A.D Sowers a,, S.P Young a, M. Grosell b,c, C.L. Browdy d, J.R. Tomasso a,e

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1 Comparative Biochemistry and Physiology, Part A 145 (2006) Hemolymph osmolality and cation concentrations in Litopenaeus vannamei during exposure to artificial sea salt or a mixed-ion solution: Relationship to potassium flux A.D Sowers a,, S.P Young a, M. Grosell b,c, C.L. Browdy d, J.R. Tomasso a,e a Department of Biological Sciences, Clemson University, Clemson, SC 29634, USA b Rosenstiel School of Marine and Atmospheric Sciences, Miami, Florida, USA c The Marine and Freshwater Biomedical Sciences Center, University of Miami, Miami, FL 33149, USA d South Carolina Department of Natural Resources, Charleston, SC 29412, USA e Department of Biological Sciences, Clemson University Clemson, SC 29634, USA Received 31 January 2006; received in revised form 6 June 2006; accepted 9 June 2006 Available online 10 June 2006 Abstract Interest in culturing the Pacific white shrimp Litopenaeus vannamei in low-salinity and brackish-well waters has led to questions about the ability of this species to osmo- and ionoregulate in environments containing low concentrations of ions and in environments with ionic ratios that differ from those found in sea water. After seven days, hemolymph osmolality and potassium, sodium and calcium values were all significantly affected by salinity (as artificial sea salt) with values decreasing with decreasing salinity. These decreases were small, however, relative to decreases in salinity, indicating iono- and osmoregulation with adjustment for gradients. The hemolymph osmolality and sodium and calcium concentrations in shrimp exposed to either 2 g/l artificial sea salt or 2 g/l mixed-ion solution (a mixture of sodium, potassium, calcium, and magnesium chlorides that approximate the concentrations and ratios of these cations found in 2 g/l dilute seawater) did not differ significantly. However, hemolymph potassium levels were significantly lower in shrimp held in the mixed-ion environment. Potassium influx rates were similar in shrimp held in either artificial sea salt or mixed ions. The results of this study indicate that salinity affects hemolymph-cation concentrations and osmolality. Further, differential potassium-influx rates do not appear to be the basis for low hemolymph potassium levels observed in shrimp held in mixed-ion environments Elsevier Inc. All rights reserved. Keywords: Ion regulation; Low salinity; Potassium flux; Shrimp 1. Introduction Pacific white shrimp Litopenaeus vannamei iono- and osmoregulate over a wide range of salinities. Iono- and osmoregulation are accomplished by a combination of active transport pathways on the gills, urine flow regulation by the antennal gland, and regulation of water uptake from the gut and across the gills (reviewed in Mantel and Farmer, 1983). Recent work on culturing shrimp in brackish well water has led to the realization that potassium concentrations are often too low in inland water supplies to support survival and growth (Boyd and Corresponding author. 509 Westinghouse Road, P.O. Box 709, Pendleton, SC 29670, USA. Tel.: ; fax: address: asowers@clemson.edu (A.D. Sowers). Thunjai, 2003; Saoud et al., 2003; McGraw and Scarpa, 2003; McNevin et al., 2004). Further work in 5 g/l mixed-ion environments designed to include the major cations found in seawater (environments that are mixtures of sodium, potassium, calcium and magnesium chlorides; Atwood et al., 2003) and 30 g/l sea salts (Zhu et al., 2004) demonstrated that the ratio of sodium to potassium was important, as well as the concentration of potassium. However, even when concentrations and ratios of sodium, potassium, calcium and magnesium in mixed-ion environments are similar to those of dilute seawater, survival and growth of L. vannamei in 2 g/l mixed-ion environments are inferior to survival and growth in dilute seawater containing the same concentrations and ratios of the four cations (Sowers et al., 2005). In order to gain a better understanding of the basis for these observations, we exposed L. vannamei to a range of salinities and to a mixed-ion /$ - see front matter 2006 Elsevier Inc. All rights reserved. doi: /j.cbpa

