Optimization of culture conditions for marine copepod Macrosetella gracilis (Dana, 1847) with emphasis on salinity and algal diets

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1 Indian Journal of Geo-Marine Sciences Vol. 44(1), October 215, pp Optimization of culture conditions for marine copepod Macrosetella gracilis (Dana, 1847) with emphasis on salinity and algal diets Nandakumar, R, B. Balaji Prasath, P. Santhanam*,, S. Ananth., T. Jayalakshmi, S. Dinesh Kumar, & A. Shenbaga Devi Marine Planktonology & Aquaculture Lab., Department of Marine Science,School of Marine Sciences, Bharathidasan University,Tiruchirappalli-62 24, Tamil Nadu. India *[ Received 25 March 214; revised 12 May 214 The effects of salinity and algal diets on the survival, egg production and population density of harpacticoid copepod, Macrosetella gracilis were studied. Seven different salinity levels (15, 2, 25, 3, 35, 4, 45 ppt) and six different algal diets (Chlorella marina (CHL), Isochrysis galbana (ISO), Dunaliella salina (DUN)., Nannochloropsis salina (NAN)., Tetraselmis suecica (TET). and mixed algal diet (combination of above mentioned microalgae)) were analyzed in this study. Highest (p <.5) nauplius production rate (NPR), survival rate (SR) and population density (PD) were achieved by copepod reared under 3-35 ppt salinity. Diet experiments showed that feed can significantly affects the NPR, SR and PD of M.gracilis. The highest NPR (66.3±4.9 nauplius female 1 day 1 ) was produced by mixed algal diet treatment followed by other feeds in descending order ISO (63.6±4.9), CHL (39±2.8), DUN (26.3±3.14), TET (26.3±2.25) and NAN (18.6±7.4 nauplius female 1 day 1 ). Among the individual feeds tested, ISO induced best survival (89%) followed by CHL (75%) and TET (69%). This study revealed that M. gracilis can be mass cultured for commercial purposes and has a great potential for use in toxicity studies due to its high reproductive performance and wide range of tolerance to local environmental conditions. [Keywords: Copepod, Macrosetella gracilis, Chlorella marina, Isochrysis galbana, Population density] Introduction Among the most abundant and important components of aquatic invertebrates, copepods receives increasing attention in the recent years due to their high calorific content and superior nutritional value. Within the various copepod suborders, harpacticoids can be considered as excellent candidates as live feeds. There are adequate evidences suggesting that harpacticoid copepods may serve as exceptional live prey for hatchery-reared fin fishes and crustaceans 1, 2. Moreover, harpacticoids have a relatively high calorific content per unit weight and superior nutritional value compared to many traditional live food sources 3, 4. The interest in culturing harpacticoid copepods arises from the fact that they have a high reproductive potential, short life cycle, fast growth, preferring a flexible diet and could tolerate a wide range of environmental conditions such as temperature and salinity, etc 5,6. Furthermore, copepod culture plays a vital role in the fundamental research fields such as phylogeny, physiology and ecology. Hence, there is a need for reliable continuous cultures for marine copepods. Studies on the effects of environmental parameters on copepod physiology are necessary to understand their tolerance limits and to provide an optimized condition for their growth under captivity. To date, no studies have been made on effect of algal diet on growth and fecundity success of this species. The harpacticoid copepod M. gracilis (Dana) is one of the dominant harpacticoid in tropical Indian waters and Muthupet lagoon in particular 7. However, no attempt so far has been made on the culture of this species. Purpose of our study was to optimize certain environmental stress factors (Salinity and algal diets) and to develop a culture system for M. gracilis which could sustain high population with least maintenance that satisfy and serve the growing demand for live feeds.

