Amy M. Regish

Distinct freshwater and seawater isoforms of Na+/K+-ATPase in gill chloride cells of Atlantic salmon

SUMMARY Gill Na+/K+-ATPase (NKA) in teleost fishes is involved in ion regulation in both freshwater and seawater. We have developed and validated rabbit polyclonal antibodies specific to the NKA α1a and α1b protein isoforms of Atlantic salmon (Salmo salar Linnaeus), and used western blots and immunohistochemistry to characterize their size, abundance and localization. The relative molecular mass of NKA α1a is slightly less than that for NKA β1b. The abundance of gill NKA α1a was high in freshwater and became nearly undetectable after seawater acclimation. NKA α1b was present in small amounts in freshwater and increased 13-fold after seawater acclimation. Both NKA isoforms were detected only in chloride cells. NKA α1a was located in both filamental and lamellar chloride cells in freshwater, whereas in seawater it was present only as a faint background in filamental chloride cells. In freshwater, NKA α1b was found in a small number of filamental chloride cells, and after seawater acclimation it was found in all chloride cells on the filament and lamellae. Double simultaneous immunofluorescence indicated that NKA α1a and α1b are located in different chloride cells in freshwater. In many chloride cells in seawater, NKA α1b was present in greater amounts in the subapical region than elsewhere in the cell. The combined patterns in abundance and immunolocalization of these two isoforms can explain the salinity-related changes in total NKA and chloride cell abundance. The results indicate that there is a freshwater and a seawater isoform of NKA α-subunit in the gills of Atlantic salmon and that they are present in distinct chloride cells.

Are we missing a mineralocorticoid in teleost fish? Effects of cortisol, deoxycorticosterone and aldosterone on osmoregulation, gill Na + ,K + -ATPase activity and isoform mRNA levels in Atlantic salmon

It has long been held that cortisol, acting through a single receptor, carries out both glucocorticoid and mineralocorticoid actions in teleost fish. The recent finding that fish express a gene with high sequence similarity to the mammalian mineralocorticoid receptor (MR) suggests the possibility that a hormone other than cortisol carries out some mineralocorticoid functions in fish. To test for this possibility, we examined the effect of in vivo cortisol, 11-deoxycorticosterone (DOC) and aldosterone on salinity tolerance, gill Na(+),K(+)-ATPase (NKA) activity and mRNA levels of NKA alpha 1a and alpha 1b in Atlantic salmon. Cortisol treatment for 6-14 days resulted in increased, physiological levels of cortisol, increased gill NKA activity and improved salinity tolerance (lower plasma chloride after a 24h seawater challenge), whereas DOC and aldosterone had no effect on either NKA activity or salinity tolerance. NKA alpha 1a and alpha 1b mRNA levels, which increase in response to fresh water and seawater acclimation, respectively, were both upregulated by cortisol, whereas DOC and aldosterone were without effect. Cortisol, DOC and aldosterone had no effect on gill glucocorticoid receptor GR1, GR2 and MR mRNA levels, although there was some indication of possible upregulation of GR1 by cortisol (p=0.07). The putative GR blocker RU486 inhibited cortisol-induced increases in salinity tolerance, NKA activity and NKA alpha 1a and alpha 1b transcription, whereas the putative MR blocker spironolactone had no effect. The results provide support that cortisol, and not DOC or aldosterone, is involved in regulating the mineralocorticoid functions of ion uptake and salt secretion in teleost fish.

Differential regulation of sodium–potassium pump isoforms during smolt development and seawater exposure of Atlantic salmon

SUMMARY Freshwater and seawater isoforms of the alpha subunit of Na+/K+-ATPase (NKA) have previously been identified in gill ionocytes of Atlantic salmon (Salmo salar). In the present study we examine the abundance and cellular localization of these isoforms during the parr–smolt transformation, a developmental process that is preparatory for seawater entry. The abundance of NKAα1a was lower in smolts than in parr, remained relatively constant during spring and decreased in summer. NKAα1b increased tenfold in smolts during spring, peaking in late April, coincident with downstream migration and increased salinity tolerance. NKAα1b increased a further twofold after seawater exposure of smolts, whereas NKAα1a decreased by 98%. The abundance of NKAα1b-positive, and NKAα1b and NKAα1a co-labeled ionocytes increased during smolt development, whereas the number of NKAα1a cells decreased. After seawater exposure of smolts, NKAα1b-positive ionocytes increased, NKAα1a-positive cells decreased, and co-labeled cells disappeared. Plasma growth hormone and cortisol increased during spring in smolts, but not in parr, peaking just prior to the highest levels of NKAα1b. The results indicate that the increase in the abundance of NKAα1b during smolt development is directly linked to the increase in salinity tolerance that occurs at this stage, but that significant changes also occur after seawater exposure. Spring increases in circulating levels of growth hormone and cortisol indicate that these hormones may be instrumental in upregulating NKAα1b during smolt development.

