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1 Available online at International Journal of Research in Fisheries and Aquaculture Universal Research Publications. All rights reserved ISSN Original Article Na + /K + -ATPase and Carbonic anhydrase activity in the tissue of Post larvae (P. monodon) acclimated to different salinities. P. N. Pallavi Dept. of Biotechnology, S. V. University, Tirupati , India toxpal@gmail.com Received 29 July 2015; accepted 23 August 2015 Abstract Tiger prawn Peneaus monodon (Fabricus) is an economically important species widely cultured in marine water Aquaculture. The experiment was conducted to study the effect of low salinity on the activity of Na + /K + - ATPase and Carbonic anhydrase activity at different salinity levels (30 to 25, 20, 15, 10, 5, 0 ppt). Objective of the study is to refined the existing post larvae production technology through the low salinity culture system during larval progression. Results is known to induce a myriad of physiological changes corresponding with active ion uptake across the gill and muscle. Na + /K + -ATPase and Carbonic anhydrase activity in the muscle and gills were measured after they were submitted to a salinity challenge in dilute seawater (0 ppt) after 7days. Enzyme activity was consistently higher in gills than in muscle. An increase in Na + /K + -ATP ase and CA activity at a reduced salinity is consistent with a strong hyper-osmoregulatory capacity in PLs, a low activity at an enhanced salinity suggests a physiological response, directed towards a reduction of Na + uptake Universal Research Publications. All rights reserved Key words: P. monodon, salinity, adaptation, Na + /K + - ATPase, Carbonic anhydrase. 1. INTRODUCTION: In recent years, tremendous developments have been made in farming of shrimps all over the world. The giant tiger shrimp, P. monodon, is the major species cultured and accounts for 60% of total shrimp production from farms worldwide (1). It has the fastest growth rate among a number of penaeid species reared in captivity (2) and is the largest species of shrimp in the world. P. monodon is markedly euryhaline and tolerates wide variations in salinity. Since culture of P. monodon in extremely high salinities of over 30 ppt may cause disease problems, particularly white spot or luminescent bacteria, more shrimp farmers keep moving towards brackish water or freshwater areas (3,4). Marine crustaceans have two general strategies for coping with salinity stress in dilute environments such as an estuary; osmoconformity and osmoregulation. Osmoconformers passively maintain hemolymph (mixture of blood and interstitial fluid) osmolality and ionic concentrations equal to the surrounding medium, while osmoregulators employ physiological pathways to actively maintain hemolymph hypertonic to the environment. The gills are extremely permeable to the environment; thus hyposaline waters lead to large-scale osmotic influxes and ionic effluxes. However, all gills in shrimps possess MRCs and are involved in osmoregulation, although some areas of individual gills remain specialized for respiration (5,6). Active salt absorption in the MRCs is accomplished via a supporting enzymes (7,8): in this context, Na+ absorption occurs via the basolateral Na +/ K + -ATPase (NKA) and cytoplasmic Carbonic anhydrase (CA). This report represents a study of the Na+/K+-ATPase and CA induction in response to low salinity exposure in the PLs of P.monodon. 2. MATERIAL AND METHODS 2.1 Experimental Animals and Design: PLs of P. monodon were obtained from a commercial farm in Muttembakam village near Nellore, Andhra Pradesh and were transferred to the laboratory in aerated plastic containers and maintained in laboratory holding tanks for a week in continuously aerated and filtered marine water (30 ppt) with a 12h light-dark cycle. During this period the prawns were fed adlibitum with commercial pelleted feed for day. After the PLs 10 mg were separately acclimated from 30ppt to six salinity levels, i.e. 25, 20, 15, 10, 5 and 0 ppt. the animals were randomly stocked into 6 tubs a density of 20 PLs per tub 115

