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Evaluation of a sulphate lake strain of Artemia as a food for larvae of the grass shrimp, Palaemonetes pugio
Aquaculture, 9 (1976) 343-350 o Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
343
EVALUATION OF A SULPHATE LAKE STRAIN OF ARTEMIA AS A FOOD FOR LARVAE OF THE GRASS SHRIMP, PALAEMONETES PUGIO
ANTHONY J. PROVENZANO Institute
of Oceanography,
Jr and JOSEPH W. GOY
Old Dominion
University,
Norfolk,
Vu. 23508
(U.S.A.)
(Received 25 May 1976; revised 4 August 1976)
ABSTRACT Provenzano Jr, A.J. and Goy, J.W., 1976. Evaluation of a sulphate lake strain of Artemia as a food for larvae of the grass shrimp, Palaemonetes pugio. Aquaculture, 9: 343-350. Artemia nauplii of a Canadian sulphate-tolerant strain from a newly available commercial source were compared with Artemia salina from two other sources for effectiveness in supporting larval development of the grass shrimp, Palaemonetes pugio. Nauplii were fed to isolated zoea larvae reared in artificial sea water of 17”/,, salinity at 25°C. Sulphate-strain Artemia gave survival at least as good as that provided by nauplii from San Francisco, and superior to that provided by nauplii from Great Salt Lake. Source of Artemia used as food had no effect on total duration of larval development nor on mean number of stages. Hatching success of the Canadian cysts was inferior to that of the other strains when 48h were allowed for hatching, and dilute sea water was the hatching medium The medium recommended by the supplier was not superior to diluted sea water, but the longer hatching time recommended may increase total hatch. If improved hatching procedures can be developed, Artemia from this source may be very satisfactory for culture work.
INTRODUCTION
It is common practice to use Artemia nauplii as a basic food for rearing many species, both for research and for commercial use. Use of Artemiu as a standard diet for culturing crustacean decapod larvae dates from the late 1950’s (Broad, 1957 a and b; Costlow and Bookhout, 1959,1968; Provenzano, 1967; Regnault, 1969). The advantages of this diet for crustaceans, as cited by Roberts (1974), are essentially similar to those for rearing other species - it is available in large quantities regardless of season, it is suitable in size for many decapod larvae and it allows complete development to the juvenile stage or beyond with reasonably consistent survival, intermolt duration and morphogenetic sequence. One of the most widely used sources, at least in the United States, is that from San Francisco Bay, California. However, increasing demand and appa-
344
rently limited supply have caused workers to seek alternative sources. Commercially available Artemia from Great Salt Lake, Utah, when used for rearing crustacean larvae, is reported to yield results inferior to those obtained using San Francisco strains (Little, 1969; Reed, 1969; Bookhout and Costlow, 1970; Wickins, 1972). When a new commercial source of Artemia from Lake Chaplin, a sulphate lake in Saskatchewan, Canada, was announced in the World Mariculture Society Newsletter of June 1975, a sample was obtained from the supplier (Pennsylvania Pet Products, Box 191, Spring City, Penn. 19475, U.S.A.) through a local distributor. A preliminary evaluation was made of the ability of these Artemiu to sustain larvae of a decapod shrimp, and effectiveness of these nauplii was compared with effectiveness of those from the two other mentioned sources. The caridean shrimp Pcduemonetes pugio was used in the test. It is closely related to larger, commercially valuable forms such as Macrobrachium, but is easier to handle in limited facilities. Larval developments of estuarine species of Puluemonetes of the eastern United States have been well documented (Broad, 1957 a; Broad and Hubschman, 1962; Hubschman and Broad, 1974). The species has been widely used experimentally (Hubschman, 1963; McFarland and Pickens, 1965; Hanson et al., 1973; Redmann, 1973; De Coursey and Vernberg, 1975) and is important in estuarine food chains (Welsh, 1975). MATERIALS
AND METHODS
Adult Puluemonetes pugio collected in the Lafayette River, Norfolk, Virginia, were induced to spawn in the laboratory by the method of Little (1968) with slight modifications. In preliminary experiments we determined that the optimum temperature for larval development of P. pugio is near 25°C when environmental salinity S varies between 15 and 20°/oo. Therefore, for hatching, the females were separated into individual 20-cm diameter culture bowls containing artificial sea water (Instant Ocean, Aquarium Systems, Inc., Eastlake, Ohio, U.S.A.) at ambient temperature (23-25°C) and 17°/o~ S. On 8 November 1975 three females hatched their eggs. The broods were combined, and individual zoeae were placed into 25 ml of 17°/oo S sea water in compartmented plastic trays. Groups of 24 zoeae from the pooled hatches of these three females were fed Artemia nauplii hatched from San Francisco, Utah, or Canadian sources and 12 zoeae were maintained unfed as a control. All zoeae were placed in a darkened incubator at 25°C receiving light only 1-2 h/day when water was changed and new food provided. To minimize possible effects of daily variation in diet concentration, an attempt was made to provide a standard density of 40 nauplii/ml. Inverted 250-ml bottles supplied with continuous aeration (Sorgeloos, 1973) were used to hatch Artemiu cysts over a 48-h period at room temperatures (approximately 23°C). Sea water of 30°/oo S was used to hatch San Francisco and Utah cysts. A formula for hatching medium to be used with the Canadian
