Influence of highly unsaturated fatty acids on the responses of white shrimp (Litopenaeus vannamei) postlarvae to low salinity

Salinity stress tests are commonly applied in shrimp hatcheries to estimate the quality of postlarvae (PL) to be used during growout. Higher larval survival during culture and to a salinity stress test in both fish and crustaceans have been reported when specimens were offered a diet containing high levels of highly unsaturated fatty acids (HUFA). However, it is not clear if increased survival is a result of better overall physiological condition resulting from the diet or a specific effect of HUFA on osmoregulatory mechanisms. This study analyzed if HUFA-rich diets could modify the fatty acid composition of membranes in gills, and if this change in composition could affect the activity of the Na⁺/K⁺ ATPase pump and carbonic anhydrase in relation to changes in salinity. One-day-old postlarvae (PL1) pooled from different spawns were fed for 20 days with Artemia sp. nauplii enriched with three levels of HUFA: low, medium and high. At PL20, survivals during culture and to salinity stress test (tap water for 30 min) were evaluated. Also at this stage, Na⁺/K⁺-ATPase and carbonic anhydrase activity, morphometric variables, and fatty acid composition in the hepatopancreas and gills were measured after they were submitted to a salinity challenge in dilute seawater (10 ppt) for 3 h. No significant differences were observed in survival rates during culture, but survival to a salinity stress test was higher and gill area was larger in PL20 fed the Artemia sp. nauplii enriched with medium HUFA levels, probably as a result of an increased 22:6n-3 content and higher 22:6n-3/20:5n-3 ratio in this diet and in the tissues of the organisms fed this diet. Na⁺/K⁺-ATPase specific activity was significantly higher in posterior gills, while the specific activity of the carbonic anhydrase was higher in anterior gills. Enzymatic activities increased significantly in PL20 submitted to a salinity challenge, and HUFA levels in the diet affected both. The proportion of fatty acids in hepatopancreas and gills were significantly affected not only by diet, but also by exposure to dilute media. This effect is discussed in relation to an increase in gill surface and changes in fatty acid composition in the phospholipids present in gill membranes, which can modify the permeability and the activity of the Na⁺/K⁺-ATPase pump. The beneficial effect of HUFA supplementation in the diet on survival to salinity stress test is partially related to modification of fatty acid composition of gills and to a larger gill area, which in turn enhances osmoregulatory mechanisms, namely Na⁺/K⁺-ATPase and carbonic anhydrase activities.     

Mechanisms of seawater acclimation in a primitive,anadromous fish,the green sturgeon

Relatively little is known about salinity acclimation in the primitive groups of fishes. To test whether physiological preparative changes occur and to investigate the mechanisms of salinity acclimation, anadromous green sturgeon, Acipenser medirostris (Chondrostei) of three different ages (100, 170, and 533 dph) were acclimated for 7 weeks to three different salinities (<3, 10, and 33 ppt). Gill, kidney, pyloric caeca, and spiral intestine tissues were assayed for Na⁺, K⁺-ATPase activity; and gills were analyzed for mitochondria-rich cell (MRC) size, abundance, localization and Na⁺, K⁺-ATPase content. Kidneys were analyzed for Na⁺, K⁺-ATPase localization and the gastro-intestinal tract (GIT) was assessed for changes in ion and base content. Na⁺, K⁺-ATPase activities increased in the gills and decreased in the kidneys with increasing salinity. Gill MRCs increased in size and decreased in relative abundance with fish size/age. Gill MRC Na⁺, K⁺-ATPase content (e.g., ion-pumping capacity) was proportional to MRC size, indicating greater abilities to regulate ions with size/age. Developmental/ontogenetic changes were seen in the rapid increases in gill MRC size and lamellar length between 100 and 170 dph. Na⁺, K⁺-ATPase activities increased fourfold in the pyloric caeca in 33 ppt, presumably due to increased salt and water absorption as indicated by GIT fluids, solids, and ion concentrations. In contrast to teleosts, a greater proportion of base (HCO₃ ⁻ and 2CO₃ ²⁻) was found in intestinal precipitates than fluids. Green sturgeon osmo- and ionoregulate with similar mechanisms to more-derived teleosts, indicating the importance of these mechanisms during the evolution of fishes, although salinity acclimation may be more dependent on body size.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

Effect of different salinity on serum osmolality,ion levels and hematological parameters of East Java strain tilapia Oreochromis niloticus

