Waterborne amino acids: uptake and functional roles in aquatic animals.

Dissolved organic matter is a ubiquitous component of freshwater and marine environments, and includes small nutrient molecules, such as amino acids, which may be available for uptake by aquatic biota. Epithelial transporters, including cotransporters, uniporters and antiporters, facilitate the absorption of dissolved amino acids (often against concentration gradients). Although there is a lack of mechanistic and molecular characterization of such transporters, pathways for the direct uptake of amino acids from the water appear to exist in a wide range of marine phyla, including Porifera, Cnidaria, Platyhelminthes, Brachiopoda, Mollusca, Nemertea, Annelida, Echinodermata, Arthropoda and Chordata. In these animals, absorbed amino acids have several putative roles, including osmoregulation, hypoxia tolerance, shell formation and metabolism. Therefore, amino acids dissolved in the water may play an important, but overlooked, role in aquatic animal nutrition.

Sub-chronic exposure to waterborne extracellular microcystin-LR impairs calcium homeostasis in rainbow trout.

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L-1 (measured values 2 and 49 µg L-1, respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L-1. A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L-1, but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown.

Hypoxia modifies calcium handling in the Pacific hagfish, Eptatretus stoutii.

Hagfishes may encounter low dissolved oxygen in their natural habitats, a consequence of association with hypoxic sediments and their feeding behaviour. In teleost fish, hypoxia exposure decreases ion uptake, speculated to be a mechanism for energy conservation. Although hagfishes osmoconform, they do regulate extracellular fluid concentrations of divalent cations such as calcium. The current study hypothesised that exposure of hagfish to hypoxia (0.4 kPA, 24 h) would reduce calcium uptake (determined via in vitro isolated skin and gut epithelial transport assays) and calcium accumulation (determined by in vivo whole animal exposures, using radiolabelled calcium (45Ca) to assess newly acquired calcium). A decrease in in vitro epidermal uptake was observed at sub-environmental calcium levels (10 µM), but not at environmental calcium levels (10 mM). No changes in gut calcium uptake were determined. Conversely, hypoxia led to a more rapid in vivo accumulation of calcium in tissues (skin, muscle, liver, heart, plasma, brain), mediated mostly by a significant increase in accumulation at the gill. These differences were only apparent after 1-h of exposure to the radiolabel (i.e., the last hour of the 24-h hypoxia exposure) and were not observed after 3 and 24 h periods of radiolabel exposure. This outcome was the opposite of the hypothesised effect. The reasons for a more rapid accumulation of calcium in hypoxic hagfish are unknown but may relate to roles for calcium in enhancing hypoxia tolerance in hagfishes or could be a consequence of changes in ventilatory frequency.

The skin of adult rainbow trout is not a significant site of ammonia clearance from the blood.

We hypothesized that the skin acts as an extrabranchial route for ammonia excretion in adult rainbow trout (Oncorhynchus mykiss) following high environmental ammonia (HEA) exposure. Trunks of control or HEA-exposed trout were perfused with saline containing 0 or 1 mmol l-1 NH4 + . Cutaneous ammonia excretion rates increased 2.5-fold following HEA exposure, however there was no difference in rates between trunks perfused with 0 or 1 mmol l-1 NH4 + . The skin is therefore capable of excreting its own ammonia load, but it does not clear circulating ammonia from the plasma.

The good, the bad and the slimy: experimental studies of hagfish digestive and nutritional physiology.

The hagfishes provide valuable insight into the physiology of feeding, digestion and nutrient absorption by virtue of unusual and unique features of their biology. For example, members of this group undergo long periods of fasting, and are the only vertebrates known to absorb organic nutrients across their epidermal surface. Such properties engender significant attention from researchers interested in feeding and feeding-related processes; however, the practical realities of employing the hagfish as an experimental organism can be challenging. Many of the key tools of the experimental biologist are compromised by a species that does not readily feed in captivity, is difficult to instrument and which produces copious quantities of slime. This Commentary provides critical insight into the key aspects of hagfish feeding and digestive processes, and highlights the pitfalls of this group as experimental organisms. We also suggest key research gaps that, if filled, will lead to better understanding of hagfishes, and we consider how this group may advance our knowledge of feeding, digestion and nutrient absorption processes.

