Pacific salmon in the Canadian Arctic highlight a range-expansion pathway for sub-Arctic fishes.

Rapid climate change is altering Arctic ecosystems at unprecedented rates. These changes in the physical environment may open new corridors for species range expansions, with substantial implications for subsistence-dependent communities and sensitive ecosystems. Over the past 20 years, rising incidental harvest of Pacific salmon by subsistence fishers has been monitored across a widening range spanning multiple land claim jurisdictions in Arctic Canada. In this study, we connect Indigenous and scientific knowledges to explore potential oceanographic mechanisms facilitating this ongoing northward expansion of Pacific salmon into the western Canadian Arctic. A regression analysis was used to reveal and characterize a two-part mechanism related to thermal and sea-ice conditions in the Chukchi and Beaufort seas that explains nearly all of the variation in the relative abundance of salmon observed within this region. The results indicate that warmer late-spring temperatures in a Chukchi Sea watch-zone and persistent, suitable summer thermal conditions in a Beaufort Sea watch-zone together create a range-expansion corridor and are associated with higher salmon occurrences in subsistence harvests. Furthermore, there is a body of knowledge to suggest that these conditions, and consequently the presence and abundance of Pacific salmon, will become more persistent in the coming decades. Our collaborative approach positions us to document, explore, and explain mechanisms driving changes in fish biodiversity that have the potential to, or are already affecting, Indigenous rights-holders in a rapidly warming Arctic.

Habitat modulates population-level responses of freshwater salmon growth to a century of change in climate and competition.

The impacts of climate change are widespread and threaten natural systems globally. Yet, within regions, heterogeneous physical landscapes can differentially filter climate, leading to local response diversity. For example, it is possible that while freshwater lakes are sensitive to climate change, they may exhibit a diversity of thermal responses owing to their unique morphology, which in turn can differentially affect the growth and survival of vulnerable biota such as fishes. In particular, salmonids are cold-water fishes with complex life histories shaped by diverse freshwater habitats that are sensitive to warming temperatures. Here we examine the influence of habitat on the growth of sockeye salmon (Oncorhynchus nerka) in nursery lakes of Canada's Skeena River watershed over a century of change in regional temperature and intraspecific competition. We found that freshwater growth has generally increased over the last century. While growth tended to be higher in years with relatively higher summer air temperatures (a proxy for lake temperature), long-term increases in growth appear largely influenced by reduced competition. However, habitat played an important role in modulating the effect of high temperature. Specifically, growth was positively associated with rising temperatures in relatively deep (>50 m) nursery lakes, whereas warmer temperatures were not associated with a change in growth for fish among shallow lakes. The influence of temperature on growth also was modulated by glacier extent whereby the growth of fish from lakes situated in watersheds with little (i.e., <5%) glacier cover increased with rising temperatures, but decreased with rising temperatures for fish in lakes within more glaciated watersheds. Maintaining the integrity of an array of freshwater habitats-and the processes that generate and maintain them-will help foster a diverse climate-response portfolio for important fish species, which in turn can ensure that salmon watersheds are resilient to future environmental change.

Body size and early marine conditions drive changes in Chinook salmon productivity across northern latitude ecosystems.

Disentangling the influences of climate change from other stressors affecting the population dynamics of aquatic species is particularly pressing for northern latitude ecosystems, where climate-driven warming is occurring faster than the global average. Chinook salmon (Oncorhynchus tshawytscha) in the Yukon-Kuskokwim (YK) region occupy the northern extent of their species' range and are experiencing prolonged declines in abundance resulting in fisheries closures and impacts to the well-being of Indigenous people and local communities. These declines have been associated with physical (e.g., temperature, streamflow) and biological (e.g., body size, competition) conditions, but uncertainty remains about the relative influence of these drivers on productivity across populations and how salmon-environment relationships vary across watersheds. To fill these knowledge gaps, we estimated the effects of marine and freshwater environmental indicators, body size, and indices of competition, on the productivity (adult returns-per-spawner) of 26 Chinook salmon populations in the YK region using a Bayesian hierarchical stock-recruitment model. Across most populations, productivity declined with smaller spawner body size and sea surface temperatures that were colder in the winter and warmer in the summer during the first year at sea. Decreased productivity was also associated with above average fall maximum daily streamflow, increased sea ice cover prior to juvenile outmigration, and abundance of marine competitors, but the strength of these effects varied among populations. Maximum daily stream temperature during spawning migration had a nonlinear relationship with productivity, with reduced productivity in years when temperatures exceeded thresholds in main stem rivers. These results demonstrate for the first time that well-documented declines in body size of YK Chinook salmon were associated with declining population productivity, while taking climate into account.

