Plasma non-esterified fatty acids of elasmobranchs: comparisons of temperate and tropical species and effects of environmental salinity.

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/n-6 ratio. n-3/n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4n-6) and docosatetraenoic acid (22:4n-6), and less docosahexaenoic acid (DHA, 22:6n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.

Light induces an increase in the pH of and a decrease in the ammonia concentration in the extrapallial fluid of the giant clam Tridacna squamosa.

The objective of this study was to examine whether 12 h of light exposure would lead to an increase in the pH of and a decrease in the concentration of total ammonia in the extrapallial fluid of the giant clam Tridacna squamosa. We also aimed to elucidate indirectly whether movements of ammonia and/or protons (H(+)) occurred between the extrapallial fluid and the outer mantle epithelium. The pH of the extrapallial fluid of T. squamosa exposed to 12 h of light was significantly higher than that of clams exposed to 12 h of darkness. Conversely, the total ammonia concentration in the extrapallial fluid of the former was significantly lower than that of the latter. In addition, the glutamine content in the mantle adjacent to the extrapallial fluid of clams exposed to 12 h of light was significantly greater than that of clams exposed to 12 h of darkness. These results suggest that in the extrapallial fluid of T. squamosa exposed to light, NH(3) combined with H(+) as NH(+)(4) and that NH(+)(4) was transported into the mantle and used as a substrate for glutamine formation. Injection of NH(4)Cl into the extrapallial fluid led to an instantaneous increase in the total ammonia concentration therein, but the total ammonia concentration decreased subsequently and returned to the control value within 1 h. This is in support of the proposition that NH(+)(4) could be transported from the extrapallial fluid to the mantle. Injection of HCl into the extrapallial fluid led to an instantaneous decrease in the pH of the extrapallial fluid. However, there was a significant increase in pH within 1 h in light or darkness, achieving a partial recovery toward the control pH value. The increase in pH within this 1-h period in light or darkness was accompanied by a significant decrease in the total ammonia concentration in the extrapallial fluid, which supports the proposition that H(+) could be transported in combination with NH(3) as NH(+)(4). Therefore, our results prompt a reexamination of the previous proposition that the removal of H(+) by NH(3) can facilitate calcification in molluscs in general and an investigation of the relationship between H(+) removal through NH(+)(4) transport and light-enhanced calcification in T. squamosa.

Some of the most interesting things we know, and don't know, about the biochemistry and physiology of elasmobranch fishes (sharks, skates and rays).

The urea-retaining strategy of elasmobranchs has shaped their biochemistry and physiology; from their metabolic organization to the structure of their membranes. It has also affected their capacity to live in freshwater. Although much new information has been uncovered in the past 30years, many unanswered questions remain. These include: a) why was urea selected as the major organic osmolyte, b) why is glutamine used as a nitrogen donor, c) why was plasma albumin lost in marine elasmobranchs, d) what membranes are involved in urea retention in the gills, e) how do urea and trimethylamine N-oxide (TMAO) affect membranes, and f) why retain urea in freshwater. Hypotheses are presented for future investigations but some questions may require a time machine to answer.

Widespread Natural Occurrence of Hydroxyurea in Animals.

Here we report the widespread natural occurrence of a known antibiotic and antineoplastic compound, hydroxyurea in animals from many taxonomic groups. Hydroxyurea occurs in all the organisms we have examined including invertebrates (molluscs and crustaceans), fishes from several major groups, amphibians and mammals. The species with highest concentrations was an elasmobranch (sharks, skates and rays), the little skate Leucoraja erinacea with levels up to 250 µM, high enough to have antiviral, antimicrobial and antineoplastic effects based on in vitro studies. Embryos of L. erinacea showed increasing levels of hydroxyurea with development, indicating the capacity for hydroxyurea synthesis. Certain tissues of other organisms (e.g. skin of the frog (64 µM), intestine of lobster (138 µM) gills of the surf clam (100 µM)) had levels high enough to have antiviral effects based on in vitro studies. Hydroxyurea is widely used clinically in the treatment of certain human cancers, sickle cell anemia, psoriasis, myeloproliferative diseases, and has been investigated as a potential treatment of HIV infection and its presence at high levels in tissues of elasmobranchs and other organisms suggests a novel mechanism for fighting disease that may explain the disease resistance of some groups. In light of the known production of nitric oxide from exogenously applied hydroxyurea, endogenous hydoxyurea may play a hitherto unknown role in nitric oxide dynamics.