This study evaluated the effect of different salinity levels on the physiology of East Java strain tilapia (Oreochromis niloticus) by measuring the serum osmolalities (SO), ion levels and hematological parameters. Their SOs above the external medium (hyper-osmotic) at 0 and 5 ppt, becoming iso-osmotic at 10 ppt and hypo-osmotic at 15 ppt. The concentrations of serum Na⁺, K⁺, Cl^? and Ca²⁺ in fish acclimated in 0 and 5 ppt were not significantly different. The levels of Na⁺, Cl^– and Ca²⁺ in fish exposed to 10 and 15 ppt were higher than those in fish acclimated at 0 and 5 ppt. In contrast, the levels of K⁺ in fish exposed to 10 and 15 ppt were lower than those in fish exposed to 0 and 5 ppt. The levels of red blood cell, hematocrit and hemoglobin of fish exposed to salinity of 0, 5, 10 and 15 ppt were not significantly different. The levels of white blood cell increased significantly at fish exposed to 10 and 15 ppt. These data provide useful information for future reference and farming practice.     

The salinity tolerance of the invasive golden apple snail (Pomacea canaliculata)

We exposed snails of an invasive species of golden apple snail (Pomacea canaliculata) to five artificial sea water treatments at salinity levels of 0, 5, 10, 15 or 20 parts per thousand (ppt) to assess their salinity tolerance. We observed the behaviour, heart rate, total haemocyte counts, haemolymph ionic concentration and Na⁺/K⁺-ATPase activity in the mantle at 0, 12, 24, 48, 72 and 96 h post salinity exposures. The heart rate declined with increasing salinity, while Na⁺/K⁺-ATPase activity in the mantle presented a reverse trend, possibly to maintain normal osmolality. A trend of rising total haemocyte count was observed from 0 ppt and 5 ppt to 10 ppt salinities, while a sudden increase in the count was observed at 15 ppt and 20 ppt salinity groups. Furthermore, haemolymph Cl^?, Na⁺ and K⁺ concentrations increased directly with elevated salinity. An additional trial was performed to assess the growth performance of the snails under exposure to low salinities. During a 1 month trial, snails grew better at 5 ppt salinity treatment. Taken together, our results demonstrate that P. canaliculata can tolerate salt stress to some extent. The finding also obviously implies a possible invasive risk to estuaries.     

Effects of low-salinity on the growth and development of spotted halibut Verasper variegatus in the larva-juvenile transformation period with reference to pituitary prolactin and gill chloride cells responses

Low-salinity adaptability was investigated in a flatfish spotted halibut Verasper variegatus during the period from late metamorphic larvae to early juveniles by a 20-day rearing experiment under different salinity regimes (1, 4, 8, 16 and 32 ppt). Effects of low-salinity on growth and development were examined and the changes in the prolactin (PRL) production level in the pituitary and the gill chloride cell morphology were examined as physiological backgrounds for low salinity adaptation. PRL cells and chloride cells were identified by immunocytochemistry with a specific antiserum for PRL₁₈₈ and Na⁺,K⁺-ATPase. Most of the fish exposed to over 4 ppt survived for 20 days, but all the fish exposed to 1 ppt died within 5 days. Fish kept in intermediate salinities (8, 16 ppt) grew significantly better than those in the control group (32 ppt). Fish exposed to 4 ppt attained almost the same body length as the control group at 20 days after transfer, although these fish showed an abnormally dark body color as well as delayed development. These results suggested that spotted halibut has a high-adaptability to low-salinity environments and prefers an intermediate salinity near iso-osmolality (about 12 ppt) from the late metamorphic larval stage, but does not completely adapt to a hypoosmotic of 4 ppt salinity or less than half of the osmolality. The percentage of PRL-cell volume to pituitary volume was significantly higher at 4 ppt than in the control group. The chloride cells in gill filaments were significantly larger at 4 ppt than in the control group. These results suggest that juveniles could adapt to a low-salinity environment due to the activation of PRL production and enlargement of chloride cells. These laboratory findings suggest that late metamorphic larvae and early juveniles of spotted halibut may utilize a low salinity environment such as estuarine tidal flats or very shallow coastal areas as their nursery grounds in the sea.     

