Carol Bucking

Toxicity of dissolved Cu, Zn, Ni and Cd to developing embryos of the blue mussel (Mytilus trossolus) and the protective effect of dissolved organic carbon.

Marine water quality criteria for metals are largely driven by the extremely sensitive embryo-larval toxicity of Mytilus sp. Here we assess the toxicity of four dissolved metals (Cu, Zn, Ni, Cd) in the mussel Mytilus trossolus, at various salinity levels while also examining the modifying effects of dissolved organic carbon (DOC) on metal toxicity. In 48 h embryo development tests in natural seawater, measured EC50 values were 6.9-9.6 microg L(-1) (95% C.I.=5.5-10.8 microg L(-1)) for Cu, 99 microg L(-1) (86-101) for Zn, 150 microg L(-1) (73-156) for Ni, and 502 microg L(-1) (364-847) for Cd. A salinity threshold of >20 ppt (approximately 60% full strength seawater) was required for normal control development. Salinity in the 60-100% range did not alter Cu toxicity. Experimental addition of dissolved organic carbon (DOC) from three sources reduced Cu toxicity; for example the EC50 of embryos developing in seawater with 20 mg C L(-1) was 39 microg Cu L(-1) (35.2-47.2) a 4-fold increase in Cu EC50. The protective effects of DOC were influenced by their distinct physicochemical properties. Protection appears to be related to higher fulvic acid and lower humic acid content as operationally defined by fluorescence spectroscopy. The fact that DOC from freshwater sources provides protection against Cu toxicity in seawater suggests that extrapolation from freshwater toxicity testing may be possible for saltwater criteria development, including development of a saltwater Biotic Ligand Model for prediction of Cu toxicity.

The alkaline tide and ammonia excretion after voluntary feeding in freshwater rainbow trout

SUMMARY We investigated the potential acid–base and nitrogenous waste excretion challenges created by voluntary feeding in freshwater rainbow trout, with particular focus on the possible occurrence of an alkaline tide (a metabolic alkalosis created by gastric HCl secretion during digestion). Plasma metabolites (glucose, urea and ammonia) were measured at various time points before and after voluntary feeding to satiation (approximately 5% body mass meal of dry commercial pellets), as was the net flux of ammonia and titratable alkalinity to the water from unfed and fed fish. Arterial blood, sampled by indwelling catheter, was examined for post-prandial effects on pH, plasma bicarbonate and plasma CO2 tension. There was no significant change in plasma glucose or urea concentrations following feeding, whereas plasma ammonia transiently increased, peaking at threefold above resting values at 12 h after the meal and remaining elevated for 24 h. The increased plasma ammonia was correlated with an increase in net ammonia excretion to the water, with fed fish significantly elevating their net ammonia excretion two- to threefold between 12 and 48 h post feeding. These parameters did not change in unfed control fish. Fed fish likewise increased the net titratable base flux to the water by approximately threefold, which resulted in a transition from a small net acid flux seen in unfed fish to a large net base flux in fed fish. Over 48 h, this resulted in a net excretion of 13 867 μmol kg–1 more base to the external water than in unfed fish. The arterial blood exhibited a corresponding rise in pH (between 6 and 12 h) and plasma bicarbonate (between 3 and 12 h) following feeding; however, no respiratory compensation was observed, as PaCO2 remained constant. Overall, there was evidence of numerous challenges created by feeding in a freshwater teleost fish, including the occurrence of an alkaline tide, and its compensation by excretion of base to the external water. The possible influence of feeding ecology and environmental salinity on these challenges, as well as discrepancies in the literature, are discussed.

Water dynamics in the digestive tract of the freshwater rainbow trout during the processing of a single meal

SUMMARY The temporal effects of feeding and digestion on chyme composition, specifically water and solid content, and net fluxes across the gastrointestinal tract, as well as plasma parameters, were examined in freshwater rainbow trout. A single meal of commercial dry pellets, incorporating ballotini beads as inert reference markers, was employed. Plasma Na+ levels increased by 15–20% at 2 h post-feeding, where Cl– levels did not change. Plasma osmolality was well regulated despite an initial chyme osmolality (775 mOsm) 2.8-fold higher than that in the blood plasma. Chyme osmolality throughout the gastrointestinal tract remained significantly higher than plasma osmolality for the duration of the 72 h period. Solid material was absorbed along the entire intestinal tract, although not in the stomach, necessitating the incorporation of an inert marker. A similar temporal pattern of transit between the ballotini beads (solid phase marker) and 3[H]-PEG 4000 (fluid phase marker), provided support for the use of ballotini beads. Large additions of water to the chyme were seen in the stomach, the largest occurring within 2 h following feeding (7.1±1.4 ml kg–1), and amounted to ∼16 ml kg–1 over the first 12 h. As the chyme entered the anterior intestine, a further large water secretion (3.5±0.5 ml kg–1) was seen. Thereafter the water fluxes into the chyme of the anterior intestine decreased steadily over time, but remained positive, whereas the mid-intestine exhibited net absorption of water at all time points, and the posterior intestine demonstrated little water handling at any time. The endogenous water that was secreted into the anterior intestine was absorbed along the tract, which showed a net water flux close to zero. However, assuming that the water secreted into the stomach was endogenous in nature, the processing of a single meal resulted in net loss of endogenous water (0.24 ml kg–1 h–1) to the environment, a beneficial consequence of the osmotic challenge offered by the food for a freshwater hyperosmotic regulator.