Feeding in Eptatretus cirrhatus: effects on metabolism, gut structure and digestive processes, and the influence of post-prandial dissolved oxygen availability.

Hagfishes are characterised by feeding behaviours that may include long intervals between meals, and a hypoxic feeding environment inside decaying carrion. The effects of feeding on metabolism (oxygen consumption rate), gut mass and morphology (gut somatic index, gut epithelium mucosal thickness), and digestive function (maltase and peptidase activity) were examined in the New Zealand hagfish, Eptatretus cirrhatus. The influence of post-prandial hypoxia on oxygen consumption rate was also investigated to replicate the immersive feeding environment. Fed hagfish displayed a 1.9-fold increase in peak oxygen consumption relative to sham controls. This elevation in post-prandial oxygen consumption continued for 72 h, during which the energy cost of digesting the meal (specific dynamic action; SDA) was 2.1 kJ. Oxygen consumption rate increased when the post-prandial environment was hypoxic, a response suggesting a lack of hypoxia tolerance in this species. Feeding did not alter gut somatic index (percentage of digesta-free gut mass to whole body mass), but there was an increase in the mucosal thickness of the gut epithelium. Maltase activity in the gut was unchanged by feeding, but the activity of gut peptidases was increased significantly, consistent with a protein-based diet. These data indicate that some postprandial responses of New Zealand hagfish are similar in nature to those seen in other animals, but this species does not exhibit the extreme post-prandial physiological and biochemical changes that are observed in other intermittently-feeding vertebrates.

Acquisition of alanyl-alanine in an Agnathan: Characteristics of dipeptide transport across the hindgut of the Pacific hagfish Eptatretus stoutii.

This study used 3 H-L -alanyl-L -alanine to demonstrate dipeptide uptake using in vitro gut sacs prepared from the hindgut of the Pacific hagfish Eptatretus stoutii. Concentration-dependent kinetic analysis resulted in a sigmoidal distribution with a maximal (± SE) uptake rate (Jmax -like) of 70 ± 3 nmol cm-2 h-1 and an affinity constant (Km -like) of 1072 ± 81 µM. Addition of high alanine concentrations to transport assays did not change dipeptide transport rates, indicating that hydrolysis of the dipeptide in mucosal solutions and subsequent uptake via apical amino acid transporters was not occurring, which was further supported by a Km distinct from that of amino acid transport. Transport occurred independent of mucosal pH, but uptake was reduced by 42% in low mucosal sodium. This may implicate cooperation between peptide transporters and sodium-proton exchangers, previously demonstrated in several mammalian and teleost species. Finally, apical L -alanyl-L -alanine uptake rates (i.e., mucosal disappearance) were significantly increased following a meal, demonstrating regulation of uptake. Overall, this examination of dipeptide acquisition in the earliest extant Agnathan suggests evolutionarily conserved mechanisms of transport between hagfish and later-diverging vertebrates such as teleosts and mammals.

Acute waterborne cadmium toxicity in the estuarine pulmonate mud snail, Amphibola crenata.

