Smaller body size under warming is not due to gill-oxygen limitation in a cold-water salmonid.

Declining body size in fishes and other aquatic ectotherms associated with anthropogenic climate warming has significant implications for future fisheries yields, stock assessments and aquatic ecosystem stability. One proposed mechanism seeking to explain such body-size reductions, known as the gill oxygen limitation (GOL) hypothesis, has recently been used to model future impacts of climate warming on fisheries but has not been robustly empirically tested. We used brook trout (Salvelinus fontinalis), a fast-growing, cold-water salmonid species of broad economic, conservation and ecological value, to examine the GOL hypothesis in a long-term experiment quantifying effects of temperature on growth, resting metabolic rate (RMR), maximum metabolic rate (MMR) and gill surface area (GSA). Despite significantly reduced growth and body size at an elevated temperature, allometric slopes of GSA were not significantly different than 1.0 and were above those for RMR and MMR at both temperature treatments (15°C and 20°C), contrary to GOL expectations. We also found that the effect of temperature on RMR was time-dependent, contradicting the prediction that heightened temperatures increase metabolic rates and reinforcing the importance of longer-term exposures (e.g. >6 months) to fully understand the influence of acclimation on temperature-metabolic rate relationships. Our results indicate that although oxygen limitation may be important in some aspects of temperature-body size relationships and constraints on metabolic supply may contribute to reduced growth in some cases, it is unlikely that GOL is a universal mechanism explaining temperature-body size relationships in aquatic ectotherms. We suggest future research focus on alternative mechanisms underlying temperature-body size relationships, and that projections of climate change impacts on fisheries yields using models based on GOL assumptions be interpreted with caution.

Differential heat shock protein responses in two species of Pacific salmon and their utility in identifying heat stress.

Rapid and accelerating warming of salmon habitat has the potential to lower productivity of Pacific salmon (Oncorhynchus species) populations. Heat stress biomarkers can indicate where warming is most likely affecting fish populations; however, we often lack clear classifications that separate individuals with and without heat stress needed to make these tools operational. We conducted a heat exposure experiment with trials lasting 12 or 36 h using juvenile Chinook salmon (Oncorhynchus tshawytscha) and coho salmon (Oncorhynchus kisutch) to validate heat stress biomarkers in white muscle. Following habituation to 13°C, individuals were exposed to water temperatures that increased to 15°C, 17°C, 19°C, 21°C or 23°C. Heat shock protein 70 abundance (HSP70 measured by ELISA) and transcription of 13 genes (mRNA measured by qPCR) including three heat shock protein genes (hsp70, hsp90, hsp27) were measured. A distinct heat stress response was apparent by 21°C in juvenile Chinook salmon and 23°C in juvenile coho salmon using HSP70. A threshold for heat stress classification in Chinook salmon of > 2 ng HSP70 mg.1 total protein identified heat stress in 100% of 21 and 23°C treated individuals compared to 4% in cooler treatments. For coho salmon, > 3 ng HSP70 mg.1 total protein identified heat stress in 100% of 23°C treated individuals compared to 4% in cooler treatments. Transcription from a panel of genes separated individuals between cooler and stressful temperature experiences (≥21°C for Chinook salmon and ≥23°C for coho salmon) with ~ 85% correct classification. Our findings indicate that juvenile Chinook salmon were more temperature-sensitive than juvenile coho salmon and support the use of a HSP70 threshold sampled from muscle for assessing heat stress in individual wild Pacific salmon with an option for non-lethal biopsies for spawning adults.

Neuroendocrine Regulation of Plasma Cortisol Levels During Smoltification and Seawater Acclimation of Atlantic Salmon.

Diadromous fishes undergo dramatic changes in osmoregulatory capacity in preparation for migration between freshwater and seawater. One of the primary hormones involved in coordinating these changes is the glucocorticoid hormone, cortisol. In Atlantic salmon (Salmo salar), cortisol levels increase during the spring smoltification period prior to seawater migration; however, the neuroendocrine factors responsible for regulating the hypothalamic-pituitary-interrenal (HPI) axis and plasma cortisol levels during smoltification remain unclear. Therefore, we evaluated seasonal changes in circulating levels of cortisol and its primary secretagogue-adrenocorticotropic hormone (ACTH)-as well as transcript abundance of the major regulators of HPI axis activity in the preoptic area, hypothalamus, and pituitary between migratory smolts and pre-migratory parr. Smolts exhibited higher plasma cortisol levels compared to parr across all timepoints but circulating ACTH levels were only elevated in May. Transcript abundance of preoptic area corticotropin-releasing factor b1 and arginine vasotocin were ~2-fold higher in smolts compared to parr in February through May. Smolts also had ~7-fold greater hypothalamic transcript abundance of urotensin 1 (uts-1a) compared to parr in May through July. When transferred to seawater during peak smolting in May smolts rapidly upregulated hypothalamic uts-1a transcript levels within 24 h, while parr only transiently upregulated uts-1a 96 h post-transfer. In situ hybridization revealed that uts-1a is highly abundant in the lateral tuberal nucleus (NLT) of the hypothalamus, consistent with a role in regulating the HPI axis. Overall, our results highlight the complex, multifactorial regulation of cortisol and provide novel insight into the neuroendocrine mechanisms controlling osmoregulation in teleosts.

