Search

Disease and parasitism cause major welfare, environmental and economic concerns for global aquaculture. In this review, we examine the status and potential of technologies that exploit genetic variation in host resistance to tackle this problem. We argue that there is an urgent need to improve understanding of the genetic mechanisms involved, leading to the development of tools that can be applied to boost host resistance and reduce the disease burden. We draw on two pressing global disease problems as case studies—sea lice infestations in salmonids and white spot syndrome in shrimp. We review how the latest genetic technologies can be capitalised upon to determine the mechanisms underlying inter- and intra-species variation in pathogen/parasite resistance, and how the derived knowledge could be applied to boost disease resistance using selective breeding, gene editing and/or with targeted feed treatments and vaccines. Gene editing brings novel opportunities, but also implementation and dissemination challenges, and necessitates new protocols to integrate the technology into aquaculture breeding programmes. There is also an ongoing need to minimise risks of disease agents evolving to overcome genetic improvements to host resistance, and insights from epidemiological and evolutionary models of pathogen infestation in wild and cultured host populations are explored. Ethical issues around the different approaches for achieving genetic resistance are discussed. Application of genetic technologies and approaches has potential to improve fundamental knowledge of mechanisms affecting genetic resistance and provide effective pathways for implementation that could lead to more resistant aquaculture stocks, transforming global aquaculture

Hydrogen peroxide (H2 O2 ) is used to treat sea lice infections of farmed salmonids in the Atlantic and Pacific Oceans and issues with resistance to this treatment, and others are a major threat to the sustainability of the industry. The objectives of this study were to determine how H2 O2 exposure affects survival and antioxidant-related gene expression in salmon lice (Lepeophtheirus salmonis) collected from the Bay of Fundy, New Brunswick. The maximum recommended dose of H2 O2 is 1,800 mg/L, while the EC50 values (with 95% CI) for the population tested were 1,486 (457, 2,515) mg/L for males and 2,126 (984, 3,268) mg/L for females. Neither temperature nor pretreatment with emamectin benzoate (EMB) impacted survival after H2 O2 exposure. RT-qPCR was performed on pre-adult sea lice exposed to H2 O2 and showed that four genes classically involved in the response to oxidative stress were unchanged between treated and control groups. Seven genes were found to be significantly upregulated in males and one in females. This is the first report on the efficacy and molecular responses of Atlantic Canada sea lice to H2 O2 treatment.

Infectious diseases are key drivers of wildlife populations and agriculture production, but whether and how climate change will influence disease impacts remains controversial. One of the critical knowledge gaps that prevents resolution of this controversy is a lack of high-quality experimental data, especially in marine systems of significant ecological and economic consequence. Here, we performed a manipulative experiment in which we tested the temperature-dependent effects on Atlantic salmon (Salmo salar) of sea lice (Lepeophtheirus salmonis)—a parasite that can depress the productivity of wild-salmon populations and the profits of the salmon-farming industry. We explored sea-louse impacts on their hosts across a range of temperatures (10, 13, 16, 19, and 22 °C) and infestation levels (zero, ‘low’ (mean abundance ± SE = 1.6 ± 0.1 lice per fish), and ‘high’ infestation (6.8 ± 0.4 lice per fish)). We found that the effects of sea lice on the growth rate, condition, and survival of juvenile Atlantic salmon all worsen with increasing temperature. Our results provide a rare empirical example of how climate change may influence the impacts of marine disease in a key social-ecological system. These findings underscore the importance of considering climate-driven changes to disease impacts in wildlife conservation and agriculture.

Treatment of infestation by the ectoparasite Lepeophtheirus salmonis relies on a small number of chemotherapeutant treatments that currently meet with limited success. Drugs targeting chitin synthesis have been largely successful against terrestrial parasites where the pathway is well characterised. However, a comparable approach against salmon lice has been, until recently, less successful, likely due to a poor understanding of the chitin synthesis pathway. Post-transcriptional silencing of genes by RNA interference (RNAi) is a powerful method for evaluation of protein function in non-model organisms and has been successfully applied to the salmon louse. In the present study, putative genes coding for enzymes involved in L. salmonis chitin synthesis were characterised after knockdown by RNAi. Nauplii I stage L. salmonis were exposed to double-stranded (ds) RNA specific for several putative non-redundant points in the pathway: glutamine: fructose-6-phosphate aminotransferase (LsGFAT), UDP-N-acetylglucosamine pyrophosphorylase (LsUAP), N-acetylglucosamine phosphate mutase (LsAGM), chitin synthase 1 (LsCHS1), and chitin synthase 2 (LsCHS2). Additionally, we targeted three putative chitin deacetylases (LsCDA4557, 5169 and 5956) by knockdown. Successful knockdown was determined after moulting to the copepodite stage by real-time quantitative PCR (RT-qPCR), while infectivity potential (the number of attached chalimus II compared with the initial number of larvae in the system) was measured after exposure to Atlantic salmon and subsequent development on their host. Compared with controls, infectivity potential was not compromised in dsAGM, dsCHS2, dsCDA4557, or dsCDA5169 groups. In contrast, there was a significant effect in the dsUAP-treated group. However, of most interest was the treatment with dsGFAT, dsCHS1, dsCHS1+2, and dsCDA5956, which resulted in complete abrogation of infectivity, despite apparent compensatory mechanisms in the chitin synthesis pathway as detected by qPCR. There appeared to be a common phenotypic effect in these groups, characterised by significant aberrations in appendage morphology and an inability to swim. Ultrastructurally, dsGFAT showed a significantly distorted procuticle without distinct exo/endocuticle and intermittent electron dense (i.e. chitin) inclusions, and together with dsUAP and dsCHS1, indicated delayed entry to the pre-moult phase. publishedVersion

This study was conducted to determine the effects of a co-infection with Moritella viscosa at different exposure levels of sea lice Lepeophtheirus salmonis in Atlantic salmon (Salmo salar). M. viscosa (1.14 × 106 cfu/ml) was introduced to all experimental tanks at 10 days post-lice infection (dpLs). Mean lice counts decreased over time in both the medium lice co-infection (31.5 ± 19.0 at 7 dpLs; 16.9 ± 9.3 at 46 dpLs) and high lice co-infection (62.0 ± 10.8 at 7 dpLs; 37.6 ± 11.3 at 46 dpLs). There were significantly higher mortalities and more severe skin lesions in the high lice co-infected group compared to medium lice co-infected group or M. viscosa-only infection. Quantitative gene expression analysis detected a significant upregulation of genes in skin from the high lice co-infection group consistent with severe inflammation (il-8, mmp-9, hep, saa). Skin lesions retrieved throughout the study were positive for M. viscosa growth, but these were rarely located in regions associated with lice. These results suggest that while M. viscosa infection itself may induce skin lesion development in salmon, co-infection with high numbers of lice can enhance this impact and significantly reduce the ability of these lesions to resolve, resulting in increased mortality.

