- Email: [email protected]
Evaluation of the rotifer Brachionus plicatilis as a substitute for Artemia in feeding larvae of Macrobrachium rosenbergii
Aquaculture,
71 (1988) 331-338 Elsevier Science Publishers B.V., Amsterdam -
331 Printed in The Netherlands
Evaluation of the Rotifer BmchionzmpZ~cati Substitute for Artemia in Feeding Larvae of
as a
Macrobrachium rosenbergii DONALD L. LOVETT and DARRYL L. FELDER
Department of Biology and Center for Crustacean Research, University of Southwestern Louisiana, P.O. Box 42451, Lafayette, LA 70504 (U.S.A.)
(Accepted 14 December 1987)
ABSTRACT Lovett, D.L. and Felder, D.L., 1988. Evaluation of the rotifer Brachionusplicatilis as a substitute for Artemia in feeding larvae of Macrobrachium rosenbergii. Aquaculture, 71: 331-338. Addition of the rotifer Brachionusplicatilis Miiller to an Artemia-based diet yielded no significant increase in larval survival or rate of larval development in Macrobrachium rosenbergii (de Man). B. plicatilis apparently contributed little to the nutrition of M. rosenbergii larvae and provided no benefit to their culture. Larvae fed on a diet restricted to Brachionus had significantly lower surviva1 and rate of development than did larvae fed on a diet that included Artemia. Larvae fed only rotifen did not have significantly different survival from that of starved larvae. Development of larvae fed only rotifers and reared in artificial sea water was arrested at a mean stage of 2.33; in natural sea water development was arrested at a mean stage of 2.98.
INTRODUCTION
The rotifer BruchionuspZicatilis Miiller has been promoted as both an initial feed and as a supplemental feed for larval crustaceans (Mock, 1971; Sorgeloos and Persoone, 1975; Solangi and Ogle, 1977). Bruchionus has been used in larviculture of penaeid shrimp as a food intermediate in size between algae and Artemiu (Kittaka, 1976; Farmer, 1979; Tseng and Cheng, 1981; Al-Hajj et al., 1983; Liao et al., 1983; Emmerson, 1984). Larvae of CuZlinectes supidus Rathbun raised exclusively on Bruchionus survive through the early zoeal stages, but do not metamorphose to the megalopa (Sulkin, 1975; Sulkin and Epifano, 1975). Ling (1967) listed rotifers among natural foods of Mucrobruchium rosenbergii (de Man) larvae. Although the use of Bruchionus in the larviculture of M. rosenbergii has been reported in a number of instances ( Adisukresno et al., 1975; Ong et al., 1977; Fontaine and Revera, 1980; Dejarme et al., 1982), there
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers B.V.
332
is no consensus as to whether Bruchionus does contribute measurably to the nutrition of M. rosenbergii. There are no published data to support assertions concerning the suitability of Bruchionus as a food for M. rosenbergii. The purpose of this study was to examine the suitability of B. plicatilis as a substitute or supplement to Artemiu nauplii in feeding larvae of M. rosenbergii. Nutritional value of Artemiu nauplii apparently decreases rapidly after hatching because high rates of metabolism and growth result in rapid utilization of yolk material (Maddox and Manzi, 1976). On the other hand, each rotifer’s gut is usually filled with bacteria and algae which could provide additional nutrition for the cultured larvae. Thus, one might predict that Bruchionus could provide a continuously high level of nutrition for cultured larvae. MATERIALS
AND METHODS
Rotifers (Brachionusplicutilis) were reared on Chlorellu sp. Inocula for both rotifer and algal cultures were obtained from the U.S. Environmental Protection Agency, Gulf Breeze, FL. These cultures were reared in pasteurized 28 ppt salinity artificial sea water (Instant Ocean@, Aquarium Systems, Inc., Eastlake, OH ) . Algae were cultured in F/2 enriched seawater medium with a technique adapted from that of Guillard (1975). Rotifers were concentrated in a plankton bucket and rinsed with clean water before being fed to larvae of M. rosenbergii. Great Salt Lake Artemiu cysts were hatched in 6 ppt salinity artificial sea water and fed to M. rosenbe.rgii 24 h after hydration. Gravid female prawns were isolated in individual aquaria and acclimatized to 6 ppt salinity artificial sea water. Within 8 h of hatching, larvae were transferred to 250-ml glass culture dishes (approximately 200 ml sea water per dish). A total of 60 larvae was placed in each dish. Treatments were assigned to each dish by a Latin-square method. Four replicates of each treatment were conducted per trial. Two trials were conducted; each trial utilized larvae from a single female. Larvae were reared with a “green water” technique (Fujimura, 1966; Wickins, 1972; Manzi et al., 1977) in either aged, pasteurized 14 ppt salinity artificial sea water or 14 ppt salinity irradiated natural sea water. Inoculum to make green water was taken from the same Chlorellu cultures as were used to feed rotifers. Density of Chlorellu in the larval culture medium was approximately 2000 cells ml-‘. The larvae were transferred daily to an acid-cleaned dish containing freshly prepared medium. Each dish was gently aerated with a glass capillary tube to prevent formation of a surface film. Larvae were reared at 28.5 +_l.O”C under a 12h:12h 1ight:dark regime. Because there is little variability in size among individuals within each of the first nine larval stages of M. rosenbergii (Uno and Kwon, 1969; Murai and Andrews, 1978)) size was not used to evaluate nutritional state. Time required for larvae to reach each stage and relative survival were used to infer the suit-
333 ability of a diet, The number of M. rosenbergii larvae alive and the ontogenetic stage of each larva were determined daily. Stage was assigned in accord with descriptions by Uno and Kwon (1969). When at least 50% of the larvae in a given dish had molted to the next stage, five specimens were collected and preserved. Some healthy larvae were found to lie on the bottom of the culture dish among dead and moribund individuals. A larva was considered to be alive if it moved appendages when nudged by a needle. Larvae that had jumped from the water and desiccated on walls of the culture dish were not counted among mortalities. Four feeding treatments were tested: (i) starved, (ii) fed Brachionus alone (approximately 70 ml-’ culture medium), (iii) fed Brachionus plus Artemia (approximately 55 ml-’ and 40 ml-‘, respectively), and (iv) fed Artemia alone (approximately 55 ml- ’ ) . As an ancillary component of the study, we examined responses of larvae to the two types of culture media currently in use in our laboratory: (i) pasteurized artificial sea water, and (ii) natural sea water which had been irradiated with a submersible ultraviolet light (Hawaiian Marine Imports, Inc., Houston, TX) for 18 h prior to use. Thus, a fifth treatment, in which larvae were reared in irradiated natural sea water and fed Brachionus alone, was included. To test whether larvae of M. rosenbergii actually consumed rotifers, four 250-ml glass culture dishes of 14ppt salinity artificial sea water without larvae were inoculated with Brachionus (approximately 70 ml-‘). Change in density of rotifers in this “control” after 24 h was compared to that in four secondstage zoea (22) cultures of M. rosenbergii (55 larvae per 250-ml dish of 14 ppt salinity artificial sea water) fed with Brachionus alone. Statistical tests were performed by means of a two-tailed t-test for comparing two sample means. Mean developmental stage was calculated with the formula Mean stage=z(SXP,) where S represents zoeal stage number and P, represents still alive at stage S.