Osmoregulation in juvenile and adult white sturgeon,Acipenser transmontanus

Synopsis Blood samples from cannulated young adult (2.5–15 kg) white sturgeon, acclimated to San Francisco Bay water (24 ppt) had plasma values of 248.8 ± 13.5 mOsm kg⁻¹ H₂O, [Na⁺] = 125 ± 8.0 mEq 1⁻¹, [K⁺] = 2.6 ± 0.8 mEq 1⁻¹ and [CL⁻] = 122 ± 3.0 mEq 1⁻¹. Freshwater acclimated sturgeon had an osmolality of 236 ± 7, [Na⁺] = 131.6 + 4.4, [K⁺] = 2.5 ± 0.7 and [CL⁻] = 110.6 ± 3.6. Freshwater acclimated fish gradually exposed to sea water (increase of 5 ppt h⁻¹) had higher plasma osmolalities than did the bay water acclimated fish. These young adult sturgeon are able to tolerate transfer from fresh water to sea water as well as gradual transfer from sea water to fresh water. Plasma electrolytes in transferred fish are regulated, but tend to differ from long term acclimated fish at the same salinities. There is a gradual increase in the upper salinity tolerance (abrupt transfer) of juvenile white sturgeon with weight: 5–10 ppt for 0.4–0.9 g fish, 10–15 ppt for 0.7–1.8 g fish, and 15 ppt for 4.9–50.0 g fish. The ability of juveniles to regulate plasma osmolality is limited. The young adult fish are able to tolerate higher salinities (35 ppt) than juvenile sturgeon but probably are also characterized by low activity of the necessary ion exchange mechanisms in the gills which permit rapid adjustment of blood electrolytes with graduate change in external salinity.     

The physiology of hyper-salinity tolerance in teleost fish: a review

Hyper-saline habitats (waters with salinity >35 ppt) are among the harshest aquatic environments. Relatively few species of teleost fish can tolerate salinities much above 50 ppt, because of the challenges to osmoregulation, but those that do, usually estuarine, euryhaline species, show a strong ability to osmoregulate in salinities well over 100 ppt. Typically, plasma Na⁺ and Cl⁻ concentrations rise slowly or not at all up to about 65 ppt. At higher salinities ion levels do rise, but the increase is small relative to the magnitude of increase in concentrations of the surrounding water. A number of adjustments are responsible for such strong osmoregulation. Reduced branchial water permeability is indicated by the observation that with the exposure to hyper-salinities drinking rates rise more slowly than the branchial osmotic gradient. Lower water permeability limits osmotic water loss and greatly reduces the salt load incurred in replacing it. Still, increased gut Na⁺/K⁺-ATPase (NAK) activity is necessary to absorb the larger gut salt load and increased HCO₃ ⁻ secretion is required to precipitate Ca²⁺ and some Mg²⁺ in the imbibed water to facilitate water absorption. All Na⁺ and Cl⁻ taken up must be excreted and increased branchial salt excreting capacity is indicated by elevated mitochondrion-rich cell density and size, gill NAK activity and expression of chloride channels. Excretion of Na⁺ and Cl⁻ occurs against a larger gradient than in seawater and calculation of the equilibrium potential for Na⁺ across the gill epithelium indicates that the trans-epithelial potential required for excretion of Na⁺ climbs with salinity up to about 65 ppt before leveling off due to the increasing plasma Na⁺ levels. During acute transition to SW or mildly hyper-saline waters, some species have shown the ability to upregulate branchial NAK activity rapidly and this may play an important role in limiting disturbances at higher salinities. It does not appear that the opercular epithelium, which in SW acts in a way that is functionally similar to the gills, continues to do so in hyper-saline waters. Little is know about the hormones involved in acclimation to hyper-salinity, but the few studies available suggest a role for cortisol, but not growth hormone and insulin-like growth factor. Despite the increased transport capacity evident in both the gill and gut in hyper-saline waters there is no clear trend toward increased metabolic rate. These studies provide a general outline of the mechanisms of osmoregulation in these species, but significant questions still remain.     

Dietary lipid composition affects the gene expression of gill Na+/K+-ATPase α1b but not the α1a isoform in juvenile fall chinook salmon (Oncorhynchus tshawytscha)