Moritella viscosa is a Gram-negative pathogen that causes large, chronic ulcers, known as winter-ulcer disease, in the skin of several fish species including Atlantic salmon. We used a bath challenge approach to profile the transcriptome responses of M. viscosa-infected Atlantic salmon skin at the lesion (Mv-At) and away from the lesion (Mv-Aw) sites. M. viscosa infection was confirmed through RNA-based qPCR assays. RNA-Seq identified 5212 and 2911 transcripts differentially expressed in the Mv-At compared to no-infection control and Mv-Aw groups, respectively. Also, there were 563 differentially expressed transcripts when comparing the Mv-Aw to control samples. Our results suggest that M. viscosa caused massive and strong, but largely infection site-focused, transcriptome dysregulations in Atlantic salmon skin, and its effects beyond the skin lesion site were comparably subtle. The M. viscosa-induced transcripts of Atlantic salmon were mainly involved in innate and adaptive immune response-related pathways, whereas the suppressed transcripts by this pathogen were largely connected to developmental and cellular processes. As validated by qPCR, M. viscosa dysregulated transcripts encoding receptors, signal transducers, transcription factors and immune effectors playing roles in TLR- and IFN-dependent pathways as well as immunoregulation, antigen presentation and T-cell development. This study broadened the current understanding of molecular pathways underlying M. viscosa-triggered responses of Atlantic salmon, and identified biomarkers that may assist to diagnose and combat this pathogen.

The objective of this study was to estimate the impact of infestation pressures on the abundance of the parasitic sea louse, Lepeophtheirus salmonis, in the Bay of Fundy, New Brunswick (NB), Canada, using the Fish-iTrends database for the years 2009-2018. Infestation pressures were calculated as time-lagged weighted averages of the abundance of adult female (AF) sea lice within a site (internal infestation pressure: IIP) and among sites (external infestation pressure: EIP). The EIP weights were calculated from seaway distances among sites and a Gaussian kernel density for bandwidths of 5 to 60 km. The EIP with a bandwidth of 10 km had the best fit, as determined with Akaike's information criterion, and historical AF sea lice abundance. This estimated dispersal distance of 10 km was similar to previous studies in Norway, Scotland and in New Brunswick. The infestation pressures estimated from empirical AF sea lice abundance within and among sites significantly increased the abundance of AF sea lice (p

Systemic phaeohyphomycosis caused by Veronaea botryosa is one of the most important emergent diseases to affect sturgeon aquaculture in North America. White sturgeon (Acipenser transmontanus) cultured at temperatures above 15 °C are at higher risk of severe disseminated disease and higher mortalities. Despite this, little is known regarding disease pathogenesis and the immune response to infection. The objective of this study was to investigate the acute (2 days post-challenge [dpc]) and chronic (32 dpc) response of white sturgeon at 13 °C and 18 °C challenged with V. botryosa via intramuscular injection, using gene expression analysis of a diverse array of soluble immune and inflammatory mediators. Significantly greater amounts of irf8 (p 0.05) and tfg-β (p 0.05) genes were detected in gills of exposed fish at 18 °C when compared to those at 13 °C 32 dpc. Transcript levels of haptoglobin, serotransferrin, serum amyloid, cathelicidin, tnf-α, and il-17 were significantly increased in splenic tissues of challenged fish maintained at 18 °C late in infection (p 0.05). However, only haptoglobin and serotransferrin transcript abundance were significantly greater in exposed fish when compared to controls 32dpc. Moreover, haptoglobin transcripts at this time point were significantly greater in exposed fish at 18 °C when compared to those challenged at 13 °C. Fewer differences were detected in fish kept at 13 °C. In agreement with transcript quantification, western blot assessment of haptoglobin showed increased levels in the challenged fish maintained at 18 °C.

The salmon louse Lepeophtheirus salmonis (Krøyer 1837) displays numerous sexually dimorphic characteristics. Insights into their underlying molecular components have only recently been explored, which serve to better understand both the basic biology of the louse, associated impacts on drug sensitivity, and evolution of resistance. Expression of 16 L. salmonis genes putatively involved in sexual dimorphism and reproduction were used to determine differences between sexes and better understand responses to mating using RT-qPCR of pre-adult and adult lice. Analysis of these genes revealed the dynamic nature of sex-biased expression across stages. However, female reception of a spermatophore did not appear to impact the expression of these particular genes. Furthermore six of these transcripts and 84 others identified previously in a large-scale louse transcriptomics experiment were used to estimate differences in evolutionary rates and codon-usage bias of sex-related genes using phylogenetic analysis by maximum likelihood (PAML) and codonW. Results suggest male-biased genes are evolving at significantly greater rates than female-biased and unbiased genes as evidenced by higher rates of non-synonymous substitutions and lower codon-usage bias in these genes. These analyses expand our understanding of interactions of sex-biased expression across the pre-adult and adult life stages and provide foundations for better understanding evolutionary differences in sex-biased genes of L. salmonis.

Skin ulcers in Atlantic salmon Salmo salar in the Canadian east coast salmon aquaculture industry lead to high mortality rates. This condition is clinically similar to winter ulcer disease in Norway with the exception that it occurs at temperatures above 10°C. Moritella viscosa is thought to be the causative agent for winter ulcer disease in Norway, and it is occasionally also isolated from skin ulcer cases in Atlantic Canada. This bacterium is known to produce cytotoxins. The objective of this study was to determine if extracellular products (ECP) from an Atlantic Canadian strain of M. viscosa could induce a tissue response similar to what is observed with M. viscosa infections in Atlantic salmon in eastern Canada. We injected fish subcutaneously with ECP and monitored the development of skin lesions. We sampled fish with early skin lesions and ulcers to describe the pathology associated with the condition. Samples were taken for histopathology, bacterial culture, and quantitative PCR (qPCR). All experimental fish expressed early skin lesions, with 5 fish (8.3%) developing deep skin ulcers after 12 d post-exposure. Our results suggest the ECP of M. viscosa from the east coast of Canada induces a similar tissue response to what is described in ulcer disease in Atlantic salmon. These extracelluar products may partially explain the pathology associated with M. viscosa.