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus).

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.

The skin of fish as a transport epithelium: a review.

The primary function of fish skin is to act as a barrier. It provides protection against physical damage and assists with the maintenance of homoeostasis by minimising exchange between the animal and the environment. However in some fish, the skin may play a more active physiological role. This is particularly true in species that inhabit specialised environmental niches (e.g. amphibious and air-breathing fish such as the lungfish), those with physiological characteristics that may subvert the need for the integument as a barrier (e.g. the osmoconforming hagfish), and/or fish with anatomical modifications of the epidermis (e.g. reduced epithelial thickness). Using examples from different fish groups (e.g. hagfishes, elasmobranchs and teleosts), the importance of fish skin as a transport epithelium for gases, ions, nitrogenous waste products, and nutrients was reviewed. The role of the skin in larval fish was also examined, with early life stages often utilising the skin as a surrogate gill, prior to the development of a functional branchial epithelium.

Adaptations to in situ feeding: novel nutrient acquisition pathways in an ancient vertebrate

During feeding, hagfish may immerse themselves in the body cavities of decaying carcasses, encountering high levels of dissolved organic nutrients. We hypothesized that this feeding environment might promote nutrient acquisition by the branchial and epidermal epithelia. The potential for Pacific hagfish, Eptatretus stoutii, to absorb amino acids from the environment across the skin and gill was thus investigated. l-alanine and glycine were absorbed via specific transport pathways across both gill and skin surfaces, the first such documentation of direct organic nutrient acquisition in a vertebrate animal. Uptake occurred via distinct mechanisms with respect to concentration dependence, sodium dependence and effects of putative transport inhibitors across each epithelium. Significant differences in the absorbed amino acid distribution between the skin of juveniles and adults were noted. The ability to absorb dissolved organic matter across the skin and gill may be an adaptation to a scavenging lifestyle, allowing hagfish to maximize sporadic opportunities for organic nutrient acquisition. From an evolutionary perspective, hagfish represent a transitory state between the generalized nutrient absorption pathways of aquatic invertebrates and the more specialized digestive systems of aquatic vertebrates.

Acid-base responses to feeding and intestinal Cl- uptake in freshwater- and seawater-acclimated killifish, Fundulus heteroclitus, an agastric euryhaline teleost.

SUMMARY Marine teleosts generally secrete basic equivalents (HCO3–) and take up Na+ and Cl– in the intestine so as to promote absorption of H2O. However, neither the integration of these functions with feeding nor the potential role of the gut in ionoregulation and acid–base balance in freshwater have been well studied. The euryhaline killifish (Fundulus heteroclitus) is unusual in lacking both an acid-secreting stomach and a mechanism for Cl– uptake at the gills in freshwater. Responses to a satiation meal were evaluated in both freshwater- and seawater-acclimated killifish. In intact animals, there was no change in acid or base flux to the external water after the meal, in accord with the absence of any post-prandial alkaline tide in the blood. Indeed, freshwater animals exhibited a post-prandial metabolic acidosis (‘acidic tide’), whereas seawater animals showed no change in blood acid–base status. In vitro gut sac experiments revealed a substantially higher rate of Cl– absorption by the intestine in freshwater killifish, which was greatest at 1–3 h after feeding. The Cl– concentration of the absorbate was higher in preparations from freshwater animals than from seawater killifish and increased with fasting. Surprisingly, net basic equivalent secretion rates were also much higher in preparations from freshwater animals, in accord with the ‘acidic tide’; in seawater preparations, they were lowest after feeding and increased with fasting. Bafilomycin (1 μmol l–1) promoted an 80% increase in net base secretion rates, as well as in Cl– and fluid absorption, at 1–3 h post-feeding in seawater preparations only, explaining the difference between freshwater and seawater fish. Preparations from seawater animals at 1–3 h post-feeding also acidified the mucosal saline, and this effect was associated with a marked rise in PCO2, which was attenuated by bafilomycin. Measurements of chyme pH from intact animals confirmed that intestinal fluid (chyme) pH and basic equivalent concentration were lowest after feeding in seawater killifish, whereas PCO2 was greatly elevated (80–95 Torr) in chyme from both seawater and freshwater animals but declined to lower levels (13 Torr) after 1–2 weeks fasting. There were no differences in pH, PCO2 or the concentrations of basic equivalents in intestinal fluid from seawater versus freshwater animals at 12–24 h or 1–2 weeks post-feeding. The results are interpreted in terms of the absence of gastric HCl secretion, the limitations of the gills for acid–base balance and Cl– transport, and therefore the need for intestinal Cl– uptake in freshwater killifish, and the potential for O2 release from the mucosal blood flow by the high PCO2 in the intestinal fluids. At least in seawater killifish, H+-ATPase running in parallel to HCO3–:Cl– exchange in the apical membranes of teleost enterocytes might reduce net base secretion and explain the high PCO2 in the chyme after feeding.

