Gill and intestinal Na+-K+ ATPase activity, and estimated maximal osmoregulatory costs, in three high-energy-demand teleosts: yellowfin tuna (Thunnus albacares), skipjack tuna (Katsuwonus pelamis), and dolphin fish (Coryphaena hippurus)

We hypothesize that the morpho-physiological adaptations that permit tunas to achieve maximum metabolic rates (MMR) that are more than double those of other active fishes should result in high water and ion flux rates across the gills and concomitant high osmoregulatory costs. The high standard metabolic rates (SMR) of tunas and dolphin fish may, therefore, be due to the elevated rates of energy expenditure for osmoregulation (i.e. teleosts capable of achieving exceptionally high MMR necessarily have SMR). Previous investigators have suggested a link between activity patterns and osmoregulatory costs based on Na⁺-K⁺ ATPase activity in the gills of active epipelagic and sluggish deep-sea fishes. Based on these observations, we conclude that high-energy-demand fishes (i.e. tunas and dolphin fish) should have exceptionally elevated gill and intestinal Na⁺-K⁺ ATPase activity reflecting their elevated rates of salt and water transfer. To test this idea and estimate osmoregulatory costs, we measured Na⁺-K⁺ ATPase activity (V _max) in homogenates of frozen samples taken from the gills and intestines of skipjack and yellowfin tunas, and the gills of dolphin fish. As a check of our procedures, we made similar measurements using tissues from hybrid red tilapia (Oreochromis mossambicus ×O. niloticus). Contrary to our supposition, we found no difference in Na⁺-K⁺ ATPase activity per unit mass of gill or intestine in these four species. We estimate the cost of osmoregulation to be at most 9% and 13% of the SMR in skipjack tuna and yellowfin tuna, respectively. Our results, therefore, do not support either of our original suppositions, and the cause(s) underlying the high SMR of tunas and dolphin fish remain unexplained. Received: 7 September 2000 / Accepted: 4 December 2000     

Branchial and intestinal osmoregulatory acclimation in the four-eyed sleeper, <Emphasis Type="Italic">Bostrychus sinensis</Emphasis> (Lacepède), exposed to seawater

Bostrychus sinensis is a facultative air breather that inhabits waters of a wide range of salinities. This study aimed to elucidate whether branchial and intestinal osmoregulatory acclimation occurred in B. sinensis transferred from 5‰ water through a progressive increase in salinities to seawater. Our results indicate that B. sinensis acted as a hyperosmotic regulator in 5‰ water, but exhibited hypoosmotic hypoionic regulation in seawater. During short- (1 day) and medium- (10 days) term acclimation to seawater, there were only minor perturbations in plasma osmolality and [Na⁺], which returned to control levels after 45 days of exposure to seawater. Branchial Na⁺/K⁺-ATPase activity was unaffected by 1, 10 or 45 days of exposure to seawater. However, prolonged (45 days) acclimation to seawater led to a significant increase in Na⁺/K⁺-ATPase α-subunit protein abundance. Taken together, these results indicate that there could be changes in the expression of Na⁺/K⁺-ATPase isoforms and/or post-translational modification of Na⁺/K⁺-ATPase in the gills of fish exposed to seawater. Immunofluorescence microscopy revealed that acclimation to seawater for 10 days only resulted in no change in branchial Na⁺/K⁺-ATPase protein expression, but there were increases in protein expression of cystic fibrosis transmembrane regulator (CFTR)-like chloride channel and Na⁺:K⁺:2Cl⁻ cotransporter (NKCC; probably NKCC1). Indeed, NKCC was undetectable in gills of fish kept in 5‰ water by Western blotting, but it became weakly detectable in fish exposed to seawater for 10 days and prominently expressed in fish exposed to seawater for 45 days. Therefore, our results indicate that branchial CFTR-like chloride channel and NKCC1 were the determining factors in the transition between hyperosmotic regulation and hypoosmotic hypoionic regulation in B. sinensis. Furthermore, the intestine of B. sinensis also served as an important osmoregulatory organ, since there were significant increases in both the activity and protein abundance of intestinal Na⁺/K⁺-ATPase in fish acclimated to seawater for 45 days. The effectiveness of branchial and intestinal osmoregulatory acclimation in B. sinensis during seawater acclimation led to only a minor increase in plasma osmolality, and thus resulted in relatively unchanged free amino acid contents in muscle and liver.     