The avermectins, emamectin benzoate (EMB) and ivermectin (IVM) have been commonly used in North America over the last two decades to control the salmon louse, Lepeophtheirus salmonis, infections in farmed Atlantic salmon. Emamectin benzoate, trade name SLICE ™, was used heavily in the Eastern Canadian industry between the years 2000–2008, due to its long lasting protection and efficacy against all parasitic life stages. However, over reliance on this drug soon resulted in reduced sensitivity in many L. salmonis populations, resulting more recently in uses of higher treatment dosages and switching to the use of IVM. For these reasons, we investigated the effects of different dosages of EMB and multiple IVM treatments on baseline immunophysiological indicators, anti-viral responses and protection against subsequent salmon lice exposure in salmon smolts. Different doses of EMB or repeated treatment with IVM did not affect feeding behaviour in salmon, however by the end of the second IVM treatment, some neurotoxicity was observed. A single (1×) EMB dose (50 μg/Kg) administered for 7 consecutive days had no significant effect on L. salmonis abundance and development, whereas triple the dosage (150 μg/Kg) significantly reduced lice development, thereby eliminating subsequent stress responses in salmon associated with lice development to pre-adult stages. Emamectin benzoate and IVM treatment did not significantly impact expression of resting antigen presentation molecules in salmon (MH class I or II), however they did inhibit short-term (6 h) induced responses to the ISA virus. The impact of gender, as previously shown, had the greatest effect on louse transcriptomic regulation, but avermectin treatment also caused perturbations in gene expression. Transcriptome differences between lice on control and 1× EMB treated fish were larger than those observed for IVM or 3× EMB. Nearly half of the transcripts differentially expressed by IVM were also affected by one of the EMB treatments. Transcriptomic results from the louse suggest a high degree of similarity and concordance within and across studies in avermectin treatment, with gender of louse and dosage of drug significantly impacting the outcomes.

The salmon louse Lepeophtheirus salmonis (Lsal) is an ectoparasitic copepod that exerts immunomodulatory and physiological effects on its host Atlantic salmon. Over 30 years of research on louse biology, control, host responses and the host-parasite relationship has provided a plethora of information on the intricacies of host resistance and parasite adaptation. Atlantic salmon exhibit temporal and spatial impairment of the immune system and wound healing ability during infection. This immunosuppression may render Atlantic salmon less tolerant to stress and other confounders associated with current management strategies. Contrasting susceptibility of salmonid hosts exists, and early pro-inflammatory Th1 type responses are associated with resistance. Rapid cellular responses to larvae appear to tip the balance of the host-parasite relationship in favour of the host, preventing severe immune-physiological impacts of the more invasive adults. Immunological, transcriptomic, genomic and proteomic evidence suggests pathological impacts occur in susceptible hosts through modulation of host immunity and physiology via pharmacologically active molecules. Co-evolutionary and farming selection pressures may have incurred preference of Atlantic salmon as a host for Lsal reflected in their interactome. Here, we review host-parasite interactions at the primary attachment/feeding site, and the complex life stage-dependent molecular mechanisms employed to subvert host physiology and immune responses.

Parasitic sea lice (e.g., Lepeophtheirus salmonis) cause costly outbreaks in salmon farming. Molecular insights into parasite-induced host responses will provide the basis for improved management strategies. We investigated the early transcriptomic responses in pelvic fins of Atlantic salmon parasitized with chalimus I stage sea lice. Fin samples collected from non-infected (i.e. pre-infected) control (PRE) and at chalimus-attachment sites (ATT) and adjacent to chalimus-attachment sites (ADJ) from infected fish were used in profiling global gene expression using 44 K microarrays. We identified 6568 differentially expressed probes (DEPs, FDR &lt, 5%) that included 1928 shared DEPs between ATT and ADJ compared to PRE. The ATT versus ADJ comparison revealed 90 DEPs, all of which were upregulated in ATT samples. Gene ontology/pathway term network analyses revealed profound changes in physiological processes, including extracellular matrix (ECM) degradation, tissue repair/remodeling and wound healing, immunity and defense, chemotaxis and signaling, antiviral response, and redox homeostasis in infected fins. The QPCR analysis of 37 microarray-identified transcripts representing these functional themes served to confirm the microarray results with a significant positive correlation (p &lt, 0.0001). Most immune/defense-relevant transcripts were downregulated in both ATT and ADJ sites compared to PRE, suggesting that chalimus exerts immunosuppressive effects in the salmon&rsquo, s fins. The comparison between ATT and ADJ sites demonstrated the upregulation of a suite of immune-relevant transcripts, evidencing the salmon&rsquo, s attempt to mount an anti-lice response. We hypothesize that an imbalance between immunomodulation caused by chalimus during the early phase of infection and weak defense response manifested by Atlantic salmon makes it a susceptible host for L. salmonis.

Drug resistance in the salmon louse Lepeophtheirus salmonis is a global issue for Atlantic salmon aquaculture. Multiple resistance has been described across most available compound classes with the exception of the benzoylureas. To target this gap in effective management of L. salmonis and other species of sea lice (e.g. Caligus spp.), Elanco Animal Health is developing an in-feed treatment containing lufenuron (a benzoylurea) to be administered prior to seawater transfer of salmon smolts and to provide long-term protection of salmon against sea lice infestations. Benzoylureas disrupt chitin synthesis, formation, and deposition during all moulting events. However, the mechanism(s) of action are not yet fully understood and most research completed to date has focused on insects. We exposed the first parasitic stage of L. salmonis to 700 ppb lufenuron for three hours and observed over 90% reduction in survival to the chalimus II life stage on the host, as compared to vehicle controls. This agrees with a follow up in vivo administration study on the host, which showed >95% reduction by the chalimus I stage. Transcriptomic responses of salmon lice exposed to lufenuron included genes related to moulting, epithelial differentiation, solute transport, and general developmental processes. Global metabolite profiles also suggest that membrane stability and fluidity is impacted in treated lice. These molecular signals are likely the underpinnings of an abnormal moulting process and cuticle formation observed ultrastructurally using transmission electron microscopy. Treated nauplii-staged lice exhibited multiple abnormalities in the integument, suggesting that the coordinated assembly of the epi- and procuticle is impaired. In all cases, treatment with lufenuron had rapid impacts on L. salmonis development. We describe multiple experiments to characterize the efficacy of lufenuron on eggs, larvae, and parasitic stages of L. salmonis, and provide the most comprehensive assessment of the physiological responses of a marine arthropod to a benzoylurea chemical., Graphical abstract Image 1, Highlights • Lufenuron is a new treatment for sea lice control in salmon aquaculture. • A 96% reduction in sea lice was observed in salmon fed lufenuron. • Salmon lice exposed to lufenuron have deformed cuticles. • Transcriptomics and metabolomics suggest that lufenuron inhibits chitin transport, organization, and/or abundance. • Novel genes related to sea lice development were discovered.

