A role for acid-sensing ion channels (ASICs) to serve as epithelial channels for Na⁺ uptake by the gill of freshwater rainbow trout was investigated. We found that the ASIC inhibitors 4′,6-diamidino-2-phenylindole and diminazene decreased Na⁺ uptake in adult rainbow trout in a dose-dependent manner, with IC₅₀ values of 0.12 and 0.96 μM, respectively. Furthermore, we cloned the trout ASIC1 and ASIC4 homologs and demonstrated that they are expressed differentially in the tissues of the rainbow trout, including gills and isolated mitochondrion-rich cells. Immunohistochemical analysis using custom-made anti-zASIC4.2 antibody and the Na⁺-K⁺-ATPase (α₅-subunit) antibody demonstrated that the trout ASIC localizes to Na⁺/K⁺-ATPase-rich cells in the gill. Moreover, three-dimensional rendering of confocal micrographs demonstrated that ASIC is found in the apical region of mitochondrion-rich cells. We present a revised model whereby ASIC4 is proposed as one mechanism for Na⁺ uptake from dilute freshwater in the gill of rainbow trout.

Arctic grayling (Thymallus arcticus) are salmonids that have a strict freshwater existence in post-glacial North America. Oil and gas development is associated with production of high volumes of hypersaline water. With planned industrial expansion into northern areas of Canada and the USA that directly overlap grayling habitat, the threat of accidental saline water release poses a significant risk. Despite this, we understand little about the responses of grayling to hypersaline waters. We compared the physiological responses and survivability of Arctic grayling and rainbow trout (Oncorhynchus mykiss) to tolerate an acute transfer to higher saline waters. Arctic grayling and rainbow trout were placed directly into 17 ppt salinity and sampled at 24 and 96 h along with control animals in freshwater at 24 h. Serum sodium, chloride and osmolality levels increased significantly in grayling at both 24 and 96 h time points, whereas trout were able to compensate for the osmoregulatory disturbance by 96 h. Sodium–potassium ATPase mRNA expression responses to salinity were also compared, demonstrating the inability of the grayling to up-regulate the seawater isoform nkaα1b. Our results demonstrated a substantially lower salinity tolerance in grayling. We also found a significant salinity-induced morphological gill remodelling by Arctic grayling, as demonstrated by the rapid growth of an interlamellar cell mass by 24 h that persisted at 96 h. We visualized and quantified the appearance of the interlamellar cell mass as a response to high salinity, although the functional significance remains to be understood fully. Compared with rainbow trout, which are used as an environmental regulatory species, Arctic grayling are unable to compensate for the osmotic stressors that would result from a highly saline produced water spill. Given these new data, collaboration between fisheries and the oil and gas industry will be vital in the long-term conservation strategies with regard to the Arctic grayling in their native habitat.

Hagfishes, the most ancient of the extant craniates, demonstrate a high tolerance for a number of unfavorable environmental conditions, including elevated ammonia. Proposed mechanisms of ammonia excretion in aquatic organisms include vesicular NH₄⁺transport and release by exocytosis in marine crabs, and passive NH₃diffusion, active NH₄⁺transport, and paracellular leakage of NH₃or NH₄⁺across the gills of fishes. Recently, an emerging paradigm suggests that Rhesus glycoproteins play a vital role in ammonia transport in both aquatic invertebrates and vertebrates. This study has identified an Rh glycoprotein ortholog from the gills of Atlantic hagfish. The hagfish Rhcg shares a 56–60% amino acid identity to other vertebrate Rhcg cDNAs. Sequence information was used to produce an anti-hagfish Rhcg (hRhcg) antibody. We have used hRhcg to localize protein expression to epithelial cells of the gill and the skin. In addition, we have quantified hRhcg expression following exposure to elevated plasma ammonia levels. Animals exposed to a 3 mmol/kg NH₄Cl load resulted in significantly elevated plasma ammonia concentrations compared with controls for up to 4 h postinjection. This correlated with net ammonia excretion rates that were also significantly elevated for up to 4 h postinjection. Rhcg mRNA expression in both the gill and skin was significantly elevated by 15 min and 1 h, respectively, and hRhcg protein expression in gills was significantly elevated at 2, 4, and 8 h postinjection. These results demonstrate a potential role for Rhcg in the excretion of ammonia in the Atlantic hagfish.

Visible implant tags are commonly used in fisheries research to mark individuals or batches of fish as part of movement and mark–recapture studies. To be effective, these tags generally need to have high retention rates and little impact on the behavior or physiology of the marked individuals. We tested the retention rates of both visible implant elastomer (VIE) and visible implant alphanumeric (VIA) tags in 80–139-mm Western Silvery Minnow Hybognathus argyritis over a 104-d period. We also measured plasma cortisol and lactate to determine the impact of tagging on stress levels. We found that VIE tags had 100% retention and did not cause a significant increase in plasma cortisol levels, though plasma lactate levels were elevated in VIE treatment groups. Overall, VIE tags were found to be suitable for marking Western Silvery Minnow and similar species. Visible implant alphanumeric tags had 36% retention over 104 d and did not cause significant increases in plasma cortisol, though plasma lactate was elevated in the treatment group with both VIE and VIA tags. Given the high retention rates and low stress effects, VIE tags appear to be more beneficial for use with Western Silvery Minnow and similar-sized cyprinids. Received April 16, 2015; accepted July 29, 2015

Freshwater fishes maintain an internal osmolality of ~300 mOsm, while living in dilute environments ranging from 0 to 50 mOsm. This osmotic challenge is met at least partially, by Na⁺/H⁺ exchangers (NHE) of fish gill and kidney. In this study, we cloned, expressed, and pharmacologically characterized fish-specific Nhes of the commercially important species Oncorhynchus mykiss. Trout (t) Nhe3a and Nhe3b isoforms from gill and kidney were expressed and characterized in an NHE-deficient cell line. Western blotting and immunocytochemistry confirmed stable expression of the tagged trout tNhe proteins. To measure NHE activity, a transient acid load was induced in trout tNhe expressing cells and intracellular pH was measured. Both isoforms demonstrated significant activity and recovered from an acute acid load. The effect of the NHE transport inhibitors amiloride, EIPA (5-(N-ethyl-N-isopropyl)-amiloride), phenamil, and DAPI was examined. tNhe3a was inhibited in a dose-dependent manner by amiloride and EIPA and tNhe3a was more sensitive to amiloride than EIPA, unlike mammalian NHE1. tNhe3b was inhibited by high concentrations of amiloride, while even in the presence of high concentrations of EIPA (500 µM), some activity of tNhe3b remained. Phenamil and DAPI were ineffective at inhibiting tNhe activity of either isoform. The current study aids in understanding the pharmacology of fish ion transporters. Both isoforms display inhibitory profiles uniquely different from mammalian NHEs and show resistance to inhibition. Our study allows for more direct interpretation of past, present, and future fish-specific sodium transport studies, with less reliance on mammalian NHE data for interpretation.