J. Natn. Sci. Coun. Sri Lanka 1995 23(4): 161-169 EFFECTS OF TEMPERATURE AND *SALINITY ON SURWAL, GROWTH AND FECUNDITY OF THE BRINE SHRIMP ARTEMIA PARTHENOGENETICA FROM SRI LANKA M.M. KURUPPU1 and S.U.K. EKARATNE2 * National Aquatic Resources Agency, Crow Island, Colombo 15. Department of Zoology, University of Colombo, Colombo 3. (Received: 18 January 1995; accepted: 03 November 1995) Abstract: Survival, growth and reproductive mode were studied in Dunaliella- fed Sri Lankan Artemia parthenogenetica raised from hatching to reproductive maturity at different salinity and temperature combinations. Survival was high in 100 and 120ppt, both a t 25" (94 and 7296, respectively) and 29OC (82 and 78%, respectively). Poor sunrival was recorded in higher salinities a t 29OC and all salinities a t 35°C. At 21°C survival decreased rapidly to near lo%, other than in 65ppt where survival was 48%.-Growth rate a t the tested salinities varied from 0.l lmm day-' to 0.16mmday1 at 2 l0e and from 0.23mm day1 to 0.34mm day1 a t 25°C. Similar growtli(0.39mm day-') occurred in 100 and 120ppt a t 29". No animals reached adult age a t 21°C whereas a t 25" and 2g°C, broodpouch development commenced by day 14 in 65,80 i d 100ppt. Reproduction occurred only a t 29°C. Ovoviviparity occurred only in the S5-and POOppt treatments on day 15 (at 68 and 56 nauplii day ', respectively). Subsequent gradual increase in salinity (4ppt day-') in the 35 and lOOppt treatments made the ovoviviparous groups to switch tooviparity a t 132ppt (at37cystsadult ')and 136ppt(at38cysts adult1), respectively. Animals reared in l2Oppt also reproduced, but without switching, showing oviparity (at 36 cysts adult-') with gradual salinity increase to 140ppt. Salinity-temperature combinations ranging from 100 to l2Oppt and 25" to 30°C are, therefore, best for survival, growth and reproduction of Sri Lankan Artemia. Key words: Artemia, cysts, Dunaliella, growth, nauplii, reproduction, salinity, survival, temperature. INTRODUCTION The brine shrimp Artemia is widely used as a larval feed'in fish and shellfish hatcheries. Natural populations of Artemia inhabit geographically isolated biotopes having specific biotic and abiotic conditions.' In Sri Lanka, a partheno- genetic strain ofArtemia inhabits solar salterns in Hambantota and Pu t t a l a~n .~?~ The abiotic cpditions ofArtemia.biotopes show substantial differences in water t empera tu reh i ~a l in i ty .~ Such differences and the commercial importance of Artemia have prompted studies into many of its known species and strains with respect to their temperature andfor salinity effects on survival,' growth and m ~ r t a l i t y ~ - ~ and maturation and fec~ndi ty .~ Results from these studies have illustrated the variation in temperature and salinity responses betweenArtemia strains. These findings have served to highlight the importance of selecting optimal ranges for known strains ofArtemia for specific aquacultural applications.' Salinity and temperature effkts on the life cycle of Sri Lankan Artemia parthenogenetica are not documented. The promotion of a wider utilization and export of local Artemia parthenogenetica requires such data that are necessary 162 M.M. Kuruppu & S. U.K. Ekaratne to determine optimum canditions for inoculatirig Artehiacttlture ponds. Field culture requires the introduction of the nauplius stage individuals directly in%o culture ponds.g Their subsequent survival, growth and reproduction under prevailing salinity and temperature conditions determine the size of the final cyst producing population. This paper reports on salinity and temperature effects on life cycle characteristics of the Sri Lankan Artemia parthenogenetica. METHODS AND MATERIALS Artemiaparthenogenetica cysts produced in Mahalewaya saltern, Hamban-tota, were used for studying