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Effect of low salinity on the growth and survival of juvenile pacific white shrimp, Penaeus vannamei: A revival
Journal Pre-proof Effect of low salinity on the growth and survival of juvenile pacific white shrimp, Penaeus vannamei: A revival Y.D. Jaffer, R. Saraswathy, M. Ishfaq, Jose Antony, D.S. Bundela, P.C. Sharma PII:
S0044-8486(19)31859-9
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734561
Reference:
AQUA 734561
To appear in:
Aquaculture
Received Date: 22 July 2019 Revised Date:
20 August 2019
Accepted Date: 1 October 2019
Please cite this article as: Jaffer, Y.D., Saraswathy, R., Ishfaq, M., Antony, J., Bundela, D.S., Sharma, P.C., Effect of low salinity on the growth and survival of juvenile pacific white shrimp, Penaeus vannamei: A revival, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734561. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Effect of low salinity on the growth and survival of juvenile Pacific white shrimp, Penaeus vannamei: a revival
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Jaffer Y.D*1, R. Saraswathy2, M. Ishfaq3, Jose Antony2 , D.S. Bundela1 and P.C. Sharma1
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ICAR-Central Soil Salinity Research Institute, Karnal, India
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ICAR-Central Institute of Brackishwater Aquaculture, Chennai, India
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3
College of Fisheries Sciences Gumla, Jharkand, India
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*
Corresponding author at ICAR-CSSRI, Karnal, India
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E-mail address: [email protected]
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Abstract
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The euryhaline white shrimp Penaeus vannamei lives in both coastal and oceanic areas and is capable of surviving over a large range of salinities. There is an ambiguity on its osmoregulation pattern and variations in its tolerance to low salinity waters. In order to determine the effects of salinity on growth, survival, osmotic regulation and ionic composition (Na+, K+, Ca++ and Mg++), the juvenilesof P. vannamei were divided into five treatments (1, 5, 7, 15 and 25 ppt) in triplicates for conducting the experiment. The experiment was carried out over a period of 3 weeks with two sampling time points (8th and 21st day). No significant differences (P>0.05) were found in feed conversion rate, survival percentage, body weight gain percentage and specific growth rate. The isosmotic point estimated was 671.3 mOsm kg−1 (21.1 ppt) which is the lowest value obtained for the genus Penaeus so far. The hemolymph osmolality was regulated within a narrow range for wide variations in salinity. Changes in hemolymph Na+ and Mg++ concentrations paralleled those of hemolymph osmolality; values were around significantly higher at 15 ppt than other treatments. Whereas, K+ and Ca++ were present at higher concentrations than Mg++ and lower than Na+ but didn’t show any trend with the hemolymph osmolality. It suggested that total calcium levels are maintained regardless of salinity and may not bear a connection with osmoregulatory mechanisms. Among the four ions determined, the sodium was found at highest concentration in hemolymph followed by calcium, potassium and magnesium. Our findings suggest that low salinity does not seem to affect osmotic regulation to the extent so that growth and survival rate will be affected in P. vannamei. If the acclimation process is followed properly, the species has excellent potential in inland saline waters at salinity as low as 1 ppt.
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Key words: Penaeus vannamei; osmotic regulation; isosmotic point; osmolaity; survival rate.
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1. Introduction
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For more than a decade, researchers have studied the osmoregulation of species completing their life cycle in different saline environments (Charmantier and Wolcott, 2001). In this
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context, estuarine environments expose the penaeids to variable saline conditions which result due to the changing pattern of tides, winds and rain action (Kumlu and Jones, 1995). Osmoregulation is the key factor that governs the adaptive capacity of an organism to cope up with different salinities and is an interesting field for understanding the physiological function of the highly economical species to improve culture conditions and production. It involves maintaining the osmotic and ionic concentrations of the extra-cellular fluid (hemolymph) at concentrations different from the external medium (most typically dilute seawater).
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Subjecting an organism towards the wide range of salinities induces adaptive responses resulting in deviation in the physiological functions, and ultimately affecting growth and survival (Young et al., 1989). Therefore, physiological adaptation to salinity has a crucial role in the recruitment of shrimp in the natural environment (Ferraris et al., 1987) and also governs its growth, survival and food consumption in rearing conditions (Jory, 1995). Wide range of salinity tolerances is being presented by penaeids, although the ideal growth conditions are fulfilled within a limited range. For a penaeid species, Penaeus monodon, 15 to 25ppt salinity (Chen et al., 1995) is considered ideal whereas Fenneropenaeus chinensis shows superior growth in the range of 20 to 25ppt (Chen et al., 1995; Zhang et al., 1999). On the other hand, the Pacific white shrimp Penaeus vannamei tolerates extensive salinity range from 1 to 50 ppt and thus is used as model species to study the mechanism of osmoregulation and salt tolerance (Pante, 1990).
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The culture of shrimp and marine fish in low salinity water is common practice in many countries throughout the world including China, Thailand, Vietnam, Ecuador, Brazil, Mexico, United States (McNevin et. al., 2004). Under adequate salinity calcium, potassium and magnesium are the major ions for shrimp survival (Davis et al., 2002; Gong et al., 2004; Araneda et al., 2008). The Pacific white shrimp, naturally distributed on the Pacific coast of the Americas from northern Mexico to northern Peru, has become one of the primary species being cultured in the East hemisphere, such as China (Cheng et al., 2006) and Thailand (Saoud et al., 2003). Recently, P. vannamei, has become a promising cultivar in most parts of the world due to its ability to grow in inland low saline waters and generating prominent economic returns (Davis et al., 2002).
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There has been a great ambiguity concerning the growth and survival of P. vannamei at low saline waters. One group of researchers found that the haemolymph osmolality of P. vannamei is isosmotic to seawater at 718 mOsm/kg and is able to osmoregulate down to salinities of 5 ppt (Castille and Lawrence, 1980). Ponce-Palafox et al. (1997) reported the highest survival of P. vannamei juveniles in salinities over 20 ppt, whereas, Briggs et al. (2004) reported the best growth between 10-15 ppt. However Diaz et al. (2001) reported the poor performance such as slow growth and low survival of this species at low salinity. Sowers et al. (2005) found that mixed salt and sea salt environments do not effect osmotic regulation in environments as low as 2 ppt TDS (total dissolved salts). On the other hand, Laramore et al. (2001) while culturing P. vannamei at 2 and 3 ppt found no significant difference in survival but the survival rate was significantly higher at 30ppt. Considering the above facts, the objective of this study was to reexamine the osmoregulatory capabilities of
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Penaeus vannamei by recording the changes in ionic composition and haemolymph osmolality at salinities of 1, 7, 15 and 25ppt over a culture period of 21 days.
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2. Materials and methods
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2.1. Experimental animals and setup
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The study was carried out at Muttakadu experimental station of Central Institute of Brackish Water Aquaculture (CIBA), Chennai, situated near to the coast where the direct seawater head is available. The experiment was carried out in oval tanks of 100-L capacity each filled 2/3rd with water. The juveniles, P. vannamei having 5.6 ±0.24 g (mean of 24 animals) average weight and 8.4 ± 0.16 cm (mean of 24 animals) average length were obtained from the Muttakadu hatchery and maintained in a fiberglass tank (500 L) under constant aeration with ambient temperature of 27–28°C and salinity 25ppt. The shrimp were distributed to 15 tanks (12 shrimp/tank) with initial salinity of 25ppt (Fig.1). Salinity was adjusted by adding bore well freshwater to reach the experimental concentrations. The shrimp were acclimated to the different treatments by lowering the salinity by 0.5ppt/2 h with a maximum rate of 5ppt/day salinity change. In this way, it took 2, 3, 4 and 5 days to reach the four treatments 1, 5, 7 and 15ppt of different salinities. Overall five treatments (1, 5, 7, 15 and 25ppt) were set in triplicates for conducting the experiment. The experiment was carried out over a period of 3 weeks with two sampling timings of 8th and 21st day. At each sampling time, the three shrimps were taken from each replicate of the treatments for calculation of different parameters. Aeration was provided 24h a day to maintain the dissolved oxygen (DO) levels near saturation with submerged air diffuser. The shrimp were fed twice daily with Vannamei Plus CIBA feed at 3% of the biomass. Siphoning was carried out after every 3–4 days to remove excess feed or faecal matter. Water was exchanged once per week (30% of the total filled volume) by supplying 2 days old aerated water of same salinity prepared in a separate tank.