345
cysts was supplied by the distributor. Preliminary investigation showed that hatch rate and hatching consistency were not as good with the formula as with sea water of 12°/oo S. Therefore the latter was used for the Canadian cysts. Each hatch of Artemia was concentrated into 25 ml and a l-ml aliquot of that concentrate was diluted to 10 ml. From this lo-ml sample three l-ml aliquots were obtained and the nauplii counted. The three counts were averaged and then multiplied by 240 to obtain an estimate of the total number of nauplii in the remaining original 24-ml concentrate, which then was adjusted to provide a concentration of 2 000 Artemia nauplii/ml. One half milliliter of this adjusted concentrate when placed in an individual compartment gave 40 f 2 nauplii/ml as the daily initial feeding concentration in 25 ml of water. Daily inspection of each Palaemonetes zoea by microscope indicated that morphological changes occurred only when all or part of an exuvium was also present in the compartment. A record of molting, mortality and sequence of larval stages was kept. The experiment was terminated after all survivors had reached the postlarval stage but before any had molted to the second postlarval stage. RESULTS
The Artemia cysts from San Francisco and Utah gave sufficiently consistent hatches to allow feeding of Palaemonetes zoeae at the desired concentration of 40 Artemia nauplii per ml per day. Trial hatchings of Canadian Artemia cysts prior to the experiment were not consistent, and the concentration fed daily varied at levels below 40 per ml per day depending upon the total hatch available on any given day. On 19 November 1975 an accident killed all Artemia nauplii being hatched and all Palaemonetes larvae received no food during the 13th and 14th days of the experiment. Source of Artemia used as food had no apparent effect on total duration of larval development. None of the starved control larvae lived beyond the 10th day. Metamorphosis in all the fed groups began on the 18th day after hatching. It was completed on the 23rd day for the Canadian-fed and San Francisco-fed larvae and on the 24th day for the group fed Utah Artemia. Diet had an apparent effect on the required number of stages prior to metamorphosis. All groups contained individuals that molted to postlarvae after nine zoeal stages, but almost all Canadian-fed larvae and a few San Franciscofed larvae metamorphosed after eight stages. None of the Utah-fed larvae metamorphosed after eight stages. One individual of the San Francisco Artemiafed group required ten zoeal stages before metamorphosis. Diet also affected survival. Percentage survival of Palaemonetes zoea from hatching through metamorphosis in the four test conditions is shown in Fig.1. A Duncan Multiple Range Test after arcsin transformation (Walpole and Myers, 1972) showed that percentage survivals for groups fed San Francisco and Canadian Artemia (63% and 71% respectively) were not statistically dif-
346
5
IO
15
20
Fig. 1. Survivorship curve for larvae of Palaemonetes pugio fed three different diets, and a starved control. o- larvae fed Canadian Artemia; a- larvae fed San Francisco Artemia; llarvae fed Utah Artemia; A- larvae receiving no food. Initial number of larvae was 12 for the starved group, 24 for each of the others.
ferent from each other but each differed significantly ((w= 0.05) from the 29% survival of larvae fed Utah Artemiu. Diet also had an apparent effect on molting. Larvae fed Canadian Artemiu showed a more regular molting cycle (Fig. 2 top) and larvae fed other strains showed more overlap in later stages (Fig. 2). The significance of this effect is unknown since metamorphosis occurred at similar times in all groups. Although occasionally there was wide variation in range of stage duration in each test group, the overall mean times to each stage for larvae fed the three test diets were not significantly different (a = 0.05). The experiment was terminated before the postlarvae molted, and duration of the first postlarval stage was not determined. DISCUSSION
Experiments reporting effects of diet variation in culture of decapod larvae are usually qualitative and often include Artemia as one of the test diets (e.g. Artemia and various algae, or Artemiu and prepared foods for shrimps: Broad, 1957 b; Regnault, 1969; Choudhury, 1971; Sandifer, 1972). Artemia, algae and various invertebrate larvae were fed to Pugurus zoeae in semi-quantitative studies by Roberts (1974). The majority of papers on decapod larval development, in which Artemiu was used as a food, report concentration of nauplii as being “in excess”. However, few workers have quantified the Artemia provided as food for larvae. Mootz and Epifanio (1974) determined the energy budget for larvae of the stone crab, Menippe mercenariu, which were fed a daily concentration of 5 nauplii/ml throughout development. Welch and Sulkin (1975) tested the effect of diet concentration on mortality and rate of development of zoeae
CANADIAN
SAN
: ,I)
I(
ARTEYIA
FED
FRANCISCO
ARTEYIA
FED
11
I : .
0 2!
2c
-*
.-.