2 A.D. Sowers et al. / Comparative Biochemistry and Physiology, Part A 145 (2006) environment and then measured osmolality and the concentrations of potassium, sodium and calcium in the hemolymph. We also compared influx of potassium for shrimp in the mixed-ion environment and dilute seawater. 2. Materials and methods 2.1. Hemolymph ion and osmolality experiments Shrimp for hemolymph-ion experiments were obtained from the Waddell Mariculture Center (South Carolina Department of Natural Resources, Bluffton, South Carolina, USA) and held in a recirculating system. The experiments were conducted at Clemson University. During the holding period, shrimp were fed a commercial shrimp ration (Rangen Inc., Angleton, TX, USA; protein 35%, fat 8%, fiber 4%). Salinity was maintained using artificial sea salt (Instant Ocean, Aquarium Systems, Mentor, OH, USA) dissolved in dechlorinated tap water (10 mg/l total hardness as CaCO 3 ). A 12-hour light/12-hour dark photoperiod was used in the holding and experimental rooms. Water quality conditions in the holding system were: total ammonia N=0.4±0.77 mg/l (mean±sd), nitrite N=0.01±0.01 mg/l, temperature=25.0±2.4 C, ph=7.3±0.3, salinity=10.7±1.87 g/ L. The water quality in the holding system and during the experiments was determined according to APHA (1989). Two experiments were conducted in which hemolymph osmolality and ion concentrations were measured. Experimental environments in the first hemolymph-ion experiment were nominal 0.5, 1, 2, 10 and 20 g/l artificial sea salt (Instant Ocean). In the second hemolymph-ion experiment, nominal environments were 2 g/l sea salt or 2 g/l mixed ions (625 mg/l Na +, 22 mg/l K +, 23 mg/l Ca ++, and 77 mg/l Mg ++, all added as chlorides). The mixed-ion environment was designed to approximate sodium, potassium, calcium and magnesium concentrations and ratios found in 2 g/l dilute sea water. Before being stocked in the experimental treatments, shrimp to be exposed to the 0.5, 1, and 2 g/l sea-salt environments and the 2 g/l mixed-ion environment were removed from the holding system and acclimated over 3 days to salinities approximately twice the salinity of their respective experimental treatment. Fig. 1. Hemolymph osmolality (N=22) of Litopenaeus vannamei (11.9±2.68 g, mean±sd) after a 7-d exposure to nominal 0.5, 1.0, 2.0, 10.0, or 20.0 g/l artificial sea salt. Osmolalities are plotted as a function of measured salinities. During the exposures two shrimp exposed to 0.5 g/l died, and one shrimp exposed to 1.0 g/l died). The dashed line represents osmotic equivalency between the hemolymph and the environment. The P value is the probability that the slope of the regression line differs from 0. On day seven of the exposure, temperature was C, ph was , total ammonia N was <2.9 mg/l, and nitrite N was <1.0 mg/l. Shrimp stocked in the 10 and 20 g/l sea-salt environments were moved directly from the holding system into the experimental treatments. The exposures were conducted in aquaria containing 30 L of constantly-aerated water. Aquaria were submerged in a water bath to maintain constant temperature. Each aquarium contained one animal. Water lost to evaporation was replaced every 48 h. Dead shrimp and molts were recorded and removed daily. Temperature, salinity, total ammonia nitrogen, nitrite nitrogen, and ph were measured at the end of each experiment. Animals were exposed for 7 days. On day 7, hemolymph was sampled from each surviving shrimp by severing the abdomen and collecting hemolymph from the posterior aorta into a 10-μL pipette. Hemolymph osmolality was determined immediately on this sample with a vapor pressure osmometer (Model 5500, Wescor Inc., Logan, UT, USA). Additional hemolymph (in a 20-μL pipet) and water samples from the aquaria were collected for potassium, sodium, calcium and magnesium analyses. Hemolymph and water samples were diluted with distilled water to bring potassium, sodium, and Table 1 Potassium, sodium and calcium concentrations in water after three days of aeration in four 30-L aquaria 2 g/l artificial sea salt 2 g/l mixed ions Nominal K + (mmol/l) Actual K + (mmol/l) K + (% nominal) 93% 114% Nominal Na + (mmol/l) Actual Na + (mmol/l) Na + (% nominal) 91% 103% Nominal Ca ++ (mmol/l) Actual Ca ++ (mmol/l) Ca ++ (% nominal) 98% 111% Two aquaria contained 2 g/l artificial sea salt (added as undried Instant Ocean) and two aquaria contained 2 g/l mixed ions (47.7 g NaCl, 1.26 g KCl, 1.91 g CaCl 2, 19.3 g MgCl 2 6H 2 O). Fig. 2. Hemolymph potassium concentrations (N=22) in Litopenaeus vannamei as a function of measured environmental potassium concentrations in the artificial sea salt after a 7-d exposure. The arrow below the x axis indicates the potassium concentration that is found in 10 g/l diluted seawater. The P values are the probabilities that the slopes of the regression lines differ from 0. Exposure details are given in the caption to Fig. 1.