2 Effect of Salinities on Na + K + - ATPase activity in the gill and muscle of PLs of P. monodon. (µmoles Pi/mg Protein/h). Data are expressed as mean ± SD of 6 observations; P>0.05. Effect of Salinities on Mg2 + ATPase activity in the gill and muscle of PLs of P.monodon. (µmoles Pi/mg Protein/h). Data are expressed as mean ± SD of 6 observations; P>0.05. with four replicates. Once a day changing at least 25% of crude homogenates were centrifuged at 1,000 g for 10min the ambient medium and tap water was aerated before at 4 C in a cooling centrifuge to sediment nuclei and tissue being added to the tub to adjust salinity. Water quality debris. Again the supernatant fractions were centrifuged at parameters including ph, salinity, temperature, dissolved 10,000 g for 10min at 4 C to obtain the clear supernatant oxygen and ammonia were monitored throughout the which was referred as sample. experimental period. 2.3 Na + /K +, Mg 2+ and Ca2 + ATPase activities: 2.2 Sample Collection: The Method of Na + /K + -ATPase activity assay were Animals were sacrificed by removing their slightly modified from (9). Na + /K + -ATPase, Mg 2+ ATPase carapace from their abdomen with a forcipes and the gills and Ca 2+ ATPase activity was determined by measuring and abdominal muscle tissues were dissected out quickly. ATP hydrolysis in a reaction medium containing ATP, 10 Tissues were washed in ice-cold normal saline (0.67%, mm MgCl 2, 100 mm NaCl, 30 mmkcl, 20 mm imidazole w/v). A 10% (w/v) homogenate of tissues were prepared at hydrochloric buffer, ph 7.2 and µg of Protein. 4 C in the homogenizing buffer (50 mm Tris- HCl, 1 mm Residual Mg 2+ ATPase Activity was assayed in the same EDTA, 0.5 mm Sucrose, 150 mm KCl ph 7.8). Tissues medium but without KCl in the presence of Ouabain were homogenized in pre-cooled mortar and pestle. The (Specific inhibitor of Na + /K + -ATPase). Na + /K + -ATPase 116

3 Effect of Salinities on Ca2 + ATPase activity in the gill and muscle of PLs of P. monodon. (µmoles Pi/mg Protein/h). Data are expressed as mean ± SD of 6 observations; P>0.05. Effect of Salinities on Carbonic anhydrase activity in the gill and muscle of PLs of P. monodon. (µmole p- nitrophenyl/mg Protein /min) Data are expressed as mean ± SD of 6 observations; P>0.05. activity was determined as the difference between the two assays. Ca 2+ ATPase Activity was assayed in the same medium but without NaCl, KCl and in the presence of 0.05mM CaCl 2. An aliquot of the corresponding samples was added to the reaction mixture and pre-incubated for 5 min at 37 0 C. The reaction was initiated by the addition of ATP. Incubation was carried out at 37ºC for 30 min. Activity was expressed in µmoles of Pi liberated per mg protein per hour. 2.4 Carbonic anhydrase activity: Carbonic anhydrase activity was measured by the method of (10,11). The assay system contained 50 mm phosphate buffer (ph 6.8), 40 µl of crude enzyme extract and 5mM p- nitrophenyl acetate to make the final volume of 2 ml. Incubation was done for 30 min at 30 0 C and absorbance was measured at 348 nm. Activity was expressed in µmoles p- nitrophenol produced per min per mg protein. 3. RESULTS 3.1 Na + /K + ATPase Activity: ATP ase activities (measure as Pi, given in moles) was significantly affected by salinity and tissue type. The activity of Na + /K + ATPase, when considering the main effect was significantly higher in gill than in muscle. The activity of Na + /K + ATPase increased by up to 35 % in gill and 11.4 % in muscle of PLS acclimated to low salinities. The activity of Mg 2+ ATPase increased by up to 17.8% in gill and 6.7% in muscle, similarly Ca 2+ ATPase increased by up to 8.5% in gill and 2.1% in muscle was significantly higher than the respective control. 3.2 Carbonic anhydrase activity: Significant increase was observed in effect of salinity for the activity of carbonic anhydrase, although a trend for a higher activity was observed at 0 ppt. The activity of carbonic anhydrase increased by up to 10% in gill and 4% in muscle was significantly higher than the respective control. 4. STATISTICAL ANALYSIS Results are presented as mean ± standard error of mean (SEM). Difference between the control and treatment means were compared by t-test, whereas difference among the control and various treatment mean were analysed by one way analysis of variance (ANOVA), followed by Duncan s new multiple range test. Differences were considered statistically significant when P < DISCUSSION Salinity is thought to act on independent aspects of physiology: osmoregulation and growth. Osmoregulation in crustaceans depends primarily on the salt pump (usually located at the gill) and low integumentary permeability to salt and water (12). Exposure to low salinity increases the activity of Na + K + -ATPase in post larvae was observed. Neuro endocrine control of osmoregulation also appears during postlarvae stages thus, an integrated series of events in post larvae links the appearance of osmoregulatory tissues. In increase of Na + K + -ATPase activity, the occurrence of hyper regulation at low salinity and an increase in salinity tolerance (13). Despite there being a single common external signal (low salinity), The two transport enzymes studied here respond differently. At 30 ppt, a salinity at which the PLs of P. monodon is an osmotic and ionic conformer, when acclimated to low salinity, usually below 15 ppt many species become osmoregulators, employing physiological mechanisms to actively maintain hemolymph osmolality and ionic concentrations at levels