食物源nC_(60)损害斑马鱼脑、鳃、肾和肝胰腺正常机能

水体中稳定存在的富勒烯纳米晶体(nC_(60))可被浮游动物滤食,并通过食物链传递到更高营养级生物。为探究食物源nC_(60)的生物效应,本试验选取携带nC_(60)的大型溞喂养斑马鱼21 d,考察了食物源nC_(60)对斑马鱼脑、鳃、肾和肝胰腺4个器官中ROS、Na~+K~+-ATPase、Ca2+-ATPase、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、谷丙转氨酶(GPT)和谷草转氨酶(GOT)活性等指标,用以评价食物源nC_(60)对斑马鱼的机能影响。暴露于食物源nC_(60)下的结果表明:斑马鱼脑ROS随时间增加而增加,暴露21 d后增加了79.17%。鳃、肾Na~+K~+-ATPase活性随暴露时间增加而降低,暴露21 d后分别降低了47.09%和51.07%;鳃、肾Ca2+-ATPase活性随暴露时间增加而减少,暴露21 d后分别降低了28.28%和35.13%。鳃、肾、肝胰腺AKP活性随时间增加而增加,暴露21 d后分别增加45.97%、26.68%和83.01%;鳃、肾、肝胰腺ACP活性随时间增加而增加,暴露21 d后分别增加38.85%、84.12%和55.77%。肝胰腺GPT和GOT活性随时间增加而降低,暴露21 d后各降低了50.05%和76.50%。本研究不但阐述了食物源nC_(60)降低高一级水生动物(斑马鱼)脑、鳃、肾和肝胰腺的正常机能,而且为进一步研究食物源nC_(60)对水生生物的生态毒理提供了部分基础数据。     

Modulation of branchial ion transport protein expression by salinity in glass eels (<Emphasis Type="Italic">Anguilla anguilla</Emphasis> L.)

The Anguillid juvenile glass eel must deal with the osmoregulatory consequences of highly variable environmental salinities on its recruitment migration from coastal to fresh waters. Changes in ionoregulatory parameters and branchial ion transport protein [Na⁺/K⁺-ATPase, Na⁺:K⁺:2Cl⁻ cotransporter (NKCC), cystic fibrosis transmembrane regulator (CFTR) anion channel, V-type proton ATPase] expression (activities, protein and/or mRNA level expression and/or cellular localization) in response to acclimation to a broad range of ionic strengths [distilled water (DW) to hypersaline water (HSW; 150%) sea water (SW 32‰)] was studied. The estuarine glass eels were very euryhaline and successfully acclimated to acute changes in environmental ionic strength from 50% SW, with high mortality only observed in HSW (51%) and sublethal osmoregulatory indicators (whole body water content and sodium levels) disturbed at the extremes (DW and HSW). Central to a high salinity acclimation were elevated branchial Na⁺/K⁺-ATPase, NKCC and CFTR expression. At lower salinity, Na⁺/K⁺-ATPase expression was maintained and NKCC and CFTR expressions were reduced. Branchial chloride cells increased in size up to SW but decreased in HSW. During hypotonic disturbance (DW), no compensatory elevation in V-ATPase or Na⁺/K⁺-ATPase expression was observed.     

Acclimation of Solea senegalensis to different ambient temperatures: implications for thyroidal status and osmoregulation

We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na⁺,K⁺-ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na⁺,K⁺-ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and non-esterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3′-triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.     

Biomarkers as tools to assess the chronic toxicity of ammonia in the juvenile Mugil cephalus

With juvenile fish as the subject, the effects of low concentration ammonia on antioxidant system were studied using Mugil cephalus. Samples of gill and liver tissue were obtained from 0.35, 0.70, 1.5 and 3?mg/L ammonia groups at 0, 5, 10, 15 and 20 days of exposure, at which times the biomarkers were measured. Results showed that gill malondialdehyde (MDA) content exhibited an initial significant increase (p?≤?0.05) at unionised ammonia concentrations of 0.70, 1.5 and 3.0?mg/L on day 5, followed by subsequent declines, while liver MDA levels exhibited significant increases (p?≤?0.05) at unionised ammonia concentrations of 1.5?mg/L starting on day 10 and at 3.0?mg/L starting on day 5. With exposure to ammonia at different concentrations, Na⁺-K⁺-ATPase activity in liver and gill decreased over time. The Na⁺-K⁺-ATPase activity was negatively related to ammonia concentration from 0.70 to 3.0?mg/L. Overall, our results show that MDA and Na⁺-K⁺-ATPase, evaluated here as potential biomarkers of ammonia exposure, exhibited responses to sublethal concentrations of ammonia that were concentration dependent.     