(I) proportion
of larvae
RESULTS Laruae reared in artificial sea water
larvae of M. rosenbergii restricted to a diet of lower survival (PC 0.01) and a lower mean stage of development (PC 0.01) than did larvae fed either a diet of Brachionus and Artemia together or a diet of Artemia alone (Fig. 1). Development of starved larvae and of larvae fed only Brachionus was arrested at a mean stage of 2.00 and 2.33, respectively. There was no significant difference Within 5 days after hatching,
Brachionus began to show significantly
334
0
4.0 1 g Q L5
STARVED
m FED ROTIFERS
30-
B
FED
0
FED ROTIFERS
ROTIFERS, N.S.W d ARTEMIA
m FED ARTEMIA
B
toog eo2 2 s
604020O-
134567
ELAPSED
DAYS
Fig. 1. Mean developmental stage (A) and cumulative larval survival (B) in Macrobrachium rosenbergii under varied feeding regimens. N.S.W. indicates cultures reared in natural sea water; all others were in artificial sea water. Vertical lines indicate positive components of the 95% confidence intervals. Values at day 2, not shown, were not significantly different from those at day 3.
(PC 0.05 ) in either survival or rate of development between those larvae fed a diet of Brachionus and Artemia together and those fed Artenia alone. For treatments in which there were substantial decreases in survival, whether in natural or artificial sea water, the decrease was preceded by an arrest in development and an increase in the number of non-swimming (but alive) larvae that accumulated on the bottom of the culture dish. Microscopic examination of these larvae revealed infestations by spirilla and bacilli bacteria and bodonid ciliates. Similar infestations were not found on larvae from any treatment in which Artemiu was included in the diet, even though cihates were present at low densities in culture media. Larvae of M. rosenbergii apparently did consume Brachionus, as dishes in which larvae were present had significantly lower numbers of rotifers after 24 h (P -=z 0.001) than did controls. Mean rate of consumption by stage 22 M. rosenbergii larvae was 207 +_8 SE rotifers larvae-’ day-‘. Larvae reared in natural sea water
Where diet was restricted to Bruchionus, development of most larvae reared in natural sea water was arrested at a mean stage of 2.98. By day 5 larvae fed
335
only Bruchionus and reared in natural sea water showed significantly greater survival (PC 0.001) and higher mean stage (PC 0.01) than larvae reared on the same diet but in artificial sea water. However, by day 7 neither survival nor mean stage of the Bruchionus-restricted cultures in natural sea water was as high (P~O.001) as that of larvae fed a diet of either Bruchionus and Artemiu together or Artemiu alone, even if reared in artificial sea water. DISCUSSION
A major problem facing hatchery operations for Mucrobruchium rosenbergii is dependence upon nauplii of Artemiu as the primary food for larvae (Hanson and Goodwin, 1977). Other feeds have been used for rearing A4. rosenbergii larvae (for example, Ling, 1967; Fujimura and Okamoto, 1970; Minamizawa and Morizane, 1970; Kloke and Potaros, 1975; Sick and Beaty, 1975; Hanson and Goodwin, 1977; Murai and Andrews, 1978; AQUACOP, 1979; Aniello and Singh, 1982). However, none of these feeds can readily replace Arteniu. Either these alternative feeds can be used only as supplements to Artemiu nauplii, or else availability of the alternative feeds is limited. The primary disadvantage to the use of Artemia as a larval food for M. rosenbergii is the expense of brine shrimp cysts, particularly in developing countries (Achmad, 1975; Ong et al., 1977; Solangi and Ogle, 1977). Cost of feeding accounts for as much as 60% of the total production costs in M. rosenbergii hatcheries (Hagood and Willis, 1976). Another disadvantage of Artemiu-based diets is that brine shrimp exuvia and shed cyst capsules accumulate in larval culture vessels. Bacterial degradation of these materials fouls the water, accumulated debris entangles larvae, and larval mortalities increase. Bruchionus plicutilis has a short life cycle, has simple dietary requirements, can be cultured in high densities, and has favorable nutritional content (Solangi and Ogle, 1977). Because it is small in size, it can be ingested completely by small decapod crustacean larvae. We have observed that individuals of M. rosenbergii in early larval stages apparently graze on the appendages of Artemiu but are not able to consume entire nauplii. We had postulated that Bruchionus, therefore, may be superior to Artemiu as a food for M. rosenbergii because early larval stages could consume entire rotifers. M. rosenbergii larvae are able to survive to stage 22 on internal food reserves alone (Uno and Kwon, 1969; Kloke and Potaros, 1975; Murai and Andrews, 1978). Because larvae fed only Bruchionus had neither greater survival nor faster development than did starved larvae, we conclude that A4. rosenbergii larvae are unable to obtain significant nutrition from Bruchionus. Furthermore, we conclude that addition of Bruchionus to the diet confers no nutritional advantage to larvae of M. rosenbergii because no significant difference in survival or rate of development was found between larvae fed only Artemiu and larvae fed both Bruchionus and Artemiu. Therefore, the rotifer Bruchionus