Background Microsporidia are highly specialized, parasitic fungi that infect a wide range of eukaryotic hosts from all major taxa. Infections cause a variety of damaging effects on host physiology from increased stress to death. The microsporidian Facilispora margolisi infects the Pacific salmon louse (Lepeophtheirus salmonis oncorhynchi), an economically and ecologically important ectoparasitic copepod that can impact wild and cultured salmonids. Results Vertical transmission of F. margolisi was demonstrated by using PCR and in situ hybridization to identify and localize microsporidia in female L. salmonis and their offspring. Spores and developmental structures of F. margolisi were identified in 77% of F1 generation copepods derived from infected females while offspring from uninfected females all tested negative for the microsporidia. The transcriptomic response of the salmon louse to F. margolisi was profiled at both the copepodid larval stage and the pre-adult stage using microarray technology. Infected copepodids differentially expressed 577 transcripts related to stress, ATP generation and structural components of muscle and cuticle. The infection also impacted the response of the copepodid to the parasiticide emamectin benzoate (EMB) at a low dose of 1.0 ppb for 24 h. A set of 48 transcripts putatively involved in feeding and host immunomodulation were up to 8-fold underexpressed in the F. margolisi infected copepodids treated with EMB compared with controls or either stressor alone. Additionally, these infected lice treated with EMB also overexpressed 101 transcripts involved in stress resistance and signalling compared to the other groups. In contrast, infected pre-adult lice did not display a stress response, suggesting a decrease in microsporidian virulence associated with lice maturity. Furthermore, copepodid infectivity and moulting was not affected by the microsporidian infection. Conclusions This study demonstrated that F. margolisi is transmitted vertically between salmon louse generations and that biological impacts of infection differ depending on the stage of the copepod host. The infection caused significant perturbations of larval transcriptomes and therefore must be considered in future studies in which impacts to host development and environmental factors are assessed. Fitness impacts are probably minor, although the interaction between pesticide exposure and microsporidian infection merits further study. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4040-8) contains supplementary material, which is available to authorized users.

Salmon lice ( Lepeophtheirus salmonis) are important ectoparasites of wild and farmed salmonids and cause major losses to the salmon farming industry throughout the Northern Hemisphere. With the emergence of resistance to several commonly used parasiticides, novel control strategies and integration of multiple treatment options are needed, including host immunostimulation. Here, we investigate the effects of a functional feed containing a peptidoglycan and nucleotide formulation on L. salmonis infection of Atlantic salmon ( Salmo salar) by characterizing lice infection levels, the expression of several host immune genes, and the parasite transcriptomic response to the immunostimulated host. Although initial infection intensities were low, the low dose (LD) immunostimulant diet reduced the total lice burden by 50% relative to controls. Immunostimulant fed hosts up-regulated interleukin-1β in the skin and spleen. This gene has been implicated in successful responses of several salmonid species to salmon lice but is typically not observed in Atlantic salmon, suggesting a favorable influence on the immune response. Lice infecting LD immunostimulated salmon overexpressed genes putatively involved in parasite immunity, including carboxylesterases, and underexpressed genes putatively involved in feeding (e.g., proteases). These lice response genes further improve the characterization of the transcriptome of the non-model parasite by identifying genes potentially involved in evading host immunity.

Syngnathidae (seahorses, seadragons and pipefish) suffer significant losses from non-tuberculous mycobacteria. However, they produce markedly different lesions in response to the disease compared to other teleost species, notably infrequent granuloma formation. This study evaluated 270 syngnathid fish, from which 92 were diagnosed with mycobacteriosis by histopathology, culture or both. Microscopic lesions variably consisted of random foci of coagulative necrosis in multiple organs, containing high numbers of free bacteria and large aggregates or sheets of macrophages with cytoplasm laden with acid-fast bacilli. Mycobacterial associated granulomas were identified in only six seahorses. Five fish had positive cultures with no observed microscopic changes. RNA-seq of the head kidney was performed to investigate the transcriptome of two infected and six non-infected lined seahorses Hippocampus erectus. Assembled and annotated putative transcripts serve to enrich the database for this species, as well as provide baseline data for understanding the pathogenesis of mycobacteriosis in seahorses. Putative components of the innate immune system (IL-1β, IL-6, TNF, NOS, Toll-like receptor 1, MHC Class I, NF-κβ, transforming growth factor beta, MyD88) were identified in the RNA-seq data set. However, a homolog for a key component in the TH1 adaptive immune response, interferon-gamma, was not identified and may underlie the unique pathologic presentation.

AquAdvantage Salmon (growth hormone transgenic female triploid Atlantic salmon) are a faster-growing alternative to conventional farmed diploid Atlantic salmon. To investigate optimal rearing conditions for their commercial production, a laboratory study was conducted in a freshwater recirculating aquaculture system (RAS) to examine the effect of rearing temperature (10.5 °C, 13.5 °C, 16.5 °C) on their antiviral immune and stress responses. When each temperature treatment group reached an average weight of 800 g, a subset of fish were intraperitoneally injected with either polyriboinosinic polyribocytidylic acid (pIC, a viral mimic) or an equal volume of sterile phosphate-buffered saline (PBS). Blood and head kidney samples were collected before injection and 6, 24 and 48 h post-injection (hpi). Transcript abundance of 7 antiviral biomarker genes (tlr3, lgp2, stat1b, isg15a, rsad2, mxb, ifng) was measured by real-time quantitative polymerase chain reaction (qPCR) on head kidney RNA samples. Plasma cortisol levels from blood samples collected pre-injection and from pIC and PBS groups at 24 hpi were quantified by ELISA. While rearing temperature and treatment did not significantly affect circulating cortisol, all genes tested were significantly upregulated by pIC at all three temperatures (except for tlr3, which was only upregulated in the 10.5 °C treatment). Target gene activation was generally observed at 24 hpi, with most transcript levels decreasing by 48 hpi in pIC-injected fish. Although a high amount of biological variability in response to pIC was evident across all treatments, rearing temperature significantly influenced transcript abundance and/or fold-changes comparing time- and temperature-matched pIC- and PBS-injected fish for several genes (tlr3, lgp2, stat1b, isg15a, rsad2 and ifng) at 24 hpi. As an example, significantly higher fold-changes of rsad2, isg15a and ifng were found in fish reared at 10.5 °C when compared to 16.5 °C. Multivariate analysis confirmed that rearing temperature modulated antiviral immune response. The present experiment provides novel insight into the relationship between rearing temperature and innate antiviral immune response in AquAdvantage Salmon.