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2.2. Growth performance
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The effect of salinity on survival, weight gain, weight gain percentage, specific growth rate and feed conversion ratio was analyzed in the treatments corresponding to the salinities of 1, 5, 7, 15 and 25ppt, with three replicates each. The sampling was done at two time points on the 8th and 21st day of culture. At the time of each sampling, mean individual weight, length and survival were determined.
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The performance of the shrimp was evaluated in terms of the following parameters
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Survival (%) = (shrimp initial number – dead shrimp number)/shrimp initial number × 100 Bodyweight gain (BWG%) = (Wf – Wi)/Wi × 100 Specific growth rate (SGR%) = (LnWf − LnWf)/t × 100 where Ln represents the natural log of individual wet weight (g); Wf is the final wet weight, Wi the initial wet weight, t is the duration in days and Feed conversion ratio (FCR) = feed intake (dry matter) (g)/weight gain (g)
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2.3. Haemolymph and water osmolality
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Haemolymph was collected randomly from animals within a tank and was pooled into a single sample. Haemolymph was collected with a 1.0-mL Insulin syringe from the junction of the cephalothorax and the abdomen by inserting the needle into the pericardial cavity; no anaesthesia was used in this experiment. The samples were transferred to 1.5-mL microcentrifuge tubes and placed in a refrigerator for clotting (4°C, 15 min.). Immediately after clotting the clot was crushed using a plastic rod, and the samples were centrifuged at 2990 g for 30min (Spinwin, MC02, Tarsons, Daihan Scientific, Seoul, Korea) to obtain serum (Tantulo and Fotedar, 2006). The water samples from each treatment were also collected at the time of sampling and stored at 4°C. Samples were placed in microcentrifuge tubes, and a 10 µl subsample was drawn with a micropipette to osmolality reading (mOsm/kg) using a cryoscopic osmometer (Osmomat® 030, Gonotec GmbH, Berlin, Germany). The isosmotic point was calculated based on the linear regression analysis between haemolymph and medium osmolality.
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2.4. Mineral analysis of water, serum and whole-body shrimp
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The mineral analysis of water, serum and whole-body shrimp was carried out by Inductively Coupled Plasma Mass Spectrometry (ICP-OES) (Agilent Technologies, 5100) which is highly sensitive and capable of multi-element trace analysis and ultra-trace analysis, often at the parts-per-trillion level (Fig. 2). However in this study, only major element like Ca++, Mg++, Na+ and K+ which play a significant role in osmoregulation were detected. The samples were acid digested in microwave digester (Anton Paar, Multiwave PRO) and diluted with double distilled water.
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2.5. Physico-chemical parameters of water
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The physicochemical parameters of water were analyzed by titration and spectrophotometer methods during the culture period. The parameters that were detected include, pH, HCO3-, alkalinity (mg CaCO3/L), total hardness (mg/L), TAN (total ammonia nitrogen) (λ=640nm), phosphorus (λ=880nm), nitrite (mg/L) (λ=540nm) and nitrate (mg/L) (λ=540nm).
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2.6. Data analysis
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Data on survival, weight gain, weight gain percentage, specific growth rate and feed conversion ratio, haemolymph and water osmolality and, mineral analysis of water, serum and whole-body shrimp were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's Multiple Range Test to determine differences among treatments. Data were tested for both salinity and sampling time (8th and 21st day) effects using two-way analyses (ANOVAs; Steel and Torrie, 1980). If statistically significant, differences were indicated at the 0.05 level, then Duncan’s Multiple Range Test was used to determine significant differences between means. The relationships between hemolymph osmolality and external osmolality; and between hemolymph ionic concentration and external ionic concentration were determined using correlation and linear regression (Statistical package SPSS 17.0 USA).
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3. Results
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3.1. Physico-chemical parameters of water
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Throughout the 3 weeks culture period, there were not many differences in water quality observed among the different experimental treatments (Table 1) except total hardness which increased with increasing salinity. The pH ranged from 7.05±0.02 to 8.10±0.28 which is under the permissible level for the culture of shrimp. No significant difference was found in nitrite and nitrate along with the treatments. Alkalinity was found highest 181±3.5 in 15 ppt on 21 days of culture. Nitrite was found within the permissible limits of culture throughout the experimental trial period. The oxygen concentration was maintained at upper levels (8-9 mg/L) by aeration pumps.
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3.2. Growth performance
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No significant differences (P>0.05) were found in feed conversion rate, survival percentage, body weight gain percentage and specific growth rate (Table 2) of the shrimp maintained at the different salinities over a period of 21 days.
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3.3. Hemolymph osmolality and ion values
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When P. vannamei was placed in diluted seawater, the osmotic concentration of the hemolymph remained same along with all the treatments except at the media concentration of 445.33 mOsm/kg (15 ppt) at which the hemolymph osmolality was significantly (P < 0.001) higher than all other treatments (Table 3). However, at the higher external media concentration 833 mOsm/kg (25 ppt), the hemolymph was significantly (P < 0.001) hypoosmotic to the seawater. At the lower media concentrations, 44.33, 197.67 and 445.33 mOsm/kg, the hemolymph was significantly (P < 0.001) hyperosmotic to the seawater. There were no significant differences between the levels on the 8th and 21st day of the experiment. In this way, this species showed hyper- and hypo- osmoregulatory behaviour in lower and in higher salinities, respectively.
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The regression analysis between hemolymph and medium osmolality (R2=0.999) used to estimate the isosmotic point can be summarized by the polynomial equation:
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Y= 0.416X2 + 22.06X + 20.76
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where, Y = haemolymph osmolality (mOsm kg−1) and X = medium osmolality (mOsm kg−1).
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The isosmotic point estimated from a regression between hemolymph and water osmolality was 671.3 mOsm kg−1, which is equivalent to salinity 21.1 ppt (Fig.1).
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3.4. Hemolymph, whole-body and medium ionic (Na+, K+, Ca++ and Mg++) concentrations at different salinities
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The concentration of hemolymph sodium and magnesium ions remained stable in all the treatments except at 15 ppt, where significantly (P < 0.001) higher concentration was found (Table 4). Potassium concentration in hemolymph was found the same in the highest (25 ppt)
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and lowest (1 ppt) salinity treatments and significantly (P < 0.001) lower than 7 and 15 ppt. Whereas, the concentration of calcium in hemolymph was found significantly (P < 0.001) higher at 25 ppt than all other treatments. Among the four ions determined, the sodium was found at the highest concentration in hemolymph followed by calcium, potassium and magnesium. Among the four ions determined, only the Ca+ didn’t vary with time. The concentration of Na+, K+ and Mg++ was found significantly (P < 0.001) higher on the 21st day of sampling than on 8th day.
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In case of shrimp whole body, the concentration of sodium followed an irregular trend where it was found highest at 7 ppt followed by 1 and 15 ppt and then lowest at 25 ppt (Table 4). The potassium, calcium and magnesium were significantly (P < 0.001) found different in all the treatments. The concentration of Ca++ was higher in the hemolymph than in the external seawater environment at all salinity treatments. There was a timely effect on all the four ions determined in whole-body of shrimp between the 8th and 21st day of sampling. It was found that the concentration of ions increased on 21st day than the 8th day of sampling significantly (P < 0.001). All the major elements in water increased at higher salinities with a significant difference. The maximum increase was found in sodium. There was no significant difference in the concentration of ions between the 8th and 21st day of sampling in the medium.
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3.5. The magnitude of the hemolymph-seawater osmotic difference
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At all the salinity concentrations, there was a positive difference between the haemolymph and water osmolality except at 25 ppt where the osmolality concentration of water was more than haemolymph. The highest and lowest osmotic difference was found at 1 and 25 ppt medium salinity. The osmotic difference was found more at lower salinities than at higher salinities.