UTAH
ARTEYlA
FED
IC
IC
s
I’6
lb DAYS
AFTER
7.b
2
5
HATCHING
Fig. 2. Sequence and frequency distribution of larval stages of Palaemonetes pugio reared at 25” C and 17”/,, S., using Artemia nauplii from three sources. Top to bottom: larvae fed Canadian, San Francisco and Utah Artemiu respectively. ZN = zoeal stage N, PL = postlarva. The more regular, better synchronized molting in the group fed Canadian Artemia is reflected in the more distinctive polygons for that group.
348
of another xanthid crab, Rhithropanopeus harrisii, using concentrations of Artemiu nauplii at 2, 5, 10, 20 and 40/ml. No differences in mortality were noticed at any of these diet concentrations. Cumulative zoeal development showed a consistently shorter development time as diet concentration increased, but a statistically significant delay in development time occurred only at a concentration of 2 nauplii/ml. Results of both those studies indicated that control of diet concentration may be an important factor in determining rates of development of crustacean decapod larvae as well as in experiments designed to measure the effect of other variables on larval development. The minimum concentration of Artemiu nauplii needed daily for Pulaemonetes pugio to complete development through metamorphosis is not known for any temperature or other specific rearing condition. Reeve (1969), working on larvae of another palaemonid shrimp, Puluemon sermtus, found no difference in survival when individual larvae in l-1 beakers were fed 40 Artemiu nauplii/l (O.O4/ml) and 2 000 nauplii/l (2/ml). Metamorphosis occurred at both feeding levels. In the present experiment, zoeae were given 40 nauplii/ml daily of San Francisco and Utah Artemiu. Since Canadian Artemia cysts could not be hatched with consistency, those nauplii were fed at densities of 5-35/ml daily depending upon the available hatch for that day. The high concentration of 40 nauplii/ml daily was selected originally to avoid any possibility of underfeeding, but subsequent studies (A.J. Provenzano Jr, J.W. Goy and K.B. Schmitz, unpublished data) have shown that 10 nauplii/ml is adequate for P. pugio. We are currently determining the minimum concentration of Artemiu required for complete larval development of this species. Despite the fact that the best survival was among shrimp larvae fed with Canadian nauplii, we do not believe the quantities fed were a factor in any of the results. On the other hand, the quality of the diets may be important. Earlier workers have shown that Artemiu from different sources may have different effects on larvae ih culture. This was demonstrated for fish (J.E. Shelboume, in Wickens, 1972, p. 151) as well as for several species of crustaceans (Little, 1969; Reed, 1969; Reeve, 1969; Bookhout and Costlow, 1970; Wickens, 1972). In particular, Artemiu from Utah have been shown in several of these studies to be inferior for larval culture and our results with P. pugio tend to confirm this. The Canadian sulphate lake strain of Artemiu did not give such unfavorable results. On the contrary, as food for the P. pugio larvae, Artemiu from this source appear to be in almost every respect at least equal in quality to the popular San Francisco brine shrimp. There may be two disadvantages with the cysts from Lake Chaplin - erratic or delayed hatchability, and the size of the supply. Instructions of the supplier indicated that the cysts from this source may require 5-6 days incubation for a complete hatch, whereas cysts from other sources usually produce an almost complete hatch within 48 h. We were using a 48-h hatching cycle, and hatches of Lake Chaplin cysts might have been improved by extending
349
the hatching time. M. Fujinaga (in Costlow, 1969, p. 356) reported 30-60% hatching rate in Artemiu from Saskatchewan, though the source of his supply may not have been Lake Chaplin. He indicated that hatching rates could be improved by treating the cysts with fresh water in advance. J.E. Shelbourne (in Costlow, 1969, p. 356) reported difficulty in getting high hatching rates from Canadian cysts. Hatches of 40% could be obtained using a rock salt and sodium carbonate monohydrate formula recommended by the supplier, but dilute sea water appeared to inhibit egg development. The formula referred to by Shelboume suggests a different source of Artemiu from that tested in the present study. The formula recommended for the Lake Chaplin stock is based largely on sodium and magnesium sulphates, without inclusion of carbonates. Even when we followed the supplier’s hatching instructions, we failed to obtain better yields in 48 h than with several alternative methods. Investigations of requirements for the most effective hatching of Lake Chaplin cysts are needed to minimize this problem. The size of the supply, believed to be only a fraction of that for the San Francisco stock, may be a greater obstacle to wider use. However, the apparent acceptability of this sulphate population in supporting decapod larval development suggests that additional populations from similar unexploited habitats might also be acceptable.
