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Environmental manipulation to stimulate rotifers in fish rearing ponds
Aquaculture, 42 (1984) 343-348 Elsevier Science Publishers B.V., Amsterdam -Printed
343 in The Netherlands
ENVIRONMENTAL MANIPULATION TO STIMULATE ROTIFERS IN FISH REARING PONDS
KAROL OPUSZYNSKI*, JEROME V. SHIREMAN, FREDERICK and ROGER W. ROTTMANN School of Forest Resources and Conservation, (U.S.A.)
J. ALDRIDGE
University of Florida, Gainesville, FL
*Permanent address: Inland Fisheries Institute, Zabieniec, 05-500 Journal Series No. 5601, Florida Agricultural Experiment
Piaseczno, Poland
Station
(Accepted 25 July 1984)
ABSTRACT Opuszynski, K., Shireman, J.V., Aldridge, F.J. and Rottmann, R.W., 1984. Experimental manipulation to stimulate rotifers in fish rearing ponds. Aquaculture, 42: 343-348. Four treatments were established in eight small ponds not stocked with fish: a control group (untreated and not fertilized) and three groups treated with organophosphate insecticide (Dylox). One group of Dylox treatment ponds was treated with chemical fertilizer, the second group with chemical and organic fertilizer, and the third group with chemical-organic fertilizer and paddlewheel water agitation. Water analysis and zooplankton samples were collected twice weekly. Rotifers increased in numbers in all treated ponds but no correlations were found between 16 hydrochemical and biological parameters and rotifer numbers. The application of rotifer management techniques for rearing larval fish is discussed.
INTRODUCTION
Successful rearing of many commercially important fish species is dependent upon providing the proper quality and quantity of live food to the fish immediately after they commence feeding. Live food is necessary because complete artificial starter feeds have yet to be developed (Dabrowski et al., in press). Rotifers are excellent food for some fish larvae when they commence active feeding (Woynarovich and Horvath, 1980). Currently rotifers are mass cultured under fully controlled conditions in Japan and their culture has contributed to the rapid development of Japanese fish culture (Hirata, 1979). The importance of rotifers for feeding small fish has been recognized in Europe and in the United States (Tamas and Horvath, 1976; Opuszynski, 1979; Geiger, 1983). Fish culturists grow rotifers in earthen ponds which are either stocked with fish larvae or the rotifers are collected from the ponds and fed to fish larvae held in rearing
0044-8486/84/$03.00
o 1984 Elsevier Science Publishers B.V.
344
facilities. Both methods yield satisfactory results provided that rotifers are grown in high numbers over the proper time period. Rotifer culture in earthen ponds is still not routinely done and high production numbers can not always be insured. For this reason we evaluated different management techniques for rotifer production in ponds. MATERIALS
AND METHODS
This experiment was conducted in eight small ponds at the Austin Cary Forest Research Facilities, University of Florida, Gainesville, FL. Pond areas ranged from 0.01 ha to 0.03 ha with an average depth of about 1 m. Ponds were not stocked with fish. Four treatments were established: Group I - control, received no treatment (Ponds 11 and 15); Group II - pesticide and chemical fertilizer treatment (Ponds 10 and 14); Group III - pesticide, chemical and organic fertilizer treatment (Ponds 12 and 17); and Group IV - same as group III with paddlewheel agitation described by Shireman et al. (1983) (Ponds 9 and 13). Dylox (80% active ingredient), an organophosphate pesticide, was applied at a concentration of 1 mg of active ingredient per 1 of pond water. Granular chemical fertilizers, 34% ammonium nitrate and 46% superphosphate were applied at a concentration corresponding to 2 mg N/l and 0.7 mg P,O,/l. Organic fertilizer (chicken manure) was applied at a rate of 2500 kg/ha wet weight (1.6 mg N/l and 1.3 mg PzO,/l). All ponds were limed with 1000 kg/ha of calcium carbonate. Water temperature, dissolved oxygen and Secchi disc measurements were taken daily at about 11 a.m. Total alkalinity, total phosphorus, total nitrogen, ammonia, calcium, magnesium, potassium, sodium, turbidity, pH, chlorophyll a and zooplankton were sampled twice a week. Temperature and oxygen were measured with a YSI-51 meter, turbidity with a Hach Model 2100 turbidimeter. Chlorophyll, total phosphorus and nitrogen were determined according to the methods described by Canfield (1983) and the remaining chemistry tests according to procedures described in Standard Methods (1975). Thirty-liter zooplankton samples consisting of three subsamples were taken from different stations within each pond. Water was strained with a 80-pm mesh plankton net. The experiment ran from 18 April to 16 May 1983. Lime, fertilizers and Dylox were applied on 19 April, 1 day after the first water chemistry and zooplankton samples were collected. Analysis of variance and Duncan’s multiple range test were used to test for significant differences (P