2 1522 INDIAN J. MAR. SCI., VOL. 44, NO. 1 OCTOBER 215 Materials and Methods Zooplankton samples were collected from the Muthupet lagoon (Lat. 1 2 N and Long E), Southeast coast of India during early morning using scoop (plankton) net with 158 µm mesh size. Copepods were identified using standard manuals 8-1. After species confirmation, gravid females of M. gracilis were isolated and stocked initially in a suitable container filled with filtered seawater. Seawater used for copepod culture was filtered through filter bags (1µm). Seawater was properly maintained 3 salinity, measured by a Hand Refractometer (ERMA, Japan). Utmost care was taken to avoid overfeeding, indicated by excess precipitation of microalgal food in the bottom of the culture tanks. Monocultures of copepod M. gracilis were started with 5-1 gravid females inoculated in 5 ml of filtered seawater. Stock cultures were maintained in the same vessel for up to three generations. Then the copepods were harvested and new stock cultures were started using gravid females from different stocks. This was done to prevent the pressure of inbreeding. Copepods harvested from stock cultures were used for mass culture at 1 L plastic containers filled with 7L of filtered seawater. Harvesting of copepod was done after 2 days. To harvest the copepods the illumination was given at the tank bottom to facilitate its upward movement to accumulate near the top of the tanks (negative phototaxis) for efficient and easy harvesting. The collected copepods in the filter mesh were detached by rinsing the mesh bag into a 1 ml beaker containing filtered seawater. When the copepod populations were sampled at the end of the culture, the vessels were drained and copepods were harvested. After harvest the copepods were counted using a Sedgewick Rafter Cell under stereo zoom microscope. Total counts of samples from the condensed populations were repeated several times for each harvest on the whole. Marine microalgal strains were obtained from the Central Institute of Brackishwater Aquaculture, Chennai, Tamil Nadu, India. Microalgal cultures (Chlorella marina (CHL), Isochrysis galbana (ISO), Dunaliella salina (DUN)., Nannochloropsis salina (NAN)., Tetraselmis suecica (TET)) were cultured in 1L capacity container at temperature between 23 and 25 o C, 3 ppt salinity and light intensity of 45-6 mmol photons/m 2 /sec. using Walney medium 11. The seawater was filtered using filter bag (1 µm). The filtered seawater was sterilized using autoclave and cooled before transferring to the culture flask. All vessels used for algal culture were sterilized and dried before use. Microalgae in exponential phase were harvested and fed to copepods. Experiments The study consisted of two experiments, salinity and diet based effects on survival, nauplius production and population density. The procedures for both the experiments were same except that mixed feed was used for salinity experiments and salinity maintained for diet experiments was 3±2 ppt. Five different feeds (CHL, DUN, NAN, ISO and TET) were used in this study. All experiments were carried out in triplicate. For survival experiment around 1 adults were placed into new 5-ml beakers filled with corresponding water of the salinity and algae, at 28-3 C and on a 12L: 12D light/dark cycle. The water in the beakers was changed every 2 days. During the experiment, algal density was maintained at 25, cells ml -1. The dead organisms and fecal pellets were removed with a pipette. Survival of the animals was determined under a stereo-microscope and recorded from the start of the experiment. For fecundity experiment individual female M. gracilis carrying first egg sacs was placed in 2 ml vial filled with filtered seawater. All the experimental vials were placed in dark, but were exposed to light for 3 to 4 hours every day during which period they were examined. Every day the copepods were checked to ascertain the time of their nauplii release and the culture media were refreshed every alternate days. Once the copepod released its nauplii it was transferred to a new 2 ml vial filled with a fresh culture medium. The copepod nauplii released from the eggs were fixed by adding a drop of 5% formalin and counted under a dissecting microscope.