The role of feeding in salt and water balance

The importance of salt and water absorption from the food by the gastrointestinal tract and its impact on iono- and osmoregulation have been largely overlooked by fish physiologists. The present review aims to correct this situation. Techniques for overcoming the practical difficulties of studying ionoregulation in feeding fish are critically assessed, and ion and water contents of a range of diets are surveyed. In freshwater fish, the quantities of most major electrolytes ingested via a normal ration far exceed those transported from the water by the gills, but net absorption rates of specific ions vary greatly with a range of influences, including complex interactions involving mucins and bile salts. The stomach plays a key role in absorption, while net secretion generally occurs in the anterior intestinal region due to biliary and pancreatic discharge. Dry commercial diets help minimize water uptake, while high NaCl diets have marked effects on plasma composition, branchial ion fluxes, and renal function. In seawater teleosts, Na + , K + , Cl − and water absorption from the food are superimposed on ion and water transport from drinking; Ca 2+ and Mg 2+ are largely excluded. Effects of feeding on the ionoregulatory functions of gills and kidney remain to be investigated. Marine elasmobranchs constitute a special case due to their urea-based osmoregulary strategy; feeding causes marked stimulation of urea synthesis, urea secretion into the chyme, and rectal gland metabolism. Future directions are highlighted.

Post-prandial metabolic alkalosis in the seawater-acclimated trout: the alkaline tide comes in.

SUMMARY The consequences of feeding and digestion on acid–base balance and regulation in a marine teleost (seawater-acclimated steelhead trout; Oncorhynchus mykiss) were investigated by tracking changes in blood pH and [HCO3–], as well as alterations in net acid or base excretion to the water following feeding. Additionally the role of the intestine in the regulation of acid–base balance during feeding was investigated with an in vitro gut sac technique. Feeding did not affect plasma glucose or urea concentrations, however, total plasma ammonia rose during feeding, peaking between 3 and 24 h following the ingestion of a meal, three-fold above resting control values (∼300 μmol ml–1). This increase in plasma ammonia was accompanied by an increase in net ammonia flux to the water (∼twofold higher in fed fish versus unfed fish). The arterial blood also became alkaline with increases in pH and plasma [HCO3–] between 3 and 12 h following feeding, representing the first measurement of an alkaline tide in a marine teleost. There was no evidence of respiratory compensation for the measured metabolic alkalosis, as PaCO2 remained unchanged throughout the post-feeding period. However, in contrast to an earlier study on freshwater-acclimated trout, fed fish did not exhibit a compensating increase in net base excretion, but rather took in additional base from the external seawater, amounting to ∼8490 μmol kg–1 over 48 h. In vitro experiments suggest that at least a portion of the alkaline tide was eliminated through increased HCO3– secretion coupled to Cl– absorption in the intestinal tract. This did not occur in the intestine of freshwater-acclimated trout. The marked effects of the external salinity (seawater versus freshwater) on different post-feeding patterns of acid–base balance are discussed.

Environmental and nutritional regulation of expression and function of two peptide transporter (PepT1) isoforms in a euryhaline teleost.

Expression and function of the oligopeptide transporter PepT1 in response to changes in environmental salinity have received little study despite the important role that dipeptides play in piscine nutrition. We cloned and sequenced two novel full-length cDNAs that encode Fundulus heteroclitus PepT1-type oligopeptide transporters, and examined their expression and functional properties in freshwater- and seawater-acclimated fish and in response to fasting and re-feeding. Phylogenetic analysis of vertebrate SLC15A1 sequences confirms the presence of two PepT1 isoforms, named SLC15A1a and SLC15A1b, in fish. Similar to other vertebrate SLC15A1s, these isoforms have 12 transmembrane domains, and amino acids essential for PepT1 function are conserved. Expression analysis revealed novel environment-specific expression of the SLC15A1 isoforms in F. heteroclitus, with only SLC15A1b expressed in seawater-acclimated fish, and both isoforms expressed in freshwater-acclimated fish. Fasting and re-feeding induced changes in the expression of SLC15A1a and SLC15A1b mRNA. Short-term fasting resulted in up-regulation of PepT1 mRNA levels, while prolonged fasting resulted in down-regulation. The resumption of feeding resulted in up-regulation of PepT1 above pre-fasted levels. Experiments using the in vitro gut sac technique suggest that the PepT1 isoforms differ in functional characteristics. An increased luminal pH resulted in decreased intestinal dipeptide transport in freshwater-acclimated fish but suggested an increased dipeptide transport in seawater-acclimated fish. Overall, this is the first evidence of multiple isoforms of PepT1 in fish whose expression is environmentally dependent and results in functional differences in intestinal dipeptide transport.