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The sensitivity of Penaeus japonicus to fenitrothion increased from larval stages to postlarvae and to juveniles. Larval stages from nauplii to mysis are the most tolerant and postlarvae are the most sensitive to fenitrothion. In juveniles kept in seawater and in diluted seawater, fenitrothion was observed to decrease the hypo- and hyper-osmoregulatory capacity in young shrimps after exposure to lethal and sub-lethal concentrations of fenitrothion in a dose and time-dependent manner.

The activity, immunolocalization, and mRNA expression of Na, K-ATPase were studied in gills of the hyper-/ hypoosmoregulating crab Pachygrapsus marmoratus acclimated to salinities of 10, 37, and 45 ppt. Enzyme activity was significantly higher in pooled posterior gills (7-9) than in anterior gills (1-6) and increased significantly upon acclimation to diluted medium, confirming previous observations with other crab species. Immunofluorescent analysis of gill sections indicated localization of Na, K-ATPase protein in basolateral membranes of branchial epithelium adjacent to the afferent blood vessel. Using degenerate primers based on Na, K-ATPase ALPHA subunit homologies published in GenBank, we amplified related sequences in cDNA transcribed from gill mRNA and employed the resulting PCR products for sequencing and mRNA expression analyses. Immunofluorescence and mRNA quantitation produced parallel results, showing high expression of Na, K-ATPase protein and mRNA in gills 6, 7, and 8 at low salinity. In gills 6 and 8, the abundance of Na, K-ATPase protein and mRNA increased dramatically with decreasing salinity. However, gill 7 showed substantial levels of Na, K-ATPase protein and mRNA even in 45 ppt seawater, suggesting that this gill may play an important role in hypoosmoregulation. Our results offer a molecular explanation for the previously observed changes in Na, K-ATPase activity in gills of euryhaline crabs adjusting to osmoregulatory demands in salinity extremes, namely the upregulation of Na, K-ATPase gene transcription and subsequent translation of Na, K-ATPase protein. Supported by NSF (IBN-9807539).

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na, K-ATPase activity in posterior gills, but it is not clear whether the response is due to activation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' untranslated region of the mRNA coding for the ALPHA subunit of Na, K-ATPase were amplified and sequenced by RT-PCR. The resulting 3,433-nucleotide cDNA encodes a putative 1,011-amino acid protein with a predicted molecular mass of 112.5 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The ALPHA subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83% identity to other arthropods and 74-77% identity to vertebrate and non-arthropod invertebrate species. Semi-quantitative PCR showed high levels of ALPHA subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na, K-ATPase ALPHA subunit protein band of the expected size. Posterior gills contained a much greater level of ALPHA subunit protein compared with anterior gills, in agreement with historical measurements of enzyme activity. Transfer of blue crabs from 35 to 5 ppt salinity was accompanied by modest increases in ALPHA subunit mRNA and protein in posterior gills, providing a molecular explanation for the enhanced Na, K-ATPase enzyme activity that accompanies the hyperosmoregulatory response. Supported by NSF (IBN-9807539).

The study tests the physiological responses of Persian sturgeon, Acipenser persicus, during the abrupt release of juveniles from freshwater ( FW) into brackish waters ( BW = 11‰) of the Caspian Sea. Fish weight at release was 2-3 g (2.55 ± 0.41 g; 8.8 ± 0.58 cm TL). Totals of 160 individuals were randomly distributed into four fiber-glass aerated tanks (volume 60-L). Two tanks served as controls ( FW groups), and two as exposure tanks for BW (Caspian Sea water = CSW). Fish were sampled at 0, 3, 6, 12, 24, 48 and 96 hr after abrupt transfer to CSW. Plasma osmolality, immunolocalization of Na+, K+ - ATPase ( NKA) and Na+/K+/2Cl- ( NKCC) Co-transporter, NKA activity and the NKA α-subunit mRNA expression were analyzed. Blood osmolality of fish transferred from FW to CSW increased significantly within hours post-transfer ( p < .05) and remained at a high level for up to 96 hr. Immunolocalization of NKCC indicated co-localization with NKA in the chloride cells in the gill epithelium. A partial sequence of the NKA α-subunit (632 bp) is described. Its expression levels were up-regulated at 12 and 48 hr following salinity transfer ( p < .05). However, NKA activity sharply increased in CSW specimens by almost 2.8-fold ( p < .05) between 48 and 96 hr after transfer. Gill NKCC co-transporter abundance increased, coinciding with increased gill NKA activity. The increased activity of NKCC during salt excretion in CSW may lead to an influx of Na+ into the chloride cells. Consequently, NKA activity increases to maintain intracellular Na+ homeostasis. [ABSTRACT FROM AUTHOR]

