Developing specific molecular biomarkers for thermal stress in salmonids.

BACKGROUND: Pacific salmon (Oncorhynchus spp.) serve as good biological indicators of the breadth of climate warming effects on fish because their anadromous life cycle exposes them to environmental challenges in both marine and freshwater environments. Our study sought to mine the extensive functional genomic studies in fishes to identify robust thermally-responsive biomarkers that could monitor molecular physiological signatures of chronic thermal stress in fish using non-lethal sampling of gill tissue. RESULTS: Candidate thermal stress biomarkers for gill tissue were identified using comparisons among microarray datasets produced in the Molecular Genetics Laboratory, Pacific Biological Station, Nanaimo, BC, six external, published microarray studies on chronic and acute temperature stress in salmon, and a comparison of significant genes across published studies in multiple fishes using deep literature mining. Eighty-two microarray features related to 39 unique gene IDs were selected as candidate chronic thermal stress biomarkers. Most of these genes were identified both in the meta-analysis of salmon microarray data and in the literature mining for thermal stress markers in salmonids and other fishes. Quantitative reverse transcription PCR (qRT-PCR) assays for 32 unique genes with good efficiencies across salmon species were developed, and their activity in response to thermally challenged sockeye salmon (O. nerka) and Chinook salmon (O. tshawytscha) (cool, 13-14 °C and warm temperatures 18-19 °C) over 5-7 days was assessed. Eight genes, including two transcripts of each SERPINH1 and HSP90AA1, FKBP10, MAP3K14, SFRS2, and EEF2 showed strong and robust chronic temperature stress response consistently in the discovery analysis and both sockeye and Chinook salmon validation studies. CONCLUSIONS: The results of both discovery analysis and gene expression showed that a panel of genes involved in chaperoning and protein rescue, oxidative stress, and protein biosynthesis were differentially activated in gill tissue of Pacific salmon in response to elevated temperatures. While individually, some of these biomarkers may also respond to other stressors or biological processes, when expressed in concert, we argue that a biomarker panel comprised of some or all of these genes could provide a reliable means to specifically detect thermal stress in field-caught salmon.

Developing molecular classifiers to detect environmental stressors, smolt stages and morbidity in coho salmon, Oncorhynchus kisutch.

Aquatic species are increasingly confronted with environmental stressors because of climate change. Although molecular technologies have advanced our understanding of how organisms respond to stressors in laboratory settings, the ability to detect physiological responses to specific stressors under complex field conditions remains underdeveloped. This research applied multi-stressor challenge trials on coho salmon, employing the "Salmon Fit-Chips" genomic tool and a random forest-based classification model to develop classifiers predictive for chronic thermal and hypoxic stress, as well as salinity acclimation, smolt stage and morbidity status. The study also examined how smolts and de-smolts (smolts not having entered SW during the smolt window) responded transcriptionally to exposure to saltwater. Using RF classifiers optimized with 4 to 12 biomarkers, we identified transcriptional signatures that accurately predicted the presence of each stressor and physiological state, achieving prediction accuracy rates between 86.8 % and 100 %, regardless of other background stressors present. Stressor recovery time was established by placing fish back into non-stressor conditions after stress exposure, providing important context to stressor detections in field applications. Recovery from thermal and hypoxic stress requires about 3 and 2 days, respectively, with >3 days needed for re-acclimation to freshwater for seawater acclimated fish. The study also found non-additive (synergistic) effects of multiple stressors on mortality risk. Importantly, osmotic stress associated with de-smolts was the most important predictor of mortality. In saltwater, de-smolts exposed to salinity, high temperature, and hypoxia experienced a 9-fold increase in mortality compared to those only exposed to saltwater, suggesting a synergistic response to multiple stressors. These findings suggest that delays in hatchery releases to support release of larger fish need to be carefully scrutinized to ensure fish are not being released as de-smolts, which are highly susceptible to additional climate-induced stressors like rising temperatures and reduced dissolved oxygen levels in the marine environment.

Patterns of selection and allele diversity of class I and class II major histocompatibility loci across the species range of sockeye salmon (Oncorhynchus nerka).

The major histocompatibility complex (MHC), an important component of the vertebrate immune system, provides an important suite of genes to examine the role of genetic diversity at non-neutral loci for population persistence. We contrasted patterns of diversity at the two classical MHC loci in sockeye salmon (Oncorhynchus nerka), MHC class I (UBA) and MHC class II (DAB), and neutral microsatellite loci across 70 populations spanning the species range from Washington State to Japan. There was no correlation in allelic richness or heterozygosity between MHC loci or between MHC loci and microsatellites. The two unlinked MHC loci may be responding to different selective pressures; the distribution of FST values for the two loci was uncorrelated, and evidence for both balancing and directional selection on alleles and lineages of DAB and UBA was observed in populations throughout the species range but rarely on both loci within a population. These results suggest that fluctuating selection has resulted in the divergence of MHC loci in contemporary populations.

