Social regulation of arginine vasopressin and oxytocin systems in a wild group-living fish.

The neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) are key regulators of social behaviour across vertebrates. However, much of our understanding of how these neuropeptide systems interact with social behaviour is centred around laboratory studies which fail to capture the social and physiological challenges of living in the wild. To evaluate relationships between these neuropeptide systems and social behaviour in the wild, we studied social groups of the cichlid fish Neolamprologus pulcher in Lake Tanganyika, Africa. We first used SCUBA to observe the behaviour of focal group members and then measured transcript abundance of key components of the AVP and OXT systems across different brain regions. While AVP is often associated with male-typical behaviours, we found that dominant females had higher expression of avp and its receptor (avpr1a2) in the preoptic area of the brain compared to either dominant males or subordinates of either sex. Dominant females also generally had the highest levels of leucyl-cystinyl aminopeptidase (lnpep)-which inactivates AVP and OXT-throughout the brain, potentially indicating greater overall activity (i.e., production, release, and turnover) of the AVP system in dominant females. Expression of OXT and its receptors did not differ across social ranks. However, dominant males that visited the brood chamber more often had lower preoptic expression of OXT receptor a (oxtra) suggesting a negative relationship between OXT signalling and parental care in males of this species. Overall, these results advance our understanding of the relationships between complex social behaviours and neuroendocrine systems under natural settings.

Cortisol enhances aerobic metabolism and locomotor performance during the transition to land in an amphibious fish.

Amphibious fishes on land encounter higher oxygen (O2) availability and novel energetic demands, which impacts metabolism. Previous work on the amphibious mangrove killifish (Kryptolebias marmoratus) has shown that cortisol becomes elevated in response to air exposure, suggesting a possible role in regulating metabolism as fish move into terrestrial environments. We tested the hypothesis that cortisol is the mechanism by which oxidative processes are upregulated during the transition to land in amphibious fishes. We used two groups of fish, treated fish (+metyrapone, a cortisol synthesis inhibitor) and control (-metyrapone), to determine the impact of cortisol during air exposure (0 and 1 h, 7 days) on O2 consumption, terrestrial locomotion, the phenotype of red skeletal muscle, and muscle lipid concentration. Metyrapone-treated fish had an attenuated elevation in O2 consumption rate during the water to air transition and an immediate reduction in terrestrial exercise performance relative to control fish. In contrast, we found no short- (0 h) or long-term (7 days) differences between treatments in the oxidative phenotype of red muscles, nor in muscle lipid concentrations. Our results suggest that cortisol stimulates the necessary increase in aerobic metabolism needed to fuel the physiological changes that amphibious fishes undergo during the acclimation to air, although further studies are required to determine specific mechanisms of cortisol regulation.

Intergenerational plasticity to cycling high temperature and hypoxia affects offspring stress responsiveness and tolerance in zebrafish.

Predicted climate change-induced increases in heat waves and hypoxic events will have profound effects on fishes, yet the capacity of parents to alter offspring phenotype via non-genetic inheritance and buffer against these combined stressors is not clear. This study tested how prolonged adult zebrafish exposure to combined diel cycles of thermal stress and hypoxia affect offspring early survival and development, parental investment of cortisol and heat shock proteins (HSPs), larval offspring stress responses, and both parental and offspring heat and hypoxia tolerance. Parental exposure to the combined stressor did not affect fecundity, but increased mortality, produced smaller embryos and delayed hatching. The combined treatment also reduced maternal deposition of cortisol and increased embryo hsf1, hsp70a, HSP70, hsp90aa and HSP90 levels. In larvae, basal cortisol levels did not differ between treatments, but acute exposure to combined heat stress and hypoxia increased cortisol levels in control larvae with no effect on larvae from exposed parents. In contrast, whereas larval basal hsf1, hsp70a and hsp90aa levels differed between parental treatments, the combined acute stressor elicited similar transcriptional responses across treatments. Moreover, the combined acute stressor only induced a marked increase in HSP47 levels in the larvae derived from exposed parents. Finally, combined hypoxia and elevated temperatures increased both thermal and hypoxia tolerance in adults and conferred an increase in offspring thermal but not hypoxia tolerance. These results demonstrate that intergenerational acclimation to combined thermal stress and hypoxia elicit complex carryover effects on stress responsiveness and offspring tolerance with potential consequences for resilience.

Lingering Effects of Legacy Industrial Pollution on Yellow Perch of the Detroit River.

