Gene expression and activity of carbonic anhydrase in salinity stressed Penaeus monodon

Carbonic anhydrase (CA) was identified by differential display PCR analysis as one of the differentially expressed genes in the gills of low salinity stressed (transferred from 25 to 3 ppt) Penaeusmonodon. To further characterize the role of CA in the regulation of salinity stress, the cDNA sequence of P.monodon carbonic anhydrase (PmCA) was attained by rapid amplification of cDNA ends and found to have a total length of 1194 bp. The deduced amino acid of PmCA shares 73% sequence identity with the CA homologue recently isolated from the crab, Callinectessapidus. Real time RT-PCR and enzymatic activity analyses were employed to determine the changes in the PmCA mRNA expression and total CA activity, respectively, after shrimps were transferred from 25 to 3 ppt salinities for up to 2 weeks. Compared to the CA level in the control group (25 ppt), PmCA mRNA was significantly increased in shrimp gills at 24 h after hypo-osmotic stress. In contrast, the epipodites and antennal gland displayed decreased levels of mRNA expression. The gross CA enzymatic activity after hypo-osmotic stress was increased in the shrimp gills but remained stable in the epipodites and antennal gland.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Differential induction of branchial carbonic anhydrase and NA(+)/K(+) ATPase activity in the euryhaline crab,Carcinus maenas,in response to low salinity exposure

The time course of induction of activity of carbonic anhydrase (CA) and Na/K ATPase, two enzymes that are central to osmotic and ionic regulation in the eyryhaline green crab, Carcinus maenas, was measured in response to a transfer from 32 to 10 ppt salinity. CA activity was low in all gills in crabs acclimated to high salinity. Activity was induced in the posterior three gills (G6-G9) starting at 96 hr following transfer to low salinity, with activity peaking at seven post-transfer. Na/K ATPase activity in posterior gills was already high in crabs acclimated to 32 ppt salinity, and it did not increase as a result of transfer to 10 ppt. Acclimation of crabs to hypersaline (40 ppt) conditions resulted in uniformly low levels of Na/K ATPase activity, and transfer from 40 ppt to 10 ppt stimulated a four-fold induction of activity in the posterior gills that was evident by seven days of low salinity exposure. Low salinity stimulates the activity of both enzymes, but a different degree of salinity change appears to be necessary to cause the induction of each enzyme. The Na/K ATPase activity is already high at a salinity (32 ppt) at which the crab is still an osmotic and ionic conformer. CA activity, however, even when expressed in low levels, is still present in excess of what is needed to supply counterions at a rate adequate to match the rate of active ion transport. It is possible that two strategies exist for the regulation of these two enzymes that coincide with the crab's intertidal and estuarine lifestyle: short-term modulation of activity of highly expressed enzyme (Na/K ATPase) and long-term modulation of enzyme concentration by changes in gene expression (CA). For all ranges of low salinity exposure, crabs undergo hemodilution, cell swelling, and subsequent cell volume readjustment as evidenced by the increase in concentration of TNPS in the hemolymph. This response takes place before the induction of enzyme activity, and it could serve as the initial signal in the induction pathway.     

Cloning and Expression of Four Aquaporin Homologs from the Chinese Black Sleeper (Bostrychus sinensis): The Effects of Salinity Acclimation

Several fish species are known to possess mechanisms that allow them to adapt to environments with different salinities. The aim of this study was to investigate the effects of salinity on the expression of aquaporins (aqp1a, aqp3a, aqp8a, and aqp9a) in the gills and intestines of Chinese black sleeper. After 30 days of acclimation, the expression of aqp1a, aqp3a, and aqp9a in the gills was significantly higher in fish transferred to 5 ppt than in those transferred to 40 ppt seawater, whereas aqp8 expression was lower. In contrast, aqp1a, aqp3a, and aqp8a expression in the intestines was higher in fish acclimated in 40 ppt than in those acclimated in 5 ppt. During abrupt salinity acclimation, the levels of aqp1a and aqp9a in the gills varied over time in fish acclimated in 5 ppt, but not in 40 ppt. The aqp3a levels in gills were higher in the 5 ppt group after 24 h than in the 40 ppt. The expression level of aqp8a in gills was higher in 40 ppt than in 5 ppt, except for that at 12 h. In the intestines, expression level of aqp1a and aqp8a were significantly upregulated from 12 to 48 h following acclimation in 40 ppt and aqp3a was higher in 40 ppt group than in 5 ppt, while aqp9a expression exhibited an opposite trend. These findings suggest that aqp1a, aqp3a, aqp8a and aqp9a may play a major osmoregulatory role in water transport in the gills and intestines during acclimation to different salinity environment.