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3.6. Ratio of Ca++ to Na+ in tissues of P. vannamei
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Average values of the Ca++/Na+ ratio in experimental seawater salinities of 1, 7, 15 and 25 ppt were 0.09, 0.04, 0.03 and 0.03 respectively. Ca++/Na+ ratio in the hemolymph decreased as the salinity increased from 1 to 25 ppt (0.09 to 0.08) whereas in the whole-body the Ca++/Na+ didn’t follow any trend, and ranged from 3.36 to 4.07.
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3.7. Comparison of major elements between medium, serum and whole-body shrimp
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There was a positive correlation between Ca++, Mg++, K+, Na+ in water and water osmolality. There was a negative correlation between Ca++ and K+ in hemolymph and Ca++ and K+ in water. In the case of whole-body shrimp, negative correlation between Na+ in body and Mg++ in water was found. Also, there was a positive correlation between Mg++, Na+ and K+ in whole body shrimp and Mg++, Na+ and K+ in hemolymph respectively.
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4. Discussion
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The studies on P. setiferus, P. aztecus, P. duorarum, showed that these species required 3–4 days for their hemolymph to stabilize (Castille and Lawrence, 1981c). In comparison, Huong et al. (2010) showed that hemolymph osmolality in P. vannamei recovered after 1–3 days of
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exposure to low salinities. Furthermore, the direct exposure of P. vannamei to the lower salinities resulted in a survival rate of only about 30% (Huong et al., 2010). Thus we had gradually acclimatized the shrimp to the lower salinity conditions by lowering the salinity from 25 ppt @ 5ppt/day, which resulted in a high survival rate of 91.7±0 to 95.8±5.9%. The lower salinities of 15, 7 and 1 ppt were achieved in 2, 3 and 5 days respectively that provided enough time to change the ionic concentrations of the body to reach the stable conditions. Jayasankar et al., (2009) reported that L. vannamei could be acclimated to a salinity of 5 ppt with a survival rate of nearly 100% using gradual acclimation procedures. Similarly, McGraw and Scarpa (2004) observed increased survival of the same species following a 72-h acclimation period to 1 ppt. The pH of the salinity treatments varied from 7.18-8.1, which is considered appropriate for good performance of penaeid shrimp (Van Wyk and Scarpa, 1999; Cohen et al., 2005). The recommended alkalinity for the culture of penaeids ≥100 mg/L CaCO3 (Van Wyk and Scarpa, 1999) was found at all salinities above than 1 ppt.
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The mean individual weights of P. vannamei were not significantly (P<0.05) influenced by the salinity. Many studies have been conducted looking on the growth and survival of P. vannamei in low salinity water. One study showed that P. vannamei prefers lower salinities over many other penaeid species, such as P. californiensis, P. brevirostris, and P. stylirostris (Mair, 1980). Another study found that P. vannamei grew better in 5 and 15 ppt seawater as opposed to higher salinities (Bray et al. 1994). There has been some conflicting research that found that P. vannamei did not survive below 2 ppt. While as Laramore et al. 2001 reported that survival did not differ significantly at 2 and 3 ppt but was significantly higher at 30 ppt. Samocha et al. (2004) and Sowers and Tomasso (2006) reported growth higher than in seawater using low saline (2 ppt) water. But in our study, the growth and survival didn’t differ at lower and higher salinities. Another study showed that P. vannamei survives and grow better when salinity is over 20 ppt TDS, but they did not test the results below 20 ppt (Ponce-Palafox et al. 1997). Based on the findings of this study, it was seen that low salinity does not seem to affect osmotic regulation to the extent so that growth and survival rate will be affected in the Pacific white shrimp.
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The growth of white shrimp at low saline waters indicates that salinity isn’t only limiting factor for its growth. Van Wyk et al. (1999) reported that at lower salinities (<0.5 ppt) the physiological stress causes a large proportion of its energy to be used in osmoregulation, therefore limiting growth and preventing it from reaching commercial sizes.
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Like most of the penaeid species, Pacific white shrimp P. vannamei exhibited hyper- and hypoosmotic regulation in salinities lower and higher than the isosmotic values respectively (Castille and Lawrence, 1981c; Dal and Smith, 1981; Cawthome et al., 1983; Ferraris et al., 1986). The difference in the ionic compositions of hemolymph in P. vannamei is due to the variation between the external concentration of ions in seawater and the need for shrimp to acclimate when exposed to lower salinity. In Rock Shrimp, for example, Sicyonia brevirostris the hemolymph is significantly hypoosmotic and hyperosmotic at the higher (944 and 1071 mOsm/kg) and lower external medium concentrations to the seawater respectively (Castille and Lawrence, 1981a).
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The isosmotic point of genus Penaeus varied from 699 mOsm/kg (24 ppt) in case of P. stylirostris (Castille and Lawrence, 1981b) to 824 mOsm/kg (26.8 ppt) in case of P. setiferus (Williams, 1960). However, in this study, the isosmotic point for P.vannamei (5.6 ±0.24 g) was found to be 671.3 mOsm/kg (21.1 ppt) which is relatively lower as found in other studies. Isosmotic point determined for P. vannamei by Castille and Lawrence, 1981c, Diaz et al., 2001 and Gong et al., 2004 lies between 25 and 26 ppt. In the case of P. monodon, hemolymph isosmotic point varied at 10g (698 mOsm) and 30g (752 mOsm) body weight (Ferraris et al 1986). In the case of Penaeus chinensis reared between 40 to 10 ppt, the isosmotic value of 707 mOsm/kg (25.0 ppt) was determined (Chen and Lin, 1994). The isosmotic point to F. subtilis was estimated at 377.07 mOsm kg−1, which is equivalent to salinity 14‰ (Silva et. al., 2010).
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The salinity of isosmotic point has been related to the superior growth performance in F. indicus, P. monodon and P. semisulcatus (Raj and Raj, 1982). However, some species show higher growth performance above (F. brasiliensis, Brito et al., 2000) and below (F. aztecus, Venkataramiah et al., 1974; L. setiferus, Rosas et al., 1999) the isosmotic point. Although P. vannamei was found to show higher growth performance (Bray et al., 1994) below the isosmotic point but in this study, no significant difference was seen in growth above or below the isosmotic point.
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Changes in hemolymph Na+ and Mg++ concentrations paralleled those of hemolymph osmolality; values were around significantly higher at 15 ppt than other treatments. Whereas, K+ and Ca++ werepresent at higher concentrations than Mg++ and lower than Na+ but didn’t show any trend with the hemolymph osmolality. The relatively higher concentration of Na+ in hemolymph is for active absorption of salt (mainly NaCl) to compensate for the passive, diffusive loss of salt (mainly NaCl) (Henry et al., 2012). Where as, Huong et al. (2010) and Castille and Lawrence (1981b) reported that only changes in sodium generally paralleled those in osmotic concentrations in response to varying low salinities. It may be due to the reason that in the case of Huong et al. (2010) the shrimp were not given enough time to stabilize with the ambient medium for the exchange of other ions.
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Hemolymph Ca++ concentration was found to be lowest at a salinity of 25ppt, whereas under all low salinities, calcium ion concentrations remained constant. Huong et al. (2010) also witnessed the similar changes with P.vannamei; however, Li and Cheng (2012) found lowest concentrations of Ca++ at 4 ppt, with higher Ca++ levels corresponding to higher salinities at each stage of the moulting cycle. The concentrations of Ca++ were higher in the hemolymph than in the external seawater environment at all salinity treatments. These results agree with the results of studies on P. vannamei (Huong et al. 2010; Li and Cheng, 2012), Macrobrachium rosenbergii by Wilder et al. (1998), where hemolymph calcium levels were higher than those of the surrounding water. Huong et al. (2010) concluded that calcium does not participate in the regulation of osmotic concentration in this species under different low salinities. Hemolymph temporarily stores the calcium absorbed from the exoskeleton to provide some Ca++ to harden the cuticle immediately after ecdysis (Greenaway, 1985) which mostly happens in lower-calcium environments. But in this study the calcium wasn’t the
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limiting factor; thus the maintenance of Ca++ concentration at constant levels at lower salinities in hemolymph indicates its role in osmoregulation that needs to be focused further.
