive extended storage. Support for this explanation comes from Tester and Turner Ž1990. who showed that the time it takes for ingested food to be converted into egg material varies among copepod species. In their study, the time ranged from 9.5 to 91 h.
4. Conclusion Copepod nauplii are the natural food of most marine fish larvae. Studies conducted to date on the effectiveness of copepods as a food for cultivated larval fish have relied on specimens collected in the wild, from enclosed ponds or reared in the laboratory. The approach reported in this study relies on laboratory culture, but cultivation of the fish larvae is no longer dependent on the simultaneous maintenance of copepod cultures. It is evident that copepod diapause eggs can be produced in the laboratory, stored, and hatched when nauplii are needed to feed larval fish. The opportunity now exists to test the nauplii as an effective food item for rearing species of fish and invertebrates that have been difficult to cultivate. If the nauplii prove to be effective for rearing species that have been problematic, then the next challenge will be to develop a large-scale system to produce greater quantities of diapause eggs. In addition, attention should be given to increasing the variety of copepods that can be reared from diapause eggs, since C. hamatus may not be suitable for tropical fish and invertebrate species that must be reared at temperatures warmer than 258C.
Acknowledgements This work was accomplished with the laboratory and technical assistance of Garry Glover, Jennifer Kelly, and Nicadia Gilles. J. Holt provided comments on the manuscript. Florida Sea Grant College Program Grant No. RrLR-A-22 to N. Marcus and J. Holt supported the research.
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