REFERENCES Bookhout, C.G. and Costlow Jr, J.D., 1970. Nutritional effects of Artemia from different locations on larval development of crabs. Helgol. Wiss. Meeresunters., 20: 435-440. Broad, A.C., 1957 a. Larval development of Pulaemonetes pugio Holthuis. Biol. Bull., 13.2 (2): 144-161. Broad, AC., 1957b. The relationship between diet and larval development of Palaemonetes. Biol. Bull., 112(2): 162-170. Broad, A.C. and Hubschman, J.H., 1962. A comparison of larval development of species of eastern U.S. Palaemonetes with special reference to the development of Palaemonetes intermedius Holthuis. Am. Zool., 2(3):172. Choudhury, P.C., 1971. Responses of larval Macrobrachium carcinus (L.) to variations in salinity and diet (Decapoda, Palaemonidae). Crustaceana, 20 : 113-l 20. Costlow, J.D. (Editor), 1969. Proceedings of the Fifth Interdisciplinary Conference on Marine Biology. Marine Biology, vol. V. Gordon C Breach, Science Publishers, New York, N.Y., 606 pp. Costlow, J.D. and Bookhout, C.G., 1959. The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol. Bull. 116(3): 373-376. Costlow Jr, J.D. and Bookhout, C.G., 1968. Larval development of the crab, Leptodius agassizii A. Milne Edwards in the laboratory (Brachyura, Xanthidae). In: Studies on Decapod Larval Development. Crustaceana, Suppl. 2: 259-270. De Coursey, P.J. and Vernberg, W.B., 1975. The effect of dredging in a polluted estuary on the physiology of larval zooplankton. Water Res., 9: 149-154. Hanson, D.J., Schimmel, SC. and Keltner Jr, J.M., 1973. Avoidance of pesticides by grass shrimp (Palaemonetes pugio). Bull. Environ. Contam. .Toxicol., 9: 129-133. Hubschman, J.H., 1963. Development and function of neurosecretory sites in the eyestalks of larval Palaemonetes. Biol. Bull., 125(l): 96-113.
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Hubschman, J.H. and Broad, A.C., 1974. The larval development of Pulaernonetes infermedius Holthuis, 1949 (Decapoda, Palaemonidae) reared in the laboratory. Crustaceana, 26(l): 89-103. Little, G., 1968. Induced winter breeding and larval development in the shrimp Pulaemonetes pugio Holthuis (Caridea; Palamonidae). In: Studies on Decapod Larval Development. Crustaceana, Suppl. 2: 19-26. Little, G., 1969. The larval development of the shrimp, Palaemon macrodactylus Rathbun, reared in the laboratory, and the effect of eyestalk extirpation on development.. Crustaceana, 17(l): 69-87. MacFarland, W.N. and Pickens, P.E., 1965. The effects of season, temperature and salinity on standard and active oxygen consumption of the grass shrimp, Palaemonetes vulgaris (Say). Can. J. Zool., 43: 571-585. Mootz, C.A. and Epifanio, C.E., 1974. An energy budget for Menippe mercenaria larvae fed Artemiu nauplii. Biol. Bull., 146(l): 44-55. Provenzano Jr, A.J., 1967. Recent advances in the laboratory culture of Decapod larvae. In: N.K. Pillai (Convener), Proceedings of a Symposium on Crustacea, Erankulam, 1965. Marine Biological Association India, Symposium Series. Part. 2, Bangalore Press, Bangalore, pp. 940-945. Redmann, G., 1973. Studies on the toxicity of mirex to the estuarine grass shrimp, Palaemonetes pugio. Gulf Res. Rep., 4: 272-277. Reed, P.H., 1969. Studies of diet and diet concentration effects on Dungeness crab zoeae. Proc. Nat. Shellfish Assoc., 59: 12. Reeve, M.R., 1969. Growth, metamorphosis and energy conversion in the larvae of the prawn, Palaemon serratus. J. Mar. Biol. Assoc. U.K., 49: 77-96. Regnault, M., 1969. Etude exphrimentale de la nutrition d’Hippolyte inermis Leach (DBcapode, Natantia) au tours de son ddveloppement larvaire, au laboratoire. Int. Rev. Gesamten Hydrobiol., 54(5): 749-764. Roberts Jr, M.H., 1974. Larval development of Pagurus Zongicarpus Say reared in the laboratory. V. Effect of diet on survival and molting. Biol. Bull., 146(l): 67-77. Sandifer, P.A., 1972. Effects of diet on larval development of Thor floridanus (Decapoda, Caridea) in the laboratory. Va. J. Sci., 23: 5-8. Sorgeloos, P., 1973. High density culturing of the brine shrimp, Artemia salina L. Aquaculture, 1: 385-391. Walpole, R.E. and Myers, R.H., 1972. Probability and Statistics for Engineers and Scientists. MacMillan, New York, N.Y., 506 pp. Welch, J. and Sulkin, S.D., 1975. Effect of diet concentration on mortality and rate of development of zoeae of the Xanthid crab, Rhithropanopeus harrisii (Gould). J. Elisha Mitchell Sci. Sot., 90(2): 69-72. Welsh, B.L., 1975. The role of the grass shrimp, Palaemonetes pugio, in a tidal marsh ecosystem. Ecology, 56(3): 513-530. Wickins, J.F., 1972. The food value of brine shrimp, Artemia sulina L., to larvae of the prawn, Palaemon serratus Pennant. J. Exp. Mar. Biol. Ecol., 10: 151-170.