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As the concentration of Ca++ decreases at low salinities, the hemolymph concentration of calcium is increased as the lowest concentration of Ca++ was found at the highest experimental salinity. However, the concentration of Ca++ was found higher in hemolymph than the external medium at all salinities. Li and Cheng (2012) also found that the concentrations of Ca++ of L. vannamei were higher in the hemolymph than in the external seawater environment. In low calcium environments, hemolymph along with gut or mid-gut gland act as storage sites for Ca++ (Neufeld and Cameron. 1992). In crustaceans during cuticle formation, the hemolymph is the second point of contact to absorb calcium after the gill epidermis in lower-calcium environments, and it can quickly transport absorbed calcium to the cuticle. Thus in order to fulfill the Ca++ requirement by P. vannamei, sources other than water like food and stored Ca++ are also used.
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The level of stress and physiological adaptation of penaeids to different salinities can be monitored through their osmoregulatory capacity, which represents the difference between the haemolymph and medium osmolality (Sang and Fotedar, 2005). All hyperosmoregulating crustaceans actively absorb salts across their gills and maintain their hemolymph osmolarity above the osmolarity of the medium (Henry et al., 2012). Hyper-regulation is the most common form of regulation found in crustaceans that mainly occurs at salinity below 26 ppt, at which point hemolymph osmolality begins to be actively maintained above that of the surrounding seawater (Henry, 2005). For salinities below 26 ppt, crustaceans behave as strong, moderate, or weak osmoregulators. The degree of tolerance depends on the magnitude of the hemolymph-seawater osmotic difference. In this study, hemolymph osmolality was regulated within a narrow range for wide variations in salinity as observed by Ferraris et al. (1986).
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5. Conclusion
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Considering the ionic composition of Penaeus vannamei, there is not much variation between 8 and 21 days of culture period indicating that the species has the capability to adjust its physiological pathway to suit the ambient medium of different salinities within a short period of time. Culturing P. vannamei at low salinity if acclimation process is followed properly, the species has excellent potential in inland saline areas since the species is able to maintain its homeostasis at lower salinities without having any effect on growth and survival. The isosmotic point for P.vannamei juveniles was found to be 671.3 mOsm/kg (21.1 ppt) is so for the lowest value of isosmotic point for the genus Penaeus. This indicates that the research on osmoregulation for the genus Penaeus and P. vannamei in particular needs more focus for the farming practices.
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Acknowledgement
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The authors are thankful to the Director of ICAR-CIBA for providing the necessary facilities for conducting the work.
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Conflict of interests
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The authors declare that they have no conflict of interest.
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Ethical statement
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The research undertaken complies with the current animal welfare laws in India. The care and treatment of animals used in this study were in accordance with the guidelines of the CPCSEA [(Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment & Forests (Animal Welfare Division), Govt of India] on care and use of animals in scientific research
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Venkataramiah, A., Lakshmi, G.J., Gunter, G., 1974. Studies on the effects of salinity and temperature on the commercial shrimp Penaeus aztecus Ives, with special regard to survival limits, growth, oxygen consumption and ionic regulation. US army Engineer Waterways. Experiment Station, Vicksburg, MS, USA Contract Report H- 74-2/AD-777-794.
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Williams, A.B., 1960. The influence of temperature on osmotic regulation in two species of estuarine shrimps (Penaeus). Biol. Bull., 119: 560-571.
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509 510
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Table 1. Physicochemical parameters of the water of P. vannamei culture at different salinities over a culture period of 21 days1. Parameters pH (pH meter)
1‰ 7.18±0.17
7‰ 8.10±0.28
15‰ 7.45±0.07
25‰ 7.54±0.23
1*
148.7±12.9
385.3±4.3
221.5±4.3
145.6±17.2
1*
98.9±10.6
120.1±5.5
145.6±3.5
165.4±14.1
1*
140.4±28.4
250.8±70.9
511.5±127.7
1003.00±0
2*
0.05±0.02
1.57±0.1
2.41±0.02
0.07±.01
2*
0.36±0.06 0.09±0
0.87±0.12 0.10±0.01
0.88±0.01 0.08±0.01
0.42±0.07 0.11±0
Nitrate (mg/L) 1.69±0 1.66±0.07 Mean of 3 replicates ±SD 1* (Titration method); 2* (Spectrophotometer)
1.65±0.01
1.71±0.01
HCO3Alkalinity (mg CaCO3/L) Total hardness (mg/L) TAN (mg/l) Phosphate Nitrite (mg/L)
2* 2* 1
Table 2. Performance of P. vannamei reared at different salinities over 21 days1 of culture period.
1
Parameters
1‰
7‰
15‰
25‰
Feed conversion ratio (FCR)
1.44±0.1
1.49±0.2
1.49±0.5
1.45±0.6
Survival (%)
95.8±5.9
91.7±0
95.5±5.9
95.8±5.9
Body weight gain (%)
29.72±1.1
29.76±2.04
28.82±2.08
30.30±01.11
Specific growth rate (%)
1.83±0.04
1.77±0.07
1.79±0.07
1.82±0.04
Mean of 3 replicates ±SD
Table 3. Mean (±SE) values of hemolymph and water osmolality (mOsm kg−1) of the shrimp P. vannamei on 8th and 21st day of culture in salinities ranging from 1 to 25‰. Salinity ‰
1
Hemolymph (n=6) Mean SE Water (n=3) Mean SE
7
15
25
8D
21D
8D
21D
8D
21D
8D
21D
643.66bc ± 6.96
629.66c ± 3.52
660.66b ± 8.09
630.00c ± 7.63
666.00b ± 15.94
738.67a ± 8.95
650.33bc ±8.95
640.00bc ± 1.52
44.33d± 2.91
41.67d± 197.67c± 194.67c 1.86 1.45 ±2.03
445.33b ±8.19
444.67b 833.00a± ±8.82 1.15
832.67a ±1.2
Table 4. Concentration (mmol/L) of ions (Ca++, Mg++, Na+ and K+) in hemolymph and wholebody of the shrimp P. vannamei at different salinities over 21 days1 of culture in salinities ranging from 1 to 25 ppt. Ca++ Salin ity (ppt)
Whole-body Hemolymph
17.03±0. 623.76±9. 189 48 16.60±0. 862.69±1 5.00 7 194 17.30±0. 828.78±8. 40 15 301 15.80±0. 532.64±1 3.80 25 682 1 Mean of 3 replicates ±SD 1
Mg++
Na+
Whole-body Hemolymph
5.72±0. 087 6.25±0. 048 7.21±0. 133 5.07±0. 261
Whole-body Hemolymph
70.48±3. 83 109.07± 1.88 106.60± 1.96 73.98±3. 21
K+
199.00± 1.47 196.72± 1.23 219.32± 2.43 176.94± 2.90
Whole-body Hemolymph
356.83±1 3.45 473.37±1 5.36 377.68±1 2.41 250.00±1 2.91
7.05±0. 163 7.15±0. 202 8.41±0. 297 5.40±0. 271
307.61±17 .49 3283.08±1 4.87 272.21±14 .62 258.71±6. 62
Fig. 1. Mutakadu Chennai experimental station of ICAR-CIBA showing the backwaters that was pumped and diluted with freshwater from bore well (left) and 100 L oval experimental tanks with aeration (right).
Fig.2. Complete process for determination of ionic compostion of whole-body shrimp samples through ICP-MS.
900
Osmolaity (mOsm/kg)
800
Y = 671.3
700 600
X= 21.1
500 400 300 200 100 0 0
5
10
15
20
25
30
Salinity (ppt)
Fig.3. Hemolymph osmolality of Penaeus vannamei (5.6 ±0.24 g, mean±SD) after a 21-d exposure to 1.0, 5.0, 7.0, 15.0, and 25.0 ppt salinity. The regression analysis between hemolymph and medium osmolality (R2=0.999) used to estimate the isosmotic point can be summarized by the polynomial equation(Y= 0.416X2 + 22.06X + 20.76).
Highlights • • • • •
Low salinity (up to 1 ppt) has no effect on feed conversion rate and specific growth rate in white shrimp. The isosmotic point of white shrimp (5.6 ±0.24 g) is 671.3 mOsm kg−1 (21.1 ppt) The hemolymph osmolality is regulated within a narrow range for wide variations in salinity. Calcium levels are maintained regardless of salinity and may not bear a connection with osmoregulatory mechanisms. Among four major ions (Na+, K+, Ca++ and Mg++) Na+ is found at highest concentration in hemolymph followed by Ca++, K+ and Mg++.