溴氰菊酯对菲律宾蛤仔体内酶活性和组织损伤的初步探索

采用不同质量浓度的溴氰菊酯(0.0070 mg·L~(-1)、0.014 mg·L~(-1)、0.020 mg·L~(-1)、0.027 mg·L~(-1))对菲律宾蛤仔进行20 d半静置染毒,测定不同时间淋巴液中乙酰胆碱酯酶(ACh E)和钠离子-钾离子-三磷酸腺苷酶(Na~+-K~+-ATPase)活性、鳃和肝脏中谷胱甘肽转硫酶(GST)活性的变化,并观察染毒20 d后鳃丝组织和消化盲囊组织的损伤情况。酶活性分析结果显示,与对照组相比,低浓度组(0.0070 mg·L~(-1))试验期间酶活性均无显著差异(P0.05);中浓度组(0.014 mg·L~(-1)、0.020 mg·L~(-1))淋巴液中ACh E和Na~+-K~+-ATPase均呈先激活后抑制的变化规律(P0.05),鳃和肝脏中GST活性均呈上升趋势(P0.05);高浓度组(0.027mg·L~(-1))淋巴液中ACh E和Na~+-K~+-ATPase、肝脏中GST活性在试验期间持续下降(P0.01),而鳃中GST活性呈先抑制后升高的趋势(P0.05)。研究表明,低中浓度的溴氰菊酯对菲律宾蛤仔体内的酶活性表现为先诱导后抑制,具有明显的时间、剂量效应;高浓度的溴氰菊酯对菲律宾蛤仔体内酶活持续抑制,且染毒浓度越高,组织细胞变异越显著,表现为鳃丝上皮细胞纤毛层萎缩、纤毛脱落,消化盲囊上皮细胞膨胀,出现包涵体样结构。     

Tissue-specific stress and hepatic DNA damage in Pelteobagrus fulvidraco caused by low concentrations of cadmium

Physiological stress and DNA damage in Pelteobagrus fulvidraco induced by continuous exposure to cadmium at concentrations of 170 and 1700 µg/L for up to 28 days was evaluated. The activities of acetylcholinesterase and monoamine oxidase in brain tissue, Na⁺-K⁺-ATPase and glutathione in gill tissue, and superoxide dismutase and catalase in liver tissue were measured. In studying random amplified polymorphic DNA to evaluate cadmium-induced hepatic genotoxicity, both the appearance of new bands and the disappearance of existing bands were observed, as well as increased levels of monoamine oxidase and Na⁺-K⁺-ATPase and decreased activities of antioxidant enzymes. The results suggest that continuous exposure to cadmium at the studied levels can induce biochemical and physiological changes and DNA damage in P. fulvidraco.     

A comparison of transport-related gill enzyme activities and tissue-specific free amino acid concentrations of Baltic Sea (brackish water) and freshwater threespine sticklebacks, Gasterosteus aculeatus, after salinity and temperature acclimation

The osmoregulatory abilities of one freshwater and two brackish water (Baltic Sea) populations of the euryhaline teleost fish Gasterosteus aculeatus were studied with respect to evolutionary physiology. Plasma osmolality, activities of Na⁺K⁺-ATPase, citrate synthase, creatine kinase in the gill and free amino acids in liver, axial muscle and pectoral fin muscle were measured. After transfer from 10 to 35 ppt at 15 °C, time-course changes of plasma osmolality and gill Na⁺K⁺-ATPase showed no significant fundamental differences between the freshwater and one of the Baltic Sea populations. In a multi-factorial experiment, each population was exposed to four different abiotic regimes. Both brackish water populations had high mortality in freshwater at 4 °C, which is discussed as a failure of osmotic regulation (reduced taurine concentrations). Freshwater specimens had higher levels of glycine in the axial and pectoral fin muscles compared to the brackish water populations. This is interpreted as a genetically based effect. In brackish (20 ppt) water of 15 °C, the freshwater population had high activities of Na⁺K⁺-ATPase, but low activities of creatine kinase, whereas both brackish water populations behaved in the opposite way. A fundamental difference between the freshwater and brackish water populations on the level of the osmoregulatory machinery was not observed. Received: 10 December 1998 / Accepted: 22 September 1999     