We assessed the effects of dietary fatty acid composition on sodium–potassium ATPase (Na⁺/K⁺-ATPase) activity and isoform expression in the gills of juvenile fall chinook salmon, Oncorhynchus tshawytscha by supplementing diets with either anchovy oil (AO) or AO blended with canola oil (CO) so that CO comprised 0% (0CO), 11% (11CO), 22% (22CO), 33% (33CO), 43% (43CO), or 54% (54CO) of the measured dietary lipid content. The effects of diet were assessed in freshwater (FW) following 104 days of diet manipulation, in response to 24-h seawater (SW) transfer at this time, and following an additional 35 days of SW acclimation. Gill Na⁺/K⁺-ATPase activity was not significantly affected by diet at any sampling time, and there were no consistent effects of diet on the expression of the Na⁺/K⁺-ATPase α1a isoform. As dietary CO increased, Na⁺/K⁺-ATPase α1b mRNA decreased in fish held in FW, with the 43CO and 54CO diet groups having significantly lower levels than fish fed the 0CO and 11CO diets. Twenty-four-hour SW challenge did not affect the expression of the Na⁺/K⁺-ATPase α1a isoform in any diet group, but this isoform was down-regulated in all diet groups following 35 days of SW acclimation. Na⁺/K⁺-ATPase α1b expression levels increased in response to 24-h SW transfer and SW acclimation only in fish fed the 54CO diet. The effects of the two extreme diets (0CO and 54CO) were also assessed at various time points during 104 days of rearing in FW. Na⁺/K⁺-ATPase α1b mRNA levels were greater in fish fed diet 0CO versus those fed diet 54CO at all times during the FW culture period. These data demonstrate that dietary fatty acid composition can influence the gill Na⁺/K⁺-ATPase isoform physiology of juvenile fall-run chinook salmon prior to SW transfer.     

Functional differentiation in the anterior gills of the aquatic air-breathing fish, <Emphasis Type="Italic">Trichogaster leeri</Emphasis>

The fish gill is a multifunctional organ responsible for gas exchange and ionic regulation. It is hypothesized that both morphological and functional differentiation can be found in the gills of the aquatic air-breathing fish, Trichogaster leeri. To test this, we used the air-breathing fish, Trichogaster leeri, to investigate various morphological/functional parameters. First, we evaluated the importance of performing the aquatic surface respiration behavior in T. leeri. A reduced survival rate was observed when fish were kept in the restrained cages in hypoxic conditions. On the gross anatomy of gills, we found evidence of both morphological and functional modification in the first and the second gills and are responsible for ionic regulation. There were large-bore arterioarterial shunts in the fourth gill arch. It is specialized for the transport of oxygenated blood and is less responsive to environmental stress. In addition, the anterior and the posterior gills differed in the Na⁺, K⁺-ATPase activity upon ionic stresses. That is, only the Na⁺, K⁺-ATPase activity of the anterior two gills was up-regulated significantly in the deionized water. Lastly, we found that the number of mitochondria-rich cells in the first and the second gills increased following ionic stress and no difference was found in the third and the fourth gills following such an exposure. These results supported the hypothesis that there are morphological and functional differences between anterior and posterior gill arches within the air-breathing Trichogaster leeri. In contrast, no significant difference was found among gills in gross anatomy, filament density and Na⁺, K⁺-ATPase activity in the non-air-breather, Barbodes schwanenfeldi.     

Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na⁺,K⁺-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na⁺,K⁺-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.     

Monitoring of Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase mRNA expression in the cinnamon clownfish, <Emphasis Type="Italic">Amphiprion melanopus</Emphasis>, exposed to an osmotic stress environment: profiles on the effects of exogenous hormone

We examined changes in the expression of Na⁺/K⁺-ATPase mRNA in the gills of the cinnamon clownfish using quantitative real-time PCR in an osmotically changing environment [seawater (35 psu; practical salinity unit, 1 psu ≈ 1‰) → brackish water (17.5 psu) and brackish water with prolactin]. The expression of Na⁺/K⁺-ATPase mRNA in gills was increased after the transfer to brackish water, and the expression was repressed by prolactin treatment. Also, activities of gill Na⁺/K⁺-ATPase and plasma cortisol levels increased after the transfer to brackish water and were repressed in brackish water with prolactin treatment. Na⁺/K⁺-ATPase-immunoreactive cells were almost consistently observed in the gill filaments, but absent from the lamella epithelia. The plasma osmolality level decreased in brackish water, but the level of this parameter increased in brackish water with prolactin treatment during salinity change. These results suggest that the Na⁺/K⁺-ATPase gene plays an important role in osmoregulation in gills, and prolactin improves the hyperosmoregulatory ability of cinnamon clownfish in a brackish water (hypoosmotic) environment.     