With respect to salmonid aquaculture, one of the most important bacterial pathogens due to high mortality and antibiotic usage is the causative agent of typical furunculosis, Aeromonas salmonicida spp. salmonicida (Asal). In Atlantic salmon, Salmo salar, the host response during infections with Asal is well-documented, with furunculosis outbreaks resulting in significant mortality in commercial settings. However, less is known about the host-pathogen interactions in the emerging aquaculture species, Arctic charr Salvelinus alpinus. Furthermore, there is no data on the efficacy or response of this species after vaccination with commonly administered vaccines against furunculosis. To this end, we examined the immunological response of S. alpinus during infection with Asal, with or without administration of vaccines (Forte Micro®, Forte Micro® + Renogen®, Elanco Animal Health). Artic charr (vaccinated or unvaccinated) were i.p.-injected with a virulent strain of Asal (106 CFUs/mL) and tissues were collected pre-infection/post-vaccination, 8, and 29 days post-infection. Unvaccinated Arctic charr were susceptible to Asal with 72% mortalities observed after 31 days. However, there was 72–82% protection in fish vaccinated with either the single or dual-vaccine, respectively. Protection in vaccinated fish was concordant with significantly higher serum IgM concentrations, and following RNA sequencing and transcriptome assembly, differential expression analysis revealed several patterns and pathways associated with the improved survival of vaccinated fish. Most striking was the dramatically higher basal expression of complement/coagulation factors, acute phase-proteins, and iron hemostasis proteins in pre-challenged, vaccinated fish. Remarkably, following Asal infection, this response was abrogated and instead the transcriptome was characterized by a lack of immune-stimulation compared to that of unvaccinated fish. Furthermore, where pathways of actin assembly and FcγR-mediated phagocytosis were significantly differentially regulated in unvaccinated fish, vaccinated fish showed either the opposite regulation (ForteMicro®), or no impact at all (ForteMicro®Renogen®). The present data indicates that vaccine-induced protection against Asal relies on the pre-activation and immediate control of humoral immune parameters that is coincident with reduced activation of apoptotic (e.g., NF-κB) and actin-associated pathways.

The role of parasitic sea lice (Siphonostomatoida; Caligidae), especially Lepeophtheirus salmonis, in the epidemiology of Infectious Salmon Anemia Virus (ISAv) has long been suspected. The epidemiological studies conducted during the 1998 major Infectious Salmon Anaemia (ISA) outbreak in Scotland demonstrated a strong correlation between sea lice presence and ISAv positive sites or subsequent clinical outbreaks of ISA. The question posed from this observation was "do sea lice infestations on Atlantic salmon make them more susceptible to viral infections?" This study investigated the role that sea lice infestations have on the severity of ISAv infections and disease mortality in experimental populations of farmed Atlantic salmon (Salmo salar). A series of experiments was carried out that investigated the potential of sea lice to modify the outcome of an ISAv infection. Experimental populations of Atlantic salmon were established that had: no lice and no ISAv, a single infection with either ISAv or lice and a co-infection with lice then ISAV. The results were quite clear, the process of infestation by the parasite prior to ISAv exposure significantly increased the mortality and death rates of Atlantic salmon, when compared to uninfected controls and ISAv infected groups only. This was consistent over two source strains of Atlantic salmon (Pennobscot and Saint John River), but the severity and timing was altered. Immunological responses were also consistent in that pro-inflammatory genes were induced in lice only and co-infected fish, whereas the anti-viral response, Mx, MH class I β, Galectin 9 and TRIM 16, 25 genes were down-regulated by lice infection prior to and shortly after co-infection with ISAv. It is concluded that the sea lice settlement on Atlantic salmon and the parasite's subsequent manipulation of the host's immune system, which increases parasite settlement success, also increased susceptibility to ISAv.

Increasing usage of non-medicinal methods (NMMs) to control sea louse infestations on salmon farms has raised questions about whether sea lice may be able to evolve tolerance of NMMs. Of particular concern is the potential for sea lice to evolve freshwater tolerance as a result of freshwater treatments. Wild trout and some juvenile salmonids swim into freshwater to control infestations and regain ionic balance after disruption by sea lice; freshwater tolerance would compromise this potentially adaptive behavior. Here we evaluated the potential for freshwater tolerance to evolve in the sea louse Lepeophtheirus salmonis. When exposed to low-salinity water, parasitic stages of sea lice are able to osmoregulate through the host, while larval planktonic stages are not. Transcriptomic work suggests that sea lice mount a costly polygenic stress response when exposed to brackish water. The population structure of sea lice is panmictic in both the Pacific and Atlantic, making it conducive to rapid evolutionary responses. It is unknown how much heritable genetic variation these panmictic populations have for freshwater treatments. While usage of freshwater treatments on wellboats is increasing, it is unclear whether the freshwater itself is a strong selective force; during the freshwater exposure, sea lice can die from physical disruption during pumping and filtration on the wellboat. Future studies are advised to quantify the heritable variation in freshwater tolerance in sea louse populations, characterize mechanisms for freshwater tolerance in planktonic and attached sea lice, and assess the risk of freshwater tolerance evolution under different management strategies Evaluating the potential for sea lice to evolve freshwater tolerance as a consequence of freshwater treatments in salmon aquaculture