Activity and inhibitor sensitivity of ATPases in the hydrothermal vent tubeworm Riftia pachyptila : a comparative approach

Phosphorylated ATPases may be involved in the effective pH regulation seen in the hydrothermal vent tubeworm Riftia pachyptila. R. pachyptila appears not only to have a large concentration of ATPases, but the main function of these ATPases seems to have shifted from other types of transport, such as Na⁺ and K⁺ movement, to the facilitation of H⁺ elimination. Plume and trophosome ATPase activity for R. pachyptila measured 646.2 ± 29.5 and 481.4 ± 32.0 μmol Pi (inorganic phosphate) g⁻¹ wet wth⁻¹, respectively. Plume tissue ATPase activity (both mass-specific and protein-specific) in R. pachyptila was higher (between 7% and 55%) than the activity measured in any tissue for 7 other shallow- and deep-living species, in this study. This supports the hypothesis that R. pachyptila regulates acid/base balance via high concentrations of H⁺-ATPases, including Na⁺/H⁺ and K⁺/H⁺ exchangers and possibly electrogenic H⁺-ATPases, as evidenced by a higher total ATPase concentration (646 μmol Pi g⁻¹ wet wt h⁻¹), lesser Na⁺/K⁺-ATPase activity (13% of the total, as compared to 20−40% found in other animals), and higher H⁺-ATPase activity (226–264 μmol Pi g⁻¹wet wt h⁻¹). Overall, R. pachyptila appears to demonstrate elevated ATPase activity, with a greater fraction of the enzymes devoted to proton elimination, in order to effectively control its extracellular pH in the face of processes acting to acidify the internal environment. Received: 9 May 2000 / Accepted: 4 October 2000     

Inhibition of Na+/K+-ATPase and of active ion-transport functions in the gills of the shore crab Carcinus maenas induced by cadmium

Inhibition of Na⁺/K⁺-ATPase from gill plasma membranes of the shore crab Carcinus maenas by cadmium was investigated and compared with inhibitory effects by known antagonists (ouabain and Ca²⁺). For comparative considerations the Cd²⁺-inhibition of the enzyme from dog kidney was also tested. Na⁺/K⁺-ATPase from dog kidney and from crab gill differed greatly in sensitivity against ouabain. The inhibition constant K _i of the dog enzyme amounted to 9.1 × 10⁻⁷ mol l⁻¹, i.e. more than 300-fold smaller than the K _i of 2.9 × 10⁻⁴ mol l⁻¹ determined for the crab enzyme. Ca²⁺ inhibited the activity of Na⁺/K⁺-ATPase from crab gill plasma membranes with a K _i of 4.3 × 10⁻⁴ mol l⁻¹. The Na⁺/K⁺-ATPase from crab gill was inhibited by Cd²⁺ with a K _i of 9.1 × 10⁻⁵ mol l⁻¹. Cd²⁺ inhibited the Na⁺/K⁺-ATPase from dog kidney with a K _i (6.4 × 10⁻⁵ mol l⁻¹) comparable to that observed in the crab gill enzyme. Under experimental conditions Cd²⁺-inhibition of Na⁺/K⁺-ATPase was irreversible. Repeated washing, centrifugation and homogenization of the plasma membranes (four times) with Cd²⁺-free buffer did not restore any activity lost in the presence of 1 × 10⁻³ mol l⁻¹ Cd²⁺. Since ouabain-insensitive (nonspecific) ATPases in the plasma membrane fraction of crab gills were inhibited by Cd²⁺ in the same way as Na⁺/K⁺-ATPase, the heavy metal is considered as an unspecific ATPase inhibitor. Comparing these results with literature data on Cd²⁺-binding to electrophoretically separated proteins suggests that Na⁺/K⁺-ATPase is a Cd²⁺-binding enzyme. The results obtained on Na⁺/K⁺-ATPase were reflected by Cd²⁺-inhibition of the branchial ion-transport functions depending on this enzyme. The transepithelial short-circuit current of isolated gill half lamellae, a direct measure of area-specific active ion uptake, and the transepithelial potential difference of isolated, perfused whole gills, also indicative of active ion uptake, were inhibited by the heavy metal in a time- and dose-dependent mode. Remarkably these inhibitions were also irreversible. These findings are ecologically and biomedically significant: even when the actual environmental or tissue concentrations measured are low, biological microstructures such as Na⁺/K⁺-ATPase may accumulate the heavy metal by tight binding over prolonged periods until the first inhibitory effects occur. Received: 25 June 1997 / Accepted: 25 August 1997     