Environment-phenotype interactions: Influences of brackish-water rearing on lake trout (<Emphasis Type="Italic">Salvelinus namaycush</Emphasis>) physiology

Fertilization and development in salmonids occurs almost exclusively within freshwater environments (< 1 ppt). A less common life history strategy in this group of fishes is the brackish-water resident life history, where entire life cycles occur in brackish water (> 1 ppt). In the present study, we tested the hypothesis that differences in rearing environment (fresh or brackish water) results in significant differences in the ability of lake trout to ionoregulate when faced with a salinity challenge later in life. To test this, genetically similar lake trout were fertilized and raised at either 0 or 5 ppt saltwater. At approximately 240 days post hatch, lake trout from both rearing environments were acutely transferred to 20 ppt salt water or their respective rearing environments as a control. Individuals were sampled at time 0, 1, 7, and 14 days post transfer. Fish raised in 5 ppt transferred to 20 ppt saltwater had significantly higher gill Na⁺ K⁺-ATPase activity, gill Na⁺ K⁺-ATPase α1b expression, and lower plasma osmolality when compared to freshwater reared lake trout transferred to 20 ppt across various time points. Additionally, the 5 ppt control treatment had greater overall aerobic scope than 0 ppt control fish and those transferred from 0 ppt to 20 ppt. These data imply that populations exhibiting a brackish-water resident life history, as has been observed in Arctic Canada, may have an advantage over freshwater reared conspecifics when foraging in marine influenced environments and colonizing new locations in coastal regions.     

Changes in Na+/K+-ATPase activity, unsaturated fatty acids and metallothioneins in gills of the shore crab Carcinus aestuarii after dilute seawater acclimation

We have assessed the activity of Na⁺/K⁺-ATPase, cAMP, free fatty acids (FFA) and metallothionein (MT) in the posterior gills of the brackish water shore crab Carcinus aestuarii during acclimation to 10 ppt dilute seawater (DSW). Following 3–18 days acclimation in DSW specific activity of Na⁺/K⁺-ATPase in native gill homogenates and partially purified membrane vesicles was progressively increased, from 1.7- to 3.9-fold. After short-term acclimation of crabs in DSW with added sucrose to make media isosmotic with the haemolymph the specific Na⁺/K⁺-ATPase activity in homogenates was not increased, relative to SW enzyme activity. Moreover, hyposmotic conditions led to depletion of cAMP in gills.In partially purified membrane vesicles isolated from posterior gills, fatty acids with compositions 16:0, 18:0, 18:1, 20:4 and 20:5 dominated in both SW- and DSW-acclimated Carcinus. During a year in which the metabolic activity of crabs was increased, the arachidonic/linoleic acids ratio (ARA/LA) for DSW-acclimated crabs was markedly increased relative to that in SW. Increased Na⁺ + K⁺-ATPase activity under hyposmotic stress may be modulated at least partially by the changed proportion of fatty acids in the purified membranes of posterior gills. Long-term acclimation of shore crabs to DSW resulted in a 2.6-fold increase in cytosolic metallothionein (MT) content in posterior gills over those in SW crabs. Assuming an antioxidant role of MT associated with intracellular zinc partitioning, the observed MT induction in posterior gills may be considered an adaptive response of C. aestuarii to hyposmotic stress.     

DDT inhibits Na<Superscript>+</Superscript>, K<Superscript>+</Superscript>, Mg<Superscript>2+</Superscript>-ATPase in the Intestinal Mucosae and Gills of Marine Teleosts

SODIUM-potassium-activated, magnesium-dependent, adenosine triphosphatase (Na⁺, K⁺, Mg²⁺-ATPase) is widely accepted as an essential factor in sodium transport¹ and observations on fish substantiate this view. There are concurrent increases, for example, of both Na⁺, K⁺, Mg²⁺-ATPase activity and osmoregulatory sodium transport², in the intestinal mucosae^3,4 and the gills^3,5 of euryhaline teleosts during adaptation to seawater. Furthermore, the gills of stenohaline seawater teleosts, which actively secrete sodium, exhibit higher Na⁺, K⁺, Mg²⁺-ATPase activity than the gills of stenohaline freshwater teleosts, which do not actively secrete sodium^3,5. Na⁺, K⁺, Mg²⁺-ATPase therefore seems to be important in maintaining tissue osmolarity well below that of seawater. It is disquieting to report therefore that Na⁺, K⁺, Mg²⁺-ATPase activity in the intestinal mucosae and gills of marine teleosts is inhibited by the organochlorine insecticide DDT. This observation may help to clarify the unexplained sensitivity of teleosts to DDT⁶.     