We used yellow perch (Perca flavescens) captured at four sites differing in legacy industrial pollution in the Lake St. Clair-Detroit River system to evaluate the lingering sublethal effects of industrial pollution. We emphasized bioindicators of direct (toxicity) and indirect (chronic stress, impoverished food web) effects on somatic and organ-specific growth (brain, gut, liver, heart ventricle, gonad). Our results show that higher sediment levels of industrial contaminants at the most downstream Detroit River site (Trenton Channel) are associated with increased perch liver detoxification activity and liver size, reduced brain size, and reduced scale cortisol content. Trenton Channel also displayed food web disruption, where adult perch occupied lower trophic positions than forage fish. Somatic growth and relative gut size were lower in perch sampled at the reference site in Lake St. Clair (Mitchell's Bay), possibly because of increased competition for resources. Models used to determine the factors contributing to site differences in organ growth suggest that the lingering effects of industrial pollution are best explained by trophic disruption. Thus, bioindicators of fish trophic ecology may prove advantageous to assess the health of aquatic ecosystems. Environ Toxicol Chem 2023;42:2158-2170. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Behavior and brain size of larval zebrafish exposed to environmentally relevant concentrations of beta-methylamino-l-alanine.

Harmful algal blooms (HABs) release toxic compounds in water and are increasing in frequency worldwide. The neurotoxin ß-methylamino-l-alanine (BMAA) is released by HABs and has garnered much attention over the past 20 years due to its association with human neurodegenerative disorders, but its effects on wildlife are still largely unknown. This study characterized the effects of chronic exposure to environmentally relevant concentrations of BMAA on the behavior and brain size of developing zebrafish (Danio rerio). Zebrafish were continuously exposed to 0, 1, 10, or 100 µg/l waterborne BMAA between 0- and 5-days postfertilization (dpf) before the onset of exogenous feeding. At 5 dpf, locomotion and responses to vibrational and visual stimuli were assessed. Following behavioral testing, larvae body and brain size were measured. Survival between 0 and 5 dpf did not differ between treatments. Moreover, BMAA exposure did not affect thigmotaxis, startle response magnitude, habituation to repeated presentation of vibrational startling stimuli, or relative brain size. A moderate increase in overall activity was observed in larvae exposed to 10 µg/l BMAA under light, but this effect was not seen in dark conditions, indicating that visual processing may have been affected by chronic BMAA exposure. Thus, passive continuous exposure to environmentally relevant concentrations of BMAA prior to first feeding in zebrafish did not affect survival or selected measures used to represent brain development, anxiety, and motor reflexes, but a limited light-dependent effect on locomotion suggests targeted neurotoxicity within the visual system.

Zebrafish parental progeny investment in response to cycling thermal stress and hypoxia: deposition of heat shock proteins but not cortisol.

The maternal match hypothesis predicts that maternal exposure to a stressor may help prepare offspring to cope with the same disturbance in later life. Although there is support for this hypothesis, the signals involved in non-genetic inheritance are unclear. In this study, we tested how adult zebrafish exposure to diel cycles of thermal stress (27-36°C), hypoxia (20-85% dissolved oxygen) or the combined treatment affects maternal and embryonic levels of cortisol and heat shock proteins (HSPs). While parental exposure to the thermal, hypoxic or combined treatment for 2 weeks did not affect whole-body cortisol levels, the combined exposure increased ovarian cortisol levels by 4-fold and reduced embryonic cortisol content by 60%. The combined treatment also elicited 3- and 19-fold increases in embryo transcripts involved in cortisol breakdown (11bhsd2) and export (abcb4), respectively. The thermal stress and combined exposure also elicited marked increases in ovary and embryo hsp70a (20- to 45-fold) and HSP70 (3- to 7-fold), and smaller increases in ovary and embryo hsp90aa and hsp47 (2- to 4-fold) and in embryo HSP90 and HSP47 (2- to 6-fold). In contrast, except for increases in ovary hsp90aa (2-fold) and embryo HSP90 (3-fold), the hypoxia treatment had little effect on HSP expression and transfer. Overall, while the embryonic deposition of HSPs largely paralleled the ovarian cellular stress response, the inverse relationship between ovary and embryo cortisol levels suggests the existence of barriers against cortisol deposition in response to environmental stressors. We conclude that the endocrine and cellular stress responses make stressor-specific and distinct contributions to non-genetic inheritance.