3 178 A.D. Sowers et al. / Comparative Biochemistry and Physiology, Part A 145 (2006) Fig. 3. Hemolymph sodium concentrations (N=22) in Litopenaeus vannamei as a function of measured environmental sodium concentrations in the artificial sea salt after a 7-d exposure. The arrow below the x axis indicates the sodium concentration that is found in 10 g/l diluted seawater. The P values are the probabilities that the slopes of the regression lines differ from 0. Exposure details are given in the caption to Fig. 1. calcium concentrations within working limits and subjected to inductively coupled plasma (ICP) spectrometry (Spectro, CIROS, Spectro Analytical Instruments Inc., Marlborough, MA). Water and hemolymph ICP results were corrected for dilution and expressed as mmol/l. Due to an error in the lab, the water samples from the mixed-ion aquaria were discarded before analysis. Table 1 provides results of ICP analyses on water treated similarly to the water in the experiment except that the water described in Table 1 was only in the aquaria for three days and no shrimp were present Potassium-influx experiment Shrimp used for the potassium-flux experiment were obtained from the Oceanic Institute (Kona, Hawaii USA). The experiment was conducted at the University of Miami. Prior to use, animals were held in constantly-aerated 100-L aquaria receiving constantly-flowing seawater, and were fed flake fish feed (Tetramin, Blacksburg, Virginia USA) daily. Shrimp were acclimated to experimental salinities during the three days immediately prior to use. Fig. 5. Potassium influx, estimated from 86 Rb uptake, in Litopenaeus vannamei (0.12 ± g, mean ± SD), as a function of environmental potassium concentrations in either dilute seawater (squares) or a mixed-ion solution (diamonds, see text for details). The arrow indicates the approximate environmental potassium concentration found in 5 g/l dilute seawater. Experiments were conducted in polystyrene beakers, each containing 700 ml of test solution and six animals. Animals were briefly rinsed in deionized water prior to stocking into beakers. During the experiment, the beakers were covered and constantly aerated. Experimental environments were 0.5, 1.0, 2.0 and 5.0 g/l total dissolved solids as either dilute seawater or the mixed-ion solution described for the previous experiment. At the initiation of the flux period, 86 Rb (Amersham, Waukesha, Wisconsin USA) was added to each beaker. The 86 Rb isotope is commonly used as a tracer for K + fluxes in the absence of a suitable K + isotope (Hamann et al., 2005). After approximately two hours (exact flux time in each beaker was measured), shrimp were removed, rinsed in deionized water for 5 min, placed in preweighed polystyrene centrifuge tubes, weighed, and then 5 ml of Soluene-350 (Perkin Elmer Life and Analytical Services) was added to each tube. The Soluene was allowed to dissolve the animal tissue overnight at 43 C. Rubidium uptake was determined by liquid scintillation counting. Potassium influx was calculated from 86 Rb activity in the animal and the 86 Rb activity and potassium concentration in the water, taking animal weight and exposure into account (Boisen et al., 2003; Tomasso Fig. 4. Hemolymph calcium concentrations (N=22) in Litopenaeus vannamei as a function of measured environmental calcium concentrations in the artificial sea salt after a 7-d exposure. The arrow below the x axis indicates the calcium concentration that is found in 10 g/l diluted seawater. The P values are the probabilities that the slopes of the regression lines differ from 0. Exposure details are given in the caption to Fig. 1. Table 2 Hemolymph ion and osmolality values of Litopenaeus vannamei (19.2±3.14 g, mean±sd) after a 7-d exposure to nominal 2 g/l artificial sea salt or 2 g/l mixed ions (chlorides of sodium, calcium, potassium and magnesium) Artificial sea salts Mixed ions P Osmolality (mosmol/kg) 494± ± Potassium (mmol/l) 10.3± ± Sodium (mmol/l) 241.2± ± Calcium (mmol/l) 8.3± ± The mixed salts approximated the concentrations of sodium, calcium, potassium and magnesium in sea water diluted to 2 g/l. During the exposures, 1 of 8 shrimp in the artificial-sea-salt treatment died, and 4 of 15 shrimp in the mixed-ion treatment died. All other shrimp were sampled. The P value is the probability that treatment affected the measured response. On day seven of the exposures, temperature was C, ph was , total ammonia N was <1.3 mg/l, and nitrite N was <1.9 mg/l.