The carbonic anhydrase of the Chinese crabEriocheir sinensis: Effects of adaption from tap to salt water

In the present investigation we studied the carbonic anhydrase (CA) in various tissues of Chinese crabEriocheir sinensis which were acclimated to different salinities (0, 10, 20, 30‰). We found only negligible CA activity in haemolymph, heart, hypodermis, antennal gland, leg muscle and digestive gland, irrespective of the acclimation medium. However, high amounts of CA activity were found in the gills. In the case of the posterior gills, a strong dependence on the acclimatization of the animals was demonstrated; the highest activities were found in those adapted to tap water. To investigate the cellular distribution of the CA in the posterior gills, the additional enzyme activities were measured in all fractions of a differential centrifugation of the gill homogenate: Na⁺/K⁺-ATP'ase (a marker for the plasmamembrane); lactate dehydrogenase (LDH; as marker for the cytosol); and succinate dehydrogenase (SDH; as marker for mitochondria). Independent of the acclimation salinity (0 or 36‰ salinity), we found about 70% of CA associated with the highest level of the Na⁺/K⁺-ATP'ase in the second 100 000 g pellet (membrane fraction), while only 15% were found in the cytosolic fractions (associated with highest levels of LDH). We conclude that the carbonic anhydrase of posterior gills of the Chinese crab is mainly membrane-bound. Furthermore, the activity of CA shows a strong dependence on the salinity of the water in which the crabs were kept.     

Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

FXYD proteins are novel regulators of Na⁺-K⁺-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.     

The importance of the olfactory rosettes in maintaining pituitary prolactin and prolactin-releasing peptide levels during hyposmotic acclimation in silver sea bream (Sparus sarba)

A potential role of the olfactory rosettes in maintaining prolactin (PRL) and prolactin-releasing peptide (PrRP) levels was examined in the euryhaline silver sea bream (Sparus sarba). The olfactory rosettes were surgically removed in silver sea bream adapted to hypo- (6 ppt) and hyper-osmotic (33 ppt) salinities and the mRNA expression of the two previously identified freshwater-adapting factors, prolactin (PRL) and prolactin-releasing peptide (PrRP), in silver sea bream was measured. The elevation of pituitary PRL and PrRP mRNA expression levels as seen in 6 ppt-adapted fish was abolished by surgical removal of the olfactory rosettes. The PRL and PrRP expression levels in fish adapted to 6 ppt were significantly lowered following olfactory rosette removal. On the other hand, hypothalamic PrRP mRNA expression in 6 ppt-adapted fish did not change. Specific signals for Na⁺-K⁺-ATPase but not CFTR mRNA expression were detected in the surface layers of olfactory epithelial cells by in situ hybridization. The mRNA abundance of CFTR and Na⁺-K⁺-ATPase α and β subunits remained unchanged in the olfactory rosette of silver sea bream adapted to 0, 6, 12, 33 and 50 ppt for 4 weeks and in fish abruptly transferred from 33 ppt to 6 ppt. Data obtained from the olfactory rosette removal experiments suggest a possible role of the olfactory system for maintaining PRL and PrRP expression during hyposmotic acclimation in sea bream.     

The effects of gill remodeling on transepithelial sodium fluxes and the distribution of presumptive sodium-transporting ionocytes in goldfish (Carassius auratus)

Goldfish, Carassius auratus, adaptively remodel their gills in response to changes in ambient oxygen and temperature, altering the functional lamellar surface area to balance the opposing requirements for respiration and osmoregulation. In this study, the effects of thermal- and hypoxia-mediated gill remodeling on branchial Na⁺ fluxes and the distribution of putative Na⁺-transporting ionocytes in goldfish were assessed. When assessed either in vitro (isolated gill arches) or in vivo at a common water temperature, the presence of an interlamellar cell mass (ILCM) in fish acclimated to 7°C clearly decreased Na⁺ efflux across the gill relative to fish maintained at 25°C and lacking an ILCM. However, loss of the ILCM in 7°C-acclimated fish exposed to hypoxia led to a decrease in Na⁺ efflux (assessed under hypoxic conditions) despite the apparent large increases in functional lamellar surface area. Goldfish possessing an ILCM were able to sustain Na⁺ uptake, albeit at a lower rate matched to efflux, owing to the re-distribution of ionocytes expressing genes thought to be involved in Na⁺ uptake [Na⁺/H⁺ exchanger isoform 3 (NHE3) and V- type H⁺-ATPase] to the edge of the ILCM where they can establish contact with the surrounding environment. NHE-expressing cells co-localized with Na⁺/K⁺-ATPase expression, suggesting a role for NHE in Na⁺-uptake in the goldfish. Implications of the ILCM on ion fluxes in the goldfish are discussed.