Significant differences were found except for water temperature (23.624.1%) and mean pH values (7.0-7.4). There were no differences in mean
345
oxygen saturation levels between the control group (8.3 mg 0,/l) and experimental groups (7.5-9.6 mg 0,/l). Mean oxygen levels in the paddlewheel ponds (7.5 mg 0,/l), however, were significantly lower than levels in ponds receiving the same treatment without paddlewheel agitation (9.6 mg 0,/l). Mean magnesium (3.1 mg/l), sodium (4.4 mg/l), phosphorus (0.05 mg/l) and chlorophyll a (0.006 mg/l) values were significantly lower in the control group, whereas mean Secchi disc (71 cm) and mean number of crustaceans (83 individuals per 1) were significantly higher. The highest average volumes for magnesium (4.9 mg/l), sodium (5.8 mg/l), and chlorophyll (0.09 mg/l) were noted in the paddlewheel ponds. The greatest turbidity (Secchi disc readings 33 cm) also occurred in paddlewheel ponds. No significant differences were found in total phosphorus concentration (0.2-0.3 mg/l) and crustacean numbers (9-14 individuals per 1) within the Dylox-treated ponds. Rotifer numbers were lowest in the control ponds during the entire experiment (Fig. 1). In all Dylox-treated ponds large numbers of rotifers were observed. However, rotifer dynamics differed within and among treat, ROTATORIA
103~~o~v/~ , CRUSTACEA
102~~~~v./~
2-
61
II
Fig. 1. Rotifer and crustacean dynamics in control ponds (11 and 16) and Dylox treatment ponds (10 and 14 - chemical fertilization; 12 and 17 - chemical and organic fertilization; 9 and 13 - chemical, organic fertilization and paddlewheel agitation).
346
ment groups. The Dylox treatment eliminated most crustaceans in all of the treated ponds from which they did not fully recover. The rotifer population in all the ponds consisted mainly of Brachionus sp.: B. calyciflorus, B. plicatilis, B. havanaensis and B. angular-is. Other rotifers such as Polyarthra sp., Filinia sp., Keratella sp., and Asplanchna sp. were occasionally found in small numbers. Copepods made up the majority of the crustacean zooplankton. The dominant species were Tropocyclops prasinus and Diaptomus sp. Cladocerans were abundant in some samples taken before Dylox treatments; Daphnia laevis was the most numerous species. Other crustaceans such as Alona rectangula, Chydorus sphaericus, Bosmina coregoni, Simocephalus vetulus, and Scapholeberis kingi occurred rarely and contributed little to the composition of the zooplankton community. There were no correlations among rotifer abundance and any of the measured parameters in Dylox-treated groups. Rotifers were positively correlated only with total alkalinity in the control group. Positive correlations existed between crustacean zooplankton abundance and sodium, calcium, chlorophyll and ammonia concentration in the control ponds, whereas crustacean number was positively correlated only with water turbidity in Dylox-treated ponds. DISCUSSION
Changes caused by Dylox treatment and fertilization could not be separated because the Dylox treatment ponds were also fertilized. Rotifer numbers and chlorophyll concentrations increased simultaneously with decreasing crustacean populations in the treatment ponds. Similar changes caused by an insecticide alone were described by Hurlbert et al. (1972). They suggested that phytoplankton population increases following treatment were caused by reduced crustacean grazing. The use of paddlewheels did not cause the same effect as was reported by Shireman et al. (1983) who found significant increases in oxygen saturation and rotifer numbers in their paddlewheel ponds. Mean oxygen levels were lower than in identically treated ponds without paddlewheel aeration. Significantly higher ammonia levels in the paddlewheel ponds might indicate that oxygen decreases were caused by more rapid organic matter decomposition. Shireman et al. (1983) did not use organic fertilizer; the mean ammonia level was 0.26 mg NH3/1 in the paddlewheel ponds. This was five times less than we measured (1.39 mg/l). In summary it can be stated that the Dylox treatment and fertilization caused an increase in rotifers. The increase was in response to the changes which occurred as the Dylox treatment exterminated cladocerans (rotifer competitors) and copepods (competitors and/or predators). Simultaneously, phytoplankton increased due to the death of algal grazers and fertilization. The development of phytoplankton depressed macrophyte growth which in turn improved conditions for rotifer growth (Hasler and Jones, 1949).