Galanin expression varies with parental care and social status in a wild cooperatively breeding fish.

As many busy parents will attest, caring for young often comes at the expense of having time to feed and care for oneself. Galanin is a neuropeptide that regulates food intake and modulates parental care; however, the relative importance of galanin in the regulation of feeding versus caring by parents has never been evaluated before under naturalistic settings. Here, we assessed how expression of the galanin system varied in two brain regions, the hypothalamus (which regulates feeding) and the preoptic area (which modulates social behaviours including care) in a wild cichlid fish, Neolamprologus pulcher. Females with young had higher hypothalamic expression of galanin receptor 1a, and the highest expression of galanin and galanin receptor 1a was observed in females that foraged the least. However, expression of five other feeding-related neuropeptides did not change while females were caring for young suggesting that changes in the hypothalamic galanin system may not have been directly related to changes in food intake. The preoptic galanin system was unaffected by the presence of young, but preoptic galanin expression was higher in dominant females (which are aggressive, regularly reproduce and care for young) compared to subordinate females (which are submissive, rarely reproduce but often help care for young). Additionally, preoptic galanin expression was higher in fish that performed more territory defense. Overall, our results indicate that galanin has brain-region-specific roles in modulating both parental care and social status in wild animals.

Social status-dependent regulation and function of the somatotropic axis in juvenile rainbow trout.

Juvenile rainbow trout (Oncorhynchus mykiss) develop social hierarchies when competing for resources in a constrained environment. Among the physiological consequences of social status are changes in organismal energy metabolism, which generally favour anabolic pathways in dominant fish and catabolic pathways in subordinate fish. The somatotropic axis is an important regulator of metabolism and growth that could be involved in mediating metabolic changes in response to social status in juvenile rainbow trout. Here we used juvenile trout housed either in dyads or individually (sham controls) to determine whether social status changes indices of somatotropic axis function. Although pituitary growth hormone expression (gh1 and gh2) did not differ among groups, circulating growth hormone (GH) increased ∼12-fold in subordinate fish compared to sham and dominant fish. Social status caused consistent differential expression of GH receptor paralogues in liver and muscle, two principal target tissues of GH. Compared to dominant and/or sham fish, ghra paralogue expression (ghra1 and ghra2) was lower, while ghrb1 expression was higher in subordinate fish. Across tissues, ghra paralogue expression was generally positively correlated with expression of insulin growth factors (igf1, igf2), while ghrb1 expression was positively correlated with transcript abundance of hormone sensitive lipase (hsl1). Because igf and hsl expression are subject to context-dependent GH control in rainbow trout, these results suggest that increased circulating GH in conjunction with differential expression of ghr paralogues may translate into prioritization of downstream catabolic lipolytic pathways in subordinate rainbow trout. These findings support a social context-dependent role for GH signalling in mediating metabolic changes in juvenile rainbow trout.

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.

Severe hypoxia exposure inhibits larval brain development but does not affect the capacity to mount a cortisol stress response in zebrafish.

Fish nursery habitats are increasingly hypoxic and the brain is recognized as highly hypoxia sensitive, yet there is a lack of information on the effects of hypoxia on the development and function of the larval fish brain. Here, we tested the hypothesis that by inhibiting brain development, larval exposure to severe hypoxia has persistent functional effects on the cortisol stress response in zebrafish (Danio rerio). Exposing 5 days post-fertilization (dpf) larvae to 10% dissolved O2 (DO) for 16 h only marginally reduced survival, but it decreased forebrain neural proliferation by 55%, and reduced the expression of neurod1, gfap and mbpa, markers of determined neurons, glia and oligodendrocytes, respectively. The 5 dpf hypoxic exposure also elicited transient increases in whole-body cortisol and in crf, uts1 and hsd20b2 expression, key regulators of the endocrine stress response. Hypoxia exposure at 5 dpf also inhibited the cortisol stress response to hypoxia in 10 dpf larvae and increased hypoxia tolerance. However, 10% DO exposure at 5 dpf for 16 h did not affect the cortisol stress response to a novel stressor in 10 dpf larvae or the cortisol stress response to hypoxia in adult fish. Therefore, while larval exposure to severe hypoxia can inhibit brain development, it also increases hypoxia tolerance. These effects may transiently reduce the impact of hypoxia on the cortisol stress response but not its functional capacity to respond to novel stressors. We conclude that the larval cortisol stress response in zebrafish has a high capacity to cope with severe hypoxia-induced neurogenic impairment.