well above ambient.the gills of marine crustaceans in general are extremely permeable to the environment; thus hyposaline waters lead to large-scale osmotic influxes and ionic effluxes (14-17). This is compounded by the fact that crustacean urine is generally iso osmotic with hemolymph; thus excretion at low-salinity results in ion loss. Marine crustaceans produce copious amounts of this urine at low salinity to volumetrically compensate for osmotic water influxes. Thus, urinary excretion contributes up to 43 and 31% of the total Na + and Cl - loss, respectively (18). Without compensatory mechanisms, these ion losses through the gill and urine would result in hemolymph dilution and cellular swelling. Marine crustaceans are able to manage cell swelling via the cellular efflux of organic osmolytes to reduce the osmotic gradient between the cell and the dilute hemolymp (19). Both osmoregulators and osmoconformers are capable of employing this strategy to reduce cell swelling; however, osmoregulators take low-salinity adaptation one step further by utilizing ionic uptake mechanisms to counteract 117

4 hemolymph dilution. Crustacean ionic uptake occurs across the gills in specialized mitochondria-rich cells (MRC). It is the concerted action of several proteins in the MRC that facilitates the transport of ions from the environment, across the apical gill membrane, into the cytoplasm, across the basolateral membrane, and finally into the hemolymph. The Na + /K + -ATPase, localized in the basolateral membrane of the gill, is believed to provide the driving force for active ion uptake (20); while CA concentrated in the cytoplasm is believed to provide H + and HCO 3 - to support Na + and Cl uptake across the apical gill membrane (21-23). Baseline levels of activity of both enzymes are found in all gills in crustaceans acclimated to low salinity and those levels increase between five- and ten-fold during acclimation to low salinity. Furthermore, both CA and the Na + /K + -ATPase have been shown to be associated with the mitochondria-rich chloride cells, the site of active ion transport in the gill, and changes in enzyme activity have been correlated with corresponding changes in chloride cell density (24,25). While changes in transport activity have been described in detail, the mechanism behind those changes remains largely unknown. It is important because hemolymph osmotic and ionic concentrations in euryhaline crustaceans stabilize within 24 h after a salinity transfer (26), and if these enzymes represent the molecular basis of ion regulation, then changes in their activities should be congruent with changes in systemic adaptations. (27) originally reported a rapid (2 h) salinity-stimulated increase in Na + /K + -ATPase activity in the gills of the blue crab, Allinectes sapidus, suggesting the activation of a preexisting pool of enzymes. For these two enzymes, the majority of the evidence supports the hypothesis that upregulation is a result of de novo synthesis of new protein (24,9). So, it appears that the onset of osmoregulation is contingent upon the activation of the specific genes responsible for the synthesis of these transport-relate proteins. Na + K + -ATPase and CA activity was low in muscle because it is secondary to hyperregulation, this provide an energy source through mobilization of lipids (28). This enzymes activity was high in gill due to this is primarily to osmo-ionoregulation. The results of low salinity adaptation further support the hypothesis that low salinity-mediated CA induction is under transcriptional regulation. The same pattern was found in other hyperregulating decapods (29), e.g. C. maenas (30,31), H.ammarus (32), Chasmagnathus granulata (33). Adenosine triphosphatases (ATPase) are involved in the maintenance of membrane permeability and energy production (34). Mg 2+ ATPase activity suggests that energy yielding processor recover more rapidly than ion fluxes across cell membranes controlled by Na + K + -ATPase. In summary, our results show that the Na + K + - ATPase and CA activity under hyperregulating conditions suggests the participation of these enzymes in response biochemical levels to varying environmental salinity. These activities in gills and muscle of PLs of P. monodon are involved in physiological processes primarily and secondary to osmo-ionoregulation. We focus on establishing the exact physiological role of these gill and muscle activities in the integrative adaptive responses of euryhaline prawns to varying environmental conditions. Conclusion and Recommendation: The survival of Post larvae (P. monodon) rearing in low salinity (0 ppt) was high and slightly different compared to those rearing larvae in salinity 30 ppt. This technique easily can be introduced to the farmers. 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