sbrvival, growth and fecundity a t different salihities and temperature-s. Artemia cysts were hatched %n inea wate'i. of 35ppt salinity a t a room temperature of 29OC under continuous illumination'and'aeration. Ratcged instar I nauplii were introduced at a density of 50.nauplii.250 in1 into glass conical flasks containing salt solutiohs of 35, 65, 80, 100 and 12Oppt. The solutions a t each of the salinitids were maintained ak tempe~ature of21,25 and 2g°C with continuous aeration. Additionally, 140 and 180ppt salinities were also used a t 29% and at 35OC. The highei- salihities weie used only a t the higher temperatures because, under field conditions inlrlrtemia ponds,-thb Mgh salinity levels occur only under increased evaporation rates associated with high1 temperatures. All experiments were conducted in triflcak. The s3llutions of different saliliities were prepared by -dissolving commercial dalt in sea water. Experiments a t 21°C and 25OC temperatures were c a n i d out; in at&nperature controlled room in thermostatically controlled water baths. Experiments a t 35OC were carried out in a constant temperature water bath (Kottermann). The feeding scEedulel Was as fol1ows:A~temia nauplii were fed daily wit'-then alga Dunaliella sp. commencing a t a drensity of 6.0 x 1Og'&id 8.4 f lo6 cells per day a t 21°C and 25OC, respectively, and a t 10.8 x lo6 cells per day a t 2g°C and 35OC, doubling the alggl densities every two days. Algal cultures were counted using a haemocytometer and tKe required concentrations for feeding were prepared after centrifugation. Faecal matter was siphoned out and 25% of the medium was renewed eve* 2d. Su'rviird was cheeked every 3d up to ldd. Body lengths were determined every 3 d f6r a random sampl'e of 20 ariimals per flask. For body length-measurements, each- animarwas transferred on to a cavit9 slide and the water was gently drawn out using a paper towel. Tfie- total lengbh was measured from the anterior margin afthe head in fronkuf the ocellus to the base of the caudal furca5 using a binocular microscope fitted with a calibrated eye- piece micrometer. ,.. > .. Ten adult animals were plated individdally in ~1~sgi ; ih l s .d 50'ml.capacity a t 2g0C in the salinities of 35,100 and l2dppt wMch were3thesal+?nities in which animals attained adult stage and feeding was coh€infted. The sa1iaity was gradually increased a t 4ppt day-Iby addition of a eoncentrated saltS6lutioa. he vials were examined daily for the presence of nauplii or cysts and counts were made of any nauplii or cysts that were produced. Physzology of the Brine Shrimp Survival: Percentage survival at different temperatures and salinities is illustrated in Figure 1 a-d. A rapid reduction in survival was seen a t 21°C in all salinity levels except in 65ppt in which s u ~ v a l had stabilized a t 48% by day 14. At 25OC survival was high in lOOppt and l2Oppt salinities. (a) 21°C 0 2 4 6 8 1 0 1 2 1 4 Days 0 2 4 6 8 1 0 1 2 1 4 Days t 3 5 p p t + 65ppt 0 - 2 4 6 - 8 10 12 14 Days + 120 p p t +-14Oppi 180 PPt Figure 1 a-d: Percentage survival in Artemia parthenogenetica at varying temperatures and salinities. At room temperature of 2g°C, 100% mortality was observed by day 5 in 140ppt and 180ppt salinities. Until day3 there was 100% survival in the salinity levels ranging from 35ppt to l2Oppt. Survival decreased rapidly from 100% a t day 3 to 48% at day 5 at 35ppt salinity. There was 68% and 52% survival a t salinities of 65ppt and 80ppt a t day 14. The highest survival (82%) a t day 14 was attained in lOOppt salinity followed by 78% survival in l2Oppt salinity. The lowest survival (37%) at room temperature was in 35ppt salinity at ddy 14. 