Migratory patterns of hatchery and stream‐reared Atlantic salmon Salmo salar smolts in the Connecticut River, U.S.A.

The timing of downstream migration and detection rates of hatchery-reared Atlantic salmon Salmo salar smolts and stream-reared smolts (stocked 2 years earlier as fry) were examined in the Connecticut River (U.S.A.) using passive integrated transponder (PIT) tags implanted into fish and then detected at a downstream fish bypass collection facility at Turners Falls, MA (river length 192 km). In two successive years, hatchery-reared smolts were released in mid-April and early May at two sites: the West River (river length 241 km) or the Passumpsic (river length 450 km). Hatchery-reared smolts released higher in the catchment arrived 7 to 14 days later and had significantly lower detection rates than smolts stocked lower in the catchment. Hatchery-reared smolts released 3 weeks apart at the same location were detected downstream at similar times, indicating that early-release smolts had a lower average speed after release and longer residence time. The size and gill Na(+) /K(+) -ATPase (NKA) activity of smolts at the time of release were significantly greater for detected fish (those that survived and migrated) than for those that were not detected. Stream-reared pre-smolts (>11·5 cm) from four tributaries (length 261-551 km) were tagged in autumn and detected during smolt migration the following spring. Stream-reared smolts higher in the catchment arrived later and had significantly lower detection rates. The results indicate that both hatchery and stream-reared smolts from the upper catchment will arrive at the mouth of the river later and experience higher overall mortality than fish from lower reaches, and that both size and gill NKA activity are related to survival during downstream migration.

Effects of elevated temperature on osmoregulation and stress responses in Atlantic salmon Salmo salar smolts in fresh water and seawater

Smolting in Atlantic salmon Salmo salar is a critical life-history stage that is preparatory for downstream migration and entry to seawater that is regulated by abiotic variables including photoperiod and temperature. The present study was undertaken to determine the interaction of temperature and salinity on salinity tolerance, gill osmoregulatory proteins and cellular and endocrine stress in S. salar smolts. Fish were exposed to rapid changes in temperature (from 14 to 17, 20 and 24°C) in fresh water (FW) and seawater (SW), with and without prior acclimation and sampled after 2 and 8 days. Fish exposed simultaneously to SW and 24°C experienced 100% mortality, whereas no mortality occurred in any of the other groups. The highest temperature also resulted in poor ion regulation in SW with or without prior SW acclimation, whereas no substantial effect was observed in FW. Gill Na+ -K+ -ATPase (NKA) activity increased in SW fish compared to FW fish and decreased with high temperature in both FW and SW. Gill Nkaα1a abundance was high in FW and Nkaα1b and Na+ -K+ -2Cl- cotransporter high in SW, but all three were lower at the highest temperature. Gill Hsp70 levels were elevated in FW and SW at the highest temperature and increased with increasing temperature 2 days following direct transfer to SW. Plasma cortisol levels were elevated in SW at the highest temperature. Our results indicate that there is an important interaction of salinity and elevated temperature on osmoregulatory performance and the cellular stress response in S. salar, with an apparent threshold for osmoregulatory failure in SW above 20°C.

Effects of ocean acidification on salinity tolerance and seawater growth of Atlantic salmon Salmo salar smolts

Human activity has resulted in increasing atmospheric carbon dioxide (CO2 ), which will result in reduced pH and higher levels of CO2 in the ocean, a process known as ocean acidification. Understanding the effects of ocean acidification (OA) on fishes will be important to predicting and mitigating its consequences. Anadromous species such as salmonids may be especially at risk because of their rapid movements between fresh water and seawater, which could minimize their ability to acclimate. In the present study, we examine the effect of future OA on the salinity tolerance and early seawater growth of Atlantic salmon Salmo salar smolts. Exposure to 610 and 1010 μatm CO2 did not alter salinity tolerance but did result in slightly lower plasma chloride levels in smolts exposed to seawater compared with controls (390 μatm). Gill Na+ -K+ -ATPase activity, plasma cortisol, glucose and haematocrit after seawater exposure were not altered by elevated CO2 . Growth rate in the first 2 weeks of seawater exposure was greater at 1010 μatm CO2 than under control conditions. This study of the effects of OA on S. salar during the transition from fresh water to seawater indicates that elevated CO2 is not likely to affect osmoregulation negatively and may improve early growth in seawater.