     

Neuroendocrine regulation of carbonic anhydrase expression in the gills of the euryhaline green crab, Carcinus maenas

The relationship between branchial carbonic anhydrase (CA) activity, CA gene expression and salinity, and potential mechanisms of regulation, was investigated in the euryhaline green crab, Carcinus maenas, acclimated to 33 ppt and transferred to 10 ppt, and the stenohaline rock crab, Cancer irroratus, acclimated to 32 ppt and transferred to 18 ppt. CA activity in green crabs acclimated to high and low salinity was a function of CA mRNA expression, with low salinity exposure resulting in an increase in both CA expression and activity. Eyestalk ablation (ESA) in green crabs acclimated to high salinity resulted in an increase in CA expression in the posterior, ion-transporting gills, in the absence of the low salinity stimulus. There were no changes in CA activity or expression in the anterior, respiratory gills. ESA also potentiated low salinity-stimulated CA induction, again, only in posterior gills. There were no changes in CA activity in any gills of Cancer irroratus, in response to either ESA or low salinity. These results suggest that CA expression in euryhaline, osmoregulating species, is under inhibitory regulation by a putative repressor found in the eyestalk, and that this mechanism is absent in stenohaline, osmoconforming species. CA expression is maintained at low, baseline levels in crabs acclimated to high salinity by the presence and action of this compound. The effects of the repressor appear to be reduced upon exposure to low salinity, allowing CA induction to occur.     

Early carbonic anhydrase induction in the gills of the blue crab, Callinectes sapidus, during low salinity acclimation is independent of ornithine decarboxylase activity

Carbonic anhydrase (CA) induction in the gills of the euryhaline blue crab, Callinectes sapidus, was measured in response to lowered environmental salinity. Simultaneous measurements of ornithine decarboxylase (ODC) activity were made in gills and nonbranchial tissues to determine whether ODC activity and the resultant synthesis of polyamines played a role in the initiation and regulation of CA induction. CA induction in the seventh gill pair (G7) was proportional to the decrease in ambient salinity, but activity in the third gill pair (G3) remained unchanged. Induction began by 24 hr after low salinity transfer, much earlier than previously reported, and peaked after 4 days. The magnitude of salinity change affected the magnitude of CA induction only, not the time course. A general cell volume regulatory response, as measured by the appearance of total ninhydrin-positive substances (TNPS) in the hemolymph, was initiated within 4 hr of low salinity transfer and was complete by 24 hr post-transfer. General cell swelling may be the initial signal in the pathway of CA induction. ODC activity in the gills of acclimated animals was not influenced by salinity. For crabs transferred from 35 to 25 ppt, ODC activity did not change significantly over the time course of acclimation. There was an early but transient increase in ODC activity in all tissues for crabs acclimated to 28 ppt and transferred to 15 ppt. Induction of ODC activity does not appear to be a precursor for CA induction; therefore, it does not appear that polyamines are substantially involved in the up-regulation of transport enzyme activity in low salinity. ODC, and resultant polyamine synthesis, may, however, have a role in cell volume regulation.     

Effect of salinity on the immune response of tiger shrimp Penaeus monodon and its susceptibility to Photobacterium damselae subsp. damselae

