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Few studies have examined changes in salmon gill ion transporter expression during the transition from seawater to freshwater, a pivotal moment in the salmonid life cycle. Seawater-acclimated Atlantic salmon were transferred to freshwater and blood and gill tissue were sampled over 30 days of acclimation. Salmon held in seawater had stable plasma osmolality and sodium and chloride levels throughout the experiment. Following freshwater exposure, plasma sodium and chloride levels and total osmolality decreased significantly before returning towards control levels over time. Gill H+-ATPase activity increased by more than 45% 14 days after exposure to freshwater, whereas H+-ATPase mRNA levels were not affected by the salinity change. Within 4 days of freshwater exposure, gill Na+/K+-ATPase activity increased ~43% over control levels, remaining significantly higher until the 30 day sampling group when it declined back to control levels. This increase in activity was associated with a more than 7-fold increase in Na+/K+-ATPase isoform ala mRNA level and a ~60% decrease in Na+/K+-ATPase isoform alb mRNA level. The mRNA levels of Na+/K+-ATPase isoforms α1c and α3 did not change as a result of freshwater exposure. The time courses for mRNA expression of the small membrane protein FXYD 11 and the β1-subunit were very similar, with levels increasing significantly 7days following freshwater exposure before subsiding back to control levels at 30 days. Taken together, these data suggest an important role for Na+/K+-ATPase in freshwater acclimation in Atlantic salmon. [ABSTRACT FROM AUTHOR]

We have explored intestinal function in the euryhaline killifish Fundulus heteroclitus after transfer from brackish water (10% seawater) to fresh water. Plasma Na+ and Cl-1 concentrations fell at 12 h post-transfer, but recovered by 7 days. Drinking rate decreased substantially at 12 h (32% of control value) and remained suppressed after 3 and 7 days in fresh water (34 and 43%). By contrast, there was a transient increase in the capacity for water absorption measured across isolated intestines in vitro (3.3- and 2.6-fold at 12 h and 3 days), which returned to baseline after 7 days. These changes in water absorption could be entirely accounted for by changes in net ion flux: there was an extremely strong correlation (R²=0.960) between water absorption and the sum of net Na+ and net Cl- fluxes (3.42±0.10 µl water µmol-1 ion). However, enhanced ion transport across the intestine in fresh water would probably not increase water uptake in vivo, because the drinking rate was far less than the capacity for water absorption across the intestine. The increased intestinal ion absorption after freshwater transfer may instead serve to facilitate ion absorption from food when it is present in the gut. Modulation of net ion flux occurred without changes in mRNA levels of many ion transporters (Na+K+-ATPase α1a, carbonic anhydrase 2, CFTR Cl- channel, Na+/K+/2Cl- cotransporter 2, and the signalling protein 14-3-3a), and before a measured increase in Na+/K+-ATPase activity at 3 days, suggesting that there is some other mechanism responsible for increasing ion transport. Interestingly, net Cl- flux always exceeded net Na+ flux, possibly to help maintain Cl- balance and/or facilitate bicarbonate excretion. Our results suggest that intestinal NaCI absorption from food is important during the period of greatest ionic disturbance after transfer to fresh water, and provide further insight into the mechanisms of euryhalinity in killifish. [ABSTRACT FROM AUTHOR]

Populations of common killifish, Fundulus heteroclitus, are distributed along the Atlantic coast of North America through a steep latitudinal thermal gradient. We examined intraspecific variation in whole-animal thermal tolerance and its relationship to the heat shock response in killifish from the northern and southern extremes of the species range. Critical thermal maxima were significantly higher in southern than in northern fish by ∼1.5°C at a wide range of acclimation temperatures (from 2–34°C), and critical thermal minima differed by ∼1.5°C at acclimation temperatures above 22°C, converging on the freezing point of brackish water at lower acclimation temperatures. To determine whether these differences in whole-organism thermal tolerance were reflected in differences in either the sequence or regulation of the heat shock protein genes (hsps) we obtained complete cDNA sequences for hsc70, hsp70-1 and hsp70-2, and partial sequences of hsp90α and hsp90β. There were no fixed differences in amino acid sequence between populations in either hsp70-1 or hsp70-2, and only a single conservative substitution between populations in hsc70. By contrast, there were significant differences between populations in the expression of many, but not all, of these genes. Both northern and southern killifish significantly increased hsp70-2 levels above control values (Ton) at a heat shock temperature of 33°C, but the magnitude of this induction was greater in northern fish, suggesting that northern fish may be more susceptible to thermal damage than are southern fish. In contrast, hsp70-1 mRNA levels increased gradually and to the same extent in response to heat shock in both populations. Hsc70 mRNA levels were significantly elevated by heat shock in southern fish, but not in northern fish. Similarly, the more thermotolerant southern killifish had a Ton for hsp90α of 30°C, 2°C lower than that of northern fish. This observation combined with the ability of southern killifish to upregulate hsc70 in response to heat shock suggests a possible role for these hsps in whole-organism differences in thermal tolerance. These data highlight the importance of considering the complexity of the heat shock response across multiple isoforms when attempting to make linkages to whole-organism traits such as thermal tolerance. [ABSTRACT FROM AUTHOR]

