Aimee Lee S. Houde

Identifying early gene expression responses to hypoxia (i.e., low dissolved oxygen) as a tool to assess the degree of exposure to this stressor is crucial for salmonids, because they are increasingly exposed to hypoxic stress due to anthropogenic habitat change, e.g., global warming, excessive nutrient loading, and persistent algal blooms. Our goal was to discover and validate gill gene expression biomarkers specific to the hypoxia response in salmonids across multi-stressor conditions. Gill tissue was collected from 24 freshwater juvenile Chinook salmon ( Oncorhynchus tshawytscha ), held in normoxia [dissolved oxygen (DO) > 8 mg L ⁻¹ ] and hypoxia (DO = 4□5 mg L ⁻¹ ) in 10 and 18°C temperatures for up to six days. RNA-sequencing (RNA-seq) was then used to discover 240 differentially expressed genes between hypoxic and normoxic conditions, but not affected by temperature. The most significantly differentially expressed genes had functional roles in the cell cycle and suppression of cell proliferation associated with hypoxic conditions. The most significant genes (n = 30) were selected for real-time qPCR assay development. These assays demonstrated a strong correlation (r = 0.88; p < 0.001) between the expression values from RNA-seq and the fold changes from qPCR. Further, qPCR of the 30 candidate hypoxia biomarkers was applied to an additional 322 Chinook salmon exposed to hypoxic and normoxic conditions to reveal the top biomarkers to define hypoxic stress. Multivariate analyses revealed that smolt stage, water salinity, and morbidity status were relevant factors to consider with the expression of these genes in relation to hypoxic stress. These hypoxia candidate genes will be put into application screening Chinook salmon to determine the identity of stressors impacting the fish.

Early marine survival of juvenile salmon is intimately associated with their physiological condition during smoltification and ocean entry. Smoltification (parr–smolt transformation) is a developmental process that allows salmon to acquire seawater tolerance in preparation for marine living. Traditionally, this developmental process has been monitored using gill Na⁺/K⁺-ATPase (NKA) activity or plasma hormones, but gill gene expression offers the possibility of another method. Here, we describe the discovery of candidate genes from gill tissue for staging smoltification using comparisons of microarray studies with particular focus on the commonalities between anadromous Rainbow trout and Sockeye salmon datasets, as well as a literature comparison encompassing more species. A subset of 37 candidate genes mainly from the microarray analyses was used for TaqMan quantitative PCR assay design and their expression patterns were validated using gill samples from four groups, representing three species and two ecotypes: Coho salmon, Sockeye salmon, stream-type Chinook salmon and ocean-type Chinook salmon. The best smoltification biomarkers, as measured by consistent changes across these four groups, were genes involved in ion regulation, oxygen transport and immunity. Smoltification gene expression patterns (using the top 10 biomarkers) were confirmed by significant correlations with NKA activity and were associated with changes in body brightness, caudal fin darkness and caudal peduncle length. We incorporate gene expression patterns of pre-smolt, smolt and de-smolt trials from acute seawater transfers from a companion study to develop a preliminary seawater tolerance classification model for ocean-type Chinook salmon. This work demonstrates the potential of gene expression biomarkers to stage smoltification and classify juveniles as pre-smolt, smolt or de-smolt.

An organism9s 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.

An organism’s ability to respond effectively to environmental change is critical to their 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 within a species. Environmental changes can induce transcriptional responses in an organism, some of which reflect generalized responses, and others are highly specific to the type of change being experienced. Thus, if transcriptional biomarkers specific to a heightened environmental stressor, even under multi-stressor impacts, can be identified, the biomarkers could be then applied in natural environments to determine when and where individuals are experiencing such stressors. Here, we validate candidate gill gene expression biomarkers by experimentally challenging juvenile Chinook salmon (Oncorhynchus tshawytscha ). A sophisticated experimental set-up (four trials) 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 up to six days during the pre-smolt, smolt, and de-smolt life stages. In addition, we also describe the changes in juvenile behaviour, plasma variables, gill Na ⁺ /K ⁺ - ATPase (NKA) activity, body size, body morphology, and skin pigmentation associated with salinity, temperature, dissolved oxygen, mortality, and smolt status. We statistically identified biomarkers specific to salinity and temperature treatments, as well as mortality across multiple stressors and life stages. Similar biomarkers for the dissolved oxygen treatment could not be identified in the data and we discuss our next steps using an RNA-seq study. This work demonstrates the unique power of gene expression biomarkers to identify a specific stressor even under multi-stressor conditions.