Addition of NaCl at 2.5% to tryptic soy broth (TSB) significantly increased the growth of Photobacterium damselae subsp. damselae. Tiger shrimp Penaeus monodon held in 25 per thousand seawater were injected with P. damsela subsp. damselae grown in TSB containing NaCl at 0.5%, 1.5%, 2.5% and 3.5% at a dose of 8.48 x 10(4)colony-forming units (cfu)shrimp(-1). Over 24-96 h, the cumulative mortality was significantly higher for the shrimp challenged with P. damselae subsp. damselae grown in 2.5% NaCl than those grown in 0.5%, 1.5% and 3.5% NaCl. In another experiment, P. monodon held in 25 per thousand were injected with TSB-grown P. damselae subsp. damselae (8.48 x 10(4)cfushrimp(-1)), and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand. After 96 h, the mortality was highest for the P. damselae subsp. damselae-injected shrimp held in 5 per thousand, and the lowest for the P. damselae subsp. damselae-injected shrimp held in 25 per thousand. In a separate experiment, P. monodon held in 25 per thousand and then transferred to 5 per thousand, 15 per thousand, 25 per thousand (control) and 35 per thousand were examined for immune parameters, and phagocytic activity and clearance efficiency of P. damselae subsp. damselae after 12-96 h. The THC, hyaline cell, phenoloxidase activity, respiratory burst, superoxide dismutase (SOD) activity, phagocytic activity and clearance efficiency decreased significantly for the shrimp held in 5 per thousand, 15 per thousand and 35 per thousand after 12h. It is concluded that tiger shrimp P. monodon transferred from 25 per thousand to low salinity levels (5 per thousand and 15 per thousand) and high salinity (35 per thousand) had reduced immune ability and decreased resistance against P. damselae subsp. damselae infection.     

The role of the antennal glands in ion and body volume regulation of cannulated Penaeus monodon reared in various salinity conditions

Urinary production rate and the osmotic and ionic concentrations in both urine and hemolymph were measured in cannulated intermolt Penaeus monodon which were either abruptly transferred from 45 ppt seawater to 15 ppt seawater (Experiment 1) or acclimated to 5, 25 and 45 ppt seawater (Experiment 2). In Experiment 1, urinary magnesium concentration fell dramatically from 228 to 30 mEq/l within 4 h post-transfer, but 8 h after transfer, U/H (urine/hemolymph) ratios stabilized at between 1.0 and 2.5. Sodium was higher in urine than in hemolymph during the first 24 h after transfer, while potassium was lower in urine than in hemolymph until 72 h after transfer, which suggests that sodium and potassium concentrations are regulated by the antennal gland after an abrupt change in media. In Experiment 2, the urinary production rate of P. monodon decreased as salinity increased, suggesting that the antennal glands also regulate body volume. In the acclimated shrimps of Experiment 2, the antennal glands did not appear to regulate osmolarity or the concentration of chloride, sodium, potassium, and calcium ions, but as salinity increased, U/H ratios of magnesium increased from 2.3 to 13.5, and active secretion by the antennal gland accounted for 57 approximately 93% of the total magnesium excretion through urine. These results suggest that active secretion of magnesium by the antennal gland enable this shrimp to maintain hypoionic levels of magnesium in the hemolymph.     

Joint action of elevated ambient nitrite and nitrate on hemolymph nitrogenous compounds and nitrogen excretion of tiger shrimp Penaeus monodon

Penaeus monodon (12.13+/-1.14 g) exposed individually to six different nitrite and nitrate regimes (0.002, 0.36 and 1.46 mM nitrite combined with 0.005 and 7.32 mM nitrate), at a salinity of 25 ppt, were examined for hemolymph nitrogenous compounds and whole shrimp's nitrogen excretions after 24 h. Nitrogen excretion increased directly with ambient nitrite and nitrate. Hemolymph nitrite, nitrate, urea and uric acid levels increased, while hemolymph ammonia, oxyhemocyanin and protein were inversely related to ambient nitrite. Exposure of P. monodon to elevated nitrite in the presence of 7.32 mM nitrate did not alter hemolymph nitrite, ammonia, uric acid, oxyhemocyanin and protein levels, but caused an increase in hemolymph nitrate and a decrease in hemolymph urea as compared to exposure to elevated nitrite only. Following exposure to elevated nitrite, nitrite was oxidized to nitrate and P. monodon showed uricogenesis and uricolysis. The shrimp also used strategies to avoid joint toxicities of nitrite and metabolic ammonia by removing ammonia or reducing ammonia production under the stress of elevated nitrite.     