We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10 g 1-1) to freshwater. Differences in Na+ regulation between each population might partially account for this difference in tolerance, because plasma Na+ was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na+/K+-ATPase mRNA expression and activity in their gills to a greater extent 1–14 days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na+ at 14 days. All observed differences in Na+ regulation were small, however, and probably cannot account for the large differences in mortality. Differences in CI- regulation also existed between populations. Plasma CI- was maintained in northern fish, but in southerns, plasma CI- decreased rapidly and remained low for the duration of the experiment. Correspondingly, net CI- loss from southern fish remained high after transfer, while northerns eliminated CI- loss altogether. Elevated CI- loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability (indicated by extrarenal clearance rate of PEG-4000) and apical crypt density in the gills (detected using scanning electron microscopy) were both higher than in northern fish. These large differences in the regulation of CI- balance probably contributed to the marked differences in mortality after freshwater transfer. Glomerular filtration rate and urination frequency were also lower in southerns. Taken together, these data suggest that northern killifish are better adapted to freshwater environments and that minimizing Cl- imbalance appears to be the key physiological difference accounting for their greater freshwater tolerance. [ABSTRACT FROM AUTHOR]

To provide insight into claudin (Cldn) tight junction (TJ) protein contributions to branchial salt secretion in marine teleost fishes, this study examined cldn-10 TJ protein isoforms of a euryhaline teleost (mummichog; Fundulus heteroclitus ) in association with salinity change and measurements of transepithelial cation selectivity. Mummichogs were transferred from freshwater (FW) to seawater (SW, 35‰) and from SW to hypersaline SW (2SW, 60‰) in a time course with transfer control groups (FW to FW, and SW to SW). FW to SW transfer increased mRNA abundance of cldn-10d and cldn -10e twofold, whilst cldn-10c and cldn -10f transcripts were unchanged. Transfer from SW to 2SW did not alter cldn-10d , and transiently altered cldn-10e abundance, but increased cldn-10c and cldn-10f fourfold. This was coincident with an increased number of single-stranded junctions (observed by transmission electron microscopy). For both salinity transfers, (1) cldn-10e mRNA was acutely responsive (i.e. after 24 h), (2) other responsive cldn-10 isoforms increased later (3-7 days), and (3) cystic fibrosis transmembrane conductance regulator ( cftr ) mRNA was elevated in accordance with established changes in transcellular Cl - movement. Changes in mRNA encoding cldn-10c and -10f appeared linked, consistent with the tandem repeat locus in the Fundulus genome, whereas mRNA for tandem cldn-10d and cldn -10e seemed independent of each other. Cation selectivity sequence measured by voltage and conductance responses to artificial SW revealed Eisenman sequence VII: Na + >K + >Rb + ∼Cs + >Li + Collectively, these data support the idea that Cldn-10 TJ proteins create and maintain cation-selective pore junctions in salt-secreting tissues of teleost fishes., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

We identified five Na+/K+-ATPase alpha-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate alpha1 isoforms (designated alpha1a, alpha1b and alpha1c), one isoform was closely related to alpha2 isoforms (designated alpha2) and the fifth was closely related to alpha3 isoforms (designated alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase alpha-isoforms were expressed in trout gills (alpha1a, alpha1b, alpha1c and alpha3). Na+/K+-ATPase alpha1c- and alpha3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase alpha1a and alpha1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPase alpha1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase alpha1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase alpha1a- and alpha1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.