343
EVALUATION OF A SULPHATE LAKE STRAIN OF ARTEMIA AS A FOOD FOR LARVAE OF THE GRASS SHRIMP, PALAEMONETES PUGIO
ANTHONY J. PROVENZANO Institute
of Oceanography,
Jr and JOSEPH W. GOY
Old Dominion
University,
Norfolk,
Vu. 23508
(U.S.A.)
(Received 25 May 1976; revised 4 August 1976)
ABSTRACT Provenzano Jr, A.J. and Goy, J.W., 1976. Evaluation of a sulphate lake strain of Artemia as a food for larvae of the grass shrimp, Palaemonetes pugio. Aquaculture, 9: 343-350. Artemia nauplii of a Canadian sulphate-tolerant strain from a newly available commercial source were compared with Artemia salina from two other sources for effectiveness in supporting larval development of the grass shrimp, Palaemonetes pugio. Nauplii were fed to isolated zoea larvae reared in artificial sea water of 17”/,, salinity at 25°C. Sulphate-strain Artemia gave survival at least as good as that provided by nauplii from San Francisco, and superior to that provided by nauplii from Great Salt Lake. Source of Artemia used as food had no effect on total duration of larval development nor on mean number of stages. Hatching success of the Canadian cysts was inferior to that of the other strains when 48h were allowed for hatching, and dilute sea water was the hatching medium The medium recommended by the supplier was not superior to diluted sea water, but the longer hatching time recommended may increase total hatch. If improved hatching procedures can be developed, Artemia from this source may be very satisfactory for culture work.
INTRODUCTION
It is common practice to use Artemia nauplii as a basic food for rearing many species, both for research and for commercial use. Use of Artemiu as a standard diet for culturing crustacean decapod larvae dates from the late 1950’s (Broad, 1957 a and b; Costlow and Bookhout, 1959,1968; Provenzano, 1967; Regnault, 1969). The advantages of this diet for crustaceans, as cited by Roberts (1974), are essentially similar to those for rearing other species - it is available in large quantities regardless of season, it is suitable in size for many decapod larvae and it allows complete development to the juvenile stage or beyond with reasonably consistent survival, intermolt duration and morphogenetic sequence. One of the most widely used sources, at least in the United States, is that from San Francisco Bay, California. However, increasing demand and appa-
344
rently limited supply have caused workers to seek alternative sources. Commercially available Artemia from Great Salt Lake, Utah, when used for rearing crustacean larvae, is reported to yield results inferior to those obtained using San Francisco strains (Little, 1969; Reed, 1969; Bookhout and Costlow, 1970; Wickins, 1972). When a new commercial source of Artemia from Lake Chaplin, a sulphate lake in Saskatchewan, Canada, was announced in the World Mariculture Society Newsletter of June 1975, a sample was obtained from the supplier (Pennsylvania Pet Products, Box 191, Spring City, Penn. 19475, U.S.A.) through a local distributor. A preliminary evaluation was made of the ability of these Artemiu to sustain larvae of a decapod shrimp, and effectiveness of these nauplii was compared with effectiveness of those from the two other mentioned sources. The caridean shrimp Pcduemonetes pugio was used in the test. It is closely related to larger, commercially valuable forms such as Macrobrachium, but is easier to handle in limited facilities. Larval developments of estuarine species of Puluemonetes of the eastern United States have been well documented (Broad, 1957 a; Broad and Hubschman, 1962; Hubschman and Broad, 1974). The species has been widely used experimentally (Hubschman, 1963; McFarland and Pickens, 1965; Hanson et al., 1973; Redmann, 1973; De Coursey and Vernberg, 1975) and is important in estuarine food chains (Welsh, 1975). MATERIALS
AND METHODS
Adult Puluemonetes pugio collected in the Lafayette River, Norfolk, Virginia, were induced to spawn in the laboratory by the method of Little (1968) with slight modifications. In preliminary experiments we determined that the optimum temperature for larval development of P. pugio is near 25°C when environmental salinity S varies between 15 and 20°/oo. Therefore, for hatching, the females were separated into individual 20-cm diameter culture bowls containing artificial sea water (Instant Ocean, Aquarium Systems, Inc., Eastlake, Ohio, U.S.A.) at ambient temperature (23-25°C) and 17°/o~ S. On 8 November 1975 three females hatched their eggs. The broods were combined, and individual zoeae were placed into 25 ml of 17°/oo S sea water in compartmented plastic trays. Groups of 24 zoeae from the pooled hatches of these three females were fed Artemia nauplii hatched from San Francisco, Utah, or Canadian sources and 12 zoeae were maintained unfed as a control. All zoeae were placed in a darkened incubator at 25°C receiving light only 1-2 h/day when water was changed and new food provided. To minimize possible effects of daily variation in diet concentration, an attempt was made to provide a standard density of 40 nauplii/ml. Inverted 250-ml bottles supplied with continuous aeration (Sorgeloos, 1973) were used to hatch Artemiu cysts over a 48-h period at room temperatures (approximately 23°C). Sea water of 30°/oo S was used to hatch San Francisco and Utah cysts. A formula for hatching medium to be used with the Canadian