Adult spawners: A critical period for subarctic Chinook salmon in a changing climate.

Concurrent, distribution-wide abundance declines of some Pacific salmon species, including Chinook salmon (Oncorhynchus tshawytscha), highlights the need to understand how vulnerability at different life stages to climate stressors affects population dynamics and fisheries sustainability. Yukon River Chinook salmon stocks are among the largest subarctic populations, near the northernmost extent of the species range. Existing research suggests that Yukon River Chinook salmon population dynamics are largely driven by factors occurring between the adult spawner life stage and their offspring's first summer at sea (second year post-hatching). However, specific mechanisms sustaining chronic poor productivity are unknown, and there is a tremendous sense of urgency to understand causes, as declines of these stocks have taken a serious toll on commercial, recreational, and indigenous subsistence fisheries. Therefore, we leveraged multiple existing datasets spanning parent and juvenile stages of life history in freshwater and marine habitats. We analyzed environmental data in association with the production of offspring that survive to the marine juvenile stage (juveniles per spawner). These analyses suggest more than 45% of the variability in the production of juvenile Chinook salmon is associated with river temperatures or water discharge levels during the parent spawning migration. Over the past two decades, parents that experienced warmer water temperatures and lower discharge in the mainstem Yukon River produced fewer juveniles per spawning adult. We propose the adult spawner life stage as a critical period regulating population dynamics. We also propose a conceptual model that can explain associations between population dynamics and climate stressors using independent data focused on marine nutrition and freshwater heat stress. It is sobering to consider that some of the northernmost Pacific salmon habitats may already be unfavorable to these cold-water species. Our findings have immediate implications, given the common assumption that northern ranges of Pacific salmon offer refugia from climate stressors.

Metabolic constraints and individual variation shape the trade-off between physiological recovery and anti-predator responses in adult sockeye salmon.

Metabolic scope represents the aerobic energy budget available to an organism to perform non-maintenance activities (e.g., escape a predator, recover from a fisheries interaction, compete for a mate). Conflicting energetic requirements can give rise to ecologically relevant metabolic trade-offs when energy budgeting is constrained. The objective of this study was to investigate how aerobic energy is utilized when individual sockeye salmon (Oncorhynchus nerka) are exposed to multiple acute stressors. To indirectly assess metabolic changes in free-swimming individuals, salmon were implanted with heart rate biologgers. The animals were then exercised to exhaustion or briefly handled as a control, and allowed to recover from this stressor for 48 h. During the first 2 h of the recovery period, individual salmon were exposed to 90 ml of conspecific alarm cues or water as a control. Heart rate was recorded throughout the recovery period. Recovery effort and time was higher in exercised fish, relative to control fish, whereas exposure to an alarm cue had no effect on either of these metrics. Individual routine heart rate was negatively correlated with recovery time and effort. Together, these findings suggest that metabolic energy allocation towards exercise recovery (i.e., an acute stressor; handling, chase, etc.) trumps anti-predator responses in salmon, although individual variation may mediate this effect at the population level.

Transcriptomic characterisation of a common skin lesion in farmed chinook salmon.

Little is known about host responses of farmed Chinook salmon with skin lesions, despite the lesions being associated with increased water temperatures and elevated mortality rates. To address this shortfall, a transcriptomic approach was used to characterise the molecular landscape of spot lesions, the most commonly reported lesion type in New Zealand Chinook salmon, versus healthy appearing skin in fish with and without spot lesions. Many biological (gene ontology) pathways were enriched in lesion adjacent tissue, relative to control skin tissue, including proteolysis, fin regeneration, calcium ion binding, mitochondrial transport, actin cytoskeleton organisation, epithelium development, and tissue development. In terms of specific transcripts of interest, pro-inflammatory cytokines (interleukin 1ß and tumour necrosis factor), annexin A1, mucin 2, and calreticulin were upregulated, while cathepsin H, mucin 5AC, and perforin 1 were downregulated in lesion tissue. In some instances, changes in gene expression were consistent between lesion and healthy appearing skin from the same fish relative to lesion free fish, suggesting that host responses weren't limited to the site of the lesion. Goblet cell density in skin histological sections was not different between skin sample types. Collectively, these results provide insights into the physiological changes associated with common spot lesions in farmed Chinook salmon.