3 NANDAKUMAR et al.: OPTIMIZATION OF CULTURE CONDITIONS FOR MACROSETELLA GRACILIS WITH EMPHASIS ON SALINITY AND ALGAL DIETS 1523 For determining the population density, an initial of 15 adults of M. gracilis were stocked into each culture bottle (1L). Adult copepods were separated from the stock culture by draining the culture water through a 158 μm sieve. The copepods were immediately placed in a petridish with a small amount of seawater. Individuals were randomly captured using a fine-tipped pipette and transferred to the experimental bottles. During the experimental period (17 days), the copepods were fed with designated diets daily. After 17 days, all the contents from the triplicates were drained through a 58 μm sieve and the total number of nauplii, copepodites and adults of M. gracilis retained were fixed in 5% formalin solution. Population counts were made using a Sedgewick Rafter counter and a high-powered microscope (Optika). Statistical analysis Data were analyzed by one-way ANOVA. If significant (P<.5) difference was found in the ANOVA test, Tukey s test was used to rank the groups. Data are presented as mean±s.e. All statistical analyses were performed using Graph Pad Prism Ver. 5. Results High and a constant production of M. gracilis were obtained in this study. For an entire period of 35 days, our system produced an average of 73,981 nauplii, 47,15 copepodids and 29,14 adults L -1 (Fig. 1). The mean production was 1, 5, 1 ind L -1 with an initial inoculum of 5-6 copepods L -1. The presently used inoculum size of 5-6 numbers was chosen to reduce the contamination. This final copepod yield can be further maximized to great numbers within a short span of time by escalating the initial adult inoculum. The influence of 5 different microalgal diets on NPR (nauplius female 1 day 1 ) of M. gracilis are shown in Figure 2. The results showed that maternal food significantly influenced M. gracilis nauplius output (p<.5). The highest NPR (66.3±4.9 nauplius female 1 day 1 ) was produced by the mixed diet, which was over 3 times higher than that of the lowest NPR (18.6±7.4 nauplius female 1 day 1 ), recorded for NAN. Among the individual feeds tested, ISO showed better NPR of 63.6±4.9 nauplius female 1 day 1. CHL, DUN and TET were the next best feeds with NPR of 39±2.8, 26.3±3.14 and 26.3±2.25 nauplius female 1 day 1, respectively. Culture density (ind L-1) Fig 1. Mean culture density of M. gracilis grown in 1L tanks Nauplius production (nauplius female 1 day 1 ) Nauplii Copepodite Adult CHL DUN ISO NAN TET MIX Fig 2. Mean nauplius production of M. gracilis fed with different diets produced by adults fed six different microalgae diets. Copepods were incubated under identical condition of 28±2 C and 3±2 ppt. Survival All tested feeds showed more than 5% mean survival. Survival of copepod was considerably poor in NAN and DUN. Among the individual feeds tested, ISO induced best survival (89%) followed by CHL and TET. The copepod survival had a sharp decline from 8% on 3 rd day to 4% on 6 th day with NAN. Further NAN was the only treatment that showed total mortality by the end of 1 th day (Fig 3).The mean total number of copepod population obtained for a period of 17 days is shown in fig. 4.

4 1524 INDIAN J. MAR. SCI., VOL. 44, NO. 1 OCTOBER 215 Survival (%) CHL DUN ISO Days Fig 3. Mean survival of M. gracilis under different algal diets Feed type was also shown to have significant effect on population density of copepods (P<.5). The total population density of M. gracilis was highest for mixed algal diet (92±98.27 ind L -1 ). In the case of individual diets ISO yielded the high result followed by CHL with population densities of 866±67.8 and 658±69.5 ind L -1, respectively. NAN feed showed the poor yield among the feeds tested with mean population being 241±51.15 ind L -1. The results showed considerable differences in the numbers of the lifestage categories, i.e. nauplii, copepodites and adults in different diet treatments. Most notably, the number of nauplii was strikingly higher for the MIX diet treatment. The ISO diet remarkably differed from the other diets in that it had substantially higher numbers of both nauplii and copepodites. Both MIX and ISO also had obviously higher numbers of adults when compared to the rest of the diet. Population growth (ind L -1 ) Nauplii Copepodite Adult CHL DUN ISO NAN TET MIX Fig 4. Mean population of M. gracilis cultured for17 days with different diets from an initial number of 15 adults and values expressed as mean ±S.E. Salinity significantly (P<.5) affected the naplius production rate of M.gracilis (Fig.5). The salinity tolerance of M. gracilis seemed to be in the range of ppt. The results showed that M. gracilis was