4 A.D. Sowers et al. / Comparative Biochemistry and Physiology, Part A 145 (2006) and Grosell, 2005). Potassium values were determined by atomic absorption flame photometry Statistical analyses Regression analysis was applied to osmolality and cation results from the shrimp exposed to the two environments. A t-test was used to compare results of the 2 g/l mixed-salt treatment with results from a 2 g/l sea salt treatment. Analysis of covariance (environment flux with environmental potassium as the covariate) was applied to the results of the flux experiment. Measured water quality values were entered into all analyses. In all analyses, a PV0.05 was considered significant. 3. Results and discussion 3.1. Hemolymph ion and osmolality experiments In the artificial sea salt experiment, hemolymph osmolality and potassium, sodium, and calcium values were all significantly affected by salinity (Figs. 1 4) with values decreasing with decreasing salinity. The decreases, however, were small compared to the decreases in environmental levels of the respective characteristic (slopes of regression lines were all 0.5), indicating osmo- and ionoregulatory activity with adjustment for environmental gradients. The osmolality values (mean value = 573 mosm/kg) and trends are similar to those reported for L. vannamei by Castille and Lawrence (1981). Decreasing hemolymph osmolality has been associated with decreasing salinity in several penaeids (McFarland and Lee, 1963; Castille and Lawrence, 1981; Ferraris et al., 1986). In all of these reported cases, the slopes of the decrease are less 0.5 indicating an adjustment in osmoregulation rather than osmoconformation. The hemolymph potassium values (mean value= 10.5 mmol/l) and trends are similar to those reported for Farfantepenaeus duorarum (Bursey and Lane, 1971) and L. setiferus (McFarland and Lee, 1963). Hemolymph sodium values (mean value = mmol/l) and trends were similar to those reported for several penaeids (McFarland and Lee, 1963; Bursey and Lane, 1971; Castille and Lawrence, 1981). Hemolymph calcium values (mean value=10.2 mmol/l) were similar to those of other penaeids in low salinity water (McFarland and Lee, 1963; Ferraris et al., 1986). However, no change in hemolymph calcium concentrations was observed in L. setiferus and L. aztecus exposed to a range of salinities (McFarland and Lee, 1963). Hemolymph potassium concentrations were lower in shrimp exposed to the mixed-ion environments compared to shrimp in 2 g/l sea salt(table 2). No significant differences were observed for the other cations measured. The lower plasma potassium concentrations in the shrimp exposed to the mixed-ion environment relative to shrimp in the sea salt environment (Table 2) provide further evidence that the need for potassium in L. vannamei environments is not simply defined by concentration of potassium or by the ratio of sodium to potassium. In our experiment, both potassium concentrations and the ratio of sodium to potassium were similar in the experimental environments (Table 1) Potassium influx experiment Potassium influx, as estimated from 86 Rb uptake, ranged from 425 nmol/g/h in the low (0.5 g/l) seawater treatment to 2,155 nmol/g/h in the high (5 g/l) mixed-ion treatment (Fig. 5). We are unaware of previous reports of K + ( 86 Rb) flux values in decapods exposed to low salinities. Overall, influx was significantly affected by potassium concentration (P =0.010) with influx increasing with increasing environmental potassium levels. However, the nature of the environment (artificial sea salt or mixed ion) had no significant effect on the rate of intake (P =0.0692). Numerically, the influx rates of the animals in the mixed-ion environments are actually higher than the rates of animals in the artificial sea salt environment. These results indicate that the basis for the lower hemolymph potassium levels inshrimpheldinthe mixed-ionenvironment (Table 2)is not a lower rate of potassium uptake at the gills. A higher efflux rate at the gills or higher urinary loss rates are other possible explanations. The current study demonstrates that the physiological basis for reduced shrimp performance in the mixed-salt environments is likely related to low hemolymph potassium levels. The basis for the low levels is not clear given that low hemolymph potassium levels develop in environments that have potassium concentrations, and sodium:potassium ratios similar to that of dilute sea water, and shrimp in these environments demonstrate potassiumuptake rates similar to shrimp in dilute seawater. Some insight into this situation might be found in a grow-out study (Sowers, Atwood, Browdy, Tomasso, Unpublished) in which one group of shrimp was grown in 2 g/l dilute seawater while a second group was grown in 1 g/l dilute seawater plus 1 g/l of the mixed-ion solution. After days, the shrimp had grown from postlarvae to 10 g. No significant differences in performance (survival, growth, feed conversion) between the treatments were observed. Apparently, 1 g/l sea salt adequately provided some other ion to the mixed-ion solution required by the animal and in some way linked to potassium homeostasis. Acknowledgments Jeff Isely provided guidance on the statistical analyses. 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