is neither a suitable substitute for Artemia nauplii nor a useful dietary supplement in larviculture of M. rosenbergii. The specific properties that make Artemia nauplii a superior food for larvae of M. rosenbergii have yet to be identified. Caloric content of rotifers per g ashfree dry weight is not significantly different from that of Artemiu nauplii (Emmerson, 1984). Sulkin (1975) reported that Artemia nauplii contain two to three times more lipid per dry weight than do rotifers. Anecdotal reports suggest that the yolk material of Artemia is nutritionally important to M. rosenbergii, but experimental data supporting such reports are lacking. It is unlikely that the introduction of rotifers to the culture medium in itself led to infestations by bacteria and ciliates in our experiment, because such infestations developed both in cultures of starved larvae and in cultures fed only Brachionus while they never developed in cultures fed both Bruchionus and Artemia. Rather, it appears that larvae lacking Artemia in the diet were unable to resist bacterial and ciliate infestations, perhaps for lack of proper nutrition. Injuries to larvae by rotifers may account for the slightly lower survival observed among larvae restricted to a diet of Brachionus than among starved larvae. Where rotifers were the sole food for the larvae, rotifers were frequently observed to consume appendages of larvae that were not healthy and active. Culture experiments involving larvae and postlarvae of many decapod crustaceans, conducted in our laboratory and in laboratories of several collaborators, have suggested that use of artificial sea water generally results in higher mortalities than those observed when high-quality natural sea water is used. In the present study, larvae fed only Brachionus and reared in natural sea water survived longer and developed to a later mean stage than did larvae fed the same diet but reared in artificial sea water. Because depressed survival and development did not occur in artificial seawater media where Artemia was included in the diet, we suggest that the depressed survival and development observed in other cases may be nutrient-related, rather than some toxicity effect of the artificial media. Larvae may obtain some nutrient from natural sea water that, outside of the Artemia diet, appears to be lacking in artificial sea water. ACKNOWLEDGEMENTS
Thanks are extended to M. Hemmer, U.S. Environmental Protection Agency, Gulf Breeze, FL, and to K. Roberts, University of Southwestern Louisiana, for assistance in development of rotifer and Chlorella culture systems. J. Domingue, Youngsville, LA, and G. Perry, Louisiana State Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA, assisted by providing gravid M. rosenbergii females. This is contribution number 13
337
from the USL Center for Crustacean Research. Partial support for this project was provided under Louisiana Sea Grant Project 169-59-5109, R/CFB-5.
REFERENCES Achmad, S., 1975. A progress report on activities related to attempts to stimulate Macrob~uchium spp. culture in Indonesia. Proc. Int. Conf. Prawn Farming, Vung Tau, Vietnam, 31 March-4 April 1975, 11 pp. Adisukresno, S., Poernomo, A., Budiona, M. and Busman, S., 1975. Preliminary experiments on mass rearing of Mucrobrachium larvae. Shrimp Culture Research Centre, Jepara, Indonesia. Proc. Int. Conf. Prawn Farming, Vung Tau, Vietnam, 31 March-4 April 1975,7 pp. Al-Hajj, A.B., James, C.M., Al-Ablani, S. and Farmer, A.S.D., 1983. Advances in hatchery production of Penaeus semisulcatus in Kuwait with particular reference to the refinement of feeding practices. J. World Maricult. Sot., 14: 64-74. Aniello, M.S. and Singh, T., 1982. Some studies on the larviculture of the giant prawn (Macrobrachium rosenbergii). In: M.B. New (Editor), Giant Prawn Farming. Elsevier, New York, NY, pp. 225-232. AQUACOP, 1979. Intensive larval culture of Mucrobrachium rosenbergii: a case study. Proc. World Maricult. Sot., 10: 429-434, Dejarme, H.E., Dominisac, J.B. and Dejarme, S.M., 1982. Notes on spawner collection methods and larval rearing of giant prawns (Macrobrachium rosenbergii de Man) at MSU-IFRD Station, Naawan, Misamis Oriental, Philippines. In: M.B. New (Editor), Giant Prawn Farming. Elsevier, New York, NY, pp. 73-82. Emmerson, W.D., 1984. Predation and energetics of Penaeus indicus (Decapoda: Penaeidae) larvae feeding on Brachionus plicatilis and Artemia