347
between rotifers and environThere are complex ecological interrelations mental factors which were not explained by our measured variables as no significant correlations existed between these variables and rotifer development. We found that neither organic fertilization nor water agitation increased rotifer numbers. The correlation found between water alkalinity and rotifer abundance might be explained by the fact that the genus Brachionus is characteristic of harder water (Edmondson, 1963). The reason that more correlations were found between crustacean abundance and the measured parameters in the control group was most likely due to the fact that crustaceans were controlled by the Dylox treatment in the experimental groups. Considerable variation was observed in rotifer abundance and the time at which peak rotifer numbers occurred in the treated ponds. In order to make this method applicable for fishery management, a set of ponds should be used simultaneously. For example, the set of six ponds used in our experiment made it possible to have rotifer densities higher than 2000 individuals per 1 in at least one pond during the 3-week period (Fig. 1). Cultured fish fry grow rapidly under favorable food conditions and in order to maintain this fast growth they must be provided with zooplankton of the proper size. The organophosphate insecticide method of zooplankton control must be coordinated so that crustacean succession occurs at approximately the time fry are able to feed upon them. Further experiments are needed in order to establish procedures, other than insecticide treatment for improving rotifer management in earthen ponds thus making it a reliable method for larval fish rearing. ACKNOWLEDGMENTS
Funding for this project was provided through a USDA/ARS Cooperative Agreement No. 58-7B30-0-777 and a grant by the U.S. Environmental Protection Agency under grant number R-807970010. The U.S. Environmental Protection Agency does not necessarily endorse any commercial products used in this study and the conclusions represent the views of the authors which do not necessarily represent the opinions, policies or recommendations of the U.S. Environmental Protection Agency.
REFERENCES Canfield, D.E., 1983. Prediction of chlorophyll a concentrations in Florida lakes: the importance of phosphorus and nitrogen. Water Resour. Bull., 2: 255-262. Dabrowski, K., Bardega, R. and Przedwojski, R., in press. Dry diet formulation study with common carp larvae. Aquaculture. Edmondson, W.T., 1963. Rotifera. In: H.B. Ward, G.C. Whipple and W.T. Edmondson (Editors), Freshwater Biology, 3rd edn. Wiley, New York, NY, pp. 420-494. Geiger, J.G., 1983. A review of pond zooplankton production and fertilization for the culture of larval and fingerling striped bass. Aquaculture, 35: 353-369.
348 Hasler, A.D. and Jones, E., 1949. Demonstration of the antagonistic action of large aquatic plants on algae and rotifers. Ecology, 3: 359-364. Hirata, H., 1979. Rotifer culture in Japan. In: Spec. Publ. 4, European Mariculture Society, pp. 361-375. Hurlbert, S.H., Mulla, M.S. and Willson, H.R., 1972. Effects of an organophosphorus insecticide on the phytoplankton, zooplankton, and insect populations of freshwater ponds. Ecol. Monogr., 42: 269-299. Opuszynski, K., 1979. Silver carp, Hypophthalmichthys molitrix (Val.), in carp ponds. II. Rearing of fry. Ekol. Pol., 27: 93-116. Shireman, J.V., Aldridge, F.J. and Rottmann, R.W., 1983. Growth and food habits of hybrid carp Ctenopharyngodon idella X Aristichthys nobilis, from aerated and nonaerated ponds. J. Fish Biol., 23: 595-604. Standard Methods, 1975. Standard Methods for the Examination of Water and Wastewater, (14th Edition). APHA, Washington, DC, 1193 pp. Tamas, G. and Horvath, L., 1976. Growth of cyprinids under optimal zooplankton conditions. Bamidgeh, 28: 50-56. Woynarovich, E. and Horvath, L., 1980. The artificial propagation of warm-water finfishes -a manual for extension. FAO Fish. Tech. Pap. 201,183 pp.