164 M.M. Kuruppu & S. U.K. Ekaratne At 35OC there was a rapid decrease in survival within two days of the experiment resulting in complete mortality by day 5 a t all salinity levels except a t the low salinity levels of 35 and 65ppt. Even a t these 35 and 65ppt salinities, 100% mortality occurred by day 8. Growth: The mean length of instar I nauplius of Sri Lankan Artemia was 475.4p.m. The mean lengths ofArtemia a t different temperatures and salinities are illustrated in Figure 2 a-c. Length measurements were discohtinued after 25 days or when there was more than 75% mortality. 5 . (a) 21°C 4 ' 3 . 0 0 5 10 15 20 25 Days,. Days 1 5 rb 1; Ib 2s Days Figure 2 a-c: Variationin meanbody length (mm) inArtemiaparthenogenetica at varying temperatures and salidties. Fecundity: Reproductive characteristics ofArtemia are summarized in Table 1. Artemia did not attain reproductive maturity a t any of the tested salinities at 21°C. A slight appearance of broodpouches was observed at 65,80 and lOOppt salinities a t 25OC. They grew only to pre-adult stage a t 25OC. At 2g°C in both 100 and 120 ppt salinity, Artemia attained reproductive maturity in 14 to 15 days within a length range of 6.9 to 7.0 mm. Physiology of the Brine Shrimp 165 Table 1: Reproduction and fecundity ofArtemiaparthenogenetica in laboratory experiments at different temperature and salinity combinations. Experimental conditions Reproduction and Fecundity T(OC) S(ppt) Age a t Naupliar production Cyst production maturity noslparent Age Salinity noslparent Age Salinity (days) (days) (ppt) (days) (ppt) n.a. = not attained T = temperature in°C d = days , n.p. = not produced S = salinity in ppt At 100 ppt salinity at 29 OC adults with broodpouches were observed.on day 14, and on day 15 five adults released nauplii a t 56 numbers adult-l (Table 1). With gradual increase in salinity, nauplii were released at 62 numbers adult1 a t 108ppt after an interval of 4 days from the initial release of nauplii. There was no release of nauplii by Artemia from 108 to 136ppt salinity. Two animals released cysts at 38 numbers adult-l and 36 numbers adult" a t 136ppt on day 25 and 140ppt on day 26, respectively. There was 100% mortality in Artemia .two days after the salinity was increased to 140ppt. In the treatments maintained at 2g°C and l2Oppt, adults with broodpouches were observed on day 15. As salinity was gradually increased a t a rate of 4ppt per day cysts were released at 140ppt at 36 cysts adult-l initially a t day 24, and 40 cysts adult-I 4.days thereafter a t the same salinity. Ovoviviparity was not observed in these adults. Artemia initially reproduced ovoviviparously in 35ppt a t 68 nauplii per female and as salinity was increased upto lOOppt, ovoviviparity re-occurred a t M.M. Kuruppu & S. U.K. Ekaratne 3 to 4 day intervals. Oviparity occurred at 132ppt and continued upto 148ppt salinity after which there was 100% mortality. Artemia did not reproduce from 100 ppt to 132ppt salinity. The highest number of nauplii (160lfemale) were produced a t 60ppt followed by 135 naupliilfemale a t 40ppt salinity. With regard to cyst production, fecundity ranged from 37 to 42 cystslfemale. DISCUSSION The best conditions for survival of Sri Lankan Artemia from hatching to maturity were a t lOOppt and 25OC with 100 and 120ppt salinities at 25O and 2g°C also supporting high survival rates. These conditions should therefore be maintained in ponds for favourable survival during the powth phase of Sri Lankan Artemia. The higher salinities (140 and 180ppt) a t 2g°C and the range of salinities from- 65 to 140ppt a t 35OC are very unfavourable to the younger stages and should be avoided a t the inoculation stage of pond culture. High mortalities a t higher temperatures that were recorded in the present study were also evident under field culture conditions where total mortality of Sri Lankan Artemia populations occurred when pond waters a t Palavi reached 330C.1° Such detrimental effects of high