Freshwater pulmonate snails are sensitive to trace metals, but to date, the sensitivity of estuarine pulmonate snails to these important environmental toxicants is undescribed. Using the estuarine mud snail Amphibola crenata, effects of a 48-h exposure to waterborne cadmium (Cd) were investigated. The 48-h median lethal concentration (LC50) was 50.4 mg L-1, a value higher than that previously reported for any gastropod mollusc. Cadmium levels in the tissues of mud snails were highest in the viscera (digestive gland and gonad), with the foot muscle and remaining tissue compartment (kidney, mantle, remaining digestive tissues and heart) displaying significantly lower concentrations. Over a Cd exposure concentration range of 0-32 mg L-1, Amphibola exhibited reduced oxygen consumption and elevated ammonia excretion in response to increasing Cd, the latter effect likely reflecting a switch to protein metabolism. This finding was supported by a declining oxygen: nitrogen ratio (O:N) as exposure Cd concentration increased. Other energy imbalances were noted, with a decrease in tissue glycogen (an effect strongly correlated with Cd burden in the viscera and foot muscle) and an elevated haemolymph glucose observed. An increase in catalase activity in the visceral tissues was recorded, suggestive of an effect of Cd on oxidative stress. The magnitude of this effect was correlated with tissue Cd burden. The induction of antioxidant defence mechanisms likely prevented an increase in levels of lipid peroxidation, which were unchanged relative to Cd exposure concentration in all measured tissues.

Effect of environmental salinity manipulation on uptake rates and distribution patterns of waterborne amino acids in the Pacific hagfish.

Among vertebrates, hagfish are the only known iono- and osmoconformers, and the only species thus far documented to absorb amino acids directly across the skin. In the current study, short-term (6h) manipulations of exposure salinities (75-125% seawater) were conducted to determine whether changes in osmotic demands influenced the uptake and tissue distribution of waterborne amino acids (alanine, glycine and phenylalanine), in the Pacific hagfish, Eptatretus stoutii. No changes in erythrocyte or muscle amino acid accumulation rates were noted, but the patterns of plasma amino acid accumulation were suggestive of regulation. Contrary to expectations, glycine transport across the skin in vitro was enhanced in the lowest exposure salinity, but no other salinity-dependent changes were demonstrated. Overall, this study indicates that uptake and distribution of amino acids varies with salinity, but not in a manner that is consistent with a role for the studied amino acids in maintaining osmotic balance in hagfish.

Behavioural, physiological and biochemical responses to aquatic hypoxia in the freshwater crayfish, Paranephrops zealandicus.

Hypoxia resulting from aquatic eutrophication threatens the population health of the New Zealand freshwater crayfish (koura), Paranephrops zealandicus. An integrated study, combining behavioural, physiological and biochemical approaches, was therefore conducted to characterise the tolerance of this species to hypoxia. When provided with a choice between water flows of high or low dissolved oxygen in short-term laboratory assays, crayfish did not preferentially inhabit waters of higher PO2. However, when an aerial refuge was provided and dissolved oxygen was progressively decreased, crayfish emersed at a PO2 of 0.56±0.03kPa, suggesting a relatively high tolerance to hypoxia. Closed-box respirometry delineated a Pcrit, the point at which crayfish transition from oxyregulating to oxyconforming, of 6.0kPa. Simultaneous measurement of heart rate showed no changes across the PO2 range. In response to 6-h exposures to fixed dissolved oxygen levels (normoxia, 19.3kPa; moderate hypoxia, 3.5kPa; and severe hypoxia, 1.7kPa), P. zealandicus showed a haemolymph PO2 that declined with the magnitude of hypoxia, and while plasma pH declined in severe hypoxia, there were no changes in plasma PCO2. Plasma glucose concentrations fell, and plasma lactate increased in both hypoxic groups. There were no changes in tissue glucose or lactate concentrations. These data indicate that P. zealandicus is relatively tolerant of hypoxia, and possesses biochemical and physiological mechanisms that facilitate survival during short-term exposures to acute hypoxia. If hypoxia is severe and/or prolonged, then this species is capable of escaping to aerial refugia.

Acute and sub-chronic effects of sub-lethal cadmium exposure on energy metabolism in the freshwater shrimp, Paratya curvirostris.