Rapid embryonic development supports the early onset of gill functions in two coral reef damselfishes.

The gill is one of the most important organs for growth and survival of fishes. Early life stages in coral reef fishes often exhibit extreme physiological and demographic characteristics that are linked to well-established respiratory and ionoregulatory processes. However, gill development and function in coral reef fishes is not well understood. Therefore, we investigated gill morphology, oxygen uptake and ionoregulatory systems throughout embryogenesis in two coral reef damselfishes, Acanthochromis polyacanthus and Amphiprion melanopus (Pomacentridae). In both species, we found key gill structures to develop rapidly early in the embryonic phase. Ionoregulatory cells appear on gill filaments 3-4 days post-fertilization and increase in density, whilst disappearing or shrinking in cutaneous locations. Primary respiratory tissue (lamellae) appears 5-7 days post-fertilization, coinciding with a peak in oxygen uptake rates of the developing embryos. Oxygen uptake was unaffected by phenylhydrazine across all ages (pre-hatching), indicating that haemoglobin is not yet required for oxygen uptake. This suggests that gills have limited contribution to respiratory functions during embryonic development, at least until hatching. Rapid gill development in damselfishes, when compared with that in most previously investigated fishes, may reflect preparations for a high-performance, challenging lifestyle on tropical reefs, but may also make reef fishes more vulnerable to anthropogenic stressors.

Survival and spawning success of American shad (Alosa sapidissima) in varying temperatures and levels of glochidia infection.

Temperature fluctuations and climate change impacts may substantially affect spawning success of fish, especially migratory species with a limited spawning window. Factors affecting American shad (Alosa sapidissima) spawning success and survival were investigated at different temperatures and periods (peak- and late-spawning periods) during the Connecticut River, USA, spawning migration in 2017. Wild caught American shad were exposed to constant temperatures regimes of 15, 18, 21, 24 and 27 °C for 2 weeks. During the peak-spawning period, an increase in temperature (15-24 °C) was shown to increase spawning success factors, including spawning probability, number of eggs, and fertilization success, but decreased egg size. Temperatures between 18 and 27 °C did not affect these factors during the late-spawning period. Glochidia infection by the alewife floater (Anodonta implicata) was much higher in the late-spawning period and significantly decreased the survival of American shad. Further research should investigate the parasite-host relationship between the alewife floater and American shad to determine annual variability of glochidia infections and how they affect American shad from physiological and passage perspectives. Higher temperatures were shown to increase spawning success of American shad during the peak-spawning period, but temperature had no effect during the late-spawning period. However, any effect during the late-spawning period may have been masked by a high level of glochidia infection.

Cortisol is an osmoregulatory and glucose-regulating hormone in Atlantic sturgeon, a basal ray-finned fish.

Our current understanding of the hormonal control of ion regulation in aquatic vertebrates comes primarily from studies on teleost fishes, with relatively little information on more basal fishes. We investigated the role of cortisol in regulating seawater tolerance and its underlying mechanisms in an anadromous chondrostean, the Atlantic sturgeon (Acipenser oxyrinchus). Exposure of freshwater-reared Atlantic sturgeon to seawater (25 ppt) resulted in transient (1-3 day) increases in plasma chloride, cortisol and glucose levels and long-term (6-14 day) increases in the abundance of gill Na+/K+/2Cl- cotransporter (NKCC), which plays a critical role in salt secretion in teleosts. The abundance of gill V-type H+-ATPase, which is thought to play a role in ion uptake in fishes, decreased after exposure to seawater. Gill Na+/K+-ATPase activity did not increase in 25 ppt seawater, but did increase in fish gradually acclimated to 30 ppt. Treatment of Atlantic sturgeon in freshwater with exogenous cortisol resulted in dose-dependent increases in cortisol, glucose and gill NKCC and H+-ATPase abundance. Our results indicate that cortisol has an important role in regulating mechanisms for ion secretion and uptake in sturgeon and provide support for the hypothesis that control of osmoregulation and glucose by corticosteroids is a basal trait of jawed vertebrates.