adaptive under varying salinity conditions, although the nauplius production decreased beyond tolerance range. NPR was found to be highest (73±4.9) when the salinity was maintained at 3 ppt followed by 35ppt with an NPR of 69± The lowest nauplius production (33.3±5.75) was recorded at 45ppt which was about two times lower than NPR at 3 and 35ppt. The higher salinities appeared to have adverse effect on nauplius production of M. gracilis as evidenced by the present results. The mean survival was above 5% at all tested salinities. Although survival rate was found maximum at the salinity ranges between 3 and 35ppt, mortality was slower at 3 ppt with final survival of 8%. Though salinity had considerable effect on survival, maximum effects were observed at higher salinities. Lowest survival of 2% was observed for copepods maintained at 45ppt (Fig 6). Nauplius production (nauplius female 1 day 1) Salinity (ppt) Fig. 5 Mean nauplius production of M. gracilis exposed to different salinities. Copepods were incubated under identical condition of 28±2 C, and 25 cells/ml of mixed algal feed Survival (%) Days Fig 6. Mean survival of M. gracilis exposed to different salinities

5 NANDAKUMAR et al.: OPTIMIZATION OF CULTURE CONDITIONS FOR MACROSETELLA GRACILIS WITH EMPHASIS ON SALINITY AND ALGAL DIETS 1525 Salinity also had significant impact on population growth of copepods (p<.5). Mean population density of M. gracilis were found highest at 3 ppt, reaching 953±93.53 ind L -1 from an initial of 15 adults. Lowest population mean (489±72.16 ind L -1 ) was obtained when copepods were maintained at 45ppt. Population density was dropped almost by 5% compared to highest mean population when salinity increased to 45ppt (Fig. 7). Copepods survived for a longer period at salinities between 3 and 35 ppt compared to other levels. Salinity had no significant effect on nauplius production. An average nauplius production of 24 nauplii female -1 day -1 was recorded for all salinity treatments. The higher tolerance span of the M. gracilis can be attributed to the fact that they are copepods that have been cultured in the laboratory for six months. Hence they could have adapted to laboratory conditions, and are able to survive better after being cultured. Population growth (ind L -1 ) Nauplii Copepodite Adult Salinity (ppt) Fig 7. Mean population of M. gracilis cultured for17 days exposed to different salinities. The initial numbers of adults were 15 and values expressed as mean ±S.E. Discussion The main objective of this study is to develop a method for the mass culture of marine harpacticoid copepod M. gracilis that is reliable, productive and labour efficient. Over 4 weeks of operation, our system produced an average of 73,981 nauplii, 47,15 copepodids and 29,14 adults L -1. We have observed that M. gracilis has a generation time of days, depending on the water temperature and preliminary trials indicated that a 2-day culture cycle was more adequate to maximize the copepod population. As cost effectiveness is the only aspect which hampers commercialization of copepod culture, the present culture method has a great significance in commercial production of live feeds. The present method showed the potential of this copepod to produce high density in low volume of seawater (5-7 L). Though culture of this copepod was not attempted in larger volumes, our trails showed that densities at lower volumes were significantly higher. Our system holds an edge in the fact that it does not require an exclusive filtration system and sophisticated infrastructure, thereby reducing a major expenditure. Furthermore, seawater exchange was not necessary, at least for a period of 4-6 months. However, inclusion of freshwater to optimize the salinity was enforced to sustain copepod culture without ciliates which was observed after a period of 4-5 weeks. Increase in number of ciliates capable of surpassing the culture host was observed when the cultures were overfed. This species (M. gracilis) was better suited to our culture method and it was able to withstand all extremes and was capable of reproducing at high densities in a smaller volume of water. There was a clear visual change in the adaptive behavior of this harpacticoid copepod where the cultures in early stages were unable to attain maximum densities which changed in a course of 6-8 generations. The culture method presented here is simple, cost effective, and reliably produces large numbers of copepods. Further studies are in progress in our laboratory to refine our technique and plan to examine the performance of ciliate-free cultures in addition to developing a technique to scale up production at densities higher than those achieved in the current system. Under natural conditions, copepods are often well adapted to deal with seasonal fluctuations in temperature and salinity 12,13,. Estuarine copepods in particular have broad tolerance ranges of salinities 14. This is a useful attribute for aquaculture, as copepods used as live feed for fish larvae will be alive and remain available in the water column under various temperature and salinity conditions that are