S0044-8486(19)31859-9
DOI:
https://doi.org/10.1016/j.aquaculture.2019.734561
Reference:
AQUA 734561
To appear in:
Aquaculture
Received Date: 22 July 2019 Revised Date:
20 August 2019
Accepted Date: 1 October 2019
Please cite this article as: Jaffer, Y.D., Saraswathy, R., Ishfaq, M., Antony, J., Bundela, D.S., Sharma, P.C., Effect of low salinity on the growth and survival of juvenile pacific white shrimp, Penaeus vannamei: A revival, Aquaculture (2019), doi: https://doi.org/10.1016/j.aquaculture.2019.734561. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
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Effect of low salinity on the growth and survival of juvenile Pacific white shrimp, Penaeus vannamei: a revival
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Jaffer Y.D*1, R. Saraswathy2, M. Ishfaq3, Jose Antony2 , D.S. Bundela1 and P.C. Sharma1
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1
ICAR-Central Soil Salinity Research Institute, Karnal, India
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2
ICAR-Central Institute of Brackishwater Aquaculture, Chennai, India
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3
College of Fisheries Sciences Gumla, Jharkand, India
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*
Corresponding author at ICAR-CSSRI, Karnal, India
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E-mail address: [email protected]
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Abstract
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The euryhaline white shrimp Penaeus vannamei lives in both coastal and oceanic areas and is capable of surviving over a large range of salinities. There is an ambiguity on its osmoregulation pattern and variations in its tolerance to low salinity waters. In order to determine the effects of salinity on growth, survival, osmotic regulation and ionic composition (Na+, K+, Ca++ and Mg++), the juvenilesof P. vannamei were divided into five treatments (1, 5, 7, 15 and 25 ppt) in triplicates for conducting the experiment. The experiment was carried out over a period of 3 weeks with two sampling time points (8th and 21st day). No significant differences (P>0.05) were found in feed conversion rate, survival percentage, body weight gain percentage and specific growth rate. The isosmotic point estimated was 671.3 mOsm kg−1 (21.1 ppt) which is the lowest value obtained for the genus Penaeus so far. The hemolymph osmolality was regulated within a narrow range for wide variations in salinity. Changes in hemolymph Na+ and Mg++ concentrations paralleled those of hemolymph osmolality; values were around significantly higher at 15 ppt than other treatments. Whereas, K+ and Ca++ were present at higher concentrations than Mg++ and lower than Na+ but didn’t show any trend with the hemolymph osmolality. It suggested that total calcium levels are maintained regardless of salinity and may not bear a connection with osmoregulatory mechanisms. Among the four ions determined, the sodium was found at highest concentration in hemolymph followed by calcium, potassium and magnesium. Our findings suggest that low salinity does not seem to affect osmotic regulation to the extent so that growth and survival rate will be affected in P. vannamei. If the acclimation process is followed properly, the species has excellent potential in inland saline waters at salinity as low as 1 ppt.
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Key words: Penaeus vannamei; osmotic regulation; isosmotic point; osmolaity; survival rate.
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1. Introduction
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For more than a decade, researchers have studied the osmoregulation of species completing their life cycle in different saline environments (Charmantier and Wolcott, 2001). In this
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context, estuarine environments expose the penaeids to variable saline conditions which result due to the changing pattern of tides, winds and rain action (Kumlu and Jones, 1995). Osmoregulation is the key factor that governs the adaptive capacity of an organism to cope up with different salinities and is an interesting field for understanding the physiological function of the highly economical species to improve culture conditions and production. It involves maintaining the osmotic and ionic concentrations of the extra-cellular fluid (hemolymph) at concentrations different from the external medium (most typically dilute seawater).
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Subjecting an organism towards the wide range of salinities induces adaptive responses resulting in deviation in the physiological functions, and ultimately affecting growth and survival (Young et al., 1989). Therefore, physiological adaptation to salinity has a crucial role in the recruitment of shrimp in the natural environment (Ferraris et al., 1987) and also governs its growth, survival and food consumption in rearing conditions (Jory, 1995). Wide range of salinity tolerances is being presented by penaeids, although the ideal growth conditions are fulfilled within a limited range. For a penaeid species, Penaeus monodon, 15 to 25ppt salinity (Chen et al., 1995) is considered ideal whereas Fenneropenaeus chinensis shows superior growth in the range of 20 to 25ppt (Chen et al., 1995; Zhang et al., 1999). On the other hand, the Pacific white shrimp Penaeus vannamei tolerates extensive salinity range from 1 to 50 ppt and thus is used as model species to study the mechanism of osmoregulation and salt tolerance (Pante, 1990).
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The culture of shrimp and marine fish in low salinity water is common practice in many countries throughout the world including China, Thailand, Vietnam, Ecuador, Brazil, Mexico, United States (McNevin et. al., 2004). Under adequate salinity calcium, potassium and magnesium are the major ions for shrimp survival (Davis et al., 2002; Gong et al., 2004; Araneda et al., 2008). The Pacific white shrimp, naturally distributed on the Pacific coast of the Americas from northern Mexico to northern Peru, has become one of the primary species being cultured in the East hemisphere, such as China (Cheng et al., 2006) and Thailand (Saoud et al., 2003). Recently, P. vannamei, has become a promising cultivar in most parts of the world due to its ability to grow in inland low saline waters and generating prominent economic returns (Davis et al., 2002).
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There has been a great ambiguity concerning the growth and survival of P. vannamei at low saline waters. One group of researchers found that the haemolymph osmolality of P. vannamei is isosmotic to seawater at 718 mOsm/kg and is able to osmoregulate down to salinities of 5 ppt (Castille and Lawrence, 1980). Ponce-Palafox et al. (1997) reported the highest survival of P. vannamei juveniles in salinities over 20 ppt, whereas, Briggs et al. (2004) reported the best growth between 10-15 ppt. However Diaz et al. (2001) reported the poor performance such as slow growth and low survival of this species at low salinity. Sowers et al. (2005) found that mixed salt and sea salt environments do not effect osmotic regulation in environments as low as 2 ppt TDS (total dissolved salts). On the other hand, Laramore et al. (2001) while culturing P. vannamei at 2 and 3 ppt found no significant difference in survival but the survival rate was significantly higher at 30ppt. Considering the above facts, the objective of this study was to reexamine the osmoregulatory capabilities of
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Penaeus vannamei by recording the changes in ionic composition and haemolymph osmolality at salinities of 1, 7, 15 and 25ppt over a culture period of 21 days.
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2. Materials and methods
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2.1. Experimental animals and setup
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The study was carried out at Muttakadu experimental station of Central Institute of Brackish Water Aquaculture (CIBA), Chennai, situated near to the coast where the direct seawater head is available. The experiment was carried out in oval tanks of 100-L capacity each filled 2/3rd with water. The juveniles, P. vannamei having 5.6 ±0.24 g (mean of 24 animals) average weight and 8.4 ± 0.16 cm (mean of 24 animals) average length were obtained from the Muttakadu hatchery and maintained in a fiberglass tank (500 L) under constant aeration with ambient temperature of 27–28°C and salinity 25ppt. The shrimp were distributed to 15 tanks (12 shrimp/tank) with initial salinity of 25ppt (Fig.1). Salinity was adjusted by adding bore well freshwater to reach the experimental concentrations. The shrimp were acclimated to the different treatments by lowering the salinity by 0.5ppt/2 h with a maximum rate of 5ppt/day salinity change. In this way, it took 2, 3, 4 and 5 days to reach the four treatments 1, 5, 7 and 15ppt of different salinities. Overall five treatments (1, 5, 7, 15 and 25ppt) were set in triplicates for conducting the experiment. The experiment was carried out over a period of 3 weeks with two sampling timings of 8th and 21st day. At each sampling time, the three shrimps were taken from each replicate of the treatments for calculation of different parameters. Aeration was provided 24h a day to maintain the dissolved oxygen (DO) levels near saturation with submerged air diffuser. The shrimp were fed twice daily with Vannamei Plus CIBA feed at 3% of the biomass. Siphoning was carried out after every 3–4 days to remove excess feed or faecal matter. Water was exchanged once per week (30% of the total filled volume) by supplying 2 days old aerated water of same salinity prepared in a separate tank.