Early marine survival of juvenile salmon is intimately associated with their physiological condition during ocean entry and especially smoltification. Smoltification is a developmental parr–smolt transformation allowing salmon to acquire the trait of seawater tolerance in preparation for marine living. Traditionally, this developmental process has been monitored using gill Na ⁺ /K ⁺ ATPase (NKA) activity or plasma hormones, but gill gene expression can be reliably used. Here, we describe the discovery of candidate genes from gill tissue for staging smoltification using comparisons of microarray studies with particular focus on the commonalities between anadromous Rainbow trout and Sockeye salmon datasets, as well as literature comparison encompassing more species. A subset of 37 candidate genes mainly from the microarray analyses was used for Taq-Man qPCR assay design and their monthly expression patterns were validated using gill samples from four groups, representing three species and two ecotypes: Coho salmon, Sockeye salmon, stream-type Chinook salmon, and ocean-type Chinook salmon. The best smoltification biomarkers, as measured by consistent changes across these four groups, were genes involved in ion regulation, oxygen transport, and immunity. Smoltification gene expression patterns (using the top 10 biomarkers) were confirmed by significant correlations with NKA activity and were associated with changes in body brightness, caudal fin darkness, and caudal peduncle length. We incorporate gene expression patterns of pre-smolt, smolt, and de-smolt trials from acute seawater transfers using a companion study to develop a preliminary seawater tolerance classification model for ocean-type Chinook salmon. This work demonstrates the potential of gene expression biomarkers to stage smoltification and classify juveniles as pre-smolt, smolt, or de-smolt.

Competition with non‐native species may impede the restoration of native species, but differences in competitive abilities among intraspecific native populations may make some populations more suitable for reintroduction than others. Here, juvenile Atlantic salmon (Salmo salar) from two allopatric populations (LaHave and Sebago) being used for reintroduction into Lake Ontario were placed into two natural stream sites differing in the presence of ecologically similar rainbow trout (Oncorhynchus mykiss). We assessed the effects of competition in the natural streams on fitness‐related traits and habitat use of the Atlantic salmon. We then compared these effects to those observed in artificial streams from a previous study. Atlantic salmon in natural streams had reduced fitness‐related traits that were associated with suboptimal microhabitats in the presence of rainbow trout, but utilised optimal microhabitats in their absence. In the presence of rainbow trout, the two Atlantic salmon populations exhibited comparable recapture proportions to each other, but the individuals from the Sebago population had better performance (body size and condition) than those from the LaHave population. Responses of both Atlantic salmon populations to competition with rainbow trout were generally similar in both direction and magnitude when compared to results from the artificial stream study. The combined results suggest that native species restoration efforts should be focused on candidate populations that are ecologically suitable to reintroduction environments, as well as on suitable habitats that do not contain exotic competitors. Moreover, this study highlights the value of controlled experiments in artificial environments for predicting fitness‐related performance in natural environments.

Competitive interactions with non‐native species can have negative impacts on the conservation of native species, resulting in chronic stress and reduced survival. Here, juvenile Atlantic salmon (Salmo salar) from two allopatric populations (Sebago and LaHave) that are being used for reintroduction into Lake Ontario were placed into semi‐natural stream tanks with four non‐native salmonid competitors that are established in Ontario streams: brown trout (S. trutta), rainbow trout (Oncorhynchus mykiss), Chinook salmon (O. tshawytscha) and coho salmon (O. kisutch). Brown trout and rainbow trout reduced the survival and fitness‐related traits of Atlantic salmon, whereas Chinook salmon and coho salmon had no impact on these traits. These data support theories on ecological niche overlap and link differences in observed aggression levels with competitive outcomes. Measurements of circulating hormones indicated that the Atlantic salmon were not chronically stressed nor had a change in social status at the 10‐month time point in the semi‐natural stream tanks. Additionally, the Sebago population was better able to coexist with the non‐native salmonids than the LaHave population. Certain populations of Atlantic salmon may thus be more suitable for some environments of the juvenile stream phase for the reintroduction into Lake Ontario.