Cadmium (Cd) is a toxic trace element enriched in waters through activities such as mining and agriculture. The freshwater shrimp Paratya curvirostris inhabits near-coastal, lowland streams potentially impacted by Cd, but nothing is known regarding its sensitivity to this metal. An acute (96h) median lethal concentration (LC50) of 405µgL-1 was derived for P. curvirostris, placing it among the most tolerant of freshwater shrimp species. Acute (4 d; 0, 50 and 100µgL-1) and sub-chronic (10 d; 0, 25 and 50µgL-1) exposures then investigated effects of Cd on energy metabolism (respiration rate, excretion rate, O:N ratio). In contrast to effects in previously studied species, Cd induced an increased respiration rate, which when coupled with an unchanged excretion rate, resulted in an increased O:N ratio. These data were explained by an increased reliance on carbohydrate and/or lipid as a metabolic substrate stimulated by increased metabolic costs of toxicant exposure. Similar effects were seen across all time-points, although the lowest effective Cd concentration decreased with increased exposure time. Overall, results suggest that Cd is unlikely to be a significant environmental stressor to P. curvirostris, except in highly contaminated freshwaters, and/or where Cd co-occurs with hypoxia.

Effects of waterborne cadmium on energy metabolism in the tropical sea cucumber, Stichopus horrens, and a comparison of tissue-specific cadmium accumulation with the temperate sea cucumber Australostichopus mollis.

The sea cucumber Stichopus horrens is an important component of near-shore ecosystems, and in the Kingdom of Tonga it also comprises an important commercial and subsistence fishery. To assess the sensitivity of this species to the toxic trace metal cadmium (Cd), adult S. horrens were exposed for 96h to an environmental (15µgL-1) or effect (765µgL-1) concentration of waterborne Cd. The respiratory tree and intestine accumulated higher concentrations of Cd than the muscle and body wall, but there were no effects of Cd on tissue ions (sodium, potassium, magnesium, calcium). For comparison, Cd accumulation was also examined in the Australasian sea cucumber Australostichopus mollis. This species displayed a similar pattern of tissue-specific accumulation to S. horrens, but exhibited lower tissue Cd burdens, likely a consequence of lower experimental temperature. Effects on gonad ion content were also seen in this species. At the highest Cd exposure concentration, S. horrens showed impaired ammonia excretion rates and an increased molar oxygen:nitrogen ratio (O:N), indicative of a decreased reliance on protein metabolism. Overall, this study suggests that S. horrens is relatively tolerant of Cd exposure, but raises concerns regarding the subsistence fishery practice of consuming the viscera of this species.

A model system using confocal fluorescence microscopy for examining real-time intracellular sodium ion regulation.

The gills of euryhaline fish are the ultimate ionoregulatory tissue, achieving ion homeostasis despite rapid and significant changes in external salinity. Cellular handling of sodium is not only critical for salt and water balance but is also directly linked to other essential functions such as acid-base homeostasis and nitrogen excretion. However, although measurement of intracellular sodium ([Na(+)]i) is important for an understanding of gill transport function, it is challenging and subject to methodological artifacts. Using gill filaments from a model euryhaline fish, inanga (Galaxias maculatus), the suitability of the fluorescent dye CoroNa Green as a probe for measuring [Na(+)]i in intact ionocytes was confirmed via confocal microscopy. Cell viability was verified, optimal dye loading parameters were determined, and the dye-ion dissociation constant was measured. Application of the technique to freshwater- and 100% seawater-acclimated inanga showed salinity-dependent changes in branchial [Na(+)]i, whereas no significant differences in branchial [Na(+)]i were determined in 50% seawater-acclimated fish. This technique facilitates the examination of real-time changes in gill [Na(+)]i in response to environmental factors and may offer significant insight into key homeostatic functions associated with the fish gill and the principles of sodium ion transport in other tissues and organisms.

Mechanisms of Nickel Toxicity in the Highly Sensitive Embryos of the Sea Urchin Evechinus chloroticus, and the Modifying Effects of Natural Organic Matter.