Enzyme activities of fish skeletal muscle and brain as influenced by depth of occurrence and habits of feeding and locomotion

Activities of lactate dehydrogenase (LDH), pyruvate kinase (PK), malate dehydrogenase (MDH) and citrate synthase (CS) were measured in the white skeletal muscle of marine fishes having different depths of occurrence and different feeding and locomotory strategies. There were significant depth-related differences in the two glycolytic enzymes, LDH and PK. LDH activity was most variable, and differed by 3 orders of magnitude between the most active shallow-living species and certain deep-sea fishes likely to have only minimal capacities for active locomotion. Superimposed on the depth-related patterns was a high degree of interspecific variation (up to 20-fold) in enzymic activity among species from any given range of depth of occurrence. Variation of both LDH and PK activities, noted for shallow- and deep-living fishes, seems to be largely accounted for by differences in feeding habits and locomotory performance. Active pelagic swimmers have much higher activities of LDH and PK than, for example, deep-living sit-and-wait predators. Benthopelagic fishes like rattails and the sablefish have the highest activities found among deep-living fishes, suggesting that these species engage in relatively active food-searching behavior compared to most other deep-sea fishes. The activity of CS, an enzyme of the citric acid cycle and an indicator of aerobic metabolism, varied little among species. Thus, the large interspecific variation in glycolytic potential (LDH and PK) among species is not associated with a similar variation in aerobic metabolism of white muscle. The much higher and more variable activity of MDH relative to CS suggests that, in addition to its function in the citric acid cycle, MDH may play an important role in redox balance in fish white muscle. In a comparison of white muscle composition between the shallow- and deep-living species, water content did not differ significantly, but protein content was significantly higher in shallow- than in deep-living fishes (211 and 130 mg g^-1 wet wt of muscle, respectively). The differences in muscle protein content are small relative to the differences between shallow- and deep-living species in LDH, PK and MDH activities. Thus, depthrelated differences in muscle enzymic activity are caused by factors other than enzyme dilution. Enzyme activities (LDH, PK and CS) in brain tissue were relatively constant among species regardless of depth of occurrence or feeding and locomotory habits. Habitat and lifestyle do not seem to influence the demands for overall metabolic function in brain. The utility of muscle enzymic activity data for making predictions about the ecological characteristics of difficult-to-observe, deep-living, fishes is discussed.     

三甲基氯化锡对斑马鱼(Danio rerio)生理生化特性的影响

为初步探讨三甲基氯化锡(trimethyltin chloride,TMT)对鱼类的毒性效应以及评价环境中TMT的安全性,采用静态鱼类急性毒性试验法测定了TMT对斑马鱼(Danio rerio)生理生化指标的影响;参考TMT的96 h-LC50值,设定3个浓度(0.39、0.78和1.17 mg·L-1)处理斑马鱼,测...     

Target-site sensitivity in a specialized herbivore towards major toxic compounds of its host plant: the Na+K+-ATPase of the oleander hawk moth (Daphnis nerii) is highly susceptible to cardenolides

The caterpillars of the oleander hawk moth, Daphnis nerii (Linnaeus, 1758) (Lepidoptera: Sphingidae) feed primarily on oleander (Nerium oleander). This plant is rich in cardenolides, which specifically inhibit the Na⁺K⁺-ATPase. Since some insects feeding on cardenolide plants possess cardenolide-resistant Na⁺K⁺-ATPases, we tested whether D. nerii also possesses this strategy for circumventing cardenolide toxicity. To do so, we established a physiological assay, which allowed direct measurement of Na⁺K⁺-ATPase cardenolide sensitivity. Using Schistocerca gregaria, as a cardenolide-sensitive reference species, we showed that D. nerii Na⁺K⁺-ATPase was extremely sensitive to the cardenolide ouabain. Surprisingly, its sensitivity is even higher than that of the cardenolide-sensitive generalist, S. gregaria. The presence or absence of cardenolides in the diet of D. nerii did not influence the enzyme’s cardenolide sensitivity, indicating that target-site insensitivity is not inducible in this species. However, despite the sensitivity of their Na⁺K⁺-ATPase, caterpillars of D. nerii quickly recovered from an injection of an excessive amount of ouabain into their haemocoel. We conclude that D. nerii possesses adaptations, which enable it to feed on a cardenolide-rich diet other than that previously described in cardenolide specialized insects, and discuss other potential resistance mechanisms.     