Growth hormone transgenesis affects osmoregulation and energy metabolism in zebrafish (Danio rerio)

Growth hormone (GH) transgenic fish are at a critical step for possible approval for commercialization. Since this hormone is related to salinity tolerance in fish, our main goal was to verify whether the osmoregulatory capacity of the stenohaline zebrafish (Danio rerio) would be modified by GH-transgenesis. For this, we transferred GH-transgenic zebrafish (T) from freshwater to 11 ppt salinity and analyzed survival as well as relative changes in gene expression. Results show an increased mortality in T versus non-transgenic (NT) fish, suggesting an impaired mechanism of osmotic acclimation in T. The salinity effect on expression of genes related to osmoregulation, the somatotropic axis and energy metabolism was evaluated in gills and liver of T and NT. Genes coding for Na⁺, K⁺-ATPase, H⁺-ATPase, plasma carbonic anhydrase and cytosolic carbonic anhydrase were up-regulated in gills of transgenics in freshwater. The growth hormone receptor gene was down-regulated in gills and liver of both NT and T exposed to 11 ppt salinity, while insulin-like growth factor-1 was down-regulated in liver of NT and in gills of T exposed to 11 ppt salinity. In transgenics, all osmoregulation-related genes and the citrate synthase gene were down-regulated in gills of fish exposed to 11 ppt salinity, while lactate dehydrogenase expression was up-regulated in liver. Na⁺, K⁺-ATPase activity was higher in gills of T exposed to 11 ppt salinity as well as the whole body content of Na⁺. Increased ATP content was observed in gills of both NT and T exposed to 11 ppt salinity, being statistically higher in T than NT. Taking altogether, these findings support the hypothesis that GH-transgenesis increases Na⁺ import capacity and energetic demand, promoting an unfavorable osmotic and energetic physiological status and making this transgenic fish intolerant of hyperosmotic environments.     

Development of a monoclonal antibody specific to yellow head virus (YHV) from Penaeus monodon

A monoclonal antibody specific to yellow head virus (YHV) was produced from a mouse immunized with gill extracts prepared from laboratory-reared Penaeus monodon dually infected with YHV and white spot syndrome virus (WSSV). One clone designated V3-2B specifically bound to native and SDS-treated viral specific antigens. Immunocytochemical studies of infected gills revealed viral specific immunoreactivities in the cytoplasm of gill tissue and in haemocytes. No antibody binding was observed in gills from non-infected shrimp. In addition, immunocytochemical examination of tissues from shrimp experimentally infected with YHV gave a positive reaction, while tissues from uninfected control shrimp or shrimp experimentally infected with WSSV did not. Western blot analysis indicated that the antibody reacted with a protein of approximately 135 kD that was present only in shrimp infected with YHV. In dot-blot indirect immunoperoxidase assays, the antibody was able to detect viral associated antigen in diluted haemolymph up to 1:50 dilution and in an ammonium sulfate precipitate of haemolymph up to 1:1000 dilution. The results suggested that this antibody might be useful for development of effective diagnostic techniques for both heavy and mild YHV infections in shrimp.     

Cloning and molecular characterization of heat shock cognate 70 from tiger shrimp (Penaeus monodon)

We cloned the complementary deoxyribonucleic acid (cDNA) of the heat shock cognate 70 (hsc70) gene of tiger shrimp (Penaeus monodon). It was 2207 bp long and included a 1959-bp coding region, a 40-bp flanking region at the 5' end, and a 208-bp flanking region at the 3' end. The deduced, 652-amino acid sequence had a molecular mass of 71 481 Da and an estimated isoelectric point (pI) of 5.2. Based on phylogenetic analysis, the gene is clustered with the hsc70 proteins of invertebrates and vertebrates. In native gel electrophoresis, recombinant P. monodon hsc70 expressed in an Escherichia coli system is tightly associated with carboxymethylated alpha-lactalbumin (CMLA), which indicates that hsc70 probably functions as a chaperone. In an in vitro adenosine triphosphatase assay, recombinant hsc70 hydrolyzed adenosine triphosphate to adenosine-5'-diphosphate and increased hydrolysis activity by binding to unfolded peptide, CMLA. In situ hybridization using an antisense riboprobe revealed that the hsc70 gene was active in most tissues of unstressed shrimp. The expression of hsc70 messenger ribonucleic acid (mRNA) in hemocytes increased 2- to 3-fold at the first hour after shrimp experienced heat shock and 0.5-hour recovery. Hsc70 mRNA decreased gradually to the background level. Cloning and characterizing the P. monodon hsc70 gene is the first, crucial step in studying the relationship of heat shock proteins with the stress or immune responses of shrimp.