Changes in cellular ultrastructure and replication associated with the dramatic digestive regulation of the Burmese python (Python molurus) with fasting and feeding were examined for gastric and intestinal epithelium. In fasted pythons, crypt cells in the gastric fundic area were packed with secretory vesicles and presented an apical tubulo- vesicular system typical of inactive cells. 12 hours post-feeding, crypts were enlarged and cell length was reduced by half due to flushing of secretory granules and formation of microvilli-like digitations within the crypt lumen. 1 day post-feeding, active cells packed with mitochondria and zymogen granules mostly appeared underneath the elongated digitations. 3 days post-feeding, cells were less active as they contained numerous cytoplasmic secretory vesicles, the apical digitations were shorter, and the apical tubulo-vesicular system had reappeared. Replicating cells at the neck region of the fundic glands were typically observed 3 days post-feeding. This was in contrast to intestinal epithelium where cellular replication was evident 1 day postfeeding. Up to 26% of epithelial cells had replicated by day 3 of digestion. Therefore, an adaptive response of pythons in coping with infrequent meals is to maintain a quiescent gut while fasting and m quickly upregulate cell function with feeding. The stomach and intestine employ different upregulatory strategies; gastric cells jump start function as soon as prey has been consumed, with cell renewal occurring later, whereas epithelial cells of the small intestine overlap the upregulation of function with replication.

The structure of the kidney and the localization of Na(+), K(+)-ATPase (NKA) immunopositive cells were examined throughout the postembryonic development of the Persian sturgeon, Acipenser persicus, from newly hatched prelarvae (10mm) to 20 days post hatch (20 DPH) larvae (31mm). Investigations were conducted through histology and immunohistochemistry by using the light and immunofluorescence microscopy. The pronephros was observed in newly hatched prelarvae. The cells lining the distal pronephric tubules and their collecting ducts showed laterally expressed NKA immunofluorescence that later extended throughout the whole cytoplasm. Mesonephrogenous placodes and pre-glomeruli were distinguished at 2 DPH along the collecting ducts posteriorly. Their tubules were formed and present in kidney mesenchyma, differentiated into neck, proximal, distal and collecting segments at 7 DPH when NKA immunopositive cells were observed. Their distal and collecting tubules showed an increasing immunofluorescence throughout their cytoplasm while the glomeruli remained unstained. From D 9 to D 17, the epithelial layer of pronephric collecting duct changed along the mesonephros to form ureters. Ureters, possessing isolated strong NKA immunopositive cells, appeared as two sac-like structures hanging under the trunk kidney. Since NKA immunopositive cells were not observed on the tegument or along the digestive tract of newly hatched prelarva, and also the gills are not formed yet, the pronephros is the only osmoregulatory organ until 4 DPH. At the larval stage, the pronephros and mesonephros are functional osmoregulatory organs and actively reabsorb necessary ions from the filtrate., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Many studies have shown that hyperosmoregulation in euryhaline crabs is accompanied by enhanced Na(+)+K(+)-ATPase activity in the posterior gills, but it remains unclear whether the response is due to regulation of pre-existing enzyme or to increased gene transcription and mRNA translation. To address this question, the complete open reading frame and 3' and 5' untranslated regions of the mRNA coding for the alpha-subunit of Na(+)+K(+)-ATPase from the blue crab Callinectes sapidus were amplified by reverse transcriptase/polymerase chain reaction (RT-PCR) and sequenced. The resulting 3828-nucleotide cDNA encodes a putative 1039-amino-acid protein with a predicted molecular mass of 115.6 kDa. Hydrophobicity analysis of the amino acid sequence indicated eight membrane-spanning regions, in agreement with previously suggested topologies. The alpha-subunit amino acid sequence is highly conserved among species, with the blue crab sequence showing 81-83 % identity to those of other arthropods and 74-77 % identity to those of vertebrate species. Quantitative RT-PCR analysis showed high levels of alpha-subunit mRNA in posterior gills 6-8 compared with anterior gills 3-5. Western blots of gill plasma membranes revealed a single Na(+)+K(+)-ATPase alpha-subunit protein band of the expected size. The posterior gills contained a much higher level of alpha-subunit protein than the anterior gills, in agreement with previous measurements of enzyme activity. Immunocytochemical analysis showed that the Na(+)+K(+)-ATPase alpha-subunit protein detected by alpha5 antibody is localized to the basolateral membrane region of gill epithelial cells. Transfer of blue crabs from 35 to 5 per thousand salinity was not accompanied by notable differences in the relative proportions of alpha-subunit mRNA and protein in the posterior gills, suggesting that the enhanced Na(+)+K(+)-ATPase enzyme activity that accompanies the hyperosmoregulatory response may result from post-translational regulatory processes.