Interaction of alcohol and kola nut on brain sodium pump activity in Wistar rats

The effect of interaction of alcohol and kola nut on sodium pump activity was studied in Wistar rats. Thirty Wistar rats were divided into six groups of five rats per group. Control received a placebo (4 mL of distilled water). Groups 2–6 were treated for a period of 21 days with 10% (v/v) alcohol, 50 mg caffeine/kg, 50 mg kola nut/kg, or a combination of 10% (v/v) alcohol + 50 mg kola nut/kg, or 10% (v/v) alcohol + 50 mg caffeine/kg, respectively. One day after the final exposure, brains were harvested and several biochemical parameters examined including activities of total ATPase, ouabain-insensitive ATPase, ouabain sensitive ATPase (Na⁺–K⁺-ATPase) and levels of nonenzymatic breakdown of ATP and inorganic phosphate (P_i) released. Results showed that the essential enzyme of the brain responsible for neuronal function, Na⁺–K⁺-ATPase, was inhibited by alcohol–kola nut co-administration relative to control, resulting in a decreased ATP production, ion transport and action potential, leading to loss of neuronal activities.     

Conductive sodium entry in gill cells of the shore crab,Carcinus maenas

Isolated posterior gills of shore crabs, Carcinus maenas, collected from the Baltic Sea, were perfused and bathed with sea water and solutions of alkali chlorides. The preparation was used to measure fluxes of sodium from the external medium across the gills into the hemolymph and to determine transepithelial potential differences (PDs). Internally negative active transport PDs resulted from perfusion and bathing the gills symmetrically with the same medium (50% sea water). Passive (asymmetry) PDs following employment of 100% sea water as internal and 50% sea water as external medium were — in contrast to symmetry PDs — insensitive to cyanide and ouabain. This result indicates that the gill recognized the desired hyperosmotic state and responded by switching off the active transport component observed under symmetry conditions. Diffusional potential differences and fluxes of Na⁺ were inhibited by the externally applied diuretic amiloride. Gradients of pure alkali chlorides between medium and blood were accompanied by cation specific PDs. These PDs and their amiloride sensitivity were inversely related to the diameter of the unhydrated cation and allowed the calculation of the permeability sequence P: Li⁺>Na⁺>K⁺>Rb⁺>Cs⁺>Cl^-. The results obtained show that the permeability of the gills to cations greatly exceeds that to anions. In addition, these findings indicate that the initial amiloride-sensitive step in Na⁺ transport across the gill is not represented by an electroneutral sodium/proton exchange but by a conductive mechanism.     

Interaction of short-term testosterone treatment with osmotic acclimation in the gilthead sea bream <Emphasis Type="Italic">Sparus auratus</Emphasis>

To assess the interaction between testosterone (T) treatment and acclimation to different salinities, seawater-acclimated gilthead sea bream (Sparus auratus) were implanted with slow-release coconut oil implants alone (control) or containing T (5 μg/g body mass). After 5 days, eight fish of control and T-treated groups were sampled. The same day, eight fish of each group were transferred to low salinity water (LSW, 6 ppt, hypoosmotic test), seawater (SW, 38 ppt, control test) and high salinity water (HSW, 55 ppt, hyperosmotic test) and sampled 9 days later. Gill Na⁺, K⁺-ATPase activity increased in HSW-acclimated fish with respect to SW- and LSW-acclimated fish in both control and T-treated groups. Kidney Na⁺, K⁺-ATPase activity was also enhanced in HSW-acclimated fish, but only in T-treated group. From a metabolic point of view, most of the changes observed can be attributed to the action of salinity and T treatment alone, since few interactions between T treatment and osmotic acclimation to different salinities were observed. Those interactions included in treated fish: in the liver, decreased capacity in using glucose in fish acclimated to extreme salinities; in the gills, decreased capacity in using amino acids in HSW; in the kidneys increased capacity in using amino acids in extreme salinities; and in the brain, decreased glycogen and acetoacetate levels of fish in LSW.     