nauplii. Aquaculture, 38: 201-209. Farmer, A.S.D., 1979. Experimental rearing of penaeid shrimps in Kuwait. Proc. World Maricult. Sot., 10: 489-502. Fontaine, C.T. and Revera, D.B., 1980. The mass culture of the rotifer Brachionusplicatilis, for use as a foodstuff in aquaculture. Proc. World Maricult. Sot., 11: 211-218. Fujimura, T., 1966. Notes on the development of a practical mass culturing technique of the giant prawn Macrobrachium rosenbergii. Proc. Indo-Pac. Fish. Count., 12th Session, Honolulu, HI, 3 pp. (IPFC/C66/WP47). Fujimura, T. and Okamoto, H., 1970. Notes on progress made in developing a mass culturing technique for Mucrobrachium rosenbergiiin Hawaii. In: T.V.R. Pillay (Editor), Coastal Aquaculture in the Indo-Pacific Region. Fishing News (Books), Farnham, Great Britain, pp. 313327. Guillard, R.R.L., 1975. Culture ofphytoplankton for feeding marine invertebrates. In: W.L. Smith and M.H. Chanley (Editors), Culture of Marine Invertebrate Animals. Plenum Press, New York, NY, pp. 29-60. Hagood, R.W. and Willis, S.A., 1976. Cost comparisons of rearing larvae of freshwater shrimp, Macrobrachium acanthurus and M. rosenbergii, to juveniles. Aquaculture, 7: 59-74. Hanson, J.A. and Goodwin, H.L., 1977. Shrimp and Prawn Farming in the Western Hemisphere. Dowden, Hutchinson, and Ross, Stroudsburg, PA, 439 pp. Kittaka, J., 1976. Food and growth of penaeid shrimp. Proc. First Int. Conf. Aquacult. Nutr., Delaware Sea Grant Program and United States-Japan Natural Resources Aquaculture Panel, October 1975, pp. 249-285. Kloke, C.W. and Potaros, M., 1975. The technology and economics of small-scale commercial prawn (Macrobrachium rosenbergii) hatcheries: a case study in Thailand. Occas. Pap. IndoPac. Fish. Count., 1975/l: 87 pp.
Liao, I.C., Su, H.M. and Lin, J-H., 1983. Larval foods for penaeid prawns. In: J.P. McVey (Editor), CRC Handbook of Mariculture, Vol. I, Crustacean Aquaculture. CRC Press, Boca Raton, FL, pp. 43-69. Ling, SW., 1967. The general biology and development of Mucrobrachium rosenbergii (de Man). FAO Experience Paper, 30: l-18 (FR:BCSP/67/E/30). Maddox, M.B. and Manzi, J.J., 1976. The effects of algal supplements on static system culture of ikcrobrachium rosenbergii (de Man) larvae. Proc. World Maricult. Sot., 7: 677-698. Manzi, J.J., Maddox, J.J. and Sandifer, P.A., 1977. Algal supplement enhancement of Mucrobrachium rosenbergii (de Man) larviculture. Proc. World Maricult. Sot., 8: 207-223. Minamizawa, A. and Morizane, T., 1970. Report on study about cultivation techniques for freshwater shrimp. Ehime Prefecture Fisheries Laboratory, Japanese Bureau of Commercial Fisheries, Office of Foreign Fisheries, U.S. Dept. Interior, Washington, DC, 55 pp. Mock, C.R., 1971. Trip Report, United States-Japan Natural Resources Aquaculture Panel Meeting, 14-19 October 1971. Galveston, TX, 34 pp. Murai, T. and Andrews, J.W., 1978. Comparison of feeds for larval stages of the giant prawn (Mucrobrachium rosenbergii). Proc. World Maricult. Sot., 9: 189-193. Ong, K.S., Ng, F.O. and Wong, Y.K., 1977. Experiences and problems in the design and operation of a Macrobrachium hatchery. Joint FAO-UNDP/SCSP and SEAFDEC Region Workshop on Aquaculture Engineering (with Emphasis on Small-scale Projects). Tigbauan, Iloilo, Philippines, 27 Nov.-3 Dec. 1977,5 pp (SCSP-SFDC/77/ACn/CP/32). Sick, L.V. and Beaty, H., 1975. Development of formula foods designed for Macrobruchium rosenbergii larval and juvenile shrimp. Proc. World Maricult. Sot., 6: 89-102. Solangi, M.A. and Ogle, J.T., 1977. A selected bibliography on the mass propagation of rotifers with emphasis on the biology and culture of Brachionusplicatilis. Gulf Res. Rep., 6 (1): 59-68. Sorgeloos, P. and Persoone, G., 1975. Technological improvements for the cultivation of invertebrates as food for fishes and crustaceans. II. Hatching and culturing of the brine shrimp, Artemia salina L. Aquaculture, 6:303-317. Sulkin, S.D., 1975. The significance of diet in growth and development of larvae of the blue crab, Callinectes sapidus Rathbun, under laboratory conditions. J. Exp. Mar. Biol. Ecol., 20: 119135. Sulkin, S.D. and Epifano, C.E., 1975. Comparison of rotifers and other diets for rearing early larvae of the blue crab, Callinectes sapidus. Estuarine Coastal Mar. Sci., 3 (1): 109-114. Tseng, W.Y. and Cheng, W.W., 1981. The artificial propagation and culture of bear shrimp, PenoeLLssemisulcatus de Haan, in Hong Kong. Proc. World Maricult. Sot., 12 (2): 260-281. Uno, Y. and Kwon, C.S., 1969. Larval development of Macrobrachium rosenbergii (de Man) reared in the laboratory. J. Tokyo Univ. Fish., 55 (2 ): 179- 190 + xii figures. Wickins, J.F., 1972. Experiments on the culture of spot prawn Pandalus platyceros Brandt and the giant freshwater prawn Macrobrachium rosenbergii (de Man). Fish. Invest., Mini& Agric. Fish.Food (G.B.),Ser.I1,27(5),23pp.