A 96 h toxicity test showed that the embryos of the New Zealand sea urchin (Evechinus chloroticus) are the most sensitive of all studied marine species to waterborne nickel (Ni), with the EC50 for the development of fully formed pluteus larvae found to be 14 µg L(-1). Failure to develop a standard larval shape suggested skeletal impairment. Whole body ions (Na, Mg) increased with Ni exposure and calcium influx was depressed. The effects of natural organic matter (NOM) on Ni accumulation and toxicity were also examined in three different seawater sources (nearshore, offshore, and near the outlet of a "brown water" stream). At low dissolved organic carbon (DOC) concentrations the brown water NOM was protective against Ni toxicity, however at higher DOC concentrations it exacerbated developmental toxicity in the presence of Ni. These results show that sea urchin development is highly sensitive to Ni via a mechanism that involves ionoregulatory disturbance, and that Ni toxicity is influenced by environmental factors such as NOM. These data will be critical for the development of water quality guidelines for Ni in the marine environment.

Iron transport across the skin and gut epithelia of Pacific hagfish: Kinetic characterisation and effect of hypoxia.

In most animals, the acquisition of the essential trace metal iron (Fe) is achieved by the gut, but in hagfishes, the skin is a nutrient absorbing epithelium, and thus may also play a role in Fe uptake. In the current study, the absorption of Fe, as Fe(II), across the intestinal and cutaneous epithelia of Pacific hagfish (Eptatretus cirrhatus) was investigated. Both epithelia absorbed Fe, with saturation at lower tested concentrations, superseded by a diffusive component at higher Fe exposure concentrations. Affinity constants (Km) of 9.4 and 137µM, and maximal Fe transport rates (Jmax) of 0.81 and 0.57nmolcm(-2)h(-1) were determined for the skin and the gut, respectively. This characterises the skin as a relatively high-affinity Fe transport epithelium. The majority of the absorbed Fe in the skin remained in the tissue, whereas in the gut, most absorbed Fe was found in the serosal fluid, suggesting distinct mechanisms of Fe handling between the two epithelia. To determine if reduced dissolved oxygen altered Fe transport, hagfish were subjected to hypoxia for 24h, before Fe transport was again assessed. Hypoxia had no effect on Fe transport across gut or skin, likely owing to the relative lack of change in haematological variables, and thus an unaltered Fe demand under such conditions. These data are the first to kinetically characterise the absorption of a nutritive trace metal across the epithelia of hagfish and add to the growing understanding of the role of the skin in nutritive transport in this group.

Novel route of toxicant exposure in an ancient extant vertebrate: nickel uptake by hagfish skin and the modifying effects of slime.

Utilizing an in vitro technique, the skin of Pacific hagfish (Eptatretus stouti) was shown to take up nickel from the water via a high affinity, low capacity transport pathway. Uptake was biphasic, with saturation occurring at low nickel exposure concentrations, superseded by linear, diffusive uptake at levels greater than 50 µM. In vivo exposures showed that nickel accumulated mainly in the gill, heart, and brain, representing a tissue distribution distinct from that found in teleosts. Slime on the epidermal surface was shown to significantly reduce the uptake of low concentrations (10 µM) of the metals zinc and nickel, but slime had no effect on organic nutrient (the amino acid l-alanine) absorption. At a higher metal exposure concentration (1 mM), slime was no longer protective, indicating saturation of metal-binding sites. This is the first study to show that metals can be taken up by the integument of hagfish. The ability of the skin to act as a transport epithelium may be of particular importance for a burrowing, benthic scavenger, such as hagfish, which are likely to be exposed to relatively enriched levels of metal toxicants through their habitat and lifestyle, and this may have consequences for human health through hagfish consumption.

Physiological and biochemical strategies for withstanding emersion in two galaxiid fishes.