A salmon louse (Lepeophtheirus salmonis salmonis) genetic linkage map was constructed to serve as a genomic resource for future investigations into the biology of this important marine parasitic copepod species, and to provide insights into the inheritance patterns of genetic markers in this species. SNP genotyping of 8 families confirmed the presence of 15 linkage groups based upon the assignment of 93,773 markers. Progeny sample size weight adjusted map sizes in males (with the exception of SL12 and SL15) ranged in size from 96.50 cM (SL11) to 134.61 cM (SL06), and total combined map steps or bins ranged from 143 (SL09) to 203 (SL13). The SL12 male map was the smallest linkage group with a weight-averaged size of 3.05 cM with 6 recombination bins. Male:female specific recombination rate differences are 10.49:1 and represent one of the largest reported sex-specific differences for any animal species. Recombination ratio differences (M:F) ranged from 1.0 (SL12) to 29:1 (SL15). The number of markers exhibiting normal Mendelian segregation within the sex linkage group SL15 was extremely low (N = 80) in comparison to other linkage groups genotyped [range: 1459 (SL12)-10206 markers (SL05)]. Re-evaluation of Mendelian inheritance patterns of markers unassigned to any mapping parent according to hemizygous segregation patterns (models presented) identified matches for many of these markers to hemizygous patterns. The greatest proportion of these markers assigned to SL15 (N increased to 574). Inclusion of the hemizygous markers revised SL15 sex-specific recombination rate differences to 28:1. Recombination hot- and coldspots were identified across all linkage groups with all linkage groups possessing multiple peaks. Nine of 13 linkage groups evaluated possessed adjacent domains with hot-coldspot transitional zones. The most common pattern was for one end of the linkage to show elevated recombination in addition to internal regions. For SL01 and SL06, however, a terminal region with high recombination was not evident while a central domain possessing extremely high-recombination levels was present. High levels of recombination were weakly coupled to higher levels of SNP variation within domains, but this association was very strong for the central domains of SL01 and SL06. From the pooled paternal half-sib lots (several virgin females placed with 1 male), only 1 or two surviving family lots were obtained. Surviving families possessed parents where both the male and female possessed either inherently low or high recombination rates. This study provides insight into the organization of the sea louse genome, and describes large differences in recombination rate that exist among individuals of the same sex, and between the sexes. These differences in recombination rate may be coupled to the capabilities of this species to adapt to environmental and pharmaceutical treatments, given that family survivorship appears to be enhanced when parents have similar recombination levels.

MicroRNAs (miRNAs) are key regulators in fish immune responses. However, no study has previously characterized the impact of polyriboinosinic polyribocytidylic acid (pIC) and formalin-killed typical Aeromonas salmonicida (ASAL) on miRNA expression in Atlantic salmon fed a commercial diet with and without immunostimulant CpG. To this end, first, we performed small RNA deep sequencing and qPCR analyses to identify and confirm pIC- and/or ASAL-responsive miRNAs in the head kidney of salmon fed a control diet. DESeq2 analyses identified 12 and 18 miRNAs differentially expressed in pIC and ASAL groups, respectively, compared to the controls. Fifteen of these miRNAs were studied by qPCR, nine remained significant by qPCR. Five miRNAs (miR-27d-1-2-5p, miR-29b-2-5p, miR-146a-5p, miR-146a-1-2-3p, miR-221-5p) were shown by qPCR to be significantly induced by both pIC and ASAL. Second, the effect of CpG-containing functional feed on miRNA expression was investigated by qPCR. In pre-injection samples, 6 of 15 miRNAs (e.g., miR-181a-5-3p, miR-462a-3p, miR-722-3p) had significantly lower expression in fish fed CpG diet than control diet. In contrast, several miRNAs (e.g., miR-146a-1-2-3p, miR-192a-5p, miR-194a-5p) in the PBS- and ASAL-injected groups had significantly higher expression in CpG-fed fish. Multivariate statistical analyses confirmed that the CpG diet had a greater impact on miRNA expression in ASAL-injected compared with pIC-injected fish. This study identified immune-relevant miRNA biomarkers that will be valuable in the development of diets to combat infectious diseases of salmon.

Cypermethrin has been administered for decades to control salmon lice (Lepeophtheirus salmonis) infestations in Atlantic salmon farming regions globally. However, resistance to cypermethrin and other available therapeutants has threatened the sustainability of this growing industry. To better understand the effects of cypermethrin on L. salmonis, a 38K oligonucleotide microarray and RT-qPCR analyses were applied to pools of copepodid larvae exposed to 1.0ppb cypermethrin or seawater controls for 24h. Phenotypic assessments and global gene expression profiles showed a significant disruption of homeostasis in copepodid L. salmonis exposed to cypermethrin. Multiple degradative enzymes were overexpressed in cypermethrin-treated lice including five trypsin-like serine proteases and three cytochrome p450s CYP3a24 (p=0.03, fold change (FC)=3.8; GenBank accession no. JP326960.1), CYP6w1 (p=0.008, FC=5.3; GenBank accession no. JP317875.1), and CYP6d4 (p=0.01; FC=7.9; GenBank accession no. JP334550.1). These enzymes represent preliminary markers for understanding the physiological response of L. salmonis to cypermethrin exposure. A general stress response was also observed in cypermethrin-treated lice which included differential expression of cell signaling genes involved in the induction of cell growth, solute transport, and metabolism. Lastly, a consensus-based analysis was completed with two previously published L. salmonis transcriptome studies revealing genes that respond to cypermethrin, emamectin benzoate (another delousing agent) and hyposalinity. This included concordant differential expression of heat shock beta-1, ammonium transporter Rh types B, and 72kDa type IV collagenase across different L. salmonis studies. This is currently the most comprehensive transcriptome assessment of chemical exposure on the first infectious stage of L. salmonis, providing novel markers for studying drug resistance and general stress in this important parasite.

To survive in changing environments fish utilize a wide range of biological responses that require energy. We examined the effect of warm acclimation on the electron transport system (ETS) enzymes and transcriptional responses to hypoxia and copper (Cu) exposure in fish. Rainbow trout (Oncorhynchus mykiss) were acclimated to cold (11 °C; control) and warm (20 °C) temperatures for 3 weeks followed by exposure to Cu, hypoxia or both for 24 h. Activities of ETS enzyme complexes I-IV (CI-CIV) were measured in liver and gill mitochondria. Analyses of transcripts encoding for proteins involved in mitochondrial respiration (cytochrome c oxidase subunits 4-1 and 2: COX4-1 and COX4-2), metal detoxification/stress response (metallothioneins A and B: MT-A and MT-B) and energy sensing (AMP-activated protein kinase α1: AMPKα1) were done in liver mitochondria, and in whole liver and gill tissues by RT-qPCR. Warm acclimation inhibited activities of ETS enzymes while effects of Cu and hypoxia depended on the enzyme and thermal acclimation status. The genes encoding for COX4-1, COX4-2, MT-A, MT-B and AMPKα1 were strongly and tissue-dependently altered by warm acclimation. While Cu and hypoxia clearly increased MT-A and MT-B transcript levels in all tissues, their effects on COX4-1, COX4-2 and AMPKα1 mRNA levels were less pronounced. Importantly, warm acclimation differentially altered COX4-2/COX4-1 ratio in liver mitochondria and gill tissue. The three stressors showed both independent and joint actions on activities of ETS enzymes and transcription of genes involved in energy metabolism, stress response and metals homeostasis. Overall, we unveiled novel interactive effects that should not be overlooked in real world situations wherein fish normally encounter multiple stress factors.