Mitochondria: aerobic and anaerobic design--lessons from molluscs and fishes.

The contributions of Peter Hochachka to the development of comparative and adaptational biochemistry are substantial. In particular, he and his academic offspring made major contributions to the understanding of the metabolism of molluscs and fishes. These two large taxonomic groups each have marine, freshwater and terrestrial/semiterrestrial representatives, and their mitochondrial metabolism has been shaped by these environmental conditions. In particular, the importance of amino acids and lipids as energy sources has interesting correlations with the environment and the osmotic strategy used. In marine molluscs, amino acids are important aerobic energy sources, and are used as osmolytes and participate in anaerobic metabolism. In marine elasmobranchs, amino acids and ketone bodies, but not lipids per se, are important energy sources in extrahepatic tissues. Marine and freshwater teleost fish by contrast use lipids as an extrahepatic energy source with minimal use of ketone bodies. Furthermore, ketone bodies are important in the metabolism of freshwater and terrestrial but not marine molluscs. The bases for these different metabolic plans may lie in the solute systems used by the different groups (e.g. amino acids in marine molluscs and urea in marine elasmobranchs). The various metabolic options used by fishes and molluscs indicate the plasticity of metabolic design in an environmental context.

Freshwater elasmobranchs: a review of their physiology and biochemistry.

Only 5% of elasmobranch species live in freshwater (FW) compared to more than 40% of known teleost species. The factors affecting the poor penetration of elasmobranchs into FW environments are currently unknown, however, an important consideration may be the high urea requirement of many proteins in marine elasmobranchs. Urea is an important osmolyte in marine elasmobranchs and must be reduced in dilute environments. There are three identifiable stages in the successful colonization of FW. The euryhaline marine species freely entering and leaving FW represent the initial stage of FW colonization. In this group, there is an apparent inability to eliminate all urea due to protein integrity issues and this results in energy and nitrogen losses that may constrain growth and reproduction. The second stage is represented by those species that live entirely in FW but must also retain some urea. This group also suffers from the same constraints as the first group. These two groups have kidneys and sensory organs that more closely resemble strictly marine forms. The third and final stage is represented by the Potamotrygonid stingrays where the need for urea in FW has been eliminated. Consequently nitrogen and energy losses are reduced and those sections of the kidney needed for urea conservation have been eliminated. The driving force for such modifications is a reduction in urea levels and the concomitant saving of energy needed for urea synthesis. Other physiological adaptations associated with survival in FW include giving birth to live young, the capacity of sperm to be activated in freshwater and modifications of the electrosensory system to function in a low conductivity environment. The need for many anatomical, metabolic and physiological modifications for FW existence may constrain the rapidity and hence the frequency of FW colonization, compared to the situation in the more advanced osmoregulating teleosts. Once optimally adapted to FW, recolonization of sea water by elasmobranchs is problematic due to the loss of urea synthetic capacity and renal structures for urea retention.

Upregulation of intracellular antioxidant enzymes in brain and heart during estivation in the African lungfish Protopterus dolloi.

The African slender lungfish, Protopterus dolloi, is highly adapted to withstand periods of drought by secreting a mucous cocoon and estivating for periods of months to years. Estivation is similar to the diapause and hibernation of other animal species in that it is characterized by negligible activity and a profoundly depressed metabolic rate. As is typically observed in quiescent states, estivating P. dolloi are resistant to environmental stresses. We tested the hypothesis that P. dolloi enhances stress resistance during estivation by upregulating intracellular antioxidant defences in brain and heart tissues. We found that most of the major intracellular antioxidant enzymes, including the mitochondrial superoxide dismutase, cytosolic superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase, were upregulated in brain tissue of lungfish that had estivated for 60 days. Several of these enzymes were also elevated in heart tissue of estivators. These changes were not due to food deprivation, as they did not occur in a group of fish that were deprived of food but maintained in water for the same period of time. We found little evidence of tissue oxidative damage in estivators. Products of lipid peroxidation (4-hydroxynonenal adducts) and oxidative protein damage (carbonylation) were similar in estivating and control lungfish. However, protein nitrotyrosine levels were elevated in brain tissue of estivators. Taken together, these data indicate that estivating P. dolloi have enhanced oxidative stress resistance in brain and heart due to a significant upregulation of intracellular antioxidant capacity.

The accumulation of methylamine counteracting solutes in elasmobranchs with differing levels of urea: a comparison of marine and freshwater species.