The galaxiid fishes of the Southern hemisphere display variable tolerance to aerial exposure. Brown mudfish (Neochanna apoda), for example, pseudoaestivate, inhabiting moist soil for months at a time, whereas inanga (Galaxias maculatus) emerse under unfavourable water conditions, but only for periods of a few hours. This study sought to identify the physiological and biochemical strategies that determine emersion tolerance in these species. Nitrogenous waste excretion was measured before and after an experimental emersion period (14 days for mudfish, 6 h for inanga). Both species showed significantly elevated ammonia "washout" upon return to water, but no increase in plasma or muscle ammonia. Post-emersion urea levels were elevated in plasma and muscle in both fish, however the extent of the accumulation did not indicate significant de novo urea production. This was supported by the lack of carbamoyl phosphate synthetase activity in tissues. Consequently, mudfish metabolism was examined to determine whether changes in parameters such as oxygen consumption, carbon dioxide excretion, and/or altered metabolic costs (represented by the key ionoregulatory enzyme Na(+), K(+)-ATPase; NKA) could explain emersion tolerance. Oxygen consumption rates, already very low in immersed mudfish, were largely maintained over the course of emersion. Carbon dioxide excretion decreased during emersion, and a small, but significant, decrease in NKA was noted. These data suggest that the extended emersion capacity of mudfish may result from a generally low metabolic rate that is maintained throughout aerial exposure via cutaneous gas exchange, and which limits the production of potentially toxic nitrogenous waste.

Development of acute and chronic sediment bioassays with the harpacticoid copepod Quinquelaophonte sp.

Reliable environmentally realistic bioassay methodologies are increasingly needed to assess the effects of environmental pollution. This study describes two estuarine sediment bioassays, one acute (96 h) and one chronic (14 d), with the New Zealand harpacticoid copepod Quinquelaophonte sp. utilising behavioural and reproductive endpoints. Spiked sediments were used to expose Quinquelaophonte sp. to three reference compounds representing important categories of estuarine chemical stressors: zinc (a metal), atrazine (a pesticide), and phenanthrene (a polycyclic aromatic hydrocarbon). Acute-to-chronic ratios (ACR) were used to further characterise species responses. Acute sediment (sandy and low total organic content) 96 h EC50 values for the sublethal inhibition of mobility for zinc, atrazine and phenanthrene were 137, 5.4, and 2.6 µg/g, respectively. The chronic EC50 values for inhibition of reproduction (total offspring) were 54.5, 0.0083, and 0.067 µg/g for zinc, atrazine, and phenanthrene, respectively. For phenanthrene, a potentially novel mode of action was identified on reproduction. Quinquelaophonte sp. was found to be more sensitive than several other estuarine species indicating choice of test organism is important to characterising the effects of environmentally relevant levels of contamination. The bioassay sediment results demonstrate the sensitivity and suitability of Quinquelaophonte sp. as a tool for the assessment use of estuarine health.

Mechanistic characterization of waterborne selenite uptake in the water flea, Daphnia magna, indicates water chemistry affects toxicity in coal mine-impacted waters.

Concentrations of selenium that exceed regulatory guidelines have been associated with coal mining activities and have been linked to detrimental effects on aquatic ecosystems and the organisms therein. Although the major route of selenium uptake in macroinvertebrates is via the diet, the uptake of waterborne selenite (HSeO3-), the prominent form at circumneutral pH, can be an important contributor to selenium body burden and thus selenium toxicity. In the current study, radiolabelled selenite (Se75) was used to characterize the mechanism of selenite uptake in the water flea, Daphnia magna. The concentration dependence (1-32 µM) of selenite uptake was determined in 1-hour uptake assays in artificial waters that independently varied in bicarbonate, chloride, sulphate, phosphate and selenate concentrations. At concentrations representative of those found in highly contaminated waters, selenite uptake was phosphate-dependent and inhibited by foscarnet, a phosphate transport inhibitor. At higher concentrations, selenite uptake was dependent on waterborne bicarbonate concentration and inhibited by the bicarbonate transporter inhibitor DIDS (4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid). These findings suggest that concentrations of phosphate in coal mining-affected waters could alter selenite uptake in aquatic organisms and could ultimately affect the toxic impacts of selenium in such waters.