Arctic charr (Salvelinus alpinus) farming is an emerging component in today's aquaculture sector; however, intensive aquaculture sites provide optimal conditions for disease. Although vaccines have been developed and tested for other major cultured salmonids, such as Atlantic salmon (Salmo salar), it is unknown if such vaccines provide protection in Arctic charr. Therefore, the present study aimed to evaluate the efficacy of two vaccines, Forte Micro® and Renogen®, administered solely or in combination, in the prevention of atypical furunculosis in Arctic charr after challenge with atypical Aeromonas salmonicida (RPC strain FFA-43). Furthermore, vaccination efficacy was assessed after short or long post-vaccination periods prior to bacterial challenge. Improved survival was observed in charr vaccinated with both single and combined vaccines in the short-term efficacy study, and this positively correlated with increased circulating IgM specific for atypical A. salmonicida. However, circulating IgM was reduced in the long-term study. Forte Micro® provided improved protection when Arctic charr were exposed to infection in the short-term vaccine efficacy compared to the long-term vaccine efficacy, where no significant protection was observed. Gene expression analysis revealed upregulation of complement, acute phase, and cellular signalling post-vaccination and post-challenge for fish in the short-term vaccine efficacy study, with similar to muted responses observed in fish in the long-term vaccine efficacy study. This study provides insight on the efficacy and molecular mechanisms of protection of two commercial salmonid vaccines in Arctic charr during infections with atypical A. salmonicida.

In Atlantic Canada, Moritella viscosa is often cultured from Atlantic salmon skin ulcers in the summer and autumn months when water temperature is > 10 °C. The objectives of our study were to: 1) describe the development of skin ulcers associated with an Atlantic Canadian M. viscosa isolate; and 2) determine if this isolate could be transmitted horizontally between fish via water under laboratory conditions. Atlantic salmon in salt water were bath-immersed with M. viscosa (5.6 × 106 cfu ml-1) at 10.9 ± 0.2 °C. After observing skin lesions, water from each bath-challenge tank was diverted to a “delayed-challenge” tank with previously unexposed fish. The initial bath-challenge fish expressed clinical signs of skin ulcers within four days of exposure. Fish that were not sacrificed during our trial fully recovered by 26 days post-challenge. No clinical signs of disease were observed in fish from the delayed-challenge tanks. Our results suggest skin lesions induced by an M. viscosa isolate from Atlantic Canada have similar pathology to winter ulcer disease described in Europe. Most bath-challenged fish were not systemically infected until the development of visible skin ulcerations. None of the delayed-challenge fish were infected with M. viscosa, which suggests horizontal transmission via the water was not sufficient to cause disease in our study.

The histozoic myxozoan parasite Kudoa thyrsites causes postmortem myoliquefaction and is responsible for economic losses to salmon aquaculture in the Pacific Northwest. Despite its importance, little is known about the host-parasite relationship, including the host response to infection. The present work sought to characterize the immune response in Atlantic salmon during infection, recovery, and reexposure to K. thyrsites . After exposure to infective seawater, infected and uninfected smolts were sampled three times over 4,275 degree-days. Histological analysis revealed infection severity decreased over time in exposed fish, while in controls there was no evidence of infection. Following a secondary exposure of all fish, severity of infection in the controls was similar to that measured in exposed fish at the first sampling time but was significantly reduced in reexposed fish, suggesting the acquisition of protective immunity. Using immunohistochemistry, we detected a population of MHIIβ + cells in infected muscle that followed a pattern of abundance concordant with parasite prevalence. Infiltration of these cells into infected myocytes preceded destruction of the plasmodium and dissemination of myxospores. Dual labeling indicated a majority of these cells were CD83 + /MHIIβ + . Using reverse transcription-quantitative PCR, we detected significant induction of cellular effectors, including macrophage/dendritic cells ( mhii / cd83 / mcsf ), B cells ( igm / igt ), and cytotoxic T cells ( cd8 / nkl ), in the musculature of infected fish. These data support a role for cellular effectors such as antigen-presenting cells (monocyte/macrophage and dendritic cells) along with B and T cells in the acquired protective immune response of Atlantic salmon against K. thyrsites .

Sea lice (Lepeophtheirus salmonis) are ectoparasitic copepods that impose a heavy financial burden on the salmon aquaculture industry. A parasiticide, emamectin benzoate (EMB; trade name SLICE®), was widely used to control sea lice before EMB resistant strains of lice emerged. Several genetic mechanisms are likely responsible for EMB resistance however these are not yet fully understood. Resistance is further complicated by sex differences in EMB tolerance with males often showing better survival upon EMB exposure compared to females. Here, candidate EMB-resistance genes were used to explore differences between sex, population, and exposure to EMB using two in vitro bioassays and an in vivo experiment. Two acetylcholine receptor subunits (nAChRα3 and nAChRα7) showed opposite expression profiles across the assays, with male and EMB-resistant lice showing significant overexpression of nAChRα7 and downregulation of nAChRα3 compared to females and EMB-sensitive lice, respectively. Furthermore, a novel gene candidate LR9 showed induced expression upon EMB exposure with the highest expressing group being EMB-resistant males. An ABC transporter, pgp, also showed highest expression in EMB-resistant males but this finding was not consistent across all experiments. Other gene candidates like CYP18A and peroxinectin did not show similar expression profiles to work completed on other populations or species of sea lice. These data have provided a consensus with other transcriptomic studies showing that neuronal acetylcholine receptor subunits are differentially regulated between sexes and populations of sea lice. This unique expression profile, alongside analysis of other EMB resistance genes, provides a detailed snapshot of mechanisms responsible for resistance in sea lice. Statement of relevance Sea lice are a major threat to salmon aquaculture and its' sustainability. With the advent of drug resistance, this problem has only become more troubling. The mechanisms of resistance are largely unknown, with a requirement on consensus findings across meta-populations of lice due to varying levels of resistance within them. This project shows consensus findings across multiple experiments for both previously explored targets as well as new, potential resistance markers.