345
cysts was supplied by the distributor. Preliminary investigation showed that hatch rate and hatching consistency were not as good with the formula as with sea water of 12°/oo S. Therefore the latter was used for the Canadian cysts. Each hatch of Artemia was concentrated into 25 ml and a l-ml aliquot of that concentrate was diluted to 10 ml. From this lo-ml sample three l-ml aliquots were obtained and the nauplii counted. The three counts were averaged and then multiplied by 240 to obtain an estimate of the total number of nauplii in the remaining original 24-ml concentrate, which then was adjusted to provide a concentration of 2 000 Artemia nauplii/ml. One half milliliter of this adjusted concentrate when placed in an individual compartment gave 40 f 2 nauplii/ml as the daily initial feeding concentration in 25 ml of water. Daily inspection of each Palaemonetes zoea by microscope indicated that morphological changes occurred only when all or part of an exuvium was also present in the compartment. A record of molting, mortality and sequence of larval stages was kept. The experiment was terminated after all survivors had reached the postlarval stage but before any had molted to the second postlarval stage. RESULTS
The Artemia cysts from San Francisco and Utah gave sufficiently consistent hatches to allow feeding of Palaemonetes zoeae at the desired concentration of 40 Artemia nauplii per ml per day. Trial hatchings of Canadian Artemia cysts prior to the experiment were not consistent, and the concentration fed daily varied at levels below 40 per ml per day depending upon the total hatch available on any given day. On 19 November 1975 an accident killed all Artemia nauplii being hatched and all Palaemonetes larvae received no food during the 13th and 14th days of the experiment. Source of Artemia used as food had no apparent effect on total duration of larval development. None of the starved control larvae lived beyond the 10th day. Metamorphosis in all the fed groups began on the 18th day after hatching. It was completed on the 23rd day for the Canadian-fed and San Francisco-fed larvae and on the 24th day for the group fed Utah Artemia. Diet had an apparent effect on the required number of stages prior to metamorphosis. All groups contained individuals that molted to postlarvae after nine zoeal stages, but almost all Canadian-fed larvae and a few San Franciscofed larvae metamorphosed after eight stages. None of the Utah-fed larvae metamorphosed after eight stages. One individual of the San Francisco Artemiafed group required ten zoeal stages before metamorphosis. Diet also affected survival. Percentage survival of Palaemonetes zoea from hatching through metamorphosis in the four test conditions is shown in Fig.1. A Duncan Multiple Range Test after arcsin transformation (Walpole and Myers, 1972) showed that percentage survivals for groups fed San Francisco and Canadian Artemia (63% and 71% respectively) were not statistically dif-
346
5
IO
15
20
Fig. 1. Survivorship curve for larvae of Palaemonetes pugio fed three different diets, and a starved control. o- larvae fed Canadian Artemia; a- larvae fed San Francisco Artemia; llarvae fed Utah Artemia; A- larvae receiving no food. Initial number of larvae was 12 for the starved group, 24 for each of the others.
ferent from each other but each differed significantly ((w= 0.05) from the 29% survival of larvae fed Utah Artemiu. Diet also had an apparent effect on molting. Larvae fed Canadian Artemiu showed a more regular molting cycle (Fig. 2 top) and larvae fed other strains showed more overlap in later stages (Fig. 2). The significance of this effect is unknown since metamorphosis occurred at similar times in all groups. Although occasionally there was wide variation in range of stage duration in each test group, the overall mean times to each stage for larvae fed the three test diets were not significantly different (a = 0.05). The experiment was terminated before the postlarvae molted, and duration of the first postlarval stage was not determined. DISCUSSION
Experiments reporting effects of diet variation in culture of decapod larvae are usually qualitative and often include Artemia as one of the test diets (e.g. Artemia and various algae, or Artemiu and prepared foods for shrimps: Broad, 1957 b; Regnault, 1969; Choudhury, 1971; Sandifer, 1972). Artemia, algae and various invertebrate larvae were fed to Pugurus zoeae in semi-quantitative studies by Roberts (1974). The majority of papers on decapod larval development, in which Artemiu was used as a food, report concentration of nauplii as being “in excess”. However, few workers have quantified the Artemia provided as food for larvae. Mootz and Epifanio (1974) determined the energy budget for larvae of the stone crab, Menippe mercenariu, which were fed a daily concentration of 5 nauplii/ml throughout development. Welch and Sulkin (1975) tested the effect of diet concentration on mortality and rate of development of zoeae
CANADIAN
SAN
: ,I)
I(
ARTEYIA
FED
FRANCISCO
ARTEYIA
FED
11
I : .
0 2!
2c
-*
.-.