71 (1988) 331-338 Elsevier Science Publishers B.V., Amsterdam -
331 Printed in The Netherlands
Evaluation of the Rotifer BmchionzmpZ~cati Substitute for Artemia in Feeding Larvae of
as a
Macrobrachium rosenbergii DONALD L. LOVETT and DARRYL L. FELDER
Department of Biology and Center for Crustacean Research, University of Southwestern Louisiana, P.O. Box 42451, Lafayette, LA 70504 (U.S.A.)
(Accepted 14 December 1987)
ABSTRACT Lovett, D.L. and Felder, D.L., 1988. Evaluation of the rotifer Brachionusplicatilis as a substitute for Artemia in feeding larvae of Macrobrachium rosenbergii. Aquaculture, 71: 331-338. Addition of the rotifer Brachionusplicatilis Miiller to an Artemia-based diet yielded no significant increase in larval survival or rate of larval development in Macrobrachium rosenbergii (de Man). B. plicatilis apparently contributed little to the nutrition of M. rosenbergii larvae and provided no benefit to their culture. Larvae fed on a diet restricted to Brachionus had significantly lower surviva1 and rate of development than did larvae fed on a diet that included Artemia. Larvae fed only rotifen did not have significantly different survival from that of starved larvae. Development of larvae fed only rotifers and reared in artificial sea water was arrested at a mean stage of 2.33; in natural sea water development was arrested at a mean stage of 2.98.
INTRODUCTION
The rotifer BruchionuspZicatilis Miiller has been promoted as both an initial feed and as a supplemental feed for larval crustaceans (Mock, 1971; Sorgeloos and Persoone, 1975; Solangi and Ogle, 1977). Bruchionus has been used in larviculture of penaeid shrimp as a food intermediate in size between algae and Artemiu (Kittaka, 1976; Farmer, 1979; Tseng and Cheng, 1981; Al-Hajj et al., 1983; Liao et al., 1983; Emmerson, 1984). Larvae of CuZlinectes supidus Rathbun raised exclusively on Bruchionus survive through the early zoeal stages, but do not metamorphose to the megalopa (Sulkin, 1975; Sulkin and Epifano, 1975). Ling (1967) listed rotifers among natural foods of Mucrobruchium rosenbergii (de Man) larvae. Although the use of Bruchionus in the larviculture of M. rosenbergii has been reported in a number of instances ( Adisukresno et al., 1975; Ong et al., 1977; Fontaine and Revera, 1980; Dejarme et al., 1982), there
0044-8486/88/$03.50
0 1988 Elsevier Science Publishers B.V.
332
is no consensus as to whether Bruchionus does contribute measurably to the nutrition of M. rosenbergii. There are no published data to support assertions concerning the suitability of Bruchionus as a food for M. rosenbergii. The purpose of this study was to examine the suitability of B. plicatilis as a substitute or supplement to Artemiu nauplii in feeding larvae of M. rosenbergii. Nutritional value of Artemiu nauplii apparently decreases rapidly after hatching because high rates of metabolism and growth result in rapid utilization of yolk material (Maddox and Manzi, 1976). On the other hand, each rotifer’s gut is usually filled with bacteria and algae which could provide additional nutrition for the cultured larvae. Thus, one might predict that Bruchionus could provide a continuously high level of nutrition for cultured larvae. MATERIALS
AND METHODS
Rotifers (Brachionusplicutilis) were reared on Chlorellu sp. Inocula for both rotifer and algal cultures were obtained from the U.S. Environmental Protection Agency, Gulf Breeze, FL. These cultures were reared in pasteurized 28 ppt salinity artificial sea water (Instant Ocean@, Aquarium Systems, Inc., Eastlake, OH ) . Algae were cultured in F/2 enriched seawater medium with a technique adapted from that of Guillard (1975). Rotifers were concentrated in a plankton bucket and rinsed with clean water before being fed to larvae of M. rosenbergii. Great Salt Lake Artemiu cysts were hatched in 6 ppt salinity artificial sea water and fed to M. rosenbe.rgii 24 h after hydration. Gravid female prawns were isolated in individual aquaria and acclimatized to 6 ppt salinity artificial sea water. Within 8 h of hatching, larvae were transferred to 250-ml glass culture dishes (approximately 200 ml sea water per dish). A total of 60 larvae was placed in each dish. Treatments were assigned to each dish by a Latin-square method. Four replicates of each treatment were conducted per trial. Two trials were conducted; each trial utilized larvae from a single female. Larvae were reared with a “green water” technique (Fujimura, 1966; Wickins, 1972; Manzi et al., 1977) in either aged, pasteurized 14 ppt salinity artificial sea water or 14 ppt salinity irradiated natural sea water. Inoculum to make green water was taken from the same Chlorellu cultures as were used to feed rotifers. Density of Chlorellu in the larval culture medium was approximately 2000 cells ml-‘. The larvae were transferred daily to an acid-cleaned dish containing freshly prepared medium. Each dish was gently aerated with a glass capillary tube to prevent formation of a surface film. Larvae were reared at 28.5 +_l.O”C under a 12h:12h 1ight:dark regime. Because there is little variability in size among individuals within each of the first nine larval stages of M. rosenbergii (Uno and Kwon, 1969; Murai and Andrews, 1978)) size was not used to evaluate nutritional state. Time required for larvae to reach each stage and relative survival were used to infer the suit-
333 ability of a diet, The number of M. rosenbergii larvae alive and the ontogenetic stage of each larva were determined daily. Stage was assigned in accord with descriptions by Uno and Kwon (1969). When at least 50% of the larvae in a given dish had molted to the next stage, five specimens were collected and preserved. Some healthy larvae were found to lie on the bottom of the culture dish among dead and moribund individuals. A larva was considered to be alive if it moved appendages when nudged by a needle. Larvae that had jumped from the water and desiccated on walls of the culture dish were not counted among mortalities. Four feeding treatments were tested: (i) starved, (ii) fed Brachionus alone (approximately 70 ml-’ culture medium), (iii) fed Brachionus plus Artemia (approximately 55 ml-’ and 40 ml-‘, respectively), and (iv) fed Artemia alone (approximately 55 ml- ’ ) . As an ancillary component of the study, we examined responses of larvae to the two types of culture media currently in use in our laboratory: (i) pasteurized artificial sea water, and (ii) natural sea water which had been irradiated with a submersible ultraviolet light (Hawaiian Marine Imports, Inc., Houston, TX) for 18 h prior to use. Thus, a fifth treatment, in which larvae were reared in irradiated natural sea water and fed Brachionus alone, was included. To test whether larvae of M. rosenbergii actually consumed rotifers, four 250-ml glass culture dishes of 14ppt salinity artificial sea water without larvae were inoculated with Brachionus (approximately 70 ml-‘). Change in density of rotifers in this “control” after 24 h was compared to that in four secondstage zoea (22) cultures of M. rosenbergii (55 larvae per 250-ml dish of 14 ppt salinity artificial sea water) fed with Brachionus alone. Statistical tests were performed by means of a two-tailed t-test for comparing two sample means. Mean developmental stage was calculated with the formula Mean stage=z(SXP,) where S represents zoeal stage number and P, represents still alive at stage S.