Novel spikey ionocytes are regulated by cortisol in the skin of an amphibious fish.

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na+-K+-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes.

Rank- and sex-specific differences in the neuroendocrine regulation of glucocorticoids in a wild group-living fish.

Individuals that live in groups experience different challenges based on their social rank and sex. Glucocorticoids have a well-established role in coordinating responses to challenges and glucocorticoid levels often vary between ranks and sexes. However, the neuroendocrine mechanisms regulating glucocorticoid dynamics in wild groups are poorly understood, making it difficult to determine the functional consequences of differences in glucocorticoid levels. Therefore, we observed wild social groups of a cooperatively breeding fish (Neolamprologus pulcher) and evaluated how scale cortisol content (an emerging method to evaluate cortisol dynamics in fishes) and expression of glucocorticoid-related genes varied across group members. Scale cortisol was detectable in ~50% of dominant males (7/17) and females (7/15)-but not in any subordinates (0/16)-suggesting that glucocorticoid levels were higher in dominants. However, the apparent behavioural and neuroendocrine factors regulating cortisol levels varied between dominant sexes. In dominant females, higher cortisol was associated with greater rates of territory defense and increased expression of corticotropin-releasing factor in the preoptic and hypothalamic regions of the brain, but these patterns were not observed in dominant males. Additionally, transcriptional differences in the liver suggest that dominant sexes may use different mechanisms to cope with elevated cortisol levels. While dominant females appeared to reduce the relative sensitivity of their liver to cortisol (fewer corticosteroid receptor transcripts), dominant males appeared to increase hepatic cortisol breakdown (more catabolic enzyme transcripts). Overall, our results offer valuable insights on the mechanisms regulating rank- and sex-based glucocorticoid dynamics, as well as the potential functional outcomes of these differences.

The use of non-lethal sampling for transcriptomics to assess the physiological status of wild fishes.

Fishes respond to different abiotic and biotic stressors through changes in gene expression as a part of an integrated physiological response. Transcriptomics approaches have been used to quantify gene expression patterns as a reductionist approach to understand responses to environmental stressors in animal physiology and have become more commonly used to study wild fishes. We argue that non-lethal sampling for transcriptomics should become the norm for assessing the physiological status of wild fishes, especially when there are conservation implications. Processes at the level of the transcriptome provide a "snapshot" of the cellular conditions at a given time; however, by using a non-lethal sampling protocol, researchers can connect the transcriptome profile with fitness-relevant ecological endpoints such as reproduction, movement patterns and survival. Furthermore, telemetry is a widely used approach in fisheries to understand movement patterns in the wild, and when combined with transcriptional profiling, provides arguably the most powerful use of non-lethal sampling for transcriptomics in wild fishes. In this review, we discuss the different tissues that can be successfully incorporated into non-lethal sampling strategies, which is particularly useful in the context of the emerging field of conservation transcriptomics. We briefly describe different methods for transcriptional profiling in fishes from high-throughput qPCR to whole transcriptome approaches. Further, we discuss strategies and the limitations of using transcriptomics for non-lethally studying fishes. Lastly, as 'omics' technology continues to advance, transcriptomics paired with different omics approaches to study wild fishes will provide insight into the factors that regulate phenotypic variation and the physiological responses to changing environmental conditions in the future.

Glucocorticoids do not promote prosociality in a wild group-living fish.

Individuals often respond to social disturbances by increasing prosociality, which can strengthen social bonds, buffer against stress, and promote overall group cohesion. Given their importance in mediating stress responses, glucocorticoids have received considerable attention as potential proximate regulators of prosocial behaviour during disturbances. However, previous investigations have largely focused on mammals and our understanding of the potential prosocial effects of glucocorticoids across vertebrates more broadly is still lacking. Here, we assessed whether experimentally elevated glucocorticoid levels (simulating endogenous cortisol responses mounted following disturbances) promote prosocial behaviours in wild groups of the cichlid fish, Neolamprologus pulcher. Using SCUBA in Lake Tanganyika, we observed how subordinate group members adjusted affiliation, helping, and submission (all forms of prosocial behaviour) following underwater injections of either cortisol or saline. Cortisol treatment reduced affiliative behaviours-but only in females-suggesting that glucocorticoids may reduce overall prosociality. Fish with elevated glucocorticoid levels did not increase performance of submission or helping behaviours. Taken together, our results do not support a role for glucocorticoids in promoting prosocial behaviour in this species and emphasize the complexity of the proximate mechanisms that underlie prosociality.