6 1526 INDIAN J. MAR. SCI., VOL. 44, NO. 1 OCTOBER 215 suitable for the culture of fish larvae 15,16. Results of our study showed that growth of M. gracilis was maximum at salinities between 3 and 35 ppt. High survival of over 8% was achieved under these salinities. Sun and Fleeger 5 stated that harpacticoid copepods grew best in the salinity regime of ppt, while it can continue to survive in the salinity range of 1 to 6 ppt. Harpacticoids in general are known to have a wide tolerance to salinity changes due to their different natural habitats with periodic exposure to salinity fluctuations. Rhodes 17 reported salinity tolerance of Nitokra lacustris ranged from 1 to 4 ppt. Recently, Zaleha and Jamaludin 18 observed the optimum condition for the maximum production of a tropical harpacticoid copepod Pararobertsonia sp. to be around 35ppt. Although M.gracilis was able to survive in a wide range of salinity levels, the overall female reproductive capacity has been observed to decrease under high salinities as agreed by many workers 19,2. In this study, the maximum growth rate peaked at 3-35 ppt and decreased at both higher and lower salinities. This may be explained by the fact that these are copepods well adapted to salinities of 3-35 ppt in our laboratory for more than a year. Further, these are salinities experienced by the copepods in natural conditions. However, it is often difficult to define a salinity threshold for estuarine species 21, as different species may adapt to different salinities at various stages of their life history due to their habitat variations. Besides salinity, feed is another important biological variable that can influence copepod growth, egg hatching success and nauplii survival 22,23. Of the six diets tested, MIX and ISO were the best diets inducing high nauplius production and population growth. In the case of survival, highest values were again recorded with the mixed diet, followed by ISO. Though MIX feed yielded the best result, ISO showed results almost close to MIX. Earlier reports have stressed use of binary algal diets for higher fecundity in copepods 18, 24,25. Although, certain workers 13 have reported better results with monoalgal (ISO) diet. The presently obtained better results in ISO may be due to the amounts of polyunsaturated fatty acids (PUFA) specifically docosahexaenoic acid. Although little is known about the fatty acid requirements of copepods, the importance of diet rich in (n-3) PUFA, especially DHA and EPA for the egg production and egg development has been emphasized in the works of Lacoste et al 26. According to Klein-Breteler et al. 27, the production of highly unsaturated fatty acids enhanced copepod egg production. The worst diet was NAN which was incapable of sustaining good survival and population density. NAN was not an optimal mono-algal diet for maximizing the rate of development of M.gracilis. Though, DUN and TET were better diets than NAN, they cannot be categorized as optimal feeds. Lower performance of these diets is likely to be a result of a deficiency of essential nutritional elements that could not be fully compensated individually. Morehead et al. 12 also found that N. oculata did not support population growth of copepods and attributed lack of DHA and other highly unsaturated fatty acids despite its high EPA for ineffectiveness of NAN diet. Payne 16, on the other hand attributed toughness and indigestibility of the cell wall for poor results associated with N.oculata. In summary, the survival and nauplius production of Macrosetella gracillis were influenced by diet and salinity. Salinity ranges of 3-35ppt can be considered optimum for growth and survival of this copepod. MIX and ISO diets aided excellent survival and resulted in high population growth. Hence the study suggests inclusion of ISO feed and salinity range of around 3ppt for effective mass culture of M.gracilis. Acknowledgement Authors are indebted to the Head, Department of Marine Science and authorities of Bharathidasan University, Tiruchirappalli, India for the facilities provided. Authors also thank the DBT and DST, Govt. of India for financial support. One of the author (RN) acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India, for Senior Research fellowship.

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