(I) proportion
of larvae
RESULTS Laruae reared in artificial sea water
larvae of M. rosenbergii restricted to a diet of lower survival (PC 0.01) and a lower mean stage of development (PC 0.01) than did larvae fed either a diet of Brachionus and Artemia together or a diet of Artemia alone (Fig. 1). Development of starved larvae and of larvae fed only Brachionus was arrested at a mean stage of 2.00 and 2.33, respectively. There was no significant difference Within 5 days after hatching,
Brachionus began to show significantly
334
0
4.0 1 g Q L5
STARVED
m FED ROTIFERS
30-
B
FED
0
FED ROTIFERS
ROTIFERS, N.S.W d ARTEMIA
m FED ARTEMIA
B
toog eo2 2 s
604020O-
134567
ELAPSED
DAYS
Fig. 1. Mean developmental stage (A) and cumulative larval survival (B) in Macrobrachium rosenbergii under varied feeding regimens. N.S.W. indicates cultures reared in natural sea water; all others were in artificial sea water. Vertical lines indicate positive components of the 95% confidence intervals. Values at day 2, not shown, were not significantly different from those at day 3.
(PC 0.05 ) in either survival or rate of development between those larvae fed a diet of Brachionus and Artemia together and those fed Artenia alone. For treatments in which there were substantial decreases in survival, whether in natural or artificial sea water, the decrease was preceded by an arrest in development and an increase in the number of non-swimming (but alive) larvae that accumulated on the bottom of the culture dish. Microscopic examination of these larvae revealed infestations by spirilla and bacilli bacteria and bodonid ciliates. Similar infestations were not found on larvae from any treatment in which Artemiu was included in the diet, even though cihates were present at low densities in culture media. Larvae of M. rosenbergii apparently did consume Brachionus, as dishes in which larvae were present had significantly lower numbers of rotifers after 24 h (P -=z 0.001) than did controls. Mean rate of consumption by stage 22 M. rosenbergii larvae was 207 +_8 SE rotifers larvae-’ day-‘. Larvae reared in natural sea water
Where diet was restricted to Bruchionus, development of most larvae reared in natural sea water was arrested at a mean stage of 2.98. By day 5 larvae fed
335
only Bruchionus and reared in natural sea water showed significantly greater survival (PC 0.001) and higher mean stage (PC 0.01) than larvae reared on the same diet but in artificial sea water. However, by day 7 neither survival nor mean stage of the Bruchionus-restricted cultures in natural sea water was as high (P~O.001) as that of larvae fed a diet of either Bruchionus and Artemiu together or Artemiu alone, even if reared in artificial sea water. DISCUSSION
A major problem facing hatchery operations for Mucrobruchium rosenbergii is dependence upon nauplii of Artemiu as the primary food for larvae (Hanson and Goodwin, 1977). Other feeds have been used for rearing A4. rosenbergii larvae (for example, Ling, 1967; Fujimura and Okamoto, 1970; Minamizawa and Morizane, 1970; Kloke and Potaros, 1975; Sick and Beaty, 1975; Hanson and Goodwin, 1977; Murai and Andrews, 1978; AQUACOP, 1979; Aniello and Singh, 1982). However, none of these feeds can readily replace Arteniu. Either these alternative feeds can be used only as supplements to Artemiu nauplii, or else availability of the alternative feeds is limited. The primary disadvantage to the use of Artemia as a larval food for M. rosenbergii is the expense of brine shrimp cysts, particularly in developing countries (Achmad, 1975; Ong et al., 1977; Solangi and Ogle, 1977). Cost of feeding accounts for as much as 60% of the total production costs in M. rosenbergii hatcheries (Hagood and Willis, 1976). Another disadvantage of Artemiu-based diets is that brine shrimp exuvia and shed cyst capsules accumulate in larval culture vessels. Bacterial degradation of these materials fouls the water, accumulated debris entangles larvae, and larval mortalities increase. Bruchionus plicutilis has a short life cycle, has simple dietary requirements, can be cultured in high densities, and has favorable nutritional content (Solangi and Ogle, 1977). Because it is small in size, it can be ingested completely by small decapod crustacean larvae. We have observed that individuals of M. rosenbergii in early larval stages apparently graze on the appendages of Artemiu but are not able to consume entire nauplii. We had postulated that Bruchionus, therefore, may be superior to Artemiu as a food for M. rosenbergii because early larval stages could consume entire rotifers. M. rosenbergii larvae are able to survive to stage 22 on internal food reserves alone (Uno and Kwon, 1969; Kloke and Potaros, 1975; Murai and Andrews, 1978). Because larvae fed only Bruchionus had neither greater survival nor faster development than did starved larvae, we conclude that A4. rosenbergii larvae are unable to obtain significant nutrition from Bruchionus. Furthermore, we conclude that addition of Bruchionus to the diet confers no nutritional advantage to larvae of M. rosenbergii because no significant difference in survival or rate of development was found between larvae fed only Artemiu and larvae fed both Bruchionus and Artemiu. Therefore, the rotifer Bruchionus