343 in The Netherlands
ENVIRONMENTAL MANIPULATION TO STIMULATE ROTIFERS IN FISH REARING PONDS
KAROL OPUSZYNSKI*, JEROME V. SHIREMAN, FREDERICK and ROGER W. ROTTMANN School of Forest Resources and Conservation, (U.S.A.)
J. ALDRIDGE
University of Florida, Gainesville, FL
*Permanent address: Inland Fisheries Institute, Zabieniec, 05-500 Journal Series No. 5601, Florida Agricultural Experiment
Piaseczno, Poland
Station
(Accepted 25 July 1984)
ABSTRACT Opuszynski, K., Shireman, J.V., Aldridge, F.J. and Rottmann, R.W., 1984. Experimental manipulation to stimulate rotifers in fish rearing ponds. Aquaculture, 42: 343-348. Four treatments were established in eight small ponds not stocked with fish: a control group (untreated and not fertilized) and three groups treated with organophosphate insecticide (Dylox). One group of Dylox treatment ponds was treated with chemical fertilizer, the second group with chemical and organic fertilizer, and the third group with chemical-organic fertilizer and paddlewheel water agitation. Water analysis and zooplankton samples were collected twice weekly. Rotifers increased in numbers in all treated ponds but no correlations were found between 16 hydrochemical and biological parameters and rotifer numbers. The application of rotifer management techniques for rearing larval fish is discussed.
INTRODUCTION
Successful rearing of many commercially important fish species is dependent upon providing the proper quality and quantity of live food to the fish immediately after they commence feeding. Live food is necessary because complete artificial starter feeds have yet to be developed (Dabrowski et al., in press). Rotifers are excellent food for some fish larvae when they commence active feeding (Woynarovich and Horvath, 1980). Currently rotifers are mass cultured under fully controlled conditions in Japan and their culture has contributed to the rapid development of Japanese fish culture (Hirata, 1979). The importance of rotifers for feeding small fish has been recognized in Europe and in the United States (Tamas and Horvath, 1976; Opuszynski, 1979; Geiger, 1983). Fish culturists grow rotifers in earthen ponds which are either stocked with fish larvae or the rotifers are collected from the ponds and fed to fish larvae held in rearing
0044-8486/84/$03.00
o 1984 Elsevier Science Publishers B.V.
344
facilities. Both methods yield satisfactory results provided that rotifers are grown in high numbers over the proper time period. Rotifer culture in earthen ponds is still not routinely done and high production numbers can not always be insured. For this reason we evaluated different management techniques for rotifer production in ponds. MATERIALS
AND METHODS
This experiment was conducted in eight small ponds at the Austin Cary Forest Research Facilities, University of Florida, Gainesville, FL. Pond areas ranged from 0.01 ha to 0.03 ha with an average depth of about 1 m. Ponds were not stocked with fish. Four treatments were established: Group I - control, received no treatment (Ponds 11 and 15); Group II - pesticide and chemical fertilizer treatment (Ponds 10 and 14); Group III - pesticide, chemical and organic fertilizer treatment (Ponds 12 and 17); and Group IV - same as group III with paddlewheel agitation described by Shireman et al. (1983) (Ponds 9 and 13). Dylox (80% active ingredient), an organophosphate pesticide, was applied at a concentration of 1 mg of active ingredient per 1 of pond water. Granular chemical fertilizers, 34% ammonium nitrate and 46% superphosphate were applied at a concentration corresponding to 2 mg N/l and 0.7 mg P,O,/l. Organic fertilizer (chicken manure) was applied at a rate of 2500 kg/ha wet weight (1.6 mg N/l and 1.3 mg PzO,/l). All ponds were limed with 1000 kg/ha of calcium carbonate. Water temperature, dissolved oxygen and Secchi disc measurements were taken daily at about 11 a.m. Total alkalinity, total phosphorus, total nitrogen, ammonia, calcium, magnesium, potassium, sodium, turbidity, pH, chlorophyll a and zooplankton were sampled twice a week. Temperature and oxygen were measured with a YSI-51 meter, turbidity with a Hach Model 2100 turbidimeter. Chlorophyll, total phosphorus and nitrogen were determined according to the methods described by Canfield (1983) and the remaining chemistry tests according to procedures described in Standard Methods (1975). Thirty-liter zooplankton samples consisting of three subsamples were taken from different stations within each pond. Water was strained with a 80-pm mesh plankton net. The experiment ran from 18 April to 16 May 1983. Lime, fertilizers and Dylox were applied on 19 April, 1 day after the first water chemistry and zooplankton samples were collected. Analysis of variance and Duncan’s multiple range test were used to test for significant differences (P