Shifts in the relationship between mRNA and protein abundance of gill ion-transporters during smolt development and seawater acclimation in Atlantic salmon ( Salmo salar )

Smolting Atlantic salmon exhibit a seasonal increase in seawater tolerance that is associated with changes in the abundance of major gill ion-transporter transcripts and proteins. In the present study, we investigate how the transcript and protein abundance of specific ion-transporter isoforms relate to each other during smolt development and seawater acclimation, and how each correlates to seawater tolerance. We show that during smolt development both mRNA and protein abundance of gill Na+/K+-ATPase α1a subunit (NKAα1a) decreased but the decrease in the mRNA was five-times greater than that of the protein. Gill NKAα1b mRNA levels increased only slightly (1.5-fold) throughout development whereas protein abundance increased 30-fold at its peak. Gill Na+/K+/2Cl- co-transporter 1 (NKCC1) increased at the mRNA and protein level (5- and 12-fold) in smolts. The abundance of a gill ion-transporters mRNA and protein changed in the same direction through development and after seawater transfer, but the changes were not always strongly correlated: NKAα1a (r = 0.768), NKAα1b (r = 0.40), and NKCC1 (r = 0.898). The maintenance of plasma chloride concentration correlated most strongly with the abundance of NKAα1a mRNA, and the ratio of NKAα1b to NKAα1a mRNA and protein. Growth performance after seawater transfer correlated most strongly with the abundance of NKAα1b protein and the ratio of NKAα1b to NKAα1a protein. Our results indicate that the abundance of ion-transporter mRNA and protein do not always correlate well and a decrease in the abundance of gill NKAα1a mRNA and increase in NKAα1b protein are strong predictors of seawater tolerance and growth performance after seawater transfer.

Exogenous thyroid hormones regulate the activity of citrate synthase and cytochrome c oxidase in warm- but not cold-acclimated lake whitefish (Coregonus clupeaformis)

Thermal acclimation is known to elicit metabolic adjustments in ectotherms, but the cellular mechanisms and endocrine control of these shifts have not been fully elucidated. Here we examined the relationship between thermal acclimation, thyroid hormones and oxidative metabolism in juvenile lake whitefish. Impacts of thermal acclimation above (19°C) or below (8°C) the thermal optimum (13°C) and exposure to exogenous thyroid hormone (60µg T4/g body weight) were assessed by quantifying citrate synthase and cytochrome c oxidase activities in liver, red muscle, white muscle and heart. Warm acclimation decreased citrate synthase activity in liver and elevated both citrate synthase and cytochrome c oxidase activities in red muscle. In contrast, induction of hyperthyroidism in warm-acclimated fish stimulated a significant increase in liver citrate synthase and heart cytochrome c oxidase activities, and a decrease in the activity of both enzymes in red muscle. No change in citrate synthase or cytochrome c oxidase activities was observed following cold acclimation in either the presence or absence of exogenous thyroid hormones. Collectively, our results indicate that thyroid hormones influence the activity of oxidative enzymes more strongly in warm-acclimated than in cold-acclimated lake whitefish, and they may play a role in mediating metabolic adjustments observed during thermal acclimation.

Evidence for episodic acidification effects on migrating Atlantic salmon Salmo salar smolts.

Field studies were conducted to determine levels of gill aluminium as an index of acidification effects on migrating Atlantic salmon Salmo salar smolts in the north-eastern U.S.A. along mainstem river migration corridors in several major river basins. Smolts emigrating from the Connecticut River, where most (but not all) tributaries were well buffered, had low or undetectable levels of gill aluminium and high gill Na(+) /K(+) -ATPase (NKA) activity. In contrast, smolts emigrating from the upper Merrimack River basin where most tributaries are characterized by low pH and high inorganic aluminium had consistently elevated gill aluminium and lower gill NKA activity, which may explain the low adult return rates of S. salar stocked into the upper Merrimack catchment. In the Sheepscot, Narraguagus and Penobscot Rivers in Maine, river and year-specific effects on gill aluminium were detected that appeared to be driven by underlying geology and high spring discharge. The results indicate that episodic acidification is affecting S. salar smolts in poorly buffered streams in New England and may help explain variation in S. salar survival and abundance among rivers and among years, with implications for the conservation and recovery of S. salar in the north-eastern U.S.A. These results suggest that the physiological condition of outmigrating smolts may serve as a large-scale sentinel of landscape-level recovery of atmospheric pollution in this and other parts of the North Atlantic region.