Transcriptomic response to elevated water temperatures in adult migrating Yukon River Chinook salmon (Oncorhynchus tshawytscha).

Chinook salmon (Oncorhynchus tshawytscha) declines are widespread and may be attributed, at least in part, to warming river temperatures. Water temperatures in the Yukon River and tributaries often exceed 18°C, a threshold commonly associated with heat stress and elevated mortality in Pacific salmon. Untangling the complex web of direct and indirect physiological effects of heat stress on salmon is difficult in a natural setting with innumerable system challenges but is necessary to increase our understanding of both lethal and sublethal impacts of heat stress on populations. The goal of this study was to characterize the cellular stress response in multiple Chinook salmon tissues after acute elevated temperature challenges. We conducted a controlled 4-hour temperature exposure (control, 18°C and 21°C) experiment on the bank of the Yukon River followed by gene expression (GE) profiling using a 3'-Tag-RNA-Seq protocol. The full transcriptome was analysed for 22 Chinook salmon in muscle, gill and liver tissue. Both the 21°C and 18°C treatments induced greater activity in genes associated with protein folding (e.g. HSP70, HSP90 mRNA) processes in all tissues. Global GE patterns indicate that transcriptomic responses to heat stress were highly tissue-specific, underscoring the importance of analyzing multiple tissues for determination of physiological effect. Primary superclusters (i.e. groupings of loosely related terms) of altered biological processes were identified in each tissue type, including regulation of DNA damage response (gill), regulation by host of viral transcription (liver) and regulation of the force of heart contraction (muscle) in the 21°C treatment. This study provides insight into mechanisms potentially affecting adult Chinook salmon as they encounter warm water during their spawning migration in the Yukon River and suggests that both basic and more specialized cellular functions may be disrupted.

Author Correction: The evolutionary consequences for seawater performance and its hormonal control when anadromous Atlantic salmon become landlocked.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The evolutionary consequences for seawater performance and its hormonal control when anadromous Atlantic salmon become landlocked.

Populations of anadromous fish have become landlocked in relatively recent geological history (<14,000 years), but the evolutionary impacts of this altered lifecycle on traits underlying seawater performance have not been established. In order to examine the effects of relaxed selection on seawater traits, anadromous and landlocked Atlantic salmon were reared under identical conditions and examined for differences in seawater performance and its underlying physiological and endocrine control during the time of spring downstream migration. Salinity tolerance, survival and initial growth in seawater were greater in anadromous than in landlocked salmon. Abundance of the seawater isoform of gill Na+/K+-ATPase increased in spring in both strains but was greater in anadromous salmon. Hormones associated with seawater acclimation (adrenocorticotropic hormone, cortisol and growth hormone) increased in spring in both strains but were higher in anadromous salmon, whereas plasma thyroid hormones did not differ. Hypothalamic urotensin I mRNA levels also increased in spring and were higher in the anadromous strain. The results provide evidence that salinity tolerance and associated physiological traits are regulated by seasonal stimulation of the hypothalamic-pituitary-interrenal axis, and that relaxed selection on seawater entry traits has decreased this stimulation in landlocked salmon.

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-transporter's 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.

Hepatic insulin-like growth-factor binding protein (igfbp) responses to food restriction in Atlantic salmon smolts.

The growth hormone (Gh)/insulin-like growth-factor (Igf) system plays a central role in the regulation of growth in fishes. However, the roles of Igf binding proteins (Igfbps) in coordinating responses to food availability are unresolved, especially in anadromous fishes preparing for seaward migration. We assayed plasma Gh, Igf1, thyroid hormones and cortisol along with igfbp mRNA levels in fasted and fed Atlantic salmon (Salmo salar). Fish were fasted for 3 or 10days near the peak of smoltification (late April to early May). Fasting reduced plasma glucose by 3days and condition factor by 10days. Plasma Gh, cortisol, and thyroxine (T4) were not altered in response to fasting, whereas Igf1 and 3,5,3'-triiodo-l-thyronine (T3) were slightly higher and lower than controls, respectively. Hepatic igfbp1b1, -1b2, -2a, -2b1 and -2b2 mRNA levels were not responsive to fasting, but there were marked increases in igfbp1a1 following 3 and 10days of fasting. Fasting did not alter hepatic igf1 or igf2; however, muscle igf1 was diminished by 10days of fasting. There were no signs that fasting compromised branchial ionoregulatory functions, as indicated by unchanged Na(+)/K(+)-ATPase activity and ion pump/transporter mRNA levels. We conclude that dynamic hepatic igfbp1a1 and muscle igf1 expression participate in the modulation of Gh/Igf signaling in smolts undergoing catabolism.

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

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.