UTAH
ARTEYlA
FED
IC
IC
s
I’6
lb DAYS
AFTER
7.b
2
5
HATCHING
Fig. 2. Sequence and frequency distribution of larval stages of Palaemonetes pugio reared at 25” C and 17”/,, S., using Artemia nauplii from three sources. Top to bottom: larvae fed Canadian, San Francisco and Utah Artemiu respectively. ZN = zoeal stage N, PL = postlarva. The more regular, better synchronized molting in the group fed Canadian Artemia is reflected in the more distinctive polygons for that group.
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of another xanthid crab, Rhithropanopeus harrisii, using concentrations of Artemiu nauplii at 2, 5, 10, 20 and 40/ml. No differences in mortality were noticed at any of these diet concentrations. Cumulative zoeal development showed a consistently shorter development time as diet concentration increased, but a statistically significant delay in development time occurred only at a concentration of 2 nauplii/ml. Results of both those studies indicated that control of diet concentration may be an important factor in determining rates of development of crustacean decapod larvae as well as in experiments designed to measure the effect of other variables on larval development. The minimum concentration of Artemiu nauplii needed daily for Pulaemonetes pugio to complete development through metamorphosis is not known for any temperature or other specific rearing condition. Reeve (1969), working on larvae of another palaemonid shrimp, Puluemon sermtus, found no difference in survival when individual larvae in l-1 beakers were fed 40 Artemiu nauplii/l (O.O4/ml) and 2 000 nauplii/l (2/ml). Metamorphosis occurred at both feeding levels. In the present experiment, zoeae were given 40 nauplii/ml daily of San Francisco and Utah Artemiu. Since Canadian Artemia cysts could not be hatched with consistency, those nauplii were fed at densities of 5-35/ml daily depending upon the available hatch for that day. The high concentration of 40 nauplii/ml daily was selected originally to avoid any possibility of underfeeding, but subsequent studies (A.J. Provenzano Jr, J.W. Goy and K.B. Schmitz, unpublished data) have shown that 10 nauplii/ml is adequate for P. pugio. We are currently determining the minimum concentration of Artemiu required for complete larval development of this species. Despite the fact that the best survival was among shrimp larvae fed with Canadian nauplii, we do not believe the quantities fed were a factor in any of the results. On the other hand, the quality of the diets may be important. Earlier workers have shown that Artemiu from different sources may have different effects on larvae ih culture. This was demonstrated for fish (J.E. Shelboume, in Wickens, 1972, p. 151) as well as for several species of crustaceans (Little, 1969; Reed, 1969; Reeve, 1969; Bookhout and Costlow, 1970; Wickens, 1972). In particular, Artemiu from Utah have been shown in several of these studies to be inferior for larval culture and our results with P. pugio tend to confirm this. The Canadian sulphate lake strain of Artemiu did not give such unfavorable results. On the contrary, as food for the P. pugio larvae, Artemiu from this source appear to be in almost every respect at least equal in quality to the popular San Francisco brine shrimp. There may be two disadvantages with the cysts from Lake Chaplin - erratic or delayed hatchability, and the size of the supply. Instructions of the supplier indicated that the cysts from this source may require 5-6 days incubation for a complete hatch, whereas cysts from other sources usually produce an almost complete hatch within 48 h. We were using a 48-h hatching cycle, and hatches of Lake Chaplin cysts might have been improved by extending
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the hatching time. M. Fujinaga (in Costlow, 1969, p. 356) reported 30-60% hatching rate in Artemiu from Saskatchewan, though the source of his supply may not have been Lake Chaplin. He indicated that hatching rates could be improved by treating the cysts with fresh water in advance. J.E. Shelbourne (in Costlow, 1969, p. 356) reported difficulty in getting high hatching rates from Canadian cysts. Hatches of 40% could be obtained using a rock salt and sodium carbonate monohydrate formula recommended by the supplier, but dilute sea water appeared to inhibit egg development. The formula referred to by Shelboume suggests a different source of Artemiu from that tested in the present study. The formula recommended for the Lake Chaplin stock is based largely on sodium and magnesium sulphates, without inclusion of carbonates. Even when we followed the supplier’s hatching instructions, we failed to obtain better yields in 48 h than with several alternative methods. Investigations of requirements for the most effective hatching of Lake Chaplin cysts are needed to minimize this problem. The size of the supply, believed to be only a fraction of that for the San Francisco stock, may be a greater obstacle to wider use. However, the apparent acceptability of this sulphate population in supporting decapod larval development suggests that additional populations from similar unexploited habitats might also be acceptable.