is neither a suitable substitute for Artemia nauplii nor a useful dietary supplement in larviculture of M. rosenbergii. The specific properties that make Artemia nauplii a superior food for larvae of M. rosenbergii have yet to be identified. Caloric content of rotifers per g ashfree dry weight is not significantly different from that of Artemiu nauplii (Emmerson, 1984). Sulkin (1975) reported that Artemia nauplii contain two to three times more lipid per dry weight than do rotifers. Anecdotal reports suggest that the yolk material of Artemia is nutritionally important to M. rosenbergii, but experimental data supporting such reports are lacking. It is unlikely that the introduction of rotifers to the culture medium in itself led to infestations by bacteria and ciliates in our experiment, because such infestations developed both in cultures of starved larvae and in cultures fed only Brachionus while they never developed in cultures fed both Bruchionus and Artemia. Rather, it appears that larvae lacking Artemia in the diet were unable to resist bacterial and ciliate infestations, perhaps for lack of proper nutrition. Injuries to larvae by rotifers may account for the slightly lower survival observed among larvae restricted to a diet of Brachionus than among starved larvae. Where rotifers were the sole food for the larvae, rotifers were frequently observed to consume appendages of larvae that were not healthy and active. Culture experiments involving larvae and postlarvae of many decapod crustaceans, conducted in our laboratory and in laboratories of several collaborators, have suggested that use of artificial sea water generally results in higher mortalities than those observed when high-quality natural sea water is used. In the present study, larvae fed only Brachionus and reared in natural sea water survived longer and developed to a later mean stage than did larvae fed the same diet but reared in artificial sea water. Because depressed survival and development did not occur in artificial seawater media where Artemia was included in the diet, we suggest that the depressed survival and development observed in other cases may be nutrient-related, rather than some toxicity effect of the artificial media. Larvae may obtain some nutrient from natural sea water that, outside of the Artemia diet, appears to be lacking in artificial sea water. ACKNOWLEDGEMENTS
Thanks are extended to M. Hemmer, U.S. Environmental Protection Agency, Gulf Breeze, FL, and to K. Roberts, University of Southwestern Louisiana, for assistance in development of rotifer and Chlorella culture systems. J. Domingue, Youngsville, LA, and G. Perry, Louisiana State Department of Wildlife and Fisheries, Rockefeller Wildlife Refuge, Grand Chenier, LA, assisted by providing gravid M. rosenbergii females. This is contribution number 13
337
from the USL Center for Crustacean Research. Partial support for this project was provided under Louisiana Sea Grant Project 169-59-5109, R/CFB-5.
REFERENCES Achmad, S., 1975. A progress report on activities related to attempts to stimulate Macrob~uchium spp. culture in Indonesia. Proc. Int. Conf. Prawn Farming, Vung Tau, Vietnam, 31 March-4 April 1975, 11 pp. Adisukresno, S., Poernomo, A., Budiona, M. and Busman, S., 1975. Preliminary experiments on mass rearing of Mucrobrachium larvae. Shrimp Culture Research Centre, Jepara, Indonesia. Proc. Int. Conf. Prawn Farming, Vung Tau, Vietnam, 31 March-4 April 1975,7 pp. Al-Hajj, A.B., James, C.M., Al-Ablani, S. and Farmer, A.S.D., 1983. Advances in hatchery production of Penaeus semisulcatus in Kuwait with particular reference to the refinement of feeding practices. J. World Maricult. Sot., 14: 64-74. Aniello, M.S. and Singh, T., 1982. Some studies on the larviculture of the giant prawn (Macrobrachium rosenbergii). In: M.B. New (Editor), Giant Prawn Farming. Elsevier, New York, NY, pp. 225-232. AQUACOP, 1979. Intensive larval culture of Mucrobrachium rosenbergii: a case study. Proc. World Maricult. Sot., 10: 429-434, Dejarme, H.E., Dominisac, J.B. and Dejarme, S.M., 1982. Notes on spawner collection methods and larval rearing of giant prawns (Macrobrachium rosenbergii de Man) at MSU-IFRD Station, Naawan, Misamis Oriental, Philippines. In: M.B. New (Editor), Giant Prawn Farming. Elsevier, New York, NY, pp. 73-82. Emmerson, W.D., 1984. Predation and energetics of Penaeus indicus (Decapoda: Penaeidae) larvae feeding on Brachionus plicatilis and