Exercise training does not affect heat tolerance in Chinook salmon (Oncorhynchus tshawytscha).

The progression of climate warming will expose ectotherms to transient heatwave events and temperatures above their tolerance range at increased frequencies. It is therefore pivotal that we understand species' physiological limits and the capacity for various controls to plastically alter these thresholds. Exercise training could have beneficial impacts on organismal heat tolerance through improvements in cardio-respiratory capacity, but this remains unexplored. Using juvenile Chinook salmon (Oncorhynchus tshawytscha), we tested the hypothesis that exercise training improves heat tolerance through enhancements in oxygen-carrying capacity. Fish were trained once daily at 60% of their maximum sustainable swim speed, UCRIT, for 60 min. Tolerance to acute warming was assessed following three weeks of exercise training, measured as the critical thermal maximum (CTMAX). CTMAX measurements were coupled with examinations of the oxygen carrying capacity (haematocrit, haemoglobin concentration, relative ventricle size, and relative splenic mass) as critical components of the oxygen transport cascade in fish. Contrary to our hypothesis, we found that exercise training did not raise the CTMAX of juvenile Chinook salmon with a mean CTMAX increase of just 0.35 °C compared to unexercised control fish. Training also failed to improve the oxygen carrying capacity of fish. Exercise training remains a novel strategy against acute warming that requires substantial fine-tuning before it can be applied to the management of commercial and wild fishes.

Olfactory behavioural and neural responses of planktivorous lacustrine sockeye salmon (Oncorhynchus nerka) to prey odours.

Fish use a variety of sensory systems when foraging. Salmonids are generally considered visual feeders. However, some species feed on zooplanktons under dark conditions, suggesting they also detect prey using nonvisual cues. Under experimental conditions, hatchery-reared rainbow trout (Oncorhynchus mykiss) have been shown to use olfaction when searching for food pellets, but olfactory foraging has not been documented in wild salmonids. In the present study, to examine their behavioural response and neural activity in the olfactory nervous system using c-fos expression as a neural molecular marker, immature wild-caught lacustrine sockeye salmon (Oncorhynchus nerka) in a flow-through aquarium were exposed to zooplanktons (Daphnia spp.) extract including zooplanktons odorant and to dimethyl sulfide. The salmon exposed to zooplanktons odour increased their total swimming distance and time, numbers of turns and ascents, and c-fos expression in the olfactory bulb, suggesting that they can detect zooplanktons extract to locate prey in the laboratory experiments. However, no response was seen in those exposed to dimethyl sulfide. The results of this study suggest that prey odour may serve as a chemosensory cue for wild immature salmonids.

Straightforward upriver migration to spawning sites by chum salmon Oncorhynchus keta homing to coastal short rivers in the Sanriku region.

In chum salmon (Oncorhynchus keta) homed to the Sanriku region, Japan, most of the fish are matured in bays and spawn near river mouths in coastal short rivers; therefore, their upriver migration is extremely short, but their behavioural characteristics have remained unknown. Upriver migration in the Otsuchi River, a typical coastal river, was evaluated from behavioural and physiological aspects. Homing salmon tracked in Otsuchi Bay held in the inner bay for less than 1 day to more than 10 days before river entry. The varied holding duration was negatively correlated with plasma 17α, 20ß-dihydroxy-4-pregnen-3-one (DHP) concentration, an indicator of maturation. After river entry, however, most fish were captured in weirs near the river mouths within 2 days regardless of the DHP concentration. Of the 34 fish released in the river, on the contrary, eighteen and five fish were seen next day in the main spawning sites located at c. 1.5 km upstream and in the branch creek, respectively, and 85% of the fish held position there until their death. The mean survival time of released fish was 5.8 days. Plasma DHP level suggested that preparations for spawning were already completed at the timing of the release. Taken together, homing salmon completed spawning preparation in the bay, and then they moved to their spawning sites immediately after river entry and spawned there during their short remaining life. This upriver migration contrasts with those of other populations, such as early migrants and long river migrants, whose maturation is completed during upriver migration.

Transhemispheric ecosystem disservices of pink salmon in a Pacific Ocean macrosystem.