Infectious agents and their physiological correlates in early marine Chinook salmon (Oncorhynchus tshawytscha).

The early marine life of Pacific salmon is believed to be a critical period limiting population-level survival. Recent evidence suggests that some infectious agents are associated with survival but linkages with underlying physiological mechanisms are lacking. While challenge studies can demonstrate cause and effect relationships between infection and pathological change or mortality, in some cases pathological change may only manifest in the presence of environmental stressors; thus, it is important to gain context from field observations. Herein, we examined physiological correlates with infectious agent loads in Chinook salmon during their first ocean year. We measured physiology at the molecular (gene expression), metabolic (plasma chemistry) and cellular (histopathology) levels. Of 46 assayed infectious agents, 27 were detected, including viruses, bacteria and parasites. This exploratory study identified.a strong molecular response to viral disease and pathological change consistent with jaundice/anemia associated with Piscine orthoreovirus,strong molecular signals of gill inflammation and immune response associated with gill agents `Candidatus Branchiomonas cysticola' and Parvicapsula pseudobranchicola,a general downregulation of gill immune response associated with Parvicapsula minibicornis complementary to that of P. pseudobranchicola.Importantly, our study provides the first evidence that the molecular activation of viral disease response and the lesions observed during the development of the PRV-related disease jaundice/anemia in farmed Chinook salmon are also observed in wild juvenile Chinook salmon.

In-field genetic stock identification of overwintering coho salmon in the Gulf of Alaska: Evaluation of Nanopore sequencing for remote real-time deployment.

Genetic stock identification (GSI) from genotyping-by-sequencing of single nucleotide polymorphism (SNP) loci has become the gold standard for stock of origin identification in Pacific salmon. The sequencing platforms currently applied require large batch sizes and multiday processing in specialized facilities to perform genotyping by the thousands. However, recent advances in third-generation single-molecule sequencing platforms, such as the Oxford Nanopore minION, provide base calling on portable, pocket-sized sequencers and promise real-time, in-field stock identification of variable batch sizes. Here we evaluate utility and comparability to established GSI platforms of at-sea stock identification of coho salmon (Oncorhynchus kisutch) using targeted SNP amplicon sequencing on the minION platform during a high-sea winter expedition to the Gulf of Alaska. As long read sequencers are not optimized for short amplicons, we concatenate amplicons to increase coverage and throughput. Nanopore sequencing at-sea yielded data sufficient for stock assignment for 50 out of 80 individuals. Nanopore-based SNP calls agreed with Ion Torrent-based genotypes in 83.25%, but assignment of individuals to stock of origin only agreed in 61.5% of individuals, highlighting inherent challenges of Nanopore sequencing, such as resolution of homopolymer tracts and indels. However, poor representation of assayed salmon in the queried baseline data set contributed to poor assignment confidence on both platforms. Future improvements will focus on lowering turnaround time and cost, increasing accuracy and throughput, as well as augmentation of the existing baselines. If successfully implemented, Nanopore sequencing will provide an alternative method to the large-scale laboratory approach by providing mobile small batch genotyping to diverse stakeholders.

Sequence analysis of MHC class I α2 from sockeye salmon (Oncorhynchus nerka).

Most studies assessing adaptive MHC diversity in salmon populations have focused on the classical class II DAB or DAA loci, as these have been most amenable to single PCR amplifications due to their relatively low level of sequence divergence. Herein, we report the characterization of the classical class I UBA α2 locus based on collections taken throughout the species range of sockeye salmon (Oncorhynchus nerka). Through use of multiple lineage-specific primer sets, denaturing gradient gel electrophoresis and sequencing, we identified thirty-four alleles from three highly divergent lineages. Sequence identity between lineages ranged from 30.0% to 56.8% but was relatively high within lineages. Allelic identity within the antigen recognition site (ARS) was greater than for the longer sequence. Global positive selection on UBA was seen at the sequence level (dN:dS = 1.012) with four codons under positive selection and 12 codons under negative selection.

Discovery and surveillance of viruses from salmon in British Columbia using viral immune-response biomarkers, metatranscriptomics, and high-throughput RT-PCR.