Significant differences were found except for water temperature (23.624.1%) and mean pH values (7.0-7.4). There were no differences in mean
345
oxygen saturation levels between the control group (8.3 mg 0,/l) and experimental groups (7.5-9.6 mg 0,/l). Mean oxygen levels in the paddlewheel ponds (7.5 mg 0,/l), however, were significantly lower than levels in ponds receiving the same treatment without paddlewheel agitation (9.6 mg 0,/l). Mean magnesium (3.1 mg/l), sodium (4.4 mg/l), phosphorus (0.05 mg/l) and chlorophyll a (0.006 mg/l) values were significantly lower in the control group, whereas mean Secchi disc (71 cm) and mean number of crustaceans (83 individuals per 1) were significantly higher. The highest average volumes for magnesium (4.9 mg/l), sodium (5.8 mg/l), and chlorophyll (0.09 mg/l) were noted in the paddlewheel ponds. The greatest turbidity (Secchi disc readings 33 cm) also occurred in paddlewheel ponds. No significant differences were found in total phosphorus concentration (0.2-0.3 mg/l) and crustacean numbers (9-14 individuals per 1) within the Dylox-treated ponds. Rotifer numbers were lowest in the control ponds during the entire experiment (Fig. 1). In all Dylox-treated ponds large numbers of rotifers were observed. However, rotifer dynamics differed within and among treat, ROTATORIA
103~~o~v/~ , CRUSTACEA
102~~~~v./~
2-
61
II
Fig. 1. Rotifer and crustacean dynamics in control ponds (11 and 16) and Dylox treatment ponds (10 and 14 - chemical fertilization; 12 and 17 - chemical and organic fertilization; 9 and 13 - chemical, organic fertilization and paddlewheel agitation).
346
ment groups. The Dylox treatment eliminated most crustaceans in all of the treated ponds from which they did not fully recover. The rotifer population in all the ponds consisted mainly of Brachionus sp.: B. calyciflorus, B. plicatilis, B. havanaensis and B. angular-is. Other rotifers such as Polyarthra sp., Filinia sp., Keratella sp., and Asplanchna sp. were occasionally found in small numbers. Copepods made up the majority of the crustacean zooplankton. The dominant species were Tropocyclops prasinus and Diaptomus sp. Cladocerans were abundant in some samples taken before Dylox treatments; Daphnia laevis was the most numerous species. Other crustaceans such as Alona rectangula, Chydorus sphaericus, Bosmina coregoni, Simocephalus vetulus, and Scapholeberis kingi occurred rarely and contributed little to the composition of the zooplankton community. There were no correlations among rotifer abundance and any of the measured parameters in Dylox-treated groups. Rotifers were positively correlated only with total alkalinity in the control group. Positive correlations existed between crustacean zooplankton abundance and sodium, calcium, chlorophyll and ammonia concentration in the control ponds, whereas crustacean number was positively correlated only with water turbidity in Dylox-treated ponds. DISCUSSION
Changes caused by Dylox treatment and fertilization could not be separated because the Dylox treatment ponds were also fertilized. Rotifer numbers and chlorophyll concentrations increased simultaneously with decreasing crustacean populations in the treatment ponds. Similar changes caused by an insecticide alone were described by Hurlbert et al. (1972). They suggested that phytoplankton population increases following treatment were caused by reduced crustacean grazing. The use of paddlewheels did not cause the same effect as was reported by Shireman et al. (1983) who found significant increases in oxygen saturation and rotifer numbers in their paddlewheel ponds. Mean oxygen levels were lower than in identically treated ponds without paddlewheel aeration. Significantly higher ammonia levels in the paddlewheel ponds might indicate that oxygen decreases were caused by more rapid organic matter decomposition. Shireman et al. (1983) did not use organic fertilizer; the mean ammonia level was 0.26 mg NH3/1 in the paddlewheel ponds. This was five times less than we measured (1.39 mg/l). In summary it can be stated that the Dylox treatment and fertilization caused an increase in rotifers. The increase was in response to the changes which occurred as the Dylox treatment exterminated cladocerans (rotifer competitors) and copepods (competitors and/or predators). Simultaneously, phytoplankton increased due to the death of algal grazers and fertilization. The development of phytoplankton depressed macrophyte growth which in turn improved conditions for rotifer growth (Hasler and Jones, 1949).