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2.2. Growth performance
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The effect of salinity on survival, weight gain, weight gain percentage, specific growth rate and feed conversion ratio was analyzed in the treatments corresponding to the salinities of 1, 5, 7, 15 and 25ppt, with three replicates each. The sampling was done at two time points on the 8th and 21st day of culture. At the time of each sampling, mean individual weight, length and survival were determined.
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The performance of the shrimp was evaluated in terms of the following parameters
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•
113
• •
114 115 116 117
•
Survival (%) = (shrimp initial number – dead shrimp number)/shrimp initial number × 100 Bodyweight gain (BWG%) = (Wf – Wi)/Wi × 100 Specific growth rate (SGR%) = (LnWf − LnWf)/t × 100 where Ln represents the natural log of individual wet weight (g); Wf is the final wet weight, Wi the initial wet weight, t is the duration in days and Feed conversion ratio (FCR) = feed intake (dry matter) (g)/weight gain (g)
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2.3. Haemolymph and water osmolality
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Haemolymph was collected randomly from animals within a tank and was pooled into a single sample. Haemolymph was collected with a 1.0-mL Insulin syringe from the junction of the cephalothorax and the abdomen by inserting the needle into the pericardial cavity; no anaesthesia was used in this experiment. The samples were transferred to 1.5-mL microcentrifuge tubes and placed in a refrigerator for clotting (4°C, 15 min.). Immediately after clotting the clot was crushed using a plastic rod, and the samples were centrifuged at 2990 g for 30min (Spinwin, MC02, Tarsons, Daihan Scientific, Seoul, Korea) to obtain serum (Tantulo and Fotedar, 2006). The water samples from each treatment were also collected at the time of sampling and stored at 4°C. Samples were placed in microcentrifuge tubes, and a 10 µl subsample was drawn with a micropipette to osmolality reading (mOsm/kg) using a cryoscopic osmometer (Osmomat® 030, Gonotec GmbH, Berlin, Germany). The isosmotic point was calculated based on the linear regression analysis between haemolymph and medium osmolality.
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2.4. Mineral analysis of water, serum and whole-body shrimp
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The mineral analysis of water, serum and whole-body shrimp was carried out by Inductively Coupled Plasma Mass Spectrometry (ICP-OES) (Agilent Technologies, 5100) which is highly sensitive and capable of multi-element trace analysis and ultra-trace analysis, often at the parts-per-trillion level (Fig. 2). However in this study, only major element like Ca++, Mg++, Na+ and K+ which play a significant role in osmoregulation were detected. The samples were acid digested in microwave digester (Anton Paar, Multiwave PRO) and diluted with double distilled water.
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2.5. Physico-chemical parameters of water
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The physicochemical parameters of water were analyzed by titration and spectrophotometer methods during the culture period. The parameters that were detected include, pH, HCO3-, alkalinity (mg CaCO3/L), total hardness (mg/L), TAN (total ammonia nitrogen) (λ=640nm), phosphorus (λ=880nm), nitrite (mg/L) (λ=540nm) and nitrate (mg/L) (λ=540nm).
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2.6. Data analysis
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Data on survival, weight gain, weight gain percentage, specific growth rate and feed conversion ratio, haemolymph and water osmolality and, mineral analysis of water, serum and whole-body shrimp were analyzed by one-way analysis of variance (ANOVA), followed by Duncan's Multiple Range Test to determine differences among treatments. Data were tested for both salinity and sampling time (8th and 21st day) effects using two-way analyses (ANOVAs; Steel and Torrie, 1980). If statistically significant, differences were indicated at the 0.05 level, then Duncan’s Multiple Range Test was used to determine significant differences between means. The relationships between hemolymph osmolality and external osmolality; and between hemolymph ionic concentration and external ionic concentration were determined using correlation and linear regression (Statistical package SPSS 17.0 USA).
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3. Results
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3.1. Physico-chemical parameters of water
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Throughout the 3 weeks culture period, there were not many differences in water quality observed among the different experimental treatments (Table 1) except total hardness which increased with increasing salinity. The pH ranged from 7.05±0.02 to 8.10±0.28 which is under the permissible level for the culture of shrimp. No significant difference was found in nitrite and nitrate along with the treatments. Alkalinity was found highest 181±3.5 in 15 ppt on 21 days of culture. Nitrite was found within the permissible limits of culture throughout the experimental trial period. The oxygen concentration was maintained at upper levels (8-9 mg/L) by aeration pumps.
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3.2. Growth performance
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No significant differences (P>0.05) were found in feed conversion rate, survival percentage, body weight gain percentage and specific growth rate (Table 2) of the shrimp maintained at the different salinities over a period of 21 days.
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3.3. Hemolymph osmolality and ion values
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When P. vannamei was placed in diluted seawater, the osmotic concentration of the hemolymph remained same along with all the treatments except at the media concentration of 445.33 mOsm/kg (15 ppt) at which the hemolymph osmolality was significantly (P < 0.001) higher than all other treatments (Table 3). However, at the higher external media concentration 833 mOsm/kg (25 ppt), the hemolymph was significantly (P < 0.001) hypoosmotic to the seawater. At the lower media concentrations, 44.33, 197.67 and 445.33 mOsm/kg, the hemolymph was significantly (P < 0.001) hyperosmotic to the seawater. There were no significant differences between the levels on the 8th and 21st day of the experiment. In this way, this species showed hyper- and hypo- osmoregulatory behaviour in lower and in higher salinities, respectively.
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The regression analysis between hemolymph and medium osmolality (R2=0.999) used to estimate the isosmotic point can be summarized by the polynomial equation:
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Y= 0.416X2 + 22.06X + 20.76
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where, Y = haemolymph osmolality (mOsm kg−1) and X = medium osmolality (mOsm kg−1).
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The isosmotic point estimated from a regression between hemolymph and water osmolality was 671.3 mOsm kg−1, which is equivalent to salinity 21.1 ppt (Fig.1).
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3.4. Hemolymph, whole-body and medium ionic (Na+, K+, Ca++ and Mg++) concentrations at different salinities
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The concentration of hemolymph sodium and magnesium ions remained stable in all the treatments except at 15 ppt, where significantly (P < 0.001) higher concentration was found (Table 4). Potassium concentration in hemolymph was found the same in the highest (25 ppt)
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and lowest (1 ppt) salinity treatments and significantly (P < 0.001) lower than 7 and 15 ppt. Whereas, the concentration of calcium in hemolymph was found significantly (P < 0.001) higher at 25 ppt than all other treatments. Among the four ions determined, the sodium was found at the highest concentration in hemolymph followed by calcium, potassium and magnesium. Among the four ions determined, only the Ca+ didn’t vary with time. The concentration of Na+, K+ and Mg++ was found significantly (P < 0.001) higher on the 21st day of sampling than on 8th day.
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In case of shrimp whole body, the concentration of sodium followed an irregular trend where it was found highest at 7 ppt followed by 1 and 15 ppt and then lowest at 25 ppt (Table 4). The potassium, calcium and magnesium were significantly (P < 0.001) found different in all the treatments. The concentration of Ca++ was higher in the hemolymph than in the external seawater environment at all salinity treatments. There was a timely effect on all the four ions determined in whole-body of shrimp between the 8th and 21st day of sampling. It was found that the concentration of ions increased on 21st day than the 8th day of sampling significantly (P < 0.001). All the major elements in water increased at higher salinities with a significant difference. The maximum increase was found in sodium. There was no significant difference in the concentration of ions between the 8th and 21st day of sampling in the medium.
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3.5. The magnitude of the hemolymph-seawater osmotic difference
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At all the salinity concentrations, there was a positive difference between the haemolymph and water osmolality except at 25 ppt where the osmolality concentration of water was more than haemolymph. The highest and lowest osmotic difference was found at 1 and 25 ppt medium salinity. The osmotic difference was found more at lower salinities than at higher salinities.