We compared levels of the major organic osmolytes in the muscle of elasmobranchs, including the methylamines trimethylamine oxide (TMAO), betaine and sarcosine as well as the beta-amino acids taurine and beta-alanine, and the activities of enzymes of methylamine synthesis (betaine and TMAO) in species with a wide range of urea contents. Four marine, a euryhaline in freshwater (Dasyatis sabina), and two freshwater species, one that accumulates urea (Himantura signifer) and one that does not (Potamotrygon motoro), were analyzed. Urea contents in muscle ranged from 229-352 micromol g-1 in marine species to 2.0 micromol g-1 in P. motoro. Marine elasmobranchs preferentially accumulate methylamines, possibly to counteract urea effects on macromolecules, whereas the freshwater species with lower urea levels accumulate the beta-amino acid taurine as the major non-urea osmolyte. A strong correlation (r2=0.84, P<0.001) with a slope of 0.40 was found between muscle urea content and the combined total methylamines plus total beta-amino acids, supporting the hypothesis that ;non-urea' osmolytes are specifically maintained at an approximately 2:1 ratio with urea in the muscle of elasmobranchs. All species examined had measurable synthetic capacity for betaine in the liver but only one species had detectable TMAO synthetic capacity. We propose a phylogenetic explanation for the distribution of TMAO synthesis in elasmobranchs and suggest that activation of liver betaine aldehyde dehydrogenase, relative to choline dehydrogenase, coincides with betaine accumulation in elasmobranchs. The latter relationship may be important in maintaining methylamine levels during periods of low dietary TMAO intake for species lacking TMAO synthesis.

Activities of antioxidant enzymes and cytochrome c oxidase in liver of Arctic and temperate teleosts.

Enhanced antioxidant status in polar fishes may occur due to high dissolved oxygen levels and membranes rich in peroxidation-sensitive polyunsaturated fatty acids. To evaluate the importance of antioxidant enzymes in polar fishes, activities of catalase (CAT), superoxide dismutase (SOD), and glutathione reductase (GR), as well as the aerobic enzyme cytochrome c oxidase (CCO), were measured at 6 degrees C and 1 degrees C in livers of confamilial Arctic and temperate teleosts: the Arctic fourhorn sculpin Triglopsis quadricornis (Cottidae) and saddled eelpout Lycodes mucosus (Zoarcidae) vs. the temperate longhorn sculpin (Myoxocephalus octodecimspinosus) (Cottidae) and ocean pout (Zoarces americanus) (Zoarcidae), respectively. At both assay temperatures, CAT activities were substantially lower in both Arctic species, SOD was similar in the cottids but lower in the Arctic zoarcid, and GR was similar in temperate and Arctic fishes. Activities at respective habitat temperatures were always significantly lower in the Arctic fishes. The lower antioxidant enzyme activities in the Arctic fishes cannot be attributed to lower aerobic status because CCO activity was similar or higher in the Arctic fishes; significant negative relationships were found between CCO and CAT and GR (but not SOD) when all species were combined, indicating that a higher apparent aerobic status does not necessarily coincide with higher antioxidant enzyme activities. Antioxidant enzyme activities may not be enhanced as part of cold adaptation in Arctic fishes, at least in the liver.

Regulation of fish gill Na(+)-K(+)-ATPase by selective sulfatide-enriched raft partitioning during seawater adaptation.

Na(+)-K(+)-ATPase is arguably the most important enzyme in the animal cell plasma membrane, but the role of the membrane in its regulation is poorly understood. We investigated the relationship between Na(+)-K(+)-ATPase and membrane microdomains or "lipid rafts" enriched in sulfatide (sulfogalactosylceramide/SGC), a glycosphingolipid implicated as a cofactor for this enzyme, in the basolateral membrane of rainbow trout gill epithelium. Our studies demonstrated that when trout adapt to seawater (33 ppt), Na(+)-K(+)-ATPase relocates to these structures. Arylsulfatase-induced desulfation of basolateral membrane SGC prevented this relocation and significantly reduced Na(+)-K(+)-ATPase activity in seawater but not freshwater trout. We contend that Na(+)-K(+)-ATPase partitions into SGC-enriched rafts to help facilitate the up-regulation of its activity during seawater adaptation. We also suggest that differential partitioning of Na(+)-K(+)-ATPase between these novel SGC-enriched regulatory platforms results in two distinct, physiological Na(+) transport modes. In addition, we extend the working definition of cholesterol-dependent raft integrity to structural dependence on the sulfate moiety of SGC in this membrane.

Regulation of a renal urea transporter with reduced salinity in a marine elasmobranch, Raja erinacea.