347
between rotifers and environThere are complex ecological interrelations mental factors which were not explained by our measured variables as no significant correlations existed between these variables and rotifer development. We found that neither organic fertilization nor water agitation increased rotifer numbers. The correlation found between water alkalinity and rotifer abundance might be explained by the fact that the genus Brachionus is characteristic of harder water (Edmondson, 1963). The reason that more correlations were found between crustacean abundance and the measured parameters in the control group was most likely due to the fact that crustaceans were controlled by the Dylox treatment in the experimental groups. Considerable variation was observed in rotifer abundance and the time at which peak rotifer numbers occurred in the treated ponds. In order to make this method applicable for fishery management, a set of ponds should be used simultaneously. For example, the set of six ponds used in our experiment made it possible to have rotifer densities higher than 2000 individuals per 1 in at least one pond during the 3-week period (Fig. 1). Cultured fish fry grow rapidly under favorable food conditions and in order to maintain this fast growth they must be provided with zooplankton of the proper size. The organophosphate insecticide method of zooplankton control must be coordinated so that crustacean succession occurs at approximately the time fry are able to feed upon them. Further experiments are needed in order to establish procedures, other than insecticide treatment for improving rotifer management in earthen ponds thus making it a reliable method for larval fish rearing. ACKNOWLEDGMENTS
Funding for this project was provided through a USDA/ARS Cooperative Agreement No. 58-7B30-0-777 and a grant by the U.S. Environmental Protection Agency under grant number R-807970010. The U.S. Environmental Protection Agency does not necessarily endorse any commercial products used in this study and the conclusions represent the views of the authors which do not necessarily represent the opinions, policies or recommendations of the U.S. Environmental Protection Agency.
REFERENCES Canfield, D.E., 1983. Prediction of chlorophyll a concentrations in Florida lakes: the importance of phosphorus and nitrogen. Water Resour. Bull., 2: 255-262. Dabrowski, K., Bardega, R. and Przedwojski, R., in press. Dry diet formulation study with common carp larvae. Aquaculture. Edmondson, W.T., 1963. Rotifera. In: H.B. Ward, G.C. Whipple and W.T. Edmondson (Editors), Freshwater Biology, 3rd edn. Wiley, New York, NY, pp. 420-494. Geiger, J.G., 1983. A review of pond zooplankton production and fertilization for the culture of larval and fingerling striped bass. Aquaculture, 35: 353-369.
348 Hasler, A.D. and Jones, E., 1949. Demonstration of the antagonistic action of large aquatic plants on algae and rotifers. Ecology, 3: 359-364. Hirata, H., 1979. Rotifer culture in Japan. In: Spec. Publ. 4, European Mariculture Society, pp. 361-375. Hurlbert, S.H., Mulla, M.S. and Willson, H.R., 1972. Effects of an organophosphorus insecticide on the phytoplankton, zooplankton, and insect populations of freshwater ponds. Ecol. Monogr., 42: 269-299. Opuszynski, K., 1979. Silver carp, Hypophthalmichthys molitrix (Val.), in carp ponds. II. Rearing of fry. Ekol. Pol., 27: 93-116. Shireman, J.V., Aldridge, F.J. and Rottmann, R.W., 1983. Growth and food habits of hybrid carp Ctenopharyngodon idella X Aristichthys nobilis, from aerated and nonaerated ponds. J. Fish Biol., 23: 595-604. Standard Methods, 1975. Standard Methods for the Examination of Water and Wastewater, (14th Edition). APHA, Washington, DC, 1193 pp. Tamas, G. and Horvath, L., 1976. Growth of cyprinids under optimal zooplankton conditions. Bamidgeh, 28: 50-56. Woynarovich, E. and Horvath, L., 1980. The artificial propagation of warm-water finfishes -a manual for extension. FAO Fish. Tech. Pap. 201,183 pp.