Pink salmon (Oncorhynchus gorbuscha) in the North Pacific Ocean have flourished since the 1970s, with growth in wild populations augmented by rising hatchery production. As their abundance has grown, so too has evidence that they are having important effects on other species and on ocean ecosystems. In alternating years of high abundance, they can initiate pelagic trophic cascades in the northern North Pacific Ocean and Bering Sea and depress the availability of common prey resources of other species of salmon, resident seabirds, and other pelagic species. We now propose that the geographic scale of ecosystem disservices of pink salmon is far greater due to a 15,000-kilometer transhemispheric teleconnection in a Pacific Ocean macrosystem maintained by short-tailed shearwaters (Ardenna tenuirostris), seabirds that migrate annually between their nesting grounds in the South Pacific Ocean and wintering grounds in the North Pacific Ocean. Over this century, the frequency and magnitude of mass mortalities of shearwaters as they arrive in Australia, and their abundance and productivity, have been related to the abundance of pink salmon. This has influenced human social, economic, and cultural traditions there, and has the potential to alter the role shearwaters play in insular terrestrial ecology. We can view the unique biennial pulses of pink salmon as a large, replicated, natural experiment that offers basin-scale opportunities to better learn how these ecosystems function. By exploring trophic interaction chains driven by pink salmon, we may achieve a deeper conservation conscientiousness for these northern open oceans.

Positive Feedback between Behavioral and Hormonal Dynamics Leads to Differentiation of Life-History Tactics.

AbstractCompetitive interaction among individuals of a single population may result in the differentiation of two or more distinct life-history tactics. For example, although they exhibit unimodal size distribution, male juveniles of salmonids differentiate into those going down to the ocean to grow and returning to the natal stream after several years to reproduce (migratory tactic) and those staying in the stream and reproducing for multiple years (resident tactic). In this study, we developed a simple mathematical model for the positive feedback between hormonal and behavioral dynamics, with the expectation of establishing multiple discrete clusters of hormone levels leading to differentiation of life-history tactics. The assumptions were that probability of winning in fighting depends both on the body size and hormone level of the two contestants. An individual with a higher hormone level would be more likely to win the competition, which further enhanced hormone production, forming a positive feedback loop between hormone level and fighting ability. If the positive feedback was strong but not excessive, discrete clusters of hormone levels emerged from a continuous distribution. In contrast, no clear clustering structure appeared in the distribution of hormone levels if the probability of winning in fighting was controlled by the body size.

The biophysical basis of thermal tolerance in fish eggs.

A warming climate poses a fundamental problem for embryos that develop within eggs because their demand for oxygen (O2) increases much more rapidly with temperature than their capacity for supply, which is constrained by diffusion across the egg surface. Thus, as temperatures rise, eggs may experience O2 limitation due to an imbalance between O2 supply and demand. Here, we formulate a mathematical model of O2 limitation and experimentally test whether this mechanism underlies the upper thermal tolerance in large aquatic eggs. Using Chinook salmon (Oncorhynchus tshawytscha) as a model system, we show that the thermal tolerance of eggs varies systematically with features of the organism and environment. Importantly, this variation can be precisely predicted by the degree to which these features shift the balance between O2 supply and demand. Equipped with this mechanistic understanding, we predict and experimentally confirm that the thermal tolerance of these embryos in their natural habitat is substantially lower than expected from laboratory experiments performed under normoxia. More broadly, our biophysical model of O2 limitation provides a mechanistic explanation for the elevated thermal sensitivity of fish embryos relative to other life stages, global patterns in egg size and the extreme fecundity of large teleosts.

A sense of place: pink salmon use a magnetic map for orientation.

The use of 'map-like' information from the Earth's magnetic field for orientation has been shown in diverse taxa, but questions remain regarding the function of such maps. We used a 'magnetic displacement' experiment to demonstrate that juvenile pink salmon (Oncorhynchus gorbuscha) use magnetic cues to orient. The experiment was designed to simultaneously explore whether their magnetic map is used to direct fish (i) homeward, (ii) toward the center of their broad oceanic range or (iii) along their oceanic migratory route. The headings adopted by these navigationally naive fish coincided remarkably well with the direction of the juveniles' migration inferred from historical tagging and catch data. This suggests that the large-scale movements of pink salmon across the North Pacific may be driven largely by their innate use of geomagnetic map cues. Key aspects of the oceanic ecology of pink salmon and other marine migrants might therefore be predicted from magnetic displacement experiments.

Reduced lactate dehydrogenase activity in the heart and suppressed sex hormone levels are associated with female-biased mortality during thermal stress in Pacific salmon.