Marine elasmobranchs retain urea and other osmolytes, e.g. trimethylamine oxide (TMAO), to counterbalance the osmotic pressure of seawater. We investigated whether a renal urea transporter(s) would be regulated in response to dilution of the external environment. A 779 bp cDNA for a putative skate kidney urea transporter (SkUT) was cloned, sequenced and found to display relatively high identity with facilitated urea transporters from other vertebrates. Northern analysis using SkUT as a probe revealed three signals in the kidney at 3.1, 2.8 and 1.6 kb. Upon exposure to 50% seawater, the levels of all three SkUT transcripts were significantly diminished in the kidney (by 1.8- to 3.5-fold). In response to environmental dilution, renal tissue osmolality and urea concentration decreased, whereas water content increased. There were no significant differences in osmolyte and mRNA levels between the dorsal-lateral bundle and ventral sections of the kidney. Taken together, these findings provide evidence that the downregulation of SkUT may play a key role in lowering tissue urea levels in response to external osmolality.

Urea transport in kidney brush-border membrane vesicles from an elasmobranch, Raja erinacea.

Marine elasmobranch fishes maintain high urea concentrations and therefore must minimize urea loss to the environment in order to reduce the energetic costs of urea production. Previous studies have identified a facilitated urea transporter in the kidney of the dogfish. We examined mechanisms of urea transport in the kidney of the little skate Raja erinacea using an isolated brush-border membrane vesicle preparation. Urea uptake by brush-border membrane vesicles is by a phloretin-sensitive, non-saturable uniporter in the dorsal section and a phloretin-sensitive, sodium-linked urea transporter (Km = 0.70 mmol l(-1), Vmax = 1.18 micromol h(-1) mg(-1) protein) in the ventral section of the kidney. This provides evidence for two separate urea transporters in the dorsal versus ventral sections of the kidney. We propose that these two mechanisms of urea transport are critical for renal urea reabsorption in the little skate.

The giant mudskipper Periophthalmodon schlosseri facilitates active NH(4)(+) excretion by increasing acid excretion and decreasing NH(3) permeability in the skin.

Periophthalmodon schlosseri is an amphibious and obligatory air-breathing teleost, which is extremely tolerant to environmental ammonia. It actively excretes NH(4)(+) in ammonia loading conditions. For such a mechanism to operate efficaciously the fish must be able to prevent back flux of NH(3). P. schlosseri could lower the pH of 50 volumes (w/v) of 50% seawater in an artificial burrow from pH 8.2 to pH 7.4 in 1 day, and established an ambient ammonia concentration of 10 mmol l(-1) in 8 days. It could alter the rate of titratable acid efflux in response to ambient pH. The rate of net acid efflux (H(+) excretion) in P. schlosseri was pH-dependent, increasing in the order pH 6.0<7.0<8.0<8.5. Net acid flux in neutral or alkaline pH conditions was partially inhibited by bafilomycin, indicating the possible involvement of a V-type H(+)-ATPase. P. schlosseri could also increase the rate of H(+) excretion in response to the presence of ammonia in a neutral (pH 7.0) external medium. Increased H(+) excretion in P. schlosseri occurred in the head region where active excretion of NH(4)(+) took place. This would result in high concentrations of H(+) in the boundary water layer and prevent the dissociation of NH(4)(+), thus preventing a back flux of NH(3) through the branchial epithelia. P. schlosseri probably developed such an 'environmental ammonia detoxification' capability because of its unique behavior of burrow building in the mudflats and living therein in a limited volume of water. In addition, the skin of P. schlosseri had low permeability to NH(3). Using an Ussing-type apparatus with 10 mmol l(-1) NH(4)Cl and a 1 unit pH gradient (pH 8.0 to 7.0), the skin supported only a very small flux of NH(3) (0.0095 micromol cm(-2) min(-1)). Cholesterol content (4.5 micromol g(-1)) in the skin was high, which suggests low membrane fluidity. Phosphatidylcholine, which has a stabilizing effect on membranes, constituted almost 50% of the skin phospholipids, with phosphatidyleserine and phsophatidylethanolamine contributing only 13% and 15%, respectively. More importantly, P. schlosseri increased the cholesterol level (to 5.5 micromol g(-1)) and altered the fatty acid composition (increased total saturated fatty acid content) in its skin lipid after exposure to ammonia (30 mmol l(-1) at pH 7.0) for 6 days. These changes might lead to an even lower permeability to NH(3) in the skin, and reduced back diffusion of the actively excreted NH(4)(+) as NH(3) or the net influx of exogenous NH(3), under such conditions.