The emergence of infectious agents poses a continual economic and environmental challenge to aquaculture production, yet the diversity, abundance, and epidemiology of aquatic viruses are poorly characterised. In this study, we applied salmon host transcriptional biomarkers to identify and select fish in a viral disease state, but only those that were negative for known viruses based on RT-PCR screening. These fish were selected for metatranscriptomic sequencing to discover potential viral pathogens of dead and dying farmed Atlantic (Salmo salar) and Chinook (Oncorhynchus tshawytscha) salmon in British Columbia (BC). We found that the application of the biomarker panel increased the probability of discovering viruses in aquaculture populations. We discovered two viruses that have not previously been characterised in Atlantic salmon farms in BC (Atlantic salmon calicivirus and Cutthroat trout virus-2), as well as partially sequenced three putative novel viruses. To determine the epidemiology of the newly discovered or emerging viruses, we conducted high-throughput reverse transcription polymerase chain reaction (RT-PCR) and screened over 9,000 farmed and wild salmon sampled over one decade. Atlantic salmon calicivirus and Cutthroat trout virus-2 were in more than half of the farmed Atlantic salmon we tested. Importantly we detected some of the viruses we first discovered in farmed Atlantic salmon in Chinook salmon, suggesting a broad host range. Finally, we applied in situ hybridisation to determine infection and found differing cell tropism for each virus tested. Our study demonstrates that continual discovery and surveillance of emerging viruses in these ecologically important salmon will be vital for management of both aquaculture and wild resources in the future.

Aquaculture mediates global transmission of a viral pathogen to wild salmon.

Global expansion of aquaculture and agriculture facilitates disease emergence and catalyzes transmission to sympatric wildlife populations. The health of wild salmon stocks critically concerns Indigenous peoples, commercial and recreational fishers, and the general public. Despite potential impact of viral pathogens such as Piscine orthoreovirus-1 (PRV-1) on endangered wild salmon populations, their epidemiology in wild fish populations remains obscure, as does the role of aquaculture in global and local spread. Our phylogeographic analyses of PRV-1 suggest that development of Atlantic salmon aquaculture facilitated spread from Europe to the North and South East Pacific. Phylogenetic analysis and reverse transcription polymerase chain reaction surveillance further illuminate the circumstances of emergence of PRV-1 in the North East Pacific and provide strong evidence for Atlantic salmon aquaculture as a source of infection in wild Pacific salmon. PRV-1 is now an important infectious agent in critically endangered wild Pacific salmon populations, fueled by aquacultural transmission.

Distribution and Phylogeny of Erythrocytic Necrosis Virus (ENV) in Salmon Suggests Marine Origin.

Viral erythrocytic necrosis (VEN) affects over 20 species of marine and anadromous fishes in the North Atlantic and North Pacific Oceans. However, the distribution and strain variation of its viral causative agent, erythrocytic necrosis virus (ENV), has not been well characterized within Pacific salmon. Here, metatranscriptomic sequencing of Chinook salmon revealed that ENV infecting salmon was closely related to ENV from Pacific herring, with inferred amino-acid sequences from Chinook salmon being 99% identical to those reported for herring. Sequence analysis also revealed 89 protein-encoding sequences attributed to ENV, greatly expanding the amount of genetic information available for this virus. High-throughput PCR of over 19,000 fish showed that ENV is widely distributed in the NE Pacific Ocean and was detected in 12 of 16 tested species, including in 27% of herring, 38% of anchovy, 17% of pollock, and 13% of sand lance. Despite frequent detection in marine fish, ENV prevalence was significantly lower in fish from freshwater (0.03%), as assessed with a generalized linear mixed effects model (p = 5.5 × 10-8). Thus, marine fish are likely a reservoir for the virus. High genetic similarity between ENV obtained from salmon and herring also suggests that transmission between these hosts is likely.

Discovery and validation of candidate smoltification gene expression biomarkers across multiple species and ecotypes of Pacific salmonids.

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.

Endangered wild salmon infected by newly discovered viruses.

The collapse of iconic, keystone populations of sockeye (Oncorhynchus nerka) and Chinook (Oncorhynchus tshawytscha) salmon in the Northeast Pacific is of great concern. It is thought that infectious disease may contribute to declines, but little is known about viruses endemic to Pacific salmon. Metatranscriptomic sequencing and surveillance of dead and moribund cultured Chinook salmon revealed a novel arenavirus, reovirus and nidovirus. Sequencing revealed two different arenavirus variants which each infect wild Chinook and sockeye salmon. In situ hybridisation localised arenavirus mostly to blood cells. Population surveys of >6000 wild juvenile Chinook and sockeye salmon showed divergent distributions of viruses, implying different epidemiological processes. The discovery in dead and dying farmed salmon of previously unrecognised viruses that are also widely distributed in wild salmon, emphasizes the potential role that viral disease may play in the population dynamics of wild fish stocks, and the threat that these viruses may pose to aquaculture.