Female-biased mortality has been repeatedly reported in Pacific salmon during their upriver migration in both field studies and laboratory holding experiments, especially in the presence of multiple environmental stressors, including thermal stress. Here, we used coho salmon (Oncorhynchus kisutch) to test whether females exposed to elevated water temperatures (18°C) (i) suppress circulating sex hormones (testosterone, 11-ketotestosterone and estradiol), owing to elevated cortisol levels, (ii) have higher activities of enzymes supporting anaerobic metabolism (e.g. lactate dehydrogenase, LDH), (iii) have lower activities of enzymes driving oxidative metabolism (e.g. citrate synthase, CS) in skeletal and cardiac muscle, and (iv) have more oxidative stress damage and reduced capacity for antioxidant defense [lower catalase (CAT) activity]. We found no evidence that a higher susceptibility to oxidative stress contributes to female-biased mortality at warm temperatures. We did, however, find that females had significantly lower cardiac LDH and that 18°C significantly reduced plasma levels of testosterone and estradiol, especially in females. We also found that relative gonad size was significantly lower in the 18°C treatment regardless of sex, whereas relative liver size was significantly lower in females held at 18°C. Further, relative spleen size was significantly elevated in the 18°C treatments across both sexes, with larger warm-induced increases in females. Our results suggest that males may better tolerate bouts of cardiac hypoxia at high temperature, and that thermal stress may also disrupt testosterone- and estradiol-mediated protein catabolism, and the immune response (larger spleens), in migratory female salmon.

Elevated incubation temperature improves later-life swimming endurance in juvenile Chinook salmon, Oncorhynchus tshawytscha.

The effect of incubation and rearing temperature on muscle development and swimming endurance under a high-intensity swimming test was investigated in juvenile Chinook salmon (Oncorhynchus tshawytscha) in a hatchery experiment. After controlling for the effects of fork length (LF ) and parental identity, times to fatigue of fish were higher when fish were incubated or reared at warmer temperatures. Significant differences among combinations of pre- and post-emergence temperatures conformed to 15-15°C > 15-9°C > 9-9°C > 7-9°C > 7-7°C in 2011 when swimming tests were conducted at 300 accumulated temperature units post-emergence and 15-9°C > (7-9°C = 7-7°C) in 2012 when swimming tests were conducted at an LF of c. 40 mm. The combination of pre- and post-emergence temperatures also affected the number and size of muscle fibres, with differences among temperature treatments in mean fibre cross-sectional area persisting after controlling for LF and parental effects. Nonetheless, neither fibre number nor fibre size accounted for significant variation in swimming endurance. Thus, thermal carryover effects on swimming endurance were not mediated by thermal imprinting of muscle structure. This is the first study to test how temperature, body size and muscle structure interact to affect swimming endurance during early development in salmon.

Effects of Hatchery Rearing Density, Handling, and Nutrition on Renibacterium salmoninarum Infection Prevalence in Juvenile Chinook Salmon.

Bacterial kidney disease, caused by Renibacterium salmoninarum (RS), is a chronic and often fatal disease of salmonid species, and can be particularly harmful to hatchery-reared Chinook Salmon Oncorhynchus tshawytscha. A considerable amount of research has focused on the prevention of vertical and horizontal transmission; however, a comparatively little amount has investigated factors that increase the prevalence of RS infection in captive environments. We evaluated the effects of three common hatchery conditions (handling, nutrition level, and rearing density) on RS infection prevalence. Fish were sampled at 30-d and 60-d postexposure to RS. Of 577 juveniles examined, 65 (11.27%) had anterior kidneys infected with RS. Using a logistic mixed model analysis, we found effects of nutrition level (P = 0.018), handling (P = 0.010), and sampling period (P = 0.003) on the prevalence of RS. The interactions of nutrition and handling (P = 0.008) and nutrition and time (P < 0.001) were also significant. When fed a standard-nutrition diet, proportionately fewer fish were infected with RS when not handled (7.16% versus 0.04%; P = 0.003). Fish in the standard-nutrition group also had a lower prevalence of RS during the second sampling period (4.08% versus 0.08%, respectively; P < 0.001). When not handled, rearing with standard nutrition (11.50% versus 0.04%; P = 0.004) resulted in a reduction in prevalence of RS infection. Additionally, nonhandled fish had a much lower prevalence of RS infection during the second sampling period (2.66% versus 0.21%; P = 0.009). While density did not affect the prevalence of RS infection (P = 0.145), fish reared at a higher density had lower RS infection when not handled (16.48% versus 0.84%, P = 0.004). For fish at a higher density, the RS prevalence was lower during the second sampling period (10.57% versus 1.40%; P = 0.002). Our results suggest that hatchery managers can reduce RS infection prevalence by maintaining an adequate nutritional regime as recommended by the manufacturer. Additionally, the prevalence of RS may be reduced if managers decrease handling of hatchery-reared Chinook Salmon if exposed to RS.