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3.6. Ratio of Ca++ to Na+ in tissues of P. vannamei
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Average values of the Ca++/Na+ ratio in experimental seawater salinities of 1, 7, 15 and 25 ppt were 0.09, 0.04, 0.03 and 0.03 respectively. Ca++/Na+ ratio in the hemolymph decreased as the salinity increased from 1 to 25 ppt (0.09 to 0.08) whereas in the whole-body the Ca++/Na+ didn’t follow any trend, and ranged from 3.36 to 4.07.
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3.7. Comparison of major elements between medium, serum and whole-body shrimp
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There was a positive correlation between Ca++, Mg++, K+, Na+ in water and water osmolality. There was a negative correlation between Ca++ and K+ in hemolymph and Ca++ and K+ in water. In the case of whole-body shrimp, negative correlation between Na+ in body and Mg++ in water was found. Also, there was a positive correlation between Mg++, Na+ and K+ in whole body shrimp and Mg++, Na+ and K+ in hemolymph respectively.
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4. Discussion
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The studies on P. setiferus, P. aztecus, P. duorarum, showed that these species required 3–4 days for their hemolymph to stabilize (Castille and Lawrence, 1981c). In comparison, Huong et al. (2010) showed that hemolymph osmolality in P. vannamei recovered after 1–3 days of
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exposure to low salinities. Furthermore, the direct exposure of P. vannamei to the lower salinities resulted in a survival rate of only about 30% (Huong et al., 2010). Thus we had gradually acclimatized the shrimp to the lower salinity conditions by lowering the salinity from 25 ppt @ 5ppt/day, which resulted in a high survival rate of 91.7±0 to 95.8±5.9%. The lower salinities of 15, 7 and 1 ppt were achieved in 2, 3 and 5 days respectively that provided enough time to change the ionic concentrations of the body to reach the stable conditions. Jayasankar et al., (2009) reported that L. vannamei could be acclimated to a salinity of 5 ppt with a survival rate of nearly 100% using gradual acclimation procedures. Similarly, McGraw and Scarpa (2004) observed increased survival of the same species following a 72-h acclimation period to 1 ppt. The pH of the salinity treatments varied from 7.18-8.1, which is considered appropriate for good performance of penaeid shrimp (Van Wyk and Scarpa, 1999; Cohen et al., 2005). The recommended alkalinity for the culture of penaeids ≥100 mg/L CaCO3 (Van Wyk and Scarpa, 1999) was found at all salinities above than 1 ppt.
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The mean individual weights of P. vannamei were not significantly (P<0.05) influenced by the salinity. Many studies have been conducted looking on the growth and survival of P. vannamei in low salinity water. One study showed that P. vannamei prefers lower salinities over many other penaeid species, such as P. californiensis, P. brevirostris, and P. stylirostris (Mair, 1980). Another study found that P. vannamei grew better in 5 and 15 ppt seawater as opposed to higher salinities (Bray et al. 1994). There has been some conflicting research that found that P. vannamei did not survive below 2 ppt. While as Laramore et al. 2001 reported that survival did not differ significantly at 2 and 3 ppt but was significantly higher at 30 ppt. Samocha et al. (2004) and Sowers and Tomasso (2006) reported growth higher than in seawater using low saline (2 ppt) water. But in our study, the growth and survival didn’t differ at lower and higher salinities. Another study showed that P. vannamei survives and grow better when salinity is over 20 ppt TDS, but they did not test the results below 20 ppt (Ponce-Palafox et al. 1997). Based on the findings of this study, it was seen that low salinity does not seem to affect osmotic regulation to the extent so that growth and survival rate will be affected in the Pacific white shrimp.
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The growth of white shrimp at low saline waters indicates that salinity isn’t only limiting factor for its growth. Van Wyk et al. (1999) reported that at lower salinities (<0.5 ppt) the physiological stress causes a large proportion of its energy to be used in osmoregulation, therefore limiting growth and preventing it from reaching commercial sizes.
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Like most of the penaeid species, Pacific white shrimp P. vannamei exhibited hyper- and hypoosmotic regulation in salinities lower and higher than the isosmotic values respectively (Castille and Lawrence, 1981c; Dal and Smith, 1981; Cawthome et al., 1983; Ferraris et al., 1986). The difference in the ionic compositions of hemolymph in P. vannamei is due to the variation between the external concentration of ions in seawater and the need for shrimp to acclimate when exposed to lower salinity. In Rock Shrimp, for example, Sicyonia brevirostris the hemolymph is significantly hypoosmotic and hyperosmotic at the higher (944 and 1071 mOsm/kg) and lower external medium concentrations to the seawater respectively (Castille and Lawrence, 1981a).
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The isosmotic point of genus Penaeus varied from 699 mOsm/kg (24 ppt) in case of P. stylirostris (Castille and Lawrence, 1981b) to 824 mOsm/kg (26.8 ppt) in case of P. setiferus (Williams, 1960). However, in this study, the isosmotic point for P.vannamei (5.6 ±0.24 g) was found to be 671.3 mOsm/kg (21.1 ppt) which is relatively lower as found in other studies. Isosmotic point determined for P. vannamei by Castille and Lawrence, 1981c, Diaz et al., 2001 and Gong et al., 2004 lies between 25 and 26 ppt. In the case of P. monodon, hemolymph isosmotic point varied at 10g (698 mOsm) and 30g (752 mOsm) body weight (Ferraris et al 1986). In the case of Penaeus chinensis reared between 40 to 10 ppt, the isosmotic value of 707 mOsm/kg (25.0 ppt) was determined (Chen and Lin, 1994). The isosmotic point to F. subtilis was estimated at 377.07 mOsm kg−1, which is equivalent to salinity 14‰ (Silva et. al., 2010).
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The salinity of isosmotic point has been related to the superior growth performance in F. indicus, P. monodon and P. semisulcatus (Raj and Raj, 1982). However, some species show higher growth performance above (F. brasiliensis, Brito et al., 2000) and below (F. aztecus, Venkataramiah et al., 1974; L. setiferus, Rosas et al., 1999) the isosmotic point. Although P. vannamei was found to show higher growth performance (Bray et al., 1994) below the isosmotic point but in this study, no significant difference was seen in growth above or below the isosmotic point.
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Changes in hemolymph Na+ and Mg++ concentrations paralleled those of hemolymph osmolality; values were around significantly higher at 15 ppt than other treatments. Whereas, K+ and Ca++ werepresent at higher concentrations than Mg++ and lower than Na+ but didn’t show any trend with the hemolymph osmolality. The relatively higher concentration of Na+ in hemolymph is for active absorption of salt (mainly NaCl) to compensate for the passive, diffusive loss of salt (mainly NaCl) (Henry et al., 2012). Where as, Huong et al. (2010) and Castille and Lawrence (1981b) reported that only changes in sodium generally paralleled those in osmotic concentrations in response to varying low salinities. It may be due to the reason that in the case of Huong et al. (2010) the shrimp were not given enough time to stabilize with the ambient medium for the exchange of other ions.
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Hemolymph Ca++ concentration was found to be lowest at a salinity of 25ppt, whereas under all low salinities, calcium ion concentrations remained constant. Huong et al. (2010) also witnessed the similar changes with P.vannamei; however, Li and Cheng (2012) found lowest concentrations of Ca++ at 4 ppt, with higher Ca++ levels corresponding to higher salinities at each stage of the moulting cycle. The concentrations of Ca++ were higher in the hemolymph than in the external seawater environment at all salinity treatments. These results agree with the results of studies on P. vannamei (Huong et al. 2010; Li and Cheng, 2012), Macrobrachium rosenbergii by Wilder et al. (1998), where hemolymph calcium levels were higher than those of the surrounding water. Huong et al. (2010) concluded that calcium does not participate in the regulation of osmotic concentration in this species under different low salinities. Hemolymph temporarily stores the calcium absorbed from the exoskeleton to provide some Ca++ to harden the cuticle immediately after ecdysis (Greenaway, 1985) which mostly happens in lower-calcium environments. But in this study the calcium wasn’t the
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limiting factor; thus the maintenance of Ca++ concentration at constant levels at lower salinities in hemolymph indicates its role in osmoregulation that needs to be focused further.