REFERENCES Bookhout, C.G. and Costlow Jr, J.D., 1970. Nutritional effects of Artemia from different locations on larval development of crabs. Helgol. Wiss. Meeresunters., 20: 435-440. Broad, A.C., 1957 a. Larval development of Pulaemonetes pugio Holthuis. Biol. Bull., 13.2 (2): 144-161. Broad, AC., 1957b. The relationship between diet and larval development of Palaemonetes. Biol. Bull., 112(2): 162-170. Broad, A.C. and Hubschman, J.H., 1962. A comparison of larval development of species of eastern U.S. Palaemonetes with special reference to the development of Palaemonetes intermedius Holthuis. Am. Zool., 2(3):172. Choudhury, P.C., 1971. Responses of larval Macrobrachium carcinus (L.) to variations in salinity and diet (Decapoda, Palaemonidae). Crustaceana, 20 : 113-l 20. Costlow, J.D. (Editor), 1969. Proceedings of the Fifth Interdisciplinary Conference on Marine Biology. Marine Biology, vol. V. Gordon C Breach, Science Publishers, New York, N.Y., 606 pp. Costlow, J.D. and Bookhout, C.G., 1959. The larval development of Callinectes sapidus Rathbun reared in the laboratory. Biol. Bull. 116(3): 373-376. Costlow Jr, J.D. and Bookhout, C.G., 1968. Larval development of the crab, Leptodius agassizii A. Milne Edwards in the laboratory (Brachyura, Xanthidae). In: Studies on Decapod Larval Development. Crustaceana, Suppl. 2: 259-270. De Coursey, P.J. and Vernberg, W.B., 1975. The effect of dredging in a polluted estuary on the physiology of larval zooplankton. Water Res., 9: 149-154. Hanson, D.J., Schimmel, SC. and Keltner Jr, J.M., 1973. Avoidance of pesticides by grass shrimp (Palaemonetes pugio). Bull. Environ. Contam. .Toxicol., 9: 129-133. Hubschman, J.H., 1963. Development and function of neurosecretory sites in the eyestalks of larval Palaemonetes. Biol. Bull., 125(l): 96-113.
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Hubschman, J.H. and Broad, A.C., 1974. The larval development of Pulaernonetes infermedius Holthuis, 1949 (Decapoda, Palaemonidae) reared in the laboratory. Crustaceana, 26(l): 89-103. Little, G., 1968. Induced winter breeding and larval development in the shrimp Pulaemonetes pugio Holthuis (Caridea; Palamonidae). In: Studies on Decapod Larval Development. Crustaceana, Suppl. 2: 19-26. Little, G., 1969. The larval development of the shrimp, Palaemon macrodactylus Rathbun, reared in the laboratory, and the effect of eyestalk extirpation on development.. Crustaceana, 17(l): 69-87. MacFarland, W.N. and Pickens, P.E., 1965. The effects of season, temperature and salinity on standard and active oxygen consumption of the grass shrimp, Palaemonetes vulgaris (Say). Can. J. Zool., 43: 571-585. Mootz, C.A. and Epifanio, C.E., 1974. An energy budget for Menippe mercenaria larvae fed Artemiu nauplii. Biol. Bull., 146(l): 44-55. Provenzano Jr, A.J., 1967. Recent advances in the laboratory culture of Decapod larvae. In: N.K. Pillai (Convener), Proceedings of a Symposium on Crustacea, Erankulam, 1965. Marine Biological Association India, Symposium Series. Part. 2, Bangalore Press, Bangalore, pp. 940-945. Redmann, G., 1973. Studies on the toxicity of mirex to the estuarine grass shrimp, Palaemonetes pugio. Gulf Res. Rep., 4: 272-277. Reed, P.H., 1969. Studies of diet and diet concentration effects on Dungeness crab zoeae. Proc. Nat. Shellfish Assoc., 59: 12. Reeve, M.R., 1969. Growth, metamorphosis and energy conversion in the larvae of the prawn, Palaemon serratus. J. Mar. Biol. Assoc. U.K., 49: 77-96. Regnault, M., 1969. Etude exphrimentale de la nutrition d’Hippolyte inermis Leach (DBcapode, Natantia) au tours de son ddveloppement larvaire, au laboratoire. Int. Rev. Gesamten Hydrobiol., 54(5): 749-764. Roberts Jr, M.H., 1974. Larval development of Pagurus Zongicarpus Say reared in the laboratory. V. Effect of diet on survival and molting. Biol. Bull., 146(l): 67-77. Sandifer, P.A., 1972. Effects of diet on larval development of Thor floridanus (Decapoda, Caridea) in the laboratory. Va. J. Sci., 23: 5-8. Sorgeloos, P., 1973. High density culturing of the brine shrimp, Artemia salina L. Aquaculture, 1: 385-391. Walpole, R.E. and Myers, R.H., 1972. Probability and Statistics for Engineers and Scientists. MacMillan, New York, N.Y., 506 pp. Welch, J. and Sulkin, S.D., 1975. Effect of diet concentration on mortality and rate of development of zoeae of the Xanthid crab, Rhithropanopeus harrisii (Gould). J. Elisha Mitchell Sci. Sot., 90(2): 69-72. Welsh, B.L., 1975. The role of the grass shrimp, Palaemonetes pugio, in a tidal marsh ecosystem. Ecology, 56(3): 513-530. Wickins, J.F., 1972. The food value of brine shrimp, Artemia sulina L., to larvae of the prawn, Palaemon serratus Pennant. J. Exp. Mar. Biol. Ecol., 10: 151-170.