Carotenoid pigmentation in salmon: variation in expression at BCO2-l locus controls a key fitness trait affecting red coloration.

Carotenoids are primarily responsible for the characteristic red flesh coloration of salmon. Flesh coloration is an economically and evolutionarily significant trait that varies inter- and intra-specifically, yet the underlying genetic mechanism is unknown. Chinook salmon (Oncorhynchus tshawytscha) represents an ideal system to study carotenoid variation as, unlike other salmonids, they exhibit extreme differences in carotenoid utilization due to genetic polymorphisms. Here, we crossed populations of Chinook salmon with fixed differences in flesh coloration (red versus white) for a genome-wide association study to identify loci associated with pigmentation. Here, the beta-carotene oxygenase 2-like (BCO2-l) gene was significantly associated with flesh colour, with the most significant single nucleotide polymorphism explaining 66% of the variation in colour. BCO2 gene disruption is linked to carotenoid accumulation in other taxa, therefore we hypothesize that an ancestral mutation partially disrupting BCO2-l activity (i.e. hypomorphic mutation) allowed the deposition and accumulation of carotenoids within Salmonidae. Indeed, we found elevated transcript levels of BCO2-l in white Chinook salmon relative to red. The long-standing mystery of why salmon are red, while no other fishes are, is thus probably explained by a hypomorphic mutation in the proto-salmonid at the time of divergence of red-fleshed salmonid genera (approx. 30 Ma).

There and back again: natal homing by magnetic navigation in sea turtles and salmon.

Diverse marine animals migrate across vast expanses of seemingly featureless ocean before returning as adults to reproduce in the area where they originated. How animals accomplish such feats of natal homing is an enduring mystery. Growing evidence suggests, however, that sea turtles and salmon imprint on the magnetic field of their home area when young and then use this information to return as adults. Both turtles and salmon have the sensory abilities needed to detect the unique 'magnetic signature' of a coastal area. Analyses have revealed that, for both groups of animals, subtle changes in the geomagnetic field of the home region are correlated with changes in natal homing behavior. In turtles, a relationship between population genetic structure and the magnetic fields that exist at nesting beaches has also been detected, consistent with the hypothesis that turtles recognize their natal areas on the basis of magnetic cues. Salmon likely use a biphasic navigational strategy in which magnetic cues guide fish through the open sea and into the proximity of the home river where chemical cues allow completion of the spawning migration. Similarly, turtles may also exploit local cues to help pinpoint nesting areas once they have arrived in the vicinity. Throughout most of the natal homing migration, however, magnetic navigation appears to be the primary mode of long-distance guidance in both sea turtles and salmon.

Salmonid gene expression biomarkers indicative of physiological responses to changes in salinity and temperature, but not dissolved oxygen.

An organism's ability to respond effectively to environmental change is critical to its survival. Yet, life stage and overall condition can dictate tolerance thresholds to heightened environmental stressors, such that stress may not be equally felt across individuals and at all times. Also, the transcriptional responses induced by environmental changes can reflect both generalized responses as well as others that are highly specific to the type of change being experienced. Thus, if transcriptional biomarkers specific to a stressor, even under multi-stressor conditions, can be identified, the biomarkers could then be applied in natural environments to determine when and where an individual experiences such a stressor. Here, we experimentally challenged juvenile Chinook salmon (Oncorhynchus tshawytscha) to validate candidate gill gene expression biomarkers. A sophisticated experimental design manipulated salinity (freshwater, brackish water and seawater), temperature (10, 14 and 18°C) and dissolved oxygen (normoxia and hypoxia) in all 18 possible combinations for 6 days using separate trials for three smolt statuses (pre-smolt, smolt and de-smolt). In addition, changes in juvenile behaviour, plasma variables, gill Na+/K+-ATPase activity, body size, body morphology and skin pigmentation supplemented the gene expression responses. We identified biomarkers specific to salinity and temperature that transcended the multiple stressors, smolt status and mortality (live, dead and moribund). Similar biomarkers for dissolved oxygen were not identified. This work demonstrates the unique power of gene expression biomarkers to identify a specific stressor even under multi-stressor conditions, and we discuss our next steps for hypoxia biomarkers using an RNA-seq study.