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As the concentration of Ca++ decreases at low salinities, the hemolymph concentration of calcium is increased as the lowest concentration of Ca++ was found at the highest experimental salinity. However, the concentration of Ca++ was found higher in hemolymph than the external medium at all salinities. Li and Cheng (2012) also found that the concentrations of Ca++ of L. vannamei were higher in the hemolymph than in the external seawater environment. In low calcium environments, hemolymph along with gut or mid-gut gland act as storage sites for Ca++ (Neufeld and Cameron. 1992). In crustaceans during cuticle formation, the hemolymph is the second point of contact to absorb calcium after the gill epidermis in lower-calcium environments, and it can quickly transport absorbed calcium to the cuticle. Thus in order to fulfill the Ca++ requirement by P. vannamei, sources other than water like food and stored Ca++ are also used.
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The level of stress and physiological adaptation of penaeids to different salinities can be monitored through their osmoregulatory capacity, which represents the difference between the haemolymph and medium osmolality (Sang and Fotedar, 2005). All hyperosmoregulating crustaceans actively absorb salts across their gills and maintain their hemolymph osmolarity above the osmolarity of the medium (Henry et al., 2012). Hyper-regulation is the most common form of regulation found in crustaceans that mainly occurs at salinity below 26 ppt, at which point hemolymph osmolality begins to be actively maintained above that of the surrounding seawater (Henry, 2005). For salinities below 26 ppt, crustaceans behave as strong, moderate, or weak osmoregulators. The degree of tolerance depends on the magnitude of the hemolymph-seawater osmotic difference. In this study, hemolymph osmolality was regulated within a narrow range for wide variations in salinity as observed by Ferraris et al. (1986).
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5. Conclusion
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Considering the ionic composition of Penaeus vannamei, there is not much variation between 8 and 21 days of culture period indicating that the species has the capability to adjust its physiological pathway to suit the ambient medium of different salinities within a short period of time. Culturing P. vannamei at low salinity if acclimation process is followed properly, the species has excellent potential in inland saline areas since the species is able to maintain its homeostasis at lower salinities without having any effect on growth and survival. The isosmotic point for P.vannamei juveniles was found to be 671.3 mOsm/kg (21.1 ppt) is so for the lowest value of isosmotic point for the genus Penaeus. This indicates that the research on osmoregulation for the genus Penaeus and P. vannamei in particular needs more focus for the farming practices.
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Acknowledgement
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The authors are thankful to the Director of ICAR-CIBA for providing the necessary facilities for conducting the work.
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Conflict of interests
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The authors declare that they have no conflict of interest.
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Ethical statement
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The research undertaken complies with the current animal welfare laws in India. The care and treatment of animals used in this study were in accordance with the guidelines of the CPCSEA [(Committee for the Purpose of Control and Supervision of Experiments on Animals), Ministry of Environment & Forests (Animal Welfare Division), Govt of India] on care and use of animals in scientific research
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Table 1. Physicochemical parameters of the water of P. vannamei culture at different salinities over a culture period of 21 days1. Parameters pH (pH meter)
1‰ 7.18±0.17
7‰ 8.10±0.28
15‰ 7.45±0.07
25‰ 7.54±0.23
1*
148.7±12.9
385.3±4.3
221.5±4.3
145.6±17.2
1*
98.9±10.6
120.1±5.5
145.6±3.5
165.4±14.1
1*
140.4±28.4
250.8±70.9
511.5±127.7
1003.00±0
2*
0.05±0.02
1.57±0.1
2.41±0.02
0.07±.01
2*
0.36±0.06 0.09±0
0.87±0.12 0.10±0.01
0.88±0.01 0.08±0.01
0.42±0.07 0.11±0
Nitrate (mg/L) 1.69±0 1.66±0.07 Mean of 3 replicates ±SD 1* (Titration method); 2* (Spectrophotometer)
1.65±0.01
1.71±0.01
HCO3Alkalinity (mg CaCO3/L) Total hardness (mg/L) TAN (mg/l) Phosphate Nitrite (mg/L)
2* 2* 1
Table 2. Performance of P. vannamei reared at different salinities over 21 days1 of culture period.
1
Parameters
1‰
7‰
15‰
25‰
Feed conversion ratio (FCR)
1.44±0.1
1.49±0.2
1.49±0.5
1.45±0.6
Survival (%)
95.8±5.9
91.7±0
95.5±5.9
95.8±5.9
Body weight gain (%)
29.72±1.1
29.76±2.04
28.82±2.08
30.30±01.11
Specific growth rate (%)
1.83±0.04
1.77±0.07
1.79±0.07
1.82±0.04
Mean of 3 replicates ±SD
Table 3. Mean (±SE) values of hemolymph and water osmolality (mOsm kg−1) of the shrimp P. vannamei on 8th and 21st day of culture in salinities ranging from 1 to 25‰. Salinity ‰
1
Hemolymph (n=6) Mean SE Water (n=3) Mean SE
7
15
25
8D
21D
8D
21D
8D
21D
8D
21D
643.66bc ± 6.96
629.66c ± 3.52
660.66b ± 8.09
630.00c ± 7.63
666.00b ± 15.94
738.67a ± 8.95
650.33bc ±8.95
640.00bc ± 1.52
44.33d± 2.91
41.67d± 197.67c± 194.67c 1.86 1.45 ±2.03
445.33b ±8.19
444.67b 833.00a± ±8.82 1.15
832.67a ±1.2
Table 4. Concentration (mmol/L) of ions (Ca++, Mg++, Na+ and K+) in hemolymph and wholebody of the shrimp P. vannamei at different salinities over 21 days1 of culture in salinities ranging from 1 to 25 ppt. Ca++ Salin ity (ppt)
Whole-body Hemolymph
17.03±0. 623.76±9. 189 48 16.60±0. 862.69±1 5.00 7 194 17.30±0. 828.78±8. 40 15 301 15.80±0. 532.64±1 3.80 25 682 1 Mean of 3 replicates ±SD 1
Mg++
Na+
Whole-body Hemolymph
5.72±0. 087 6.25±0. 048 7.21±0. 133 5.07±0. 261
Whole-body Hemolymph
70.48±3. 83 109.07± 1.88 106.60± 1.96 73.98±3. 21
K+
199.00± 1.47 196.72± 1.23 219.32± 2.43 176.94± 2.90
Whole-body Hemolymph
356.83±1 3.45 473.37±1 5.36 377.68±1 2.41 250.00±1 2.91
7.05±0. 163 7.15±0. 202 8.41±0. 297 5.40±0. 271
307.61±17 .49 3283.08±1 4.87 272.21±14 .62 258.71±6. 62
Fig. 1. Mutakadu Chennai experimental station of ICAR-CIBA showing the backwaters that was pumped and diluted with freshwater from bore well (left) and 100 L oval experimental tanks with aeration (right).
Fig.2. Complete process for determination of ionic compostion of whole-body shrimp samples through ICP-MS.
900
Osmolaity (mOsm/kg)
800
Y = 671.3
700 600
X= 21.1
500 400 300 200 100 0 0
5
10
15
20
25
30
Salinity (ppt)
Fig.3. Hemolymph osmolality of Penaeus vannamei (5.6 ±0.24 g, mean±SD) after a 21-d exposure to 1.0, 5.0, 7.0, 15.0, and 25.0 ppt salinity. The regression analysis between hemolymph and medium osmolality (R2=0.999) used to estimate the isosmotic point can be summarized by the polynomial equation(Y= 0.416X2 + 22.06X + 20.76).
Highlights • • • • •
Low salinity (up to 1 ppt) has no effect on feed conversion rate and specific growth rate in white shrimp. The isosmotic point of white shrimp (5.6 ±0.24 g) is 671.3 mOsm kg−1 (21.1 ppt) The hemolymph osmolality is regulated within a narrow range for wide variations in salinity. Calcium levels are maintained regardless of salinity and may not bear a connection with osmoregulatory mechanisms. Among four major ions (Na+, K+, Ca++ and Mg++) Na+ is found at highest concentration in hemolymph followed by Ca++, K+ and Mg++.