Artemia nauplii. Aquaculture, 38: 201-209. Farmer, A.S.D., 1979. Experimental rearing of penaeid shrimps in Kuwait. Proc. World Maricult. Sot., 10: 489-502. Fontaine, C.T. and Revera, D.B., 1980. The mass culture of the rotifer Brachionusplicatilis, for use as a foodstuff in aquaculture. Proc. World Maricult. Sot., 11: 211-218. Fujimura, T., 1966. Notes on the development of a practical mass culturing technique of the giant prawn Macrobrachium rosenbergii. Proc. Indo-Pac. Fish. Count., 12th Session, Honolulu, HI, 3 pp. (IPFC/C66/WP47). Fujimura, T. and Okamoto, H., 1970. Notes on progress made in developing a mass culturing technique for Mucrobrachium rosenbergiiin Hawaii. In: T.V.R. Pillay (Editor), Coastal Aquaculture in the Indo-Pacific Region. Fishing News (Books), Farnham, Great Britain, pp. 313327. Guillard, R.R.L., 1975. Culture ofphytoplankton for feeding marine invertebrates. In: W.L. Smith and M.H. Chanley (Editors), Culture of Marine Invertebrate Animals. Plenum Press, New York, NY, pp. 29-60. Hagood, R.W. and Willis, S.A., 1976. Cost comparisons of rearing larvae of freshwater shrimp, Macrobrachium acanthurus and M. rosenbergii, to juveniles. Aquaculture, 7: 59-74. Hanson, J.A. and Goodwin, H.L., 1977. Shrimp and Prawn Farming in the Western Hemisphere. Dowden, Hutchinson, and Ross, Stroudsburg, PA, 439 pp. Kittaka, J., 1976. Food and growth of penaeid shrimp. Proc. First Int. Conf. Aquacult. Nutr., Delaware Sea Grant Program and United States-Japan Natural Resources Aquaculture Panel, October 1975, pp. 249-285. Kloke, C.W. and Potaros, M., 1975. The technology and economics of small-scale commercial prawn (Macrobrachium rosenbergii) hatcheries: a case study in Thailand. Occas. Pap. IndoPac. Fish. Count., 1975/l: 87 pp.
Liao, I.C., Su, H.M. and Lin, J-H., 1983. Larval foods for penaeid prawns. In: J.P. McVey (Editor), CRC Handbook of Mariculture, Vol. I, Crustacean Aquaculture. CRC Press, Boca Raton, FL, pp. 43-69. Ling, SW., 1967. The general biology and development of Mucrobrachium rosenbergii (de Man). FAO Experience Paper, 30: l-18 (FR:BCSP/67/E/30). Maddox, M.B. and Manzi, J.J., 1976. The effects of algal supplements on static system culture of ikcrobrachium rosenbergii (de Man) larvae. Proc. World Maricult. Sot., 7: 677-698. Manzi, J.J., Maddox, J.J. and Sandifer, P.A., 1977. Algal supplement enhancement of Mucrobrachium rosenbergii (de Man) larviculture. Proc. World Maricult. Sot., 8: 207-223. Minamizawa, A. and Morizane, T., 1970. Report on study about cultivation techniques for freshwater shrimp. Ehime Prefecture Fisheries Laboratory, Japanese Bureau of Commercial Fisheries, Office of Foreign Fisheries, U.S. Dept. Interior, Washington, DC, 55 pp. Mock, C.R., 1971. Trip Report, United States-Japan Natural Resources Aquaculture Panel Meeting, 14-19 October 1971. Galveston, TX, 34 pp. Murai, T. and Andrews, J.W., 1978. Comparison of feeds for larval stages of the giant prawn (Mucrobrachium rosenbergii). Proc. World Maricult. Sot., 9: 189-193. Ong, K.S., Ng, F.O. and Wong, Y.K., 1977. Experiences and problems in the design and operation of a Macrobrachium hatchery. Joint FAO-UNDP/SCSP and SEAFDEC Region Workshop on Aquaculture Engineering (with Emphasis on Small-scale Projects). Tigbauan, Iloilo, Philippines, 27 Nov.-3 Dec. 1977,5 pp (SCSP-SFDC/77/ACn/CP/32). Sick, L.V. and Beaty, H., 1975. Development of formula foods designed for Macrobruchium rosenbergii larval and juvenile shrimp. Proc. World Maricult. Sot., 6: 89-102. Solangi, M.A. and Ogle, J.T., 1977. A selected bibliography on the mass propagation of rotifers with emphasis on the biology and culture of Brachionusplicatilis. Gulf Res. Rep., 6 (1): 59-68. Sorgeloos, P. and Persoone, G., 1975. Technological improvements for the cultivation of invertebrates as food for fishes and crustaceans. II. Hatching and culturing of the brine shrimp, Artemia salina L. Aquaculture, 6:303-317. Sulkin, S.D., 1975. The significance of diet in growth and development of larvae of the blue crab, Callinectes sapidus Rathbun, under laboratory conditions. J. Exp. Mar. Biol. Ecol., 20: 119135. Sulkin, S.D. and Epifano, C.E., 1975. Comparison of rotifers and other diets for rearing early larvae of the blue crab, Callinectes sapidus. Estuarine Coastal Mar. Sci., 3 (1): 109-114. Tseng, W.Y. and Cheng, W.W., 1981. The artificial propagation and culture of bear shrimp, PenoeLLssemisulcatus de Haan, in Hong Kong. Proc. World Maricult. Sot., 12 (2): 260-281. Uno, Y. and Kwon, C.S., 1969. Larval development of Macrobrachium rosenbergii (de Man) reared in the laboratory. J. Tokyo Univ. Fish., 55 (2 ): 179- 190 + xii figures. Wickins, J.F., 1972. Experiments on the culture of spot prawn Pandalus platyceros Brandt and the giant freshwater prawn Macrobrachium rosenbergii (de Man). Fish. Invest., Mini& Agric. Fish.Food (G.B.),Ser.I1,27(5),23pp.