Fish expend significant amounts of energy to handle the numerous potentially stressful biotic and abiotic factors that they commonly encounter in aquatic environments. This universal requirement for energy singularizes mitochondria, the primary cellular energy transformers, as fundamental drivers of responses to environmental change. Our study probed the interacting effects of thermal stress, hypoxia-reoxygenation (HRO) and copper (Cu) exposure in rainbow trout to test the prediction that they act jointly to impair mitochondrial function. Rainbow trout were acclimated to 11 (controls) or 20°C for 2 months. Liver mitochondria were then isolated and their responses in vitro to Cu (0-20μM) without and with HRO were assessed. Sequential inhibition and activation of mitochondrial electron transport system (ETS) enzyme complexes permitted the measurement of respiratory activities supported by complex I-IV (CI-IV) in one run. The results showed that warm acclimation reduced fish and liver weights but increased mitochondrial protein indicating impairment of energy metabolism, increased synthesis of defense proteins and/or reduced liver water content. Whereas acute rise (11→20°C) in temperature increased mitochondrial oxidation rates supported by CI-IV, warm acclimation reduced the maximal (state 3) and increased the basal (state 4) respiration leading to global uncoupling of oxidative phosphorylation (OXPHOS). HRO profoundly inhibited both maximal and basal respiration rates supported by CI-IV, reduced RCR for all except CII and lowered CI:CII respiration ratio, an indication of decreased OXPHOS efficiency. The effects of Cu were less pronounced but more variable and included inhibition of CII-IV maximal respiration rates and stimulation of both CI and CIII basal respiration rates. Surprisingly, only CII and CIII indices exhibited significant 3-way interactions whereas 2-way interactions of acclimation either with Cu or HRO were portrayed mostly by CIV, and those of HRO and Cu were most common in CI and II respiratory indices. Our study suggests that warm acclimation blunts sensitivity of the ETS to temperature rise and that HRO and warm acclimation impose mitochondrial changes that sensitize the ETS to Cu. Overall, our study highlights the significance of the ETS in mitochondrial bioenergetic dysfunction caused by thermal stress, HRO and Cu exposure.

Inshore winter flounder ( Pseudoplueronectes americanus ) populations in NY, USA have reached record low numbers in recent years, and recruitment into the fishery appears to be limited by survival of post-settlement juvenile fish. In order to identify cellular pathways associated with site-specific variation in condition and mortality, we examined differential mRNA expression in juvenile winter flounder collected from six different bays across a gradient in human population density and sewage inputs. Illumina sequencing of pooled samples of flounder from contrasting degraded sites and less impacted sites was used to guide our choice of targets for qPCR analysis. 253 transcripts of > 100 bp were differentially expressed, with 60% showing strong homology to mostly teleost sequences within the NCBI database. Based on these data, transcripts representing nine genes of interest associated with contaminant exposure, immune response and glucose and glycogen metabolism were examined by qPCR in individual flounder from each site. Statistically significant site-specific differences were observed in expression of all but one gene, although patterns in expression were complex with only one (vitellogenin), demonstrating a west to east gradient consistent with known loadings of municipal sewage effluent. Principal components analysis (PCA) identified relationships among the genes evaluated. Our data indicate that juvenile winter flounder are responding to estrogenic chemicals in more urbanized coastal bays, and suggests potential mechanistic links between immune response, contaminant exposure and energy metabolism.

pleurocidin was significantly higher in fin than liver, but was highly variable among individuals. Some statistically significant differences in pleurocidin expression among sites were observed, although elevated levels were not associated with degree of urbanization. Expression was related in part to fish size: a positive correlation between expression and total length ( LT) of fish was observed with the largest LT class (125 mm) exhibiting significantly elevatedpleurocidin expression as compared with fish in the smaller LT class. This indicates that immune competency may increase with age. No site-specific differences in cyp1a expression were observed. These data suggest that exposure to aromatic hydrocarbon contaminants is fairly widespread throughout the study area and that any differences in pleurocidin expression in YOY P. americanus are probably due to other factors. Antimicrobial activity was also measured as a functional indicator of immune response. Activity was highly variable, showing no significant site-specific differences, and no significant correlation to pleurocidin expression. The lack of correlation between pleurocidin expression and antimicrobial activity indicates that other antimicrobial peptides may be active against the bacteria tested or that other factors are influencing antimicrobial activity. This is the first quantitative evaluation of pleurocidin expression in YOY P. americanus from an urban area. Further work is needed to characterize factors controlling pleurocidin expression, as well as other indicators of immune response in young fish.

Antiparasitic drugs such as emamectin benzoate (EMB) are relied upon to reduce the parasite load, particularly of the sea louse Lepeophtheirus salmonis, on farmed salmon. The decline in EMB treatment efficacy for this purpose is an important issue for salmon producers around the world, and particularly for those in the Atlantic Ocean where widespread EMB tolerance in sea lice is recognized as a significant problem. Salmon farms in the Northeast Pacific Ocean have not historically experienced the same issues with treatment efficacy, possibly due to the relatively large population of endemic salmonid hosts that serve to both redistribute surviving lice and dilute populations potentially under selection by introducing naive lice to farms. Frequent migration of lice among farmed and wild hosts should limit the effect of farm-specific selection pressures on changes to the overall allele frequencies of sea lice in the Pacific Ocean. A previous study using microsatellites examined L. salmonis oncorhynchi from 10 Pacific locations from wild and farmed hosts and found no population structure. Recently however, a farm population of sea lice was detected where EMB bioassay exposure tolerance was abnormally elevated. In response, we have developed a Pacific louse draft genome that complements the previously-released Atlantic louse sequence. These genomes were combined with whole-genome re-sequencing data to design a highly sensitive 201,279 marker SNP array applicable for both subspecies (90,827 validated Pacific loci; 153,569 validated Atlantic loci). Notably, kmer spectrum analysis of the re-sequenced samples indicated that Pacific lice exhibit a large within-individual heterozygosity rate (average of 1 in every 72 bases) that is markedly higher than that of Atlantic individuals (1 in every 173 bases). The SNP chip was used to produce a high-density map for Atlantic sea louse linkage group 5 that was previously shown to be associated with EMB tolerance in Atlantic lice. Additionally, 478 Pacific louse samples from farmed and wild hosts obtained between 2005 and 2014 were also genotyped on the array. Clustering analysis allowed us to detect the apparent emergence of an otherwise rare genotype at a high frequency among the lice collected from two farms in 2013 that had reported elevated EMB tolerance. This genotype was not observed in louse samples collected from the same farm in 2010, nor in any lice sampled from other locations prior to 2013. However, this genotype was detected at low frequencies in louse samples from farms in two locations reporting elevated EMB tolerance in 2014. These results suggest that a rare genotype present in Pacific lice may be locally expanded in farms after EMB treatment. Supporting this hypothesis, 437 SNPs associated with this genotype were found to be in a region of linkage group 5 that overlaps the region associated with EMB resistance in Atlantic lice. Finally, five of the top diagnostic SNPs within this region were used to screen lice that had been subjected to an EMB survival assay, revealing a significant association between these SNPs and EMB treatment outcome. To our knowledge this work is the first report to identify a genetic link to altered EMB efficacy in L. salmonis in the Pacific Ocean.