Corticotropin-releasing factor protects against ammonia neurotoxicity in isolated larval zebrafish brains.

The physiological roles of corticotropin-releasing factor (CRF) have recently been extended to cytoprotection. Here, to determine whether CRF is neuroprotective in fish, the effects of CRF against high environmental ammonia (HEA)-mediated neurogenic impairment and cell death were investigated in zebrafish. In vivo, exposure of 1 day post-fertilization (dpf) embryos to HEA only reduced the expression of the determined neuron marker neurod1 In contrast, in 5 dpf larvae, HEA increased the expression of nes and sox2, neural progenitor cell markers, and reduced the expression of neurog1, gfap and mbpa, proneuronal cell, radial glia and oligodendrocyte markers, respectively, and neurod1 The N-methyl-d-aspartate (NMDA) receptor inhibitor MK801 rescued the HEA-induced reduction in neurod1 in 5 dpf larvae but did not affect the HEA-induced transcriptional changes in other neural cell types, suggesting that hyperactivation of NMDA receptors specifically contributes to the deleterious effects of HEA in determined neurons. As observed in vivo, HEA exposure elicited marked changes in the expression of cell type-specific markers in isolated 5 dpf larval brains. The addition of CRF reversed the in vitro effects of HEA on neurod1 expression and prevented an HEA-induced increase in cell death. Finally, the protective effects of CRF against HEA-mediated neurogenic impairment and cell death were prevented by the CRF type 1 receptor selective antagonist antalarmin. Together, these results provide novel evidence that HEA has developmental time- and cell type-specific neurotoxic effects, that NMDA receptor hyperactivation contributes to HEA-mediated impairment of determined neurons, and that CRF has neuroprotective properties in the larval zebrafish brain.

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.

Temporal profiles of cortisol accumulation and clearance support scale cortisol content as an indicator of chronic stress in fish.

The development of chronic stress indicators for fish is of great interest, but appropriate non-invasive methods are lagging those used in terrestrial vertebrates. Here, we explore the possibility that levels of the stress hormone cortisol in scales could be used as a chronic stress indicator. Three experiments were conducted to assess the temporal profiles of cortisol rise and fall in plasma and scales of goldfish (Carassius auratus) in response to stressors of varying intensity and duration. Results show that a single acute air emersion stressor does not influence scale cortisol content. In contrast, relative to plasma levels, the fall in scale cortisol content following a high-dose cortisol implant is delayed by at least 8 days, and the rise and fall in scale cortisol content in response to unpredictable chronic stress are delayed by at least 7 days. Also, scale cortisol content is spatially heterogeneous across the body surface of goldfish. Overall, since high and sustained circulating cortisol levels are needed to influence scale cortisol content and the rates of cortisol accumulation and clearance are much slower in scales than in plasma, our results show that scales can provide an integrated measure of cortisol production and serve as a chronic stress indicator.

Cross Talk without Cross Tolerance: Effect of Rearing Temperature on the Hypoxia Response of Embryonic Zebrafish.

Environmental stressors, such as warm temperatures and hypoxia, can interact and pose a threat to aquatic species. Cross talk between the hypoxia and heat stress cellular pathways can lead to enhanced cross tolerance between these environmental stressors. In this study, we questioned whether elevated temperatures (from 27° to 32°C) during rearing would enhance the hypoxia-inducible transcription factor-1 (HIF-1)-mediated transcriptional response to hypoxia (5% dissolved O2) in early stages of zebrafish development and whether these differences would be associated with enhanced larval tolerance and survival to hypoxia. We found that embryos reared at 32°C had an enhanced cellular HIF-1 response (elevated hif-1ab and insulin-like growth factor binding-protein mRNA level) and that acute hypoxia (4 h) activated the heat-shock response (elevated hsp70a and hsp90aa mRNA levels). Elevated rearing temperatures and hypoxia exposure also induced precocious hatching, but neither environmental stressor had an effect on the hypoxia tolerance (critical O2 tension) of 4-d-old larvae and did not protect larvae against the lethal effects of a second acute hypoxia exposure. Overall, during early zebrafish development, cross talk between the hypoxia and heat stress cellular pathways at the gene expression level did not confer cross tolerance at the whole-animal level with respect to hypoxia stress.

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.