temperature on survival dictate that culture conditions should be manipulated so as to avoid pond waters reaching high temperature values, which tends to occur particularly during the later periods of the dry season. Survival studies on a variety of other Artemia strains have shown increased survival to occur over a wider range of temperatures than reported by us, with survival in excess of 90% being reported over a 20 to 2g°C temperature range in the Indian Tuticorin Artemia and survival decreasing to 50% with increase in temperature to 32 to 33OC.'Artemia from Iraq also displayed higher survival a t lower temperatures of 15 to 25OC but became motionless a t 35OC and further temperature increases (to 42 to 44OC) resulted in total mortality." For the Indian Tuticorin strain, the preferred temperature was a t 27OC and optimal survival occurred a t 30 to 40ppt salinity range.12 Reports indicate a wide range of variability in the survival and growth of Artemia a t different salinities and temperatures, e.g. A. franciscana from Lake Grassmere, New Zealand, achieved over 90% survival and fastest growth a t 20 to 28OC in 100 to 170ppt salinity range8; 50 to lOOppt salinity range for increased growth in Tuticorin Artemia13; a higher growth rate in Artemia from Sambhar Lake, India reared a t 12.5% (corresponding to 125ppt) salinity than a t a low salinity of 6.5%6 (corresponding to 65ppt). These reports indicate that the extent to which growth is influenced by the salinity of the external medium varies with the stock and sex of A r t e m i ~ . ~ Even though it had been found that reproductive maturity in parthenogenetic females from Sri Lanka was attained in 15 to 17 days when reared a t 35ppt and a t 140ppt a t 25°C,5 in the present study only a slight development ofbroodpouches was observed in Sri Lanka Artemia a t 25OC a t the low salinities tested. Artemia from Sambhar Lake, India, became reproductively mature in 15 to 17 days when Physiology of the Brine Shrimp reared a t 125ppt salimty while those reared a t a low salinity of 6.5% became reproductively mature i n 20 to 25 days.6 The tolerance threshold ofdrtemia is strain-dependant and as far as optimum temperature is concerned it has been observed that there are probably as many temperature optima as there are Artemia habitats4 In the present study Artemia reproduced ovoviviparously at 100ppt and oviparous reproduction occurred later when salinity was increased gradually to 132ppt. Similarly i t had been found that the first brood of offspring in Artemia was ovoviviparous both in the wild and in the laboratory.14 Ovoviviparous reproduction, however, was not observed in Sri Lankan Artemia cultured in 120ppt salinity or at increase of salinity from 120 to 140ppt since these animals produced cysts at 136ppt without prior nauplii production. Under field conditions at Mahalewaya saltern Artemia commenced cyst production a t a similar salinity of 1 3 2 ~ p t . ~ Observations on cyst production reported here point to the possibility of manipulating salinity values in the salterns for triggering cyst production. Reports have indicated that in parthenogenetic populations, the percent of offspring born viviparously was higher than encysted offspring.15 Fecundity observations from the present study lend support to this viewpoint. The salinity range of 100 to l2Oppt observed in the Mahalewaya salterns as carrying the highest populations of Sri Lankan Artemia relate favourably to the observations made under laboratory conditions.1° A salinity range of 100 to 120ppt in combination with a temperature range of 25 to 2g°C are recommended as optimal ranges for these two parameters for survival, growth and reproduction of Artemia from Sri Lanka. Acknowledgments This work was funded by the Natural Resources, Energy and Science Authority (RG/87/B/3). Laboratory facilities were provided by the Department of Zoology, University of Colombo and the National Aquatic Resources Agency. References 1. Vanhaecke P., Siddall S. E. & Sorgeloos P. (1984). International study on Artemia. =I. Combined effects of temperature and salinity on the survival of Artemia of various geographical origin. J o u m l of Experimental Marine Biology and Ecology 80:259-275. 