Reduced hypoosmoregulatory ability and alteration in gill chloride cell distribution in mature chum salmon (Oncorhynchus keta) migrating upstream for spawning

Osmoregulatory ability of mature chum salmon (Oncorhynchus keta) during spawning migration was examined by following the changes in gill Na⁺, K⁺-ATPase activity and in the distribution and morphology of chloride cells. Mature chum salmon caught in Otsuchi Bay, northern Honshu Island, Japan, died within 5 d in seawater (SW) in association with a marked increase in plasma osmolality, whereas the fish transferred to fresh water (FW) maintained plasma osmolality efficiently. Gill Na⁺, K⁺-ATPase activity decreased in both SW-maintained and FW-transferred fish. Well-developed chloride cells, identified by immunocytochemical staining specific for Na⁺, K⁺-ATPase, were present mainly in the filament epithelium of immature fish caught in the ocean. In mature fish caught in the bay, however, additional chloride cells were also found in the lamellar epithelium. The number of filament chloride cells decreased markedly in the mature fish both in SW and in FW, whereas the number of lamellar chloride cells was maintained. These results suggest that the loss of hypoosmoregulatory ability in mature chum salmon may be attributable to the decrease in filament chloride cells and associated decrease in gill Na⁺, K⁺-ATPase activity, and also that appearance of lamellar chloride cells may be preparatory to the forthcoming upstream migration. Received: 14 April 1997 / Accepted: 5 May 1997     

Morphological adaptations to chronic hypoxia in deep-sea decapod crustaceans from hydrothermal vents and cold seeps

Animals inhabiting hydrothermal vents and cold seeps face conditions that are challenging for survival. In particular, these two habitats are characterized by chronic hypoxia, sometimes reaching complete anoxia. The characteristics of the scaphognathite and gills were studied in four species of shrimp and three species of crabs from hydrothermal vents and cold seeps, in order to highlight potential adaptations that could enhance oxygen acquisition in comparison with shallow-water relatives. All the vent and seep species studied here exhibit significantly larger scaphognathites, likely allowing more water to flow over their gills per stroke of this appendage. This is probably more energetically efficient that prolonged hyperventilation. In contrast to annelids, vent and seep decapods usually do not possess enlarged gills, a phenomenon likely due to the physical limitations imposed by the size of the gill chamber. In the vent shrimp Rimicaris exoculata and the vent crab Bythograea thermydron, however, there is a significantly higher specific gill surface area linked to a higher number of lamellae per gram of gill. Again in contrast to annelids, the diffusion distance through the gills is not strikingly different between the vent shrimp Alvinocaris komaii and the shallow-water species Palaemon spp. This may indicate that the epithelium and cuticle of the decapod gills are already optimized for oxygen uptake and that reducing the thickness of these compartments is not physically possible without affecting the physical integrity of the gills.     

The effect of cadmium on antioxidant responses and the susceptibility to oxidative stress in the hydrothermal vent mussel Bathymodiolus azoricus

Hydrothermal vents are a unique environment of extreme physical–chemical characteristics and biological species composition. Cd is a toxic non-essential metal present in high concentrations in the hydrothermal vent environment, contrary to those found in marine coastal areas. Cd toxicity has been related, among other things, with reactive oxygen species production, even though this is a non-redox metal. Bathymodiolus azoricus is a deep-sea Mytilid bivalve very common in the Mid Atlantic Ridge (MAR) hydrothermal vent fields and very little is known about the antioxidant defence system in this specie. Because lethal Cd concentration in B. azoricus is unknown, the aim of this study was to assess the effects of a Cd concentration higher than that found in the hydrothermal vents on oxidative stress biomarkers, such as antioxidant enzymes. Mussels were exposed to 100 μg l⁻¹ Cd during 24, 48 and 144 h, respectively, in a pressurized aquarium (IPOCAMP). Superoxide dismutase (SOD), catalase (CAT), glutathione peroxidases (GPx), total oxyradical scavenging capacity (TOSC), metallothionein (MT) and lipid peroxidation (LPO) were measured in the gills and mantle of B. azoricus. The results indicate that gills are first affected by Cd toxicity. This may be due to different physiological functions of the tissues and by the presence of thio and methanotrophic symbiotic bacteria in the gills. The SOD and CAT are inhibited during the first day of exposure in the gills, although TOSC and MT concentrations were the same in control and exposed mussels. In the mantle, enzymatic activation only occurred after 6 days, and no significant differences in MT concentrations were found in the control and exposed mussels during the first day, as observed in the gills.