The osmotic response of the Asian freshwater stingray (Himantura signifer) to increased salinity: a comparison with marine (Taeniura lymma) and Amazonian freshwater (Potamotrygon motoro) stingrays.

The white-edge freshwater whip ray Himantura signifer can survive in freshwater (0.7 per thousand ) indefinitely or in brackish water (20 per thousand ) for at least two weeks in the laboratory. In freshwater, the blood plasma was maintained hyperosmotic to that of the external medium. There was approximately 44 mmol l(-1) of urea in the plasma, with the rest of the osmolality made up mainly by Na(+) and Cl(-). In freshwater, it was not completely ureotelic, excreting up to 45% of its nitrogenous waste as urea. Unlike the South American freshwater stingray Potamotrygon motoro, H. signifer has a functional ornithine-urea cycle (OUC) in the liver, with hepatic carbamoylphosphate synthetase III (CPS III) and glutamine synthetase (GS) activities lower than those of the marine blue-spotted fan tail ray Taeniura lymma. More importantly, the stomach of H. signifer also possesses a functional OUC, the capacity (based on CPS III activity) of which was approximately 70% that in the liver. When H. signifer was exposed to a progressive increase in salinity through an 8-day period, there was a continuous decrease in the rate of ammonia excretion. In 20 per thousand water, urea levels in the muscle, brain and plasma increased significantly. In the plasma, osmolality increased to 571 mosmol kg(-1), in which urea contributed 83 mmol l(-1). Approximately 59% of the excess urea accumulated in the tissues of the specimens exposed to 20 per thousand water was equivalent to the deficit in ammonia excretion through the 8-day period, indicating that an increase in the rate of urea synthesis de novo at higher salinities would have occurred. Indeed, there was an induction in the activity of CPS III in both the liver and stomach, and activities of GS, ornithine transcarbamoylase and arginase in the liver. Furthermore, there was a significant decrease in the rate of urea excretion during passage through 5 per thousand, 10 per thousand and 15 per thousand water. Although the local T. lymma in full-strength sea water (30 per thousand ) had a much greater plasma urea concentration (380 mmol l(-1)), its urea excretion rate (4.7 micromol day(-1) g(-1)) was comparable with that of H. signifier in 20 per thousand water. Therefore, H. signifer appears to have reduced its capacity to retain urea in order to survive in the freshwater environment and, consequently, it could not survive well in full-strength seawater.

A comparison of the effects of environmental ammonia exposure on the Asian freshwater stingray Himantura signifer and the Amazonian freshwater stingray Potamotrygon motoro.

The white-edge whip tail ray Himantura signifer inhabits a freshwater environment but has retained the capability to synthesize urea de novo through the arginine-ornithine-urea cycle (OUC). The present study aimed to elucidate whether the capacity of urea synthesis in H. signifer could be upregulated in response to environmental ammonia exposure. When H. signifer was exposed to environmental ammonia, fairly high concentrations of ammonia were accumulated in the plasma and other tissues. This would subsequently reduce the net influx of exogenous ammonia by reducing the NH(3) partial pressure gradient across the branchial and body surfaces. There was also an increase in the OUC capacity in the liver. Since the ammonia produced endogenously could not be excreted effectively in the presence of environmental ammonia, it was detoxified into urea through the OUC. In comparison, the South American freshwater stingray Potamotrygon motoro, which has lost the capability to synthesize urea de novo, was unable to detoxify ammonia to urea during ammonia loading. No increase in glutamine was observed in the various tissues of H. signifer exposed to environmental ammonia despite a significant increase in the hepatic glutamine synthetase activity. These results indicate that the excess glutamine formed was channelled completely into urea formation through carbamoyl phosphate synthetase III. It has been reported elsewhere that both urea synthesis and urea retention were upregulated in H. signifer exposed to 20 per thousand water for osmoregulatory purposes. By contrast, for H. signifer exposed to environmental ammonia in freshwater, the excess urea formed was excreted to the external medium instead. This suggests that the effectiveness of urea synthesis de novo as a strategy to detoxify ammonia is determined not simply by an increase in the capacity of urea synthesis but, more importantly, by the ability of the animal to control the direction (i.e. absorption or excretion) and rate of urea transport. Our results suggest that such a strategy began to develop in those elasmobranchs, e.g. H. signifer, that migrate into a freshwater environment from the sea but not in those permanently adapted to a freshwater environment.