We examined the ontogeny of the osmoregulatory sites of the branchial cavity in embryonic and early postembryonic stages of the European lobster Homarus gammarus through transmission electron microscopy, immunofluorescence microscopy, and immunogold electron microscopy using a monoclonal antibody IgGalpha(5) raised against the avian alpha-subunit of the Na(+),K(+)-ATPase. In mid-late embryos, Na(+),K(+)-ATPase was located along the pleurites and within the epipodite buds. In late embryos just before hatching, the enzyme was confined to the epipodite epithelia. After hatching, slight differentiations of ionocytes occured in the epipodites of larval stages. Na(+),K(+)-ATPase was also located in the ionocytes of the epipodites of larvae exposed to seawater (35.%o) and to dilute seawater (22.1 %o). After metamorphosis, the inner-side branchiostegite epithelium appeared as an additional site of enzyme location in postlarvae held in dilute seawater. Within the ionocytes, Na(+),K(+)-ATPase was mostly located along the basolateral infoldings. These observations are discussed in relation to the physiological shift from osmoconforming larvae to slightly hyper-regulating (in dilute seawater) postmetamorphic stages. The acquisition of the ability to hyper-osmoregulate probably originates from the differentiation, on the epipodites and mainly along the branchiostegites, of ionocytes that are the site of ion pumping as evidenced by the location of Na(+),K(+)-ATPase.

The localization of Na+,K(+)-ATPase in epithelia of the organs of the branchial cavity of Homarus gammarus exposed to seawater and dilute seawater was examined by immunofluorescence microscopy and immunogold electron microscopy with a monoclonal antibody IgG alpha 5 raised against the avian alpha-subunit of the Na-,K(+)-ATPase. In juveniles held in seawater, fluorescent staining was observed only in the epithelial cells of epipodites. In juveniles held in dilute seawater, heavier immunoreactivity was observed in the epithelial cells of epipodites, and positive immunostaining was also observed along the inner-side epithelial layer of the branchiostegites. No fluorescent staining was observed in the gill epithelia. At the ultrastructural level, the Na+,K(+)-ATPase was localized in the basolateral infolding systems of the epipodite and inner-side branchiostegite epithelia of juveniles held in dilute seawater, mostly along the basal lamina. The expression of Na+,K(+)-ATPase therefore differs within tissues of the branchial cavity and according to the external salinity. These and previous ultrastructural observations suggest that the epipodites, and to a lesser extent the inner-side epithelium of the branchiostegites, are involved in the slight hyper-regulation displayed by lobsters at low salinity. Enhanced Na+,K(+)-ATPase activity and de novo synthesis of Na+,K(+)-ATPase within the epipodite and branchiostegite epithelia may be key points enabling lobsters to adapt to low salinity environments.