2. Sunderam R. I. M. & Royan J. P. (1984). Note on the occurrence ofArtemia in Sri Lanka. Journal of the National Aquatic Resources Agency 31:123-125. 3. Kuruppu M. M. & Ekaratne S. U. K. (1989). Nutritional evaluation of Sri Lankan ArYemia parthenogenetica for use in larval rearing. In: Fish Nutrition Research in Asia. (Ed. S. S. De Silva) pp. 112-117. Proceedings of the Third Asian Fish Nutrition Network Meeting. Asian Fisheries Society Special Publication 4. Asian Fisheries Society, Manila, Philippines. 168 M.M. Kuruppu & S. U.K. Ekaratne 4. Persoone G. & Sorgeloos P. (1980). General aspects of the ecology and biogeography of Artemia. In: The Brine Shrimp Artemia. Vol. 3. Ecology, culturing, use in aquaculture. (Eds. G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers) pp. 3-24. Universa Press, Wetteren, Belgium. 5. Gilchrist B. M. (1960). Growth and form of the brine shrimp Artemia salina (L.). Proceedings of the Zoological Society, London 134(2):221-235. 6. Baid I. C. (1963). The effect of salinity on the growth and form ofArtemia salina (L.). Journal of Experimental Zoology 153: 279-283. 7. Wear R.G. & Haslett S. J . (1986). Effects of temperature and salinity on the biology ofArtemia franciscana Kelloggfrom Lake Grassmere, New Zealand. 1. Growth and mortality. Journal ofExperimenta1 Marine Biology and Ecology 98:153-166. 8. Wear R.G., Haslett S.J. &Alexander N.L. (1986). Effects oftemperature and salinity on the biology ofArtemia franciscana Kellogg f r ~ m Lake Grassmere, New Zealand. 2. Maturation, fecundity and generation times. Journal of Experimental Marine Biology and Ecology 98:167-183. 9. Sorgeloos P., Lavens P., Leger Ph., Tackaert W. & Versichele D. (1986). Manual for the culture and use of brine shrimp Artemia in aquaculture. pp. 319. Universa Press, Wetteren, Belgium. 10. Kuruppu M.M. & Ekaratne S.U.K. (.1994). Culture potential of Artemia parthenogenetica in Sri Lanka. Proceedings of the First Annual Scientific Sessions ofthe National Aquatic Resources Agency, 1993. National Aquatic Resources Agency, Colombo 15, Sri Lanka. pp. 25-36. 11. Al-Uthman H.S. (1971). Ecological observations on Artemia salina (L.) (Anostraca: Artemiidae) in Iraq. Bulletin of the Biological Research Centre 5:49-65. 12. Royan J.P. (1980). Laboratory and field studies on an Indian strain of the brine shrimp Artemia. In: The Brine Shrimp Artemia. Vol. 3. Ecology, culturing, use in aquaculture. (Eds. G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers) pp. 223-230. Universa Press,Wetteren, Belgium. 13. Balasunderam C. & Kumaraguru A. K. (1987). Laboratory studies on growth and reproduction ofArtemia (Tuticorin strain). 1n:Artemia Research and its applications. Vo1.3. Ecology, culturing, use in aquaculture. (Eds. P. Sorgeloos, D.A. Bengtson, W. Decleir and E. Jaspers) pp. 331-338. Universa Press, Wetteren, Belgium. Physiology of the Brine Shrimp 169 14. D'Agostino A. (1980). The vital requirements of Artemia : physiology. and nutrition. In: The Brine Shrimp Artemia. Vol. 2. Physiology, biochemistry, molecular biology. (Eds. G. Persoone, P. Sorgeloos, 0. Roels and E. Jaspers) pp. 55-82. Universa Press, Wetteren, Belgium. 15. Browne R.A., Sgllee S.E., Grosch D.S., Segreti W.O. & Purser S.M. (1984). Partitioning gebetic aqd environmental components of reproduction and lifespan in ~ r t e h i a . Eqology 65: 949-960. 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