Molecular indices of viral disease development in wild migrating salmon†.

Infectious diseases can impact the physiological performance of individuals, including their mobility, visual acuity, behavior and tolerance and ability to effectively respond to additional stressors. These physiological effects can influence competitiveness, social hierarchy, habitat usage, migratory behavior and risk to predation, and in some circumstances, viability of populations. While there are multiple means of detecting infectious agents (microscopy, culture, molecular assays), the detection of infectious diseases in wild populations in circumstances where mortality is not observable can be difficult. Moreover, if infection-related physiological compromise leaves individuals vulnerable to predation, it may be rare to observe wildlife in a late stage of disease. Diagnostic technologies designed to diagnose cause of death are not always sensitive enough to detect early stages of disease development in live-sampled organisms. Sensitive technologies that can differentiate agent carrier states from active disease states are required to demonstrate impacts of infectious diseases in wild populations. We present the discovery and validation of salmon host transcriptional biomarkers capable of distinguishing fish in an active viral disease state [viral disease development (VDD)] from those carrying a latent viral infection, and viral versus bacterial disease states. Biomarker discovery was conducted through meta-analysis of published and in-house microarray data, and validation performed on independent datasets including disease challenge studies and farmed salmon diagnosed with various viral, bacterial and parasitic diseases. We demonstrate that the VDD biomarker panel is predictive of disease development across RNA-viral species, salmon species and salmon tissues, and can recognize a viral disease state in wild-migrating salmon. Moreover, we show that there is considerable overlap in the biomarkers resolved in our study in salmon with those based on similar human viral influenza research, suggesting a highly conserved suite of host genes associated with viral disease that may be applicable across a broad range of vertebrate taxa.

Infectious disease, shifting climates, and opportunistic predators: cumulative factors potentially impacting wild salmon declines.

Emerging diseases are impacting animals under high-density culture, yet few studies assess their importance to wild populations. Microparasites selected for enhanced virulence in culture settings should be less successful maintaining infectivity in wild populations, as once the host dies, there are limited opportunities to infect new individuals. Instead, moderately virulent microparasites persisting for long periods across multiple environments are of greatest concern. Evolved resistance to endemic microparasites may reduce susceptibilities, but as barriers to microparasite distributions are weakened, and environments become more stressful, unexposed populations may be impacted and pathogenicity enhanced. We provide an overview of the evolutionary and ecological impacts of infectious diseases in wild salmon and suggest ways in which modern technologies can elucidate the microparasites of greatest potential import. We present four case studies that resolve microparasite impacts on adult salmon migration success, impact of river warming on microparasite replication, and infection status on susceptibility to predation. Future health of wild salmon must be considered in a holistic context that includes the cumulative or synergistic impacts of multiple stressors. These approaches will identify populations at greatest risk, critically needed to manage and potentially ameliorate the shifts in current or future trajectories of wild populations.

The salmonid MHC class I: more ancient loci uncovered.

An unprecedented level of sequence diversity has been maintained in the salmonid major histocompatibility complex (MHC) class I UBA gene, with between lineage AA sequence identities as low as 34%. The derivation of deep allelic lineages may have occurred through interlocus exon shuffling or convergence of ancient loci with the UBA locus, but until recently, no such ancient loci were uncovered. Herein, we document the existence of eight additional MHC class I loci in salmon (UCA, UDA, UEA, UFA, UGA, UHA, ULA, and ZE), six of which share exon 2 and 3 lineages with UBA, and three of which have not been described elsewhere. Half of the UBA exon 2 lineages and all UBA exon 3 lineages are shared with other loci. Two loci, UGA and UEA, share only a single exon lineage with UBA, likely generated through exon shuffling. Based on sequence homologies, we hypothesize that most exchanges and duplications occurred before or during tetraploidization (50 to 100 Ma). Novel loci that share no relationship with other salmonid loci are also identified (UHA and ZE). Each locus is evaluated for its potential to function as a class Ia gene based on gene expression, conserved residues and polymorphism. UBA is the only locus that can indisputably be classified as a class Ia gene, although three of the eight loci (ZE, UCA, and ULA) conform in three out of four measures. We hypothesize that these additional loci are in varying states of degradation to class Ib genes.